International Review of Cytology, Vol. 41 0123643414, 9780123643414, 9780080585789

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INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME41

ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN W. BERNHARD GARY G. BORISY ROBERT W. BRIGGS R. COUTEAUX

B. DAVIS N. B. EVERETT DON FAWCETT MICHAEL FELDMAN WINFRID KRONE K. KUROSUMI MARIAN0 LA VIA

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GIUSEPPE MILLONIG MONTROSE J. MOSES ANDREAS OKSCHE VLADIMIR R. PANTIC LIONEL I. REBHUN JEAN PAUL REVEL WILFRED STEIN ELTON STUBBLEFIELD H. SWIFT J. B. THOMAS TADASHI UTAKOJI ROY WIDDUS A. L. YUDIN

INTERNATIONAL

Review of Cytology

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EDITED BY

G. H. BOURNE

J. F. DANIELLI

Yerkes Regional Primate Research Center Emo y University Atlanta, Georgia

Center f o r Theoretical Biology State University of New York a t Buflalo Buffalo, New York

ASSISTANT EDITOR K. W. JEON Department of Zoology University of Tennessee Knoxville, Tennessee

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VOLUME41

ACADEMIC PRESS New York San Francisco London 1975 A Subsidiary of Harcourt Brace ]ovanovich, Publishers

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COPYRIGHT 0 1975. BY ACADEMIC PRESS,INC.

ALL RIGHTS RESERVED. N O PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

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111 Fifth Avenue,New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London N W l

LIBRARY OF CONGRESS CATALOG CARD NUMBER: 52-5203 ISBN 0-12-364341 -4 PRINTED IN T H E UNITED STATES OF AMERICA

zyx zyxwvutsrq zyxwvut Contents

LIST OF CONTRIBUTORS .

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ix

The Attachment of the Bacterial Chromosome to the Cell Membrane PAUL

J . LEIROWITZAND MOSELIO SCHAECHTEH

. . . . . . . . . . . . I . Introduction . . . . . . . . . I1 . Morphological Considerations . . . . . 111. The Mode of Segregation of the Cell Membrane . . . . . IV . Polarization in the Segregation of Chromosomes . . . . . V . Methodological Considerations of Cell Fractionation VI . Is the Chromosome Attached to a Special Region of the Membrane? . . VII . Is the Origin of DNA Replication Attached to the Membrane? . . . . VIII Does DNA Replication Take Place on the Membrane? . . . . . . IX . Is the Chromosome Attached at Many Sites? . . X The Nature of the Binding of the Chromosome to the Membrane . XI The Role of the Membrane in Maintaining the Compactness of the . . . . . . . . . . . . Nucleoid . XI1 . Conclusions . . . . . . . . . . . . . References . . . . . . . . . . . . .

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Regulation of the Lactose Operon in Escherichia coli by CAMP G . CARPENTERAND B . H . SELLS

. . . . . . I . Introduction. I1 CAMP Formation and Catabolite Repression 111. In Vioo Evidence of Site of cAMP Action . . . . IV cAMP Action in Vitro . V Additional Aspects of Lac Operon Regulation References . . . . . . .

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Regulation of Microtubules in Tetrahymena

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NORMAN E WILLIAMS

I . Introduction.

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I1. Regulatory Patterns and the Cell Cycle .

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. 111. Microtubule Stability and Regression IV. The Dynamic Nature of Formed Microtubules . . V . Control of Microtubule Formation . . .

VI * Epilog: The Cell Cycle Revisited . . . . References . V

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59 60 68 74 77 83 84

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Cellular Receptors and Mechanisms of Action of Steroid Hormones SHUTSUNG LIAO

I . Introduction.

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I1. Steroid-Binding Proteins in Blood

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111 IV. Cytoplasmic-Nuclear Interaction of Steroid Receptors

Gene Expression and Steroid Receptor Concluding Remarks . . . . References . . . . . .

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87 90 92 127 139 151 157

173 177 205 234 235

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A Cell Culture Approach to the Study of Anterior Pituitary Cells A . TIXIEH.VIDAL. D . GOURDJI. AND c . TOUGAHD I . Introduction .

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I1. Characteristics of Anterior Pituitary Cells Grown in Vitro

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Reactivity to Specific Regulating Agents

IV. Conclusion . References

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Immunohistochemical Demonstration of Neurophysin in the Hypothalamoneurohypophysial System

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W B . WATKINS

. . . . . . . . . . . . I . Introduction . I1 . Relationship between Neurosecretory Material and Neurophysin . . . . . . . . . 111. Methods of Extraction of Neurophysin . . . . . . . IV. Purification of Neurophysin Antigens V. Production of Antibodies against Neurophysin . . . . . . VI . General Considerations of Antibody Production and Detection . . . . . . . . VII . Immunohistochemical Techniques . VIII . Demonstration of Neurophysin in the Hypothalamoneurohypophysial System Using Cross-Species Reactive Antineurophysin . . . . Ix. Use of Species-Specific Antisera for the Demonstration of Neurophysin . . . . . . . . . . . . . X . Conclusions . References . . . . . . . . . . . . .

241 243 244 246 250 255 256 260 279 280 28 1

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CONTENTS

The Visual System of the Horseshoe Crab

Limulus polyphemus

WOLF H . FAHRENBACH

I. I1 111. IV V VI VII VIII . IX

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Introduction. . . . . Dioptric Structures . . Pigment Cells . . . Neuroglial Cells . . Receptor Cells . . . Basal Lamina and Hemoc:eel Axons and Plexus . . Optic Nerves . . . Optic Centers . . . Miscellaneous Aspects . Vision and Behavior . References . . . .

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SUBJECT INDEX . . . . . CONTENTS OF PREVIOUS VOLUMES .

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285 287 296 304 306 318 321 333 335 338 341 344

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zyxwv zyxwvu zyxwvut zyxwvu List of Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

G. CARPENTER* (29), Laboratories of Molecular Biology, Faculty of Medicine, Memorial University of Newfoundland, S t . John’s, Newfoundland, Canada

WOLF H. FAHRENBACH (285), Laboratory of Electron Microscopy, Oregon Regional Primate Research Center, Beaverton, Oregon

D. GOURDJI(173), Groupe de Neuroendocrinologie Cellulaire, Laboratoire de Physiologie Cellulaire, Collbge d e France, Paris, France PAULJ. LEIBOWITZ (l),Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts

SHUTSUNG LIAO (87), The Ben May Laboratory for Cancer Research and the Department of Biochemistry, T h e University of Chicago, Chicago, I11inois

MOSELIO SCHAECHTER(l),Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts B. H. SELLS(29), Laboratories of Molecular Biology, Faculty of Medicine, Memorial University of Newfoundland, S t . John’s, Newfoundland, Canada

C. TOUGARD (173), Groupe d e Neuroendocrinologie Cellulaire, Laboratoire de Physiologie Cellulaire, Collhge de France, Paris, France A. TIXIER-VIDAL (173), Groupe de Neuroendocrinologie Cellulaire,

Laboratoire de Physiologie Cellulaire, Collhge de France, Paris, France

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W. B. WATKINS (241), Postgraduate School of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand NORMAN E. WILLIAMS (59), Department

Iowa City, Iowa

of Zoology, University of Zowa,

’ Present address: Department of Biochemistry, Vanderbilt University, Nashville, Tennessee. ix

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The Attachment of the Bacterial Chromosome to the Cell Membrane

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PAUL J. LEIBOWITZAND MOSELIO SCHAECHTER Department of Molecular Biology and Microbiology, TUBSUniversity School of Medicine, Boston, Massachusetts

. . . . . . . The Nucleoid . . . . . The Membrane . . . .

I. Introduction

11. Morphological Considerations

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IV. V. VI. VII. VIII. IX. X. XI.

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A. B. C. Morphological Evidence for the Association between Nucleoids and the Membrane . . . . . . . The Mode of Segregation of the Cell Membrane. . . . Polarization in the Segregation of Chromosomes . . , Methodological Considerations of Cell Fractionation . . Is the Chromosome Attached to a Special Region of the Membrane? . . . . . . . . . . . . Is the Origin of DNA Replication Attached to the Membrane? . . . . . . . . . . . . . Does DNA Replication Take Place on the Membrane?. . Is the Chromosome Attached at Many Sites? . . . . The Nature of the Binding of the Chromosome to the Membrane . . . . . . . . . . . . . The Role of the Membrane in Maintaining the Compactness of the Nucleoid . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . References . . . . . . . . . . . . .

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I. Introduction

Prokaryotic cells are small, do not contain organelles limited by independent membrane systems and, instead of a complex nucleus, possess a pleomorphic central region of condensed DNA, the nucleoid. Nucleoids do not divide by mitosis and, at least in Escherichia coli, consist of single chromosomes. The DNA is in the form of a circular duplex molecule, and its replication takes place in both directions from a distinct starting point. Bacteria have the ability to respond to changes in environmental conditions by grossly altering their size and macromolecular composition. Thus, as a response to changes in the kind of nutrients provided, growing bacteria may vary in size or in RNA content by a factor of 10 or more. They change from one physiological state to another in a remarkably efficient and rapid manner. A review of 1

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P. J. LEIBOWITZ AND M. SCHAECHTER

these attributes appears in a book by Maalfle and Kjeldgaard (1966). Bacteria are efficient and structurally simple; consequently they make multiple uses of cellular structures. In this vein, the synthesis and regulation of several macromolecules have been shown to be related to the behavior of the cell membrane. The subject of this article is one of these relationships, the connection of the bacterial chromosome with the cell membrane. Concern for this subject originated with a proposal by Jacob, Brenner, and Cuzin for the control of DNA replication in bacteria. They formally termed the unit of DNA replication the replicon, and proposed that initiation of replication is controlled by diffusible gene products (Jacob et al., 1963; Jacob and Brenner, 1963): A structural gene produces an initiator which acts upon a region of the chromosome at a specific site, the origin. Replication begins at the origin and proceeds linearly until the entire chromosome has been duplicated. Included in the model is the proposition that the chromosome is attached to the bacterial membrane. The model suggests that the DNA-synthesizing complex is fixed to the bacterial membrane and that the DNA moves through this complex. The membrane is thought to provide a mechanism for the segregation of the daughter replicons by growth of the cell surface between their sites of attachment. The bacterial membrane would thereby perform the function of the mitotic apparatus of higher organisms, as well as being the site of DNA synthesis. This model predicts the existence of membrane componer,ts that recognize specific sites on the chromosome. The replicon model stimulated a search for the association between the bacterial chromosome and the cell membrane. In this article we present morphological, genetic, and biochemical evidence for this association. We attempt to provide tentative state-of-the-art answers to the following questions: (1) Is DNA attached to the membrane? (2) If so, at how many sites? (3) If there are several sites, are they alike in function? (4) Is attachment possible along any region of the genome? ( 5 ) Is the membrane unique at the site of attachment? Answers to these questions are tentative, because the necessary methodology is in an early state of development. In very few cases have findings been confirmed by unrelated techniques. Even more difficult are the following questions: (6) Do all attachment points exist at the same time? (7)At what time in the cell cycle are attachment points born? (8) Do attachment points remain at their site of birth? Many researchers have interpreted their data in terms of a connection between the bacterial chromosome and the cytoplasmic mem-

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BACTERIAL CHROMOSOME AND CELL MEMBRANE

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brane. We feel that much of this work is only tangentially related to the subject of this article. For this reason we have selected evidence that comes closest to providing answers to the questions listed above, and which we feel deals directly with the issue of whether or not attachment exists. In addition, we have omitted work that is relevant to this field but which appears to lie beyond the conceptual framework of this article. The special topic of the attachment of bacteriophage DNA to the bacterial cell membrane has been reviewed recently (Siege1 and Schaechter, 1973).

zyx zyxwvu 11. Morphological Considerations

A. THE NUCLEOID

Perhaps the most convincing proof for the existence of nucleoids comes from observations of living E. coli with the phase-contrast microscope. Nucleoids can be seen when cells are grown in media of high refractive index. In time-lapse motion pictures nucleoids are first seen to change in conformation as they divide, and then to segregate into daughter cells prior to the completion of cell division (Adler et al., 1969). There has never been any demonstration of a membrane separating nucleoids from the cytoplasm, nor is there evidence of any of the elements of a mitotic apparatus. The DNA of E. coli consists of a single circular duplex molecule about 1100 pm in length (Cairns, 1963). Since the apparent volume of the nucleoid of this cell is about 0.1 pm3, it follows that the chromosome must be folded on itself and exist in a phase state quite unlike that of DNA in solution. Although this represents a very high concentration of DNA, the nucleoid is considerably less dense than its surrounding cytoplasm. The degree of condensation of the nucleoid observed in the electron microscope varies with the method of fixation. Freeze-etched preparations of unfixed bacteria reveal no clear distinction between the nucleoid and cytoplasm, but typical nucleoids are seen occasionally if cells are fixed with osmium tetroxide (Nanninga, 1968). The Ryter-Kellenberger (R-K) procedure is currently the most widely used fixation method for ultrathin sectioning of bacteria. It employs osmium tetroxide for both prefixation and fixation. With this fixation nucleoids are most frequently seen in the central portion of the cell, and they show bundles of fibers with dimensions similar to those of DNA (Kellenberger et al., 1958). Prefixation in glutaraldehyde followed by fixation with osmium tetroxide (G-0 fixa-

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tion) causes the nucleoplasm to appear in a dispersed configuration rather than as a centrally located body (Margaretten et al., 1966; McCandless et al., 1968). Recent work by M. L. Higgins and L. Daneo-Moore (personal communication) suggests that degradation of RNA, which takes place during R-K but not G - 0 fixation, may lead to further condensation of the nucleoids. Despite the uncertainties introduced by these studies, R-K fixation results in sections which conform to what is expected from studies on living bacteria with the phase microscope.

B. THE MEMBRANE The cytoplasm of bacteria is bounded by a trilaminar membrane, about 8-10 nm thick. Outside this membrane is the cell wall and outside it, in gram negative cells, is a membranelike outer layer. The inner membrane has intracytoplasmic involutions termed mesosomes, which vary in complexity among taxonomic groups (FitzJames, 1960; van Iterson, 1961; Glauert et al., 1961; Glauert, 1962).A comprehensive review of these structures has recently appeared (Reusch and Berger, 1973). The mesosomes of gram-positive bacteria appear as extensions of the cytoplasmic membrane forming saclike structures (outer mesosomal membranes) filled with vesicles, tubules, and/or lamellae (internal mesosomal membranes) (e.g., Bacilli. Ryter and Jacob, 1966, van Iterson, 1961, 1965, Fitz-James, 1960; Holt and Leadbetter, 1969; Listeria monocytogenes: Edwards and Stevens, 1963; Mycobacteria: Imaeda and Ogura, 1963; Streptomyces: Glauert, 1962). These internal structures are considered in turn to be invaginations of the sac, or the outer mesosomal membrane (Fitz-James, 1960; Ryter and Jacob, 1966). Mesosomes are also found in gram-negative bacteria (E. coli: Kaye and Chapman, 1963; Steed and Murray, 1966; Pseudomonas aemginosa: H o h a n n et al., 1973; Spirillum serpens: Steed and Murray, 1966; Caulobacter: Stove Poindexter and Cohen-Bazire, 1964). Ultrathin sections usually reveal that these mesosomes are uncomplicated structures, most often containing lamellae which apparently result from delicate foldings of the plasma membrane. Mesosomes in gram-negative bacteria are probably devoid of tubules. As with gram-positive bacteria, there is variation in structure among taxonomic groups. There is disagreement on the true morphology of mesosomes and on their number and location in the cell (Remsen, 1968; Nanninga, 1968; Highton, 1969, 1970a,b; Burdett and Rogers, 1970; Rogers, 1970). It must be emphasized that the morphology of the mesosome

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BACTERIAL CHROMOSOME AND CELL MEMBRANE

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is altered markedly by the conditions of fixation (e.g., Burdett and Rogers, 1970). In ultrathin sections mesosomes are seen to be touching the nucleoids of dividing cells, and to be continuous with division septa. There is evidence to implicate these structures in DNA replication (Higgins and Daneo-Moore, 1972), in the segregation of chromosomes (Ryter and Jacob, 1963), in the location of membrane and cross-wall synthesis and prespore septation (Ellar et al., 1967; Steed and Murray, 1966; Chapman and Hillier, 1953; Fitz-James, 1960, 1967; Freese, 1973), in subcellular degradative activities (lysosomal functions) (Reusch and Berger, 1972), and in oxidative function (van Iterson and Leene, 1964; Ferrandes et al., 1966). The morphological development of mesosomes was followed in synchromously dividing Bacillus megateriiurn by Ellar et al. (1967). Mesosomes develop by an initial concentric infolding of the cytoplasmic membrane and eventually assume a saclike shape. Cross wall formation begins at the base of these mesosomes which are located at the center of the cells. This implicates them in the initiation of cross wall synthesis. Later, the mesosome is seen on both sides of the developing cross wall, which suggests that it is also involved in the synthesis of cross walls. These central mesosomes are often associated with nucleoids, as are other mesosomes located at the poles. From these morphological considerations it seems likely that mesosomes are responsible for thickening of the cell wall prior to cell separation, and for initiation and synthesis of the cross wall. This has not yet been borne out by fractionation studies, since mesosomes have been found not to be particularly rich in enzymes and precursors involved in membrane or wall synthesis (Patch and Landman, 1971; Reusch and Berger, 1972). However, Nanninga (1968) showed differences in the freeze-etched surface structure of mesosomes and cytoplasmic membranes and concluded that mesosomes may in fact differ from the rest of the cytoplasmic membrane. This subject has been reviewed by Reusch and Berger (1973).

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MORPHOLOGICALEVIDENCEFOR THE ASSOCIATION BETWEEN NUCLEOIDSAND THE MEMBRANE In ultrathin sections the nucleoid is located in a central region of the cell and is not in obvious contact with the peripheral membrane. For this reason the morphological association between them escaped detection for many years. Upon closer examination the nuclear regions and mesosomes of both gram-positive and gram-negative

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FIG. 1. Ultrathin sections of growing B . subtilis showing the association of mesosomes (M)with nucleoids (N). Fixation by the R-K method. (From Jacob et al., 1966, reproduced with permission from the publishers, The Royal Society, and the authors.)

BACTERIAL CHROMOSOME AND CELL MEMBRANE

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FIG. 2. Ultrathin section of a mesosome in B. subtilis after prefixation with the G O method. x90,OOO. (From Ryter, 1968, reproduced with permission from the publishers, The American Society for Microbiology, and the author.)

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FIG. 3. Mesosomes (M) of E . coli, which appear as delicate folds of membrane in contact with the bacterial nucleoid. X85,600. (From Ryter and Jacob, 1966, reproduced with permission from the publishers, Masson et Cie., Editeurs, and the authors.)

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cells can nearly always be shown to be touching (see Figs. 1-3). They usually have considerable surface contact and often penetrate one another (van Iterson, 1961; Ryter and Jacob, 1964, 1966; Ellar et al., 1967; Pontefract et al., 1969; Remsen, 1968; Hoffmann et al., 1973). The contact is less dramatic in most gram-negative cells, because their mesosomes are smaller. In fact, in a mutant of E. coli which forms extensive intracytoplasmic membranes, the contact of the DNA with these membranes is readily evident (Altenburg and Suit, 1970; Altenberg et al., 1970). Offhand it should not be surprising to find that the pleomorphic nuclear region occasionally makes contact with the mesosome. If the association were fortuitous, however, one would expect great disparity among individual cells. Ryter and Jacob (1964) determined that the nucleoid and mesosomes of Bacillus subtilis were visibly linked in each of 20 serially sectioned cells which included all stages of the cell cycle. The nucleoid was associated with either one or two mesosomes, depending on the stage of growth (Ryter, 1968). Smaller nucleoids appeared to be attached to one mesosome, while larger ones were often attached to two. Consequently, it was possible to arrange the three-dimensional constructs in an order thought to reflect the cell cycle, Initially, the two nucleoids in each cell are seen attached to two separate mesosomes; as the chromosome replicates, mesosomes seem to split in two, each maintaining contact with one of the two newly formed nucleoids; segregation of nucleoids is accomplished by the growth of the membrane between them; after this segregation process begins, the cell septum starts to form. There is considerable disagreement with this model, at least in its simplest form. Several investigators have found that mesosomes do not arise by division but are formed de n o w at the site of septum formation. This was reported for B . rnegaterium by Ellar et al. (1967), Streptococcus faecalis by Higgins and Shockman (1970a, 1971), and E. coli by Pontefract et al. (1969). There is evidence that the nucleoid is always associated both with a polar mesosome and with the newly synthesized, septa1 mesosome (Ellar et al., 1967; Pontefract et al., 1969). Mesosomes that form at septa become polar mesosomes in daughter cells. Since two polar mesosomes within one cell arise during different cell division cycles, it should be possible in future work to distinguish between an old segregation apparatus and a new one. There are also indications that nucleoids may not always be associated with mesosomes. For instance, Highton (1970b)found that

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multinucleated B . subtilis cells contain fewer mesosomes than nucleoids. There is evidence that in s.faecalis mesosomes may not participate in nucleoid segregation. This spherical bacterium has an equatorial band on the external surface of the wall which marks the site of new cross wall synthesis and the boundary between old and new wall. Upon initiation of wall synthesis these bands split, double in number, and move to a subequatorial position. Each daughter cell has an equatorial band from the preceding generation which marks the initiation site of wall growth for that generation. Mesosomes are usually seen just beneath an equatorial wall band on the cell surface, and are attached to the base of the septal membrane by a membranous stalk (Higgins and Shockman, 1970b, 1971; see Fig. 4). Mesosomes located near the septum are most often seen penetrating the nuclear mass. Mesosome formation precedes cross wall formation. The mesosome appears to maintain direct contact with the septum only during the early part of the cell cycle. The septal connection is lost prior to the completion of the cross wall at the time the nucleoid appears to have divided into two masses. Two new mesosomes are now found beneath wall bands in the developing daughter cell (Higgins and Shockman, 1971; Higgins and DaneoMoore, 1972). The effect of selective inhibition of DNA, RNA, and protein synthesis on the development of mesosomes was studied in S. faecalis by Higgins and Daneo-Moore (1972). It has been shown that the cross-sectional area of mesosomes increases rapidly during amino acid starvation (Higgins and Shockman, 1970b). These authors proposed that the increase in mesosome size might be related to continued DNA synthesis since, during amino acid starvation, RNA synthesis is shut off and the rate of protein synthesis decreases (Ziegler and Daneo-Moore, 1971).They suggest that the termination of DNA replication might result in activation of the regions of the envelope involved in segregation to form a site for the formation of a new mesosome. They suggest that mesosomes in this organism are necessary for the initiation of the cross wall and for DNA replication, but not for cross wall formation or nuclear segregation. Segregation would take place through direct attachment to the cytoplasmic membrane. It is not known if these discrepancies in the behavior of mesosomes are due to differences among various species of bacteria. It is likely that they are due to a combination of many factors, including fixation artifacts and differences in the physiological state of the

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Wall Band

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62 New Wall Synthesis

FIG.4. Diagrammatic representation of the cell division cycle for Streptococcus faecalis. The model proposes that linear wall elongation is a unitary process which results from wall synthetic activity at the leading edges of the nascent cross wall. The diplococcus in A is in the process of growing new wall at its cross wall and segregating its nuclear material to the two nascent daughter cocci. In rapidly growing exponential phase cultures before completion of the central cross wall, new sites of wall elongation are established at the equators of each of the daughter cells at the junction of old, polar wall (stippled) and new equatorial wall beneath a band of wall material that encircles the equator (B).Beneath each band a mesosome is formed while the nucleoids separate and the mesosome at the central site is lost. The mesosome appears to be attached to the plasma membrane by a thin membranous stalk (BI). Invagination of the septa1 membrane appears to be accompanied by centripetal cross wall penetration (BZ).A notch is then formed at the base of the nascent cross wall which creates two new wall bands (B3). Wall elongation at the base of the cross wall pushes newly made wall outward. At the base of the cross wall, the new wall peels apart into peripheral wall, pushing the wall bands apart (B4). When sufficient new wall is made so that the wall bands are pushed to a subequatorial position (e.g., from C to A to B) a new cross wall cycle is initiated. Meanwhile the initial cross wall centripetally penetrates into

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cells. We feel that precise knowledge of the number and location of mesosomes awaits a detailed analysis of synchronously dividing cells growing at different rates. Linkage of the nucleoid to mesosomes has been reported to persist throughout the first stages of sporulation (Ryter and Jacob, 1964). In the first stage the contact appears to be mediated through one of the polar mesosomes which eventually participates in the development of the spore membrane. Later, the nucleoid migrates to a peripheral position in the spore cytoplasm, the mesosome disappears, and vesicles suggestive of mesosome tubules are found along the spore membrane. In the last stage of sporulation, the nucleoid is connected directly to the spore membrane. It is not clear if this represents the initial contact between the nucleoid and mesosome seen in the first stage of sporulation, or if a new contact point is formed. The sequence of events in nuclear division has also been studied in spore germination (Ryter, 1967). Early in germination of B. subtilis spores, the nucleoid assumes a central position, becomes an axial filament, and is connected to the spore membrane by a huge mesosome. Later, the number and size of mesosomes vary, and they are not always in contact with the nucleoid. Nonetheless, the nucleoid is linked to the membrane at two sites, either through mesosomes, by direct attachment to the membrane, or both. The distance between the attachment points increases as the cell elongates, but the distance from each attachment point to the pole of the cell seems to remain the same. This suggests that the membrane grows by the deposition of new material at the equator of the cell. Occasional sections reveal the presence of small nonmesosome structures in the membrane to which the nuclear fibrils are attached (Ryter, 1967). Mesosomes are evaginated when cells are plasmolyzed in hypertonic medium or when spheroplasts (wall-less or wall-deficient cells) are prepared. In such cases the nucleoid is found at the periphery of the cell, as if it had been dragged toward the cell surface by its attachment to the membrane (Ryter and Landman, 1964, 1967; Ryter and Jacob, 1964, 1966). One would expect that upon extrusion of the mesosome the chromosome would be linked to the portion of the membrane that was the cytoplasmic surface of the mesosome. In the cell, dividing it into two daughter cocci. At all times the body of the mesosome appears to be associated with the nucleoid. Doubling of the number of mesosomes seems to precede completion of the cross wall by a significant interval. Nucleoid shapes and the position of mesosomes are based on projections of reconstructions of serially sectioned cells. (From Higgins and Shockman, 1971, with permission of CRC Press, Inc., and by courtesy of the authors.)

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some sections of spheroplasts, the cytoplasmic membrane is markedly invaginated toward the nucleoid. In B . subtilis spheroplasts remnants of mesosome tubules are seen attached to the outer surface of the membrane in contact with nuclear fibrils (see Fig. 5). This suggests that the association is not fortuitous. Spheroplasts of B . subtilis do not contain mesosomes and cannot divide unless transferred to special reversion media. Here the protoplasm apparently divides without the intervention of mesosomes (Ryter and Landman, 1964). Direct contact of the nucleoid with the membrane persists throughout reversion, but is not readily observed in the newly reverted bacillary form. During reversion mesosomes are morphologically atypical. In these cells contact with the membrane appears to be mediated not through mesosomes, but through a small vesicular attachment apparatus similar to that found in germinating spores by Ryter (1967), Ryter and Landman (1967), and Landman et al. (1968).These investigators proposed that mesosomes are not required for reversion of spheroplasts and may not be essential for any cellular function, since their absence has no noticeable effect on DNA replication, chromosome segregation, or cell division. They point out the need for a thorough investigation of the small attachment apparatus often seen connecting the nucleoid to the membrane in the absence of obvious mesosomes.

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111. The Mode of Segregation of the Cell Membrane

We now consider how the membrane of bacteria “grows,” that is, where on its surface new material is deposited. It is important to consider the various models of membrane growth because each makes different predictions on the role of the membrane in chromosome segregation. If the cell membrane were synthesized solely at the equator of the cell, its lateral displacement would lead directly to segregation of chromosomes attached to it. Two newly replicated chromosomes would separate from each other by the intercalation of new membrane material between their points of attachment. If, however, the membrane is synthesized at many sites on its surface, its role in chromosome segregation would be either passive or more complicated. FIG.5. Protoplasts of B . subtilis showing remnants of mesosomal tubules (M) still attached to the outer surface of membrane. Nuclear fibrils are in contact with this portion of the membrane, believed to be the outer membrane of the mesosome prior to extrusion. Top, X80,OOO; bottom, X 120,000. (From Ryter and Jacob, 1966, reproduced with permission from the puhlishers,Masson et Cie., Editeurs, and the authors.)

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Only a few experiments have been reported that deal with this point, and they are not conclusive. There is only one report of a direct attempt to determine the site of synthesis by pulse-labeling cells with specific membrane precursors and determining the location of newly synthesized material by radioautography. These investigators found that newly made lipids were not localized at specific membrane sites (Mindich and Dales, 1972). Most of the relevant studies dealt with this question by determining the pattern of segregation of the membrane. They are based on the following reasoning. If there is one site of membrane synthesis and it is at the equator of the cell, then new and old membrane may be distributed among daughter cells in a semiconseruative pattern. Conversely, if there are many sites of synthesis, old and new membrane will be dispersed among progeny cells. Several investigators have used the fact that bacterial flagella are attached to the membrane to follow the pattern of membrane segregation. Thus Ryter (1971) used a mutant of B. subtilis which synthesizes flagella at 37°C but not at 46°C. When this mutant was shifted from 37" to 46"C, the old flagella were not distributed randomly among the daughter cells but followed the distribution expected from an equatorial deposition of new membrane material. This result differs from an earlier one by Quadling and Stocker (1962) obtained with Salmonella typhimurium. However, the assumption that the distribution of flagella reflects that of the membrane site to which they are connected may not be equally valid for both gram-positive and gram-negative bacteria. Analogous experiments were carried out by following the distribution of membrane lipids when cells labeled with specific precursors were grown in unlabeled medium. The distribution of label in individual cells was followed by density gradient centrifugation of membrane fragments (Wilson and Fox, 1971), or by radioautography of whole cells (Lin et al., 1971; Green and Schaechter, 1972). It was found that the label became dispersed over the progeny population, suggesting that new membrane was deposited at many sites. This interpretation is clouded, however, by the possibility that the membrane is sufficiently fluid to permit the rapid lateral movement of newly made pieces. The pattern of segregation would then not correspond to the pattern of membrane synthesis. One kind of membrane protein, the permease involved in the transport of P-galactosides, has been reported to segregate semiconseruatively (Kepes and Autissier, 1972).When E. coli is grown under conditions that prevent the synthesis of new permease, old permease

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molecules are not segregated randomly but are conserved in a few of the progeny cells. It is not clear at present whether the discrepancy between this result and those described above reflects differences in the behavior of various membrane constituents or in the methods employed.

IV.

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Polarization in the Segregation of Chromosomes

If the progeny of a single cell could be lined up in a row that reflects their genealogy, would the original DNA strands always be found in certain cells, or would they segregate randomly? In other words, is the segregation process polarized? This question is relevant to our concerns because polarization in segregation suggests that DNA is attached to the membrane. Perhaps the simplest mechanism to explain polarization is that some time after replication the parental strands of each daughter chromosome become permanently attached to two different sites on the membrane. The relevant experiments have been done by growing cells whose DNA had been previously labeled in unlabeled medium under conditions in which the genealogical relationships are spatially maintained. This can be achieved by growth on agar in the presence of methylcellulose which restrains cell movement so that progeny cells are maintained as chains. The position of original parental DNA can then be determined by radioautography. Early work done by this or by analogous methods gave contradictory evidence. Thus Chai and Lark (1967, 1970) and Eberle and Lark (1966)found that the pattern of chromosome segregation was nonrandom in Lactobacillus acidophilus, B . subtilis, and E . coli. Ryter et al. (1968),Ryter (1968),and Lin et al. (1971)found the opposite, that the parental DNA was dispersed randomly over the progeny cells. To a large extent this discrepancy may be attributed to a physiological peculiarity of rodshaped bacteria, namely, that the number of nuclei per rod is dependent on the growth rate (e.g., Maalge and Kjelgaard, 1966). Moreover, the number of points on each chromosome where replication takes place also varies with the growth rate. Thus the number of “units of DNA conservation” in the original cell, that is, the number of labeled individual DNA strands, varies from a minimum of 2 at very slow growth rates, to 8 or 16 at fast growth rates. A high number of labeled strands in the original cell obscures segregation data, making the analysis of such experiments intricate and laborious. Perhaps the most thorough study of this point has been that of Pierucci and Zuchowski (1973),who reported that chromosome seg-

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regation is indeed not random. They studied E. coli B/r grown at slow growth rates, Experimental and theoretical distribution patterns were determined for the location of labeled cells in chains. They found that all cells became unlabeled with unequal frequency, and concluded that segregation is polarized. They considered various models in detail, and the data appear to fit best the following. Upon replication the newly made strand does not immediately attach to the segregation structure, but becomes stably bound at the time it is first used as a template, namely, one round of replication later. Furthermore, only one strand of the duplex is capable of attachment. This conclusion differs from that of Lark and co-workers, who proposed that either strand of the duplex is capable of attachment to the membrane the first time it is used as a template. These data obtained by Pierucci and Zuchowski may fit other models of segregation not yet conceived, and it is not clear to us if the fit of their data is sufficiently good to be taken as proof of any one model. In a particularly interesting experiment, Chai and Lark (1967) determined that the segregation of the DNA of L. acidophilus is coupled to the segregation of the envelope of the cell. They did this by exposing the cells to tritiated thymidine and to fluorescent antibody made against the cell envelope. Upon subsequent growth in medium not containing either of these markers, they determined the proportion of cells containing both original DNA and original envelope material. The fraction of such cells was in fact much higher than expected from independent segregation of both components. It appears therefore from all considerations, that segregation of the bacterial chromosome is a polarized event, a fact that can best be explained by its attachment to the membrane. The proposition that the chromosome is attached to the membrane has been tested by determining whether different replicons contained within the same cell are distributed randomly or not. This has been done by comparing the segregation pattern of the chromosome and of an extrachromosomal element, the F' episome. This plasmid, in addition to carrying genes that impart fertility on E. coZi, also carries other markers that can be used to detect its presence or to turn on or off its replication selectively. This allows a large set of ingenious experiments to show whether or not the chromosome and the episome cosegregate into the progeny cells. It was shown that under a variety of circumstances these replicons do not segregate randomly and that a mechanism must exist to insure unilateral distribution (Cuzin and Jacob, 1965; Hohn and Korn, 1969). Since the two genomes are not known to be directly linked to one another, it

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follows that they may both be linked to the cell membrane which becomes the agent of their joint segregation. This was among the earliest evidence for the replicon model and for the suggestion that bacterial DNA is attached to the membrane.

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V. Methodological Considerations of Cell Fractionation At present it is not possible to describe the properties of the site of the membrane to which DNA is attached. It is not known how large this site is and whether it differs structurally from the rest of the membrane. Therefore operational criteria for the fractionation and isolation of a DNA-membrane complex do not exist. If one includes as many relevant constituents as possible and perhaps isolates the whole membrane and its attached DNA, the fraction may contain much extraneous material, some of which may be entrained artifactually. However, it may be thought desirable to isolate only the region of the membrane that is in direct apposition to the DNA. This is not easy to do, since it is impossible, a priori, to tell when a complex contains only and all the relevant constituents. The obvious problems include how to determine if a complex exists in the cells or is formed during breakage, and how to tell if some relevant components may have become detached by mechanical or enzymic action. In general, artifacts of aggregation that arise after cell breakage may be assessed by reconstruction experiments. Fractionation of labile cell components should only be carried out after breaking bacteria in a gentle manner. For this reason almost all the methods described below deal with spheroplasts, bacteria that lack some wall constituents and are sensitive to detergents, osmotic shock, or relatively weak mechanical forces. There are four classes of techniques in current use for the retrieval of DNA-membrane complexes:

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1. Rapid sedimenting complexes (RSCs). Since DNA-membrane complexes are heavy relative to other cell constituents, they can be retrieved by centrifugation through sucrose gradients. Usually, such gradients are made over a cushion of cesium chloride or denser sucrose, and material that pellets to this interface may be called a RSC. Free DNA released from the membrane by shearing or nuclease action remains at the top of the gradient. In many of the reports that describe this technique (in several variations), the RSC contains much membrane material and relatively little of the total cell DNA. Under the conditions used membrane

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fragments probably vesiculate, which may lead to the mechanical entrainment of DNA in membrane vesicles. While exogenously added purified DNA is not entrained in RSCs, it is not known if DNA as it exists in a cell with its normal ligands may in fact be entrained. 2. Complexes obtained by isopycnic centrifugation or electrophoresis. DNA-membrane complexes can be expected to possess buoyant densities different from those of other cell constituents. Ivarie and P h e (1970) separated newly synthesized from uniformly labeled DNA on linear renografin density gradients. An advantage of this technique is the short time required for the membrane-DNA complex to reach its equilibrium and for its separation from the other cellular components. The complex they isolated is enriched for markers near the origin and terminus. Daniels (1971)fractionated the cell membrane into various portions, one of which contains DNA and can be banded in equilibrium gradients. Since the DNA is found in only one or two of nine fractions, it appears unlikely that it is entrained nonspecifically in membrane vesicles. In an analogous fashion, DNA-membrane complexes can be expected to have a distinct mobility in an electric field and may be retrieved by electrophoresis. This has been done recently by Olsen et al. (1974). 3. M bands. When cell lysates are mixed with crystals obtained by adding magnesium salts to Sarkosyl (the detergent sodium lauroyl sarcosinate), a characteristic band is formed after centrifugation through sucrose gradients. This band (M band) contains crystals to which membrane-DNA complexes adhere. There are reasons to believe that this adhesion is due to an affinity between the hydrophobic surface of the crystals and the membrane. Membranes alone attach to the crystals, while native DNA does not. Therefore it is assumed that DNA is found in M bands because it is bound to the membrane (Tremblay et al., 1969; Earhart et al., 1968). It is possible to increase the proportion of membrane in the M band by letting the lysate-crystal mixture stand or to decrease it by using Triton X-100 as the lysing detergent. Nearly all the DNA of the cell is attached to as little as 4% of the total membrane (Ballesta et al., 1972). Hence different portions of the membrane appear to be heterogeneous in their affinities for the crystals, with the DNA-bearing portion having a very high affinity. 4. Zsolated nucleoids. When lysis of E. coli is carried out in the presence of 1 M sodium chloride, the DNA does not become extended but remains in a folded, compact state. These nuclear bodies can be isolated by centrifugation through sucrose gradients. Unlike DNA in solution, they do not contribute significantly to the viscosity

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of the medium and have a high sedimentation coefficient. They contain a small amount of protein, nascent RNA, and some membrane constituents (Stonington and Pettijohn, 1971). They can be isolated as membrane-attached bodies containing either as much as 20% of the cell membrane or nearly free of membrane material. These compact nucleoids contain nearly all the RNA polymerase found in the cell. They sediment to positions in sucrose gradients, which apparently correspond to their stage of replication at the time of preparation (Worcel and Burgi, 1972).

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VI. Is the Chromosome Attached to a Special Region of the Membrane?

This is a difficultquestion, since there are no satisfactory means for the chemical characterization of different portions of the bacterial membrane. Thus mesosomes, the likely candidates for the membrane region to which DNA is bound, are not decisively different from the rest of the membrane. One of the recognizable membrane components in “membraneattached” nuclear bodies (Stonington and Pettijohn, 1971)is a peptide which is found in the outer layer of E. coli (Worcel and Burgi, 1974). Other membrane components may be present, but are not sufficiently distinct for identification. This finding suggests that under certain conditions the DNA-membrane complex contains both inner and outer membrane components. These two layers are in close apposition at about 200 places over the cell surface (Bayer, 1968a,b), and it seems possible that these sites of contact may play a special role in DNA binding. The membrane component of M bands or of fractions isolated by an analogous method have a different phospholipid composition than the average membrane (Ballesta et al., 1972; Daniels, 1971). They are richer in phosphatidylethanolamine and contain less phosphatidylglycerol and cardiolipin, the other two major phospholipids of E. coli or B . megaterium. In rebanding experiments the portion of the membrane contained in M bands was shown to have a higher affinity for magnesium-Sarkosyl crystals than the rest of the membrane (Ballesta et al., 1972). Daniels (1971) did not find that the DNAbearing fraction had different proteins than the rest of the membrane when examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. However, the large number of peptides seen in these preparations made it difficult to determine the presence of unique components.

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VII. Is the Origin of DNA Replication Attached to the Membrane?

The best evidence comes from an analysis of the genetic markers found in DNA-membrane complexes. Sueoka and Quinn (1968)used transformation in B . subtilis to show that RSCs containing 5-10% of the total DNA were substantially enriched in markers located at the origin of chromosome replication in this organism. As expected from the pattern of DNA replication, this enrichment was greater in cells growing at fast rates (O’Sullivan and Sueoka, 1972). These results were confirmed by Snyder and Young (1969), Ivarie and PBne (1970), and Yamaguchi et al. (1971), and extended to E. coli (Fielding and Fox, 1970) where the origin of replication was located by radioactive labeling. Several investigators also reported that the membrane-bound fraction is enriched in markers located at the terminus of DNA replication in B . subtilis (Sueoka and Quinn, 1968; Snyder and Young, 1969; Ivarie and PBne, 1970). A unique, but as yet uncharacterized region of the DNA of Mycoplasma gallisepticum is permanently attached to the membrane (Quinlan and Maniloff, 1973).

VIII. Does DNA Replication Take Place on the Membrane? While this question precedes many others historically, a definitive answer is not yet available. The strongest hint came from the finding that in bacterial conjugation genetic markers are transferred at the time of their synthesis (Jacob et al., 1963). This implies that in the donor cell DNA replicates at or near the site on the membrane involved in the formation of the conjugal bridge. Several investigators have reported the isolation of RSCs enriched in newly made DNA (Ganesan and Lederberg, 1965; Smith and Hanawalt, 1967; Ivarie and PBne, 1970; Fuchs and Hanawalt, 1970; Yamagcchi et al., 1971; Quinlan and Maniloff, 1972; Fujita et al., 1973). The extent of enrichment in newly made DNA in RSCs is not very great. In fact, in most experiments the enrichment factor:

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Newly made DNA in RSC/Total DNA in RSC Newly made DNA not in RSC/Total DNA not in RSC

vanes from unity (no enrichment) as reported by Yamaguchi et al. (1971), to a value of 2 to 4 in other reports. The value rarely approaches that expected if DNA were attached to the membrane uniquely at its point of replication. However, before concluding that

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replication does not take place on the membrane, it should be pointed out that a number of experimental complications do not permit a rigorous analysis of this question. Thus Yamaguchi et al. (1971) found that the extent of enrichment in newly made DNA varied according to the method of preparation of RSCs. Among the difficulties encountered may be the possibility that the conditions of fractionation release the replicating complex from the membrane. It is possible in fact to isolate what appears to be a replicating complex in a fraction devoid of membrane material (Fuchs and Hanawalt, 1970). Likewise, the content of DNA polymerase I in RSCs depends on ionic conditions (Ivarie and PBne, 1970). Newly made DNA has unusual properties which may influence its behavior in cell fractionation. According to current views, DNA is replicated in short stretches (Okazaki fragments) using an RNA primer (Okazaki et al., 1973). It is rapidly detached from this RNA and is linked to the older portion of the chromosome. The physical properties of newly made DNA can be expected to differ from those of the rest of the chromosome. The nature of these differences is not known, but is relevant to the problem of membrane attachment. We have observed that, in E. coli infected with bacteriophage T4,DNA labeled for very short periods is not found in the M band, while DNA labeled for a longer time is (Leibowitz and Schaechter, unpublished data). At least some of this DNA is in the form of Okazaki fragments which ostensibly do not remain attached to their complementary strand under the conditions of fractionation. This result can give the false impression that DNA is not synthesized on the membrane. When an experiment is carried out with the M-band technique, the finding is open to this interpretation, since M bands contain virtually all the DNA. However, this is not obvious when an RSC method is used, since the membrane fraction usually contains only a fraction of the total DNA. These difficulties may not exist for all systems, since in Pneumococcus newly made DNA of the size of Okazaki fragments is found exclusively in the M band (Firshein, 1972). Conversely, instead of being excluded, newly made DNA may be artifactually entrained in RSCs. Newly made DNA can be easily denatured (Kidson, 1960), a fact that may be quite relevant because denatured DNA tends to stick to surfaces, including cell membranes (Dworsky and Schaechter, 1973). Another factor that would lead to an apparent decrease in the enrichment in newly made DNA in RSCs is that DNA may be attached at many sites, perhaps including those where replication takes place. If so, what remains bound to the

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membrane after shearing is a large amount of DNA that is not the replicating region. This may influence the detection of enrichment in newly made DNA.

IX. Is the Chromosome Attached at Many Sites?

The replicon model proposes that the chromosome is attached to the membrane at the site of DNA replication. There are several reports that this is not the only site of attachment, and that the chromosomes of E. coEi and €4. subtilis are attached at many places. These data are derived from experiments in which a given number of breaks are introduced in the DNA and the proportion of DNA released from the membrane is measured. The number of breaks, estimated from molecular weight measurements, required to release a given amount of DNA allows one to calculate the number of attachment sites. Breaks can be introduced by x-ray or y-ray irradiation or by shearing lysates. The number of attachment sites reported from experiments of this type is in the order of 10 to 30 for E. coli (Rosenberg and Cavalieri, 1968; Dworsky and Schaechter, 1973) and as many as 70 to 90 in B. subtilis (Ivarie and P h e , 1973). Different methods give different numbers (Ivarie and Phne, 1973; Dworsky and Schaechter, 1973). These estimates are computed on the assumption that the DNA loops between two adjacent attachment sites are similar in length. If a few loops are very long and many are very short, their total number would be seriously underestimated. At present, the available data do little more than suggest that the likely number of attachment sites is large but not as large as the number of folds required to pack the bacterial chromosome inside the bacteria. The E. coli chromosome is about 1100 pm in length (Cairns, 1963) and must be folded well over lo00 times to fit inside the cell. Likewise, the number of attachment sites is considerably smaller than the number of RNA polymerase molecules present in E . coli growing under conditions similar to those of these experiments (it has been estimated that the total number of RNA polymerase molecules is about 7000; Matzura et al., 1973). However, the number of attachment sites is larger than that of origins of replication or sites where replication takes place. The actual numbers would depend on the rate of growth (Helmstetter and Cooper, 1968), but for the cells in question would vary between about one and three and one and seven, respectively. Since the frequency of initiation of RNA synthesis (Bremer and Yuan, 1968) and DNA synthesis (Helmstetter and Cooper, 1968) is dependent on the growth rate, one would anticipate

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that the absolute numbers of attachment points would be determined by the growth medium. For these reasons it is unlikely that the attachment of DNA to the membrane is related to a single function. There is in fact an indication that these attachment sites are of more than one kind. When cells are treated with the antibiotic rifampin, which inhibits transcription by binding to free RNA polymerase, the number of attachment sites decreases four- to fivefold (Dworsky and Schaechter, 1973).This suggests that there are functional or structural differences between the attachment sites that are destroyed by rifampin treatment and those that remain after such treatment. Since the estimated number of rifampin-resistant sites was two to five, it is tempting to think that they may represent the origins of DNA replication. The sites that are sensitive to rifampin (8 to 20 in number) depend on some property of RNA polymerase, since they are unaffected by treatment of a mutant whose RNA polymerase is insensitive to the drug. It is tempting to ascribe to these sites a role in transcription. This would have to be a special process, since it is likely that transcription takes place at many more sites than 8 to 20. Perhaps the repeated transcription of some cistrons (e.g., the rRNA cistrons) differs from the less frequent transcription of other cistrons, and may occur in contact with the membrane. These notions are totally unproved but can be subjected to experimental scrutiny.

X. The Nature of the Binding of the Chromosome to the Membrane In this area we are limited entirely to speculations. There are only hints and few facts, since there are no enzymes or chemicals known that remove the entire bacterial chromosome from the membrane in a manner that is nondestructive to either component. Membranes and DNA may be easily dissociated from one another, suggesting that they are not bound through covalent bonds. However, such bonds could easily escape detection. There is a suggestion that some of the attachment may be due to the adherence to the membrane of denatured regions of the chromosome. In reconstruction experiments it was shown that, while native DNA does not stick to membrane, heat-denatured DNA does (Dworsky and Schaechter, 1973).It is not known if extensive regions of denaturation exist in the chromosomes of growing bacteria. These may be found, for example, at the sites of replication, repair, recombination, or transcription. It has been shown that transcription may

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not require denaturation over more than a few base pairs (Saucier and Wang, 1972),but special regions of frequent transcription may in fact result in the formation of larger “bubbles” of denatured DNA. Such regions may correspond to the attachment sites that are destroyed by rifampin treatment (see Section IX). RNA may play a role in the attachment of DNA to the membrane since Earhart et al. (1973) found that RNase liberates both E. coli and T4 phage DNA from the membrane. However, this effect may be indirect since it requires the presence of endonuclease I and is not seen in mutants that lack this enzyme. It is likely that at sites other than the origin of replication DNA is bound in a dynamic sense and, rather than being permanently attached, rapidly moves through the site. This is necessarily true if replication takes place on the membrane, and is likely to be the case if the attachment is at sites of special types of transcription. For this reason the involvement of single-stranded DNA is particularly alluring, since DNA denaturation brings about a readily reversible change which has great consequences for the physical properties of the molecule.

XI, The Role of the Membrane in Maintaining the Compactness of the Nucleoid Highly compact nucleoids can be isolated by lysing cells in the presence of 1M sodium chloride (Stonington and Pettijohn, 1971). It has been proposed by Worcel and Burgi (1972) that their compactness is the result of supercoiling of the DNA. These investigators found that the entire chromosome does not act as a single supercoiled circle but rather as if it were separated into 10 to 80 individual supercoils. These loops are thought to be held in their individual position by a “core” of RNA, because thev are unfolded upon treatment with RNase (Stonington and Pettijohn, 1971). It is tempting to relate this to the finding obtained by in vivo treatment with the drug rifampin, which induces both the unfolding of the nucleoids and the elimination of most of the attachment sites of the DNA to the membrane (Dworsky and Schaechter, 1973). It seems possible that the separation of the supercoiled loops is due to their attachment to the membrane, and that the RNA core is located on the membrane and may consist of a rifampin-sensitive structure. In favor of this notion is the coincidence of the estimated number of loops (10 to 80) and the rifampin-sensitive attachment sites (about 8 to 20),and the analogous action of rifampin in vivo and RNase in vitro. However, the amount of membrane found in isolated nucleoids prepared at room temperature is as little as 0.1% of the total. This amount, while very small,

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may constitute an important portion of the membrane, since these purified nucleoids will form M bands (Wright and Michaelis, personal communication). The notion that the chromosome exists in the cell in a compact state is being challenged (see Section 11,B). The generally accepted picture of a central compact body may turn out to be inaccurate. Yet it seems unlikely that the bacterial chromosome exists in a random state within cells. Whatever its organization, it seems likely that the attachment to the membrane plays an important role.

XII. Conclusions It should be evident from what we have written above that we believe that work in this field is not at a conclusive stage. Therefore we believe that it is not yet possible to give an accurate picture of the role of membrane attachment in different functions of the bacterial chromosome. However, many of the available data are suggestive and promise further developments. We would like to summarize what is known to date, and what to us is physiologically plausible. The available evidence suggests that the replicon model is correct in postulating that nuclear segregation in bacteria takes place by virtue of the attachment of daughter chromosomes to different sites on the membrane. The relevant conclusions are the origin of DNA replication appears to be attached to the membrane, perhaps in a specific manner, and that daughter chromosomes probably segregate in a polarized fashion. Missing is the evidence that membrane growth takes place in a semiconservative manner which would immediately enable it to function as the segregation apparatus. However, the evidence to the contrary is not conclusive. The evidence on the behavior of mesosomes, the most likely sites of DNA attachment, is particularly confusing. It is not clear whether they split in two or arise de novo. As described in Section VIII, the evidence that DNA replication takes place at the membrane is not strong. The biochemistry of DNA replication is not fully elucidated and is certainly complex. It is not surprising that in vitro systems do not help determine if the membrane is involved in this process. There are better indications that transcription may be involved in membrane attachment, perhaps by creating special regions of DNA denaturation (Sections X and XI). In conclusion, there seem to be good reasons to believe that the bacterial chromosome is attached to the cell membrane. It is not quite as easy to assign to this attachment a role in chromosome segregation, replication, or transcription. The methodological dif-

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ficulties appear great but not impossible, and it can be expected that further work will elucidate many of the current questions. It is tempting to think that the relative structural simplicity of bacteria necessitates that its structures be used in several ways, allowing multiple regulatory connections between various functions. If so, the bacterial cell membrane is a busy place.

zyx zyxwvuts ACKNOWLEDGMENTS

The helpful comments of B. Beck, P. Dworsky, L. Daneo-Moore, C. Earhart, J. Fox, S. Guterman, M. Higgins, M. Inouye, L. A. McNicol, J. T. Park, A. Ryter, and G. Shockman are gratefully acknowledged. Work from the authors’ laboratory was supported by grants A1 05103 and A1 09465 from the United States Public Health Service. P.J. Leibowitz is a postdoctoral research fellow of the Public Health Service.

Note added in pro05 Since the submission of this review at least two important papers have appeared. D. L. Parker and D. A. Glaser [(1974),J. Mol. B i d . 87,153-1681 found that both the origin and the site of chromosome replication in E. coli are membrane-bound. This study represents a thorough application of available techniques. C. E. Helmstetter [(1974),J. Mol. Biol. 84, 1-19,21-36] has proposed that the synthesis of a special region of the membrane serves as a signal for initiation of chromosome replication in E . coli. REFERENCES Adler, H. I.. Fisher, W. D., and Hardigree, A. A. (1969).Trans. N. Y. Acad. Sci. 31,

1050-1070. Altenburg, B. C., and Suit, J. C. (1970).J. Bacteriol. 103,227-237. Altenburg, B. C., Suit, J. C., and Brinkley, B. R. (1970).J. Bacteriol. 104, 529-555. Bdesta, J. P.. CundlifFe, E., Daniels, M. J., Silverstein, J. L., Susskind, M.M., and Schaechter, M. (1972).J. Bacteriol. 112,195-199. Bayer, M. E. (1968a).J . Gen. Mlcrobiol. 53,395-404. Bayer, M. E. (1968b).J . Vtrol. 2,346-356. Bremer, H.,and Yuan, D. (1968).]. Mol. Blol. 38, 163-180. Burdett, I. D. J., and Rogers, H. J. (1970).J. Ultrastruct. Res. 30,354-367. Cairns, J. (1963).Cold Spring Harbor Symp. Quant. Bid. 28,43-46. Chai, N., and Lark, K. G. (1967).J . Bacteriol. 94,415-421. Chai, N., and Lark, K. G. (1970).J . Bacteriol. 104,401-409. Chapman, G. B., and Hillier, J. (1953).J. Bactertol. 66,362-373. Cuzin, F., and Jacob, F. (1965).C.R. Acad. Sci. 260,5411-5414. Daniels, M. J. (1871).Btochem. J. 122, 197-207. Dworsky. P., and Schaechter, M. (1973).J. Bacteriol. 116, 1364-1374. Earhart, C. F., Sauri, C. J., Fletcher, G., and Wolff, J. (1973).J. Vtrol. 11, 527-534. Earhart, C. F., Tremblay, G. Y.,Daniels, M. J., and Schaechter, M. (1968).Cold Spring Harbor Symp. Quant. B i d . 33,707-710. Eberle, H., and Lark, K. G. (1966).J . Mol. Btol. 22, 187-191. Edwards, M.R.,and Stevens, R. W. (1963).J . Bacteriol. 86,414-428. Ellar, D. J., Lundgren, D. G., and Slepecky, R.A. (1967).]. Bactertol. 94, 1189-1205.

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27

Ferrandes, B., Chaix, P., and Ryter, A. (1966).C.R. Acad. Sci. 263,1632-1635. Fielding, P., and Fox, C. F. (1970).Biochem. Biophys. Res. Commun. 41, 157-162. Firshein, W. (1972).J . Mol. Biol. 70,383-397. Fitz-James, P.C. (1960).J . Biophys. Biochem. Cytol. 8,507-528. Fitz-James, P.C. (1967).In “Symposium on Microbial Protoplasts, Spheroplasts, and L-Forms” (L. B. Guze, ed.), pp. 124-143.Williams & Wilkins, Baltimore, Maryland. Freese, E. B. (1973).J . Gen. Microbiol. 75, 187-190. Fuchs, E.,and Hanawalt, P. (1970).J . Mol. B i d . 52,301-322. Fujita, H., Komano, T., and Maruyama, Y. (1973).Biochem. Biophys. Res. Commun. 52,1361-1367. Ganesan, A. T.,and Lederberg, J. (1965).Biochem. Biophys. Res. Commun. 18, 824-835. Glauert, A. M. (1962).Brit. Med. Bull. 18,245-250. Glauert, A. M., Brieger, E. M., and Allen, J. M. (1961).E r p . Cell Res. 22, 73-85. Green, E. W.,and Schaechter, M. (1972).Proc. Nat. Acad. Sci. US.69,2312-2316. Helmstetter, C. W., and Cooper, S. (1968).J . Mol. B i d . 31,507-518. Higgins, M.L., and Daneo-Moore, L. (1972).J . Bacteriol. 109, 1221-1231. Higgins, M. L., and Shockman, G. D. (1970a).J. Bacteriol. 101,643-648. Higgins, M. L., and Shockman, G. D. (1970b).J. Bacteriol. 103,244-254. Higgins, M. L., and Shockman, G. D. (1971).Chem. Rubber Co.Critical Reo. Microb i d . 1,29-72. Highton, P. J. (1969).J . Ultrastruct. Res. 26, 130-147. Highton, P. J. (1970a).J . Ultrastruct. Res. 31,247-259. Highton, P. J. (1970b).J . Ultrastruct. Res. 31,260-271. H o h a n n , H., Geftic, S. G., Heymann, H., and Adair, F. W. (1973).J.Bacteriol. 114, 434-438. Hohn, B., and Kom, D. (1969).J . Mol. Biol. 45,385-395. Holt, S. C., and Leadbetter, E. R. (1969).Bacteriol. Rev. 33,346-378. Imaeda, T.,and Ogura, M. (1963).J . Bacteriol. 85,151-163. Ivarie, R. D.,and PBne, J. J. (1970).J. Bacteriol. 104,839-850. Ivarie, R. D., and PBne, J. J. (1973).J . Bacteriol. 114,571-576. Jacob, F., and Brenner, S. (1963).C. R . Acad. Sci. 256,298-300. Jacob, F., Brenner, S., and Cuzin, F. (1963).Cold Spring Harbor Symp. Quant. B i d . 28,329-348. Jacob, F., Ryter, A., and Cuzin, F. (1966).Proc. Roy. Soc., Ser. B . 164, 267-278. Kaye, J. J., and Chapman, G. B. (1963).J. Bacteriol. 86,536-543. Kellenberger, E . , Ryter, A., and SBchaud, J. (1958).J . Biophys. Biochem. Cytol. 4, 671-678. Kepes, A., and Autissier, F. (1972).Biochim. Biophys. Acta 265,443-469. Kidson, C. (1966).J . Mol. Biol. 17, 1-9. Landman, 0.E.,Ryter, A., and FrBhel, C. (1968).J. Bacteriol. 96,2154-2170. Lin, E.C. C., Hirota, Y., and Jacob, F. (1971).J. Bacteriol. 108,375-385. Maal@e,O., and Kjeldgaard, N. 0. (1966).In “Control of Macromolecular Synthesis” (B. Davis, ed.). Benjamin, New York. McCandless, R. G., Cohen, M., Kalmanson, G., and Guze, L. B. (1968).J. Bacteriol. 96, 1400-1412. Margaretten, W.,Morgan, C., Rosenkranz, H. S., and Rose, H. M. (1966).J. Bacteriol. 91,823-833. Matzura, H., Hansen, B. S., and Zeuthen, J. (1973).J . Mol. B i d . 74,9-20. Mindich, L., and Dales, S. (1972).J . Cell B i d . 55,32-41. Nanninga, N. (1968).J. Cell B i d . 39,251-263.

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Okazaki, R.,Sugino, A,, Hirose, S., Okazaki, T., Imae, Y., Kainuma-Kuroda, R.,Ogawa, T., Arisawa, M., and Kurosawa, Y. (1973).In “DNA Synthesis In Vitro” (R.D. Wells and R. B. Inman, eds.), pp. 83-106. University Park Press, Baltimore, Maryland, Olsen, W. L., Heidrich, H.-G., Hofschneider, P. H., and Hannig, K. (1974). J . Bacteriol. 118,646-653.

O’Sullivan, M. A., and Sueoka, N. i1972).J . Mol. Biol. 69,237-248. Patch, C. T.,and Landman, 0. E. (1971).J . Bacteriol. 107,345-357. Pierucci, 0.. and Zuchowski, C.(1973).J . Mol. B i d . 80,477-503. Pontefract, R. D.,Bergeron, G., and Thatcher, F. S. (1969).J. Bacteriol. 97, 367-375. Quadling, C.,and Stocker, B. A. D. (1962).J . Gen. Mfcrobiol. 28,257-270. Quinlan, D.C.,and Maniloff, J. (1972).J . Bacteriol. 112,1375-1379. Quidan, D. C., and Maniloff, J. (1973).J. Bacterlol. 115,117-120. Remsen, C.C.(1968).Arch. Mikrobfol. 61,4047. Reusch, V. M., and Berger, M. M. (1972).Fed. Proc., Fed. Amer. SOC. Erp. Biol. 31, 413. Reusch, V. M., and Berger, M.M. (1973).Biochim. Biophys. Acta 300,79-104. Rogers, H. J. (1970).Bactetiol. Reo. 34,194-214. Rosenberg, B. H., and Cavalieri, L. F. (1968).Cold Spring Harbor Symp. Quant. Biol. 33,65-72. Ryter, A. (1967).Folfa Mimbiol. (Prague) 12,283-290. Ryter, A. (1968).Bacteriol. Reu. 32,39-54. Ryter, A. (1971).Ann. Inst. Pasteur, Paris 121,271-288. Ryter, A,, and Jacob, F. (1963).C. R. Acad. Scf. 257,3060-3063. Ryter, A,, and Jacob, F. (1964).Ann. Inst. Pasteur, Paris 107,384400. Ryter, A,, and Jacob, F. (1966).Ann. Inst. Pasteur, Paris 110,801-812. Ryter, A,, and Landman, 0. E. (1964).J . Bacteriol. 88,457-467. Ryter, A., and Landman, 0. E. (1967).In “Symposium on Microbial Protoplasts, Spheroplasts, and L-Foms” (L. B. Guze, ed.), pp. 110-123. Williams & Wilkins, Baltimore, Maryland. Ryter, A., Hirota, Y.. and Jacob, F. (1968).Cold Spring Harbor Symp. Quant. Biol. 33, 669-676. Salton, M. R.J. (1971).Chem. Rubber Co.Critical Reo. Mfcrobiol. I, 29-72. Saucier, J. M., and Wang, J. C. (1972).Nature (London). New Biol. 239, 167-170. Siegel, P. J., and Schaechter. M. (1973).Annu. Reo. Microbiol. 27,261-282. Smith, D. W., and Hanawalt, P. C. (1967).Btochim. Biophys. Acta 149, 519-531. Snyder, R. W., and Young, F. E. (1969).Biochem. Biophys. Res. Commun. 35, 354-362. Steed, P., and Murray, R. G. E.(1966).Can. J . Microbiol. 12,263-270. Stonington, 0. G., and Pettijohn, D. E. (1971).Proc. Nut. Acad. Sci. U.S.68,6-9. Stove Poindexter, J. L., and Cohen-Bazire, G. (1964). J . Cell Biol. 23,587-607. Sueoka, N.. and Quinn, W. G. (1968).Cold Spring Harbor Symp. Quant. Biol. 33, 695-705. Tremblay, G. Y., Daniels, M. H., and Schaechter, M. (1969).J . Mol. Biol. 40,65-76. van Iterson, W. (1961).J . Biophys. Bfochem. Cytol. 9, 183-192. van Iterson, W. (1965).Bmteriol. Reo. 29,299-325. van Iterson, W., and Leene, W. (1964).J . Cell Biol. 20,361-375. Wilson, G., and Fox, C. F. (1971).Biochem. Biophys. Res. Commun. 44, 503-509. Worcel, A., and Burgi, E. (1972).J. Mol. Biol. 71,127-147. Worcel, A., and Burgi, E. (1974).J. Mol. Biol. 82,Ql-105. Yamaguchi, K., Murakawi, S., and Yoshikawa, H. (1971).Biochem. Biophys. Res. Commun. 44,1559-1565. Ziegler, R. J., and Daneo-Moore, L. (1971).J . Bacteriol. 105, 190-199.

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Regulation of the Lactose Operon in Escherichia coli by cAMP G. CARPENTER'AND B. H. SELLS

zy zy

zyxwvu

Laboratories of Molecular Biology, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada

. . . . . . . . . . . . . . . . . .

I. Introduction. A. Role of CAMP. . . . . . . . . . . . . . . . . B. Organization of the Lac Operon . . . . . . . . . . C. Nature of the Inducer 11. cAMP Formation and Catabolite Repression A. cAMP and the Catabolite Repression of &Calactosidase . . B. Cellular Concentrations of cAMP C. Enzymes Involved in cAMP Metabolism. . . . . . . D. Control of Catabolite Repression of fl-Galactosidase Synthesis. . . . . . . . . . . . . . . . . . . 111. In Vioo Evidence of Site of CAMP Action. . . . . . . . . A. Transcriptional or Translational Control. B. cAMP and Lac Promoter Mutants . . . . . . . . . . IV. cAMPActioninVitro. . . . . . . . . . . . . . . . A. In Vitro Synthesis of /ibGalactosidase. . . . . . . . . B. Mediation of cAMP Action C. Properties of CAMP-Receptor Protein . . . . . . . . D. Initiation of Lac mRNA Synthesis in Vitro . . . . . . V. Additional Aspects of Lac Operon Regulation . . . . . . . References

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. . . . . . . . . . . . . . . . . . . I. Introduction

29

30

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34 36 36 37 39

42 43 43 45 46 46 47 49 51

54 55

Over the past two decades, significant progress has been made in explaining the general mechanism involved in the synthesis of protein from information encoded in the polydeoxynucleotide sequences in DNA. How synthesis of a particular protein is controlled has been resolved only in a few instances. The system that has received the greatest attention and which is best understood is the lactose (lac) operon in Escherichia coli. This article, which deals with a description of the lac operon, examines the various points of control in the synthesis of the enzyme pgalactosidase. Protein synthesis in its broadest sense consists of two processes -transcription and translation. The evidence currently avail-

' Present address: Department of Biochemistry, Vanderbilt University, Nashville, Tennessee. 29

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G. CARPENTER AND B. H. SELLS

able concerning pgalactosidase formation suggests that most, if not all, the control of its expression occurs at the level of transcription. This control involves regulation by several components which interact with genetic material at distinct locations within the lac operon. This interaction is influenced by several low molecular weight compounds whose concentration modulates the expression of the operon. The model described for pgalactosidase synthesis may provide a prototype for understanding how production of other specific proteins is regulated. Following the pioneering work of Jacob and Monod on enzyme induction, a discovery which greatly aided in our understanding of the mechanisms that regulate pgalactosidase synthesis, was the recognition that the metabolite cyclic AMP (adenosine 3’,5’-monophosphate) (CAMP)is involved in the process. This cyclic nucleotide, which has an influence on a variety of biological systems, is discussed specifically in terms of its effect on the lac operon.

zyxwvu

A. ROLE OF cAMP Since 1957, when Sutherland and co-workers (Rall and Sutherland, 1958; Rall et al., 1957; Sutherland and Rall, 1957, 1958) identified CAMP as the biological factor that mediates the stimulatory effect of epinephrine and glucagon on glycogenolysis in rat liver, the nucleotide has been implicated in the regulation of many diverse physiological processes. The role of cAMP in mediating hormone action was explained by Sutherland et al. (1965) in their second messenger hypothesis. This hypothesis postulates that many polypeptide hormones interact with the cytoplasmic membranes of target cells and increase the activity of the membrane-bound enzyme adenyl cyclase which catalyzes the synthesis of cAMP from ATP. Increased intracellular levels of cAMP then bring about the appropriate biochemical response. For his discovery of cAMP and his identification of its physiological role, Sutherland was awarded the Nobel prize for medicine in 1971. Detailed review articles concerned with the role of cAMP in mammalian cells have been published (Jost and Rickenberg, 1971; Pastan and Perlman, 1971; Robinson et al., 1968). 1. Presence of CAMP in Bacteria The presence of this cyclic nucleotide in bacteria was first reported by Makman and Sutherland (1963), who identified cAMP in extracts of E. cold. The same year, Okabayashi et al. (1963) identified the nucleotide in extracts of Breuibactedum liquefuciens. Two years

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zy zyx zyxwv REGULATION OF THE LACTOSE OPERON

31

later, Makman and Sutherland (1965) noted a correlation between the intracellular concentration of cAMP and the amount of glucose in the culture medium. As the medium became depleted of glucose, the intracellular level of cAMP increased sharply. When a suspension of E . coli was grown in excess glucose and transferred to a phosphate buffer without glucose, the concentration of cAMP increased from lo-' to M within 60 minutes. The addition of glucose to the phosphate buffer completely prevented the rise in cAMP concentration. Although it had been known for many years that glucose exerts a repressive effect on the synthesis of many enzymes in microorganisms, it was not until 1968 that Perlman and Pastan (1968a)and U11mann and Monod (1968) made the initial discovery that cAMP can overcome the glucose repression of pgalactosidase synthesis. This finding implicated this cyclic nucleotide in the regulation of enzyme synthesis in prokaryotic cells. During the past 5 years, many investigators have succeeded in elaborating the mechanism by which cAMP controls pgalactosidase synthesis. This system now represents one of the best understood in terms of regulation at the gene level. 2. Biological Events Efiected b y cAMP Recently, several reviews concerning the role of cAMP in enzyme synthesis in prokaryotic cells have been published (Pastan and Perlman, 1970; Perlman and Pastan, 1971, Reznikoff, 1972; Zubay and Chambers, 1971). As shown in Table I, cAMP is involved in the regulation of a large and diverse number of biological processes in bacteria. The list is undoubtedly incomplete, and the involvement of cAMP in processes other than enzyme synthesis may be indirect and reflect a requirement for the synthesis of a particular protein. With the exception of glutamate synthetase and glutaminase, cAMP regulates enzyme synthesis in a positive manner. Since the role of cAMP is best understood in the case of p g a l a c tosidase synthesis, we consider in this article the regulatory mechanisms by which cAMP controls the formation of this enzyme. B. ORGANIZATION OF THE LAC OPERON PGalactosidase is one of three enzymes coded for by a segment of DNA on the E. coli genome known as the lac operon. On the addition of lactose to the growth medium, the intracellular levels of these enzymes increased more than 1000-fold within a few minutes, enabling the bacteria to adapt to a new nutritional environment rapidly. PGalactosidase, which is coded for by the z cistron of the lac operon,

32

zyxwvu G. CARPENTER AND B. H. SELLS

TABLE I EFFECTSOF CAMP ON BACTERIAL METABOLISM

zyx zyxw zyxwvu

Process Enzyme synthesis SGalactosidase Lac permease Galactokinase

Glycerol kinase a-Glycerolphosphate permease Arabinase operon regulator protein L-Arabinose permease LArabinose isomerase Fructose enzyme I1 (phosphotransferase) Tryptophanase DSerine deaminase Thymidine phosphorylase Threonine deaminase Pseudouridine kinase Pseudouridylate synthetase Chloramphenicol acetyl transferase GMP reductase Glutamine synthetase Glutamate dehydrogenase Glutaminase A Cytochromes Streptomycin adenylcyl transferase Oxidative phosphorylation Lysogeny Flagellar formation Bioluminescence Transformation Plasmid DNA replication Colicin DNA replication Protein kinase activity

Reference

Perlman and Pastan (1Q68a); Ullmann and Monod (1963) de Crombrugghe et 01. (196Qb) Tao h Schweiger (1970); de Crombrugghe et al. (1Q69b); Nissley et al. (1971); Park et al. (1971); Wetekam et al. (1971) de Crombrugghe et al. (lQ6Qb) de Crombrugghe et al. (1969b) Yang and Zubay (1973) de Crombrugghe et al. (186913) Nakazawa and Yokota (1973) de Crombrugghe et al. (1969b) del Camp0 et al. (1970); Perlman and Pastan (1988a); Ramirez et al. (1072) de Crombrugghe et al. (1869b); McFall (1973) de Crombrugghe et al. (1QBQb) Shizuta and Hayaishi (1970) Perlman and Pastan (1971) Perlman and Pastan (1971) de Crombrugghe et al. (1972); Harwood and Smith (1971) Benson et al. (1971) Prusiner et al. (1972) Prusiner et al. (1972) Prusiner et al. (1972) Broman and Dobrogosz (1972) Harwood and Smith (1971) Hempfling and Breman (1971) Grodzicker et al. (1972) Yokota and Cots (1970) Nealson et 01. (1972) Wise et al. (1973) Katz et al. (1973) Nakazawa and Tamada (1972) Khandelwal et al. (1973); Kuo and Greengard (1869)

REGULATION OF THE LACTOSE OPERON

zyx zy 33

hydrolyzes lactose to glucose and galactose. The enzymology of p galactosidase has been discussed by Zabin and Fowler (1970). The molecular weight of the active enzyme is 540,000 daltons, and the enzyme is a tetramer composed of four identical polypeptide chains. The product of the z cistron therefore, is a polypeptide of 135,000 daltons. Although the synthesis of active enzyme is dependent on subunit assembly, Adamson et al. (1970)have reported that subunit assembly is not a rate-limiting factor during Pgalactosidase induction. The y cistron of the lac operon codes for a membrane-localized protein, referred to as the lac permease or M protein, which is responsible for the transport of pgalactosides such as lactose into intact cells. The lac permease is composed of subunits which have a molecular weight of 30,000 daltons; however, the active form of this enzyme is not known. Kennedy (1970) and Kepes (1971) have reviewed various aspects of the lac permease system. The third enzyme of the lac operon, thiogalactoside transacetylase, is a product of the a cistron of this operon. The gene product of this cistron is a 32,000-dalton polypeptide. Apparently, the active enzyme exists as a dimer of two identical subunits. Although thiogalactoside transacetylase activity can be detected in crude extracts by the transfer of a radioactive acetyl group from acetyl CoA to a thiogalactoside acceptor, the role of this enzyme in lactose metabolism remains obscure. Strains carrying a deletion of the a cistron grow as well as wild-type strains on lactose (Fox et al., 1966). The enzyme may be involved in a secondary pathway or side reaction during lactose metabolism, and has been discussed at length by Zabin and Fowler (1970)and Kennedy (1970). The arrangement of the z, y, and a cistrons in the lac operon, together with the regulatory sites that control the expression of these cistrons, is shown in Fig. 1. The i cistron, which is located just to the left of the lac operon,

zyxw

FIG.1. Diagramatic arrangement of components of lac operon (not drawn to scale).

34

zyxwvuts zyx G. CARPENTER AND B. H. SELLS

codes for the lac repressor. This protein is thought to be constitutively synthesized, and has a low level of production as indicated by the fact that there are approximately 10 molecules of active repressor per haploid genome. The active repressor is a tetrameric molecule composed of four identical subunits. The product of the i cistrons is then a polypeptide with a molecular weight of 38,000 daltons. The nature of the repressor has been discussed in detail by Gilbert and Miiller-Hill (1970)and Bourgeois (1971). The repressor prevents the synthesis of pgalactosidase, lac permease, and transacetylase by binding to the operator ( 0 )region of the lac operon. The p region is the promoter of this operon, and is the site on the DNA to which RNA polymerase binds and at which the initiation of transcription takes place. The binding of repressor between the promoter and the z cistron blocks the transcription of the z, y, and a cistrons and constitutes a negative type of regulation. Synthesis of the enzymes of the lac operon occurs when an inducer is added to the system. The inducer combines with the repressor and decreases the affinity of the repressor for the operator site on the DNA. When the repressor is removed from the operator, transcription of the z, y, and a cistrons takes place. Although the effect of added inducer is commonly referred to as induction of the lac operon, the term derepression is more technically correct. We follow the current usage, however, and refer to induction of pgalactosidase by inducer.

C. NATURE OF THE INDUCER In the presence of lactose, the intracellular levels of the enzymes of the lac operon are increased over 1000-fold within a matter of minutes. Although valuable insights into the mechanism of the induction process have been obtained since the original discoveries of Jacob and Monod, the identity of the natural inducer of the lac operon was only recently established. The effectiveness of synthetic inducers such as isopropylthiogalactoside(IPTG) and thiomethylgalactoside (TMG) in early work obviated the necessity for identifying the natural inducer. Investigations by Burstein et al. (1965) suggested that the lactose molecule itself is not the natural inducer of the lac operon. This conclusion was based on the observation that strains lacking Pgalactosidase are unable to produce transacetylase in the presence of lactose. These workers demonstrated that the presence of some pgalactosidase is necessary to form inducer by a transgalactosidation reaction. Miiller-Hill et al. (1964)had previously observed that allolactose (1,6-O-P-D-galaCtOpyranOSyl-D-glUCOSe),

zyx

REGULATION OF THE LACTOSE OPERON

35

an isomer of lactose (1,4O-~-~galactopyranosyl-~-glucose) was an effective inducer of Pgalactosidase in uiuo. Recently, Jobe and Bourgeois (1972)presented strong evidence identifying allolactose as the natural inducer of the lac operon. These investigators showed that allolactose, when added to crude extracts, is the only sugar bound to subsequently purified repressor, and that allolactose acts as an efficient inducer in uitro by destabilizing the operator-repressor complex. They further extended the results of other workers by showing that allolactose is an effective inducer of the lac operon in uiuo. From their studies they also demonstrated that P-galactosidase catalyzes allolactose formation by a rearrangement reaction which converts the 1-4linkage of lactose to a 1-6linkage. The fact that active P-galactosidase is required to produce the inducer ensures that inactive protein forms of the lac operon enzymes are not made and energy wasted as a result of mutations in the z gene or cellular conditions unfavorable to P-galactosidase activity. A mechanism that prevents the expenditure of cellular energy on the synthesis of inactive proteins is illustrated by studies demonstrating that lactose acts as an antiinducer of the lac operon. Jobe and Bourgeois (1973)showed that high concentrations of lactose inhibit the induction of the lac operon in uiuo. That lactose acts as an antiinducer was shown by experiments in uitro,which demonstrated that lactose competitively inhibits the binding of inducers to purified repressor and that lactose stabilizes the repressor-operator complex. These results indicate that E. coli has evolved mechanisms to ensure: (1)that only enzymically active molecules of Pgalactosidase are induced, and (2)that in the absence of active P-galactosidase induction of the entire lac operon is prevented. A model of the effect of lactose and allolactose upon the repressor-operator binding constants is shown in Fig. 2.

z

zyxwvut

FIG.2. Interaction of lactose and allolactose with the lac repressor. (From Jobe and Bourgeois, 1973.)

36

zyxwvu zyxwv zyx G. CARPENTER A N D B. H. SELLS

11. CAMP Formation and Catabolite Repression

Although the inhibitory effect of metabolites, particularly glucose, on the production of enzymes in bacteria has long been recognized, an understanding of this process, initially termed catabolite repression by Magasanik (1961), has only recently been uncovered. The reader is referred to the review by Paigen and Williams (1970) for extensive information concerning catabolite repression and various aspects of bacterial metabolism. A. CAMP AND OF

THE CATABOLITE REPRESSION

~GALACTOSIDASE

The studies of Makman and Sutherland (1965)provided the initial hint that eventually led to an understanding of the mechanism of glucose repression of enzyme synthesis. In 1965,these workers demonstrated a significant effect of glucose on intracellular concentrations of CAMP. The addition of glucose to a suspension of bacteria was shown to reduce markedly the intracellular levels of CAMP, although the physiological role of CAMP was not recognized at that time.

0

1;'be 0.03

0.04 I

0.105

0.06 I

Optical density 560 n m I FIG.3. EfFect of glucose and CAMPon the rate of synthesis of @gdactosidase. All samples contained IPTC. Glucose (closed circles) or glucose and CAMP (5 mM) (squares) was added at the arrow. Glycerol (open circles). (From Pastan and Perlman, 1970.) Copyright 1970 by the American Association of the Advancement of Science.

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37

In 1968, the experiments of Perlman and Pastan ( 1 W a ) and Ullmann and Monod (1968)demonstrated that the addition of cAMP to E , coli growing in a glucose medium relieved the repressive effect of glucose on pgalactosidase synthesis (Fig. 3). The relationship of glucose, CAMP,and pgalactosidase synthesis raises two broad questions. How do metabolites such as glucose affect the intracellular levels of CAMP?What is the mechanism by which cAMP regulates the synthesis of pgalactosidase? Investigations pertinent to the former question are discussed in the remaining portion of this s e e tion, while studies concerning the latter question are considered in Section IV.

B. CELLULAR CONCENTRATIONS OF cAMP Buettner et al. (1973) have studied the intracellular concentrations of cAMP in strains of E. coli sensitive or resistant to catabolite repression. The catabolite repression-resistant (CR-) strains examined by these investigators either possess little cAMP phosphodiesterase activity (Crookes strain) or are phosphodiesterase-negative (strain AB257Pe') mutants. Phosphodiesterase is the enzyme that degrades cAMP to AMP. Presumably, these two strains are resistant to catabolite repression because their intracellular levels of cAMP are higher than those of strains that possess active phosphodiesterase and are sensitive to catabolite repression (CR+ strains). The data in Table I1 show the relationships among growth on different carbon sources, intracellular cAMP concentrations, and pgalactosidase levels. These results indicate that there is an inverse correlation between cell growth rate and intracellular cAMP levels. The CRstrain had slightly higher levels of cAMP than the CR+ strains at most growth rates. During growth on glucose the difference in cAMP concentration was approximately twofold. The results indicate, however, that catabolite repression of pgalactosidase formation is not mediated solely by CAMP. While pgalactosidase specific activity is approximately 300- to 400-fold higher in CR- strains compared to CR+strains during growth on a mixture of glucose and gluconate, the intracellular levels of cAMP are nearly equal in the different strains. Furthermore, the specific activity of pgalactosidase in the CR+ strain increases 20-fold when transferred from a medium containing a mixture of glucose and gluconate to a medium containing glucose. A possible explanation for these inconsistent relationships is that carbohydrates that repress pgalactosidase synthesis may interfere with the entry of inducers into intact cells (Kepes, 1960; Magasanik, 1970). Experiments using constitutive strains would obviate this difficulty

38

G. CARPENTER AND B. H. SELLS

zyxw zy zyxw

TABLE I1 CELLULAR CONCENTRATION OF CAMP AND ~GALACTOSIDASE ACTIVITY DURING EXPONENTIAL GROWTH’

Shin

AB257 (CR+)

AB257”-’ (CR-)

Crookes (CR-)

Source of carbonb Glucose plus gluconate Glucose Glycerol Succinate Acetate L-Proline Glucose plus gluconate Glucose Glycerol Succinate Glucose plus gluconate Glucose Glycerol Succinate

Generation time (minutes) 57

56 96 170 270 218 58 72 66 157 48

Intracellular cAMP (10-5 M )

/?-Galactosidase specific activity‘

1.2

10

1.2 4.3 5.0 5.5 24.0 0.8

200 12,500 14,500 19,800 43.800 4,000

2.3 10.2 9.3 0.8

11,000 13,000 13,400 3,500

zyxwvutsr 53 59 86

2.2 4.5 6.2

11,000 13,000 15,400

a From Buettner et ol. (1973). with permission of the American Society for Microbiology. b Concentrations were: glucose and gluconate, 10 mM each; glycerol, 20 mM; succinate, 15 mM; acetate, 30 mM; L-Proline, 30 mM. Specific pgalactosidase activity is given as nanomoles of o-nitrophenol formed per minute per milligram of bacterial protein at 37°C.

and provide a better model to study the correlation between Pgalactosidase levels and intracellular CAMP. Pastan et al. (1969) showed that the addition of high concentrations of cAMP to a glucosegluconate medium reverses the catabolite repression encountered in this medium. Numerous workers (Buettner et al., 1973; Monod et aZ., 1970; Peterkofsky and Gazdar, 1971) repeated the initial observations of Makman and Sutherland (1965), which demonstrated that cellular concentrations of cAMP increase quickly when the E. coli medium is depleted of glucose. A similar observation has been made with yeast (Sy and Richter, 1972). This change in cAMP concentration in E. coli takes place rapidly when the glucose in the medium is exhausted. Within a 15-minute period the intracellular levels of cAMP rose 1000-fold. Since addition of chloramphenicol to the medium does not

z z

z zyx zyxwvutsrq -

REGULATION OF THE LACTOSE OPERON

-\

GIUWS~

ATp

adeny cyclasc

f .......

Intermediary metabolism

...............:.............

..",................."..............

e i E

'\

39

'"'

Phospho--,/-

' ' AMP

CATABOUTE REPRESSOR .,

./'

diesterase

-m

LAC OPERON

.".......................

---------

"

inhibition stimulation

FIG.4. Indirect control of the lac operon by a catabolite repressor.

zyxwvu

prevent the increase in cAMP upon removal of glucose, it is unlikely that new protein synthesis is required for this change to occur. Several investigators (Makman and Sutherland, 1965; Peterkofsky and Gazdar, 1971) have suggested that excretion of CAMP into the medium is an important mechanism in the regulation of CAMP levels. The results of Buettner et al. (1973) indicate, however, that the intracellular concentration of cAMP bears little relationship to the extracellular concentration. They also demonstrated that the high intracellular levels of cAMP produced in intact cells by depleting the medium of glucose can be dramatically lowered by the addition of glucose to the cells. In this case, the decrease in intracellular cAMP is not reflected by the accumulation of cAMP in the medium. These observations suggest that the activities of adenyl cyclase and cAMP phosphodiesterase play a larger role than the export mechanism in controlling the intracellular concentration of CAMP.

C. ENZYMES INVOLVED IN cAMP METABOLISM 1. Aden yl Cyclase Adenyl cyclase catalyzes the conversion of ATP to cAMP and pyrophosphate in bacterial and eukaryotic cells. The importance of this enzyme to carbohydrate metabolism is demonstrated by the isolation of a mutant of E. coli deficient in adenyl cyclase activity (Perlman and Pastan, 1969). In the absence of exogenous CAMP,this mutant is unable to grow on lactose, maltose, arabinose, mannitol, or glycerol, and grows poorly on glucose, fructose, or galactose. Peterkofsky and Gazdar (1973)devised a procedure to measure the

40

zyxwvuts zyxwvu zyxw G . CARPENTER AND B. H. SELLS

activity of adenyl cyclase in uiuo. This method involves labeling cAMP in uiuo from precursor adenosine, followed by quantitative analysis of the labeled nucleotide. When the labeling period is short and the specific activity of the ATP pool is measured, the rate of incorporation of radioactivity corresponds to a determination of adenyl cyclase activity in uioo. The results indicate that increases in cAMP levels that occur when glucose in a growth medium is exhausted are accompanied by an equivalent increase in the activity of adenyl cyclase. Measurements in uitro of adenyl cyclase activity in extracts prepared from yeast cells at various stages of growth in a glucose medium also showed that the enzyme activity increases when the medium is depleted of glucose (Sy and Richter, 1972). Abou-Sabe’ and Nardi (1973)recently isolated a catabolite repression-resistant mutant of E . cold which synthesizes cAMP during growth on glucose at a rate equivalent to that obtained during growth on glycerol. During growth on a glucose medium, synthesis of both Pgalactosidase and cAMP was stimulated. When the adenyl cyclase of this strain was isolated in a membrane fraction, the specific activity of the enzyme was increased by the addition of glucose to the reaction mixture. Solubilization of the adenyl cyclase resulted in an enzyme preparation which was insensitive to stimulation by glucose. These results suggest that glucose may interact directly or indirectly with the membrane-bound adenyl cyclase in E. colt and thereby regulate the intracellular levels of CAMP. This situation is strikingly similar to hormonal interactions with membrane-bound adenyl cyclase in eukaryotic cells. Purification of E . colt adenyl cyclase has proved difficult because of its association with particulate matter and instability after solubilization (Kepes, 1960).Although Ide (1969)was unable to extract the enzyme with detergents, Tao and Lipmann (1969)and Tao and Huberman (1970)found that it could be removed from the particulate fraction by washing with buffer. Both groups observed that the enzyme requires Mg+*and that its activity is inhibited by pyridoxal phosphate, oxaloacetate, pyruvate, malate, and ribose 5phosphate. The significance of these inhibitory compounds is questionable, as ATP was shown to be degraded by competing enzymes when pyruvate, ribose $phosphate, or oxaloacetate was added to the reaction mixture. The molecular weight of the adenyl cyclase was estimated at 110,OOO by sucrose density gradient centrifugation (Tao and Huberman, 1970). The adenyl cyclase of B. Ztquefaciens was studied by Hirata and Hayaishi (1965,1967) and Ide and co-workers (1967). In con-

zyxw

zyx zyxwvu zyxwvu REGULATION OF THE LACTOSE OPERON

41

bast to the E . coZi enzyme, they found that adenyl cyclase in B . Ziquefuciens is not associated with the particulate fraction but is found in the cell sap. Furthermore, the enzyme is dependent on pyruvate. Ide (1971) surveyed the adenyl cyclases of 21 strains of bacteria and reported that, in bacteria in which adenyl cyclase was found in the supernatant fraction, the enzyme was activated by pyruvate. In strains in which adenyl cyclase was located in the particulate fraction, the enzyme was not activated by pyruvate. Khandelwal and Hamilton (1971, 1972) purified 3200-fold the adenyl cyclase from Streptococcus suZiuurius and studied the effect of various metabolites on the purified enzyme. The enzyme was inhibited by ADP and various nucleoside triphosphates in a competitive manner. Inhibition by these compounds was also reported in E. coZi (Tao and Huberman, 1970) and B. Ziquefuciens (Hirata and Hayaishi, 1967). Inhibition of the S. suZiuurius enzyme was also observed when various diphosphate glucose nucleosides were added to the reaction mixture. An examination of the effect of individual glycolytic intermediates on adenyl cyclase activity showed that glucose 6-phosphate, glucose 1-phosphate, 2-phosphoglycerate, and pyruvate stimulated the enzyme up to 40%. Citrate and lactate inhibited the enzyme up to 50%. In the presence of fructose 6phosphate, fructose l,&diphosphate, glyceraldehyde 3-phosphate, and phosphoenolpyruvate, enzyme activity was either enhanced or inhibited, depending on the concentration of the compound present. Khandelwal and Hamilton (1972) have discussed the relationship between the adenyl cyclase system and the activity of various glycolytic enzymes.

2. Phosphodiesteruse Ide (1971)demonstrated the presence of CAMPphosphodiesterase activity in a variety of bacterial species. The enzyme from Serrutiu murcescans was purified over 1000-fold; however, the possible regulation of enzyme activity by metabolic effectors was not reported (Okabayashi and Ide, 1970). Aboud and Burger (1971b) have reported that the phosphodiesterase activity of E. cold is much lower in cells grown on glucose than in cells grown on glycerol. When cells were grown on glucose plus CAMP,the levels of phosphodiesterase increased to that obtained with cells grown in a glycerol-containing medium. Although the mechanisms by which phosphodiesterase activity was lowered during growth on glucose were not determined, these investigators suggested that the differences were due to the rate of enzymes synthesis. The hypothesis that synthesis of phospho-

42

zyxwvu

G. CARPENTER AND B. H. SELLS

diesterase is controlled by the type of carbohydrate present and/or intracellular cAMP levels, however, can be inferred only from measurements of enzyme activity. Peterkofsky and Gazdar (1971)did not find a correlation between the amount of glucose in the medium and the activity of phosphodiesterase. Monod, Janecek and Rickenberg (1970) purified phosphodiesterase from E. coli. They found that the enzyme was composed of two protein components, separable by differential centrifugation, and a third dialyzable component. Since the activity of the dialyzed enzyme was increased by the addition of phosphorylated hexoses or pentoses, they suggested that the dialyzable factor is a metabolic intermediate. Subsequently, Monod and Rickenberg (1971) reported that one of the protein components (C-11) bound cAMP but did not hydrolyze it. The other protein component ((2-1)catalyzed the reduo tion of glutathione in the presence of NADPH or NADH. They reported that the cAMP hydrolysis took place when C-I and C-I1 were mixed with NADPH, NADH, or a thiol. These investigators proposed that the concentration of cellular reducing equivalents regulates cAMP phosphodiesterase activity. Monod and Rickenberg also reported that a mutant resistant to catabolite repression was defective in component C-I.

zyxw

D. CONTROLOF CATABOLITE REPRESSION OF ~GALACTOSIDASE SYNTHESIS Studies on the control of intracellular levels of cAMP by adenyl cyclase and phosphodiesterase activity suggest a possible mechanism by which a catabolite repressor molecule(s) indirectly regulates the synthesis of enzymes such as pgalactosidase. This model predicts that, during growth on glucose or other carbohydrates that give rise to catabolite repression, metabolites are generated which influence the intracellular concentration of cAMP by interacting with adenyl cyclase and/or phosphodiesterase (de Crombrugghe et al., 1969a; Goldenbaum et al., 1970; Monod et al., 1969). The ability of various metabolites to activate or inhibit these enzymes in oitro suggests that the formation of cAMP may be controlled by intermediary metabolism. This model is illustrated in Fig. 4. Buettner et al. (1973) showed that the transient catabolite repression of pgalao tosidase synthesis that occurs during the initial exposure of a suspension of glycerol-grown E. coli to glucose is not accompanied by a detectable change in the intracellular concentration of CAMP.The basis of transient catabolite repression is obscure, but these results indicate that transient repression may influence the expression of the

zyxw zyxwvu

REGULATION OF THE LACTOSE OPERON

43

lac operon by means other than alteration of cAMP levels. Although interaction of the hypothetical catabolite repressor with the lac permease to prevent entry of inducer molecules into the system is a possible explanation for transient repression, this possibility appears unlikely since constitutive mutants also are sensitive to transient catabolite repression (Tyler and Magasanik, 1969). Magasanik (1970) has suggested that the catabolite repressor may act directly on the initiation of transcription of the lac operon. However, no evidence is available to support this proposal.

zyxw zyxwvut zyxw

111. In V i m Evidence of Site of cAMP Action

A. TRANSCRIPTIONAL OR TRANSLATIONAL CONTROL

Essential to the understanding of the mechanism by which cAMP regulates pgalactosidase synthesis are experiments demonstrating whether or not this cyclic nucleotide exerts its effect at the level of mRNA transcription or translation. Conclusive evidence of a direct effect of cAMP on one or the other depends on experiments in which the two processes can be separated experimentally. This is a difficult condition to achieve in uiuo, since transcription and translation may be coupled processes. These processes may be physically coupled either physiologically, as suggested by Stent (1964), or in the sense of being controlled by regulatory molecules common to both. Although the relationship of transcription to translation has been widely studied, the crucial question whether or not the two processes occur independently in d u o has not been resolved. Most of the evidence obtained with intact cells has supported the suggestion that cAMP acts at the transcriptional level. Nakada and Magasanik (1964), Sells (1965), and Kepes (1963) performed experiments in which transcription of the lac operon was dissociated from translation by exposure of cells to inducer for a very short period of time, followed by removal of the inducer by filtration or dilution. These experiments showed that the presence of glucose inhibited transcription of lac mRNA but did not affect its translation. These results suggest strongly that the site of catabolite repression is the transcription of mRNA. This observation was substantiated by Perlman and Pastan (1968b), who reported that cAMP increased the transcription of mRNA for pgalactosidase in the presence of glucose when protein synthesis was inhibited by chloramphenicol or amino acid starvation. The amount of mRNA accumulated in these studies was judged by the formation of active Pgalactosidase on the removal

44

zyxwvu

zy zyxwvut zyxw C. CARPENTER AND B. H. SELLS

of inducer, glucose, CAMP, and protein synthesis inhibition. These experiments, however, do not measure mRNA molecules made and degraded during the inhibition of protein synthesis. Evidence has been presented implying that cAMP influences the translation of pgalactosidase (Paigen and Williams, 1970; Parks et al., 1971) and tryptophanase (Pastan and Perlman, 1968a). These conclusions were based on studies in which proflavin was used to inhibit transcription. Although this dye has no effect on protein synthesis, Conde et al. (1971) reported that cAMP interferes with its inhibition of RNA synthesis. Similar experiments using rifampicin to inhibit transcription have shown, however, that tryptophanase synthesis (del Camp0 et al., 1970; Ramirez et al., 1972) and Bgalao

"[

zyxw 5O0r

A

SECONDS

zy

FIG. 5. Kinetics of accumulation of labeled lac mRNA. Escherkhia coll was grown to log phase. Thirty milliliters of this culture were placed in each of three

M).and IPTC, glucose, and CAMP flasks. IPTG (5x lo-' M),IPTG and glucose (lo-*M )were added simultaneously to the cultures 5% minutes before labeling. Specimens were removed after 5 minutes for pgalactosidase assay, and 30 seconds later (time zero) 1.5 mCi of uridine3H was added simultaneously to each flask. Samples of 10 ml each were then removed at 30,60,and 180 seconds for RNA extraction. Following measurement of optical density at 260 nm, acid-precipitable counts in 0.5 pg of RNA were determined for each preparation; the specific activities were computed and plotted in (A). RNA species, 0.5 pg each, were then hybridized for 20 hours at 75% with filters containing 0.3 pg of Ah80 or AMOdlac W-labeled in the presence of 22 pg of unlabeled R N A from a lac deletion strain. Counts bound to the hh80 filters were subtracted from counts bound to the kh8Odlac filters, and the differences were assumed to measure lac specific counts in each preparation. These values are plotted in (B)as a function of time. Induced culture (triangles); glucose-repressed culture (circles); CAMP-treated culture (squares). (From Varmus et al., 1970.)

REGULATION OF THE LACTOSE OPERON

zyx 45

zyxwv zy

tosidase synthesis (Jacquet and Kepes, 1969)are controlled at the transcriptional level by glucose and CAMP. The most conclusive evidence that cAMP regulates Pgalactosidase synthesis at the transcriptional level has been provided by experiments that titrate the level of lac mRNA by the DNA-RNA hybridization (Varmus et al., 1970).The effect of glucose and CAMP on the formation of lac mRNA is shown in Fig. 5. From studies by Varmus et al. (1970),which demonstrated that glucose and cAMP do not alter the rate of degradation of lac mRNA, it was concluded that cAMP and glucose monitor the rate of synthesis of mRNA. Also, Miller et al. (1971)showed that the synthesis of mRNA from the galactose (gal) operon is regulated by cAMP and glucose in a manner similar to the control of lac operon mRNA.

B. CAMP AND LAC PROMOTER MUTANTS Following the discovery that cAMP stimulates pgalactosidase synthesis by increasing the level of transcription, attempts were made to define the precise site of action of this cyclic nucleotide. Ullmann and Monod (1968)reported that the effect of cAMP is distinct from the repressor-operator control of the lac operon, since cAMP is active in mutants of both repressor and operator loci. This observation prompted investigation of the promoter region of the lac operon as a possible site of action of CAMP. To initiate these studies, experiments were performed to investigate pgalactosidase synthesis in promoter mutants of the lac operon. Examination of these mutants, originally isolated by Scaife and Beckwith (1966),led to the conclusion that the promoter is distinct from the operator. In support of this belief is the demonstration that promoter mutants are insensitive to catabolite repression, whereas operator constitutive mutations have no effect on catabolite repression. Mapping experiments indicate that the promoter region is located between the lac repressor cistron (i locus) and the lac operator (Ippen et aZ., 1968; Miller et al., 1968). Several laboratories have now concluded that the promoter region is the site of action of catabolite repression and cAMP (Pastan and Perlman, 1968b; Perlman et al., 1969; Silverstone et al., 1969, 1970;Yudkin, 1970). Although data have been presented indicating that catabolite repressors and cAMP do not interfere with lac repressor-operator interaction (Miller et al., 1968;Tao and Huberman, 1970;Yudkin and Reddy, 1971),it is by no means clear from studies in uiuo that the promoter and operator regions are not overlapping areas (Beckwith et aZ., 1972; Smith and Sadler, 1971).Studies by Beckwith et al.

46

zyxwvut zyxwv C. CARPENTER AND B. H. SELLS

(1972) indicate, however, that there are two distinct sites on the promoter. One of these is a site that normally promotes a low level of lac transcription, possibly by RNA polymerase holoenzyme alone. The second site is one through which cAMP and its binding protein stimulate lac transcription. Since much information pertinent to the function of cAMP and the promoter has been obtained from experiments with cell-free systems, the promoter function is discussed in more detail in Section IV.

zyxw

IV. CAMPAction in Vitro

A. In Vitro SYNTHESISOF ~GALACTOSIDASE

Although studies of cAMP action in uiuo have provided considerable insight concerning the regulation of pgalactosidase activity, the validity of conclusions has been limited by the complexity of the intact cell. A detailed analysis of Pgalactosidase synthesis was made possible by the development by Lederman and Zubay (1968) of a DNA-directed system capable of synthesizing active Pgalactosidase molecules in uitro. Although synthesis of pgalactosidase in this system is very inefficient compared to synthesis in d u o , nearly all the effects, that is, repression, induction, observed in uiuo can also be observed in uitro. Whether the effects observed in uitro are produced by similar mechanisms cannot be affirmed beyond doubt, since the conditions present in uiuo may not be faithfully replicated in uitro. A cell-free system has been developed to provide optimal conditions specifically for the synthesis of pgalactosidase. Several modifications of the reaction system have increased the efficiency of /3galactosidase synthesis to a larger extent than the synthesis of total protein, as judged by the incorporation of radioactive amino acid into acid-insoluble material. The most significant advance in the development of this system was the use of bacteriophage DNA which by genetic recombination had E. coli lac DNA incorporated into its genome. Since the phage genome is considerably smaller than the E. coli genome, the use of phage DNA in uitro meant considerable enrichment (1Wfold) of the lac operon in the system. A crude soluble fraction (S-30) of E. coli was used in this system to provide the necessary RNA and protein components required for transcription and translation. The S-30 was prepared from a strain of E. coli carrying a deletion of the lac operon, and thus addition of pgalactosidase to the assay system was avoided.

zy

zyxwvu zy REGULATION OF THE LACTOSE OPERON

47

Shortly after cAMP stimulation of Pgalactosidase synthesis in intact cells was first reported, Chambers and Zubay (1969; Zubay and Chambers, 1969) examined the effect of this nucleotide on enzyme synthesis in uitro. They showed that addition of 1 mM cAMP to a cell-free system increased the synthesis of Pgalactosidase 30-fold in uitro. At the same time, it also increased the efficiency of repression in oitro by the lac repressor from 50 to 95%. The effect on repression was explained by assuming that the presence of cAMP produces a higher percentage of transcriptional starts at the promoter site. The irrepressible synthesis observed in the absence of cAMP was thought to result from incorrect starts, probably near the beginning of the z cistron. The repression observed in this in uitro system was responsive to the inducer IPTG. In the presence of repressor, cAMP had little effect on Pgalactosidase synthesis. This fact, coupled with the increased efficiency of repression, suggested that cAMP increases the correct initiation of lac mRNA synthesis at the promoter site. That cAMP increases the amount of mRNA synthesized in uitro was demonstrated by de Crombrugghe et al. (1970). Using a DNARNA hybridization technique to detect lac mRNA, they showed that cAMP increased the rate of synthesis of lac mRNA 10-fold. Since cAMP did not increase the overall rate of RNA synthesis (the lac operon accounted for only 5% of the DNA), this constitutes evidence for a specific effect on lac mRNA synthesis.

zyxw

B. MEDIATION OF cAMP ACTION Although cAMP was observed to increase specifically the rate of synthesis of catabolite-repressible enzymes such as Pgalactosidase and galactokinase in uiuo and in uitro, it was difficult to imagine how this mononucleotide was able to recognize a specific DNA region. That another molecule might mediate the cAMP effect was indicated by an analysis of mutants unable to metabolize several carbohydrates, such as lactose, arabinose, and maltose (Perlman and Pastan, 1969). These mutants were divided into two groups based on their response to exogenous CAMP.In the presence of CAMP,one group of mutants was able to utilize the carbon compounds that were not metabolized in the absence of CAMP.The supression of the phenotype of these mutants by exogenous cAMP suggested that these strains are deficient in their ability to synthesize CAMP. Subsequent studies showed that many of these strains do not possess appreciable adenyl cyclase activity. A second class of mutants unable to synthesize catabolite-repressible enzymes did not respond to exogenous cAMP

48

zyxwv zyxw

G. CARPENTER AND B. H. SELLS

zyxwv

and appeared to have active adenyl cyclase systems. This observation suggested that the defect in this group of mutants might be in a molecule that mediates the action of cAMP on lac DNA.

1. Discoueqj of CAMP-Receptor Protein Further understanding of this system was provided by the discovery of a protein in the extract of wild-type cells that bound cAMP as reported by Zubay et al. (1970a) and Emmer et al. (1970). The mutants described above that were not responsive to added cAMP were tested for the ability of their cell-free extracts to promote pgalactosidase synthesis in oitro. The soluble fraction (S-30)prepared from these mutants was inactive in uitro, even in the presence of CAMP. However, when the purified CAMP binding protein, designated CAP or CPR, was added to these extracts together with CAMP,the synthesis of /3-galactosidase was stimulated fivefold. No stimulation was observed in the absence of CAMP.If extracts from wild-type cells were used, addition of the cAMP binding protein did not increase the amount of pgalactosidase synthesized in uitro. These results indicated that the mutants unable to synthesize pgalactosidase in the presence of cAMP were defective in this binding protein which was essential for cAMP action.

zy

2. Eflect of the Receptor Protein on CAMPAction in Vitro Perlman et al. (1970) and Arditti and co-workers (1970) demonstrated that the addition of cAMP and its binding protein increased the transcription of lac mRNA in uttro. The cell-free lac system was refined by de Crombrugghe et al. (1971b,c) and Eron et al. (1971; Eron and Block, 1971). The crude extract previously used for pgalactosidase synthesis was eliminated and replaced by purified components which were required for the transcription of lac mRNA. The essential elements of this purified system were double-stranded lac DNA, RNA polymerase containing sigma factor, and CAMP. In this system, transcription of RNA from the DNA template occurred on both strands of the double-stranded DNA template. However, the correct information for the lac operon is contained on only one strand, referred to as the correct strand, and mRNA synthesized from the other strand does not direct the synthesis of active enzymes. Both groups of investigators demonstrated that the addition of cAMP and its binding protein to the reaction mixture greatly increased the percentage of lac mRNA transcribed from the correct strand of the DNA template. Eron et a2. (1971) demonstrated that the sigma factor of

REGULATION OF THE LACTOSE OPERON

49

RNA polymerase was also necessary for stimulation of lac transcription. Sigma factor (Losick, 1972)is a small protein which affects the initiation of transcription of mRNA from certain promoters. The requirement for sigma factor in the lac system suggests that cAMP and its binding protein are not transcriptional factors that replace sigma factor to alter the specificity of RNA polymerase for catabolitesensitive promoters. However, this observation does not preclude the possibility that cAMP and its binding protein may modify RNA polymerase in a way other than replacement of sigma factor. An obvious possibility is that the cAMP binding protein may have a CAMPdependent protein kinase activity. Although protein kinase activity has been demonstrated in E. coli (Kuo and Greengard, 1969),no evidence has been reported indicating that the cAMP binding protein has kinase activity. Nevertheless, these studies have demonstrated that the action of cAMP on the lac operon is mediated by a protein molecule, and that the transcription of lac mRNA is stimulated in uitro by cAMP and its binding protein. It should be realized, however, that these results do not eliminate the possibility that cAMP also influences the translation of lac mRNA, as only transcription occurs in this system. Parks et al. (1971)and Nissley et al. (1971)showed that synthesis of galactokinase in uitro and transcription of the gal operon in uitro are also dependent on cAMP and the same binding protein required for control of the lac operon.

zyxw zyxw

C. PROPERTIESOF CAMP-RECEPTORPROTEIN

1. Physical Properties Investigations have been reported by several workers on the characterization of the cAMP binding protein. Anderson et al. (1971)and Riggs et ul. (1971)purified the protein to apparent homogeneity, and from sedimentation equilibrium studies concluded that its molecular weight is 45,000daltons. The protein is composed of two identical subunits as judged by sedimentation equilibrium studies in 6 M guanidine, disc gel electrophoresis in sodium dodecyl sulfate and identical amino terminal amino acid sequences. The binding protein is a basic molecule with an isoelectric point of 9.12and binds cAMP liters mole-'. Although with an association constant of 1.1 X studies suggest that there is one binding site per dimer, they do not rule out the possibility of two sites and a cooperative binding mechanism in which binding at the first site decreases binding affinity at the second site. Emmer et al. (1970)and Zubay et ul. (1970a,b)have

zyxw zyx

50

zyxwvuts zyxwvu zyxw G . CARPENTER AND B. H. SELLS

indicated that cAMP is reversibly bound and does not undergo chemical alteration during its attachment to the receptor protein. Based on the results of purification of the binding protein, Anderson et al. (1971) estimated that it constitutes 0.10% of the total protein in E. coli. This suggests that there are approximately 1300 molecules of binding protein per cell in E. coli. Recently, Saunders and McGeoch (1973) isolated a mutant of E. coli that appears to contain an altered cAMP binding protein. This altered binding protein stimulates pgalactosidase synthesis in vitro and in uiuo in the presence of cGMP. Although cGMP has been detected in E. coli (Hardman et al., 1971), no information has been presented defining its biological role. The antagonistic effect of cGMP on several CAMPdependent systems in eukaryotes has recently been discussed (Kolata, 1973). It remains to be determined whether cGMP is physiologically important in controlling CAMP-dependent systems, such as the lac operon, in bacteria. Nissley et al. (1972) investigated the effect of various cAMP analogs on the binding of cAMP to the receptor protein, and on the activity of receptor protein-dependent transcription of the gal operon in uitro. Their results iiidicated that the purine base, the ribose moiety, and the cyclic phosphate group are involved in the binding of the nucleotide to its receptor protein. Thus there is a marked specificity for cAMP to elicit the correct conformational change in the binding protein in order to stimulate transcription. 2. Binding to DNA Interaction of the receptor protein with other macromolecules in the presence and absence of cAMP has been studied. Binding of the receptor protein to RNA polymerase has not been detected (Nissley et al., 1972; Zubay et al., 1970b).The binding of the CAMP-receptor protein to DNA was studied by Riggs and co-workers (1971) and by Nissley et al. (1972). These workers demonstrated that CAMPreceptor protein binds to DNA and that the binding is dependent on the presence of CAMP.The binding is inhibited by cGMP, a competitive inhibitor of CAMP. It has not yet been possible to demonstrate specific binding to catabolite repression-sensitive genes or promoters. Binding has been observed with DNAs from salmon sperm, calf thymus, and chicken blood. The CAMP-receptor protein complex also binds to poly (dAT), denatured DNA, and single-stranded DNA. The binding is specific for DNA, however, as no binding was observed with rRNA or tRNA. Krakow and Pastan (1973) studied the effect of proteolysis on the binding of the receptor protein to DNA.

z

zyx zyxwv

REGULATION OF THE LACTOSE OPERON

51

They found that receptor protein binds to DNA in a CAMP-dependent manner at pH 8, but in a CAMP-independent manner at pH 6. These investigators suggest that CAMP-independent binding at pH 6 is the result of an increase in the net positive charge of the receptor protein. As mentioned previously, the isoelectric point of the receptor protein is 9.12. If the receptor protein is treated with a proteolytic enzyme (subtilisin, trypsin, or chymotrypsin) in the absence of CAMP,DNA binding at pH 8 or 6 is not affected. However, if proteolysis occurs in the presence of CAMP,binding to DNA at pH 8 is abolished, but the binding at pH 6 is not affected. Gel electrophoresis in sodium dodecyl sulfate indicates that the 22,500dalton subunit of the binding protein is reduced to a 12,500-dalton fragment by proteolysis. Treatment with proteases in the absence of cAMP had no effect on the 22,500-dalton subunit. Krakow and Pastan suggest that cAMP induces a conformational change in the receptor protein which is necessary for DNA binding and which accounts for susceptibility to the action of proteolytic enzymes. Studies of the interaction of cAMP with its receptor protein and the interaction of this complex with DNA in uitro will undoubtedly provide considerable information on the mechanism of gene expression.

zyxw zyxwv

D. INITIATIONOF

LAC

mRNA SYNTHESISin Vitro

1. Effect of Lac Promoter Mutations Eron and Block (1971) showed that when DNA prepared from bacteriophage DNA harboring promoter mutations of the lac operon is used as a template for lac transcription in vitro, lac mRNA is synthesized in a manner that reflects, qualitatively, the effect of these promoter mutations on Pgalactosidase synthesis in uiuo. When a preparation of DNA carrying a partial deletion of the lac promoter was used, 3-to 10-fold less lac mRNA was produced compared to wildtype lac DNA. The level of lac RNA was not increased by the addition of cAMP or cAMP binding protein to the reaction mixture. In viuo this mutation results in a 100-fold reduction in Pgalactosidase synthesis. These results suggest that the deletion in this mutant, L1, may have removed a portion of the lac promoter involved in the binding of CAMP-receptor protein. Eron and Block (1971) also examined the effect in oitro of promoter mutations that increase the synthesis of Pgalactosidase in viuo. When used to direct the transcription of lac RNA in vitro, these “superpromoter” mutations yielded increased levels of mRNA for the lac operon.

52

zyxwv zyx zyxw

G. CARPENTER AND B. H. SELLS

These results indicate that the levels of mRNA from the lac operon are regulated by the nucleotide sequence of the promoter region and the CAMP-receptor protein complex in uitro as well as in uiuo.

zyxw

2. Formation of Rifampicin-Resistant Znitiation Complexes

De Crombrugghe et al. (1971b)have provided evidence indicating that CAMP and the receptor protein are required for the binding of RNA polymerase to the lac promoter and the formation of a rifampicin-resistant transcription initiation complex. Rifampicin is known to bind to the beta subunit of E. coli RNA polymerase and thereby prevent the initiation of RNA synthesis. In the absence of rifampicin and nucleotides, however, RNA polymerase binds to DNA and forms an initiation complex capable of transcribing RNA when the nucleotides are added together with rifampicin. De Crombrugghe et al. (1971b) found that, when CAMPand the receptor protein were added together with RNA polymerase to DNA, transcription of RNA increased approximately 30-fold when the nucleotides and rifampicin were added to the system. This indicates that the cAMP-receptor protein complex increases the transcription of lac RNA by increasing the binding of RNA polymerase to DNA. It is possible that the CAMP-receptor protein complex may increase the binding of RNA polymerase to DNA by either of two mechanisms. The effector complex might interact with the polymerase in such a manner as to increase the affinity of the polymerase for proper DNA nucleotide sequences. This is unlikely, however, as no physical or enzymic interactions between the effector complex and RNA polymerase have been detected. As previously mentioned, the CAMP-receptor protein complex binds to DNA. This suggests that the effector complex may act by affecting the DNA binding sites for RNA polymerase in a positive manner. De Crombrugghe et al. (1971a) analyzed the binding of RNA polymerase and the effector complex to DNA by varying the concentrations of molecules in the formation of rifampicin-resistant initiation complexes. When the RNA polymerase concentration was held constant and the amount of DNA vaned, the concentration of CAMPreceptor protein required for maximal lac transcription was directly proportional to the amount of DNA. This suggests that the CAMPreceptor protein complex binds to DNA in a manner independent of RNA polymerase. De Crombrugghe et al. (1971a) also investigated the binding of RNA polymerase and CAMP-receptor protein to wild-type lac DNA and to DNA carrying a superpromoter mutation in the lac operon. In

REGULATION OF THE LACTOSE OPERON

53

the presence of high levels of CAMP-receptor protein and a fixed amount of DNA, the concentration of RNA polymerase needed for maximal transcription from each template was nearly identical, although twice as much RNA was transcribed from the template carrying the promoter mutation. However, when the levels of polymerase and DNA were held constant, higher levels of transcription of lac RNA from the DNA with the promoter mutation were achieved at a significantly lower concentration of CAMP-receptor protein than was required with the DNA template carrying a wild-type promoter. Based on these observations de Crombrugghe and co-workers (1971a) have proposed that the following sequence of steps occurs during the initiation of lac transcription:

z

zyxwvu zyxw zyxwvu

+

1. CAMP CRP + CAMP-CRP 2. CAMP-CRP lac DNA + CAMP-CRP-lac DNA 3. CAMP-CRP-lac DNA RNA polymerase + CAMP-CRP-lac DNA-RNA polymerase

+

+

The third step is probably a complicated process involving several reactions, and additional steps may be required prior to the actual formation of nucleotide bonds. The molecular mechanisms by which the CAMP-receptor protein alters the conformation of DNA so as to increase the binding of polymerase are not known. Beckwith and co-workers (1972) have presented genetic evidence suggesting that the promoter region of the lac operon contains two sites. They suggest that an operator distal site in the promoter, defined by the promoter deletion L1, is involved in the binding of the CAMP-receptor protein complex to the DNA template. An operator proximal site in the promoter is proposed to be the binding site for RNA polymerase. This proposal predicts that promoter mutants exist which affect the binding of RNA polymerase to the promoter but which do not affect the binding of CAMP-receptor protein to the DNA. To date, no such mutants have been reported.

3. Effect of the Repressor on Lac Transcription An important consideration in the regulation of the lac operon is the possible overlap of the operator and promoter regions. As pointed out by Beckwith et al. (1972), promoter mutants analyzed to date are those that affect the CAMP-receptor protein binding site in the promoter, and therefore cannot be used to define the operator proximal portion of the promoter. Smith and Sadler (1971) isolated

54

zyxwvuts zyxwvu zyxw zyxwv G . CARPENTER AND B. H. SELLS

presumed operator mutants that affect the level of lac operon expression. This suggests a possible overlap between the operator and promoter regions of the E . coZi lac operon. Chen et d.(1971) studied the effect of the lac repressor on the binding of RNA polymerase to lac DNA in uitro. Their results indicate that the repressor and polymerase bind to DNA independently. However, competitive binding between repressor and polymerase occurred when DNA with a superpromoter mutation in the lac operon was used. Since a single mutation can bring about an overlap of repressor and polymerase binding sites, these two sites must be very close if not directly adjacent to each other.

V. Additional Aspects of Lac Operon Regulation Although the CAMP-controlled initiation of mRNA synthesis at the lac promoter undoubtedly plays a major role in regulating the expression of the lac operon, it should be pointed out that other mechanisms may be involved in this complex process of gene activity. To date these additional processes are not well understood, and their physiological significance remains to be established. Therefore these areas are not within the scope of this article and are mentioned only briefly. Sequence studies of the mRNAs from the trp (Bronson et al., 1973) and lac (Maizels, 1973) operon have been reported. These studies indicate that the translation initiation sites of both messengers are preceded by large nucleotide sequences. It is not known whether these sequences have a role in regulating the expression of these operons. Numerous investigators have examined the proposed coupling of the transcription and translation processes in E. co2i. These studies of metabolic integration have not yielded conclusive results as to the possible regulatory role of protein synthesis in the synthesis of mRNA. Obviously, if the two processes are coupled in a physical manner as proposed by Stent (1964), or coupled by common regulatory elements, any process that affects protein synthesis exerts an important influence on transcription. The mechanism by which the expression of the lac operon results in a natural polarity of enzyme synthesis is not known (Miller, 1970). Polarity refers to the fact that equal amounts of enzyme are not synthesized from the three structural genes of the lac operon. The amount of enzyme synthesized decreases as the distance from the

zyx

zyx

zyx zyx zyxwv

REGULATION OF THE LACTOSE OPERON

55

site of RNA initiation increases. Since the structural genes are transcribed as one unit of RNA, this polar effect must be due to a differential translation of different segments of the mRNA. This could result from the secondary structure of the RNA, increased degradation of the 3’ end of the message compared to the 5’ end, or termination of transcription within the operon. Evidence has been presented from various systems to substantiate each of these explanations. However, the evidence does not yet appear conclusive. REFERENCES

Aboud, M., and Burger, M. (1970). Biochem. Biophys. Res. Commun. 38,1023. Aboud, M., and Burger, M. (1971a).Biochem. J. 122,219. Aboud, M., and Burger, M. (1971b). Biochem. Biophys. Res. Commun. 43,174. Abou-Sabe’, M. A. (1973). Nature (London),New Biol. 243, 182. Abou-Sabe’, M. A., and Nardi, R. (1973). Biochem. Biophys. Res. Commun. 51, 551. Adamson, L.,Gross, C., and Novick, A. (1970).In “The Lactose Operon” (J.R. Beckwith and D. Zipser, eds.), Dp. 27-47. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Anderson, W. B., Schneider, A. B., Emmer, M., Perlman, R. L.,and Pastan, I. (1971).J. Biol. Chem. 246,5929. Arditti, R. R.,Eron, L.,Zubay, C., Tocchini-Valentini, G.,Conway, S., and Beckwith, J. (1970). Cold Spring Harbor Symp. Quant. Biol. 35,419. Beckwith, J . R. (1970). In “The Lactose Operon” (J. R. Beckwith and D. Zipser, eds.), pp. 5-26. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Beckwith, J. R., Grodzickev, T., and Arditti, R. (1972).J . Mol. Biol. 69, 155. Benson, C. E., Brehmeyer, B. A,, and Gots, J. S . (1971). Biochem. Biophys. Res. Commun. 43,1089. Bourgeois, S. (1971). Cum Top. Cell. Regul. 4,39. Broman, R. B., and Dobrogosz, W. J. (1972). Bacteriol. Proc. p. 176. Bronson, M. J., Squires, C., and Yanofsky, C. (1973). Proc. Nut. Acad. Sci. U . S . 70,

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2335.

Buettner, M. J., Spitz, E., and Rickenberg, H. V. (1973).J . Bacteriol. 114,1068. Burstein, C., Cohn, M., Kepes, A., and Monod, J. (1965). Biochim. Biophys. Acta 95, 634. Chambers, D. A., and Zubay, G. (1969).Proc. Nut. Acad. Sci. U . S . 63,118. Chen, B., de Crombrugghe, B., Anderson, W. B., Gottesman, M.E.,Perlman, R. L., and Pastan, I. (1971). Nature (London),New Biol. 233,67. Conde, F., del Campo, F. F., and Ramirez, J. M. (1971). FEBS (Fed. Eur. Biochem. SOC.), Lett. 16, 156. de Crombrugghe, B., Perlman, R. L.,and Pastan, I. (1969a).]. Biol. Chem. 244,5828. de Crombrugghe, B., Perlman, R. L., Vannus, H. E., and Pastan, I. (1969b).J. Biol. Chem. 244,5828. de Crombrugghe, B., Varmus, H.E., Perlman, R. L., and Pastan, I. (1970). Biochem. Biophys. Res. Commun. 38,894. de Crombrugghe, B.. Chen, B., Anderson, W. B., Gottesman, M. E., Perlrhan, R. L., and Pastan, I. (1971a).J . Biol. Chern. 246, 7343. de Crombrugghe, B., Chen, B., Anderson, W. B., Nissley, P., Gottesman, M. E., Pastan, I., and Perlman, R. L. (1971b). Nature (London),New Biol. 231,139.

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de Crombrugghe, B., Chen, B., Gottesman, M., Pastan, I., Varmus, H. E., Emmer, M., and Perlman, R. L. (1971~). Nature (London),New Biol. 230,37. de Crombrugghe, B., Shaw, B., Rosner. J., and Pastan, I. (1972).Nature (London), New Biol. 241,237. del Campo, F. F., Ramirez, J. M., and Canovas. J. L. (1970).Biochem. Biophys. Res. Commun. 40,77. Emmer, M., de Crombrugghe, B., Pastan. I., and Perlman, R. L. (1970).Proc. Nut. Acad. Sci. U.S. 66,480. Eron, L. and Block, R. (1971).Proc. Nut. Acad. Sci. U.S. 68,1828. Eron, L., Arditti, R.,Zubay, G., Connaway, S.,and Beckwith, J. R. (1971).Proc. Nut. Acad. Sci. U.S. 68,215. Fox, C. F., Beckwith, J. R., Epstein, W., and Signer, E. R. (1966).J. Mol. Biol. 19,576. Gilbert, W.,and Muller-Hill. B. (1970).In “The Lactose Operon” (J. R. Beckwith and D. Zipser, eds.), pp. 93-109. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Goldenbaum, P. E., Broman, R. L., and Dobrogosz. W. J. (1970)./. Bacteriol. 103,663. Crodzicker, T.,Arditti, R. R.,and Eisen, H. (1972).Proc. Nut. Acad. Sci. U.S. 69,366. Hardman, J,, Robinson, G., and Sutherland, E. (1971).Annu. Reo. Physbl. 33, 311. Harwood, J., and Smith, D. H.(1971).Biochem. Bfophyr. Res. Commun. 42,57. Hempfling, W. P., and Breman, D. K. (1971).Biochem. Biophys. Res. Commun. 45, 924. H h t a . M., and Hayaishi, 0. (1965). Biochem. Biophys. Res. Commun. 21,361. Hirata, M., and Hayaishi, 0.(1967).Biochim. Biophys. Acta 149, 1. Ide, M. (1969).Biochem. Biophys. Res. Commun. 36,42. Ide, M. (1971).Arch. Biochem. Biophys. 144,262. Ide, M., Yoshimoto, A., and Dkabayashi, T.(1967).J.Bacteriol. 94,317. Ippen, K., Miller, J. H., Scaife, J., and Beckwith, J. (1968).Nature (London) 217, 825. Jacquet, M., and Kepes, A. (1969).Biochem. Biophys. Res. Commun. 36,84. Jobe, A,, and Bourgeois, S. (1972).J. Mol. Biol. 69,397. Jobe, A., and Bourgeois, S. (1973).J . Mol. Biol. 75,303. Jost, J., and Rickenberg, H. V. (1971).Annu. Reu. Biochem. 40,771. Kak, L., Kingsburry, D. T.,and Helinski, D. R. (1973).J . Bacterfol. 114,577. Kennedy, E. P. (1970).In “The Lactose Operon” (J.R. Beckwith and D. Zipser, eds.), pp. 49-92. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Kepes. A. (1960). Biochim. Biophys. Acta 40,70. Kepes, A. (1963).Biochim. Biophys. Acta 76,293. Kepes, A. (1971).J . Membrane Biol. 4,87. Khandelwal, R. L., and Hamilton, I. R. (1971).J . Biol. Chem. 426,3297. Khandelwal, R. L., and Hamilton, I. R. (1972).Arch. Biochem. Biophys. 151,75. Khandelwal, R. L.. Spearman, T. N., and Hamilton, I. R. (1973).FEBS (Fed. Eur. Biochem. SOC.), Lett. 31,246. Kolata, G. B. (1973).Science 182,149. Krakow, J. S., and Pastan, 1. (1973).Proc. Nut. Acad. Sci. U.S. 70,2529. Kuo, J. F., and Greengard, P. (1969).J . Biol. Chem. 244,3417. Lederman, M., and Zubay. G. (1968).Biochem. Biophys. Res. Commun. 32,710. Losick, R. (1972).Annu. Reo. Biochem. 41,409. McFall. E. (1973).J . Bacteriol. 113,781. Magasanik, B. (1961).Cold Spring Harbor Symp. Quant. Biol. 26,249. Magasanik, B. (1970).In ‘The Lactose Operon” (J.R. Beckwith and D. Zipser, eds.), pp. 189-219.Cold Spring Harbor Lab., Cold Spring Harbor, New York.

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Maizels, N. M. (1973). Proc. Nut. Acad. Scf. U.S . 70,3585. Makman, R. S., and Sutherland, E. W. (1963). Fed. Proc., Fed. Amer. Soc. E r p . Biol. 22, 470.

Makman, R. S., and Sutherland, E. W. (1965). J . Biol. Chem. 240,1309. Miller, J. H.(1970). In “The Lactose Operon” (J. R.Beckwith and D. Zipser, eds.), pp. 173-188. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Miller, J. H., Ipper, K, Scaife, J. G., and Beckwith, J. R. (1968). J . Mol. B i d . 38,413. Miller, Z., Vannus, H. E., Parks, J. S., Perlman, R. L., and Pastan, I. (1971). J. Biol. Chem. 246,2898. Monod, D., and Rickenberg, H. V. (1971). Bacteriol. Proc., p. 155. Monod, D., Janecek, J., and Rickenberg, H. V. (1969). Biochem. Biophys. Res. Commun. 35,584. Monod, D., Janecek, J., and Rickenberg, H.V. (1970). In “The Lactose Operon” (J. R. Beckwith and D. Zipser, eds.), pp. 393-400. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Miiller-Hill, B., Rickenberg, H. V., and Wallenfels, K. (1964). J. Mol. Biol. 10, 303. Nakada, D., and Magasanik, B. (1964). Biochtm. Biophys. Acta 61,835. Nakazawa, A., and Tamada, T. (1972). Biochem. Biophys. Res. Commun. 49, 977. Nakazawa, T., and Yokota, J. (1973). J . Bacteriol. 113, 1412. Nealson, K. H., Eberhard, A., and Hastings, J. W. (1972). Proc. Nut. Acad. Sci. U.S . 69, 1073.

Nissley, S. P., Anderson, W. B., Gottesman, M. E., Perlman, R. L., and Pastan, I. (1971). J . Biol. Chem. 246,2419. Nissley, P., Anderson, W.B., Gallo, M., Pastan, I., and Perlman, R. L. (1972). J . Biol. Chem. 247,4264. Okabayashi, T., and Ide, M. (1970). Biochim. Biophys. Acta 220,116. Okabayashi, T., Yoshimoto, A., and Ide, M. (1963). J . Bacteriol. 86,930. Paigen, K., and Williams, B. (1970). Adoan. Microbial Physiol. 4,252. Parks, J. S., Gottesman. M., Perlman, R. L., and Pastan, 1. (1971). J . Biol. Chem. 246, 2,419.

Pastan, I., and Perlman, R. L. (1968a). J . Biol. Chem. 244,2226. Pastan, I., and Perlman, R. L.(1968b). Proc. Nut. Acad. Sci. U.S . 61, 1336. Pasta, I., and Perlman, R. L. (1970). Science 169,339. Pastan, I., and Perlman, R. L. (1971). Nature (London),New Biol. 229,s. Perlman, R. L., and Pastan, I. (1968a). Biochem. Biophys. Res. Commun. 30, 656. Perlman, R. L., and Pastan, I. (1968b).J. Biol. Chem. 243,5420. Perlman, R. L., and Pastan, I. (1969). Biochem. Biophys. Res. Commun. 37,151. Perlman, R. L., and Pastan, I. (1971). Cum. Top. Cell. Regul. 3, 117. Perlman, R. L., de Crombrugghe, B., and Pastan, I. (1969). Nature (London) 223,810. Perlman, R. L., Chen, B., de Crombrugghe, B., Emmer, M., Gottesman, M., Varmus, H., and Pastan, I. (1970). Cold Spring Harbor Symp, Quant. Biol. 35,419. Peterkofsky, A,, and Gazdar, C. (1971). Proc. Nut. Acad. Sci. U.S . 68,2794. Peterkofsky, A,, and Gazdar, C. (1973). Proc. Nut. Acad. Sci. U.S . 70,2149. Prusiner, S., Miller, R. E., and Valentine, R. C. (1972). Proc. Nut. Acad. Sci. U.S . 69, 2922.

Rall, T. W., and Sutherland, E. W. (1958). J . Biol. Chem. 232, 1065. Rall, T. W., Sutherland, E. W., and Berthet, J. (1957). J . Biol. Chem. 224,463. Ramirez, J. M., Conde, F., and del Campo, F. F. (1972). Eur. J . Biochem. 25, 471. Reznikoff, W. S. (1972). Annu. Reu. Genet. 6, 133. Riggs, A. D., Reiness, G., and Zubay, G. (1971). Proc. Nut. Acad. Sci. U . S . 68, 1222.

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471. Yang, H. L., and Zubay, G. (1973).Mol. Gen. Genet. 122,131. Yokota, T., and Cots, J. S. (1970).J. Bacteriol. 103,513. Yudkin, M. D.(1970).Biochem. J. 118,741. Yudkin, M. D.,and Reddy, S. (1971).Mol. Gen. Genet. 113,297. Zabin, I., and Fowler, A. V. (1970).In “The Lactose Operon” (J. R. Beckwith and D. Zipser, eds.), pp. 27-47. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Zubay, G., and Chambers, D. A. (1969).Cold Spring Harbor Symp. Quant. Bid. 34,

753. Zubay, G., and Chambers, D. A. (1971).In “Metabolic Regulation” (H. J. Vogel, ed.) Metabolic Pathways, Vol. 5, pp. 297-347. Academic Press, New York. Zubay, G., Schwartz, D., and Beckwith, J. R. (1970a).Proc. Nat. Acad. Sci. U. S. 66,

104. Zubay, G., Schwartz, D., and Beckwith, J. R. (1970b).Cold Spring Harbor Symp. Quant. Biol. 1,433.

zyx zy

Regulation of Microtubules in Tetrahymena'

zyxwvu zyxwvu NORMAN E. WILLIAMS

Department of Zoology, Unioersity of Iowa, Zowa City, Zowa

I. Introduction . . . . . . . . . . . . . 11. Regulatory Patterns and the Cell Cycle A. The Somatic Ciliature in the Cell Cycle . . . . . B. The Oral Apparatus in the Cell Cycle. C. Nuclear Microtubules . . . . . . . . D. Cycle-Independent Formation and Regression of Micro. . . . . . . . . . . . . tubules. 111. Microtubule Stability and Regression A. Somatic and Oral Microtubules . . . . . . . B. Nuclear Microtubules . . . . . . . . . . C. Conclusion. IV. The Dynamic Nature of Formed Microtubules . . . . A. Evidence in Tetrahymena . . . . . . . . . B. Turnover and Subunit Exchange . . . . . . . V. Control of Microtubule Formation. A. Tubulin Synthesis in Relation to Microtubule Formation B. Synthesis-Dependent Assembly of Preexisting Tubulin VI. Epilog: The Cell Cycle Revisited. References

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59 60 60 63

65 66 68 69 72 73 74 74 75 77

77 79 83 84

I. Introduction The recent development of in uitro systems for the study of microtubule assembly (Weisenberg, 1972; Borisy and Olmsted, 1972; Shelanski et d., 1973) offers an exciting and promising approach to the study of problems having fundamental significance in cell biology. However, in uiuo studies of microtubules should continue to be of importance for the total solution of many of these problems. Living cells possess the ability to form microtubules in specific places at specific times, and also to resorb them in a similarly controlled fashion. A combined approach, using both in uiuo and in vitro studies, will probably be required to develop a satisfactory understanding of the mechanisms by which cells regulate microtubules and microtubule complexes. This discussion is a review of the information available concerning the regulation of microtubules in the ciliate Tetrahymena pyraformis. The formation and regression of micro-

' Supported by

grant number GB-41389 from the National Science Foundation. 59

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zyxwvu zyxwv zyx NORMAN E. WILLIAMS

tubules in Tetruhymena is detailed first within the context of the cell cycle. Following this, general problems are discussed in relation to microtubule regulation in other systems. 11. Regulatory Patterns and the Cell Cycle

zyxwvu A. THE SOMATIC CILIATUREIN

THE

CELL CYCLE

The term somatic ciliature refers to the total body ciliature exclusive of the oral apparatus, or feeding structure. This system is extensive in Tetruhyrnenu, as is seen in Figs. 1 and 2. It is a major repository of microtubules. Each somatic cilium, containing the familiar 9 2 array of microtubules, extends outward from a basal body, or kinetosome, located beneath the surface within the cell cortex. Here

+

zyxwvuts

FIG.1. Scanning electron micrograph of T. pyrffonnfs showing the oral apparatus and somatic cilia, The bar indicates 10 q. Micrograph provided by J. J. Ruffolo, Jr.

REGULATION OF MICROTUBULES IN

zyxw Tetmhymena

61

zyxwv zyxwv

FIG.2. Tetruhyrnenu late in the cell cycle, showing somatic cilia, the anterior oral apparatus, and a newly forming oral apparatus (lower left) with many short cilia. Micrograph provided by J. J. Ruffolo, Jr.

each basal body is associated in a regular way with other structural elements, including precisely oriented bundles of additional cortical microtubules. A basal body, together with its attached cilium and associated cortical elements, is called a ciliary unit. A study by Allen (1967) provides a useful three-dimensional reconstruction of the somatic cortex of Tetrahymena.

1. Number and Distribution of Ciliary Units There are typically 17 to 19 longitudinal rows of somatic cilia (meridians or kineties) in T. pyrifomis, as can best be seen in the denuded cell in Fig. 4. Two of these, the ones abutting on the oral apparatus, are shorter than the remaining ones. Cells within a given strain may have fewer or more than 17 to 19 ciliary rows. This type of variation within a population increases in response to certain (usually deleterious) environmental changes. However, most cells

62

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return to near 18 rows (the stability center) under optimal growing conditions. Although the stability center is likely under genic control in Tetrahymena, as it is in the ciliate Euplotes (Heckmann and Frankel, 1968; Frankel, 1973), a genetic analysis of deviations from the stability center suggests that mechanisms of cortical inheritance operate as well in the determination of ciliary row number (Nanney, 1966). The first estimate of the total number of somatic ciliary units in Tetrahymena, by Furgason (1940), was close to 800. The strain he used is no longer available. An estimate in the commonly used strain GL can be made from the counts of Williams and Scherbaum (1959); here the total appears to progress from about 600 to 1200 over the cell cycle. Data on a strain in syngen 1by Nanney (1971a)provide an estimate of from about 450 to 900 over the cell cycle. Counts of basal bodies in cells at a common stage in the cell cycle in 24 strains representing 12 syngens (Nanney and Chow, 1974) show that considerable variation in total basal body number exists among strains. A point of great interest is the finding that the total number of ciliary units is apparently regulated independently of the number of ciliary rows. Nanney (1971b) has shown that cells of a single strain with different row numbers (from 16 to 25) show nearly equivalent numbers of total ciliary units.

2. Pattern of Increase and Segregation New ciliary units are added adjacent to old units within the rows. Basal bodies appear first, immediately anterior to old basal bodies. Next, they move anteriorly to occupy a position midway between old ciliary units, and then sprout cilia (Williams and Scherbaum, 1959). The process has been described at the ultrastructural level by Allen (1969). Probasal bodies form in what is now considered classic fashion, elongate, and tilt to contact the cell surface with their distal ends. Other cortical elements which are part of the ciliary unit begin to form during tilting. Ciliary outgrowth is the final event. An early morphological study of the pattern of increase in somatic ciliary units in Tetrahgmena (Williams and Scherbaum, 1959) showed that developing units are found in all regions of the body and at all times in the cell cycle. Detailed quantitative analyses have been carried out subsequently by Nanney (1971a) and Perlman (1973). The conclusion that emerges from these studies is that, to a first approximation, the pattern of increase is uniform in time (throughout the cell cycle) and space (all ciliary rows and regions within rows). There appears to be, however, a rate change in two of

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the rows near the new mouth during oral apparatus formation, and a slight overall reduction in the rate of increase within the cell at this time. Light microscopic observations show that equatorial breaks occur in the somatic ciliary rows beginning about three-quarters of the way through the cell cycle (Fig. 3); this has not yet been characterized at the ultrastructural level. The fission furrow forms subsequently, and the half-rows are segregated into the daughter cells.

B. THE ORAL APPARATUS I N THE CELL CYCLE Another 160 to 170 basal bodies, also associated with additional microtubules and fibrous material intracellularly, are found in the oral apparatus of T. pydfomis (Nilsson and Williams, 1966;Forer et ul., 1970; reviewed in Elliott and Kennedy, 1973). These basal bodies, most of them ciliated, occur in four groups, thus constituting the four compound ciliary structures of membranous nature from which Tetruhymenu derives its name. Three of the oral ciliary groups, called membranelles, are found along the (cell’s) left wall of

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FIG. 3. Outline of major events in the cell cycle of T.p y z i f o n i s GL. Formation of the oral apparatus for the posterior division product is indicated in the cell outlines at the top. The three membranelles (M) and the single undulating membrane (UM) of the oral apparatus are designated on the left. Macronuclear development is outlined in the lower figures. The approximate times in the cell cycle at which the given configurations occur are indicated by the bar at the bottom. The transition point (T.P.), which occurs in strain GL at about 78% of the way through the cell cycle, is the time of an abrupt transition from high to low sensitivity to a wide range of chemical and physical agents (discussed further in the text). The shaded zone indicates the part of the cell cycle during which oral primordium resorption can readily be induced.

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the oral cavity (M in Fig. 3).Each consists of three tightly packed rows of ciliated basal bodies; the outside membranelle (Ml)has about 57 cilia, the middle one (M2)has about 42,and the inner one (M3)has about 15.Membranellar cilia can be seen in the center of the old oral apparatus in Fig. 1, and in the new oral apparatus in Fig. 2.It is the undulating membrane that has nonciliated basal bodies. This structure is found along the right margin of the oral cavity, as indicated in Fig. 3 (labeled UM). It consists of a single row of about 27 ciliated basal bodies (to the viewer’s left in Figs. 1 and 4)and an adjacent row of about 27 nonciliated basal bodies next to the row bearing the cilia. The extensive tubular and filamentous interconneo tions within the oral apparatus at the level of the basal bodies (see Elliott and Kennedy, 1973,Fig. 14)permit its isolation as a cell fraction.

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1. Changes in the Old Oral Apparatus

No growth or morphological change occurs in the old oral apparatus of T . pyrffomis during the first three-quarters of the cell cycle. Although there have been no detailed quantitative studies, casual observation suggests that the overall size of the oral apparatus does not change in the cell cycle, nor does it change even during starvation (cf. Frankel, 1970, Figs. 10 and 12). Constant size is also suggested by the fact that the basal body counts that have been made (see above), although not numerous, have been fairly uniform. If any change occurs, it is probably slight. Extensive alterations occur in the old mouth, however, during the last quarter of the cell cycle. The old oral apparatus comes to lie on the surface of the cell, loses some fibers and the undulating membrane cilia, and shows a shortening of the membranellar cilia (see Frankel and Williams, 1973;Buhse et al., 1973).These remarkable regressive changes bring the old mouth to the precise state of the newly forming mouth in the cell at this time. Subsequently, during cleavage, the old oral apparatus redevelops the missing components in synchrony with completion of development of the new oral apparatus. The adaptive significance of these changes in the old oral apparatus during division is a mystery; perhaps there is some undetected growth, or deterioration, during the cell cycle which must be compensated for.

2. Formation of the New Oral Apparatus The old oral apparatus is inherited by the anterior division product, whereas the new oral apparatus is formed for the posterior

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division product in the midequatorial region of the parent cell cortex (Figs. 2 and 3). The first sign of oral apparatus formation is the appearance of a stomatogenic field of unciliated basal bodies. This occurs just prior to the half-way point in the cell cycle. The site of formation is nearly always adjacent to the right member of the pair of somatic ciliary rows which abut on the old mouth, in the manner shown in Fig. 3. This row is the so-called stomatogenic meridian. Ciliary growth begins early. Next, the nascent ciliary units move into double files to form three membranellar anlagen; the third row in each membranelle is added later. Undulating membrane formation starts later than membranelle formation, but the two processes overlap. The development described thus far occurs on the surface of the cell. The oral cavity is formed subsequently, along with various associated structures. Electron micrographs of the developing oral apparatus can be found in a recent article by Williams and Frankel (1973);the reader is referred to Frankel and Williams (1973) for further details of stomatogenesis and references to the literature.

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C. NUCLEARMICROTUBULES Tetrahymena typically have two different nuclei. One of the two types, the macronucleus, is hyperpolyploid, yet shows no visible chromosomes. This nucleus is found in all viable cells. The other, the micronucleus, is diploid, and shows chromosomes in both mitosis and meiosis, yet is absent in many strains. Unfortunately, most studies of microtubules and the cell cycle have been made with amicronucleate strains. Such a strain is GL, shown in Fig. 3. Most workers have failed to find microtubules in the interphase macronucleus (Elliott and Kennedy, 1973). Macronuclear microtubules appear in abundance, however, during macronuclear elongation. This process begins just prior to the onset of cytoplasmic cleavage, proceeds rapidly, and ends in the segregation of daughter nuclei. Macronuclear microtubules were first seen by Roth and Minick (1961) who described them as fibrillar elements in the dividing nucleus. Later workers showed them to be tubular fibers, and described their orientation. They have been described by Falk et al. (1968)and It0 et al. (1968)as occurring singly or in bundles, with a tendency to occur peripherally and show attachment to the inner nuclear membrane. Attachments to chromatin and nucleoli were also frequent, which led both groups of investigators to suggest that the microtubules may serve to anchor these structures to the nuclear membrane, Both groups also suggested that this might in some way assure the independence and/or segregation of diploid subnuclei.

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The membrane attachment of nuclear microtubules has been confirmed by Wunderlich and Speth (1970); Tamura et al. (1969) have noted that the tubules tend to be oriented along the long axis of the dividing macronuclei. The correlation between the appearance of microtubules and the elongation of the macronucleus suggests a possible role of microtubules in this process, and there is some experimental evidence for this (see Section 111).Other postulated functions remain, as do many things about the ciliate macronucleus, highly conjectural. The micronucleus, when present, divides mitotically within the persisting micronuclear membrane. Micronuclear and macronuclear division are well separated in time; the former begins earlier, about two-thirds of the way into the cell cycle, and is completed about the time macronuclear division begins. Elliott and Kennedy (1973) showed the presence of microtubules within the dividing micronucleus of Tetruahymena. There are few descriptive (and no experimental) data available on these tubules.

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AND REGRESSION OF D. CYCLE-INDEPENDENTFORMATION MICROTUBULES

Ciliary regeneration and oral replacement are two processes involving formation of microtubules which can occur in nongrowing cells, In addition, oral replacement always involves regression of microtubules, and ciliary regeneration may be preceded by ciliary resorption in some situations. Both processes, described in Section D, may be regarded as programs for microtubule regulation which are simplified by being uncoupled from the division cycle. As such, they offer certain advantages for experimental inquiry into the minimal mechanisms involved in formation and regression of microtubules. 1. Ciliary Regeneration

Tetrahgmenu can be divested of their cilia under conditions that leave the cells viable and capable of regeneration (Child, 1965; Rosenbaum and Carlson, 1969). The method involves concentrating the cells in a solution of appropriate ionic composition in the cold at pH 6.0, subjecting them to multiple shearings with a glass syringe and an 18-gauge needle, and then placing them in a recovery medium. Used first with strain W,the procedure works equally well, without modification, with strain GL (Nelsen, 1974; Rannestad, 1974). Partial deciliation can be accomplished by omitting shearing (Rannestad, 1974).

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Some cells may retain their oral cilia subsequent to the amputation procedure, but these drop off after about 10 minutes in the recovery medium. Regenerating cilia can first be detected by scanning electron microscopy (SEM)about 12 minutes after deciliation. The SEM observations of Rannestad (1974) and Nelsen (1974) have confirmed the general pattern of regeneration suggested by the original light microscope observations of Rosenbaum and Carlson (1969). The oral cilia appear and elongate prior to the majority of somatic cilia (Fig. 4). Ciliary growth within the mouth is synchronous, unlike the situation in oral apparatus development. The appearance and elongation of somatic cilia is asynchronous; also, early cilia tend to be distributed throughout all regions of the body (Fig. 4). The average cell regains motility about 45 minutes after amputation, but does not

FIG.4. Ciliary regeneration in Tetrahymena. This is a scanning electron micrograph of a cell 30 minutes after deciliation. Note that all the oral cilia have begun to grow out, but only a few scattered somatic cilia are visible. Micrograph provided by E. M. Nelsen.

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have the full complement of mature cilia until 100 minutes after deciliation.

2. Oral Replacement Members of the genus Tetrahymena can form an oral apparatus in the anterior region of the cell, as well as at the equator (reviewed by Frankel and Williams, 1973).In this case the new mouth replaces the old one which is resorbed. In T. pyriformis, oral replacement occurs in nondividing cells, and the new mouth is apparently identical to the one that is resorbed. The adaptive significance of this is not immediately apparent. Oral replacement occurs typically in T . pyrtfomnis after a nutritional shift-down. It has been found that the process can be synchronized in mass populations by administering the classic heat-shock program for induced division synchrony, after first starving the cells for amino acids (Frankel, 1970).As far as we know, the new oral apparatus forms in exactly the same way it does during division (see Section II,B), only the location is different. The replacement primordium forms immediately posterior to the old mouth, and then moves anteriorly to take the latter's place when resorption is complete. The simultaneous resorption of one mouth and formation of another, separated by only a few micrometers, is a dramatic demonstration of the precision of the spatial controls that operate in the ciliate cortex. Electon microscope study of oral replacement has shown that old oral cilia are resorbed either in situ, or after withdrawal into the cytoplasm; both methods operate simultaneously within each cell (Williams and Frankel, 1973).Cilia withdrawn into the cytoplasm show no associated membrane material, neither their own nor those of autophagic vacuoles. The absence of autophagic vacuoles suggests disassembly without degradation to amino acids. 111. Microtubule Stability and Regression

Microtubules are classified as stable or labile (Behnke, 1970)on the basis of their response to cold, colchicine, and high hydrostatic pressure. Labile tubules are directly disrupted by these agents, whereas stable tubules are not. These agents block the formation of both kinds of tubules, however. It will be seen in the present section that most Tetrahgmena microtubule systems, and perhaps all, either survive these treatments or are lost as an indirect consequence of them. The conditions under which these and other agents bring about microtubule regression in Tetrahymena are discussed,

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together with the information available on the possible mechanisms involved.

A. SOMATICAND ORALMICROTUBULES Cold, colchicine, and pressure all block the formation of microtubules in Tetrahymena, as is true in other systems. Regarding disruption of microtubules, the results are more complex. The oral apparatus is discussed first because there is more information about this system.

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1. Oral Structures Prior to the Transition Point The responses of both the anterior oral apparatus and the developing oral primordium differ on either side of the transition point (T.P. in Fig. 3). The transition point, which occurs at approximately 78% of the way through the cell cycle, is the time of transition from sensitivity to relative insensitivity in the cell cycle. Treatments by a wide variety of agents prior to the transition point result in excess division delays, that is, delays in excess of the duration of the treatment. Treatments after the transition point do not produce significant excess division delays (see Zeuthen and Rasmussen, 1972). The anterior oral apparatus does not regress as a result of treatment with any of the three agents under consideration if exposed prior to the transition point. The developing primordium does not regress, at least to any significant degree, when treated prior to the onset of membranellar organization. This occurs at about 62% of the way through the cell cycle (Fig. 3). From this time until the transition point, however, the developing oral primordium responds to treatment with cold (Frankel, 1962),colchicine (Nelsen, 1970),and pressure (Simpson and Williams, 1970)with a dramatic regression of all structural elements, including microtubules. This period of sensitivity of the developing primordium is indicated by the shaded zone in Fig. 3. Inducible regression notwithstanding, the oral primordium microtubules during this sensitive period should probably not be regarded as labile in the sense that this term has been applied to mitotic or other cytoplasmic microtubules. The reason is that there is considerable evidence suggesting that oral primordium resorption is an indirect consequence of treatment with cold, colchicine, or pressure. For example, it is well known that the loss of labile microtubules under the influence of these agents is rapid, reversible, and energy-independent. Oral primordium resorption, however, is slower, of an all-or-none character, and energy-dependent (Frankel, 1967).Partic-

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ularly illustrative of the fundamental difference between the two types of regression is the response to cold. Labile microtubules disappear during the cold treatment, whereas the oral primordium disappears only after the cold treatment is over and the cells have been returned to normal temperatures. Ultrastructural analysis has shown that every identifiable microtubular component of the developing oral apparatus is intact after 60 minutes at 2°C (R. J. Williams, unpublished). Loss of tubules follows only upon rewarming the cells. It must therefore be concluded that the loss of primordium microtubules is not due to a simple equilibrium shift toward dissociation of subunits, but rather to a process of active cellular degradation, possibly of enzymic nature, as originally concluded by Frankel (1967).It is known, furthermore, that the resorption mechanism can be triggered by heat shocks and a wide variety of metabolic inhibitors (see Frankel and Williams, 1973), as well as agents that affect microtubules. It thus appears that the period just before the transition point is a period of general cellular sensitivity, such that any perturbation of critical cellular processes leads to the initiation of oral primordium resorption, as well as to very long excess division delays (see Section VI for further discussion).

2. Oral Structures Following the Transition Point The situation after the transition point is very different. First, the anterior oral apparatus is now resorption-susceptible, probably because it too is undergoing developmental changes at this time (see Section 11,B).Second, metabolic inhibitors do not cause excess division delays, nor do cold, colchicine, or pressure. Finally, metabolic inhibitors and cold shocks do not trigger oral resorption, but colchicine and pressure do (in both oral apparatuses). The application of the latter two agents after the transition point therefore has severe consequences for the next generation; it produces daughter cells with nonfunctional mouths. The cells recover, however. Resorption of the old structures occurs, and new mouths form to take their place. Progress toward division, and development of both mouths, are halted by cold treatments initiated after the transition point. Upon rewarming the cells all development resumes and normal daughter cells are produced with no significant excess division delays and no oral resorption (Frankel, 1962). This establishes that blocking formation of microtubules per se is insufficient to trigger resorption, at least after the transition point.

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If this is true, and oral microtubules are stable, what are the mechanisms by which colchicine and pressure initiate oral resorption after the transition point? Nelsen (1970) showed that colchicine stops development of both oral apparatuses while permitting cell division to continue. In this case, unlike the situation with cold shocks, there is a dif5erentiaZ effect on cell development. It is perhaps the resulting imbalance that may be viewed as the specific trigger for posttransition oral resorption. Pressure must operate differently, because there is no differential blocking effect; both cleavage and oral development stop during treatment. Upon release of pressure, however, cleavage goes forward and oral development goes “backward.” An electron microscope analysis carried out on posttransition cells fixed under pressure may provide the answer. Moore (1972) reported no loss of microtubules, but what may be described as microtubule displacements were abundant. For example, ciliated basal bodies were found in the endoplasm, and noncylindrical associations of basal body triplets were seen. Perhaps such disruptions of microtubule positioning represent the specific stimulus for pressure-induced resorption, rather than tubule breakdown, or the blocking (or differential blocking) of microtubule formation in posttransition development. As Moore points out, however, the alterations after 10 minutes under 7000 psi pressure could also represent the first stages in the resorption process rather than the direct influences of pressure. Ultrastructural descriptions of oral regression are given by Moore (1972)and Williams and Frankel (1973).Cilia are resorbed in situ or intracytoplasmically. The ciliary doublets first lose the outer wall of the B tubule, then the inner wall, and last the A tubule. Doublet regression is asynchronous within a cilium. Basal bodies seem to disconnect triplets and then disintegrate into piles of granular material. All intracytoplasmic breakdown stages occur free in the cytoplasm; no relation to autophagic vacuoles has been seen in Tetrahymena.

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3. Somatic Microtubules Regression of microtubules of the somatic ciliary units in Tetrahymena has not been reported in connection with treatment by cold or colchicine. There is a report, however, that notes some loss of certain cortical microtubules following the application of 7500 psi of pressure for 10 minutes (Kennedy and Zimmerman, 1970). A similar study, using 7000 psi for 10 minutes, failed to note these effects (Moore, 1972). If confirmed, the somatic regressions may be due either to direct pressure effects or, as in the case of the oral appara-

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tus, to the initiation of active resorption mechanisms. Further work is required before it can be determined whether or not there are any labile microtubules in the cortex of Tetrahyrnena.

B. NUCLEARMICROTUBULES Macronuclear microtubules form during macronuclear elongation (Fig. 3), which occurs only after the transition point Kennedy (1969) and Wunderlich and Speth (1970) showed that the application of colchicine relatively early in the cell cycle prevents the formation of nuclear microtubules. This is undoubtedly correct. However, the usefulness of this demonstration is diminished considerably by the fact that the same result is almost certain to be obtained with the application of cycloheximide, or any of several agents, prior to the transition point. Colchicine, and all the rest, simply prevent the cell from entering the phase of the cell cycle to which nuclear microtubule formation is restricted. These studies therefore accomplish the dubious feat of nonspecifically preventing the formation of nuclear microtubules with an agent that specifically blocks microtubule formation. This criticism is circumvented in a precise study by Tamura et al. (1969),in which colchicine was added to synchronized cells after the transition point. They found that colchicine applied at this time had no blocking or delaying effect on either macronuclear or cell division. However, nuclear cleavage was typically unequal. Many daughter cells were produced with macronuclei that were either smaller or larger than normal, and some had no nuclei at all. On the basis of these results and their electron microscope observations, Tamura et al. (1969) suggested that colchicine (1) blocks the formation of microtubules required for macronuclear elongation, and (2) causes the loss of existing macronuclear microtubules. It is possible that a block in the formation of tubules alone is sufficient to cause unequal nuclear divisions, thus the evidence for loss of preexisting tubules is entirely ultrastructural. Their electron microscope observations were not extensive, however, and it is possible that the nuclei they observed were cleaved nuclei; microtubules disappear normally very soon after nuclear division, which is completed well before cytoplasmic cleavage is over. For the present, the suggestion that the microtubules of the nucleus are colchicine-labile should perhaps be regarded as tentative. What is more certain is that colchicine blocks the formation of macronuclear microtubules required for nuclear elongation, which in turn leads to unequal macronuclear divisions. Studies of the effects of cold and high hydrostatic pressure

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on macronuclear microtubules have not been reported. No information is available regarding micronuclear microtubules.

CONCLUSION Most, and perhaps all, of the microtubules in Tetrahymena are stable, that is, they are not directly dissociated by cold shocks, colchicine, or high hydrostatic pressure. The possible exceptions are nuclear microtubules and certain somatic tubules. All three agents, as well as others, can lead to the loss of microtubules in Tetrahymena, however. This effect is mediated by an active resorption mechanism which can be triggered within the cell. Whether resorption is initiated depends on the agent, the microtubule system in question, and the position in the cell cycle. Thus the resorption mechanism is highly discriminatory, both spatially and temporally. The control of resorption is one of the most important problems in the in uiuo regulation of microtubules. It is important in Tetrahymena, and probably other cell types as well. It is even conceivable that some of the “labile” microtubules in other systems may, upon closer examination, prove to be stable microtubules which are regulated by resorption mechanisms. The studies with Tetrahymena suggest several possible activating circumstances for the resorption of stable microtubules, all or only some of which may actually apply. C.

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1. Subtle damage to microtubules may be caused by some agents, particularly high hydrostatic pressure. Tetrahymena microtubules tend to persist intact under pressure, but some sort of subtle intratubule damage cannot be ruled out as a trigger for the subsequent microtubule resorption process. 2. Microtubule displacements produced by some agents may represent a specific stimulus for resorption. This is suggested by the fact that microtubules in abnormal locations have been seen soon after the application of high hydrostatic pressure in some instances, whereas the tubules themselves appeared to be intact. 3. Blocking the development of microtubules per se may lead to the resorption of others already present. Although this may be true in some cases, it has been shown not to apply to cold-arrested oral primordia after the transition point. 4. An imbalance created by blocking microtubule formation without arresting other cell cycle developmental processes may be a specific trigger for resorption. This is suggested above in the case of colchicine-blocked oral primordium development after the transition

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point, and may apply as well to this system prior to the transition point. 5. An imbalance created by arresting progress in the cell cycle without blocking microtubule formation may also constitute a specific stimulus for microtubule resorption in some cases. Perhaps the resorption of the oral primordium that follows the application of diverse metabolic inhibitors prior to the transition point is brought about in this manner. 6. Abnormal patterns of microtubules, such as those formed after prolonged exposure to high temperature or colchicine, may trigger the resorption mechanism (see Frankel and Williams, 1973). 7. Specific microtubule resorptions may be included in the program for development in the cell cycle. Examples from Tetrahymena are the regression that occurs in the anterior oral apparatus after the transition point, and the disappearance of nuclear microtubules after division.

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IV. The Dynamic Nature of Formed Microtubules A. EVIDENCEIN Tetrahymena Ciliary and oral apparatus proteins have been shown to be turning over in nondeveloping, or morphostatic, structures. This was first demonstrated in an autoradiographic study of the oral apparatus (Williams et al., 1969). By using synchronized cells it was possible to show that pulse-administered radioactive amino acids are incorporated into proteins which can be localized within the nondeveloping anterior oral apparatus of growing cells. Furthermore, the rate was fairly high; incorporation into the developing oral primordium was greater by less than a factor of 2. A relatively rapid decline in specific activity of prelabeled anterior oral apparatus protein was also demonstrated following a chase with unlabeled amino acids. These results establish the dynamic character of the nondeveloping oral apparatus; proteins are in a constant state of flux within the system. Moreover, the flux rate is high enough to create problems of interpretation in certain types of experiments dealing with biogenesis. The flux of microtubule proteins, specifically, has been demonstrated within the morphostatic oral apparatus. In one experiment (Nelsen and Williams, unpublished), prelabeled cells were washed and suspended in fresh medium containing no labeled amino acids. Microtubule proteins (tubulins) were prepared from oral apparatuses isolated near the beginning of the chase and again after 9-13 hours

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(the development of new oral apparatuses was prevented during this period by intermittent heat shocks). Specific activity measurements from the tubulins of the two samples indicated a decline in specific activity of about 2.5% per hour (subtracting the heat periods, which also block turnover). These data, as well as previously published information (Williams and Nelsen, 1973), reflect the dynamic nature of nondeveloping oral apparatus microtubules. So far, the discussion pertains to the intracytoplasmic components of the oral apparatus only (of which basal bodies constitute the major fraction); this is because the oral cilia are removed during the oral apparatus isolation procedure. Nelsen (1974) investigated turnover of the microtubule proteins of morphostatic cilia in Tetrahymena, however, and presents evidence for its occurrence there as well. In these studies cells were pulse-labeled with radioactive amino acids and then washed into a chase medium incapable of supporting growth. It was found that the microtubule proteins of ciliary axonemes show a 2% per hour decline in specific activity which cannot be attributed to morphogenesis. Furthermore, it has been found that the microtubule proteins in nondeveloping cilia become labeled in pulse experiments at fully one-half the rate of labeling during ciliary regeneration (Nelsen, unpublished).

B. TURNOVER AND SUBUNIT EXCHANGE It appears from the above considerations that the protein subunits of nondeveloping microtubules in Tetrahymena are in a constant state of flux, entering and leaving the structures, both in growing and nongrowing cells. This process is referred to here as subunit exchange, or exchange. It has been referred to previously as turnover; however, this term is commonly used for the simultaneous synthesis and degradation of protein that occurs extensively in nongrowing cells. A certain amount of ambiguity is introduced therefore by referring to the flux of proteins within a structure as turnover, particularly when radioactive amino acids are used in nongrowing cells to demonstrate it. A terminology that distinguishes exchange from turnover and synthesis also focuses more clearly on questions of the interrelationships between these processes. For example, how do rate changes or blocks in synthesis or turnover affect subunit exchange, and vice versa? Answers to questions of this type would be greatly facilitated by the development of methods for isolating soluble subunits from the cytoplasm. This has not yet been reported for Tetrahymena tubulin. This view of subunit exchange has implications for the interpreta-

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tion of various types of labeling experiments. For example, it is recognized that drawing conclusions about the rate at which exchange occurs from the rate of incorporation of labeled amino acids into the macromolecular subunits isolated from structures is not justified unless it is known that the rate of synthesis, or turnover, is not limiting. In other types of experiments, independent information on subunit exchange may be of consequence for the interpretation of experiments dealing with such things as pool sizes or induced synthesis. For instance, a ratio of incorporation of labeled amino acids into the proteins of regenerating versus nonregenerating structures of, say, 20 :1, is suggestive of induced synthesis during regeneration. This result can also be obtained with no induced synthesis during regeneration, however, in a system that has a large precursor pool of macromolecular subunits which turns over rapidly but which exchanges with morphostatic structures at a low rate. These examples are not intended to represent actualities, but to illustrate the possibilities that must be considered until more is known about the relationships between synthesis, turnover and subunit exchange. The apparently high exchange rate of tubulin in Tetrahymena ciliary and oral microtubules may not be typical of all systems. In another ciliate, Eupbtes, Ruffolo (1970)showed that labeled proteins are differentially retained in the original oral apparatus, that is, the one that was formed during the pulse, for at least three generations of growth and division under chase conditions. G. W. Grimes (personal communication) had showed the same thing in Oxytricha oral cilia over eight generations. This type of relatively fixed localization of labeled proteins does not occur in Tetrahymena (Williams et al., 1969), and it suggests that there may be very different subunit exchange rates in the oral systems of these hypotrichous ciliates and Tetrahymena. The oral microtubules of ciliates in general are stable to cold, colchicine, and high hydrostatic pressure. It has been suggested that this type of stability may be due to low turnover (exchange) of microtubule subunits in these microtubules (Behnke, 1970; Raff et al., 1971; Olmsted and Borisey, 1973).The high exchange rates of stable microtubules in Tetrahymena, and the lack of correspondence of exchange rates with degrees of stability in the ciliates mentioned above, do not appear to support this idea. The site, mechanism, and adaptive significance of subunit exchange are all unclear at the present time. Subunits may in theory exchange within microtubules in restricted zones, or at sites distributed randomly throughout. The radioautographic data of Rosenbaum

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and Child (1967) are more suggestive of the latter possibility, although this has not been established. The mechanism of exchange within microtubules is also unknown, and there is little to suggest at the present time what its adaptive significance might be. One possibility is that it may provide a mechanism for repairing microtubules without resorbing and re-forming them.

V. Control of Microtubule Formation A. TUBULINSYNTHESIS IN RELATION TO MICROTUBULEFORMATION

Studies with inhibitors have shown that protein synthesis is required for the formation of the microtubule systems of Tetrahymena in most instances, but not in all. Protein synthesis is required for ciliary regeneration (Rosenbaum and Carlson, 1969; Nelsen, 1974; Rannestad, 1974), oral replacement (Frankel, 1970), and for oral (and probably somatic) microtubule formation in the cell cycle prior to the transition point (see Frankel and Williams, 1973). Protein synthesis is not required for somatic, oral, or macronuclear microtubule formation in the cell cycle after the transition point. This information, together with the fact that microtubules are composed primarily, if not exclusively, of tubulin, readily suggests the hypothesis that tubulin synthesis may be limiting for microtubule formation, that is, the assembly of microtubules may be controlled by the supply of tubulin subunits. According to this hypothesis, microtubule formation during oral replacement, ciliary regeneration, and development in the cell cycle before the transition point would involve de nouo synthesis of tubulin; microtubule formation after the transition point would proceed in the absence of protein synthesis, presumably because of the attainment of a threshold store of tubulin sufficient to insure the completion of cell and nuclear division. This hypothesis has been tested by exploring the relationship between the synthesis of tubulin and the formation of microtubules during oral replacement (Williams and Nelsen, 1973) and ciliary regeneration (Nelsen, 1974). Both studies used nongrowing cells in which the absence of net synthesis of protein provides an optimal background for detecting specific synthesis associated with morphogenesis. The experimental design used in both studies can be formulated in general terms as in Fig. 5. The cells were prelabeled by growth in labeled amino acids (solid half-circles in Fig. 5). Radioactive tubulin was then synthesized (solid circles), and both ciliary

zyx

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zyxwvu zyxw zyxwvutsrqp NORMAN E. WILLIAMS

A

Smil

assembly

T T

FIG.5. Experimental design used to test for induced synthesis of tubulin during ciliary regeneration and oral replacement in Tetahymena. Half-circles represent amino acids, complete circles represent tubulin, and aligned circles represent microtubules. Solid symbols denote labeled, and open symbols unlabeled, amino acids and tubulin. Result Cz indicates induced synthesis of tubulin during regeneration, whereas result C1 indicates the use of preexisting tubulin with little or no induced synthesis of tubulin during regeneration. Result C, was obtained in both oral replacement and ciliary regeneration. Further discussion is presented in the text.

and oral microtubules (aligned circles) were formed from labeled tubulin. The cells were next washed and transferred to a medium incapable of supporting growth, which also contained an excess of unlabeled amino acids (open half-circles). The old structures were then caused to regress or were physically removed (B); synchronous oral replacement was induced in one study, and the cells were deciliated in the other. The structures were then allowed to regenerate (C).If the new microtubules are formed from tubulin synthesized de nouo, they should contain predominantly unlabeled tubulin (C,). If, however, the new microtubules contain predominantly labeled tubulin (C,), their formation must be supported primarily by a pool of previously synthesized tubulin. This pool might be the old oral apparatus in the case of oral replacement (reutilization), but would necessarily be a soluble fraction in the case of ciliary regeneration because the old cilia were discarded. The critical information obtained was the relation between the specific activities of tubulin from the prelabeled (A) and regenerated (C, and C,) microtubules. The data in both studies conform to result C , in Fig. 5. The measurements indicate that about 6% of the tubulin of the oral basal bodies and associated microtubules, and less than 10% of the tubulin in ciliary microtubules, were synthesized during regeneration of the structures. It was concluded that the immediate requirement for protein synthesis during microtubule formation is not for bulk supply of tubulin subunits. Rather, the assembly of preexisting tubulin subunits must be controlled by the synthesis of some other factor (Fig. 6).

zy zy

zyxw z

REGULATION OF MICROTUBULES IN Tetrahymena

/\Assembly

79

r=u n

t2 w e s

Factor

Tubulin

zy

FIG.6. Scheme for the regulation of tubulin assembly in Tetruhyrnena.Ciliary and oral microtubules appear to be formed largely from preexisting stores of tubulin (see Fig. 5), yet de nouo synthesis of protein is required for assembly. The protein that must be synthesized is designated assembly factor in this figure. Assembly factor(s) may be a structural component of cilia, because ciliary regeneration can be obtained in the absence of protein synthesis if resorption of old cilia is induced. See text for further discussion.

Further studies suggest that this view of tubulin synthesis and assembly also applies to growing Tetrahymena. Bieber and Stone (1972)found that vinblastine (VLB) blocks the formation of microtubules in growing Tetrahymena but does not block synthesis of tubulin. By using appropriate pulse and pulse-chase experiments, they showed that ciliary microtubules formed during one doubling in cell number after release from a VLB block contained more tubulin synthesized during the block than after the block. Furthermore, the tubulin pool present at the end of the VLB block was unable to assemble into microtubules in the presence of cycloheximide. These data support the general conclusion that microtubule formation in Tetrahymena may involve extensive use of stored cytoplasmic tubulin, and that the assembly of this preexisting tubulin is synthesisdependent. The use of proteins from a previously synthesized pool was also shown in oral primordium development in growing cells by Williams et al. (1969). The chase experiments of Rannestad and Williams (1971)showing a decline in specific activity of oral apparatus tubulin of 50% per generation probably indicate a balanced increase in both pool and oral apparatus tubulin, rather than the lack of a cytoplasmic tubulin pool as originally suggested.

zyxwv

B.

SYNTHESIS-DEPENDENT

ASSEMBLYOF

PREEXISTING TUBULIN 1. Nature of the Synthesis Requirement What is the nature of the gene product presumed to regulate tubulin assembly (assembly factor in Fig. 6), and how does it operate? There may be more than one protein involved of course, and more than one mode of control. This appears to be probable, at

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least in ciliates which have many microtubule systems showing somewhat independent patterns of regulation. Given that there are pools of unassembled tubulin within the cell, and considering the thermodynamics of assembly (InouB, 1964), microtubule self-assembly is to be expected, unless there is either an assembly repressor (negative control) or some catalytic or structural component that must interact with tubulin before it can assemble (positive control). There is evidence for the latter type of control in other systems. For example, Mason and Schatz (1973) present evidence that mitochondrially synthesized cytochrome-c oxidase subunits regulate the assembly of the cytoplasmically synthesized subunits of this enzyme complex. A control mechanism for regulating the biogenesis of structures in which preexisting stores of proteins may be induced to assemble by the temporally regulated synthesis of one required component was predicted by King and Mykolajewcz (1973) on the basis of their studies of T4 assembly, although this does not occur in phage. The studies in Tetrahyrnena reviewed above suggest that it might be possible to identify tubulin assembly factors by their pattern of regulation. It has been found that exposure to a sublethal heat shock blocks the assembly of tubulin into microtubules (both net and via exchange), but does not block synthesis of total cell protein (Williams and Nelsen, 1973). Similarly, it has been shown that VLB blocks assembly of microtubules but does not block the synthesis of tubulin (Bieber and Stone, 1972). Both procedures may block the synthesis of tubulin assembly factors differentially, because the inability of tubules to form after these treatments in the absence of protein synthesis implies that there is no accumulation of these factors during the blocks. Alternatively, heat and VLB might impose an instability upon these proteins differentially. If the postulated tubulin assembly proteins are elements that are incorporated into structure, they should be easier to identify than agents acting catalytically or by means of negative control mechanisms. There is some indication, discussed in Section By2, that the proteins regulating ciliary regeneration are structural components of cilia, and that they might be identified by their pattern of de novo synthesis during regeneration.

zyx zyxwvu

2. ControZ of Microtubule Formation in Tetrahymena Evidence suggesting that the protein factor required for tubulin assembly during ciliogenesis may be a structural component of cilia comes from the work of Rannestad (1974), in which an exception to

REGULATION OF MICROTUBULES IN

zyxw z zyxw Tetrahymena

81

the rule that ciliary regeneration requires de nouo protein synthesis is reported. The permissive condition is that the cells resorb some of their preexisting cilia. This was found to occur after partial deciliation of Tetrahymena cells, which can be accomplished by eliminating the shearing step in the deciliation procedure. Quantitative study of a timed series of scanning electron micrographs has established that the cilia remaining attached to the body are not cast off, but are instead withdrawn into the cytoplasm within the first 20 minutes after partial deciliation. Regeneration of new cilia begins before resorption of the old cilia is complete. This regeneration, unlike regeneration in fully deciliated cells, is not dependent on protein synthesis. Thus it appears that the protein factor controlling ciliary regeneration can either be synthesized de nouo, or obtained by resorbing old cilia (Fig. 6). The measurements show that complete regeneration of body cilia does not occur in cycloheximide in partially deciliated cells. However, significantly more ciliary material is formed than is resorbed. Rannestad has suggested that the difference comes from cytoplasmic stores, and that material from the resorbed cilia in some nonstoichiometric way potentiates utilization of pool material. If the synthesis of a regulatory protein does control the assembly of preexisting tubulin, the former should have a much higher specific activity than the latter in experiments in which labeled amino acids are provided during ciliary regeneration. If, additionally, the regulatory protein is a structural component of cilia, as suggested by Rannestad's experiments, it should be possible to isolate it from cilia and to identify it by its relatively high specific activity in this type of experiment. Following this approach, Nelsen (1974)has suggestive evidence that a high-molecular-weight component of cilia isolated on SDS gels may contain the regulatory protein(s). The method of fractionation makes it impossible to say whether this material is a component of microtubules or of some other part of the cilium. In theory it could be either; if tubulin does not assemble without it, a critical interaction with tubulin is implied whether or not it is assembled into microtubules. However, it may be significant that microtubules assembled in uitro from assembly-purified tubulin apparently also contain some high-molecular-weight material (Granett et al., 1973). Some peculiarities of the oral apparatus system in Tetrahymena suggest that it may be regulated differently. It has been found in Tetrahymena (Nelsen, 1974),and also in Stentor (Plapp and Burchill, 1972),that oral cilia can regenerate, at least to a considerable extent, in the absence of protein synthesis. This is something that somatic

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NORMAN E. WILLIAMS

cilia cannot do. Either there are different assembly regulators for the two types of cilia, or at least oral cilia have first priority for limited supplies of a common factor. Another difference is that oral apparatus formation cannot be supported in the absence of protein synthesis by the resorption of old structures. The model in Fig. 6 can be modified to accommodate this difference by imagining that the assembly factor for oral microtubules is synthesized in conjunction with a protein that inhibits its function. Oral development would occur only when de nouo synthesis provides for neutralization of the inhibitor. Such a mechanism of negative control could account for the inability of the resorption of previous oral structures alone to promote oral development; resorption would make assembly factor available but would not inactivate the assembly inhibitor. These considerations perhaps do little more than emphasize the complexity that will be required to explain the total regulation of microtubules within a single cell. They lead additionally, however, to possible explanations for the change from synthesis-dependent to synthesis-independent microtubule formation, which occurs at the transition point in the cell cycle of Tetrahymena. According to the model in Fig, 6, transition may be correlated with the accumulation of enough assembly factor to ensure completion of the somatic, oral, and macronuclear microtubule formation required for cell and nuclear division. According to the negative control mechanism discussed above, transition could be achieved only if the assembly inhibitor were also inactivated simultaneously.

zy

zy

3. Assembly Factor Models Applied to Microtubules in Other Cell TY Pes There is evidence that mechanisms of the general sort discussed above may operate in several other cell types. Shubert et aZ. (1971) have established that neurotubule assembly during formation of cell processes in neuroblastoma cells in culture does not occur in the absence of protein synthesis. As in Tetrahymena, the required synthesis appears not to be of tubulin. Morgan and Seeds (1973) found no increase in the amount of tubulin during neurite formation, and Yamada and Wessels (1971) report the same for neutite production by cultured dorsal root ganglia under the influence of nerve growth hormone. Tubulin was measured by the colchicine-binding assay in both studies. Both mitotic spindle formation and ciliogenesis in sea urchin embryos require de nouo synthesis of protein, which in neither case appears to be tubulin. It was first shown by Hultin (1961), and later by

REGULATION OF MICROTUBULES IN

zyxw z Tetrahymena

83

Wilt et al. (1967),that protein synthesis is required for formation of the cleavage spindle in sea urchin eggs. Borisy and Taylor (1967) then discovered that unfertilized eggs contain large quantities of tubulin, and Raff et al. (1971)subsequently demonstrated large cytoplasmic tubulin pools from fertilization through the gastrula state. It is generally agreed that the formation of the mitotic spindle and the appearance of cilia at the blastula stage must both be controlled in some manner other than by the induced synthesis of tubulin. Stephens (1972)studied the synthesis of ciliary proteins during ciliogenesis in sea urchin blastula and has reported that at least six minor components can be identified that are synthesized at relatively rapid rates during ciliogenesis. He concludes that bulk ciliary proteins, including tubulin, are made prior to ciliation in considerable excess, and suggests that the morphogenetic process is marked by a round of de nouo synthesis of minor, but critical, structural components. Stephens (1972)further suggests that the regeneration of cilia reported to occur in the absence of protein synthesis in older embryos (Auclair and Siegel, 1966)may be supported by the earlier synthesis of multiple rounds of limiting proteins in this case. Regeneration of flagella in Chlamydomonas may be controlled in a similar manner. As in Tetrahymena, protein synthesis is required for regeneration (Rosenbaum et al., 1969),yet there is evidence that the synthesis of tubulin is not limiting (Rosenbaum, personal communication). Resorption of old flagella in Chlamydomonas can also support regeneration of new flagella in the absence of protein synthesis (Rosenbaum et al., 1969;Coyne and Rosenbaum, 1970).

zyxwv VI. Epilog: The Cell Cycle Revisited

The relation between the regulation of microtubules and the mechanism of heat-induced division synchrony in Tetrahymena requires a brief comment. Zeuthen and co-workers have shown that heat shocks produce excess division delays (setbacks) prior to the transition point, which are greater in older cells and lesser in younger cells (see Zeuthen and Rasmussen, 1972). By this means cells are brought together in time, and then progress in synchrony toward division after the heat-shock treatment. A recent hypothesis of the mechanism by which setbacks are induced visualizes the progressive development of structural entities within the cell, which are destroyed by the heat shocks (Zeuthen and Williams, 1969; Zeuthen, 1971; Zeuthen and Rasmussen, 1972). The question then arises whether microtubule systems in Tetrahymena constitute such

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zyxwvut zyxw zyx NORMAN E. WILLIAMS

developing structural entities. Certainly the developing oral primordium is set back, that is, resorbed, if treated during the membranellar organizing period (see Section 111). However, it is unlikely that excess division delays, and therefore induced division synchrony, result from direct effects on microtubular systems in Tetruhymenu. The major reason for this conclusion is the lack of a strict correlation between the resorption of microtubules and the production of excess division delays. On the one hand, a variety of agents, including colchicine (Nelsen, 1970) and VLB (Williams and Stone, unpublished), produces excess division delays at all times prior to the transition point yet, as far as we know, leads to a loss of microtubules only in the oral primordium, and here only part of the time. On the other hand, the oral primordium can be set back (caused to resorb) without attendant excess division delays, for example, by the application of high hydrostatic pressure after the transition point (see Section 111). In addition, the dissociability of microtubule formation from the cell cycle (see Section I1,D) argues against the notion of microtubules as central to the control of the cell cycle. Rather, the mechanisms of microtubule biogenesis and regression should perhaps be regarded as quasi-autonomous, showing integration with fundamental cell cycle control mechanisms only at certain times and in specific regions. ACKNOWLEDGMENTS The author thanks Dr. E. Marlo Nelsen, Dr. Joseph Frankel, and Ruth Jaeckel Williams for helpful discussions and a critical review of the article. I also thank Dr. John J. Ruffolo, Jr., for the micrographs reproduced in Fig. 1 and 2,and Dr. E. Marlo Nelsen for the micrograph reproduced in Fig. 4.

zyxwvu REFERENCES

Allen, R. D. (1967).J . Protozool. 14,553. Allen, R. D. (1969).1. Cell Biol. 40,716. Auclair, W . ,and Siege], B. W.(1966).Science 154,913. Behnke, 0.(1970).Int. Reo. E x p . Pathol. 9,1. Bieber, R. W..and Stone, G. E. (1972)./.Cell B i d . 55,lQa. Borisy, G.G.,and Olmsted, J. B. (1972).Science 177,1196. Borisy, G . G.,and Taylor, E. W. (1967). J. Cell Biol. 34,535. Buhse, H. E., Stamler, S. J., and Corliss. J. 0. (1973).‘frans.Amer. Microsc. SOC. 92,

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Child, F. M. (1965).J. Cell Biol. 21, Ma. Coyne, B., and Rosenbaum, J. L.(1970).J. Cell Biol. 47,777. Elliott, A. M.,and Kennedy, J. R. (1973).In “The Biology of Tetruhymena” (A. M. Elliott, ed.), pp. 57-87.Dowden, Hutchinson, & Ross, Stroudsburg. Pennsylvania.

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Falk, H., Wunderlich, F., and Franck, W. W. (1968).J . Protozool. 15,776. Forer, A., Nilsson, J. R., and Zeuthen, E. (1970).C. R. Trau. Lab. Carlsberg 38, 67. Frankel, J. (1962).C. R. Trau. Lab. Carlsberg 33, 1. Frankel, J. (1967).J . E r p . Zool. 164,435. Frankel, J. (1970).J . E r p . Zool. 173,79. Frankel, J. (1973).J . E r p . Zool. 183,71. Frankel, J., and Williams, N. E. (1973). In “The Biology of Tetruhymena” (A. M. Elliott, ed.), pp. 375-409. Dowden, Hutchinson, & Ross, Stmudsberg, Pennsylvania. Furgason, W. H. (1940).Arch. Protistenk. 94,224. Granett, S., Dentler, W., Whitman, G. B., and Rosenbaum, J. L. (1973).J . Cell Biol. 59, 119, Heckmann, E, and Frankel, J. (1968).J . E r p . Zool. 168, 11. Hultin, T. (1961).Erperientia 17,410. Inoub, S . (1964). In “Primitive Motile Systems in Cell Biology” (R.D. Allen and N. Kamiya, eds.), pp. 549-594. Academic Press, New York. Ito, J., Lee, Y. C., and Scherbaum, 0. H. (1968).Exp. Cell Res. 53,85. Kennedy, J. R. (1969).In “The Cell Cycle: Gene Enzyme Interactions” (G. M. Padilla, I. L. Cameron, and G. L. Whitson, eds.), pp. 227-248. Academic Press, New York. Kennedy, J. R., and Zimmerman, A. M. (1970).J. Cell Biol. 47,568. King, J., and Mykolajewycz, N. (1973).J. Mol. Biol. 75,339. Mason, T. H., and Schak, G. (1973).J. Biol. Chem. 248,1355. Moore, K. C. (1972).J. Ultrastruct. Res. 41,499. Morgan, J. L., and Seeds, N. W. (1973).J.Cell Biol. 59,233a. Nanney, D. L. (1966).Genetics 54,955. Nanney, D. L. (1971a).Deuelop. Biol. 26,296. Nanney, D. L. (1971b).J . Exp. Zool. 178, 177. Nanney, D. L., and Chow, M. (1974).Amer. Natur. 108,125. Nelsen, E. M. (1970).J . E r p . Zool. 175,69. Nelsen, E. M. (1974).Ph.D. Thesis, University of Iowa, Iowa City. Nilsson, J. R., and Williams, N. E. (1966).C. R. Trau. Lab. Carlsberg 35, 119. Olmsted, J. B., and Borisy, G. G. (1973).Annu. Reo. Bfochem. 42,507. Perlman, B. S. (1973).J . Exp. Zool. 184,365. Plapp, F. V., and Burchill, B. R. (1972).J. Protozool. 19,663. RaE, R. A., Greenhouse, G., Gross, K. W., and Gross, P. R. (1971).J . Cell Biol. 50,516. Rannestad, J. (1974).J . Cell Biol. In press. Rannestad, J., and Williams, N. E. (1971).J . Cell Biol. 50, 709. Rosenbaum, J. L., and Carlson, K. (1969).J . Cell Biol. 40,415. Rosenbaum, J. L., and Child, F. M. (1967).J . Cell Biol. 34,345. Rosenbaum, J. L., Moulder, J. E., and Ringo, D. L. (1969). J . Cell Biol. 41, 600. Roth, L. E., and Minick, 0. T. (1961).J . Protozool. 8, 12. Ruffolo, J. J., Jr. (1970).J . Protozool. 17, 115. Schubert, D., Humphreys, S.,DeVitry, F., and Jacob, F. (1971).D e v e l o p . Biol. 25,514. Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973).Proc. Nat. Acad. Sci. U.S. 70, 765. Simpson, R. E., and Williams, N. E. (1970).J. E r p . Zool. 175,85. Stephens, R. E. (1972). Biol. Bull. 142,489. Tamura, S., Tsuruhara, T., and Watanabe, Y. (1969).E x p . Cell Res. 55,351. Weisenberg, R. C. (1972).Science 177,1104. Williams, N. E., and Frankel, J. (1973).J . Cell Biol. 56,441. Williams, N . E., and Nelsen, E. M. (1973).J. Cell B i d . 56,458.

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Williams, N. E.,and Scherbaum, 0.(1950).J. Embryol. Erp. Morphol. 7,241. Williams, N. E.,Michelsen, O., and Zeuthen, E. (1969).J . Cell Sci. I, 143. Wilt, F. H.,Sakai, H.,and Mazia, D. (1867).J . Mol. Biol. 27, 1. Wunderlich, F.,and Speth, V. (1970).Protoplasma 70,139. Yamada, K M.,and Wessels, N. K. (1971).Erp. Cell Res. 66,346. Zeuthen, E.(1971).Aduon. Cell Biol. 5111. Zeuthen, E.,and Rasmussen, L. (1972).In “Research in Protozoology” (T. T. Chen, ed.), Vol IV,pp. 9-145. Pergamon, Oxford. Zeuthen, E.,and Williams, N. E. (1969).In “Nucleic Acid Metabolism, Cell Differentiation and Cancer Growth” (E.V. Cowdry and S. Seno, eds.), pp, 203-217. Pergamon, Oxford.

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Cellular Receptors and Mechanisms of Action of Steroid Hormones

zyxwvuts SHUTSUNGLIAO

The Ben May Laboratory for Cancer Research and the Department of Biochemist y.The Unioersity of Chicago. Chicago. Zllinofs

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I. Introduction 87 90 I1 Steroid-Binding Proteins in Blood 92 I11. Steroid Receptors in Target Tissues A Estrogen Receptors . . . . . . . . . . . . . . . 92 B. Androgen Receptors. 101 C. Progestin Receptors 109 D. Glucocorticoid Receptors 113 E . Mineralocorticoid Receptors 120 F. Steroid Receptors in Brains . . . . . . . . . . . . 123 125 C . Receptor and Steroid Dependency of Cancer IV. Cytoplasmic-Nuclear Interaction of Steroid Receptors . . . . 127 A . Transformation and Nuclear Retention of Cytoplasmic Receptors . . . . . . . . . . . . . . 127 131 B. Cytoplasm-Independent Nuclear Receptors 132 C. Chromatin Acceptor Sites for Receptors. D . Ribonucleoprotein Binding of Receptors . . . . . . . 137 E . Intracellular Recycling of Receptors . . . . . . . . . 139 V. Gene Expression and Steroid Receptor 139 A . RNA Synthesis and Protein Induction . . . . . . . . 139 B. Hypothetical Models . . . . . . . . . . . . . . 144 C In Vitro Experimental Approaches . . . . . . . . . . 147 VI . Concluding Remarks . . . . . . . . . . . . . . . . 151 A. ReceptorandUptakeofSteroidbyCells . . . . . . . . 151 B. Natural Forms of Steroid Receptors . . . . . . . . . 151 C . Nature of Receptor-Steroid Interaction . . . . . . . . 152 D. Insect Hormones and Vitamin D3 . . . . . . . . . . 154 E . Cyclic AMP and Steroid Hormones . . . . . . . . . 155 References . . . . . . . . . . . . . . . . . . . 157

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I

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Introduction

One of the most significant developments in the study of the molecular process of steroid hormone action in the last decade was the discovery of proteins that can selectively bind active steroids in target cells . Although the role of these proteins in hormone action has not been clearly established. they exhibit various properties one 87

z

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zyx

can expect to be characteristic of the functional receptors in target tissues. Interest in the study of steroid receptor molecules in a variety of target tissues has gained momentum in the last 5 years, the annual number of publications on the subject increasing from a few dozen to a few hundred during that time. This article covers those aspects related to the action of estrogens, androgens, progestins, glucocorticoids, and mineralocorticoids in vertebrates. The search for receptor proteins for these steroid hormones in most instances follows a unique pattern which includes (1) study of the uptake and retention of a radioactive hormone; (2) identification of the hormone presumed to be the active form; (3) detection and isolation of a specific protein that binds an active steroid but not an inactive steroid with a high affinity, and exists in larger amounts in target cells than in insensitive cells; and (4) demonstration that steroid antagonists can interfere with receptor binding of an active steroid. Studies on steroid receptors have been recently extended to several more biodynamic aspects such as qualitative and quantitative analysis of receptor proteins and their relation to biological responses of target cells and to some clinical situations. Concentrated efforts, however, are being made to discover the molecular mechanism whereby a steroid receptor complex may participate in the regulation of gene expression of the target cells. For this reason extensive studies have been carried out on the interaction of the steroid-receptor complex and the nuclear components of target cells. Numerous articles and books are available for readers interested in more comprehensive descriptions of the effects of various steroid hormones on cellular metabolic activities, including gene transcription (RNA synthesis) and translation (protein synthesis) (WilliamsAshman, 1965; Tata, 1966; Litwack, 1970, 1972; Smellie, 1971; McKern, 1971, 1974; Rasp&, 1971; Pasqualini and Scholler, 1972; Karlson and Sekeris, 1973; O’Malley and Means, 1973; Pitot and Yatvin, 1973; Rickenberg, 1974; Niu and Segal, 1974). This article reviews only those aspects that have been discussed in relation to the function of steroid receptors. Since many methodological aspects of the hormone receptor study can be found in a forthcoming volume of Methods in Enzymology (Academic Press), they are not included. For convenience, the chemical structures of many representative steroid hormones, natural or synthetic, and their antagonists are shown in Fig. 1. Since steroid hormones are transported from their production sites to many target tissues, a brief summary of the steroid-binding proteins in blood is presented in the next section.

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CELLULAR RECEPTORS FOR STEROID HORMONES NATURAL

SYNTHETIC

aP

0’-

5o-Dihydrotestosterone

89

ANTAGONISTIC

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Cyproterone

Z-Oxa-1Io-methyl-17~3-hydroxyestra-4.9.Il-trien-3-one

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CH30&

Diethylstilbestrol

iln-Eth~yl-I!l-nortestosterone

Progesterone

Nafoxidine

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CHZOH

CH20H LO

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Dexmethasone

Cortisol

HO

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0-CH

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90- Fluorocortisol

Cortexolone

SDirolactone

FIG. 1. Chemical structures of natural and synthetic steroid hormones and their antagonists often used in the study of steroid receptors in target tissues.

90

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11. Steroid-Binding Proteins in Blood

The dynamics of steroid hormone distribution in the body are very complex. The process involves biosynthesis, distribution by means of the blood, interaction with blood and tissue components, metabolic alterations, and excretion. Each of these factors in turn is also affected by many other factors, including individual and diurnal variations. Biochemically, these conditions ultimately govern the availability of the active forms of the steroid to the cellular receptor sites, and the duration of hormonal effects on the target cells. The blood steroid concentrations appear to be of the order of 10-100 nM for androgens (Liao and Fang, 1969), glucocorticoids, and progestins, and of the order of 1 nM or lower for estrogens and mineralocorticoids (Diczfalusy, 1970). Most of the blood steroids are disposed of continuously and rapidly, so that to maintain the hormonal status for long periods requires large amounts of steroids. For example, in young adult male mammals the plasma concentration of testosterone is of the order of 5 nglml, but the production rate for androgens is 2-10 mg per day in a human being and 25-160 mg per day in a bull (Mann, 1964). The binding of steroids to serum proteins has been reviewed in detail by Westphal(l970, 1971), Liao and Fang (1969),and others in published symposia (Pincus et al., 1966; Diczfalusy, 1970). Serum albumin, the most abundant protein constituent of plasma, has a low affinity and low steroid specificity, but a high capacity for steroid binding. More specific steroid-binding globulins are present in lesser amounts (-1%)but, because of their high affinity, large portions of blood steroids may be strongly bound to them if total blood steroid levels are low. Many blood steroid-binding proteins have been purified and characterized (Table I). Their affinity constant is in general of the order of lo* M-' or even higher. Many of them do not have very rigid steroid specificities and in many cases appear to recognize limited portions of the steroid molecules such as special functional groups on ring A or D. These properties distinguish them from the cellular receptors that have a high affinity for very specific groups of steroids. Their presence, however, often complicates the study of cellular steroid receptors. The biological function of the plasma steroid-binding globulins is not clear. To emphasize its possible importance in transport, a plasma globulin was called transcortin (Sandberg et al., 1966). In several cases the steroid-binding proteins appear to be responsible for slower metabolic clearance (Sandberg et al., 1966; Baird et

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TABLE I RELATIVE STEROID BINDING AFFINITY Steroid Testosterone Dihydrotestosterone Estradiol Estrone Progesterone Cortisol Corticosterone Aldosterone K a ir M-I

Human SAb Human CBG'

34

-

100 28 56 4 6 2 1.6 x 105

+

-

+

(+)

70 60 100

FOR

Human SBGd Human AAG'

33 100 20

-

2 1 1

(+)

-

1.0 x 109

1.2 x 109

67

-

11

-

100 2 11 8.9 x 105

SERUM PROTEINS'

Rabbit DBGf Guinea pig PBG'

Rat fetus EBGh

33 100 5 6

5 15 0

1

100

0 54 100 12

-

-

14 X loR

2.6 x loR

1

-

6.2 X

loR

0

0 0

9

-

15

Fr

s*

9

En B

cd

32

" For human SA, CBG, and AAG, the ratios of the affinity constants are compared using the strongest binder as 100. For others, they

Y

are standardized from the data shown in the references cited. L, SA, serum albumin (Westphal, 1970). CBG, Corticoid-binding globulin; +, moderately active; (+), very weakly bound. Existing in numerous species. Increased by estrogens and decreased by corticoids (Chader and Westphal, 1968a,b; Sandberg et al., 1966). SBG, Sex steroid-binding globulin. Elevated during pregnancy or by estrogens (Pearlman and Crepy, 1967; Vermeulen and Verdonck, 1970; Mercier-Bodard et al., 1970). AAG, &,-acid glycoprotein (Kerkay and Westphal, 1968). DBG, Dihydrotestosterone-binding globulin (Mahoudeau and Corval, 1973). PBG, Progesterone-binding globulin. Existing in pregnant guinea pig but not in unpregnant guinea pig. Cannot be induced by estrogen or progesterone. Not present in pregnant rats or women (Milgrom et al., 1973a; Lea, 1973a,b). EBG, Estrogen-binding globulin, but does not bind diethylstilbestrol (Swartz et al., 1973; Raynaud et al., 1971).A similar estrogenbinding protein is also found in the plasma of pregnant rats but not in normal and castrated adults. The estrogen binding is not inhibited by antiestrogens (Soloff et al., 1971). K,, Apparent affinity constants (4°C) for the strongest binders shown.

!a

0

2M

8 x

I?

0

3m

z

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al., 1969; Mahoudeau et al., 1973). This may partially explain, for example, the high level of blood progesterone in pregnant animals (Illingworth et al., 1970; Milgrom et al., 1973a), or high blood retention of estrogens in immature rats (Weisz and Gunsalus, 1973; Raynaud et al., 1971). In addition, blood steroid-binding globulins may protect steroids from degradation (Sandberg and Slaunwhite, 1963; DeHertogh et al., 1970; Corvol and Bardin, 1973). There is, however, no strong evidence that blood proteins can indeed act as a reservoir from which intact steroids can be fed gradually to target organs. In fact, the biological activity of steroids in plasma is probably exerted by the unbound fraction, which is only a small percentage of the total concentration. This view is supported by several experiments showing the suppression of hormonal activities by a prior globulin binding of the steroid hormones that are to be administered to experimental animals (Sandberg et al., 1966; Gala and Westphal, 1967). Whether any significant portions of globulin-bound steroids ever become active under normal circumstances in uioo, or whether steroid binding by blood proteins is involved in regulation of the ratios of various steroids with different biological activities (Burke and Anderson, 1973), still needs further study. The latter possibility is indicated by the fact that the blood content of many of these globulins is influenced by the endocrine status of the animals (Table I). Whatever the function of the steroid-binding proteins in blood may be, it should be noted that not all animal species contain similar sets of steroid-binding globulins in the blood. It is therefore apparent that these proteins are not directly involved in the action of steroid hormones as functional receptors at cellular sites.

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111. Steroid Receptors in Target Tissues

A.

ESTROGENRECEPTORS

1. Identification and Properties of Uterine Receptors The existence of receptor molecules for steroid hormones in target tissues was first indicated in a study of the uptake and retention of radioactive estrogens by mammalian tissues (Jensen and Jacobson, 1962). For example, immediately after the injection of a physiological dose of radioactive estradiol (17 pestradiol) into immature rats, uptake of the radioactive steroid by various tissues appears to reflect blood estrogen concentrations. The radioactivity then disappears

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rapidly from the blood and tissues less sensitive to estrogens (kidney, liver, muscle, and diaphragm), but only slowly from target tissues such as the uterus and vagina (Fig. 2). The retention, but not the total uptake of estrogen, can be saturated by increasing the estrogen doses to the animals, indicating the presence of a limiting number of specific binding sites with a high affinity for estrogens (Jensen et al., 1967a,b). The importance of estrogen retention is also emphasized by the finding that only estrogenic compounds, including diethylstilbestrol, but not nonestrogenic compounds can compete with estradioL3H for uterine binding, and that the process is antagonized by many antiestrogenic compounds (Callantine et al., 1968; Kahwanago et al., 1970; Geynet et al., 1972; Jensen et al., 1972a). The use of various estrogen antagonists such as ethamoxytriphetol, clomiphene, nafoxidine, and Parke-Davis (21-628 has been valuable in distinguishing specific estrogen binding from nonspecific steroid association by tissues in uiuo and in uitro. pM’MG

1

DRY TISSUE

/o

\

2

4

5

6

16

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FIG.2. Uptake and retention of radioactive estradiol by various tissues of immature rats receiving a single subcutaneous injection of 0.098 pg (11.5pCi) of estradiol3H. Total radioactivity is expressed as disintegrations per minute per milligram of dry tissue or disintegrations per minute per 5 p1 of blood. Since blood contains a mixture of radioactive metabolites but uterus and vagina incorporate only estradiol, the ratio of estradiol concentration between uterus and blood is about 500:l. (Details in Jensen and Jacobson, 1960, 1962.)

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The uterus of the immature rat (Jensen and Jacobson, 1962) or mouse (Stone, 1964) can retain estradiol firmly, but not other free or conjugated metabolites which are readily formed in liver and present in blood. Hexestrol, diethylstilbestrol, 17a-methylestradiol, 17aethynylestradiol, and estriol can also be retained by target tissues, all without prior chemical transformation (Jensen et al., 1967a,b; Laumas et al., 1970). Free l‘lphydroxyl and phenolic groups appear to be needed for uterine retention of estradiol (Noteboom and Gorski, 1965).Estrone retention by target tissues is less distinct than retention of estradiol. A portion of the estrone administered is reduced to estradiol and is retained. Autoradiographic and cell fractionation studies have shown that e ~ t r o g e n - ~localizes H in the nucleus as well as in the cytoplasm. Most of the radioactive estrogen retained by the uterus of immature rats is normally found in the cell nuclei (Talwar et al., 1964, 1968; Noteboom and Gorski, 1965; Jensen et al., 1968).Autoradiographic pictures show that nucleoli contain very little radioactive estrogen (Stumpf, 1969), but Arnaud et al. (1971) used a fractionation technique to demonstrate that the nucleolus can be a major site of estradiol retention. The presence of estradiol-binding protein(s) in the high-speed supernatant (cytosol) was first indicated by the failure of Sephadex gel, which excludes proteins but retains free steroids, to retain radioactive estrogen (Talwar et al., 1964). Toft and Gorski (1966) then found that, on ultracentrifugation in a sucrose gradient, the estradiolprotein complex sediments as a discrete band with a sedimentation coefficient of 9.5s. The protein appeared to be identical with the 8s components reported later by other investigators (Erdos, 1968; Rochefort and Baulieu, 1968; Korenman and Rao, 1968). These workers found that, when KCl or NaCl is present in the sample and gradient at concentrations of 0.2 M or higher, the 8s complex is reversibly transformed into a lighter complex which migrates slightly more slowly than bovine plasma albumin, or at about 4s. Addition of calcium ions and salt to uterine cytosol, prepared in the presence of EDTA, yields a “stabilized” 4s binding unit which after removal of salt does not revert to the 8s or larger aggregates (DeSombre et al., 1969; Jensen et al., 1969).Puca et al. (1971, 1972) have reported that the 8.6s form actually sediments as 5.3s in high concentrations of KC1 (>0.2 M).They consider 8.6s to be a dimer of the 5.3s form which may have a molecular weight of about 118,000. They have also presented evidence (Puca et al., 1972; Bresciani et al., 1973) showing that the 4.5s calcium-stabilized form (MW 61,000)

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95

is formed from the 5.3s complex by a Ca2+-activated “receptortransforming factor.” The factor appears to be a protease which promotes transverse cleavage of the 5.3s complex into two equimolecular fragments. Estradiol retained by the uterine nuclear fraction appears to be associated with a protein that can be solubilized by extraction with 0.3-0.4 M KCl at pH 7.4-8.5 (Jungblut et al., 1967; Puca and Bresciani, 1968). The solubilized estradiol-receptor complex sediments at about 5S, somewhat more rapidly than bovine plasma albumin, and therefore can be clearly distinguished from the 4 s cytosol complex (Jensen et al., 1969). The 5s complex, like the cytosol 4 s unit, can aggregate to the 8-9s form if salt is removed (Korenman and Rao, 1968). According to Steggles and King (1970), the uterus of mature rats contains a specific 4 s estrogen receptor that does not associate to form 8s. This 4 s complex is not found in ovariectomized rats, and therefore is not identical with the 4 s complex described by other workers. Such a qualitative difference in the uterine 4 s entity in mature, in ovariectomized, or in immature animals has not been detected in other laboratories (Chamness and McGuire, 1972; Vondehaar et al., 1970). Rochefort and Baulieu (1972), however, have reported the presence of the 4s form in uterine or nontarget tissue nuclei previously incubated with estradiol-labeled uterine cytosol. The nuclear 4s complex also does not revert to 8 s and differs from the cytosol 4 s and nuclear 5 s forms. DeHertogh et al. (1973a) have described a 3.5s estradiol-protein complex that can be extracted from crude uterine nuclei by tris-EDTA buffer (without KC1). The reported association constant ( K a ) for the cytosol complex from uteri of various species has been generally in the range of 10y-lO1o M-’ if analyzed by Sephadex gel filtration (Giannopoulos and Gorski, 1971b; Lee and Jacobson, 1971), charcoal absorption (Korenman, 1970),sucrose gradient centrifugation (Toft et al., 1967), equilibrium dialysis (Truong and Baulieu, 1971), or other techniques (Steggles and King, 1970; Notides, 1970) that separate bound from unbound steroid. Hahnel and Twadale (1973) have estimated the K, of the estradiol-receptor complex in human breast cancer to be 4 X 1OI2M-I. K, for the nuclear complex (by gel filtration method) has been shown to be about lo9M-’(Puca and Bresciani, 1969).Alberga et al. (1971), however, have claimed the presence of a nuclear receptor with an exceptionally high affinity for estradiol (K, l O I 4 A 4 - I ) in the nonhistone chromatin protein fraction. Techniques involving kinetic analyses for steroid association and dissociation have also indicated K, val-

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ues of 101’-1012M-’ (Best-Belpomme et al., 1970; Jensen and DeSombre, 1972). Such a high affinity has also been predicted from the study of estrogen uptake and retention in uiuo (DeHertogh et al., 1971) and in uitro (Alberga and Baulieu, 1968). The purification of receptor proteins for various steroid hormones generally faces two difficulties, instability and tendency toward aggregation. In addition, the quantities of receptor protein in target tissues are very low, and it is necessary to purify 100,000-fold to achieve a pure state. In earlier unsuccessful efforts, affinity chromatographic techniques involving estradiol-linked benzylcellulose (Jungblut et al., 1967; Jensen et al., 1967b) and to poly(viny1-Nphenylene maleimide) (Vonderhaar and Mueller, 1969) were employed. Sica et al. (1973a,b) also found that various agarose derivatives containing estradiol covalently bound through linkages with the A ring of the steroid molecules are ineffective as adsorbents for receptors. These investigators, however, found that agarose derivatives containing 19-nortestosterone 17-hemisuccinate can selectively bind estradiol receptors. The most useful adsorbents were prepared by attaching 17pestradiol 17-hemisuccinate to agarose derivatives containing albumin or the branched copolymer of poly(L-lysine) as a backbone and poly(DL-alanine) as side arms. These macromolecular “leashes” presumably allow the attached ligand to stay very distant from the agarose and also, by possessing many functional groups, permit a high degree of ligand substitution. By using this new group of adsorbents, the cytosol estradiol receptor can be purified between 10,OOO- and 100,000-fold with an overall yield of 30-50% in a single step. The purified Ca”+-stabilized receptor sedimented as the 4.5s form. The electrofocusing pattern showed two major peaks at pH 6.6 and 6.8. The estradiol-binding activity was destroyed by heating for 5 minutes at 65°C. Truong et al. (1973) also employed somewhat different affinity columns to purify partially the Ca2+-stabilized4 s estradiol-receptor complex of calf uteri. A specific activity of the purified material reached 167 pmoleslmg protein, but the recovery from the column (10-40%) was low. Using more conventional techniques involving ammonium sulfate precipitation, Sephadex G-200 gel filtration, and DEAE-cellulose chromatography, DeSombre et al. (1969, 1971) purified the Ca2+stabilized 4 s complex about 5000-fold (ca. 5% pure). Further purification by acrylamide gel electrophoresis yielded a material showing a single radioactive protein band by amido black staining (De Sombre et al., 1971).The nuclear form of the cytosol complex (or the transformed cytosol receptor; see Section IV,A) has also been puri-

CELLULAR RECEPTORS FOR STEROID HORMONES

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fied in a similar manner to a homogeneous component by gel electrophoresis and analytical ultracentrifugation. The purified complex sediments at 4.8s in sucrose gradients, with or without salt, and shows an isoelectric point (PI) of 5.8 and a Stokes radius of 36.5,indicating a molecular weight of about 72,000. It separates on gel electrophoresis from the Ca2+-stabilizedform of the cytosol receptor. Its amino acid composition has been determined (Gore11 et al., 1974).

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2. Biodynamic Aspects In rat uterus the receptor protein for estradiol is present as early as the first day of life. The estradiol-receptor complex also can be retained by uterine nuclei at this time. A sudden induction of the receptor proteins therefore is not the trigger for puberty (McGuire and Lisk, 1971). Somjen et al. (1973b) have shown that the receptor content reaches its peak at the age of 10 days (see also Clark and Gorski, 1970). The specific estrogen-induced protein found by Gorski (see Section V,A) is not found in 5-day old rats, but its rate of synthesis (after an injection of estradiol) at 10 days is about twothirds of that observed at 20 days. Since the rapid estrogen stimulation of amino acid incorporation into protein fractions does not occur until the fifteenth day, Somjen et al. (1973a) believe that the nuclear binding of the estradiol receptor and the synthesis of the induced protein may be necessary but not sufficient conditions for some trophic action of estradiol. In the immature rat uterus, there are about 100 fmoleslmg cytosol protein of 8s estradiol-receptor complex (Jensen et al., 1968). Estradiol can enhance the synthesis of its receptor as the general protein-synthesizing activity of the uterine cells increase. Such an effect is a relatively slow process compared to the more rapid effects of estradiol, which occur well within the first 30 minutes (see Section V,A). There is, furthermore, no rapid loss of receptor protein after the animals are deprived of estrogens. SarfF and Gorski (1971) estimated the half-life of the receptor proteins as about 138 hours. Estradiol bound to the receptors in the cytoplasm or nuclei appears to be able to exchange rapidly with the circulating hormone, as DeHertogh et al. (1973a,b,c) have indicated by a careful infusion technique. Whether this is secondary to a recycling of the receptor molecule is not clear. Estrogen treatment of the uterus in vivo or in vitro is known to promote rapid depletion of cytoplasmic receptors, which is accompanied by a parallel increase in the nuclear estradiol receptor content (Jensen et al., 1968; Giannopoulos and Gorski, 1971a). Ac-

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cording to Somjen et al. (1973a,b), cell nuclei of rat uterus during postnatal development contain about 3000 to 7000 receptor binding sites per diploid cell nucleus. Estradiol injection increases this number to as much as 38,000. The concentration of the nuclear estrogen-receptor complex in rat uterine cells closely parallels the level of ovarian estrogen during the estrus cycle (Lee and Jacobson, 1971). Calculation from the data of Clark et al. (1972) shows that in each diploid cell the number of cytoplasmic estradiol receptors increases moderately from 5580 sites in metestrus to 8100 sites in proestrus. The changes in the number of receptors retained by uterine nuclei are more dramatic: 1000 (estrus and metestrus), 3500 (diestrus), and 5OOO (proestrus) sites per diploid nucleus. (The number of molecules in each nucleus can be calculated by assuming 2.5 pg DNA per diploid nucleus for chick cells and 6 pg DNA per diploid nucleus for a mammalian cell. If there are a femtomoles per microgram DNA and each nucleus contains b picograms DNA, the number of molecules per nucleus is a X b X 600.) Since estrogenic responses are significantly higher in proestrus than in metestrus or diestrus, the fluctuations in the nuclear receptor contents appear to correlate well with biological activities. In the hypothalamus, pituitary, and uterus of rat and hamster (Lisk et al., 1972), estradiol retention is also low just prior to ovulation when the animal is producing much estrogen, and high early in the cycle when very little estrogen is present in the system.

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3. Universality Estradiol receptors have been most extensively studied in the uterus of experimental animals such as the rat, calf, and pig (Little et al., 1972), and also in human beings (Siiteri et al., 1972, 1973). Similar proteins have also been found in other estrogen-sensitive tissues such as the vagina, ovary, mammary gland, pituitary, and hypothalamus, as well as in the kidney, chick liver, pancreas (Sandberg et al., 1973), and testis (Brinkman et al., 1972). As noted later, it is debatable whether all the known estrogenic actions involve the same receptor mechanism. In this connection, it is important to note that King and Thompson (1974),after reviewing many estrogen-sensitive systems, concluded that there is no reason to suspect that different end-organ responses to estrogens are due to different forms of active estrogens. They pointed out that in most systems estradiol is the most potent natural estrogen. Jensen and Jacobson (1962) suggested that the action of estrone is due to its conversion to estradiol. Other studies in uiuo also show

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CELLULAR RECEPTORS FOR STEROID HORMONES

99

that estrone retained by the mouse uterus (Stone and Martin, 1964) or vagina (Martin and Baggett, 1964a) declines more rapidly than estradiol. In the vagina most of the radioactivity retained at 25 minutes after local application of e ~ t r o n e - ~ can H also be identified as estradioh3H (Martin and Baggett, 1964b). It has further been shown that e ~ t r o n e - ~can H bind to the same receptor that binds e~tradiol-~H, but that, at the low concentrations of steroids under which the estradi~l-~H-receptorcomplex can be transformed to the one retainable by uterine nuclei (see Section IV,A), the e~trone-~Hreceptor complex is not retained by the same nuclei. Whether estrone itself has estrogenic activity in target cells has been reinvestigated recently by Ruh et al. (1973).This study measured the relative activity of the three estrogens in uitm in stimulating the synthesis of an estrogen-inducible protein (IP) during incubation of uteri. Quantitatively, the induction for all three steroids closely paralleled the specific uptake of the steroid for cytoplasmic estrogen-binding protein (estradiol > estriol > estrone). Since during the incubation estradiol was not formed to any significant amount from estriol or estrone, it was concluded that each of these estrogens in its own right is a biologically active estrogen. Ruh et al. (1973) were not able to detect the formation of a nuclear estrone-protein complex, but this was thought to be due to rapid dissociation and not to the inability of estrone to form a biologically active complex. Geynet et al. (1972), however, have been able to find estrone and estriol-receptor complexes (both as 4 and 5s forms) in the extract of uterine nuclei previously incubated with cytosol and radioactive estrone or estriol. For human uterus, translocation of the cytoplasmic estradiol- or estrone-receptor complex to the nuclei can be demonstrated in uitro (Siiteri et al., 1972, 1973). One property often considered unique is that estriol, which is as active as estradiol in stimulating uterine water imbibition, is much less potent than estradiol in increasing uterine dry weight. Anderson et al. (1973a), however, believe that there is a good correlation between the amount of nuclear estrogen receptor in the uterine cells and the extent of water imbibition and increase in uterine weight, regardless of which estrogen (estradiol, estrone, or estriol) is used, Another interesting case is the glucose oxidation response, which can be elicited rapidly by quantities of estrone, estradiol, and estriol that are insufficient to produce maximal quantities of nuclear estrogen receptor in the uterus. This observation may indicate that the nuclear estrogen receptor complex is not involved, or that the quantity of the receptor molecules required is only a fraction of the total

100

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SHUTSUNG LIAO

number available (Anderson et al., 1973a). Sodium retention occurs during pregnancy, while plasma estrogen levels are high and estrogen at high doses can decrease the daily sodium excretion rate in humans and dogs (Johnson et al., 1970) and in adrenalectomized male rats (DeVries et al., 1972). Based on a study of steroid uptake and exchange, rat kidney appears to have specific and high-affinity (K,6.1 X 1OloW1)receptors for estrogens. The binding capacity appears to be low, the amounts being less than 10%of that found in rat uterus. A unique type of estrogen retention has been considered by Szego’s group, who showed that lysosome organelles of target organs of rats can retain estradioL3H very rapidly in uivo or in cell-free systems. Since protein-bound estradiol can be liberated from lysosomes, it was suggested that the formation of specific estrogen-protein complexes occurs in lysosomes and is involved in certain actions of estrogens (Szego, 1972; Szego and Seeler, 1973). By acridine orange fluorescent microscopy, it was observed that lysosomal components were present in high concentrations in nuclear fractions of preputial glands (and uteri) of rats pretreated with estradiol, diethylstilbestrol, or testosterone, but essentially absent in preparations obtained from animals injected with control (no hormone) or with 17aestradiol (nonestrogenic) solutions. The estradiol-induced response was reported to occur within 1 minute of hormone administration and was not observed in preparations from lung. Enzymic analysis also showed an increased release of hydrolytic enzymes from lysosomes, and nuclear metachromasy, upon hormone injection, suggesting that the nuclear surface andlor the intranuclear constituents might be modified. These effects were reported to be inhibited when the animals were treated with cortisol. Szego (1971, 1972) has proposed that alteration of the lysosomal membrane and the subsequent release of active agents such as vasoactive amines and hydrolases are responsible for multiple cellular events. She also believes that the primary effect of the intracellular action of estrogen receptors is reorientation of the cytostructure of the involved membranes. Such an effect on the plasma membrane is presumed to cause alteration of the cellular levels of cyclic AMP (see Section V1,E). Another type of estrogen retention presented by Tchernitchin (1973) and Tchernitchin and co-workers (1973) suggests that estrogens are retained somewhat specifically by rat uterine eosinophils. These investigators believe that the binding of estrogens by receptor molecules in uterine eosinophils is responsible for some of the early estrogenic responses, such as water imbibition, histamine

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CELLULAR RECEPTORS FOR STEROID HORMONES

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release, and estrogen-priming effects, and is different from the cytosol-nuclear (8s-5s)system. Williams, Rabin, and their co-workers (Sunshine et al., 1971) have also described an unusual observation in which estradiol in males and testosterone in females promoted binding of polysomes to smooth microsomal membranes of rat liver in uitro. Such a sex-specific effect was reported to accompany selective binding of these steroid hormones to microsomal membranes (Blyth et al., 1971, 1972). The possibility that there are multiple sets of receptor systems involved in different types of steroid hormone action deserves more study. The interesting observations made with lysosomes, eosinophils, and microsomal membranes need confirmation and further characterization before their significance in estrogen action can be properly evaluated. B. ANDROGEN RECEPTORS

1. Zdenti$cation and Properties of Prostate Receptor Earlier attempts to show selective retention of androgen by target tissues was rather difficult, principally because of the need to inject into experimental animals large quantities of androgens with a low specific radioactivity (see a review by Liao and Fang, 1969). Nevertheless, studies in rats by Pearlman and Pearlman (1961) and Harding and Samuels (1962) had indicated clearly that, while large amounts of conjugated metabolites could be found in the blood and liver, the prostate could accumulate unconjugated androgen metabolites. The uptake and retention of radioactive androgen by various androgen-sensitive tissues were later reinvestigated most extensively by a Norwegian group (Tveter and Attramadal, 1968; Tveter and Aakvaag, 1969) and also by other groups (Anderson and Liao, 1968; Fang et al., 1969; Bruchovsky and Wilson, 1968a; Belham et al., 1969; Mainwaring, 1969a). In these studies rats were injected with highly radioactive testosterone, and clear retention was observed for ventral and dorsal prostates, seminal vesicles, and coagulating gland ?h-3 hours from the time of test~sterone-~H injection. The radioactivity in the blood, spleen, lung, thymus, and diaphragm is rapidly cleared within the first hour. In the male sex accessory organs, radioactive steroids can be detected even 16 hours after injection. Two or three hours after the injection, however, essentially all the radioactivity retained can be identified as dihydrotestoset al., 1969). The prolonged retention of dihydrotest e r ~ n e - ~(Fang H tosterone by these tissues is due mainly to the selective retention of

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SHUTSUNG LIAO

this androgen by the cell nuclei of prostate (but not liver, thymus, or diaphragm), a phenomenon detectable within minutes after testoster~ne-~H reaches the male accessory glands (Bruchovsky and Wilson, 1968a; Anderson and Liao, 1968). These findings made by biochemical fractionation techniques agree well with autoradiographic observations by Tveter and Attramadal (1969)and Sar et al. (1970). In addition, the selective retention of dihydrotestosterone by cell nuclei of target tissues can be demonstrated by incubating minced tissue and radioactive androgens (Anderson and Liao, 1968; Fang et al., 1969), which provides a convenient system for in uitro study. The discovery of selective dihydrotestosterone retention by prostate cell nuclei immediately raised the possibility that dihydrotestosterone may be an active form of cellular androgen (Wilson and Gloyna, 1970; Liao and Fang, 1969). Earlier data showing that dihydrotestosterone is more active than testosterone in several bioassay systems, including the growth of rat prostates (cf. Liao and Fang, 1969), is also consistent with this proposal. Strong support comes from the finding that potent antiandrogens such as cyproterone (Fang and Liao, 1969) and flutamide (Peets et al., 1974; Liao et al., 1974a) can inhibit the retention of dihydrotestosterone by prostate tissues or nuclei in uiuo or in uitro. The dihydrotest~sterone-~H associated with prostate cell nuclei is tightly bound to nuclear proteins (Bruchovsky and Wilson, 1968b; Liao, 1968) and can be extracted with 0.4-1.0 M KCl solutions (Bruchovsky and Wilson, 1968b; Fang and Liao, 1969; Fang et al., 1969; Mainwaring, 1969a). The nuclear dihydrotestosterone-protein complex migrates as 3s in 0.4 M KC1 media (Fang and Liao, 1969; Fang et al., 1969). At low concentrations of radioactive androgens, cytosol dihydrotestosterone is exclusively bound to a high-affinity, lowcapacity protein and forms a complex which migrates as 3.5s either in 0.4 M KCI or in a medium with low ionic strength. An 8-9s complex can be also identified in the cytosol fraction exposed to dihydrotest~sterone-~H in uitro or obtained from rats injected with test~sterone-~H (Unhjem et al., 1969; Mainwaring, 1969b; Fang et al., 1969; Tveter et al., 1971; Baulieu and Jung, 1970). The 8s complex dissociates into components of about 3-4s in 0.4 M KCl. In the cytosol of rat ventral prostate, there are at least two proteins that bind dihydrotestosterone. One of them can be precipitated by the addition of ammonium sulfate to 40% saturation in respect to the salt (Liao and Fang, 1970). The protein ( p protein) exhibits an extremely high affinity (our recent data show K, 10l2 M - l ) and

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CELLULAR RECEPTORS FOR STEROID HORMONES

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specificity toward dihydrotestosterone and several synthetic androgens such as 7a,l7a-dimethyl-19-nortestosteroneand 2-oxa-17amethyl-l7/3-hydroxyestra-4,9,1 l-triene3-one, but not toward inactive steroids with related structures (5pdihydrotestosterone, 17a-dihydrote~tosterone)~ estrogens, progesterone, or corticosteroids Other natural androgens such as andros(Liao et al., 1972, 1973~). tenedione and 3p, 17P-dihydroxy-5a-androstanealso do not bind firmly to p protein, but they can be converted to dihydrotestosterone in the prostate (Bruchovsky, 1971). Another protein (called a protein) in the prostate cytosol fraction sediments at the ammonium sulfate concentration of 55-70% saturation. This protein binds dihydrotestosterone as well as testosterone, progesterone, and estradiol, but not cortisol (Fang and Liao, 1971). For convenience, the complexes of dihydrotestosterone and a or fl protein are designated complex I or complex I1 (Liao and Fang, 1970). In 0.4 M KCl solution, both cytosol complexes have sedimentation coefficients of about 3.5s. In the absence of KCl, complex I1 (but not complex I), gradually aggregates to larger forms. The aggregation of the small binding unit (3.5s)appears to involve other cellular components, since recentrifugation or partial purification of complex I1 tends to minimize the extent of aggregation (Liao et al., 1971b). In whole cytosol or in a crude ammonium sulfate preparation, the sedimentation properties of complex I1 vary in media with different salt concentrations but, more importantly, with different pH. Our recent study shows that, if the pH of the medium at 2°C is raised from 7.0 to 9.0, the amount of aggregates decreases and large 8s and small 3.5s forms emerge. At pH 9.5 only a 7s form is clearly present. At a higher pH most of the steroid is released from the proteins, whereas if the pH is lowered from 7.5 to 6.0 more aggregates appear with nearly complete loss of the 8 and 3.5s peaks. As shown later, only complex I1 (but not complex I) can be the precursor of the 3s steroid-receptor complex retained by prostate nuclei. The formation of complex I1 and nuclear retention of the complex in an in vivo or in vitro cell-free system can be diminished by antiandrogens such as cyproterone (Fang and Liao, 1969,1971) or by flutamide (Peets et al., 1974; Liao et al., 1974a). Other workers also have found that other antiandrogens can interfere with the binding of dihydrotestosterone to specific androgen-binding protein (Tveter and Aakvaag, 1969; Baulieu and Jung, 1970; Mangan and Mainwaring, 1972). These observations, steroid specificity and highaffinity binding as well as tissue specificity (e.g., complex I1 is not present in the liver, spleen, thymus, diaphragm, or blood, where

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androgens have very limited effects), suggest strongly that complex I1 is the specific androgen-receptor complex possibly functioning in the cells. Whether complex I is a precursor or a degradation product of complex I1 has not been determined. Various analytical properties of the dihydrotestosterone receptor proteins of rat ventral prostate have been well studied by Mainwaring and his associates. Using DNA-cellulose chromatography and isoelectric focusing, Mainwaring and Irving (1973) purified the cytosol receptor protein about 2000-fold or higher. The purified dihydrotestosterone-3H-receptor complex still had a sedimentation coefficient of 8s and, more importantly, was retained by prostate nuclear chromatin (see Section IV,C). The same investigators also employed the same procedure for the partial purification of the nuclear dihydrotestosterone-receptor complex.

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2. Biodynamic Aspects The amount of receptor protein for dihydrotestosterone in the prostate cells diminishes after rats are castrated (Mainwaring, 1970; Baulieu and Jung, 1970; Mainwaring and Mangan, 1973).This loss is gradual (Liao et al., 1971b) and closely follows the rate of regression of the prostate after animals have been deprived of androgens (Sullivan and Strott, 1973). The immediate action of dihydrotestosterone after it reaches a target cell is therefore likely to depend on the existing cellular receptor rather than on a specific dihydrotestosteronedependent induction of the receptor protein. Our estimate of the half-life of the prostate receptor protein for dihydrotestosterone is about 3-4 days, which is similar to that for estradiol receptor in rat uterus (see Section III,A,2) and progesterone receptor in the uterus of the guinea pig (see Section III,C,2). The receptor contents of the ventral prostate of rats of different ages have been studied by Shain and Axelrod (1973). In 60-day-old rats, the amount of cytosol receptor (based on the appearance of the 10-11s form) was estimated to be of the order of 30 fmoleslmg protein, whereas in 14- to 23-month-old rats no receptor was detected. Our estimate of dihydrotestosterone receptor (complex 11) in the cytoplasm, including that bound to microsomal fractions (30%), in many groups of adult rats is 100 h o l e s +40 fmoleslmg protein, corresponding to about 4000 to 10,OOO binding sites per cell. The prostate nuclei, in oioo and in oitro, have the ability to retain 2000 to 6000 receptors per cell nucleus (Liao and Fang, 1970; Liao et al., 1971b, 1972; Mainwaring and Peterken, 1971; Rennie and Bruchovsky, 1972). Since not all the cell nuclei of rat ventral prostate re-

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tain equal amounts of dihydrotestosterone, the maximum capacity of certain nuclei to retain receptor may be considerably higher than the values shown. Because testosterone, the major testicular androgen in blood, may be converted to dihydrotestosterone before receptor binding of the androgen (and thus function) can occur in the prostate, the levels of both the androgens and the converting enzyme (including NADPHdependent A4-3-ketosteroid-5~oxidoreductase; Shimazaki et al., 1965; Ofner, 1968; Moore and Wilson, 1972), at the local areas of target tissues can affect androgenic activity. Wilson and Gloyna (1970)concluded, from an assay of reductase activity in the accessory sexual tissues of many species and in human skin from a variety of anatomical sites, that dihydrotestosterone-forming activity is correlated with androgenic activity in many cases but is not clearly an obligatory feature of all androgen actions. In humans the dihydrotestosterone content has been found to be significantly greater in hypertrophic than in normal prostates, but the differences are not directly related to variations in the rate of dihydrotestosterone-forming activity. In the periurethral urea, where prostatic hypertrophy usually commences, the dihydrotestosterone content is reported to be two or three times greater than in the outer regions of the gland. In some cases of prostatic carcinoma in humans (Giorgi et al., 1971, 1972) and in the dog (Gloyna et al., 1970), the conversion of testosterone to dihydrotestosterone is indeed found to be small. It should be pointed out that the blood level of dihydrotestosterone has been shown to be significant, being 10-30% of that of blood testosterone in adult humans (It0 and Horton, 1970; Tremblay et al., 1970). Therefore in many target tissues the local concentration of dihydrotestosterone may be determined by the receptor proteins; and even in the tissues that lack reductase, significant androgenic action can occur. In a series of very careful studies, Bruchovsky and his associates examined the cellular concentrations in rat ventral prostate of dihydrotestosterone originating from various natural androgens. It appears that in these animals, dihydrotestosterone is formed from several androgenic precursors (Bruchovsky, 1971), and that more dihydrotestosterone is found in the prostate with androgens of high potency than with androgens of low potency. The intracellular distribution kinetics of androgens in the prostate have been studied by a pulse-chase method (Rennie and Bruchovsky, 1973). The relationship between androgen-binding and androgen insensitivity in the testicular feminization (Tfm) syndrome has been studied in recent years. In male pseudohermaphroditic rats, the cell nuclei of

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the preputial gland (Bullock and Bardin, 1970), liver, and kidney ( R i t z h et al., 1972) appear to have reduced ability to concentrate dihydrotestosterone. In mice the amount of dihydrotestosteronebinding protein in the cytoplasm and nuclei of the kidney has been found to be distinctly less in Tfm than in normal mice (Gehring et al., 1971). However, the uptake of radioactive androgen by the kidney (Bullock et al., 1971) and by the submandibular gland (Goldstein and Wilson, 1972) in the Tfm animals appears to be normal. In fact, there may be more androgen-binding protein in the submandibular glands of Tfm animals than in the same glands of normal male mice (Wilson and Goldstein, 1972; Dunn et al., 1973). Nevertheless, a defect in the cytosol androgen receptor in the kidney of Tfm mice is possible, since 7.5s is found in normal male or female mice but not in Tfm animals. Such a defect may be responsible for the decreased nuclear uptake of androgens. The androgen-dependent mouse mammary carcinoma (Shionogi 115 tumor grown in males; Yamaguchi et aZ., 1971) also contains androgen-binding proteins. By transferring the androgen-dependent cells to female mice, Bruchovsky and Meakin (1973) obtained androgen-insensitive cells. They found that four times more androgen (mainly testosterone) was bound to the receptor in the cytosol of androgen-dependent cells than in the autonomous tumor. An identical finding was made by Mainwaring and Mangan (1973),who studied the dihydrotestosterone-binding component with a sedimentation coefficient of 8s. Both groups indicated that the reduced concentration of cytoplasmic receptors appeared to be responsible for the impaired incorporation of androgens into nuclei of the insensitive cells.

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3. Universality The suggestion that dihydrotestosterone receptor protein is involved in the actions of androgens in wide ranges of target tissues is supported by the finding of similar proteins in many androgen-sensitive tissues such as seminal vesicles (Tveter and Unhjem, 1969; Stem and Eisenfeld, 1969; Liao et al., 1971b), hair follicles (Fazekas and Sandor, 1973), sebaceous and preputial glands (Adachi and Kano, 1972; Eppenberger and Hsia, 1972; Bullock and Bardin, 1970; Mainwaring and Mangan, 1973), uterus (Jungblut et al., 1971), kidney (Gehring et aZ., 1971; RitzCn et al., 1972), submandibular gland (Goldstein and Wilson, 1972; Dunn et al., 1973), brain (see Section II1,F) and androgen-sensitive tumors (see Section III,B,2). The uptake of radioactive androgens (Gorgi et al., 1971; Hansson and

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Tveter, 1971)and the presence of a specific receptor for dihydrotestosterone in human prostate have been indicated (Hansson et al., 1971, 1972; Geller and Worthman, 1973; Mainwaring and Milroy, 1973; Fang, 1973). The dihydrotestosterone-receptor complex of human prostate nuclei sediments as 3s in the medium containing 0.4 M KCl (Castaiieda and Liao, 1974b). Rat epididymis cytosol contains at least two dihydrotestosteronebinding proteins ( R i t z h et al., 1971).One of these appears to originate in the testis and is transported to the epididymis in efferent duct fluid. The other component appears to be formed in the epididymis. The nuclear uptake and binding of dihydrotest~sterone-~H can occur at a time when the testicular binding component is no longer present in the epididymis (Tindall et al., 1972).The cytosol dihydrotestoster~ne-~H-binding protein has been described as a 3-5s unit by Hansson and Djoseland (1972)and Tindall et al. (1972),but the presence of a 8.5s form has been detected (Blaquier and Calandra, 1973). Rat testis contains, in addition to the androgen-binding protein that binds both testosterone and dihydrotestosterone, a specific dihydrotestosterone-binding protein in the cytosol and nuclei. The protein is similar to that reported for ventral prostate (Hansson et al., 1973). No clear-cut evidence has yet been obtained for the existence of a dihydrotestosterone receptor in muscles which are also androgen target tissues. Jung and Baulieu (1972)found an 8s testosteronebinding protein that also binds dihydrotestosterone to some extent in the rat levato ani muscle. They speculate that the testosteronereceptor complex may be associated with myotrophic activities, whereas the dihydrotestosterone-receptor complex is involved in androgenic activities. Giannopoulos (1973a) also detected testosterone-binding protein(s) in the cytosol and nuclei of immature rat uterus. The protein(s) also bind dihydrotestosterone, but not progesterone or cortisol. The binding is antagonized slightly by estradiol, but not by antiandrogen (cyproterone acetate). Cellular testosteronebinding proteins that also bind dihydrotestosterone well have been found in the ventral prostate (Fang et al., 1969; Rennie and Bruchovsky, 1972) and the kidney (RitzBn et al., 1972). Testosteronebinding proteins have also been detected in rat anterior hypophysis and bovine spermatozoan preparations (Wester and Foote, 1972) as well as chick oviduct (Harrison and Toft, 1973). Further study is needed before these proteins can be identified as the specific cellular receptors for active androgens. Arguing from the retention-competition data for rabbit prostate, Kasuya and Wolff (1973)suggested that each of the three androgens 17P-hydroxy-5a-androstan-17a-

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TABLE I1 ANDROGEN ACTIONS NOT ATTRIBUTED TO DIHYDROTESTOSTERONE (DHT)" System Vagina, epithelial cells

Uterus

Prostate organ culture Seminal vesicle

Muscle Sexual behavior

Observation

Reference

3a-Hydroxy and 3-keto-androstanes stimulate the production of mucus by the superficial cells; 3p-hydroxy steroids affect deeper layers* Testosterone, but not DHT, stimulates glandular secretion and increases the height of the luminal epithelium' DHT and not 3p-androstanediol increases cell proliferation, but both androgens maintain epithelial cell growth The secretory output of fructose and citric acid is stimulated more by testosterone than by DHT No DHT receptor protein has been detectede.d Testosterone is active, but DHT is not in some speciesf

Gonzalez-Diddi et al. (1972) Baulieu et al. (1968); Gittinger and Lasnitzki (1972) Mann et al. (1971)

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Blood cell

Erythropoiesis may be affected by 5p-dihydrotestosterone

Anovulatory sterility

Testosterone is active, but DHT is not Differentiation is induced by testosterone when 5a-reductase activity is absent

Wolffian ducts

Huggins et al. (1954)

Aakvaag et al. (1972)

Whalen and Luttge (1971a); Beyer et al. (1973) Kappas and Granick (1968); Gordon et al. (1970) Whalen and Luttge (1971b) Wilson (1973)

Modified from Liao and Liang (1974). proteins for 3-hydroxy androstanes have been isolated from rat vagina (Shao et al., 1975). Receptorlike proteins which bind testosterone preferentially over DHT have been found in these tissues (see text). The testosterone action may be due to its aromatization to phenolic estrogens that can activate male sexual behavior in male (.Feder et al., 1974).

* Binding

methyZJ4C, 17~-hydro~y-5a-androst-2-en-17a-methyZ-~~C, and dihydrotestosterone, binds to a different receptor site, although no androgen receptor protein has been isolated from this tissue, The possibility that some androgen actions may not be attributed to the receptor mechanism involving dihydrotestosterone alone is in fact strong. Some of the observations supporting, but not necessarily proving, this view are summarized in Table 11.

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Further studies on these systems may show that varieties of androgen receptor molecules function either in the same or different cells in somewhat different manners.

zyxwvu C. PROGESTINRECEPTORS

1. Identification, Properties, and Universality

The biological effects of progestational compounds are often dependent on prior estrogenization. In line with this fact, estrogen has been reported to increase the uptake of progesterone in the vagina of the ovariectomized mouse (Podratz and Katzman, 1968), and in the uterus of the hamster (Reuter et al., 1970; Leavitt and Blaha, 1972), guinea pig (Milgrom et al., 1970; Falk and Bardin, 1970), rabbit (Wiest and Rao, 1971), and rat (Davies and Ryan, 1972; Safian et al., 1973), and in chick oviduct (O’Malley et al., 1969; Toft and O’Malley, 1972). Administered pr~gesterone-~H undergoes transformation in the animals to 5a-pregnane-3,20-dione and other polar metabolites (Falk and Bardin, 1970; Wichmann, 1967; Armstrong and King, 1970) but, 1-3 hours after the injection of proge~terone-~H, 80% or more of the radioactivity retained by the uterus of an estrogen-primed guinea pig (Falk and Bardin, 1970) and rabbit (Wiest and Rao, 1971) can be identified as progesterone. In ovariectomized rats, however, estrogen-insensitive accumulation of a polar metabolite has been observed (McGuire and DeDella, 1971). Wiest (1971) has carefully reviewed the distribution and metabolism of progesterone in the uterus and their possible relationship to the progestational response. He concludes that the capacity to elicit the response resides in the progesterone molecule itself and supports the suggestion (Munck, 1968) that progesterone metabolism represents a destruction of potency. Although Armstrong and King (1970)have suggested that specific intranuclear reduction of progesterone may be a possible reaction mechanism, such a proposal remains unsubstantiated. Whether the broad spectrum of biological activity can be said to be exclusively due to progesterone cannot be determined from the limited information available so far. Nevertheless, the suggestion that progesterone is a universal progestin that can act without metabolic transformation is supported by the finding of specific progesterone-binding proteins in a variety of target tissues. a. Chick Ouiduct. Progesterone receptor molecules have been studied most extensively in the chick oviduct by O’Malley and his associates (1971a,b, 1972). The binding components of oviduct cytosol that bind pr~gesterone-~H in uitro and in uiuo have been isolated

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and characterized by sucrose gradient centrifugation, polyacrylamide gel electrophoresis, and gel filtration on agarose. The radioactive steroid in the isolated complex was identified as progesterone. The steroid affinity appears in the order: progesterone > testosterone > 20a-hydroxy-4-pregnene-3-one > estradiol > cortisol > estrone > androstenedione. It is thus closely related to their relative potencies (Sherman et al., 1970). Progesterone-binding components can be clearly separated from the corticosteroid-binding globulin of chick plasma. In the absence of KCI, the major components sediment as 5 and 8S, and have molecular weights of about 100,OOO and 360,000, respectively. In the presence of 0.3 M KCl the pr~gesterone-~H-receptor complex sediments as a single peak at about 4 s (Sherman et al., 1970). On a DEAEcellulose ion-exchange column, two progesterone-binding components (A and B) were identified. They have identical steroid specificity and binding kinetics (dissociation constant Kd 0.8 nM). It has been suggested that they may be subunits of the progesteronereceptor complex. However, A and B alone, or together, are not able to reconstruct the 8s components under a variety of conditions (Schrader and O'Malley, 1972). The progesterone receptor formed in uiuo or in oitro can be extracted from nuclei by 0.3 M KCI. The nuclear binding complex on gradient centrifugation (with 0.3 M KCI) sediments closely with the cytosol complex as 4s. b. Guinea Pig Uterus. A progesterone-binding protein that does not bind nonprogestogenic steroids has been found in the estrogenprimed guinea pig uterus but not in nontarget organs. In low salt solutions it sediments as a 7 s unit which is converted to a 3.5-4.5s form in high-salt media (Milgrom et al., 1970; Corvol et al., 1972). By competition and on gradient centrifugation, the progesterone binder can be distinguished from plasma proteins that bind progesterone. MacLaughlin et al. (1972) also found a 5.5s progesterone-binding unit that sediments considerably more heavily than the progesterone-binding globulin. Faber et al. (1972a,b) also reported a 7.6s receptor for the (presumably) same progesterone receptor; but in 0.3 M KCl, the complex is said to be inactivated rather than dissociated into a 3.5-5.08 subunit. According to Corvol et al. (1972), the affinity constant of the progesterone-binding proteins is 2.3 x 10" M-I. Kontula et al. (1972) described a progesterone-binding protein from pregnant guinea pig uterus, which was different from that found in the blood plasma of pregnant animals and from that induced by estrogen priming in uteri of nonpregnant guinea pigs. The protein binds progesterone and 5a-pregnane-3, 20-dione equally well, but

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not pregnenolone, 20(a or P)-hydroxy-4-pregnan-3-one,or cortisol. Significant binding was, however, observed with deoxycorticosterone. The progesterone-protein complex sediments as 5 s in a lowsalt medium as well as in high ionic media. c. Rabbit Uterus. The cytosol and nuclear fractions of rabbit uterus also contain specific progesterone-binding proteins (McGuire and DeDella, 1971; Wiest and Rao, 1971). Prior to estrogen priming, bound progesterone in cytosol is associated only with a 4s unit, but estrogen pretreatment results in the appearance of an 8s form which dissociates into a 4 s unit in 0.4 M KC1. The receptor molecule of rabbit uterus (and human endometrium) does not bind testosterone, estradiol, corticosterone, or progesterone metabolites firmly, and can be separated from corticosteroid-binding globulin by ammonium sulfate fractionation. The protein has a K d of about 0.2 nM. The mass ratio (an indication of the degree of purification needed to obtain completely pure material) of progesterone receptor to other proteins in the cytosol of an atrophic rabbit uterus has been estimated to be 1 :320,000 when the receptor molecular weight is taken as 60,000 (4s). Estrogen treatment increases the relative mass concentration of the receptor to 1 :35,000 (Rao e t al., 1973). McGuire et al. (1974) recently studied the structural requirement for steroids to bind to rabbit uterine progestogen receptor. d. Other Target Tissues. The cytosol of rat uteri, like that of rabbit uteri, contains a progesterone-binding protein which binds progesterone and cortisol well and has properties similar to plasma corticosteroid-binding globulin (Milgrom and Baulieu, 1970).This protein is not present in appreciable amounts in the cytosol of kidney, intestine, or muscle, and appears to be an intracellular component of the uterus rather than an experimental contaminant of the plasma or interstitial fluid. A similar protein is also found in human myometrium (Kontula e t al., 1973). Rat uteri, however, contain a different protein, which binds progesterone firmly (Kd 0.1 nM) but not nonprogestational compounds, and is not present in either blood or the cytosol fraction of nontarget tissues (McGuire and DeDella, 1971; McGuire and Bariso, 1972). Feil et al. (1972)and Philibert and Raynaud (1973) showed that the protein migrates as 4 and 7s. The progesterone-binding protein of mouse uterus has been studied by several investigators (Stone and Baggett, 1965; Smith et al., 1970; Feil et al., 1972; Philibert and Raynaud, 1973). Similar proteins, probably related to corticosteroid-binding globulins have been also found in rat lymphosarcoma (Hollander and Chiu, 1966) and hepatoma tissue culture cells (Gardner and Tomkins, 1969).

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Pearlman et al. (1973) found a specific progesterone-binding component in human breast cyst fluid. The protein has a low binding affinity for estradiol, testosterone, cortisol, and corticosterone, and is distinctly different from corticosteroid-binding globulins. Verma and Laumas (1973)also have described a progesterone-binding protein in human endometrium and myometrium.

2. Biodynamic Aspects In the chick oviduct or uterine systems described above, the amounts of specific progesterone-binding proteins in the target tissues of immature or ovariectomized animals are increased by estrogen injection, which also enhances the biological effects of progesterone, In a thorough study of this aspect, Milgrom et al. (1973b) showed that, 6 hours after estradiol injection in guinea pigs, the content of 4.5s uterine progesterone-binding receptor alone (either per cell or per milligram of protein) is doubled. The heavy form (6.7s) is found at about 20 hours after injection. Since the effects of the estrogen are prevented by inhibitors of RNA and protein synthesis, a new synthesis of the receptor or protein factors may be involved. It is of interest that progesterone administered 20 hours after estrogen provokes a rapid fall in receptor concentration to less than 20% within 1 day. Freifeld et al. (1973) have reported that such an effect can be observed within 3 hours of progesterone administration. These effects of estradiol and progesterone or progesterone receptor content in the uterus are compatible with the cyclic changes observed physiologically in intact animals. For example, progesterone receptors in guinea pig uterus vary during the estrous cycle (Milgrom et al., 1972a,b). Their concentration peaks to about 40,000 binding sites per cell at proestrus, and then falls rapidly during estrus and postestrus to about 2500 sites at diestrus. At proestrus, the heavy form (6.7s) predominates, whereas only the light form (4.5s) is observed at diestrus. Davies and Ryan (1972, 1973) showed that in rat uterus there is a causal relationship between the appearance in pregnancy or disappearance prior to parturition of the uptake of progesterone and of the progesterone-binding protein. The progesterone-binding sites in myometrical cytosol increase (20,000 fmolelmg protein) at the beginning of pregnancy, but decrease to a minimum (5000 fmoleslmg protein) during the last week of gestation. These changes also appear to play a major role in regulating progesterone concentration in the myometrium. The half-life of the progesterone receptor in the uterus of the

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CELLULAR RECEPTORS FOR STEROID HORMONES

guinea pig has been estimated to be about 5 days (Milgrom et al., 1973b). The rat uterine receptor probably also has such a long halflife since, after ovariectomy, the 7s receptor diminishes slowly and lasts a week or longer. The complete recovery of the receptor level is seen 4-9 days after estrogen injection (Feil et al., 1972). In the castrated rabbit uterine progesterone-binding sites have been shown to increase from 550 sites to about 3500 sites per cell after estrogen treatment. The progesterone-binding sites in uterine cytosol of the guinea pig and rabbit have been estimated to be about 5000 fmoles (Corvol et al., 1972) and 10,000 fmoles (McGuire and Bariso, 1972) per milligram of protein. In the chick oviduct the progesterone receptor binds various steroids. The binding affinity appears to have the same order of effectiveness in potency as avidin inducer for these steroids. The fact that both avidin induction and progesterone-binding sites are stimulated by a prior treatment of the chicks with estrogens indicates a functional role for the progesterone-binding protein.

D. GLUCOCORTICOID RECEPTORS 1. Identification, Properties, and Universality Demonstration of the existence of a specific glucocorticoid receptor in animal tissues has often been complicated by extensive metabolism of the hormone in the animal (especially in the liver) and the presence of the multiple species of proteins that bind natural corticosteroids. One of the most practical techniques is to study the protein binding of potent synthetic glucocorticoids, since many corticosteroid-binding proteins that apparently are not cellular receptors do not bind to some of them. a. Liver. As a major organ of steroid catabolism, the liver, a glucocorticoid target tissue, contains large amounts (as much as 90% of the hepatic radioactivity) of the metabolites formed from injected cortisoL3H. However, 20 minutes after the injection of c~rtisol-~H, only the macromolecular fraction of the liver cytosol and the nucleus contain unmetabolized cortisoL3H (Beato et al., 1969; Morey and Litwack, 1969; Singer and Litwack, 1971a; Litwack et al., 1971). In addition, c ~ r t i s o l - ~itself H is virtually the only ~ t e r o i d - ~bound H to the cell nucleus (Beato et al., 1969). The relative unimportance of metabolites of cortisol is also apparent from the fact that although there is a marked sex difference in the metabolic patterns of steroid in liver, the corticosteroid induction of tyrosine aminotransferase in

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this tissue is equally effective in females and in males (Singer and Litwack, 1971b). Two cortisol-binding proteins were originally identified in liver. One of them (B protein, MW 64,000) is very similar if not identical to the corticosteroid-binding globulin (transcortin) of the serum, and can be precipitated with antibodies to serum proteins. Another binder (A protein, MW 51,000) appears to be present only in the liver cytosol and has a higher affinity for natural glucocorticoids than B protein or purified transcortin (Beato et al., 1972). Kinetic experiments indicate that in uiuo cortisol may be first bound to B protein and then transferred to A protein, but other explanations are possible. The biological importance of A and B proteins is not obvious, since neither of them binds potent synthetic glucocorticoids such as 9wfluorocortiso1, triamcinolone, or dexamethasone. Later, a third binder (G protein), which can be clearly separated by column chromatography on Sephadex gels and distinguished from A and B protein, was found in liver cytosol (Beato and Feigelson, 1972; Koblinsky et aZ., 1972). G protein binds cortisol as well as synthetic glucocorticoids including dexamethasone. Unlike A and B proteins, which in sucrose gradients sediment as 4 s units independently of ionic strength, G protein sediments in low-salt sucrose gradients mostly as a heavy complex, 7s (MW 200,000), but as the 4 s form (MW 66,000) in the presence of 0.3 M KCl. The K, of G protein for dexamethasone at 0°C (2.7-7.3 X lo* M-') is somewhat lower than the binding of cortisol by A or B protein, but at physiological temperature the K, of G protein ( lo8A 4 - I ) is one order of magnitude higher than the K, for A or B proteins. Because of its proper steroid specificity, affinity, and resemblance in sedimentation properties to other steroid receptors, G protein is considered the glucocorticoid receptor in liver. Using chromatographic separation on DEAE-Sephadex gels, Litwack and co-workers (1972) also isolated four macromolecules in rat liver cytosol, which bind corticosteroids and their anionic metabolites. Binder I (MW 20,000), called ligandin (Litwack et al., 1971; Singer and Litwack, 1971a), binds different groups of steroids, their anionic metabolites, and bilirubin. It also binds azo dye carcinogens and 3-methylcholanthrene covalently as well as noncovalently. Binder I11 (MW 7000) has a high 260 nm/280 nm absorbance ratio, appears to contain nucleotides, and binds preferentially in uiuo a corticosteroid anion which is possibly a monosulfate derivative of a reduced metabolite (Morey and Litwack, 1969). Binder IV (50,000) has been identified as transcortin (CBG). From the ligand

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specificity, the three binders do not appear to be the specific glucocorticoid receptor protein. Binder I1 (MW 67,000) has high-affinity binding and appropriate glucocorticoid-binding specificity: dexamethasone > corticosterone > cortisol S- cortisone + deoxycorticosterone. But it also binds progesterone and other sex hormones to some extent. The Kd for dexamethasone is about 0.6 nM, and for cortisol, in the range of 10 nM. An antibody has been prepared to binder 11. The antibody does not cross-react with other corticosteroid-binding proteins (Litwack et al., 1973). Binder I1 apparently can transfer to nuclear sites. From the liver nuclei exposed to corticosteroid in uiuo or in uitm, a macromolecular complex having characteristics (including PI 6.7) similar to cytosol binder I1 can be extracted. G proteins, which is obviously identical with binder 11, can also carry glucocorticoid-3H into nuclei where it is retained. The nuclear complex in 0.4 M KCl migrates as a 4 s form (Beato et al., 1973; Kalimi et al., 1973). b. Thymocytes. The most clear-cut evidence that cortisol can act without chemical alteration is seen in experiments with isolated thymus cells that do not metabolize cortisol (Munck and BrinckJohnsen, 1968). Specific binding of cortisol by thymus cells was originally characterized by the fact that cortisol bound to the cells dissociates relatively slowly compared to the more rapid steroid dissociation from nonspecific binding sites (Munck and Brinck-Johnsen, 1968). Glucocorticoid-binding proteins can be isolated from both the nucleus and the cytosol of disrupted cells. They have a higher affinity for dexamethasone than for cortisol and therefore are different from corticosteroid-binding globulins (Wira and Munck, 1970). Bell and Munck (1973) thoroughly studied the steroid-binding properties, especially those related to ligand-binding kinetics and stability of the cytosol glucocorticoid receptors of rat thymus cells. The K, for complexes with cortisol, dexamethasone, and triamicinolone acetonide at 3°C were estimated to be 3.3 X lo7W *1.3 ,x lo8M-I, and 2.7 x loyM - l , respectively. acetonideKaiser et al. (1973) showed that the triam~inolone-~H protein complexes of rat thymocyte cytosol sediment as 3.5 and 7s. The 7 s unit is transformed to the 4 s form by incubation at 37°C or by increasing the salt concentration. In 0.15 M KC1 only the 4 s form can be seen. The nuclear pellet fraction also contains the 4 s form which can be extracted by 0.15-0.4 M KC1. It is of interest that the cytosol of mouse thymocytes also contains the 7 s complex but not the 3.5s

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form. Antiglucocorticoid, corte~olone-~H, which competes for the specific binding of corticoids by thymus (Munck e t al., 1972), forms a 3.5scomplex which is unaffected by changes in temperature and salt concentration. c. Hepatoma Cells. Rat hepatoma tissue culture (HTC) cells provide an excellent system for the study of steroid hormone actions, since glucocorticoids induce several proteins, including tyrosine aminotransferase. Baxter and Tomkins (1971a,b) showed that, after incubation of HTC cells with de~amethasone-~H at 3 7 C , radioactivity is specifically bound to macromolecules in both the nuclear and cytoplasmic fractions. Since, in cell-free incubations, most of the binding activity is in the cytoplasmic fraction, nuclear localization of the radioactive steroid appears to occur only after initial binding in the cytoplasm. The receptor proteins isolated by these investigators sediment near 4 s at 0.5 M KC1 and near 8s at low ionic strength. The & has been estimated as 0.74 nM. The occasional finding of multiple components that migrate between 2 and 10s has also been described. The specific HTC cell receptor differs from transcortin in its steroid specificity and binding affinity. The binding reaction is reversible and is characteristic of second-order kinetics of binding and firstorder kinetics of dissociation. d. Lung. Glucocorticoids accelerate lung development and precocious appearance of pulmonary surfactant in fetal rabbit and lamb. Ballard and Ballard (1972), in a study of de~amethasone-~H binding to various tissues of the fetal animal, found proteins that bind glucocorticoid with a specificity that fits well with that shown by biological activity in HTC cells. The finding strongly suggests that glucocorticoid acts directly on lung. The & of the cytosol steroid-protein complex has been estimated as 2.7 nM. Nuclei exposed to the dexametha~one-~H-receptor complex retain the complex firmly. acetonideToft and Chytil (1973)found that the triam~inolone-~H receptor complex in the cytosol of rabbit fetal lung migrates as 7s in low salt. Giannopoulos (1973b) also has shown specific 7s dexamethasone-binding protein in rabbit fetal lung. In 0.4 M KCl, most of the binding is at the 4 s region. Cortisol inhibits formation of the 7s dexamethasone-protein complex, but by itself binds to the 4 s unit and does not form a 7s complex. It is suggested that synthetic and natural corticosteroids may bind to different conformational forms of the same binding protein. This indicates that the 7s form is not required for cortisol action. e. Other Target Cells. There is evidence that glucocorticoids act

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directly in the uterus by inhibiting certain estrogen actions (fluid imbibition, growth). Giannopoulos (1973~)has described an 8s dexamethasone-binding macromolecule (Kd 0.27 nM) in the cytosol fraction of rabbit uteri. Cortisol and corticosterone, but not progesterone, testosterone, or estradiol, compete for binding. The KC1-extractable nuclear complex sediments at 4s. Specific dexamethasone binding, however, was not found by the same investigator in the rat uterus. Cellular proteins that specifically bind corticosteroids have been found in the placenta (Wong and Burton, 1973), brain (McEwen et al., 1972; Chytil and Toft, 1972), muscle, small intestine, skin (Giannopoulos et al., 1973a), kidney, heart (Funder et al., 1973c), fibroblasts (Hackney and Pratt, 1971), lymphoid cells, leukemic cells, and lymphosarcoma (Schaumburg, 1970; Wira and Munck, 1970; Kirkpatrick et al., 1972; Gailani et al., 1973; Werthamer et al., 1973; Simonsson, 1972; Lippman et al., 1973). If one assumes that these binders are indeed functional receptors, their existence may indicate that glucocorticoids act directly at many sites not normally considered target tissues.

2. Biodynamic Aspects Several lines of evidence support the suggestion that dexamethasone-binding proteins are indeed the cellular receptors involved in glucocorticoid action. One of the best studies of this subject was carried out with HTC cells. In this system the correlation between biological activity and receptor binding of steroids was examined in detail (Baxter and Tomkins, 1971a; Rousseau et al., 1972a). First, the kinetics of dexamethasone-binding and dissociation at 37°C is found to be rapid enough to account for the rapid kinetics of induction and reinduction of tyrosine aminotransferase. Second, the extent of protein induction and of steroid binding as a function of dexamethasone concentrations are very similar. This is apparently also true for other corticosteroids (cortisol, aldosterone) if their metabolic rates are taken into consideration. Third, the binding characteristics of several steroids (including inducers, antiinducers, and inactive steroids) parallel their biological activity. It is of interest that these steroid specificities for biological activities in turn are very similar to those reported for steroid binding by de~amethasone-~Hbinding proteins in liver and HTC cells (Beato et al., 1972; Rousseau et al., 1972a; Van Der Meulen and Sekeris, 1973), thymus cells (Munck et al., 1972), lungs (Ballard and Ballard, 1972; Giannopoulos, 1973b), and most of the other tissues described above. Strong support for the involvement of specific binding proteins in

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glucocorticoid action in target cells comes from the work of Rosenau et al. (1972), who studied cultured mouse lymphoid cells killed by exposure to glucocorticoids. These cells also contain cytosol dexamethasone-binding protein (430 fmoleslmg protein; 3000 sites per cell) which can be retained by isolated nuclei. In variant cell lines

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that resist the steroid killing effect, dexamethasone-binding capacity is found to be only 10%of that of sensitive cells. The apparent Kd for cytosol receptors of sensitive cells (20 nM) also differs from that of resistant cells (4.8 nM). The nuclei from both cell types are equally effective in retaining cytosol receptors in a cell-free system, but in intact cells only the nuclei of the sensitive line retain the dexamethasone-receptor complex, It is therefore concluded that the defect in the cytoplasmic receptor is responsible for the insensitivity of resistant cell lines to glucocorticoids. The importance of glucocorticoid-receptor complexes in the earliest steps in the action of glucocorticoids on thymus cells has been elegantly elaborated on by Munck et al. (1972). Cortisol and other glucocorticoids added at physiological concentrations to rat thymus cells in uitro at 37°C begin to inhibit glucose transport after about 15 minutes. Using antiglucocorticoid (cortexolone), actinomycin D, and cycloheximide to block receptor binding and RNA and protein synthesis, and to analyze their effects on glucose transport or protein synthesis, they visualized the time course of the steroid effect on thymus cells at 37°C as shown in Fig. 3. Within the first minute, cortisol is bound to the cytoplasmic receptor. The complex is then transferred to the cell nuclei, a process that occurs more effectively at 37°C than at 3°C. Within 5 minutes sufficient specific RNA for the hormonal effect is apparently made, and thereafter removal of cortisol (and steroid receptor) from the cell nuclei (andlor inhibition of RNA synthesis) does not abolish the hormonal effect. Next, there is a temperature-sensitive step, which is blocked at 20°C. After this gap the synthesis of specific proteins and inhibition of glucose metabolism start to appear at 15-20 minutes. General inhibition of protein synthesis then becomes clear at 40 minutes and is followed by cell lysis. Van Der Meulen and Sekeris (1973)studied glucocorticoid activity in stimulating RNA synthesis in vivo and dexamethasone-binding activity in the liver of postnatal rats. Both activities appear to be low during the first 10 days after birth, but increase nearly 10 times in the next 10 days and reach a maximal value at 20 days. Singer and Litwack (1971b),however, showed high corticosteroid binding by a receptor (binder 11) at the time of birth, but also that there is binding

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FIG.3. Time course in rat thymus cell suspensions at 37°C of cortisol-receptor complex formation, cortisol-induced inhibition of glucose transport, and inhibition of protein synthesis. Cross-hatched segments of the horizontal bars in the lower part of the figure indicate roughly the periods (on the time scale above) during which emergence of the cortisol effect on glucose metabolism can be blocked by treatment with cortexolone (which displaces cortisol from glucocorticoid receptors), actinomycin D, and cyclohexirnide, and delayed by lowering the temperature. Open bars indicate periods during which these treatments have no effect. At the top of the figure is given the sequence of steps by which it is hypothesized that the cortisol-receptor complex leads to synthesis of a specific protein which inhibits glucose transport. (From Munck et al., 1972.)

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activity by another small, nucleotide-containing, steroid-binding protein (binder 111),which increases as the enzyme-inducing ability of the animals increases between 15 and 40 days. Whether binder I11 is needed for the function of binder I1 (considered to be the cellular receptor) or whether binder I11 alone is involved in enzyme induction is not clear. In the lung the cytoplasmic receptor is present at 18 days of gestation. The concentration of receptor sites for de~amethasone-~H in fetal rabbit lung is about 430 fmoleslmg of cytosol protein, which is two to five times greater than that in fetal skin, kidney, heart, liver, thymus, and other tissues. It is estimated that there are 9500 nuclear binding sites and 12,000 cytoplasmic receptor sites per fetal lung cell. Giannopoulos (197313) detected the same receptor molecule in the fetal lung of rat, guinea pig, and human, but not in the adult lungs of these species. He suggests therefore that in some animals glucocorticoid action in the lung may be limited to certain periods of

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the development. Toft and Chytil (1973), however, found the receptor molecule in the lung of adult adrenalectomized rats. E. MINEFULOCORTICOIDRECEPTORS

Aldosterone is the principal and most potent mineralocorticoid in vertebrates, although many glucocorticoids also show mineralocorticoid activity. Mineralocorticoids are normally characterized by their ability to regulate salt balance in vertebrates by increasing active Na+ transport across many epithelial tissues such as urinary, kidney, intestinal tract, and anuran skin and heart. Studies showing the counteractive effects of a variety of inhibitors of RNA and protein synthesis strongly support the induction hypothesis according to which mineralocorticoid presumably increases the concentration of induced proteins, which may alter the rate-limiting steps in Na+ transport (DeWeer and Crabbd, 1968; Sharp and Komack, 1971; Lahav et al., 1973; Lifschitz et aZ., 1973). Additional support for this view comes from the finding that aldosterone increases ribosomal capacity for protein synthesis (Trachewsky et al., 1972) and RNA polymerase activity (Liew et al., 1972). One direct action of aldosterone in the isolated toad bladder is indicated by its recovery as the unmetabolized steroid after the hormonal effect on Na+ transport is completed (Crabbd, 1963). Aldosterone obviously binds noncovalently to cellular binding sites, since the unchanged steroid is extracted readily from target tissues with organic solvents (Edelman et al., 1963; Sharp et al., 1966). Using a displacement binding technique, it is possible to show a saturable aldosterone-binding site in cytosol and nuclei of toad bladder epithelial cells (2700 sites diploid per nucleus) (Sharp and Alberti, 1971) and rat kidney (Edelman, 1971). Specific displacement of aldost e r ~ n e - ~is H seen in these systems, with nonradioactive d-aldosterone or other mineralocorticoids such as 9a-fluorocortisol, deoxycorticosterone, and cortisol in the approximate order predicted from their activity in the tissue. Very weak displacement was found with estradiol, but no interference was seen with inactive steroids like cholesterol and testosterone. The importance of the binding phenomena is underlined by the ability of mineralocorticoid antagonists including spirolactone. Autoradiographic techniques have also been used to substantiate the cellular localization of aldosterone-3H in both cytoplasm (perinuclear areas) and nuclei of toad bladder epithelium (Porter et al., 1964). Data from ald~sterone-~H uptake-retention studies indicate the presence of heterogenous aldosterone-binding sites in the bladder

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and renal systems (Fanestil and Edelman, 1966; Alberti and Sharp, 1969; Swaneck et al., 1970). This is best illustrated by the study of Edelman and his co-workers on rat kidney corticosteroid-binding proteins. Using tritiated aldosterone, dexamethasone, and corticosterone, and analyzing the protein binding of each of the radioactive steroids in the presence of other nonradioactive competitors in vivo and during in vitro tissue slice incubation, they identified three different proteins showing different binding affinity for various steroids. Type I appears to be the mineralocorticoid receptor, since it has a high affinity for ald~sterone-~H (& at 3 7 T , 0.5 nM; 30 fmoleslmg cytosol protein), and desoxycorticosterone (a potent steroid), but not for corticosterone which has very low mineralocorticoid activity in the rat (Funder et al., 1973a). Type I1 has a high affinity for dexamethasone-3H (& at 25T, 5 nM; 160 fmoles/mg cytosol protein). The affinity for various steroids is in the order: dexamethasone > corticosterone > desoxycorticosterone 2 aldosterone 3 cortisol > progesterone >>> estradiol and dihydrotestosterone. At 25"C, but not at O T , de~amethasone-~H receptor is transferred from the cytoplasm to the nuclei (Funder et al., 1973b). Type I11 has a high affinity for corticosterone (& at 2 5 T , 3 nM; 800 holeslmg cytosol protein), and its affinity for other steroids is: corticosterone > cortisol > deoxycorticosterone > progesterone > aldosterone > dexamethasone. The order of steroid affinity exhibited by type I11 is identical to that of corticosteroid-binding globulin (CBG). However, type I11 sediments as 8s and also as 4s forms in low-Ca2+media, but CBG sediments only as a 4s unit. Type I11 also serves as a donor for nuclear cortic~sterone-~H uptake; the KC1-extractable nuclear complex sediments as 3s (Feldman et al., 1973). Although the affinity of the mineralocorticoid receptor (type I) for aldosterone is much higher than for corticosterone in rat kidney, the physiological plasma concentration of corticosterone is much higher than that of aldosterone. The net effect of the differences in affinities and plasma concentrations indicates that cellular mineralocorticoid receptor sites may be predominantly and inappropriately occupied by corticosterone. Funder et al. (1973a), however, on the basis of a series of in vivo infusion experiments, found that the corticosterone effect on mineralocorticoid binding of aldosterone was in fact smaller than was expected. They have suggested that the ability of aldosterone to occupy the receptors under physiological conditions is a function of the extensive preferential plasma binding of corticosterone. Specific aldosterone-protein complexes have been detected in the

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cytosol fraction of rat kidney (Herman et al., 1968; Robinson and Fanestil, 1970),duodenal mucosa, spleen, liver and brain (Swaneck et al., 1969),and salivary gland (Funder et al., 1972).The kidney cytosol receptor bound to aldo~terone-~H sediments as two broad peaks at 4.5 and 8.5s. Addition of 0.4 M KCl shifted the 8.5s peak to 4.5S, and addition of 6 mM CaC1, resulted in a peak at 3.5s with a shoulder at 4.5s (Marver et al., 1972). Two forms of the aldosterone receptor have been characterized from the renal nuclei of rats. The first, a 0.1 M tris-extractable complex, has a sedimentation coefficient of 3s; the second, a 0.4 M KC1-extractable complex, sediments at 4 s and is tightly bound to chromatin. The nuclear binding of both receptors appears to require intact DNA. Kinetic studies and mixing experiments with ald~sterone-~H-labeledcytosol and unlabeled nuclei suggest that, in uiuo, cytoplasmic receptor entering the nucleus (probably in the 4.5s form) is first converted to the 3s unit and then bound to chromatin as the 4 s form (Swaneck et al., 1970; Funder et al., 1972; Marver et al., 1972). Funder et al. (1974) recently studied the ability of a series of 24 spirolactone analogs to compete for aldosterone-3H binding to the cytosol receptor during the incubation of rat kidney slices. Among some interesting effects of structural modification are a decrease in the affinity by ring-B unsaturation at the C-6 and C-7 positions, and an increase in esterification or thioesterification at the C-7 position, also in ring B. Forte (1972) studied the effect of mineralocorticoid agonists and antagonists on the binding of aldosterone-gH to rat kidney plasma membranes. Mineralocorticoid receptors can be found in fetal kidney of the guinea pig at 25-40 days of gestation (Pasqualini et al., 1972). More than 50% of the total aldosterone receptors were found in the nucleus. Most of them are tightly bound to the chromatin fraction and can be extracted by 1 M NaCl but not by 0.1 M tris buffer. The nuclear complexes sediment as 2.5-3.5s and 4-5S, whereas a 8-9s form can be detected in the cytosol fraction. Pasqualini et al. (1972) also provide evidence that aldosterone bound to macromolecules may be metabolized to a tetrahydroaldosterone-macromolecule complex without dissociation. It should be noted again that the glucocorticoid receptor also binds aldosterone to some extent. In many cells, such as HTC cells (Rousseau et al., 1972b),aldosterone and dexamethasone appear to bind to a single class of sites with affinities that correspond to their potencies in glucocorticoid activity rather than in mineralocorticoid activity.

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F. STEROIDRECEPTORS IN BRAINS Many reports are now available to show that estrogen in the female or androgen in the male can be preferentially retained by the hypothalamus, an apparent site of hormone action in the regulation of gonadotrophin production or sexual behavior (Eisenfeld and Axelrod, 1966; Kato and Villee, 1967; McEwen et al., 1970; Tuohimaa, 1971; Maurer and Woolley, 1971; Pkrez-Palacios et al., 1973; Plapinger and McEwen, 1973). No such sex difference in the selective retention of sex steroids appears to exist in pre- and neonatal rats before sexual differentiation of the hypothalamus (Tuohimaa and Niemi, 1972a). Androgenization of the female rat also results in loss of the capacity of the hypothalamus and anterior pituitary to accumulate estradiol (Flerkb et al., 1969; Tuohimaa and Johansson, 1971; Vertes et aZ., 1973), possibly explaining the loss of estrogen action in these animals. The feminization of male rats by neonatal antiandrogen (cyproterone) treatment (Neumann et al ., 1967) induces a cyclic output of pituitary gonadotrophins and a female type of sex steroid (estrogen) retention by the hypothalamus (Tuohimaa and Niemi, 1972b). The distribution of androgen-concentrating neurons can be seen in specific areas of the brain by autoradiographic techniques (Sar and Stumpf, 1972). Selective retention of androgen occurs in areas of the preoptic parolfactory region, the hypothalamus, the hippocampus, and the amygdala, as well as in cells of the anterior pituitary. The topographic distribution of androgen in the brain agrees well with areas that have been associated with the regulation of gonadotropin secretion and male sexual behavior (Sar and Stumpf, 1973a,b). In the pituitary the nuclei of a small number of anterior lobe cells (about 15%) concentrate radioactivity 1 hour after te~tosterone-~H injection. The cells of the intermediate and posterior lobes did not retain radioactivity. The labeling of anterior pituitary cells is confined to a 60% area of gonadotrophs (Sar and Stumpf, 1973a,b). However, estradioPH in male and female rats showed nuclear estrogen concentration not only in gonadotrophs but also in other areas of anterior pituitary cells (Stumpf, 1971). The differences in the topographic distribution of radioactivity in the brain and pituitary indicate that not all the action of androgen in the pituitary is due to the conversion of testosterone to estrogen (see Table I1 footnote). These studies are in accord with other findings, showing that estrogen and androgen can act directly on the pituitary gland in addition to a negative feedback effect on the hypothalamus (Debeljuk et al., 1972). The quantity of

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receptor in the pituitary has been estimated to be severalfold greater than in the hypothalamus. Hypothalamic receptors, however, are confined to the basal medial region, and the concentration in this region (1.1 fmolelpg DNA) is similar to the concentration in the pituitary (1.3 finolelpg DNA) (Anderson et al., 1973b; cf. Leavitt et al., 1973). Studies on isolated sex steroid receptors in the hypothalamus and pituitary have been very limited, but both the cytoplasmic and nuclear fractions of these tissues appear to have estrogen receptor molecules similar to those in uterus (Kahwanago et al., 1970; Clark et al., 1972; Payne et al., 1973; Kato et al., 1974).Testosterone-binding proteins are also found in the cytoplasm and in nuclear preparations of the anterior pituitary (Jouan et al., 1971). It has been shown that most of the protein-bound androgens can be identified as testosterone (Jouan et al., 1973). The formation of dihydrotestosterone from testosterone, however, occurs in the pituitary (Kniewald et al., 1969) or hypothalamus (Kniewald et al., 1971), and direct action of the dihydrotestosterone-receptor complex is also possible. Kato and Onouchi (1973) recently isolated an 8.6s dihydrotestosteronebinding component from rat hypothalamic cytosol. The number of binding sites is estimated to be 9 fmoleslmg cytosol protein. The dissociation constant of the complex is reported to be approximately 0.7 nM. An autoradiographic study in songbirds has shown that androgenconcentrating cells are in a midbrain area from which vocalizations can be electrically stimulated, suggesting that androgen acts on this particular site to affect avian vocal behavior (Zigmond et al., 1973). The possibility that progesterone inhibits the expression of an androgen-dependent courtship display of the male ring dove by blocking the hypothalamic accumulation of testosterone has been discussed by Stem (1972). Corticosteroids affect ACTH secretion and behavior in animals, apparently by functioning through the hypothalamus and other areas of brain (Bohus, 1970; Pfaff et al., 1971). The uptake and binding of radio-active corticosteroids, as seen by autoradiographic techniques and by biochemical analysis of rat tissue fractions, appear to occur preferentially in the neuron cell bodies of the hippocampus. Much of the labeled steroid appears in the cell nuclei, and the steroid-protein complex can be extracted from them by 0.4 M salt solutions (McEwen et al., 1972).Rat brain cytosol also contains corticosterone3H-binding macromolecules which are different from serum-binding

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proteins. The protein binds natural and synthetic corticosteroids (cortisol, triamicinolone, dexamethasone, and corticosterone) and progesterone, but not estradiol, testosterone, or dihydrotestosterone (Chytil and Toft, 1972; McEwen et al., 1972; Grosser et al., 1973; McEwen and Wallach, 1973).The greatest concentration of the binding protein appears to be in the hippocampus. Watanabe et al. (1974) recently showed that mouse pituitary tumor cells contain glucocorticoid receptors in the cytosol and nuclear fractions. The cytosol 4-5s complex appears to translocate to the cell nuclei by a temperature-dependent process. The uptake of progestins by the brain has been studied, but specific or selective retention has not been clearly demonstrated (Whalen and Luttge, 1971c; Wade and Feder, 1972). G. RECEPTORAND STEROID DEPENDENCY O F CANCER Lactating and tumorous mammary tissues in experimental animals also contain 8 and 4S, high-affinity (K, lo9 M - l ) cytosol receptors, and 4-5s nuclear receptors for estrogens (Jensen et al., 1971a; Harris et al., 1971; McGuire and Julian, 1971; Wittliff et al., 1972; Boylan and Wittliff, 1973; Bresciani et al., 1973). The concentration of cytoplasmic receptor in mammary tissue appears to vary with differentiation of the mammary gland (Wittliff et al., 1972) and increases markedly during lactation. At the tenth day of lactation, there are about 5000 receptor sites per cell. This increase is not accompanied by a corresponding increase in the concentration of the nuclear estrogen-receptor complex. Since in many cases an injection of exogenous estradiol can result in the appearance of the nuclear receptor, the low nuclear receptor content may be simply due to the low blood level of estrogen during lactation. (Shyamala and Nandi, 1972; Hsueh et al., 1973; Gardner and Wittliff, 1973). However, in some DMBA-induced rat or mouse mammary tumors that fail to regress after ovariectomy, estradiol is unable to accumulate in the nuclei (McGuire and Julian, 1971; Shyamala, 1972; McGuire and Chamness, 1973). McGuire et al. (1972)believe that this difference is not due to the inability of nuclear chromatin to retain the cytoplasmic estradiol-receptor complex. Binding analysis revealed (McGuire et al., 1971) a similar affinity for cytoplasmic estradiol binding (Kd 0.29 nM) in both, but the number of binding sites in the hormone-dependent tumor (26 fmoleslmg cytosol protein) was much higher than that in the hormone-independent tumor (2.4 fmoles/mg cytosol protein).

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Human mammary carcinoma also contains estradiol-binding protein (8 and 4 s ) of high affinity (K,109-1012M-'),The content of estradiol binding sites in patients varies greatly, with a majority at 30-100 fmoleslmg protein, but some patients have as much as 600 finoles/mg protein (Hahnel and Twaddle, 1973; Spaeren et al., 1973; McGuire, 1973; McGuire and Delagarza, 1973; Hilf et al., 1973; Jensen et aZ., 1973; Brooks et aZ., 1973). The possibility that estrogen- or progesterone-insensitive mammary cancers of humans contain very few or no receptors for sex steroids has been studied in many laboratories, since this may provide an important means of screening patients who may not respond to hormone therapy and therefore can be spared unnecessary major surgery such as adrenalectomy. In an urgent need to clarify the feasibility of the method for therapeutic guidance, a Breast Cancer Cooperative Group has decided on a standard assay based on a charcoal adsorption technique and agar gel electrophoresis as an optional method (Heuson, 1973). The most reliable although more laborious method, however, is to assay for the formation of 8s receptor. Recently we developed a simple and rapid method of estimating a specific steroid receptor in the presence of nonspecific steroid-binding proteins. [The technique involves the use of an antibody to a steroid to remove the 3H-labeled steroid bound to blood and nonreceptor cellular steroid-binding proteins. Since 3H-labeled steroid bound to receptors is not dissociated in the presence of the antibody, it can be separated from the antibody-bound 3H-labeled steroid by gradient centrifugation or simply by the use of insolubilized antibody. The latter method can be used to assay multiple samples within 3-4 hours (Castaiieda and Liao, 1974a).] From the limited data available, encouraging results have been reported to show that there is a good correlation between the cytosol receptor content of tumors and the success or failure of endocrine treatment (Jensen et al., 1971a, 1973; Maass et al., 1972). More information is needed to establish whether quantitative estimation of estrogen receptors in mammary cancers can be used for the definite prediction of individual response to endocrine treatment. Factors such as age of the patient, histological type of tumor (Spaeren et al., 1973),and receptors for progesterone (Terenius, 1973) and other hormones may have to be considered. As described in Section III,B, specific androgen receptors have been demonstrated in human prostate tissue. Whether or not the receptor assay can assist in selecting the appropriate therapy for patients with prostate carcinoma is being studied (Mobbs et d., 1974).

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IV. Cytoplasmic-Nuclear Interaction of Steroid Receptors

AND NUCLEARRETENTION OF CYTOPLASMIC A. TRANSFORMATION

RECEPTORS It is now clear that in most steroid target tissues (with the exceptions noted below), nuclear retention of steroids and receptors is dependent on prior formation of steroid-receptor complexes in the cytoplasm. The “two-step” mechanism was put forth independently by Jensen and Gorski. An indication for this came from autoradiographic and cell fractionation studies showing that, when uterine tissue is incubated with estradioL3H at Oo-2”C, more than 70% of the radioactive steroid is seen in the extranuclear region on autoradiography but, when the tissue is warmed to 3 7 T , the nuclear bound steroid becomes predominant (Jensen et al., 1968). During such redistribution in uitro, the level of cytosol receptor identifiable as 8s rapidly diminishes, with a concomitant appearance of the nuclear complex (Jensen et al., 1968, 1969; Gorski et al., 1968; Shyamala and Gorski, 1969; Giannopoulos and Gorski, 1971a). Such a process appears to operate in uiuo, since injection of a physiological dose of estradiol can result in a progressive fall in the level of cytosol receptor for about 4 hours, after which the receptor content is gradually restored (Jensen et al., 1969; Sarff and Gorski, 1971). The requirement for cytosol in formation of the 5s nuclear complex is also evident from the fact that no 5s complex can be obtained if estradioL3H is incubated directly with uterine nuclei or a nuclear extract of estrogen-deprived animals. In the presence of a cytosol fraction, however, the nuclear estrogen receptor complex is readily detected in the extract of reisolated nuclei. The fact that the receptor proteins for estradiol are not found in the cell nuclei of uteri deprived of estrogens implies that nuclear retention of the cytosol receptor protein is dependent on prior interaction of steroids and receptors. Initially, it was assumed that the 5s nuclear complex was formed in the nucleus from the 4s cytosol complex (Jensen et al., 1969), but subsequent studies showed that incubation of uterine cytosol alone in the presence of (but, importantly, not in the absence of) estradiol yields a 5s complex which can be retained by the uterine nuclei. The transformation takes place only slowly in the cold, proceeds readily at 25 to 3 7 C , and is accelerated with increasing pH over the range 6.5-8.5 and by the presence of salt. EDTA, Ca2+,Mgz+,or Mn2+retards the transformation. Besides estradiols, other estrogens such as diethylstilbestrol, estriol, and hex-

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estrol also promote the 4s-to-5S transformation. Estrone binds to the 4 s unit but does not promote the process (Jensen et al., 1971b, 1972b) unless the concentration is raised to a higher level than that required for estradiol (cf. Ruh et al., 1973). It is suggested that the estrogendependent transformation of the 4 s complex to the 5s form involves a steroid-dependent change in the conformation of the receptor protein. [Jensen’s group (Gore11 et d . , 1974) recently named the untransformed cytosol (extranuclear) receptor estrophilin-I, and the transformed (the active form that can be retained by the nucleus) estrophilin-11.1 It should be noted that in 4 M urea both the cytosol and the nuclear estradiol receptor complexes have sedimentation coefficients (3.6s) identical with ovalbumin (Stance1 et al., 1973b), suggesting that no gross alteration of the basic chemical nature of the receptor protein occurs during the transformation. A somewhat different view has been presented by an Italian group (Puca et al., 1972; Bresciani et al., 1973). As described in Section III,A, they believe that estradiol binds to a 5.3s cytosol receptor, but that the complex is cleaved by a proteolytic factor to 4.5s forms retained by the uterine cell nucleus. Besides the receptor transformation, a temperature-dependent alteration of the cell nuclei may also facilitate receptor binding. In a study of the ability of the nuclear chromatin fraction of mammary tumors to retain the uterine estradiol-receptor complex, McGuire et al. (1972) showed that preincubation of the estrogen-receptor complex (but not the chromatin) alone at 21°C can result in a significant but small increase in binding, whereas much more binding occurs when the complex and chromatin are incubated together at 21°C. Chatkoff and Julian (1973) have also suggested that the chromatin acceptor” sites may be under dynamic control. They found that progesterone treatment (3 days) of ovariectomized rabbits increases the number of uterine chromatin acceptor sites and the binding affinity for the retention of estradiol receptor complexes. The temperature-dependent increase in retention of the dihydrotest~sterone-~H-receptorcomplex by prostate nuclei was originally observed in tissue incubation experiments using cell fractionation (Fang et al., 1969) and in autoradiographic study (Sar et al., 1970). Indication that specific dihydrotestosterone binding by prostate cell nuclei is dependent on the cytosol fraction is clear, since the salt extracts of the prostate nuclei of castrated rats contain very few proteins that bind dihydrotestosterone tightly. Incubation of isolated nuclei with radioactive androgen also does not result in significant formation of the nuclear androgen receptor complex. The nuclear complex, 66

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however, can be obtained readily if the whole homogenate is used or a cytosol fraction is incubated with the nuclei (Fang et al., 1969; Liao and Fang, 1969). The need for prior interaction of dihydrotestosterone with the cytosol receptor (p protein) in nuclear retention of the receptor protein has been shown by Fang and Liao (1971). In the absence of androgen, p protein is not retained by isolated prostate cell nuclei. Another dihydrotestosterone-binding protein (aprotein) binds androgen but is not retained by nuclei. The levels of nuclear retention of other proteins that bind testosterone, estradiol, progesterone, and cortisol are not significant (Liao and Fang, 1970). In a cell-free system, the binding of dihydrotestosterone-receptor complex by prostate nuclei proceeds better at 20°C than at 4°C (Fang and Liao, 1971; Liao and Liang, 1974). Mainwaring and Peterken (1971) also observed the same temperature effect using prostate nuclear chromatin. A brief warming of the 8s cytosol dihydrotestoster~ne-~H-receptor complex before its incubation with nuclei markedly accelerated the rate but not the overall extent of the transfer of the complex into prostate chromatin. The temperature-dependent

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FIG. 4. Temperature-dependent transformation of steroid-receptor complexes. The estradiol-receptor complex in the cytosol(3.8S) of calf uterus can be transformed, by incubation at 25"C, to a 5.3s form which can be retained by uterine cell nuclei (left). Similarly, the cytosol dihydrotestosterone-receptor complex (3.8s) of rat ventral prostate can be transformed, by incubation at 20°C for 20 minutes, to a 2.9s form which is indistinguishable from the one retained by prostate cell nuclei (right). Gradient solutions containing 0.4 M KCI were used in the sedimentation studies. Arrows show where bovine serum albumin (4.6s) and ovalbumin (3.6s) sediment. The left figure is taken from DeSombre et al. (1972).The right figure is based on our experiment.

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alteration of the androgen-receptor complex is accompanied by a pronounced decrease in sedimentation coefficient from 8 to 4.2s and, more interestingly, the isoelectric point declines from 5.8 to 6.5 (Mainwaring and Irving, 1973). Our study indicates that the 8s-to-4S transformation is not dihydrotestosterone-dependent and thus may differ in essence from the estradiol-dependent transformation of the 4 s unit to a 5 s form which enters uterine nuclei. The temperaturedependent transformation of the cytosol receptor to a nuclear form is probably best shown by one of our recent observations. As shown earlier, the prostate cytosol receptor (3.5s) sediments slightly faster than the nuclear receptor (3s)in 0.4 M KCl (Fang and Liao, 1969) or in 2 M urea (Liao, unpublished observation). Under conditions favoring the nuclear retention, the cytosol 3.5s form can be transformed to the 3s form by incubating at 20°C for 10-20 minutes (Fig. 4). As in the case of the estradiol-uterus and dihydrotestosteroneprostate systems, little or no salt-extractable progesterone-binding activity can be detected in oviduct nuclei prior to progesterone administration. Following injection of pr~gesterone-~H in vivo or incubation of oviduct slices in vitro with pr~gesterone-~H, a progressive increase in nuclear binding is observed. This is accompanied by a concomitant depletion of cytoplasmic receptor, also supporting the hypothesis that the nuclear steroid receptor arises by a hormonedependent transfer of the cytoplasmic receptor complex to the nucleus (O’Malley and Toft, 1971; O’Malley et al., 1971, 1972). Specific association of glucocorticoid with cell nuclei in the presence of cytosol has been studied in many systems. With thymus cells, Munck et al. (1972) showed that cytosol receptor complex prepared at 3°C is transferred to isolated nuclei at 3°C only if it has been prewarmed to 25°C. Bell and Munck (1973) present two possible mechanisms involved in the temperature-dependent transformation of the complex. One is an equilibrium mechanism in which the effect of the steroid is to change the position of equilibrium of the two forms of the receptor so as to favor the form with high affinity for the nucleus. The other mechanism is a kinetic one in which the steroid accelerates the rate of the temperature-sensitive transformation. In the equilibrium mechanism the association constant for the interaction of steroid with the transformed receptor must be greater, whereas in the kinetic mechanism the association constant is not expected to change. In rat liver the activation process also appears to occur only if the receptor is bound and is accelerated by an increase in temperature

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and ionic strength (Kalimi et al., 1973; Beato et al., 1974). It is interesting that Milgrom et al. (1973~)found that the affinity of activated dexamethasone-receptor complex is raised not only for liver nuclei, but also for various polyanions (homologous and heterologous DNA, RNA, and even carboxymethyl and sulfopropyl Sephadex). Caz+ markedly inhibited the “acidiophilic activation” of the receptors. Rousseau et al. (1973)showed that binding of dexamethasone-3H by the HTC cells results in a loss of most of the cytoplasmic receptor, and retention of an equivalent number of steroid molecules in the nucleus. When the steroid is removed from the culture, it dissociates from the nucleus, while the level of cytoplasmic receptor returns to normal, even if protein or RNA synthesis is inhibited. Evidence showing retention of cytosol glucocorticoid-receptor complexes by nuclei has also been presented for rabbit fetal lung (Ballard and Ballard, 1972; Giannopoulos et al., 1973b), rat thymocytes (Kaiser e t al., 1973), and rat brain (McEwen and Wallach, 1973). The nuclear retention of mineralocorticosteroid in kidney also proceeds in a similar manner; the nuclear uptake of aldo~terone-~Hreceptor in rat kidney can amount to 60% of the receptor content lost from the cytosol fraction. Data from the time course of generation are consistent with the mechanism: cytosol (8.5 or 4.5s) + tris-soluble nuclear (3s)+ chromatin-bound (4s). However, the 3 and 4 s nuclear complexes may bind to independent nuclear sites. The formation of the chromatin-bound species is seen at 37°C but not at 0°C (Marver et al., 1972; Funder et al., 1972). Numerous examples are available to suggest that the two-step mechanism originally proposed for the nuclear retention of estrogen appears to be a general phenomenon. The mechanism involves a key step: steroid-dependent activation of steroid-receptor complexes to forms that can bind to nuclear sites of target cells. Whether the activation involves enzymic modification or conformational change in the complex is yet to be proven but, as Jensen and DeSombre (1973) believe, the step may represent the transformation of specific steroid-receptor proteins to biochemically functional forms.

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B. CYTOPLASM-INDEPENDENT NUCLEAR RECEPTORS In some studies formation of the specific steroid-receptor complex is possible with nuclei incubated with radioactive steroids without the addition of cytosol proteins. For example, cortisol-protein complexes can be extracted from purified rat thymus nuclei incubated with ~ortisol-~H (Munck and Wira, 1971; Abraham and Sekeris, 1973). Pasqualini et al. (1972) found that nuclear ald~sterone-~H-

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receptor complexes can be obtained after direct incubation of radioactive steroid with purified kidney nuclei. This differs from the suggestion that in the kidney of adult adrenalectomized rats the nuclear aldosterone-receptor complexes are derived from the cytosol complexes (see Section IV,A). In our laboratory, Shao and Castaiieda found tissue- and steroidand 3p, specific formation of 3/3,17p-dihydro~y-A~-androstene-~H 17~-dihydro~yandrostane-~H-protein complexes in vaginal nuclei in vioo. These complexes migrate as 4s complexes in 0.4 M KCl, but similar complexes in the cytosol fraction have not been detected. Vaginal cytosol is not required for formation of the nuclear complex during the incubation of purified nuclei and radioactive steroids. Cell nuclei from liver, prostate, brain, thymus, and kidney are not able to form the complex. The complex is distinguishable from the estradiol receptor complex in the vagina (Shao et al., 1975). Mester and Baulieu (1972) also suggested the presence of an estrogen-binding protein in the KC1-insoluble nuclear aggregate of chick liver. Ozon and Belle (1973) detected a 4s complex in liver nuclear extracts of chicken and toad. Although estrogen stimulates phosphoprotein synthesis in chick liver, no cytosol receptor could be detected in this organ by either group of workers. Arias and Warren (1971), however, have described an estrophilic macromolecule in chick liver cytosol. More studies must be made before the above examples can be regarded as exceptional cases in which hormone action is not dependent on the cytoplasmic receptor. For example, the apparent lack of steroid-protein complex in the cytosol fractions may be due to instability of the receptor protein and to insensitivity of the detection methods. Moreover, in hormone-deprived animals there may be some endogenous steroids that can facilitate the binding of the cytosol receptor to the cell nuclei. Nevertheless, it is possible that some receptors are bound to cellular components in the cytoplasm as well as in the nuclei and simply await activation by the arrival of steroid molecules. During such a process the interaction of a steroid hormone with a receptor protein may weaken the binding of the receptor with the cellular sites and thus facilitate its translocation.

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CHROMATIN ACCEPTOR SITES FOR RECEPTORS The number of receptor binding sites in target cell nuclei has been estimated to be in the order of 2000 to 10,000 per diploid genome, assuming one site for one receptor containing one steroid molecule. Whether all these bindings represent biologically important interac-

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tions is open to question, It is also not clear whether more than one type of specific binding is involved in different functions in U ~ U O . Studies in this area have been hindered by difficulty in distinguishing between specific and nonspecific binding. One of the earliest observations showing the possibility that nuclear retention of the cytosol-receptor complex is nuclear-specific came from the study of nuclear retention of the dihydrotestosteronereceptor complex in cell-free systems (Liao and Fang, 1970; Fang and Liao, 1971; Liao et al., 1971b). To eliminate nonspecific association the amount of dihydrotestosterone-receptor complex retained in the specific manner was estimated by measuring the portion of the retained complex that could be extracted from the nuclei by 0.4 M KCl and sedimented as 3s (as is the complex retained in uiuo). By this technique it was shown that nuclei from prostate, but not liver, thymus, or diaphragm, can retain significant amounts of the dihydrotestosterone-receptor complex, and that nuclear binding sites can be saturated by the androgen-receptor complex. Specific retention was not observed with other steroid-protein complexes. Heating of nuclei at temperatures higher than 40T,or treatment of nuclei by proteolytic enzymes resulted in loss of specific binding, but also in an increase in nonspecific association of the steroid-receptor complex with the aggregated chromatin. It was suggested therefore that prostate nuclei contain limiting numbers of tissue and steroid receptor specific binding sites and that certain heat-labile protein factors (defined as acceptor proteins) are necessary for formation of the ternary complex. Subsequent studies showed that tissue and receptor specificity could be demonstrated by using chromatin fractions prepared from prostate nuclei (Mainwaring and Peterken, 1971; Steggles et a1., 1971), or with deoxyribonucleoprotein complexes reconstructed with purified DNA and salt-extractable proteins of prostate nuclei (Tymoczko and Liao, 1971). In a reconstructed system employing Millipore membrane filtration, the acceptor proteins appear to be heat-labile nonhistone proteins and require native calf thymus DNA for its receptor binding activity. Heat-denatured DNA does not appear to be functional, but binds the steroid-receptor complex in a nonspecific manner. Poly A and poly G, but not poly U or poly C, can substitute for native DNA in showing acceptor activity. A similar study using a reconstructed system for the retention of estradiol-receptor and progesteronereceptor complexes by uterine nuclei (Liang and Liao, 1972) has indicated the presence of similar heat-labile acceptor proteins in the 0.4 M KCl extract of female target tissue. In support of this view,

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Thompson and King (1974) recently showed that uterine chromatin exposed to 0.4 M KCI binds less estradiol-receptor complex than that not exposed to 0.4 A4 KCl. In contrast, preextraction of rat kidney nuclei with KC1 did not destroy their ability to accept the aldosterone-,H-receptor complex, indicating that the acceptor factor is not solubilized in 0.4 M KCl (Marver et al., 1972). The presence of acceptor proteins was also proposed independently by O’Malley and co-workers in chick oviduct systems. They first demonstrated that the progesterone-receptor complex can be retained by cell nuclei of the oviduct but not of the spleen, lung, intestine, or liver (O’Malley et al., 1971b). Similar results were obtained when nuclear chromatin fractions were compared (Steggles et al., 1971; Spelsberg et al., 1971). Using chromatin from which histones were removed selectively by 2 M NaCl and 5 M urea and reconstructed by the addition of various nuclear protein fractions, they showed that an acidic protein fraction (AP,) was necessary for chromatin to bind the progesterone-receptor complex (O’Malley et al., 1972). It is interesting that, of the two progesterone-protein complexes (Schrader and O’Malley, 1972), only receptor component B binds to chromatin containing AP3. Component A, however, binds to DNA but not to chromatin (Schrader et al., 1972).These observations are analogous to the finding that in prostate only dihydrotestosterone coupling with @protein (but not with a-protein) can be retained by prostate cell nuclei (Fang and Liao, 1971). AP, appears to contain an acceptor protein very similar to that of rat ventral prostate. Whether the oviduct protein is heat-labile is not known, however. A similar view on the nature of the nuclear binding of receptor has been expressed by King et al. (1971).Since all types of DNA bind estradiol receptor without showing specificity, but nuclei or chromatin of uterine origin bind more receptor than those of spleen or liver, they believe that tissue specificity is the result of different histonenonhistone protein compositions which determine the accessibility of DNA (the primary acceptor) to the receptor in different cells. The involvement of DNA is shown by the fact that DNase can release bound estradiol from uterine nuclei (Shyamala, 1971; King and Gordon, 1972). Since the removal of histone or other chromatin proteins enhances DNA binding of receptor, King and Gordon (1972) have warned that any attempt to purify acceptor protein factors must prove that the sites involved in the reconstructed system are identical with those in the intact chromatin. Furthermore, in the aforementioned studies involving rat ventral prostate and chick oviduct, there are many reports showing that the nuclear acceptor sites that bind a steroid-receptor complex in a

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defined manner can be saturated if the steroid-receptor complex is present in excess. From such studies the apparent Kd values for ternary complexes of the nuclear acceptor and estradiol receptor in rat uterus (King and Gordon, 1972; Higgins et al., 1973b), dexamethasone receptor in HTC cells (Higgins et al., 1973a) and lung (Ballard and Ballard, 1972), and dihydrotestosterone receptor in rat ventral prostate (our data) have been estimated to be about 0.2-1 nM. A careful study by Higgins et al. (197313) has shown that different steroid-receptor complexes may bind to different types of nuclear acceptor sites. In this study hepatoma nuclei were found to have a fixed number of glucocorticoid receptor binding sites (3850 per haploid genome), but did not have specific sites for the estrogenreceptor complex of rat uterus. In the rat uterus it has been estimated that there are about 2150 nuclear sites for the hepatoma glucocorticoid-receptor complex and 3350 sites for the uterine estradiol receptor per haploid genome. The binding of one class of the radioactive steroid-receptor complex is shown to be inhibited only by the same class of nonradioactive complex and not by the other type, suggesting that the nuclear sites for the two types of complexes have different specificities. As additional evidence for the difference, Baxter et al. (1972) showed that DNase can inhibit nuclear binding activity for the glucocorticoid receptor but not for the estrogen receptor. They also estimated that purified DNA contains 6.7 pmoleslmg DNA binding sites for binding of glucocorticoid-receptor complex; in isolated nuclei there are only 0.6-2.2 pmoleslmg DNA available for binding of the same complex. This observation is not in agreement with other reports that nuclear bound estradiol-receptor complex in the uterus can be released by DNase and that the glucocorticoid receptor of rat liver does not bind purified DNA absorbed to cellulose if dexamethasone is also present (Beato et al., 1973). In correlating steroid induction of the synthesis of ovalbumin, conalbumin, ovomucoid, and lysozyme (Palmiter, 1972; Palmiter and Haines, 1973) with steroid receptor binding, Palmiter et al. (1973) studied the retention of dihydrotestosterone, progesterone, and estradiol-receptor complexes by the chromatin of magnum explants during tissue incubation. Since these steroids have synergistic effects on the production of egg-white proteins but do not compete for chromatin binding, it is suggested that they have distinct acceptor sites. The number of acceptor sites in the magnum from withdrawn chicks has been estimated as 1070, 2320, and 6400 sites per cell, respectively, for receptors of dihydrotestosterone, progrsterone, and estradiol. Mainwaring and Mangan (1971) were the first to use DNA-

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cellulose column chromatography to study the specificity of DNA binding of the dihydrotestosterone-receptor complex of rat ventral prostate. DNA prepared from rat prostate was shown to be more effective than DNA of rat liver, kidney, or Escherichia coli in retaining the androgen-receptor complex (see also Mainwaring and Peterken, 1971). Using the same technique, Clemens and Kleinsmith (1972) and Toft (1973) reported that the estradiol-receptor complex of rat uterus was able to bind more efficiently to rat DNA than to salmon DNA or E . coli DNA. Component A of the chick oviduct progesterone receptor was also shown to bind to chick oviduct DNA (Kd 0.3 nM) more firmly than to BacilZus subtilis DNA (O’Malley et al., 1973). In most of these studies, whether the observed differences are due to artifactual alteration of DNA during its preparation has not been carefully examined. It would be premature therefore to conclude that in steroid-sensitive cells there are tissue- or speciesspecific DNA sequences that bind specific steroid-receptor complexes. The binding of steroid hormones to DNA has been examined in the past decade. Huggins and Yang (1962) and Dannenberg (1963) pointed out the similarity in the molecular geometry of steroids and the base pairs of the DNA helix. T’so and Lu (1964), however, found that single-stranded DNA could bind steroids much more firmly than duplex DNA. More recently, Kidson et al. (1971)showed that the relative affinity of various steroids toward denatured bovine spleen DNA is in the order: progesterone = dihydrotestosterone > testosterone > estradiol > corticosterone = cis-testosterone (17a-hydroxy) = aldosterone. Of many polynucleotides tested, high affinity was observed with dG- or G-containing polymers (but not dG:dC), suggesting the involvement of 2-amino groups of guanine (see also Conen et al., 1969). It is interesting to note that Goldberg and Atchley (1966) indicated that certain steroid hormones could weaken the DNA intrastrand bonds holding the double helix. This action, however, has not been reproducible in our laboratory. Gottfried (1972) has proposed a model in which steroid-carbohydrate (carbosteroid) polymers can interact with DNA strands rich in certain bases. However, steroid receptors in uiuo bind unconjugated steroids, and there is no evidence for the existence of carbosteroidlike substances in the cell nuclei of target tissues. An interesting observation in this regard was made by Sluyser (1966a,b,c), who showed that, in rats injected with radioactive testosterone, radioactive steroid binds preferentially to the lysine-rich histone fraction of ventral prostate rather than to the lysine-poor histone

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fractions. He claimed that testosterone could diminish the ability of prostatic lysine-rich histone to keep the two DNA chains from separating at raised temperatures. Cortisol, however, was shown to associate mostly with the lysine-poor or arginine-rich histones of rat liver (Sluyser, 1966c, 1969). The suggestion that there is a limited number of specific acceptor sites on the nucleus of a target cell has not been generally accepted. For example, Bresciani et al. (1973) estimated that as many as 100,000 estradiol-receptor complexes of either the native or Ca2+stabilized form can bind to certain basic proteins in the uterine cell nucleus. From a careful examination of the distribution of the estradiol-receptor complex in the uterine cytoplasm and the nuclei, Williams and Gorski (1972a,b) concluded that receptor distribution behaves like a unimolecular system, not in accord with the concept that nuclear interaction involves a limited number of acceptor sites. Chamness et al. (1974) also showed that uterine estradioL3Hreceptor complex binds to cell nuclei of the uterus as well as to nontarget tissues, and that the extent of binding is proportional to the concentration of the receptor, no saturation occurring when total radioactivity bound to nuclei is measured. This finding is consistent with the observations of Clark and Gorski (1969), Fang and Liao (1971), and Milgrom et al. (1973~) that nonspecific nuclear association of the steroid-receptor complex occurs readily and that the specific binding sites cannot be estimated unless an additional selective process is employed (cf. Clark et al., 1973).

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D. RIBONUCLEOPROTEIN BINDINGOF RECEPTORS In mammalian cell nuclei, 20-30% of the total nucleic acid content is RNA. Nuclear RNAs (see a review by Weinberg, 1973) include precursors of cytoplasmic RNA (rRNA, tRNA, and mRNA), as well as those that remain in the nuclei. A major portion of the nuclear RNA appears to form complexes with nuclear proteins and exists as ribonucleoprotein (RNP) particles. Besides RNP particles that eventually become ribosomes (Burdon, 1971; Kumar and Warner, 1972), some of them may participate in the regulation of gene transcription (Paul, 1971; Britten and Davidson, 1969)or form informosomelike (or informoferlike) particles and become involved in transport of mRNA (Spirin, 1969; Samarina et al., 1968). To explore the possibility that some steroid hormones may be involved in the functions of various RNP particles, Liao, Liang, and Tymoczko (1973a) initiated a study on the nature and properties of the binding in estradiol-receptor and dihydrotestosterone-receptor

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complexes to nuclear and cytoplasmic RNP particles of the uterus and prostate. By using gradient centrifugation these steroidreceptor complexes were found to associate readily with the nuclear particles from the respective target tissues but to a lesser extent with those from liver or other less responsive tissues. The ternary complexes of steroid-receptor-RNP sediment at 60-80S, although in the uterine system distinct 50 and 80s peaks can be observed. The binding phenomena can be abolished by prior heating of RNP particles or by treatment with proteases or RNases (but not with DNase I), suggesting the involvement of RNA and heat-sensitive protein components of the particles. The steroid-receptor complex can be dissociated reversibly from the ternary complex by 0.4 M KCl. The receptor-binding sites on the particles can be saturated, and under such conditions less than 10-20% of the isolated nuclear RNP particles can bind to the steroid-receptor complex. It is possible that only those RNP particles with heat-labile acceptor factors can associate with the steroid-receptor complex. About 30-50% of the total cytosol steroid-receptor complexes present can bind to RNP particles, indicating that other steroid-protein complexes may be structurally not compatible with the RNP acceptor sites. The cytoplasmic fractions of uterus and prostate also contain RNP particles (40-60s) which bind estradiol-receptor and dihydrotestosterone-receptor complexes in a cell-free system. However, cytoplasmic polysomes or 80s monosome forms of ribosomes do not bind either of the steroid-receptor complexes. Since, with rats injected with 3H-labeled sex steroids, a radioactive steroid-protein complex can be found to associate with RNP particles, RNP binding of the steroid receptor may indeed occur in uioo. 3H-Labeled sex steroids added in a free form or as steroid-receptor complexes but inactivated by heating (SOT, 10 minutes) do not associate with either the nuclear or the cytoplasmic RNP particles of uterus or prostate. In addition, in the absence of steroid hormones, RNP particles do not appear to have the ability to retain receptor proteins which can later be released and bind radioactive steroids. This is analogous to the current belief that receptor proteins are not retained by cell nuclei without prior binding of the specific steroids (Tymoczko and Liao, 1974; Liang and Liao, 1974a). Binding of the dihydrotestosterone-receptor complex to various polyribonucleotides has been studied recently (Tymoczko and Liao, 1974). With polymers having a sedimentation coefficient of about 4 f l S , poly A, poly U, and to some extent poly G, gave distinct radioactive peaks at 50-70s. Such a significant shift in the sedimen-

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tation coefficient was not observed with poly C, or tFtNA (liver or E. coli.).

E. INTRACELLULAR RECYCLING OF RECEPTORS In a study of glucocorticoid receptor of thymus cells in culture, Munck and Brinck-Johnson (1968) found that there is a correlation between ATP concentration and magnitude of specific cortisol binding. Thus cytoplasmic fractions from anaerobically incubated cells have reduced steroid-binding capacity. Since ATP added to the cytosol cannot increase the binding, and also the possibility of receptor degradation and resynthesis can be excluded, the energy-sensitive step is considered to be at the generation of active receptor. It was suggested (Munck et al., 1972) that ATP directly or indirectly is necessary for the conversion of the receptor from a form in which it is unable to bind cortisol into an active form. Bell and Munck (1973) suggested that ATP may supply the free energy for maintenance of a cycle in which the receptor forms a complex with the hormone, becomes inactivated after reaching the nucleus, and emerges to be reactivated and form a new complex. Ishii et al. (1972), from studies of the energy dependence of the binding of triamcinolone acetonides to cultured mouse fibroblasts (L cells), also have proposed that release of the receptor from the particulate fraction (nucleus) and its regeneration ( t l l z30 minutes) to a form capable of binding steroid again are energy-dependent. The possibility that nuclear RNP particles may be involved in steroid receptor recycling in target cells, and that such a recycling may be functionally related to gene expression, is being considered (Liao and Fang, 1969; Liao et al., 1973a,b), and is discussed in Section V,B. The fact that various steroid hormones can promote rapid transfer of cytoplasmic receptors to the cell nuclei (within minutes), and that the receptors appear to have a long half-life of about 3-5 days, also suggests that receptors are not rapidly consumed or destroyed during their action and very likely are shuttled between the nucleus and cytoplasm in target cells. V. Gene Expression and Steroid Receptor A. RNA SYNTHESISAND PROTEININDUCTION

Numerous studies have shown the importance of RNA and protein production in the early stages of steroid hormone action. They include the demonstration of (1)specific enzyme or protein induction

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by steroid hormones; (2) prevention of the effects of steroid hormones by inhibitors of RNA or protein synthesis; (3) rapid stimulation (or inhibition in thymocytes) of RNA synthesis in uiuo or nuclear RNA polymerase activities; and (4) ability of RNA extracted from steroid hormone-treated cells to elicite certain hormone responses in hormone-deficient target cells (see references cited in Section I). It is not clear, however, whether or not steroid hormones actually act directly on the main machinery of genetic transcription or translation. RNA synthesis in mammalian nuclei has often been studied as two separate entities: nucleolar RNA synthesis, which generally represents rRNA synthesis; and nucleoplasmic RNA synthesis, which presumably includes mRNA production. Polymerase complexes I and I1 are assumed, respectively, to be responsible for the types of RNA synthesized in nucleolar and nucleoplasmic areas of the nuclei. Since actinomycin D at low concentrations selectively inhibits nucleolar RNA synthesis by polymerase I, whereas a-amanitin inhibits polymerase I1 action of nucleoplasm, these inhibitors have been used effectively in the elucidation of hormonal action on nuclear RNA synthesis. The involvement of rRNA synthesis during the early effect of steroid hormones has been demonstrated for androgens in prostate (Liao et al., 1965, 1966; Liao and Fang, 1969), estrogens in uterus (see below) glucocorticoids in liver (Yu and Feigelson, 1969), and aldosterone in kidney (Chu and Edelman, 1972). There are strong indications that an increase in rRNA (or nucleolar RNA) synthesis in liver (Yu and Feigelson, 1971, 1972) and in prostate (Mainwaring et al., 1971) systems is due to an increase in active enzyme content rather than a change in overall template activity. Whether there is direct action of the steroid-receptor complex on nucleolar RNA synthesis is not known but, in several cases steroid-dependent induction of enzymes appears to be independent of rRNA synthesis (Gelehrter and Tomkins, 1967; Wicks, 1968; Jost et al., 1973). The manner in which a steroid hormone may affect rRNA synthesis by regulating nucleoplasmic polymerase activity is best illustrated in the studies by many investigators who traced the early steps of estradiol action in uterus. Within 1-4 hours after estradiol injection in estrogen-deprived rats, nuclear RNA polymerase activity is stimulated (Mueller et al., 1958; Gorski, 1964; Hamilton et al., 1965), predominantly at nucleolar sites (Barton and Liao, 1967; Tata, 1968; Hamilton, 1968; Billing et al., 1969). Since inhibitors (puromycin or cycloheximide) of protein synthesis suppress the increase in RNA

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polymerase activity, continuous protein synthesis appears to be needed for the estrogen effect (Mueller et al., 1961; Gorski et al., 1965; Nicolette and Mueller, 1966). In fact, a group of proteins, including one acidic protein which can be identified electrophoretically, is formed in response to estrogen injection (Notides and Gorski, 1966) or in uitro estrogen treatment (DeAngelo and Gorski, 1970; Katzenellenbogen and Gorski, 1972) of uteri. The acidic protein, often called induced protein (IP) can be detected 30 minutes after estradiol enters uterine cells. Whether IP is responsible for the more general stimulation of RNA polymerase activity detectable later has not been proved. The synthesis of IP, however, is inhibited by actinomycin D and cr-amanitin (Baulieu et al., 1972a), suggesting a new synthesis of mRNA for IP. By pulse-labeling techniques an increase in in uiuo synthesis of labeled RNA can be observed within 30 minutes after estrogen administration (Hamilton, 1968; Wira and Baulieu, 1972). Glasser et al. (1972) also showed that a transient increase in a-amanitin-sensitive RNA polymerase activity can be detected within 10-15 minutes after estrogen treatment of animals. The simplest and currently accepted conclusion that can be drawn from the above studies is that estrogen (with or without receptor) enhances the synthesis of an mRNA that codes for specific protein(s). The protein(s) is then required for the subsequent increase in production of rRNA, and maybe also for other mRNA. In rat liver the need for a supply of protein(s) for rRNA synthesis is shown by the finding of Yu and Feigelson (1972) that in the nucleolus the turnover of RNA polymerase activity is rapid (tllP1.3 hours). Since rat liver cytosol and nuclei contain large amounts of wamanitin-insensitive (our recent observation) polymerase in soluble forms (Liao et al., 1968), the protein(s) required are probably regulatory factor(s) rather than catalytic portions of the enzyme complex. An alternative suggestion that the RNA product of a-amanitin-sensitive polymerase action may be directly involved in nucleolar RNA synthesis (Sekeris and Schmid, 1973) has not been explored. Several workers studied the effect of hormones on the template activity of nuclear chromatin of target tissues. Nuclear chromatins were prepared from hormone-deficient and also from hormonetreated animals and assayed for their capacity to support RNA synthesis in the presence of excess amounts of purified RNA polymerase. Cortisone (for liver of adrenalectomized rats, see Dahmus and Bonner, 1965; Beato et al., 1970a), estradiol (for uterus of ovariectomized rats, see Barker and Warren, 1966; Teng and Hamilton, 1968; Glasser et ul., 1972; for rabbit uterus, see Church and

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McCarthy, 1970; for chick oviduct, see Cox et al., 1973), thyroxine (for tadpole liver, see Kim and Cohen, 1966), and progesterone (for chick oviduct, see O’Malley et al., 1969) were found to enhance the template activity of isolated chromatin to a significant level in a matter of a few hours to several days. These observations are in accord with the finding that template activities of nuclear chromatin isolated from cells highly active in RNA synthesis in general are higher than those from cells showing a low rate of RNA synthesis (Bonner et al., 1968). Under the conditions that stimulate RNA synthesis, however, no significant increase in chromatin template activity is observed with androgens in rat ventral prostate (Liao and Lin, 1967), with estrogens in mouse uterus (Dati and Maurer, 1971), and with aldosterone in rat kidney (Trachewsky and Cheah, 1971). Caution in interpreting chromatin template activity as assayed in uitro has been expressed by many workers (Liao and Fang, 1969; Dati and Maurer, 1971; Glasser et al., 1972; Cox et al., 1973). Factors such as hydrolytic enzymes (nucleases, nucleotidases, proteolytic enzymes), which may affect template activity, have not been studied carefully in most instances. Whether the chromatin fractionated from target nuclei still represents the native status of all or selective regions of chromatin can also be questioned. More seriously, the RNA polymerase (mostly bacterial) used in these studies may not produce the same type of RNA as in hormone-stimulated nuclei in uiuo or in an isolated nuclear system (Liao and Lin, 1967; Butterworth et al., 1971). In addition, if only a few genes are activated, changes in template activity or an increase in incorporation of radioactive precursor into the RNA fraction probably may be very small and may not be detected. Alteration of DNA-associated chromatin components by hormones can be probed by measuring the DNA sites available for a c tinomycin-D binding in intact nuclei. The method initially employed for rat prostate suggests that there is no androgen-induced gross unmasking of DNA (Liao and Lin, 1967; Barton, 1967; cf. Seligy and Lurquin, 1973). Aldosterone, in rat kidney, results in lower actinomycin-D binding which appears to be correlated with an alteration in chromatin components (Trachewsky et al., 1972). Cortisol, within 10-20 minutes, enhances about 15% of actinomycin-D binding capacity and template activity of rat liver nuclei in uiuo and in uitro, suggesting that some chromosomal proteins are removed from the DNA surface (Beato et al., 1970a). Estradiol (Teng and Hamilton, 1970) and cortisol (Shelton and Allfrey, 1970) have been shown to

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play a regulatory role in the synthesis of nonhistone nuclear protein in target tissues. Similarly, steroid-induced changes in nuclear protein components in rat prostate by androgen (Chung and Coffey, 1971; Couch and Anderson, 1973; Anderson et al., 1973), and in rat kidney and heart by aldosterone (Liew et al., 1973), have been seen. In some instances chromatin fractions with high template activities have been found to have high concentrations of acetyl (Pogo et al., 1968) or phosphate esters (Allfrey et al., 1966). An increase in histone acetylation has been reported in liver and thymus of adrenalectomized rats after treatment with cortisol (Allfrey et al., 1966), in kidney of adrenalectomized rats treated with aldosterone (Libby, 1972a; Liew et al., 1973; Trachewsky and Lawrence, 1972), and in rat uterus treated with estradiol (Libby, 1972b). In rat uterus the effect was seen between 5 and 10 minutes after estrogen administration, but not at a later period (20 minutes). Since actinomycin D and cycloheximide were not inhibitors, the synthesis of new proteins or RNA was not considered to be required (Libby, 1972b). A confirmation of this important study is still lacking (cf. Anderson and Gorski, 1971). Hormone specificity has often been considered to be due to the specific activation of certain genes. This is supported by the fact that many hormones can selectively increase production of the amounts of specific enzymes or proteins in target cells. The most exciting achievements in this regard came from the laboratories of Shimke and of O’Malley and co-workers, who by isolating mRNA and translating it in uitro supplied clear evidence showing an estrogen-dependent increase in the mRNA for ovalbumin in chick oviduct (Rhoads et al., 1971; Palmiter and Schimke, 1973; Comstock et al., 1972; Chan et al., 1973). A similar study was made in Feigelson’s laboratory on the glucocorticoid control of mRNA for hepatic trytophan oxygenase (Schutz et al., 1973). One of the interesting features of the oviduct system is that the induction of ovalbumin mRNA is seen only with oviduct previously treated with estrogens for several days. During the primary period estrogens may be considered to act at cellular sites other than those directly involved in ovalbumin mRNA production. Whether or not different estrogen receptors are involved in these two stages of estrogen action is not known. Elucidation of the roles of steroid hormones in such a system and in others in which cell differentiation is involved may be tied directly to a better understanding of the mechanisms by which a cell can switch from one type of gene program to another.

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B. HYPOTHETICALMODELS

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Immediately after our first realization that genetic formation is transmitted from DNA by mRNA to direct the synthesis of a specific protein, the “hormone-gene theory” was proposed (Zalokar, 1961; Liao and Williams-Ashman, 1962; Williams-Ashman, 1965). With the appearance of the original operon theory initially postulated for bacterial systems, and the suggestion that induction of some bacterial enzymes is the result of the removal of gene repressors to allow mRNA synthesis, it was proposed that hormones might act by incapacitating certain gene repressor molecules (Karlson, 1963). While this is still one of the most attractive hypotheses, it is now clear that the regulation of genetic expression can be achieved in a variety of ways at different sites: synthesis of RNA on DNA template (transcriptional control), processing of RNA, protein synthesis (translational control), or degradation of RNA and proteins. Taking into account some of the newer information, several hypothetical models (Fig. 5) have been proposed to show how steroid hormones may act on the molecular processes of gene expression. The incapacitation of a gene (or transcriptional) repressor may be achieved by the interaction of a steroid with a repressor. The repressor itself then is the ultimate receptor for the steroid. If this is so, 1. NEOATNE CONTROL A . Transcrptional Repressor

N-{t-+y,PRESSOR N-&+y,PRESSOR

,

I

STFROID RECEPTOR STFRAID RECEPTOR

’I

INACTIVATION

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STEROID RECEPTOR [\RNA

FIG.5. Hypothetical models showing how steroid hormones and their receptors may regulate gene expression in target cells. See text for explanation. Double helix at the left side of each model represents DNA.

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the need for nuclear retention of the cytosol steroid-receptor complex is puzzling. It is possible that the cytosol steroid-binding protein is merely a transport protein which supplies a specific steroid to a specific nuclear locus where it functions (Beato et al., 1970b), possibly by transferring the steroid to another nuclear receptor (Baulieu et aZ.,197213). Alternatively, the steroid receptor and not the steroid alone may interact with the repressor. Repressor activity can also be abolished if its synthesis is inhibited by a steroid or steroid-receptor complex (Fig. 5, 1.A). Tomkins et aZ. (1970, 1972) in a study of the HTC cell system, described a “paradoxic” increase in the rate of tyrosine aminotransferase activity (superinduction) following actinomycin-D addition to glucocorticoid-treated cells. They proposed the existence of a labile posttranscriptional repressor which inhibits translation of specific mRNA and enhances mRNA degradation. Superinduction is thought to be produced by actinomycin-D inhibition of the synthesis of a translational repressor, although a more traditional model based on transcriptional control has been offered to explain similar results (Reel and Kenny, 1968; Palmiter and Schimke, 1973). From these studies, Tomkins et al. (1970) suggested that the steroid-receptor complex inhibits translational repressor activity by direct interaction with the repressor or by inhibiting its synthesis (Fig. 5, 1.B left). In a study of enzyme induction in prostate, Ohno has suggested that the receptor protein for an androgen may act as a “translational block” by binding to certain mRNAs and preventing them from being translated by ribosomes (Fig. 5, 1.B right). Androgen is assumed to bind to the suppressive protein. As a result, the mRNAs are released and utilized for the production of specific enzymes. Ohno has speculated further that the same androgen-receptor complex enters the prostate nuclei and activates nucleolar RNA polymerase (Ohno, 1971). It has been suggested that RNA polymerase may be present in excess in mammalian cells (Liao and Lin, 1967; Liao et aZ., 1968), and that the restricted distribution of certain nucleotide sequences may serve an important function in directing polymerase to a specific area of the DNA template or in regulating the rate of RNA synthesis. It was suggested that the action of steroid hormones may involve a confonnational fit of molecules required for the initiation of RNA synthesis. In recent years regulatory factors of this type that are necessary for specific synthesis of RNAs by polymerase have been described in bacterial systems. As distinct from “negative control” involving repressor-depression action, the role of these factors is to

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specify the DNA sites where RNA synthesis is to occur and therefore can be categorized as “positive control” (Fig. 5, I1 left). The possibility that steroid hormones are involved in posttranscriptional events has not been well investigated. Among some of the pertinent observations are that cortisol can affect the processing of 45s RNA in rat liver (Jacob et al., 1969) and that, in the uterus, the percentage of nuclear RNA transported to the cytoplasm (Church and McCarthy, 1970) and the maturation of ribosomal precursor RNA or particles (Luck and Hamilton, 1972) can be stimulated by estrogen treatment of the animals. Whether or not these occurrences are due to a direct effect of steroid hormones has not been determined. From the finding that androgen- and estrogen-receptor complexes can associate with certain nuclear RNP particles in target cells, Liao et al. (1973a) have recently proposed that the steroidreceptor complexes may be involved in the processing of nuclear RNA or in the protein synthesis (possibly initiation). In the hypothetical model (Fig. 5, I1 right), a steroid hormone forms a complex with a receptor protein in the cytoplasm. After a conformational change, the complex enters the cell nucleus and becomes involved in the regulation (possibly initiation) of RNA synthesis. During such a process the steroid-receptor complex and other protein factors (including an acceptor) may recognize and bind to certain sequences of DNA initially, but then to the specific RNA product (Liao and Fang, 1969). Steroid-receptor-bound RNP may be processed to a mature form and enter the cytoplasm and participate in protein synthesis. One can visualize the role of the steroid-receptor complex as providing structural specificity needed for the formation (i.e., selecting specific RNA species from a large RNA pool), processing, and/or functioning of RNP (cf. Kwan and Brawerman, 1972; Blobel, 1973; Weinberg, 1973). In the above model the receptor protein may lose its ability to bind to RNP at various stages of processing and utilization, especially if the steroid hormone of the cell is depleted. Both the receptor proteins and the acceptor factors may reassociate with these RNP particles when the steroid hormone is replenished. Thus the recycling process and its functions may be reinitiated by the steroid hormone at many different points (in the nucleus or in the cytoplasm) in the receptor cycle (Liao et al., 1973a,b; Liao, 1974). This suggests that the importance of gene transcription (RNA synthesis) in relation to gene translation (protein synthesis) for the overall functioning of a steroid hormone in target cells may be dependent on the amount of RNP particles at different stages of processing, and on their RNA and

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protein constituents in the target cells at the time the hormone is supplied. If the target cells contain sufficient amounts of RNA and protein constituents of RNP, the early actions of the hormone may be simply dependent on the processing and utilization (or the activity) of RNP, and not upon RNA synthesis. If this is so, steroid hormone actions under some conditions may not be actinomycin-D-sensitive (cf. Talwar et al., 1965; Frieden et al., 1968). Recently, Whelly and Barker (1974)showed that 1 hour after estradiol administration in the ovariectomized mature rat there is a transient increase in the rate of peptide elongation in isolated uterine ribosomes. This effect was reported not to be inhibited by actinomycin D. Liang and Liao (1974b) also found that androgen injected into castrated rats can, within 10 minutes, stimulate the ability of the prostate cytosol fraction to sustain GTP-dependent binding of an initiator tRNA labeled with m e t h i ~ n i n e - ~ % to prostate ribosomal particles. The finding indicates the possibility that androgen may be involved in the regulation of the activities of the initiation factors in protein synthesis, C. In Vitro EXPERIMENTAL APPROACHES A clear understanding of the molecular mechanism involved in steroid hormone action must be dependent on demonstration of the a hormonal effect in a cell-free system. Attempts by numerous investigators to show a change in RNA-synthesizing activity by in vitro addition of physiological levels of steroids to isolated cell nuclei generally have failed. At high concentrations (> 10 testosterone and cortisol were reported to enhance (by 10%) RNA synthesis of isolated liver cell nuclei (Lukacs and Sekeris, 1967). Seshadri and H Warren (1968)also reported that RNA synthesis from g ~ a n i n e - ~by rat uterine nuclei was enhanced by 2 nM to 1 pM estrone. Estradiol was reported to be less active. Barker and Warren (1966, 1968) also reported that DNA template activity for RNA synthesis of uterine chromatin can be increased by incubation with 5 estrone. Beato et al. (1968) reported a similar effect of cortisol on liver nuclear systems. It has been also claimed that dihydrotestosterone but not testosterone, in the absence of added cytoplasmic protein, can stimulate the incorporation of radioactive nucleosides into RNA fractions by cell nuclei of rat ventral prostate (Bashirelahi et al., 1969; Bashirelahi and Villee, 1970). Most of these observations, however, have not been confirmed by other investigators. Probably the first experimental report to describe a steroid hormone as an inactivator of the repressor activity of steroid-binding

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protein came from Talwar et al. (1964), who found that in the absence of estradiol the cytosol fraction of rat uterus was inhibitory for RNA synthesis directed by purified calf thymus DNA and E. cold RNA polymerase. It was claimed that the injection of estradiol 1 hour prior to sacrifice, or direct addition of a minute amount of estradiol, abolished the inhibitory action of the cytosol preparation. DeSombre et al. (1966), after extensive and careful studies, obtained a similar cytosol preparation which inhibited in uiuo RNA synthesis, but they could not demonstrate reversal of the inhibition by estradiol in uitro or in uiuo. In close accord with Talwar’s report, Wacker (1965) has reported that a soluble macromolecular protein fraction obtained from extracts of Pseudomonas testosteroni also inhibited RNA synthesis in the presence of sperm DNA and purified E . coli RNA polymerase. The protein fraction was less inhibitory when it was isolated from cells grown in the presence of testosterone, an inducer of several steroid-transforming enzymes. They also claimed to observe a partial reversal of the inhibition by addition of low levels of testosterone in uitro. Similar observations were reported by the same group on Streptomyces hydrogenans (Wacker et al., 1965b) and E. coli (Wacker et al., 1965a). Shikita and Talalay (1967) reinvestigated the P . testosteroni system in great detail. They found that the inhibitory effect resides largely in a heat-stable component, but they were unable to confirm that inducer steroids had any direct effect on the inhibitory properties, or any quantitative differences in the inhibitory power of fractions derived from steroid-induced or noninduced cells. Raynaud-Jammet and Baulieu (1969) reported that the ability of the nuclei from heifer endometrium to incorporate radioactive nucleotides into RNA is enhanced manyfold when they are first incubated with a mixture of estradiol and uterine cytosol. Since estradiol or cytosol alone was not effective, interaction of the hormone and receptor protein in the cytosol was considered to be responsible for the stimulative activity. Mousseron-Canet and her co-workers also reported that an increase in RNA polymerase activity of heifer endometrium nuclei, or of the enzyme preparation from these nuclei, could be produced by the direct addition of certain uterine fractions to the polymerase assay system (Amaud et d.,1971; Andress et al., 1973). Studies by Jensen and co-workers (1972b) showed that nuclei from immature rat uteri have much less ability to incorporate labeled nucleotide into RNA than do kidney or liver nuclei. After incubation

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(25°C for 30 minutes) with uterine cytosol containing estradiol, the RNA polymerase activity of the reisolated uterine nuclei was reported to be increased nearly three-fold. Cytosol or estradiol alone was not effective. There was no enhancement of the already high activity of liver or kidney nuclei, whether incubation was in the uterine cytosol or in their own cytosols. Most importantly, these investigators observed that nuclei from calf endometrium were activated by incubation with estradiol and endometrial cytosol at 25"C, but not at 0°C. However, the stimulation can be seen with nuclei incubated at 0°C if the estradiol-cytosol mixture is first warmed to 25°C. Since the receptor transformation from the 4 s to the 5s form (see Section IV,A) takes place readily at 25°C but not at O"C, and the 5s form, but not the 4 s complex, can associate with the uterine nuclei, it was suggested that the 5s complex is responsible for the effect. In accord with this view, estrone, which binds to the 4 s form but does not readily induce its transformation to 5s at the concentration used, did not cause nuclear stimulation. Since estradiol cytosol-dependent stimulation was also seen with RNA polymerase activity solubilized from treated nuclei and assayed with purified DNA, the steroid receptor effect was thought to be at least in part on the enzyme itself (Mohla et a2., 1972; DeSombre et al., 1972; Jensen and DeSombre, 1973). According to Beato et al. (1970b), liver nuclei, in the presence of cytosol, responded with increased RNA synthesis to cortisol in the range of 0.1-0.01 pM, which is far lower than the amounts needed for glucocorticoids to stimulate the nuclei (10 /AM). They believe that steroid-binding proteins facilitate the transport of hormones into the nucleus, indicating support for the traditional mechanism (Fig. 5, 1.A) involving the interaction of a steroid, rather than the receptor protein, with RNA-synthesizing machinery. Abraham and Sekeris (1971) also reported that cortisol (10 /AM) could inhibit rRNA synthesis of isolated rat thymus nuclei. However, when receptor protein was extracted from the nuclei, no cortisol effect was observed. The addition of the receptor back into the system was effective in restoring sensitivity to cortisol (Van Der Meulen et al., 1972). The effects of cortisol on liver and thymus described above were also shown by Bottoms et a2. (1972). Using a fractionated cortisolbinding protein from rat liver cytosol, Gopalakrishnan and Sadgopal (1972) and Beato et al. (1970b) detected changes in the template activity of liver chromatin due to the presence of cortisol hemisuccinate (10 pM) or the cortisol-protein complex. Ribarac-Stepic et al.

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(1973) also obtained a cytosol preparation of cortisol-protein complex which could enhance the incorporation of radioactivity into the RNA fraction during the incubation of liver cell nuclei and orthop h ~ s p h a t e - ~ ~While P . Gopalakrishnan and Sadgopal (1972) showed that the active cortisol-protein complex was salted out at 50-70% ammonium sulfate saturation, Beato et al. (1970b) and RibaracStepic et al. (1973) reported that the active complex is salted out at 20-40% ammonium sulfate saturation. Davies et al. (1972), working on rat and dog prostate nuclei, observed an increase in the incorporation of radioactive isotope from %-labeled ATP into the RNA fraction by 40 pA4 of dihydrotestosterone and 5a-androstane-3p7 l7/3-diol in the absence of cytosol. Several other related steroids were inactive, while many estrogens were inhibitory. At 4 pA4, the effect was observed only in the presence of a cytosol preparation (Davies and Griffiths, 1973a,b). The steroid effect was observed with fractionated cytosol receptor (8 or 3s) or nuclear receptor (4.5s). Both dihydrotestosterone-protein complexes (I and 11)as originally described by Fang and Liao (1971) appear to be active. When RNA polymerase was isolated from prostate nuclei and assayed in the presence of prostate chromatin as exogenous template, the extent of stimulation was most distinct when the polymerase was the nucleolar (polymerase I) form (50 to 140%increase). The nucleoplasmic (polymerase 11)form gave a marginal effect of about 10%.An insignificant increase was observed when calf thymus DNA or liver chromatin was employed. The direct effect on nucleolar enzyme activity is interesting, since nucleolar RNA polymerase activity is particularly sensitive to androgen action in uivo (Liao and Lin, 1967; Liao and Stumpf, 1968).This view, however, does not conform with the previous suggestion that the enhancement of nucleolar RNA synthesis is secondary to a stimulation of nucleoplasmic RNA polymerase activity. It is possible, however, that the RNA polymerase activity purported to be nucleolar may indeed be responsible for the synthesis of rRNA as well as some mRNA (Liao and Fang, 1969). An in uitro demonstration that RNA synthesis can be affected by steroid hormones or the steroid-receptor complex appears strongly to support the current concept that nuclear chromatin is the ultimate cellular site for hormone action. Although our understanding of the chromatin activity involved in gene transcription is very limited and the results of the in uitro effects described so far can be subjected to severe criticism for their ambiguity, further study in this area may unfold the mystery ofthe molecular processes involved in steroid function. Many other possibilities, however, should also be considered.

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VI. Concluding Remarks A. RECEPTORAND UPTAKE

OF

STEROID

BY

CELLS

Peck et al. (1973) examined the uptake and retention of estradiol3H by uterine and diaphragm tissue under initial velocity and equilibrium conditions, respectively. The rate of uptake of estradiol by the nontarget tissue, diaphragm, which possesses no estrogen receptor, is the same as that for uterus. Inhibitors of estrogen binding to receptor also do not alter the rate of uptake of estradiol in either tissue. These results also indicate that the estrogen receptor is not involved in the movement of estradiol into uterine tissue. The cell membranes of thymus cells are also freely permeable to cortisol, since the rate of dissociation of selectively retained cortisol from thymus cells with a time constant of 3 minutes at 37°C is essentially identical to that of glucocorticoid from the isolated cortisol-receptor complex (Munck and Wira, 1971; Bell and Munck, 1973). The possibility that a protein-mediated process is involved in estrogen entrance into rat uterine cells is suggested by Milgrom et al. (1973d), who showed that sulfhydryl-blocking agents inhibit the uptake of radioactive estrogens by target cells. The estrogen receptor is not considered to be the protein, since the uptake of diethylstilbestrol, which binds to the receptor, is not blocked significantly.

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B. NATURAL FORMS OF STEROIDRECEPTORS Essentially, in all the systems described in this article, cytosol receptors carefully prepared at low temperatures (0°-2"C) can form complexes that sediment as 7-12s and 3-5s units. The larger forms can be transformed to the smaller ones by incubating at 20°-37"C or by making the salt concentrations 0.4 M KC1. Since many conditions are clearly not physiological, it is not clear whether these forms reflect an in viuo situation or are products of the factitious association of receptor themselves or receptors with other cellular materials, To mimic natural physiological conditions, cytosol estradiol-receptor complexes have been studied in 0.15 M KCl and found to be about 6s (Giannapoulos and Gorski, 1971b; Chamness and McGuire, 1972). It is interesting that, when the estrogen-receptor complex was pressed directly from the uteri with no added buffer and sedimented in sucrose gradients prepared in the same press juice (but deproteinized), only the 6s form was detectable (Reti and Erdos, 1971). These observations indicate, but do not prove, the possibility that the 6s form is the predominate one in the target cytoplasm.

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Several investigators have also pointed out the danger in the misuse of gradient centrifugation in identifying natural forms of receptor molecules. For example, Harris (1971) showed that by adjusting the concentration of the polyanion heparin, Polytak-RNA, or dextran sulfate, the 4-5s estradiol-receptor complex of rat uterine nuclei can sediment at 8s in a low-salt sucrose gradient. A similar study made by Chamness and McGuire (1972) also demonstrated that one can deliberately cause the cytosol or nuclear estrogenreceptor complex to assume any form sedimenting between 4 and 9s by altering the heparin concentration. In salt concentrations ranging from no KC1 to 0.4 M KC1, the estradiol-receptor complex has been observed in forms with sedimentation coefficients from 3.8 to 9s. These values appear to result from a slow aggregation of estradiolbinding proteins following tissue homogenization. Since the timedependent aggregation can be minimized by working with dilute solutions, Stance1 et al. (1973a) concluded that in uivo the uterine estradiol receptor may exist as a 3.8-4.8s species, rather than as 8s as normally assumed. The extreme instability of 8s complexes at temperatures between 10" and 37°C may indicate that such a complex, if formed in uiuo, is very rapidly altered to the 3-5s form. Unless the process is tied directly to receptor function, the usefulness of forming and degrading the 8s complex can be questioned. Nevertheless, the fact that 8s forms are found in the extracts of virtually all steroid target cells remains an interesting puzzle deserving more study. As described above, different receptors for different groups of steroid hormones may exist in the same target cell. In various responsive tissues, steroid action may be dependent on heterogeneous receptors specific for different steroids. The presence of heterogeneous receptors for the same steroid hormone in a target cell, and thus different ways of functioning, is a distinct possibility but has not been clearly proven. In rat uterus a majority (80%) of the cytosol estradiol-receptor complexes can enter the cell nuclei. In rat ventral prostate only about 50% of the cytosol receptor can be retained by nuclei or the nuclear RNP particles (Fang and Liao, 1971; Liao et al., 1973a). Whether the remaining portion of the receptor represents another type or altered form of receptor is not clear. Similar considerations can be given to other steroid-receptor complexes described.

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c.

NATURE O F RECEPTOR-STEROID INTERACTION

In the past attempts have been made to use a semiempirical approach by comparing chemical structure and end-point activity to

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predict the way androgens might interact with hypothetical receptors (see reviews by Liao and Fang, 1969).Suggestions were made for the binding of androgens by receptors from &face, @face, and peripheral attachments. It was also indicated that the steric and not the electronic characteristics of the steroid are the most important in eliciting a biological response (Wolff and Zanati, 1970, and references cited therein). These earlier predictions are well in line with the conclusions of Liao et al. (1972, 1973b), who studied the structural requirements for steroids to bind to the defined androgen receptor of rat ventral prostate. It is suggested that the bulkiness and flatness of the steroid molecule play a more important role in receptor binding than the detailed electronic structure of the steroid nucleus. The role of the A4-3-keto-5a-oxidoreductaseis apparently to convert testosterone to a flatter molecule which fits better to the receptor binding site and not simply to eliminate the double bond on ring A of the steroid. In fact, potent androgens with conjugated double bonds extending from rings A and B to ring C (such as 2-oxa-17~-hydroxyestra-4,9,1l-trien%one) are indeed very flat molecules and bind to the androgen receptor very firmly. These and other studies using methylated androgens suggest that the receptor binds simultaneously at multiple sides of an androgenic steroid as if the steroid molecule were being enveloped. This is in marked contrast to steroid-metabolizing enzymes or blood steroid-binding proteins which generally recognize only a portion of the steroid molecule. This conclusion is in line with our recent observation that steroids bound to various blood proteins can be removed by various steroid antibodies, while steroids bound to various cellular receptors are not affected (Section IV,A). The localization of steroid-binding sites well inside receptor proteins may be responsible for the very high affinity constants for receptor binding of steroids, the extremely slow rates of association and dissociation of steroids and receptor proteins (many hours) at low temperatures, the acceleration of rates of exchange of unbound steroids with bound steroids by freezing and thawing (Fang and Liao, 1971), and the inability of ethanol (30%)and detergents (2% Triton X-100 or desoxycholate) to free steroids from receptors in the cold. Some careful analyses of glucorticoid-binding properties have also enabled several workers to suggest the nature of the interaction of steroids and receptors. Using thymus cells, Munck et al. (1972) showed that the association rates of cortisol, dexamethasone, and cortexolone are very similar, and that differences in their binding constants are largely determined by dissociation rates (Bell and Munck, 1973). They suggest that the groups that distinguish these steroids,

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particularly the llp-hydroxyl and the 9CY-fluor0, do not come into play until the steroid has entered the steroid-binding site of a receptor. These and other findings on structural requirements for receptor binding also allow these investigators to conclude that both the a and psides of the steroid interact with the binding sites, which are probably located in a hydrophobic pocket in the receptor protein. Koblinsky et al. (1972) also showed that, while the interaction of cortisol with liver A and B proteins, and transcortin-binding of corticoids, are accompanied by a negative entropy change dexamethasone interaction with the G protein gives a positive entropy change (cf. Westphal, 1971). The finding is interesting in that the entropy change may be due to the displacement of water molecules (Warner, 1965) as well as to a local relaxation in the structure of the protein, which leads to an increased degree of freedom. This is probably necessary if the “enveloping” of a steroid by a hydrophobic region of a protein inside a protein (cf. Engle, 1967) is to occur. The enveloping of a steroid in the hydrophobic pockets of a receptor protein is likely to invoke the reorientation of certain flexible polypeptide chains. This may result in a conformational change in the protein, which is necessary before the steroid-receptor complex can interact with acceptor complexes and trigger hormone action. It is interesting that antiglucocorticoids such as progesterone-,H in HTC cells (Rousseau et al., 1973) and corte~olone-~H in rat thymocytes (Kaiser et al., 1972) bind to cytosol glucocorticoid receptors, but the complexes are not retained by the nuclei of these target cells, apparently because of their structural incompatibility. The concept of steroid enveloping suggests that receptor proteins, and not specific functional groups of steroids, participate in the key events leading to hormone action. This may explain why many different steroid hormones exhibit crossover hormonal activities.

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D. INSECTHORMONES AND VITAMIN D, It is now apparent that cellular receptor proteins for steroid hormones in vertebrate animals have many properties in common and may function in similar ways in target cells. This generalization can also be extended to some steroidal insect hormones (Gilbert and King, 1973) and, most interestingly, to vitamin D. Vitamin D3 appears to be metabolized in the liver and other tissues to 25-hydroxycholecalciferol, and subsequently in the kidney to 1,25-dihydroxycholecalciferol, before it can act in initiating intestinal calcium transport (Haussler et al., 1971; Boyle et al., 1972; Wong et al., 1972). There is evidence that the active (hormonal) form of vitamin D,

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binds to the chromatin protein of target cells (Tsai et al., 1972; Chen and DeLuca, 1973),and that nuclear binding is dependent on cytosol receptor protein (Tsai and Norman, 1973; Brumbaugh and Haussler, 1973).

E. CYCLICAMP AND STEROIDHORMONES Many peptide hormones employ cyclic AMP as a “second messenger” for their actions (Robison et al., 1971).The suggestion that some steroid hormones may use cyclic nucleotides as second messengers has not gained general support (Hechter and Soifer, 1971; Major and Kilpatrick, 1972). In the uterus, Szego and Davis (1967) reported acute elevation of cyclic AMP level by estradiol in vivo in 15 seconds, and cyclic AMP was seen to mimic certain effects of estrogen including amino acid and nucleotide incorporation into macromolecular fractions (Hechter et al., 1967; Griffin and Szego, 1968; Sharma and Talwar, 1967). Korenman et al. (1973) reported recently that estradiol did not stimulate cyclic AMP in vivo or in vitro. Singhal et al. (1971) showed that the administration of cyclic AMP produces testosteronelike induction of certain enzyme activities in rat prostate. The adenyl cyclase activities of various prostate preparations, however, are not affected by castration or androgens in vivo or in vitro (Rosenfeld and O’Malley, 1970; Liao et al., 1971a). Similarly, cortisol does not appear to influence the adenyl cyclase activity of rat liver cells (Soifer and Hechter, 1971). Although Ahmed (1971) has reported that androgens in vivo enhance the ability of prostate nuclei to phosphorylate nuclear proteins in vitro by “P-labeled ATP, Ichii et al. (1973)showed that androgens can decrease cyclic AMP-dependent protein kinase activity in prostate. Besides the well-known action of cyclic AMP on protein kinase, it is known that in bacterial systems cyclic AMP can bind to a specific protein and that the complex formed can promote the production (by binding to genome) of specific mRNA which codes for specific enzymes (Zubay et d., 1970; Pastan and Perlman, 1970). Since peptide hormone-sensitive adenyl cyclase appears to be present in the cell nuclei of rat liver (Soifer and Hechter, 1971) and rat ventral prostate (Liao et al., 1971a), a similar mechanism may be operating in mammalian cells. Mangan et al. (1973), however, reported that nuclear RNA polymerase activities that are enhanced by androgens are not stimulated by administration of cyclic AMP. It is intriguing to point out that cyclic AMP and steroid hormones are similar in many aspects. For example, their production is under the influence of some peptide hormones. Each of them can conform

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FIG. 6. Molecular models of steroid hormones and cyclic AMP. Corey-Pauling atomic models with Koltun connectors were used to construct estradiol (top left), cortisol (top right), dihydrotestosterone (bottom left) and cyclic AMP (bottom right). For steroids, they are side views with ring-A oxygens at the left and the a faces of the steroid facing downward. For cyclic AMP, the adenine base is at the left and the phosphate group at the far right. White balls are hydrogens, caged balls are hydroxyl oxygen, and black blocks are carbon atoms. For construction of models, the following articles were consulted: Cooper et uZ. (1969), Norton (1965), Cooper and Duax (1969), and Watenpaugh et aZ. (1968).

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to rather compact structures which are very similar in general geometric dimensions (Fig. 6). Both appear to function in target cells by binding noncovalently to specific receptor proteins. In a bacterial system cyclic AMP, like steroids, appears to enhance the affinity of the receptor protein (receptor transformation) for genome (Riggs et al., 1971; Anderson et al., 1972) and to promote the production of specific species of RNA. In calf ovary cells there is now experimental evidence indicating a cyclic AMP-dependent translocation of cytoplasmic cyclic AMP-binding protein and possibly of cytoplasmic protein kinase to nuclear acceptor sites (Jungmann et aZ., 1974). In rat ventral prostate cyclic AMP-binding protein can be clearly distinguished from dihydrotestosterone receptor protein by an isoelectric focusing technique (Mangan et al., 1973). Cyclic AMP and androgen do not compete for the protein-binding sites. The complexes resemble each other in that they are found in the cytosol as well as in the nuclei (extracted by 0.4 M KCl), and they sediment in

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the vicinity of 5s and 3s (Liao and Liang, 1974; Liao et aZ., 1974b). Our study has revealed that the cytosol cyclic AMP-binding protein can be retained by prostate cell nuclei, but this transfer is not dependent on formation of the cytosol dihydrotestosterone-receptor complex. The similarity between the corticosteroid-binding protein (binder 11) and cyclic AMP-binding proteins in rat liver is more obvious. They have several similar properties including molecular weight, isoelectric point, and comigration as one distinct band on polyacrylamide disc gel electrophoresis. These binding proteins in rat liver, however, can be distinguished from the regulating subunit or the protein inhibitor of phosphoprotein kinase (Filler and Litwack, 1973). ACKNOWLEDGMENTS

Research carried out in the author’s laboratory was supported by grants from the U S . National Institute of Health and the American Cancer Society, Inc. The author thanks Ms. Cassandra M. Black, Ms. Pamela A. Chudzinski, and Ms. Diane K. Howell

for their help in preparation of the manuscript. REFERENCES

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A Cell Culture Approach to the Study of Anterior Pituitary Cells

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A. TIXIER-VIDAL,D. GOURDJI,AND C. TOUGARD Groupe de Neuroendocrinologie Cellulaire, Laboratoire de Physiologie Cellulaire, CollBge de France, Paris, France

I. Introduction . . . . . . . . . . . . . . . . . . . 11. Characteristics of Anterior Pituitary Cells Grown in Vitro. . . . . . . . . . . . . . . . . . . . . A. Hormonal' Secretion . . . . . . . . . . . . . . . B. Morphological Features . . . . . . . . . . . . . 111. Reactivity to Specific Regulating Agents . . . . . . . . . A. Primary Cultures. . . . . . . . . . . . . . . . B. Effects of Regulating Agents on Continuous Cell Lines . IV. Conclusion . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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I. Introduction The anterior pituitary cells offer fascinating material for the cell biologist. They belong to an endocrine gland, the adenohypophysis, which plays a prominent role in numerous endocrine as well as nonendocrine functions of the organism, and whose activity is closely regulated by substances originating either from the central nervous system or from target endocrine glands. At the level of the whole endocrine gland, several biochemical functions are expressed which correspond to the well-known adenohypophysial hormones. Three of them are polypeptides: corticotropic hormone (ACTH, 39 amino acids), the melanophorotropic hormones (a-MSH, 13amino acids, and P-MSH, 18 amino acids), and lipotropic hormone (PLPH); all three possess important common amino acid sequences. Two are proteins: somatotropic or growth hormone (STH or GH) and mammotropic hormone or prolactin (MTH or PRL), whose primary structure only is known (MW from 20,000 to 25,000) and which possess some common amino acid sequences. Three are glycoproteins (MW -30,000): thyrotropic hormone (TSH) and the two gonadotropic hormones -follicle-stimulating hormone (FSH) and luteinizing hormone (LH). These three glycoprotein hormones share important common features. They have a quaternary structure, and each of them consists of two subunits, a and P. Peptide sequences of these two subunits have been established in several mammalian species. These studies revealed that the a subunits are 173

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similar for the three hormones, and carry the zoological specificity, while the p subunits have more distinct primary structures, and carry the biological specificity (see review by Jutisz and D e La Llosa,

1972). To this biochemical heterogeneity of function, corresponds the

cellular heterogeneity of the glandular tissue. This has been demonstrated by 50 years of morphological research using more and more elaborate techniques (see reviews by Herlant, 1964; Purves, 1966), from light microscopy to electron microscopy, and from tinctorial to cytochemical and immunocytochemical methods. As in many other endocrine cells, the fundamental feature of pituitary cells is the ability to store secretory products within secretory granules. Two types of consequences result from this feature. From a cytological point of view, this allowed workers to distinguish several glandular cell types according to the chemical or immunochemical nature of their secretory products. Finally, this led to the notion that one glandular cell type. corresponds to each pituitary hormone. In some cases, nevertheless, for example, LH and FSH, ACTH, and MSH, the existence of either a common or a dual cell type is still debated. Important physiological features of the pituitary cell are also related to its storage ability. One may assume on the basis of autoradiographic and biochemical studies that pituitary protein hormones are synthesized on the ribosomes and then follow a migratory pathway within the endoplasmic reticulum, through the Golgi zone to the secretory granules ,(see reviews by Farquhar, 1971; Farquhar et al., 1974; Labrie et al., 1973). The hormones are then released out of the cell mainly, if not only, by exocytosis. There is therefore a space and time discontinuity between synthesis and release of the hormone. In addition, an intracellular mechanism allows degradation of undischarged secretory granules, via lysosomes (Farquhar, 1971). This brief survey of the fundamental properties of anterior pituitary cells raises the problem of mechanisms that regulate (1)the rate of their secretory activity, and (2) the biochemical nature of their secretory product, in other words, that control both quantitatively and qualitatively the expression of their genetic differentiation. An impressive amount of physiological and biochemical research deals with regulation of the secretion of anterior pituitary hormone (see review by Kraicer, 1974). Two types of agents play this role: neurohormones and hormones from pituitary target glands, that is, gonads, thyroid, and adrenals. The neurohormones are polypeptides synthesized within the hypothalamus and carried to the anterior pituitary cells via the hypothalamohypophysial portal vessel system,

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They have been isolated and chemically characterized on the basis of their specific stimulating effect on the release of one anterior pituitary hormone. Therefore one hypothalamic releasing factor (RF) or hormone (RH) corresponds to each pituitary hormone. The structure of several of these neurohormones has been elucidated and their synthesis effected, for example: MSH-RH, GH-RH, TSH-RH, and LH-RH. Moreover, progress in research led to a discovery at the same time of the existence of a dual hypothalamic control, stimulating and inhibiting, for several pituitary hormones such as MSH, GH, PRL, the inhibiting control prevailing in uivo for PRL. In addition, some synthetic hypothalamic hormones now appear to be less specific in their effects than initially presumed, since TSH-RH (TRH) acts not only on TSH release but also on PRL, and LH-RH (LRH) stimulates the release of both LH and FSH. These findings might question the specificity of hypothalamic hormones as regards anterior pituitary hormones. Sex steroids, corticosteroids, and thyroid hormones (thyroxine and triiodothyronine) form the other class of regulating agents known to act directly at the pituitary cell level in addition to their action through the hypothalamus (see review by Stumpf et al., 1974). Biochemically, they evidently differ from hypothalamic polypeptides. They inhibit the secretion of their corresponding pituitary hormone by a well-known feedback effect. But again the specificity of this action may be questioned since, for example, thyroxine and estradiol have been found to exert a direct stimulating action on the activity of PRL cells (Nicoll and Meites, 1963; Meites et al., 1963). The mechanism that controls differentiation of the anterior pituitary cells has been far less studied than those related to the control of their secretory activity. If it is assumed that all cells of a multicellular organism possess an identical genome, their differentiation within functionally specialized cell populations appears as the result of epigenetic events leading to the expression of one function only. In the case of the anterior pituitary gland, which contains an heterogeneous population of glandular cells, two steps must be distinguished in the course of their differentiation: (1) their being “programmed” as glandular cells able to synthesize and to secrete a group of seven proteins only, and (2) their being specialized within several cell lines, each one being able to synthesize only one of the seven proteins. The first step seems to occur during the first stages of fetal life: the pituitary anlage derived from the Rathke pouch follows its differentiation as adenohypophysial tissue when isolated at an early stage of the nervous infundibular floor, that is, 48 hours in the

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chick embryo (Ferrand, 1972) and 10 days in the mouse embryo (Ferrand and Nanot, 1968). The mechanisms involved in this first step are still unknown, and cell cultures do not seem to be a useful approach to their study. The second step may be considered during a later stage of fetal life, as well as during adult life. During fetal life electron microscope studies in the chick embryo (Guedenet et al., 1970; Mikami et al., 1973) and in the fetal rat (Yoshida, 1966; Daikoku et aZ., 1973; Dupouy and Magre, 1973) show at the beginning small primordial and agranular cells. Thereafter membrane-bound secretory granules appear progressively within an increasing number of cells. Finally, several cell types may be identified which look like adult pituitary cell types, although with fewer secretory granules. At the same time, some primitive cells still persist, together with follicular cells which seem to constitute an independent stem line (see review by Olivier et aZ., 1974). Immunocytochemical methods allowed GH cells, PRL cells, LH cells, and TSH cells to be observed at different stages in bovine fetus (Dubois, 1971a,b) and in rat fetus (S6td6 and Nakane, 1972). These morphological data agree with physiological studies demonstrating the appearance of several pituitary functions during fetal life (see review by Jost, 1966). Is differentiation of the pituitary cells definitely achieved after birth? In fact, several facts observed in adult pituitary do not fit in with this statement. Although the adenohypophysis is known as an organ of low mitosis (Leblond and Walker, 1956), dividing pituitary cells are seen even in steady-state conditions (Nouet and Kujas, 1973). Besides, when the production of one hormone is triggered by a modification of endocrine homeostasis, rapid changes in the cell population occur: e.g., cellular hypertrophy and hyperplasy of gonadotropic cells after castration, and of thyrotropic cells after thyroidectomy. The mechanism of such an adaptation of the glandular tissue is still unknown, although it has fascinated many pituitary cytologists. In their recent review, Olivier et al. (1974) discuss three main hypothesis which have been proposed and examined by several workers. The first one stipulates the persistence in the adult gland of stem cells, that is, multipotent cells that have retained the ability to divide and to differentiate into one specialized type when the production of one hormone is stimulated. The second hypothesis involves the mitosis of fully differentiated cells which have been observed in some cases and some species. The last hypothesis involves cellular transformation of one cell type to another. On the whole, the present studies do not allow one to decide definitely among these

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three hypotheses, mainly because of difficulties in determining if one cell is functionally differentiated by morphological criteria only. Indeed, several features of the adenohypophysis hamper analysis at the cellular level of mechanisms involved in the control of differentiation and of secretory activity of pituitary cells. The main difficulties reside in the cellular heterogeneity of the gland and its anatomical position in one of the less accessible regions of the head. In this respect pituitary cell cultures offer an invaluable approach which has been used increasingly in recent years. In this chapter we concentrate our attention on pituitary cell cultures which allow work on dispersed pituitary cells maintained in uitro for months or years. They differ from pituitary organ cultures in which tissue organization is maintained, and from tissue culture starting from small fragments of tissue which grow and can display selection in some cell populations (see review by Tixier-Vidal, 1974). Among cell cultures we must distinguish (1) primary cultures that start from normal adult fully differentiated anterior pituitary cells, and (2) continuous cell lines that consist of homogeneous populations of pituitary glandular cells which continuously grow in uitro. In Section 11 we report the functional and morphological features of pituitary cells grown in these two situations. In Section 111 the effects on these models of factors that regulate the secretion of anterior pituitary hormones are analyzed and discussed with a view toward answering the main question raised by pituitary cells.

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11. Characteristics of Anterior Pituitary Cells Grown in Vitro

A.

HORMONALSECRETION

1. Primary Cultures of Normal Pituitary Cells

Primary cultures are initiated from normal anterior pituitary cells (man, monkey, rat) previously dispersed by enzymic treatment according to a large variety of techniques proposed within recent years (Portanova et aZ., 1970; Vale et al., 1972a; Hymer et al., 1973; TixierVidal et aZ., 1973; Hopkins and Farquhar, 1973). The cell suspension is then inoculated into plastic petri dishes or bottles, using an appropriate medium always enriched with adult and fetal serums. Such cultures can be maintained for weeks or months. Young adult pituitaries were generally used, with the exception of human fetal glands (Reusser et al., 1962; Gailani et al., 1970). This means that generally monolayers are started from adult, fully dif-

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A. TIXIER-VIDAL, D. COURDJI AND C. TOUCARD

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ferentiated cells. The major problem to be studied therefore concerns the evolution in culture of the biochemical and morphological parameters of their differentiation. Hormonal secretion has been followed as a function of culture time by measuring the hormonal content of the medium more frequently and the hormonal content of the cells less frequently. The sensitivity of the hormonal assay limits conclusions concerning the maintenance or disappearance of hormonal activity in a culture. In this respect great progress has been obtained as a result of radioimmunoassays, which have been applied to cell cultures only for the last 3 or 4 years. Two major features characterize the evolution in cell culture of the secretion of anterior pituitary hormones.

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1. There is an important rise in medium PRL content. In fact such an increase in PRL secretion in uitro was first observed in pituitary tissue cultures (man and rat: Pasteels, 1961, 1963) and organ cultures (rat: Meites et al., 1961, 1963), and then widely confirmed in mammals (see review by Tixier-Vidal, 1974). Our observations (see Table I) show that the same phenomenon also occurs in cell culture. A very high level of medium PRL content is already observed after 5 days of culture (see Table I). This high level is maintained for 3 or 4 weeks, and thereafter a very slight decrease is observed. Since a similar hyperactivity of PRL cells has been observed in uivo after interruption of the hypothalamohypophysial anatomical link (see reviews by Pasteels, 1963; Meites et al., 1963), this cannot be considered an artifact of culture methods and represents an original feature of PRL cells. 2. For the other pituitary hormones, the medium hormonal content decreases with time in culture. This has been found for ACTH in rat (Stark et al., 1965), for GH in human adult (Batzdorf et al., 1971) or fetus (Gailani et al., 1970), for LH and FSH in rat (Vale et al., 1972a; Steinberger e t al., 1973; Tixier-Vidal et al., 1973) and in human fetus (Gailani et al., 1970), and for TSH in rat (Vale et al., 1972a). Depending on the investigator, this decrease leads either to the maintenance of a low level or to a more-or-less precocious disappearance, that is, an undetectable level. In our experiments on LH and FSH secretion, which involved more than 30 cultures, there was in this respect intrinsic variability from one experiment to another. In some cases gonadotropic hormones disappeared after 15 days, but in numerous series they were maintained for 50 days or more. In all cases the FSH/LH ratio increased with culture time,

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CELL CULTURE O F ANTERIOR PITUITARY CELLS

TABLE I

SD 26

AGAINST

Age of monolayer

5 days

14 days

21 days

28 days

35 days

56 days

ANTISERA OVINELHan

SERIES- NUMBER OF CELLS IMMUNOREACTIVE WITH

OVINEFSH, OVINELH, OVINELHP,

Positive cells (a)

FSH LH LHP LHa FSH LH LHP LHa FSH LH LHP LHa FSH LH LHP LHa FSH LH LHP LHa FSH LH LHP LHa

1.7 4.4