Recent Progress in Hormone Research: Proceedings of the 1975 Laurentian Hormone Conference

EDWARD C. REIFENSTEIN, JR.

Even before Ed Reifenstein was born, it was ordered that he would have a long and productive career in medicine. His father practiced medicine until he was almost ninety years old and he retired then only at the gentle and diplomatic insistence of his three doctor sons. What was not ordered, but came about by good fortune, was that Ed’s medical career would be in investigative medicine.

Edward Conrad Reifenstein, Jr., was born in Syracuse, New York in 1908, graduated from the University of Syracuse in 1930 and from the University of Syracuse Medical School (magna cum laude) in 1934. After a rotating internship and a medical residency, he went into psychiatry and became interested in the use of drugs in psychiatric disorders, specifically in exploring the use of the then new amphetamines in the treatment of alcoholism. While doing some research in this area (the subject of some thirteen publications), he began to think of postdoctoral study and research as a career. He was, however, loath to disappoint his father, who fondly hoped that his son would join him in his large practice of internal medicine and psychiatry. Moreover, he had married Esther Tilden at the end of his fourth year of medical school and their son Edward Reifenstein 3rd was born in 1937 while Ed was still a resident in the Syracuse Psychopathic Hospital. Since interns were paid $100 per month in those days and married interns were unheard of, Ed decided on the practical course of action and practiced medicine with his father from 1937 until 1940. In the summer of 1939, however, he took two weeks off to come to Boston with his pregnant wife and his son Edward to take Fuller Albright’s course in clinical endocrinology. That was when the science of endocrinology was in its childhood. Born in Europe, it was growing up in Boston, and just beginning its adolescent growth spurt. It was Ed’s sure and shrewd sense of what would prove to be important which sent his application for postgraduate training in endocrinology to Fuller Albright’s office and lodged it in the Prospective folder. He had given clinical practice, and his father’s suggestions, a good trial and knew that he did not want to be a practitioner.

The Prospective folder was a fat folder. When a space in Albright’s small group became available, the folder was pulled from the file, the letters were ruffled through, and, guided by no-one-ever-knew-what, Albright would pull one out and instruct his secretary to Tell him to come if he can get his own money. If the applicant cared more for intellectual than material sustenance, and if, like Ed, he had a loyal and loving wife who was willing to scrimp and sacrifice, he would drop whatever other plans he might be considering and come. That is how Ed’s keen mind and his great capacity for hard and well-organized work came to be applied in a most fortunate milieu, where they soon resulted in excellent and sustained achievement.

Within the year the United States was at war. The energies of the Albright department were turned toward metabolic aspects of convalescence including bone and wound healing as were the energies of endocrine departments in Baltimore, New York, and Montreal. The Macy Foundation sponsored regular conferences at which the several groups met in New York and reported their progress. It fell to Ed, not only to do much of the work of carrying out the metabolic experiments and of administering the metabolic ward, but to edit the proceedings of the Macy Foundation meetings. The results of these labors are historic. The paper by Reifenstein, Albright, and Wells, entitled The Accumulation, Interpretation and Presentation of Data pertaining to Metabolic Balances, notably those of Calcium, Phosphorus and Nitrogen, describes the meticulous technique of a good metabolic balance study and shows how to analyze its results intelligently. The fourteen volumes of the Macy Foundation’s reports on metabolic aspects of convalescence present, in exceptionally lucid and readable form, most of what we know today about the adrenal response to stress and its metabolic consequences, and about the hormonal control of anabolism. The nitrogen and electrolyte losses caused by stress were measured, as were the opposed anabolic actions of testosterone. The separate contributions of stress and of immobilization to atrophy of bone were studied. Totally intravenous feeding (Ed would not have used the term hyperalimentation) was explored. Ward IV became a model for metabolic wards all over the country. A less tangible, but no less important, result of the Macy meetings was the tradition they established among all their participants, of freely sharing unpublished ideas and findings. Ed never wavered in this morality, and one of the joys of talking with him at meetings in the years which followed was the candor and generosity with which he shared his immense store of information.

During the war Ed had been declared by Selective Service to be indispensible to the Massachusetts General because of the importance to the armed services of the work in which he was engaged, and the obvious inability of Fuller Albright, with his severe physical handicap, to carry it out without him. By the time the war had ended and the Macy Meetings had been discontinued, Ed and Fuller had started on another project of great interest and importance. The Parathyroids and Metabolic Bone Disease by Albright and Reifenstein is one of the most remarkable and valuable medical texts written in our time. In an era when medical texts are seldom expected to survive three years without revision, this classic text was reprinted unaltered and widely read for twenty years. (One of the few things Ed had to regret was that because Albright died, and his collaborators were scattered and otherwise occupied, he was not able to revise and update that book). Work on the book was largely finished by 1946, and, the war being over, Albright was able to find able assistants. Ed was 38 years old and ready to cease being an assistant and seek an independent career. He did not leave that department, however, before being immortalized by a new syndrome. In the clinic Ed had observed a patient (Mr. K.) with an unusual kind of testicular defect. After patiently tracking down and examining a large number of Mr. K’s relatives, he described the rare genetic disorder, Reifenstein’s syndrome, which has proved to be of great theoretical interest.

By that time the importance of endocrinology to many branches of medicine had gained recognition, and the Sloan-Kettering Institute, perceiving its relevance to the understanding and treatment of cancer, wanted an endocrinology department and a metabolic ward. Ed became the chief of the endocrine unit and remained there until 1950. During that time he became a consultant to Ayerst McKenna and Harrison, and in 1949 became the director of their division of medical research. Always interested in pharmacology, and particularly in the development and applications of the steroid hormones, he found that the work suited him ideally. Except for three years when he served as director of the Medical Research Institute in Oklahoma City, he continued to do similar work for a time with the Schering Co., but chiefly at the Squibb Medical Research Institute, until his retirement in 1974.

It was during those twenty years that the adrenal and gonadal steroids became available in pure form for clinical use and began to assume their important place in medicine. Just before Ed had left the Massachusetts General Hospital, desoxycorticosterone, estradiol, and testosterone had become available in pellet form. Ed had learned the technique of inserting pellets and before leaving had purchased the equipment and taught his colleagues how to use it. The gonadal steroids, their anabolic actions, their use in the menopause and old age, and later on, their importance as contraceptives remained Ed’s chief interest, although far from his only interest, for the rest of his life. His knowledge of the field was quite extraordinary. He subscribed to twenty-six journals and read and abstracted every one.

Ed’s other interests were climbing mountains, which was reserved largely for summer vacations, preparing spectacular collections of color slides of his trips, and playing the piano, which he did at the end of the day to relax after work, or to enliven evenings with his friends. His home, secluded in the country, was a happy one and he liked to be in it as much as possible. His study, like his mind, was peaceful and orderly: he considered it the most important room in the house, and spent a good deal of time in it. The big desk is near a window which looks out on trees and a lake. The shelves of bound journals reach to the ceiling. A large adjoining closet is filled with filing cabinets and slide cabinets. In the strict physical sense, as well as mentally, Ed kept a vast store of information so ordered that he could instantly find any part of it.

His writing was as well ordered as his mind—scholarly, lucid, and accurate. The same qualities which made him a good organizer and director made him a good editor; they were solid reliability and unshakable integrity, combined with a very shrewd perception of what was sound, what was important, and what was practicable. It is not necessary to tell the readers of Recent Progress how much they and the members of the Laurentian meetings have profited from these abilities of Ed’s. (No one who attended will forget how devotedly Ed worked at these meetings, his central role in the Committee on Arrangements, or his gentle persistence in calling for delinquent revisions of typescripts.)

One might digress briefly to recall how these qualities were formed (and why we may not soon see them again). The shrewdness was native, but the integrity and morality with which it was implemented were the gifts of his family and of the era in which he was trained. The traditional medical career, at the time the Reifenstein family became committed to it, was one which was rewarded much more by respect than by wealth. Before World War II, one could say the same for medical research. The field was uncrowded, research grants were small, laboratory equipment was simple, and assistants worked for a pittance. Idealism was the rule, and ethics were taken for granted. Cooperation among investigators and between individual investigators was assumed; secretiveness and selfish competition were frowned upon. The entry of government into medical research and the subsequent enormous increase, both in the amount of money required for research and in the number of people competing for that money, have changed the atmosphere greatly, but Ed never changed. He was always conservative, always businesslike and practical, always open and ready to share results and ideas: he remained so without budging a hairsbreadth in morality. He remained as true to his principles as to his friends, and he commented that he was almost glad to be retiring from a profession which had grown too large for close team work and was tending to lose the disinterested devotion that he had found in, and given to, academic medicine. He also welcomed retirement, because he hoped it would give him more time to work. He envisioned remaining at his desk, reading and writing and serving as an editor and consultant. He was accustomed to being a pillar of strength, reliability and integrity in these capacities; by his many friends and colleagues he will be remembered and he will be missed.

ANNE P. FORBES and FREDERIC C. BARTTER

Tribute to Gordon Tomkins

ISIDORE S. EDELMAN

On July 25 of this year the San Francisco Chronicle carried a story that began with the following paragraph:

Dr. Gordon M. Tomkins of the University of California, one of America’s most distinguished biochemists and a pioneer researcher in the field of hormone activity died in New York City on July 22, 1975. He was 49 years of age and had been ill since undergoing difficult brain surgery last June.

The facts of Gordon’s life are that he was born on June 4, 1926, in Chicago; his forty-ninth birthday passed while he was in a coma. He was a superb student, graduating at the age of 19 from the University of California, Los Angeles, with high honors, received an M.D. degree from Harvard Medical School at the age of 23, again with high honors, and completed a Ph.D. degree at the age of 27 at the University of California, Berkeley, under the guidance of Professor I. L. Chaikoff. In addition to an internship at the Peter Bent Brigham Hospital and postdoctoral fellowships, Gordon worked at the National Institutes of Health for more than a decade and in the last seven years of his stay, served as Chief of the Laboratory of Molecular Biology where he established a truly distinguished biochemical laboratory. Since 1969 he was on the faculty at the University of California in San Francisco. Gordon authored or co-authored 187 papers of remarkable impact on contemporary endocrine biochemistry. Gordon received many honors: He was the recipient of the prize in Molecular Biology of the Washington Academy of Sciences. He was awarded a Mider Lectureship at the Nationl Institutes of Health, a Jesup Lectureship at Columbia University, a Harvey Society Lectureship, and a Prather Lectureship at Harvard. He was elected to the American Academy of Arts and Sciences, and at the University of California, San Francisco, he received the highest recognition that we could give him in naming him the Faculty Lecturer for 1973. I think most of you are familiar with Gordon’s many distinguished contributions–of remarkable scope–in the fields of enzymology, steroid biosynthesis, and molecular biology of regulatory processes, particularly with respect to hormone action. Recently he was deeply involved in formulating a highly original proposition with respect to the evolution of regulatory systems, particularly hormonal systems. The written record of Gordon’s contributions to contemporary biology and medicine tell only part of the story, however. Gordon was a remarkably gifted man. While in college, he played as a professional jazz musician with Stan Kenton’s orchestra and with Charlie Barnett. Moreover, he was an excellent classical musician. He was widely read in a range of fields that were quite remarkable, but beyond all that, he had the ability to give both friendship and enthusiasm that is almost unparalleled in a society of scientists. Gordon’s favorite word was terrific and he applied the word not to his own work but to the work of others. He had this incredible quality of being able to be deeply involved in his own work and deeply interested in what everybody else was doing. Consequently he made numerous contributions to the work of others that will never appear as a part of the record. For me, Gordon’s death was a great personal loss; and, of course an even greater one for his wife, Millicent, a highly talented artist and musician in her own right, and their lovely and musically gifted daughters, Leslie and Tanya. Beyond the personal circles of family and friends, Gordon will be sorely missed because of his special qualities as a scientist. His originality, creativeness, and ability to inspire students and colleagues was awesome. His death leaves a permanent void in our community.

Genetic Approaches to Steroid Hormone Action

KEITH R. YAMAMOTO and MARTHA R. STAMPFER,     Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California

ULRICH GEHRING¹

CAROL H. SIBLEY²

Publisher Summary

Current biochemical evidence favors a common molecular mechanism for all the steroid hormones. The steroid binds tightly and specifically to a cytoplasmic receptor protein, thereby increasing the affinity of the receptor for some chromosomal component. This reaction triggers events that directly affect the expression of certain specific genes. Thus, steroid receptors are the most experimentally accessible candidates for eukaryotic transcriptional regulatory proteins. However, biochemical studies are alone limited to a descriptive or correlative role in characterizing events as they occur in vivo. A full understanding of some regulatory mechanisms in the simplest prokaryotic systems has been achieved through the combined use of biochemistry and genetics. Although such manipulation of eukaryotic cells is complex, recent advances in somatic cell suggest that this strategy can provide valuable information in these systems as well. Therefore, it seems desirable to seek these biological approaches to verify existing results and get more detailed insight into the interactions between receptor–steroid complexes and nuclear binding sites. This chapter presents some cell biological studies to determine the molecular actions of steroid hormone receptors and a general class of eukaryotic gene regulatory elements and reviews their results.

I Introduction

Present biochemical evidence favors a common molecular mechanism for all steroid hormones in which the steroid binds tightly and specifically to a cytoplasmic receptor protein, thereby increasing the affinity of the receptor for some chromosomal component. This reaction is then presumed to trigger events that directly affect the expression of certain specific genes (for a review of this and other viewpoints, see Gorski and Gannon, 1976). Thus, steroid receptors appear to be the most experimentally accessible candidates for eukaryotic transcriptional regulatory proteins (Yamamoto and Alberts, 1976). However, biochemical studies alone are somewhat limited to a descriptive or correlative role in characterizing events as they occur in vivo. Only through the combined use of biochemistry and genetics has a full understanding of some of the regulatory mechanisms in even the simplest prokaryotic systems been achieved. Although such manipulation of eukaryotic cells is complex, recent advances in somatic cell genetics (Thompson and Baker, 1973; Chasin et al., 1974; Fenwick and Caskey, 1975) suggested that this strategy may provide valuable information in these systems as well. Thus, in order to verify existing results andget more detailed insight into the interactions between receptor–steroid complexes and nuclear binding sites, it seemed desirableto seek these more biological approaches.

Recognizing the advantages of a genetic system for studying mammalian gene regulatory mechanism, Gordon Tomkins chose a few years ago to exploit certain properties of a cultured line of glucocorticoid-responsive mouse lymphoma cells. We recount here some of thescientific fruits of his inspiration, enthusiasm, guidance, and support. It seems certain that systems such as this one, as well as others that are described below, will be important in ultimately determining the molecular actions of steroid hormone receptors and perhaps a general class of eukaryotic gene regulatory elements.

II Cell Biological Studies

Steroid action can be conceptualizedas a series of discrete steps, arranged, in the simplest case, in a linear pathway (Fig. 1). By this view, a defect at any step in the pathway would result in a failure of the cellto display the hormone response. Ideally, then, selection of a large number of clonal populations, each lacking the specific response, would yield a family of defective cell lines which together represent lesions at each step in the pathway. Such lines might then be usedto define the reactions actually involved in the response, and how they relate to one another at the molecular level.

FIG. 1 Hypothetical linear pathway for steroid hormone action. S, steroid; R, receptor protein; N, nuclear binding site.

A SELECTION OF VARIANTS

Clearly, the complexity of eukaryotic systems requires highly selective conditions if specific lesions are to be detected. In the mouse, as well as in some other vertebrates, certain types of immunocytes are killed by physiological doses of glucocorticoids (Claman, 1972). Although the actual mechanism of the killing is not well defined (see below), such a biological phenomenon provides the necessary conditions for isolation of nonresponding variants.

S49 cells are a cloned line derived from a mineral oil-induced lymphoma in BALB/cmice (Horibata and Harris, 1970). Chromosome analysis reveals a stable pseudodiploid karyotype of 40 acrocentric chromosomes, although a few spontaneous pseudotetraploid subclones have appeared. The cells grow in suspension culture with a generation time of 14–18 hours; the presence of θ and TL antigens suggests that they are of thymic origin and have retained at least some of their differentiated properties. Moreover, they remain sensitive to the cytotoxic action of glucocorticoids (Harris, 1970; Baxter et al., 1971; Rosenau et al., 1972).

When S49 cells are plated in soft agar on mouse embryo fibroblast feeder layers (Sibley and Tomkins, 1974a), they clone with an efficiency close to 100%. If, however, the medium contains the synthetic glucocorticoid dexamethasone (dex), the cloning efficiency is greatly diminished (Fig. 2). Whereas the vast majority of cells cannot form colonies in agar supplemented with dex, an occasional steroid-resistant clone is seen. When these colonies are isolated and recloned, increasing steroid concentrations have no effect on their colony-forming ability (Fig. 2). The efficiency of plating of these cells was the same in the presence and absence of the steroid, evenafter growth in the absence of dex for more than a year. Thus, resistance to steroid killing is a stable heritable trait.

FIG. 2 ) clones were plated in the presence of the indicated concentrations of dexamethasone according to the procedure described by Sibley and Tomkins (1974a). Briefly, a mouse embryo fibroblast feeder layer was seeded at low density (5 to 10 × 10⁴ cells per 60-mm petri dish) and overlayed with 5 ml of growth medium containing 0.51% agar. A second overlay (1.2 ml of medium at 0.31% agar) contained the lymphoma cells. The number of clones per plate was counted at the end of 10 days. The efficiency of plating shown is the average of five identical plates. From Sibley et al. (1974).

B ORIGIN OF STEROID-RESISTANT CELLS

A fluctuation test (Luria and Delbrück, 1943) was used to calculate the rateof appearance of steroid-resistant cells in asteroid-sensitive population. In these experiments, 45 separate steroid sensitive clones were isolated and immediately recloned in the presence and in the absence of 5 × 10–7 M dex. The number of steroid-resistant subclones was normalized to the plating efficiency (0.4–1.0). With these data (not shown), we used the minimal deviation method of Lea and Coulson (1949) to calculate the rate ofoccurrence of steroid-resistant cells, accounting both for the appearance of new resistantcells in each generation and for their replication in subsequent generations. By this method, we estimated that the rate of developmentof new resistant cells is 3.5 × 10–6 per cell per generation (Sibley and Tomkins, 1974a).

The stability and frequency of occurrence of the resistant phenotype is similar to that found with heritable changes in other mammalian somatic cells which are thought to be due to true genetic alterations (Thompson and Baker, 1973). However, a second possibility is that the transition is a stable phenotypic change, perhaps induced by steroids in a small fraction of all sensitive cells. This possibility seemed particularly plausible since immature steroid-sensitive mouse thymocytesbecome steroid resistant during their differentiation into mature immunocompetent T cells (Claman, 1972). However, two types of experimental results imply that this explanation is incorrect. First, the fluctuation test (Luriaand Delbrück, 1943) showed that the transition is a random event, rather than occurring uniformly at the time of steroid treatment. Second, treatment of the cells with any ofthree agents known to be mutagenic in other systems (9-aminoacridine, N-methyl-N′-nitro-N-nitrosoguanidine, and γ-irradiation) increased the frequency of steroid resistance 2–60-fold (Sibley and Tomkins, 1974a). Thus, although definitive proof of a mutational event will require demonstration of an altered nucleic acid and protein sequence, our data are consistent with the idea that the dex-resistant variants have a mutational origin.

C PRELIMINARY PHENOTYPIC SCREENING OF VARIANTS

If the selection for dex-resistant variants indeed yields a series of clones withlesions at each step in the mechanism of steroid action (Fig. 1), the different clones should be distinguishable in their ability to bind radioactive steroid. For example, steroid-resistant cells defective in hormone penetration or hormone–receptor association should exhibit diminished cellular retention of [³H] dex. In contrast, cellswith defects in subsequent steps in the pathway would take up wild-type levels of steroid but might display abnormal transfer of the steroid-receptor complex to the nucleus. Thus, in order to rapidly screen relatively large numbers of resistant clones, we developed a method for quantitation of whole-cell uptake (Fig. 3A) as well as localizationof bound dex in crude cell fractions (Fig. 3B).

FIG. 3 Outline of the method used for estimating specific steroid binding in whole cells and in crude cell fractions. Duplicate cell suspensions (1 to 3 × 10⁷ cells) of each clone to be tested were centrifuged at 800 g for 5 minutes. The pellets were resuspended in 0.5 ml of Dulbecco’s Modified Eagle’s medium supplemented with 10% horse serum and 4 × 10–8 M [³H] dexamethasone (35 Ci/mole). To one cell suspension of each clone was also added a 500–1000-fold excess of unlabeled dex. Specific binding of [³H] dex is defined as the difference in [³H] dex cpm bound in the presence and absence of unlabeled dex. After incubation at 37°C for 40 minutes in a humidified atmosphere of 10% CO2–90% air, the cells were centrifuged, washed with 5 ml of phosphate-buffered saline (PBS) at 25°C, and resuspended in 0.5 ml of cold PBS. A 100-µl aliquot was counted to determine specific retention of [³H] dex by whole cells. The remaining suspension was centrifuged and resuspended in 0.4 ml of cold hypotonic buffer (20 mM tricine, 2 mM CaCl2, 1 mM MgCl2, pH 8.0 at 0°C). The cells were lysed by freezing and thawing, and crude nuclear and soluble fractions were separated by centrifugation at 800 g for 5 minutes. A 200-µl aliquot of the cytosol was counted directly. The nuclear pellet was washed once with 5 ml of cold hypotonic buffer, then counted. From Sibley and Tomkins (1974b).

The whole-cell binding assay revealed that the majority of dex-resistant clones retained little or no steroid. Figure 4summarizes such an analysis of a representative series of 200 clones, showing that about 55% of the isolates failed to bind any hormone above background levels and 70–75% retain <30% of the wild-type levels of specifically bound steroid. The remaining clones exhibit varying levels of binding ranging up to about twice the wild-type levels.

FIG. 4 Specific retention of [³H] dex by steroid-resistant clones. A series of 200 steroid-resistant clones was assayed for specific retention of [³H] dex in whole cells as described in Fig. 3.

Clearly, whole-cell binding of steroid reflects complex phenomena at a very grosslevel without regard to mechanisms. For example, diminished steroid retention could resultfrom at least three different defects: impaired penetration of the hormone into the cell, decreased concentrations of intracellular receptor protein, or a reduced affinity of the receptor for steroid. Lesions affecting receptor dosage and hormone affinity could be differentiated by analysis of binding data with Scatchard plots (Scatchard, 1949), although we have not yet carried out an extensive series of these tests. Defective hormone transport is distinguished from these latter possibilities by carrying out cell-free steroid binding on soluble extracts of individual clones. Figure 5demonstrates that wild-type extracts show specific and saturable binding of [³H] dex, as measured by retention of [³H] dex receptor complexes on DEAE filters (Santi et al., 1973). Likewise, resistant clones that bind dex normally in the whole-cell assay exhibit normal bindingin extracts. In contrast, Fig. 5 shows that cells which fail to retain steroid are also devoid of dex-binding activity in extracts. Thus, in the case of the clones illustrated in Fig. 5, and indeed for all clones tested, cells lacking binding activity appear to have receptor lesions rather than steroid transport defects.

FIG. 5 Cell-free steroid binding activity of steroid-sensitive cells and two classes of steroid-resistant clones. Cell extracts were prepared as described in Baxter and Tomkins (1971). Briefly, cells were washed with cold PBS, disrupted in hypotonic buffer using a Teflon-glass tissue homogenizer, and centrifuged at 100,000 g for 1 hour. The supernatant fractions were incubated in duplicate 75-µl aliquots for 90 minutes at 0°C, then in the indicated concentrations of [³H] dex. Specifically bound steroid was assayed using the DEAE-cellulose filter method of Santi et al. (1973). Protein concentrations were assayed according to Lowry et al. , ×, two independently isolated steroid-resistant receptor-activity-deficient clones. From Sibley and Tomkins (1974b).

Previous experiments (Baxter et al., 1971; Rosenau et al., 1972) had suggested that nuclear localization of the receptor–steroid complex occurs following steroid binding in S49 lymphoma cells, just as in other hormone-responsive systems. Therefore, clonesdisplaying steroid binding in the whole cell assay were further characterized with respectto their ability to translocate steroid–receptor complex to the nucleus, using the method diagrammed in Fig. 3B. Under the conditions we adopted for that assay, about 50% of the [³H] dex-labeled receptors localize in the nuclear fraction. Receptor-containing variants, on the other hand, showa broad range in their ability to localize receptors in the nucleus. Some clones transferred <10% of labeled receptors into the nuclearcompartment, some appeared to transfer receptor normally, and others localized virtually all their receptors in the nucleus. Figure 6summarizes the results of nuclear translocation experiments with 70 receptor-containing clones.

FIG. 6 Nuclear localizations of [³H] dex by wild-type and steroid-resistant receptor-containing cultures. Intracellular distribution of the bound [³H] dex was determined by the procedure outlined in Fig. 3. Nuclear localization was defined as in Table I. Open bar, steroid-resistant clones; stippled bar, wild-type clones.

From these crude but simple assays, all dex-resistant variants isolated can be placed in one of four phenotypic classes (TableI): r–, receptor activity deficient³; nt–, nuclear transfer deficient; d–, the so-called deathless phenotype which appears to be normal inits steroid- and nuclear-binding characteristics; nti, increased nuclear transfer. These phenotypes have subsequently been subjected to the more detailed biochemical and genetic analyses described below.

TABLE I

Intracellular Distribution of Bound[³H] Dexamethasone after in Vivo Labelinga

aWild-type and steroid-resistant clones were assayed for specific retention of [³H] dex as described in Fig. 3. The percentage of nuclear-localized [³H] dex was determined as the specifically bound [³H] dex in the nuclear pellet divided by the sum of specifically bound [³H] dex cpm in the nuclear and cytosol fractions. Values given are the mean and range of 2–4 determinations.

III Physical Properties of Crudeand Partially Purified Receptors

Although it seemed reasonable that the receptor protein itself might be defectivein the r– cells, the lesions responsible for the other phenotypes were not easily predicted. As a preliminary experiment the nt– and nti variants were tested in heterologous cell-free nuclear binding experiments to determine whether the defective components were located in the nuclear or cytoplasmic compartments. For example, Table II shows that when [³H] dex-labeled extracts from wild-type and nt– cells are incubated with either wild-type or nt– nuclei, the nuclear retention of receptors is characteristic of the source of the labeled soluble extract, regardless of the source of the nuclei. Likewise, the nti defect also appears to reside in the cytoplasm (datanot shown).

TABLE II

IIn Vitro Binding of [³HJDexamethasone-Receptor Complexes to Nuclei from Wild-Type and nt− Cellsa

aExtracts were labeled with [³H] dexamethasone and incubated for 60 minutes at 20°C with 10⁸ nuclei. The incubations contained 3.8 mg of cytosol protein, corresponding to 0.87 and 0.63 pmole of specifically bound dexamethasone per milligram of protein in the wild-type and nt– extracts, respectively. Data are from Gehring and Tomkins (1974).

Thus, at least three of the four variant phenotypes display cytoplasmic lesions, perhaps in the receptor protein itself. These results suggested that it might be possible to detect differences inthe physical properties of the receptors fromwild-type and variant lines. For these preliminary experiments, physical studies were carried out under nondenaturing conditions by monitoring the [³H] dexreceptor complex in neutral sucrose gradients and gel permeation chromatography.

A SUCROSE GRADIENT CENTRIFUGATION

Table III summarizes sedimentation data for extracts from wild-type cells and some selected representatives of nt–, nti, and d– clones. The 5 to 20% sucrose gradients were prepared in buffers containing either 50 mM or 250 mM NaCl. Under these conditions, crude extracts containing wild-type receptors sediment at 5.5 S and 4.0 S, respectively. In contrast, the nt– and nti extractsshow small but reproducible differences from the wild-type receptors, ranging from 0.4 to 0.9 Svedberg unit (S), depending on the cloneand ionic conditions used. The d– receptors, however, are indistinguishable from wild type.

TABLE III

Sedimentation Properties of [³H]Dexamethasone-Labeled Receptorsa

aFrozen cell pellets were homogenized in TEGNO5 (10 mM Tris, pH 8.1; 1 mM EDTA; 10% (v/v) glycerol; 50 mM NaCl; 1 mM β-mercaptoethanol; 100 µg of bovine serum albumin per milliliter) and subjected to ultracentrifugation (90 minutes at 160,000 g); the supernatant receptors were labeled in the presence of 5 × 10–8 M dex. Where indicated, the receptors were purified 10²–10³-fold by the DNA-cellulose procedure described in the text. Extracts were adjusted to the desired NaCl concentration, and 100-µl aliquots (5 to 15 × 10³ cpm as receptor-bound [³H] dex) were sedimented through 5 to 20% sucrose gradients containing the same buffer as the sample applied. Sedimentation was at 234,000 g for 17–24 hours, and Svedberg units were calculated relative to the enzyme activity peak of a 6.2 S internal standard (Escherichia coli alkaline phosphatase) in each gradient. Values given are the average of 2–5 determinations.

The low salt and high salt forms of the receptors arefreely interconvertible in crude extracts (data not shown). However, as shown in Table III, receptors that have been purified 10²–10³-fold by DNA-cellulose chromatography (see below) remain in their more slowly sedimenting high salt forms even when they are sedimented in 50 mM NaCl. When intermediate ionic strengths are tested, intermediate sedimentation behavior is observed, whereas more elevated salt concentrations (up to at least 750 mM) do not affect the behavior of the form observed at 250 mM NaCl. Moreover, addition of unlabeled crude extracts to partially purifiedreceptors at 50 mM NaCl causes the labeled receptors to return to the more heterogeneously sedimenting low salt forms (K. R. Yamamoto, unpublished). Taken together, these results suggest that the sedimentation behavior of these receptors at <250 mM NaCl reflects aggregation of receptors with nonreceptor components. This complicates the interpretation of sedimentation differences detected under low ionic strength conditions. Although the aggregation products could be specific and biologically significant, their heterogeneity implied to us that they are nonspecific innature and probably represent artifacts of cell homogenization. Other investigators have come to similar conclusions in their studies with other receptor systems (Chamness and McGuire, 1972; Stancel et al., 1973).

B GEL PERMEATION CHROMATOGRAPHY

The detectable difference in sedimentation properties encouraged us to characterize the receptors further by means of gel permeation columns. For example, Fig. 7A compares the elution profiles of partially purified wild-type and nti receptors, chromatographed on separate Bio-Gel A 0.5M columns. It can be seen that the receptors display striking differences when assayed by this procedure: wild-type receptor elutes before a catalase standard, whereas the nti receptor does not appear until after the hemoglobin standard. Figure 7B shows that this difference is not an artifact of different contaminating components in the two receptor preparations. In this experiment, the two receptors were mixed, then chromatographed on a single Bio-Gel column. The bound [³H] dex eluted as two distinct peaks at precisely the locations of the independently chromatographed receptors.

FIG. 7 Gel permeation chromatography of partially purified S49 receptors. Receptors were prepared, labeled, and partially purified as described in Table III. Extracts were adjusted to 0.50 M NaCl, and 500-µl aliquots were loaded onto 1 × 45 cm columns of Bio-Gel A 0.5M. Elution was regulated by pumping at 2 ml/hour; 350-µl fractions were collected, and a 100-µl aliquot of each fraction was counted. Peaks at fractions 14–15 represent aggregated receptors at the excluded volume of the columns, and peaks at fractions 68–69 are unbound [³H] dex marking the included volume. Vertical arrows denote internal protein standards: c, beef liver catalase; ap, Escherichia coli ) chromatographed on separate columns. Lower panel: wild-type and nti receptors mixed and chromatographed on a single column.

C ESTIMATION OF MOLECULAR WEIGHT AND SHAPE

Using the data from both sucrose gradient sedimentation and gel permeation chromatography, one can calculate estimates for the daltons true molecular weight and shape of the receptors (Siegel and Monty, 1966). Thus, the wild-type receptor is approximately 90,000 daltonswith an axial ratio (prolate) of about 8:1, while the nti receptor testedis 50,000 daltons with a 4:1 axial ratio. In contrast nt– receptors examined by these methods were only slightly altered from wild-type behavior and the d– receptors were identicalto wild type (not shown).

One interpretation of these data is that the nti and nt– clones tested are products ofmutational events in the structural gene for the receptor. Obviously, such a conclusion is only preliminary, and further substantiation will require characterization of highly purified receptors under denaturing conditions. Nevertheless, it is clear that cell-free extracts from wild-type and some of the variant lines display detectable differences. Thus, we proceeded to further exploit these differences to investigate other aspects of the steroid response.

IV DNA Is the Major Nuclear Binding Site in Vivo

Although the phenomenon of nuclear localization of steroid–receptor complexes is well established, the actual nuclear component to which the receptors bind in vivo is unknown. In vitro approaches to this question have been limited by the fact that steroid receptors appear to interact with many nuclear components under the appropriate conditions. Thus, various investigators have reported binding of radioactive steroid–receptorcomplexes to chromatin preparations (Maurer and Chalkley, 1967; O’Malley et al., 1971), to nuclear membranes (Jackson and Chalkley, 1974), to specific nuclear acidic (O’Malley et al., 1972) and basic proteins (Puca et al., 1974), and toDNA (Baxter et al., 1972; King and Gordon, 1972; Yamamoto and Alberts, 1972). However, it is difficult to evaluate the significance of these phenomena in vitro because correlation with observations in vivo is either indirect or lacking altogether.

The nt– and nti lymphoma variants were therefore obvious choices for further in vitro study: they carry receptors with functional hormone binding sites but are aberrant in their in vivo nuclear binding properties. We reasoned that if the receptors interact with DNA in the whole cell, then in vitro conditions might be found in which the ability of receptors to bind pure DNA reflects their nuclear binding potential. On the other hand, if receptors actually bind only to chromosomal proteinsor nuclear membrane, a direct relationship with DNA binding would not be expected.

A RECEPTOR BINDING TO DNA REQUIRES ACTIVATION

To assess the binding of the variousreceptors to DNA, cell extracts were preparedin buffer containing 50 mM NaCl and chromatographed on native calf thymus DNA-cellulose columns. Only a minor proportion of the labeled receptor loaded onto the column (an average of 8% in wild type) bound to the DNA if the extract was kept cold throughout the procedure (Table IV). However, if either the wild-type or variant extracts were incubated at 20°C for 35 minutes prior to chromatography at 4°C, the DNA-binding of receptor–steroid complex increased by about an order of magnitude. This phenomenon has been termed activation and has been shown to stimulate cell-free binding of receptor–steroid complexes to intact nuclei and DNA (Higgins et al., 1973; Milgrom et al., 1973; Yamamoto et al., 1974). The physical changes occurring upon activation are not understood; preliminary experimentsshow that the 20°C incubation does not result in a detectable change in the sedimentation properties of the receptors (K. R. Yamamoto, unpublished), unlike the 4 S to 5 S conversion observed with estradiol receptors under similar conditions.

TABLE IV

Binding of Receptors to DNAa

aInput [³H] dexamethasone ([³H] dex)-receptor complex was quantitated by subjecting an aliquot of extract either to treatment with activated charcoal to remove free [³H] dex or to sucrose gradient sedimentation. Extracts containing 10,000–50,000 cpm of [³H] dex-labeled receptor in 0.5 ml of TEGNO5 buffer were loaded onto 1-ml DNA-cellulose columns as previously described (Yamamoto et al., 1974) and pumped at a constant rate of 3–4 column volumes per hour. After the unbound material was rinsed out with 6 column volumes of TEGNO5, bound receptor was eluted with buffer containing 250 mM NaCl. Fractions (200–300 µl) were collected directly into counting vials. Values given are the mean and range of at least two determinations.

The observation that the 20°C incubation stimulates DNA binding in not only the wild-type, but also the variant, extracts suggests that none of the resistant clones isdefective in the activation step. Furthermore, activation-dependent DNA-binding was exploited for partial purification of receptors. Extracts are chromatographed on DNA-cellulose without prior incubation at 20°C. The column flowthrough, containing receptors and proteins that do not bind DNA, is then incubated at 20°C and chromatographed on a second DNA-cellulose column. Receptors eluted from this second column are purified 10²−10³-fold in 50–75% yield, remain in the slowly sedimenting high salt form even at 50 mM NaCl, and are stableto storage at −70°C (data not shown). The term partially purified receptor in this report refers to extracts which have been subjected to this DNA-cellulosepurification regimen.

B DNA-BINDING AND ELUTION OF VARIANT RECEPTORS

Table IV shows that under conditionswhere 75–100% of the input receptors from wild-type, nti, and d– lines bind DNA, only 10–15% of the nt– receptors bind. This small fraction bound appears to be an equilibrium figure, since it does not increase as the duration of contact between the extract and the DNA-cellulose is lengthened from 20 minutes to 4 hours. Furthermore, when both bound and unbound fractions were reloaded onto new DNA-cellulose columns, about the same low percentage binding occurred with each, showing that the binding observed is not due merely to heterogeneity in the receptor population (data not shown). Moreover, when the bound receptors are eluted from DNA-cellulose with a gradient of increasing ionicstrength, the variant receptors elute from DNA-cellulose at 110–120 mM NaCl, significantly lower than the 170 mM NaCl required to elute wild-type receptors (Fig. 8A).

FIG. 8 Elution of receptors bound to DNA-cellulose with 50–300 mM , NaCl gradient. (A) and (C) are from Yamamoto et al. (1974). After the columns were rinsed free of unbound material, bound receptors in the two columns were eluted with a NaCl gradient drawn from a single mixing chamber.

The nti receptors, which show abnormally high nuclear localization in whole cells, bind like wild type, almost quantitatively to DNA-cellulose columns after 20°C activation(Table IV). However, these variant receptors required 210 mM NaCl to be eluted from the DNA, a significantly higher concentration than that needed for elution of wild-type receptor (Fig. 8C).

In contrast, the DNA-binding and elution properties of the d– receptors are indistinguishable from wildtype (Fig. 8B). Thus, according to both physical and binding criteria, d– cells contain normal receptor molecules. Table V summarizes these data, showing that there is a direct relationship between the ability of a receptor to associate with nuclei in vivo, and the ionic strength required to elute it from purified DNA in vitro. Although the order of elution in a gradient of increasing ionic strength isnot a rigorous measure of the relative affinities of the receptors for DNA, these results strongly support the notion that the receptors from the nt– variants bind to DNA with affinities lower than wildtype, whereas those from the nti cells bind with higher affinity than theparental line.

TABLE V

Relationship of in Vivo Nuclear Binding and in Vitro DNA Bindinga

aConcentrations of NaCl required to elute receptors from DNA-cellulose were determined with linear elution gradients as described in Fig. 8. In vivo nuclear binding data, included for comparison, are from Table I.

Using sedimentation partition chromatography (Yamamoto and Alberts, 1974), the receptor-DNA interaction could be directly studied under equilibrium conditions. This technique employs a specially constructed sucrose gradient in which a large layer of DNA (sheared to 15–20 S in size) at a constant concentration is sedimented through a narrow band of partially purified 4 S [³H] dex-labeled receptors. After sedimentation, gradient fractions are collected normally. Any DNA–receptor interaction results in a partitioning of the receptors into the DNA layer of the gradient, and the degree of partitioning is a function of the equilibrium binding constant of receptor for DNA. Figure 9 depicts a comparison of wild type and nt– receptorsbinding to DNA. Calculation of equilibrium dissociation constants using this technique (Yamamoto and Alberts, 1974) shows that the interaction of the nt– receptor with DNA is characterized by an affinity which is about 70-fold lower than that for the wild-type receptor. Analogous experimentscomparing wild-type and nti receptors indicate that the latter bind DNA with affinities one to two orders of magnitude greater than wild-type (data not shown).

FIG. 9 Estimation of affinities of receptors for DNA as analyzed by sedimentation partition chromaotography. Receptors were prepared and partially purified as described in Table III, then subjected to sedimentation partition chromatography in TEGNO5. As described in detail elsewhere (Yamamoto and Alberts, 1974), this technique involves layering a narrow zone containing the receptor into the middle of a 5 to 20% sucrose gradient, the remainder of the gradient containing 15 µg of 15–20 S native DNA per milliliter. The gradients are centrifuged in a swinging-bucket rotor (15 hours at 114,000 g), causing the DNA zone to move continuously through the receptor zone. The sedimentation rate of this receptor increases as a function of its affinity for DNA, and can be used to calculate the dissociation constant (K). Arrow denotes the fraction in both gradients containing peak of alkaline phosphatase marker activity; stippling denotes DNA-containing fractions in the experimental gradient after sedimentation. Left panel: wild-type receptor, calculated KRD = 1.3 × 10–5 M. Right panel: nt– receptor, calculated KRD = 9.2 × 10–4 M.

Previous experiments with various steroid receptors have provided indirect evidence that DNA binding in vitro may be related to nuclear localization in vivo. First, the receptors must be complexed with hormone in order to bind either nuclei (Rousseau et al., 1973) or DNA (Yamamoto and Alberts, 1972, 1975; Rousseau et al., 1975). Second, a time- or temperature-dependent activation process required for nuclear transfer is also required for DNA-binding (Gorski et al., 1968; Jensen et al., 1968; Yamamoto, 1974). Third, the activation of both glucocorticoid and estradiol receptors gives increased affinities for DNA, but not for RNA (Yamamoto and Alberts, 1974; Rousseau et al., 1975). Finally, suboptimal- or anti-inducer compounds, which bind to receptors but do not promote their nuclear localization, also do not stimulate the receptor–DNA interaction (Baxter et al., 1972; Yamamoto and Alberts, 1972).

The wild-type and variant populations of S49 cells allow comparisons to be made more directly between in vivo and in vitro phenomena. Using these cell lines, our results demonstrate a direct relationship between nuclear binding in intact cells and changes in the apparent affinity of the receptors for purified DNA in cell-free extracts. Of course, it is conceivable that the variant receptors are also modified in their affinities for nuclear components other than DNA. Although this possibility has not been ruled out, the altered DNA-binding properties reported here would, by themselves, change the whole-cell nuclear localization characteristics in the ways observed, since sites are certainly available for protein-DNA interaction in the intact nucleus. Thus, in the simplest interpretation, these results strongly support the contention that DNA is the primary nuclear binding site for glucocorticoid receptors in these mouse lymphoma cells in vivo.

Using the methods described here, wehave not been able to approach the question of whether the receptor can recognize a specific DNA base sequence and bind to it with high affinity. In general, DNA-cellulose chromatography is not suited to the detection of specific site binding, owing to the high relative concentrations of nonspecific DNA sequences. Thus, even proteins withvery high affinities for specific sequences bind readily to nonspecific DNA under these conditions (Lin and Riggs, 1972).

Nevertheless, it is of interest that the requirement for activation, and the hierarchy of whole-cell nuclear binding observed for wild-type and variant receptors is faithfully preserved in cell-free binding to nonhomologous DNA. One interpretation of this resultis that the binding observed in whole cells may itself be to nonspecific sequences. Indeed, in vivo nuclear binding of estrogen (Williams and Gorski, 1972) and glucocorticoid receptors (Simons et al., 1976) appears to be nonsaturable and of low affinity. Similarly, certain results concerning in vitro transcription following steroid administration (Glasser et al., 1972) are most easily explained if receptors bind to many loci nonspecifically (Yamamoto and Alberts, 1976).

At present, we cannot exclude the possibility that steroid action requires only interaction of the receptors with any DNA sequence. Alternatively, steroid receptors might function by binding to only a few high-affinity chromosomal sites whose specificity is masked by the vast excess of low-affinity binding (Yamamoto and Alberts, 1974, 1975). The existence of the nti variant class implies that the first model, in its mostextreme case, is incorrect. Moreover, the apparently paradoxical binding behavior of the nti receptors can be easily explained in terms of the second model. That is, these receptors may have an increased affinity for biologically nonfunctional genome binding sites, resulting in increased nuclear binding together with loss of the hormone response. It is interesting to note that a certain class of mutants in the E. coli lac repressor appears to confer partial constitutivity of lac operon expression by a similar mechanism (Pfahl, 1976).

V Dominance and Complementation Tests

It is intriguing that each of the seven independently isolated nti clones that we have characterized has receptors indistinguishable from those in other nti clones as judged by sedimentation rate, elution from gel permeation columns, and apparent DNA-binding affinity. Thecalculated molecular weight differences between the wild-type and nti receptors is 40,000 daltons. One explanation forthese results is that the wild-type receptor is composed of two or more dissimilar subunits, and that nti cells contain normal levels of the steroid-binding subunits(s), but bind to DNA aberrantly due to the lack of a 40,000 dalton subunit. Although there is no direct biochemical evidence supporting such a model for the glucocorticoid receptor, activated estradiol receptors do appear to contain a 40,000–50,000 dalton nonsteroid-binding component (Yamamoto and Alberts,1972; Notides and Nielsen, 1974; Yamamoto, 1974).

According to these ideas, then, supplying such defective cells with the appropriate nonreceptor components might result in recovery of the sensitive phenotype. Indeed, the genetic approach to steroid action would be most useful if various combinations of resistant phenotypes could be shown to regain a wild-type characteristic inmixed extracts or dex sensitivity in somatic cell hybrids. From such procedures one could estimate the number of complementation groups, and therefore the number of functional elements, involved in the overall interaction of glucocorticoids with these target cells. Moreover, if in vivo complementation could be verified in cell-free systems, the in vitro assays would be useful for the purification and functional analysis of the active components. Complementation assays of this type have been successfully developed in prokaryotic systems(Barry and Alberts, 1972).

Another reason to carry out mixing experiments is to establish the dominance relationships between the wild-type and variants lines. Such data might suggest or rule out certain mechanistic possibilities. For example, the results we have described thus far are consistent with the idea that nt– and nti have defective receptors. However, an alternative explanation for the DNA-binding properties of the receptors from variant lines is that inhibitory substances, present in the normal cell extracts, are more active in nt– and less active in nti extracts. Still another possibility is that components that stimulate receptor binding might act in the nti and wild-type extracts, but not in the nt– extracts.

A DNA BINDING IN MIXED EXTRACTS

Two types of cell-free complementation experiments were carried out to determine whether such stimulatory or inhibitory activities could be detected. In one experiment, wild-type and nt– extracts were prepared and the receptors were associated with either [³H] dex or unlabeled dex. They were mixed by pairs in the combinations shown in Table VI, incubated at 20°, and chromatographed on DNA-cellulose. The results, shown in Table VI, demonstrate that the extracts behave independently, with no inhibition of wild-type binding by nt–, and no stimulation of nt– binding by wild-type.

TABLE VI

DNA Binding in Wild-Type X nt− Mixed Extractsa,b

aFrom Yamamoto et al. (1974).

bExtracts of a wild-type and a nuclear transfer-deficient clone were prepared in buffer containing 80 mM NaCl (to maximize the differential binding of the two receptors), divided, and treated with radioactive or nonradioactive dex. Aliquots of extracts containing 15,000 cpm of [³H] dex-labeled receptor were mixed with an equal volume of unlabeled extract as indicated, incubated 35 minutes at 20°C, and chromatographed on DNA-cellulose as described in the legend to Table IV.

The second type of experiment was carried out with wild-type and nti extracts, both ofwhich were labeled with [³H]dex. Aliquots of the extracts were mixed, either before or after the 20°C incubation, then loaded onto DNA-cellulose columns; the bound components were gradient-eluted. The results, depicted in Fig. 10, show that substances present in one extract do not affect the DNA-binding properties of the other, regardless of whether the two extracts are mixed before or after the 20°C incubation.

FIG. 10 DNA binding of mixed extracts from wild-type and nti cultures. Extracts were prepared as described in ) incubation at 20°C. The mixed extracts were then loaded onto DNA-cellulose columns, and the bound material was gradient-eluted as described in Fig. 8. [³H] dex = ³H-labeled dexamethasone.

Clearly, there are many interpretations of these negative results. However, if taken at face value, the data lend further support to the notion that the aberrant DNA-binding properties of the variant receptors are due to alterations in the receptor molecules themselves, rather than to alterations in other soluble components in the extracts.

B SOMATIC CELL HYBRIDS

In order to examine the questions ofdominance and complementation under in vivo conditions, several types of somatic cell hybrids have been constructed. The parental line had originally been selected for growth either in bromodeoxyuridineor 6-thioguanine, thus yielding parental subclones with presumptive defects in thymidine kinase (TK–) or hypoxanthine–guanine phosphoribosyl transferase (HPRT–). Thus each dex-resistant clone also carries one of these defects in its pyrimidine–purine salvage pathways. When the appropriate cells are fused using inactivated Sendai virus (Harris and Watkins, 1965), viable hybrids can be isolated with HAT selective medium (Szybalski et al., 1962; Littlefield,1964), in which the drug aminopterin is used to kill cells lacking either salvage pathway. Because the hybrids complement these defects, only they survive in the presence of the drug. The karyotype of each resulting hybrid clone eventually becomes stable; the number of chromosomes ranges from 65 and 80 in the various clones.

We first determined whether cells with distinguishable receptors continued to synthesize both receptor types upon hybridization. For example, labeled extracts of (wild type × nti) hybrids were loaded onto DNA-cellulose columns, and the bound receptors were gradient-eluted. Figure 11 shows that extracts from these hybrids yielded two peaks of radioactivity eluting from the columns at precisely the ionic strengths characteristic of each parent. In contrast, controls such as (wild type × wild type) (not shown) or (r– × nti) (Fig. 11) showed onlya single elution peak at the expected ionic strengths for wild-type and nti receptors, respectively. In addition to providing further proof that we have constructed true hybrid cells, these results show that the presence of one type of receptor does notextinguish the ability to synthesize another. Furthermore, no intermediate or other aberrant forms of receptors appear in the hybrids.

FIG. 11 ) were prepared as described in Table III and loaded onto separate DNA-cellulose columns; the bound material was gradient-eluted as described in Fig. 8. Vertical arrows denote the positions at which the parental receptors from wild-type and nti cultures elute. [³H] dex = ³H-labeled dexamethasone.

Dominance relationships were tested by comparing the relative cloning efficiency, in the presence and in the absence of dex, of(wild type × wild type), (wild type × variant), and (variant × variant) hybrids. A similar type of experiment had been previously reported (Gehring et al., 1972) using hybrids between the mouse lymphoma line EL4 (Gorer, 1950) andRPC5-CL4, a mouse myeloma cell line (Mohit and Fan, 1971). Both of these cell types carry glucocorticoid receptors, but only RPC5-CL4 cells exhibit a cytolytic response to the hormone. We subsequently showed (K. R. Yamamoto and P. Jones, unpublished) that the EL4 receptors bind to DNA with the characteristics of an S49 nt– receptor. Gehring et al. (1972) found that the EL4 × RPC5-CL4 hybrids exhibited a dex sensitivity indistinguishable from that of the sensitive RPC5-CL4 parent. This apparent dominance of the sensitive phenotype implied that dex may function by inducing synthesis of some macromolecule responsible for the killing effect and makes unlikely the possibility that steroid resistance in EL4 is due to intracellular inhibitors of the dex response.

In our experiments with S49 cell hybrids, we monitored relative cloning efficiency in soft agar, an assay that we found to be more sensitive than cell survival in suspension cultures (M. R. Stampfer, K. R. Yamamoto, and P. Coffino, unpublished). In contrast to the clear dominance of the wild-type phenotype, we found that the killing of S49 (wild-type × variant) hybrids was codominant, i.e., intermediate to the efficient killing of (wild-type × wild-type) hybrids and the complete resistance of (variant × variant) hybrids (Table VII). These results are