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Characterization of the heat shock proteins in cultured trout cells Mach II Burgess, Elizabeth Anne 1984

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CHARACTERIZATION OF THE HEAT SHOCK PROTEINS IN CULTURED TROUT CELLS Machll bv Elizabeth Anne Burgess B.Sc. The University of Manitoba, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF i DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Biochemistry We accept this thesis as conforminq to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 1984 Elizabeth Anne Burgess, 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Sin sA The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 >E-6 (3/81) ( i i ) Abstract Despite considerable investigation, the function of the heat shock proteins (hsps) remains obscure. In this thesis, the heat shock proteins of Salmo gairdnerii RTG-2 cells were examined by two-dimensional gel electrophoresis, and the induction of the 70,000 dalton hsp (hsp70) was studied. As in Drosophila melanogaster each trout hsp detected by one dimensional analysis separated into several isoelectric variants following two-dimensional gel electrophoresis. Post-translational modification of a single polypeptide could have given rise to charge variants similar to those observed here, but no evidence for either the phosphorylation of the hsps or for changes in the phosphorylation of other proteins was obtained. Messenger RNA was isolated from heat shocked cells and translated in the in vitro rabbit reticulocyte system.. Most of the isoelectric variants were detected in the in vitro translation suggesting that either different transcripts encode the isoelectric variants or that post-translational modification is occurring in the in vitro system. Heat shock mRNA was isolated by hybridization to a cDNA clone of the 70K hsp and translated in vitro. Two-dimensional gel electrophoresis of the translation products revealed that a l l 70K isoelectric variants were present. The induction of hsp70 shortly after the addition of sodium arsenite was examined using an antibody to chick hsp70. Some cross-reaction was observed with a polypeptide of 70,000 in ( i i i ) u n s h o c k e d c e l l s , w h i c h c o u l d i n d i c a t e t h e p r e s e n c e o f h s p 7 0 i n n o n - s h o c k e d c e l l s . T h i s i s n o t c o n c l u s i v e s i n c e t h e a n t i b o d y wa s p o l y c l o n a l . I t w a s s t i l l p o s s i b l e t o d e t e c t t h e i n d u c t i o n o f h s p 7 0 a b o v e t h i s b a c k g r o u n d . A s h a r p i n c r e a s e i n t h e l e v e l o f a n t i b o d y b i n d i n g w a s d e t e c t e d a f t e r 1 5 m i n u t e s . T h e r e l a t i o n s h i p o f h i s t o n e a c e t y l a t i o n t o h s p i n d u c t i o n w a s a l s o e x a m i n e d u s i n g s o d i u m b u t y r a t e u n d e r c o n d i t i o n s w h i c h c a u s e a h i g h d e g r e e o f h i s t o n e h y p e r a c e t y l a t i o n . S y n t h e s i s o f t h e h e a t s h o c k p r o t e i n s w a s n e i t h e r i n d u c e d n o r b l o c k e d . T h e r e f o r e , t h e s t a t e o f h i s t o n e a c e t y l a t i o n d o e s n o t a f f e c t t h e h e a t s h o c k r e s p o n s e . (iv) Table of Contents ABSTRACT i i TABLE OF CONTENTS ....iv FIGURES . v i i i TABLE xi ACKNOWLEDGEMENTS xii ABBREVIATIONS x i i i INTRODUCTION 1 I. The heat shock response ...1 A. Introduction 1 B. ' Regulation of the heat shock response 3 1. Regulation of transcription 3 2. Regulation of translation ....4 3. Role of hsp70 in recovery from heat shock 6 -4. Evolutionary conservation of the transcriptional regulation 7 5. Effect of heat shock on chromatin structure 8 6. Other effects of heat shock on chromatin 9 C. Heat shock proteins 10 1. Evolutionary conservation of heat shock proteins 10 2. Isoelectric variants of the heat shock proteins 11 3. Sub-cellular localization of the heat shock proteins 14 \ ( v ) 4. Heat shock and thermotolerance 15 5. Developmental regulation of the heat shock response 18 D. The response of RTG-2 cells to heat or NaAsC>2 treatment... 19 II. Histone acetylation and heat shock 22 A. Histone acetylation 22 B. The effect of sodium butyrate on histone acetylation 24 III. The present investigation 26 EXPERIMENTAL PROCEDURES ....29 I. Materials , 29 II. Methods 30 A. Cell line and culture conditions.... 30 B. Induction with sodium arsenite, incubation at high temperature and treatment with sodium butyrate 30 1. Sodium arsenite induction 30 2. Induction by incubation at 27°C 31 3. Sodium butyrate induction 31 C. Radio-isotope labelling conditions 32 35 1. S-methionine labelling 32 32 2. P-ortho-phosphate labelling 33 D. Polyacrylamide gel electrophoresis 33 1. Sample preparation.......... 33 2. Two-dimensional gel electrophoresis 33 3. One-dimensional SDS acrylamide gel electrophoresis 34 4. Acid-urea polyacrylamide gel electrophoresis 35 ( v i ) 5. Staining and autoradiography of the gels after electrophoresis . 35 E. Isolation of RNA 36 F. Hybridi zation-select ion 37 G. In vitro translation 39 H. Protein blotting 39 I. Iodination of goat anti-rabbit IgG 40 J. Extraction of histones 40 RESULTS.... 42 I. Characterization of the heat shock proteins of RTG-2 cells...42 A. Two-dimensional gel electrophoresis of heat shock proteins...42 B. Post-translational modifications of the heat shock proteins..50 1. The effect of sodium arsenite treatment on phosphorylation of the hsps 50 2. In vitro translation of heat shock mRNA 53 3. Hybrid-selection of hsp70 mRNA 55 C. Induction of hsp70 57 II. The effect of histone acetylation on the induction of hsps by sodium arsenite 63 A. Sodium butyrate-induced histone acetylation 63 B. The effect of sodium butyrate on cellular protein synthesis..66 C. The effect of sodium butyrate on the heat shock response 66 DISCUSSION 70 ( v i i ) I. The response of trout RTG-2 cells to heat or sodium arsenite.70 A. Two-dimensional gel electrophoresis of trout heat shock proteins 70 B. The nature of the isoelectric variants of the heat shock proteins , 73 1. Post-translational modification of the heat shock proteins 73 2. Many genes code for heat shock proteins 74 3. Hybrid-selection of heat shock messenger RNA 75 4. Genetic polymorphism of the heat shock genes 76 C. Induction of hsp70..-. 77 II. The effect of histone acetylation on the response to sodium arsenite 80 A. The effect of sodium butyrate on trout cells 80 B. The effect of sodium butyrate on the response to sodium arsenite 81 REFERENCES 83 (viii) F i g u r e s Figure 1 Comparison of the predicted amino acid sequence of hsp70 from trout, D. melanogaster and yeast 12 Figure 2 Two-dimensional fluorograms of proteins from RTG-2 / cells 43 Figure 3 Two-dimensional fluorograms of proteins separated on 8% polyacrylamide gels in the second dimension 46 Figure 4 Fluorograms of proteins from heat shocked cells separated by non-equilibrium pH gradient gel electrophoresis 48 Figure 5. Heat shock proteins from cells treated with either NaAs02 or heat 49 Figure 6 Two-dimensional gels showing polypeptides 14 to 17 from cells treated in different ways 51 Figure 7 One-dimensional analysis of phosphorylated polypeptides from control and heat shocked cells..52 Figure 8 Two-dimensional analysis of phosphorylated (ix) polypeptides 54 Figure 9 Fluorograms of proteins from in vitro translations of mRNA isolated from untreated cells or from those treated with NaAsC>2 56 Figure 10 Restriction map of the M13 clone used in the hybridization-selection of heat shock mRNA 58 Figure 11 Fluorogram of proteins from in vitro translations of mRNA isolated by hybrid-selection to an hsp70 M13 clone... 59 Figure 12 Cross-reaction of the antibody to chick hsp70 with trout hsp70 60 Figure 13 Induction jpf hsp70 62 Figure 14 The effect of different concentrations of sodium butyrate on histone acetylation 64 Figure 15 Time .course of histone acetylation induced by sodium butyrate 65 Figure 1.6 The effect of sodium butyrate on total cellular protein synthesis as determined,by two-(x) dimensional gel electrophoresis 67 Figure 17 The effect of sodium butyrate on the induction of heat shock proteins in RTG-2 cells by sodium arsenite 69 l \ (xi) T a b l e Table 1 Molecular weights and isoelectric points (IEP) of heat shock proteins in trout RTG-2 cells 45 l - x i i -Acknowledgements I would l i k e to f i r s t express my g r a t i t u d e to my s u p e r v i s o r , Peter Candido, f o r h i s support and encouragement. I would a l s o l i k e to thank the many people who c o n t r i b u t e d to my knowledge and understanding o f biochemistry. Among these, I wish to thank p a r t i c u l a r l y members of my l a b : J e f f Hewitt, C h r i s K r e i s , C o l i n Hay, Rashmi Kothary, Roland Russnak, Rob Kay and Don Jones. S p e c i a l thanks i s due a l s o to those o u t s i d e o f my l a b whose advice and h e l p made many p a r t s o f t h i s t h e s i s p o s s i b l e . These include Dave H i c k s , Ross M a c G i l l i v r a y and Joan MacPherson. F i n a l l y , mention should be made of the warm and f r i e n d l y atmosphere o f t h i s department which made completion o f t h i s t h e s i s a pleasant task. (xiii) A b b r e v i a t i o n s . . x A C S : A q u e o u s c o u n t i n g s c i n t i l l a n t . b i s - a c r y l a i r i i d e : N , N * - m e t h y l e n e - b i s - a c r y l a m i d e . B S A : B o v i n e s e r u m a l b u m i n . DNA : D e o x y r i b o n u c l e i c a c i d . D N A a s e I : D e o x y r i b o n u c l e a s e I . EDTA : ( E t h y l e n e d i n i t r i l o ) t e t r a c e t i c a c i d . E G T A : E t h y l e n e g l y c o l - b i s - ( 2 - a m i n o e t h y l e t h e r ) N , N ' - t e t r a c e t i c a c i d . H E P E S : N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l p h o n i c a c i d . H s p : H e a t s h o c k p r o t e i n . H s p 3 0 : 3 0 , 0 0 0 d a l t o n h e a t s h o c k p r o t e i n . H s p 7 0 : 7 0 , 0 0 0 d a l t o n h e a t s h o c k p r o t e i n . H s p 8 7 : 8 7 , 0 0 0 d a l t o n h e a t s h o c k p r o t e i n . H s p l O O : 1 0 0 , 0 0 0 d a l t o n h e a t s h o c k p r o t e i n . N P - 4 0 : N o n i d e t P 4 0 . P B S : P h o s p h a t e b u f f e r e d s a l i n e , 8 . g N a C l , 0 . 2 g K H 2 P C > 4 , 13 g N a „ H P 0 - 7 H 0 p e r l i t e r . 2 4 2 < R N A : R i b o n u c l e i c a c i d . R N A a s e : R i b o n u c l e a s e . R N P : R i b o n u c l e a r p r o t e i n p a r t i c l e . S a r c o s y l : N - l a u r y l s a r c o s i n e ( s o d i u m s a l t ) (xiv) Standard saline citrate, 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0 TEMED: N,N,N*,N'-Tetramethylethylenediamine. Tris: Tris(hydroxymethyl)aminomethane. ( 1 ) I N T R O D U C T I O N P a r t I . T h e H e a t S h o c k R e s p o n s e . A . I n t r o d u c t i o n . T h e p h e n o m e n o n o f h e a t s h o c k h a s b e e n e x t e n s i v e l y s t u d i e d i n r e c e n t y e a r s . When t h e g r o w t h t e m p e r a t u r e o f a n o r g a n i s m i s e l e v a t e d , n o r m a l p r o t e i n s y n t h e s i s d e c r e a s e s a n d s y n t h e s i s o f a d i s c r e t e s e t o f p o l y p e p t i d e s b e g i n s (1) . R i t o s s a (2) f i r s t o b s e r v e d t h e h e a t s h o c k r e s p o n s e i n t h e p o l y t e n e c h r o m o s o m e s o f D r o s o p h i l a m e l a n o g a s t e r . T h e p u f f i n g p a t t e r n o f s a l i v a r y g l a n d c h r o m o s o m e s f r o m h e a t s h o c k e d f l i e s w a s d i f f e r e n t f r o m t h e p a t t e r n o f c h r o m o s o m e s f r o m f l i e s w h i c h h a d n o t b e e n s h o c k e d : n i n e n e w p u f f s a r e i n d u c e d u p o n h e a t s h o c k , w h i l e a l l o t h e r p u f f s r e g r e s s . L a t e r , h e a t s h o c k w a s s h o w n t o i n d u c e t h e s y n t h e s i s o f n i n e p o l y p e p t i d e s (3) . T h e h e a t s h o c k r e s p o n s e i s u b i q u i t o u s ; s i m i l a r s e t s o f p o l y p e p t i d e s a r e s y n t h e s i z e d i n o r g a n i s m s r a n g i n g f r o m E . c o l i t o man (4 ,5) . T h e s e p o l y p e p t i d e s , c a l l e d h e a t s h o c k p r o t e i n s ( h s p s ) , c a n b e i n d u c e d b y a v a r i e t y o f s t r e s s o r s , s u c h a s m e t a b o l i c i n h i b i t o r s , u n c o u p l e r s o f o x i d a t i v e p h o s p h o r y l a t i o n a n d r e c o v e r y f r o m a n o x i a . A l t h o u g h t h e r e s p o n s e t o t h e d i f f e r e n t a g e n t s i s n o t a l w a y s i d e n t i c a l , t h e t e r m " h e a t s h o c k " h a s b e e n a p p l i e d t o t h e e f f e c t s o f t h e s e v a r i o u s i n d u c e r s . B e c a u s e s o m a n y c o m p o u n d s i n d u c e t h e h s p s , a p r o t e c t i v e r o l e d u r i n g t i m e s \ ( 2 ) of stress has been proposed for the hsps (63). However, despite much investigation, the actual functions of these polypeptides remain unclear. Besides the interest in the hsps themselves, the regulation of the heat shock response has provoked much investigation. Temperature elevation causes a rapid shift in protein synthesis such that, in D. melanogaster, over 70% of a l l protein synthesis is devoted to the synthesis of heat shock proteins after 1 hour of heat treatment (6). This shift to heat shock protein synthesis is caused by both transcriptional and translational regulation (2,7). In D. melanogaster, heat shock induces a unique set of puffs on the salivary gland chromosomes, which suggests that transcription is occurring at these sites (1). Translational control is also important. Under certain conditions, the translation of normal cellular mRNA is repressed, although the messages remain stable in the cytoplasm and can be translated in in vitro systems (7,8). These effects are fully reversible; upon return to normal - temperature, heat shock protein synthesis decreases and normal protein synthesis resumes. Thus, the heat shock phenomenon is of interest from several points of view. The functions and modes of action of the polypeptides themselves remain to be elucidated and the regulation of the response itself is unclear. Finally, the heat shock phenomenon provides an excellent model system for studies of gene activation. (3) B . R e g u l a t i o n o f t h e h e a t s h o c k r e s p o n s e . 1 . R e g u l a t i o n o f t r a n s c r i p t i o n . A f t e r h e a t t r e a t m e n t o f c e l l s o r t i s s u e s , t r a n s c r i p t i o n o f t h e h e a t s h o c k g e n e s i s m a r k e d l y i n c r e a s e d , w h i l e c o n t r o l g e n e t r a n s c r i p t i o n i s r e p r e s s e d . L a t e r , d u r i n g r e c o v e r y , t h i s p r o c e s s i s r e v e r s e d a n d c o n t r o l g e n e s a r e a c t i v a t e d w h i l e h e a t s h o c k g e n e s a r e r e p r e s s e d . I n t h i s t h e s i s , g e n e s o t h e r t h a n t h e h e a t s h o c k g e n e s a n d t h e i r t r a n s c r i p t s a n d p r o d u c t s w i l l b e r e f e r r e d t o a s " c o n t r o l " g e n e s . T h e t r a n s c r i p t i o n a l c h a n g e s a c c o m p a n y i n g h e a t s h o c k h a v e b e e n d e m o n s t r a t e d i n s e v e r a l w a y s . I n D . m e l a n o g a s t e r , i n w h i c h t h e h e a t s h o c k r e s p o n s e h a s b e e n e x t e n s i v e l y s t u d i e d , a c t i v a t i o n o f t h e h e a t s h o c k g e n e s i s r e f l e c t e d i n t h e p u f f i n g p a t t e r n o f t h e p o l y t e n e c h r o m o s o m e s ( 2 ) . A u n i q u e s e t o f p u f f s i s i n d u c e d b y e l e v a t i o n o f t h e t e m p e r a t u r e t o 3 5 ° C w h i l e p u f f s p r e s e n t a t t h e n o r m a l t e m p e r a t u r e , 2 5 ° C , 3 r e g r e s s . T h e h e a t s h o c k p u f f s i n c o r p o r a t e H - u r i d i n e i n d i c a t i n g t h a t RNA s y n t h e s i s i s o c c u r r i n g a t t h e s e s i t e s ( 3 , 9 ) . N e w l y s y n t h e s i z e d RNA , i s o l a t e d f r o m h e a t s h o c k e d c e l l s , h y b r i d i z e s t o t h e h e a t s h o c k p u f f s ( 1 0 ) . I n c u b a t i o n w i t h a c t i n o m y c i n D p r e v e n t s t h e a c c u m u l a t i o n o f h e a t s h o c k RNA a n d a l s o p r e v e n t s t h e i n d u c t i o n o f t h e h e a t s h o c k p u f f s . S i n c e , i n t e r \ — — — • a l i a , a c t i n o m y c i n D i n h i b i t s R N A s y n t h e s i s , t h i s s u g g e s t s t h a t h e a t s h o c k i n d u c e s t r a n s c r i p t i o n o f t h e g e n e s l o c a t e d a t t h e s i t e s o f t h e h e a t s h o c k p u f f s a n d t h a t RNA s y n t h e s i s c a u s e s (4 ) puffing. In agreement with this, immunological studies have shown that, during heat shock, RNA polymerase II migrates out of control puffs and accumulates in heat shock puffs (11,12). Finally, isolated genes have been used to detect the changes in transcription after heat shock (13). RNA was pulse-labelled with 3 H-uridine, isolated and hybridized to either a cloned actin gene or a cloned heat shock gene. While the levels of actin mRNA were reduced during heat shock, the levels of the heat shock mRNA were increased. Upon recovery, the levels of heat shock mRNA decreased and actin mRNA transcription resumed. Thus, during heat shock, transcription of heat shock genes is activated while that of other genes is repressed. 2. Regulation of translation. A severe heat shock almost completely stops control protein synthesis (3,10). However, actinomycin D treatment after heat shock does not prevent general protein synthesis during recovery (14) and if mRNA is isolated and translated in vitro, control proteins are synthesized although they were not made in vivo (15) . This means that mRNA translation is also controlled during heat shock. Heat shock mRNA is translated while other transcripts are not. The control mRNA is not degraded, but remains in a form which is functional in vitro. Lysates made from heat shocked D. melanogaster cells translate heat shock messages (6,7) but not other mRNA. Lysates from unshocked cells can translate a l l I C5) mRNA, as does the cell itself during recovery. Heat shock lysates (16) have been fractionated in an attempt to isolate the factors responsible for the discrimination between heat shock and control transcripts, but the factors are not soluble and seem to be bound to the ribosomes. Thus, in D. melanogaster at least, the heat shock response is maximized by translational control: transcription of heat shock genes is rapidly induced while translation of other genes is repressed such that only hsps are made. Since heat shock results in the disappearance of pre-existing polysomes and the formation of new polysomes (10,15), translational control may be mediated by changes in the polysomes. On sucrose gradients, polysomes from unshocked cells have a unimodal distribution of 12 to 13 ribosomes per mRNA. The distribution of polysomes from heat shocked cells on the other hand, is bimodal, with peaks at 9 ribosomes per mRNA and 20 ribosomes per mRNA. However when actin and^-tubulin genes are hybridized to RNA from heat shock polysomes, control transcripts are detected (17). Each control transcript carries the expected number of ribosomes, but only 1/3 to 1/2 as many ribosomes are associated with the control polysomes as in the unshocked cells. Therefore, polysomes containing control transcripts are present in heat shocked cells but at a lower level than in unshocked cells. Since these polysomes, which are indistinguishable from polysomes in control cells, are present in lower amounts in heat shocked cells, heat shock must block translation of control mRNA at an (.6) initiation step. Basal protein synthesis in heat shocked cells is 5% to 10% that of control cells. Since there are 1/3 to 1/2 as many ribosomes with control mRNA in heat shocked cells as in control cells, elongation steps must also be blocked. Whether these inhibitions are caused by the same factors which block translation of control transcripts in heat shocked lysates is unknown. 3. Role of hsp70 in recovery from heat shock. Both transcriptional control (6) and translational control (18) are dependent on the length and severity of the shock. Transcription increases more rapidly with a severe shock than with a mild shock while the translation of control mRNA is not repressed during a mild shock. In addition, each heat shock protein is induced and repressed with different kinetics (6). Control proteins appear to behave as a cohort with identical induction and repression kinetics. Studies on the induction of the 70,000 dalton heat shock protein (hsp70) in both D. melanogaster (19) and E. coli (20) have shown that the recovery of basal protein synthesis correlates with the synthesis of hsp70. In addition, an active hsp70 is required for repression ^  of the heat shock response,during recovery. Thus, i f cells are incubated with amino acid analogues, such as canavanine, which are incorporated into hsp70, and thereby inactivate i t , heat shock transcription is not repressed after a return to normal (7) temperature, and normal protein synthesis does not resume (19). Hence, hsp70 regulates its own synthesis and promotes the recovery of normal protein synthesis. 4. Evolutionary conservation of the transcriptional regulation. The isolation of genes coding for the hsps has made possible the identification of sequences controlling the expression of these genes. The cloned gene coding for the D. melanogaster hsp70 has been transfected into'monkey cells (21), rat cells (22), mouse cells (23) and Xenopus oocytes (24,25). In a l l cases, the transcription of the gene is induced by heat shock and, in oocytes, arsenite treatment will also induce the gene. This means that the system regulating the transcription of heat shock genes has been conserved in a l l of these organisms. Hsp70 genes with various deletions were injected into a monkey cell line (21) to determine the sequences reguired for heat-regulated expression of the gene. If nucleotide sequences from -47 to -66 are deleted, (nucleotide 1 is the transcription start site) then the gene is no longer expressed upon incubation at increased temperature. This region is conserved in a l l heat shock genes and lies within an inverted repeat. The consensus sequence is CTGGMTNTTCTAGA and i t may be located a variable distance upstream from the TATA box, usually within 28-35 nucleotides of i t . However, although transcriptional control of the heat shock genes depends upon ra certain sequence which is highly conserved among different (8) v organisms, translational control appears to be species specific. In Xenopus oocytes, the transcription of the D. melanogaster hsp70 gene increases upon heat shock, but the D. melanogaster transcript is not translated at high temperature (25). Rather, i t behaves as a control mRNA and its translation is repressed. This indicates that the translational control is not dependent upon some highly conserved sequence in the heat shock transcript. 5. Effect of heat shock on chromatin structure. The cloned heat shock genes have also been used as probes for chromatin structure. As in .many other genes, DNAase I hypersensitive sites are located at the 5' end of the genes for hsp70 (26,27), hsp83 (26,27,28), and the small hsps of D. melanogaster (29). The hypersensitive sites are present whether or not the gene is active, although the coding region of the gene becomes much more sensitive to DNAase I when the gene is activated. Similar studies have been performed using micrococcal nuclease (30). Again, when the gene is activated, the coding region becomes much more sensitive to the nuclease although i t is more resistant than the surrounding, non-transcribed regions, when repressed. It has recently been suggested that this is because of removal of the histones from the coding region during transcription. Karpov et a l . (31) cross-linked histones to' DNA to show that the coding region of hsp70 is depleted of histones when activated. Micrococcal nuclease appears to cleave the same (9) sites as DNAase I, but this is difficult to determine because of the preference of micrococcal nuclease for certain sequences (30). These nuclease sensitive sites appear to be required but not sufficient for transcription (30). Their presence at the 5' end of inactive heat shock genes may reflect the state of these genes prior to activation and the speed with which they can be activated. ; 5. Other effects of heat shock on chromatin. Although nucleosome structure and histone modification will be discussed further under Part II below, some observations on chromosomal proteins in relation to heat shock are presented here. Levinger and Varshavsky (32) have examined the protein content of nucleosomes containing the hsp70 gene in D. melanogaster. As with other transcribed genes, one out of two nucleosomes of the hsp70 gene contain ubiquitin-H2A while the content of ubiquitin-H2A is greatly reduced in the chromatin of non-transcribed satellite DNA. In the latter, nucleosomes are enriched in a non-histone chromosomal protein, DI. DI is not present in the nucleosomes associated with the hsp70 genes or other transcribed genes. Again, the presence of ubiquitin-H2A and absence of DI suggests that the hsp70 gene is maintained ready for transcription. Heat shock also affects the synthesis of chromosomal proteins. In D. melanogaster, the synthesis of H2B is three-Cio) fold induced while the synthesis of the other histones is decreased 2 to 10-fold (33). Normally, histone synthesis is coordinately regulated and i t is not known whether the increase in H2B synthesis is because of transcriptional or translational activation. The effect of heat shock on histone edification will be discussed below. C. Heat shock proteins. 1. Evolutionary conservation of heat shock proteins. In D. melanogaster, where these polypeptides have been studied extensively, nine major hsps are observed, with the following approximate molecular weights: 83,000, 72,000, 70,000, 68,000, 34,000, 27,000, 26,000, 23,000 and 22,000 (1). Five polypeptides of molecular weights: 87,000, 70,000, 42,000, 32,000, and 30,000 are induced in trout RTG-2 cells, a fibroblast cell line, by heat treatment (36). If trout cells are exposed to sodium arsenite, polypeptides of 62,000 and 100,000 daltons are induced in addition to the five observed upon heat shock. The 70,000 dalton heat shock protein (hsp70) is most strongly induced in both trout and D. melanogaster. This protein is highly conserved in organisms ranging from E. coli to man (4). Antibodies to chick hsp70 cross-react with proteins of similar molecular weight in a large number of organisms (5) while peptide maps produced by V8 proteolysis of hsp70 from D. melanogaster, (11) chick and man have many peptides in common (37). Peptide maps of the 83,000 dalton polypeptide (hsp83) from these same animals show even greater similarities. Conservation is also observed at the level of the DNA sequence; a D. melanogaster genomic clone of hsp70 will hybridize with mouse heat shock mRNA (38) and the predicted amino acid sequence of the yeast hsp70 gene is 72% homologous with the D. melanogaster gene (39). Fig. 1 shows part of the amino acid sequences of hsp70 from D. melanogaster, yeast and trout. Non-homologous regions are boxed and i t is quite ,clear that hsp70 is highly conserved in these organisms. A polypeptide of 30,000 daltons is also strongly induced in trout. This protein probably corresponds to the small molecular weight heat shock proteins of D. melanogaster, which are also highly induced. 2. Isoelectric variants of the heat shock proteins. Recently, two-dimensional gel electrophoresis of the D.  melanogaster heat shock proteins has revealed a response of greater complexity than expected (40). Over 70 polypeptides in 14 different size classes are detected in both heat shocked salivary glands and heat shocked D. melanogaster tissue culture cells. The manner in which these polypeptides arise remains unclear. In D. melanogaster, five genes code for the hsp70 polypeptide, but ( 1 2 ) . Figure 1. Comparison of the predicted amino acid sequences of hsp70 from trout, D. melanogaster and yeast. THS70.7 is derived from a cDNA of trout hsp70 mRNA isolated by R. Kothary. The D. melanogaster sequence is taken from Ingolia et al. (39). The yeast sequence is from the YG100 gene, also of Ingolia et al (39). Regions compared are amino acids number 128 to 406, based on the D. melanogaster hsp70 numbering. Mismatches in the sequences have been boxed in. The single dashes represent deleted amino acids relative to the compared sequence. The solid line in the yeast sequence represents information not yet available. THS70.7 Dr.HS70 YG100 128 _ A E A Y L G 2 K V S N A V I AEftYLGESITDAVI TVPAYFNDSQRQATKDAGH AESYLGAKVNDAVVTVPAYFNDSQRQATKDAGTI TVPAYFNDSQRQATKDAG|V|I A G L N V L R I I N E P T A A S I A G L N V L R I I A G L N V L R I I NEPTAAIA: N E P T A A A L A Y G L D K N L K G E R N V L I AYGLDKKGKEEH AYGWHlGMSR|E|R|NVLI FDLGGGTF FDLGGGTF -VLI FDLGGGTF THS70.7 DVSff Dr.HS70 DVSI YGIOO DVSL LTI LP LF ED3-I EDG-I FEVKATAGDTHLGGEDFDNRLVSHFVEEFKRKHKKD D E 3 S L F E V R S T A G D T H L G G E D F D N R L V T H L A E E F K R K Y K K D L R S N P R A L R R L R T A A E R A K R T L SSSTE FEVKATAGDTHLGGEDFDNRLVNHFIQEFKRKNKKDLSINQRALRRLRTACESQENFVSS-ISCNKRALRRLRTACERAKRTLSSS SQ AQ THS70.7 Dr. HS70 YGIOO ASI ATI TSVj EIDSLFEG EIDALFEG EIDSKNEG 2DFYTKVSRARFEELCANLFRNTL2PVEKALNbAKMDK3QI DF YT|SITjRARFEEyC|AD LFR S TI^PVEr^LgDAKyDKgQ VDEI DFYT|STT]RARFEE|MJC|SI^ H D I V L V G G S T R I PKVQSLLQJE JFF V L V G G S T R I THS70.7 |NfepElLN^SINPDEAVJGr?GEA[l|QAAI L S G E J K J S — Dr. HS70 lHGktJLN[LlsiNPDEAv|AkdAAMQAAILSGD|Q|s—|GKI|QD|V|LL[VD|V'APLSLG YGIOO 406 ENVjQD|L(LL|LD|VAPLSLG IETAGG IETAGG GlVAPLSLGIETA-G (13) upon two-dimensional gel electrophoresis, 40 different isoelectric variants are observed. Post-translational modifications could explain some of these isoelectric variants. For instance, chick and hamster hsp70 and hsp87 are methylated at arginyl and lysyl residues (41,42), while the 87,000 dalton protein is ADP-ribosylated (43). Phosphorylation of the hsp70 protein of Dictyostelium disco ideum has also been reported (44), but similar studies on the D. melanogaster hsp70 show no phosphorylation (45). If post-translational modification accounts for the isoelectric variants observed on two-dimensional gel electrophoresis, i t seems unlikely that a l l of the modifications would be seen when mRNA isolated from heat shocked cells is translated in a heterologous system. In fact, a l l isoelectric variants are observed in gels of in vitro translations of heat shock mRNA from D. melanogaster (40). Unless post-translational modification is occurring in the in vitro translation system, individual transcripts must be present for each isoelectric variant. In support of this, Wadsworth (46) has isolated mRNA by hybridization to a genomic clone of the hsp70 gene from the 87C locus of D. melanogaster and translated the isolated mRNA in  vitro. On one-dimensional SDS gel electrophoresis, four polypeptides of approximately 70,000 daltons were observed, which indicated that the cloned hsp70 gene had hybridized to different homologous transcripts. . These polypeptides were also observed in vivo and partial proteolysis mapping revealed that the polypeptides are related. Deletions spanning both the 87A and 87C (14) heat shock loci, which are known to code for hsp70, result in mutants which cannot produce the 70,000 dalton protein but which s t i l l synthesize the three polypeptides of slightly larger molecular weight (46). This means that the genes for these polypeptides must reside outside of the known heat shock loci. Three additional genes in D. melanogaster (48) and nine in yeast (39) which are homologous with the hsp70 gene of D.  melanogaster have been identified recently. However, most of these genes are expressed in cells at normal temperature and hence cannot explain the many isoelectric variants observed in heat shocked cells. Those genes homologous to hsp70 which are expressed at normal temperature are called cognate genes (48). Additional genes coding for heat shock proteins may s t i l l be found outside of regions known to code for hsps, thereby accounting for some of the isoelectric variants. 3. Subcellular localization of the heat shock proteins. To gain further insight into the function of the hsps, . sub—cellular fractionation, electron microscope autoradiography and immunofluorescent technigues have been used to determine the localization of the hsps. Hsp83 of D. melanogaster is always located in the cytoplasm and may be membrane bound (49,50). While hsp70 and the small hsps of D. melanogaster are distributed throughout the ce l l , a portion of these hsps is concentrated in the nucleus during heat shock (49-53,125). (15) However, when the cells are shifted back to their normal growth temperature, the hsps leave the nucleus and enter the cytoplasm (45,53). Within the nucleus, hsp70 and hsp30 appear to be bound to the nuclear matrix. The nuclear matrix is functionally defined as the structure remaining after the extraction of lipids and • histones and digestion by nucleases in a defined sequence (124). Various nuclear functions such as RNA transcription and DNA replication have been associated with the nuclear matrix (124). The nuclear hsps cannot be extracted from chromatin by 2M NaCl (50) and are present in nuclear scaffold preparations (54). Levinger and Varshavsky (50) therefore suggested that the hsps may protect chromatin structure during periods of stress, but this fails to account for the cytoplasmic subset of these hsps. Recently, Kloetzel and Bautz (55) irradiated cells with ultraviolet light and showed that the hsps are cross-linked to RNA. Hsps are found in both cytoplasmic and nuclear RNP particles, which may explain their observed localization. In addition, the small hsps of D. melanogaster can be isolated as an aggregate with the characteristics of RNP particles. 4. Heat shock and thermotolerance. Because the heat shock response occurs in a l l organisms and is highly conserved, the function of the hsps must be important. Since a wide variety of compounds el i c i t the heat shock response, a protective role during times of stress,has been proposed for the (.16) heat shock proteins. Studies on thermotolerance in mammalian cell lines support the suggestion that hsps function in stress resistance. Thermotolerance develops in cells given a non-lethal heat, treatment before receiving a second, lethal heat treatment. The initial mild heat shock protects the cells from the second, severe treatment which would k i l l untreated cells. In hamster cells', thermotolerance has been correlated with heat shock protein synthesis: maximal thermotolerance only occurs with maximal hsp synthesis (56-60). In addition, arsenite, ethanol and recovery from anoxia, which a l l induce hsp synthesis also induce thermotolerance (61,62). A severe heat treatment before ,the protective heat treatment delays the acquisition of thermotolerance; at the same time, protein synthesis is repressed and recovery, which begins with hsp synthesis, is delayed. Thus, thermotolerance does not occur until the cell has synthesized a given quantity of hsps. Another example of thermotolerance occurs in D. melanogaster: phenocopy induction by heat treatment of embryos can be blocked by an earlier heat treatment (63). Phenocopy occurs when wild type D. melanogaster embryos develop into adult animals with phenotypes identical to those of known mutants after being stressed, for example, by heat treatment, during development. Presumably, the mutant phenotype results from the inactivation of a gene product required for normal development. Phenocopy protection correlates with heat shock protein synthesis as well. During thermotolerance, the cell continues protein synthesis throughout the second heat shock, (17) allowing expression of the functions required to prevent phenocopy. Protein synthesis which occurs during thermotolerance is a consequence of translational regulation and is correlated with hsp synthesis (64). Control transcripts are stable in the cell after the ini t i a l heat treatment, but are only translated during recovery. A second, more severe heat treatment will not block the recovered translation of control mRNAs. This means that, after a mild heat shock, the translational apparatus becomes more resistant to temperature elevation. Recently, Lee et a l . have identified adenylylated nucleotides which accumulate during heat shock in E. coli and S. typhimurium. Exposure of S. typhimurium to oxidizing quinones also induces the adenylylated nucleotides which include 1 4 ' 1 3 P ,P -diadenosine-5 '-tet raphosphate , P - (adenosine-5')-P -(guanosine-5 ') -triphosphate and P"*"- (adenosine-5') -P"^ -(guanosine-3'-diphosphate-5')-triphosphate (65)., The adenylylated nucleotides accumulate during heat shock, even in mutant cells which do not make the heat shock proteins. This indicates that the heat shock proteins are not responsible for the synthesis of the adenylylated nucleotides. Lee et a l . proposed that these compounds are alarmones, or compounds which signal a specific metabolic stress (66). Specifically, the adenylylated nucleotides accumulate during oxidation stress and may trigger the heat shock response. Heat shock is induced by many oxidizing agents, such as hydrogen peroxide, menadione and diamide (1). Other compounds, such as sodium arsenite react with sulfhydryls which, in the form (.18) of glutathione, are known to be important in the maintenance of the cellular oxidation-reduction state. In line with this, levels of glutathione within the cell increase during thermotolerance (67). Thus, the heat shock phenomenon may be a response to oxidation stress and may be induced by alarmones in the form of adenylylated nucleotides. 5. Developmental regulation of the heat shock response. Heat shock protein synthesis is also regulated developmentally. One of the first proteins synthesized by the developing mouse embryo is the 70 K hsp. Bensaude et al . (68) have shown that hsp70 is synthesized upon activation of the embryonic genome at the two cell stage. This is similar.to recovery from heat shock in which synthesis of the heat shock proteins precedes transcription of other genes. Similarly, transcription from chromosomal site 67B in D. melanogaster is developmentally regulated (69). This region codes for two unidentified transcripts as well as for the small molecular weight hsps. A l l are expressed in normal development during the third instar and early pupal stages. One possibility is that products of these transcripts protect the animal from some stress during development. For example, anoxia could occur if the permeability of the pupal case limits oxygen diffusion. Alternatively, the developmentally regulated expression could be caused by some other factor. The third instar stage is CI 9) characterized by high titers of the hormone ecdysterone and the small heat shock proteins are induced in D. melanogaster tissue culture cells treated with ecdysterone (70). The response of embryos to heat shock also varies depending upon the developmental stage of the embryo. Sea urchin embryos (71) can only induce hsps upon heat treatment after hatching while D. melanogaster (72) w i l l only synthesize hsps after the blastoderm stage. In both cases, the embryo cannot respond to heat shock until i t is transcriptionally active. Not surprisingly, the ability of the embryo to withstand heat treatment depends on its ability to synthesize heat shock proteins. D. The response of RTG-2 cells to heat or sodium arsenite  treatment. Trout RTG-2 cells are a fibroblast cell line derived from mixed gonadal tissue of male and female rainbow trout (95). Kothary and Candido (36) have reported that heat shock occurs when RTG-2 cells are incubated at 27°C as opposed to 22°C, the normal growth temperature. As in other organisms, the , response to heat shock varies depending on the length and severity of the stress. Five polypeptides of molecular weight 87,000, 70,000, 42,000, 32,000 and 30,000 may be induced by v temperatures ranging from 27°C to 37°C: hsp87 and hsp42 ( 2 0 ) are only observed below 29°C and incubation of the cells above 34°C is lethal unless exposure time is short. Synthesis of the hsps reaches its maximal levels after 5 hours of incubation at 27°C and is maintained at that level for at least 16 hours of treatment. Upon return to 22°C, heat shock protein synthesis decreases non-coordinately. Synthesis of hsp70 and hsp42 declines f i r s t , followed by a decline in the synthesis of hsp87 and hsp30. Non-coordinate induction and repression kinetics have been observed for the hsps of D.  melanogaster (6). The heat shock response of RTG-2 cells differs from that of other organisms in that control protein synthesis does not cease during heat shock. A similar response to the heat shock response can be induced in RTG-2 cells by treatment with sodium arsenite, which reacts with sulfhydryl groups. Kothary and Candido (36) have shown that 5 proteins of identical molecular weight to the heat shock proteins are induced by NaAsO^ treatment. In addition, polypeptides of 62,000 and 100,000 daltons are synthesized after arsenite treatment. The polypeptides induced by sodium arsenite treatment do not show identical induction kinetics. Rather, each hsp is optimally induced and repressed with different kinetics after removal of the arsenite. Unlike heat shock, arsenite treatment of RTG-2 cells results in the repression of non-heat shock protein synthesis. As in other organisms the block in control protein synthesis is probably at the level of translation since mRNA from cells treated with sodium arsenite can program (.2 1.) the synthesis of control proteins in the in vitro rabbit reticulocyte system. Hence, sodium arsenite elicits a response in RTG-2 cells which is similar, but not identical to the heat shock response. .v ( 2 2 ) Part II. Histone acetylation and heat shock. A. Histone acetylation. The DNA of a l l eukaryotes is packaged into nucleoprotein particles called nucleosomes (73). Besides DNA, each nucleosome contains five basic proteins called histones. Histones H2A, H2B, H3 and H4 form the core particle and are referred to as the "core histones", while HI is attached to the linker DNA between nucleosomes. DNA replication and transcription seem to occur on DNA which is packaged in nucleosomes. As part of the process of gene activation, i t has been proposed that post-translational1 modification of histones may alter nucleosome structure in such a way as to permit transcription (74). Histones can be post-translationally modified in a number of ways, including methylation, phosphorylation, ADP-ribosylation and acetylation (74). Some evidence exists that histone acetylation may be involved in gene activation. / Active genes are sensitive to digestion with DNAase I (75). If nuclei are digested briefly with DNAase I, a procedure that should release nucleosomes carrying transcribed sequences, the resulting released nucleosomes contain hyperacetylated histones (76,77). This suggests that nucleosomes from active genes have hyperacetylated histones, but the nucleosomes released by DNAase I digestion are not necessarily homogeneous. Two or more nucleosome populations could be released by DNAase I and the nucleosomes (2 3) containing transcribed sequences are not necessarily identical to those containing hyperacetylated histones. Histones may be acetylated in two ways. Histones HI, H2A and H4 are acetylated on the <*-amino group of the amino terminal seryl residue shortly after synthesis. This modification occurs in the cytoplasm and is irreversible. In addition, the f-amino groups of specific lysyl residues in the core histones may be acetylated reversibly in the nucleus (78,79). H2B, H3 and H4 contain four sites of acetylation in their N-terminal regions (residues 1-30), whereas H2A contains only a single lysyl residue which is subject to this modification. Acetylation neutralizes the basic charge of the histone molecule by+1 for each residue so modified. In a population of histone molecules, a given site is usually found in the modified and unmodified states. Histones H^ B, H^  and H^  can therefore exist in multi-acetylated forms with charges ranging from +5 to -1 in their N-terminal residues. It has been suggested that neutralization of the positive charge by acetylation could reduce the ionic interactions between the histones and the DNA, thereby leading to an altered nucleosome conformation which permits transcription (73,74). Heat shock affects histone modifications. Acetylation of the histones is reduced during heat shock (34 ) while the methylation patterns change in a complex way (35). Methylation of H2B increases during heat shock while methylation of H3 decreases. Sodium arsenite causes methylation of H2B as well, but does not ' (24) affect H3 methylation. The significance of these alterations is not known, but histone modifications may affect the transcriptional changes that occur during heat shock. B. The effect of sodium butyrate on histone acetylation. Studies of histone acetylation have been greatly facilitated by the use of sodium butyrate. By inhibiting the histone deacetylase, sodium butyrate causes massive hyperacetylation of the histones (80-82). The term "hyperacetylation" here refers to increased steady-state levels of acetylation at the normal in vivo sites of this modification. Pulse-label studies of butyrate-treated cells have shown that two populations of acetylated histones exist (83): most of the histones are acetylated and deacetylated slowly, with a half-life of three hours, whereas a small subset of the histones is rapidly acetylated and deacetylated with a half-life o£ three minutes. The significance of these two populations remains a matter of speculation. Although histone hyperacetylation caused by sodium butyrate is reversible, prolonged treatment can inhibit the histone acetylase, resulting in unacetylated histones (84). If histone acetylation is involved in gene activation, then treatment with sodium butyrate might be expected to activate repressed genes. Two-dimensional gel electrophoresis of proteins from butyrate treated and untreated cells has yielded conflicting results. In Friend erythroleukemic cells, a number of (25) polypeptides are present in butyrate treated cells that are not present in untreated cells (85). This is not true for Chinese hamster ovary cells (86), where butyrate causes no change in protein synthetic patterns. These contradictory results may be explained by the fact that butyrate, treatment causes differentiation of the Friend erythroleukemic cells into mature haemoglobin producing cells (87). No changes in cell morphology or differentiated state are observed for the Chinese hamster cells upon butyrate treatment and this may explain the absence of new polypeptide synthesis in these cells. In addition to the induction of haemoglobin in erythroleukemic cells, sodium butyrate has a number of effects on cultured cells. Among these are the induction of certain enzymes (84-86) and the inhibition of cell division (87). On the other hand, estrogen induction of ovalbumin in chick oviduct (88) and the corticosteroid induction of the enzyme tyrosine transaminase (89) in hamster cells are blocked in the presence of butyrate. Induction of five other proteins in hamster cells by glucocorticoids is also inhibited i f butyrate is present (90). In the case of ovalbumin, butyrate has been shown to decrease the level of transcription. This effect may be mediated via the inhibition of histone deacetylase, since analogues of butyrate such as propionate and isobutyrate which are less effective at inhibiting ovalbumin induction are also less effective at causing the hyperacetylation of the histones. (26) Part III. The present investigation. Although the heat shock proteins are synthesized in a l l organisms during times of stress, l i t t l e is known about the actual functions of these proteins. This thesis is mainly concerned with characterization of the heat shock proteins of trout RTG-2 cells by two-dimensional gel electrophoresis. As in D. melanogaster, each trout hsp detected by one-dimensional analysis separates into several isoelectric variants upon two-dimensional gel electrophoresis. Since the pattern of charge variants resembles the pattern expected for proteins which are post-translationally 32 modified, cells were incubated with P-ortho-phosphate to see if the hsps were phosphorylated. No evidence for either phosphorylation of the hsps or for changes in the phosphorylation of other proteins was obtained. Messenger RNA was isolated from heat shocked cells and translated in the in vitro rabbit reticulocyte system. Most of the isoelectric variants could be detected in in vitro translations, suggesting that either post-translational modifications are occurring in the cell-free system, or that different transcripts encode the isoelectric variants. Heat shock mRNA was also hybridized to a cDNA clone coding for a portion of the 70 K heat shock protein. The mRNA isolated by this procedure was translated in vitro. A l l 70 K isoelectric variants were present, which again suggests that multiple transcripts encode the 70 K proteins. A minor portion of this thesis is concerned with induction of (2 7) the heat shock proteins. This was studied using an antibody to chick hsp70 which cross-reacts with the trout hsp70. A protein of identical molecular weight to the heat shock 70 K protein which cross-reacts with the antibody to chick hsp70 exists at low levels in cells which have not been shocked. However, a rapid and massive increase in the amount of hsp70 could be demonstrated in stressed cells using the chick anti-hsp antibody. The effect of histone acetylation on the heat shock response was also examined using sodium butyrate. Sodium butyrate was used at concentrations that cause a high degree of histone hyperacetylation, but synthesis of the heat shock proteins was neither induced nor blocked. Therefore, although heat shock causes some deacetylation of the histones in some systems (33), the state of histone acetylation does not appear to affect the heat shock response in RTG-2 cells. (27a) Leaf 28 omitted i n numbering. (29) Experimental Procedures I. Materials. All chemicals were obtained commercially and were of the highest purity or reagent grade. Special reagents were obtained as follows: RTG-2 cells were obtained from the American Type Culture Collection; Earle's salts, foetal bovine sera, nonessential amino acids, penicillin/streptomycin and the GIBCO Selectamine kit were a l l obtained from GIBCO; RNAase A (E.C.3.1.4.22) from BRL, micrococcal nuclease (E.C.3.1.4.7) from Sigma, DNAase I (E.C.3.1.4.6) from Boehringer Mannheim, BSA from Calbiochem, TEMED, acrylamide, bis-acrylamide and ammonium persulphate a l l from Bio-Rad Laboratories, ultrapure Urea from 35 32 125 Schwartz/Mann, S-methionine, P-orthophosphate and I-sodium iodide a l l from New England Nuclear, EnHance from New England Nuclear, Cronex intensifying screen from Dupont, unlabelled goat antirabbit IgG from Miles Laboratories, Whatman 541 f i l t e r paper from Fisher, Sephadex-G-50 from Pharmacia, guanidinium hydrochloride from Schwartz/Mann, Rabbit reticulocyte in vitro translation kit from New England Nuclear. r (30) I I . Methods o A. Cell line and culture conditions. ' RTG-2 cells are a fibroblast-like line derived from mixed gonadal tissue of male and female rainbow trout, Salmo  gairdnerii (95) . Cells were grown at 22°C in disposable polystyrene flasks in Eagle's minimum essential medium containing Earle's salts, non-essential amino acids, 100 U/ml of penicillin-streptomycin and 10% fetal calf serum. B. Induction with sodium arsenite, incubation at high temperature and treatment with sodium butyrate. 1. Sodium arsenite induction. i For induction with sodium arsenite, the medium was made 50 f uM in NaAsCX, and the cultures were incubated at 22°c, the normal growth temperature, for various times. To allow the cells to recover, the medium containing NaAsO^ was removed and replaced with medium lacking arsenite. Recovery was for 2 hours. It has been found in most systems that the heat shock polypeptides are synthesized at a maximal rate after a recovery period following exposure of the cells to the inducing stimulus. (31) 2. Induction by incubation at 27°C. Heat treatment was accomplished by immersing the flask of cells in a water bath maintained at 27°C for the desired length of time. The flask was then removed and placed at 22°C for 2 hours to allow cell recovery prior to labelling. 3. Sodium butyrate induction. For sodium butyrate treatment, 5 M sodium butyrate (pH 7.5) was added to the medium to give the desired final concentration. 35 Cells were labelled with S-methionine in the presence of sodium butyrate to prevent any deacetylation of the histones during the labelling period. To study the effect of histone acetylation on the heat shock response, the medium was made 5mM in sodium butyrate for 24 hours. NaAsO,, was either added ini t i a l l y and induction was for 24 hours or arsenite was added 21 hours after the butyrate and induction was for 3 hours. (32) Radio-isotope labelling conditions. 35 1. S-methionine labelling. Cells were labelled in medium lacking unlabelled methionine (Gibco Selectamine kit) and containing 50 uCi/ml of 35 S-methionine (New England Nuclear, 1000 Ci/mmole). Labelling was carried out at 22°C for 2 hours. Incorporation was terminated by removing the labelling medium and washing the cells with icercold saline-EDTA (137 mM NaCl, 0.5 mM Na EDTA, 2.7 mM KC1, 8.1 mM Na HPO , 1.5 mM KH PO , 1 mM glucose). Cells were removed from the flask with a gentle stream of saline-EDTA from a Pasteur pipette and centrifuged briefly in a bench-top centrifuge. Pellets were immediately frozen at -70°C. Harvesting was performed as quickly as possible to avoid inadvertant induction of heat shock proteins. 32 2. P-ortho-phosphate labelling. Cells were labelled in medium buffered at pH 7.3 by 5mM HEPES 32 instead of phosphate, and containing 1 mCi/ml (0.3 uM) of P-ortho-phosphate (New England Nuclear, 3000 Ci/nmole), for 4 hours. Cells were either induced with 50 uM NaAsC>2 for 3 hours before 32 32 P-labelling or at the same time as labelling with P. (33) D. Polyacrylamide gel electrophoresis. 1. Sample preparation. For two dimensional gel electrophoresis, cell pellets were resuspended in either 0'Farrell sample buffer (96) (9.5 M Urea, 2% NP-40, 2% ampholines, 5% 2-mercaptoethanol) or 10 mM Tris-HCl, pH 7.5, 5 mM EDTA and freeze-thawed rapidly five times. Those samples in 10 mM Tris-HCl, pH 7.5, 5 mM EDTA were further hand homogenized and incubated for 15 min. on ice with 50 ug/ml of RNAase A and 50 ug/ml of DNAase I. Solid urea was then added to.a final concentration of 9 M, followed by an equal volume of 0'Farrell sample buffer. Samples for one dimensional SDS polyacrylamide gel electrophoresis were added to an equal volume of loading buffer (0.1 M Tris-HCl, pH 6.8, 2.0% SDS, 0.32 M 2-mercaptoethanol, 15% sucrose) and boiled for two min. , Lyophilized histones for acid-urea polyacrylamide gel electrophoresis were solubilized in loading buffer consisting of 2.5 M urea, 0.9 M acetic acid, 0.5 M 2-mercaptoethanol and 10% sucrose. 2. Two dimensional gel electrophoresis. Equilibrium two dimensional gel electrophoresis was performed as described by O'Farrell (96). The pH range of the isoelectric (34) focussing gels was from pH 4.5 to pH 6.8 and was determined by slicing a gel without sample into 1 cm lengths. The slices were incubated in 2 ml of degassed distilled water for 20 min before determining the pH. Electrophoresis on the isoelectric focussing gels was carried out for 17 hours at 400 V. The second dimension SDS gel consisted of a 5 to 22% exponential acrylamide gradient (0.08 cm x 19 cm x 14 cm) and electrophoresis was performed for 15 hours at 80 V. To obtain better resolution of the high molecular weight region, 8% acrylamide gels were used as the second dimension. Electrophoresis of these gels was carried out for 5 hours at 200 V. Non-equilibrium pH gradient gel electrophoresis was performed as described by 0'Farrell et al . (97) using pH 3 -10 ampholines. The first dimension was. subjected to electrophoresis for 4 hours at 400 V and resulted in a pH gradient extending from pH 4.0 to pH 9.5. The second dimension consisted of an exponential acrylamide gradient gel as described above. 3. One dimensional SDS acrylamide gel electrophoresis. One dimensional slab gel electrophoresis was performed using a modification (98) of the discontinuous buffer system of Laemmli (99). The separating gels (0.08 cm x 7.5 cm x 10 cm) contained 10% acrylamide (acrylamide:bisacrylamide ratio of 30:0.8,w/w), 0.375 M Tris-HCl, pH 8.8, 0.1% SDS, 0.03% TEMED, and 0.1% ammonium persulphate. The stacking gel contained 3.0% acrylamide (acrylamide: bisacrylamide ratio of 30:0.8,w/w), 0.125 M Tris-HCl, pH 6.8, 0.1% (35) SDS, 0.05% TEMED and 0.1% ammonium persulphate. Electrophoresis was performed in 0.05 M Tris-HCl, pH about 8.3, 0.38 M glycine and 0.1% SDS for 2 hours at 120 V. 4. Acid-urea polyacrylamide gel electrophoresis. -Histones were separated on acid-urea gels by a modification (100) of the procedure of Panyim and Chalkley (101). The separating gels (0.08 cm x 7.5 cm x 10.0 cm) contained 15% acrylamide (acrylamide:bisacrylamide ratio of 30:0.8,w/w), 2.5 M urea, 0.9 M acetic acid, 0.5% TEMED and 0.125% ammonium persulphate. i Before the stacking gel was poured, the separating gel was prerun for 2 hours at 200 V at 4°C in running buffer of 0.9 M acetic acid. The stacking gel was composed of 4.5% acrylamide (acrylamide: bisacrylamide ratio of 30:0.8, w/w), 2.5 M urea, 0.9 M acetic acid, 0.375 M potassium acetate, pH 4.0, 1% TEMED and 0.125% ammonium persulphate. Electrophoresis of these gels was in running buffer of 0.9 M acetic acid at 100 V for 6 hours at 4°C. 5. Staining and autoradiography of the gels after  electrophoresis. / All gels were stained with 0.25% Coomassie blue in methanol: glacial acetic acid:water (2:1:5,v/v) and destained in methanol: acetic acid:water (2:1:5,v/v). At this point, one dimensional gels were scanned on a Beckman DU-8 spectrophotometer. (36) Alternatively, densitometric tracings were obtained from 35 auto radiograms. Gels containing S-labelled proteins were treated with EnHance (New England Nuclear) as described by the manufacturer. Dried gels were fluorographed at -70°C using 32 Kodak X-OMAT AR film. Gels containing P-labelled proteins were dried without pretreatment and autoradiographed at -70°C using a Dupont Cronex intensifying screen. E. I s o l a t i o n of RNA. Cells were grown to confluence in roller bottles, induced with arsenite and scraped off the bottle in saline-EDTA. RNA was isolated from the resulting c e l l s as described (102). Briefly, the c e l l s were collected by centrifugation and homogenized on ice in 6 M guanidinium chloride (Gu'HCl) , 20 mM sodium acetate, 0.1 M 2-mercaptoethanol, pH 5.0. The homogenate was centrifuged'at 4 °c for 15 min at 12,000 x g. Crude RNA was precipitated from the supernatant with 0.5 volumes of 95% ethanol (-20°C). The precipitate was pelleted by centrifugation at 12,000 x g for 15 min at -10°C, resuspended i n 7.5 M Gu'HCl, 25 mM sodium citrate, 50 mM 2-mercaptoethanol, pH 7.0 and reprecipitated as before. After washing with 95% ethanol, the resulting pellet was dried and extracted three times with d i s t i l l e d water. The resulting RNA was precipitated with ethanol and redissolved in d i s t i l l e d water (37) i at a concentration of 2 mg/ml. All glassware was treated with 0.1% diethylpyrocarbonate and baked before use. Solutions were prepared in distilled water which was treated with 0.1% diethylpyrocarbonate and autoclaved. Polyadenylated RNA (poly A + RNA) was prepared by chromatography through an oligo-dT cellulose column (Collaborative Research Inc., Type 2) using the procedure of Aviv and Leder (103). Oligo-dT cellulose (0.1 g) was suspended in NETS buffer (10 mM Tris-HCl, pH 7.5, 1 mM Na EDTA , 0.25 M NaCl, 0.5% SDS) and loaded into a 1.0 ml syringe plugged with glass wool. Total cellular RNA was diluted with NETS buffer and heated at 90°C for 5 min. After rapid cooling on ice, the RNA was loaded onto the column and recirculated for l/2^hour. The column was eluted with ETS buffer (10 mM Tris-HCl, pH 7.5, 1 mM Na^ EDTA, 0.5% SDS). Carrier E. c o l i tRNA was added to the poly A + RNA to a final concentration of 100 ug/ml and the RNA was precipitated with ethanol at -20°C. F. Hybridization Selection. An M13 clone containing 690 base pairs of the coding region of a trout hsp70 cDNA was obtained from R. Kothary. The bacteriophage were grown on E. c o l i JM101 for 8 hours and the cells pelleted by centrifugation at 12,000 x g for 10 min. The supernatant was made 3.7% in polyethylene glycol-and 0.5 M in NaCl, incubated for 15 min at room temperature and centrifuged (38) at 12,000 x g for 10 min. The resulting pellet was dissolved in 20 mM Tris-HCl, pH 7.5/ 20 mM NaCl, 1 mM EDTA. The solution was extracted once with phenol and twice with phenol rCHCl^ (1:1) • The DNA was precipitated with ethanol, redissolved, and reprecipitated again with ethanol. The pellet, consisting of the plus strand of the M13 recombinant DNA and containing the region complementary to part of the hsp70 mRNA, was resuspended in 400 ul of sterile diethylpyrocarbonate-treated water. Ten ug of the plus, strand was bound to nitrocellulose (Schleicher and Schuell, B-6) using a millipore sintered glass filtration unit as described by Young et al. (104). The fi l t e r was first washed with about 50 ml of 2xSSC. The DNA was diluted to 10 ml in 3xSSC and passed through the fi l t e r at approximately 0.75 ml/min. The fi l t e r was baked for 2 hours before hybridization to 10 ug of poly A + RNA as described by Tilghman et al. (105). The f i l t e r was incubated in hybridization buffer (50% formamide, 0.75 M NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM Na^ EDTA) containing 10 ug of poly A + RNA for 20 hours at o o • 43 C. After washing with hybridization buffer at 43 C, bound RNA was released by incubating the f i l t e r in 90% formamide, 10 mM Tris-HCl,, pH 7.5, 1 mM EDTA at 45°C for one hour. Carrier E. coli tRNA was added before the RNA was precipitated with ethanol and lyophilized to remove any traces of formamide. / (39) G . I n v i t r o t r a n s l a t i o n ; RNA was t r a n s l a t e d i n a r a b b i t r e t i c u l o c y t e s y s t e m (New 35 E n g l a n d N u c l e a r ) u s i n g S - m e t h i o n i n e a s t h e l a b e l ( 1 0 6 ) . Two ug o f t o t a l RNA i n 1 u l was t r a n s l a t e d i n t h e r a b b i t r e t i c u l o c y t e l y s a t e c o n t a i n i n g magnes ium, s p e r m i d i n e , c r e a t i n e p h o s p h a t e , d i t h i o t h r e i t o l and g u a n o s i n e t r i p h o s p h a t e a s d e s c r i b e d b y t h e m a n u f a c t u r e r . A f t e r t r a n s l a t i o n , 50 ug/ml o f RNAase was a d d e d , a 5 u l a l i q u o t o f t h e t r a n s l a t i o n m i x was m i x e d w i t h 5 u l o f Laemml i s amp le b u f f e r (99) f o r one d i m e n s i o n a l g e l e l e c t r o p h o r e s i s , and t h e r e s t o f t h e sample was p r e c i p i t a t e d w i t h 6 vo lumes o f 95% e t h a n o l . The p e l l e t s were r e s u s p e n d e d i n O ' F a r r e l l s amp l e b u f f e r and s e p a r a t e d o n i s o e l e c t r i c f o c u s s i n g g e l s a s d e s c r i b e d a b o v e . H . P r o t e i n B l o t t i n g . Cyanogen b r o m i d e - a c t i v a t e d - p a p e r was p r e p a r e d a s d e s c r i b e d by C l a r k e e t a l . ( 1 0 8 ) . P r o t e i n s w e r e s e p a r a t e d on a 10% p o l y a c r y l a m i d e SDS g e l a s d e s c r i b e d a b o v e . A f t e r e l e c t r o p h o r e s i s , t h e g e l was washed 3 t i m e s f o r 5 min ea ch i n 0 .1 M s o d i u m p h o s p h a t e , pH 6 . 5 c o n t a i n i n g 0 . 1 % SDS, and 3 t i m e s i n 20 mM s o d i u m p h o s p h a t e , pH 6 . 5 w i t h o u t SDS. P r o t e i n s were t r a n s f e r r e d e l e c t r o p h o r e t i c a l l y t o C N B r - a c t i v a t e d pape r i n 20 mM s o d i u m ( 4 0 ) phosphate, pH 6.8 using a Bio-Rad electroblot apparatus. Transfer was for 4 hours at 1 A or 15 hours at 100 mA. After transfer, the CNBr-activated paper was incubated 4 times for 15 min each in quench buffer (0.1 M Tris-HCl, pH 8.0, 1% glycine, 0.1% BSA, 10% ethanolamine) to block any remaining reactive sites. The paper was then incubated at 37°C for 1 hour in 5 ml of Buffer A (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 5 mM Na EDTA, 0.1% BSA and 0.05% NP-40) containing 10 ul of the rabbit antibody to chick hsp70. After washing 4 times for 15 min in Buffer B (50 mM Tris-HCl, pH 7.4, 1.0 M NaCl, 5 mM Na^DTA, 0.1% BSA and 0.4% (w/v) sarcosyl) the paper was incubated in 5 ml of Buffer A containing 1 x 10 dpm/ml of 125 I-labelled goat anti-rabbit IgG. The paper was again washed in Buffer B and fluorographed using a Cronex screen. I. Iodination of goat anti-rabbit IgG. Goat anti-rabbit IgG was labelled by the chloramine T method of Greenwood et al . (109). Goat anti-rabbit IgG (1 mg/ml) was 125 incubated with 1 mCi/ml of Na I (New England Nuclear, 17.4 Ci/mg) and 400 ug/ml chloramine T in PBS for 10 min. Then 3 mg/ml of Na„S0 and 200 ug of BSA were added and the sample 2 5 was chromatographed through a Sephadex G-50 column (1.5 cm x 20 cm) in 50 mM sodium phosphate, pH 7.4 plus 0.1% BSA. The totally excluded fractions were pooled. J. Extraction of histones. (41) Histones were isolated at 4°C by a modification of the procedure of Marushige et a l . (110). Ce l l pel lets were homogenized in TMKS with 0.5% Triton-X-100 in a glass-teflon homogenizer. The sample was centrifuged at 3000 x g for 10 min and the crude nuclear pel let was washed once in TMKS. The pel let was then homogenized in 10 mM Tris-HCl, pH 7.4, and centrifuged at 12,000 x g for 15 min. The resulting chromatin pel let was extracted with 0.4 N H SO„ for 20 min on ice and 2 4 , centrifuged for 20 min at 12,000 x g . The supernatant was added to four volumes of 95% ethanol (-20°C) and the histones were allowed to precipitate overnight at -20°C. M l buffers contained 1 mM PMSF, 10 mM 2-mercaptoethanol and lOmM sodium butyrate. (42) R E S U L T S Part I . Characterization of the heat shock proteins of RTG-2  c e l l s . A. Two-dimensional g e l e l e c t r o p h o r e s i s of heat shock proteins. Sodium arsenite was chosen to induce the heat shock proteins i n RTG-2 c e l l s . Although the e f f e c t of NaAsC>2 i s not i d e n t i c a l to the e f f e c t of elevated temperature (36,45), i t i s more straightforward and more precise to use a chemical inducer than to increase the temperature of the c e l l s as a period of e q u i l i b r a t i o n must occur a f t e r the s h i f t before the c u l t u r e temperature reaches that of the environment. I n i t i a l l y , . a n a r s e n i t e treatment protocol was chosen which caused a strong induction of a l l heat shock proteins as detected by one-dimensional g e l electrophoretic analysis (36). C e l l s were t r e a t e d with 50 uM NaAs0 2 for 3 hours 35 and allowed to recover f o r 2 hours p r i o r to l a b e l l i n g with S-methionine. F i g . 2 shows a representative fluorogram of the protein pattern obtained from c e l l s treated i n t h i s way. The i s o e l e c t r i c focussing g e l consisted of a pH gradient extending from pH 4.5 to pH 6.8, while the second dimension was a 5% to 20% polyacrylamide gradient. A l l two dimensional-gel electrophoresis was performed under these conditions unless otherwise s p e c i f i e d . C l e a r l y , the heat shock response, i s of much greater complexity ( 4 3 ) ure 2. Two-dimensional fluorograms of proteins from RTG-2 cells. The first dimension was a pH gradient ranging from pH 4.5 to pH 6.8 and the second dimension was a 5% to 20% polyacrylamide gradient. The left end is the acidic end. Molecular weight markers were: phosphorylase b (94,000), BSA (67,000), ovalbumin (43,000), soybean trypsin inhibitor (20,100) and °<-lactalbumin (14,400). Marker positions are indicated on the left side of the gel. a) cells were untreated, b) cells were treated with 50 uM NaAsO,, for 3 hours and allowed to recover in medium without NaAsC^ for 2 35 hours before labelling with S-methionine as described in the text. Heat shock proteins are numbered and the protein labelled A was tentatively identified as actin. Exposure times were for 10 days and 500,000 cpm were loaded in a) and 300,000 cpm were loaded in b). (43*) ( 4 4 ) than is revealed by one-dimensional SDS gel electrophoresis alone. In a l l , over 20 polypeptides are detected which are not present in control cells or are present at low levels. These results are summarized in Table I. Five size classes of polypeptides are synthesized, each corresponding to a polypeptide band observed on a one-dimensional gel, and each consisting of at least four isoelectric variants. However, no polypeptides corresponding to the hsp of 87,000 daltons are present on the two-dimensional fluorogram. To obtain better separation of the region of the gel where hsp87 should be located, 8% polyacrylamide gels were used as the second dimension. Examples of the separations obtained on 8% polyacrylamide gels are shown in Fig. 3. The lower percentage of polyacrylamide expands the region of the gel where an 87,000 dalton polypeptide should run as shown by the increased separation of the 67,000 and 94,000 molecular weight markers. Many polypeptides are apparent on the gel of the arsenite treated sample (Fig. 3b) which are not obvious on the-gel of the untreated sample (Fig. 3a). However, the control gel in Fig. 3a is underexposed and most of the polypeptides in the arsenite treated sample can be faintly detected in the control sample. To ensure that none of these polypeptides corresponded to hsp87, the molecular weights of polypeptides in the arsenite sample migrating between the 67,000 dalton marker and the 94,000 dalton marker were determined. This was done by measuring the mobility from the origin for each polypeptide. No polypeptides had molecular (45) Table 1. Molecular weights and i s o e l e c t r i c p o i n t s (IEP) of heat shock p r o t e i n s i n Trout RTG-2 c e l l s . S i z e P r o t e i n IEP M r Treatment  Cl a s s 50uM NaAs0 2 1^ 50yM NaAsOa 27°C f o r 1 ) i n v i t r o 2 ^ f o r 3 h f o r 24 h 24 h t r a n s l a t i o n 100K 1 ND 3) + + _ + 2 ND + + • - + 3 ND + + - + 4 ND + + - + 70K 5 5 .91 67,000 + + + + 6 5 .97 67,000 + + + + 7 5 .98 69,800 + + + + 8 5 .98 71,100 + + + + 9 6 .06 70,000 + + + + 60K 10 5. .67 59,000 + + + -11 5. .80 60,000 + + + -12 5 .82 6 2,000 + + + -13 5. .89 62,000 + + + — 40K 14 5, .13 43,000 + + - -- 15 5. .23 44,000 + + - -16 5. .39 44,000 + + - -17 5. .61 44,000 + - -18 4. ,77 40,000 + - -19 4. 93 40,000 + + - -3 OK 20 5. 27 31,000 + + + + 21 5. 46 31,000 + + + + 22 5. 29 29,000 + + + + 23 5. 41 28,000 + + + + 24 5. 55 29,000 + + + + 25 5. 59 29,000 + + + + 1) A l l treatments were as d e s c r i b e d i n M a t e r i a l s and Methods. 2) mRNA was i s o l a t e d from c e l l s t r e a t e d w ith 50 pM NaAs02 f o r 6 h and t r a n s l a t e d in^ v i t r o as d e s c r i b e d i n the t e x t . 3) Not determined. (46) ure 3. Two-dimensional fluorograms of proteins separated on 8% polyacrylamide gels in the second dimension. Cells were induced and labelled as described in the text, a) proteins from untreated cells. The gel contained 100,000 cpm and was exposed for 21 days, b) proteins from cells treated with 50 uM NaAsC>2 for 3 hours and allowed to recover for 2 hours. The gel contained 500,000 cpm and was exposed for 14 days. Molecular weight markers are indicated along the left side of the gel. / (47) weights of 87,000. 'Since the first dimension gel had a limited pH gradient of pH 4.5 to pH 6.8, i t was possible that the 87K hsp might be too basic for these gels. Non-equilibrium pH gradient gel electrophoresis was performed to establish whether hsp87 has a basic isoelectric point. These gels had pH gradients ranging from pH 3 to pH 10. Fig. 4 shows that, although the other hsps are present, hsp87 is clearly not present in the basic region of the gel. The analysis is more difficult because most cellular proteins have acidic rather than basic isoelectric points causing a high density of spots in the acidic region of the gel. However, careful examination reveals that, as in the equilibrium pH gradient gels, no polypeptides of 87,000 daltons are present in the heat or arsenite treated sample which are not present in the control sample. Similar patterns were observed for fluorograms derived from cells treated with arsenite for varying times or from cells incubated at 27°C, as shown in Fig. 5. It may be noted from Fig. 5 that heat treatment did not inhibit normal protein synthesis to the same extent as arsenite treatment. As expected, there are no 62,000 or 100,000 dalton polypeptides in the heat treated sample, since treatment at 27°C does not induce these polypeptides (36). Heat shock polypeptides 14 t o 17 (the 42,000 dalton polypeptides) are also not detected in gels from heat-treated samples. However, the presence of these proteins is somewhat variable. Polypeptides 14 to 17 are not (48) Figure 4. Fluorograms of proteins from heat shocked cells separated by non-equilibrium pH gradient gel electrophoresis. Cells were induced and labelled as described in the text, a) proteins from control cells. The gel contained 700,000 cpm and was exposed for 2 days, b) proteins from cells incubated at 27°C for 4 hours. The gel contained 100,000 cpm and was exposed for 15 days, c) proteins from cells treated with 50 uM NaAs02 for 24 hours. The gel contained 25,000 cpm and was exposed for 45 days, d) proteins from-' cells treated with 50 uM NaAsC>2 for 3 hours and allowed to recover for 2 hours. The gel contained 110,000 cpm and was exposed for 17 days. Molecular weight markers are indicated, on the left sid£ of the gel. (486.) (49) Figure 5. Heat shock proteins from cells treated with either NaAsO^  or heat. Cells were induced and labelled as described in the text, a) proteins from untreated cells. The gel contained 300,000 cpm and was exposed for 10 days, b) proteins from cells treated with 50 uM NaAsC^ for 3 hours. The gel contained 300,000 cpm and was exposed for 7 days, c) proteins from cells treated with 50 uM NaAs02 for 24 hours. The gel contained 120,000 cpm and was exposed for 27 days, d) proteins from cells incubated at 27°C for 24 hours. The gel contained 100,000 cpm and was exposed for 21 days. Molecular weight markers are indicated on the left side of the gel. (50) present in in vitro translations of heat shock mRNA, nor are they present in samples that have been incubated on ice for 15 minutes in 10 mM Tris-HCl, pH 7.4, 5 mM MgCl^ Representative gels of these samples are shown in Fig. 6. The other treatment protocols resulted in the synthesis of the expected hsp size classes, each composed of several isoelectric variants. B. Post-translational modifications of the heat shock proteins. 1. The effect of sodium arsenite treatment on phosphorylation of  the hsps. The isoelectric variants separate on the two-dimensional gels in a manner expected for polypeptides which are post-translationally modified. One commonly occurring post-translational modification is phosphorylation. To determine i f the hsps are phosphorylated cells were incubated in phosphate-32 free medium with P-orthophosphate. Sodium arsenite was added 32 either 3 hours before the p-orthophosphate or at the same time 32 as the p-orthophosphate. This procedure should also detect changes in the phosphorylation of other cellular proteins caused by heat shock. Upon one-dimensional gel electrophoresis, as shown in Fig. 7, no phosphorylation of the heat shock proteins was detected and no changes in phosphorylated polypeptides were observed in, heat shocked cells under the conditions used. To ensure that the phosphorylation of minor polypeptides was not (51) Figure 6. Two dimensional gels showing polypeptides 14 to 17 from cells treated in different ways. Cells were induced and labelled as described in Methods, a) proteins from cells treated with 50 uM NaAs02 for 3 hours. The gel contained 275,000 cpm and was exposed for 7 days, b) proteins from cells incubated at 27° for 24 hours. The gel contained 100,000 cpm and was exposed for 25 days, c) proteins from cells treated with 50 uM NaAsC>2 for 3 hours. The gel contained 100,000 cpm and was exposed for 25 days. This sample was suspended in 10 mM Tris-HCl, pH 7.4, 5 mM MgCl2 and incubated on ice for 15 min; d) proteins resulting from in vitro translation of mRNA isolated from cells treated with 50 uM NaAs02 for 6 hours. The gel contained 116,000 cpm and was exposed for 28 days. The mRNA was isolated and translated as described in the text. Each size class of the hsps is identified. Molecular weight markers are indicated on the left side for gels a and c. (51*.) (52) ure 7. One dimensional analysis of phosphorylated polypeptides from control and heat shocked cells. Cells were induced and labelled as described in the text, a) proteins from non-heat shocked cells; b) proteins from cells treated with 50 uM 32 NaAs02 for 4 hours while labelling with p-orthophosphate; c) proteins from cells treated with 50 uM 32 NaAs02 for 4 hours before labelling with p-orthophosphate for 4 hours. Molecular weight markers are indicated on the left side of the gels. C 5 3 ) ; masked by the presence of major phosphorylated proteins in both heat shocked and control cells, two-dimensional gel electrophoresis was performed also. Fig. 8 shows examples of two-dimensional gels of phosphorylated polypeptides. None of the phosphorylated polypeptides migrate in the positions expected for heat shock proteins. Therefore, the phosphorylated polypeptides are cellular proteins which were synthesized before arsenite treatment and phosphorylated in the presence of NaAsO.^ . Some polypeptides are present in the control gel but not in the arsenite gel. Different exposure times can account for some of these polypeptides which can be detected faintly in the arsenite sample. Others were not observed consistently in the various unshocked samples. Therefore, under these labelling conditions, arsenite treatment does not affect the phosphorylation of either the heat shock proteins or cellular proteins. 2. In vitro translation of heat shock mRNA. Another possible explanation for the charge heterogeneity of the hsps would be the existence of several related but distinct genes coding for each protein size class. This possibility is supported by the fact that, in D. melanogaster, at least five genes code for hsp70 (47). To determine whether different transcripts exist for each hsp size class, RNA was isolated from cells treated with 50 uM NaAsO„ and translated in vitro in the ( 5 4 ) ure 8. Two-dimensional analysis of phosphorylated polypeptides. Cells were labelled as described in Methods, a) proteins from untreated cells labelled for 32 4 hours with P-orthophosphate. The gel contained g 3 x 10 cpm and was exposed for 6 hours, b) proteins from cells treated with 50 uM NaAsC>2 at the same 32 time as labelling with P-orthophosphate for 4 hours. The gel contained 1 x 10 6 cpm and was exposed for 3 hours. Molecular weight markers are indicated on the left side of the gels. (55) rabbit reticulocyte system. In the simplest case, if the reticulocyte system is incapable of the same post-translational modification(s) , isoelectric variants should only be observed if variant mRNAs are present. Fluorograms obtained from in vitro translations are presented in Fig. 9. Polypeptides 10 to 13 (the 62,0000 dalton hsps) and polypeptides 14 to 17 (the 42,000 dalton hsps) are clearly not present on these fluorograms, as shown in Table 1. The lack of polypeptides 14 to 17 is difficult to explain, but as mentioned previously (Fig. 6), these proteins are also absent from gels of samples which have been incubated in 10 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 on ice for 15 min and from gels of cells which have been incubated at 27°C. The small spots to the right of hsp70 could represent incomplete translation products from degraded hsp70 mRNA. All isoelectric variants are present in the in vitro translation mix, meaning that either post-translational modification is occurring in the translation system or that more than one transcript exists for these polypeptides. 3. Hybrid-selection of hsp70 mRNA. A cDNA clone complementary to hsp70 mRNA of trout has recently been isolated and used to investigate the number of genes coding for this protein in trout (R. Kothary, personal communication). A fragment of this cDNA was subcloned into M13 and the strand complementary to the hsp70 transcript was used (56) (+) • (-; 30*-2 0 ^ Figure 9. Fluorograms of p r o t e i n s from i n v i t r o translations of mRNA i s o l a t e d from untreated c e l l s or from those treated with NaAsC^* Messenger RNA was i s o l a t e d and translated from c e l l s treated as described i n Methods, a) mRNA from control c e l l s . The ge l contained 250,000 cpm and was exposed for 13 days, b) mRNA from c e l l s t r eated w i t h 50 uM NaAsC>2 for 6 hours. The g e l contained 160,000 cpm and was exposed for 19 I days. Heat shock proteins are l a b e l l e d with arrows. Molecular weight markers are in d i c a t e d on the l e f t side of the g e l s . (57) in hybrid-selection experiments to determine whether the transcripts encoding the different hsp70 isoelectric variants hybridize to this sequence. Fig. 10 shows the M13 clone used in the hybrid-selection. Single' stranded phage DNA was bound to nitrocellulose and annealed under stringent conditions to poly A + mRNA isolated from cells treated with arsenite. Bound RNA was eluted from the immobilized DNA and translated in vitro. The gel pattern from the in vitro translation mixture is shown in Fig. 11. A streak is observed corresponding to the 70 K variants. It should be noted that discrete spots are not usually observed for trout hsp70 in the in vitro translation system, as i f the proteins aggregate in the IEF dimension under these conditions. Again, this result suggests either the presence of several transcripts or that post-translationax modification occurs in the in vitro translation system. D. Induction of hsp70. The induction of hsp70 after short arsenite treatments was studied using an antibody to chick hsp70 which was kindly supplied by Dr. M. J. Schlesinger (Department of Microbiology and Immunology, Washington University School of Medicine, St. Louis, Mo.). This antibody cross-reacts with trout hsp70 as shown in Fig. 12. Slight reaction is observed with proteins from cells which have not been shocked. This may mean that the cells are sufficiently stressed during harvesting to begin heat ( 5 8 ) \ J T 1 T 1 B i : F i g u r e 1 0 . R e s t r i c t i o n map o f t h e M13 c l o n e u s e d i n t h e h y b r i d i z a t i o n s e l e c t i o n o f h e a t s h o c k mRNA. A 6 9 0 b a s e p a i r P s t I f r a g m e n t f r o m t h e c D N A c l o n e T H S 7 0 . 7 w a s s u b c l o n e d i n t o t h e P s t I s i t e o f M13 mp8 a n d g e n e r o u s l y s u p p l i e d b y R . K o t h a r y . T h e ' t r o u t c l o n e c o r r e s p o n d s t o t h e r e g i o n c o d i n g f o r a m i n o a c i d s 128 t o 348 o f t h e D . m e l a n o g a s t e r h s p 7 0 s e q u e n c e a n d i s r e p r e s e n t e d b y t h e o p e n b o x . T h e s o l i d l i n e s r e p r e s e n t M 1 3 s e q u e n c e . T h e r e s t r i c t i o n s i t e s a r e : P , P s t I ; T , T a g I a n d B , Bam H I . (59) (+) • H a Figure 11. Fluorogram of proteins from in vitro translations of mRNA isolated by hybrid-selection to an hsp70 M13 clone. Poly A RNA from c e l l s treated with 50 uM NaAs0 2 for 5 hours was hybridized to an hsp70 M13 clone (Fig. 10) as described in Methods. Hybridization was for 16 hours at 43°c in 50% formamide. a) Two-dimensional fluorogram of selected mRNA. The gel contained 50,000 cpm and was exposed for 25 days, b) 70 K region of a fluorogram resulting from the translation of total mRNA from ce l l s treated with 50 uM NaAsC>2 for 6 hours. The gel contained 160,000 cpm and was exposed for 19 days. The arrow indicates the hsp70 polypeptides. Molecular weight markers are indicated on the l e f t side of the gels. (60) ure 12. Cross-reaction of the antibody to chick hsp70 with trout hsp70. Protein blotting was performed as described in Methods, a) proteins from cells treated with 50 uM NaAs02 for 3 hours and allowed to recover for 2 hours, b) proteins from untreated cells. (61) shock protein synthesis. To study hsp70 induction, cells were treated with 50 uM NaAs02 for times ranging from 5 min to 2 hours before harvesting. Harvesting was performed as quickly as possible to minimize induction of the heat shock proteins. As can be seen in Fig. 13, lane a, the antibody s t i l l reacts with a protein of about 70,000 daltons in unshocked cells. With increasing times of arsenite treatment, the antibody reacts with increasing amounts of the 70,000 dalton protein. An increase in antibody binding is detectable after just 5 min of induction. (62) ure 13. induction of hsp70. Cells were induced for various times and the proteins separated on one-dimensional SDS gels and transferred to CNBr-activated paper as described in the methods, a) proteins from unshocked cells; b) proteins from cells treated with 50 uM NaAsC>2 for 5 min; c) 15 min; d) 30 min; e) one hour; f) 2 hours; and g) 3 hours. In a l l cases, cells were allowed to recover for 2 hours before protein extraction. The arrow indicates hsp70. (63) Part II. The effect of histone acetylation on the induction of hsps by sodium arsenite. ———————————— i A. Sodium butyrate induced histone acetylation. RTG-2 cells were treated with varying concentrations of butyrate for 24 hours to establish the best conditions to achieve maximum acetylation. Histones were isolated from cells treated with different concentrations of the fatty acid for 24 hours and separated on acid-urea gels. These gels separate basic proteins according to their charge. In the acid-urea system used here, only the acetylated forms of H4 are completely, resolved from the other histones. However, the extent of histone acetylation can s t i l l be determined using this gel system by monitoring the acetylation of H4. For this reason, Fig. 14 and Fig. 15 show only the H4 region of the gel. Fig. 14 shows the effect of treatment with different concentrations of sodium butyrate on histone acetylation in RTG-2 cells. Comparison of the unacetylated and the monoacetylated forms of H4 shows that acetylation is maximal after 24 hours of treatment with 5 mM sodium butyrate. In the untreated sample, the mono-acetylated form of H4 represents less than one-third of the total H4 while 24 hours of butyrate treatment causes the mono-acetylated form to increase until i t almost equals the acetylated form. A time course of butyrate treatment is shown in Fig. 15. Cells were treated with 5 mM sodium butyrate for different times and the histones isolated and separated on \ (64) ure 14. The effect of different concentrations of sodium butyrate on histone acetylation. Cells were induced with different concentrations of sodium butyrate for 24 hours. The histones were isolated as described in Methods, separated on acid-urea gels, stained with Coomassie blue and the region of the gel containing H4 was scanned at 545 nm with a Beckman DU-8 spectrophotometer. A q is the unacetylated form, is the mono-acetylated form, A^ is the di-acetylated form and A^ is the tri-acetylated form. The absorbance range was 1. . \ 50 m M 5 m M 3 m M control • ( - ) (65) ure 15. Time course of histone acetylation induced by sodium butyrate. Cells were induced with 5 mM sodium butyrate for the indicated times. The histones were isolated as described in the methods,separated on acid-urea gels and the region of the gel containing H4 scanned at 545 nm. The acetylated forms of the histones are labelled as in Fig.14. The absorbance range is 1. (65B.) (6 6.) acid-urea gels. Histone acetylation was maximal after 15 hours of butyrate treatment. Since butyrate is known to block cell division at the stage, subsequent experiments were performed in 5 mM sodium butyrate for 24 hours or less to achieve maxima^  acetylation with minimal effects on cell division. B. The effect of sodium butyrate on cellular protein synthesis. To examine the effects of sodium butyrate on overall patterns of protein synthesis, cells were treated with 5 mM butyrate for 35 either 3 hours or for 24 hours and then labelled with S-methionine prior to two-dimensional gel electrophoresis. If acetylation increases transcriptional activity, then an increase in the synthesis of certain polypeptides might be expected to occur. There is no apparent difference in the protein synthetic patterns of the untreated sample and in the sample treated with butyrate for 24 hours (Fig. 16a and 16c). However, changes are observed in the protein synthetic- pattern of the sample treated with sodium butyrate for 3 hours (Fig. 16b). Two polypeptides of 53,000 and 67,000 daltons increase in amount, while a third of 53,000 decreases. By 24 hours, the pattern has returned to that observed for control cells. Clearly, sodium butyrate is not causing drastic changes in the protein synthetic patterns of the RTG-2 cells. (67) Figure 16. The effect of sodium butyrate on total cellular protein synthesis as determined by two-dimensional gel electrophoresis. RTG-2 cells were treated with 5 mM butyrate 35 for 3 hours or 24 hours prior to labelling with S-methionine and separation of the total cellular proteins on two-dimensional gels. Portions of the gels in which differential protein synthesis is observed are shown. Panels a, control; b, butyrate treatment for 3 hours; c, butyrate treatment for 24 hours. The arrowhead indicates the induced proteins. The large gel was treated with 5 mM butyrate for three hours and molecular weight markers are indicated. (67cX) (+) • • H a b c (68) C. The effect of sodium butyrate on the response to sodium  arsenite. Since sodium butyrate can block the hormonal induction of certain -enzymes (87,89) , the effect of sodium butyrate on heat shock protein synthesis was examined. The effect of histone acetylation on the heat shock response was examined by treating cells with 5 mM butyrate for 24 hours before or during arsenite induction. The sodium arsenite was either added simultaneously with the butyrate (24 hour induction of the hsps), or for the final 3 hours of the butyrate treatment (3 hour induction of hsps). Despite the changes in acetylation caused by the different butyrate treatments, the heat shock proteins are s t i l l induced by sodium arsenite (Fig. 17). (69) a b c d e f Figure 17. The effect of sodium butyrate on the induction of heat shock proteins in RTG-2 cells by sodium arsenite. Cells were treated with 5 mM sodium butyrate or 50 uM sodium arsenite or both before labelling with "^S-methionine and separation of the proteins on SDS gels. Lanes a) sodium butyrate for 24 hours; b) sodium butyrate for 24 hours with sodium arsenite added for the final 3 hours; c) sodium arsenite for 3 hours; d) butyrate and arsenite for 24 hours; e) sodium arsenite alone for 24 hours; f) untreated cells. Arrows indicate the heat shock proteins. (70) D i s c u s s i o n Part I. The response of RTG-2 c e l l s to heat or sodium arsenite. A. Two-dimensional gel electrophoresis of trout heat shock proteins. Two-dimensional gel electrophoresis of trout RTG-2 c e l l heat shock proteins reveals a complex pattern. The five size classes of proteins separate into more than 20 isoelectric variants. Buzin and Petersen observed similar results in D. melanogaster (40). As in trout, the heat shock proteins separate into large numbers of isoelectric variants upon two-dimensional gel electrophoresis. In trout, the isoelectric points of the hsps range from pH 4.7 to pH 6.8 for hsp70. The D. melanogaster hsps have a greater range of isoelectric points: some of the isoelectric variants of the small hsps have isoelectric points of pH 8.0 to pH 10.0 (40). A l l of the trout polypeptides detected by one-dimensional gel electrophoresis are present on the two-dimensional gels except for the 87,000 dalton hsp. Hsp87 i s not observed when the second dimension gel is an 8% gel, nor is i t observed upon non-equilibrium pH gradient gel electrophoresis. The 8% gel should allow better separation of higher molecular weight polypeptides while the non-equilibrium pH gradient gel electrophoresis should allow separation of polypeptides with isoelectric points ranging from pH 4.5 to pH 10.0. The corresponding protein in D. melanogaster, hsp83, has an O i) isoelectric point of pH 5.33 (40). Thus, if the trout hsp87 is at a l l similar to hsp83 in D. melanogaster, then i t should be detected in these gel systems. It is unlikely that the isoelectric point of hsp87 is outside the range of the non-equilibrium pH gradient gels. A plausible explanation is that hsp87 might be undetected in these systems due to the presence of extensive charge heterogeneity which spreads the label over a large number of components. As hsp87 is induced in smaller quantities ,than some of the other hsps (36), i t may be difficult to detect the presence of numerous charge variants by two-dimensional gel electrophoresis. Since carbamylation of amino groups in proteins by isocyanate derived from urea can lead to charge heterogeneity, the possibility that the isoelectric variants observed might be due to this reaction must be considered. All precautions suggested by O'Farrell to avoid this effect (96) were followed in these experiments. Samples were either suspended in sample buffer and loaded on gels immediately o or they were frozen at -70 C. Sample buffer ^ contained protective ampholines and samples were not allowed to warm in the presence of urea. Ultrapure urea solutions were either freshly made each time or stored frozen in aliquots at -20°C. Finally, isoelectric focussing gels were pre-run to remove isocyanate. Furthermore, multiple spots are not observed on control gels, nor for many of the non-hsp components of gels containing heat shock proteins. Also, a high degree of charge complexity in hsps has been observed in other systems (15,40,43,52). For these reasons, i t can ( 7 2 X be concluded that the observed heterogeneity is a characteristic feature of the hsps themselves. ^ In agreement with the data of Kothary and Candido, (36) the 100,000 dalton hsps and the 62,000 dalton hsps are not observed in heat shocked cells. However, surprisingly, the 42,000 dalton isoelectric variants are also not detected in heat shocked cells but are observed after a 24 hour arsenite treatment. The reverse was observed by Kothary and Candido (36): hsp42 was present in heat shocked cells, but not in cells which had been treated with arsenite for 24 hours. These hsps are not detectable in in vitro translations of mRNA isolated from arsenite-treated cells or in samples which have been incubated on ice for 15 minutes (Fig. 6). Thus, i t appears as though these polypeptides are labile and that their presence or absence may be dependent on the preparation of the sample. This could explain the difference between the results presented here and those in reference 36. Another unexpected result is the absence of the 62,000 dalton isoeletric variants in the in vitro translation products of mRNA isolated from arsenite-treated cells. This suggests that these polypeptides may result from proteolytic degradation of larger polypeptides, perhaps the hsp70 variants. (73) i B. The nature of the iso-electric variants of the heat shock  proteins. 1. Post-translational modification of the heat shock proteins. The nature of the heat shock isoelectric variants is unknown. No evidence for the phosphorylation of trout heat shock proteins was found, although phosphorylation of Dictyostelium discoideum hsp70 under similar conditions has been reported (44). Phosphorylation of hsp70 may be unique to D. discoideum however, as analogous experiments have not revealed phosphorylation of the D. melanogaster hsp70 (45) . , Phosphorylation may also be dependent on the agent employed to induce the heat shock proteins. Sodium arsenite may f a i l to affect steps of the phosphorylation process affected by heat or the two perturbants may affect different steps in.the process. Vincent and Tanguay (45) have observed the phosphorylation of a cellular protein after heat treatment, but not after arsenite treatment. Further, the detection of hsp phosphorylation could be dependent on the labelling conditions. For example, the hsps would not be labelled under conditions used in the present study i f the turnover of phosphate groups was very slow. Other post-translational modifications of heat shock proteins have been shown to occur. Hsp70 and Hsp87 are methylated in chick and hamster cells (41,42) and hsp87 is ADP-ribosylated (43). These modifications may account for some of the charge (74) heterogeneity observed upon two-dimensional gel electrophoresis, but i f the variants are caused predominantly by post-translational modification then similar modifications must occur in the c e l l -free rabbit reticulocyte system, for heat shock mRNA from both D.  melanogaster (40) and trout results in multiple isoelectric variants upon translation in this system. Ovalbumin (113) and actin (114) are acetylated at their N-terminals in the in vitro rabbit reticulocyte system and other post-translational modifications may well occur in the cell-free system although they have not been reported to date. 2. Many genes code for heat shock proteins. Alternatively, the heterogeneity of the hsps couid be due to the existence of multiple amino acid sequence variants of these proteins. Such -heterogeneity of amino acid sequence could be generated at the DNA level by the existence of several genes. In trout, the analysis of genomic DNA by Southern hybridization to an hsp70 cDNA clone reveals the existence of a small number (perhaps two or three) or closely homologous sequences, and several weakly hybridizing sequences (R. Kothary, personal communication). Five genes code for hsp70 in D. melanogaster, although recently three other genes with 70% homology to the hsp70 gene have been identified (48). A similar gene family consisting of 10 homologous genes exists in yeast (39). However1, not a l l of these genes are expressed during heat shock. Some, the cognate genes, are expressed under normal conditions, and (75) hence cannot account for the charge variants observed upon heat shock. 3. Hybrid-selection of heat shock messenger RNA. Since hybridization of heat shock mRNA to an hsp70 cDNA clone isolates mRNA which is translated into several charge variants, either different transcripts code for the different isoelectric variants, or post-translational modification is occurring in the in vitro translation system. Hybridization xof heat shock mRNA from D.  melanogaster to a genomic hsp70 gene isolates not only the transcript coding for the 70 K hsp, but also transcripts corresponding to minor hsps of 72,000, 73,000 and 75,000 daltons (46). Peptide mapping of these polypeptides shows that they are" closely related. However,, deletions spanning the 87C and 87A heat shock loci result in mutants which do not make hsp70, but do synthesize the three slightly larger polypeptides (47). The genes coding for hsp70 are known to exist at the heat shock loci 87A and 87C. Therefore, the genes coding for the minor proteins which are closely related to hsp70 must reside outside the known heat shock loci. In soybean, over 20 polypeptides of 15,000 to 18,000 daltons are induced upon heat shock ( 1 1 5 ) A cDNA coding for one of the 16,000 dalton proteins has been isolated (116). These numerous polypeptides constitute a large gene family since many bands are present on Southern blots of soybean genomic DNA probed with the cDNA clone. The in vitro translation of the messenger RNA which hybridizes to the (76) 16 K hsp cDNA clone results in a series of isoelectric variants similar to those observed in this thesis. If the hybridization-selection is^ performed at higher stringency, then some of the charge variants in the in vitro translation disappear. In other words, the cDNA clone coding for the 16,000 dalton hsp hybridizes to a number of transcripts and the charge variants observed in this system are not caused by post-translational modification in the in vitro translation system. If the variation in sequence is/small and the transcript large, as would be expected for isoelectric variants of a 70,000 dalton polypeptide, then i t may be difficult to distinguish the different transcripts by hybridization conditions. In the experiments described here, although the stringency of hybridization was high, increasing the stringency s t i l l further might result in the selection of only one transcript. 4. Genetic polymorphism of the heat shock genes. Some of the complexity of the hsps of D. melanogaster can also be explained by the existence of genetic polymorphism in the fly population. Sequencing of the small hsp genes from the Oregon R strain has revealed a number of restriction site changes attributed to genetic polymorphism (117). Since the RTG-2 cell line was derived from pooled trout gonadal tissue (95), genetic polymorphism may also account for some of the complexity of the trout hsps. It is likely that a l l of the above factors contribute in some measure to the charge heterogeneity seen* in these proteins. (77) C. Induction of hsp70. To study hsp70 induction, immunological techniques were employed because of their high sensitivity. An antibody to chick hsp70 which cross-reacts with trout hsp70 was used. This cross-reaction of the antibody to the chick protein with the trout protein is another example of the conservation of hsp70 structure in different species. In addition to strong reaction with hsp70 in heat shocked cells, slight but significant binding to a protein of 70,000 daltons is detectable in control cells. It is possible that hsp70 is constitutively present at low levels in control cells, or that the cells are sufficiently stressed during harvesting to begin making heat shock proteins. Similar observations have^been reported in other systems (5,15,38). This cross-reaction could also have resulted if proteins similar to hsp70 are expressed in control cells (e.g. the cognate proteins), or, since the chick antibody is not a monoclonal antibody, from contamination by control proteins of the antigen used for immunization. The hsp70 used to innoculate the rabbits was isolated by Sepharose 6B and DEAE-cellulose chromatography (5). Purity of the hsp70 was assayed by one-dimensional gel electrophoresis. However,'the two-dimensional gels presented in this thesis clearly show the presence of proteins of similar size (78) and charge as hsp70 in control cells. Such control proteins which could result from the expression of the cognate genes, may have contaminated the hsp70 preparation used in immunization. Proteins coded for by the cognate genes may carry similar antigenic determinants to those of hsp70 since the cognate genes of D.  melanogaster and yeast are 70% homologous to hsp70 at the amino acid level (39,48). Recently, Lindquist et al. (118) have isolated a monoclonal antibody to hsp70 which does not cross-react with proteins from unshocked cells. Thus, although a number of groups have reported the presence of hsp70 in control cells, the protein detected in control cells is *probably transcribed from a cognate gene and is not identical to hsp70. Although the antibody to chick hsp70 cross-reacts with proteins in the control cells to a small degree, the induction of hsp70 synthesis is s t i l l clearly detectable. A sharp increase in antibody binding is detectable after 5 min of treatment with 50 uM NaAsC>2 indicating the synthesis of hsp70 upon heat shock. However, the corresponding RNA transcript is only detected by RNA dot-blot analysis"after 15 min of treatment ( (R. Kothary, personal communication). The disparity between the' appearance of the protein and the presence of the transcript may reflect the sensitivity of the two techniques. Each transcript can be translated many times to generate a large amount of protein which is then detectable by immunological techniques. In addition, more than one gene may code for the trout hsp70 and transcripts from other genes may not hybridize'to the hsp70 cDNA (79) clone under conditions of high stringency. Since the antibody is polyclonal, i t will likely bind to several variant hsp70s. In D. melanogaster, the transcript is detectable 5 min after shift to 35°C (13) while, the protein is detectable after 10 min of incubation at 35°C (18). These induction times are comparable to the results described in this thesis. However, as mentioned previously, comparisons between different organisms are not very meaningful because the induction of the hsps is affected by the severity of the stress. (_8 0 ) Part II. The effect of histone acetylation on the response  to sodium arsenite. A. The effect of sodium butyrate on trout cells. Maximal levels of acetylation of RTG-2 histones are achieved by the treatment of RTG-2 cells with 5 mM sodium butyrate for 15 hours. Further acetylation is not achieved by the use of higher concentrations of butyrate or longer treatment times. Similar results have been obtained for rat, mouse, hamster and Xenopus cells (80) . After 24 hours of butyrate treatment, no changes in protein synthesis, as determined by two-dimensional gel electrophoresis, are apparent. However, treatment for shorter lengths of time from 3 hours to 6 hours results in an increase in the amounts of two polypeptides and a decrease in a third. It is possible that the level of acetylation achieved after 3 hours of butyrate treatment is sufficient to affect the expression of these proteins whereas further acetylation does not alter their original pattern of expression. Since butyrate is known to change the distribution of the cell population across the cell cycle, i t is more likely that these proteins are cell-cycle specific. Sodium butyrate blocks mammalian cells in G^  phase (91). In C h i n e s e hamster ovary cells, butyrate has also been shown to have l i t t l e effect on protein synthesis (86), but in Friend erythroleukemic cells, butyrate has been shown to induce a large number of proteins (85). This difference may be explained by the (81) f a c t t h a t b u t y r a t e c a u s e s d i f f e r e n t i a t i o n o f F r i e n d e r y t h r o l e u k e m i c c e l l s i n t o m a t u r e c e l l s t h a t p r o d u c e h a e m o g l o b i n ( 8 7 ) . H e n c e , a n u m b e r o f new p o l y p e p t i d e s a r e s y n t h e s i z e d i n F r i e n d e r y t h r o l e u k e m i c c e l l s w h e r e a s i n C h i n e s e h a m s t e r c e l l s a n d R T G - 2 c e l l s , w h i c h a r e r e l a t i v e l y u n a f f e c t e d b y b u t y r a t e , s y n t h e s i s , o f o n l y a f e w p o l y p e p t i d e s i s i n d u c e d . B . T h e e f f e c t o f s o d i u m b u t y r a t e o n t h e r e s p o n s e t o s o d i u m  a r s e n i t e . H y p e r a c e t y l a t i o n o f t h e h i s t o n e s h a s n o e f f e c t o n t h e i n d u c t i o n o f h e a t s h o c k p r o t e i n s b y s o d i u m a r s e n i t e . C e l l s w e r e e i t h e r t r e a t e d w i t h v b u t y r a t e b e f o r e a r s e n i t e t r e a t m e n t o r w e r e t r e a t e d s i m u l t a n e o u s l y w i t h b o t h b u t y r a t e a n d N a A s C ^ . T h e h e a t s h o c k p r o t e i n s w e r e i n d u c e d i n a l l c a s e s . T r e a t m e n t w i t h s o d i u m b u t y r a t e f o r t h e s e t i m e - p e r i o d s ( g r e a t e r t h a n 3 h o u r s ) c a u s e s b o t h b u l k h i s t o n e a c e t y l a t i o n a n d a c e t y l a t i o n o f t h e r a p i d l y a c e t y l a t e d s u b s e t o f t h e h i s t o n e s . T h e e x t e n t o f b o t h t y p e s o f h i s t o n e a c e t y l a t i o n i n c r e a s e s d r a m a t i c a l l y u n d e r t h e s e c o n d i t i o n s . I t i s d i f f i c u l t t o i m a g i n e h o w t h e i n d u c t i o n o f t h e h e a t s h o c k p r o t e i n s c o u l d r e m a i n u n a f f e c t e d i f t h e s t a t e o f h i s t o n e a c e t y l a t i o n i s i m p o r t a n t - i n t h e h e a t s h o c k r e s p o n s e . A r r i g o ( 34 ) h a s r e p o r t e d t h a t h e a t s h o c k c a u s e s d e a c e t y l a t i o n o f h i s t o n e s i n D .  m e l a n o g a s t e r c u l t u r e d c e l l s . I f t h i s i s t r u e , t h e n i t a p p e a r s t o b e a c o n s e q u e n c e . o f t h e h e a t t r e a t m e n t r a t h e r t h a n p a r t o f t h e i n d u c t i o n m e c h a n i s m f o r h s p s s i n c e h y p e r a c e t y l a t i o n o f t h e (82) histones does not block heat shock protein synthesis. The ability of butyrate to block the activation of steroid-inducible genes (92-94) does not appear to apply to al l inducible genes. In particular, i t has no effect on the induction of heat shock proteins in the system described here. This is perhaps not surprising, since different control regions are probably involved in these-two classes of inducible genes, and they may act via different mechanisms. The heat shock genes are activated virtually immediately upon exposure of cells to an appropriate inducer, whereas steroid induction typically requires several hours (122,123). 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