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Inducibility of heat shock polypeptides in cells containing hyperacetylated histones
Short notes 273
SHORT NOTE Inducibility of Heat Shock Polypeptides in Cells Containing Hyperacetylated Histones ELIZABETH
A. BURGESS, RASHMIKANT K. KOTHARY and E. PETER M. CANDID0 Department of Biochemistry, University of British Columbia, Vancouver,
BC Canada V6Tl W5
We have examined the effect of sodium butyrate on the levels of histone acetylation, the pattern of protein synthesis and the inducibility of heat shock polypeptides (hsps) in cultured trout fibroblasts. Maximal levels of histone acetylation are achieved upon treatment of these cells with 5 mM butyrate for 24 h. No significant changes in the pattern of protein synthesis, as detected by two-dimensional gel electrophoresis, are apparent under these conditions, although changes in the levels of three polypeptides are seen at shorter times of exposure to butyrate. Heat shock polypeptides are inducible at normal levels in butyrate-treated cells. This is in contrast to the ability of butyrate to inhibit the activation of steroid-inducible genes in some systems. 0 1984 Academic RUSS. hc.
Sodium butyrate has many effects on cultured cells. Among these are the induction of certain enzymes [l-3], induction of hemoglobin in Friend erythroleukemic cells [4], and inhibition of cell division [5]. Since butyrate is a competitive inhibitor of histone deacetylase [&8] it causes massive hyperacetylation of the histones [9], which might then result in some or even all of the effects described above. Histone acetylation has been correlated broadly with transcription [lO-121, although specific changes in chromatin structure which accompany transcription are as yet unknown. A useful approach to this problem is to study the effects of alterations in chromatin structure on the activity of specific genes. For example, the induction of ovalbumin by estrogen in chick oviduct [ 131and of the enzyme tyrosine amino transferase by corticosteroids in hamster cells [14] is blocked in the presence of butyrate. In addition, the induction of five other proteins in hamster cells by glucocorticoids is inhibited if butyrate is present [ 151. 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 histones. In view of the ability of butyrate to prevent the induction of several steroidinducible genes, we have examined its effect on another group of inducible genes, namely those coding for the heat shock polypeptides in a trout cell line. Elevation of the normal growth temperature of all organisms studied to date results in the induction of certain polypeptides, called heat shock proteins (hsps) [16]. This induction occurs at the level of transcription and can also be caused by other external factors, including exposure to metabolic inhibitors such as sodium Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/84 $03.00
274 Short notes
b 24h 15h
3h
control
(+) -
(-1
Fig. I. The effect of sodium butyrate on histone acetylation in RTG-2 cells. Cells were treated with varying concentrations of sodium butyrate for different times. Histones were isolated as described under Materials and Methods, separated on acid-urea gels and scanned. Scans of the H4 region of acid-urea gels from cells treated with (a) varying concentrations of butyrate for 24 h; (h) 5 mM butyrate for varying times.
arsenite. This report demonstrates that butyrate has little or no effect on the induction of hsps in trout cells. Materials and Methods RTG-2 cells are a fibroblast-like line derived from mixed gonadal tissue of male and female rainbow trout. S&no gairdnerii [l7]. Cells were grown at 22°C 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 (FCS) (all from Gibco Ltd). When required, sodium butyrate and sodium arsenite were added to final concentrations of 5 mM and 50 PM. respectively. Cultures were labelled with 50 uCi/ml of [35S]methionine (I 000 Ci/mmole, New England Nuclear) for 2 h in medium without methionine (Gibco Selectamine kit). Incorporation was terminated by removing the labelling medium and washing the cells with ice-cold saline-EDTA (137 mM NaCI, 0.5 mM Na,EDTA, 2.7 mM KCI, 8. I mM Naz HPO,, I .5 mM KH2P04, I. I 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. Histones were isolated by a modification of the procedure of Marushige et al. [IS]. Cell pellets were homogenized in TMKS (50 mM Tris-HCI, pH 7.4. 5 mM MgCl*, 25 mM KCI, 0.25 M sucrose) with 0.5 % Triton X-100 in a glass teflon homogenizer. The sample was centrifuged at 3 000 g for 10 min and washed once in TMKS. The pellet was homogenized in 10 mM Tris-HCl, pH 7.4 and centrifuged at 12000 g for 15 min. The chromatin pellet was extracted with 0.4 N HzS04 for 20 min on ice and centrifuged for 20 min at 12000 g. The supernatant was added to 4 vol of 95% ethanol (-20°C) and histones allowed to precipitate overnight at -20°C. All buffers contained I mM PMSF, IO mM P-mercaptoethanol and IO mM sodium butyrate. Histones were separated on acid-urea gels (0.08x7.5x10 cm) by a modification [19] of the procedure of Panyim & Chalkley 1201. After electrophoresis, gels were stained with 0.25% Coomassie Blue, destained and scanned with a Beckman DU-8 spectrophotometer. Two-dimensional gel electrophoresis was performed as described [2l]. The pH range of the isoelectric focusing gels was from pH 4.5 to 6.8. The second dimension (0.08x 14.0x 19.0 cm) consisted of a 5-22 % exponential acrylamide gradient. One-dimensional SDS gels (0.08~7.5~ IO cm) were a modification [22] of the discontinuous buffer system of Laemmli [23]. After electrophoresis, gels were stained with 0.25 % Coomassie Blue and destained. Gels were then treated with EnHance (New England Nuclear) according to the manufacturer’s instructions. Dried gels were fluorographed using Kodak X-Omat AR film.
Results To determine the effect of butyrate on histone acetylation in RTG-2 cells, cells were treated with varying concentrations of the fatty acid for 24 h. Histones were
276 Short notes butyrate for 3 h, while a third decreases. By 24 h, the pattern has returned to that observed for control cells. Clearly, butyrate is not causing a massive induction of new protein synthesis in these cells. Heat shock proteins were induced by treating cells with 50 yM sodium arsenite for 3 h before labelling with [35S]methionine. This protocol which has been shown to result in the strong induction of all hsps in these cells [24]. The effect of butyrate was examined by treating cells with 5 mM butyrate for 24 h prior to the addition of arsenite as above. As shown in fig. 3, no significant effect on the induction of the hsps is observed. Butyrate neither induces these proteins itself, nor inhibits their induction by sodium arsenite. Discussion Maximal levels of acetylation of RTG-2 histones are achieved upon treatment of the cells with 5 mM butyrate for 24 h. At this time, no changes in protein synthesis, as determined by 2D gel electrophoresis, are apparent. However, treatment for shorter lengths of time results in an increase in the amount of two polypeptides and a decrease in a third. The cause of this transient effect is unknown but might be due, e.g., to changes in the distribution of the cell population across the cell cycle. Butyrate has been shown to block mammalian cells in Gl phase [5]. Hyperacetylation of the histones has no effect on the induction of heat shock proteins by sodium arsenite. Recently, Arrigo [25] reported that heat shock causes deacetylation of histones in Drosophila cultured cells. If this is true, then it appears to be a consequence of the heat shock rather than part of the induction mechanism for hsps, since hyperacetylation of the histones does not block heatshock protein synthesis. The ability of butyrate to block the activation of steroidinducible genes uoes not appear to apply to all inducible genes. In particular, it has no effect on the induction of hsps 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 [26, 271. Thus, while the heat shock genes are primed for immediate transcription, the activation of hormone-inducible genes seems to require additional processes, at least one of which is susceptible to inhibition by sodium butyrate. This work was supported by grants from the MRC of Canada and the B.C. Health Care Research Foundation. E. A. B. was supported by a MRC of Canada Studentship.
References 1. Prasad, K N & Sinha, P K, In vitro 12 (1976) 125. 2. Fishman, P H & Brady, R 0, Science 194 (1979) 906. 3. Griffin, M J, Price, G H, Bazzell, K L, Cox, R P & Ghash, N K, Arch biochem biophys 164(1974) 619. Exp Cell Res 155 (1984)
Short notes
275
3 Fig. 2. The effect of sodium butyrate on total cellular protein synthesis as determined by twodimensional gel eiectrophoresis. RTG-2 cells were treated with 5 mM butyrate for 3 or 24 h prior to labelling with [35S]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. I a, 2 a, Control; I b, 2 b, butyrate treatment for 3 h; I c, 2 c, butyrate treatment for 24 h. Fig. 3. 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 yM sodium arsenite or both before labelling with [%]methionine and separation of the proteins on SDS gels. Sodium butyrate for n, 24 h; b, 24 h with sodium arsenite added for the final 3 h; c, sodium arsenite for 3 h; d, butyrate and arsenite for 24 h; e, sodium arsenite alone for 24 h;f, control. Arrows indicate the heat shock proteins.
then isolated and separated on acid-urea gels. Fig. 1 shows that the maximal effect on histone acetylation occurs at a concentration of 5 mM butyrate. A time course (fig. 1 b) showed that acetylation was complete by 15 h in 5 mM butyrate. Since butyrate is known to inhibit cell division, subsequent experiments were performed in 5 mM butyrate for 24 h or less to achieve maximal acetylation with minimal effects on cell division. To examine the effects of sodium butyrate on overall patterns of protein synthesis, cells were treated with 5 mM butyrate for 3 or 24 h and labelled with [35S]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. Fig. 2 shows that little or no difference is detected between patterns observed for control cells and those treated for 24 h with 5 mM butyrate. However, two polypeptides do increase in cells treated with Exp Cell Res 155 (1984)
Short
notes
277
4. Leder, A & Leder, P, Cell 5 (1975) 319. 5. Hagopian, H K, Riggs, M G, Swartz, L A & Ingram, V M, Cell 12 (1979) 855. 6. Candido, E P M, Reeves, R & Davie, J R, Cell 14 (1978) 105. 7. Vidali, G, Boffa, L C, Bradbury, E M & Allfrey, V G, Proc natl acad sci US 75 (1978) 2239. 8. Sealy, L & Chalkley, R, Cell 14 (1978) 115. 9. Riggs, M G, Whittaker, R G, Neumann, J R & Ingram, V M, Nature 268 (1977) 462. 10. Mathis, D, Oudet, P & Chambon, P, Prog nucleic acid res mol biol 24 (1980). 11. Davie, J R & Candido, E P M, Proc natl acad sci US 75 (1978) 3574. 12. Nelson, D, Covault, J & Chalkley, R, Nucleic acids res 8 (1980) 1745. 13. McKnight, G S, Hager, L & Palmiter, R D, Cell 28 (1980) 499. 14. Tichonicky, Z, Defer, N, Kruh, J, Santana-Calderon, M A, Griesen, E M & Bick, G, Eur j biochem 120 (1981) 427. 15. Plesko, M M, Hargrove, J L, Granner, D K & Chalkley, R, J biol them 258 (1983) 13738. 16. Schlesinger, M J, Ashbumer, M & Tissieres, A, Heat shock: from bacteria to man. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). 17. Wolf, K & Quimby, M C, Science 135 (1962) 1065. 18. Marushige, K & Bonner, J, J mol biol 15 (1966) 160. 19. Davie, J R, Anal biochem 120 (1982) 276. 20. Panyim, S & Chalkley, R, Arch biochem biophys 130 (1969) 337. 21. O’Farrell, P, J biol them 250 (1975) 4007. 22. Matsudaira, P T & Burgess, P R, Anal biochem 87 (1978) 386. 23. Laemmli, U K, Nature 227 (1970) 680. 24. Kothary, R K & Candido, E P M, Can j biochem 60 (1982) 347. 25. Amigo, A-P, Nucl acids res 11 (1983) 1389. 26. Palmiter, R N, J biol them 248 (1973) 8260. 27. Thompson, E B, Tomkins, G M & Curran, J F, Proc natl acad sci US 56 (1966) 296. Received May 17, 1984
Printed
in Sweden
Exp Cell Res 15.5 (1984)
SHORT NOTE Inducibility of Heat Shock Polypeptides in Cells Containing Hyperacetylated Histones ELIZABETH
A. BURGESS, RASHMIKANT K. KOTHARY and E. PETER M. CANDID0 Department of Biochemistry, University of British Columbia, Vancouver,
BC Canada V6Tl W5
We have examined the effect of sodium butyrate on the levels of histone acetylation, the pattern of protein synthesis and the inducibility of heat shock polypeptides (hsps) in cultured trout fibroblasts. Maximal levels of histone acetylation are achieved upon treatment of these cells with 5 mM butyrate for 24 h. No significant changes in the pattern of protein synthesis, as detected by two-dimensional gel electrophoresis, are apparent under these conditions, although changes in the levels of three polypeptides are seen at shorter times of exposure to butyrate. Heat shock polypeptides are inducible at normal levels in butyrate-treated cells. This is in contrast to the ability of butyrate to inhibit the activation of steroid-inducible genes in some systems. 0 1984 Academic RUSS. hc.
Sodium butyrate has many effects on cultured cells. Among these are the induction of certain enzymes [l-3], induction of hemoglobin in Friend erythroleukemic cells [4], and inhibition of cell division [5]. Since butyrate is a competitive inhibitor of histone deacetylase [&8] it causes massive hyperacetylation of the histones [9], which might then result in some or even all of the effects described above. Histone acetylation has been correlated broadly with transcription [lO-121, although specific changes in chromatin structure which accompany transcription are as yet unknown. A useful approach to this problem is to study the effects of alterations in chromatin structure on the activity of specific genes. For example, the induction of ovalbumin by estrogen in chick oviduct [ 131and of the enzyme tyrosine amino transferase by corticosteroids in hamster cells [14] is blocked in the presence of butyrate. In addition, the induction of five other proteins in hamster cells by glucocorticoids is inhibited if butyrate is present [ 151. 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 histones. In view of the ability of butyrate to prevent the induction of several steroidinducible genes, we have examined its effect on another group of inducible genes, namely those coding for the heat shock polypeptides in a trout cell line. Elevation of the normal growth temperature of all organisms studied to date results in the induction of certain polypeptides, called heat shock proteins (hsps) [16]. This induction occurs at the level of transcription and can also be caused by other external factors, including exposure to metabolic inhibitors such as sodium Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/84 $03.00
274 Short notes
b 24h 15h
3h
control
(+) -
(-1
Fig. I. The effect of sodium butyrate on histone acetylation in RTG-2 cells. Cells were treated with varying concentrations of sodium butyrate for different times. Histones were isolated as described under Materials and Methods, separated on acid-urea gels and scanned. Scans of the H4 region of acid-urea gels from cells treated with (a) varying concentrations of butyrate for 24 h; (h) 5 mM butyrate for varying times.
arsenite. This report demonstrates that butyrate has little or no effect on the induction of hsps in trout cells. Materials and Methods RTG-2 cells are a fibroblast-like line derived from mixed gonadal tissue of male and female rainbow trout. S&no gairdnerii [l7]. Cells were grown at 22°C 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 (FCS) (all from Gibco Ltd). When required, sodium butyrate and sodium arsenite were added to final concentrations of 5 mM and 50 PM. respectively. Cultures were labelled with 50 uCi/ml of [35S]methionine (I 000 Ci/mmole, New England Nuclear) for 2 h in medium without methionine (Gibco Selectamine kit). Incorporation was terminated by removing the labelling medium and washing the cells with ice-cold saline-EDTA (137 mM NaCI, 0.5 mM Na,EDTA, 2.7 mM KCI, 8. I mM Naz HPO,, I .5 mM KH2P04, I. I 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. Histones were isolated by a modification of the procedure of Marushige et al. [IS]. Cell pellets were homogenized in TMKS (50 mM Tris-HCI, pH 7.4. 5 mM MgCl*, 25 mM KCI, 0.25 M sucrose) with 0.5 % Triton X-100 in a glass teflon homogenizer. The sample was centrifuged at 3 000 g for 10 min and washed once in TMKS. The pellet was homogenized in 10 mM Tris-HCl, pH 7.4 and centrifuged at 12000 g for 15 min. The chromatin pellet was extracted with 0.4 N HzS04 for 20 min on ice and centrifuged for 20 min at 12000 g. The supernatant was added to 4 vol of 95% ethanol (-20°C) and histones allowed to precipitate overnight at -20°C. All buffers contained I mM PMSF, IO mM P-mercaptoethanol and IO mM sodium butyrate. Histones were separated on acid-urea gels (0.08x7.5x10 cm) by a modification [19] of the procedure of Panyim & Chalkley 1201. After electrophoresis, gels were stained with 0.25% Coomassie Blue, destained and scanned with a Beckman DU-8 spectrophotometer. Two-dimensional gel electrophoresis was performed as described [2l]. The pH range of the isoelectric focusing gels was from pH 4.5 to 6.8. The second dimension (0.08x 14.0x 19.0 cm) consisted of a 5-22 % exponential acrylamide gradient. One-dimensional SDS gels (0.08~7.5~ IO cm) were a modification [22] of the discontinuous buffer system of Laemmli [23]. After electrophoresis, gels were stained with 0.25 % Coomassie Blue and destained. Gels were then treated with EnHance (New England Nuclear) according to the manufacturer’s instructions. Dried gels were fluorographed using Kodak X-Omat AR film.
Results To determine the effect of butyrate on histone acetylation in RTG-2 cells, cells were treated with varying concentrations of the fatty acid for 24 h. Histones were
276 Short notes butyrate for 3 h, while a third decreases. By 24 h, the pattern has returned to that observed for control cells. Clearly, butyrate is not causing a massive induction of new protein synthesis in these cells. Heat shock proteins were induced by treating cells with 50 yM sodium arsenite for 3 h before labelling with [35S]methionine. This protocol which has been shown to result in the strong induction of all hsps in these cells [24]. The effect of butyrate was examined by treating cells with 5 mM butyrate for 24 h prior to the addition of arsenite as above. As shown in fig. 3, no significant effect on the induction of the hsps is observed. Butyrate neither induces these proteins itself, nor inhibits their induction by sodium arsenite. Discussion Maximal levels of acetylation of RTG-2 histones are achieved upon treatment of the cells with 5 mM butyrate for 24 h. At this time, no changes in protein synthesis, as determined by 2D gel electrophoresis, are apparent. However, treatment for shorter lengths of time results in an increase in the amount of two polypeptides and a decrease in a third. The cause of this transient effect is unknown but might be due, e.g., to changes in the distribution of the cell population across the cell cycle. Butyrate has been shown to block mammalian cells in Gl phase [5]. Hyperacetylation of the histones has no effect on the induction of heat shock proteins by sodium arsenite. Recently, Arrigo [25] reported that heat shock causes deacetylation of histones in Drosophila cultured cells. If this is true, then it appears to be a consequence of the heat shock rather than part of the induction mechanism for hsps, since hyperacetylation of the histones does not block heatshock protein synthesis. The ability of butyrate to block the activation of steroidinducible genes uoes not appear to apply to all inducible genes. In particular, it has no effect on the induction of hsps 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 [26, 271. Thus, while the heat shock genes are primed for immediate transcription, the activation of hormone-inducible genes seems to require additional processes, at least one of which is susceptible to inhibition by sodium butyrate. This work was supported by grants from the MRC of Canada and the B.C. Health Care Research Foundation. E. A. B. was supported by a MRC of Canada Studentship.
References 1. Prasad, K N & Sinha, P K, In vitro 12 (1976) 125. 2. Fishman, P H & Brady, R 0, Science 194 (1979) 906. 3. Griffin, M J, Price, G H, Bazzell, K L, Cox, R P & Ghash, N K, Arch biochem biophys 164(1974) 619. Exp Cell Res 155 (1984)
Short notes
275
3 Fig. 2. The effect of sodium butyrate on total cellular protein synthesis as determined by twodimensional gel eiectrophoresis. RTG-2 cells were treated with 5 mM butyrate for 3 or 24 h prior to labelling with [35S]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. I a, 2 a, Control; I b, 2 b, butyrate treatment for 3 h; I c, 2 c, butyrate treatment for 24 h. Fig. 3. 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 yM sodium arsenite or both before labelling with [%]methionine and separation of the proteins on SDS gels. Sodium butyrate for n, 24 h; b, 24 h with sodium arsenite added for the final 3 h; c, sodium arsenite for 3 h; d, butyrate and arsenite for 24 h; e, sodium arsenite alone for 24 h;f, control. Arrows indicate the heat shock proteins.
then isolated and separated on acid-urea gels. Fig. 1 shows that the maximal effect on histone acetylation occurs at a concentration of 5 mM butyrate. A time course (fig. 1 b) showed that acetylation was complete by 15 h in 5 mM butyrate. Since butyrate is known to inhibit cell division, subsequent experiments were performed in 5 mM butyrate for 24 h or less to achieve maximal acetylation with minimal effects on cell division. To examine the effects of sodium butyrate on overall patterns of protein synthesis, cells were treated with 5 mM butyrate for 3 or 24 h and labelled with [35S]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. Fig. 2 shows that little or no difference is detected between patterns observed for control cells and those treated for 24 h with 5 mM butyrate. However, two polypeptides do increase in cells treated with Exp Cell Res 155 (1984)
Short
notes
277
4. Leder, A & Leder, P, Cell 5 (1975) 319. 5. Hagopian, H K, Riggs, M G, Swartz, L A & Ingram, V M, Cell 12 (1979) 855. 6. Candido, E P M, Reeves, R & Davie, J R, Cell 14 (1978) 105. 7. Vidali, G, Boffa, L C, Bradbury, E M & Allfrey, V G, Proc natl acad sci US 75 (1978) 2239. 8. Sealy, L & Chalkley, R, Cell 14 (1978) 115. 9. Riggs, M G, Whittaker, R G, Neumann, J R & Ingram, V M, Nature 268 (1977) 462. 10. Mathis, D, Oudet, P & Chambon, P, Prog nucleic acid res mol biol 24 (1980). 11. Davie, J R & Candido, E P M, Proc natl acad sci US 75 (1978) 3574. 12. Nelson, D, Covault, J & Chalkley, R, Nucleic acids res 8 (1980) 1745. 13. McKnight, G S, Hager, L & Palmiter, R D, Cell 28 (1980) 499. 14. Tichonicky, Z, Defer, N, Kruh, J, Santana-Calderon, M A, Griesen, E M & Bick, G, Eur j biochem 120 (1981) 427. 15. Plesko, M M, Hargrove, J L, Granner, D K & Chalkley, R, J biol them 258 (1983) 13738. 16. Schlesinger, M J, Ashbumer, M & Tissieres, A, Heat shock: from bacteria to man. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). 17. Wolf, K & Quimby, M C, Science 135 (1962) 1065. 18. Marushige, K & Bonner, J, J mol biol 15 (1966) 160. 19. Davie, J R, Anal biochem 120 (1982) 276. 20. Panyim, S & Chalkley, R, Arch biochem biophys 130 (1969) 337. 21. O’Farrell, P, J biol them 250 (1975) 4007. 22. Matsudaira, P T & Burgess, P R, Anal biochem 87 (1978) 386. 23. Laemmli, U K, Nature 227 (1970) 680. 24. Kothary, R K & Candido, E P M, Can j biochem 60 (1982) 347. 25. Amigo, A-P, Nucl acids res 11 (1983) 1389. 26. Palmiter, R N, J biol them 248 (1973) 8260. 27. Thompson, E B, Tomkins, G M & Curran, J F, Proc natl acad sci US 56 (1966) 296. Received May 17, 1984
Printed
in Sweden
Exp Cell Res 15.5 (1984)