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Developmental and tissue specific control of the heat shock induced 70 kDa related proteins in the flesh fly, Sarcophaga crassipalpis
J. Insrrr Physiol. Vol.36, No. 4. pp. 239-249, Printed in Great Britain. All rights reserved
1990 Copyright
0022-1910/90 $3.00 + 0.00 Q 1990 Pergamon Press plc
DEVELOPMENTAL AND TISSUE SPECIFIC CONTROL OF THE HEAT SHOCK INDUCED 70kDa RELATED PROTEINS IN THE FLESH FLY, SARCOPHAGA CRASSIPALPIS KARL H. JOPLINand DAVIDL. DENLINGER Department of Entomology, The Ohio State University, 1735 Neil Avenue, Columbus, OH 43210-1220,U.S.A. (Received 11 September 1989; revised 22 Noclember1989)
shock (4G43”C) induces two proteins in Sarcophaga crassipalpis that are immunologically related to the heat shock 70 protein family in Drosophila. These Sarcophaga heat shock proteins, M, of 65 and 72 kDa, show developmental and tissue-specificexpression. Heat shock protein 65 is expressed in the brain and integument of third-instar larvae at 43°C. Expression of heat shock protein 65 in both tissues ceases at pupariation and heat shock protein 72 is the protein induced at 43°C throughout the rest of Abstract-Heat
development. Tissue specificity for the expression of these two heat shock proteins can be observed in 3-day-old adult males: brain and integument express heat shock protein 72 while the terminalia and flight muscle express heat shock protein 65. Both a developmental and temperature switch can be seen in the male terminalia: day-l terminalia express heat shock protein 72 at either 40 or 43°C on day-2 heat shock protein 72 is expressed at 40°C but heat shock protein 65 is expressed at 43°C thereafter the terminalia express primarily heat shock protein 65 at either heat shock temperature. Control of heat shock protein expression in S. crassipalpis is thus considerably more complex than the Drosophila literature suggests. Key Word Index:
Sarcophaga; heat shock protein; developmental
protein expression; tissue-specific
protein expression
INTRODUCTION High temperatures and a number of other forms of biological stress elicit the rapid synthesis of a set of heat shock proteins and a concurrent suppression of proteins normally expressed during nonstressed conditions (Ashburner and Bonner, 1979; Schlesinger et al., 1982; Craig, 1985; Lindquist, 1986). This limited set of heat shock proteins is thought to be expressed, with minor exceptions, in all tissues and all developmental stages of a stressed organism. Our study examines this concept in the flesh fly, Surcophuga crassipalpis.
A heat shock response has been observed in all organisms studied (Kelley and Schlesinger, 1978; McAlister et al., 1979; Schlesinger et al., 1982) and appears to be a conserved physiological response to stress. The heat shock 70 protein family is the most highly conserved of the heat shock proteins and is induced to the highest levels during heat shock (Moran et nl., 1983). The genetic loci consist of a small multigene family of heat shock induced proteins and a set of heat shock related proteins that are expressed at normal temperatures but are not induced under heat shock (Ingolia and Craig, 1982; Craig et al., 1983). The heat shock response in S. crassipalpis was examined by in vitro pulse labelling of proteins in specific tissues and at different developmental stages. The data demonstrate that Sarcophugn heat shock protein expression differs from that reported in Drosophila, particularly in the control of the heat
induced 70 kDa related protein expression. Two heat shock proteins expressed in Sarcophaga tissues, proteins 65 and 72, are immunologically related to the Drosophila heat shock 70 family of proteins, and we demonstrate that these two proteins display developmental and tissue specific switches of expression during heat shock. MATERIALS
AND METHODS
Colony maintenance
Colonies of S. crassipalpis were maintained in standard laboratory nondiapause inducing conditions of 15 h light-9 h dark at 25°C (Denlinger, 1972). Liver was supplied for larviposition on day 10 after eclosion. Eighty to 100 larvae were raised on liver in 2 x 4 in folded aluminium foil boats. Larvae and pupae were reared under the above conditions. Organ culture conditions
Male flies were used in all adult tissue studies. Tissues from red eye pharate adults and wandering larvae were used from unsexed cohorts. Wandering larvae were selected after the gut contents were purged and pharate adults were selected at the beginning of the red eye stage (around 6 days after pupariation at 25°C). The in vitro cultures were prepared by dissecting the tissue in saline (Ephrussi and Beadle, 1936). Imaginal discs, fat body and retinal tissue were removed from the excised organs, excess saline was 239
240
KARL H. JOPLINand DAVIDL. DENLINGER
removed and the tissues were incubated in 30 ~1 Grace’s medium lacking methionine (Grace, 1962). The brain cultures were from five individuals. Terminalia were prepared by cutting off the ninth tergum from five adult males and separating the post-abdominal segments and the aedeagus from the abdominal segments at the fifth tergum of the fly (Seguy, 1951). This structure appears to consist primarily of muscles used to move the genitalia. Integument cultures consisted of the abdominal integument of one adult from which the internal organs and tissues were removed, leaving the cuticle. epidermis and intersegmental muscles. Flight-muscle cultures were prepared from the thorax of one adult fly. Microfuge tubes containing each tissue preparation were incubated at either 25.40 or 43’C for 1 h before the ‘?3-methionine was added. Each culture received I pl (IO /(C/p I) Tran3S-label (Tran35S-labelT M, 1259 Ciimmol, ICN Radiochemicals) and incubation was continued for an additional hour at the same temperature. Protein electrophoresis and detection Cultures were washed twice with 1 ml saline, suspended in 20-30~1 sample buffer (0.06% Tris, 10% glycerol, 2% SDS, 10% /?-mercaptoethanol, bromophenol blue). placed in a boiling water bath for 5 min. centrifuged and frozen at - 70°C until used. The amount of incorporation of “S was determined by trichloroacetic acid precipitation (Mans and Novelli. 1961). Polyacrylamide gel electrophoresis (PAGE) was performed using equal counts from each culture and run on a 0.75 mm thick. 10% polyacrylamide discontinuous gel (Laemmli, 1970) with a 4% stacking gel. Gels were run at 20mAjgel. stained with Coomassie blue, de-stained with acetic methanol, saturated with I M sodium salicylate (Chamberlain, 1979) for 30 min, dried on Whatman filter paper and exposed to Kodak X-Omat X-ray film at -70’C.
Tissues
were
isolated and labelled as described sections. After labelling at the test temperature for I h the culture medium was removed and the tissues washed in saline. Tissues were suspended in 100 ~1 RIPA buffer (IO mM Tris-HCI pH 8.5. 0. I5 M NaCI, 5 mM EDTA. I % Triton X- 100, I % sodium deoxycholate, 0.1% SDS; Simmen er (II., 1984) in a microfuge tube and homogenized with a plastic disposable pestle. The suspension was microfuged for 2min and the supernatant stored at -70 c. lmmunoprecipitation was carried out with either a primary rabbit polyclonal antibody which recognizes the heat shock cognate 70 protein of Drosophila (Dm hsc 70) or a rabbit polyclonal antibody which recognizes the heat shock protein 83 of Drosophila, both provided by Dr Robert Tanguay, Centre Hospitalier Universite Laval, Quebec. Canada. A IO ~1 aliquot of the protein extract was incubated with 5 ~1 of the primary antibody and incubated at room temperature for 1 h. At the end of the incubation the volume was increased to 100 ~1 with RIPA and 10 mg of a 100 mgiml slurry of Sepharose protein A (Sigma) in the previous
was added. The suspension was incubated for 1 h at 4°C. After this second incubation, 1 ml RIPA with 0.1% SDS was added and the precipitate was pelleted in a microfuge for 2 min. The supernatant was aspirated and the pellet was carefully washed 3 times with RIPA + 0.1% SDS. The pellet was suspended with SDS-PAGE sample buffer, boiled for 5 min and electrophoresed on a 10% polyacrylamide gel. The gel was then prepared for autoradiography as previously described.
RESULTS In
vitro heat shock proteins
The expression of heat shock proteins was examined in tissues heat shocked and labelled in vitro. The in oivo heat shock response in pharate adults of S. crassipalpis was elicited between 40 and 43°C (data not shown), and this is the same temperature range that elicited heat shock proteins in tissues treated and labelled in vitro. Protein expression from tissues of 3-day-old adult males labelled at 43°C for 1 h is shown in Fig. I. The integument and brain expressed heat shock proteins at 92, 72, 30, 26, 25 and 25 kDa (lanes A and F). Unexpectedly, heat shock proteins labelled in terminalia and flight muscle were different from the heat shock proteins expressed in the integument and brain. Of special interest is heat shock protein 72 which is expressed in the integument and brain at 43 C but appears to be missing in the terminalia and flight muscle, where a 65 kDa protein is expressed instead. Thus, flight muscle and terminalia express a 65 kDa protein while the brain and integument express a 72 kDa protein. Faint proteins of MW 65 are also expressed in fat body and testis (data not shown). Identl$cation of’ heat shock proteins by immunoprecipitation The identity of both heat shock proteins 65 and 72 was established by immunoprecipitation of proteins from in vitro labelled heat shock induced tissue by a polyclonal antibody raised against Drosophila heat shock 70 cognate proteins which reacts with heat shock 70 proteins from a variety of sources (Dr Tanguay, personal communication). The heavily induced heat shock 65 protein seen in the 3-day male terminalia, wandering larvae brain and integument treated at 43’C was the major band precipitated by the Dmhsc 70 antibody (Fig. 2, lanes F, J, K). The 25~C control yielded no precipitated bands and no proteins were precipitated when the primary antibody was not added (lanes H and E). The heat shock protein 72 band from adult heat shocked brain was also immunoprecipitated with the same antibody (lane B), thus demonstrating that both heat shock proteins 65 and 72 of S. crassipafpis are immunologically related to the Drosophila heat shock protein 70 family. We have repeated these experiments in S. bullata and demonstrated the presence of both heat shock proteins 65 and 72 in this related species (data not shown). Heat shock protein 92 from S. crassipalpis is also immunologically related to the Drosophila heat shock protein 83 (lane C). The
72 65
Fig. 1. Heat shock proteins of in aitro labelled tissues of 3-day-old male S. crassipalpis. Tissues were dissected, placed at the test temperature for 1 h before addition of 10 pCi/pl Trans3WabelrM for an additional hour. Integument and flight-muscle samples were from single individuals; brains and terminalia proteins were pooled from 5 individuals. (A) Integument, 43°C; (B) Flight muscle. 43°C; (C) Terminalia. 25, C; (D) Terminalia, 43°C; (E) Brain, 25°C; (F) Brain, 43°C. Indicated molecular weights of the 3 heat shock bands discussed in the text.
241
A
B
C
D
E
F
H
I
J
K
-
92
-
65
72
Fig. 2. Immunoprecipitation of S. crussipalpis heat shock proteins. Tissues were heat shocked and labeiied as in Fig. I. Immunoprecipitation described in text. Lanes are from multiple gels and lane I is a shorter exposure of the same gel. (A) 3-day adult male total labelled protein, 43°C; (B) Proteins from A immunoprecipitated with Drosophila hsc 70 antibody: (C) Proteins from A immunoprecipitated with Drosopl~ilu heat shock protein 83 antibody: (D) 3-day-old adult male terminalia total protein, 43°C; (E) Proteins from D with no primary antibody added; (F) Proteins from D with Drosophila hsc 70 antibody; (G) Terminalia labelled at 25°C; (H) Proteins from G immunoprecipitated with Drosophila hsc 70 antibody; (I) Total proteins from wandering third-instar larval brains labelled at 43°C; (J) Proteins from I immunoprecipitated with Drosophila hsc 70 antibody; (K) Proteins from wandering third-instar larval integument labelled at 43’C and immunoprecipitated with Drosophila hsc 70 antibody.
242
ABCDEFGH
I
72 65
Fig. 3. Prcsteins from in vitro heat shocked brains at different stages of development in S. crusfsipalpiis. Five brains per treatment were labelled as in Fig. I from stages (A) third-instar wandering larva e; 09 on the day oi- Pulpariation; (C) 1, (D) 2, (E) 3, and (F) 4 days after pupariation; (G) red eye phara lte adt tit; (H) day- I and (I) day-2 post eclosion adults. Numbers indicate molecular weights of the 3 heat bands discussed in the text.
243
A
0
C
D
F
F
-
72
Fig. 4. Proteins from S. crassipalpis integument heat shocked in Ntro at different developmental stages Integument treated and labelled as in Fig. 1 from (A) third-instar wandering larvae: (B) red eye pharate adult: (C) day-l, (D) -2. (E) -4. (F) -8. and (G) -10 post eclosion adult males. Numbers indicate molecular weights of the 3 heat shock bands discussed in the text.
244
A
B
C
D
E
F
G
-
72
-
65
Fig. 5. Proteins from male terminalia of S. crassipnlpis labelled in vitro on different days after adult eclosion. Five terminalia per treatment were labelled as in Fig. 1 (A, B) day 1, 25 and 43°C; (C-E) day 2, 25, 40 and 43°C; (F, G) day 5, 25 and 43°C. Numbers indicate molecular weights of the 3 heat shock bands discussed in the text.
245
Heat shock proteins in Surcophuga
identity of the smaller protein bands immunoprecipitated by Dmhsc 70 antibody in the integument of third stage wandering larvae (lane K) is unknown. Developmental expression of heat shock proteins
Heat shock protein expression was examined in the brain during a range of developmental stages from wandering phase of third-instar larvae to lo-day-old adults, Fig. 3. Brains from the wandering larvae expressed heat shock protein 65 in response to heat shock but a developmental switch occurs on the day of pupariation, and thereafter only heat shock protein 72 was expressed. Heat shock protein expression of integument at different stages of development exhibited a similar pattern to that Seen in the brain (Fig. 4): a switch from heat shock protein 65 to heat shock protein 72 occurred between the wandering larval stage and the red eye pharate adult. Terminalia from cohorts of adult male flies labelled at various times after eclosion also show a switch in heat shock protein expression during adult development, Fig. 5. Terminalia from l-day-old males expressed heat shock protein 72. Tenninalia from 2-day-old males incubated at 40°C also expressed heat shock protein 72 but at 43°C the expression switched to heat shock protein 65. Terminalia from subsequent days expressed primarily heat shock protein 65 at either 40 or 43°C. The switch from heat shock protein 72 to heat shock protein 65 is a tissue specific developmental switch, since the brain and integument continued to express heat shock protein 72 during this period (Figs 3 and 4) and this 72-65 switch is the reverse of the 65-72 developmental switch seen in brains and integument on the day of pupariation. Neither the brain nor the integument exhibited a switch when exposed to either of these two temperatures during any day of adult development (data not shown). DISCUSSION
Many aspects of heat shock in Sarcophaga are similar to the heat shock responses seen in other organisms; a limited set of new proteins are induced at elevated temperatures with the concomitant repression of proteins expressed in the normal temperature range. The temperature range for heat shock induction, 4043.C. is higher than the 3537°C range in Drosophila, but is similar to the heat shock temperatures in other insects such as Manduca sexta, 42°C (Fittinghoff and Riddiford, 1988), Locusta migratoria, 45,-C (Walker et al., 1986) and Papilio glaucus 40-42’ C (Joplin, personal observation). One difference in the heat shock response between Sarcophaga and Drosophila is that in Sarcophaga many proteins continue to be expressed in tissues labelled in vitro during heat shock. Repression of normal proteins is a common aspect of the heat shock response, but translational control appears to be more complete in cell culture (Lindquist, 1980b, 198I, 1986). Drosophiia tissue labelled in vitro express a more complex set of proteins during heat shock than do cells in culture (Tissieres et al., 1974; Lewis et al., 1975; Ish-Horowitch et al., 1979). This lessthan-complete control of protein expression during heat shock has been reported in other insects such as
Manduca
241
sexta where the integument continues to express epidermal proteins (Fittinghoff and Riddiford, 1988) and in Locusta migratoria where vitellogenin synthesis persists at heat shock temperatures (Walker et al., 1986). The number of proteins expressed during heat shock is unresolved (Buzin and Petersen, 1982) although the most studied heat shock proteins are considered to belong to three separate gene families. Heat shock 70 protein is the most conserved of the heat shock proteins and in Drosophila consists of 5 stress-activated genes (Craig et al., 1979; Livak et al., 1978; Spradling et al., 1975) and related proteins expressed at normal temperature but not during heat shock (Ingolia and Craig, 1982; Craig et al., 1983). These cognate genes bring the number of heat shock 70 protein loci to 9 (Wermer-Washburn et al., 1987). Expression of Drosophila heat shock proteins have different optimal temperatures, suggesting that independent regulation occurs (Lindquist, 1980a). The relationship of expression for this family of proteins has not been worked out in Drosophila, but a family of heat shock 70 related proteins in yeast displays a complex interaction of expression between the various proteins (Craig et al., 1987; Wermer-Washburn et al., 1987). The interactions in yeast suggest that different proteins are functionally similar but are expressed in response to different conditions. In our examination of proteins from different tissues of Sarcophaga after heat shock treatment two apparently different forms of the heat shock 70 type proteins, heat shock proteins 65 and 72, were observed, and these two heat shock proteins were differentially expressed in adult tissues and during development. The Drosophila heat shock 70 proteins have not been reported to exhibit tissue or developmentally specific expression during heat shock. In contrast, our results in Sarcophaga show that the two heat shock 70 related proteins exhibit a complex series of switches in expression. There is a developmental switch in both brain and integument from heat shock protein 65 to heat shock 72 at the time of pupariation. This switch occurs both in tissues that retain their integrity during metamorphosis (brain) as well as in tissues that undergo histolysis and regeneration (integument). In addition to the developmental switches occurring at pupariation another type of switch in expression is exhibited in the adult terminalia. This switch is opposite to that observed in the brain and integument at pupariation: the terminalia expresses heat shock protein 72 on day 1 but switches to heat shock protein 65 on day 2 after eclosion. In contrast, the brain and integument do not switch during adult life but consistently express only heat shock protein 72. A further control on heat shock protein expression identified in Sarcophaga is the temperature dependent switch that transiently operates only on day-2 terminalia. At low heat shock temperature (40°C) heat shock protein 72 is expressed while at high heat shock temperature (43°C) heat shock protein 65 is primarily expressed. This differential temperature control is restricted to day-2 terminalia and is not observed in older or younger terminalia nor in brain or integument (data not shown). Studies in
248
KARL H. JOPLIN and DAVID L. DENLINGER
Drosophila
have shown that expression of different heat shock proteins have different optimal temperatures, but switches of expression have not been reported (Lindquist, 1980a). Our study demonstrates that heat shock protein 65 and 72 are both immunologically related to the Drosophila heat shock protein 70 family. We have also identified heat shock protein 92 as related to Drosophila heat shock protein 83. Heat shock protein 65 was previously reported in another flesh fly, S. buffata (Bultmann, 1986). We have confirmed the presence of heat shock protein 65 and report that S. bullata also expresses heat shock protein 72 in other developmental stages and tissues. Thus, at least two members of the Sarcophaga clade express both forms of the heat shock 70 related protein. This complexity in control of heat shock protein expression may also occur in Drosophila, though it has not been detected or recognized in previous studies. If the proteins seen in Sarcophaga were more similar in size the observed controls on expression would not have been detected. Such complexity has possibly been overlooked in Drosophila because the protein expression reported in the early heat shock protein literature was from tissue of a single stage and a comprehensive developmental study on multiple tissues was not reported (Tissieres et al., 1974; Lewis et al., 1975). Most recent work on heat shock proteins has focused on established cultured cells and this approach would preclude the discovery of such switches in the control of protein expression. Although our study does not resolve the function of the apparent tissue and developmental complexity of differential heat shock protein expression. it is possible that the organism uses these controls to ensure that some form of heat shock protein 70 is expressed in all tissues during heat shock. A similar scenario has been suggested for the developmental control of the alcohol dehydrogenase gene in Drosophila (Savakis et al., 1986). Alternatively, each member of the heat shock protein 70 family may have different functions as suggested by work on the yeast heat shock protein 70 family (Werner-Washburne et al., 1987). Characterization of constitutively expressed heat shock protein mutations in Drosophila revealed that all the mutants had different tissue and developmental patterns of heat shock protein expression (Parker-Thornburg and Bonner. 1987). This tissue distribution may be due to heat shock protein induction caused by cell-specific metabolic mutations as seen during aberrant actin production (Hiromi et a/., 1986). It is possible, however, that some of the mutants were detecting cell and developmental specific control products or promotor sites affecting heat shock expression. We are addressing these questions in Sarcophaga by isolating genomic clones corresponding to both heat shock protein 65 and 72. Such clones will enable us to examine the transcriptional capability of different mRNA and then identify genomic sequences for promotors of heat shock protein expression. This would make Sarcophaga a valuable model system for probing the complexity of heat shock protein regulation. Acknowledgements-This study was supported by USDACRGO grant No. 88-37153-3473. We thank Dr Robert
Tanguay for generously supplying the Drosophila heat shock antibodies.
REFERENCES Ashburner M. and Bonner J. J. (1979) The induction ofgene activity in Drosophila by heat shock. Cell 17, 241-254. Bultmann H. (1986) Heat shock responses in polytene footpad cells of Sareophaga bullara. Chromosoma 93, 347-357. Buzin C. H. and Petersen N. S. (1982) A comparison of the multiple Drosophila heat shock proteins in cell lines and larval salivary glands by two-dimensional gel electrophoresis. J. Mol. Biol. 158, 181-201. Chamberlain J. P. (1979) Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Analyf. Biochem. 98, 132-135. Craig E. A. (1985) The heat shock response. CRC Crit. Rev. Biochem. 18, 239-280. Craig E. A., Ingolia T. D. and Manseau L. J. (1983) Expression of Drosophila heat-shock cognate genes during heat shock and development. Deo. Biol. 99,418426. Craig E. A.. Kramer J. and Kosic-Smithers J. (1987) SSCl. a member of the 70-kDa heat shock protein multigene family of Saccharomyces cerevisiae. is essential for growih. Proc. natn. Acad. Sci. U.S.A. 84, 41564160. Craig E. A.. McCarthv B. J. and Wadsworth S. C. (1979) Sequence organization of two recombinant plasmids con: taining genes for the major heat shock induced protien of Drosophila melanogaster. Cell 16, 575-589. Denlinger D. L. (1972) Induction and termination of pupal diapause in Sarcophuga (Diptera: Sarcophagidae). Biol. Bull. 142, I I-24. Ephrussi B. and Beadle G. W. (1936) A technique of transplantation for Drosophila. Am. Nat. 70, 218-225. Fittinghoff G. M. and Riddiford L. M. (1988) Effect of heat shock on nonheat shock proteins of the insect integument (abstract). J. Cell Biochem. 12D, 268. Grace T. D. C. (1962) Establishment of four strains of cells from insect tissues grown in vitro. Nature 195, 788-789. Hiromi Y., Okamoto H., Gehring W. J. and Hotta Y. (1986) Germ line transformation with Drosophila mutant actin genes induces constitutive expression of heat shock genes. Cell 44, 293-301. lngolia T. D. and Craig E. A. (1982) Drosophila gene related to the major heat shock-induced gene is transcribed at normal temperatures and not induced by heat shock. Proc. natn. Acad. Sri. U.S.A. 79, 525-529. Ish-Horowitz D., Pinchin S. M., Gausz J.. Gyurkovics H., Bencze G., Goldschmidt-Clermont M. and Holden J. J. (1979) Deletion mapping of two D. melanogaster loci that code for the 70.000 dalton heat-induced protein. Cell 17, 565-571. Kelley P. M. and Schlesinger M. J. (1978) The effects of amino acid analogs and heat shock on gene expression in chicken embryo fibroblasts. Cell 15, 1277-1286. Laemmli I-J. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lewis M., Helmsing P. J. and Ashburner M. (1975) Parallel changes in puffing activity and patterns of protein synthesis in salivary glands of Drosophila. Proc. natn. Acad. Sri. U.S.A. 72, 36043608. Lindquist S. (1980a) Varying patterns of protein synthesis in Drosophila during heat shock: implications for regulation. Dev. Biol. 77, 463479. Lindquist S. (1980b) Translational efficiency of heatinduced messages in Drosophila melanogaster cells. J. Mol. Biol. 137, 151-158. Lindquist S. (1981) Regulation of protein synthesis during heat shock. Nature 293, 31 I-314. Lindquist S. (1986) The heat-shock response. Ann. Rev. Biochem. 55, 1151-1191.
Heat shock proteins in Sarcophuga Livak K. F.. Freund R., Schweber M., Wensink P. L. and Meselson M. (1978) Sequence organization of transcription of two heat shock loci in Drosophila. Proc. natn. Acad. Sci. U.S.A. 15, 5613-5617.
McAlister L., Strauskey S., Kulaga A. and Finkelstein D. B. (1979) Altered patterns of protein synthesis induced by heat shock of yeast. Curr. Genet. 1, 63-74. Mans R. J. and Novelli G. D. (1961) Measurement of the incorporation of radioactive amino acids into protein by a filter-paper disk method, Archs Biochem. Biophys. 94, 48-53.
Moran L. A., Chauvin M., Kennedy M. E., Korri M., Lowe D. G., Nicholson R. C. and Perry M. D. (1983) The major heat-shock protein (hsp70) gene family: related sequences in mouse, Drosophila and yeast. Can. J. Biochem. Cell Biol. 61, 488499.
Parker-Thornburg J. and Bonner J. J. (1987) Mutations that induce the heat shock response of Drosophila. Cell 51, 763-772.
Savakis C., Ashburner M. and Willis J. H. (1986) The expression of the gene coding for alcohol dehydrogenase during the development of Drosophila melanogaster. Den. Biol. 114, 194-207. Schlesinger M. J., Ashburner M. and Tissieres A. (eds)
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(1982) Heat Shock: From Bacteria to Man. Cold Spring Harbor Laboratory, Cold Spring Harbor. Seguy E . (1951) Ordre des Dipteres. Traite de Zoologie 10, 499-744. Simmen F. A., Schultz T. Z., Headon D. R., Wright D. A., Carnenter G. and O’Mallev B. W. (1984) Translation in Xenapus oocytes of messenger RNA from A431 cells for human epidermal growth factor receptor proteins. DNA 3, 393-399.
Spradling A.. Penman S. and Pardue M. L. (1975) Analysis of Drosophila mRNA by in situ hybridization: sequences transcribed in normal and heat shocked cultured cells. Cell 4, 395404.
Tissieres A., Mitchell H. K. and Tracy U. M. (1974) Protein synthesis in salivary glands of Drosophila melanogaster: relation to chromosome puffs. J. Mol. Biol. 84, 389-398.
Walker V. K., Whyard S., Wyatt G. R. and Kanest M. R. (1986) Heat shock and gene transfer in locusts (abstract) J. Cell. Biochem. lOC, 56.
Werner-Washburn M., Stone D. E. and Craig E. A. (1987) Complex interations among members of an essential subfamily of hsp70 genes in Saccharomyes cereeesiae. Mol. Cell. Biol. 7, 2568-2577.
1990 Copyright
0022-1910/90 $3.00 + 0.00 Q 1990 Pergamon Press plc
DEVELOPMENTAL AND TISSUE SPECIFIC CONTROL OF THE HEAT SHOCK INDUCED 70kDa RELATED PROTEINS IN THE FLESH FLY, SARCOPHAGA CRASSIPALPIS KARL H. JOPLINand DAVIDL. DENLINGER Department of Entomology, The Ohio State University, 1735 Neil Avenue, Columbus, OH 43210-1220,U.S.A. (Received 11 September 1989; revised 22 Noclember1989)
shock (4G43”C) induces two proteins in Sarcophaga crassipalpis that are immunologically related to the heat shock 70 protein family in Drosophila. These Sarcophaga heat shock proteins, M, of 65 and 72 kDa, show developmental and tissue-specificexpression. Heat shock protein 65 is expressed in the brain and integument of third-instar larvae at 43°C. Expression of heat shock protein 65 in both tissues ceases at pupariation and heat shock protein 72 is the protein induced at 43°C throughout the rest of Abstract-Heat
development. Tissue specificity for the expression of these two heat shock proteins can be observed in 3-day-old adult males: brain and integument express heat shock protein 72 while the terminalia and flight muscle express heat shock protein 65. Both a developmental and temperature switch can be seen in the male terminalia: day-l terminalia express heat shock protein 72 at either 40 or 43°C on day-2 heat shock protein 72 is expressed at 40°C but heat shock protein 65 is expressed at 43°C thereafter the terminalia express primarily heat shock protein 65 at either heat shock temperature. Control of heat shock protein expression in S. crassipalpis is thus considerably more complex than the Drosophila literature suggests. Key Word Index:
Sarcophaga; heat shock protein; developmental
protein expression; tissue-specific
protein expression
INTRODUCTION High temperatures and a number of other forms of biological stress elicit the rapid synthesis of a set of heat shock proteins and a concurrent suppression of proteins normally expressed during nonstressed conditions (Ashburner and Bonner, 1979; Schlesinger et al., 1982; Craig, 1985; Lindquist, 1986). This limited set of heat shock proteins is thought to be expressed, with minor exceptions, in all tissues and all developmental stages of a stressed organism. Our study examines this concept in the flesh fly, Surcophuga crassipalpis.
A heat shock response has been observed in all organisms studied (Kelley and Schlesinger, 1978; McAlister et al., 1979; Schlesinger et al., 1982) and appears to be a conserved physiological response to stress. The heat shock 70 protein family is the most highly conserved of the heat shock proteins and is induced to the highest levels during heat shock (Moran et nl., 1983). The genetic loci consist of a small multigene family of heat shock induced proteins and a set of heat shock related proteins that are expressed at normal temperatures but are not induced under heat shock (Ingolia and Craig, 1982; Craig et al., 1983). The heat shock response in S. crassipalpis was examined by in vitro pulse labelling of proteins in specific tissues and at different developmental stages. The data demonstrate that Sarcophugn heat shock protein expression differs from that reported in Drosophila, particularly in the control of the heat
induced 70 kDa related protein expression. Two heat shock proteins expressed in Sarcophaga tissues, proteins 65 and 72, are immunologically related to the Drosophila heat shock 70 family of proteins, and we demonstrate that these two proteins display developmental and tissue specific switches of expression during heat shock. MATERIALS
AND METHODS
Colony maintenance
Colonies of S. crassipalpis were maintained in standard laboratory nondiapause inducing conditions of 15 h light-9 h dark at 25°C (Denlinger, 1972). Liver was supplied for larviposition on day 10 after eclosion. Eighty to 100 larvae were raised on liver in 2 x 4 in folded aluminium foil boats. Larvae and pupae were reared under the above conditions. Organ culture conditions
Male flies were used in all adult tissue studies. Tissues from red eye pharate adults and wandering larvae were used from unsexed cohorts. Wandering larvae were selected after the gut contents were purged and pharate adults were selected at the beginning of the red eye stage (around 6 days after pupariation at 25°C). The in vitro cultures were prepared by dissecting the tissue in saline (Ephrussi and Beadle, 1936). Imaginal discs, fat body and retinal tissue were removed from the excised organs, excess saline was 239
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KARL H. JOPLINand DAVIDL. DENLINGER
removed and the tissues were incubated in 30 ~1 Grace’s medium lacking methionine (Grace, 1962). The brain cultures were from five individuals. Terminalia were prepared by cutting off the ninth tergum from five adult males and separating the post-abdominal segments and the aedeagus from the abdominal segments at the fifth tergum of the fly (Seguy, 1951). This structure appears to consist primarily of muscles used to move the genitalia. Integument cultures consisted of the abdominal integument of one adult from which the internal organs and tissues were removed, leaving the cuticle. epidermis and intersegmental muscles. Flight-muscle cultures were prepared from the thorax of one adult fly. Microfuge tubes containing each tissue preparation were incubated at either 25.40 or 43’C for 1 h before the ‘?3-methionine was added. Each culture received I pl (IO /(C/p I) Tran3S-label (Tran35S-labelT M, 1259 Ciimmol, ICN Radiochemicals) and incubation was continued for an additional hour at the same temperature. Protein electrophoresis and detection Cultures were washed twice with 1 ml saline, suspended in 20-30~1 sample buffer (0.06% Tris, 10% glycerol, 2% SDS, 10% /?-mercaptoethanol, bromophenol blue). placed in a boiling water bath for 5 min. centrifuged and frozen at - 70°C until used. The amount of incorporation of “S was determined by trichloroacetic acid precipitation (Mans and Novelli. 1961). Polyacrylamide gel electrophoresis (PAGE) was performed using equal counts from each culture and run on a 0.75 mm thick. 10% polyacrylamide discontinuous gel (Laemmli, 1970) with a 4% stacking gel. Gels were run at 20mAjgel. stained with Coomassie blue, de-stained with acetic methanol, saturated with I M sodium salicylate (Chamberlain, 1979) for 30 min, dried on Whatman filter paper and exposed to Kodak X-Omat X-ray film at -70’C.
Tissues
were
isolated and labelled as described sections. After labelling at the test temperature for I h the culture medium was removed and the tissues washed in saline. Tissues were suspended in 100 ~1 RIPA buffer (IO mM Tris-HCI pH 8.5. 0. I5 M NaCI, 5 mM EDTA. I % Triton X- 100, I % sodium deoxycholate, 0.1% SDS; Simmen er (II., 1984) in a microfuge tube and homogenized with a plastic disposable pestle. The suspension was microfuged for 2min and the supernatant stored at -70 c. lmmunoprecipitation was carried out with either a primary rabbit polyclonal antibody which recognizes the heat shock cognate 70 protein of Drosophila (Dm hsc 70) or a rabbit polyclonal antibody which recognizes the heat shock protein 83 of Drosophila, both provided by Dr Robert Tanguay, Centre Hospitalier Universite Laval, Quebec. Canada. A IO ~1 aliquot of the protein extract was incubated with 5 ~1 of the primary antibody and incubated at room temperature for 1 h. At the end of the incubation the volume was increased to 100 ~1 with RIPA and 10 mg of a 100 mgiml slurry of Sepharose protein A (Sigma) in the previous
was added. The suspension was incubated for 1 h at 4°C. After this second incubation, 1 ml RIPA with 0.1% SDS was added and the precipitate was pelleted in a microfuge for 2 min. The supernatant was aspirated and the pellet was carefully washed 3 times with RIPA + 0.1% SDS. The pellet was suspended with SDS-PAGE sample buffer, boiled for 5 min and electrophoresed on a 10% polyacrylamide gel. The gel was then prepared for autoradiography as previously described.
RESULTS In
vitro heat shock proteins
The expression of heat shock proteins was examined in tissues heat shocked and labelled in vitro. The in oivo heat shock response in pharate adults of S. crassipalpis was elicited between 40 and 43°C (data not shown), and this is the same temperature range that elicited heat shock proteins in tissues treated and labelled in vitro. Protein expression from tissues of 3-day-old adult males labelled at 43°C for 1 h is shown in Fig. I. The integument and brain expressed heat shock proteins at 92, 72, 30, 26, 25 and 25 kDa (lanes A and F). Unexpectedly, heat shock proteins labelled in terminalia and flight muscle were different from the heat shock proteins expressed in the integument and brain. Of special interest is heat shock protein 72 which is expressed in the integument and brain at 43 C but appears to be missing in the terminalia and flight muscle, where a 65 kDa protein is expressed instead. Thus, flight muscle and terminalia express a 65 kDa protein while the brain and integument express a 72 kDa protein. Faint proteins of MW 65 are also expressed in fat body and testis (data not shown). Identl$cation of’ heat shock proteins by immunoprecipitation The identity of both heat shock proteins 65 and 72 was established by immunoprecipitation of proteins from in vitro labelled heat shock induced tissue by a polyclonal antibody raised against Drosophila heat shock 70 cognate proteins which reacts with heat shock 70 proteins from a variety of sources (Dr Tanguay, personal communication). The heavily induced heat shock 65 protein seen in the 3-day male terminalia, wandering larvae brain and integument treated at 43’C was the major band precipitated by the Dmhsc 70 antibody (Fig. 2, lanes F, J, K). The 25~C control yielded no precipitated bands and no proteins were precipitated when the primary antibody was not added (lanes H and E). The heat shock protein 72 band from adult heat shocked brain was also immunoprecipitated with the same antibody (lane B), thus demonstrating that both heat shock proteins 65 and 72 of S. crassipafpis are immunologically related to the Drosophila heat shock protein 70 family. We have repeated these experiments in S. bullata and demonstrated the presence of both heat shock proteins 65 and 72 in this related species (data not shown). Heat shock protein 92 from S. crassipalpis is also immunologically related to the Drosophila heat shock protein 83 (lane C). The
72 65
Fig. 1. Heat shock proteins of in aitro labelled tissues of 3-day-old male S. crassipalpis. Tissues were dissected, placed at the test temperature for 1 h before addition of 10 pCi/pl Trans3WabelrM for an additional hour. Integument and flight-muscle samples were from single individuals; brains and terminalia proteins were pooled from 5 individuals. (A) Integument, 43°C; (B) Flight muscle. 43°C; (C) Terminalia. 25, C; (D) Terminalia, 43°C; (E) Brain, 25°C; (F) Brain, 43°C. Indicated molecular weights of the 3 heat shock bands discussed in the text.
241
A
B
C
D
E
F
H
I
J
K
-
92
-
65
72
Fig. 2. Immunoprecipitation of S. crussipalpis heat shock proteins. Tissues were heat shocked and labeiied as in Fig. I. Immunoprecipitation described in text. Lanes are from multiple gels and lane I is a shorter exposure of the same gel. (A) 3-day adult male total labelled protein, 43°C; (B) Proteins from A immunoprecipitated with Drosophila hsc 70 antibody: (C) Proteins from A immunoprecipitated with Drosopl~ilu heat shock protein 83 antibody: (D) 3-day-old adult male terminalia total protein, 43°C; (E) Proteins from D with no primary antibody added; (F) Proteins from D with Drosophila hsc 70 antibody; (G) Terminalia labelled at 25°C; (H) Proteins from G immunoprecipitated with Drosophila hsc 70 antibody; (I) Total proteins from wandering third-instar larval brains labelled at 43°C; (J) Proteins from I immunoprecipitated with Drosophila hsc 70 antibody; (K) Proteins from wandering third-instar larval integument labelled at 43’C and immunoprecipitated with Drosophila hsc 70 antibody.
242
ABCDEFGH
I
72 65
Fig. 3. Prcsteins from in vitro heat shocked brains at different stages of development in S. crusfsipalpiis. Five brains per treatment were labelled as in Fig. I from stages (A) third-instar wandering larva e; 09 on the day oi- Pulpariation; (C) 1, (D) 2, (E) 3, and (F) 4 days after pupariation; (G) red eye phara lte adt tit; (H) day- I and (I) day-2 post eclosion adults. Numbers indicate molecular weights of the 3 heat bands discussed in the text.
243
A
0
C
D
F
F
-
72
Fig. 4. Proteins from S. crassipalpis integument heat shocked in Ntro at different developmental stages Integument treated and labelled as in Fig. 1 from (A) third-instar wandering larvae: (B) red eye pharate adult: (C) day-l, (D) -2. (E) -4. (F) -8. and (G) -10 post eclosion adult males. Numbers indicate molecular weights of the 3 heat shock bands discussed in the text.
244
A
B
C
D
E
F
G
-
72
-
65
Fig. 5. Proteins from male terminalia of S. crassipnlpis labelled in vitro on different days after adult eclosion. Five terminalia per treatment were labelled as in Fig. 1 (A, B) day 1, 25 and 43°C; (C-E) day 2, 25, 40 and 43°C; (F, G) day 5, 25 and 43°C. Numbers indicate molecular weights of the 3 heat shock bands discussed in the text.
245
Heat shock proteins in Surcophuga
identity of the smaller protein bands immunoprecipitated by Dmhsc 70 antibody in the integument of third stage wandering larvae (lane K) is unknown. Developmental expression of heat shock proteins
Heat shock protein expression was examined in the brain during a range of developmental stages from wandering phase of third-instar larvae to lo-day-old adults, Fig. 3. Brains from the wandering larvae expressed heat shock protein 65 in response to heat shock but a developmental switch occurs on the day of pupariation, and thereafter only heat shock protein 72 was expressed. Heat shock protein expression of integument at different stages of development exhibited a similar pattern to that Seen in the brain (Fig. 4): a switch from heat shock protein 65 to heat shock protein 72 occurred between the wandering larval stage and the red eye pharate adult. Terminalia from cohorts of adult male flies labelled at various times after eclosion also show a switch in heat shock protein expression during adult development, Fig. 5. Terminalia from l-day-old males expressed heat shock protein 72. Tenninalia from 2-day-old males incubated at 40°C also expressed heat shock protein 72 but at 43°C the expression switched to heat shock protein 65. Terminalia from subsequent days expressed primarily heat shock protein 65 at either 40 or 43°C. The switch from heat shock protein 72 to heat shock protein 65 is a tissue specific developmental switch, since the brain and integument continued to express heat shock protein 72 during this period (Figs 3 and 4) and this 72-65 switch is the reverse of the 65-72 developmental switch seen in brains and integument on the day of pupariation. Neither the brain nor the integument exhibited a switch when exposed to either of these two temperatures during any day of adult development (data not shown). DISCUSSION
Many aspects of heat shock in Sarcophaga are similar to the heat shock responses seen in other organisms; a limited set of new proteins are induced at elevated temperatures with the concomitant repression of proteins expressed in the normal temperature range. The temperature range for heat shock induction, 4043.C. is higher than the 3537°C range in Drosophila, but is similar to the heat shock temperatures in other insects such as Manduca sexta, 42°C (Fittinghoff and Riddiford, 1988), Locusta migratoria, 45,-C (Walker et al., 1986) and Papilio glaucus 40-42’ C (Joplin, personal observation). One difference in the heat shock response between Sarcophaga and Drosophila is that in Sarcophaga many proteins continue to be expressed in tissues labelled in vitro during heat shock. Repression of normal proteins is a common aspect of the heat shock response, but translational control appears to be more complete in cell culture (Lindquist, 1980b, 198I, 1986). Drosophiia tissue labelled in vitro express a more complex set of proteins during heat shock than do cells in culture (Tissieres et al., 1974; Lewis et al., 1975; Ish-Horowitch et al., 1979). This lessthan-complete control of protein expression during heat shock has been reported in other insects such as
Manduca
241
sexta where the integument continues to express epidermal proteins (Fittinghoff and Riddiford, 1988) and in Locusta migratoria where vitellogenin synthesis persists at heat shock temperatures (Walker et al., 1986). The number of proteins expressed during heat shock is unresolved (Buzin and Petersen, 1982) although the most studied heat shock proteins are considered to belong to three separate gene families. Heat shock 70 protein is the most conserved of the heat shock proteins and in Drosophila consists of 5 stress-activated genes (Craig et al., 1979; Livak et al., 1978; Spradling et al., 1975) and related proteins expressed at normal temperature but not during heat shock (Ingolia and Craig, 1982; Craig et al., 1983). These cognate genes bring the number of heat shock 70 protein loci to 9 (Wermer-Washburn et al., 1987). Expression of Drosophila heat shock proteins have different optimal temperatures, suggesting that independent regulation occurs (Lindquist, 1980a). The relationship of expression for this family of proteins has not been worked out in Drosophila, but a family of heat shock 70 related proteins in yeast displays a complex interaction of expression between the various proteins (Craig et al., 1987; Wermer-Washburn et al., 1987). The interactions in yeast suggest that different proteins are functionally similar but are expressed in response to different conditions. In our examination of proteins from different tissues of Sarcophaga after heat shock treatment two apparently different forms of the heat shock 70 type proteins, heat shock proteins 65 and 72, were observed, and these two heat shock proteins were differentially expressed in adult tissues and during development. The Drosophila heat shock 70 proteins have not been reported to exhibit tissue or developmentally specific expression during heat shock. In contrast, our results in Sarcophaga show that the two heat shock 70 related proteins exhibit a complex series of switches in expression. There is a developmental switch in both brain and integument from heat shock protein 65 to heat shock 72 at the time of pupariation. This switch occurs both in tissues that retain their integrity during metamorphosis (brain) as well as in tissues that undergo histolysis and regeneration (integument). In addition to the developmental switches occurring at pupariation another type of switch in expression is exhibited in the adult terminalia. This switch is opposite to that observed in the brain and integument at pupariation: the terminalia expresses heat shock protein 72 on day 1 but switches to heat shock protein 65 on day 2 after eclosion. In contrast, the brain and integument do not switch during adult life but consistently express only heat shock protein 72. A further control on heat shock protein expression identified in Sarcophaga is the temperature dependent switch that transiently operates only on day-2 terminalia. At low heat shock temperature (40°C) heat shock protein 72 is expressed while at high heat shock temperature (43°C) heat shock protein 65 is primarily expressed. This differential temperature control is restricted to day-2 terminalia and is not observed in older or younger terminalia nor in brain or integument (data not shown). Studies in
248
KARL H. JOPLIN and DAVID L. DENLINGER
Drosophila
have shown that expression of different heat shock proteins have different optimal temperatures, but switches of expression have not been reported (Lindquist, 1980a). Our study demonstrates that heat shock protein 65 and 72 are both immunologically related to the Drosophila heat shock protein 70 family. We have also identified heat shock protein 92 as related to Drosophila heat shock protein 83. Heat shock protein 65 was previously reported in another flesh fly, S. buffata (Bultmann, 1986). We have confirmed the presence of heat shock protein 65 and report that S. bullata also expresses heat shock protein 72 in other developmental stages and tissues. Thus, at least two members of the Sarcophaga clade express both forms of the heat shock 70 related protein. This complexity in control of heat shock protein expression may also occur in Drosophila, though it has not been detected or recognized in previous studies. If the proteins seen in Sarcophaga were more similar in size the observed controls on expression would not have been detected. Such complexity has possibly been overlooked in Drosophila because the protein expression reported in the early heat shock protein literature was from tissue of a single stage and a comprehensive developmental study on multiple tissues was not reported (Tissieres et al., 1974; Lewis et al., 1975). Most recent work on heat shock proteins has focused on established cultured cells and this approach would preclude the discovery of such switches in the control of protein expression. Although our study does not resolve the function of the apparent tissue and developmental complexity of differential heat shock protein expression. it is possible that the organism uses these controls to ensure that some form of heat shock protein 70 is expressed in all tissues during heat shock. A similar scenario has been suggested for the developmental control of the alcohol dehydrogenase gene in Drosophila (Savakis et al., 1986). Alternatively, each member of the heat shock protein 70 family may have different functions as suggested by work on the yeast heat shock protein 70 family (Werner-Washburne et al., 1987). Characterization of constitutively expressed heat shock protein mutations in Drosophila revealed that all the mutants had different tissue and developmental patterns of heat shock protein expression (Parker-Thornburg and Bonner. 1987). This tissue distribution may be due to heat shock protein induction caused by cell-specific metabolic mutations as seen during aberrant actin production (Hiromi et a/., 1986). It is possible, however, that some of the mutants were detecting cell and developmental specific control products or promotor sites affecting heat shock expression. We are addressing these questions in Sarcophaga by isolating genomic clones corresponding to both heat shock protein 65 and 72. Such clones will enable us to examine the transcriptional capability of different mRNA and then identify genomic sequences for promotors of heat shock protein expression. This would make Sarcophaga a valuable model system for probing the complexity of heat shock protein regulation. Acknowledgements-This study was supported by USDACRGO grant No. 88-37153-3473. We thank Dr Robert
Tanguay for generously supplying the Drosophila heat shock antibodies.
REFERENCES Ashburner M. and Bonner J. J. (1979) The induction ofgene activity in Drosophila by heat shock. Cell 17, 241-254. Bultmann H. (1986) Heat shock responses in polytene footpad cells of Sareophaga bullara. Chromosoma 93, 347-357. Buzin C. H. and Petersen N. S. (1982) A comparison of the multiple Drosophila heat shock proteins in cell lines and larval salivary glands by two-dimensional gel electrophoresis. J. Mol. Biol. 158, 181-201. Chamberlain J. P. (1979) Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Analyf. Biochem. 98, 132-135. Craig E. A. (1985) The heat shock response. CRC Crit. Rev. Biochem. 18, 239-280. Craig E. A., Ingolia T. D. and Manseau L. J. (1983) Expression of Drosophila heat-shock cognate genes during heat shock and development. Deo. Biol. 99,418426. Craig E. A.. Kramer J. and Kosic-Smithers J. (1987) SSCl. a member of the 70-kDa heat shock protein multigene family of Saccharomyces cerevisiae. is essential for growih. Proc. natn. Acad. Sci. U.S.A. 84, 41564160. Craig E. A.. McCarthv B. J. and Wadsworth S. C. (1979) Sequence organization of two recombinant plasmids con: taining genes for the major heat shock induced protien of Drosophila melanogaster. Cell 16, 575-589. Denlinger D. L. (1972) Induction and termination of pupal diapause in Sarcophuga (Diptera: Sarcophagidae). Biol. Bull. 142, I I-24. Ephrussi B. and Beadle G. W. (1936) A technique of transplantation for Drosophila. Am. Nat. 70, 218-225. Fittinghoff G. M. and Riddiford L. M. (1988) Effect of heat shock on nonheat shock proteins of the insect integument (abstract). J. Cell Biochem. 12D, 268. Grace T. D. C. (1962) Establishment of four strains of cells from insect tissues grown in vitro. Nature 195, 788-789. Hiromi Y., Okamoto H., Gehring W. J. and Hotta Y. (1986) Germ line transformation with Drosophila mutant actin genes induces constitutive expression of heat shock genes. Cell 44, 293-301. lngolia T. D. and Craig E. A. (1982) Drosophila gene related to the major heat shock-induced gene is transcribed at normal temperatures and not induced by heat shock. Proc. natn. Acad. Sri. U.S.A. 79, 525-529. Ish-Horowitz D., Pinchin S. M., Gausz J.. Gyurkovics H., Bencze G., Goldschmidt-Clermont M. and Holden J. J. (1979) Deletion mapping of two D. melanogaster loci that code for the 70.000 dalton heat-induced protein. Cell 17, 565-571. Kelley P. M. and Schlesinger M. J. (1978) The effects of amino acid analogs and heat shock on gene expression in chicken embryo fibroblasts. Cell 15, 1277-1286. Laemmli I-J. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lewis M., Helmsing P. J. and Ashburner M. (1975) Parallel changes in puffing activity and patterns of protein synthesis in salivary glands of Drosophila. Proc. natn. Acad. Sri. U.S.A. 72, 36043608. Lindquist S. (1980a) Varying patterns of protein synthesis in Drosophila during heat shock: implications for regulation. Dev. Biol. 77, 463479. Lindquist S. (1980b) Translational efficiency of heatinduced messages in Drosophila melanogaster cells. J. Mol. Biol. 137, 151-158. Lindquist S. (1981) Regulation of protein synthesis during heat shock. Nature 293, 31 I-314. Lindquist S. (1986) The heat-shock response. Ann. Rev. Biochem. 55, 1151-1191.
Heat shock proteins in Sarcophuga Livak K. F.. Freund R., Schweber M., Wensink P. L. and Meselson M. (1978) Sequence organization of transcription of two heat shock loci in Drosophila. Proc. natn. Acad. Sci. U.S.A. 15, 5613-5617.
McAlister L., Strauskey S., Kulaga A. and Finkelstein D. B. (1979) Altered patterns of protein synthesis induced by heat shock of yeast. Curr. Genet. 1, 63-74. Mans R. J. and Novelli G. D. (1961) Measurement of the incorporation of radioactive amino acids into protein by a filter-paper disk method, Archs Biochem. Biophys. 94, 48-53.
Moran L. A., Chauvin M., Kennedy M. E., Korri M., Lowe D. G., Nicholson R. C. and Perry M. D. (1983) The major heat-shock protein (hsp70) gene family: related sequences in mouse, Drosophila and yeast. Can. J. Biochem. Cell Biol. 61, 488499.
Parker-Thornburg J. and Bonner J. J. (1987) Mutations that induce the heat shock response of Drosophila. Cell 51, 763-772.
Savakis C., Ashburner M. and Willis J. H. (1986) The expression of the gene coding for alcohol dehydrogenase during the development of Drosophila melanogaster. Den. Biol. 114, 194-207. Schlesinger M. J., Ashburner M. and Tissieres A. (eds)
249
(1982) Heat Shock: From Bacteria to Man. Cold Spring Harbor Laboratory, Cold Spring Harbor. Seguy E . (1951) Ordre des Dipteres. Traite de Zoologie 10, 499-744. Simmen F. A., Schultz T. Z., Headon D. R., Wright D. A., Carnenter G. and O’Mallev B. W. (1984) Translation in Xenapus oocytes of messenger RNA from A431 cells for human epidermal growth factor receptor proteins. DNA 3, 393-399.
Spradling A.. Penman S. and Pardue M. L. (1975) Analysis of Drosophila mRNA by in situ hybridization: sequences transcribed in normal and heat shocked cultured cells. Cell 4, 395404.
Tissieres A., Mitchell H. K. and Tracy U. M. (1974) Protein synthesis in salivary glands of Drosophila melanogaster: relation to chromosome puffs. J. Mol. Biol. 84, 389-398.
Walker V. K., Whyard S., Wyatt G. R. and Kanest M. R. (1986) Heat shock and gene transfer in locusts (abstract) J. Cell. Biochem. lOC, 56.
Werner-Washburn M., Stone D. E. and Craig E. A. (1987) Complex interations among members of an essential subfamily of hsp70 genes in Saccharomyes cereeesiae. Mol. Cell. Biol. 7, 2568-2577.