The expression of heat shock genes during normal development in <i>Drosophila melanogaster</i> (heat shock/abundant transcripts/developmental regulation)

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The expression of heat shock genes during normal development in Drosophila melanogaster (heat shock/abundant

transcripts/developmental regulation)

MASON, Philip John, HALL, Lucinda M. C., GAUSZ, Janos

Abstract

Drosophila melanogaster cells and tissues respond to heat shock by dramatically altering their pattern of transcription and translation, leading to the rapid synthesis of a small number of polypeptides, the heat shock proteins (hsps). By using cloned hsp DNA we have detected sequences complementary to heat shock genes in RNA prepared from non-heat-shocked animals of different developmental stages. Hsp 83 mRNA is present at high levels in all stages examined. Hsp 68 and 70 mRNAs are present at very low levels in most stages and at slightly higher concentration in pupae. Hsp 26 and 27 mRNAs are detected in embryos. Hsp 23, 26 and 27 mRNA are barely detectable in early third instar larvae but are major components of late third instar and early pupal RNA. Hsp 22 mRNA is also detected in early pupae. Later in development the levels of the small hsp mRNAs decrease but a further peak in abundance of hsp 26 and 27 mRNAs is found in mature adult females.

MASON, Philip John, HALL, Lucinda M. C., GAUSZ, Janos. The expression of heat shock genes during normal development in Drosophila melanogaster (heat shock/abundant

transcripts/developmental regulation). Molecular and General Genetics , 1984, vol. 194, no.

1-2, p. 73-78

DOI : 10.1007/BF00383500

Available at:

http://archive-ouverte.unige.ch/unige:128114

Disclaimer: layout of this document may differ from the published version.

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The expression of heat shock genes

during normal development in Drosophila melanogaster

(heat shock• abundant transcripts/developmental regulation)

P.J. Mason 1'*, L.M.C. HalP, and J. Gausz 1'2"**

1 Department of Molecular Biology, University of Geneva, 30, quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland 2 Department of Animal Biology, 154, rte de Malagnou, CH-1224 Chane-Bougeries, Switzerland

Summary. Drosophila melanogaster cells and tissues respond to heat shock by dramatically altering their pattern of transcription and translation, leading to the rapid synthesis of a small number of polypeptides, the heat shock proteins (hsps). By using cloned hsp DNA we have detected sequences complementary to heat shock genes in RNA prepared from non-heat-shocked animals of different developmental stages. Hsp 83 mRNA is present at high levels in all stages examined. Hsp 68 and 70 mRNAs are present at very low levels in most stages and at slightly higher concentration in pupae. Hsp 26 and 27 mRNAs are detected in embryos. Hsp 23, 26 and 27 mRNA are barely detectable in early third instar larvae but are major components of late third instar and early pupal RNA. Hsp 22 mRNA is also detected in early pupae. Later in development the levels of the small hsp mRNAs decrease but a further peak in abundance of hsp 26 and 27 mRNAs is found in mature adult females.

Introduction

Exposure of Drosophila cells or tissues to elevated temperatures, or to a number of other experimental stresses, induces a drastic change in their pattern of protein synthesis (re- viewed by Ashburner and Bonner 1979). While most of the RNA and protein synthesis active before the heat shock is repressed the production of a small number of polypeptides, the heat shock proteins (hsps) is strongly induced (Tissi~res et al. 1974; McKenzie et al. 1975). The major Drosophila melanogaster hsps named according to their mo- lecular weight in kilodaltons are hsp 83, 70, 68, 27, 26, 23 and 22. The genes coding for all of these proteins have been cloned and most have been sequenced (Ingolia et al.

1980; T6r6k and Karch 1980; Karch et al. 1981 ; Holmgren et al. 1981 ; Ingolia and Craig 1982b; Southgate et al. 1983).

Nevertheless the function of the heat shock proteins remains unclear. In several different organisms there is evidence that they protect cells from damage at higher temperatures (Schlesinger et al. 1982) but the mechanism of this protection is not known.

* Present address: Imperial Cancer Research Fund, Mill Hill Laboratories, Burtonhole Lane, London, NW7 lAD, England

** Permanent address: Institute of Genetics, Hungarian Academy of Sciences, P.O.B. 521, Szeged, Hungary

Offprint requests to: L.M.C. Hall

An important question concerns the possible role of these proteins in normal metabolism or development. Kel- ley and Schlesinger (1982) found proteins which cross reacted with chicken hsp 70 and hsp 89 antibodies in a variety of non-heat-shocked animal tissues. Sirotkin and Davidson (1982) reported that hsp 22 and hsp 26 mRNA were abundant in non-heat-shocked late third instar larvae and early pupae of Drosophila. The Drosophila hsp 83 and its mRNA are abundant in non-heat-shocked tissue culture cells (Storti et al. 1980; O'Connor and Lis 1981). We have undertaken a study of the pattern of expression of the major heat shock genes in Drosophila melanogaster. In this paper we report upon the detection of heat shock protein mRNA sequences in total RNA from non-heat-shocked animals at various developmental stages.

Materials and methods

Plasmids and enzymes. Restriction enzymes were obtained from New England Biolabs Inc. (Beverly, Mass., USA) and B6hringer (Mannheim, FRG) and used according to the manufacturers recommendations. Construction of recombinant plasmids containing heat shock genes have been described previously in the references given in the legend to Fig. 1 except for pPW227BH1 which consists of the BamHI to HindIII fragment ofpPW227 (Holmgren et al. 1979) sub- cloned into pBR322 using standard techniques. Plasmid DNAs were prepared by CsC1 banding from cleared lysates according to standard procedures.

Selection of staged animals'. Flies of the Oregon-R stock of Drosophila melanogaster were raised at 20 ° C. Embryos were collected on food trays spread with fresh, living yeast paste. For our embryo sample we took all the embryos collected during 4 h. The larvae were raised at 20°C and in a 12 h light-dark cycle. Four-day-old, early third instar larvae were collected by removing the food from vials and adding a 20% sucrose solution. The larvae from the surface of the solution were taken and those of larger body size excluded. Late third instar larvae were collected as those which had left the food and were on the walls of the vials.

All pupal samples were synchronised as white pre-pupae and kept at 20 ° C, early pupae were collected after a further 16 h, midpupae after 60 h and late pupae after 120 h.

Freshly eclosed adult males and females were collected within 2 h after eclosion and another sample was taken after 4 days.

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74

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Fig. 1. A Restriction maps of recombinant DNAs containing heat shock genes used in this study. Filled sections represent DNA complementary to the various heat shock mRNAs. The arrows above the map show the direction of transcription of the genes. The ^data are from Holmgren et al. (1981) for hsp 68 and hsp 83; Goldschmidt-Clermont (1980) for hsp 70; and Southgate et al. (1983) for hsps 22, 23, 26 and 27. Hatched regions show the positions of non-heat shock genes included in the gel shown in B; these are taken from O'Connor and Lis (1981) for T1 and Sirotkin and Davidson (1982) for T2 and T3. Only restriction enzyme sites used in our experiments, and those used in the construction of the subclones, are shown. An EcoRI site was created adjacent to the J(baI site near the start of the hsp 27 gene during subcloning (Southgate et al. 1983). B An ethidium bromide stained 0.8% agarose gel showing the separation of the transcribed regions from the heat shock loci. The tracks are a, 301.1 digested with HincII; b, 122 x 14 BamHI, XbaI; c, pPW227BH1 BamHI, HindlII; d, 17955 HindIII, EcoRI; e, 17920 EeoRI, BamHI, J(baI and f, 179F4, PstI, XbaI. The key on the right shows which bands contain the 3' regions of heat shock genes and TI, T2 and T3

Preparation of RNA and cDNA. RNA was isolated from whole animals of the various stages essentially as described by Kemp et al. (1978) except that we included a precipita- tion from 3 M sodium acetate pH 6.0 to remove DNA.

To prepare high specific activity cDNA 12 lag RNA was incubated for 1 h at 42 ° C in a solution containing 50 mM Tris-HC1 pH 8.0, 10 mM MgC12, 30 mM fl-mercaptoetha- nol, 70 mM KC1, 0.33 mM dATP, dGTP and TTP, 40 laCi c~-3zP dCTP (2-3,000 Ci/mmol), 0.5 lag oligo (dT) and 10 U reverse transcriptase (Life Sciences Inc.). After hydrolysis of the RNA, unincorporated nucleotides were removed on Sephadex G-75.

Filter hybridisation. DNA was transferred from agarose gels to nitrocellulose filters essentially as described by Southern (1975). The filters were pre-hybridised for 12 h at 65°C in 4xSSC (SSC is 0.3 M NaC1; 0.03 M Na citrate) 0.1%

Ficoll, 0.1% polyvinyl pyrrolidone, 0.1% bovine serum al- bumin, 0.1% SDS, 0.1% NacP207 and 50 lag/ml denatured calf thymus DNA. Hybridisation was carried out in the same solution containing 210 x 106 dpm cDNA for 36 h

at 65 ° C. Following hybridisation, filters were washed thrice in 3 x SSC, 0.1% SDS, and twice in 0.2x SSC, 0.1% SDS, all at 65 ° C. Filters were exposed to X-ray film (Kodak) at -70 ° C with an intensifying screen.

Results

In the experiments presented here we have assayed RNA preparations from various developmental stages of D. mela- nogaster for the presence of hsp mRNA. To achieve this we have used recombinant plasmids containing the genes for hsp 83 and hsp 68 (Holmgren etal. 1981) hsp 70 (Goldschmidt-Clermont 1980) and hsps 22, 23, 26 and 27 (Voellmy et al. 1981 ; Southgate et al. 1983). Restriction digests of these plasmids were designed to produce an array of fragments on an agarose gel representing all of the genes.

The clones and enzymes that we used are shown in Fig. 1.

The gels were blotted onto nitrocellulose and the filters hybridised with radioactive oligo-(dT)-primed cDNA co- pied from total RNA preparations from staged animals.

After stringent washing, the amount of cDNA hybridising

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Table 1. The concentration of heat shock mRNA in RNA populations from various stages of normal development of

Drosophila

hsp83 hsp70 hsp68 hsp27 hsp26 hsp23 hsp22

Embryo + + + + / - + / - + + + + - -

Early 3rd instar + + + + / - + / - + / - + / - + / - -

Late 3rd instar + + + + / - + / - + + + + + + + + + + -

White prepupa + + + + / - + / - + + + + + + + + + + + + -

Early pupa + + + + + + + + + + + + + + + + +

Mid-pupa + + + + + + + + + + + + + + +

Late pupa + + + + + + + + -

Freshly eclosed c~ + + + + / - + / - + / - + / - - -

Freshly eclosed ~2 + + + + / - + / - + + + + -

4-day-old ~ + + + + / - + / - + / - + / - - -

4-day-old 9 + + + + / - + / - + + + + + - -

The results were derived from three series of experiments, examples of which are shown in Fig. 2. Hybridisation of labelled cDNA to various heat shock genes was determined by densitometry and is shown relative to the hsp 83 signal, as explained in the text

- not detected, + / - barely detectable, + detected at <30% hsp 83 + + 30%-70% hsp 83, + + + 70%-130% hsp 83, + + + +

>130% hsp 83

to each restriction fragment was determined by autoradiog- raphy and densitometry. Under the conditions that we used the mass of cDNA hybridising to each band should be proportional to the concentration of cDNA in the hybridisation solution (Biessmann et al. 1979) and also to the amount of D N A in the band. Care was taken to load equi- molar amounts of the different plasmids onto each gel and to use the same restriction digests for a set of experiments comparing the various R N A populations. To compare ac- curately m R N A concentrations between different stages the ideal standard would be a cloned D N A coding for a polyadenylated m R N A which is present at the same concentration throughout development. Because we do not know of a gene which satisfies this criterion we used hsp 83 R N A as our internal standard. This R N A gives a consistently strong signal at all stages of development. Our results are therefore expressed relative to hsp 83 m R N A and are sum- marised in Table 1.

To show that the relative signal of any two bands is independent of total cDNA concentration we performed controls in which cDNA concentration was varied by 100-fold, and initial R N A concentration was varied by 10-fold. In these experiments the proportion of hsp 23 R N A relative to hsp 83 R N A was measured for late third instar larvae. When the results were compiled, the standard devia- tion was found to be 20% of the mean value (5.25+_ 1.06), which is in the same range as the variability between dupli- cated experiments.

The m R N A for hsp 83 then, is present in all stages we have examined. In some experiments, cloned D N A from the actin gene at chromosome interval 5C (Fyrberg et al.

1981) was present on the same filter as hsp 83 D N A (not shown). Hsp 83 m R N A was about two to three-fold less abundant than actin m R N A in all stages from embryos to late pupae but considerably more abundant in adults.

Since actin mRNAs are amongst the most abundant mRNAs in many developmental stages and tissues (Fyrberg et al. 1980) we conclude that hsp 83 m R N A is present as a relatively abundant R N A throughout the life cycle of

Drosophila.

A transcript encoded at 1 kb from hsp 83 (O'Connor and Lis 1981) is also abundant at some stages (TI in Fig. 2).

Our results show that some of the genes for the small heat shock proteins are also expressed as major mRNAs during normal development. In embryos we detect significant levels of hsp 26 and hsp 27 mRNAs while in early third instar larvae we can detect only low levels of hsp 23, 26 and 27 mRNAs. In late third instar larvae the concentration of these three mRNAs is dramatically increased and in white prepupae exceeds that of hsp 83 m R N A (Fig. 2A). Through puparium formation and in young pupae the levels of these transcripts remains high and we also detect low levels of hsp 22 mRNA. The mRNAs are still present at about the same levels after 60 h (at 20 ° C) of pupal development but their concentration is much lower in late pupae and young adults (hsp 23 m R N A being more abundant than the others in young females). In 4-day-old male flies transcripts of the small heat shock genes are not detected but in female flies of the same age we see a significant accumulation of hsp 26 and hsp 27 mRNAs while those for hsps 22 and 23 are not detected (Fig. 2B).

We do not detect hsp 68 and hsp 70 mRNAs at the high concentrations found for hsp 23, 26, 27 and 83 but we do find a low level of hybridisation in most stages and a small peak in early pupae. Hsp 68 and hsp 70 are members of a family of related genes that contain some members, hsp 70 cognates, which are expressed during normal development (Craig et al. 1982). However, at the stringency we used in washing the filters (0.2 x SSC, 65 ° C) hybrids between hsp cognate cDNAs and hsp 70 and 68 DNAs (which show about 70% homology) should not be stable.

Discussion

We have presented evidence that several of the hsp RNAs are present as abundant polyadenylated RNAs at some stages of normal development. Although we have not dem- onstrated that these sequences are present as mature, cyto- plasmic, translatable messengers several studies have shown the presence of heat shock proteins under non-heat-shock conditons. For example hsp 83 is a major component of 25°C tissue culture cells (Mirault et al. 1978; Storti et al.

1980) and of a variety of larval tissues (Storti et al. 1980).

Low levels of hsp 70 have been detected in 25 ° C tissue

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embryos earty 3rd instar white prepupae rate

a b c def ab c def abc de f abc pupae d e f

A

adul.t femates adutt femal.es adul.t mates

(newl,y ecl,osed) (4-day) (4-day)

ab c def ab de f a bd ef

B

Fig. 2A, B. Hybridization of azP-labelled cDNA prepared from total RNA of various developmental stages to restriction fragments of DNA from heat shock loci. DNA was transferred from agarose gels such as that shown in Fig. 1 B to nitrocellulose filters. The filters were hybridised, washed and exposed to X-ray film as described in Materials and methods. Tracks are named as in Fig. 1, and at each stage all tracks derive from a single filter. A Illustrates the detection of hsp 26 and 27 mRNAs in embryos, the increase in the concentration of hsp 23, 26 and 27 mRNAs between early third instar larvae and white prepupae and their lower concentration in late_ pupae. B Shows the high concentration of hsp 26 and 27 mRNAs in female relative to male flies and the absence of hsp 23 mRNA in 4-day-old, relative to freshly eclosed females. (Track d in adult males contains some contaminating DNA, giving rise to a smear, which was eliminated in other experiments). The upper bands in tracks a and e do not contain heat shock genes (see Fig. t) and are not discussed in detail in this paper

culture cells (Velazquez et al. 1983) and Ireland et al. (1982) detect small heat shock proteins in imaginal discs in vivo and after ecdysterone treatment of imaginal discs or cultured cells.

The results indicate that hsp 83 mRNA in all stages, and hsp 23, 26 and 27 mRNA at some stages, are amongst the most abundant transcripts in the cell (as can be deduced by comparison with the abundant actin transcripts). In support of this conclusion Sirotkin and Davidson (1982) isolated DNA from locus 67B in experiments designed to puri- fy the most abundantly expressed genes of young pupae.

In a study of the transcripts from 315 kb of DNA in the rosy-Ace region of the D. melanogaster genome the majority of transcripts detected were 10-100 times less abundant than this actin species (Hall et al. 1983).

The four small heat shock genes are arranged in a single cluster within 11 kb of DNA at locus 67B (Corces et al.

1980; Craig and McCarthy 1980; Voellmy et al. 1981). The nucleotide sequences of the genes and the derived amino acid sequences suggest that the proteins are, at least to some extent, functionally related and the genes probably have a common evolutionary origin (Ingolia and Craig 1982b; Southgate et al. 1983). The four genes, however, show three distinct patterns of expression during development. The transcripts for hsp 26 and hsp 27 are detected in embryos, are abundant in late third instar larvae and throughout most of pupal development; in late pupae and freshly eclosed adults their concentration is low but it is again increased in 4-day-old female, but not male flies. Hsp 23 mRNA is also abundant in late third instar larvae and throughout pupal development; in freshly eclosed female flies we detect this transcript at higher levels than hsp 26 or 27 whilst in four-day-old female and male flies it is unde- tectable. Hsp 22 also accumulates in early to mid-pupae

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but at a much lower level than the others and is not detected in adults.

Ireland and Berger (1982) and Ireland etal. (1982) showed that the four genes for the small hsps are transcribed in tissue culture cells in response to treatment with ecdysterone. They noted that this is consistent with the increase in abundance of some small hsp mRNAs in late third instar larvae (Sirotkin and Davidson 1982) when ecdysterone titres are high (Handler 1982). We confirmed that a dramatic increase in concentration of hsp 23, 26 and 27 mRNA takes place between early and late third instar larvae. Ireland et al. (1982) went on to show that imaginal discs isolated from pupariating larvae could be stimulated to produce small heat shock proteins in vitro by treatment with ecdysterone. In vivo the response is probably not re- stricted to imaginal discs; Cheney and Shearn (1983) found that hsp 23 is abundant in the larval fat body.

We investigated if there was any correlation between changes in ecdysterone titre and small hsp mRNA accumulation at other stages in the life cycle. We examined heat shock mRNA levels in synchronised pupae 16 h (20 ° C) after puparium formation, when ecdysterone titres are low, and at 60 h when they are high. Apart from a generally lower level of small hsp transcripts in the 60 h pupae there was no great difference between the two stages. However, the biological significance of the mid-pupal ecdysterone peak is not clear.

A different pattern of small hsp gene expression is ob- served after eclosion. In four-day-old females hsp 26 and 27 mRNAs accumulate in the absence of hsp 23 mRNA.

This finding and that of hsp 26 and 27 in embryos are in agreement with the data of Zimmerman et al. (1983) who detected these two mRNAs in ovaries and showed that in embryos these mRNAs were of maternal original. (Since we separated only adults according to their sex we cannot exclude the possibility of sex-specific gene expression at ear- lier stages).

The heat shock cluster at 67B, therefore, is activated at two distinct stages of development to produce two different sets of hsps. While there is evidence that ecdysterone induces the expression of the small hsps during puparium formation it is unlikely that the hormone is involved in the regulation of hsp synthesis in adults. Firstly a different set of genes is expressed and secondly there appear to be no detectable differences in ecdysterone levels between male and female flies (Handler 1982). The relationship between ecdysterone and the small hsps may be clarified by studying hsp synthesis in temperature-sensitive mutants deficient in ecdysterone production.

We found high levels of hsp 83 mRNA in total RNA from all stages that we examined, and used the hsp 83 signal as a standard with which to compare the abundance of other transcripts. We should emphasize that we have only looked at polyadenylated hsp 83 mRNA in these experiments; O'Connor and Lis (1981) showed that in 25 ° C tissue culture cells a significant proportion of hsp 83 mRNA is not polyadenylated.

Our finding of the ubiquity of hsp 83 mRNA is consistent with the data of Kelley and Schlesinger (1982) who found the analogous chicken protein in every embryonic tissue that they examined. Hsp 83 is located almost exclu- sively in the cytoplasm (Mitchell and Lipps 1975; Arrigo 1980; Storti etal. 1980) and is very highly conserved throughout evolution (Kelley and Schlesinger 1982;

Voellmy et al. 1983; see also Schlesinger et al. 1982). These facts, taken together, suggest the protein may have a structural role in the cytoplasm.

We have found only very low levels of hsp 70 and hsp 68 mRNA in any stage. However these transcripts were detectable, especially in pupae, at about the same concentration found for other "rare" mRNAs in the rosy-Ace region, some of which are associated with vital functions (Hall et al. 1983; L.M.C. Hall, unpublished observations).

Hsp 70 and hsp 68 are two members of a small family of related genes, only some of which are induced by heat shock (Ingolia and Craig 1982a; Wadsworth 1982) whilst the others, the hsp 70 cognates, are transcribed at normal temperatures (Craig et al. 1982). Related vertebrate proteins are abundant at physiological temperatures (Kelley and Schlesinger 1982) and appear to be associated with the cytoskeleton (Wang et al. 1981; Hughes and August 1982). Assuming the hsp 70 cognates are related in function to hsp 70, this function is required in normal development as well as during the response to stress.

Acknowledgements. We thank M. Meselson for gifts of plasmids pPW227 and p30lA and R. Southgate for plasmids containing small hsp genes. We thank H. Gloor and A. Tissi6res for support and A. Tissi6res for his continued help, advice and encouragement P.J. Mason thanks A. and P. Spierer for their hospitality. L.M.C.

Hall and P.J. Mason were supported by long term fellowships from E.M.B.O. This work was supported by the Swiss National Science Foundation (grant No. 3.079.81 to A. Tissi~res).

References

Arrigo AP (1980) Investigation of the function of the heat shock proteins in Drosophila melanogaster tissue culture cells. Mol Gen Genet 178:517-524

Ashburner M, Bonner JJ (1979) The induction of gene activity in Drosophila by heat shock. Cell 17:241-254

Biessmann H, Craig EA, McCarthy BJ (1979) Rapid quantitation of individual RNA species in a complex population. Nucl Acids Res 7:981-996

Cheney CM, Shearn A (1983) Developmental regulation of Dro- sophila imaginal disc proteins : synthesis of heat shock proteins under non-heat shock conditions. Dev Biol 95 : 325-330 Corces V, Holmgren R, Freund R, Morimoto R, Meselson M

(1980) Four heat shock proteins of Drosophila melanogaster coded within a 12-kilobase region in chromosome subdivision 67B. Proc Natl Acad Sci USA 77:5390-5393

Craig EA, McCarthy BJ (1980) Four Drosophila heat shock genes at 67B: characterization of recombinant plasmids. Nucl Acids Res 8 : 44414457

Craig E, Ingolia T, Slater M, Manseau L, Bardwell J (1982) Dro- sophila and yeast multigene families related to the Drosophila heat-shock genes. In: Schlesinger MJ (ed) Heat shock from bacteria to man. Cold Spring Harbor Laboratory, New York, pp 11-18

Fyrberg E, Kindle K, Davidson N, Sodja A (1980) The actin genes of Drosophila: a dispersed multigene family. Cell 19 : 365-378 Fyrberg EA, Bond B J, Hershey DN, Mixter KS, Davidson N

(1981) The actin genes of Drosophila: protein coding regions are highly conserved but intron positions are not. Cell 24:107-116

Goldschmidt-Clermont M (1980) Two genes for the major heat- shock protein of Drosophila melanogaster arranged as an in- verted repeat. Nucl Acids Res 8:235-252

Hall LMC, Mason PJ, Spierer P (1983) Transcripts, genes and bands in 315,000 base-pairs of Drosophila DNA. J Mol Biol 169 : 83-96

(7)

78

Handler AM (1982) Ecdysteroid titers during pupal and adult development in Drosophila melanogaster. Dev Biol 93 : 73-82 Holmgren R, Livak K, Morimoto R, Freund R, Meselson M

(1979) Studies of cloned sequences from four Drosophila heat shock loci. Cell 18:1359-1370

Holmgren R, Corces V, Morimoto R, Blackman R, Meselson M (1981) Sequence homologies in the 5' regions of four Drosophila heat-shock genes. Proc Natl Acad Sci USA 78: 3775-3778 Hughes EN, August TJ (1982) Coprecipitation of heat shock pro-

teins with a cell surface glycoprotein. Proc Natl Acad Sci USA 79 : 2305-2309

Ingolia TD, Craig EA (1982a) Drosophila gene related to the major heat shock-induced gene is transcribed at normal temperatures and non induced by heat shock. Proc Natl Acad Sci USA 79: 525-529

Ingolia TD, Craig EA (1982b) Four small Drosophila heat shock proteins are related to each other and to mammalian a-crystal- fin. Proc Natl Acad Sci USA 79:2360-2364

Ingolia TD, Craig EA, McCarthy BJ (1980) Sequence of three copies of the gene for the major Drosophila heat shock induced protein and their flanking regions. Cell 21 : 669-679

Ireland RC, Berger EM (1982) Synthesis of low molecular weight heat shock peptides stimulated by ecdysterone in a cultured Drosophila cell line. Proc Natl Acad Sci USA 79:855-859 Ireland RC, Berger E, Sirotkin K, Yund MA, Osterbur D, Fristrom

J (1982) Ecdysterone induces the transcription of four heat- shock genes in Drosophila $3 cells and imaginal discs. Dev Biol 93 : 498-507

Karch F, TSr6k I, Tissibres A (1981) Extensive regions of homology in front of the two hsp 70 heat shock variant genes in Dro- sophila melanogaster. J Mol Biol 148:219-230

Kelley PM, Schlesinger MJ (1982) Antibodies to two major chicken heat shock proteins cross react with similar proteins in widely divergent species. Mol Cell Biol 2:267-274

Kemp DJ, Thomson JA, Peacock VJ, Higgins TJV (1978) Mes- senger RNA for the insect storage protein calliphorine: in vitro translation and chromosomal hybridization analysis of a 20S poly(A)-RNA fraction. Biochem Genet 16: 355-371

McKenzie SL, Henikoff S, Meselson M (1975) Localization of RNA from heat-induced polysomes at puff sites in Drosophila melanogaster. Proc Natl Acad Sci USA 72:1117-1121

Mirault ME, Goldschmidt-Clermont M, Arrigo PA, Tissi6res A (1978) The effect of heat shock on gene expression in Drosophila melanogaster. Cold Spring Harbor Symp Quant Biol 42:819-827

Mitchell HK, Lipps LS (1975) Rapidly labelled proteins on the salivary gland chromosomes of Drosophila melanogaster. Bio- chem Genet 13:585-602

O'Connor D, Lis JT (1981) Two closely linked transcription units

within the 63B heat shock puff locus of D. melanogaster display strikingly different regulation. Nucl Acids Res 9:5075-5092 Schlesinger MJ, Ashburner M, Tissirres A, eds (1982) Heat shock

from bacteria to man. Cold Spring Harbor Laboratory, New York

Sirotkin K, Davidson N (1982) Developmentally regulated transcription from Drosophila melanogaster site 67B. Dev Biol 89:196-210

Southern E (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98 : 503-517

Southgate R, Ayme A, Voellmy R (1983) Nucleotide sequence analysis of the Drosophila small heat shock gene cluster at locus 67B. J Mol Biol 165:35-37

Storti RV, Scott MP, Rich A, Pardue ML (1980) Translational control of protein synthesis in response to heat shock in D.

melanogaster cells. Cell 22: 825-834

Tissirres A, Mitchell HK, Tracy UM (1974) Protein synthesis in salivary glands of Drosophila melanogaster: relation to chromosome puffs. J Mol Biol 84:389-398

Trrrk I, Karch F (1980) Nucleotide sequence of heat shock activated genes in Drosophila melanogaster. I. Sequences in the region of the 5' and 3' ends of the hsp 70 gene in the hybrid plasmid 56H8. Nucl Acids Res 8:3105-3123

Velazquez JM, Sonoda S, Bugaisky G, Lindquist S (1983) Is the major Drosophila heat shock protein present in cells that have not been heat shocked. J Cell Biol 96:286-290

Voellmy R, Goldschmidt-Clermont M, Southgate R, Tissirres A, Levis R, Gehring WJ (1981) A DNA segment isolated from chromosomal site 67B in D. melanogaster contains four closely linked heat-shock genes. Cell 23 : 261-270

Voellmy R, Bromley P, Kocher HP (1983) Structural similarities between corresponding heat-shock proteins from different eu- caryotic cells. J Biol Chem 258:3516-3522

Wadsworth SC (1982) A family of related proteins is encoded by the major Drosophila heat shock gene family. Mol Cell Biol 2: 286-292

Wang C, Gomer RH, Lazarides E (1981) Heat shock proteins are methylated in avian and mammalian cells. Proc Natl Acad Sci USA 78:3531-3535

Zimmerman JL, Petri W, Meselson M (1983) Accumulation of a specific subset of D. melanogaster heat shock mRNAs in normal development without heat shock. Cell 32:1161-1170 Communicated W. Gehring

Received October 14, 1983