Response to heat shock of gene <i>1</i>, a <i>Drosophila melanogaster</i> small heat shock gene, is developmentally regulated

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Reference

Response to heat shock of gene 1 , a Drosophila melanogaster small heat shock gene, is developmentally regulated

VAZQUEZ, Julio

Abstract

The expression of gene 1, a member of the small heat shock gene family from the Drosophila melanogaster chromosomal locus 67B was studied. In contrast to the other heat shock genes, the response of gene 1 to stress was modulated during development. In the absence of stress, gene 1 was expressed at the beginning of pupation, and at a very low level in adult males. Expression of gene 1 was substantially increased by heat shock in pupae, but was one to two orders of magnitude lower in adults or in embryos. Under the same conditions, hsp70 or hsp26 were induced to similar levels in all stages. This developmental effect could be mimicked in cultured Drosophila cells: expression of gene 1 was stimulated by heat shock in the presence, but not in the absence, of the moulting hormone ecdysterone, while the level of expression of hsp26 and hsp70 in response to heat shock was independent of the presence of the hormone. Thus, the presence and activity of the heat shock transcription factor are not sufficient for the maximal response of gene 1 to stress. These results suggest that the heat shock activator protein requires additional [...]

VAZQUEZ, Julio. Response to heat shock of gene 1 , a Drosophila melanogaster small heat shock gene, is developmentally regulated. Molecular and General Genetics , 1991, vol. 226, no. 3, p. 393-400

DOI : 10.1007/BF00260651

Available at:

http://archive-ouverte.unige.ch/unige:141163

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Response to heat shock of gene 1, a Drosophila melanogaster

small heat shock gene, is developmentally regulated

Julio Vazquez

D~partement de Biologie Mol6culaire, Universit~ de Gen~ve, Sciences II, 30, Quai Ernest Ansermet, 1211 Gen~ve 4, Switzerland Recieved July 27, 1990

Summary. The expression of gene 1, a member of the small heat shock gene family from the Drosophila mela- nogaster chromosomal locus 67B was studied. In contrast to the other heat shock genes, the response of gene 1 to stress was modulated during development. In the absence of stress, gene 1 was expressed at the beginning of pupation, and at a very low level in adult males.

Expression of gene I was substantially increased by heat shock in pupae, but was one to two orders of magnitude lower in adults or in embryos. Under the same conditions, hsp70 or hsp26 were induced to similar levels in all stages. This developmental effect could be mimicked in cultured Drosophila cells: expression of gene 1 was stimulated by heat shock in the presence, but not in the absence, of the moulting hormone ecdysterone, while the level of expression of hsp26 and hspTO in response to heat shock was independent of the presence of the hormone. Thus, the presence and activity of the heat shock transcription factor are not sufficient for the maximal response of gene 1 to stress. These results suggest that the heat shock activator protein requires additional factors, which are developmentally regulated, to activate transcription of gene 1. Furthermore, $1 nuclease mapping analysis revealed several gene 1 mRNA species, which are generated by the use of alternative polyadenylation sites and by the use of differentially regulated transcriptional initiation sites.

Key words: Heat shock gene - Drosophila melanogaster - Ecdysterone - Developmental regulation

Introduction

The heat shock genes of D. melanogaster have for many years provided a system of choice to study both inducible and developmentally regulated gene expression. Sev- Present address and address for correspondence: Department of Mo- lecular Biology, Princeton University, Princeton, NJ 08544, USA

en of the heat shock genes are clustered in a 15 kb DNA region at chromosomal locus 67B (Ayme and Tissi6res 1985). They include the four small heat shock protein genes hsp22, hsp23, hsp26, and hsp27 (Southgate et al.

1983), as well as two related genes, genes 1 and 3, plus gene 2 which has a completely different structure (Ayme and Tissi&es 1985; Pauli and Tonka 1987; Pauli et al.

1988). The proteins encoded by genes 1, 2 and 3 have yet to be identified.

All seven genes at locus 67B respond to a heat shock by an increase in the amount of their respective mRNAs and, for the four small heat shock protein genes, a paral- lel increase in the abundance of their protein products (Tissi6res etal. 1974; Spradling etal. 1975, 1977;

McKenzie et al. 1975; Ayme and Tissi&es 1985; Pauli and Tonka 1987; Pauli etal. 1988, 1989). In addition to their activation by heat shock, the seven genes show a complex pattern of expression during development.

All are expressed, though at different levels, at the beginning of the pupal peroid. In addition, hsp26 is expressed in adults in a complex tissue-specific pattern, hsp27 is expressed in the ovaries, gene 3 is expressed in mid-embryogenesis, and gene 2 is expressed during embryogenesis and in the pupal and adult male reproductive organs (Sirotkin and Davidson 1982; Zimmermann et al. 1983;

Mason et al. 1984; Ayme and Tissi6res 1985; Glaser et al. 1986; Pauli and Tonka 1987; Pauli et al. 1988).

Little is known about the distribution of the products of these genes, but different heat shock proteins may have different intracellular and tissue localizations (Zim- merman et al. 1983; Glaser et al. 1986; Arrigo and Pauli 1988).

Extensive analysis of the promoters of the heat shock genes has revealed that their activation in response to heat shock is due to a particular sequence, the heat shock element (HSE), originally defined as the 12 bp sequence CtnGAAnnTTCnAG (Pelham 1982), which is now believed to consist of the nGAAn motif repeated in alter- nating orientations and at regular intervals (Amin et al.

1988; Xiao and Lis 1988). The heat shock element is the target for the heat shock transcription factor (HSTF)

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394

or the heat shock activator protein (Parker and Topol 1984a, b; Wu 1984; Topol etal. 1985; Perisic etal.

1989). In Drosophila, the HSTF is present in unstressed cells, but is able to specifically bind the HSE and induce transcription only after stress-mediated activation (Wu et al. 1987; Zimarino and Wu 1987). Thus, response of the D. melanogaster heat shock genes to stress is mediated by similar promoter sequences and a unique trans- acting factor.

Much less is known about the sequences and factors responsible for the developmentally regulated expression of the small heat shock genes. There is evidence, however, that developmental and heat shock-induced expression of these genes are mediated by different cis-acting sequences and trans-acting factors (Cohen and Meselson 1985; Pauli et al. 1986; Glaser et al. 1986; Hoffman and Corces 1986; Klemenz and Gehring 1986; Mestril et al.

1986; Riddihough and Pelham 1986; Glaser and Lis 1990). Studies with Drosophila cultured cells or isolated imaginal discs, and analysis of an ecdysteroid-deficient mutant, indicate that the molting hormone ecdysterone is involved in the developmental regulation of the heat shock genes (Ireland and Berger 1982; Ireland etal.

1982; Cheney and Shearn 1983; Vitek and Berger 1984;

Lawson et al. 1985; Thomas and Lengyel 1986).

If the transcriptional induction of the heat shock genes by heat shock is due to the interaction of a unique trans-acting factor with functionally similar promoter sequences, one would postulate that the heat shock genes should be co-ordinately induced during heat shock. I have studied the expression of gene 1 in different developmental stages and in Drosophila tissue culture cells, and have been able to show that the response of this particular gene to heat shock in whole organisms changes dramatically according to the stage of development. In cultured cells, response of gene i to heat shock is modulated by ecdysterone. These results suggest that developmentally regulated factors interact with the heat shock activatory protein to induce the expression of gene 1.

Materials and methods

Selection of staged animals. The Oregon-R stock of D.

melanogaster was raised at 20 ° C and 70% relative hu- midity. Embryos were collected on 2% agar, 1% sucrose plates supplemented with live yeast, and dechorionated with 50% commercial bleach for 3 rain before RNA extraction. Later stages were raised on cornmeal-agar on a 12 h light-dark cycle. White prepupae were collected within 1 h after puparium formation. Early pupae were collected within 12 h after puparium formation. Adult samples, males and females, were collected within 6 days after eclosion.

Cell cultures. The D. melanogaster tissue culture cell line used was Kc161, adapted to grow in suspension at 25 ° C in D22 medium supplemented with 2% foetal calf serum (Gibco) (Echalier and Ohanessian 1970). The cells were grown at densities between 2 x 10 6 and 8 x 10 6 cells/ml.

The generation time was about 15 h. Ecdysterone (20- OH-ecdysone, Sigma) was added to a final concentration of 1 gM.

Heat shocks. Embryos, larvae and pupae from D. mela- nogaster were transferred to 1.5 ml Eppendorf tubes and immersed in a water bath equilibrated at 37 ° C for 1 h.

Adults were transferred to plastic vials or glass bottles previously equilibrated to 37 ° and maintained in a 37 ° C incubator for 1 h. Kc161 cells were transferred to 100 ml plastic bottles, heated to 36 ° by immersion in a 55 ° C water bath under manual agitation, and the bottles were transferred to a 37 ° C bath for 1 h.

RNA extraction. Total nucleic acids were extracted from staged animals or cultured ceils as follows. Ceils were collected by centrifugation at 700 x g for 10 rain at 4 ° C.

The cell pellets or staged organisms were resuspended in 100 mM TRIS-HC1, pH 9.0, 100 mM NaC1, 20 mM EDTA, 1% Sarkosyl, and homogenized in a Dounce homogenizer. After lysis, the suspension was extracted three times with a mixture of phenol-chloroform-isoamyl alcohol (50:50:1 by volume) and ethanol-precipitated.

Total nucleic acids were collected by centrifugation and resuspended in distilled water at concentrations of 2- 10 mg/ml. Poly(A) + RNA was prepared using oligo-dT chromatography following standard procedures (Mania- tis et al. 1982).

Northern analysis. Total nuclei acids or affinity-purified poly(A) + RNA were electrophoresed on 1.2% agarose gels containing 2.2 M formaldehyde as described (Man- iatis et al. 1982), and transferred to nitrocellulose filters (Schleicher and Schuell) by capillary action using 20 x SSC as the transfer medium (1 x SSC is 0.15 M NaC1, 0.015 M sodium citrate, pH 7.0). The filters were baked for 2 h at 80 ° C under vacuum and prehybridized for 6 h at 42 ° C in 5 x SSPE, 0.1% sodium pyrophosphate, 0.4% SDS, 50% formamide, 8% dextran sulphate and 250 gg/ml denatured salmon sperm DNA (1 x SSPE is 180mM NaC1, 10raM NaHzPO4, pH7.4, 1 mM EDTA). The appropriate radiolabelled probes were added and the filters were incubated for 16 h. Washings were performed at 65 ° C for 1-2h in 0.1 x SSC, 0.1% SDS.

The filters were exposed to Kodak XOmat XS5 film at -70 ° C with an intensifying screen.

A single-stranded DNA probe specific for gene 1 was synthesized with the Klenow fragment of E. coli DNA polymerase using a single-stranded M13 subclone containing a PvuII-SalI DNA fragment covering the 5' untranslated leader and the first 130 nucleotides of the gene 1 coding region (Ayme and Tissi6res 1985). High specific activity of the probe was obtained using [e- 32p]dGTP and [c~-32p]dTTP (3000Ci/mmol, Amer- sham) as radiolabelled precursors. A riboprobe specific for hsp70 was synthesized with T3 RNA polymerase, suing [c~-32p]UTP (400 Ci/mmol, Amersham) as the radiolabelled precursor. The template was an XbaI-SaII restriction fragment containing the entire transcription unit of an hsp70 gene from locus 87C1 (Karch et al.

1981) inserted in the pBS+ vector (Stratagene). The

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plasmid was linearized and an RNA probe was synthesized and purified according to the recommendations of the Stratagene manual. The abundance and integrity of RNA on the filters was checked by staining the filters with 0.05% methylene blue for 10 rain followed by extensive washing in distilled water.

S1 nuclease mapping analysis. SI nuclease mapping analysis was performed using double-stranded, end-labelled DNA probes, as described by Favaloro et al. (1980). The PstI-ClaI and EcoRI-ClaI fragments of plasmid 179P2 (Ayme and Tissi6res 1985) were used to map the 5' end of gene 1 mRNAs. The AccI-XbaI fragment of plasmid 1795 (Ayme and Tissi6res 1985) was used to map the 3' end of gene 1 transcripts. To detect hsp26 transcripts, the SacI-BamHI fragment of plasmid 179209 (Southgate et al. 1983) was used. The plasmids were digested with the appropriate restriction enzymes and the fragments separated on 1% agarose gels. The desired fragments were electroeluted and end-labelled to specific activities of about 5 x 106 cpm/gg either by filling-in with the Klenow fragment of E. coli DNA polymerase for mapping of 3' termini or by phosphorylation using T4 polynucleotide kinase and [7-32p]ATP (5000 Ci/mmol, Amersham) for the mapping of 5' termini, using standard procedures (Maniatis et al. 1982).

Fifty nanograms of end-labelled probe were hybridized at 54 ° C for 16 h with up to 100 gg of total RNA or 10 gg of poly(A)+RNA in a final volume of 20 gl of 40 mM Pipes, pH 6.4, 400 mM NaC1, 1 mM EDTA, 80% formamide. Hybridization was stopped by adding 300 gl of cold S1 buffer (280 mM NaCI, 30 mM sodium acetate, pH 4.4, 4.5 mM zinc acetate, containing 100 un- its/ml of nuclease S1, BRL) and single-stranded nucleic acids were digested at 30 ° C for 1 h. The reaction was stopped with 50 gl of 5M ammonium acetate, 50 mM EDTA and ethanol-precipitated. The reaction products were analysed on 5% acrylamide, 7 M urea, thin squenc- ing gels.

cDNA cloning and sequencing. Gene 1 cDNA clones were isolated from a pupal cDNA library (Poole et al. 1985).

After restriction analysis, three cDNAs were subcloned in phage M 13 and sequenced using the dideoxy nucleotide chain-termination technique (Sanger et al. 1977, 1980).

Results

Induction of gene 1 by heat shock during Drosophila development

Gene 1 was first identified and cloned by virtue of its preferential expression in early pupae (Sirotkin and Da- vidson 1982). Cross-hybridization studies and sequence analysis revealed that this gene was homologous to the small heat shock protein coding genes (Ayme and Tissi6res 1985). Northern analysis showed that expression ofgene 1 was regulated during development, in a manner analogous to the expression of the small heat shock

hsp27 hsp23 ] gene h sp26 hsp22ge~ege3ne IK&~u

f f f

O ~ - ' ~ ~ C

b ~

Fig. 1. Map of locus 67B of Drosophila melanogaster. The four small heat shock protein genes are depicted as filled boxes and the three additional developmentally regulated heat shock genes as open boxes with the direction of transcription indicated. An expanded map of gene 1 is shown below, with the major restriction sites. The coding region is indicated by a heavy line. The arrows indicate the major start site of transcription and the two polyadenylation sites. The PstI-ClaI (a), EeoRI-ClaI (b) and AecI-XbaI (c) fragments used as probes in the $1 nuclease experiments are also shown

genes, and that this gene was also heat-inducible (Ayme and Tissi6res 1985). Curiously, the quantity of gene 1 mRNAs that accumulated after a heat shock was consid- erably higher in early pupae than in the other developmental stages tested. Since this observation may be of interest for the understanding of the mechanisms involved in heat shock gene expression, the pattern of expression of gene i was characterized further.

Figure 1 shows the location of gene 1 in locus 67B, with its major cap site and 3' processing sites. The probes used in the S1 nuclease mapping experiments are also indicated. Stress-induction of gene 1 was studied at different stages of D. melanogaster development by North- ern analysis, using a single-stranded DNA probe specific for gene 1 (Fig. 2). In the absence of stress, expression of gene 1 could be detected in prepupae and early pupae, but was barely detectable in the other developmental stages. Heat shock led to a significant increase in the amount of gene 1 mRNAs in prepupae and early pupae.

In post-blastoderm embryos, first instar and early third instar larvae, or adults, however, the amount of gene I mRNAs after heat shock remained low (at least 10- 20 times less than in pupae). In contrast, the major heat shock gene hsp70 was strongly induced throughout development (Fig. 2b). One exception, however, was the apparent low level of induction of hsp70 in larvae (first instar and early third instar). A more detailed analysis revealed that a heat shock of I h at 37 ° C was lethal to those stages. When heat shock was performed at 35.5 ° C, however, hsp70 was strongly induced while the levels of gene 1 mRNAs remained extremely low (data not shown). Thus, gene 1 shows a developmental stage- dependent response to heat shock.

A more sensitive analysis by S1 nuclease mapping was used to study expression of gene 1 in adults (Fig. 3).

Gene 1 transcripts were identified using an end-labelled EcoRI-CIaI fragment which hybridizes with their 5' ends (see Fig. 1). In the absence of stress, gene 1 was expressed at moderate levels in pupae (about 20% the level of hsp26). In adult males, expression of gene 1 was one to two orders of magnitude lower than in pupae, and expression was almost undetectable in adult females.

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396

0 I 2 3 4 5 6 7 8 9 I0 II 12 I

0 3 /4 5 6 7

b

Fig. 2a-e. Developmental stage-specific heat shock induction of gene I. Ten micrograms of total RNA from 12- to 16-h-old embryos (lanes 1, 2), first instar larvae (lanes 3, 4), early third instar larvae (lanes 5, 6), white prepupae (lanes 7, 8), early pupa (lanes 9, 10) and adults (lanes 11, 12) were electrophoresed on a 1.2% agarose-formaldehyde gel and transferred to a nitrocellulose membrane for Northern analysis. RNA in lanes 2, 4, 6, 8, 10 and 12 was extracted from heat shocked animals. The filter was probed with a labelled PvuII-Sail single-stranded DNA fragment specific for gene 1 (a), a riboprobe specific for hsp70 (b) or stained with methylene blue to show total RNA loading (e). Exposure was for 6 days (a) or 3 h (b) both with an intensifying screen

Heat shock led to a significant increase in the amount of gene i mRNAs in pupae (4- to 5-fold). In adults, the abundance of gene i mRNAs after heat shock was about 10- to 20-fold lower than in pupae. In males, where a low level of gene 1 mRNAs could be detected in the absence of stress, the increase in abundance due to heat shock was about 2-fold.

As a control, Fig. 3 b shows the expression of hsp26, another heat shock gene from locus 67B. hsp26 is subject to developmental regulation in the absence of heat shock, being expressed at a high level in pupae and adult females, and at a very low level in adult males (Mason et al. 1984; Glaser et al. 1986; Glaser and Lis 1990).

In contrast to gene 1, hsp26 mRNAs accumulated to approximately identical levels in heat shocked pupae, adult males and adult females, despite very different levels of expression in the absence of stress. These results indicate that although the different developmental stages of Drosophila are competent to induce the major heat shock genes in response to stress, gene 1 is preferentially induced during puparium formation.

Induction of gene 1 in cultured cells

Expression of the small heat shock genes during the early pupal period is mediated by the moulting hormone ecdysterone (20-OH-ecdysone). This is supported by sever-

Fig. 3a and b. SI mapping analysis of gene I expression in adults.

a Fifty micrograms of total RNA from early pupae (lanes 1, 2), adult males (lanes 3, 4), adult females (lanes 5, 6) or the same amount of yeast tRNA (lane 7) were hybridized with the EcoRI- ClaI fragment (see Fig. 1), 5' end-labelled with T4 polynucleotide kinase. RNA in lanes 2, 4 and 6 was from heat shocked animals.

The hybrids were digested with SI nuclease and the protected fragments were run on a 5% polyacrylamide-7 M urea thin sequencing gel. The star indicates the full-length probe, and the arrowhead the fragment protected by gene 1 transcripts. The abundant, larger protected fragments observed in pupae are due to a migration artefact, b Fifty micrograms of total RNA were hybridized with a 3' end-labelled probe specific for the hsp26 gene and subjected to S1 analysis as in a. Exposure times were a 90 h and b 12 h

al observations. First, maximal levels of expression of these genes correlate with the peaks in the concentration of the hormone. Second, expression of the small heat shock genes is impaired in an ecdysteroid-deficient mutant and can be rescued by the addition of hormone (Thomas and Lengyel 1986). Finally, expression of the small heat shock genes can be induced by ecdysterone in cultured cells or isolated imaginal discs (Ireland and Berger 1982; Ireland et al. 1982; Vitek and Berger 1984;

Lawson et al. 1985). Since maximal induction of gene i by heat shock is achieved in early pupae, it was interesting to ask whether the response of this gene to stress could be modulated by ecdysterone.

To test this hypothesis, the induction of gene 1 by heat shock was analysed in cells incubated in the presence or the absence of the hormone (Fig. 4A). Gene 1 mRNAs were undetectable by Northern analysis in cells grown in the absence of the hormone, but accumulated to a low level in cells grown for 48 h in the presence of I ~tM ecdysterone. Heat shock led to a substantial increase in the abundance of gene 1 mRNAs in cells grown in the presence of ecdysterone, but not in the absence of ecdysterone. In contrast to gene 1, hsp70 was identically induced by heat shock in cells incubated with

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A

t C

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(I

Oh 4.8h

HS C HS

b

B

Oh lh 5h 24h

'- HS"- HS"- HS"- HS'

Fig. 4A and B. Ecdysterone-dependent induction of gene 1 by heat shock. A RNA was extracted from non-heat shocked (C) or heat shocked (HS) Kcl61 cells grown in the absence (0 h) or the presence (48 h) of the 1 laM ecdysterone for 48 h. Two micrograms of the poly(A) + (+) or the poly(A)- ( - ) fraction were subject to North- ern analysis. The filter was probed for a gene 1 or b hsp70 as in Fig. 2. Exposure was for a 2 days or b 3 h. B Kcl61 cells were grown for the indicated times in the presence of ecdysterone and then submitted (HS) or not submitted ( - ) to heat shock. RNAs were isolated and 20 lag were subjected to Northern analysis using a probe specific for gene 1. Exposure was for 7 days

121

I 2 3 4 5 6

"X"

910

655 659

Fig. 5a and b. S1 analysis of gene 1 transcripts in Kc161 cells.

a Cells were grown in the absence (lanes 1, 2) or the presence of 1 laM ecdysterone for 12 h (lanes 3, 4) or 48 h (lanes 5, 6) and then submitted (lanes 2, 4 and 6) or not submitted to heat shock.

Poly(A) + RNA was purified and 12 lag were hybridized to the EcoRI--ClaI fragment, 5' end-labelled with T4 polynucleotide kinase. S1 analysis was performed as in Fig. 3. The star indicates the full-length probe and the arrowhead shows the fragment protected by the gene 1 mRNAs initiated at the major cap site. b

$1 analysis was performed with 4 lag of poly(A) + RNA using a 3' end-labelled probe specific for hsp26. Exposure was for a 7 days o r b l 2 h

the hormone and in untreated cells (Fig. 4A). The two bands detected in this experiment with a probe specific for gene 1 mRNAs correspond to two families of transcripts differing in the lengths of their T-untranslated sequences (see below).

The effect of ecdysterone on the heat shock response of gene 1 is not immediate. Figure 4 B shows that gene ! mRNAs were almost undetectable in untreated cells. In cells that were heat shocked after incubation with ecdysterone for 1 or 5 h, heat shock did not lead to any significant increase in the abdundance of gene 1 mRNAs.

However, induction by heat shock was observed in cells incubated with the hormone for 24 h.

A more sensitive $1 nuclease mapping assay (Fig. 5 a) shows that gene 1 was weakly induced by heat shock in untreated cells, but was induced to at least 10-fold higher levels in cells incubated in the presence of hormone. In contrast, hsp26 transcripts accumulated to approximately equal amounts in cells grown in the presence or in the absence of ecdysterone (Fig. 5 b). In cells grown for 24 h in the presence of the hormone, however, the level of hsp26 mRNAs was already very high, and no further increase in their abundance could be observed after heat shock. Two classes of gene i transcripts were observed in Fig. 5. The first class represents the correctly initiated mRNAs (see below); synthesis of these RNAs was induced by ecdysterone, and showed an ecdysterone-dependent response to heat shock. The second class consists of transcripts initiating about 60 bp upstream from the major cap site. These transcripts were found only in heat-shocked cells. Transcription from this site in response to heat shock was independent of the presence of ecdysterone. An additional m R N A species initiating approximately 350 bp upstream was also detected.

Both species were also present at very low levels in pupae (Fig. 3).

Structure of gene 1 transcripts

Northern analysis of RNAs isolated from Kc cells revealed two major polyadenylated transcripts of gene i (see Fig. 4). Both transcripts were also occasionally observed when RNAs were extracted from whole organisms. The sizes of the transcripts were estimated to be 1.6 and 1.9 kb, by hybridizing the filter with probes specific for mRNAs of known sizes. $1 nuclease protection analysis was used to map the transcripts (Fig. 6). One major cap site was identified which corresponds to that already described (Ayme and Tissi6res 1985). A minor initiation site was found approximately 20 bp downstream. Minor upstream initiation sites were occasionally observed at about 60 and 350 nucleotides upstream from the major cap site (see Figs. 3 and 5; the strong, diffuse bands observed in pupae in Fig. 3 were never observed in other experiments and probably represent a migration artefact). A trinucleotide 5 ' - C A G - 3 ' , which is present at the cap site of many Drosophila genes, could be found at all four positions (Ayme and Tissi6res 1985).

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398

A B

M 1 2 3 4. 5 6 7 8 1 2 3 4. M

1566 - I101 ^-

6 2 ( - ...

5 2 7 -

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Fig. 6A and B. Structure of gene 1 transcripts. A The cap site of gene 1 mRNAs was determined by $1 mapping. One hundred mi- crogams of yeast tRNA (lanes 2, 6), or total RNA from heat shocked pupae (lanes 3, 7) or from control pupae (lanes 4, 8) were hybridized with an EcoR[- ClaI (lanes 2, 3 and 4) or a PstI- Clal (lanes 6, 7 and 8) dNA fragment 5' end-labelled with T4 polynucleotide kinase (see Fig. 1). The hybrids were digested with $1 nuclease and the protected fragments were run on a 5% polyacrylamide- 7 M urea thin sequencing gel. Lanes 1 and 5 were loaded with a sample of the EcoRI- ClaI and PstI- ClaI probes respectively.

Plasmid pBR322 was digested with MspI and the end-labelled restriction fragments were used as size markers (M). B St mapping of the polyadenylation site. One hundred micrograms of yeast tRNA (lane 1), total RNA from control pupae (lane 2), heat shocked pupae (lane 3) or 5 gg of poly(A) + RNA from Kcl61 cells heat shocked after incubation in 1 gM ecdysterone for 48 h (lane 4) were hybridized with an AccI-XbaI DNA fragment (see Fig. 1), 3' end-labelled by filling-in with the Klenow fragment of Escherichia eoli DNA polymerase. $1 analysis was as in A. The bands corresponding to the upstream (black arrow) or downstream poly(A) site (white arrow) are indicated

Two major polyadenylation sites were found (Fig. 6b) corresponding to the two major bands seen by Northern analysis. The larger mRNAs were predomi- nant in Kc cells and organisms. The use of two polyadenylation sites was confirmed by the sequencing of three cDNA clones (data not shown). The upstream site was mapped at position + 1330 of the sequence of Ayme and Tissi~res (1985). The downstream site was mapped at position -590 of the hsp26 gne (Southgate et al.

1983), which is approximately 420 nucleotides upstream from the hsp26 cap site. This polyadenylation site was also reported by Cohen and Meselson (1985). Both polyadenylation sites map close to polyadenylation consensus sequences (Southgate et al. 1983; Ayme and Tissi6re 1985). cDNA sequencing also confirmed the existence of transcripts initiating upstream from the major cap site (data not shown). Thus, several gene 1 mRNA species coexist in organisms or cultured cells. It is likely, although the formal proof is lacking, that transcripts initiated at each of the four identified cap sites may terminate at either polyadenylation site.

Discussion

A major element of the heat shock response is the increased expression of a small set of genes, the heat shock

genes. The heat shock response is believed to take place in most cells of an organism and during the whole of its development (for review see Nover 1984; Craig 1985;

Lindquist 1986). In Drosophila, the early stages of embryogenesis are the only known exceptions (Graziosi et al. 1980: Dura 1981). In addition, one trans-acting activator protein, the heat shock transcription factor, and functionally similar promoter sequences are believed to be responsible for the co-ordinate expression of the heat shock genes following a heat shock.

In this report is described the expression of gene 1, a small heat shock gene from locus 67B. In the absence of stress this gene was expressed during the early pupal stage and at a very low level in adult males. Heat shock led to a substantial increase in the amount of gene 1 mRNAs in pupae, but had little effect when applied at other stages, in particular in post-blastoderm embryos and adults. In contrast, hsp70 or hsp26 mRNAs accumulated to similar levels in all developmental stages. These results confirm and extend earlier observations (Ayme and Tissi6res 1985). Thus, response of gene 1 to heat shock appears to be modulated in a developmental stage-specific manner. The response of gene 1 to heat shock in Drosophila cultured cells was modulated by the molting hormone ecdysterone; induction of gene 1 by heat shock in cells incubated in the presence of the hormone was 10 to 20 times stronger than in untreated cells. The effect was not immediate: several hours of hormone treatment were required to affect the response to heat shock, suggesting that this was a secondary rath- er than a primary effect of the hormone.

Sequence analysis has revealed the presence of homologies to the heat shock consensus element in the promoter region of gene 1 (Ayme and Tissi6res 1985). Thus, it appears likely that response of this gene to heat shock is mediated by the same cis-acting sequences and trans- acting factors as the other heat shock genes. The ques- tion then arises of why this gene shows this peculiar mode of expression. The homologies to the heat shock consensus sequence in the 5'-region of gene 1 are very weak. The promoter of gene 1 could hence have a low affinity for the heat shock transcription factor. Binding of the HSTF might be enhanced if additional factors are already bound to DNA in the promoter region.

In support of this hypothesis is the finding that one heat shock element alone is not sufficient to confer full heat inducibility (Dudler and Travers 1985: Amin et al.

1985; 1988; Simon etal. 1985), suggesting that more than one heat shock factor must bind to a heat shock promoter to induce a high level of transcription. Fur- thermore, binding of the heat shock factor to a low affinity site was shown to be facilitated by a second factor bound to a neighbouring site (Topol et al. 1985), and interactions between several factors increase the level of expression (Cohen and Meselson 1988). In the case of gene 1, binding of HSTF to the promoter may be inefficient, or the bound HSTF molecules may not be sufficient to active transcription to detectable levels. The presence of additional factors could facilitate the binding of HSTF to the heat shock elements, or cooperate with the bound HSTF molecules to activate transcription.

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Both types of mechanisms have indeed been shown to play important roles in the regulation of transcriptional initiation (see for example Lichtsteiner and Schibler 1989; Tronche et al. 1989; Lin et al. 1990). Supporting this idea, Glaser and Lis (1990) have reported that full induction of hsp26 by heat shock required a sequence without homology to the heat shock element.

In a detailed analysis of DNAse I hypersensitivity at locus 67B, Kelly and Cartwright (1989) observed a major alteration of chromatin structure in the 5' regulatory region of gene 1 following exposure of Drosophila cultured cells to ecdysterone, but not after heat shock. Their results suggest that the HSTF alone is unable to bind to its regulatory sites on the gene i promoter region, or to modify the chromatin structure, in contrast to the other heat shock genes. The opening of the chromatin structure in response to ecdysterone treatment may facilitate the access of HSTF molecules to their binding sites.

Another interesting possibility is that heat shock-induced transcription of gene ! is mediated by remote heat shock elements, for example those that normally control the expression of the divergently transcribed hsp26 gene, which would in this case act as enhancers. It has indeed been shown that heat shock elements can act as enhancers (Bienz and Pelham 1986). This would require the gene 1 promoter to be in a responsive state, which would best be achieved at certain developmental stages or in the presence of ecdyterone, when gene 1 is expressed.

The results presented here do not rule out the possibility that the particular pattern of expression of gene ! is achieved at the post-transcriptional level. It has indeed been shown that heat shock can stabilize heat shock mRNAs (Petersen and Lindquist 1988). However, several transcriptional initiation sites were shown to be used in cultured cells. The abundance of the heat shock-induced transcripts initiated at the (major) downstream site was greatly affected by the presence of ecdysterone, while the abundance of the minor transcripts initiated at the upstream sites was similar in hormone-treated and in untreated cells. These results argue for a regulatory mechanism that operates at the level of transcription.

Another difference between gene 1 and the other heat shock genes is its relatively low level of expression in cultured cells as compared to organisms. Accumulation of hsp26 mRNAs, for example, is very similar in Kc161 cells and in whole organisms. Gene 1, however, is expressed at a very low level in Kc cells after ecdysterone induction (about 2% the level of hsp26), similar to that observed in adults, but at least one order of magnitude lower than in pupae, where gene 1 is expressed at about 20% the level of hsp26. Thus, some factor(s) present in pupae but absent or inactive in the other stages and in cultured cells must be responsible for the elevated level of expression of gene l in pupae, and for the enhanced response to heat shock. Such a factor or factors must be acting at least at the transcriptional level, since transcriptional activity at gene 1 could be detected by run-on analysis in nuclei isolated from pupae, but not in nuclei isolated from embryos or Kc cells (J. Vazquez, unpublished data). Structural and functional analysis of

the gene 1 regulatory sequences may be ultimately required to understand the mechanism involved in its particular mode of expression.

Acknowledgements. I am particularly grateful to Alfred Tissi6res for his continuous interest and support; and to Alfred Tissi6res, Pierre Spierer and Martina Buck for critical reading of the manu- script. I am also greatiy indebted to Fabienne Bujard and Otto Jenni for excellent artwork and photography. This work was supported by a grant from the Swiss National Science Foundation to A. Tissi6res, and by the Canton Geneva.

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