Deficient activation of heat shock gene transcription in embryonal carcinoma cells

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Deficient Activation of Heat Shock Gene Transcription in Embryonal Carcinoma Cells VALBRIEMEZGER,'OLIVIERBENSAUDE,ANDMICHELMORANGE Laboratoire de Biologie Molbculaire du Stress, Dkpartement de Biologie Molbulaire, Institut Pasteur, 25 rue du Dr Roux, Y5Y24Paris Cedex 15, France Received May 18, 1987; accepted in revised fomn August 3, 1987 Heat shock protein (HSP) synthesis cannot be induced by stress in the cleavage stage embryos of many different species. For instance, no HSP synthesis can be induced in the mouse embryo before the formation of the blastocyst. Similarly, HSP synthesis is not stress inducible in some embryonal carcinoma (EC) cell lines such as PCC4 and PCC7-S-1009 (1009). We show that RNAs coding for the major stress inducible murine heat shock protein, HSP68, do not accumulate in PCC4 or 1009 EC cells in response to a stress. Using an in vitro nuclear transcription assay, we demonstrate that the transcription of the corresponding genes is not activated after a stress. A specific gene switch-off due to DNA methylation or chromatin conformation is unlikely to account for this result. Indeed, stress does not promote the activation of the heterologous Drosophila HSP’IO heat shock promoter in transfection assays of these cells. In contrast, the same promoter, like endogenous HSP synthesis, becomes stress inducible in 1009 cells after in vitro differentiation. This suggests that, in contrast to differentiated cells, these EC cells, and maybe the very early mouse embryonic cells, could lack a transacting activating transcription factor or contain a repressor. o 1987 Academic PWS, h.

with a conserved sequence (HSE) found upstream the A wide range of stresses (heat shock, anoxia, arsenite, TATA box (Top01 et ah, 1985; Wu, 1984). The induction etc.) induces the synthesis of a very conserved set of mechanism is highly conserved. A Drosophila sequence polypeptides in eucaryotic and procaryotic cells: the which contains both promoter and regulatory upstream heat shock proteins (HSPs) (Schlesinger, 1986). How- elements of the HSP70 genes, can confer heat inducibilever, induction of HSPs cannot be triggered by any ity upon any gene located downstream from it, in stress in very early (cleavage stages) embryos (Bienz, transfected mammalian cells. A translational control is found in Xenopus oocytes 1985; Heikkila and Schultz, 1984). In the mouse, expres(Bienz and Gurdon, 1982) and chicken reticulocytes sion of the zygotic genome initiates at the two-cell stage (Banerji et ah, 1984). During oogenesis and reticulocyte with a spontaneous synthesis of HSP68 and HSP’IO (the 68 and 70kDa HSP) (Bensaude et al., 1983). However, differentiation, HSP70 encoding mRNAs accumulate in synthesis of HSP68 is transient and restricted to the significant amounts but are not translated into protein. A heat shock is required to trigger the corresponding two-cell stage. At the eight-cell and early morula HSP70 synthesis. In the cleaving mouse embryo, it stages, no spontaneous HSP68 synthesis is observed might be questioned whether the absence of heat inand stress does not induce its synthesis (Wittig et al., duction of HSP68 was due to a lack of transcriptional 1983; Morange et al., 1984; Muller et ab, 1985). Induction activation of the corresponding genes, or whether the of HSP68 by stress initiates at the blastocyst stage and RNAs coding for HSP68 are synthesized but are deis a general property of almost all differentiated cell graded or remain untranslated. types (except the plasmacytoma cells; Aujame and Mouse embryonal carcinoma cell lines (EC) display Morgan, 1985). HSP68 is the major heat-induced mubiochemical, immunological, and some of the differenrine HSP. tiation properties of the cells from the preimplantation In most cells, activation of HSP gene transcription is mouse embryo, and are thus used as a model system to required for induction of HSP synthesis. It is correlated study gene expression during early embryogenesis (Niwith an alteration in the metabolism of ubiquitin (for colas et al, 1976; Pfeiffer et al, 1981; Strickland, 1981). reviews, see Pelham, 1985; Munro and Pelham, 1985; Induction of HSPs is never obtained in some EC cell Lindquist, 1986; Schlesinger, 1986). In Drosophila, a lines, such as PCC4 or PCC7-S-1009 (referred to as 1009 transcription factor (HSTF) has been shown to interact in the text) when propagated under the undifferentiated state (Wittig et al., 1983; Morange et al., 1984). ’ To whom correspondence should be addressed. After in vitro differentiation, a usual pattern of inducINTRODUCTION

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tion is observed. In another EC cell line, F9, HSP68 is strongly stress inducible even in the nondifferentiated state. A mouse genomic DNA fragment has been isolated by hybridization with a Drosophila HSP70 gene by Hunt and Morimoto (to be published). We show that this fragment corresponds to a gene coding for HSP68. This probe has been used to examine RNA accumulation (Northern blots) or transcription rate (nuclear run-on assays). Stress fails to induce HSP68 gene transcription in PCC4 cells. Transfection experiments with a fusion gene containing the heat shock promoter of Drosophila HSP70 gene show that heat shock promoter is not active in PCC4 or 1009 EC cells, while it is in 3T6 or differentiated 1009 cells. These results suggest that in such EC cells (and perhaps in the very early embryo), the transacting factors required for heat shock gene transcription enhancement are absent or inactive, or that there is a repressor. However, the normal positive control of heat shock gene transcription would be established during differentiation.

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Samples of labeled proteins were analysed by electrophoresis through 10% polyacrylamide gels containing 0.1% SDS (Laemmli, 1970). Fluorography was carried out according to Bonner and Laskey (1974). Plasmids

Plasmid pM1.8 was provided by R. Morimoto. It contains a genomic DNA fragment isolated from a murine genomic lambda phage library by heterologous hybridization with the Drosophila HSP70 gene. The genomic fragment is inserted at the EcoRI and BamHI sites of pBR322. It contains 1.4 kbp of coding sequence, 120 bp of transcribed but untranslated sequence, and about 600 bp upstream of the transcription initiation site (Hunt and Morimoto, to be published). Plasmid pAL41 was provided by S. Alonso. It was isolated from a mouse lymphocyte cDNA library and contains a 1.15 kb p nonmuscle actin cDNA fragment (Alonso et al., 1986). Plasmid HSP-CAT was constructed by P. Herbomel. It contains the fragment from nucleotide -440 to nuMATERIALS AND METHODS cleotide 171 of the distal Drosophila HSP70 gene of the locus 87Cl (provided by H. Pelham), inserted between Cell Culture, Stress, and Labeling the Sal1 and Hind111 sites of plasmid pSB1 (Herbomel EC cells were grown at 37°C in tissue culture dishes et al., 1984). with Dulbecco’s modified Eagle’s medium (DMEM) Plasmid pCHll0 expresses the bacterial P-galactosisupplemented with 15% fetal calf serum in a 12% COZ dase under the control of the SV40 early promoter (Hall atmosphere. F9 EC cells were plated on gelatin-coated et al., 1983). tissue culture dishes. F9 and PCC4-Aza Rl are derived from the same teratocarcinoma OTT6050 (obtained by RNA Analysis transplantation of a 6.5-day-old embryo). PCC7-S-1009 is derived from a spontaneous teratocarcinoma in testes RNAs were prepared according to Aufl’ray and Rouof a 129xC57B/6J mouse. For in vitro differentiation geon (1980). The hybridization selection technique of experiments, retinoic acid (Sigma Chemical Co.) was Ricciardi et al., (1979) with modifications suggested by added at 0.2 j&f final concentration and 1 day later 1 Fyrberg (1981) was followed. The plasmid pM1.8 was mM dibutyryl CAMP was added for 4 more days with a linearized by EcoRI digestion. Ten micrograms of plasmedium change every 48 hr. 3T6 cells were grown with mid was hybridized to 100 pg of total RNAs isolated from arsenite-treated F9 cells 3 hr after the end of the 10% FCS. For heat shock, sealed culture dishes were immersed treatment. The hybrid-selected RNAs and total RNAs in a 45°C waterbath for 10 min. Recovery times at 37°C were in vitro translated in a rabbit reticulocyte lysate are specified in the figure legends. system as described in the Amersham procedure. For Arsenite treatment was performed by exposing the Northern blot analysis, total RNAs were separated cells for 1 hr to 150 &kf sodium arsenite followed by a through 7% formaldehyde, 1.4% agarose gel and transmedium change. Recovery times are specified in the ferred onto a nitrocellulose filter (Schleicher and &hull, BA-85) according to Maniatis et al. (1982). Prefigure legends. Cells were labeled for 1 hr with 100 &i/ml of y5S]- hybridization and hybridization were performed as demethionine (1200 Ci/mmole; Amersham Corp.) in me- scribed. Filters were washed in 2X SSC (0.3 M NaCl, thionine-free medium at 37°C. After labeling, cells were 0.03 M sodium citrate), 0.1% SDS at room temperature, washed twice with serum-free medium and then har- then in 1X SSC, 0.1% SDS at 65”C, and finally in 0.2X vested with a rubber policeman into Laemmli sample SSC, 0.1% SDS at 65°C. Autoradiography was perbuffer (Laemmli, 1970) for polyacrylamide gel electro- formed at -70°C with a Kodak X-OMAT film and an intensifying screen. phoresis analysis.

DEVELOPMENTALBIDLOGY

546 Nuclear Run-on Transcription

Assay

Nuclei preparation was based on methods described by Groudine et al. (1981), Dony et al. (1985), Greenberg and Ziff (1984), and Linial et al. (1985). Subconfluent culture dishes (lo* cells) were rinsed twice with cold phosphate-buffered saline, lysed, and scraped into 2 ml of lysis buffer (0.01 M Tris, pH 7.4; 0.01 M NaCl; 3 mM MgClz; 7.5 mM DTT) containing 0.5% Nonidet-P40 (NP-40) for 3T6 nuclei, or without NP-40 for PCC4 nuclei. Cell membranes were disrupted in a Dounce homogenizer, and nuclei were pelleted at 1OOOgfor 4 min and resuspended in 1 ml of lysis buffer containing 10 pg ml-’ of RNase A. After 30 min on ice the nuclei were centrifuged through a 3-ml cushion of 30% (w/v) sucrose in lysis buffer. The pellet, which contained the nuclei, was resuspended twice in 1 ml of storage buffer (40% glycerol; 50 mMTris, pH 8.3; 5 mM MgClz; 0.1 mM EDTA; 10 mMDTT) and centrifuged at 2500 rpm for 4 min. Nuclei were stored in 100 ~1 of the same buffer at -70°C for further use. One hundred microliters of nuclear suspension was mixed with 100 ~1 of a solution containing 10 mM MnC12, 10 mM Tris, pH 8.0,2.5 mM MgClz, 300 mM KCl, 0.5 mM ATP, 0.25 mM GTP, 0.25 mM CTP, 100 &i [a32P]UTP (3000 Ci mmole -l; Amersham), 20 m&f phosphocreatine (Sigma), and 100 pg creatine phosphokinase (Sigma). After 20 min at 3O”C, nuclei were pelleted at 2500 rpm for 4 min, lysed in an equal volume of high-salt buffer (0.6 M NaCl, 0.06 M MgClz, 12 mM Tris, pH 7.4), 20 ~1 of RQl DNase (1 U/& Promega Biotec), and 1 ~1 of ribonuclease inhibitor for 2 min at room temperature. The reaction was stopped by adding 5 ~1of 0.5 M EDTA and 10 ~1 SDS 10% (w/v). RNAs were then extracted with phenol:chloroform (l:l), followed by a chloroform extraction and precipitation with ammonium acetate (2.5 M) and 2.5 vol of ethanol. The precipitate was resuspended in 400 ~1 of hybridization buffer (50% deionized formamide, 1% SDS, 3~ SSC, 5X Denhardt’s solution, 1 mM sodium pyrophosphate, pH 7.0, 10 mM sodium phosphate, pH 7.0, 50 pg/ml of yeast tRNA). Typical experiments gave between 2 X lo6 and 4 X 10’ cpm for total incorporation of [cY~~P]UTP.Filters were prepared as follows: pM1.8 was digested by EcoRI and BamHI in order to generate 5 pugof the 2-kb insert. pAL41 was digested by PstI in order to generate 5 pg of the 1.15 kb P-actin insert. The two reaction mixtures were mixed together and the different fragments were separated by a 1% agarose gel electrophoresis. The gel was blotted onto a N-Hybond membrane (Amersham). Prehybridization was performed in hybridization buffer for 48 to 72 hr at 42°C. After a 48-hr hybridization at 42°C filters were washed in 2X SSC for 1 hr at

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65”C, and in 2X SSC, 10 pg/ml RNase A (Sigma) for 30 min at room temperature. Filters were autoradiographed. Densitometer scannings of the autoradiograms provided an estimate of the relative transcription levels. Transfection

Experiments

The 3T6, PCC4, or 1009 cells (subconfluent cultures of 60-mm dishes) were cotransfected with 2 pg of pCHll0 and 8 pg of HSP-CAT according to the procedure described by Graham and van der Eb (1973) and modified by Parker and Stark (1979). Twenty-four hours after transfection, cells were submitted to a heat shock (10 min at 45°C) and analyzed (as described by Willer (1972)) for ,&galactosidase activity after 5 hr (1009 cells) or 6 hr (PCC4 and 3T6 cells) which are the times at which the activity was found to be highest. CAT expression assay was performed as described by Gorman et al. (1982) and the amount of cell extract utilized was normalized for @-galactosidase activity. Variations between a heat-shocked extract and a control cell extract were found to be insignificant when extracts were isolated 5 hr after stress. RESULTS

The Amount of HSPSS RNA Does Not Increase after Stress in PCC& EC Cells

The pM1.8 plasmid, isolated by hybridization with a HSP70 gene, recognizes strongly heat-inducible RNAs in mouse fibroblasts (Hunt and Morimoto, to be published). In F9 cells, where HSP68 synthesis is strongly stress inducible, pM1.8 recognizes a set of strongly inducible RNAs as determined by Northern blot analysis (Fig. 1). This suggests that pM1.8 corresponds to a gene coding for HSP68. RNAs from stressed F9 cells were hybrid selected on pM1.8 and translated in a reticulocyte lysate system. The only protein detected in the translation products of these RNAs was a 68 kDa protein (Fig. 3, lane 2) as shown by comparison with the in vitro translation pattern of total RNAs isolated from arsenite-treated F9 cells (Fig. 2, lane l), and with the in vivo protein synthesis pattern of arsenite-treated and control F9 cells (Fig. 2, lanes 3 and 4). Therefore pM1.8 appears to recognize RNAs coding for HSP68. We were unable to detect RNAs recognized by pM1.8 in stressed PCC4 EC cells. In contrast, large amounts of RNAs hybridized with pM1.8 in arsenite-treated F9 cells (2.7 and 3.3 kb) and in heat-shocked 3T6 cells (Fig. 1).

Drosophila

MEZGER,BENSAUDE, ANDMORANGE

A. Fg CASC

PCC4

B. 3T6 HS

C

3.3 kb 2.7 kb’

FIG. 1. Northern blot analysis of RNAs isolated from (A) F9 and PCC4 cells, and (B) 3T6 cells. mP-Labeled pM1.8 was used in hybridization. C, control cells; AS, arsenite-treated cells allowed to recover for 3 hr; HS, cells heat shocked for 10 min at 44°C and allowed to recover for 3 hr at 37°C.

Northern blot analyses with pM1.8 thus did not reveal any increase in the amount of HSP68 RNA present in PCC4 EC cells after a stress. The Transcription Rate of the HSP68 Genes Is Not Increased by Stress in PCCJ Cells

In order to determine whether the absence of HSP68 RNAs in arsenite-treated or heat-shocked PCC4 cells was due to a failure in transcription, the transcription rate of the HSP68 genes was estimated using the pM1.8 probe in a nuclear run-on assay. Transcription of actin genes was used as an internal control. In run-on assays, transcription cannot be initiated but transcripts that have been initiated in vivo are elongated, thus giving an estimate of the transcription rate at the time of cell lysis. We analyzed the variation of gene transcription in samples containing the same number of nuclei. In nuclei from 3T6 control cells, the transcription level of the HSP68 genes corresponded to about 20% that of the actin genes (Fig. 3a). The general transcription level was severely affected for several hours after a heat shock. Nuclei isolated from 3T6 cells between 0 and 30 min after the end of the heat shock showed a progressive and strong increase in the transcription of HSP68 genes, while the transcription of the actin genes decreased considerably. The transcription of the HSP68 genes was stimulated more than 9 times 30 minutes after the end of the shock and was still high 2 hr after heat shock (1.5fold that of actin gene transcription; Fig. 3b). In contrast, immediately after the end of the shock, the transcription of the actin genes was reduced 10 times and recovered slowly: 30 min after heat shock, it was expressed at only 30% of its value before stress,

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and it was not fully recovered 2 hr after the end of the shock (not shown). The level of transcription of the HSP68 genes was 25% of the actin gene transcription in nuclei isolated from PCC4 control cells. No stimulation of the HSP68 genes was detected after a heat shock (Fig. 3a). The HSP68 gene transcription followed that of the actin genes (Fig. 3b): both genes showed a decrease in their transcription rates. Thus, HSP68 gene transcription in PCC4 cells behaved like that of a nonheat shock gene and followed the general variation in the level of total RNA transcription (Fig. 3b). The Cat Gene under the Control of the Regulatory Region of the Drosophila HSP70 Gene Is Not Inducible in PCC4 EC and Undifferentiated 1009 EC Transfected Cells

The defect of transcription activation of HSP68 genes by stress could result from the specific switch-off of these genes in EC cells (by DNA methylation or chromatin conformation, for example). Although the transacting transcription factors required for induction are present and active, HSP68 gene transcription would not be increased by stress, whereas the transcription of an indicator gene under the control of an unmodified heat shock promoter would be activated in transfected EC cells after a stress. Alternatively, the heat shock promoter could be inactive in PCC4 and 1009 EC cells. To test these hypotheses, 3T6 and PCC4 cells were cotransfected with plasmids pCHll0 and HSP-CAT (see Materials and Methods) and gene expression was 0

12

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70 6&l=

FIG. 2. Identification of the coding properties of RNAs hybridizing with pM1.8 plasmid. Gel electrophoresis analysis. Lanes 0 to 2, in vitro translation products; (0) no RNA added; (1) total RNAs from arsenite-treated F9 cells; (2) RNAs from arsenite-treated F9 cells hybridselected with pM1.8. Lanes 3 and 4, [%]methionine incorporation in proteins of F9 cells after in wivo labeling; (3) arsenite-treated cells; (4) control cells. Numbers indicate the position of 68 and 70 kDa HSPs.

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(a) Autoradiograms of the hybridization of 32P-labeled run-on transcripts to FIG. 3. Run-on assay of the actin and HSP68 gene transcription. PBR322 sequences, pAL41 p-a&in cDNA, and pM1.8 HSP68 probe. Nuclei were isolated from 3T6 or PCC4 cells without heat shock or at various times (0,15,30, 60 min) after a 15-min heat shock at 45°C. (b) Ratio of HSP68 gene transcription to p-actin gene transcription. (m) PCCI. (0) 3T6.

assayed with or without heat shock. The P-galactosidase gene was expressed in PCC4 cells as well as in 3T6 cells. The different samples were normalized to the same value of P-galactosidase activity to correct for the variations in DNA uptake. 3T6 or PCC4 transfected control cells did not show more CAT activity than mock-transfected cells (Figure 4a, lanes C compared to lanes M). In 3T6 transfected cells, CAT activity was markedly increased after a heat shock (by about 100 times as determined by densitometer scanning of the A.

B.

3T6 .w- PCC4 - -~~ 0 CATM C HS M C HS 0 I,

a ic

autoradiogram; Figure 4a, 3T6, lane HS). In contrast, no induction of CAT activity was detected in PCC4 transfected EC cells after heat shock when compared to control cells (Fig. 4a, PCC4, lanes C and HS). PCC4 cells do not differentiate in vitro. The defect in heat shock response might be linked with the undifferentiated state of PCC4 cells or be due to a mutation having occurred during the establishment of this cell line. We looked for another EC cell line which displays a similar phenotype relative to the heat shock response

1009

c- 1009 DIF CAT 0 M C HS

CAT 0

M C HS

FIG. 4. CAT expression under the control of the Drosophila HSP70 promoter after transfection. (a) In 3T6 cells and in PCC4 cells. (b) In undifferentiated 1009 EC cells. (c) In differentiated 1009 cells. 0, No extract added; CAT, 0.01 U of CAT (P. L. Biochemicals); M, mock-transfected cells; C, control transfected cells; HS, heat-shocked transfected cells (10 min at 45°C). The amount of extracts used for each point was normalized to the same P-galactosidase activity.

MEZGER,BENSAUDE,AND MORANCE

and which can differentiate in vitro. HSP synthesis was not stress inducible in 1009 EC cells (Morange et aZ., 1984) and the Drosophila HSP’IO promoter did not confer heat inducibility on the CAT gene in transfected cells (Fig. 4b). In the differentiated 1009 cells, however, CAT expression was as strongly stress inducible as in fibroblasts (Fig. 4~). DISCUSSION

Run-on transcription experiments did not reveal any increase in the HSP68 gene transcription in PCC4 cells in response to a stress. This lack of induction could be explained by different mechanisms. For instance, one could imagine that the HSP68 genes in PCC4 cells are specifically switched off and maintained in a chromatin conformation that does not allow interactions with transcriptional factors involved in the induction (such as HSTF). This hypothesis seems unlikely since, (i) HSP68 is not the only heat shock protein to be uninducible in early embryonic cells, the 105 kDa (described bv Subieck and Sciandra. 1982) is not induced bv stress either IMorange et a& i984),‘and recent unpiblished data from our laboratory indicate that ubiquitin RNAs are stress inducible in fibroblasts but not in PCC4 cells; (ii) run-on transcription assays showed a low but significant level of transcription for HSP68 genes in both control and stressed PCC4 cells. This suggests that these genes are in an open conformation. Another hypothesis could be that heat shock promoters in PCC4 and 1009 EC cells are inactive because of the lack of transacting activating factors or the presence of a repressor. Transfection experiments support this hypothesis: the heterologous promoter from a Drosophila HSP70 gene which confers efficient stress inducibility upon the CAT gene in 3T6 fibroblasts does not direct transcription in PCC4 stressed cells. The 1009 cells display the same phenotype, and the Drosophila heat shock promoter does not confer heat inducibility to the CAT gene after transfection in these cells. In contrast, both endogenous HSP68 and HSP105 (Morange et aZ.,1984) and the exogenous CAT gene become strongly inducible upon 1009 cell differentiation. Thus, a functional induction system is apparently established during 1009 differentiation. Although the hypothesis of a repressor cannot be excluded, one explanation could be that PCC4 or 1009 EC cells fail to produce an active form of HSTF or some other factor required for the transcription of HSP genes in response to heat shock, whereas the level of active factor in 3T6 cells or in differentiated 1009 cells would be sufficient to trigger the transcription of HSP genes. Generalizing our results obtained with EC cells, we propose a model to explain what happens during early

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embryogenesis. In the two-cell embryo, genes coding for HSP68 are heavily transcribed in a stress-independent manner (Bensaude et ah, 1983). At this stage, a specific activation mechanism might be involved, or transcription factors from maternal origin might be used. At the eight-cell and early morula stages, HSP68 is no longer synthesized, even after stress. The lack of HSP68 synthesis might be due to the degradation of material from maternal origin which has taken place before this stage of development (Flach et aL, 1982). A sequential appearance in the inducibility of the different HSP genes is observed during the formation of the blastocyst. During this process, the zygotic genome would contribute to a gradually increasing level of functional heat shock gene activator. We are much indebted to Dr. Richard Morimoto for providing us with plasmid pM1.8. We also thank Philippe Herbomel for plasmid HSP-CAT. We thank Tony Pugsley for reading the manuscript, Marc Dreyfus, Philippe Duprey, Jean-Jacques Panthier, Francois Jacob, and all the members of the laboratory of Biologie Molbculaire du Stress for fruitful discussions. This work was supported by grants from the Direction des Recherches Etudes et Techniques (DC16gation G&&ale pour 1’Armement) (n” 84-1’74) and from the Association pour la Recherche contre le Cancer (ARC 6250). REFERENCES ALONSO, S., MINTY, A., BOURLET, Y., and BUCKINGHAM, M. (1986). Comparison of the three actin-coding sequences in the mouse: Evolutionary relationships between the actin genes of warm-blooded vertebrates, J. Mel Evol. 23, 11-22. AUFFRAY, C., and ROUGEON,F. (1980). Purification of mouse immunoglobulin heavy-chain messenger RNA from total myeloma tumor RNA. Eur. J. Biochem 107,303-314. AUJAME, L., and MORGAN,C. (1985). Nonexpression of a major heat shock gene in mouse plasmacytoma MPC-11. Mol. Cell. Biol. 5, 1780-1783. BANERJI, S. S., THEODORAKIS,N. G., and MORIMOTO,R. I. (1984). Heat shock-induced translational control of HSP 70 and globin synthesis in chicken reticulocytes. MoL Cell Biol 4,2437-2448. BENSAUDE, O., BABINET, C., MORANGE, M., and JACOB, F. (1983). Heat-shock proteins, first major products of zygotic gene activity in mouse embryo. Nature (London) 305,331-333. BIENZ, M., and GURDON,J. B. (1982). The heat-shock response is selfregulated at both transcriptional and post-transcriptional levels. Cell 29.811-819. BIENZ, M. (1985). Transient and developmental activation of heatshock genes. Trends Biochem Sci. 10,157-161. BONNER, W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. B&hem

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HALL, C. V., JACOB,P. E., RINGOLD,G. M., and LEE, F. (1983). Expression and regulation of Escherichia Coli lo& gene fusions in mammalian cells. J. Mol. Appl. Genet. 2, 101-109. HEIKKILA, J. J., and SCHULTZ,G. A. (1984). Different environmental stresses can activate the expression of a heat shock gene in rabbit blastocysts. Gamete Res. 10,45-56. HERBOMEL,P., BOURACHOT,B., and YANIV, M. (1984). Two distinct enhancers with different cell specificity coexist in the regulatory region of polyoma. Cell 39, 653-662. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (W) 227, 680-685. LINIAL, M., GUNDERSON,N., and GROUDINE, M. (1985). Enhanced transcription of c-myc in bursal lymphoma cells requires continuous protein synthesis. Science 230,1126-1132. LINDQUIST,S. (1986). The heat shock response. Annu. Rev. B&hem. 55,1151-1191. MANIATIS, T., FRITSCH, E. F., and SAMBROOK,J. (1982). “Molecular Cloning. A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MORANGE,M., DIU, A., BENSAUDE,O., and BABINET, C. (1984). Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells. Mol. Cell. BioL 4, 730-735. MULLER, W. U., LI, G. C., and GOLDSTEIN,L. S. (1985). Heat does not induce synthesis of heat shock proteins or thermotolerance in the earliest stage of mouse embryo development. Int. J Hyperthermia 1,97-102.

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