The expression of microinjected chimeric genes containing Drosophila hsp 70 and Xenopus hsp 70 and hsp 30 promoters linked to the reporter gene coding for bacterial chloramphenicol acetyltransferase (CAT) was examined during early development of Xenopus laevis. Heat-inducible expression of fusion genes containing either the Drosophila hsp 70 promoter (1100 bp) or the Xenopus hsp 70 promoter (750 bp) was first detectable after the midblastula stage of development. This coincides with the embryonic stage at which the endogenous hsp 70 gene is first heat-inducible. A Xenopus hsp 30/CAT fusion gene containing 350 bp of promoter sequences was also heat-inducible after the midblastula stage unlike the endogenous hsp 30 genes which were not heat-inducible until the early tailbud stage (stage 23– 24). Sequences that are present within either the coding or 3’ region of the hsp 30 clone do not cause the microinjected hsp 30 gene to be developmentally regulated in a normal manner. Additionally, microinjected hsp 30 gene sequences have no effect on the developmental regulation of endogenous hsp 30 genes which continue to be activated at the tailbud stage of development. Our data suggest, that an inhibitory system, which may control the expression of the endogenous hsp 30 gene during development, does not regulate the expression of the injected hsp 30 gene.

Exposure of prokaryotic and eukaryotic cells to environmental stress such as elevated temperatures, heavy metals or arsenite results in the synthesis of a highly conserved set of proteins known as heat-shock proteins (hsps; reviewed by Nover, 1984; Atkinson & Walden, 1985; Craig, 1985; Burdon, 1986; Lindquist, 1956). Developmental stage-dependent regulation of hsp gene expression during early embryogenesis has been demonstrated in a variety of animal systems (reviewed by Heikkila et al. 1985b, 1986). For example, during Drosophila development, heat-induced hsp synthesis was not observed until embryos reached the blastoderm stage (Graziosi et al. 1980; Dura, 1981). Also, it has been shown that mouse and rabbit embryos are incompetent for heat-induced hsp synthesis during the early cleavage stages of development but do synthesize hsp 70 at the blastocyst stage (Heikkila & Schultz, 1984; Heikkila et al. 1985b). During early embryogenesis of Xenopus laevis, heat-inducible expression of hsp 70, hsp 87 and ubiquitin genes is first detectable in embryos which have reached the midblastula stage of development (Bienz, 1984a; Heikkila et al. 1985a, 1987; Nickells & Browder, 1985; Ovsenek & Heikkila, 1988), a point that coincides with transcriptional activation of the embryonic genome (Newport & Kirschner, 1982a,b). In contrast, Xenopus hsp 30 genes are not heat-inducible until the tailbud stage of development (Krone & Heikkila, 1988).

An effective technique for the examination of cis-acting sequences involved in developmentally regulated transcription is the introduction of reporter gene constructions into developing embryos. This approach has proven effectual in the identification of sequences involved in the developmental regulation of a number of genes in Drosophila (Spradling & Rubin, 1983; Goldberg et al. 1983), sea urchin (Flytzanis et al. 1987; Hough-Evans et al. 1987; Franks et al. 1988) and mouse (Palmiter & Brinster, 1985; Krumlauf et al. 1985). The fate and expression of DNA microinjected into developing Xenopus embryos has been extensively studied (Etkin et al. 1984; Etkin et al. 1987; Etkin & Pearman, 1987; Marini et al. 1988) and has recently been used to identify an enhancer element (Krieg & Melton, 1985; 1987) as well as putative inhibitory sequences (Steinbeisser et al. 1988) implicated in the developmental regulation of several Xenopus genes.

In the present study, we have sought to identify sequences involved in heat-inducible and developmental’ regulation of hsp 70 and hsp 30 genes during Xenopus embryogenesis. We have found that fusion genes containing either 1100 bp of the Drosophila hsp 70 promoter or 750 bp of the Xenopus hsp 70 promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) gene are first heat-inducible after the mid-blastula stage in microinjected Xenopus embryos. Interestingly, an hsp 30/CAT fusion gene containing 350 bp of promoter also becomes heat-inducible at the midblastula stage, which is earlier during development than is found for the expression of the endogenous hsp 30 genes. Sequences that are present within the hsp 30 coding sequence or in the region downstream of the gene do not confer proper developmental regulation on a microinjected Xenopus hsp 30 gene. Furthermore, microinjected Xenopus hsp30 gene sequences have no effect on the developmental regulation of endogenous hsp 30 genes, which continue to be activated at the tailbud stage of development. Our data suggest that an inhibitory mechanism that may control expression of the endogenous hsp 30 genes during development does not regulate the expression of the injected hsp 30 gene.

Plasmid constructions

To minimize variability that may result from the plasmids containing different bacterial vector sequences, all microinjected constructs used in this study were derived from pUC 13. DNA fragments used for cloning were purified by excision from low melting point agarose following electrophoresis. The 2·1 kb BamHl/Hmdlll fragment from pXS43B (gift of Dr M. Bienz, Zoological Institute, University of Zurich-Irchel, Zurich, Switzerland), which contains an entire hsp 30 gene (Bienz, 1984b), was cloned into BamHI/Hmdllldigested pUC 13 to give the plasmid p3013. A promoterless hsp 30 construct (pX303) was produced by cloning of the 1·6 kb Pstl/Hindlll fragment from p3013 containing the coding and 3’ regions of the hsp 30 gene into pUC 13. Plasmid pCA13 was constructed by insertion of the 780 bp Hzndlll fragment from pCM 7 containing the bacterial CAT gene (Close & Rodriguez, 1982; Pharmacia) into pUC 13. The 500 bp hsp 30 promoter sequence from p3013 was excised by digestion with BamHI and PstI and cloned into BamHI/Bstldigested pCA 13 to give pXL30CA. In order to clone the SV40 polyadenylation site downstream of the CAT gene, the 750 bp BomHI/EcoRI fragment from pXL30CA containing the hsp 30 promoter as well as the first 250 bp of the CAT gene was inserted between the BamIII and EcoRI sites of pUC 13. The rest of the CAT gene as well as the SV40 polyadenylation site contained in the 2·2 kb EcoRI fragment of Drosophila hsp 70/CAT (gift of Dr I. Dawid, NIH, Bethesda, MD.; DiNocera & Dawid, T983) was cloned into the EcoRI site of the resulting construct to give pXL30CB. This construct contains approximately 350 bp of DNA upstream of the hsp 30 transcriptional start site as well as 143 bp of untranslated leader sequence.

In order to construct a Xenopus hsp 70/CATfusion gene, a 750 bp hsp 70B promoter fragment containing 33 bp of untranslated leader sequence was excised from the plasmid pXL16P (gift of Dr M. Bienz; see Bienz, 1984b) by Hin dII /Bg/HI double digestion. A promoterless CAT/SV40 construct (pCB 13) was produced by digestion of pXL30CB with Efindlll. The protruding ends of both digestion products were filled in with Klenow DNA polymerase I before the Xenopus hsp 70 promoter sequence was blunt-end ligated into the promoterless CAT/SV40 vector to give pXL70CB.

For the synthesis of antisense hsp 30/CAT RNA, the 750 bp BamHl/EcoRI fragment from pXL30CB, which spans the transcriptional start site of the hsp 30 promoter, was cloned into the vector pGEM-2 (Promega) to give pG230CB. The 750 nucleotide antisense RNA probe produced by SP6 RNA polymerase transcription of this construct protects a 402 nucleotide correctly initiated hsp 30/CAT transcript as well as a 143 nucleotide portion of endogenous hsp 30 mRNA. For the analysis of hsp 70/CAT mRNA, the lkb PstI/EcoRI fragment from pXL70CB was cloned into pGEM-2 to give the plasmid pG270CB. The 1000 nucleotide antisense probe produced by SP6 RNA polymerase transcription of this construct protects 288 nucleotides of a correctly initiated hsp 70/CAT transcript.

Embryo and tadpole manipulation

Xenopus laevis eggs were obtained, fertilized and dejellied by the methods of Heikkila et al. (1985a). Embryos were staged by external criteria according to Nieuwkoop & Faber (1967). For microinjection experiments, dejellied embryos were transferred to Steinberg’s solution containing 4% Ficoll (Krieg & Melton, 1985) and microinjected with 20 nl of DNA solution at the 1-to 2-cell stage using the apparatus described by Hitchcock & Friedman (1980). Unless otherwise stated, embryos were injected with supercoiled plasmid DNA prepared by an alkaline lysis procedure and further purified by excision from low-melting-point agarose after electrophoresis. Following microinjection, embryos were maintained in Steinberg’s solution containing 4 % Ficoll until stage 7 at which point they were transferred to Steinberg’s solution containing 2% Ficoll. Stage 8 embryos were transferred to Steinberg’s solution that lacked Ficoll.

Chloramphenicol acetyltransferase enzyme assay

CAT enzyme assays were performed essentially as described by Gorman et al. (1982). Briefly, 20 frozen embryos were homogenized in 100μl of 250 mm-Tris-HCl (pH 7·8) and cell debris was pelleted by centrifugation. Samples of the supernatant equivalent to 5 embryos were incubated with 0·5μCi of [14C]chloramphenicol and 4/μl of 4mm-acetyl coenzyme A at 37°C for 90min. Reaction products were extracted with ethyl acetate and separated by thin layer chromatography. Quantification of CAT activity levels was accomplished by determining the radioactivity of spots on TLC plates by liquid scintillation counting.

RNA isolation

Total lithium-chloride-precipitable RNA was isolated according to the method of Auffrey & Rougeon (1980) as modified by Mohun et al. (1984). Briefly, frozen embryos were homogenized in 3m-lithium chloride, 6m-urea, 0 ·5% SDS, 70mm-β-mercaptoethanol, lOmm-sodium acetate (pH 5·0) and precipitated overnight at 4°C. The RNA was pelleted by centrifugation, resuspended in 0·2% SDS, lOOmm-sodium acetate (pH5·0) and extracted twice with phenol/chloroform (1:1) and twice with chloroform. The aqueous phase was adjusted to 0·3m-sodium acetate and the RNA was ethanol precipitated at —20°C overnight. RNA was again pelleted by centrifugation, dissolved in 200 μl of 40 mm-Tris-HCl (pH7·9), 6mm-MgC12 and contaminating DNA was removed by digestion with RNase-free DNase (Promega). RNA was extracted once with phenol/chloroform (1:1) and once with chloroform before being precipitated as described above.

SP6 RNA polymerase transcription and RNase protection analysis

SP6 RNA polymerase transcription and RNase mapping were carried out essentially as described by Melton et al. (1984). Preceding transcription, the plasmids pG230CB and pG270CB were linearized by digestion with BamHI and PstI, respectively. The antisense RNA probe that was used for detection of endogenous hsp 70 RNA was generated by SP6 RNA polymerase transcription of the plasmid pGI70H (gift of Dr J. Shuttleworth, University of Warwick, Coventry, UK; see Horrel et al. 1987) that had been linearized with EcoRI. 10 μg of total embryo RNA was hybridized to 5 ×105 cts min-1 of labelled antisense probe in 30 μ\ of 80 % formamide, 40 ITIm-Pipes-KOH (pH6-7), 400mm-NaCl, and Imm-EDTA at 45°C for 15 h. RNase digestion was carried out at 30°C for 30 min following the addition of 350 μl of RNase digestion mixture containing 300mm-NaCl, lOmm-Tris (pH7·5), 5mm-EDTA, 40 μg ml-’ RNase A, and 2^g ml-1 RNase Tr Following digestion, 5 μi of 10 mg ml-1 Proteinase K and 10 μl of 20% SDS were added and the incubation was continued at 42°C for an additional 15 min. The reaction mixture was extracted with an equal volume of phenol/chloroform (1:1) and the RNA precipitated at —70°C for Ih following the addition of 2μg of carrier tRNA and 1ml of ethanol. Following denaturation at 90°C for 5 min in 80% formamide loading buffer, the RNA was analyzed by polyacrylamide/urea gel electrophoresis and autoradiography.

DNA isolation and Southern hybridization

DNA was isolated by homogenizing 10 embryos in 200 μl of a buffer containing 100mm-Tris-HCl (pH7 ·4), Imm-EDTA, and 1 % SDS. Extracts were digested with 50 μg of proteinase K at 37°C for 90 min and extracted twice with phenol and once each with phenol/chloroform (1:1) and chloroform. The aqueous phase was adjusted to 0·3m-sodium acetate and precipitated overnight at —20°C with ethanol. For each sample, 5 ug of DNA was digested with a restriction enzyme which linearized the injected plasmid, electrophoresed on a 0·8% agarose gel and transferred to nitrocellulose as described by Maniatis et al. (1982). Blots were hybridized against the nick-translated CAT insert from pCM 7 and washed as described previously (Heikkila et al. 1987). Autoradiography was performed using Kodak XAR-5 film at —70°C. Densitométrie measurements on appropriately exposed autoradiograms (within the linear range of the film) were performed on a Bio-Rad Model 1650 Scanning Densitometer.

Microinjected Drosophila hsp 70/CAT and Xenopus hsp 70/CAT fusion genes are heat-inducible after the midblastula stage of Xenopus development

In previous studies, it has been reported that heatinducible expression of hsp 70 genes during Xenopus embryogenesis does not occur until after the midblastula stage of development (Bienz, 1984a; Heikkila et al. 1985a; 1987; Nickells & Browder, 1985). In order to examine the mechanisms associated with the developmental stage-dependent expression of hsp 70 genes, microinjected chimeric genes containing either the Drosophila or Xenopus hsp 70 promoters fused to the bacterial CAT gene (Fig. 1, panels A and B) were microinjected into fertilized eggs of Xenopus laevis. Expression of the microinjected genes during development was monitored by means of CAT enzyme assays. Control experiments revealed only background CAT activity (less than 0 · 1% conversion of [‘4C]chloramphenicol to acetylated forms) in control or heatshocked uninjected embryos or embryos injected with buffer, pUC 13 or the promoterless CAT/SV40 construct, pCB 13, at all developmental stages examined in this study (data not shown). Southern blot analysis of injected DNA isolated from embryos revealed that the ratio of plasmid DNA to genomic DNA remained constant until the late gastrula stage of development. The levels of the injected DNA then declined to relatively low levels by the late tailbud stage (data not shown). As shown in Fig. 2, very little CAT activity (less than 0· 1% conversion of [14C]chloramphenicol to acetylated forms) was detectable in control or heatshocked cleavage stage embryos injected with 400 pg of either the Drosophila or Xenopus hsp 70/CAT genes. However, heat shock resulted in at least a 100-fold increase in CAT activity levels in late blastula and gastrula stage embryos injected with either construct. In contrast to blastula and gastrula stage embryos, neurula stage embryos that were transformed with Xenopus hsp 70/CAT exhibited a constitutive level of CAT activity (15 % conversion of [14C]chloramphenicol to acetylated forms), which increased approximately threefold upon heat shock. A similar finding was observed in neurula stage embryos microinjected with the Drosophila hsp 70/CAT fusion gene (data not shown). Also, heatinducible Xenopus hsp 70/CAT expression in postmidblastula embryos was observed when as little as 50pg of plasmid DNA was injected (data not shown).

Fig. 1.

Plasmids used for hsp 70/CAT microinjection experiments. (A) Drosophila melanogaster hsp70/CAT fusion gene containing1·1kb of DNA upstream of the transcription initiation site as well as 65 bp of untranslated leader sequence (DiNocera & Dawid, 1983). (B) Xenopus laevis hsp 70/CAT (pXL70CB) containing 720 bp of upstream promoter sequence as well as 33 bp of DNA downstream of the transcription initiation site.(C) Antisense RNA probe utilized for RNase protection analysis of Xenopus hsp 70/CAT transcripts. The 1 kb Psti/EcoRI fragment from Xenopus hsp 70/CAT which spans the transcriptional start site of the hsp 70 promoter was cloned into pGEM-2 to give the plasmid pG270CB. The 1000 nucleotide antisense RNA probe that is generated by SP6 RNA polymerase in vitro transcription of this construct protects 288 nucleotides of a correctly initiated hsp 70/CAT transcript.

Fig. 1.

Plasmids used for hsp 70/CAT microinjection experiments. (A) Drosophila melanogaster hsp70/CAT fusion gene containing1·1kb of DNA upstream of the transcription initiation site as well as 65 bp of untranslated leader sequence (DiNocera & Dawid, 1983). (B) Xenopus laevis hsp 70/CAT (pXL70CB) containing 720 bp of upstream promoter sequence as well as 33 bp of DNA downstream of the transcription initiation site.(C) Antisense RNA probe utilized for RNase protection analysis of Xenopus hsp 70/CAT transcripts. The 1 kb Psti/EcoRI fragment from Xenopus hsp 70/CAT which spans the transcriptional start site of the hsp 70 promoter was cloned into pGEM-2 to give the plasmid pG270CB. The 1000 nucleotide antisense RNA probe that is generated by SP6 RNA polymerase in vitro transcription of this construct protects 288 nucleotides of a correctly initiated hsp 70/CAT transcript.

Fig. 2.

CAT activity in Xenopus embryos microinjected with either the Drosophila hsp 70/CAT fusion gene (A) or the Xenopus hsp 70/CAT fusion gene (B). CAT enzyme assays were performed essentially as described by Gorman et al. (1982) using extracts from embryos maintained at either 22°C (C) or 33°C (H) for 1 · 5 h at the cleavage (Cleav.), blastula (Blast.) or gastrula (Gast.) or neurula (Neur.) stages. Positive control was 0-05 units CAT. Acetylated forms of chloramphenicol (CM) are indicated by arrows.

Fig. 2.

CAT activity in Xenopus embryos microinjected with either the Drosophila hsp 70/CAT fusion gene (A) or the Xenopus hsp 70/CAT fusion gene (B). CAT enzyme assays were performed essentially as described by Gorman et al. (1982) using extracts from embryos maintained at either 22°C (C) or 33°C (H) for 1 · 5 h at the cleavage (Cleav.), blastula (Blast.) or gastrula (Gast.) or neurula (Neur.) stages. Positive control was 0-05 units CAT. Acetylated forms of chloramphenicol (CM) are indicated by arrows.

RNase protection analysis was performed to ensure that the transcription of microinjected Xenopus hsp 70/CAT DNA was initiated correctly. The Pstl/EcoRI fragment from pXL70CB which spans the transcriptional start site of the Xenopus hsp 70 promoter, was cloned into pGEM-2 to give the plasmid pG270CB (Fig. 1, panel C). The 1000 nucleotide antisense hsp 70/CAT RNA produced by SP6 RNA polymerase transcription of this construct allowed us to detect a 288 nucleotide protected fragment of correctly initiated hsp 70/CAT mRNA in heat-shocked blastula and gastrula stage embryos (Fig. 3, lanes 4 and 6). By contrast, hsp 70/CAT transcripts were not detectable in heat-shocked cleavage stage embryos (lane 2) or in cleavage, blastula and gastrula stage embryos that were maintained at control temperatures (22°C; lanes 1, 3, and 5) which correlates with the lack of CAT activity. Therefore, the heat-induced increase in CAT activity observed in blastula and gastrula stage embryos injected with the Xenopus hsp 70/CAT fusion construct is the result of the accumulation of correctly initiated hsp 70/CAT transcripts. Hsp 70/CAT transcripts were also detected in heat-shocked neurula stage embryos (lane 8). Interestingly, the undetectable level of hsp 70/CAT transcript in control neurulae was in contrast to the constitutive CAT activity present in these embryos (Fig. 2B).

Fig. 3.

RNase protection analysis of RNA isolated from control (22°C for T5h) or heat-shocked (33°C for 1·5 h) embryos microinjected with the Xenopus hsp 70/CAT fusion gene. 10 μg of total RNA was hybridized against the hsp 70/CAT antisense RNA probe, digested with RNase A and RNase Tj and the digestion products separated on a 4% polyacrylamide-urea denaturing gel. The 288 nucleotide protected fragment of hsp 70/CAT mRNA is indicated by an arrow. Lane 1, cleavage, control; Lane 2, cleavage, heat shock; Lane 3, late blastula, control; Lane 4, late blastula, heat shock; Lane 5, gastrula, control; Lane 6, gastrula, heat shock; Lane 7, neurula, control; Lane 8, neurula, heat shock.

Fig. 3.

RNase protection analysis of RNA isolated from control (22°C for T5h) or heat-shocked (33°C for 1·5 h) embryos microinjected with the Xenopus hsp 70/CAT fusion gene. 10 μg of total RNA was hybridized against the hsp 70/CAT antisense RNA probe, digested with RNase A and RNase Tj and the digestion products separated on a 4% polyacrylamide-urea denaturing gel. The 288 nucleotide protected fragment of hsp 70/CAT mRNA is indicated by an arrow. Lane 1, cleavage, control; Lane 2, cleavage, heat shock; Lane 3, late blastula, control; Lane 4, late blastula, heat shock; Lane 5, gastrula, control; Lane 6, gastrula, heat shock; Lane 7, neurula, control; Lane 8, neurula, heat shock.

A microinjected Xenopus hsp 30/CAT fusion gene is heat-inducible at an earlier developmental stage than endogenous hsp 30 genes

We have recently shown that heat-inducible hsp 30 gene expression is first detectable at the tailbud stage (stage 30 –32) of Xenopus embryogenesis (Krone & Heikkila, 1988) and not at the tadpole stage (stage 42) as was previously reported (Bienz, 1984a). A Xenopus hsp 30/CAT fusion gene was constructed to determine if sequences that are required for the correct developmental regulation of endogenous hsp 30 gene expression are present in the promoter region of an hsp 30 genomic clone. The resultant construct, pXL30CB, contains approximately 350 bp of promoter sequence upstream of the transcription initiation site as well as 143 bp of untranslated leader sequence (Fig. 4, panel A). In preliminary’ experiments, we found that this chimeric gene was strongly heat-inducible in microinjected oocytes (data not shown). Fertilized eggs were microinjected with 400 pg of the supercoiled hsp 30/CAT gene and expression of the injected constructs during embryogenesis was monitored by CAT enzyme assays. Surprisingly, heat shock induced a 50-to 100 fold increase in CAT activity levels in late blastula and gastrula stage embryos relative to controls (Fig. 5). CAT activity was barely detectable (less than 0·1% conversion of [l4C]chloramphenicol to acetylated forms) in cleavage stage embryos that were maintained at either control or heat shock temperatures. Tailbud embryos exhibited a low level of constitutive CAT activity (1 % conversion of [14C]chloramphenicol to acetylated forms) which was increased only threefold upon heat shock. The pattern of expression of hsp 30/CAT was not due to promoter-like elements that might be present in the vector DNA upstream of the hsp 30 promoter since microinjection of hsp 30/CAT linearized at the unique Sall site in the pUC 13 polylinker immediately upstream of the hsp 30 promoter sequences was also heat-inducible in embryos after the midblastula stage of development (data not shown). The low level of CAT activity (4% conversion of [14C]chloramphenicol to acetylated forms) in nonheat-shocked postmidblastula stage embryos was not due to a stress response resulting from the microinjection procedure or the presence of the hsp 30/CAT plasmid since we did not detect an increase in the levelsm of endogenous hsp 70 mRNA by RNase protection analysis (data not shown).

Fig. 4.

Diagram of the plasmids pXL30CB and pG230CB. (A) Xenopus laevis hsp 30/CAT fusion gene (pXL30CB). This construct contains promoter sequences to 350 bp upstream of the transcription initiation site as well as 143 bp of downstream DNA. (B) Antisense RNA probe utilized for RNase protection analysis of hsp 30/CAT and endogenous hsp 30 transcripts. The 750 bp BamHI/EcoRI fragment from hsp 30/CAT spanning the transcriptional start site of the hsp 30 promoter was cloned into pGEM-2 to give the plasmid pG230CB. SP6 RNA polymerase in vitro transcription of this construct results in a 750 nucleotide antisense RNA probe which protects a 402 nucleotide correctly initiated hsp 30/CAT mRNA fragment as well as a 143 nucleotide portion of endogenous hsp 30 mRNA.

Fig. 4.

Diagram of the plasmids pXL30CB and pG230CB. (A) Xenopus laevis hsp 30/CAT fusion gene (pXL30CB). This construct contains promoter sequences to 350 bp upstream of the transcription initiation site as well as 143 bp of downstream DNA. (B) Antisense RNA probe utilized for RNase protection analysis of hsp 30/CAT and endogenous hsp 30 transcripts. The 750 bp BamHI/EcoRI fragment from hsp 30/CAT spanning the transcriptional start site of the hsp 30 promoter was cloned into pGEM-2 to give the plasmid pG230CB. SP6 RNA polymerase in vitro transcription of this construct results in a 750 nucleotide antisense RNA probe which protects a 402 nucleotide correctly initiated hsp 30/CAT mRNA fragment as well as a 143 nucleotide portion of endogenous hsp 30 mRNA.

Fig. 5.

CAT activity in Xenopus embryos microinjected with the hsp 30/CATfusion gene. Cleavage (Cleav.), blastula (Blast.), gastrula (Gast.), and tailbud embryos were maintained at either 22°C (C) or 33°C (H) for 1 ·5 h. Acetylated forms of chloramphenicol (CM) are indicated by arrows. Positive control was 0 ·05 units CAT.

Fig. 5.

CAT activity in Xenopus embryos microinjected with the hsp 30/CATfusion gene. Cleavage (Cleav.), blastula (Blast.), gastrula (Gast.), and tailbud embryos were maintained at either 22°C (C) or 33°C (H) for 1 ·5 h. Acetylated forms of chloramphenicol (CM) are indicated by arrows. Positive control was 0 ·05 units CAT.

RNase protection analysis of hsp 30/CAT mRNA was performed to determine whether the observed increases in CAT activity were the result of increased levels of correctly initiated hsp 30/CAT transcription. A 750 nucleotide fragment from pXL30CB, which spans the transcriptional start site of the hsp 30 promoter, was cloned into pGEM-2 to give the plasmid pG230CB (Fig. 4, panel B). In RNase protection assays, the 750 nucleotide antisense probe protected a 402 nucleotide fragment of correctly initiated hsp 30/CAT mRNA as well as a 143 nucleotide portion of endogenous hsp 30 mRNA. In agreement with the CAT assay data (Fig. 3), heat-shock treatment enhanced the accumulation of correctly initiated hsp 30/CAT mRNA in blastula, gastrula, and tailbud stage embryos (Fig. 6, lanes 4, 6, and 8). No hsp 30/CAT mRNA was detectable in either control or heat-shocked cleavage stage embryos (lanes 1 and 2). Furthermore, heat-inducible accumulation of endogenous hsp 30 mRNA was not detectable in the microinjected embryos until the early tailbud stage of development (stage 23–24; lane 8). Compared to blastula stage embryos, the amount of hsp 30/CAT mRNA in heat-shocked tailbud stage embryos (Fig. 6, compare lanes 4 and 8) was much higher than the corresponding CAT activity would indicate (Fig. 5). At present, it is not known if this is due to a specific translational block of hsp 30/CAT mRNA at this stage.

Fig. 6.

RNase protection analysis of hsp 30/CAT and endogenous hsp 30 mRNA in embryos microinjected with the hsp 30/CAT fusion gene. 10 fig of total RNA was isolated from either control (22°C for 1·5 h) or heat-shocked (33°C for 1·5 h) embryos, subjected to RNase protection analysis and separated on a 4% polyacrylamide-urea denaturing gel. Arrows indicate the positions of the 402 nucleotide hsp 30/CAT mRNA and 143 nucleotide endogenous hsp 30 mRNA protected fragments. Lane 1, cleavage, control; Lane 2, cleavage, heat shock; Lane 3, late blastula, control; Lane 4, late blastula, heat shock; Lane 5, gastrula, control; Lane 6, gastrula, heat shock;Lane 7, early tailbud, control; Lane 8, early tailbud, heat shock.

Fig. 6.

RNase protection analysis of hsp 30/CAT and endogenous hsp 30 mRNA in embryos microinjected with the hsp 30/CAT fusion gene. 10 fig of total RNA was isolated from either control (22°C for 1·5 h) or heat-shocked (33°C for 1·5 h) embryos, subjected to RNase protection analysis and separated on a 4% polyacrylamide-urea denaturing gel. Arrows indicate the positions of the 402 nucleotide hsp 30/CAT mRNA and 143 nucleotide endogenous hsp 30 mRNA protected fragments. Lane 1, cleavage, control; Lane 2, cleavage, heat shock; Lane 3, late blastula, control; Lane 4, late blastula, heat shock; Lane 5, gastrula, control; Lane 6, gastrula, heat shock;Lane 7, early tailbud, control; Lane 8, early tailbud, heat shock.

Developmental regulation of hsp 30/CATexpression is not affected by plasmid copy number

It was conceivable that a high copy number of hsp 30/CAT plasmid DNA may overload the regulatory mechanism(s) controlling hsp 30 gene expression and allow for premature transcription of the construct in pretailbud stage embryos. To address this question, we microinjected fertilized eggs with 50, 200 or 400 pg of hsp 30/CAT and determined by densitométrie analysis of autoradiograms of Southern blots that the quantity of injected DNA remaining in gastrula stage embryos was equivalent to approximately 25–30% of the quantity injected (data not shown). Most of the DNA was lost immediately after injection followed by replication of the remaining DNA at a rate similar to that observed for chromosomal DNA. By direct comparison with known quantities of hsp 30/CAT DNA, we found that embryos injected with 400, 200, or 50 pg of hsp 30/CAT DNA contain approximately 50– 60, 20 – 30, and 3 – 5 copies of plasmid DNA, respectively, per haploid genome equivalent at the gastrula stage. However, even at the low level of 3–5 copies of hsp 30/CAT per haploid genome equivalent, heat-inducible CAT activity as well as accumulation of correctly initiated hsp 30/CAT transcripts was apparent (Fig. 7). Also, heatinducible expression of hsp 30/CAT was detectable when as little as 10pg (i.e. less than 1 copy per haploid genome equivalent at the gastrula stage) of plasmid had been injected.

Fig. 7.

Effect of the amount of injected plasmid DNA on hsp 30/CAT gene expression. (A) CAT activity in gastrula stage embryos maintained at either 22°C (C) or 33°C heat-shocked (H) which had been microinjected with either 50, 200 or 400 pg of hsp 30/CAT at the 1-cell stage. Acetylated forms of chloramphenicol (CM) are indicated by arrows. (B) RNase protection analysis of hsp 30/CAT mRNA in control (C) and heat-shocked (H) gastrula stage embryos microinjected at the 1-cell stage with 50, 200 or 400 pg of hsp 30/CAT. Arrows indicate the position of the 402 nucleotide hsp 30/CAT mRNA protected fragment.

Fig. 7.

Effect of the amount of injected plasmid DNA on hsp 30/CAT gene expression. (A) CAT activity in gastrula stage embryos maintained at either 22°C (C) or 33°C heat-shocked (H) which had been microinjected with either 50, 200 or 400 pg of hsp 30/CAT at the 1-cell stage. Acetylated forms of chloramphenicol (CM) are indicated by arrows. (B) RNase protection analysis of hsp 30/CAT mRNA in control (C) and heat-shocked (H) gastrula stage embryos microinjected at the 1-cell stage with 50, 200 or 400 pg of hsp 30/CAT. Arrows indicate the position of the 402 nucleotide hsp 30/CAT mRNA protected fragment.

Reexamination of endogenous hsp 30 gene expression during early Xenopus development

Given our finding that the hsp 30/CAT constructs were expressed in pretailbud stages, it was possible that the inability to detect endogenous hsp 30 gene transcription in uninjected embryos (Krone & Heikkila, 1988) was a consequence of the limited sensitivity of the Northern hybridization assay. This question was addressed by using antisense RNA probes for both hsp 70 and hsp 30 mRNA in RNase protection assays. The 506 nucleotide antisense hsp 70 RNA probe produced by SP6 RNA polymerase transcription of pG170H was used to protect a 476 nucleotide region of transcripts that are derived from the hsp 70B gene and a 368 nucleotide segment of hsp 70A mRNA. Samples from the same total RNA samples were used in both hsp 30 and hsp 70 RNase protection assays for each developmental stage examined. As shown in Fig. 8, transcripts arising from the hsp 70 genes were first detectable in embryos heat-shocked during the midblastula transition (panel B, lane 4) with greater quantities accumulating in gastrula, neurula, tailbud, and tadpole stage embryos (lanes 6, 8, 10, and 12). However, hsp 30 gene expression was not detectable until the embryos had reached the tailbud stage of development (panel A, lane 10). A relatively larger heat-shock response was observed in embryos at the tadpole stage (lane 12). These results suggest that heat-inducible endogenous hsp 30 gene expression does not occur in neurula or earlier stage embryos. We have subjected as much as 100 μg of total RNA from control and heat-shocked gastrula stage embryos to RNase protection analysis and were unable to detect hsp 30 transcripts. In independent RNase protection experiments, low levels of hsp 30 mRNA were first detectable in heat-shocked early tailbud embryos at stage 23– 24 (data not shown) w’hich is earlier during development than previously reported (Krone & Heikkila, 1988).

Fig. 8.

RNase protection analysis of endogenous hsp 30 (panel A) and hsp 70 (panel B) mRNA levels during early Xenopus development. Total RNA was isolated from embryos that had been maintained at either 22°C or 33°C for 1·5 h and 10 μg of this RNA was subjected to RNase protection analysis. Hsp 30 transcripts were detected with the hsp 30/CAT antisense RNA probe as described in the legend of Fig. 4. The riboprobe that was used for detection of hsp 70 transcripts was obtained by SP6 RNA polymerase transcription of pGl 70H (Horrel et al. 1987). This 506 nucleotide antisense probe (P) protects a 476 nucleotide region of transcripts derived from the hsp 70B gene as well as a 368 nucleotide region of mRNA transcribed from the hsp 70A gene. Protected fragments were separated on either a 6% (hsp .30) or 4% (hsp 70) polyacrylamide-urea denaturing gel. U, undigested hsp 70 probe; Lane 1, cleavage, 22°C; Lane 2, cleavage, 33°C; Lane 3, midblastula, 22°C; Lane 4, midblastula, 33°C; Lane 5, gastrula, 22°C; Lane 6, gastrula, 33°C; Lane 7, neurula, 22°C; Lane 8, neurula, 33°C; Lane 9, tailbud (stage 30–32), 22°C; Lane 10, tailbud (stage 30-32), 33°C; Lane 11, tadpole (stage 42), 22°C; Lane 12, tadpole (stage 42), 33°C.

Fig. 8.

RNase protection analysis of endogenous hsp 30 (panel A) and hsp 70 (panel B) mRNA levels during early Xenopus development. Total RNA was isolated from embryos that had been maintained at either 22°C or 33°C for 1·5 h and 10 μg of this RNA was subjected to RNase protection analysis. Hsp 30 transcripts were detected with the hsp 30/CAT antisense RNA probe as described in the legend of Fig. 4. The riboprobe that was used for detection of hsp 70 transcripts was obtained by SP6 RNA polymerase transcription of pGl 70H (Horrel et al. 1987). This 506 nucleotide antisense probe (P) protects a 476 nucleotide region of transcripts derived from the hsp 70B gene as well as a 368 nucleotide region of mRNA transcribed from the hsp 70A gene. Protected fragments were separated on either a 6% (hsp .30) or 4% (hsp 70) polyacrylamide-urea denaturing gel. U, undigested hsp 70 probe; Lane 1, cleavage, 22°C; Lane 2, cleavage, 33°C; Lane 3, midblastula, 22°C; Lane 4, midblastula, 33°C; Lane 5, gastrula, 22°C; Lane 6, gastrula, 33°C; Lane 7, neurula, 22°C; Lane 8, neurula, 33°C; Lane 9, tailbud (stage 30–32), 22°C; Lane 10, tailbud (stage 30-32), 33°C; Lane 11, tadpole (stage 42), 22°C; Lane 12, tadpole (stage 42), 33°C.

3’ coding and noncoding regions are not involved in the expression of the hsp 30 genomic clone in microinjected embryos

The expression of globin genes in a number of different organisms is regulated by an enhancer element present in the 3’ flanking region (Choi & Engel, 1986; Hesse et al. 1986; Behringer et al. 1987; Kollias et al. 1987; Trudel & Costantini, 1987; Antoniou et al. 1988). Also, the 3’ end of a chicken / β -actin gene has been implicated in its down-regulation during myogenic differentiation (Lohse & Arnold, 1988). In this context, a civ-acting regulatory element that is responsible for normal developmental expression may be located in the coding region or 3’ end of the isolated hsp 30 genomic clone. Since such an element may interact with sequences present elsewhere on the gene and, moreover, correct spacing between such sequences may be crucial for proper developmental regulation, the expression of the entire hsp 30 genomic clone in Xenopus embryos following microinjection into fertilized eggs was examined. As shown in Fig. 9, the transcription of the microinjected intact hsp 30 genomic clone was detected in heat-shocked gastrula stage embryos (lane 6). RNase protection studies have shown that the endogenous hsp 30 genes are not expressed at this stage; however, it was possible that the injected genes may somehow activate the expression of the endogenous hsp 30 genes. This does not appear to be the case since microinjection of the hsp 30 promoter/CAT fusion gene construct has no effect on the developmental regulation of the endogenous hsp 30 genes (Fig. 6). Furthermore, microinjection of (i) a construct containing a sequence further downstream from those present in pXL30CB (pX303) including 500 bp of 3’ flanking region or (ii) coinjection of both 5’ and 3’ regions does not prematurely activate transcription of the endogenous genes (lane 1 to 4). Thus, neither hsp 30 coding sequences nor 3’ flanking sequences that are present on the hsp 30 genomic clone appear to be responsible for correct developmental regulation.

Fig. 9.

3’-coding and noncoding regions are not involved in the expression of the hsp 30 genomic clone in microinjected embryos. In order to examine the involvement of the 3’-coding and noncoding regions in the developmental regulation of hsp 30 gene expression, fertilized eggs were microinjected with the intact hsp 30 gene cloned into pUC 13 and allowed to develop to the gastrula stage. RNase protection analysis revealed the presence of hsp 30 transcripts in heat-shocked gastrula stage embryos (33 °C for 1·5 h; lane 6) but not in those maintained at control temperatures (22°C; lane 5) for the same length of time. Given the possibility that the 3’-coding and noncoding regions may be activating expression of the endogenous genes, fertilized eggs were injected with the plasmid pX303 which contains the hsp 30 gene sequences downstream of the PstI site found in the untranslated leader sequence of the hsp 30 gene (lanes 1 and 2) or coinjected with pX303 and the hsp 30/CAT fusion gene (lanes 3 and 4). Embryos were again allowed to develop to the gastrula stage and then maintained at either control (22°C; lanes 1 and 3) or heat shock (33°C; lanes 2 and 4) temperatures for 1·5 h. Hsp 30 mRNA was not detectable by RNase protection analysis of total RNA isolated from these samples.

Fig. 9.

3’-coding and noncoding regions are not involved in the expression of the hsp 30 genomic clone in microinjected embryos. In order to examine the involvement of the 3’-coding and noncoding regions in the developmental regulation of hsp 30 gene expression, fertilized eggs were microinjected with the intact hsp 30 gene cloned into pUC 13 and allowed to develop to the gastrula stage. RNase protection analysis revealed the presence of hsp 30 transcripts in heat-shocked gastrula stage embryos (33 °C for 1·5 h; lane 6) but not in those maintained at control temperatures (22°C; lane 5) for the same length of time. Given the possibility that the 3’-coding and noncoding regions may be activating expression of the endogenous genes, fertilized eggs were injected with the plasmid pX303 which contains the hsp 30 gene sequences downstream of the PstI site found in the untranslated leader sequence of the hsp 30 gene (lanes 1 and 2) or coinjected with pX303 and the hsp 30/CAT fusion gene (lanes 3 and 4). Embryos were again allowed to develop to the gastrula stage and then maintained at either control (22°C; lanes 1 and 3) or heat shock (33°C; lanes 2 and 4) temperatures for 1·5 h. Hsp 30 mRNA was not detectable by RNase protection analysis of total RNA isolated from these samples.

Previous studies have shown that heat shock-induced accumulation of hsp 70 mRNA does not occur until after the midblastula stage of Xenopus embryogenesis (Bienz, 1984a; Heikkila et al. 1985a; 1987; Nickells & Browder, 1985). In the present study, we have examined the regulation of both a Drosophila and a Xenopus hsp 70/CAT fusion gene following microinjection into developing embryos of Xenopus laevis and found that both constructs behaved in a manner similar to the endogenous hsp 70 genes. Heat-induced transcription from either of the injected promoters was activated only after embryos reached the midblastula stage of development (Fig. 2). Additionally, we have shown by RNase protection analysis that transcription of the Xenopus hsp 70/CAT construct was correctly initiated (Fig. 3). Recently, a similar result regarding the developmentally regulated expression of a Xenopus hsp 70/CAT gene has been reported by Harland & Misher (1988) using Northern hybridization analysis. The mechanism of the onset of transcriptional activity after the midblastula stage is not fully understood. Newport et al. (1985) have proposed that the lack of transcriptional activity during cleavage stages may be due to the overabundance of either cytoplasmic maturation-promoting factor or mitosis-initiation factors which prevent a G phase in the cell cycle by rapidly triggering the onset of mitosis after S phase. Mechanisms responsible for transcriptional activation of the zygotic genome may also be responsible for allowing heat-inducible hsp 70 gene transcription in postmidblastula stage embryos.

In blastula and gastrula stage embryos that had been transformed with the Xenopus hsp 70/CAT gene, no detectable CAT enzyme activity or hsp 70/CAT transcripts were detectable at the control temperature (22°C; Figs 2 and 3). In contrast, recent preliminary studies in our laboratory have shown that this construct was constitutively expressed in Xenopus oocytes (Krone & Heikkila, unpublished results). This is in agreement with other studies examining the expression of Xenopus hsp 70 genomic clones in oocytes (Bienz, 19846; Horrell et al. 1987). Furthermore, Bienz (1986) reported that the constitutive expression of a Xenopus hsp 70 genomic clone in oocytes is dependent on the presence of both a CCAAT box and a heat-shock element in the promoter region. Thus, it is possible that oocyte-specific irarw-acting factors interacting with these and other elements may account for the strong constitutive expression of the hsp 70/CAT gene in Xenopus oocytes. In the present study, we also observed CAT enzyme activity in control neurulae which was increased upon heat shock (Fig. 2). Since hsp 70/CAT transcripts were undetectable in control neurulae (Fig. 3), it is possible that CAT enzyme activity present in these embryos may be due to an accumulation of stable CAT enzyme between the gastrula and neurula stages resulting from either a continuous basal level of transcription or a transient burst of constitutive hsp 70/CAT gene expression.

We have previously demonstrated by Northern blot analysis that heat-shock-induced expression of hsp 30 genes does not occur until Xenopus embryos reach the late tailbud stage of development (stage 30–32; Krone & Heikkila, 1988). In the present study, we have shown by the more powerful technique of RNase protection analysis that hsp 30 gene expression is first detectable in heat-shocked early tailbud embryos (stage 23 –24). In contrast to the developmental regulation of endogenous hsp 30 genes, constitutive and heat-inducible expression of a microinjected hsp 30/CAT fusion gene containing 350 bp of hsp 30 promoter sequence was detectable only after the midblastula stage of development (Figs 5 and 6). We have also shown that the 3’ coding and noncoding regions of the hsp 30 genomic clone are not responsible for proper developmental regulation (Fig. 9). Expression of the endogenous hsp 30 genes at the tailbud stage may occur either by means of a positive regulator or removal of an inhibitory system. The fact that the injected hsp 30 gene was capable of being expressed in both control and heat-shocked pretailbud stage embryos suggests that an inhibitory system may regulate the expression of the endogenous genes. Recently, Steinbeisser et al. (1988) have reported the presence of a putative negative regulatory element within 680 bp of promoter region responsible for correct developmental regulation of the Xenopus skeletal muscle actin gene. A negative regulatory element has also been shown to reside between 120 and 603 nucleotides upstream of the transcription initiation site of the chicken ol-crystallin gene (Borras et al. 1988). Furthermore, the 3’ end and 3’ flanking sequence of the chicken /J-actin gene appear to be necessary for its down-regulation during myogenic differentiation (Lohse & Arnold, 1988). Although our results suggest that a negative regulatory element seems unlikely within the 5’ and 3’ flanking regions or within the coding sequence of the particular hsp 30 genomic clone that was used in the present study, they do not rule out the presence of such elements further upstream or downstream of the gene. To address this possibility, we are presently screening a Xenopus genomic library to isolate clones containing more extensive flanking sequences.

Recently, the clustered arrangement of the chicken globin gene family has been reported to be involved in the developmental regulation of the different globin genes (Choi & Engel, 1988). For example, inactivation of the embryonic e-globin gene in definitive erythroid cells was directly due to the activation of the adult /J-globin gene which is located immediately upstream. In preliminary work, screening of a Xenopus genomic library revealed the presence of several different clones which contain more than one hsp 30 gene (Krone & Heikkila, unpublished results). Thus, it appears that the Xenopus hsp 30 genes may be present as a tightly linked cluster. Such a clustered structure could be involved in the developmental regulation of the hsp 30 genes given the similar linked arrangement of globin genes as well as the Drosophila small heat-shock genes, which also exhibit a complex pattern of developmental regulation (Dura, 1981; Sirotkin & Davidson, 1982; Zimmerman et al. 1983; Mason et al. 1984). This type of clustered formation may be an efficient mechanism by which expression of the estimated 5-10 copies of the hsp 30 gene (Bienz, 1984b) could be coordinately regulated. Alternatively, the regulation of hsp 30 gene expression could be controlled by methylation (for review see Cedar, 1988) or a change in the replication timing of the genes during the cell cycle (Goldman, 1988).

This research has been supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to J.J.H. P.H.K. is the recipient of an NSERC postgraduate scholarship. The authors would like to thank Dr J. J. Pasternak for helpful suggestions throughout the course of this work and the preparation of the manuscript.

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We apologize to the author for the delay in transmission of this manuscript to the Cambridge Office