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Isolation and characterization of a sea urchin hsp 70 gene segment
Cell Differentiation, 24 (1988) 97-104 Elsevier Scientific Publishers Ireland, Ltd.
97
CDF 00509
Isolation and characterization of a sea urchin hsp 70 gene segment G. Sconzo 1, M. La Rosa 1, M. La Farina 1, M.C. Roccheri 1, D. Oliva 1 and G. Giudice 1,2 Dipartimento di Biologia Cellulare e dello Soiluppo and 2 Istituto di Biologia dello Soiluppo del CNR, Via Archirafi n. 2 0 - 22, 90123 Palermo, Italy
(Accepted 15 February 1988)
Three clones containing Paracentrotm lividus sea urchin DNA sequences which cross-hybridize to Drosophila heat shock protein (hsp) 70 gene were isolated. The sequence arrangements in the three cloned DNA inserts were compared by restriction and cross-hybridization analysis. The results showed that they contain four different genes related to one Drosophila hsp 70 gene. One of these genes was subcloned, and two of the isolated fragments were shown to hybridize to genomic DNA and to RNA from heat-treated sea urchin embryo. Heat shock protein 70, Sea urchin; DNA sequence; Cross hybridization
Introduction
The response to heat shock with the production of heat shock proteins (hsp) is a well-known phenomenon, common to most, if not all, organisms (Loomis and Wheeler, 1980; Storti et al., 1980; Kelley and Schlesinger, 1982; Schlesinger et al., 1982; Yamamori and Yura, 1982; Craig et al., 1983; Bardwell and Craig, 1984; Velazques and Lindqttist, 1984; Atkinson and Walden, 1985; Heikkila et al., 1985a; O'Malley et al., 1985; Lindquist, 1986; Schlesinger, 1986). This response in sea urchin embryos is specific to the developmental stage considered: when exposed to heat shock, embryos between fertilization and early blastula are unable to synthesize the heat shock proteins, and degenerate and die. On the other hand, when
Correspondence address: G. CAudice, Istituto di Biologia dello Sviluppo del CNR, Via Archirafi n. 20-22, 90123 Palermo, Italy.
subjected to heat from the hatching stage until the pluteus stage, they synthesize the heat shock proteins and develop normally (Giudice et al., 1980; Roccheri et al., 1981a, b, 1986; Sconzo et al., 1983, 1985, 1986). Sea urchin embryos therefore represent, in this respect, a suitable model for the study of cell differentiation at a molecular level during the course of embryonic development. In sea urchins, the two major hsp, of 72.5 and 70 kDa, are abundant (the 70 kDa comigrates, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with Drosophila 70 kDa hsp; Sconzo, unpublished data), and their production is regulated at the transcriptional level (Roccheri et al., 1982). As an approach to investigating the activation mechanism of the hsp 70 gene during different developmental stages, we isolated and characterized three clones, in which there are four different genes cross-hybridizing to the Drosophila hsp 70 gene. One of these, subcloned into the pUC8 vector, was used as a hybridization probe in the analysis
0045-6039/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland, Ltd.
98 of the genomic DNA. It was able to hybridize only to one electrophoretic band of RNA from non-heated gastrula embryos and to two electrophoretic bands of RNA from heat-shocked gastrula embryos. Materials and Methods
Embryo culture The embryos were allowed to develop in Millipore filtered sea water at 20°C as previously described (Giudice and Mutolo, 1967) up to the desired stage and then divided into two parts: one part was subjected to heat shock at 31°C for 1 h (heat-treated embryos) and the other part was left to develop at 20 °C (untreated embryos). Genomic DNA The genornic DNA was isolated from a single male (Paracentrotus lividus), as described by Kedes et al. (1975). One part was partially digested with Sau3A enzyme to prepare a genomic library. The other part was used for Southern blot analysis (Southern, 1975). Genomic library A genomic library from P. lividus sperm DNA was constructed in EMBL3 phage using standard methods (Maniatis et al., 1982). The genomic DNA, prepared as described previously, was partially digested with S a u 3 A enzyme and fractionated on sucrose gradient. Fragments of the size selected, 13-20 kb, were ligated into BamHI/EcoRI cut EMBL3 phage (courtesy of Dr. Spinelli). The ligation mixture was in vitro packaged, using extracts from a kit form (packaging system for lambda DNA, Amersham), and about 105 plaques//~g DNA were obtained upon plating onto selective strain Q359 (Karn et al., 1980). More than 5 x 105 plaques were screened (Benton and Davis, 1977) by low stringency hybridization in a solution containing 5 × SSC (0.75 M sodium chloride and 0.075 M sodium citrate) and 5 × Denhardt solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), using as a probe 10 ng/ml of the 2.2 kb SalI fragment containing the coding sequence of a hsp 70 Drosophila gene prepared from
the 56H8 clone (Moran et al., 1979). This DNA fragment was nick-translated using standard methods (Maniatis et al., 1982), reaching a value of about 2.5 × 108 cpm//tg. Three positive clones were isolated and after plaque purification the recombinant DNA was prepared from lysates (Frischauf, 1983). These clones have been named XPLH1, XPLH3 and XPLH9.
Restriction mapping, gel electrophoresis and gel blotting Single or double restriction enzyme digestions of the three clones were performed in order to obtain the restriction maps under the conditions recommended by the suppliers (Amersham and Boehringer). Restriction fragments were separated by 0.6-1.0% agarose gel electrophoresis. After the Southern blot, the filters were hybridized in 5 x SSC and 5 × Denhardt solution, containing 10 ng/ml of radiolabeled fragments (at about 2.5 x 10 s cpm/~tg). Fragments of 1.3 kb or 0.9 kb were prepared by BamHI enzyme digestion from the 2.2 kb SalI fragment of the hsp 70 Drosophila gene. The filters were therefore exhaustively washed in 2 × SSC. Subclones In order to obtain the gene regions contained within the inserts, the first insert, XPLH1, was digested with SalI enzyme and the resulting three fragments were ligated to pUC8 vector (Vieira and Messing, 1982) to generate P l l , P16 and P17 plasmids. Each of these was digested with the suitable restriction enzymes EcoRI (E), HindlII (H), BamHI (B), AccI (A), Sail (S), PstI (P), to insert the resulting fragments in the pUC8 vector. The recombinant plasmids PAl6, PB16 and PC16 were derived from P16 plasmid; P1735, P1718, P1716, P1707 and P1711 from P17 plasmid; and Pl104, Pll06 from P l l plasmid. Genomic DNA and Southern blot analysis Genomic DNA was digested with both EcoRI and Sail enzymes, separated on 0.8% agarose gel electrophoresis and, after Southern blot, hybridized by high stringency hybridization in a solution containing 2 x SSC and 5 x Denhardt's, using as a probe the radiolabeled fragment (at
99 about 2.5 × 108 c p m / # g ) isolated from the P1716 plasmid. Bacteriophage ~, D N A - H i n d l I I and pBR322-HinfI were used as markers.
R N A isolation and Northern blot analysis Total R N A was extracted from heat-treated embryos or untreated embryos, at the stages of morula and gastrtfla, by a procedure modified from Holmes and Bonner (1973). The embryos were lysed in 7 M urea, 2% SDS, 0.35 M sodium chloride, 10 m M Tris-HC1 (pH 8.0) and extracted four times with equal volume phenol-chloroform. After precipitation the pellet was resuspended in 0.5% SDS, 20 m M sodium chloride, 1 m M EDTA, extracted four more times and precipitated twice. The purified total R N A was treated with 50 # g / m l D N A s e I (Miles) and fractionated by methylmercury agarose gel electrophoresis (Bailey and Davidson, 1986), using bacteriophage fl m R N A s (4 and 1.45 kb) as markers. Our R N A markers were visualized by hybridizing R N A extracted from fl infected E. coli to a probe derived from sequences of gene IV of that phage as described in La Farina and Model (1983). Total H I
E i
S E I I
Ell II
E S S ~P"~"~"J"~Jllml
a
R N A was transferred onto a nylon membrane (Hybond, Amersham) fixed by UV irradiation and hybridized to the radiolabeled fragments isolated either from P1716 plasmid or P1718 plasmid.
Autoradiography Autoradiography was done at - 7 0 ° C K o d a k X - O m a t film and an intensifier.
with
Results and Discussion We have constructed a genomic library of P. lividus sperm D N A in EMBL3 phage and have screened more than 5 x 105 plaques using, as a hybridization probe, the 2.2 kb Sail fragment of hsp 70 Drosophila gene, derived from 56H8 clone (courtesy of Dr. W. Gehring). Three clones carrying sequences cross-hybridizing to hsp 70 Drosophila probe were isolated by low stringency hybridization. After plaque purification, the restriction mapping and the Southern blot analysis of these recombinant D N A s showed that the isolated clones carried three different segments of P. lividus D N A . The organization and restriction
•~ PLH1
b
E
E
B
S
BS
SEE PLH3
c
1Kb I--t
d
BS
SEE
'E I
ESE I I I
,~PLH9
e
3' 1.3Kb 0.9Kb
Fig. 1. Restriction maps of the three DNA inserts from ~PLH1, XPLH3 and XPLH9 clones. Restriction maps of the XPLH3 and XPLH9 inserts were obtained by single or double digestion with BamHI (B), Sail (S), EcoRI (E) enzyme; and restriction map of the XPLH1 with the same enzymes and also with HindlII (H). The gene regions cross-hybridizingto 2.2 kb Sail of the hsp 70 Drosophila gene are represented by the boxes. The dashed boxes hybridize mainly to the 1.3 kb fragment of the hsp 70 Drosophila gene corresponding to the 5' terminus (see inset). The shadowed boxes hybridize to the 0.9 kb fragment of the hsp 70 Drosophila gene corresponding the 3' terminus. The inset shows the two fragments, used as probes, of 1.3 kb and 0.9 kb, prepared from the 2.2 kb Sail fragment of the hsp 70 Drosophila gene derived from 56H8 clone by digestion with BamHI enzyme.
100 mapping analysis of these three different isolated segments are depicted in Fig. 1. The first insert, ?~PLH1, is 14 kb in length and contains two different gene regions (a and b) separated by nearly 9500 nucleotides. The positions of the 5' and 3' termini in the cross-hybridizing regions were defined by using as hybridization probes 5' or 3' fragments, of the Drosophila hsp 70 probe, obtained by digesting it with BarnHI enzyme (see inset in Fig. 1). The second segment, kPLH3, about 17.5 kb in length, also contains two gene regions (c and d) related to the Drosophila hsp 70 gene, separated by 7000 nucleotides. The comparison of the organization of these two DNA inserts shows that the genes differ from one another. The third insert, ~ PLH9, about 18 kb in length, contains only one gene region (e) at one end. Regions e and d show the same restriction map. The portions of the three inserts which are outside of the gene show restriction maps which are different from each other. We infer that the three isolated clones contain four different genes related to the Drosophila hsp 70 gene; three, b, c, d = e, are complete and the fourth, a, is about one half gene, containing the 3' end. In order to purify the gene regions contained within one of the inserts, the first insert, )tPLH1, was divided into three fragments by SalI restriction enzyme and subcloned into the pUC8 vector. Each of these subclones, P16, P17, P l l , was further subcloned. Fig. 2 shows the total map of the 13 subclones. One of these subclones, P1716, containing the 1.5 kb Eco-Sal insert, was found in the Southern blot experiments to hybridize, with high stringency, to a 1.5 kb band of the genomic Eco-Sal restricted DNA (Fig. 3). This is a further proof that this subclone is actually constitutively well represented in the sea urchin genome. The autoradiogram also shows another expected hybridization band, i.e. that of 6.3 kb corresponding to the EcoRI-SalI segment of XPLH3. In order to understand whether the isolated gene is a heat-shock gene transcribed at a specific stage of development, we hybridized the P1716 probe to the total RNA prepared from heat-treated embryos and untreated embryos of two different
IKb
SE
H
~
H
E
I
I
E I
E I
AE I
SPS }~ P L H 1 P16
k F
PA 16
4
PB16 PC16 P17 P1735
I
P1718
!
P1711
F------I
P1707 I
P1716
I
Pll P1106 H
P1104
Fig. 2. Subcloning of )~PLH1 DNA insert. The restriction map of hPLH1 DNA insert (top) and schematic representation of the fragments subcloned into the pUC8 vector (below). The boxes represent the cross-hybridizing regions to 2.2 kb SalI fragment of Drosophila hsp 70 gene. The P16, P17, P l l were generated directly from )tPLH1 insert by SalI restriction enzyme digestion. Further subcloning from P16, P17 and P l l gave rise to three plasmids (PAl6, PB16, PC16), to five plasraids (P1735, P1718, P1716, P1711, P1707) and two plasmids (Pl106 and P1104), respectively.
stages: morula (unresponsive to heat shock) and gastrula (responsive to heat shock). We found two hybridization bands in the total RNA from heat-treated gastrula embryos (Fig. 4D). The two bands may be related to RNA of the major 72.5 and 70 kDa hsp of sea urchin embryos, suggesting a strong cross-homology between them, like that found in Drosophila between hsp 70 and 68 genes (Holmgren et al., 1981). By contrast, the RNA of the untreated gastrula embryos shows only one weaker hybridization band (Fig. 4C), corresponding to the slower RNA band of the treated gastrula embryos. The weaker band of the untreated gastrula embryos may suggest that the hsp 72.5 gene is weakly expressed constitutively also in the absence of heat shock at the gastrula stage. No hybridization is present in the total RNA prepared from heat-treated and untreated embryos of unresponsive morula stage (Fig. 4B and A). This confirms our previous experiments (Roccheri et al., 1982) on cell-free system protein synthesis, in which the two major hsp (72.5 and 70
101
would expect for the corresponding 72.5 and 70 kDa hsp. In another sea urchin species (A. punctulata) Heikkila et al. (1985b) found that the heterologous hsp 70 probe of Drosophila hybridized to polyadenylate RNA, prepared from heat-treated blastula embryos, larger than 0.8 kb with respect to the Drosophila hsp 70 mRNA. These results suggest the presence of an extensive untranslated region (about 1 kb) within these RNAs. There is a similar situation with respect to mRNA size in sea urchin embryos: actin mRNA contains an untranslated region of about 1 kb (Bushman and Crain, 1983). In Drosophila the hsp 70 mRNA also has an untranslated leader mRNA
A ¸
S~
C ¸
D
Fig. 3. Autoradiogram of a Southern blot of the genomic DNA of P. lividus. DNA was digested with EcoRI/SalI restriction enzymes, electrophoresed on 0.8% agarose gel, transferred onto nitrocellulose paper and hybridized to radiolabeled EcoRI/Sall fragment derived from P1716 plasmid. The hybridization was performed by high stringency (2 × SSC and 2 × Denhardt's). XDNA-HindIII and pBR322-Hin fI were used as markers (not shown).
kDa) were synthesized in the presence of RNA extracted from heat-treated gastrula embryos, whereas, in the presence of RNA from untreated gastrula embryos, only a slight synthesis of the 72.5 kDa hsp was observed. The hybridization bands are approx. 3200 and 3000 nucleotides in length (using mRNAs of the fl phage as markers). They therefore represent RNAs larger than one
Fig. 4. Autoradiogram of a Northern blot of P. lividus total RNAs hybridized to P1716 fragment. Total RNAs prepared from the heat-treated morula (M) and gastrula (G) embryos (31°C for 1 h), slot B and D, and untreated morula and gastrula embryos (20 o C), slot A and C, were fractionated on methylmercury agarose gel electrophoresis, transferred (Northern blot) onto hybond paper and hybridized to radiolabeled P1716 fragment by high stringency hybridization. The mRNA markers (fl) of bacteriophage fl are described in Materials and Methods.
102
A
B
shows that there is a different hybridization homology in the two RNA bands. The fact that the upper band shows a somewhat weaker hybridization may be explained by assuming that this band, corresponding to the hsp 72.5 gene, diverges to some extent in the upstream sequences from those upstream of the hsp 70 gene. Again the RNA from non-heated gastrula embryos shows a weak hybridization restricted to the upper band (Fig. 5A), whereas the absence of the slower band in untreated embryos and its presence in heat-treated embryos suggest that the slower RNA band corresponding to the hsp 70 gene is actually a heat-shock RNA band synthesized only after a heat shock.
Conclusions
Fig. 5. Autoradiogram of Northern blot of P. lividus total RNAs hybridized to P1718 fragment. Total RNAs prepared from the heat-treated gastrnla embryos (31°C for 1 h), slot B, and untreated gastrula embryos (20°C), slot A, were fractionated on methylmercury agarose gel electrophoresis, transferred (Northern blot) onto hybond paper and hybridized to radiolabeled P1718 fragment by high stringency hybridiza-
sequence of 250 nucleotides in length (Moran et al., 1979). In order to investigate whether the bands of the Fig. 4D actually contain some other RNA regions corresponding to a portion of the genome adjacent to the open reading frame of the hsp, we hybridized the RNA from heated gastrula embryos to the P1718 subclone, which corresponds to the genomic region upstream of that cross-hybridizing to the Drosophila hsp 70 gene. The autoradiogram of Fig. 5B shows that this probe hybridized again to the two bands, thus indicating that these also contain sequences corresponding to DNA sequences upstream of those hybridizing to the hsp 70 gene of Drosophila. The autoradiogram also
We have isolated from a Paracentrotus sperm DNA library four genes cross-hybridizing with the Drosophila hsp 70 gene. Their restriction map was then analyzed and their polarity determined. One such gene was subcloned and shown to hybridize only weakly to one electrophoretic band of RNA from non-heated gastrula embryos and to two electrophoretic bands of RNA from heated gastrula embryos. The absence of hybridization in the unresponsive morula stage indicates that this gene is transcribed almost exclusively at the specific stage of development and under heat shock conditions; this fact together with its homology to the Drosophila hsp 70 gene is a further indication that the subcloned gene actually is one hsp gene. The fact that the other three genes also cross-hybridize with the Drosophila hsp 70 gene suggests that there are at least 4 hsp 70 genes. The possibility, however, that they may be cognate genes has to be explored.
Acknowledgements The invaluable technical help of Mr. D. Cascino is acknowledged. This work was supported in part by funds of the MPI (40% and 60%) to G.G. and from the CNR of Italy (IBS).
103
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La Farina, M. and P. Model: Transcription in Bacteriophage fl-infected Escherichia coli messenger population in the infected cell J. Mol. Biol. 164, 377-393 (1983). Lindquist, S.: The heat-shock response. Armu. Rex,. Biochem. 55, 1151-1191 (1986). Loomis, W.F. and S. Wheeler: Heat shock response of Dictyostelium. Dev. Biol. 79, 399-408 (1980). Maniatis, T., E.F. Fritsch and J. Sambrook: Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, New York) (1982). Moran, L., M.E. Mirault, A. Tissi6res, J. Lis, P. Schedl, S. Artavanis-Tsakonas and W.J. Gehring: Physical map of two Drosophila melanogaster DNA segments containing sequences coding for 70000 Dalton heat shock protein. Cell 17, 1-8 (1979). O'Malley, K., A. Mauron, J.D. Barchas and L. Kedes: Constitutively expressed rat mRNA encoding a 70 kilodalton heat shock-like protein. Mol. Cell. Biol. 5, 3476-3483 (1985). Roccheri, M.C., M.G. Di Bernardo and G. Giudice: Synthesis of heat shock proteins in developing sea urchins. Dev. Biol. 83, 173-177 (1981a). Roccheri, M.C., G. Sconzo, M.G. Di Bernardo, I. Albanese, M. Di Carlo and G. Giudice: Heat shock proteins in sea urchin embryos, territorial and intracellular location. Acta Embryol. Morphol. Exp. 2, 91-99 (1981b). Roccheri, M.C., G. Sconzo, M. Di Carlo, M.G. Di Bernardo, A.M. Pirrone, R. Gambino and G. Giudice: Heat shock proteins in sea urchin embryos, transcriptional and posttranscriptional regulation. Differentiation 22, 175-178 (1982). Roccheri, M.C., G. Sconzo, M. La Rosa, D. Oliva, A. Abrignani and G. Giudice: Response to heat shock of different sea urchin species. Cell Differ. 18, 131-135 (1986). Schlesinger, M.J.: Heat shock proteins: the search for functions. J. Cell Biol. 103, 321-325 (1986). Schlesinger, M.J., M. Ashburner and A. Tissi6res: Heat Shock, From Bacteria to Man (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) (1982). Sconzo, G., M.C. Roccheri, M. Di Carlo, M.G. Di Bernardo and G. Giudice: Synthesis of heat shock proteins in dissociated sea urchin embryonic cells. Cell Differ. 12, 317-320 (1983). Sconzo, G., M.C. Roached, D. Oliva, M. La Rosa and G. Giudice: Territorial localization of heat shock mRNA production in sea urchin gastrulae. Cell Biol. Int. Rep. 9, 877-881 (1985). Sconzo, G., M.C. Roccheri, M. La Rosa, D. Oliva, A. Abrignani and G. Giudice: The acquisition of thermotolerance in sea urchin embryos is correlated with the synthesis and the age of the heat shock proteins. Cell Differ. 19, 173-177 (1986). Southern, E.M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517 (1975). Storti, R., M.P. Scott, A. Rich and M.L. Pardue: Translational control of protein synthesis in response to heat shock in Drosophila melanogaster cells. Cell 22, 825-834 (1980). Velazques, T.M. and S. Lindquist: Hsp 70 nuclear concentra-
104 tion during environmental stress and cytoplasmic storage recovery. Cell 36, 655-662 (1984). Vieira, J. and J. Messing: The pUC plasmids, and M13 mp7derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259-268 (1982).
Yamamori, T. and T. Yura: Genetic control of heat shock protein synthesis and its bearing on growth and thermal resistance in Escherichia coli K 12. Proc. Natl. Acad. Sci. USA 79, 860-864 (1982).
97
CDF 00509
Isolation and characterization of a sea urchin hsp 70 gene segment G. Sconzo 1, M. La Rosa 1, M. La Farina 1, M.C. Roccheri 1, D. Oliva 1 and G. Giudice 1,2 Dipartimento di Biologia Cellulare e dello Soiluppo and 2 Istituto di Biologia dello Soiluppo del CNR, Via Archirafi n. 2 0 - 22, 90123 Palermo, Italy
(Accepted 15 February 1988)
Three clones containing Paracentrotm lividus sea urchin DNA sequences which cross-hybridize to Drosophila heat shock protein (hsp) 70 gene were isolated. The sequence arrangements in the three cloned DNA inserts were compared by restriction and cross-hybridization analysis. The results showed that they contain four different genes related to one Drosophila hsp 70 gene. One of these genes was subcloned, and two of the isolated fragments were shown to hybridize to genomic DNA and to RNA from heat-treated sea urchin embryo. Heat shock protein 70, Sea urchin; DNA sequence; Cross hybridization
Introduction
The response to heat shock with the production of heat shock proteins (hsp) is a well-known phenomenon, common to most, if not all, organisms (Loomis and Wheeler, 1980; Storti et al., 1980; Kelley and Schlesinger, 1982; Schlesinger et al., 1982; Yamamori and Yura, 1982; Craig et al., 1983; Bardwell and Craig, 1984; Velazques and Lindqttist, 1984; Atkinson and Walden, 1985; Heikkila et al., 1985a; O'Malley et al., 1985; Lindquist, 1986; Schlesinger, 1986). This response in sea urchin embryos is specific to the developmental stage considered: when exposed to heat shock, embryos between fertilization and early blastula are unable to synthesize the heat shock proteins, and degenerate and die. On the other hand, when
Correspondence address: G. CAudice, Istituto di Biologia dello Sviluppo del CNR, Via Archirafi n. 20-22, 90123 Palermo, Italy.
subjected to heat from the hatching stage until the pluteus stage, they synthesize the heat shock proteins and develop normally (Giudice et al., 1980; Roccheri et al., 1981a, b, 1986; Sconzo et al., 1983, 1985, 1986). Sea urchin embryos therefore represent, in this respect, a suitable model for the study of cell differentiation at a molecular level during the course of embryonic development. In sea urchins, the two major hsp, of 72.5 and 70 kDa, are abundant (the 70 kDa comigrates, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with Drosophila 70 kDa hsp; Sconzo, unpublished data), and their production is regulated at the transcriptional level (Roccheri et al., 1982). As an approach to investigating the activation mechanism of the hsp 70 gene during different developmental stages, we isolated and characterized three clones, in which there are four different genes cross-hybridizing to the Drosophila hsp 70 gene. One of these, subcloned into the pUC8 vector, was used as a hybridization probe in the analysis
0045-6039/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland, Ltd.
98 of the genomic DNA. It was able to hybridize only to one electrophoretic band of RNA from non-heated gastrula embryos and to two electrophoretic bands of RNA from heat-shocked gastrula embryos. Materials and Methods
Embryo culture The embryos were allowed to develop in Millipore filtered sea water at 20°C as previously described (Giudice and Mutolo, 1967) up to the desired stage and then divided into two parts: one part was subjected to heat shock at 31°C for 1 h (heat-treated embryos) and the other part was left to develop at 20 °C (untreated embryos). Genomic DNA The genornic DNA was isolated from a single male (Paracentrotus lividus), as described by Kedes et al. (1975). One part was partially digested with Sau3A enzyme to prepare a genomic library. The other part was used for Southern blot analysis (Southern, 1975). Genomic library A genomic library from P. lividus sperm DNA was constructed in EMBL3 phage using standard methods (Maniatis et al., 1982). The genomic DNA, prepared as described previously, was partially digested with S a u 3 A enzyme and fractionated on sucrose gradient. Fragments of the size selected, 13-20 kb, were ligated into BamHI/EcoRI cut EMBL3 phage (courtesy of Dr. Spinelli). The ligation mixture was in vitro packaged, using extracts from a kit form (packaging system for lambda DNA, Amersham), and about 105 plaques//~g DNA were obtained upon plating onto selective strain Q359 (Karn et al., 1980). More than 5 x 105 plaques were screened (Benton and Davis, 1977) by low stringency hybridization in a solution containing 5 × SSC (0.75 M sodium chloride and 0.075 M sodium citrate) and 5 × Denhardt solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), using as a probe 10 ng/ml of the 2.2 kb SalI fragment containing the coding sequence of a hsp 70 Drosophila gene prepared from
the 56H8 clone (Moran et al., 1979). This DNA fragment was nick-translated using standard methods (Maniatis et al., 1982), reaching a value of about 2.5 × 108 cpm//tg. Three positive clones were isolated and after plaque purification the recombinant DNA was prepared from lysates (Frischauf, 1983). These clones have been named XPLH1, XPLH3 and XPLH9.
Restriction mapping, gel electrophoresis and gel blotting Single or double restriction enzyme digestions of the three clones were performed in order to obtain the restriction maps under the conditions recommended by the suppliers (Amersham and Boehringer). Restriction fragments were separated by 0.6-1.0% agarose gel electrophoresis. After the Southern blot, the filters were hybridized in 5 x SSC and 5 × Denhardt solution, containing 10 ng/ml of radiolabeled fragments (at about 2.5 x 10 s cpm/~tg). Fragments of 1.3 kb or 0.9 kb were prepared by BamHI enzyme digestion from the 2.2 kb SalI fragment of the hsp 70 Drosophila gene. The filters were therefore exhaustively washed in 2 × SSC. Subclones In order to obtain the gene regions contained within the inserts, the first insert, XPLH1, was digested with SalI enzyme and the resulting three fragments were ligated to pUC8 vector (Vieira and Messing, 1982) to generate P l l , P16 and P17 plasmids. Each of these was digested with the suitable restriction enzymes EcoRI (E), HindlII (H), BamHI (B), AccI (A), Sail (S), PstI (P), to insert the resulting fragments in the pUC8 vector. The recombinant plasmids PAl6, PB16 and PC16 were derived from P16 plasmid; P1735, P1718, P1716, P1707 and P1711 from P17 plasmid; and Pl104, Pll06 from P l l plasmid. Genomic DNA and Southern blot analysis Genomic DNA was digested with both EcoRI and Sail enzymes, separated on 0.8% agarose gel electrophoresis and, after Southern blot, hybridized by high stringency hybridization in a solution containing 2 x SSC and 5 x Denhardt's, using as a probe the radiolabeled fragment (at
99 about 2.5 × 108 c p m / # g ) isolated from the P1716 plasmid. Bacteriophage ~, D N A - H i n d l I I and pBR322-HinfI were used as markers.
R N A isolation and Northern blot analysis Total R N A was extracted from heat-treated embryos or untreated embryos, at the stages of morula and gastrtfla, by a procedure modified from Holmes and Bonner (1973). The embryos were lysed in 7 M urea, 2% SDS, 0.35 M sodium chloride, 10 m M Tris-HC1 (pH 8.0) and extracted four times with equal volume phenol-chloroform. After precipitation the pellet was resuspended in 0.5% SDS, 20 m M sodium chloride, 1 m M EDTA, extracted four more times and precipitated twice. The purified total R N A was treated with 50 # g / m l D N A s e I (Miles) and fractionated by methylmercury agarose gel electrophoresis (Bailey and Davidson, 1986), using bacteriophage fl m R N A s (4 and 1.45 kb) as markers. Our R N A markers were visualized by hybridizing R N A extracted from fl infected E. coli to a probe derived from sequences of gene IV of that phage as described in La Farina and Model (1983). Total H I
E i
S E I I
Ell II
E S S ~P"~"~"J"~Jllml
a
R N A was transferred onto a nylon membrane (Hybond, Amersham) fixed by UV irradiation and hybridized to the radiolabeled fragments isolated either from P1716 plasmid or P1718 plasmid.
Autoradiography Autoradiography was done at - 7 0 ° C K o d a k X - O m a t film and an intensifier.
with
Results and Discussion We have constructed a genomic library of P. lividus sperm D N A in EMBL3 phage and have screened more than 5 x 105 plaques using, as a hybridization probe, the 2.2 kb Sail fragment of hsp 70 Drosophila gene, derived from 56H8 clone (courtesy of Dr. W. Gehring). Three clones carrying sequences cross-hybridizing to hsp 70 Drosophila probe were isolated by low stringency hybridization. After plaque purification, the restriction mapping and the Southern blot analysis of these recombinant D N A s showed that the isolated clones carried three different segments of P. lividus D N A . The organization and restriction
•~ PLH1
b
E
E
B
S
BS
SEE PLH3
c
1Kb I--t
d
BS
SEE
'E I
ESE I I I
,~PLH9
e
3' 1.3Kb 0.9Kb
Fig. 1. Restriction maps of the three DNA inserts from ~PLH1, XPLH3 and XPLH9 clones. Restriction maps of the XPLH3 and XPLH9 inserts were obtained by single or double digestion with BamHI (B), Sail (S), EcoRI (E) enzyme; and restriction map of the XPLH1 with the same enzymes and also with HindlII (H). The gene regions cross-hybridizingto 2.2 kb Sail of the hsp 70 Drosophila gene are represented by the boxes. The dashed boxes hybridize mainly to the 1.3 kb fragment of the hsp 70 Drosophila gene corresponding to the 5' terminus (see inset). The shadowed boxes hybridize to the 0.9 kb fragment of the hsp 70 Drosophila gene corresponding the 3' terminus. The inset shows the two fragments, used as probes, of 1.3 kb and 0.9 kb, prepared from the 2.2 kb Sail fragment of the hsp 70 Drosophila gene derived from 56H8 clone by digestion with BamHI enzyme.
100 mapping analysis of these three different isolated segments are depicted in Fig. 1. The first insert, ?~PLH1, is 14 kb in length and contains two different gene regions (a and b) separated by nearly 9500 nucleotides. The positions of the 5' and 3' termini in the cross-hybridizing regions were defined by using as hybridization probes 5' or 3' fragments, of the Drosophila hsp 70 probe, obtained by digesting it with BarnHI enzyme (see inset in Fig. 1). The second segment, kPLH3, about 17.5 kb in length, also contains two gene regions (c and d) related to the Drosophila hsp 70 gene, separated by 7000 nucleotides. The comparison of the organization of these two DNA inserts shows that the genes differ from one another. The third insert, ~ PLH9, about 18 kb in length, contains only one gene region (e) at one end. Regions e and d show the same restriction map. The portions of the three inserts which are outside of the gene show restriction maps which are different from each other. We infer that the three isolated clones contain four different genes related to the Drosophila hsp 70 gene; three, b, c, d = e, are complete and the fourth, a, is about one half gene, containing the 3' end. In order to purify the gene regions contained within one of the inserts, the first insert, )tPLH1, was divided into three fragments by SalI restriction enzyme and subcloned into the pUC8 vector. Each of these subclones, P16, P17, P l l , was further subcloned. Fig. 2 shows the total map of the 13 subclones. One of these subclones, P1716, containing the 1.5 kb Eco-Sal insert, was found in the Southern blot experiments to hybridize, with high stringency, to a 1.5 kb band of the genomic Eco-Sal restricted DNA (Fig. 3). This is a further proof that this subclone is actually constitutively well represented in the sea urchin genome. The autoradiogram also shows another expected hybridization band, i.e. that of 6.3 kb corresponding to the EcoRI-SalI segment of XPLH3. In order to understand whether the isolated gene is a heat-shock gene transcribed at a specific stage of development, we hybridized the P1716 probe to the total RNA prepared from heat-treated embryos and untreated embryos of two different
IKb
SE
H
~
H
E
I
I
E I
E I
AE I
SPS }~ P L H 1 P16
k F
PA 16
4
PB16 PC16 P17 P1735
I
P1718
!
P1711
F------I
P1707 I
P1716
I
Pll P1106 H
P1104
Fig. 2. Subcloning of )~PLH1 DNA insert. The restriction map of hPLH1 DNA insert (top) and schematic representation of the fragments subcloned into the pUC8 vector (below). The boxes represent the cross-hybridizing regions to 2.2 kb SalI fragment of Drosophila hsp 70 gene. The P16, P17, P l l were generated directly from )tPLH1 insert by SalI restriction enzyme digestion. Further subcloning from P16, P17 and P l l gave rise to three plasmids (PAl6, PB16, PC16), to five plasraids (P1735, P1718, P1716, P1711, P1707) and two plasmids (Pl106 and P1104), respectively.
stages: morula (unresponsive to heat shock) and gastrula (responsive to heat shock). We found two hybridization bands in the total RNA from heat-treated gastrula embryos (Fig. 4D). The two bands may be related to RNA of the major 72.5 and 70 kDa hsp of sea urchin embryos, suggesting a strong cross-homology between them, like that found in Drosophila between hsp 70 and 68 genes (Holmgren et al., 1981). By contrast, the RNA of the untreated gastrula embryos shows only one weaker hybridization band (Fig. 4C), corresponding to the slower RNA band of the treated gastrula embryos. The weaker band of the untreated gastrula embryos may suggest that the hsp 72.5 gene is weakly expressed constitutively also in the absence of heat shock at the gastrula stage. No hybridization is present in the total RNA prepared from heat-treated and untreated embryos of unresponsive morula stage (Fig. 4B and A). This confirms our previous experiments (Roccheri et al., 1982) on cell-free system protein synthesis, in which the two major hsp (72.5 and 70
101
would expect for the corresponding 72.5 and 70 kDa hsp. In another sea urchin species (A. punctulata) Heikkila et al. (1985b) found that the heterologous hsp 70 probe of Drosophila hybridized to polyadenylate RNA, prepared from heat-treated blastula embryos, larger than 0.8 kb with respect to the Drosophila hsp 70 mRNA. These results suggest the presence of an extensive untranslated region (about 1 kb) within these RNAs. There is a similar situation with respect to mRNA size in sea urchin embryos: actin mRNA contains an untranslated region of about 1 kb (Bushman and Crain, 1983). In Drosophila the hsp 70 mRNA also has an untranslated leader mRNA
A ¸
S~
C ¸
D
Fig. 3. Autoradiogram of a Southern blot of the genomic DNA of P. lividus. DNA was digested with EcoRI/SalI restriction enzymes, electrophoresed on 0.8% agarose gel, transferred onto nitrocellulose paper and hybridized to radiolabeled EcoRI/Sall fragment derived from P1716 plasmid. The hybridization was performed by high stringency (2 × SSC and 2 × Denhardt's). XDNA-HindIII and pBR322-Hin fI were used as markers (not shown).
kDa) were synthesized in the presence of RNA extracted from heat-treated gastrula embryos, whereas, in the presence of RNA from untreated gastrula embryos, only a slight synthesis of the 72.5 kDa hsp was observed. The hybridization bands are approx. 3200 and 3000 nucleotides in length (using mRNAs of the fl phage as markers). They therefore represent RNAs larger than one
Fig. 4. Autoradiogram of a Northern blot of P. lividus total RNAs hybridized to P1716 fragment. Total RNAs prepared from the heat-treated morula (M) and gastrula (G) embryos (31°C for 1 h), slot B and D, and untreated morula and gastrula embryos (20 o C), slot A and C, were fractionated on methylmercury agarose gel electrophoresis, transferred (Northern blot) onto hybond paper and hybridized to radiolabeled P1716 fragment by high stringency hybridization. The mRNA markers (fl) of bacteriophage fl are described in Materials and Methods.
102
A
B
shows that there is a different hybridization homology in the two RNA bands. The fact that the upper band shows a somewhat weaker hybridization may be explained by assuming that this band, corresponding to the hsp 72.5 gene, diverges to some extent in the upstream sequences from those upstream of the hsp 70 gene. Again the RNA from non-heated gastrula embryos shows a weak hybridization restricted to the upper band (Fig. 5A), whereas the absence of the slower band in untreated embryos and its presence in heat-treated embryos suggest that the slower RNA band corresponding to the hsp 70 gene is actually a heat-shock RNA band synthesized only after a heat shock.
Conclusions
Fig. 5. Autoradiogram of Northern blot of P. lividus total RNAs hybridized to P1718 fragment. Total RNAs prepared from the heat-treated gastrnla embryos (31°C for 1 h), slot B, and untreated gastrula embryos (20°C), slot A, were fractionated on methylmercury agarose gel electrophoresis, transferred (Northern blot) onto hybond paper and hybridized to radiolabeled P1718 fragment by high stringency hybridiza-
sequence of 250 nucleotides in length (Moran et al., 1979). In order to investigate whether the bands of the Fig. 4D actually contain some other RNA regions corresponding to a portion of the genome adjacent to the open reading frame of the hsp, we hybridized the RNA from heated gastrula embryos to the P1718 subclone, which corresponds to the genomic region upstream of that cross-hybridizing to the Drosophila hsp 70 gene. The autoradiogram of Fig. 5B shows that this probe hybridized again to the two bands, thus indicating that these also contain sequences corresponding to DNA sequences upstream of those hybridizing to the hsp 70 gene of Drosophila. The autoradiogram also
We have isolated from a Paracentrotus sperm DNA library four genes cross-hybridizing with the Drosophila hsp 70 gene. Their restriction map was then analyzed and their polarity determined. One such gene was subcloned and shown to hybridize only weakly to one electrophoretic band of RNA from non-heated gastrula embryos and to two electrophoretic bands of RNA from heated gastrula embryos. The absence of hybridization in the unresponsive morula stage indicates that this gene is transcribed almost exclusively at the specific stage of development and under heat shock conditions; this fact together with its homology to the Drosophila hsp 70 gene is a further indication that the subcloned gene actually is one hsp gene. The fact that the other three genes also cross-hybridize with the Drosophila hsp 70 gene suggests that there are at least 4 hsp 70 genes. The possibility, however, that they may be cognate genes has to be explored.
Acknowledgements The invaluable technical help of Mr. D. Cascino is acknowledged. This work was supported in part by funds of the MPI (40% and 60%) to G.G. and from the CNR of Italy (IBS).
103
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