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Cloning of the cDNA encoding rat homologue of the mismatch repair gene MSH2 and its expression during spermatogenesis
Gene 185 (1997) 19–26
Cloning of the cDNA encoding rat homologue of the mismatch repair gene MSH2 and its expression during spermatogenesis R. Geeta Vani a, M.R.S. Rao a,b,* a Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India b Jawaharlal Nehru Centre for Advanced Scientific Research, Indian Institute of Science, Bangalore 560 012, India Received 7 May 1996; revised 29 July 1996; accepted 29 July 1996
Abstract A rat cDNA clone encoding the mismatch repair protein MSH2 has been isolated and characterized. The cDNA has an open reading frame of 2802 nucleotides in length coding for a protein of 933 amino acids (100 kDa). It shows significant homology to human and mouse MSH2. Northern blot analysis of rat MSH2 in the testes of rats of different ages showed maximum expression at 20 days, at which time the germ cells are undergoing premeiotic DNA replication. We observed down-regulation in the expression of rat MSH2 beyond 25 days by which time the germ cells have entered meiotic prophase. Keywords: RT-PCR; Sequence; Northern blot; Testis
1. Introduction The mismatch repair system plays a pivotal role in maintaining the fidelity of DNA replication (Modrich, 1991). The best studied mismatch repair pathway is the MutHLS pathway of Escherichia coli. The MutS gene product recognizes and binds to mismatches that arise during DNA replication. MutH, a latent endonuclease, is activated by MutL and MutS-DNA complex and nicks at the unmethylated GATC site in the newly synthesized strand. Identification of several eukaryotic MutS and MutL homologues has lead to understanding the mechanism of mismatch repair in eukaryotic cells. MutS homologues (MSH1 to 6) (Reenan and Kolodner, 1992a; New et al., 1993; Ross-Macdonald and Roeder, 1994; Hollingsworth et al., 1995; Marsischky et al., 1996) and MutL homologues (MLH1, MLH2 and PMS1) have been identified in Saccharomyces cerevisiae ( Kramer et al., 1989; Prolla et al., 1994a,b). In humans, MutS homologues MSH2 and GTBP/p160 and MutL * Corresponding author at address a. Tel. +91 80 3092547; Fax +91 80 3341683; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; kb, kilobase(s) or 1000 bp; MLH1, mutL homologue; nt, nucleotide(s); PCR, polymerase chain reaction; rMSH2, rat mutS homologue; RT-PCR, reverse transcription PCR; UTR, untranslated region(s). 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 6 22 - 1
homologues hMLH1, hPMS1 and hPMS2 have been identified and characterized and mutations in hMSH2, hMLH1, hPMS1 and hPMS2 have been linked to hereditary non-polyposis colon cancer ( Fishel et al., 1993; Leach et al., 1993; Bronner et al., 1994; Papadopoulos et al., 1994; Nicolaides et al., 1994; Drummond et al., 1995; Palombo et al., 1995). Knock out mice for MSH2 develop normally but are susceptible for lymphoid tumor providing strong evidence for its association with pathogenesis of cancer (de Wind et al., 1995; Reitmar et al., 1995). Recombinant MSH2 protein from humans and yeast binds to insertion/deletion loops of 1–14 nt in length ( Fishel et al., 1994a,b; Alani et al., 1995). MSH2 mutants of S. cerevisiae are defective in mismatch repair, show an increase in mitotic mutation rate, postmeiotic segregation events, microsatellite instability and homologous recombination (Reenan and Kolodner, 1992b; Strand et al., 1993; Alani et al., 1994; and Selva et al., 1995). MSH2 deficient mouse cells show loss of mismatch binding activity, mutator phenotype and an increase in homologous recombination ( Reitmar et al., 1995; de Wind et al., 1995). MSH2 homologues have also been identified in Xenopus and mouse ( Varlet et al., 1994). The mismatch repair system, in addition to having a key role in maintaining the integrity of nucleotide sequences in the genome, also plays an important role in providing a barrier to interspecies recombination by
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its antirecombinogenic role in homologous recombination (Rayssiguier et al., 1989; Radman and Wagner, 1993; Matic et al., 1995). Mammalian spermatogenesis offers a unique system wherein germ cells undergo several rounds of mitotic division of spermatogonial cells after which they get committed to meiotic division. A unique event characteristic of meiotic division is recombination between paired homologous chromosomes in primary spermatocytes. Hence it is interesting to see the temporal expression of MSH2 during spermatogenesis. Towards this objective, we have cloned and sequenced the rat homologue of MSH2 and studied its expression in the testes of rats of different age groups.
2. Materials and methods 2.1. Materials Restriction endonucleases and DNA modifying enzymes were obtained from New England Biolabs, Amersham International, and Bangalore Genie, India. Biochemicals were obtained from Sigma Chemical Company. Oligonucleotide primers were synthesized in the oligonucleotide synthesis facility, Indian Institute of Science, and Bangalore Genie, India. T sequencing kit 7 was obtained from Pharmacia Biotech, Sweden and rat testes lZap(II ) cDNA library was obtained from Stratagene, USA. [a-32P]dATP and [35S]dATP were obtained from Board of Radiation and Isotope Technology, India. 2.2. Methods 2.2.1. Cloning of rMSH2/2.8 kb The construction of various clones is described below. (1) prMSH2/350 bp. RT-PCR was performed using degenerate oligos synthesized for the aa sequences TGPNM and FATH(F/Y ). The sequences of oligonucleotides were 5∞-CGCGGATCCAC(G/A/T/C ) GG(GA/T/C ) CC(G/A/T/C ) AA ( T/C ) ATG-3∞ (primer C ) and 5∞-CGCAAGCTT(G/A) (A/T )A (G/A)TG(G/A/T/C )GT(G/A/T/C )GC(G/A)AA-3∞ (primer E ). RT-PCR was carried out using these primers and 30 ng of first strand cDNA synthesized from poly(A)+RNA of rat testes (40 days of age) and Taq DNA polymerase (Bangalore Genei). PCR was done with cycling conditions of 94°C for 1 min, 50°C for 2 min, and 72°C for 1 min for 30 cycles. The expected size PCR product (350 bp) was eluted from the agarose gel and subjected to another round of PCR amplification. The PCR product was digested with BamHI and HindIII and cloned into pBluescript II (SK ). The clones that hybridized to human 350 bp MSH2 product were sequenced by
the method of Sanger et al. (1977) using a T 7 sequencing kit (Pharmacia). (2) prMSH2/1.2 kb. The human MSH2 cDNA was digested with NcoI and EcoRI and a 1.5 kb fragment corresponding to the 3∞ end of hMSH2 was gel purified and used as a probe to screen the rat testes lZap(II ) cDNA library (Stratagene). About 3×105 plaques were screened with hybridization at 42°C in 6×SSC, 5×Denhardt’s solution, 0.5% SDS, 100 mg/ml denatured salmon sperm DNA and denatured probe (5×106 cpm/ml ). The filters were washed in 1×SSC, 0.1% SDS at 50°C and exposed for autoradiography at −70°C with an intensifying screen. The positive signal obtained after primary screening was purified and subjected to secondary screening using similar conditions. The positive plaques were purified and recombinant pBluescript (SK ) plasmid containing the insert was excised using the Exassit Excision system according to the manufacturer’s (Stratagene) protocol. The insert was released by EcoRI and sequenced using KS, SK, T , T and insert specific primer (primer D). 3 7 (3) prMSH2/2.0 kb. To obtain cDNA coding for the 5∞ end of rMSH2, RT-PCR was done using an upstream degenerate oligonucleotide synthesized for mouse aa sequence (MAVAP) 5∞-CCGAATTCGCATATG CG(G/A/T/C ) GT (G/A/TC ) CA (G/A) CC (G/A/T/C ) AA-3∞ (primer A) and a downstream oligo specific for rMSH2 sequence 5∞-CCGTCTGACGGATGTATGTTG-3∞ (primer B) and rat testes cDNA prepared from 20-day-old rats. A 2.0 kb PCR product was obtained which was cloned into pMOS-Blue T vector (Amersham) and the clone was completely sequenced using insert specific (primer F ) 5∞-(TAATCTGTTTGCCAGGGTCC-3∞) and making various deletions using internal restriction sites for EcoRI, HindIII, PstI which were subcloned into pBluescript II SK or pUC 18. (4) prMSH2/2.8 kb. The complete cDNA coding for the ORF of rMSH2 was constructed using overlapping PCR. The insert of prMSH2/2.0 kb was amplified using primers A and B which gave a product insert of 2050 bp. The clone prMSH2/1.2 was amplified using primers C and 5∞-CTCCGTCGACCGGAGCCTTTACCCGTGA-3∞ (primer D), giving a product of 800 bp. This was done in order to eliminate the 5∞ end 168 nt of clone prMSH2/1.2 (shown in hatched box in Fig. 1), since this 168 nt sequence was an artifact of reverse transcription as revealed by sequence comparison. A 2.8 kb cDNA was obtained upon PCR with the 2.0 kb and 0.8 kb PCR products using primers A and D, since there is an overlap of 50 nt between the two PCR products. This 2.8 kb cDNA has the ORF coding for
R. Geeta Vani, M.R.S. Rao/Gene 185 (1997) 19–26
rMSH2, and has been cloned into pMOS Blue-T vector. 2.2.2. Genomic Southern blot analysis Rat genomic DNA was isolated from spleen (Sambrook et al., 1989). 15 mg of genomic DNA was digested with BamHI, EcoRI, HindIII, XbaI and SfiI, and electrophoresed on a 0.8% agarose gel and blotted onto Hybond Nylon membrane by vacuum blotting in 10×SSC. The DNA was UV crosslinked to the membrane and prehybridized in 6×SSC, 5×Denhardt’s, 0.5% SDS, 100 mg/ml denatured salmon sperm DNA at 65°C for 2 h. Hybridization was done for 16 h, in the presence of random hexamer primed and labeled rMSH2 cDNA at (1×107 cpm/ml ) at 65°C. Filters were washed at a final stringency of 0.1×SSC/0.1% SDS at 65°C and exposed to autoradiography with an intensifying screen at −70°C. The sizes of the fragments hybridizing to rMSH2 cDNA were calculated by the SEQAID program. 2.2.3. Northern blot analysis Total RNA was isolated from rat testes of various age groups using the acid guanidinium isothiocyanate phenol-chloroform method (Chomczynski and Sacchi, 1987). 30 mg of total RNA was electrophoresed on a 1% agarose gel in the presence of 2.2 M formaldehyde and blotted onto Hybond Nylon membrane (Amersham) in 20×SSC after an initial alkali denaturation in 0.05 N NaOH for 20 min. The filter was UV crosslinked and prehybridized with 6×SSC, 0.5% SDS, 5×Denhardt’s, 50% formamide, 100 mg/ml denatured
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salmon sperm DNA at 42°C for 2 h. Hybridization was done under the same conditions, with an a-32P-labeled rMSH2 cDNA probe at approx. 1×107 cpm/ml for 16 h at 42°C. Filters were washed at 0.1×SSC/0.1% SDS at 65°C and exposed to autoradiography. The radiolabeled rat glyceraldehyde 3-phosphate dehydrogenase cDNA was hybridized under similar conditions.
3. Results and discussion 3.1. Cloning of rat MSH2 (rMSH2) cDNA To obtain rat cDNA coding for the most conserved region of known MutS homologues a degenerate primer driven PCR was employed. Two degenerate oligonucleotide primers for the conserved aa sequences TGPNM (upstream oligo) and FATH (F/Y ) (downstream oligo) were used for the RT-PCR reaction using rat testes cDNA prepared from testes poly(A)+RNA as the template. The expected size of the PCR product (350 bp) was obtained which was cloned into pBluescript II (SK )+ plasmid and subsequently sequenced. One of the clones showed 87% identity to the corresponding region in human MSH2 cDNA. This cloned PCR product was then used as a probe to screen the rat testes lZap(II ) cDNA library (Stratagene). However, during this screening we observed considerable background hybridization, possibly due to hybridization of the probe with the endogenous E. coli mutS gene. To circumvent this problem, we used the 3∞ 1.5 kb EcoRI fragment of human MSH2 cDNA (which encompasses the highly conserved
Fig. 1. Schematic representation of various clones used for the construction of rMSH2 cDNA. rMSH2/2.0 kb and rMSH2/350 bp were obtained by RT-PCR, rMSH2/1.2 kb was obtained by screening and rMSH2/2.8 kb was constructed by overlapping PCR as described in Section 2.2. Various primers used for PCR and sequencing are also indicated. Deletion constructs for sequencing of prMSH2/2.0 kb are also depicted. The hatched box in prMSH2/1.2 kb represents the 168 bp which are an artifact of reverse transcription.
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NTP and DNA binding domain) as a heterologous probe to screen the lZap(II ) cDNA library under low stringency hybridization conditions. One positive clone was identified, having an insert of 1.2 kb in length, which upon sequencing showed homology to the human MSH2 cDNA. This clone had an ORF followed by a
stop codon and 3∞-UTR. In this 1.2 kb clone, the 5∞ 168 nt showed homology to the human MSH2 sequence in the complementary strand as opposed to the rest of the coding region, and in the reverse orientation (Fig. 1). This could have arisen due to an artifact of reverse transcription during cDNA synthesis. This clone was
Fig. 2. cDNA sequence and deduced aa sequence of rMSH2. The cDNA sequence has an ORF from nt 1 to 2802, coding for a protein of 933 aa. The 3∞-UTR extends from nt 2803 to 3002, having a putative polyadenylation signal AATAAA which is underlined. The various primers used for sequencing and PCR are underlined. The unique EcoRI seen only in the rMSH2 site is also underlined. The EMBL accession number for rMSH2 is X93591.
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partial, missing the 5∞-coding region. To obtain cDNA having a full-length ORF, the same library was rescreened using the 1.2 kb rat MSH2 cDNA as a probe. However, no full-length clone could be identified. Hence as an alternative strategy RT-PCR was used, using a degenerate oligonucleotide primer designed for the first 5 aa of mouse MSH2 sequence as the upstream primer and a specific downstream oligonucleotide primer based on the sequence of 1.2 kb rMSH2 cDNA. RT-PCR was done using these two sets of primers and rat testes cDNA of 20-day-old rats as the template. Twenty-dayold rat testes RNA was used, as the preliminary Northern data with 1.2 kb rMSH2 clone indicated that the level of expression was high at that age. A PCR product of the expected size of 2.0 kb was obtained which was subsequently cloned into pMOS-Blue T vector (Amersham) and sequenced. This clone had an ORF coding for the rat MSH2 having high homology to human and mouse MSH2 proteins (Fig. 1). Several clones were sequenced to rule out PCR generated errors.
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3.2. Construction of a full-length rMSH2 clone, and sequence analysis The two clones coding for rMSH2 cDNA were partial, one with respect to the 5∞ end (2.0 kb) and the other to the 3∞ end (1.2 kb). A cDNA having a complete ORF was constructed using overlapping PCR, from the PCR products obtained from each of the clones (Fig. 1). While constructing the full-length cDNA, PCR was done for the 1.2 kb clone, using primers so as to eliminate the 5∞ 168 nt which were showing a discrepancy in homology, due to an artifact of reverse transcription. The complete nt sequence of the rMSH2 was obtained by sequencing various deletion clones and using a number of specific primers (Fig. 1). A total sequence of 3002 nt was obtained. This cDNA has an ORF of 2802 nt starting from ATG at position 1 and coding for a protein of 933 aa (Fig. 2). The 3∞-UTR extends from 2803 to 3002 nt and has a putative polyadenylation signal. This clone does not have a 5∞-UTR since the
Fig. 3. Comparison of the deduced aa sequences of rat, mouse, human and Xenopus MSH2 sequences. rMSH2, aa sequence from rat, MMSH2 Gen is from mouse, HSO391 is from human and XELMSH2 A from Xenopus. * indicates identical aa and · indicates conserved substitutions. A–D are NTP binding motifs and HTH is helix-turn-helix motif, which are underlined. The alignment was done using the CLUSTAL program.
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primer used for the RT-PCR starts with the ATG codon. The homology comparison of rMSH2 with other MSH2 sequences revealed 90.4, 86.1 and 72.5% with respect to mouse, human and Xenopus respectively at the nt level while it was 94, 91.7 and 77.5% at the aa level. The highest level of conservation among all the known MutS homologues is in the C-terminal part of the protein which has a NTP binding domain and a DNA binding helix-turn-helix motif ( Fig. 3). 3.3. Genomic organization of rMSH2 Rat genomic DNA was digested with various restriction endonucleases and hybridized to rMSH2 cDNA probe after transfer to the Hybond membrane. Hybridization of rMSH2 cDNA to BamHI digest revealed fragments of 9.2 kb and 4.0 kb. Hybridization to EcoRI digest revealed fragments of 11 kb, 7.9 kb and 5.6 kb, and that of HindIII digest revealed fragments of 8.9 kb, 3.3 kb and 0.6 kb. XbaI digest showed hybridization to fragments of 10 kb, 7.6 kb, 5.01 kb, 3.5 kb, 2.8 kb and 0.9 kb. SfiI digest revealed hybridization to fragments greater than 23 kb. An idea of the genomic fragments hybridizing to rMSH2 cDNA will help the future studies in identifying the rMSH2 gene. The estimate of the rat genomic fragment encompassing the rMSH2 sequence is approx. 30 kb in length (a minimum estimate). The actual genomic fragment encompassing the entire rMSH2 transcription unit would be longer than this size since the cDNA probe used in this experiment does not contain the 5∞-UTR sequence and lacks the 3∞-UTR sequence. Furthermore, intronic sequences flanked by restriction sites for the enzymes used in this Southern blot will also not be detected. The human MSH2 gene has been localized to chromosome 2 (2p.22-21), and the genomic locus of hMSH2 has been isolated and characterized. The gene is approx. 70 kb in length and is split into 16 exons ( Kolodner et al., 1994) ( Fig. 4). 3.4. Expression of rMSH2 during spermatogenesis Earlier studies have shown that MSH2 does not have a tissue specific expression and is expressed in almost all the tissues suggesting that it functions as a house keeping gene ( Varlet et al., 1994). However, a recent study by Wilson et al. (1995) on the expression of rMSH2 in various normal human tissues shows that its expression is highest in thymus and testis. We have examined the expression of rMSH2 during various stages of spermatogenesis by isolating total RNA from the testes of rats of different ages and carrying out a Northern blot analysis with rMSH2 cDNA as a probe. By this study we can correlate the expression of rMSH2 with several events associated with the germ cell development and differentiation during spermatogenesis. A
Fig. 4. Rat genomic Southern blot with rMSH2 as a probe. Rat genomic DNA digested with various restriction enzymes was probed with rMSH2 cDNA.
single transcript (approx. 3.5 kb) was observed ( Fig. 5A). The results also reveal that the expression of rMSH2 mRNA is highest at day 20, beyond which its level decreases with increasing age of the rat. Equal amounts of total RNA was loaded in each of the lanes as judged by the intensities of 28S and 18S rRNA bands ( Fig. 5B). Hybridization with rat GAPDH cDNA probe was used as an internal control (Fig. 5C ). Mammalian spermatogenesis is asynchronous and new waves of germ cells start before the first round of germ cells complete their maturation process. However, the approximate time scale of the appearance of a specific germ cell at different stages of their differentiation in the first wave of spermatogenesis with respect to the age of the rat is known (Meistrich et al., 1978). At 10 days of age, the predominant germ cells in the testes are spermatogonial cells. These cells enter premeiotic S-phase at the age of 18–20 days wherein there is active premeiotic DNA synthesis. Subsequently these cells enter meiotic prophase among which pachytene spermatocytes have the longest lifetime of 8–10 days (25–35 days of age in the rat). It is generally believed that the recombination between the paired homologous chromosomes takes place at the pachytene interval. Haploid spermatids in the rat are generated between the 35th and 38th day. It is interesting to note that the expression of rMSH2 is highest at 20 days of age in the rat during which time the germ cells are engaged in premeiotic DNA synthesis. A more interesting observation is that rMSH2 is down-regulated during the meiotic prophase. As mentioned in Section 1, the mismatch repair system also functions as antirecombinogenic effector. Hence a
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Fig. 5. Northern blot analysis of rMSH2 during spermatogenesis. (A) Hybridization of rat testes total RNA (30 mg) from 10–60-day-old rats with rat MSH2 cDNA. (B) Total RNA from rat testes of 10–60-day-old rats. (C ) Hybridization with glyceraldehyde phosphate dehydrogenase cDNA.
down-regulation of rMSH2 during meiotic prophase may be physiologically relevant to allow recombination between mammalian homologous chromosomes containing many of the repeat sequence families like microsatellites and minisatellites. It may also be pertinent to point out here that additional MutS homologues, MSH4 and MSH5, have been discovered in S. cerevisiae (RossMacdonald and Roeder, 1994; Hollingsworth et al., 1995). MSH4 is expressed in a meiotic specific manner during sporulation of yeast and MSH5 gene acts in meiosis. Their gene products have been implicated in processing specifically the recombination intermediates. It is noteworthy that MSH4 and MSH5 mutants are not defective in DNA repair but show an increase in non-disjunction of homologous chromosomes at meiosis. It remains to be seen whether similar MSH2 homologues are conserved in mammals and if so, what would be their pattern of expression during spermatogenesis in comparison to MSH2.
4. Conclusions (1) We have cloned a cDNA coding for the rat homologue of mismatch repair gene MSH2. (2) The rMSH2 shows very high homology to the known eukaryotic MSH2 at both nucleic acid and amino acid level. (3) The genomic locus coding for rMSH2 is approximately 30 kb in length. (4) The expression of rMSH2 shows down-regulation during later stages of spermatogenesis. The expression is highest at 20 days of age in the rat associated with germ cells undergoing premeiotic DNA replication. Subsequently there is a down-regulation of the level of MSH2 mRNA with the progress of meiotic prophase and spermiogenesis.
Acknowledgement This work was financially supported by the Department of Science and Technology, Government of India, New Delhi. We would like to thank Dr. S.K. Brahmachari, Convenor of the Oligonucleotide Facility of the Indian Institute of Science for the oligonucleotides used for PCR and sequencing. The help rendered by the Bioinformatics group IISc. is gratefully acknowledged. R.G.V. is a Senior Research Fellow of the University Grants Commission, New Delhi.
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Papadopoulos, N., Nicolaides, N.C., Wei, Y.-F., Ruben, S.M., Carter, K.C., Rosen, C.A., Haseltine, W.A., Fleischmann, R.D., Fraser, C.M., Adams, M.D., Venter, J.C., Hamilton, S.R., Petersen, G.M., Watson, P., Lynch, H.T., Peltomati, P., Mecklin, J.P., De la Chapelle, A., Kinzler, K.W. and Vogelstein, B. (1994) Mutation of a mut L homolog in hereditary colon cancer. Science 263, 1625–1629. Prolla, T.A., Christie, D.M. and Liskay, R.M. (1994a) Dual requirement in yeast DNA mismatch repair for MLH1 and PMS1, two homologs of the bacterial mut L gene. Mol. Cell. Biol. 14, 407–415. Prolla, T., Panga., Alani, E., Kolodner, R.D. and Liskay, R.M. (1994b) MLH1, PMS1 and MSH2 interactions during the initiation of DNA mismatch repair in yeast. Science 265, 1091–1093. Radman, M. and Wagner, R. (1993) Mismatch recognition in chromosomal interactions and speciation. Chromosoma 102, 369–373. Rayssiguier, C., Thaler, D.S. and Radman, M. (1989) The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch repair mutants. Nature 342, 396–401. Reenan, R.A.G. and Kolodner, R.D. (1992a) Isolation and characterization of two Saccharomyces cerevisiae genes encoding homologs of the bacterial Hex A and Mut S mismatch repair proteins. Genetics 132, 963–973. Reenan, R.A.G. and Kologner, R.D. (1992b) Characterization of insertion mutations in the Saccharomyces cerevisiae MSH 1 and MSH 2 genes: evidence for separate mitochondrial and nuclear functions. Genetics 132, 975–985. Reitmar, A.H., Schmits, R., Ewel, A., Bapat, B., Redston, M., Mitri, A., Waterhouse, P., Mittrucker, H.W., Wakeham, A., Liu, B., Thomason, A., Griesser, H., Gallinger, S., Balljausen, W.G., Fishel, R. and Mak, T.W. (1995) MSH2 deficient mice are viable and susceptible to lymphoid tumors. Nat. Genet. 11, 64–70. Ross-Macdonald, P. and Roeder, G.S. (1994) Mutation of a meiosis specific Mut S homolog decreases crossing over but not mismatch correction. Cell 79, 1069–1080. Sambrook, J., Fritsch, C.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467. Selva, E.M., New, L., Crouse, F.G. and Lahue, R.S. (1995) Mismatch correction acts as a barrier to homologous recombination in Saccharomyces cerevisiae. Genetics 139, 1175–1188. Strand, M., Prolla, T.A., Liskay, R.M. and Petes, T.D. (1993) Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature 365, 274–276. Varlet, I., Pallard, C., Radman, M., Moreau, J. and de Wind, N. (1994) Cloning and expression of Xenopus and mouse MSH2 DNA mismatch repair genes. Nucleic Acids Res. 22, 5723–5728. Wilson, T.M., Ewel, A., Duguid, R.J., Eble, N.J., Lescoe, M.K., Fishel, R. and Kelley, M.R. (1995) Differential cellular expression of the human MSH2 repair enzyme in small and large intestine. Cancer Res. 55, 5146–5150.
Cloning of the cDNA encoding rat homologue of the mismatch repair gene MSH2 and its expression during spermatogenesis R. Geeta Vani a, M.R.S. Rao a,b,* a Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India b Jawaharlal Nehru Centre for Advanced Scientific Research, Indian Institute of Science, Bangalore 560 012, India Received 7 May 1996; revised 29 July 1996; accepted 29 July 1996
Abstract A rat cDNA clone encoding the mismatch repair protein MSH2 has been isolated and characterized. The cDNA has an open reading frame of 2802 nucleotides in length coding for a protein of 933 amino acids (100 kDa). It shows significant homology to human and mouse MSH2. Northern blot analysis of rat MSH2 in the testes of rats of different ages showed maximum expression at 20 days, at which time the germ cells are undergoing premeiotic DNA replication. We observed down-regulation in the expression of rat MSH2 beyond 25 days by which time the germ cells have entered meiotic prophase. Keywords: RT-PCR; Sequence; Northern blot; Testis
1. Introduction The mismatch repair system plays a pivotal role in maintaining the fidelity of DNA replication (Modrich, 1991). The best studied mismatch repair pathway is the MutHLS pathway of Escherichia coli. The MutS gene product recognizes and binds to mismatches that arise during DNA replication. MutH, a latent endonuclease, is activated by MutL and MutS-DNA complex and nicks at the unmethylated GATC site in the newly synthesized strand. Identification of several eukaryotic MutS and MutL homologues has lead to understanding the mechanism of mismatch repair in eukaryotic cells. MutS homologues (MSH1 to 6) (Reenan and Kolodner, 1992a; New et al., 1993; Ross-Macdonald and Roeder, 1994; Hollingsworth et al., 1995; Marsischky et al., 1996) and MutL homologues (MLH1, MLH2 and PMS1) have been identified in Saccharomyces cerevisiae ( Kramer et al., 1989; Prolla et al., 1994a,b). In humans, MutS homologues MSH2 and GTBP/p160 and MutL * Corresponding author at address a. Tel. +91 80 3092547; Fax +91 80 3341683; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; kb, kilobase(s) or 1000 bp; MLH1, mutL homologue; nt, nucleotide(s); PCR, polymerase chain reaction; rMSH2, rat mutS homologue; RT-PCR, reverse transcription PCR; UTR, untranslated region(s). 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 6 22 - 1
homologues hMLH1, hPMS1 and hPMS2 have been identified and characterized and mutations in hMSH2, hMLH1, hPMS1 and hPMS2 have been linked to hereditary non-polyposis colon cancer ( Fishel et al., 1993; Leach et al., 1993; Bronner et al., 1994; Papadopoulos et al., 1994; Nicolaides et al., 1994; Drummond et al., 1995; Palombo et al., 1995). Knock out mice for MSH2 develop normally but are susceptible for lymphoid tumor providing strong evidence for its association with pathogenesis of cancer (de Wind et al., 1995; Reitmar et al., 1995). Recombinant MSH2 protein from humans and yeast binds to insertion/deletion loops of 1–14 nt in length ( Fishel et al., 1994a,b; Alani et al., 1995). MSH2 mutants of S. cerevisiae are defective in mismatch repair, show an increase in mitotic mutation rate, postmeiotic segregation events, microsatellite instability and homologous recombination (Reenan and Kolodner, 1992b; Strand et al., 1993; Alani et al., 1994; and Selva et al., 1995). MSH2 deficient mouse cells show loss of mismatch binding activity, mutator phenotype and an increase in homologous recombination ( Reitmar et al., 1995; de Wind et al., 1995). MSH2 homologues have also been identified in Xenopus and mouse ( Varlet et al., 1994). The mismatch repair system, in addition to having a key role in maintaining the integrity of nucleotide sequences in the genome, also plays an important role in providing a barrier to interspecies recombination by
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its antirecombinogenic role in homologous recombination (Rayssiguier et al., 1989; Radman and Wagner, 1993; Matic et al., 1995). Mammalian spermatogenesis offers a unique system wherein germ cells undergo several rounds of mitotic division of spermatogonial cells after which they get committed to meiotic division. A unique event characteristic of meiotic division is recombination between paired homologous chromosomes in primary spermatocytes. Hence it is interesting to see the temporal expression of MSH2 during spermatogenesis. Towards this objective, we have cloned and sequenced the rat homologue of MSH2 and studied its expression in the testes of rats of different age groups.
2. Materials and methods 2.1. Materials Restriction endonucleases and DNA modifying enzymes were obtained from New England Biolabs, Amersham International, and Bangalore Genie, India. Biochemicals were obtained from Sigma Chemical Company. Oligonucleotide primers were synthesized in the oligonucleotide synthesis facility, Indian Institute of Science, and Bangalore Genie, India. T sequencing kit 7 was obtained from Pharmacia Biotech, Sweden and rat testes lZap(II ) cDNA library was obtained from Stratagene, USA. [a-32P]dATP and [35S]dATP were obtained from Board of Radiation and Isotope Technology, India. 2.2. Methods 2.2.1. Cloning of rMSH2/2.8 kb The construction of various clones is described below. (1) prMSH2/350 bp. RT-PCR was performed using degenerate oligos synthesized for the aa sequences TGPNM and FATH(F/Y ). The sequences of oligonucleotides were 5∞-CGCGGATCCAC(G/A/T/C ) GG(GA/T/C ) CC(G/A/T/C ) AA ( T/C ) ATG-3∞ (primer C ) and 5∞-CGCAAGCTT(G/A) (A/T )A (G/A)TG(G/A/T/C )GT(G/A/T/C )GC(G/A)AA-3∞ (primer E ). RT-PCR was carried out using these primers and 30 ng of first strand cDNA synthesized from poly(A)+RNA of rat testes (40 days of age) and Taq DNA polymerase (Bangalore Genei). PCR was done with cycling conditions of 94°C for 1 min, 50°C for 2 min, and 72°C for 1 min for 30 cycles. The expected size PCR product (350 bp) was eluted from the agarose gel and subjected to another round of PCR amplification. The PCR product was digested with BamHI and HindIII and cloned into pBluescript II (SK ). The clones that hybridized to human 350 bp MSH2 product were sequenced by
the method of Sanger et al. (1977) using a T 7 sequencing kit (Pharmacia). (2) prMSH2/1.2 kb. The human MSH2 cDNA was digested with NcoI and EcoRI and a 1.5 kb fragment corresponding to the 3∞ end of hMSH2 was gel purified and used as a probe to screen the rat testes lZap(II ) cDNA library (Stratagene). About 3×105 plaques were screened with hybridization at 42°C in 6×SSC, 5×Denhardt’s solution, 0.5% SDS, 100 mg/ml denatured salmon sperm DNA and denatured probe (5×106 cpm/ml ). The filters were washed in 1×SSC, 0.1% SDS at 50°C and exposed for autoradiography at −70°C with an intensifying screen. The positive signal obtained after primary screening was purified and subjected to secondary screening using similar conditions. The positive plaques were purified and recombinant pBluescript (SK ) plasmid containing the insert was excised using the Exassit Excision system according to the manufacturer’s (Stratagene) protocol. The insert was released by EcoRI and sequenced using KS, SK, T , T and insert specific primer (primer D). 3 7 (3) prMSH2/2.0 kb. To obtain cDNA coding for the 5∞ end of rMSH2, RT-PCR was done using an upstream degenerate oligonucleotide synthesized for mouse aa sequence (MAVAP) 5∞-CCGAATTCGCATATG CG(G/A/T/C ) GT (G/A/TC ) CA (G/A) CC (G/A/T/C ) AA-3∞ (primer A) and a downstream oligo specific for rMSH2 sequence 5∞-CCGTCTGACGGATGTATGTTG-3∞ (primer B) and rat testes cDNA prepared from 20-day-old rats. A 2.0 kb PCR product was obtained which was cloned into pMOS-Blue T vector (Amersham) and the clone was completely sequenced using insert specific (primer F ) 5∞-(TAATCTGTTTGCCAGGGTCC-3∞) and making various deletions using internal restriction sites for EcoRI, HindIII, PstI which were subcloned into pBluescript II SK or pUC 18. (4) prMSH2/2.8 kb. The complete cDNA coding for the ORF of rMSH2 was constructed using overlapping PCR. The insert of prMSH2/2.0 kb was amplified using primers A and B which gave a product insert of 2050 bp. The clone prMSH2/1.2 was amplified using primers C and 5∞-CTCCGTCGACCGGAGCCTTTACCCGTGA-3∞ (primer D), giving a product of 800 bp. This was done in order to eliminate the 5∞ end 168 nt of clone prMSH2/1.2 (shown in hatched box in Fig. 1), since this 168 nt sequence was an artifact of reverse transcription as revealed by sequence comparison. A 2.8 kb cDNA was obtained upon PCR with the 2.0 kb and 0.8 kb PCR products using primers A and D, since there is an overlap of 50 nt between the two PCR products. This 2.8 kb cDNA has the ORF coding for
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rMSH2, and has been cloned into pMOS Blue-T vector. 2.2.2. Genomic Southern blot analysis Rat genomic DNA was isolated from spleen (Sambrook et al., 1989). 15 mg of genomic DNA was digested with BamHI, EcoRI, HindIII, XbaI and SfiI, and electrophoresed on a 0.8% agarose gel and blotted onto Hybond Nylon membrane by vacuum blotting in 10×SSC. The DNA was UV crosslinked to the membrane and prehybridized in 6×SSC, 5×Denhardt’s, 0.5% SDS, 100 mg/ml denatured salmon sperm DNA at 65°C for 2 h. Hybridization was done for 16 h, in the presence of random hexamer primed and labeled rMSH2 cDNA at (1×107 cpm/ml ) at 65°C. Filters were washed at a final stringency of 0.1×SSC/0.1% SDS at 65°C and exposed to autoradiography with an intensifying screen at −70°C. The sizes of the fragments hybridizing to rMSH2 cDNA were calculated by the SEQAID program. 2.2.3. Northern blot analysis Total RNA was isolated from rat testes of various age groups using the acid guanidinium isothiocyanate phenol-chloroform method (Chomczynski and Sacchi, 1987). 30 mg of total RNA was electrophoresed on a 1% agarose gel in the presence of 2.2 M formaldehyde and blotted onto Hybond Nylon membrane (Amersham) in 20×SSC after an initial alkali denaturation in 0.05 N NaOH for 20 min. The filter was UV crosslinked and prehybridized with 6×SSC, 0.5% SDS, 5×Denhardt’s, 50% formamide, 100 mg/ml denatured
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salmon sperm DNA at 42°C for 2 h. Hybridization was done under the same conditions, with an a-32P-labeled rMSH2 cDNA probe at approx. 1×107 cpm/ml for 16 h at 42°C. Filters were washed at 0.1×SSC/0.1% SDS at 65°C and exposed to autoradiography. The radiolabeled rat glyceraldehyde 3-phosphate dehydrogenase cDNA was hybridized under similar conditions.
3. Results and discussion 3.1. Cloning of rat MSH2 (rMSH2) cDNA To obtain rat cDNA coding for the most conserved region of known MutS homologues a degenerate primer driven PCR was employed. Two degenerate oligonucleotide primers for the conserved aa sequences TGPNM (upstream oligo) and FATH (F/Y ) (downstream oligo) were used for the RT-PCR reaction using rat testes cDNA prepared from testes poly(A)+RNA as the template. The expected size of the PCR product (350 bp) was obtained which was cloned into pBluescript II (SK )+ plasmid and subsequently sequenced. One of the clones showed 87% identity to the corresponding region in human MSH2 cDNA. This cloned PCR product was then used as a probe to screen the rat testes lZap(II ) cDNA library (Stratagene). However, during this screening we observed considerable background hybridization, possibly due to hybridization of the probe with the endogenous E. coli mutS gene. To circumvent this problem, we used the 3∞ 1.5 kb EcoRI fragment of human MSH2 cDNA (which encompasses the highly conserved
Fig. 1. Schematic representation of various clones used for the construction of rMSH2 cDNA. rMSH2/2.0 kb and rMSH2/350 bp were obtained by RT-PCR, rMSH2/1.2 kb was obtained by screening and rMSH2/2.8 kb was constructed by overlapping PCR as described in Section 2.2. Various primers used for PCR and sequencing are also indicated. Deletion constructs for sequencing of prMSH2/2.0 kb are also depicted. The hatched box in prMSH2/1.2 kb represents the 168 bp which are an artifact of reverse transcription.
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NTP and DNA binding domain) as a heterologous probe to screen the lZap(II ) cDNA library under low stringency hybridization conditions. One positive clone was identified, having an insert of 1.2 kb in length, which upon sequencing showed homology to the human MSH2 cDNA. This clone had an ORF followed by a
stop codon and 3∞-UTR. In this 1.2 kb clone, the 5∞ 168 nt showed homology to the human MSH2 sequence in the complementary strand as opposed to the rest of the coding region, and in the reverse orientation (Fig. 1). This could have arisen due to an artifact of reverse transcription during cDNA synthesis. This clone was
Fig. 2. cDNA sequence and deduced aa sequence of rMSH2. The cDNA sequence has an ORF from nt 1 to 2802, coding for a protein of 933 aa. The 3∞-UTR extends from nt 2803 to 3002, having a putative polyadenylation signal AATAAA which is underlined. The various primers used for sequencing and PCR are underlined. The unique EcoRI seen only in the rMSH2 site is also underlined. The EMBL accession number for rMSH2 is X93591.
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partial, missing the 5∞-coding region. To obtain cDNA having a full-length ORF, the same library was rescreened using the 1.2 kb rat MSH2 cDNA as a probe. However, no full-length clone could be identified. Hence as an alternative strategy RT-PCR was used, using a degenerate oligonucleotide primer designed for the first 5 aa of mouse MSH2 sequence as the upstream primer and a specific downstream oligonucleotide primer based on the sequence of 1.2 kb rMSH2 cDNA. RT-PCR was done using these two sets of primers and rat testes cDNA of 20-day-old rats as the template. Twenty-dayold rat testes RNA was used, as the preliminary Northern data with 1.2 kb rMSH2 clone indicated that the level of expression was high at that age. A PCR product of the expected size of 2.0 kb was obtained which was subsequently cloned into pMOS-Blue T vector (Amersham) and sequenced. This clone had an ORF coding for the rat MSH2 having high homology to human and mouse MSH2 proteins (Fig. 1). Several clones were sequenced to rule out PCR generated errors.
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3.2. Construction of a full-length rMSH2 clone, and sequence analysis The two clones coding for rMSH2 cDNA were partial, one with respect to the 5∞ end (2.0 kb) and the other to the 3∞ end (1.2 kb). A cDNA having a complete ORF was constructed using overlapping PCR, from the PCR products obtained from each of the clones (Fig. 1). While constructing the full-length cDNA, PCR was done for the 1.2 kb clone, using primers so as to eliminate the 5∞ 168 nt which were showing a discrepancy in homology, due to an artifact of reverse transcription. The complete nt sequence of the rMSH2 was obtained by sequencing various deletion clones and using a number of specific primers (Fig. 1). A total sequence of 3002 nt was obtained. This cDNA has an ORF of 2802 nt starting from ATG at position 1 and coding for a protein of 933 aa (Fig. 2). The 3∞-UTR extends from 2803 to 3002 nt and has a putative polyadenylation signal. This clone does not have a 5∞-UTR since the
Fig. 3. Comparison of the deduced aa sequences of rat, mouse, human and Xenopus MSH2 sequences. rMSH2, aa sequence from rat, MMSH2 Gen is from mouse, HSO391 is from human and XELMSH2 A from Xenopus. * indicates identical aa and · indicates conserved substitutions. A–D are NTP binding motifs and HTH is helix-turn-helix motif, which are underlined. The alignment was done using the CLUSTAL program.
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primer used for the RT-PCR starts with the ATG codon. The homology comparison of rMSH2 with other MSH2 sequences revealed 90.4, 86.1 and 72.5% with respect to mouse, human and Xenopus respectively at the nt level while it was 94, 91.7 and 77.5% at the aa level. The highest level of conservation among all the known MutS homologues is in the C-terminal part of the protein which has a NTP binding domain and a DNA binding helix-turn-helix motif ( Fig. 3). 3.3. Genomic organization of rMSH2 Rat genomic DNA was digested with various restriction endonucleases and hybridized to rMSH2 cDNA probe after transfer to the Hybond membrane. Hybridization of rMSH2 cDNA to BamHI digest revealed fragments of 9.2 kb and 4.0 kb. Hybridization to EcoRI digest revealed fragments of 11 kb, 7.9 kb and 5.6 kb, and that of HindIII digest revealed fragments of 8.9 kb, 3.3 kb and 0.6 kb. XbaI digest showed hybridization to fragments of 10 kb, 7.6 kb, 5.01 kb, 3.5 kb, 2.8 kb and 0.9 kb. SfiI digest revealed hybridization to fragments greater than 23 kb. An idea of the genomic fragments hybridizing to rMSH2 cDNA will help the future studies in identifying the rMSH2 gene. The estimate of the rat genomic fragment encompassing the rMSH2 sequence is approx. 30 kb in length (a minimum estimate). The actual genomic fragment encompassing the entire rMSH2 transcription unit would be longer than this size since the cDNA probe used in this experiment does not contain the 5∞-UTR sequence and lacks the 3∞-UTR sequence. Furthermore, intronic sequences flanked by restriction sites for the enzymes used in this Southern blot will also not be detected. The human MSH2 gene has been localized to chromosome 2 (2p.22-21), and the genomic locus of hMSH2 has been isolated and characterized. The gene is approx. 70 kb in length and is split into 16 exons ( Kolodner et al., 1994) ( Fig. 4). 3.4. Expression of rMSH2 during spermatogenesis Earlier studies have shown that MSH2 does not have a tissue specific expression and is expressed in almost all the tissues suggesting that it functions as a house keeping gene ( Varlet et al., 1994). However, a recent study by Wilson et al. (1995) on the expression of rMSH2 in various normal human tissues shows that its expression is highest in thymus and testis. We have examined the expression of rMSH2 during various stages of spermatogenesis by isolating total RNA from the testes of rats of different ages and carrying out a Northern blot analysis with rMSH2 cDNA as a probe. By this study we can correlate the expression of rMSH2 with several events associated with the germ cell development and differentiation during spermatogenesis. A
Fig. 4. Rat genomic Southern blot with rMSH2 as a probe. Rat genomic DNA digested with various restriction enzymes was probed with rMSH2 cDNA.
single transcript (approx. 3.5 kb) was observed ( Fig. 5A). The results also reveal that the expression of rMSH2 mRNA is highest at day 20, beyond which its level decreases with increasing age of the rat. Equal amounts of total RNA was loaded in each of the lanes as judged by the intensities of 28S and 18S rRNA bands ( Fig. 5B). Hybridization with rat GAPDH cDNA probe was used as an internal control (Fig. 5C ). Mammalian spermatogenesis is asynchronous and new waves of germ cells start before the first round of germ cells complete their maturation process. However, the approximate time scale of the appearance of a specific germ cell at different stages of their differentiation in the first wave of spermatogenesis with respect to the age of the rat is known (Meistrich et al., 1978). At 10 days of age, the predominant germ cells in the testes are spermatogonial cells. These cells enter premeiotic S-phase at the age of 18–20 days wherein there is active premeiotic DNA synthesis. Subsequently these cells enter meiotic prophase among which pachytene spermatocytes have the longest lifetime of 8–10 days (25–35 days of age in the rat). It is generally believed that the recombination between the paired homologous chromosomes takes place at the pachytene interval. Haploid spermatids in the rat are generated between the 35th and 38th day. It is interesting to note that the expression of rMSH2 is highest at 20 days of age in the rat during which time the germ cells are engaged in premeiotic DNA synthesis. A more interesting observation is that rMSH2 is down-regulated during the meiotic prophase. As mentioned in Section 1, the mismatch repair system also functions as antirecombinogenic effector. Hence a
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Fig. 5. Northern blot analysis of rMSH2 during spermatogenesis. (A) Hybridization of rat testes total RNA (30 mg) from 10–60-day-old rats with rat MSH2 cDNA. (B) Total RNA from rat testes of 10–60-day-old rats. (C ) Hybridization with glyceraldehyde phosphate dehydrogenase cDNA.
down-regulation of rMSH2 during meiotic prophase may be physiologically relevant to allow recombination between mammalian homologous chromosomes containing many of the repeat sequence families like microsatellites and minisatellites. It may also be pertinent to point out here that additional MutS homologues, MSH4 and MSH5, have been discovered in S. cerevisiae (RossMacdonald and Roeder, 1994; Hollingsworth et al., 1995). MSH4 is expressed in a meiotic specific manner during sporulation of yeast and MSH5 gene acts in meiosis. Their gene products have been implicated in processing specifically the recombination intermediates. It is noteworthy that MSH4 and MSH5 mutants are not defective in DNA repair but show an increase in non-disjunction of homologous chromosomes at meiosis. It remains to be seen whether similar MSH2 homologues are conserved in mammals and if so, what would be their pattern of expression during spermatogenesis in comparison to MSH2.
4. Conclusions (1) We have cloned a cDNA coding for the rat homologue of mismatch repair gene MSH2. (2) The rMSH2 shows very high homology to the known eukaryotic MSH2 at both nucleic acid and amino acid level. (3) The genomic locus coding for rMSH2 is approximately 30 kb in length. (4) The expression of rMSH2 shows down-regulation during later stages of spermatogenesis. The expression is highest at 20 days of age in the rat associated with germ cells undergoing premeiotic DNA replication. Subsequently there is a down-regulation of the level of MSH2 mRNA with the progress of meiotic prophase and spermiogenesis.
Acknowledgement This work was financially supported by the Department of Science and Technology, Government of India, New Delhi. We would like to thank Dr. S.K. Brahmachari, Convenor of the Oligonucleotide Facility of the Indian Institute of Science for the oligonucleotides used for PCR and sequencing. The help rendered by the Bioinformatics group IISc. is gratefully acknowledged. R.G.V. is a Senior Research Fellow of the University Grants Commission, New Delhi.
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