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Isolation of a cDNA encoding mouse DNA topoisomerase III which is highly expressed at the mRNA level in the testis
Biochimica et Biophysica Acta 1396 Ž1998. 127–131
Short sequence-paper
Isolation of a cDNA encoding mouse DNA topoisomerase III which is highly expressed at the mRNA level in the testis Takahiko Seki a , Masayuki Seki b, Toshiaki Katada a , Takemi Enomoto a
b,)
Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, UniÕersity of Tokyo, Tokyo 113, Japan b Department of Molecular Cell Biology, Faculty of Pharmaceutical Sciences, Tohoku UniÕersity, Sendai 980-77, Japan Received 26 August 1997; revised 13 October 1997; accepted 21 October 1997
Abstract A cDNA Ž mTOP3 . encoding mouse DNA topoisomerase III Žtopo III. was cloned using the human TOP3 cDNA as a probe. The deduced amino acid sequence of mouse topo III showed 86.8% identity with that of human topo III. Mouse TOP3 mRNA was highly expressed in the testis in comparison with other tissues. The TOP3 mRNA level in the testis increased slightly 14 days after birth and showed a marked increase in 17 days, times when the cells in pachytene phase begin to appear and increase in number. q 1998 Elsevier Science B.V. Keywords: Topoisomerase III; Expression in the testis; Pachytene
Topoisomerases Žtopo. are classified into type I and type II. Type I enzymes introduce a break into one strand of DNA for strand passage, and type II enzymes introduce a breakage into a double strand DNA and passing another DNA double strand through the break before resealing w1,2x. Eukaryotic topo III, which is classified as a type I enzyme, was discovered in yeast in 1989 w3x. The gene encoding the yeast enzyme was originally identified by its ability to suppress mitotic recombination between repetitive sequences. Thus, yeast cells having mutations in the gene encoding topo III protein Ž TOP3 . show a hyperrecombinogenic phenotype. In addition, the mutant cells show slow growth phenotype and a sporulation defect w3x. The slow growth
)
Corresponding author. Fax: q81-22-217-6873; E-mail: [email protected]
phenotype of top3 mutants is suppressed by introducing mutations into the SGS1 Žslow growth suppressor 1. gene, which encodes a protein with homology to E. coli RecQ protein w4x. Topo III and Sgs1p also appear to physically interact. Recently, a cDNA encoding human topo III was isolated but little is known about the function of this molecule w5x. In human, the genes encoding E. coli RecQ homologues were cloned as DNA helicase Q1 w6xrRECQL w7x, BLM and WRN w8,9x. The latter 2 genes were isolated by positional cloning to identify genes responsible for Bloom’s syndrome Ž BLM . and Werner’s syndrome Ž WRN .. However, the interaction of these RecQ homologues and topo III has not been reported. As the first step to investigate the function of topo III in higher eukaryotic cells, we cloned a cDNA encoding mouse topo III and examined the expression of TOP3 mRNA in various tissues, especially in the testis after birth in which spermatogene-
0167-4781r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 7 . 0 0 1 9 2 - 9
128
T. Seki et al.r Biochimica et Biophysica Acta 1396 (1998) 127–131
Fig. 1. Molecular cloning of a cDNA encoding for mouse DNA topoisomerase III. ŽA. The schematic depiction of isolated cDNAs. ŽB. Nucleotide and amino acid sequences of mTOP3. Underlines indicate the sequences used for PCR primer. The double underline indicates polyadenylation signal sequence. The nucleotide sequence has been deposited in DDBJrEMBLrGenBank under accession number AB006074.
T. Seki et al.r Biochimica et Biophysica Acta 1396 (1998) 127–131
sis occurs fairly synchronously and meiotic recombination occurs during spermatogenesis. Human topo III cDNA was obtained by polymerase chain reaction ŽPCR. using a human fetal brain cDNA library and primers, 5X-ATGATCTTTCC T G T C G C C C G -3 X and 5 X -T G T T C T G A G GACAAAAGGGAC-3X. The PCR product was used to make a digoxigenin-labeled probe by random priming ŽDIG DNA Labeling Kit, Boehringer, Manheim.. The probe thus obtained was used to screen a mouse spermatocyte lgt11 cDNA library. Thirty-two positive clones obtained from approximately 1 = 10 6 clones were subjected to the second round of screening. The clones confirmed to be positive by Southern blotting, were subcloned into pBluescript II KSŽy. at EcoRI site. Finally, 3 positive clones were isolated, and sequencing analysis indicated that these clones encoded mouse topo III but lacked the 5X and 3X terminal regions ŽFig. 1Ž A.. . To obtain the 5X and 3X
129
terminal regions, PCR was performed using a mouse spermatocyte cDNA library and primers, 5XCAGCAAATGTCCAGAGACTG-3X ŽFig. 1Ž B.. and 5X-TTGACACCAGACCAACTGGTAATG -3X Ž sequence derived from lgt11. for the 5X terminus, and using a mouse T-cell line cDNA library and primers, 5X-GAGTTTGTCGGCTGCATAGG-3X Ž Fig. 1Ž B.. and 5X-CAGTTGAAGTGAACTTGCGG-3X Ž sequence derived from the vector. for the 3X terminus. To generate the full-length cDNA, 3 fragments were denatured, annealed, and extended by PCR without primers for 15 cycles. Then, the products were used as templates for PCR using Pwo polymerase. The sequence of the resultant DNA was determined by primer-walking. In addition, RT-PCR was performed on total RNA prepared from testis of BALBrc mice by using primers, 5X-GGGAGTTGTAGTTCAAGGCTGC-3X and 5X-AGCTATGGGTGTCCTAGATGGAG-3X to generate full-length cDNA, and the PCR
Fig. 2. Amino acid alignment of mouse and human topo III. Asterisks between the lines indicate identical amino acids. Boxes indicate the putative active site of type I topoisomerase and the 6 cysteines corresponding to the C-terminal proximal 6 cysteines in the tetracystein motif of E. coli DNA topoisomerase I. Underlines indicate the C-terminal 28-residue repeats.
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T. Seki et al.r Biochimica et Biophysica Acta 1396 (1998) 127–131
product was sequenced directly. The complete DNA and deduced amino acid sequence is shown in Fig. 1ŽB.. Two consensus translation initiation signals ŽGGGA ATGATC and TGCC ATGGAA. w10x were identified near the 5X end of the sequence as the case of human TOP3 w5x. Thus, the cDNA encodes a polypeptide consisting of 1004 or 979 amino acids. The 3X untranslated region contained a polyadenylation signal sequence ŽAATAAA. w11x. The overall nucleotide sequence showed 78.8% identity with that of human TOP3, and the sequence corresponding the open reading frame showed 85.0% identity. The deduced amino acid sequence showed 86.8% identity with human topo III. The alignment of amino acid sequences of mouse and human topo IIIs is presented in Fig. 2. The putative active site region of type I topoisomerase and the 6 cysteines corresponding to the C-terminal proximal 6 cysteines in the tetracys-
tein motif of E. coli DNA topoisomerase I are conserved in the mouse and human genes. The carboxyl-terminal 28-residue repeats, which are considered to be the result of duplication events, are present in both topo IIIs, suggesting that the event occurred before evolutionary separation of human and mouse. We examined the expression of TOP3 mRNA in various mouse tissues and found that a ; 3.8 kb transcript was strongly expressed in the testis but was weakly or not expressed in other tissues ŽFig. 3.. Spermatogenesis occurs fairly synchronously in the mouse testis. Thus, we examined the expression of TOP3 mRNA using RNA prepared from testis of mice at various ages. TOP3 mRNA level increased slightly 14 days after birth and showed a marked increase in 17 days, times when cells in the pachytene phase begin to appear and increase in number ŽFig. 4.. The increase in TOP3 mRNA level in 17 days
Fig. 3. Tissue distribution of mTOP3 mRNA. Total RNA samples prepared from various tissues of BALBrc mice were analyzed by Northern blotting. Aliquots of 40 mg of total RNA extracted with guanidine isothiocyanate were electrophoresed in formaldehyde gels and transferred onto Hybond N q membranes ŽAmersham. with a vacuum blotter ŽPharmacia.. The blots were hybridized with w32 Px-labeled mTOP3 Ž2214–3028 bp. probe in hybridization solution Ž5 = SSPE, 10 = Denhardt’s solution, 2% SDS, 50% formamide. at 428C. After hybridization, blots were washed in buffer 1 Ž2 = SSC, 0.05% SDS. twice at room temperature, followed by washing in buffer 2 Ž0.1 = SSC, 0.1% SDS. once at 508C and visualized by autoradiography on X-ray film.
T. Seki et al.r Biochimica et Biophysica Acta 1396 (1998) 127–131
131
days may be due to expression in spermatogonia andror sertoli cells. This work was supported by Grants-in-Aid for Scientific Research, for Scientific Research on Priority Areas, and for JSPS Fellows from The Ministry of Education, Science, Sports and Culture of Japan.
References
Fig. 4. Northern blotting of total RNA from testis of ICR mice at various ages. Aliquots of 40 mg of total RNA per lane were electrophoresed, followed by hybridization and washing as described in Fig. 3.
was observed reproducibly. These results suggest that topo III is involved in meiotic recombination because this process occurs in pachytene cells. The low level expression of TOP3 mRNA in the testis before 12
w1x J.C. Wang, Biochim. Biophys. Acta 909 Ž1987. 1–9. w2x J.C. Wang, J. Biol. Chem. 266 Ž1991. 6659–6662. w3x J.W. Wallis, G. Chrebet, G. Brodsky, M. Rolfe, R. Rothstein, Cell 58 Ž1989. 409–419. w4x S. Gangloff, J.P. McDonald, C. Bendixen, L. Arthur, R. Rothstein, Mol. Cell. Biol. 14 Ž1994. 8391–8398. w5x R. Hanai, P.R. Caron, J.C. Wang, Proc. Natl. Acad. Sci. U.S.A. 93 Ž1996. 3653–3657. w6x N.A. Ellis, J. Groden, T.Z. Ye, J. Straughen, D.J. Lennon, S. Ciocci, M. Proytcheva, J. German, Cell 83 Ž1995. 655– 666. w7x M. Seki, H. Miyazawa, S. Tada, J. Yanagisawa, T. Yamaoka, S. Hoshino, S.K. Ozawa, T. Eki, M. Nogami, K. Okumura, H. Taguchi, F. Hanaoka, T. Enomoto, Nucleic Acids Res. 22 Ž1994. 4566–4573. w8x K.L. Puranam, P.J. Blackshear, J. Biol. Chem. 269 Ž1994. 29838–29845. w9x C.E. Yu, J. Oshima, Y.H. Fu, E.M. Wijsman, F. Hisama, R. Alisch, S. Matthews, J. Nakura, T. Miki, S. Ouais, G.M. Martin, J. Mulligan, G.D. Schellenberg, Science 272 Ž1996. 258–262. w10x M. Kozak, Nucleic Acids Res. 15 Ž1987. 8125–8148. w11x N. Proudfoot, Cell 64 Ž1991. 671–674.
Short sequence-paper
Isolation of a cDNA encoding mouse DNA topoisomerase III which is highly expressed at the mRNA level in the testis Takahiko Seki a , Masayuki Seki b, Toshiaki Katada a , Takemi Enomoto a
b,)
Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, UniÕersity of Tokyo, Tokyo 113, Japan b Department of Molecular Cell Biology, Faculty of Pharmaceutical Sciences, Tohoku UniÕersity, Sendai 980-77, Japan Received 26 August 1997; revised 13 October 1997; accepted 21 October 1997
Abstract A cDNA Ž mTOP3 . encoding mouse DNA topoisomerase III Žtopo III. was cloned using the human TOP3 cDNA as a probe. The deduced amino acid sequence of mouse topo III showed 86.8% identity with that of human topo III. Mouse TOP3 mRNA was highly expressed in the testis in comparison with other tissues. The TOP3 mRNA level in the testis increased slightly 14 days after birth and showed a marked increase in 17 days, times when the cells in pachytene phase begin to appear and increase in number. q 1998 Elsevier Science B.V. Keywords: Topoisomerase III; Expression in the testis; Pachytene
Topoisomerases Žtopo. are classified into type I and type II. Type I enzymes introduce a break into one strand of DNA for strand passage, and type II enzymes introduce a breakage into a double strand DNA and passing another DNA double strand through the break before resealing w1,2x. Eukaryotic topo III, which is classified as a type I enzyme, was discovered in yeast in 1989 w3x. The gene encoding the yeast enzyme was originally identified by its ability to suppress mitotic recombination between repetitive sequences. Thus, yeast cells having mutations in the gene encoding topo III protein Ž TOP3 . show a hyperrecombinogenic phenotype. In addition, the mutant cells show slow growth phenotype and a sporulation defect w3x. The slow growth
)
Corresponding author. Fax: q81-22-217-6873; E-mail: [email protected]
phenotype of top3 mutants is suppressed by introducing mutations into the SGS1 Žslow growth suppressor 1. gene, which encodes a protein with homology to E. coli RecQ protein w4x. Topo III and Sgs1p also appear to physically interact. Recently, a cDNA encoding human topo III was isolated but little is known about the function of this molecule w5x. In human, the genes encoding E. coli RecQ homologues were cloned as DNA helicase Q1 w6xrRECQL w7x, BLM and WRN w8,9x. The latter 2 genes were isolated by positional cloning to identify genes responsible for Bloom’s syndrome Ž BLM . and Werner’s syndrome Ž WRN .. However, the interaction of these RecQ homologues and topo III has not been reported. As the first step to investigate the function of topo III in higher eukaryotic cells, we cloned a cDNA encoding mouse topo III and examined the expression of TOP3 mRNA in various tissues, especially in the testis after birth in which spermatogene-
0167-4781r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 7 . 0 0 1 9 2 - 9
128
T. Seki et al.r Biochimica et Biophysica Acta 1396 (1998) 127–131
Fig. 1. Molecular cloning of a cDNA encoding for mouse DNA topoisomerase III. ŽA. The schematic depiction of isolated cDNAs. ŽB. Nucleotide and amino acid sequences of mTOP3. Underlines indicate the sequences used for PCR primer. The double underline indicates polyadenylation signal sequence. The nucleotide sequence has been deposited in DDBJrEMBLrGenBank under accession number AB006074.
T. Seki et al.r Biochimica et Biophysica Acta 1396 (1998) 127–131
sis occurs fairly synchronously and meiotic recombination occurs during spermatogenesis. Human topo III cDNA was obtained by polymerase chain reaction ŽPCR. using a human fetal brain cDNA library and primers, 5X-ATGATCTTTCC T G T C G C C C G -3 X and 5 X -T G T T C T G A G GACAAAAGGGAC-3X. The PCR product was used to make a digoxigenin-labeled probe by random priming ŽDIG DNA Labeling Kit, Boehringer, Manheim.. The probe thus obtained was used to screen a mouse spermatocyte lgt11 cDNA library. Thirty-two positive clones obtained from approximately 1 = 10 6 clones were subjected to the second round of screening. The clones confirmed to be positive by Southern blotting, were subcloned into pBluescript II KSŽy. at EcoRI site. Finally, 3 positive clones were isolated, and sequencing analysis indicated that these clones encoded mouse topo III but lacked the 5X and 3X terminal regions ŽFig. 1Ž A.. . To obtain the 5X and 3X
129
terminal regions, PCR was performed using a mouse spermatocyte cDNA library and primers, 5XCAGCAAATGTCCAGAGACTG-3X ŽFig. 1Ž B.. and 5X-TTGACACCAGACCAACTGGTAATG -3X Ž sequence derived from lgt11. for the 5X terminus, and using a mouse T-cell line cDNA library and primers, 5X-GAGTTTGTCGGCTGCATAGG-3X Ž Fig. 1Ž B.. and 5X-CAGTTGAAGTGAACTTGCGG-3X Ž sequence derived from the vector. for the 3X terminus. To generate the full-length cDNA, 3 fragments were denatured, annealed, and extended by PCR without primers for 15 cycles. Then, the products were used as templates for PCR using Pwo polymerase. The sequence of the resultant DNA was determined by primer-walking. In addition, RT-PCR was performed on total RNA prepared from testis of BALBrc mice by using primers, 5X-GGGAGTTGTAGTTCAAGGCTGC-3X and 5X-AGCTATGGGTGTCCTAGATGGAG-3X to generate full-length cDNA, and the PCR
Fig. 2. Amino acid alignment of mouse and human topo III. Asterisks between the lines indicate identical amino acids. Boxes indicate the putative active site of type I topoisomerase and the 6 cysteines corresponding to the C-terminal proximal 6 cysteines in the tetracystein motif of E. coli DNA topoisomerase I. Underlines indicate the C-terminal 28-residue repeats.
130
T. Seki et al.r Biochimica et Biophysica Acta 1396 (1998) 127–131
product was sequenced directly. The complete DNA and deduced amino acid sequence is shown in Fig. 1ŽB.. Two consensus translation initiation signals ŽGGGA ATGATC and TGCC ATGGAA. w10x were identified near the 5X end of the sequence as the case of human TOP3 w5x. Thus, the cDNA encodes a polypeptide consisting of 1004 or 979 amino acids. The 3X untranslated region contained a polyadenylation signal sequence ŽAATAAA. w11x. The overall nucleotide sequence showed 78.8% identity with that of human TOP3, and the sequence corresponding the open reading frame showed 85.0% identity. The deduced amino acid sequence showed 86.8% identity with human topo III. The alignment of amino acid sequences of mouse and human topo IIIs is presented in Fig. 2. The putative active site region of type I topoisomerase and the 6 cysteines corresponding to the C-terminal proximal 6 cysteines in the tetracys-
tein motif of E. coli DNA topoisomerase I are conserved in the mouse and human genes. The carboxyl-terminal 28-residue repeats, which are considered to be the result of duplication events, are present in both topo IIIs, suggesting that the event occurred before evolutionary separation of human and mouse. We examined the expression of TOP3 mRNA in various mouse tissues and found that a ; 3.8 kb transcript was strongly expressed in the testis but was weakly or not expressed in other tissues ŽFig. 3.. Spermatogenesis occurs fairly synchronously in the mouse testis. Thus, we examined the expression of TOP3 mRNA using RNA prepared from testis of mice at various ages. TOP3 mRNA level increased slightly 14 days after birth and showed a marked increase in 17 days, times when cells in the pachytene phase begin to appear and increase in number ŽFig. 4.. The increase in TOP3 mRNA level in 17 days
Fig. 3. Tissue distribution of mTOP3 mRNA. Total RNA samples prepared from various tissues of BALBrc mice were analyzed by Northern blotting. Aliquots of 40 mg of total RNA extracted with guanidine isothiocyanate were electrophoresed in formaldehyde gels and transferred onto Hybond N q membranes ŽAmersham. with a vacuum blotter ŽPharmacia.. The blots were hybridized with w32 Px-labeled mTOP3 Ž2214–3028 bp. probe in hybridization solution Ž5 = SSPE, 10 = Denhardt’s solution, 2% SDS, 50% formamide. at 428C. After hybridization, blots were washed in buffer 1 Ž2 = SSC, 0.05% SDS. twice at room temperature, followed by washing in buffer 2 Ž0.1 = SSC, 0.1% SDS. once at 508C and visualized by autoradiography on X-ray film.
T. Seki et al.r Biochimica et Biophysica Acta 1396 (1998) 127–131
131
days may be due to expression in spermatogonia andror sertoli cells. This work was supported by Grants-in-Aid for Scientific Research, for Scientific Research on Priority Areas, and for JSPS Fellows from The Ministry of Education, Science, Sports and Culture of Japan.
References
Fig. 4. Northern blotting of total RNA from testis of ICR mice at various ages. Aliquots of 40 mg of total RNA per lane were electrophoresed, followed by hybridization and washing as described in Fig. 3.
was observed reproducibly. These results suggest that topo III is involved in meiotic recombination because this process occurs in pachytene cells. The low level expression of TOP3 mRNA in the testis before 12
w1x J.C. Wang, Biochim. Biophys. Acta 909 Ž1987. 1–9. w2x J.C. Wang, J. Biol. Chem. 266 Ž1991. 6659–6662. w3x J.W. Wallis, G. Chrebet, G. Brodsky, M. Rolfe, R. Rothstein, Cell 58 Ž1989. 409–419. w4x S. Gangloff, J.P. McDonald, C. Bendixen, L. Arthur, R. Rothstein, Mol. Cell. Biol. 14 Ž1994. 8391–8398. w5x R. Hanai, P.R. Caron, J.C. Wang, Proc. Natl. Acad. Sci. U.S.A. 93 Ž1996. 3653–3657. w6x N.A. Ellis, J. Groden, T.Z. Ye, J. Straughen, D.J. Lennon, S. Ciocci, M. Proytcheva, J. German, Cell 83 Ž1995. 655– 666. w7x M. Seki, H. Miyazawa, S. Tada, J. Yanagisawa, T. Yamaoka, S. Hoshino, S.K. Ozawa, T. Eki, M. Nogami, K. Okumura, H. Taguchi, F. Hanaoka, T. Enomoto, Nucleic Acids Res. 22 Ž1994. 4566–4573. w8x K.L. Puranam, P.J. Blackshear, J. Biol. Chem. 269 Ž1994. 29838–29845. w9x C.E. Yu, J. Oshima, Y.H. Fu, E.M. Wijsman, F. Hisama, R. Alisch, S. Matthews, J. Nakura, T. Miki, S. Ouais, G.M. Martin, J. Mulligan, G.D. Schellenberg, Science 272 Ž1996. 258–262. w10x M. Kozak, Nucleic Acids Res. 15 Ž1987. 8125–8148. w11x N. Proudfoot, Cell 64 Ž1991. 671–674.