Abstract
The mei-3 gene of Neurospora crassa encodes a homolog of the Escherichia coli RecA and Saccharomyces cerevisiae Rad51 proteins, which are required for recombination and repair of DNA double-strand breaks. To determine the molecular function of MEI3 protein, anti-MEI3 antibody was prepared and used in Western blot analysis. The antibody cross-reacted only with crude extracts prepared from perithecia, the fruiting bodies of Neurospora. The molecular weight of the MEI3 protein was estimated to be 38 kDa. Transformation experiments showed that a DNA fragment longer than previously reported was needed to complement the mei-3 mutation. On sequencing cDNA and genomic DNA, one open reading frame (ORF) was found, which consists of three exons interrupted by two small introns. This ORF encoded a MEI3 protein of 353 amino acids, and the inferred MW of 38 kDa is in good agreement with the results from Western blot analysis. Comparisons of MEI3 with other Rad51 homologs indicated that MEI3 protein contains the two conserved core domains (I and II) generally observed in Rad51 homologs in eukaryotes. Northern blot analysis showed that expression of mei-3 was raised remarkably after UV-irradiation or methyl methanesulfonate (MMS)-treatment. The transcript size was 1.6 kb and this was also larger than was reported previously.
Similar content being viewed by others
References
Akaboshi E, Inoue Y, Ryo H (1994) Cloning of the cDNA and genomic DNA that correspond to the recA-like gene of Drosophila melanogaster. Jpn J Genet: 663–670
Basile G, Aker M, Mortimer RK (1992) Nucleotide sequence and transcriptional regulation of the yeast recombinational repair gene RAD51. Mol Cell Biol 12:3235–3246
Bezzubova O, Shinohara A, Mueller RG, Ogawa H, Buerstedde JM (1993) A chicken RAD51 homologue is expressed at high levels in lymphoid and reproductive organs. Nucleic Acids Res 21:1577–1580
Cheng R, Baker TI, Cords CE, Radloff RJ (1993) mei-3, recombination and repair gene of Neurospora crassa, encodes a RecA-like protein. Mutat Res 294:223–234
Clark AB, Margulies AN (1965) Isolation and characterization of recombination-deficient mutants of Escherichia coli K12. Proc Natl Acad Sci USA 53:451–459
Cole GM, Mortimer RK (1989) Failure to induce a DNA repair gene RAD54, in Sacccharomyces cerevisiae does not affect DNA repair or recombination phenotypes. Mol Cell Biol 9:3314–3322
Cole GM, Schild D, Lovett ST, Mortimer RK (1987) Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage. Mol Cell Biol 7:1078–1084
Edelmann SE, Staben C (1994) A statistical analysis of sequence features within genes from Neurospora crassa. Exp Mycol 18:70–81
Guan KL, Dixon JE (1991) Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion protein with glutathione S-transferase. Anal Biochem 192:262–267
Morita T, Yoshimura Y, Yamamoto A, Murata K, Mori M, Yamamoto H, Matsusiro A (1993) A mouse homolog of the Escherichia coli recA and Saccharomyces cerevisiae RAD51 gene. Proc Natl Acad Sci USA 90:6577–6580
Newmeyer D, Galeazzi DR (1977) The instability of Neurospora duplication Dp(IL → IR)H4250 and its genetic control. Genetics 85:461–487
Newmeyer D, Galeazzi DR (1981) A meiotic UV-sensitive mutant that causes deletion of duplication in Neurospora. Genetics 89:245–269
Ogawa T, Shinohara A, Nabetani A, Ikeya T, Yu X, Egelman EH, Ogawa H (1993a) RecA-like recombination proteins in eukaryotes: functions and structures of RAD51 genes. Cold Spring Harbor Symp 58:567–576
Ogawa T, Yu X, Shinohara A, Egelman EH (1993b) Similarity of the yeast Rad51 filament to the bacterial RecA filament. Science 256:1896–1899
Raju NB, Perkins DD (1978) Barren perithecia in Neurospora crassa. Can J Genet Cytol 20:41–59
Resnick MA (1987) Investigating the genetic control of biochemical events in meiotic recombination. In: Moens PB (ed) Meiosis. Academic Press, Orlando, pp 157–210
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Habor Laboratory, Cold Spring Habor, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schroeder AL (1986) Chromosome instability in mutagen sensitive mutants of Neurospora. Curr Genet 10:381–387
Schroeder AL, Olson LD (1983) Mutagen sensitivity of Neurospora meiotic mutants. Can J Genet Cytol 25:16–25
Shinohara A, Ogawa H, Ogawa T (1992) Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell 69:457–470
Shinohara A, Ogawa H, Matsuda Y, Ushio N, Ikeo K, Ogawa T (1993) Cloning of human, mouse and fission yeast recombination genes homologous to RAD51 and recA. Nature Genet 4:239–243
Sokolovsky V, Kaldenhoff R, Ricci M, Russo VEA (1990) Fast and reliable mini-prep RNA extraction from Neurospora crassa. Fungal Genet Newslett 37:41–43
Tomita H, Soshi T, Inoue H (1993) The Neurospora uvs-2 gene encodes a protein which has homology to yeast Rad18 with unique zinc finger motifs. Mol Gen Genet 238:225–233
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proc Natl Acad Sci USA 76:4350–4354
Vollmer SJ, Yanofsky C (1986) Efficient cloning of Neurospora crassa. Proc Natl Acad Sci USA 83:4869–4873
West SC (1992) Enzymes and molecular mechanisms of genetic recombination. Annu Rev Biochem 61:603–640
Yoshimura Y, Morita T, Yamamoto A, Matsushiro A (1993) Cloning and sequence of the human recA-like gene cDNA. Nucleic Acids Res 21:1665
Author information
Authors and Affiliations
Additional information
Communicated by M. Sekiguchi
Rights and permissions
About this article
Cite this article
Hatakeyama, S., Ishii, C. & Inoue, H. Identification and expression of the Neurospora crassa mei-3 gene which encodes a protein homologous to Rad51 of Saccharomyces cerevisiae . Molec. Gen. Genet. 249, 439–446 (1995). https://doi.org/10.1007/BF00287106
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00287106