Identification and expression of the Neurospora crassa mei-3 gene which encodes a protein homologous to Rad51 of Saccharomyces cerevisiae

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.