Sakuraba Y, Schroeder A L, Ishii C, Inoue H
Department of Regulation-Biology, Faculty of Science, Saitama University, Urawa, Japan.
Mol Gen Genet. 2000 Nov;264(4):392-401. doi: 10.1007/s004380000342.
We isolated a Neurospora crassa cDNA that encodes a Rad52 homologue (ncRAD52) by PCR, using degenerate primers. RFLP mapping demonstrated that the cloned gene is located close to the ro-4 locus on the right arm of linkage group V (LGVR). In a second experiment, we used sib selection to identify a cosmid clone containing the mus-11 gene in a N. crassa genomic library. Fine-scale mapping of the mus-11 mutant showed the gene order on LGVR near ro-4 to be: ad-7 - (9.5 mu) - pab-2 (7.8 mu) - mus-11 - (3.7 mu) - inv. The nucleotide sequence of the mus-11 gene matched that of the ncRAD52 cDNA. Thus, the mus-11 gene encodes the Rad52 homologue. The deduced amino acid sequence of the MUS11 protein shows 32.0% and 27.5% overall identity to the Schizosaccharomyces pombe Rad22 protein and the human hRad52 protein, respectively, and a higher level of identity (55-66%) within the conserved N-terminal region (141 residues). The MUS11 protein shows homology to Rad52 from budding yeast only within the N-terminal region (53.2% identity over 141 amino acids) which is conserved among Rad52 homologues. Yeast two-hybrid analysis reveals that the MUS11 protein binds to both the MEI-3 protein, a Rad51 homologue, and to itself in vivo. An ncRAD52 mutant obtained by the RIPping procedure showed the same sensitivity as the original mus-11 mutant to the following mutagens and chemicals: UV light, 4NQO (4-nitroquinoline 1-oxide), MMS (methyl methanesulfonate), EMS (ethyl methanesulfonate), MNNG (N-methyl-N'-nitro-N-nitrosoguanidine), TBHP (tert-butyl hydroperoxide), HU (hydroxyurea) and histidine. Unlike the RAD52 transcript in Saccharomyces cerevisiae, the mus-11 transcript could not be detected in mycelium under normal growth conditions, but expression of the gene was induced by UV irradiation or treatment with MMS.
我们使用简并引物通过聚合酶链反应（PCR）从粗糙脉孢菌中分离出一个编码Rad52同源物（ncRAD52）的互补DNA（cDNA）。限制性片段长度多态性（RFLP）图谱显示，克隆的基因位于第五连锁群右臂（LGVR）上靠近ro - 4基因座的位置。在第二个实验中，我们利用同胞选择在粗糙脉孢菌基因组文库中鉴定出一个包含mus - 11基因的黏粒克隆。对mus - 11突变体的精细定位表明，LGVR上靠近ro - 4的基因顺序为：ad - 7 - （9.5厘摩） - pab - 2（7.8厘摩） - mus - 11 - （3.7厘摩） - inv。mus - 11基因的核苷酸序列与ncRAD52 cDNA的序列匹配。因此，mus - 11基因编码Rad52同源物。推导的MUS11蛋白氨基酸序列与粟酒裂殖酵母Rad22蛋白和人hRad52蛋白的总体一致性分别为32.0%和27.5%，在保守的N端区域（141个残基）内一致性更高（55 - 66%）。MUS11蛋白仅在N端区域与芽殖酵母的Rad52具有同源性（141个氨基酸上的一致性为53.2%），该区域在Rad52同源物中是保守的。酵母双杂交分析表明，MUS11蛋白在体内既能与Rad51同源物MEI - 3蛋白结合，也能与自身结合。通过重复诱导点突变（RIPping）程序获得的一个ncRAD52突变体，对以下诱变剂和化学物质表现出与原始mus - 11突变体相同的敏感性：紫外线、4 - 硝基喹啉1 - 氧化物（4NQO）、甲基磺酸甲酯（MMS）、甲基磺酸乙酯（EMS）、N - 甲基 - N' - 硝基 - N - 亚硝基胍（MNNG）、叔丁基过氧化氢（TBHP）、羟基脲（HU）和组氨酸。与酿酒酵母中的RAD52转录本不同，在正常生长条件下，在菌丝体中检测不到mus - 11转录本，但该基因的表达可由紫外线照射或MMS处理诱导。
