Genetic and molecular characterization of proteins essential for DNA repair and recombination in Saccharomyces cervisiae

9417573 Ramirez本研究资助的主要目标是克隆和鉴定酿酒酵母的Rec1和Rec3基因，这两个基因被认为在有丝分裂重组和DNA修复的重叠过程中发挥功能。Rec1-和rec3-菌株被证明在有丝分裂重组中存在缺陷，并具有温度敏感表型rec 1-是在39{符号176\f“符号”}处致死的，而Rec1-和rec3-在36{符号176\f“符号”}上都是关于有丝分裂重组、X射线敏感性和产孢量的ts。此外，这两个突变都定义了不同的基因，这些基因定位在11号染色体上。酵母基因组文库将被用于转化酿酒酵母Rec1-和rec3-菌株，以便通过互补的方式克隆相应的野生型基因。以前的研究旨在克隆REC3基因，结果分离出了rec3-RPD3的抑制子。(RPD3被认为编码了编码在染色体XIV上的多效性转录调节因子)。因此，我们将集中精力对与Rec1-和rec3-突变互补的克隆进行鉴定，并在凝胶分离的整个染色体的Southern杂交上鉴定与染色体Vll DNA特异杂交的克隆。为了加速REC3的克隆，我们还将测试与REC3基因座映射紧密的基因克隆(即包含与AR02基因相邻的遗传物质的克隆)，以确定它是否包含补充rec3-缺陷的序列。克隆后，将对Rec1和REC3克隆进行测序。所得到的遗传序列将与其他序列遗传序列数据库进行比较，以深入了解这些基因编码的蛋白质的功能。克隆的Rec1和REC3 DNA也将用于创建零突变，以提供明确的图谱信息，确定致命性，并通过Northern杂交检测基因表达。由于我们已经发现RPD3抑制rec3-缺陷，本建议的另一个目的是确定RPD3在重组中可能发挥的作用。这将通过测试RPD-菌株对电离辐射、甲基甲烷磺酸(MMS)的敏感性和测量重组速率来测试。这个基础研究项目将有助于我们对真核生物中重组和DNA修复的调控和/或酶学的了解。***

9417573 Ramirez The primary objectives of this research starter grant are to clone and characterize the genes REC1 and REC3 of Saccharomyces cerevisiae that are thought function in the overlapping processes of mitotic recombination and DNA repair. rec1- and rec3- strains have been shown to be defective in mitotic recombination and possess temperature sensitive phenotypes rec 1- is a ts-lethal at 39{SYMBOL 176 \f "Symbol"}, while both rec1- and rec3- are ts with respect to mitotic recombination, X-ray sensitivity and sporulation at 36{SYMBOL 176 \f "Symbol"}. Additionally, both these mutations define distinct genes which map to chromosome 11. A yeast genomic library will be used to transform rec1- and rec3- strains of S. cerevisiae in order to clone the corresponding wild-type genes by complementation. Previous studies aimed at cloning the REC3 gene resulted in the isolation of a suppressor of rec3-, RPD3. (RPD3 is thought to encode pleiotropic transcriptional regulator encoded on chromosome XIV). Hence, we will focus our efforts on characterizing clones which complement the rec1- and rec3- mutations and which specifically hybridize to chromosome Vll DNA on a Southern blot of electrophoretically separated whole chromosomes. To accelarate cloning of REC3, we will also test a genetic clone that maps closely to the REC3 locus (i.e., a clone containing genetic material adjacent to the AR02 gene) to determine if it contains sequences which complement the rec3- defect. Once cloned, the REC1 and REC3 clones will be sequenced. The resulting genetic sequence will be compared to other sequences genetic sequence data bases to gain insight into the function of the proteins encoded by these genes. Cloned REC1 and REC3 DNA will also be used to create null mutations to provide definitive mapping information, to determine lethality, and to assay gene expression by Northern hybridization. Since we have found that RPD3 suppresses the rec3- defect, an additional objecti ve of this proposal is to determine the possible role played by RPD3 in recombination. This will be tested by testing the sensitivity of rpd- strains to ionizing radiation, methyl methane sulfonate (MMS), and measuring rates of recombination. %%% The information generated frorm, this basic research project will contribute to our knowledge of the regulation and/or enzymology of recombination and DNA repair in eukaryotes. ***