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

9417573 Ramirez这项研究启动资金的主要目标是克隆和表征酿酒酵母的基因REC 1和REC 3，它们被认为在重叠的 有丝分裂重组和DNA修复的过程。rec 1-和rec 3-菌株已被证明 在有丝分裂重组中有缺陷并具有温度敏感表型 rec 1-在39{符号176 \f“Symbol”}处是ts-致死的，而rec 1-和rec 3-在有丝分裂方面都是ts 重组，X射线敏感性和孢子形成在36{符号176 \f“符号”}。此外，这两种突变 定义了定位于11号染色体的不同基因。将使用酵母基因组文库 转化rec 1-和rec 3-菌株。酿酒酵母，以克隆相应的 野生型基因互补。先前的研究旨在克隆REC 3基因 导致rec 3-的抑制子RPD 3的分离。(RPD3被认为编码了 在染色体XIV上编码的多效性转录调节子）。因此，我们将重点 我们致力于表征补充rec 1-和rec 3-突变的克隆， 其特异性地与染色体Vll DNA杂交。 分离整个染色体。为了加速REC 3的克隆，我们还将测试与REC 3基因座紧密定位的遗传克隆（即，含有遗传物质的克隆体 与AR 02基因相邻的序列），以确定其是否含有与AR 02基因互补的序列。 rec 3-缺陷。克隆后，将对REC 1和REC 3克隆进行测序。所得 基因序列将与其他基因序列数据库进行比较， 深入了解这些基因编码的蛋白质的功能。克隆的REC 1和 REC 3 DNA还将用于创建无效突变，以提供确定性作图 信息，以确定致死率，并通过北方分析基因表达 杂交方法由于我们已经发现RPD 3抑制rec 3缺陷，因此额外的 该建议的目的是确定RPD 3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、甲基甲磺酸盐（MMS）的敏感性和测量重组率来进行测试。 %这个基础研究项目产生的信息将有助于我们的知识 真核生物中重组和DNA修复的调控和/或酶学。 ***

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. ***