MOLECULAR MECHANISMS OF GENETIC RECOMBINATION IN YEAST

我们研究的长期目标是了解 基因重组的分子机制。 在下一个 项目期间，研究将集中在两个方面：（1）研究 重组刺激序列HOT 1，来源于酵母 rRNA基因簇，和（2）分离和表征 减数分裂交换缺陷的酵母突变体。 HOT 1对应于启动位点和增强子， 通过RNA聚合酶I转录。 当插入小说时 在酵母基因组中，HOT 1刺激有丝分裂 相邻序列中的重组。 HOT 1作用的研究 对染色体内和染色体间交换的影响表明，HOT 1促进的 重组事件起始于双链断裂（DSB）。 为了探索DSB的作用，（i）rad 52突变的影响 (that（ii）在第（1）款所述的情况下， 将尝试物理地证明HOT 1诱导的DSB， 和（iii）HOT 1促进的交流将与事件进行比较 由HO内切核酸酶诱导的DSB促进。 此外该 HOT 1启动的转录与重组之间的关系 这些研究将涉及建设 顺式作用HOT 1突变体，然后测量其 重组刺激能力和转录能力。 最后，HOT 1促进重组的能力， rDNA阵列将通过构建和检查一个小型的 rDNA重复序列的阵列在HOT 1活性上有缺陷。 将分离出减数分裂重组缺陷的酵母突变体 通过诱变单倍体，染色体III的二体，和 筛选或显示染色体间减少的突变体 交易所 这些突变体将受到一系列的遗传 旨在识别缺陷步骤的测试。 被认为在交换意愿启动方面有缺陷的突变体 测试促进染色体内 在电子显微镜下检查重组是否存在 的联会复合体，并在体外测定， 测量质粒间重组。 步骤缺陷突变体 启动后，将在体外重组中进行检查 测定和物理测定中， 体内重组限制性片段。 突变体被认为 将在物理测定中检查错配修复的改变， 异源双链体DNA和用于错配校正的体外测定。 一些减数分裂重组基因将被克隆， 测序;克隆基因的表达将通过 lacZ融合基因的构建与分析。

The long term goal of our research is an understanding of the molecular mechanisms of genetic recombination. During the next project period, research will focus in two areas: (1) studies of a recombination-stimulating sequence, HOT1, derived from the yeast rRNA gene cluster, and (2) the isolation and characterization of yeast mutants defective in meiotic exchange. HOT1 corresponds to the initiation site and enhancer of transcription by RNA polymerase I. When inserted at novel locations in the yeast genome, HOT1 stimulates mitotic recombination in adjacent sequences. Studies of the effect of HOT1 on intra- and inter-chromosomal exchange suggest that HOT1-promoted recombination events initiate with a double-strand break (DSB). To explore the role of DSBs, (i) the effect of a rad52 mutation (that confers a defect in DSB repair) will be examined, (ii) attempts will be made to physically demonstrate HOT1-induced DSBs, and (iii) HOT1-promoted exchanges will be compared with events promoted by DSBs induced by the HO endonuclease. In addition, the relationship between HOT1-promoted transcription and recombination will be examined; these studies will involve the construction of cis-acting HOT1 mutants followed by measurements of their recombination-stimulating ability and transcriptional capacity. Finally, the ability of HOT1 to promote recombination within the rDNA array will be tested by construction and examining a mini- array of rDNA repeats defective on HOT1 activity. Yeast mutants defective in meiotic recombination will be isolated by mutagenizing a haploid, disomic for chromosome III, and screening or mutants that display reduced inter-chromosomal exchange. These mutants will be subjected to series of genetic tests designed to identify the step at which they are defective. Mutants thought to be defective in the initiation of exchange will be tested for the ability to promote intra-chromosomal recombination examined in the electron microscope for the presence of synaptonemal complex, and assayed in an in vitro assay the measures inter-plasmid recombination. Mutants defective in steps after initiation will be examined in the in vitro recombination assay and in a physical assay that measure the production of recombinant restriction fragments in vivo. Mutants believed to be altered in mismatch repair will be examined in a physical assay for heteroduplex DNA and in an in vitro assay for mismatch correction. Some of the meiotic recombination genes will be cloned and sequenced; expression of the cloned genes will be examined through the construction and analysis of lacZ fusion genes.