YEAST MITOCHONDRIAL DNA RECOMBINATION

这项拟议的研究代表了遗传和生物化学的综合 线粒体DNA在酿酒酵母中重组的研究特例 重点分析了其分子特征 21S rRNA基因座上的非互易重组。证据将是 从初步数据中寻找一个开放阅读框架的建议 在其中一个21S rRNA等位基因的1.1 kb内含子内编码 非互惠重组所需的反式作用因子。我们假设 这个因子，可能与其他可能与之相关的蛋白质 它介绍了在无内含子的21S中的一种位点特异性双链切割 并且这种双链切割是一种中间产物 非互惠交换。这一命题将通过分析加以检验 21S基因两种等位基因的突变体，体外生化研究 复制体内现象的各个方面，免疫学方法 确定和表征假定的ORF产品，并通过比较 酿酒酵母21S基因座的非互易交换特性 在线粒体的其他基因座上发生了相互重组 基因组。建议进行更多的研究，以利用方法论 我们已经开发出将高效地导入酿酒酵母细胞中， 各种工业物种的线粒体基因组中蕴藏着有用的 多态和遗传特性。这些研究包括测量 与内含子相关的侧翼序列共转化的梯度 21S rRNA基因在哪些菌株中的转移和序列变异 缺乏内含子，但在异极杂交中产生非典型输出，影响 那里的重组活动。最后，提出了有待发展的研究方向。 具有一般缺陷的核突变体的获得和鉴定方法 线粒体DNA重组，并开始研究线粒体DNA的分子机制 Var1重组--酵母线粒体基因组上唯一的另一个基因座 这是众所周知的经历了非互惠交换。

The proposed research represents an integrated genetic and biochemical approach to mtDNA recombination in Saccharomyces cervisiae. Particular emphasis will be given to the analysis of the molecular features of non-reciprocal recombination at the 21S rRNA locus. Evidence will be sought for the suggestion from preliminary data that an open reading frame within a 1.1 kb intron of one of the 21S rRNA alleles encodes a trans-acting factor required for non-reciprocal recombination. We posit that this factor, perhaps with other proteins that may be associated with it, introduces a site specific double-strand cut in the intron-less 21S allele and that this double-strand cut is an intermediate in the non-reciprocal exchange. This proposition will be examined by the analysis of mutants of both alleles of the 21S gene, in vitro biochemical studies to reproduce aspects of the in vivo phenomenon, immunological approaches to identify and characterize the putative ORF product, and by comparisons of the properties of non-reciprocal exchange at the 21S locus in S. cerevisiae with reciprocal recombination occurring at other loci in the mitochondrial genome. Additional studies are proposed to take advantage of methodology we have developed to introduce productively into S. cerevisiae cells, the mitochondrial genomes of various industrial species that harbor useful polymorphisms and genetic properties. These studies include measurements of the gradient of flanking sequence co-conversion associated with intron transfer and how sequence variations in the 21S rRNA gene in strains which lack the intron, but yield atypical outputs in heteropolar crosses, affect recombinational events there. Finally, studies are proposed to develop methods to obtain and characterize nuclear mutants with defects in general mtDNA recombination and to initiate studies on the molecular mechanisms of var1 recombination--the only other locus on the yeast mitochondrial genome that is known to undergo non-reciprocal exchange.