YEAST MITOCHONDRIAL DNA RECOMBINATION

酵母线粒体 DNA 重组

• 批准号: 3288395
• 负责人: RONALD A BUTOW
• 金额: $ 27.37万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-05 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288395
• 关键词: Saccharomyces; alleles; cell type; endonuclease; fungal genetics; genetic manipulation; genetic mapping; genetic markers; genetic recombination; immunoprecipitation; mitochondrial DNA; molecular cloning; molecular genetics; nucleic acid probes; nucleic acid sequence; plasmids; ribosomal RNA

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.

拟议的研究代表了一个综合的遗传和生物化学 酿酒酵母线粒体DNA重组的研究进展 特别 重点将是分析的分子特征， 在21 S rRNA基因座处的非相互重组。 证据将被 从初步数据中寻求建议，即开放式阅读框架 在21 S rRNA等位基因之一的1.1 kb内含子内编码一个 非相互重组所需的反式作用因子。 我们断定 这个因素，也许与其他蛋白质， 它在无内含子的21 S中引入位点特异性双链切割， 等位基因，这双链切割是一个中间体， 非互惠交换。 这一命题将通过分析来检验 的突变体的两个等位基因的21 S基因，在体外生化研究， 再现体内现象的各个方面， 鉴定和表征推定的ORF产物，并通过比较 S. 21 S位点的非相互交换特性。酿酒酵母 在线粒体的其它位点发生相互重组 基因组 建议进行更多的研究，以利用方法学 我们已经开发出有效地引入S。酿酒酵母细胞 各种工业物种的线粒体基因组， 多态性和遗传特性。 这些研究包括测量 与内含子相关的侧翼序列共转换的梯度 转移和如何在21 S rRNA基因序列变异的菌株， 缺乏内含子，但在异极杂交中产生非典型输出，影响 重组事件。 最后，建议开展研究， 获得和表征具有缺陷的核突变体的方法 线粒体DNA重组，并开始研究的分子机制， var 1重组--酵母线粒体基因组上唯一的另一个位点 这是已知的进行非互惠交换。