Analysis of Revertants of a Yeast Mitochondrial Promoter Mutant

酵母线粒体启动子突变体的回复体分析

• 批准号: 8916078
• 负责人: Vicki Cameron
• 金额: $ 9.8万
• 依托单位: Ithaca College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-15 至 1993-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8916078&HistoricalAwards=false
• 关键词: Analysis; Revertants; Yeast; Mitochondrial; Promoter

The objective of the research described in this grant is to understand the transcriptional process for genes encoded on mitochondrial DNA in yeast. A yeast mutant defective in transcription of subunit II of cytochrome c oxidase has been isolated. Subunit II DNA of the mutant contains a single nucleotide change in the transcriptional promoter. The mutation apparently interferes with recognition of the sequence as a transcriptional promoter. We have isolated nine revertants of the mutant which have recovered the ability to respire. Some of these revertants have not recovered normal growth rates so recovery of function cannot be the consequence of restoration of the normal sequence but must involve compensatory changes. Classical genetic techniques will be used to determine if the compensatory changes are found on nuclear or mitochondrial DNA. For mitochondrial revertants, genetic mapping experiments will localize the specific region of mitochondrial DNA carrying the compensatory change. The region will be sequenced and compared to the normal region to identify the specific alteration. For nuclear revertants, the altered genes responsible for recovery of function will be identified by fragmenting DNA from the revertant strains, cloning those fragments and introducing the clones into the original mutant. Clones which restore respiration must contain the altered gene and will be molecularly characterized to understand how restoration of function occurred. Analysis of the revertants will provide information about other sequences able to function as promoters of yeast mitochondrial transcription and about other gene products required for the process.

本补助金所述研究的目的是 了解基因编码的转录过程， 酵母中的线粒体DNA 一种酵母突变体， 细胞色素c氧化酶亚基II的转录已经被 与世隔绝 突变体的亚基II DNA含有一个 转录启动子中的核苷酸变化。 突变 显然干扰了序列作为 转录启动子 我们已经分离出了九个 恢复呼吸能力的突变体 一些 这些回复突变体还没有恢复正常的生长速率， 功能恢复不能是恢复的结果， 正常序列，但必须涉及补偿性变化。 经典的遗传技术将被用来确定是否 在细胞核或线粒体DNA上发现了代偿性变化。 对于线粒体回复突变体，遗传作图实验将 定位线粒体DNA的特定区域， 补偿性变化 该区域将被测序和比较 正常区域以识别特定的改变。 为 核回复突变体，改变的基因负责恢复 功能将通过将DNA片段化来鉴定。 回复突变株，克隆这些片段，并引入 克隆到原来的突变体中 恢复的克隆 呼吸必须包含改变的基因， 以了解功能恢复是如何发生的为特征。 对回复突变体的分析将提供有关其他 能够作为酵母线粒体启动子的序列 转录和其他基因产物所需的 过程