Analysis of Revertants of a Yeast Mitochondrial Promoter Mutant

这项研究的目的是为了了解酵母中线粒体DNA编码基因的转录过程。分离到一株细胞色素C氧化酶亚基II转录缺陷的酵母突变株。该突变体的亚基II DNA在转录启动子中包含单核苷酸变化。该突变显然干扰了该序列作为转录启动子的识别。我们已经分离出9个突变体，它们已经恢复了呼吸能力。其中一些逆转株没有恢复正常的生长速度，因此功能的恢复不能是正常序列恢复的结果，而必须涉及代偿性变化。经典的基因技术将被用来确定是否在核或线粒体DNA上发现了代偿性变化。对于线粒体回复突变体，遗传图谱实验将定位携带代偿变化的线粒体DNA的特定区域。将对该区域进行测序，并与正常区域进行比较，以确定具体的变化。对于核回复菌株，负责功能恢复的改变基因将通过从回复菌株中分离DNA，克隆这些片段，并将克隆引入原始突变菌株中来识别。恢复呼吸的克隆必须包含改变的基因，并将进行分子特征分析，以了解功能恢复是如何发生的。对回复突变体的分析将提供能够作为酵母线粒体转录启动子的其他序列的信息，以及这一过程所需的其他基因产物的信息。

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.