Sherman F, Stewart J W, Jackson M, Gilmore R A, Parker J H
Genetics. 1974 Jun;77(2):255-84. doi: 10.1093/genetics/77.2.255.
A medium containing chlorolactate has been devised to enrich for mutants that are unable to utilize lactate for growth, and therefore that may be defective in cytochrome c. Complementation tests of 6,520 chlorolactate-resistant mutants that were obtained spontaneously or induced with UV, ICR-170, or nitrosoimidazolidone resulted in the identification of 195 mutations at the cyc1 locus, which controls the primary structure of iso-1-cytochrome c. These 195 mutants, with 16 cyc1 mutants previously isolated, were examined for total cytochrome c by spectroscopic methods, growth on lactate medium, suppressibility by defined nonsense suppressors, mutational sites by x-ray-induced recombination, ability to revert, and in 86 cases, whether intragenic revertants contain altered iso-1-cytochrome c. Except for the deletion mutant cyc1-1, all of the mutants appeared to contain single-site mutations that could be assigned to at least 35 different sites within the gene. The cyc1 mutants either completely lacked iso-1-cytochrome c or contained iso-1- cytochromes c that were completely or partially nonfunctional. In spite of the fact that the cyc1 mutants obtained by the chlorolactate procedure were selected on the basis of defective function, 68% appeared to completely lack iso-1-cytochrome c. The remaining cyc1 mutants contained below normal amounts of iso-1-cytochromes c. Studies at several incubation temperatures indicated that these nonfunctional iso-1-cytochromes c were thermolabile. It is suggested that the predominant means for abolishing iso-1-cytochrome c by mutations are either through a complete loss, such as produced by chain terminating codons, or impairments through drastic changes of tertiary structure which lead to instability and thermolability.
已设计出一种含有氯乳酸盐的培养基，用于富集那些无法利用乳酸盐进行生长、因而可能在细胞色素c方面存在缺陷的突变体。对通过自发产生或用紫外线、ICR - 170或亚硝基咪唑烷酮诱导获得的6520个抗氯乳酸盐突变体进行互补测试，结果鉴定出在cyc1基因座上有195个突变，该基因座控制同工型-1-细胞色素c的一级结构。将这195个突变体与之前分离出的16个cyc1突变体一起，通过光谱法检测总细胞色素c、在乳酸盐培养基上的生长情况、特定无义抑制子的抑制能力、X射线诱导重组的突变位点、回复能力，并且在86个案例中检测基因内回复体是否含有改变的同工型-1-细胞色素c。除了缺失突变体cyc1 - 1外，所有突变体似乎都含有单点突变，这些突变可定位到该基因内至少35个不同位点。cyc1突变体要么完全缺乏同工型-1-细胞色素c，要么含有完全或部分无功能的同工型-1-细胞色素c。尽管通过氯乳酸盐程序获得的cyc1突变体是基于功能缺陷进行筛选的，但68%的突变体似乎完全缺乏同工型-1-细胞色素c。其余的cyc1突变体所含的同工型-1-细胞色素c低于正常水平。在几个培养温度下的研究表明，这些无功能的同工型-1-细胞色素c是热不稳定的。有人提出，通过突变消除同工型-1-细胞色素c的主要方式要么是完全丧失，如由链终止密码子产生的情况，要么是通过三级结构的剧烈变化导致不稳定和热不稳定性从而造成功能受损。
