Hampsey M, Na J G, Pinto I, Ware D E, Berroteran R W
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport 71130.
Biochimie. 1991 Dec;73(12):1445-55. doi: 10.1016/0300-9084(91)90177-3.
The cycl-362 allele contains a point mutation that generates an aberrant AUG codon upstream of the normal CYC1 translation initiation codon. Mutants containing this allele express only about 2% of normal iso-1-cytochrome c, presumably due to translation initiation at the upstream AUG, termination at a UAA sequence six codons downstream, and failure to reinitiate at the normal AUG codon two nucleotides later. Both intragenic and extragenic revertants of cycl-362, expressing elevated levels of iso-1-cytochrome c, have been isolated simply by selecting for growth on lactate medium. Here we describe an improved method for isolating and readily distinguishing cis- from trans-acting suppressors of the upstream AUG. Eight different genes, designated sua1-sua8, are represented in our current collection of extragenic suppressors; all are recessive and enhance iso-1-cytochrome c levels to 10-60% of normal. None of the sua genes is allelic to SUI2 or sui3, which encode eIF-2 alpha and eIF-2 beta, respectively, or to SUI1. Many of the suppressors exhibit pleiotropic phenotypes, including slow growth, cold (16 degrees C) and heat (37 degrees C) sensitivity. These phenotypes have been exploited to clone the SUA5, SUA7 and SUA8 genes, which are presently being characterized. The structure of cyc1-362 and the number of sua genes already uncovered suggest that the SUA genes are likely to encode factors affecting several different cellular processes, including translation initiation, mRNA stability and possibly transcription start site selection.
cycl - 362等位基因包含一个点突变，该突变在正常CYC1翻译起始密码子上游产生一个异常的AUG密码子。含有此等位基因的突变体仅表达约2%的正常异 - 1 - 细胞色素c，推测是由于在上游AUG处起始翻译，在下游六个密码子处的UAA序列处终止，并且未能在两个核苷酸后的正常AUG密码子处重新起始翻译。通过选择在乳酸培养基上生长，已经分离出了cycl - 362的基因内和基因外回复突变体，它们表达升高水平的异 - 1 - 细胞色素c。在这里，我们描述了一种改进的方法，用于分离并容易地区分上游AUG的顺式作用和反式作用抑制子。在我们目前收集的基因外抑制子中代表了八个不同的基因，命名为sua1 - sua8；它们都是隐性的，并且将异 - 1 - 细胞色素c水平提高到正常水平的10% - 60%。没有一个sua基因与分别编码eIF - 2α和eIF - 2β的SUI2或sui3等位，也不与SUI1等位。许多抑制子表现出多效性表型，包括生长缓慢、对冷（16℃）和热（37℃）敏感。这些表型已被用于克隆目前正在进行表征的SUA5、SUA7和SUA8基因。cyc1 - 362的结构以及已经发现的sua基因数量表明，SUA基因可能编码影响几种不同细胞过程的因子，包括翻译起始、mRNA稳定性以及可能的转录起始位点选择。
