Molecular cloning of the yeast OPI3 gene as a high copy number suppressor of the cho2 mutation.
Molecular cloning of the yeast OPI3 gene as a high copy number suppressor of the cho2 mutation.
复制标题
酵母 OPI3 基因的分子克隆，作为 cho2 突变的高拷贝数抑制子。
DOI：
10.1007/bf00352006
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发表时间：
1993
期刊：
影响因子：
2.5
通讯作者：
Kohlwein,SD
中科院分区：
文献类型：
--
作者：
Preitschopf,W;Lückl,H;Summers,E;Henry,SA;Paltauf,F;Kohlwein,SD
通过对acdg1、cho2双突变体的胆碱的营养缺陷型需求进行功能互补，通过用高拷贝数质粒中的基因组DNA文库进行转化，鉴定出两种不同类型的互补DNA插入片段。先前已表明一种类型的插入片段代表 CHO2 结构基因。在本报告中，我们描述了第二种补充活性的分子和生化特征。克隆基因编码的转录物长度约为 1000 nt，并根据可溶性磷脂前体、肌醇和胆碱进行调节。基因破坏导致在 23°C 或 30°C 下没有明显的生长表型，但在单甲基乙醇胺存在的情况下在 37°C 下缺乏生长。无效突变体表现出肌醇分泌表型，表明脂质生物合成途径中存在突变。互补分析、体内磷脂甲基化途径的生化分析以及克隆基因与已发表序列的限制模式的比较，明确鉴定克隆基因为OPI3基因，编码酵母中的磷脂-N-甲基转移酶。当以多个拷贝存在时，OPI3 基因可有效抑制 acho2 突变的磷脂甲基化缺陷。由于磷脂酰胆碱合成受损，用高拷贝数质粒上的OPI3基因转化的incho2突变体消除了INO1失调表型。综合起来，这些数据表明OPI3基因产物对于从头Ptd-Cho生物合成途径中的三个连续磷脂甲基化反应具有显着重叠的特异性。
By functional complementation of the auxotrophic requirements for choline of acdg1, cho2double-mutant, by transformation with a genomic DNA library in a high copy number plasmid, two different types of complementing DNA inserts were identified. One type of insert was earlier shown to represent theCHO2structural gene. In this report we describe the molecular and biochemical characterization of the second type of complementing activity. The transcript encoded by the cloned gene was about 1000-nt in length and was regulated in response to the soluble phospholipid precursors, inositol and choline. A gene disruption resulted in no obvious growth phenotype at 23°C or 30°C, but in a lack of growth at 37°C in the presence of monomethylethanolamine. Null-mutants exhibited an inositol-secretion phenotype, indicative of mutations in the lipid biosynthetic pathway. Complementation analysis, biochemical analysis of the phospholipid methylation pathway in vivo, and comparison of the restriction pattern of the cloned gene to published sequences, unequivocally identified the cloned gene as theOPI3gene, encoding phospholipid-N-methyltransferase in yeast. When present in multiple copies theOPI3gene efficiently suppresses the phospholipid methylation defect of acho2mutation. As a result of impaired synthesis of phosphatidylcholine, theINO1-deregulation phenotype is abolished incho2mutants transformed with theOPI3gene on a high copy number plasmid. Taken together, these data demonstrate a significantly overlapping specificity of theOPI3gene product for three sequential phospholipid methylation reactions in the de novo Ptd-Cho biosynthetic pathway.