Hjelmstad R H, Bell R M
J Biol Chem. 1987 Mar 15;262(8):3909-17.
A colony autoradiographic assay for the sn-1,2-diacylglycerol cholinephosphotransferase activity in Saccharomyces cerevisiae was developed. Twenty-two mutants defective in cholinephosphotransferase activity were isolated. Genetic analysis revealed that all of these mutations were recessive, and three complementation groups were identified. The cholinephosphotransferase activities in membranes prepared from cpt1 mutants were reduced 2-10-fold compared to wild-type activity. The cholinephosphotransferase activities of two cpt1 isolates differed from wild-type activity with respect to their apparent KM for CDP-choline. The residual cholinephosphotransferase activities of cpt1 isolates were more sensitive to inhibition by CMP than the wild-type activity. The CPT1 gene was cloned by genetic complementation of cpt1 using a yeast genomic library. In strains transformed with the CPT1-bearing plasmid, a 5-fold overproduction of cholinephosphotransferase activity with wild-type kinetic properties was observed. The CPT1 gene was localized to a 1.2-2.4-kilobase region of DNA by transposon Tn5 mutagenesis and deletion mapping. An insertional mutant of the CPT1 gene was constructed and introduced into the chromosome by integrative transformation. The resulting cpt insertional mutant fell into the cpt1 complementation group. The cholinephosphotransferase activity in membranes prepared from the cpt1 insertional mutant was reduced 5-fold and exhibited CMP sensitivity. The sn-1,2-diacylglycerol ethanolaminephosphotransferase activities in membranes from all of the cpt1 isolates including the insertional mutant were normal. The data indicate that the cloned CPT1 gene represents the yeast cholinephosphotransferase structural gene, that the yeast choline- and ethanolaminephosphotransferase activities are encoded by different genes, and that the CPT1 gene is nonessential for growth.
开发了一种用于酿酒酵母中sn-1,2-二酰基甘油胆碱磷酸转移酶活性的菌落放射自显影测定法。分离出22个胆碱磷酸转移酶活性有缺陷的突变体。遗传分析表明，所有这些突变都是隐性的，并鉴定出三个互补群。与野生型活性相比，从cpt1突变体制备的膜中的胆碱磷酸转移酶活性降低了2至10倍。两种cpt1分离株的胆碱磷酸转移酶活性在其对CDP-胆碱的表观KM方面与野生型活性不同。cpt1分离株的残余胆碱磷酸转移酶活性比野生型活性对CMP抑制更敏感。通过使用酵母基因组文库对cpt1进行遗传互补克隆了CPT1基因。在用携带CPT1的质粒转化的菌株中，观察到胆碱磷酸转移酶活性有5倍的过量产生，具有野生型动力学特性。通过转座子Tn5诱变和缺失图谱分析，CPT1基因定位于1.2至2.4千碱基的DNA区域。构建了CPT1基因的插入突变体，并通过整合转化将其引入染色体。所得的cpt插入突变体属于cpt1互补群。从cpt1插入突变体制备的膜中的胆碱磷酸转移酶活性降低了5倍，并表现出对CMP的敏感性。包括插入突变体在内的所有cpt1分离株的膜中的sn-1,2-二酰基甘油乙醇胺磷酸转移酶活性正常。数据表明，克隆的CPT1基因代表酵母胆碱磷酸转移酶结构基因，酵母胆碱和乙醇胺磷酸转移酶活性由不同基因编码，并且CPT1基因对于生长不是必需的。
