Cho Y W, Kim H G, Park E H, Fuchs J A, Lim C J
Division of Life Sciences, College of Natural Sciences, Kangwon National University, Chuncheon, South Korea.
Biochim Biophys Acta. 2000 Dec 15;1517(1):171-5. doi: 10.1016/s0167-4781(00)00242-6.
The genomic DNA encoding thioltransferase was isolated from Schizosaccharomyces pombe using the polymerase chain reaction. The amplified DNA fragment was confirmed by Southern hybridization, completely digested with HindIII and BamHI, and then ligated into the yeast-Escherichia coli shuttle vector pRS316, which resulted in plasmid pEH1. The insert of plasmid pEH1 was transferred into the multi-copy vector YEp357 to generate plasmid pYEH1. The determined nucleotide sequence harbors an open reading frame consisting of four exons and three introns, which encodes a polypeptide of 101 amino acids with a molecular mass of 11261 Da. Thioltransferase activity was increased 1.6-fold in Saccharomyces cerevisiae containing plasmid pYEH1, and 1.8- and 2.7-fold in S. pombe containing plasmid pEH1 and pYEH1, respectively. The upstream sequence and the region encoding the N-terminal six amino acids were fused into promoterless beta-galactosidase gene of the shuttle vector YEp357R to generate the fusion plasmid pYEHR1. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by zinc and NO-generating S-nitroso-N-acetylpenicillamine.
利用聚合酶链反应从粟酒裂殖酵母中分离出编码硫醇转移酶的基因组DNA。扩增的DNA片段经Southern杂交确认，用HindIII和BamHI完全消化，然后连接到酵母-大肠杆菌穿梭载体pRS316中，得到质粒pEH1。将质粒pEH1的插入片段转移到多拷贝载体YEp357中，产生质粒pYEH1。测定的核苷酸序列包含一个由四个外显子和三个内含子组成的开放阅读框，其编码一个101个氨基酸、分子量为11261 Da的多肽。在含有质粒pYEH1的酿酒酵母中，硫醇转移酶活性提高了1.6倍，在含有质粒pEH1和pYEH1的粟酒裂殖酵母中，分别提高了1.8倍和2.7倍。将上游序列和编码N端六个氨基酸的区域与穿梭载体YEp357R的无启动子β-半乳糖苷酶基因融合，产生融合质粒pYEHR1。发现锌和产生NO的S-亚硝基-N-乙酰青霉胺可增强融合质粒中β-半乳糖苷酶的合成。
