Cloning, expression and regulation of Schizosaccharomyces pombe gene encoding thioltransferase.
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.
Cho
Y W
YW
Division of Life Sciences, College of Natural Sciences, Kangwon National University, Chuncheon, South Korea.
Kim
H G
HG
Park
E H
EH
Fuchs
J A
JA
Lim
C J
CJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002712:Chlorides / Q000494:pharmacology; D018076:DNA, Complementary / Q000302:isolation & purification; D005696:Galactosidases / Q000096:biosynthesis; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D054477:Glutaredoxins; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics*; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011490:Protein Disulfide Reductase (Glutathione); D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment; D017967:Zinc Compounds / Q000494:pharmacology
D002712:Chlorides; D018076:DNA, Complementary; D054477:Glutaredoxins; C110881:S-nitro-N-acetylpenicillamine; D017967:Zinc Compounds; C016837:zinc chloride; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); D005696:Galactosidases; D010396:Penicillamine