|Budget Amount *help
¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1994 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1993 : ¥1,200,000 (Direct Cost : ¥1,200,000)
The Na^+-translocating NADH-quinone reductase purified from the marine bacterium Vibrio alginolyticus is composed of three subunits, alpha, beta, and gamma. Each subunit was purified by SDS-polyacryl-amide gel electrophoresis and each N-terminal amino acid sequence was determined. Each subunit was also partially digested with V8 protease, and the N-terminal amino acid sequence of the purified polypeptide fragment was determined. From these informations, oligonucleotides corresponding to forward and reverse primers for each gene (NQR A,B and C) encoding alpha, beta and gamma subunits, respectively, were synthesized. Using these primers, a part of each gene was amplified from the chromosomal DNA of V.alginolyticus by a PCR method, and the PCR products were used for the cloning of NQR gene in lambda phage. Among the subclones selected by the probe C,the expression beta-subunit as a gene product was detected in Escherichia coli membranes by the activity staining and by Western blotting analyzes.