Isolation, overexpression and disruption of a Saccharomyces cerevisiae YNK gene encoding nucleoside diphosphate kinase.
Nucleoside diphosphate kinase (NDPK) is the major enzyme responsible for the synthesis of all nucleoside triphosphates except ATP. A gene (YNK) encoding NDPK was isolated from the yeast Saccharomyces cerevisiae. The coding region consists of 459 bp encoding 153 amino acid (aa) residues. The M(r) of NDPK, calculated from the deduced aa sequence, is 17,166. Yeast NDPK was 59% and 58% identical to the rat NDPK alpha and beta, respectively. Overexpression of YNK in yeast showed high NDPK activity. The overproduced NDPK cross-reacted with anti-NDPK antibody raised against rat NDPK by Western blot analysis. Despite the fact that NDPK has features of a housekeeping enzyme, disruption of the YNK locus in a haploid strain was neither lethal nor significantly affected phenotypic behaviors such as growth rate, spore formation, mating ability and morphology. Yeast with a defective YNK still possessed NDPK activity to approximately 10% of the wild-type level. Possible sources of the remaining enzyme activity are discussed.
Fukuchi
T
T
Department of Experimental Biology, Tokyo Metropolitan Institute of Gerontology, Japan.
Nikawa
J
J
Kimura
N
N
Watanabe
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016415:Sequence Alignment
D009838:Oligodeoxyribonucleotides; D009701:Nucleoside-Diphosphate Kinase