[Cloning of the GSH1 and GSH2 genes complementing the defective biosynthesis of glutathione in the methylotrophic yeast Hansenula polymorpha].
The cloning of 7.2- and 9.6-kbp fragments of the methylotrophic yeast Hansenula polymorpha DNA restored the wild-type phenotype Gsh+ in the glutathione-dependent gsh1 and gsh2 mutants of this yeast defective in glutathione (GSH) synthesis because of a failure of the gamma-glutamylcysteine synthetase reaction. The 9.6-kbp DNA fragment was found to contain a 4.3-kbp subfragment, which complemented the Gsh- phenotype of the gsh2 mutant. The Gsh+ transformants of the gsh1 and gsh2 mutants, which bear plasmids pG1 and pG24 with the 7.2- and 4.3-kbp DNA fragments, respectively, had a completely restored wild-type phenotype with the ability to synthesize GSH and to grow in GSH-deficient synthetic media on various carbon sources, including methanol, and with acquired tolerance to cadmium ions. In addition, the 4.3-kbp DNA fragment borne by plasmid pG24 eliminated pleiotropic changes in the gsh2 mutants associated with methylotrophic growth in a semisynthetic (GSH-supplemented) medium (poor growth and alterations in the activity of the GSH-catabolizing enzyme gamma-glutamyltransferase and the methanol-oxidizing enzyme alcohol oxidase).
Ubiĭvovk
V M
VM
Institute of Cell Biology, National Academy of Sciences of Ukraine, ul. Dragomanova 14/16, Lviv, 79005 Ukraine. ubivovk@biochem.lviv.ua
Nazarko
T Iu
TIu
Stasyk
E G
EG
Sibirnyĭ
A A
AA
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media; D018076:DNA, Complementary / Q000096:biosynthesis / Q000737:chemistry; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D005721:Glutamate-Cysteine Ligase / Q000235:genetics* / Q000378:metabolism; D005978:Glutathione / Q000096:biosynthesis / Q000172:deficiency; D005981:Glutathione Synthase / Q000235:genetics*; D000432:Methanol / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D017354:Point Mutation; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D003470:Culture Media; D018076:DNA, Complementary; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D005723:gamma-Glutamyltransferase; D005721:Glutamate-Cysteine Ligase; D005981:Glutathione Synthase; D005978:Glutathione; D000432:Methanol