Isolation of a Cytochrome P-450 Structural Gene from 'Saccharomyces cerevisiae'

摘要

A Saccharomyces cerevisiae host with an S. cerevisiae genomic library contained in the shuttle vector YEp24 was transformed and the resultant transformants were screened for resistance to ketoconazole (Kc), an inhibitor of the cytochrome P-450 (P-450) enzyme lanosterol 14 alpha-demethylase. Two plasmids were isolated which transformed yeast to both increased resistance to Kc and increased levels of total P-450. Hybrid-selection and immunoprecipitation experiments showed that these plasmids, pVK1 and pVK2, contained the structural gene for an S. cerevisiae P-450. This conclusion was confirmed by the nucleotide sequence of a portion of pVK2, which revealed an open reading frame encoding a characteristic P-450 heme-binding region.