Isolation of the CAR1 gene from Saccharomyces cerevisiae and analysis of its expression

Abstract

We isolated the CAR1 gene from Saccharomyces cerevisiae on a recombinant plasmid and localized it to a 1.58-kilobase DNA fragment. The cloned gene was used as a probe to analyze polyadenylated RNA derived from wild-type and mutant cells grown in the presence and absence of an inducer. Wild-type cells grown without the inducer contained very little polyadenylated RNA capable of hybridizing to the isolated CAR1 gene. A 1.25-kilobase CAR1-specific RNA species was markedly increased, however, in wild-type cells grown in the presence of inducer and in constitutive, regulatory mutants grown without it. No CAR1-specific RNA was observed when one class of constitutive mutant was grown in medium containing a good nitrogen source, such as asparagine. Two other mutants previously shown to be resistant to nitrogen repression contained large quantities of CAR1 RNA regardless of the nitrogen source in the medium. These data point to a qualitative correlation between the steady-state levels of CAR1-specific, polyadenylated RNA and the degree of arginase induction and repression observed in the wild type and in strains believed to carry regulatory mutations. Therefore, they remain consistent with our earlier suggestion that arginase production is probably controlled at the level of gene expression.