Analysis of full-length cDNA clones carryingGALIofSaccharomyces cerevisiae:a model system for cDNA expression

Abstract

A cDNA cloning vector that allows expression in Saccharomyces cerevisiae has been developed using the plasmid primer approach described by Okayama and Berg [Mol. Cell. Biol. 2:161-170(1982)]. The vector contains ARS1 and TRP1 for plasmid maintenance in yeast and the ADC1 or GAL1 promoter and the TRP5 terminator for expression of the cloned cDNA. Using this system, several recombinants with nearly full-length GAL1 cDNA inserts in a cDNA library made with galactose-induced yeast mRNA were identified. By measurement of galactokinase mRNA and its protein, the expression of GAL1 cDNA was shown to be under the control of the promoter placed upstream of the cDNA insert. Nucleotide sequence analysis revealed that the 3'-ends of the GAL1 cDNA inserts were not unique, indicating that polyA tails were added to GAL1 transcripts at multiple sites in the GAL1 gene. Genetic complementation of appropriate yeast mutants permitted the isolation of clones containing the coding sequences for GAL1, HIS3, and LEU2 from the same cDNA library.