Screening and identification of yeast sequences that cause growth inhibition when overexpressed

Abstract

To isolate genes that negatively regulate cell growth, we constructed a galactose-inducible expression library with partially digested Saccharomyces cerevisiae genomic DNA fragments inserted downstream of the GAL10 promoter. In all, 240,000 yeast transformants were screened for lethality on galactose medium. From 17 such transformants identified, 16 nonoverlapping DNA sequences were obtained. Restriction mapping and determination of DNA sequences adjacent to the GAL10 promoter indicated that the inserts encoded part or all of the URA2, RBP1, TPK3, SAC7, BOI1, and BNI1 genes, and also open reading frames (ORFs) from chromosomes IV, V, IX, XI, and XIII. Some of the identified sequences lacked the amino-terminal sequences of the ORFs, suggesting that truncated forms of the proteins might be necessary for growth inhibition. The sequence of the pGA108 insert was highly homologous to the telomeric X-element and contained an ARS consensus sequence, suggesting a possible growth inhibitory effect of an RNA molecule. Overexpression of the BNI1 deltaN and BOI1 deltaN genes, which lacked amino-terminal sequences, was associated with phenotypes similar to those of mutants defective in bud formation. Overexpression of the GIN4 and GIN12 sequences induced elongated buds and a G2/M arrest-like phenotype, respectively. The phenotypes induced by the overexpression of our cloned sequences could result from either a dominant-positive or a dominant-negative effect and, unexpectedly, in one case from an effect of an RNA.