Cloning and characterization of SKT5, a Saccharomyces cerevisiae gene that affects protoplast regeneration and resistance to killer toxin of Kluyveromyces lactis

Abstract

Abstract Saccharomyces cerevisiae strains carrying a mutation in the SKT5 gene are resistant to the killer toxin of Kluyveromyces lactis and are defective in protoplast regeneration. To understand the role of the SKT5 gene in toxin sensitivity and protoplast regeneration, we initiated a molecular study of this gene. A DNA fragment that complemented both the skt5 phenotypes was isolated from a yeast genomic library constructed in a single copy vector YCp50. Deletion mapping revealed that the entire complementing activity was carried on a 2.0 kilobase fragment. The DNA sequence of this fragment was determined and a long open reading frame encoding 533 amino acids with a calculated molecular weight of 59,165 daltons was found in the DNA sequence. In a homology search of the GenBank and EMBL data bases, this gene product showed no significant sequence similarity to any known protein coding sequence. Chromosomal mapping by pulsed field gradient gel electrophoresis and Southern hybridization analyses revealed that the SKT5 gene was a single copy gene located on chromosome II. The 2.0 kilobase transcript of this gene was detected by Northern analysis. A genomic disruption of the SKT5 gene was constructed by the insertion of a 2 kilobase LEU2 fragment. Disruption of this gene was not lethal; cells carrying the disrupted skt5 allele ( skt5 : : LEU2 ) were viable and had phenotypes similar to those of cells carrying the original skt5 allele. Thus, the 59 kilodalton protein deduced from the DNA sequence analysis is indeed a product of the SKT5 gene and is unlikely to be essential for the growth of yeast cells.