Mutagenesis and Gene Cloning in Schizosaccharomyces pombe Using Nonhomologous Plasmid Integration and Rescue

Abstract

Genes are commonly cloned in yeasts and bacteria by plasmid complementation, where the introduction of the gene of interest into a host strain carrying a recessive mutation in that gene suppresses the host's mutant phenotype. However, a lack of low copy cloning vectors in the fission yeast Schizosaccharomyces pombe can complicate this approach especially when overexpression of one gene may suppress a defect in another gene or when overexpression of the desired gene is detrimental, if not lethal, to the cell. We describe here a method of identifying mutations in S. pombe that allows for the rapid and direct cloning of the defective gene. This involves the nonhomologous integration of a marked plasmid into the yeast genome and its subsequent rescue into Escherichia coli, so that DNA at the site of insertion is incorporated into the recovered plasmid. As two of three insertions obtained in this study occurred outside of the affected gene's open reading frame, this method should be applicable to cloning both essential genes and nonessential genes.