Mutagenesis and gene cloning in Schizosaccharomyces pombe using nonhomologous plasmid integration and rescue

Genes are commonly cloned in yeasts and bacteria by plasmid complementation , where the introduction of the gene of interest into a host strain carryin g a recessive mutation in that gene suppresses the host's mutant phenotype. However a lack of low copy cloning vectors in the fission yeast Schizosacc haromyces pombe can complicate this approach especially when overexpression of one gene may suppress a defect in another gene or when overexpression o f 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 rapi d and direct cloning of the defective gene. This involves the nonhomologous integration of a marked plasmid into the yeast genome and its subsequent r escue into Escherichia coli, so that DNA at the site of insertion is incorp orated into the recovered plasmid. As two of three insertions obtained in t his study occurred outside of the affected gene's open reading frame, this method should be applicable to cloning both essential genes and nonessentia l genes.