Use of gap repair in fission yeast to obtain novel alleles of specific genes.
We have adapted a method for making libraries of mutations in any specific gene for use in the fission yeast Schizosaccharomyces pombe . This elegant and simple method consists of PCR amplification of the gene of interest, followed by co-transformation of fission yeast with the PCR fragment and a linearized plasmid vector prepared such that the ends of the vector share DNA sequence with the ends of the PCR fragment. Homologous recombination between the vector and the PCR fragment occurs at a high frequency and results in a collection of yeast transformants, most harboring a mutated allele of the original gene within the vector of choice. This library can then be screened or selected for phenotypes of interest.
Kostrub
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Department of Genetics, Harvard Medical School, Warren Alpert Building, 200 Longwood Avenue, Boston, MA 02115, USA. kostrub@rascal.med.harvard.edu
Lei
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EP
Enoch
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D000483:Alleles; D018797:Cell Cycle Proteins / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D015723:Gene Library; D005800:Genes, Fungal / Q000235:genetics*; D016296:Mutagenesis; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins; D014170:Transformation, Genetic
D018797:Cell Cycle Proteins; D029702:Schizosaccharomyces pombe Proteins; C106332:hus1 protein, S pombe