Nature of abortive transformation in Saccharomyces cerevisiae.
Disruption mutagenesis by homologous recombination in Saccharomyces cerevisiae is carried out by transforming-DNA fragments containing the target gene disrupted by a selectable marker. A large number of transient (abortive) transformants are often formed that may hinder the isolation of integrants containing the gene disruption. We show that abortive transformants result from re-circularization of the linear transforming-DNA in vivo. Their number was greatly reduced when the cut DNA could not readily re-ligate, either by digestions that gave non-compatible or blunt ends, or by de-phosphorylation. In addition, true integrants could be readily distinguished from abortive transformants through replica plating onto selective media. Enhanced disruption-mutagenesis was also observed when non-compatible ends were generated in an ARS-containing insertion vector.
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Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, MA 02115, USA.
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D019747:Aldose-Ketose Isomerases; D004251:DNA Transposable Elements; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D016296:Mutagenesis; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic
D004251:DNA Transposable Elements; D004271:DNA, Fungal; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D019747:Aldose-Ketose Isomerases; C490353:TRP1 protein, S cerevisiae