NATURE OF ABORTIVE TRANSFORMATION IN SACCHAROMYCES-CEREVISIAE

Disruption mutagenesis by homologous recombination in Saccharomyces ce revisiae is carried out by transforming-DNA fragments containing the t arget gene disrupted by a selectable marker. A large number of transie nt (abortive) transformants are often formed that may hinder the isola tion of integrants containing the gene disruption. We show that aborti ve transformants result from re-circularization of the linear transfor ming-DNA in vivo. Their number was greatly reduced when the cut DNA co uld not readily re-ligate, either by digestions that gave non-compatib le or blunt ends, or by de-phosphorylation. In addition, true integran ts 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-co ntaining insertion vector.