A general screening procedure has been devised for the selection of in vivo-generated deletions in haploid Saccharomyces cerevisiae cells. It is based on the introduction into a cyh2 host (resistant to the drug cycloheximide) of a tandemly duplicated CYH2 gene (a dominant allele, conferring sensitivity to cycloheximide), and subsequent selection for Cyhr derivatives. The duplicated CYH2 gene has been introduced on CEN ARS plasmids or integrated into chromosome II. A variable but significant proportion of the Cyhr derivatives of such transformants were deletion mutants in which both CYH2 copies had suffered deletions. Some of the deletions extended into sequences outside the tandemly duplicated CYH2 gene. A total of 61 independently selected deletions ranged in length from 3.1 to over 20 kilobases and had no obvious preferred endpoints. Restriction analysis showed that other frequently isolated Cyhr derivatives appeared to retain one of the two CYH2 copies. Such single-copy derivatives of CEN ARS plasmids did not contain a functional CYH2 gene. The frequency of true deletions in CEN ARS plasmids, of approximately 10(-7) per viable cell, was comparable in RAD52 and rad52 strains. Chromosomal deletions, which occurred at a frequency of approximately 10(-8) per viable cell, were observed only in rad52 hosts. Derivatives exhibiting an additional altered phenotype, such as the inactivation of a neighboring gene or, less frequently, the transcriptional activation of a previously silent gene, were isolated by screening deletion mutants. These results show that the method described can be used for in vivo deletion mapping or for the generation of gene fusions.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Bitoun R, Zamir A

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