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Disruption of six Saccharomyces cerevisiae ORFs on chromosome XII results in three lethal disruptants
Author(s) -
Alloush Habib M.,
Edwards Thomas A.,
ValleLisboa Virginia,
Wheals Alan E.
Publication year - 2001
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.808
Subject(s) - orfs , biology , plasmid , genetics , gene , saccharomyces cerevisiae , locus (genetics) , selectable marker , complementation , homology (biology) , chromosome , ploidy , microbiology and biotechnology , open reading frame , phenotype , peptide sequence
Six ORFs of unknown function from the left arm of chromosome XII of Saccharomyces cerevisiae were chosen for a reverse genetic approach to provide materials to assist in assignment of function. A two‐step PCR using long‐flanking homology was employed to amplify disruption cassettes consisting of a kanMX gene as selectable marker flanked by 250–350 bp long regions homologous to the target gene. The diploid strains FY1679 and CEN.PK2 were transformed with the replacement cassettes and transformants were selected for geneticin (G418) resistance. Correct targeting of the replacement cassettes at the genomic locus was verified by Southern blot analysis with the kanMX gene as a probe. Disruption cassettes were cloned in pUG7 plasmid for systematic gene inactivation in other yeast strains and the cognate genes were cloned in pRS416 plasmid for gene complementation studies. Sporulation and tetrad analysis of heterozygous disruptants showed that three of the six ORFs [ YLR141w ( RRN5 ), YLR145w and YLR147c ( SMD3 )] were essential genes that were complemented by their cognate genes. ylr146c Δ ( spe4 ) homozygous diploids showed enhanced sporulation efficiency, whereas ylr147c Δ heterozygous diploids failed to sporulate in the FY1679 but not in the CEN.PK2 genetic background. The other two disruptants [ ylr143w and ylr144c ( acf2 )] gave no phenotype. Copyright © 2002 John Wiley & Sons, Ltd.