Functional Characterization of the <i>S. cerevisiae</i> Genome by Gene Deletion and Parallel AnalysisThe functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.
HeterologousHIS3 Marker and GFP Reporter Modules for PCR-Targeting inSaccharomyces cerevisiaeWe have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and Schizosac-charomyces pombe to the strong TEF gene promotor of the filamentous fungus Ashbya gossypii. Both chimeric modules and the cognate S. kluyveri HIS3 gene were tested in transformations of his3 S. cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences. The 1.4 kb chimeric Sz. pombe module (HIS3MX6) performed best. With less than 5% incorrectly targeted transformants, it functions as reliably as the widely used geniticin resistance marker kanMX. The rare false-positive His+ transformants seem to be due to non-homologous recombination rather than to gene conversion of the mutated endogenous his3 allele. We also cloned the green fluorescent protein gene from Aequorea victoria into our pFA-plasmids with HIS3MX6 and kanMX markers. The 0.9 kb GFP reporters consist of wild-type GFP or GFP-S65T coding sequences, lacking the ATG, fused to the S. cerevisiae ADH1 terminator. PCR-synthesized 2.4 kb-long double modules flanked by 40-45 bp-long guide sequences were successfully targeted to the carboxy-terminus of a number of S. cerevisiae genes. We could estimate that only about 10% of the transformants carried inactivating mutations in the GFP reporter.
PCR-based gene targeting in the filamentous fungus Ashbya gossypii5 PCR-Based Gene Targeting in Saccharomyces cerevisiaeHeterologous HIS3 Marker and GFP Reporter Modules for PCR‐Targeting in Saccharomyces cerevisiaeWe have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and Schizosac-charomyces pombe to the strong TEF gene promotor of the filamentous fungus Ashbya gossypii. Both chimeric modules and the cognate S. kluyveri HIS3 gene were tested in transformations of his3 S. cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences. The 1·4 kb chimeric Sz. pombe module (HIS3MX6) performed best. With less than 5 % incorrectly targeted transformants, it functions as reliably as the widely used geniticin resistance marker kanMX. The rare false-positive His+ transformants seem to be due to non-homologous recombination rather than to gene conversion of the mutated endogenous his3 allele. We also cloned the green fluorescent protein gene from Aequorea victoria into our pFA-plasmids with HIS3MX6 and kanMX markers. The 0·9 kb GFP reporters consist of wild-type GFP or GFP-S65T coding sequences, lacking the ATG, fused to the S. cerevisiae ADH1 terminator. PCR-synthesized 2·4 kb-long double modules flanked by 40–45 bp-long guide sequences were successfully targeted to the carboxy-terminus of a number of S. cerevisiae genes. We could estimate that only about 10 % of the transformants carried inactivating mutations in the GFP reporter.? 1997 John Wiley