Gene disruption and basic phenotypic analysis of nine novel yeast genes from chromosome XIV.
In this work, we describe the disruption of nine ORFs of S. cerevisiae (YNL123w, YNL119w, YNL115c, YNL108c, YNL110c, YNL124w, YNL233w, YNL232w and YNL231c) in two genetic backgrounds: FY1679 and CEN.PK2. For the construction of the deletant strains, we used the strategy of short flanking homology (SFH) PCR. The SFH-deletion cassette was made by PCR amplification of the KanMX4 module with primers containing a 5' region of 40 bases homologous to the target yeast gene and with a 3' region of 20 bases homologous to pFA6a-KanMX4 MCS. Sporulation and tetrad analysis of heterozygous deletants revealed that YNL110c, YNL124w and YNL232w are essential genes. The subcellular localization of the protein encoded by the essential gene YNL110c was investigated using the green fluorescent protein (GFP) approach, revealing a nuclear pattern. Basic phenotypic analysis of the non-essential genes revealed that the growth of ynl119w delta haploid cells was severely affected at 37 degrees C in N3 medium, indicating that this gene is required at high temperatures with glycerol as a non-fermentable substrate. The ynl233w delta haploid cells also showed a particular phenotype under light microscopy and were studied in detail in a separate work.
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Dipartimento di Biologia, Università degli Studi di Padova, via G. Colombo 3, I-35121 Padova, Italy.
Sartorello
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Dal Pero
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D'Angelo
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Vezzi
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Campanaro
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Valle
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D015825:Chromosomes, Fungal; D017353:Gene Deletion; D005800:Genes, Fungal; D018613:Microscopy, Confocal; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid