DISRUPTION OF 6 NOVEL YEAST GENES REVEALS 3 GENES ESSENTIAL FOR VEGETATIVE GROWTH AND ONE REQUIRED FOR GROWTH AT LOW-TEMPERATURE

We describe here the construction of six deletion mutants and their ba sic phenotypic analysis. Six open reading frames (ORFs) from chromosom e X, YJR039w, YJR041c, YJR043c, YJR046w, YJR053w and YJR065c, were dis rupted by deletion cassettes with long (LFH) or short (SFH) flanking r egions homologous to the target locus. The LFH deletion cassette was m ade by introducing into the kanMX4 marker module two polymerase chain reaction (PCR) fragments several hundred base pairs (bp) in size homol ogous to the promoter and terminator regions of a given ORF. The SFH g ene disruption construct was obtained by PCR amplification of the kanM X4 marker with primers providing homology to the target gene. The regi on of homology to mediate homologous recombination was about 70 bp. Sp orulation and tetrad analysis revealed that ORFs YJR041c, YJR046w and YJR065c are essential genes. Complementation tests by corresponding co gnate gene clones confirmed this observation. The non-growing haploid segregants were observed under the microscope. The yjr041c Delta haplo id cells gave rise to microcolonies comprising about 20 to 50 cells. M ost yjr046w Delta cells were blocked after one or two cell cycles with heterogeneous bud sizes. The yjr065c Delta cells displayed an unbudde d spore or were arrested before completion of the first cell division cycle with a bud of variable size. The deduced protein of ORF YJR065c, that we named Act4, belongs to the Arp3 family of actin-related prote ins. Three other ORFs, YJR039w, YJR043c and YJR053w are non-essential genes. The yjr043c Delta cells hardly grew at 15 degrees C, indicating that this gene is required for growth at low temperature. Complementa tion tests confirmed that the disruption of YJR043c is responsible for this growth defect. In addition, the mating efficiency of yjr043c Del ta and yjr053w Delta cells appear to be moderately affected. (C) 1997 John Wiley & Sons, Ltd.