ISSN:
0749-503X
Keywords:
gene disruption
;
functional analysis
;
chromosome X
;
Saccharomyces cerevisiae
;
Life and Medical Sciences
;
Genetics
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
Notes:
We describe here the construction of six deletion mutants and their basic phenotypic analysis. Six open reading frames (ORFs) from chromosome X, YJR039w, YJR041c, YJR043c, YJR046w, YJR053w and YJR065c, were disrupted by deletion cassettes with long (LFH) or short (SFH) flanking regions homologous to the target locus. The LFH deletion cassette was made by introducing into the kanMX4 marker module two polymerase chain reaction (PCR) fragments several hundred base pairs (bp) in size homologous to the promoter and terminator regions of a given ORF. The SFH gene disruption construct was obtained by PCR amplification of the kanMX4 marker with primers providing homology to the target gene. The region of homology to mediate homologous recombination was about 70 bp. Sporulation and tetrad analysis revealed that ORFs YJR041c, YJR046w and YJR065c are essential genes. Complementation tests by corresponding cognate gene clones confirmed this observation. The non-growing haploid segregants were observed under the microscope. The yjr041cΔ haploid cells gave rise to microcolonies comprising about 20 to 50 cells. Most yjr046wΔ cells were blocked after one or two cell cycles with heterogeneous bud sizes. The yjr065cΔ cells displayed an unbudded 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 proteins. Three other ORFs, YJR039w, YJR043c and YJR053w are non-essential genes. The yjr043cΔ cells hardly grew at 15°C, indicating that this gene is required for growth at low temperature. Complementation tests confirmed that the disruption of YJR043c is responsible for this growth defect. In addition, the mating efficiency of yjr043cΔ and yjr053wΔ cells appear to be moderately a ffected. © 1997 John Wiley & Sons, Ltd.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
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