Kelly R, Miller S M, Kurtz M B
Department of Microbial Biochemistry and Genetics, Squibb Institute for Medical Research, Princeton, NJ 08543-4000.
Mol Gen Genet. 1988 Sep;214(1):24-31. doi: 10.1007/BF00340174.
The Candida albicans LEU2 gene was disrupted by substituting lambda DNA for a small deletion within the LEU2 gene. Cotransformation with a selectable URA3 ARS vector was used to introduce a linear fragment containing the disruption into the genome of a C. albicans ura3 deletion mutant. Cotransformants containing the lambda DNA were identified by colony hybridization and the URA3 plasmid was subsequently cured. Leu2 disrupted heterozygotes were detected by Southern hybridization and one disruptant was subsequently treated with UV irradiation. Only one leu2 ura3 mutant (SGY-484) was isolated out of 11,000 mutagenized cells. SGY-484 was transformed to Leu+ with either the C. albicans or Saccharomyces cerevisiae LEU2 gene. Southern hybridization analysis revealed that the mutant is not homozygous for the disruption; the leu2 mutation reverts and is most likely a point mutation. Unexpectedly, an ade2 ura3 mutant was isolated from the same mutagenesis.
通过用λDNA取代白色念珠菌LEU2基因内的一个小缺失片段，破坏了该基因。使用可选择的URA3 ARS载体进行共转化，将包含该破坏片段的线性片段导入白色念珠菌ura3缺失突变体的基因组中。通过菌落杂交鉴定含有λDNA的共转化体，随后去除URA3质粒。通过Southern杂交检测到Leu2破坏杂合子，随后对一个破坏体进行紫外线照射处理。在11,000个诱变细胞中仅分离出一个leu2 ura3突变体（SGY-484）。用白色念珠菌或酿酒酵母的LEU2基因将SGY-484转化为亮氨酸营养型（Leu+）。Southern杂交分析表明，该突变体并非破坏位点的纯合子；leu2突变回复，很可能是一个点突变。出乎意料的是，在同一诱变过程中分离出了一个ade2 ura3突变体。
