Isogenic strain construction and gene mapping in Candida albicans.
Isogenic strain construction and gene mapping in Candida albicans.
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DOI：
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发表时间：
1993-06
期刊：
影响因子：
3.3
通讯作者：
D. Irwin;W. Fonzi;D. Ho;S. Sag
中科院分区：
文献类型：
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作者：
D. Irwin;W. Fonzi;D. Ho;S. Sag
白念珠菌的二倍体基因组和无性生活史限制了其遗传操作。当在随机诱变过程中引入的杂合和不需要的突变不能通过遗传回交去除时，隐性突变不表达。为了避免这些问题，我们开发了一种基因型筛选，允许在URA 3基因座的杂合隐性突变的鉴定。通过靶向诱变、转化DNA的同源整合引入突变，以避免引入外源突变。通过第二轮转化使ura 3突变纯合，从而产生与亲本临床分离物等基因的Ura-菌株。Ura-菌株的后续突变通过使用URA 3基因作为选择标记的靶向诱变来实现。通过在URA 3基因的侧翼插入鼠伤寒沙门氏菌hisG基因的同向重复序列，重复使用URA 3选择来顺序引入突变。侧翼重复序列之间的自发染色体内重组切除了URA 3基因，恢复了Ura-表型。在含有5-氟乳清酸的培养基上选择这些Ura-分离子并用于下一轮诱变。为了允许破坏基因的物理作图，将内切核酸酶I-SceI的18-bp识别序列掺入hisG重复序列中。用I-SceI对染色体进行位点特异性切割，揭示了整合序列的位置。
Genetic manipulation of Candida albicans is constrained by its diploid genome and asexual life cycle. Recessive mutations are not expressed when heterozygous and undesired mutations introduced in the course of random mutagenesis cannot be removed by genetic back-crossing. To circumvent these problems, we developed a genotypic screen that permitted identification of a heterozygous recessive mutation at the URA3 locus. The mutation was introduced by targeted mutagenesis, homologous integration of transforming DNA, to avoid introduction of extraneous mutations. The ura3 mutation was rendered homozygous by a second round of transformation resulting in a Ura- strain otherwise isogenic with the parental clinical isolate. Subsequent mutation of the Ura- strain was achieved by targeted mutagenesis using the URA3 gene as a selectable marker. URA3 selection was used repeatedly for the sequential introduction of mutations by flanking the URA3 gene with direct repeats of the Salmonella typhimurium hisG gene. Spontaneous intrachromosomal recombination between the flanking repeats excised the URA3 gene restoring a Ura- phenotype. These Ura- segregants were selected on 5-fluoroorotic acid-containing medium and used in the next round of mutagenesis. To permit the physical mapping of disrupted genes, the 18-bp recognition sequence of the endonuclease I-SceI was incorporated into the hisG repeats. Site-specific cleavage of the chromosome with I-SceI revealed the position of the integrated sequences.