Castano Irene, Kaur Rupinder, Pan Shihjung, Cregg Robert, Penas Alejandro De Las, Guo Nini, Biery Matthew C, Craig Nancy L, Cormack Brendan P
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Genome Res. 2003 May;13(5):905-15. doi: 10.1101/gr.848203. Epub 2003 Apr 14.
We describe and characterize a method for insertional mutagenesis of the yeast pathogen Candida glabrata using the bacterial transposon Tn7. Tn7 was used to mutagenize a C. glabrata genomic fosmid library. Pools of random Tn7 insertions in individual fosmids were recovered by transformation into Escherichia coli. Subsequently, these were introduced by recombination into the C. glabrata genome. We found that C. glabrata genomic fragments carrying a Tn7 insertion could integrate into the genome by nonhomologous recombination, by single crossover (generating a duplication of the insertionally mutagenized locus), and by double crossover, yielding an allele replacement. We were able to generate a highly representative set of approximately 10(4) allele replacements in C. glabrata, and an initial characterization of these shows that a wide diversity of genes were targeted in the mutagenesis. Because the identity of disrupted genes for any mutant of interest can be rapidly identified, this method should be of general utility in functional genomic characterization of this important yeast pathogen. In addition, the method might be broadly applicable to mutational analysis of other organisms.
我们描述并表征了一种使用细菌转座子Tn7对酵母病原体光滑念珠菌进行插入诱变的方法。Tn7被用于诱变光滑念珠菌基因组fosmid文库。通过转化到大肠杆菌中回收单个fosmid中随机Tn7插入的文库。随后，通过重组将这些文库导入光滑念珠菌基因组。我们发现携带Tn7插入的光滑念珠菌基因组片段可通过非同源重组、单交换（产生插入诱变位点的重复）和双交换整合到基因组中，从而实现等位基因替换。我们能够在光滑念珠菌中产生一组高度代表性的约10⁴个等位基因替换，对这些替换的初步表征表明诱变靶向了多种基因。由于可以快速鉴定任何感兴趣突变体中被破坏的基因的身份，该方法在这种重要酵母病原体的功能基因组表征中应具有普遍实用性。此外，该方法可能广泛适用于其他生物体的突变分析。
