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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Disruption of gene mg218 of Mycopiasma genitalium through homologous recombination leads to an adherence- deficient phenotype
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Disruption of gene mg218 of Mycopiasma genitalium through homologous recombination leads to an adherence- deficient phenotype

机译：通过同源重组破坏Mycopiasma genitalium基因mg218导致粘附缺陷型

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Although the complete genome of Mycoplasma genitalium has been sequenced, the functional identification of various genes, including those involved in virulence, has not been accomplished. Further compounding these difficulties has been the failure to develop genetic tools in mycoplasmas that permit the assessment of gene and operon function and regulation. To dstermine whether homologous recombination could be devel- oped as a tool to analyze the function of genes in M genitalium, a plasmid that replicates in Escherichia coli but not in M. genitalium was constructed to disrupt the cytadherence-related gene mg218 of M. genitalium. The clectroporation of this disrup- tion plasmid into wild-type hemadsorption-positive (HA~+) M. genitalium cells permitted the isolation of HA~- (strain JB1) and partial HA~+ (strains JB2 and JB20) transformants. Analysis of the transformants by Southern hybridization indicated that homologous recombination occurred at the mg218 locus by single-crossover events in JB1 and JB2 and by a double-crossover event in JB20. While integration of the disruption construct abolished the expression MG218 in JB1, strains JB2 and JB20 exhibited a truncated MG218 protein (160 kDa), possibly be- cause of in-frame fusion of the disrupted mg218 gene with sequences downstream of the gentamycin-resistance gene present in the disruption construct. Strain JB1, which lacked MG218, displayed a post-translational defect being unable to maintain the structural integrity of the major adhesin P140 and its operon-related protein P110, in contrast to JB2 and JB20. It appears th
机译：尽管已对生殖支原体的完整基因组进行了测序，但尚未完成对各种基因（包括与毒力有关的基因）的功能鉴定。使这些困难更加复杂的是未能在支原体中开发遗传工具，该工具无法评估基因和操纵子的功能和调控。为了确定是否可以将同源重组开发为分析生殖器支原体中基因功能的工具，构建了一种在大肠杆菌中而不在生殖器中复制的质粒，以破坏生殖器支原体中与细胞粘附相关的基因mg218。 。将这种分散质粒选集到野生型生殖器出血热（HA〜+）生殖生殖支原体细胞中，可以分离出HA〜-（JB1株）和部分HA〜+（JB2和JB20株）转化子。通过Southern杂交对转化体的分析表明，通过JB1和JB2中的单交换事件和JB20中的双交换事件，在mg218基因座发生同源重组。尽管整合了破坏构建体后，JB1中的MG218表达消失，但菌株JB2和JB20却显示出MG218蛋白被截短（160 kDa），这可能是由于破坏的mg218基因与庆大霉素抗性基因下游序列在框内融合所致。存在于破坏结构中。与JB2和JB20相比，缺少MG218的菌株JB1显示出翻译后缺陷，无法维持主要粘附素P140及其操纵子相关蛋白P110的结构完整性。看来

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