Revealing the essentiality of multiple archaeal pcna genes using a mutant propagation assay based on an improved knockout method.
Revealing the essentiality of multiple archaeal pcna genes using a mutant propagation assay based on an improved knockout method.
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DOI：
10.1099/mic.0.042523-0
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发表时间：
2010-11
期刊：
影响因子：
1.5
通讯作者：
Changyi Zhang;Li Guo;Ling Deng;Yuanxin Wu;Yunxiang Liang;Li Huang;Q. She
中科院分区：
文献类型：
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作者：
Changyi Zhang;Li Guo;Ling Deng;Yuanxin Wu;Yunxiang Liang;Li Huang;Q. She
Crenarchaeota谱系的生物体含有三个增殖细胞核抗原（PCNA）亚基，而Euryarchaeota只有一个亚基。为了研究古细菌滑动夹的机制，我们试图产生每个pcna基因的islandicus，一个超嗜热crenarcheon的敲除，但失败了两个传统的敲除方法。然后，开发了一种新的敲除方案，称为标记插入和靶基因缺失（MID），利用该方案获得每个pMID-pcna质粒的转化体。我们发现，在反向选择条件下，pMID-pcna 3和pMID-araS-pcna 1转化体孵育期间，突变细胞持续存在于传代培养物中。通过缺失的靶基因等位基因（Δ pcna 1或Δ pcna 3）的半定量PCR分析研究突变细胞的繁殖，发现突变细胞不能再繁殖，表明这些pcna基因是宿主细胞生存力绝对需要的。因为该测定的唯一先决条件是产生MID引物，所以该方法通常可应用于任何精通同源重组的微生物。
Organisms belonging to the Crenarchaeota lineage contain three proliferating cell nuclear antigen (PCNA) subunits, while those in the Euryarchaeota have only one, as for Eukarya. To study the mechanism of archaeal sliding clamps, we sought to generate knockouts for each pcna gene in Sulfolobus islandicus, a hyperthermophilic crenarchaeon, but failed with two conventional knockout methods. Then, a new knockout scheme, known as marker insertion and target gene deletion (MID), was developed, with which transformants were obtained for each pMID-pcna plasmid. We found that mutant cells persisted in transformant cultures during incubation of pMID-pcna3 and pMID-araS-pcna1 transformants under counter selection. Studying the propagation of mutant cells by semiquantitative PCR analysis of the deleted target gene allele (Δpcna1 or Δpcna3) revealed that mutant cells could no longer be propagated, demonstrating that these pcna genes are absolutely required for host cell viability. Because the only prerequisite for this assay is the generation of a MID transformant, this approach can be applied generally to any micro-organisms proficient in homologous recombination.