An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae
An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae
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DOI：
10.1128/aem.67.11.5190-5196.2001
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发表时间：
2001-11-01
影响因子：
4.4
通讯作者：
Morrison, DA
中科院分区：
文献类型：
--
作者：
Sung, CK;Li, H;Morrison, DA
天然遗传转化提供了一种直接途径，通过该途径可以将合成基因构建体置于肺炎链球菌的单个环状染色体中。然而，缺乏一般的负选择标记阻碍了不赋予可选择表型的构建体的引入。构建包含卡那霉素（Kn）抗性标记（kan）和反向选择性rpsL（+）标记的1.3-kb盒。该盒在S.肺炎。它被证明可以用于一个两步转化过程中，将任意序列的DNA放置在一个选定的靶位点。第一次转化为Sm抗性菌株使用该盒通过同源重组在染色体上标记靶基因，同时赋予重组体Kn抗性但Sm敏感性。在第二次转化期间用任意DNA片段替换盒恢复了Sin抗性（和Kn敏感性），允许构建沉默突变和缺失或缺乏可选择表型的其他基因替换。研究还表明，基因转换发生在两个rpsL等位基因之间的一个过程中，依赖于recA，并且容易通过错配修复进行校正。
Natural genetic transformation offers a direct route by which synthetic gene constructs can be placed into the single circular chromosome of Streptococcus pneumoniae. However, the lack of a general negative-selection marker has hampered the introduction of constructs that do not confer a selectable phenotype. A 1.3-kb cassette was constructed comprising a kanamycin (Kn) resistance marker (kan) and a counterselectable rpsL(+) marker. The cassette conferred dominant streptomycin (Sm) sensitivity in an Sm-resistant background in S. pneumoniae. It was demonstrated that it could be used in a two-step transformation procedure to place DNA of arbitrary sequence at a chosen target site. The first transformation into an Sm-resistant strain used the cassette to tag a target gene on the chromosome by homologous recombination while conferring Kn resistance but Sm sensitivity on the recombinant. Replacement of the cassette by an arbitrary segment of DNA during a second transformation restored Sin resistance (and Kn sensitivity), allowing construction of silent mutations and deletions or other gene replacements which lack a selectable phenotype. It was also shown that gene conversion occurred between the two rpsL alleles in a process that depended on recA and that was susceptible to correction by mismatch repair.