A conjugation-based system for genetic analysis of group II intron splicing in Lactococcus lactis
A conjugation-based system for genetic analysis of group II intron splicing in Lactococcus lactis
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DOI：
10.1128/jb.186.7.1991-1998.2004
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发表时间：
2004-04-01
影响因子：
3.2
通讯作者：
Dunny, GM
中科院分区：
文献类型：
--
作者：
Klein, JR;Chen, YQ;Dunny, GM
来自乳酸乳球菌的接合元件pRS 01编码推定的松弛酶蛋白LtrB。ItrB基因被功能组11内含子LI. ItrB中断。两个ItrB外显子的精确剪接是合成编码LtrB接合松弛酶的mRNA和随后的质粒转移所必需的。开发了基于缀合的遗传测定以鉴定影响剪接的LI.ItrB突变。在该测定中，使用非剪接的、转移缺陷的pRS 01衍生物（pM1014）和携带ItrB区（包括L1.ItrB内含子）的穿梭载体（pCOM 9）。pCOM9提供pM1014的转移缺陷的剪接依赖性互补。在pCOM9的背景下，在催化RNA中或在内含子编码的蛋白质基因ItrA中，在L1.ItrB内产生定点突变。当在基于缀合的测定中测试这些突变体时，观察到交配显著减少。体内剪接活性的定量分子分析证实，所观察到的交配缺陷导致减少剪接。一旦该系统被验证为工程突变体，随机诱变的内含子，然后遗传和分子筛选剪接缺陷导致识别点突变，影响剪接。
The conjugative element pRS01 from Lactococcus lactis encodes the putative relaxase protein LtrB. The ItrB gene is interrupted by the functional group 11 intron LI.ItrB. Accurate splicing of the two ItrB exons is required for synthesis of the mRNA encoding the LtrB conjugative relaxase and subsequent plasmid transfer. A conjugation-based genetic assay was developed to identify LI.ItrB mutations that affect splicing. In this assay a nonsplicing, trans fer-defective pRS01 derivative (pM1014) and a shuttle vector carrying the ItrB region, including the LI.ItrB intron (pCOM9), are used. pCOM9 provides splicing-dependent complementation of the transfer defect of pM1014. Site-directed mutations within LI.ItrB, either in the catalytic RNA or in the intron-encoded protein gene ItrA, were generated in the context of pCOM9. When these mutants were tested in the conjugation-based assay, significantly reduced mating was observed. Quantitative molecular analysis of in vivo splicing activity confirmed that the observed mating defects resulted from reduced splicing. Once the system was validated for the engineered mutants, random mutagenesis of the intron followed by genetic and molecular screening for splicing defects resulted in identification of point mutations that affect splicing.