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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >UNMARKED GENE INTEGRATION INTO THE CHROMOSOME OF MYCOBACTERIUM SMEGMATIS VIA PRECISE REPLACEMENT OF THE PYRF GENE
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UNMARKED GENE INTEGRATION INTO THE CHROMOSOME OF MYCOBACTERIUM SMEGMATIS VIA PRECISE REPLACEMENT OF THE PYRF GENE

机译：通过PYRF基因的精确置换，将未标记的基因整合到耻垢分枝杆菌的染色体中

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After integration into the bacterial chromosome an exogenous gene may be stably expressed without continued selection for the recombinant locus. However, chromosomal integration events occur infrequently, requiring the concomitant integration of a drug resistance marker in order to identify colonies of recombinant cells. The generation of a drug-resistant recombinant strain can both reduce the in vivo applicability of the strain and preclude the use of recombinant vectors which use the same drug resistance marker. We have constructed a plasmid, pINT-Delta, which allows recombination of exogenous genes onto the Mycobacterium smegmatis chromosome. The exogenous gene completely replaces the pyrF gene and the resultant strain lacks any exogenous drug resistance marker. The methodologies described herein are general and applicable even to those bacteria for which extrachromosomal plasmids are not available. Using pINT-Delta we integrated the lacZ gene into the M. smegmatis chromosome via a precise exchange of lacZ and pyrF. The resultant strain was used to demonstrate that the expression of genes integrated at the pyrF locus is repressed twofold by inclusion of uracil in the growth medium. In addition, we used pINT-Delta to construct an M. smegmatis strain with a precise deletion of its pyrF locus. This strain, TSm-627, grows normally in rich medium but does not grow in medium lacking uracil. TSm-627 cells allow the pyrF gene to be used as a selectable marker for growth on medium lacking uracil. In TSm-627 cells, the pyrF gene is also useful as a counterselectable marker on complete medium containing 5'-fluoroorotic acid and uracil. Two pyrF-containing plasmids, designed to exploit the new Delta pyrF strain, have been constructed and their possible applications to problems in mycobacteriology are discussed. (C) 1997 academic Press. [References: 18]
机译：整合入细菌染色体后，外源基因可以稳定表达而无需继续选择重组基因座。然而，染色体整合事件很少发生，需要伴随药物抗性标记的整合以鉴定重组细胞的集落。耐药重组菌株的产生既可以降低菌株的体内适用性，又可以排除使用具有相同耐药标记的重组载体。我们已经构建了一个质粒pINT-Delta，该质粒可以将外源基因重组到耻垢分枝杆菌染色体上。外源基因完全取代了pyrF基因，所得菌株缺乏任何外源抗药性标记。本文描述的方法是通用的，甚至适用于那些无法获得染色体外质粒的细菌。使用pINT-Delta，我们通过lacZ和pyrF的精确交换将lacZ基因整合到耻垢分枝杆菌染色体中。所得菌株用于证明在pyrF基因座整合的基因的表达通过在生长培养基中包含尿嘧啶而被抑制了两倍。此外，我们使用pINT-Delta构建了耻垢分枝杆菌菌株，并精确删除了其pyrF基因座。该菌株TSm-627在富营养培养基中正常生长，但在缺乏尿嘧啶的培养基中不生长。 TSm-627细胞可以使pyrF基因用作在缺乏尿嘧啶的培养基上生长的选择标记。在TSm-627细胞中，pyrF基因还可用作包含5'-氟乳清酸和尿嘧啶的完全培养基上的反选择标记。已构建了两个旨在开发新的ΔryrF菌株的含pyrF的质粒，并讨论了它们对分枝杆菌学问题的可能应用。 （C）1997学术出版社。 [参考：18]

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