Belitskiĭ B R, Shakulov R S
Genetika. 1988 Aug;24(8):1333-42.
The gpp gene involved in the pppGpp conversion into ppGpp in Escherichia coli cells was cloned and localized within the multicopy pBR322 plasmid. Amplification of the gpp gene leads to the decline of the intracellular level of pppGpp, which implies enhanced activity of the corresponding enzyme, guanosine pentaphosphatase. To inactivate the cloned gene, a fragment of the pUC4K plasmid containing the kan gene was inserted within the gpp gene. The functional chromosomal allele of the gpp gene was replaced by its inactivated gpp::kan allele, taking advantage of homologous recombination during the transformation of recBC, sbcB cells with the intact hybrid plasmid. This procedure is accompanied by plasmid elimination and may be used for the replacement of other loci of bacterial chromosome with appropriate cloned alleles.
在大肠杆菌细胞中参与将pppGpp转化为ppGpp的gpp基因被克隆并定位在多拷贝pBR322质粒内。gpp基因的扩增导致细胞内pppGpp水平下降，这意味着相应的酶——鸟苷五磷酸酶的活性增强。为使克隆基因失活，将含有kan基因的pUC4K质粒片段插入gpp基因内。利用recBC、sbcB细胞与完整杂交质粒转化过程中的同源重组，gpp基因的功能性染色体等位基因被其失活的gpp::kan等位基因取代。此过程伴随着质粒消除，可用于用合适的克隆等位基因替换细菌染色体的其他位点。
