1887

Abstract

survives and multiplies inside macrophages of its host by modulating the expression of several genes essential for survival. An expression system has been developed, based on green fluorescent protein and kanamycin resistance, to identify genes which appear to be up-regulated in infected macrophages. A promoter-trap shuttle vector, pLL192, was constructed, containing a streptomycin resistance gene as selection marker and an artificial bicistronic operon composed of the promoterless green fluorescent protein () gene, followed by the kanamycin resistance gene. A unique HI site upstream of the gene allowed for insertion of promoter libraries. The vector was validated by the use of known regulated or constitutive promoters. In addition, an genomic DNA library was inserted into pLL192 and then introduced into BCG. The recombinant BCG cells were then used to infect the J774A.1 murine macrophage-like cell line in the presence of kanamycin. Several recombinant BCG cells were thereby selected that were resistant to kanamycin within infected macrophages, but were sensitive to kanamycin when grown . The kanamycin resistance phenotype was paralleled by the fluorescence phenotype. After nucleotide sequencing, the corresponding genes were identified as , PE_PGRS63(RV3097c), Rv2232, Rv1026, Rv1635c, , Rv2231(C) and Rv0997. Real-time PCR analysis using RNA isolated at various time points from and BCG grown and within macrophages, confirmed the up-regulation of these genes. The level of up-regulation varied from 2- to 40-fold in macrophages compared to growth .

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2007-03-01
2019-10-17
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