1887

Abstract

The survival of within the human host after infection, especially within macrophages, is likely to require the activation of a number of mycobacterial genes. To identify such genes, a promoter-probe library was constructed in which fragments of H37Rv DNA were inserted upstream of a reporter gene, using an –mycobacterial shuttle vector. Bacille Calmette–Guérin (BCG) was subsequently transformed with this library and 4800 BCG clones were arrayed in a 96-well microtitre format, enabling the testing of individual clones for promoter activity under a variety of conditions. From preliminary screening, 41 clones were selected that exhibited upregulation of expression when subjected to acidified sodium nitrite. Subsequent sequence analyses identified 26 of these clones as containing potential promoters. After measuring expression in BCG clones recovered from a THP-1 macrophage cell line, three genes were selected for assessment of their expression in during macrophage infection, by real-time RT-PCR. Two of these genes, Rv1265 (with unknown function) and Rv2711 (encoding the iron-dependent repressor protein IdeR), showed evidence of being upregulated within macrophages.

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2002-05-01
2020-04-04
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