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• W1826672138 abstract "With the completion of the Mycobacterium tuberculosis genome sequence, the focus of research has turned to functional characterization of genes, and, in particular, identification of virulence factors. The inability to identify double-crossover events was a major impediment in the study of M. tuberculosis pathogenicity. The first mutant of the M. tuberculosis complex made through allelic replacement was a M. bovis BCG mutant, created using the ureC gene as a target. A major factor in the small number of mutants obtained using suicide plasmids was the efficiency of transformation. This difficulty is avoided by using replicating plasmids, although there is the new problem of ensuring plasmid loss. The first counterselectable marker to be described in mycobacteria was streptomycin sensitivity. This system takes advantage of the fact that the S12 ribosomal protein is the target of streptomycin. A major drawback when using replicating plasmids was that it was necessary to isolate plasmid free cells. To induce efficient plasmid loss at will, a thermosensitive replicon was isolated. Marked mutants, in which an antibiotic resistance gene is used to interrupt the gene of interest, are the most straightforward to make. Even when the gene studied is essential, studying the phenotype of a conditional mutant can be useful because it can help to unravel the function of the gene. In conclusion, many genetic tools are now available for studying mycobacteria. It is possible to select directly for allelic exchange in a single-step strategy or to construct an unmarked mutation in a two-step strategy." @default.
• W1826672138 created "2016-06-24" @default.
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• W1826672138 date "2005-01-01" @default.
• W1826672138 modified "2023-09-27" @default.
• W1826672138 title "Gene Replacement Systems†" @default.
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• W1826672138 doi "https://doi.org/10.1128/9781555817657.ch12" @default.
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