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SWTTING: Demonstrating that a gene is essential is always difficult, but this is particularly true for a slow-growing organism such as Mycobacterium tuberculosis. One method currently used is to show that homologous recombination leading to gene inactivation only occurs in the presence of a second copy of the gene, but obtaining statistically significant data can be prohibitively difficult. L5-based integrating plasmids have been widely used in the genetic analysis of mycobacteria. The L5 excisionase has been used in Mycobacterium smegmatis to excise and recover these plasmids from chromosome.OBJECTIVE: Our aims were to establish whether the L5 excisionase could function in M. tuberculosis to remove an L5-based integrated plasmid and, if so, to use this technology as the basis for an improved method for determining whether a gene is essential.DESIGN: We took two strains of M. tuberculosis carrying the essential gene glnE integrated into the chromosome on an L5-based plasmid, one of which lacked the functional chromosomal copy of the gene. We transformed these with vectors expressing the L5 excisionase and looked for loss of the integrated plasmid.RESULTS: We obtained efficient excision of an integrated vector from the wild-type strain. However, when the integrated vector carried the only functional copy of the essential gene glnE, the numbers of colonies recovered were reduced to background levels.CONCLUSION: The L5 excisionase does function in M. tuberculosis and can be used to confirm the essentiality of a gene. This technology also allows further analysis of essential genes that is difficult Ray Ban Stockists Liverpool or impossible using current methods.Copyright 2001 Harcourt Publishers Ltd.
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