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Genetics and Molecular Biology |
Unité de Génétique Mycobactérienne, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France1
Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag No. 6, Newtown, NSW, 2042, Australia2
Author for correspondence: James A. Triccas. Tel: +61 2 9565 6163. Fax: +61 2 9565 6101. e-mail: J.Triccas{at}centenary.usyd.edu.au
The natural fluorescence of the Aequoria victoria green fluorescent protein was exploited to isolate strong expression signals of Mycobacterium tuberculosis. Mycobacterium bovis bacille Calmette–Guérin harbouring M. tuberculosis fragments driving high levels of gfp expression were isolated by fluorescence-activated cell sorting (FACS). DNA sequencing and subsequent comparison with the M. tuberculosis genome sequence revealed that a total of nine postulated promoters had been identified. The majority of the promoters displayed activity that was greater than or equal to the Mycobacterium fortuitum ß-lactamase promoter, one of the strongest mycobacterial promoters characterized to date. Two of the promoters corresponded to proteins predicted to be involved in calcium and magnesium utilization, the importance of such functions for cell physiology suggesting why these two genes are controlled by strong transcription signals. The seven other promoters corresponded to genes encoding proteins of unknown function. Promoter activity was maintained after prolonged incubation within macrophages, implying that these promoters could be used to drive sustained foreign gene expression in vivo. The strength of these expression signals identified could be employed for the overexpression of foreign genes in mycobacteria to aid protein purification and vaccine vector development. Furthermore, this study demonstrated that FACS provides a sensitive and efficient technique to measure and select strong mycobacterial expression signals.
Keywords: strong promoters, green fluorescent protein, genome, macrophage
Abbreviations: BCG, bacille Calmette–Guérin; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein
a Present address: Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag No 6, Newtown, NSW, 2042, Australia.
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