Li, Ming-Shi, Monahan, Irene M, Waddell, Simon J., Mangan, Joseph A, Martin, Steve L, Everett, Martin J and Butcher, Philip D (2001) cDNA-RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular Mycobacterium bovis BCG. Microbiology, 147 (8). pp. 2293-2305. ISSN 1350-0872

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Abstract

Identifying genes that are differentially expressed by Mycobacterium bovis BCG after phagocytosis by macrophages will facilitate the understanding of the molecular mechanisms of host cell-intracellular pathogen interactions. To identify such genes a cDNA-total RNA subtractive hybridization strategy has been used that circumvents the problems both of limited availability of bacterial RNA from models of infection and the high rRNA backgrounds in total bacterial RNA. The subtraction products were used to screen a high-density gridded Mycobacterium tuberculosis genomic library. Sequence data were obtained from 19 differential clones, five of which contained overlapping sequences for the gene encoding mycocerosic acid synthase (mas). Mas is an enzyme involved in the synthesis of multi-methylated long-chain fatty acids that are part of phthiocerol dimycocerosate, a major component of the complex mycobacterial cell wall. Northern blotting and primer extension data confirmed up-regulation of mas in intracellular mycobacteria and also revealed a putative extended -10 promoter structure and a long untranslated upstream region 5' of the mas transcripts, containing predicted double-stranded structures. Furthermore, clones containing overlapping sequences for furB, groEL-2, rplE and fadD28 were identified and the up-regulation of these genes was confirmed by Northern blot analysis. The cDNA-RNA subtractive hybridization enrichment and high density gridded library screening, combined with selective extraction of bacterial mRNA represents a valuable approach to the identification of genes expressed during intra-macrophage residence for bacteria such as M. bovis BCG and the pathogenic mycobacterium, M. tuberculosis.

Item Type:	Article
Keywords:	High-density gridded genomic library, macrophage, mycobacterial mRNA microarray
Schools and Departments:	Brighton and Sussex Medical School > Clinical and Experimental Medicine Brighton and Sussex Medical School > Global Health and Infection
Subjects:	Q Science > Q Science (General) Q Science > QR Microbiology > QR0075 Bacteria
Depositing User:	Simon Waddell
Date Deposited:	03 Jan 2012 16:10
Last Modified:	02 Jul 2019 18:03
URI:	http://sro.sussex.ac.uk/id/eprint/7647
Google Scholar:	32 Citations

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