| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Genetics and Molecular Biology |
Department of Medical Microbiology, St George’s Hospital Medical School, University of London, Cranmer Terrace, London SW17 0RE, UK1
Glaxo Wellcome Research and Development, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK2
Author for correspondence: Philip D. Butcher. Tel: +44 20 8725 5721. Fax: +44 20 8672 0234. e-mail: butcherp{at}sghms.ac.uk
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.
Keywords: high-density gridded genomic library, macrophage, mycobacterial mRNA microarray
Abbreviations: MBN, mung-bean nuclease
a Present address: Department of Paediatrics, Imperial College School of Medicine, St Mary’s Hospital, Norfolk Place, London W2 1PG, UK.
This article has been cited by other articles:
|
|
 |
|
  A. H. Li, W. L. Lam, and R. W. Stokes Characterization of genes differentially expressed within macrophages by virulent and attenuated Mycobacterium tuberculosis identifies candidate genes involved in intracellular growth Microbiology, August 1, 2008; 154(8): 2291 - 2303. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Gazdik and K. A. McDonough Identification of Cyclic AMP-Regulated Genes in Mycobacterium tuberculosis Complex Bacteria under Low-Oxygen Conditions J. Bacteriol., April 15, 2005; 187(8): 2681 - 2692. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. D. Sirakova, A. M. Fitzmaurice, and P. Kolattukudy Regulation of Expression of mas and fadD28, Two Genes Involved in Production of Dimycocerosyl Phthiocerol, a Virulence Factor of Mycobacterium tuberculosis J. Bacteriol., December 15, 2002; 184(24): 6796 - 6802. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
