title: cDNA-RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular Mycobacterium bovis BCG.
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.
authors.affiliation: Department of Medical Microbiology, St George's Hospital Medical School, University of London, Cranmer Terrace, London SW17 0RE, UK1.
authors.forename: Ming-Shi
authors.initials: MS
authors.lastname: Li
authors.affiliation: Department of Medical Microbiology, St George's Hospital Medical School, University of London, Cranmer Terrace, London SW17 0RE, UK1.
authors.forename: Irene M
authors.initials: IM
authors.lastname: Monahan
authors.affiliation: Department of Medical Microbiology, St George's Hospital Medical School, University of London, Cranmer Terrace, London SW17 0RE, UK1.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Waddell
authors.affiliation: Department of Medical Microbiology, St George's Hospital Medical School, University of London, Cranmer Terrace, London SW17 0RE, UK1.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Mangan
authors.affiliation: Glaxo Wellcome Research and Development, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK2.
authors.forename: Steve L
authors.initials: SL
authors.lastname: Martin
authors.affiliation: Glaxo Wellcome Research and Development, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK2.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Everett
authors.affiliation: Department of Medical Microbiology, St George's Hospital Medical School, University of London, Cranmer Terrace, London SW17 0RE, UK1.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Butcher
mesh_terms: D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D015964:Gene Expression Regulation, Bacterial; D015698:Genomic Library; D006801:Humans; D008264:Macrophages / Q000382:microbiology*; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D009169:Mycobacterium tuberculosis / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D012329:RNA, Bacterial; D012333:RNA, Messenger; D000217:Acyltransferases; C044110:mycocerosic acid synthase