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Volume 147, Issue 8

cDNA–RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular BCG Free

Abstract

Identifying genes that are differentially expressed by 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 genomic library. Sequence data were obtained from 19 differential clones, five of which contained overlapping sequences for the gene encoding mycocerosic acid synthase (). 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 in intracellular mycobacteria and also revealed a putative extended −10 promoter structure and a long untranslated upstream region 5′ of the transcripts, containing predicted double-stranded structures. Furthermore, clones containing overlapping sequences for , , and 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 BCG and the pathogenic mycobacterium,

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): high-density gridded genomic library , macrophage , MBN, mung-bean nuclease and mycobacterial mRNA microarray
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2001-08-01
2024-04-25
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cDNA–RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular Mycobacterium bovis BCG
Microbiology 147, 2293 (2001); https://doi.org/10.1099/00221287-147-8-2293
/content/journal/micro/10.1099/00221287-147-8-2293
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