ABSTRACT
The possibility to visualize small bacterial RNAs inside macrophages infected with Mycobacterium tuberculosis was demonstrated for the first time. A macrophage cell line was infected with the M. tuberculosis strain expressing small noncoding mycobacterial RNA MTS1338 fused with an RNA aptamer, which could bind a fluorophore and trigger its fluorescence. As a result, treatment of the infected macrophages with the DFHBI-1T fluorophore allowed fluorescence-based detection of the aptamer-labeled MTS1338 both in mycobacteria and in the host cell cytoplasm. This system can significantly aid in revealing the role of small M. tuberculosis RNAs in the pathogenesis of tuberculosis through identification of their secretion routes and eukaryotic targets and elucidation of the associated molecular pathways.
Subject(s)
Aptamers, Nucleotide/genetics , Fluorescent Dyes/metabolism , Green Fluorescent Proteins/genetics , Macrophages/microbiology , Mycobacterium tuberculosis/genetics , RNA, Small Untranslated/genetics , Animals , Aptamers, Nucleotide/metabolism , Cells, Cultured , Fluorescence , Green Fluorescent Proteins/metabolism , Macrophages/metabolism , Mice , Mycobacterium tuberculosis/isolation & purification , Mycobacterium tuberculosis/metabolism , RNA, Small Untranslated/metabolismABSTRACT
Epigenetic elements of the genome, i.e. elements that determine stably inherited changes in gene expression without changes in the genomic DNA sequence, are essential tools of genetic regulation in higher eukaryotes. The complete sequencing of the human and other genomes allowed studies to be started on positioning of these elements within long multigenic regions of the genome, which is a prerequisite for a comprehensive functional annotation of genomes. This mini-review considers some recent experimental approaches to the high-throughput identification and mapping of epigenetic elements of mammalian genomes, including the mapping of methylated CpG sites, open and closed chromatin regions, and DNase I hypersensitivity sites.
