The recent increase in the incidence of tuberculosis (TB) in the United States (due at least in part to the HIV epidemic) has focused renewed interest on this disease and the physiology of its causative organism. Regulation of gene expression in pathogenic mycobacteria such as M. tuberculosis is incompletely characterized. The investigators have identified a 36 nucleotide sequence, rpt-1, occurring both as single units and as 2-, 3- or 4-unit tandem repeats in 30 M. tuberculosis sequences. Sequences similar to rpt-1 were also found to occur in M. bovis BCG and M. leprae, suggesting a broader role of repetitive DNA elements in regulating mycobacterial gene expression. rpt-1 sequences occurred upstream and in the same orientation to annotated ORFs in 27 of 30 cases; in the remaining cases, rpt-1 was located between two ORFs read in opposite directions. Among the genes preceded by rpt-1 sequences were a putative DNA helicase, a putative transcriptional regulator and several putative response regulators. Tandem repeats of rpt-1 units were separated by 20-23 intervening nucleotides, close to the number of nucleotides comprising two turns of the DNA helix so that tandem rpt-1 sequences lie on the same side of the DNA helix. The regulatory role of rpt-1 or its ability to interact with mycobacterial proteins is completely unknown. The investigators' preliminary studies using electrophoretic mobility shift assays and radiolabeled rpt-1 probes indicated that rpt-1 bound specifically to distinct protein(s) in cleared BCG cytosolic lysates. Unexpectedly, specific DNA-binding activity for rpt-1 was also present in nuclear extracts from eukaryotic HeLa and MCF-7 human breast cancer cell lines. The existence of mycobacterial DNA recognition sites in eukaryotic transcription factors could have important implications for pathogenesis of TB and other chronic intracellular infections. The long-term goal of this project is to characterize the role of regulators of mycobacterial gene expression in the pathogenesis of TB. The present project will test the hypothesis that rpt-1 is important for regulating mycobacterial gene expression.
The Specific Aims of the project are: 1) characterize nucleotide motifs involved in binding of rpt-1 to mycobacterial cytosolic proteins; 2) characterize the protein(s) binding to rpt-1 biochemically using microsequencing and cloning and immunochemically; and 3) examine the role of rpt-1 as a regulatory element in M. tuberculosis gene expression under various environmental conditions and in reporter gene fusions preceded by rpt-1 sequences.
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| Cobb, A; Moore, L; Godfrey, H P et al. (2001) The use of hairless (IAF/HA-HO) guinea pigs for the determination of delayed-type hypersensitivity to tuberculin. Int Immunopharmacol 1:349-53 |
