This proposal is an extension of ongoing research designed to provide basic information on the structure and function of the Mycobacterium tuberculosis. One goal is to understand how the insertion sequence is integrated into the genome of the M. tuberculosis complex and to evaluate its mobility. Previously identified repeats will be characterized and other insertion sequences will be isolated. Insertion sequences may be important in promoting,rearrangements in the chromosome and in altering gene expression. Regardless of their function, repeated sequences provide insight into the evolutionary relationship of these organisms. Another goal is to examine-genetic differences between M. tuberculosis and M. bovis to understand the significance of IS6110 copy number. A third goal is to isolate putative virulence genes from M. tuberculosis H37Ra.
The specific aims are: 1) to demonstrate that IS6110 functions as a mobile element by showing that it can transpose, and to determine the frequency with which such insertions occur. 2) To determine if the sites of IS6110 insertion are conserved within individual strains, species, and across species line and if the IS6110 insertion is target site specific. This will involve isolating DNA flanking IS6110, using these flanking sequences to probe genomic blots, and DNA sequencing of the flanking regions. 3) To characterize two previously identified repeated sequences and several of the insertion elements. 4) To analyze genetic differences between M. tuberculosis and M. bovis by fingerprinting the rRNA genes and DNA gyrase B genes to determine the basis for the difference in copy number of IS6110 in these organisms, and to compare phenotypic characteristics with copy number. 5) To determine whether the IS6110 insertions in M. tuberculosis H37Ra correlated with the loss of virulence. This will include searching for the uninterrupted genes in M. tuberculosis H37Rv, transforming H37Ra with these putative virulence genes, demonstrating virulence in mice, and identifying gene products.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI027284-06
Application #
2063786
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1989-02-01
Project End
1996-07-31
Budget Start
1994-08-01
Budget End
1996-07-31
Support Year
6
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Arkansas for Medical Sciences
Department
Pathology
Type
Schools of Medicine
DUNS #
City
Little Rock
State
AR
Country
United States
Zip Code
72205
Smith, K C; Starke, J R; Eisenach, K et al. (1996) Detection of Mycobacterium tuberculosis in clinical specimens from children using a polymerase chain reaction. Pediatrics 97:155-60
Cave, M D; Eisenach, K D; Templeton, G et al. (1994) Stability of DNA fingerprint pattern produced with IS6110 in strains of Mycobacterium tuberculosis. J Clin Microbiol 32:262-6
Eisenach, K D (1994) Use of an insertion sequence for laboratory diagnosis and epidemiologic studies of tuberculosis. Ann Emerg Med 24:450-3
Mazurek, G H; Cave, M D; Eisenach, K D et al. (1991) Chromosomal DNA fingerprint patterns produced with IS6110 as strain-specific markers for epidemiologic study of tuberculosis. J Clin Microbiol 29:2030-3
Eisenach, K D; Sifford, M D; Cave, M D et al. (1991) Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction. Am Rev Respir Dis 144:1160-3
Cave, M D; Eisenach, K D; McDermott, P F et al. (1991) IS6110: conservation of sequence in the Mycobacterium tuberculosis complex and its utilization in DNA fingerprinting. Mol Cell Probes 5:73-80