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Title: Towards the development of molecular genetic approaches for the study of mycobacterial pathogenesis
Authors: Kalpana, Ganjam V.
Keywords: Microbiology.
Molecular biology.
Issue Date: 1991
Publisher: ProQuest Dissertations & Theses
Citation: Source: Dissertation Abstracts International, Volume: 52-04, Section: B, page: 1846.;Advisors: Barry R. Bloom.
Abstract: Mycobacteria, particularly Mycobacterium tuberculosis, M. leprae and M. avium are major pathogens of man. While insertional mutagenesis has been an invaluable genetic tool for analyzing the mechanisms of microbial pathogenesis, it has not yet been applied to the Mycobacteria. To overcome the intrinsic difficulties in directly manipulating the genetics of slow-growing mycobacteria, including M. tuberculosis, and Bacille Calmette-Guerin (BCG) vaccine strains, a system for random shuttle mutagenesis was developed. A genomic library of M. smegmatis was subjected to transposon mutagenesis with Tn5 seq1, a derivative of Tn5, in E. coli and these transposon-containing fragments were reintroduced into mycobacterial chromosome via. homologous recombination. This system has allowed us to isolate several random auxotrophic mutants of M. smegmatis. To extend this strategy to M. tuberculosis and BCG, and to optimize the system for homologous recombination in these species, targeted mutagenesis was performed using a cloned BCG methionine gene that was subjected to Tn5 seq1 mutagenesis in E. coli and reintroduced into the mycobacteria. Surprisingly for prokaryotes, both BCG and M. tuberculosis were found to incorporate linear DNA fragments into illegitimate sites throughout the mycobacterial genomes, at a frequency of {dollar}\approx{dollar}10{dollar}\sp{lcub}-5{rcub}{dollar} to 10{dollar}\sp{lcub}-4{rcub}{dollar} relative to autonomously replicating vectors. The efficient illegitimate recombination of linear DNA fragments now provides the basis for an insertional mutagenesis system for M. tuberculosis.;Another approach we took to understand the mycobacterial pathogenesis was to compare the genomic organization of closely related, virulent and avirulent strains of mycobacteria. We began macro-restriction analysis of the mycobacterial genomes utilizing pulsed-field gel electrophoresis (PFG). We demonstrated that restriction enzymes with an AT-rich recognition site, such as DraI were useful in such analysis. To order the contiguous macro-restriction fragments, we developed a method for isolating the linking clones of macro-restriction fragments, using SupF tRNA as marker. During these studies we isolated an E. coli mutant that enabled efficient isolation of the linking clones. We were able to determine the linkage of several fragments using this method. We also found the existence of a DraI RFLP between set of virulent and avirulent M. tuberculosis strains, H37Rv and H37Ra. Linking clone analysis revealed that this RFLP was generated by the insertion of an IS-element. Analyzing the region spanning the RFLP might contribute to the understanding of pathogenesis of M. tuberculosis.
Appears in Collections:Albert Einstein College of Medicine: Doctoral Dissertations

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