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Title: Development of systems for the genetic study of mycobacteria: Towards a recombinant BCG multivaccine
Authors: Snapper, Scott Brian
Keywords: Microbiology.
Molecular biology.
Issue Date: 1990
Publisher: ProQuest Dissertations & Theses
Citation: Source: Dissertation Abstracts International, Volume: 52-05, Section: B, page: 2415.;Advisors: Barry R. Bloom; William R. Jacobs.
Abstract: Tuberculosis and leprosy continue to inflict disease and death upon millions of people, primarily in the developing world. A significant increase in mycobacterial infections worldwide has been associated with the AIDS pandemic. The aim of this work was to develop a genetic system in mycobacteria that would allow the transfer, mutation and expression of specific genes. These techniques are essential for a detailed understanding of mycobacterial pathogenesis and for the development of improved mycobacterial vaccines.;A phage-based system to introduce and express genes in mycobacteria was developed from the temperate mycobacteriophage L1. Recombinant phage vectors, termed shuttle phasmids, which replicate in Escherichia coli as plasmids and in mycobacteria as phage, were constructed by inserting an E. coli cosmid into mycobacteriophage L1. A gene that confers kanamycin resistance in E. coli was cloned into an L1-shuttle phasmid and introduced into Mycobacterium smegmatis and BCG. Stable kanamycin-resistant lysogens were obtained, establishing kanamycin as the first useful selectable marker in mycobacteria.;With an effective selectable marker, a plasmid transformation system was developed for mycobacteria to further facilitate genetic analyses and to complement the phage-based system. M. fortuitum/E. coli hybrid shuttle plasmids containing mycobacterial and E. coli replicons and a kanamycin-resistance gene were constructed and successfully introduced into M. smegmatis and M. bovis-BCG vaccine substrains by electroporation. Moreover, M. smegmatis mutants were isolated that could be transformed with plasmid DNA by electroporation at efficiencies five orders of magnitude greater than the prototype wild type M. smegmatis mc{dollar}\sp2{dollar}6. The high efficiency of electrotransformation of these mutants greatly facilitated the genetic analysis of mycobacteria, as demonstrated by the rapid mapping of the essential region(s) of replication of mycobacterial plasmids.;Finally, the gene encoding the immunodominant M. leprae 65-kDa heat shock protein was cloned into a shuttle plasmid. This cloned foreign antigen was expressed in both M. smegmatis and M. bovis-BCG transformants as demonstrated by western analysis with an antibody that recognizes an M. leprae immunospecific epitope. The ability to express foreign genes in BCG should allow for the development and testing of recombinant BCG vaccines.
Appears in Collections:Albert Einstein College of Medicine: Doctoral Dissertations

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