Publication Date

1994

Document Type

Dissertation/Thesis

First Advisor

Kuhl, Steven

Degree Name

M.S. (Master of Science)

Department

Department of Biological Sciences

LCSH

Whooping cough||Bordetella pertussis

Abstract

Initial investigations into the molecular biology of the RecA protein and its associated DNA-repair and recombinational functions in the respiratory pathogen Bordetella pertussis required the construction of knockout mutations in the recA gene. Antibiotic drug-resistance cassettes (DRC’s) were utilized to insertionally inactivate the wild-type B. pertussis recA gene cloned into a suicide vector. The mutant allele was mobilized by conjugal gene transfer from E. coli strain SM10 into different genetic backgrounds of B. pertussis. Southern hybridization studies showed that a selected mutant, of the RecA' phenotype, contained a DRC which had integrated exclusively within a recA gene containing Clal genomic DNA fragment. Selected mutants were assayed to quantify recombinational and DNA- repair defeciencies. These were shown to be highly sensitive to both chemically- induced and physically-induced DNA damage. Gene transfer studies also indicated that the RecA mutants were highly deficient at intergenic recombination. To further characterize recA and associated gene activity, a lexA homolog was cloned from a B. pertussis genomic library utilizing a "killer" vector system. When expressed in the heterologous host E. coli, the cloned gene was able to promote survival. This was presumed to occur by repression of the sulA gene, thus exhibiting LexA-like repressor functions. This gene was further subcloned, and isolated to a 1.6-Kb SaB fragment which retained its ability to promote survival within a sulA+, /ex4(Def) genetic background of E coli. The results of this investigation corroborate previous studies with the cloned B. pertussis recA gene, and demonstrate that activities of this gene in the original host promotes both DNA repair and recombination. The existence of a LexA repressor and the phenotypes of RecA mutants in this organism are consistent with SOS repair.

Comments

Includes bibliographical references (pages [42]-47)

Extent

iv, 47 pages

Language

eng

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

Media Type

Text

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