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UBC Theses and Dissertations
Identification of genomic alterations and altered expression profiles using novel baterial genome display techniques Malloff, Chad Alexander
Abstract
The efficacy of antimicrobial agents against common bacterial pathogens has been diminishing significantly in recent years. Numerous clinical isolates and lab strains with altered virulence and drug resistances have been identified. The identification of novel and acquired genes involved in pathogenesis will be imperative in the development of new drugs. Large scale genomic and expression profiling of pathogenic phenotypes will prove invaluable for gene identification. We have developed three whole genome-scanning techniques to aid in the identification of acquired and novel genes. First, two-dimensional bacterial genomic display (2DBGD) was developed, from current two-dimensional DNA electrophoresis (2DDE) techniques, for the high-throughput comparison of bacterial genomes. This technique was used to generate high-resolution displays that enable the direct comparison of > 800 genomic fragments simultaneously. 2DBGDs allowed the identification of large insertions, encoding for antibiotic resistance, in strains of the respiratory pathogens B. pertussis and P. aeruginosa, and a 7 nucleotide substitution in a 20 kbp plasmid. 2DBGDs are capable of detecting acquired DNA, however, only in very closely related strains. If used to compare more distantly related bacteria (different species) numerous small changes (ie. small deletions and point mutations), unrelated to the interesting phenotype, would encumber the comparison of 2DBGDs. For this reason a second method, bacterial comparative genomic hybridization (BCGH), was developed to directly compare the genomes of more distantly related bacteria to identify gain or loss of genomic DNA. The utility of BCGH was also evaluated by comparing two strains of the opportunistic pathogen P. aeruginosa. Detection of a single copy gene insertion responsible for gentamicin resistance validates this method. Hybridization of the BCGH template with cDNA probes generated from the comparison strains was also used to confirm the identity of the gentamicin resistance gene. Lastly we used a bacterial artificial chromosome (BAC) library of B. pertussis to generate a template of BAC fingerprints spanning 87.2% of the genome. This template was used for the comparison of cDNA populations that represent different virulence phases in B. pertussis. Known virulence genes were identified and numerous candidate genes demonstrated alternate expression. These novel genome wide scanning techniques will enable the identification of novel and acquired genes that may be used as targets for combating the reoccurrence of bacterial pathogens.
Item Metadata
Title |
Identification of genomic alterations and altered expression profiles using novel baterial genome display techniques
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
2002
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Description |
The efficacy of antimicrobial agents against common bacterial pathogens has been
diminishing significantly in recent years. Numerous clinical isolates and lab strains with
altered virulence and drug resistances have been identified. The identification of novel and
acquired genes involved in pathogenesis will be imperative in the development of new drugs.
Large scale genomic and expression profiling of pathogenic phenotypes will prove invaluable
for gene identification. We have developed three whole genome-scanning techniques to aid
in the identification of acquired and novel genes. First, two-dimensional bacterial genomic
display (2DBGD) was developed, from current two-dimensional DNA electrophoresis
(2DDE) techniques, for the high-throughput comparison of bacterial genomes. This
technique was used to generate high-resolution displays that enable the direct comparison of
> 800 genomic fragments simultaneously. 2DBGDs allowed the identification of large
insertions, encoding for antibiotic resistance, in strains of the respiratory pathogens B.
pertussis and P. aeruginosa, and a 7 nucleotide substitution in a 20 kbp plasmid. 2DBGDs
are capable of detecting acquired DNA, however, only in very closely related strains. If used
to compare more distantly related bacteria (different species) numerous small changes (ie.
small deletions and point mutations), unrelated to the interesting phenotype, would encumber
the comparison of 2DBGDs. For this reason a second method, bacterial comparative
genomic hybridization (BCGH), was developed to directly compare the genomes of more
distantly related bacteria to identify gain or loss of genomic DNA. The utility of BCGH was
also evaluated by comparing two strains of the opportunistic pathogen P. aeruginosa.
Detection of a single copy gene insertion responsible for gentamicin resistance validates this
method. Hybridization of the BCGH template with cDNA probes generated from the
comparison strains was also used to confirm the identity of the gentamicin resistance gene.
Lastly we used a bacterial artificial chromosome (BAC) library of B. pertussis to generate a
template of BAC fingerprints spanning 87.2% of the genome. This template was used for the
comparison of cDNA populations that represent different virulence phases in B. pertussis.
Known virulence genes were identified and numerous candidate genes demonstrated alternate
expression. These novel genome wide scanning techniques will enable the identification of
novel and acquired genes that may be used as targets for combating the reoccurrence of
bacterial pathogens.
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Extent |
11406470 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-08-17
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0090336
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
2002-05
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Item Media
Item Citations and Data
Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.