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  Partner: UNT Libraries
 Decade: 2000-2009
 Degree Discipline: Molecular Biology
 Collection: UNT Theses and Dissertations
Development of a Real-time Pcr Assay for the Detection of Campylobacter Jejuni and Campylobacter Coli.

Development of a Real-time Pcr Assay for the Detection of Campylobacter Jejuni and Campylobacter Coli.

Date: May 2009
Creator: Lewis, Sally
Description: Campylobacter organisms are the most commonly reported bacterial causes of foodborne infection in the world, with Campylobacter jejuni and Campylobacter coli responsible for over 99% of reported infections. Traditionally, Campylobacter species detection is an arduous process, requiring a special incubation environment as well as specific growth media for an extended growth period. The development of a rapid and reliable diagnostic tool for the detection of Campylobacter species would be a valuable aid to the medical diagnostic decision process, especially to rule out Campylobacter infection during the enteric pre-surgical time period. Improved patient outcomes would result if this rapid assay could reduce the number of enteric surgeries. Assays performed during this dissertation project have demonstrated that both SYBR® green and hydrolysis probe assays targeting an 84 nucleotide portion of cadF, a fibronectin-binding gene of Campylobacter jejuni and Campylobacter coli, were able to detect from 101 to 108 copies of organism from stool specimens, did not detect nonspecific targets, and exhibited a coefficient of variation (CV) of 1.1% or less. Analytical validation of sensitivity, specificity and precision, successfully performed in these studies, warrants additional clinical validation of these assays.
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Linkage of a nitrilase-containing Nit1C gene cluster to cyanide utilization in Pseudomonas fluorescens NCIMB 11764.

Linkage of a nitrilase-containing Nit1C gene cluster to cyanide utilization in Pseudomonas fluorescens NCIMB 11764.

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Date: May 2009
Creator: Ghosh, Pallab
Description: Pseudomonas fluorescens NCIMB 11764 (Pf11764) is uniquely able to grow on the poison cyanide as its sole nitrogen source. It does so by converting cyanide oxidatively to carbon dioxide and ammonia, the latter being assimilated into cellular molecules. This requires a complex enzymatic machinery that includes nitrilase and oxygenase enzymes the nature of which are not well understood. In the course of a proteomics analysis aimed at achieving a better understanding of the proteins that may be required for cyanide degradation by Pf11764, an unknown protein of 17.8 kDa was detected in cells exposed to cyanide. Analysis of this protein by ESI-coupled mass spectrometry and bioinformatics searches gave evidence of strong homology with a protein (Hyp1) of unknown function (hypothetical) present in the bacterium Photorhabdus luminescens subsp. laumondii TTO1 (locus plu_1232). A search of available microbial genomes revealed a number of Hyp1 orthologs the genes of which are found in a conserved gene cluster known as Nit1C. Independent studies revealed that in addition to Hyp1, Pf11764 possesses a gene (nit) specifying a nitrilase enzyme whose closest homologue is a nitrilase found in Nit1C gene clusters (77% amino acid identity). DNA sequence analysis has further revealed that indeed, hyp1Pf11764 and nitPf11764 ...
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A regulatory role for N-acylethanolamine metabolism in Arabidopsis thaliana seeds and seedlings.

A regulatory role for N-acylethanolamine metabolism in Arabidopsis thaliana seeds and seedlings.

Date: May 2009
Creator: Teaster, Neal D.
Description: N-Acylethanolamines (NAEs) are bioactive acylamides that are present in a wide range of organisms. Because NAE levels in seeds decline during imbibition similar to ABA, a physiological role was predicted for these metabolites in Arabidopsis thaliana seed germination and seedling development. There is also a corresponding increase of AtFAAH (fatty acid amide hydrolase), transcript levels and activity, which metabolizes NAE to ethanolamine and free fatty acids. Based on whole genome microarray studies it was determined that a number of up-regulated genes that were responsive to NAE were also ABA responsive. NAE induced gene expression in these ABA responsive genes without elevating endogenous levels of ABA. It was also determined that many of these NAE/ABA responsive genes were associated with an ABA induced secondary growth arrest, including ABI3. ABI3 is a transcription factor that regulates the transition from embryo to seedling growth, the analysis of transcript levels in NAE treated seedlings revealed a dose dependent, inverse relationship between ABI3 transcript levels and growth, high ABI3 transcript levels were associated with growth inhibition. Similar to ABA, NAE negatively regulated seedling growth within a narrow window of early seedling establishment. When seedlings are exposed to NAE or ABA within the window of sensitivity, ...
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Applications of Molecular Genetics to Human Identity.

Applications of Molecular Genetics to Human Identity.

Date: December 2008
Creator: Turnbough, Meredith A.
Description: The primary objectives of this project were: 1. to develop improved methods for extraction of DNA from human skeletal remains, 2. to improve STR profiling success of low-copy DNA samples by employing whole genome amplification to amplify the total pool of DNA prior to STR analysis, and 3. to improve STR profiling success of damaged DNA templates by using DNA repair enzymes to reduce the number/severity of lesions that interfere with STR profiling. The data from this study support the following conclusions. Inhibitory compounds must be removed prior to enzymatic amplification; either during bone section pretreatment or by the DNA extraction method. Overall, bleach outperformed UV as a pretreatment and DNA extraction using silica outperformed microconcentration and organic extraction. DNA repair with PreCR™ A outperformed both whole genome amplification and repair with PreCR™ T6. Superior DNA extraction results were achieved using the A6 PMB columns (20 ml capacity column with 6 layers of type A glass fiber filter), and DNA repair with PreCR™ A led to an overall improvement in profile quality in most cases, although whole genome amplification was unsuccessful. Rapid, robust DNA isolation, successful amplification of loci from the sample-derived DNA pool, and an elimination of DNA damage ...
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Genetic and Cellular Analysis of Anoxia-Induced Cell Cycle Arrest in Caenorhabditis elegans

Genetic and Cellular Analysis of Anoxia-Induced Cell Cycle Arrest in Caenorhabditis elegans

Date: December 2008
Creator: Hajeri, Vinita A.
Description: The soil-nematode Caenorhabditis elegans survives oxygen deprivation (anoxia < 0.001 kPa of O2, 0% O2) by entering into a state of suspended animation during which cell cycle progression at interphase, prophase and metaphase stage of mitosis is arrested. I conducted cell biological characterization of embryos exposed to various anoxia exposure times, to demonstrate the requirement and functional role of spindle checkpoint gene san-1 during brief anoxia exposure. I conducted a synthetic lethal screen, which has identified genetic interactions between san-1, other spindle checkpoint genes, and the kinetochore gene hcp-1. Furthermore, I investigated the genetic and cellular mechanisms involved in anoxia-induced prophase arrest, a hallmark of which includes chromosomes docked at the nuclear membrane. First, I conducted in vivo analysis of embryos carried inside the uterus of an adult and exposed to anoxic conditions. These studies demonstrated that anoxia exposure prevents nuclear envelope breakdown (NEBD) in prophase blastomeres. Second, I exposed C. elegans embryos to other conditions of mitotic stress such as microtubule depolymerizing agent nocodazole and mitochondrial inhibitor sodium azide. Results demonstrate that NEBD and chromosome docking are independent of microtubule function. Additionally, unlike anoxia, exposure to sodium azide causes chromosome docking in prophase blastomeres but severely affects embryonic viability. ...
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Expression analysis of the fatty acid desaturase 2-4 and 2-3 genes from Gossypium hirsutum in transformed yeast cells and transgenic Arabidopsis plants.

Expression analysis of the fatty acid desaturase 2-4 and 2-3 genes from Gossypium hirsutum in transformed yeast cells and transgenic Arabidopsis plants.

Date: August 2008
Creator: Zhang, Daiyuan
Description: Fatty acid desaturase 2 (FAD2) enzymes are phosphatidylcholine desaturases occurring as integral membrane proteins in the endoplasmic reticulum membrane and convert monounsaturated oleic acid into polyunsaturated linoleic acid. The major objective of this research was to study the expression and function of two cotton FAD2 genes (the FAD2-3 and FAD2-4 genes) and their possible role in plant sensitivity to environmental stress, since plants may increase the polyunsaturated phospholipids in membranes under environmental stress events, such as low temperature and osmotic stress. Two FAD2 cDNA clones corresponding to the two FAD2 genes have been isolated from a cotton cDNA library, indicating both genes are truly expressed in cotton. Model yeast cells transformed with two cotton FAD2 genes were used to study the chilling sensitivity, ethanol tolerance, and growth rate of yeast cells. The expression patterns of the two FAD2 genes were analyzed by reverse transcription polymerase chain reactions (RT-PCR) and Western blot analyses in cotton plants under different treatment conditions. The coding regions of both FAD2 genes were inserted downstream from the CaMV 35S promoter in the pMDC gateway binary vector system. Five different FAD2/pMDC constructs were transformed into the Arabidopsis fad2 knockout mutant background, and multiple potential transgenic Arabidopsis plant ...
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Physical Map between Marker 8O7 and 146O17 on the Medicago truncatula Linkage Group 1 that Contains the NIP Gene

Physical Map between Marker 8O7 and 146O17 on the Medicago truncatula Linkage Group 1 that Contains the NIP Gene

Date: December 2007
Creator: Lee, Yi-Ching
Description: The Medicago truncatula NIP gene is located on M. truncatula Linkage Group 1. Informative recombinants showed crossovers that localize the NIP gene between markers 146O17 and 23C16D. Marker 164N9 co-segregates with the NIP gene, and the location of marker 164N9 is between markers 146O17 and 23C16D. Based upon data from the Medicago genome sequencing project, a subset of the model legume Medicago truncatula bacterial artificial chromosomes (BACs) were used to create a physical map on the DNA in this genetic internal. BACs near the potential NIP gene location near marker 164N9 were identified, and used in experiments to predict the physical map by a BAC-by-BAC strategy. Using marker 164N9 as a center point, and chromosome walking outward, the physical map toward markers 146O17 and 23C16D was built. The chromosome walk consisted of a virtual walk, made with existing sequence of BACs from the Medicago genome project, hybridizations to filters containing BAC DNA, and PCR reactions to confirm that predicted overlapping BACs contained DNA that yielded similar PCR products. In addition, the primers which are made for physical mapping via PCR could be good genetic markers helpful in discovering the location of the NIP gene. As a result of efforts repotted ...
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Evaluation of Zinc Toxicity Using Neuronal Networks on Microelectrode Arrays: Response Quantification and Entry Pathway Analysis

Evaluation of Zinc Toxicity Using Neuronal Networks on Microelectrode Arrays: Response Quantification and Entry Pathway Analysis

Date: August 2007
Creator: Parviz, Maryam
Description: Murine neuronal networks, derived from embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate zinc toxicity at concentrations ranging from 20 to 2000 mM total zinc acetate added to the culture medium. Continual multi-channel recording of spontaneous action potential generation allowed a quantitative analysis of the temporal evolution of network spike activity generation at specific zinc acetate concentrations. Cultures responded with immediate concentration-dependent excitation lasting from 5 to 50 min, consisting of increased spiking and enhanced, coordinated bursting. This was followed by irreversible activity decay. The time to 50% and 90% activity loss was concentration dependent, highly reproducible, and formed linear functions in log-log plots. Network activity loss generally preceded morphological changes. 20% cell swelling was correlated with 50% activity loss. Cultures pretreated with the GABAA receptor antagonists bicuculline (40 mM) and picrotoxin (1 mM) lacked the initial excitation phase. This suggests that zinc-induced excitation may be mediated by interfering with GABA inhibition. Partial network protection was achieved by stopping spontaneous activity with either tetrodotoxin (200 nM) or lidocaine (250 mM). However, recovery was not complete and slow deterioration of network activity continued over 6 hrs. Removal of zinc by early medium changes showed irreversible, catastrophic ...
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Analysis of the Expression Profiles of Two Isoforms of the Antifungal Protein Osmotin from Gossypium hirsutum

Analysis of the Expression Profiles of Two Isoforms of the Antifungal Protein Osmotin from Gossypium hirsutum

Date: May 2007
Creator: Spradling, Kimberly Diane
Description: The expression of two cotton osmotin genes was evaluated in terms of the mRNA and protein expression patterns in response to chemical inducers such as ethylene, hydrogen peroxide, and sodium chloride. Reverse transcriptase-polymerase chain reactions (RT-PCR) indicated that osmotin mRNAs are expressed constitutively in root tissues of cotton plants, and that they are rapidly induced in leaf and stem tissues upon ethylene treatment. Real time RT-PCR indicated that osmotin transcript levels were induced 2 to 4 h after treatment with ethephon. The osmotin mRNA levels appear to increase 12 h after treatment, decrease, and then increase again. The osmotin protein expression patterns were analyzed in Western blot analyses using an anti-osmotin antibody preparation. A 24-KDa protein band was detected from cotton plants treated with the inducers. The 24-KDa osmotin proteins were induced 4 h after treatment with ethephon, while down-regulated 96 h after treatment. Multiple osmotin isoforms were observed to be induced in cotton plants upon treatment with ethephon by two-dimensional gel electrophoresis. One goal of this dissertation research was to genetically engineer two cotton osmotin genes to routinely overproduce their antifungal proteins in transgenic Arabidopsis and cotton plants as a natural defense against fungal infections, using co-cultivation with Agrobacterium ...
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Map-based cloning of the NIP gene in model legume Medicago truncatula.

Map-based cloning of the NIP gene in model legume Medicago truncatula.

Date: May 2007
Creator: Morris, Viktoriya
Description: Large amounts of industrial fertilizers are used to maximize crop yields. Unfortunately, they are not completely consumed by plants; consequently, this leads to soil pollution and negative effects on aquatic systems. An alternative to industrial fertilizers can be found in legume plants that provide a nitrogen source that is not harmful for the environment. Legume plants, through their symbiosis with soil bacteria called rhizobia, are able to reduce atmospheric nitrogen into ammonia, a biological nitrogen source. Establishment of the symbiosis requires communication on the molecular level between the two symbionts, which leads to changes on the cellular level and ultimately results in nitrogen-fixing nodule development. Inside the nodules hypoxic environment, the bacterial enzyme nitrogenase reduces atmospheric nitrogen to ammonia. Medicago truncatula is the model legume plant that is used to study symbiosis with mycorrhiza and with the bacteria Sinorhizobium meliloti. The focus of this work is the M. truncatula nodulation mutant nip (numerous infections and polyphenolics). The NIP gene plays a role in the formation and differentiation of nodules, and development of lateral roots. Studying this mutant will contribute knowledge to understanding the plant response to infection and how the invasion by rhizobia is regulated. Previous genetic mapping placed NIP ...
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Identification and characterization of an incomplete root hair elongation (IRE)-like gene in Medicago truncatula (L.) root nodules.

Identification and characterization of an incomplete root hair elongation (IRE)-like gene in Medicago truncatula (L.) root nodules.

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Date: May 2006
Creator: Pislariu, Catalina Iulia
Description: Cloning and molecular characterization of new genes constitutes a useful approach in studying the symbiotic interactions between the model plant Medicago truncatula and Synorhizobium meliloti. Large numbers of expressed sequence tags (ESTs) available for Medicago truncatula, along with numerous cDNA, oligonucleotides, and Affimetrix DNA microarray chips, represent useful tools for gene discovery. In an attempt to identify a new gene that might be involved in the process of nodulation in Medicago truncatula, preliminary data reported by Fedorova et al. (2002), who identified 340 putative gene products or tentative consensus sequences (TCs) expressed only in nodules, was used. This research was focused on TC33166 (TC103185), which has 3 ESTs in the TC, and whose strongest BLASTX hit of TC103185 is the incomplete root hair elongation (IRE) protein kinase-like protein (NP_192429) from Arabidopsis thaliana. The Arabidopsis IRE gene is required for normal root hair growth, and a role in apical growth was suggested (Oyama et al., 2002). Infection thread growth can be looked at as an inward growth of the root hair. Thus, TC103185 was a good candidate for identifying a gene that may be involved in early events of nodulation. MtIRE (GenBank accession AC122727) is organized in 17 exons and 16 ...
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Regulation of pyrimidine biosynthesis and virulence factor production in wild type, Pyr- and Crc- mutants in Pseudomonas aeruginosa.

Regulation of pyrimidine biosynthesis and virulence factor production in wild type, Pyr- and Crc- mutants in Pseudomonas aeruginosa.

Date: May 2006
Creator: Asfour, Hani
Description: Previous research in our laboratory established that pyrB, pyrC or pyrD knock-out mutants in Pseudomonas aeruginosa required pyrimidines for growth. Each mutant was also discovered to be defective in the production of virulence factors. Moreover, the addition of exogenous uracil did not restore the mutant to wild type virulence levels. In an earlier study using non-pathogenic P. putida, mutants blocked in one of the first three enzymes of the pyrimidine pathway produced no pyoverdine pigment while mutants blocked in the fourth, fifth or sixth steps produced copious quantities of pigment, just like wild type P. putida. The present study explored the correlation between pyrimidine auxotrophy and pigment production in P. aeruginosa. Since the pigment pyoverdine is a siderophore it may also be considered a virulence factor. Other virulence factors tested included casein protease, elastase, hemolysin, swimming, swarming and twitching motilities, and iron binding capacity. In all cases, these virulence factors were significantly decreased in the pyrB, pyrC or pyrD mutants and even in the presence of uracil did not attain wild type levels. In order to complete this comprehensive study, pyrimidine mutants blocked in the fifth (pyrE) and sixth (pyrF) steps of the biosynthetic pathway were examined in P. aeruginosa. ...
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Impaired virulence factor production in a dihydroorotate dehydrogenase mutant (pyrD) of  Pseudomonas aeruginosa.

Impaired virulence factor production in a dihydroorotate dehydrogenase mutant (pyrD) of Pseudomonas aeruginosa.

Date: December 2005
Creator: Ralli, Pooja
Description: Previous research in our laboratory showed that when knockout mutations were created in the pyrB and pyrC genes of the pyrimidine pathway in Pseudomonas aeruginosa, not only were the resultant mutants auxotrophic for pyrimidines but they were also impaired in virulence factor production. Such a correlation had not been previously reported for P. aeruginosa, a ubiquitous opportunistic pathogen in humans. In an earlier study it was reported that mutants blocked in one of the first three enzymes of the pyrimidine pathway in the non-pathogenic strain P. putida M produced no pyoverdin pigment while mutants blocked in the later steps produced copious amounts of pigment, just like the wild type. This study probed for the same connection between pyrimidine auxotrophy and pigment production applied in P. aeruginosa. To that end a knockout mutation was created in pyrD, the fourth step in the pyrimidine pathway which encodes dihydroorotate dehydrogenase. The resulting mutant required pyrimidines for growth but produced wild type pigment levels. Since the pigment pyoverdin is a siderophore it may also be considered a virulence factor, other virulence factors were quantified in the mutant. These included casein protease, hemolysin, elastase, swimming, swarming and twitching motility, and iron binding capacity. In all ...
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Isolation and Characterization of Polymorphic Loci from the Caribbean Flamingo (Phoenicopterus ruber ruber): New Tools for Wildlife Management

Isolation and Characterization of Polymorphic Loci from the Caribbean Flamingo (Phoenicopterus ruber ruber): New Tools for Wildlife Management

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Date: December 2005
Creator: Preston, E. Lynn
Description: Methods to determine genetic diversity and relatedness within populations are essential tools for proper wildlife management. Today the approach of choice is polymerase chain reaction-based microsatellite analysis. Seven new polymorphic loci were isolated from a microsatellite-enriched Caribbean flamingo genomic library and used to characterize survey populations of Caribbean and African greater flamingos. In addition, four of these loci were used to verify parentage relationships within a captive-breeding population of African greater flamingos. Parentage predictions based upon gamekeeper observations of breeding and nesting did not always agree with genetic-based parentage analyses of the nine suggested family groups. Four family groups were supported (groups I, II, III and VI) by there results. However, an analysis of the remaining five suggested groups, with a total of eight offspring/dam and eight offspring/sire suggested relationships, yielded seven exclusions of the suggested dam and six exclusions of the suggested sire. This put the overall suggested dam exclusion rate at 35% and exclusion rate for suggested sires at 29%. Although the keeper observation data for our family groups must be considered a variable of concern at this time, these findings are certainly suggestive that more carefully controlled studies may reveal that flamingos are not monogamous as long ...
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Microsatellite-based genetic profiling for the management of wild and captive flamingo populations.

Microsatellite-based genetic profiling for the management of wild and captive flamingo populations.

Date: December 2005
Creator: Kapil, Richa
Description: Flamingo species generate tremendous interest whether they are small captive groups or wild populations numbering in the thousands. Genetic pedigrees are invaluable for maintaining maximum genetic diversity in captive, as well as wild, populations. However, presently there is a general lack of genetic data for flamingo populations. Microsatellites are loci composed of 2-6 base pair tandem repeats, scattered throughout higher eukaryotic genomes, often exhibiting high levels of polymorphism and heterozygosity. These loci are thus important genetic markers for identity, parentage and population studies. Here, six microsatellite loci were isolated from a microsatellite-enriched Caribbean flamingo partial genomic library. Two are compound complex repeats and four are perfect trinucleotide repeats. Each locus was amplified from Caribbean, African greater, Chilean and lesser flamingo genomic DNAs. Heterozygosity frequencies were calculated for Caribbean (range 0.12-0.90) and African greater flamingos (range 0.23-0.94) loci. All six microsatellite loci were found to be in Hardy-Weinberg equilibrium and linkage disequilibrium analyses did not suggest linkage for any pair of two greater flamingo subspecies (African and Caribbean) loci. At least five of the loci also exhibit polymorphism in Chilean and lesser flamingos, but due to small sample numbers, relevant allele/heterozygosity frequency calculations could not be estimated. Nucleotide sequence comparisons of ...
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Characterization of Infection Arrest Mutants of Medicago Truncatula and Genetic Mapping of Their Respective Genes.

Characterization of Infection Arrest Mutants of Medicago Truncatula and Genetic Mapping of Their Respective Genes.

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Date: May 2005
Creator: Veereshlingam, Harita
Description: In response to compatible rhizobia, leguminous plants develop unique plant organs, root nodules, in which rhizobia fix nitrogen into ammonia. During nodule invasion, the rhizobia gain access to newly divided cells, the nodule primordia, in the root inner cortex through plant-derived cellulose tubes called infection threads. Infection threads begin in curled root hairs and bring rhizobia into the root crossing several cell layers in the process. Ultimately the rhizobia are deposited within nodule primordium cells through a process resembling endocytosis. Plant host mechanisms underlying the formation and regulation of the invasion process are not understood. To identify and clone plant genes required for nodule invasion, recent efforts have focused on Medicago truncatula. In a collaborative effort the nodulation defect in the lin (lumpy infections) mutant was characterized. From an EMS-mutagenized population of M. truncatula, two non-allelic mutants nip (numerous infections with polyphenolics) and sli (sluggish infections) were identified with defects in nodule invasion. Infection threads were found to proliferate abnormally in the nip mutant nodules with only very rare deposition of rhizobia within plant host cells. nip nodules were found to accumulate polyphenolic compounds, indicative of a host defense response. Interestingly, nip was also found to have defective lateral root ...
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Callus Development and Organogenesis in Cultured Explants of Cowpea (Vigna unguiculata (L.) Walp

Callus Development and Organogenesis in Cultured Explants of Cowpea (Vigna unguiculata (L.) Walp

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Date: December 2004
Creator: Omwenga, George Isanda
Description: Cowpea, Vigna unguiculata (L.) Walp is an excellent source of protein, vitamins and minerals and a major food crop many parts of Africa. Optimal production levels are hampered by insect pests and diseases. Biotechnological techniques such as tissue culture and genetic engineering can aid in the development of varieties with resistance to insect pests and diseases. The objective of this study was to investigate conditions necessary for the development of a reproducible tissue culture system that can be applied to regenerate transformed cells from culture. The in vitro manipulation of cowpea using Murashige and Skoog (MS) medium, auxins and cytokinins resulted in the formation of callus and rhizogenesis. Calli that were formed were separated into six classes based on color and texture. Yellowish friable callus, yellowish compact, soft yellowish callus and green and white were composed of largely vacuolated cells and were non-regenerative. Friable green callus was the most prevalent callus type and could form of roots in some hormone combinations. Green spots were formed on hard compact green callus. The green spots became nodular, forming root primordia and ultimately giving rise to roots. None of the six calli types gave rise to the formation of shoots. Embryogenic callus was ...
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Isolation of a  Pseudomonas aeruginosa Aspartate Transcarbamoylase Mutant and the Investigation of Its Growth Characteristics, Pyrimidine Biosynthetic Enzyme Activities, and Virulence Factor Production

Isolation of a Pseudomonas aeruginosa Aspartate Transcarbamoylase Mutant and the Investigation of Its Growth Characteristics, Pyrimidine Biosynthetic Enzyme Activities, and Virulence Factor Production

Date: December 2004
Creator: Hammerstein, Heidi Carol
Description: The pyrimidine biosynthetic pathway is an essential pathway for most organisms. Previous research on the pyrimidine pathway in Pseudomonas aeruginosa (PAO1) has shown that a block in the third step of the pathway resulted in both a requirement for exogenous pyrimidines and decreased ability to produce virulence factors. In this work an organism with a mutation in the second step of the pathway, aspartate transcarbamoylase (ATCase), was created. Assays for pyrimidine intermediates, and virulence factors were performed. Results showed that the production of pigments, haemolysin, and rhamnolipids were significantly decreased from PAO1. Elastase and casein protease production were also moderately decreased. In the Caenorhabditis elegans infection model the nematodes fed the ATCase mutant had increased mortality, as compared to nematodes fed wild type bacteria. These findings lend support to the hypothesis that changes in the pyrimidine biosynthetic pathway contribute to the organism's ability to effect pathogenicity.
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Purification and Characterization of Proteolytic Aspartate Transcarbamoylase (ATCase) from  Burkholderia cepacia 25416 and Construction of a  pyrB1 Knock-out Mutant

Purification and Characterization of Proteolytic Aspartate Transcarbamoylase (ATCase) from Burkholderia cepacia 25416 and Construction of a pyrB1 Knock-out Mutant

Date: December 2004
Creator: Kim, Seongcheol
Description: Burkholderia cepacia is a common soil bacterium of significance in agriculture and bioremediation. B. cepacia is also an opportunistic pathogen of humans causing highly communicable pulmonary infections in cystic fibrosis and immunocompromized patients. The pyrB gene encoding ATCase was cloned and ATCase was purified by the glutathione S-transferase gene fusion system. The ATCase in B. cepacia has been previously classified as a class A enzyme by Bethell and Jones. ATCase activity gels showed that B. cepacia contained a holoenzyme pyrBC complex of 550 kDa comprised of 47 kDa pyrB and 45 kDa pyrC subunits. In the course of purifying the enzyme, trimeric subunits of 140 kDa and 120 kDa were observed as well as a unique proteolysis of the enzyme. The 47 kDa ATCase subunits were cleaved to 40 kDa proteins, which still demonstrated high activity as trimers. The proteolysis site is between Ser74 and Val75 residues. To confirm this, we converted the Ser74 residue to an Ala and to an Arg by site-directed mutagenesis. After this primary sequence changed, the proteolysis of ATCase was not observed. To further investigate the characteristics of B. cepacia pyrB gene, a pyrB knock-out (pyrB-) was constructed by in vitro mutagenesis. In the assay, ...
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Bacterial Cyanide Assimilation: Pterin Cofactor and Enzymatic Requirements for Substrate Oxidation

Bacterial Cyanide Assimilation: Pterin Cofactor and Enzymatic Requirements for Substrate Oxidation

Date: May 2004
Creator: Dolghih, Elena
Description: Utilization of cyanide as the sole nitrogen source by Pseudomonas fluorescens NCIMB 11764 (Pf11764) occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated oxygenolytically by an enzyme referred to as cyanide oxygenase (CNO), which exhibits properties of a pterin-dependent hydroxylase. The pterin requirement for Pf11764 CNO was satisfied by supplying either the fully (tetrahydro) or partially (dihydro) reduced forms of various pterin compounds at catalytic concentrations (0.5 µM). These compounds included, for example, biopterin, monapterin and neopterin, all of which were also identified in cell extracts. A related CNO-mediated mechanism of cyanide utilization was identified in cyanide-degrading P. putida BCN3. This conclusion was based on (i) the recovery of CO2 and NH3 as enzymatic reaction products, (ii) the dependency of substrate conversion on both O2 and NADH, and (iiii) utilization of cyanide, O2 and NADH in a 1:1:1 reaction stoichiometry. In contrast to findings reported for Pf11764, it was not possible to demonstrate a need for exogenously added pterin as a cofactor for the PpBCN3 enzyme system. However, results which showed that cells of PpBCN3 contained approximately seven times the amount of pterin as Pf11764 (of which a significant portion ...
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Cyanide Assimilation in Pseudomonas Fluorescens: Characterization of Cyanide Oxygenase as a Pterin-Dependent Multicomponent Enzyme Complex

Cyanide Assimilation in Pseudomonas Fluorescens: Characterization of Cyanide Oxygenase as a Pterin-Dependent Multicomponent Enzyme Complex

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Date: May 2004
Creator: Fernandez, Ruby
Description: Cyanide utilization in Pseudomonas fluorescens NCIMB 11764 occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated by an enzyme referred to as cyanide oxygenase (CNO), previously shown to require components in both high (H) (>30 kDa) and low (L) (<10 kDa) molecular weight cell fractions. In this study, tetrahydrobiopterin (H4biopterin) was identified as a cofactor in fraction L, thus making CNO appear as a pterin- dependent hydroxylase. CNO was purified 150-fold (specific activity 0.9 U/mg) and quantitatively converted cyanide to formate and ammonia as reaction products. When coupled with formate dehydrogenase, the complete enzymatic system for cyanide oxidation to carbon dioxide and ammonia was reconstituted. CNO was found to be an aggregate of known enzymes that included NADH oxidase (Nox), NADH peroxidase (Npx), cyanide dihydratase (CynD) and carbonic anhydrase (CA). A complex multi-step reaction mechanism is proposed in which Nox generates hydrogen peroxide which in turn is utilized by Npx to catalyze the oxygenation of cyanide to formamide accompanied by the consumption of one and two molar equivalents of oxygen and NADH, respectively. The further hydrolysis of formamide to ammonia and formate is thought to be mediated by CynD. The ...
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Analysis of a Cotton Gene Cluster for the Antifungal Protein Osmotin

Analysis of a Cotton Gene Cluster for the Antifungal Protein Osmotin

Date: December 2003
Creator: Wilkinson, Jeffery Roland
Description: Three overlapping genomic clones covering 29.0 kilobases of cotton DNA were found to encompass a cluster of two presumptive osmotin genes (OSMI and OSMII) and two osmotin pseudogenes (OSMIII and OSMIV). A segment of 16,007 basepairs of genomic DNA was sequenced from the overlapping genomic clones (GenBank Accessions AY303690 and AF304007). The two cotton osmotin genes were found to have open reading frames of 729 basepairs without any introns, and would encode presumptive osmotin preproteins of 242 amino acids. The open reading frames of the genes are identical in sequence to two corresponding cDNA clones (GenBank Accessions AF192271 and AY301283). The two cDNA inserts are almost full-length, since one lacks codons for the four N-terminal amino acids, and the other cDNA insert lacks the coding region for the 34 N-terminal amino acids. The cotton osmotin preproteins can be identified as PR5 proteins from their similarities to the deduced amino acid sequences of other plant osmotin PR5 preproteins. The preproteins would have N-terminal signal sequences of 24 amino acids, and the mature 24 kilodalton isoforms would likely be targeted for extracellular secretion. Prospective promoter elements, including two ethylene response elements, implicated as being positive regulatory elements in the expression of a ...
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Influence of Cholesterol Import on  Aspergillus fumigatus Growth and Antifungal Suscepibility

Influence of Cholesterol Import on Aspergillus fumigatus Growth and Antifungal Suscepibility

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Date: December 2003
Creator: Hassan, Saad A.
Description: Invasive pulmonary aspergillosis is a life-threatening fungal infection commonly observed in immunocompromised patients and has a mortality rate approaching 100% once the disease is disseminated. Aspergillus fumigatus is the most common pathogen. Early diagnosis improves the prognosis but is very difficult since most signs and symptoms are nonspecific. Antifungal therapy, usually based on sterol biosynthesis inhibitors, is also of limited efficacy. In my attempts to discover a diagnostic sterol marker for aspergillosis, I observed that A. fumigatus incorporates large amounts of cholesterol from serum-containing medium. This observation suggested the hypothesis that exogenous cholesterol from the host can be imported by A. fumigatus and used as a substitute for ergosterol in the cell membrane. This proposed mechanism would reduce the efficacy of antifungal drugs that act as sterol biosynthesis inhibitors. Experiments to test this hypothesis were designed to determine the effects of serum-free and serum-containing medium on growth of A. fumigatus in the presence and absence of azole antifungal agents. The results showed a marked increase in growth in the presence of human serum. Cultures in media containing cholesterol but no serum also showed enhanced growth, a result indicating that a non-cholesterol component of serum is not primarily responsible for the ...
Contributing Partner: UNT Libraries
Pyrimidine Genes in  Pseudomonas Species

Pyrimidine Genes in Pseudomonas Species

Date: December 2003
Creator: Roush, Wendy A.
Description: This thesis is a comparative study of gene arrangements in Pseudomonas species, and is organized into three major sections. The first section compares gene arrangements for different pathways in Pseudomonas aeruginosa PAO1 to determine if the gene arrangements are similar to previous studies. It also serves as a reference for pyrimidine gene arrangements in P. aeruginosa. The second part compares the physical, and genetic maps of P. aeruginosa PAO1 with the genome sequence. The final section compares pyrimidine gene arrangements in three species of Pseudomonas. Pyrimidine biosynthesis and salvage genes will be aligned for P. aeruginosa PAO1, P. putida KT2440, and P. syringae DC3000. The whole study will gives insight into gene patterns in Pseudomonas, with a focus on pyrimidine genes.
Contributing Partner: UNT Libraries
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