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  Partner: UNT Libraries
 Degree Discipline: Molecular Biology
 Degree Level: Doctoral
 Collection: UNT Theses and Dissertations
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. ...
Contributing Partner: UNT Libraries
Construction of a  Pseudomonas aeruginosa Dihydroorotase Mutant and the Discovery of a Novel Link between Pyrimidine Biosynthetic Intermediates and the Ability to Produce Virulence Factors

Construction of a Pseudomonas aeruginosa Dihydroorotase Mutant and the Discovery of a Novel Link between Pyrimidine Biosynthetic Intermediates and the Ability to Produce Virulence Factors

Date: August 2003
Creator: Brichta, Dayna Michelle
Description: The ability to synthesize pyrimidine nucleotides is essential for most organisms. Pyrimidines are required for RNA and DNA synthesis, as well as cell wall synthesis and the metabolism of certain carbohydrates. Recent findings, however, indicate that the pyrimidine biosynthetic pathway and its intermediates maybe more important for bacterial metabolism than originally thought. Maksimova et al., 1994, reported that a P. putida M, pyrimidine auxotroph in the third step of the pathway, dihydroorotase (DHOase), failed to produce the siderophore pyoverdin. We created a PAO1 DHOase pyrimidine auxotroph to determine if this was also true for P. aeruginosa. Creation of this mutant was a two-step process, as P. aeruginosa has two pyrC genes (pyrC and pyrC2), both of which encode active DHOase enzymes. The pyrC gene was inactivated by gene replacement with a truncated form of the gene. Next, the pyrC2 gene was insertionally inactivated with the aacC1 gentamicin resistance gene, isolated from pCGMW. The resulting pyrimidine auxotroph produced significantly less pyoverdin than did the wild type. In addition, the mutant produced 40% less of the phenazine antibiotic, pyocyanin, than did the wild type. As both of these compounds have been reported to be vital to the virulence response of P. aeruginosa, ...
Contributing Partner: UNT Libraries
Zebrafish Von Willebrand Factor

Zebrafish Von Willebrand Factor

Date: August 2012
Creator: Carrillo, Maira M.
Description: In humans, von Willebrand factor (vWF) is a key component in hemostasis and acts as a 'cellular adhesive' by letting the circulating platelets bind to exposed subendothelium. It also acts as a carrier and stabilizer of factor VIII (FVIII). A dysfunction or reduction of vWF leads to von Willebrand disease (vWD), resulting in bleeding phenotype which affects 1% of the population. Currently there are a variety of animal models used for the study of vWF and vWD; however, they do not possess the advantages found in zebrafish. Therefore, we set out to establish zebrafish as a model for the investigation of vWF and vWD through the use of bioinformatics and various molecular techniques. Using bioinformatics we found that the vWF gene is located on chromosome 18, that the GPIb? protein sequence is conserved. Confirmation of vWF production was shown by means of immunostaining and by RT-PCR, in thrombocytes as well as in veins and arteries. Evidence of vWF involvement in hemostasis and thrombosis was shown using MO and VMO technology to produce a vWD like phenotype, resulting in an increase in TTO and TTA, as well as a reduction in FVIII when blood was tested using the kPTT assay, coinciding ...
Contributing Partner: UNT Libraries
Subcloning and Nucleotide Sequence of Two Positive Acting Regulatory Genes, xy1R and xy1S, from the Pseudomonas putida HS1 TOL Plasmid PDK1

Subcloning and Nucleotide Sequence of Two Positive Acting Regulatory Genes, xy1R and xy1S, from the Pseudomonas putida HS1 TOL Plasmid PDK1

Date: May 1992
Creator: Chang, Teh-Tsai
Description: TOL plasmids of Pseudomonas putida encode enzymes for the degradation of toluene and related aromatics. These genes are organized into two operons regulated by the Xy1R and Xy1S transcriptional activators. Previous analysis of the TOL pDK1 catechol-2,3-dioxygenase gene (xy1E) and a comparison of this gene to xy1E from the related TOL plasmid pWW0, revealed the existance of a substantial level of sequence homology (82%).
Contributing Partner: UNT Libraries
Cyanide Assimilation in  Pseudomonas fluorescens NCIMB 11764: Characterization of Cyanide Oxygenase as a Pterin-Dependent Multicomponent Enzyme Complex

Cyanide Assimilation in Pseudomonas fluorescens NCIMB 11764: Characterization of Cyanide Oxygenase as a Pterin-Dependent Multicomponent Enzyme Complex

Access: Use of this item is restricted to the UNT Community.
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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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. ...
Contributing Partner: UNT Libraries
Influence of Cholesterol Import on  Aspergillus fumigatus Growth and Antifungal Suscepibility

Influence of Cholesterol Import on Aspergillus fumigatus Growth and Antifungal Suscepibility

Access: Use of this item is restricted to the UNT Community.
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
Construction of a Cloning Vector Based upon a Rhizobium Plasmid Origin of Replication and its Application to Genetic Engineering of Rhizobium Strains

Construction of a Cloning Vector Based upon a Rhizobium Plasmid Origin of Replication and its Application to Genetic Engineering of Rhizobium Strains

Date: May 1992
Creator: Jeong, Pyengsoo
Description: Rhizobia are Gram-negative, rod-shaped, soil bacteria with the ability to fix atmospheric nitrogen into ammonia as symbiont bacteroids within nodules of leguminous plant roots. Here, resident Rhizobium plasmids were studied as possible sources of components for the construction of a cloning vector for Rhizobium species.
Contributing Partner: UNT Libraries
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 ...
Contributing Partner: UNT Libraries
Multiple Activities of Aspartate Transcarbamoylase in Burkholderia cepacia: Requirement for an Active Dihydroorotase for Assembly into the Dodecameric Holoenzyme

Multiple Activities of Aspartate Transcarbamoylase in Burkholderia cepacia: Requirement for an Active Dihydroorotase for Assembly into the Dodecameric Holoenzyme

Date: December 2010
Creator: Kim, Hyunju
Description: The aspartate transcarbamoylase (ATCase) was purified from Burkholderia cepacia 25416. In the course of purification, three different ATCase activities appeared namely dodecameric 550 kDa holoenzyme, and two trimeric ATCases of 140 kDa (consists of 47 kDa PyrB subunits) and 120 kDa (consists of 40 kDa PyrB subunits) each. The 120 kDa PyrB polypeptide arose by specific cleavage of the PyrB polypeptide between Ser74 and Val75 creating an active polypeptide short by 74 amino acids. Both the 40 and 47 kDa polypeptides produced active trimers. To compare the enzyme activity of these trimers, an effector assay using nucleotides was performed. The 140 kDa trimer showed inhibition while the 120 kDa polypeptide showed less inhibition. To verify the composition of the pyrBC holoenzyme complex, B. cepacia dihydroorotase (DHOase, subunit size of 45 kDa) was purified by the pMAL protein fusion and purification system and holoenzyme reconstruction was performed using purified ATCase and DHOase. Both the 140 kDa and the 120 kDa trimers could produce holoenzymes of 550 kDa and 510 kDa, respectively. The reconstructed ATCase holoenzyme from cleaved ATCase showed better reconstruction compared to that from uncleaved ATCase in the conventional ATCase activity gel assay. To characterize the relationship between pyrimidine pathway ...
Contributing Partner: UNT Libraries
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