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  Partner: UNT Libraries
 Degree Discipline: Molecular Biology
 Collection: UNT Theses and Dissertations
Investigating the Ability of Pseudomonas Aeruginosa Pyre Mutants to Grow and Produce Virulence Factors

Investigating the Ability of Pseudomonas Aeruginosa Pyre Mutants to Grow and Produce Virulence Factors

Date: December 2014
Creator: Niazy, Abdurahman A
Description: Pseudomonas aeruginosa are medically important bacteria that are notorious for causing nosocomial infections. To gain more knowledge into understanding how this organism operates, it was decided to explore the pyrimidine biosynthetic pathway. Pyrimidine synthesis, being one half of the DNA structure, makes it a very important pathway to the organism’s survivability. With previous studies being done on various genes in the pathway, pyrE has not been studied to the fullest extent. To study the function of pyrE, a site directed mutagenesis was done to completely knock out pyrE, which encodes the protein orotate phosphoribosyl transferase that is responsible for converting orotate into orotate monophosphate (OMP). A mutation in this step leads to accumulation and secretion of orotate into the medium. Analyzing virulence factors produced by the mutant and comparing to the wild type, some intriguing features of the mutant were discovered. One of the findings was the over expression of virulence factors pyoverdin and pyocyanin. Pyocyanin over expression, based on the results of this study, is due to the accumulation of orotate while over production of pyoverdin is due to the accumulation of dihydroorotate. The other virulence factors studied were motility assays, exoproducts, and growth analysis. All virulence factor production ...
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Comparison of Aspartate Transcarbamoylase Activity Between Pseudomonas Aeruginosa Which Has One Chromosome and Burkholderia Cepacia Which Has Three Chromosomes

Comparison of Aspartate Transcarbamoylase Activity Between Pseudomonas Aeruginosa Which Has One Chromosome and Burkholderia Cepacia Which Has Three Chromosomes

Date: August 2012
Creator: Nusair, Arwa Y.
Description: The pyrimidine biosynthetic pathway is essential and similar in all bacteria. The pathway from Pseudomonas is regulated by nucleotides which bind to the upstream region of the pyrBC’ gene complex. Work in our lab mapped the genes and showed that the pyrB and pyrC’ were part of an overlap complex. The Pseudomonas aeruginosa has one circular chromosome. A former Pseudomonas now called Burkholderia cepacia is similar to P. aeruginosa except that it contains three circular chromosomes (CI, CII, CIII) and one large plasmid. The primary chromosome named CI contains the pyrBC’. To our knowledge there has been no report of the activity of ATCase in Pseudomonas and contrasted with that of Burkholderia. Here, we compare the activity of ATCase in P. aeruginosa and B .cepacia. Cells of both organisms were grown in Pseudomonas minimal medium and in Enriched medium. The ATCase was extracted and partially purified from each sample. It is hypothesized that the B. cepacia has greater activity for ATCase than do the Pseudomonas.
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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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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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The regulatory roles of PyrR and Crc in pyrimidine metabolism in  Pseudomonas aeruginosa

The regulatory roles of PyrR and Crc in pyrimidine metabolism in Pseudomonas aeruginosa

Date: August 2001
Creator: Patel, Monal V.
Description: The regulatory gene for pyrimidine biosynthesis has been identified and designated pyrR. The pyrR gene product was purified to homogeneity and found to have a monomeric molecular mass of 19 kDa. The pyrR gene is located directly upstream of the pyrBC' genes in the pyrRBC' operon. Insertional mutagenesis of pyrR led to a 50- 70% decrease in the expression of pyrBC', pyrD, pyrE and pyrF while pyrC was unchanged. This suggests that PyrR is a positive activator. The upstream regions of the pyrD, pyrE and pyrF genes contain a common conserved 9 bp sequence to which the purified PyrR protein is proposed to bind. This consensus sequence is absent in pyrC but is present, as an imperfect inverted repeat separated by 11 bp, within the promoter region of pyrR. Gel retardation assays using upstream DNA fragments proved PyrR binds to the DNA of pyrD, pyrE, pyrF as well as pyrR. This suggests that expression of pyrR is autoregulated; moreover, a stable stem-loop structure was determined in the pyrR promoter region such that the SD sequence and the translation start codon for pyrR is sequestered. β-galactosidase activity from transcriptional pyrR::lacZ fusion assays, showed a two-fold in increase when expressed in a ...
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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.

Access: Use of this item is restricted to the UNT Community.
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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Structural analysis of the TOL pDK1 xylGFJQK  region and partial characterization of the xylF and xylG gene products

Structural analysis of the TOL pDK1 xylGFJQK region and partial characterization of the xylF and xylG gene products

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Date: December 1999
Creator: Poulter, Melinda D.
Description: TOL plasmids encode enzymes responsible for utilization of toluene and related aromatic compounds by Pseudomonas putida, ultimately converting them to central metabolic intermediates. The nucleotide sequence for the 5.6 kb xylGFJQK region of the pDK1 TOL meta operon was determined. DNA sequence analysis revealed the presence of five open reading frames corresponding to xylG (1458 bp), xylF (846 bp), xylJ (783 bp), xylQ (936 bp) and xylK (1047 bp), encoding predicted protein products of 51.6, 31.3, 27.8, 32.8, and 36.6 kDa in size, respectively. The average G+C content of the xylLTEGFJQK region was 65.7%, somewhat higher than the 58.9% seen in the immediately upstream xylXYZ region and substantially more than the 50% G+C content reported for the upper TOL operon of this plasmid. Homology comparisons were made with genes and proteins of related catabolic plasmids. The dmpCDEFG and pWWO xylGFJQK regions exhibit consistently high levels of nucleotide and amino acid homology to pDK1 xylGFJQK throughout the entire region. In contrast, although the nucleotide sequence homology of the Acinetobacter atdCDE region to xylGFJ is high, the homology of atdFG to xylQK is markedly less. Such radical changes in homology between corresponding regions of different operons, combined with variable base and codon ...
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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

Access: Use of this item is restricted to the UNT Community.
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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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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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