You limited your search to:

  Partner: UNT Libraries
 Degree Discipline: Molecular Biology
Comparative Mitochondrial DNA Sequence Diversity in Isolated and Open Populations of Southern Flying Squirrels

Comparative Mitochondrial DNA Sequence Diversity in Isolated and Open Populations of Southern Flying Squirrels

Access: Use of this item is restricted to the UNT Community.
Date: August 1999
Creator: Cook, Melaney Birdsong
Description: Three populations of Southern flying squirrels were studied in the Ouachita Mountains of Arkansas to assess the impact of population subdivision-due to island formation--on the population genetics of Glaucomys volans. One island, one mainland, and one open population were investigated. A 367 nucleotide hypervariable region of mitochondrial DNA was sequenced in individuals from each population. Individuals and populations were compared to assess relatedness. Higher sequence diversity was detected in the open and island populations. One island individual shared characters with both the island and mainland populations. Results support the hypothesis that the mainland population may have reduced gene flow. Also, the island population may have been originally founded by at least two maternal lineages.
Contributing Partner: UNT Libraries
Cassette Systems for Creating Intergeneric Hybrid ATCases

Cassette Systems for Creating Intergeneric Hybrid ATCases

Date: December 1999
Creator: Simpson, Luci N.
Description: Cassette systems for creating intergeneric hybrid ATCases were constructed. An MluI restriction enzyme site was introduced at the carbamoylphosphate binding site within the pyrB genes of both Pseudomonas putida and Escherichia coli. Two hybrids, E. coli pyrB polar domain fused with P. putida pyrB equatorial domain and P. putida pyrB polar domain fused with E. coli pyrB equatorial domain, are possible. The intergeneric E. coli-P. putida hybrid pyrB gene was constructed and found to encode an active ATCase which complemented an E. coli Pyr- strain. These hybrids are useful for kinetic and expression studies of ATCase in E. coli.
Contributing Partner: UNT Libraries
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

Access: Use of this item is restricted to the UNT Community.
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 ...
Contributing Partner: UNT Libraries
Mutation Rate Analysis of the Human Mitochondrial D-loop and its Implications for Forensic Identity Testing

Mutation Rate Analysis of the Human Mitochondrial D-loop and its Implications for Forensic Identity Testing

Date: May 2000
Creator: Warren, Joseph E.
Description: To further facilitate mitochondrial DNA (mtDNA) sequence analysis for human identity testing, a better understanding of its mutation rate is needed. Prior to the middle 1990's the mutation rate applied to a forensic or evolutionary analysis was determined by phylogenetic means, This method involved calculating genetic distances as determined by amino acid or DNA sequence variability within or between species. The mutation rate as determined by this method ranged from 0.025-0.26 nucleotide substitutions/ site/ myr (million years). With the recent advent of mtDNA analysis as a tool in human identity testing an increased number of observations have recently come to light calling into question the mutation rate derived from the phylogenetic method. The mutation rate as observed from forensic analysis appears to be much higher than that calculated phylogenetically. This is an area that needs to be resolved in human identity testing. Mutations that occur within a maternal lineage can lead to a possible false exclusion of an individual as belonging to that lineage. A greater understanding of the actual rate of mutation within a given maternal lineage can assist in determining criteria for including or excluding individuals as belonging to that lineage. The method used to assess the mutation ...
Contributing Partner: UNT Libraries
Isolation and analysis of cotton genomic clones encompassing a fatty acid desaturase (FAD2) gene

Isolation and analysis of cotton genomic clones encompassing a fatty acid desaturase (FAD2) gene

Date: May 2001
Creator: Kongcharoensuntorn, Wisatre
Description: Polyunsaturated fatty acids are major structural components of plant chloroplast and endoplasmic reticulum membranes. Two fatty acid desaturases (designated FAD2 and FAD3) desaturate 75% of the fatty acids in the endoplasmic reticulum. The w -6 fatty acid desaturase (FAD2) may be responsible for cold acclimation response, since polyunsaturated phospholipids are important in helping maintain plant viability at lowered temperatures. To study regulation of FAD2 gene expression in cotton, a FAD2 gene was isolated from two genomic libraries using an Arabidopsis FAD2 hybridization probe and a cotton FAD2 5¢ -flanking region gene-specific probe, respectively. A cotton FAD2 gene was found to be in two overlapping genomic clones by physical mapping and DNA sequencing. The cloned DNA fragments are identical in size to cotton FAD2 genomic DNA fragments shown by genomic blot hybridization. The cotton FAD2 coding region has 1,155 bp with no introns and would encode a putative polypeptide of 384 amino acids. The cotton FAD2 enzyme has a high identity of 75% with other plant FAD2 enzymes. The enzyme has three histidine-rich motifs that are conserved in all plant membrane desaturases. These histidine boxes may be the iron-binding domains for reduction of oxygen during desaturation. To confirm that this FAD2 ...
Contributing Partner: UNT Libraries
A Novel Mechanism for Site-Directed Mutagenesis of Large Catabolic Plasmids Using Natural Transformation

A Novel Mechanism for Site-Directed Mutagenesis of Large Catabolic Plasmids Using Natural Transformation

Date: August 2001
Creator: Williamson, Phillip C.
Description: Natural transformation is the process by which cells take up DNA from the surrounding medium under physiological conditions, altering the genotype in a heritable fashion. This occurs without chemical or physical treatment of the cells. Certain Acinetobacter strains exhibit a strong tendency to incorporate homologous DNA into their chromosomes by natural transformation. Transformation in Acinetobacter exhibits several unique properties that indicate this system's superiority as a model for transformation studies or studies which benefit from the use of transformation as an experimental method of gene manipulation. Pseudomonas putida is the natural host of TOL plasmids, ranging between 50 kbp and 300 kbp in size and encoding genes for the catabolism of toluene, meta-toluate, and xylene. These very large, single-copy plasmids are difficult to isolate, manipulate, or modify in vitro. In this study, the TOL plasmid pDKR1 was introduced into Acinetobacter calcoaceticus strains and genetically engineered utilizing natural transformation as part of the process. Following engineering by transformation, the recombinant DNA molecule was returned to the native genetic background of the original host P. putida strain. Specific parameters for the successful manipulation of large plasmids by natural transformation in Acinetobacter were identified and are outlined. The effects of growth phase, total ...
Contributing Partner: UNT Libraries
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 ...
Contributing Partner: UNT Libraries
A Possible Role of Ascorbate in Boron Deficient Radish (Raphanus sativa L. cv. Cherry Belle)

A Possible Role of Ascorbate in Boron Deficient Radish (Raphanus sativa L. cv. Cherry Belle)

Date: August 2001
Creator: Sedlacek, Theresa D.
Description: The most apparent symptom of boron deficiency in higher plants is a cessation of growth. Deficiency causes a reduction in ascorbate concentration and the absorption of nutrient ions. Addition of ascorbate temporarily relieves deficiency symptoms. In boron sufficient plants the addition of ascorbate to media causes an increased uptake of nutrients. In an attempt to discover if ascorbate addition to deficient plants causes increased ion uptake, radish plants were grown hydroponically in four different strengths of boron solution. A colorimetric assay for phosphorus was performed both before and after supplementation. Results, however, were inconclusive.
Contributing Partner: UNT Libraries
Purification of Aspartate Transcarbamoylase from  Moraxella (Branhamella) catarrhalis

Purification of Aspartate Transcarbamoylase from Moraxella (Branhamella) catarrhalis

Date: August 2001
Creator: Stawska, Agnieszka A.
Description: The enzyme, aspartate transcarbamoylase (ATCase) from Moraxella (Branhamella) catarrhalis, has been purified. The holoenzyme has a molecular mass of approximately 510kDa, harbors predominantly positive charges and is hydrophobic in nature. The holoenzyme possesses two subunits, a smaller one of 40 kDa and a larger one of 45 kDa. A third polypeptide has been found to contribute to the overall enzymatic activity, having an approximate mass of 55 kDa. The ATCase purification included the generation of cell-free extract, streptomycin sulfate cut, 60 °C heat step, ammonium sulfate cut, dialysis and ion, gel-filtration and hydrophobic interaction chromatography. The enzyme's performance throughout purification steps was analyzed on activity and SDS-PAGE gradient gels. Its enzymatic, specific activities, yield and fold purification, were also determined.
Contributing Partner: UNT Libraries
Characterization of  Moraxella bovis Aspartate Transcarbamoylase

Characterization of Moraxella bovis Aspartate Transcarbamoylase

Access: Use of this item is restricted to the UNT Community.
Date: December 2001
Creator: Hooshdaran, Sahar
Description: Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in the pyrimidine biosynthetic pathway. Bacterial ATCases have been divided into three classes, class A, B, and C, based on their molecular weight, holoenzyme architecture, and enzyme kinetics. Moraxella bovis is a fastidious organism, the etiologic agent of infectious bovine keratoconjunctivitis (IBK). The M. bovis ATCase was purified and characterized for the first time. It is a class A enzyme with a molecular mass of 480 to 520 kDa. It has a pH optimum of 9.5 and is stable at high temperatures. The ATCase holoenzyme is inhibited by CTP > ATP > UTP. The Km for aspartate is 1.8 mM and the Vmax 1.04 µmol per min, where the Km for carbamoylphosphate is 1.05 mM and the Vmax 1.74 µmol per min.
Contributing Partner: UNT Libraries
Molecular cloning and analysis of the genes for cotton palmitoyl-acyl carrier protein thioesterase (PATE) and Δ-12 fatty acid desaturase (FAD2-3) and construction of sense and anti-sense PATE plasmid vectors for altering oilseed composition of transgenic cotton plants.

Molecular cloning and analysis of the genes for cotton palmitoyl-acyl carrier protein thioesterase (PATE) and Δ-12 fatty acid desaturase (FAD2-3) and construction of sense and anti-sense PATE plasmid vectors for altering oilseed composition of transgenic cotton plants.

Date: May 2002
Creator: Nampaisansuk, Mongkol
Description: A cotton PATE cDNA clone has a 1.7-kb insert with an coding region for 410 amino acids, lacking codons for the three N-terminal amino acids. The predicted amino acid sequence of the PATE preprotein has a characteristic stromal-targeting domain and a 63% identity to the Arabidopsis FatB1 thioesterase sequence. A cotton genomic clone containing a 17.4-kb DNA segment was found to encompass a palmitoyl-ACP thioesterase (FatB1) gene. The gene spans 3.6 kb with six exons and five introns. The six exons are identical in nucleotide sequence to the open reading frame of the corresponding cDNA, and would encode a preprotein of 413 amino acids. The preprotein is identified as a FatB thioesterase from its deduced amino acid sequence similarity to those of other FatB thioesterase preproteins. A 5'-flanking region of 914 bp was sequenced, with the potential promoter/enhancer elements including basic helix-loop-helix elements (E box). Alkaline blot hybridization of cotton genomic DNA suggests the presence at least two FatB1 thioesterase genes in cotton. Four plasmid constructs for both constitutive and seed-specific anti-sense RNA suppression and gene-transgene co- suppression of PATE gene expression were successfully generated. Two overlapping cotton genomic clones were found to encompass a Δ-12 fatty acid desaturase (FAD2-3) ...
Contributing Partner: UNT Libraries
Dna Profiling of Captive Roseate Spoonbill (Ajaia Ajaja) Populations As a Mechanism of Determining Lineage in Colonial Nesting Birds.

Dna Profiling of Captive Roseate Spoonbill (Ajaia Ajaja) Populations As a Mechanism of Determining Lineage in Colonial Nesting Birds.

Date: May 2002
Creator: Sawyer, Gregory M.
Description: Roseate spoonbills are colonial nesting birds with breeding grounds extending from the United States Gulf coast to the pampas of Argentina. The U.S. population suffered a severe bottleneck from 1890 to 1920. The population's recovery was slow and partially credited to migrations from Mexican rookeries, but a gene pool reduction would be expected. Five polymorphic Spoonbill autosomal short tandem repeat (STR) loci [three (GAT)n, one (AAAG)n and one (GT)n] and one Z/W-linked microsatellite exhibiting sex-specific dimorphism were isolated and characterized. The Z/W-linked STR locus accurately confirmed the sex of each bird. Allelic profiles for 51 spoonbills obtained from Dallas (Texas), Fort Worth (Texas) and Sedgwick County (Kansas) zoos revealed a non-continuous distribution of allele frequencies, consistent with the effects of a population bottleneck. Allelic frequencies also differed significantly between the isolated zoo populations. Although extra-pair copulations were suspected and difficult to document, zoos commonly used observational studies of mating pairs to determine familial relationships among adults and offspring. STR parentage analysis of recorded family relationships excluded one or both parents in 10/25 cases studied and it was further possible to identify alternative likely parents in each case. Mistaken familial relationships quickly lead to the loss of genetic variability in captive ...
Contributing Partner: UNT Libraries
Characterization of cDNA and Genomic Clones for a Palmitoyl-acyl Carrier Protein Thioesterase and an Osmotin-Like PR5 Protein in Gossypium Hirsutum.

Characterization of cDNA and Genomic Clones for a Palmitoyl-acyl Carrier Protein Thioesterase and an Osmotin-Like PR5 Protein in Gossypium Hirsutum.

Access: Use of this item is restricted to the UNT Community.
Date: May 2002
Creator: Yoder, David W.
Description: Putative cotton cDNA clones and cognate genomic clones for a palmitoyl-acyl carrier protein (ACP) thioesterase (PATE) and an osmotin-like pathogenesis-related 5 (PR5) protein have been isolated and characterized. PATE is a class B fatty acid thioesterase with specificity for saturated long-chain fatty acids such as palmitate, and is implicated as a key enzyme to be targeted for regulation of fatty acid synthesis in order to alter cotton seed oil profiles. A nearly full-length 1.7-kb cDNA clone was isolated using a hybridization probe derived from an Arabidopsis PATE cDNA clone designated TE 3-2. A 17-kb genomic segment encompassing the PATE gene was also isolated, which has six exons and five introns with high sequence identity with other FatB cDNA/gene sequences. The deduced PATE preprotein amino acid sequence of 413 residues has putative signal sequences for targeting to the chloroplast stroma. PR5 proteins called osmotins are made in response to fungal pathogen stress or osmotic stress (water deprivation or salt exposure). Osmotins may actually form pores in fungal membranes, leading to osmotic rupture and destruction of the fungal cells. A cotton osmotin-like PR5 cDNA insert of 1,052 base-pairs was isolated and shown to encode a preprotein of 242 amino acids and is ...
Contributing Partner: UNT Libraries
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 ...
Contributing Partner: UNT Libraries
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 ...
Contributing Partner: UNT Libraries
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 ...
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
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
Structure-Function Studies on Aspartate Transcarbamoylase and Regulation of Pyrimidine Biosynthesis by a Positive Activator Protein, PyrR in Pseudomonas putida

Structure-Function Studies on Aspartate Transcarbamoylase and Regulation of Pyrimidine Biosynthesis by a Positive Activator Protein, PyrR in Pseudomonas putida

Access: Use of this item is restricted to the UNT Community.
Date: December 2003
Creator: Kumar, Alan P.
Description: The regulation of pyrimidine biosynthesis was studied in Pseudomonas putida. The biosynthetic and salvage pathways provide pyrimidine nucleotides for RNA, DNA, cell membrane and cell wall biosynthesis. Pyrimidine metabolism is intensely studied because many of its enzymes are targets for chemotheraphy. Four aspects of pyrimidine regulation are described in this dissertation. Chapter I compares the salvage pathways of Escherichia coli and P. putida. Surprisingly, P. putida lacks several salvage enzymes including nucleoside kinases, uridine phosphorylase and cytidine deaminase. Without a functional nucleoside kinase, it was impossible to feed exogenous uridine to P. putida. To obviate this problem, uridine kinase was transferred to P. putida from E. coli and shown to function in this heterologous host. Chapter II details the enzymology of Pseudomonas aspartate transcarbamoylase (ATCase), its allosteric regulation and how it is assembled. The E. coli ATCase is a dodecamer of two different polypeptides, encoded by pyrBI. Six regulatory (PyrI) and six catalytic (PyrB) polypeptides assemble from two preformed trimers (B3) and three preformed regulatory dimers (I2) in the conserved 2B3:3I2 molecular structure. The Pseudomonas ATCase also assembles from two different polypeptides encoded by pyrBC'. However, a PyrB polypeptide combines with a PyrC. polypeptide to form a PyrB:PyrC. protomer; six ...
Contributing Partner: UNT Libraries
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 ...
Contributing Partner: UNT Libraries
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 ...
Contributing Partner: UNT Libraries
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.
Contributing Partner: UNT Libraries
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, ...
Contributing Partner: UNT Libraries
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

Access: Use of this item is restricted to the UNT Community.
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 ...
Contributing Partner: UNT Libraries
FIRST PREV 1 2 3 4 NEXT LAST