UNT Theses and Dissertations - 34 Matching Results

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Expression of Granulocyte-Macrophage Colony-Stimulating Factor Gene in Insect Cells by a Baculovirus Vector
The focus of this research is to describe the production and characterization of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in insect cells, using Autographa californica buclear polyhedrosis virus (AcNPV) as an expression vector. All three forms of biological activity of hGM-CSF. Following N-glycanase treatment, the two glycosylated hGM-CSF proteins (15.5 and 16.5 KDa) which bound to Concanavalin A affinity column ran as a 14.5-15.5 KDa band on SDS-PAGE. Western blot analysis of expression in Sf9 cells treated with tunicamycin revealed only the presence of the 14.5 KDa species. The N-terminal amino acid sequence of the recombinant hGM-CSF was identical to that of natural hGM-CSF deduced from cDNA. These results demonstrate that baculovirus-produced hGM-CSF could be N-glycosylated in Sf9 cells, the signal peptide of recombinant hGM-CSF could be recognized and cleaved by infected insect cells and the resultant molecule secreted into the medium.
Electrophysiological and Morphological Analyses of Mouse Spinal Cord Mini-Cultures Grown on Multimicroelectrode Plates
The electrophysiological and morphological properties of small networks of mammalian neurons were investigated with mouse spinal cord monolayer cultures of 2 mm diameter grown on multimicroelectrode plates (MMEPs). Such cultures were viewed microscopically and their activity simultaneously recorded from 2 of any 36 fixed recording sites. The specific aims achieved were: development of techniques for production of functional MMEPs and maintenance of mini-cultures, characterization of the spontaneous activity of mini-cultures, application of inhibitory and disinhibitory agents, development of staining methods for cultured neurons and initial light microscopic analysis with correlation of electrophysiological and morphological characteristics.
Physical Characterization and Restriction Mapping of the Sal Plasmid From Pseudomonas Putida
Physical and restriction mapping of the salicylate catabolic plasmid SAL from Pseudomonas putida strain PpG 2119 was carried out by standard multiple restriction analysis and by cross hybridization studies using radioactively labeled restriction fragment probes. The total numbers of fragments produced, their respective sizes, the arrangement of the restriction fragments on the plasmid and the map locations of the enzyme recognition sites for Hpal, Xhol, Dral and Smal are given.
How Do Enzymes Wear Out? Effects of Posttranslational Modifications on Structure and Stability of Proteins; The Triosephosphate Isomerase Model
Triosephosphate isomerase (EC 5.3.1.1., TPI) undergoes specific posttranslational modifications (deamidation and oxidation) which are believed to initiate protein turnover by destabilization of the dimer. The crystal structures, amino acid sequences, and aging related changes of TPI from various species have been independently characterized by several laboratories. TPI has thus become the prototype enzyme for examining the initial steps in protein turnover. The binding of substrate enhances the specific deamidation of the mammalian enzyme, and a general mechanism of 'molecular wear and tear' [Gracy, R. W., Yiiksel, K. 0., Chapman, M. L., and Dimitrijevich, S. D. (1990) in Isozymes-Structure, Function and Use in Biology and Medicine (Ogita, Z-I., and Markert, C. L., Eds) pp. 787-817, Wiley-Liss, New York] has been proposed to explain how enzymes may wear out.
Investigating the Ability of Pseudomonas aeruginosa pyrE Mutants to Grow and Produce Virulence Factors
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 was reduced significantly in the mutant compared to the wild type. The casein protease assay showed absolutely no production of proteases in the mutant. The conclusion is that orotate accumulation leads to a significant reduction in virulence factor production in Pseudomonas aeruginosa. In addition to that, it was found that excess orotate in the wild type led to a decrease in quorum sensing regulated products.
Biochemical Systematics of the Genus Sophora
Three unusual amino acids, y-amino-n-butyric acid, pipecolic acid, and 4-hydroxypipecolic acid, and an uncommon dipeptide, y-glutamyltyrosine, have been isolated and characterized from the seeds of members of the genus Sophora. Structural proof of these compounds was carried out by paper chromatography, thin-layer chromatography, column chromatography on amino acid analyzer, infrared, nuclear magnetic resonance, mass spectrometry, and C, H, N analysis. The presence and absence of these compounds was used as a criterion for the classification of 23 species of the genus Sophora. A phylogenetic classification which seems to follow the morphological taxonomy of this genus was carried out on the basis of seeds that contained pipecolic acid, those which did not contain pipecolic acid, and plants which contained both pipecolic acid and 4-hydroxypipecolic acids. Another chemical classification was also introduced based on the presence and absence of y-amino-n-butyric acid and y-glutamyltyrosine.
A New LC Column for the Separation and the Quantitation of Nucleotides
A new column, Dionex AS4A, (polystyrenedivinylbenzene matrix) used for the separation of ribonucleotides and deoxyribonucleotides for the first time, and previously used for ion analysis was found superior to conventional silica columns because it separates ribonucleotides and deoxyribonucleotides. Resolution of dGTP was not possible with the Dionex column and CTP and GDP often co-eluted. Using conventional silica columns, monophosphates separated from diphosphates and diphosphates from triphosphates. Using the new Dionex column resolves all three simultaneously. The Dionex column resolved nucleotides with sharper peaks than silica columns, and the longer its retention time the better was the resolution. This Dionex column is stable, with 80 runs possible without cleaning while resolving ribonucleotides and deoxyribonucleotides to the picomole level.
Origin and Role of Factor Viia
Factor VII, the initiator of the extrinsic coagulation cascade, circulates in human plasma mainly in its zymogen form, Factor VII and in small amounts in its activated form, Factor VIIa. However, the mechanism of initial generation of Factor VIIa is not known despite intensive research using currently available model systems. Earlier findings suggested serine proteases Factor VII activating protease, and hepsin play a role in activating Factor VII, however, it has remained controversial. In this work I estimated the levels of Factor VIIa and Factor VII for the first time in adult zebrafish plasma and also reevaluated the role of the above two serine proteases in activating Factor VII in vivo using zebrafish as a model system. Knockdown of factor VII activating protease did not reduce Factor VIIa levels while hepsin knockdown reduced Factor VIIa levels. After identifying role of hepsin in Factor VII activation in zebrafish, I wanted to identify novel serine proteases playing a role in Factor VII activation. However, a large scale knockdown of all serine proteases in zebrafish genome using available knockdown techniques is prohibitively expensive. Hence, I developed an inexpensive gene knockdown method which was validated with IIb gene knockdown, and knockdown all serine proteases in zebrafish genome. On performing the genetic screen I identified 2 novel genes, hepatocytes growth factor like and prostasin involved in Factor VII activation.
In Situ Hybridization of 70 kD Heat Shock Protein mRNA in a Rat Model of Ethanol Self-Administration
Sucrose fading was used to initiate self-administration of ethanol on an FR4 schedule in male Fischer 344 rats. Rats showed low response rates for ethanol alone. After administration of liquid diet containing ethanol, ethanol intake increased over levels prior to administration of the liquid diet. In situ hybridization compared mRNA for the inducible or constitutive 70 kD heat shock proteins in ethanol and nonethanol rats. Both inducible and constitutive mRNAs were found in nonethanol and ethanol tissues. In peripheral organs, radiolableling was higher in ethanol tissue. In brain regions, nonethanol tissues showed higher radiolabeling.
Posttranslational Modification of Proteins by ADP-ribosylation
This work presents the development of a highly sensitive and selective chemical assay for mono(ADP-ribose) residues covalently bound to proteins in vivo. An extensive review of the literature is presented in the introduction of this work. The physiological.functions of mono(ADP-ribosyl)transferase activities associated with certain bacterial toxins (e.g., diphtheria, cholera and pertussis toxins) are well established. However, the roles of endogenous vertebrate transferases are unknown. The elucidation of the roles of these cellular transferases will likely require identification of the physiologically relevant target proteins. Toward this end, it will also be important to identify the types of (ADP-ribose)-protein linkages present in vivo. ADP-ribosylation reactions catalyzed by the different bacterial and vertebrate transferases are specific for different amino acid acceptors in vitro. However, the vertebrate transferases that have been characterized thus far are NAD:arginine mono(ADP-ribosyl)transferases. The work presented here describes the development of a chemical assay for the detection of in vivo modified, ADP-ribosylated proteins containing N-glycosylic linkages to arginine. The assay was applied to the analysis of ADP-ribose residues in adult rat liver. The strategy employed for detection of protein-bound ADP-ribose residues eliminated potential artifacts arising from trapped nucleotides (or their degradation products), since the acid-insoluble material was completely dissolved in a strongly denaturing solution and freed of non-covalently bound nucleotides prior to chemical release from proteins. Thus, the studies presented here demonstrate the unambiguous detection and quantification of protein-bound ADP-ribose residues in adult rat liver. "Arginine-linked" mono(ADP-ribose) residues (31.8 pmol/mg protein) were present in vivo at a level almost 400-fold higher than poly(ADP-ribose). A minor fraction (23%) of the ADP—ribose residues detected were bound via a second more labile linkage with chemical properties very similar to those described previously for carboxlylate esterlinked ADP-ribose. After fractionation of rat liver proteins by gel filtration HPLC, the major peak of "arginine-linked" ADP-ribose residues …
Subcloning and Nucleotide Sequence of the xylO/PUWCMA Region from the Pseudomonas putida TOL Plasmid pDK1
The TOL plasmids of Pseudomonas putida encode enzymes required for the oxidation of toluene and other related aromatic compounds. These genes are organized into two operons, the xylUWCMABN operon (upper), and the xylXYZLTEGFJQKIH operon (lower). Here we report the nucleotide sequence of a 7107 bp segment of the TOL pDK1 plasmid encoding the region just upstream of the "upper" operon through the genes encoding xylUWCMA. Sequence analysis, comparison of base-usage patterns, codon-usage patterns, and intergenic distances between genes help support the idea that the "upper" and "lower" operons have evolved independently in different genetic backgrounds and have only more recently been brought together in TOL and related catabolic plasmids.
Cloning of Carbonic Anhydrase from Cotton (Gossypium hirsutum L.)
Carbonic anhydrase is a ubiquitous zinc-metalloenzyme that catalyzes the interconversion of carbon dioxide and carbonate and has been found to play a wide range of roles in animals, plants and bacteria. Cotton genomic and cDNA libraries were screened for the plastidial isoform of carbonic anhydrase. The nucleotide sequences of two 1.2 Kb partial cDNA clones were determined. These clones exhibit high homology to carbonic anhydrases from other dicot plants and possess all the expected peptide motifs. For example, serine and threonine rich chloroplastic targeting peptide and conserved zinc binding residues are both present. These clones were utilized to isolate two carbonic anhydrase genes that were shown to encode different isoforms by PCR and RFLP analysis.
Nucleotide Sequence Determination, Subcloning, Expression and Characterization of the xy1LT Region of the Pseudomonas putida TOL Plasmid pDK1
The complete nucleotide sequence of the region encoding the DHCDH function of the pDK1 lower operon was determined. DNA analysis has shown the presence of two open reading frames, one gene consisting of 777 nucleotides encoding a polypeptide of 27.85 kDa and another gene of 303 nucleotides encoding a polypeptide of 11.13 kDa. The results of enzymatic expression studies suggest that DHCDH activity is associated only with xy1L. However although the addition of xy1T cell-free extracts to xy1L cell-free extracts does not produce an increase in DHCDH activity, subclones carrying both xy1L and xy1T exhibit 300- 400% more DHCDH activity than subclones carrying only xy1L.
Cell-Free Recovery and Isotopic Identification of Cyanide Degrading Enzymes from Pseudomonas Fluorescens
Cell-free extracts from Pseudomonas fluorescens NCIMB 11764 catalyzed the degradation of cyanide into products that included C02, formic acid, formamide and ammonia. Cyanide-degrading activity was localized to cytosolic cell fractions and was observed at substrate concentrations as high as 100 mM. Two cyanide degrading activities were identified by: (i) the determination of reaction products stoichiometries, (ii) requirements for NADH and oxygen, and (iii) kinetic analysis. The first activity produced CO2 and NH3 as reaction products, was dependent on oxygen and NADH for activity, and displayed an apparent Km for cyanide of 1.2 mM. The second activity generated formic acid (and NH3) pfus formamide as reaction products, was oxygen independent, and had an apparent Km of 12 mM for cyanide. The first enzymatic activity was identified as cyanide oxygenase whereas the second activity consists of two enzymes, a cyanide nitrilase (dihydratase) and putative cyanide hydratase. In addition to these enzymes, cyanide-grown cells were also induced for formate dehydrogenase (FDH), providing a means of recycling NADH utilized by cyanide oxygenase.
Changes in Gene Expression Levels of the Ecf Sigma Factor Bov1605 Under Ph Shift and Oxidative Stress in the Sheep Pathogen Brucella Ovis
Brucella ovis is a sexually transmitted, facultatively anaerobic, intracellular bacterial pathogen of sheep (Ovis aries) and red deer (Cervus elaphus). Brucella spp. infect primarily by penetrating the mucosa and are phagocytized by host macrophages, where survival and replication occurs. At least in some species, it has been shown that entry into stationary phase is necessary for successful infection. Brucella, like other alphaproteobacteria, lack the canonical stationary phase sigma factor ?s. Research on diverse members of this large phylogenetic group indicate the widespread presence of a conserved four-gene set including an alternative ECF sigma factor, an anti-sigma factor, a response regulator (RR), and a histidine kinase (HK). The first description of the system was made in Methylobacterium extorquens where the RR, named PhyR, was found to regulate the sigma factor activity by sequestering the anti-sigma factor in a process termed "sigma factor mimicry." These systems have been associated with various types of extracellular stress responses in a number of environmental bacteria. I hypothesized that homologous genetic sequences (Bov_1604-1607), which are similarly found among all Brucella species, may regulate survival functions during pathogenesis. To further explore the involvement of this system to conditions analogous to those occurring during infection, pure cultures of B. ovis cells were subjected to environments of pH (5 and 7) for 15, 30, and 45 minutes and oxidative (50mM H2O2) stress, or Spermine NONOate for 60 minutes. RNA was extracted and converted to cDNA andchanges in transcript levels of the sigma factor Bov1605 were measured using qPCR. Preliminary results indicate that under the exposure to Spermine NONOate there was little change in expression, but under oxidative stress expression of the sigma factor Bov1605 was 4.68-fold higher than that expressed under normal conditions. These results suggest that the sigma factor Bov1605 may be involved in oxidative stress defense during …
Purification of Cyanide-Degrading Nitrilase from Pseudomonas Fluorescens NCIMB 11764.
Cyanide is a well known toxicant that arises in the environment from both biological and industrial sources. Bacteria have evolved novel coping mechanisms for cyanide and function as principal agents in the biosphere for cyanide recycling. Some bacteria exhibit the unusual ability of growing on cyanide as the sole nitrogen source. One such organism is Pseudomonas fluorescens NCIMB 11764 (Pf11764) which employs a novel oxidative mechanism for detoxifying and assimilating cyanide. A unique complex of enzymes referred to as cyanide oxygenase (CNO) is responsible for this ability converting cyanide to ammonia which is then assimilated. Because one component of the four member CNO complex was previously shown to act on cyanide independent of the other members, its characterization was sought as a means of gaining a better understanding of the overall catalytic mechanism of the complex. Preliminary studies suggested that the enzyme belonged to a subset of nitrilase enzymes known as cyanide dihydratases (CynD), however, a cynD-like gene in Pf11764 could not be detected by PCR. Instead, a separate nitrilase (Nit) linked to cyanide metabolism was detected. The corresponding nit gene was shown to be one of a conserved set of nit genes traced to a unique cluster in bacteria known as Nit1C. To determine whether the previously described CynD enzyme was instead Nit, efforts were undertaken to isolate the enzyme. This was pursued by cloning and expressing the recombinant enzyme and by attempting to isolate the native enzyme. This thesis is concerned with the latter activity and describes the purification of a Nit-like cyanide-degrading nitrilase (NitCC) from Pf11764 to ~95% homogeneity. Purification was greatly facilitated by the discovery that fumaronitrile, as opposed to cyanide, was the preferred substrate for the enzyme (20 versus 1 U/mg protein, respectively). While cyanide was less effective as a substrate, the specificity for cyanide …
Multiple Activities of Aspartate Transcarbamoylase in Burkholderia cepacia: Requirement for an Active Dihydroorotase for Assembly into the Dodecameric Holoenzyme
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 and virulence factor production, motility tests and biofilm assays were conducted using pyrC- mutant. Even though no significant difference in growth rates was observed, there were significant differences between the wild type and mutant in the production of biofilm and virulence factors. This study will help us to understand the structure and regulation of ATCase holoenzyme with DHOase, and facilitate the use of B. cepacia as an applicable bio-tool. Additionally, we can potentially pursue more efficient drug targets for B. cepacia.
Photoactivatable Quantum Dots in Super-Resolution Microscopy of Muscle
Super-resolution 3D imaging was achieved using newly synthesized photoactivatable quantum dot (PAQ dot) probes. Quantum dots were modified with a novel quencher system to make them photoactivatable. The unique properties of these PAQ dots enable single-fluorophore localization in three dimensions using a confocal microscopy optical sectioning method. Myosin and tropomyosin of rabbit myofibrilar bundles were specifically labeled with the newly synthesized PAQ dot. A sufficient number of single quantum dots were photoactivated, localized and reduced to their centroid and then reconstructed to a super-resolution image. The acquired super-resolution image shows a lateral and an axial sub-diffraction resolution and demonstrates ultrafine striations with widths less than 70 nm that are not evident by conventional confocal microscopy. The striations appear to be related to nebulin thin filament binding protein. This newly developed imaging system is cutting edge for its high resolution and localization as well its simplicity and convenience.
Applications of Molecular Genetics to Human Identity.
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 and inhibitors may assist in providing sufficient genetic information from cases that might otherwise lie on the fringe of what is possible to obtain today.
Genetic and Cellular Analysis of Anoxia-Induced Cell Cycle Arrest in Caenorhabditis elegans
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. Finally, to identify the genetic mechanism(s) of anoxia-induced prophase arrest, I conducted extensive RNA interference (RNAi) screen of a subset of kinetochore and inner nuclear membrane genes. RNAi analysis has identified the novel role of 2 nucleoporins in anoxia-induced prophase arrest.
Influence of Cholesterol Import on Aspergillus fumigatus Growth and Antifungal Suscepibility
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 increased growth. However, sterol analysis of A. fumigatus cultured in the absence of inhibitors showed little or no change in ergosterol levels. This result suggested that the imported cholesterol was not being used as membrane sterol. However, in parallel experiments using Itraconazole™, an antifungal agent that attenuates sterol biosynthesis by inhibiting the sterol 14a-demethylase (ERG11), ergosterol levels decreased with increasing doses of inhibitor. Moreover, serum-containing medium partially rescued A. fumigatus from the effects of Itraconazole™, and a similar rescue effect was observed with serum-free media containing cholesterol. From the preceding results, it can be concluded that human serum enhances A. …
Physical Map between Marker 8O7 and 146O17 on the Medicago truncatula Linkage Group 1 that Contains the NIP Gene
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 here, gap in physical map between marker 164N9 and 146O17 was closed.
Isolation and Characterization of Polymorphic Loci from the Caribbean Flamingo (Phoenicopterus ruber ruber): New Tools for Wildlife Management
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 accepted, but rather socially monogamous or even promiscuous. Thus we have now been able to both characterize and demonstrate the utility of our polymorphic microsatellite loci. We hope these results will interest additional wildlife facilities in further parentage and behavioral studies that will collectively aid to improve monitoring and maintenance of genetic diversity, and as provide better insight into breeding habits of both wild and captive populations.
Impaired virulence factor production in a dihydroorotate dehydrogenase mutant (pyrD) of Pseudomonas aeruginosa.
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 cases these virulence factors were significantly decreased in the mutant. Even supplementing with uracil did not attain wild type levels. Starvation of the pyrimidine mutant for uracil caused increased specific activity of the pyrimidine enzymes, suggesting that regulation of the pyrimidine pathway occurred at the level of transcription. This effect has also been reported for P. oleovorans. The present research consolidates the idea that pyrimidine auxotrophs cause decreased pathogenicity in P. aeruginosa. Such a finding may open the search for chemotherapy targets in cystic fibrosis and burn victims where P. aeruginosa is an infecting agent.
Microsatellite-based genetic profiling for the management of wild and captive flamingo populations.
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 the amplicons derived from the four flamingo groups reveal a high level of sequence conservation at all loci. Although small sample numbers again limit the data for lesser flamingos and to some degree for the Chilean birds, the sequences of the two greater flamingo subspecies were identical and the number of nonconserved nucleotides appears to be higher for lesser/greater comparisons than for Chilean/greater comparisons. This is consistent with Chilean flamingos being a different species within the same genus as the greater flamingos, while lesser flamingos belong to a separate genus. Parentage analyses on suggested African greater flamingo family groups from …
Callus Development and Organogenesis in Cultured Explants of Cowpea (Vigna unguiculata (L.) Walp
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 induced from cowpea explants cultured on MS medium supplemented with dicamba and picloram. Embryogenic suspension cultures were initiated from callus induced on MS supplemented with 3.0 mg/L dicamba or picloram and conditions for maintenance of embryogenic suspension cultures were evaluated. Somatic embryos were formed in suspension cultures. Attempts to convert and germinate the somatic embryos resulted in the formation of callus or formation of appendages on the somatic embryos or in the death of the embryos. The appendages formed roots on prolonged culture. Further research is needed to determine appropriate optimal conditions for embryo conversion and germination and ultimately plant …
Purification and Characterization of Proteolytic Aspartate Transcarbamoylase (ATCase) from Burkholderia cepacia 25416 and Construction of a pyrB1 Knock-out Mutant
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, the 550 kDa holoenzyme and 140 kDa and 120 kDa trimers disappeared and were replaced with a previously unseen 480 kDa holoenzyme pyrB- strain. The results suggest that B. cepacia has two genes that encode ATCase. ATC1 is constitutive and ATC2 is expressed only in the absence of ATC1 activity. To check for the virulence of these two strains, a eukaryotic model virulence test was performed using Caenorhabditis elegans (C. elegans). The pyrB1+pyrB2+ (wild type) B cepacia killed the nematode but pyrB1-pyrB2+ B. cepacia had lost its virulence against C. elegans. This suggests that ATC1 (pyrB1) is involved in virulence …
Isolation of a Pseudomonas aeruginosa Aspartate Transcarbamoylase Mutant and the Investigation of Its Growth Characteristics, Pyrimidine Biosynthetic Enzyme Activities, and Virulence Factor Production
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.
Analysis of a Cotton Gene Cluster for the Antifungal Protein Osmotin
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 number of PR-proteins, occur in the 5'-flanking regions. The mature osmotin proteins accumulate in cotton plants treated with the inducers ethephon and hydrogen peroxide. Thus, the two cotton osmotin genes encode osmotin proteins. The coding regions of the two genes have been expressed and isolated as fusion polypeptides in a bacterial expression system. Binary constructs containing the open reading frames of the two osmotin genes under the control of the 35S CaMV promoter have been generated for eventual production of transgenic Arabidopsis and cotton plants for potential constitutive expression of the osmotin proteins for increased resistance against fungal pathogens.
Structure-Function Studies on Aspartate Transcarbamoylase and Regulation of Pyrimidine Biosynthesis by a Positive Activator Protein, PyrR in Pseudomonas putida
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 of these assemble into a dodecamer of structure 2B3:3C'2. pyrC' encodes an inactive dihydroorotase with pyrB and pyrC' overlapping by 4 bp. Chapter III explores how catabolite repression affects pyrimidine metabolism. The global catabolite repression control protein, Crc, has been shown to affect pyrimidine metabolism in a number of ways. This includes orotate transport for use as pyrimidine, carbon and nitrogen sources. Orotate is important because it interacts with PyrR in repressing the pyr genes. Chapter IV describes PyrR, the positive activator of the pyrimidine pathway. As with other positive activator proteins, when pyrimidine nucleotides are depleted, PyrR binds to …
Pyrimidine Genes in Pseudomonas Species
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.
Characterization of Moraxella bovis Aspartate Transcarbamoylase
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.
Structural Analysis of the TOL pDK1 xylGFJQK Region and Partial Characterization of the xylF and xylG Gene Products
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 usage patterns within and between operons, provides additional support for the idea that the upper and lower operons encoding enzymes of aromatic pathways have evolved independently of one another and that these operons have continued to exchange genetic material with homologous expression units through a series of recombination events. Recombinant plasmids were constructed for individual expression of each of the xylGFJQK genes. HMSD (XylG) and HMSH (XylF) were partially purified and characterized with respect to substrate specificity and kinetic mechanism. Evidence was obtained suggesting that the HMSD reaction occurs via a steady state ordered mechanism or a random mechanism where …
Cassette Systems for Creating Intergeneric Hybrid ATCases
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.
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