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Regulation, Evolution, and Properties of the ato Qperon and its Gene Products in Escherichia coli

Description: The regulation of short chain fatty acid metabolism has been examined. Metabolism of acetoacetate, and short chain fatty acids such as butyrate and valerate, is predicated upon the expression of genes of the ato operon. Acetoacetate induces expression of a CoA transferase (encoded by the atoDA genes) and expression of a thiolase (encoded by the atoB gene). Metabolism of saturated short chain fatty acids requires the activities of the transferase and thiolase and enzymes of 6-oxidation as well. Spontaneous mutant strains were isolated that were either constitutive or that were inducible by valerate or butyrate instead of acetoacetate.
Date: August 1993
Creator: Chen, Chaw-Yuan

Isolation and Characterization of the Operon Containing Aspartate Transcarbamoylase and Dihydroorotase from Pseudomonas aeruginosa

Description: The Pseudomonas aeruginosa ATCase was cloned and sequenced to determine the correct size, subunit composition and architecture of this pivotal enzyme in pyrimidine biosynthesis. During the course of this work, it was determined that the ATCase of Pseudomonas was not 360,000 Da but rather present in a complex of 484,000 Da consisting of two different polypeptides (36,000 Da and 44,000 Da) with an architecture similar to that of E. coli ATCase, 2(C3):3(r2). However, there was no regulatory polypeptide found in the Pseudomonas ATCase.
Date: May 1993
Creator: Vickrey, John F. (John Fredrick), 1959-

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

Description: TOL plasmids of Pseudomonas putida encode enzymes for the degradation of toluene and related aromatics. These genes are organized into two operons regulated by the Xy1R and Xy1S transcriptional activators. Previous analysis of the TOL pDK1 catechol-2,3-dioxygenase gene (xy1E) and a comparison of this gene to xy1E from the related TOL plasmid pWW0, revealed the existance of a substantial level of sequence homology (82%).
Date: May 1992
Creator: Chang, Teh-Tsai

Cloning of Carbonic Anhydrase from Cotton (Gossypium hirsutum L.)

Description: 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.
Date: December 1998
Creator: Local, Andrea

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

Description: Rhizobia are Gram-negative, rod-shaped, soil bacteria with the ability to fix atmospheric nitrogen into ammonia as symbiont bacteroids within nodules of leguminous plant roots. Here, resident Rhizobium plasmids were studied as possible sources of components for the construction of a cloning vector for Rhizobium species.
Date: May 1992
Creator: Jeong, Pyengsoo

Nucleotide Sequence Determination, Subcloning, Expression and Characterization of the xy1LT Region of the Pseudomonas putida TOL Plasmid pDK1

Description: 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.
Date: December 1992
Creator: Baker, Ronald F. (Ronald Fredrick)

Cassette Systems for Creating Intergeneric Hybrid ATCases

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.
Date: December 1999
Creator: Simpson, Luci N.

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

Description: Cyanide utilization in Pseudomonas fluorescens NCIMB 11764 occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated by an enzyme referred to as cyanide oxygenase (CNO), previously shown to require components in both high (H) (>30 kDa) and low (L) (<10 kDa) molecular weight cell fractions. In this study, tetrahydrobiopterin (H4biopterin) was identified as a cofactor in fraction L, thus making CNO appear as a pterin- dependent hydroxylase. CNO was purified 150-fold (specific activity 0.9 U/mg) and quantitatively converted cyanide to formate and ammonia as reaction products. When coupled with formate dehydrogenase, the complete enzymatic system for cyanide oxidation to carbon dioxide and ammonia was reconstituted. CNO was found to be an aggregate of known enzymes that included NADH oxidase (Nox), NADH peroxidase (Npx), cyanide dihydratase (CynD) and carbonic anhydrase (CA). A complex multi-step reaction mechanism is proposed in which Nox generates hydrogen peroxide which in turn is utilized by Npx to catalyze the oxygenation of cyanide to formamide accompanied by the consumption of one and two molar equivalents of oxygen and NADH, respectively. The further hydrolysis of formamide to ammonia and formate is thought to be mediated by CynD. The role of H4biopterin and of the enzyme CA in the proposed process remains unclear, but the involvement of each in reactive oxygen and radical chemistry is consistent with the proposed formation of such species in the catalytic process. H4biopterin may additionally serve as a protein stabilizing agent along with a protein co-purifying with CynD identified as elongation factor Tu, a known chaperone. At least two of the CNO components (Nox and CynD) are complex oligomeric proteins whose apparent association with Npx and CA appears to be favored in bacterial cells induced with cyanide allowing their purification in toto as a ...
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Date: May 2004
Creator: Fernandez, Ruby

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

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

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

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 populations. Here, a decreased heterozygosity (HO) in 2nd generation captive-bred birds was observed at 3 out of 4 loci evaluated. Although these results could not be statistically validated because of the small number of individuals available for study (15 wild birds with no offspring vs. eight 2nd generation captive birds), they are considered biologically important, as decreased HO is an indicator of inbreeding and this apparent decrease occurred within two generations of removal from the wild. Collectively, the evidence obtained from this study suggests that captive spoonbill populations are experiencing rapid loss of diversity from an already depleted wild gene ...
Date: May 2002
Creator: Sawyer, Gregory M.

Genetic Characterization of Central and South American Populations of Scarlet Macaw (Ara macao)

Description: The wild populations of the Scarlet Macaw subspecies native to southern Mexico and Central America, A. m. cyanoptera, have been drastically reduced over the last half century and are now a major concern to local governments and conservation groups. Programs to rebuild these local populations using captive bred specimens must be careful to reintroduce the native A. m. cyanoptera, as opposed to the South American nominate subspecies (A. m. macao) or hybrids of the two subspecies. Molecular markers for comparative genomic analyses are needed for definitive differentiation. Here I describe the isolation and sequence analysis of multiple loci from 7 pedigreed A. m. macao and 14 pedigreed A. m. cyanoptera specimens. The loci analyzed include the 18S rDNA genes, the complete mitogenome as well as intronic regions of selected autosomally-encoded genes. Although the multicopy18S gene sequences exhibited 10% polymorphism within all A. macao genomes, no differences were observed between any of the 21 birds whose genomes were studied. In contrast, numerous polymorphic sites were observed throughout the 16,993 bp mitochondrial genomes of both subspecies. Although much of the polymorphism was observed in the genomes of both subspecies, subspecies-specific alleles were observed at a number of mitochondrial loci, including 12S, 16S, CO2 and ND3. Evidence of possible subspecies-specific alleles were also found in three of four screened nuclear loci. Collectively, these mitochondrial and nuclear loci can be used as the basis to distinguish A. m. cyanoptera from the nominate subspecies, A. m. macao, as well as identify many hybrids, and most importantly will contribute to further reintroduction efforts.
Date: May 2016
Creator: Kim, Tracy

A New LC Column for the Separation and the Quantitation of Nucleotides

Description: 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.
Date: December 1987
Creator: Brock, Patricia C. (Patricia Charlene)

Regulation of Colony-Stimulating Factor-1 Biosynthesis

Description: Recent studies suggest that synthesis of the Colony-stimulating factor (CSF) is a well regulated process. However, the molecular mechanisms of the signal transduction of the various inducers of CSF such as monokines and lymphokines are not well understood. Using Interleukin 1 (IL-1) stimulation of CSF-1 in the MIA PaCa-2 cell line as a model system, the involvement of G-protein has been studied. The IL-1 induction of CSF-1 synthesis can be inhibited by both Pertussis toxin and Cholera toxin, which are known to modify the Gᵢ and Gₛ proteins respectively, thus activating adenylate cyclase to release more cAMP. The toxin inactivation can be prevented by inhibitors of the ADP-ribosylation such as, benzamide and MBAMG. Addition of dibutyryl-cAMP inhibits the IL-1 induced CSF production. Both Theophylline and Forskolin which increase cAMP by inhibiting phosphodiesterase and stimulating adenylate cyclase respectively, also inhibit CSF-1 production. Results from these studies have shown that cAMP level inversely regulates the biosynthesis of CSF-1. Preincubation of MIA PaCa-2 cells with IL-1 and 5'- guanylylimidodiphosphate (GppNHp) prevents the inhibitory effect of pertussis toxin on CSF-1 production. These data are consistent with the hypothesis that IL-1 binds to its receptor and couples to Gᵢ∝ resulting in the inhibition of adenylate cyclase and reducing cAMP level. Lowering of the' cAMP level leads to the activation of CSF-1 gene expression. The activity of another inducer of CSF-1 production in this system, 12-0-tetradecanoylphorbol-13-acetate (TPA), can be abolished by 1- (5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), which is a specific inhibitor of protein kinase C. However, H-7 failed to inhibit IL-1 stimulated CSF-1 production. Other known activators of protein kinase C namely, Ca²⁺ and L-α-l-oleoyl-2-acetoyl-sn- 3-glycerol (OAG), also increase CSF production. On the other hand, Indomethacin which is known to inhibit prostaglandin E (PGE), stimulates CSF-1 production in MIA PaCa-2 cells. These data suggest that different mechanisms ...
Date: May 1990
Creator: Ku, Chun-Ying

Structural Analysis of the TOL pDK1 xylGFJQK Region and Partial Characterization of the xylF and xylG Gene Products

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 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 ...
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Date: December 1999
Creator: Poulter, Melinda D.

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

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 network failure to develop in a concentration-dependent time window between 50% and 90% activity loss. Investigation of entry routes suggested the L-type but not N-type calcium channels to be the main entry pathway for zinc. Data are presented implicating the chloride channel to be an additional entry route.
Date: August 2007
Creator: Parviz, Maryam

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

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 rate in this study was to compare mtDNA sequences derived from the HVI and HVII regions of the D-loop from several different maternal lineages. The sequence information was derived from five unrelated families consisting of thirty-five individuals. One intergenerational mutational event was found. This derives to approximately 1.9 nucleotide substitutions/ site/ myr. This mutation rate was very consistent with several other similar studies. This increased mutation rate needs to be considered by forensic testing laboratories performing mtDNA sequence analysis prior to formulating any conclusive results.
Date: May 2000
Creator: Warren, Joseph E.

Mutagenized HLA DNA Constructs: Tools for Validating Molecular HLA Typing Methodologies

Description: This study describes the development and validation of mutagenized cloned DNA constructs, which correspond to the polymorphic regions of the class II region of the HLA complex. The constructs were used to verify the allelic specificity of primers and probes in polymerase chain reaction (PCR)-based HLA typing assays such as Sequence Specific Primers (SSP) and Sequence Specific Oligonucleotide Probes (SSOP). The constructs consisted of the entire polymorphic region of exon 2 of class II HLA allele sequences that included primer annealing sites or probe hybridization sites. An HLA allele sequence was inserted into a plasmid, cloned, then mutagenized to match a specific HLA allele, and finally, the correct clone was verified by bidirectional sequencing of the insert. Thus, the construct created a cloned reference DNA sample for any specific allele, and can be used to validate the accuracy of various molecular methodologies.
Date: May 1999
Creator: Schulte, Kathleen Q.

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

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.
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Date: August 1999
Creator: Cook, Melaney Birdsong

A Computer Assisted Micro-Dye Uptake Interferon Assay System

Description: A new rapid computer assisted micro-titer plate interferon assay system was developed and characterized for use in high capacity clinical and research applications. The biological aspect of the assay was a modification of the assay methods of Finter, Armstrong and McManus. It was an application of spectrophotometric quantification of the reduction of viral cytopathic effect (CPE) as reflected by neutral red dye uptake by viable cells. A computer program was developed for the extrapolation of raw data to reference interferon units.
Date: August 1981
Creator: Duvall, John C.

Biochemical Systematics of the Genus Sophora

Description: 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.
Date: December 1973
Creator: Izaddoost, Mohamed

Stress Response by Alternative σ-factor, RpoH, and Analysis of Posttranslational Modification of the Heat Shock Protein, Dnak, in Escherichia coli

Description: Bacteria have developed specialized responses that involve the expression of particular genes present in a given regulon. Sigma factors provide regulatory mechanisms to respond to stress by acting as transcriptional initiation factors. This work focuses on σ32 during oxidative stress in Escherichia coli. The differential response of key heat shock (HS) genes was investigated during HS and oxidative stress using qPCR techniques. While groEL and dnaJ experienced increases in transcriptional response to H2O2 (10 mM), HS (42°C), and paraquat (50 mM) exposure, the abundance of dnaK over the co-chaperones was apparent. It was hypothesized that DnaK undergoes oxidative modification by reactive carbonyls at its Lys-rich C-terminus, accounting for the differential response during oxidative stress. A σ32-mediated β-galactosidase reporter was devised to detect the activity of wild-type DnaK and DnaKV634X modified to lack the Lys-rich C-terminus. Under unstressed conditions and HS, σ32 was bound at the same rate in both strains. When subjected to H2O2, the WT DnaK strain produced significantly higher β-galactosidase than DnaKV634X (one-tailed Student’s t test p=0.000002, α=0.05) and approached the same level of output as the lacZ positive control. The β-galactosidase assay indicates that DnaK undergoes Lys modification in the WT strain, preventing the protein from binding σ32, increasing the activity of σ32, and resulting in higher β-galactosidase activity than the DnaKV634X strain. In the DnaKV634X strain DnaK continues to bind σ32 so that σ32 could not promote the production of β-galactosidase. These findings demonstrate how DnaK is oxidatively modified, hindering the interaction with σ32 in manner distinct from HS.
Date: May 2015
Creator: Martinez, Sarah N.

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

Description: Pseudomonas fluorescens NCIMB 11764 (Pf11764) is uniquely able to grow on the poison cyanide as its sole nitrogen source. It does so by converting cyanide oxidatively to carbon dioxide and ammonia, the latter being assimilated into cellular molecules. This requires a complex enzymatic machinery that includes nitrilase and oxygenase enzymes the nature of which are not well understood. In the course of a proteomics analysis aimed at achieving a better understanding of the proteins that may be required for cyanide degradation by Pf11764, an unknown protein of 17.8 kDa was detected in cells exposed to cyanide. Analysis of this protein by ESI-coupled mass spectrometry and bioinformatics searches gave evidence of strong homology with a protein (Hyp1) of unknown function (hypothetical) present in the bacterium Photorhabdus luminescens subsp. laumondii TTO1 (locus plu_1232). A search of available microbial genomes revealed a number of Hyp1 orthologs the genes of which are found in a conserved gene cluster known as Nit1C. Independent studies revealed that in addition to Hyp1, Pf11764 possesses a gene (nit) specifying a nitrilase enzyme whose closest homologue is a nitrilase found in Nit1C gene clusters (77% amino acid identity). DNA sequence analysis has further revealed that indeed, hyp1Pf11764 and nitPf11764 are contained in a cluster that includes also a gene specifying an oxygenase. Given the possible connection of Nit1C-endoded nitrilase and oxygenase enzymes to enzymatic cyanide degradation, there is strong reason for thinking that the genes specifying these enzymes contribute to bacterial growth on cyanide in those bacteria containing the Nit1C cluster. Because the biological function of the Hyp1 protein is currently unknown, it was cloned and the protein expressed in E. coli so that its properties could further be explored. Unfortunately, the expression of the protein in an insoluble form complicated these analyses. However, at least two lines of ...
Date: May 2009
Creator: Ghosh, Pallab

9-Lipoxygenase Oxylipin Pathway in Plant Response to Biotic Stress

Description: The activity of plant 9-lipoxygenases (LOXs) influences the outcome of Arabidopsis thaliana interaction with pathogen and insects. Evidence provided here indicates that in Arabidopsis, 9-LOXs facilitate infestation by Myzus persicae, commonly known as the green peach aphid (GPA), a sap-sucking insect, and infection by the fungal pathogen Fusarium graminearum. in comparison to the wild-type plant, lox5 mutants, which are deficient in a 9-lipoxygenase, GPA population was smaller and the insect spent less time feeding from sieve elements and xylem, thus resulting in reduced water content and fecundity of GPA. LOX5 expression is induced rapidly in roots of GPA-infested plants. This increase in LOX5 expression is paralleled by an increase in LOX5-synthesized oxylipins in the root and petiole exudates of GPA-infested plants. Micrografting experiments demonstrated that GPA population size was smaller on plants in which the roots were of the lox5 mutant genotype. Exogenous treatment of lox5 mutant roots with 9-hydroxyoctadecanoic acid restored water content and population size of GPA on lox5 mutants. Together, these results suggest that LOX5 genotype in roots is critical for facilitating insect infestation of Arabidopsis. in Arabidopsis, 9-LOX function is also required for facilitating infection by F. graminearum, which is a leading cause of Fusarium head blight (FHB) disease in wheat and other small grain crops. Loss of LOX1 and LOX5 function resulted in enhanced resistance to F. graminearum infection. Similarly in wheat, RNA interference mediated silencing of the 9-LOX homolog TaLpx1, resulted in enhanced resistance to F. graminearum. Experiments in Arabidopsis indicate that 9-LOXs promote susceptibility to this fungus by suppressing the activation of salicylic acid-mediated defense responses that are important for basal resistance to this fungus. the lox1 and lox5 mutants were also compromised for systemic acquired resistance (SAR), an inducible defense mechanism that is systemically activated throughout a plant in response to a ...
Date: May 2012
Creator: Nalam, Vamsi J.

Purification of Cyanide-Degrading Nitrilase from Pseudomonas Fluorescens NCIMB 11764.

Description: 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 ...
Date: December 2010
Creator: Chou, Chia-Ni