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Effects of a Methylcholanthrene-Induced Lymphosarcoma on the Blood of DBA/1J Mice

Description: This investigation was concerned with characterizing a tumor line induced and maintained in this laboratory. Various chemical assays, cell counts, and electron microscopy were the methods employed to characterize the blood of mice bearing the tumor at days 3, 6, 9, and 12 after injection of the 1.2 x 10^8 tumor cells.
Date: May 1972
Creator: Lindsey, Jerri Kay

Effects of a Methylcholanthrene-Induced Lymphosarcoma on Various Tissues of DBA/1J and Swiss White Mice

Description: This investigation was concerned with characterizing effects of this tumor line on lipid metabolism in DBA/lJ mice and serum protein levels and cellular changes in DBA/lJ and Swiss white mice. Total lipids, lipid phosphorus, neutral lipids, and changes in fatty acids were determined in liver, spleen, skin, and tumor of DBA/lJ mice bearing the lymphosarcoma at various days after injection of tumor cells.
Date: May 1973
Creator: Lindsey, Terri Jay

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

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

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

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

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)

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-

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

Cell-Free Recovery and Isotopic Identification of Cyanide Degrading Enzymes from Pseudomonas Fluorescens

Description: 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.
Date: December 1995
Creator: Wang, Chien-Sao

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.

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.

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

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 enzyme is functional, a plasmid construct containing the cotton FAD2 coding region was transformed into Saccharomyces cerevisiae. The transformed yeast cells were able to catalyze the conversion of oleic acid (C18:1) into linoleic acid (C18:2). The FAD2 gene contains an intron of 2,967 bp in its 5¢ -flanking region, 11 bp upstream from the initiation codon. The intron could be essential for transcriptional regulation of FAD2 gene expression. Several putative promoter elements occur in the 5¢ -flanking region of this gene. A potential TATA basal promoter element occurs at 41 bp upstream from the cap site. Two presumptive helix-loop-helix (bHLH) ...
Date: May 2001
Creator: Kongcharoensuntorn, Wisatre

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

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 transforming DNA concentration, transforming DNA conformation, and gene dosage on transformation efficiency are presented. Addition of Acinetobacter plasmid DNA sequences to the manipulated constructs did not have an effect on transformation rates. Results suggest that a broadly applicable and efficient method to carry out site-directed genetic manipulations of large plasmids has been identified. The ability to easily reintroduce the recombinant DNA molecules back into the original host organism was maintained.
Date: August 2001
Creator: Williamson, Phillip C.

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

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 pyrR- strain compared to the isogenic pyrR+ strain. Thus, pyrR is negatively regulated while the other pyr genes (except pyrC) are positively activated by PyrR. That no regulation was seen for pyrC is in keeping with the recent discovery of a second functional pyrC that is not regulated in P. aeruginosa. Gel filtration chromatography shows the PyrR protein exists in a dynamic equilibrium, and it is proposed that PyrR functions as a monomer in activating pyrD, pyrE and pyrF and as a dimeric repressor for pyrR by binding to the inverted repeat. A related study discovered that the catabolite repression ...
Date: August 2001
Creator: Patel, Monal V.

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

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 predicted to be secreted to the extracellular matrix as a neutral isoform. The deduced amino acid sequence has 16 cysteine residues that are highly conserved in osmotin-like proteins and are important in stabilizing the three-dimensional structure seen in thaumatin, zeamatin, and PR5-d. The intronless cognate cotton genomic clone has two putative ethylene response elements (GCC boxes) found in other PR5 gene promoter regions, as well as several tentative promoter/enhancer elements possibly involved in spatial/temporal gene expression.
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Date: May 2002
Creator: Yoder, David W.

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.

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.

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) gene. The continuous FAD2-3 coding region is 1,155 bp and would encode a protein of 384 amino acids. The FAD2-3 gene has one large intron of 2,967 bp entirely within its 5'-untranslated region. Several potential promoter/enhancer elements, including several light responsive motifs occur in the 5'-flanking region. Yeast cells transformed with a plasmid construct containing the cotton FAD2-3 coding region accumulate an appreciable amount of linoleic acid (18:2), not normally present in wild-type yeast cells, indicating that the gene encodes a functional FAD2 enzyme.
Date: May 2002
Creator: Nampaisansuk, Mongkol

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

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, we decided to test the ability of the DHOase mutant strain to produce other virulence factors as well. Here we report that a block in the conversion of carbamoyl aspartate (CAA) to dihydroorotate significantly impairs the ability of P. aeruginosa to affect virulence. We believe that the accumulation of CAA in the cell is the root cause of this observed defect. This research demonstrates a potential role for pyrimidine intermediates in the virulence response of P. aeruginosa and may lead to novel targets for chemotherapy against P. aeruginosa infections.
Date: August 2003
Creator: Brichta, Dayna Michelle

Analysis of a Cotton Gene Cluster for the Antifungal Protein Osmotin

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 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.
Date: December 2003
Creator: Wilkinson, Jeffery Roland

Influence of Cholesterol Import on Aspergillus fumigatus Growth and Antifungal Suscepibility

Description: Invasive pulmonary aspergillosis is a life-threatening fungal infection commonly observed in immunocompromised patients and has a mortality rate approaching 100% once the disease is disseminated. Aspergillus fumigatus is the most common pathogen. Early diagnosis improves the prognosis but is very difficult since most signs and symptoms are nonspecific. Antifungal therapy, usually based on sterol biosynthesis inhibitors, is also of limited efficacy. In my attempts to discover a diagnostic sterol marker for aspergillosis, I observed that A. fumigatus incorporates large amounts of cholesterol from serum-containing medium. This observation suggested the hypothesis that exogenous cholesterol from the host can be imported by A. fumigatus and used as a substitute for ergosterol in the cell membrane. This proposed mechanism would reduce the efficacy of antifungal drugs that act as sterol biosynthesis inhibitors. Experiments to test this hypothesis were designed to determine the effects of serum-free and serum-containing medium on growth of A. fumigatus in the presence and absence of azole antifungal agents. The results showed a marked increase in growth in the presence of human serum. Cultures in media containing cholesterol but no serum also showed enhanced growth, a result indicating that a non-cholesterol component of serum is not primarily responsible for the 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. ...
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Date: December 2003
Creator: Hassan, Saad A.

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

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 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 ...
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Date: December 2003
Creator: Kumar, Alan P.

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

Callus Development and Organogenesis in Cultured Explants of Cowpea (Vigna unguiculata (L.) Walp

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 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 ...
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Date: December 2004
Creator: Omwenga, George Isanda