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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.

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

Analysis of the Expression Profiles of Two Isoforms of the Antifungal Protein Osmotin from Gossypium hirsutum

Description: The expression of two cotton osmotin genes was evaluated in terms of the mRNA and protein expression patterns in response to chemical inducers such as ethylene, hydrogen peroxide, and sodium chloride. Reverse transcriptase-polymerase chain reactions (RT-PCR) indicated that osmotin mRNAs are expressed constitutively in root tissues of cotton plants, and that they are rapidly induced in leaf and stem tissues upon ethylene treatment. Real time RT-PCR indicated that osmotin transcript levels were induced 2 to 4 h after treatment with ethephon. The osmotin mRNA levels appear to increase 12 h after treatment, decrease, and then increase again. The osmotin protein expression patterns were analyzed in Western blot analyses using an anti-osmotin antibody preparation. A 24-KDa protein band was detected from cotton plants treated with the inducers. The 24-KDa osmotin proteins were induced 4 h after treatment with ethephon, while down-regulated 96 h after treatment. Multiple osmotin isoforms were observed to be induced in cotton plants upon treatment with ethephon by two-dimensional gel electrophoresis. One goal of this dissertation research was to genetically engineer two cotton osmotin genes to routinely overproduce their antifungal proteins in transgenic Arabidopsis and cotton plants as a natural defense against fungal infections, using co-cultivation with Agrobacterium tumefaciens cells harboring pCAMBIA 2301 vector constructs containing the osmotin genes. Many transgenic Arabidopsis and cotton plants were generated. However, genomic blotting analyses indicated the absence of the osmotin transgenes, but the presence of GUS genes from the vector cassette. Alkaline blot analyses of the vector DNAs from transformed Agrobacterium cells confirmed that an anomalous DNA structural rearrangement or aberrant recombination event probably occurred in the Agrobacterium cells, interdicting the integration of osmotin transgenes into the Arabidopsis and cotton plants. This research provides crucial baseline information on expression of cotton osmotin mRNAs and proteins.
Date: May 2007
Creator: Spradling, Kimberly Diane

Applications of Molecular Genetics to Human Identity.

Description: 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.
Date: December 2008
Creator: Turnbough, Meredith A.

Bacterial Cyanide Assimilation: Pterin Cofactor and Enzymatic Requirements for Substrate Oxidation

Description: Utilization of cyanide as the sole nitrogen source by Pseudomonas fluorescens NCIMB 11764 (Pf11764) occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated oxygenolytically by an enzyme referred to as cyanide oxygenase (CNO), which exhibits properties of a pterin-dependent hydroxylase. The pterin requirement for Pf11764 CNO was satisfied by supplying either the fully (tetrahydro) or partially (dihydro) reduced forms of various pterin compounds at catalytic concentrations (0.5 µM). These compounds included, for example, biopterin, monapterin and neopterin, all of which were also identified in cell extracts. A related CNO-mediated mechanism of cyanide utilization was identified in cyanide-degrading P. putida BCN3. This conclusion was based on (i) the recovery of CO2 and NH3 as enzymatic reaction products, (ii) the dependency of substrate conversion on both O2 and NADH, and (iiii) utilization of cyanide, O2 and NADH in a 1:1:1 reaction stoichiometry. In contrast to findings reported for Pf11764, it was not possible to demonstrate a need for exogenously added pterin as a cofactor for the PpBCN3 enzyme system. However, results which showed that cells of PpBCN3 contained approximately seven times the amount of pterin as Pf11764 (of which a significant portion was protein-bound) were interpreted as indicating that sufficient bound CNO-cofactor exists, thus eliminating any need for a supplemental source.
Date: May 2004
Creator: Dolghih, Elena

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

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.

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

Changes in Gene Expression Levels of the Ecf Sigma Factor Bov1605 Under Ph Shift and Oxidative Stress in the Sheep Pathogen Brucella Ovis

Description: 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 ...
Date: December 2012
Creator: Kiehler, Brittany Elaine

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

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

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

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.

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

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

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 Degradation as an Indicator of Post-Mortem Interval

Description: The question of post-mortem interval (PMI) or time since death is often the most sought after piece of information associated with a medical death investigation. Based on the observation that DNA degradation disproportionately affects the analysis of larger genetic loci, it was proposed that DNA degradation, as a result of autolysis or putrefaction, could prove suitable as a potential rate-of-change indicator of PMI. Nine randomly amplified polymorphic DNA (RAPD) analysis primers and three sets of directed amplification primers were evaluated to determine their suitability for use in assessing the degree of DNA fragmentation in tissue samples. They were assessed for amplicon specificity, total DNA target sensitivity, allele monomorphism and the observance of degradation-based profile changes. Markers meeting the requisite criteria were then used to assess a range samples degraded under controlled and uncontrolled conditions. Tissue samples collected from seven domestic pigs (Sus scrofa) were incubated under controlled laboratory or uncontrolled field conditions to produce samples simulating those potentially collected in a forensic case. DNA samples isolated from these specimens were then analyzed at those loci which had been determined to meet the requisite criteria. Collectively, data generated from these analyses indicate that genetic profiles generated by this approach can provide information useful for estimating the post-mortem interval, with the locus and amplicons used being most useful during the first 72 hours after death.
Date: August 2010
Creator: Watson, William H.

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.

DNA Typing of HLA-B by PCR with Primer Mixes Utilizing Sequence-Specific Primers

Description: The aim of this study was to design a resolution typing system for the HLA-B gene. This technique involves a one-step PCR reaction utilizing genomic DNA and sequence-specific primers to determine the specificity of each allele and to produce a larger primer data base ideal for serological analysis. The application of this technique to serological analysis can improve serology detection which is currently hindered by antibody cross-reactivity and the unavailability of useful typing reagents.
Date: August 1997
Creator: Chiu, Angela Chen-Yen

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

Engineered Microbial Consortium for the Efficient Conversion of Biomass to Biofuels

Description: Current energy and environmental challenges are driving the use of cellulosic materials for biofuel production. A major obstacle in this pursuit is poor ethanol tolerance among cellulolytic Clostridium species. The first objective of this work was to establish a potential upper boundary of ethanol tolerance for the cellulosome itself. The hydrolytic function of crude cellulosome extracts from C. cellulolyticum on carboxymethyl cellulose (CMC) with 0, 5, 10, 15, 20 and 25% (v/v) ethanol was determined. Results indicated that the endoglucanase activity of the cellulosome incubated in 5% and 10% ethanol was significantly different from a control without ethanol addition. Furthermore a significant difference was observed in endoglucanase activity for cellulosome incubated in 5%, 10%, 15%, 20% and 25% ethanol in a standalone experiment. Endoglucanase activity continued to be observed for up to 25% ethanol, indicating that cellulosome function in ethanol will not be an impediment to future efforts towards engineering increasing production titers to levels at least as high as the current physiological limits of the most tolerant ethanologenic microbes. The second objective of this work was to study bioethanol production by a microbial co-culture involving Clostridium cellulolyticum and a recombinant Zymomonas mobilis engineered for the utilization of oligodextrans. The recombinant Z. mobilis ZM4 pAA1 and wild type ZM4 were first tested on RM medium (ATCC 1341) containing 2% cellobiose as the carbon source. Ethanol production from the recombinant Z. mobilis was three times that observed from the wild type Z. mobilis. Concomitant with ethanol production was the reduction in OD from 2.00 to 1.580, indicating the consumption of cellobiose. No such change in OD was observed from the wild type. The recombinant ZM4 was then co-cultured with C. cellulolyticum using cellobiose and microcrystalline cellulose respectively as carbon sources. Results indicate that the recombinant ZM4 acted synergistically with C. cellulolyticum ...
Date: August 2014
Creator: Anieto, Ugochukwu Obiakornobi

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

Expression analysis of the fatty acid desaturase 2-4 and 2-3 genes from Gossypium hirsutum in transformed yeast cells and transgenic Arabidopsis plants.

Description: Fatty acid desaturase 2 (FAD2) enzymes are phosphatidylcholine desaturases occurring as integral membrane proteins in the endoplasmic reticulum membrane and convert monounsaturated oleic acid into polyunsaturated linoleic acid. The major objective of this research was to study the expression and function of two cotton FAD2 genes (the FAD2-3 and FAD2-4 genes) and their possible role in plant sensitivity to environmental stress, since plants may increase the polyunsaturated phospholipids in membranes under environmental stress events, such as low temperature and osmotic stress. Two FAD2 cDNA clones corresponding to the two FAD2 genes have been isolated from a cotton cDNA library, indicating both genes are truly expressed in cotton. Model yeast cells transformed with two cotton FAD2 genes were used to study the chilling sensitivity, ethanol tolerance, and growth rate of yeast cells. The expression patterns of the two FAD2 genes were analyzed by reverse transcription polymerase chain reactions (RT-PCR) and Western blot analyses in cotton plants under different treatment conditions. The coding regions of both FAD2 genes were inserted downstream from the CaMV 35S promoter in the pMDC gateway binary vector system. Five different FAD2/pMDC constructs were transformed into the Arabidopsis fad2 knockout mutant background, and multiple potential transgenic Arabidopsis plant lines harboring the cotton FAD2 genes were generated. The cotton FAD2 genes were amplified by the polymerase chain reaction (PCR) from the genomic DNAs isolated from the transgenic Arabidopsis T1 plant lines. Complementation of the putative transgenic Arabidopsis plants with the two cotton FAD2 genes was demonstrated by gas chromatography analyses of the fatty acid profiles of leaf tissues. The cellular localization of cotton FAD2-4 polypeptides with N-terminal green fluorescence protein (GFP) was visualized by confocal fluorescence microscopy. The phenotype of transgenic Arabidopsis plants transformed with the cotton FAD2-4 gene was compared to Arabidopsis knockout fad2 mutant plants and wild ...
Date: August 2008
Creator: Zhang, Daiyuan

Genetic Analysis of Development and Behavior in Hypoxia and Cellular Characterization of Anoxia Induced Meiotic Prophase Arrest in Caenorhabditis Elegans

Description: It was hypothesized that chronic hypoxia will affect various biological processes including developmental trajectory and behavior. To test this hypothesis, embryos were raised to adulthood in severe hypoxic environments (0.5% O2 or 1% O2, 22°C) and analyzed for survival rate, developmental progression, and altered behaviors. Wildtype hermaphrodites survive chronic hypoxia yet developmental trajectory is slowed. The hermaphrodites raised in chronic hypoxia had different phenotypes in comparison to the normoxic controls. First, hermaphrodites exposed to chronic hypoxia produced a significantly lower number of embryos and had a slight increase in male progeny. This suggests that chronic hypoxia exposure during development affects the germline. Second, animals raised in chronic hypoxia from embryos to young adults have a slight increase in lifespan when re-exposed to a normoxic environment, indicating that chronic hypoxia does not negatively decrease lifespan. Finally, hermaphrodites that were raised in hypoxia will lay the majority of their eggs on the area of the agar plate where the bacterial lawn is not present. This is in contrast to animals in normoxia, which lay the majority of their eggs on the bacterial lawn. One hypothesis for this hypoxia-induced egg-laying behavior is that the animal can sense microenvironments in hypoxia. To examine if various pathways are involved with chronic-hypoxia responses RNAi and assayed genetic mutants were used. Specifically, genetic mutations affecting oxygen sensing (egl-9), aerotaxis (npr-1), TFG-ß signaling (dbl-1, daf-7) and predicted oxygen-binding proteins (globin-like genes) were phenotypically analyzed. Results indicate that mutations in several of these genes (npr-1, dbl-1) resulted in a decrease in hypoxia survival rate. A mutation in egl-9 also had a detrimental affect on the viability of an animal raised in chronic hypoxia. However, a similar phenotype was not observed in the vhl-1 mutation indicating that the phenotype may not be due to a mere increase in HIF-1 levels, ...
Date: August 2011
Creator: Little, Brent Ashley