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QTL and Candidate Genes for Growth Traits in Pinus Taeda L

Description: The reason for the project is to find the genetic factors which control growth at ages closer to commercial harvest (also known as QTL detection). To date, efforts to find genetic factors which control growth have been limited to seedlings. Because tree breeders want to find molecular markers which are linked to traits of direct economic value, finding linkage to factors controlling older-tree growth is more critical than seedling growth. Our current research interest includes both absolute height at ages 10-13 years but also growth trajectory or the rate of growth from seedling to half-rotation.
Date: October 1, 2002
Creator: Williams, Claire G.
Partner: UNT Libraries Government Documents Department

Activities of Human Gene Nomenclature Committee

Description: The objective of this project, shared between NIH and DOE, has been and remains to enable the medical genetics communities to use common names for genes that are discovered by different gene hunting groups, in different species. This effort provides consistent gene nomenclature and approved gene symbols to the community at large. This contributes to a uniform and consistent understanding of genomes, particularly the human as well as functional genomics based on comparisons between homologous genes in related species (human and mice).
Date: July 16, 2002
Partner: UNT Libraries Government Documents Department

Progress Report: DE-FG03-97ER20274, ''Microbial Production of Isoprene''

Description: We have discovered that microorganisms produce and emit the hydrocarbon isoprene (2-methyl-1,3-butadiene), and have suggested that if isoprene-producing enzymes and their genes can be harnessed, useful hydrocarbon-producing systems might be constructed. The main goal of the proposed work is to establish the biochemical mechanism and regulation of isoprene formation in the bacterial system, Bacillus subtilis. Specific objectives of the proposed work are the following: (A) to characterize the physiological regulation of isoprene formation in B. subtilis; (B) to characterize mutations in B. subtilis 168 that suppress isoprene formation, clone these genes, and determine how isoprene and isoprenoid carbon flow are regulated; and (C) to test ''overflow'' and ''signaling'' models for Bacillus isoprene formation. We are also pursuing the isolation and cloning of B. subtilis isoprene synthase, which we believe may be a regulatory enzyme.
Date: March 13, 2002
Creator: Fall, Ray
Partner: UNT Libraries Government Documents Department

Adaptive dimension reduction for clustering high dimensional data

Description: It is well-known that for high dimensional data clustering, standard algorithms such as EM and the K-means are often trapped in local minimum. many initialization methods were proposed to tackle this problem, but with only limited success. In this paper they propose a new approach to resolve this problem by repeated dimension reductions such that K-means or EM are performed only in very low dimensions. Cluster membership is utilized as a bridge between the reduced dimensional sub-space and the original space, providing flexibility and ease of implementation. Clustering analysis performed on highly overlapped Gaussians, DNA gene expression profiles and internet newsgroups demonstrate the effectiveness of the proposed algorithm.
Date: October 1, 2002
Creator: Ding, Chris; He, Xiaofeng; Zha, Hongyuan & Simon, Horst
Partner: UNT Libraries Government Documents Department

Nuclear apoJ: A low dose radiation inducible regulator of cell death. Final report for period September 15, 1998 - September 14, 2001

Description: This project was based on preliminary data that was published by Dr. Boothman (Yang et al. 2000) which indicated a strong induction of apoJ gene expression, increased secretion of the protein, and accumulation of an apparently somewhat different form of the apoJ protein in the nucleus of MCF-7 breast carcinoma cells undergoing response to DNA damage. A clone expressing apoJ protein was isolated that was capable of interacting with Ku80, a component of the double strand break repair complex that is essential for the successful repair of rearranging immunoglobulin and T-cell receptor genes as evidenced by failure to produce mature B and T cells in the absence of Ku70. ApoJ clones isolated and characterized by Dr. Boothman bound strongly to a Ku-70 ''bait'' protein. Over-expression of these same clones in a cell line was capable of killing the cell. ApoJ is very strongly induced in many instances of programmed cell death and has been proposed repeatedly to play some sort of effector role in the process. Our principle hypothesis for this study was that the strong induction of the apoJ gene and the particular expression of a nuclear form of the protein was potentially a causal factor in the decision point made by the cell as it attempts to repair double-strand breakage based DNA damage. The hypothesis was that if sufficiently high damage occurred, it would be deleterious to maintain the cell's viability through continued DNA repair. One method to inhibit DNA repair might be by inhibiting proteins such as Ku-70 that are necessary for double-strand break repair. If apoJ does play a critical role in tipping the decision balance over to cell death, we reasoned that deficiency of apoJ would cause increased accumulation of cells with DNA damage and that this might decrease cell death in response to DNA ...
Date: April 19, 2002
Creator: Aronow, Bruce J.
Partner: UNT Libraries Government Documents Department

Mechanisms and Determinants of RNA Turnover: Plant IRESs and Polycistrons for Metabolic Engineering

Description: There is a strong need for tools that allow multiple transgenes to be expressed in genetically engineered plants. For the last 30 years it has been believed that nearly all eukaryotic mRNAs were monocistronic, with ribosomes entering at the 5' end and scanning through the 5'UTR to the first AUG codon. It is now clear that perhaps 3% of vertebrate and yeast mRNAs utilize IRESs (Internal Ribosome Entry Sites) within their 5'UTRs to promote the internal entry of ribosomes to mRNAs and subsequent translation of protein without scanning. The working hypothesis behind this proposal is that IRES sequences function in plants and can be used to engineer the efficient co-expression of multiple proteins from polycistronic transcripts. Our goal was to translate multiple proteins from single polycistroic mRNAs. We cloned four IRESs from the following sources: CrTMV (plant virus), EMCV (human encephalomyocarditis virus), eIF4G (human), and c-myc (human). All four IRES were cloned into a specially designed test vector with the strong constitutive ACT2 actin regulatory sequences and flanked by multicloning sites for two reporter genes. These four IRESs were tested in three different test systems with strong paired reporter activities: two fluorescent proteins, two mercury resistance enzymes, and two biosynthetic enzymes making thiolpeptides. All of the four IRES constructs with the fluorescent protein reporter genes were tested for transient expression after particle gun bombardment of tobacco BY2 cells. Three of the IRESs gave reasonable activity (10%-40%) for the second cistron fluorescent reporter (DsRFP) relative to the first cistron reporter (GFP). As a control, translational blocking sequence placed at the 5' end of duplicate constructs had little effect on activity from the second cistron, but blocked the first cistron. These initial positive data lead us to examine the four IRES constructs with three pairs of reporters in hundreds of transgenic Arabidopsis ...
Date: August 1, 2002
Creator: Meagher, Richard B.
Partner: UNT Libraries Government Documents Department

Chlorinated Hydrocarbon Degradation in Plants: Mechanisms and Enhancement of Phytoremediation of Groundwater Contamination

Description: Several varieties of transgenic poplar containing cytochrome P-450 2E1 have been constructed and are undergoing tests. Strategies for improving public acceptance and safety of transgenic poplar for chlorinated hydrocarbon phytoremediation are being developed. We have discovered a unique rhizobium species that lives within the stems of poplar and we are investigating whether this bacterium contributes nitrogen fixed from the air to the plant and whether this endophyte could be used to introduce genes into poplar. Studies of the production of chloride ion from TCE have shown that our present P-450 constructs did not produce chloride more rapidly than wild type plants. Follow-up studies will determine if there are other rate limiting downstream steps in TCE metabolism in plants. Studies of the metabolism of carbon tetrachloride in poplar cells have provided evidence that the native plant metabolism is due to the activity of oxidative enzymes similar to the mammalian cytochrome P-450 2E1.
Date: June 1, 2002
Creator: Strand, Stuart E.
Partner: UNT Libraries Government Documents Department

SNPs in putative regulatory regions identified by human mouse comparative sequencing and transcription factor binding site data

Description: Genome wide disease association analysis using SNPs is being explored as a method for dissecting complex genetic traits and a vast number of SNPs have been generated for this purpose. As there are cost and throughput limitations of genotyping large numbers of SNPs and statistical issues regarding the large number of dependent tests on the same data set, to make association analysis practical it has been proposed that SNPs should be prioritized based on likely functional importance. The most easily identifiable functional SNPs are coding SNPs (cSNPs) and accordingly cSNPs have been screened in a number of studies. SNPs in gene regulatory sequences embedded in noncoding DNA are another class of SNPs suggested for prioritization due to their predicted quantitative impact on gene expression. The main challenge in evaluating these SNPs, in contrast to cSNPs is a lack of robust algorithms and databases for recognizing regulatory sequences in noncoding DNA. Approaches that have been previously used to delineate noncoding sequences with gene regulatory activity include cross-species sequence comparisons and the search for sequences recognized by transcription factors. We combined these two methods to sift through mouse human genomic sequences to identify putative gene regulatory elements and subsequently localized SNPs within these sequences in a 1 Megabase (Mb) region of human chromosome 5q31, orthologous to mouse chromosome 11 containing the Interleukin cluster.
Date: January 1, 2002
Creator: Banerjee, Poulabi; Bahlo, Melanie; Schwartz, Jody R.; Loots, Gabriela G.; Houston, Kathryn A.; Dubchak, Inna et al.
Partner: UNT Libraries Government Documents Department

Phytoremediation of ionic and methylmercury pollution

Description: Our long-term objective is to enable highly productive plant species to extract, resist, detoxify, and/or sequester toxic organic and heavy metal pollutants (Meagher, 2000) applying scientific strategies and technologies from a rapidly developing field called phytoremediation. The phytoremediation of toxic elemental and organic pollutants requires the use relatively different approaches (Meagher, 2000). Our current specific objectives are to use transgenic plants to control the chemical species, electrochemical state, and aboveground binding of mercury to (a) prevent methylmercury from entering the food-chain, (b) remove mercury from polluted sites, and (c) hyperaccumulate mercury in aboveground tissues for later harvest. Various parts of this strategy are being critically tested by examining different genes in model plants and field species and comparing the results to control plants as we recently reviewed (Meagher et al., 2000; Rugh et al., 2000). A positive spin-off from this work on mercury has been a strategy for the phytoremediation of arsenic (Dhankher et al., 2002) and cadmium.
Date: June 1, 2002
Creator: Meagher, Richard B.
Partner: UNT Libraries Government Documents Department

Cyr61 promotes breast tumorigenesis and cancer progression

Description: Cyr61, a member of the CCN family of genes, is an angiogenic factor. We have shown that it is overexpressed in invasive and metastatic human breast cancer cells and tissues. Here, we investigated whether Cyr61 is necessary and/or sufficient to bypass the ''normal'' estrogen (E2) requirements for breast cancer cell growth. Our results demonstrate that under E2-depleted condition, Cyr61 is sufficient to induce MCF-7 cells grow in the absence of E2. MCF-7 cells transfected with Cyr61 (MCF-7/Cyr61) became E2-independent but still E2-responsive. On the other hand, MCF-7/vector cells remain E2-dependent. MCF-7/Cyr61 cells acquire an antiestrogen-resistant phenotype, one of the most common clinical occurrences during breast cancer progression. MCF-7/Cyr61 cells are anchorage-independent and capable of forming Matrigel outgrowth patterns in the absence of E2. ERa expression in MCF-7/Cyr61 cells is decreased although still functional. Additionally, MCF-7/Cyr61 cells are tumorigenic in ovariectomized athymic nude mice. The tumors resemble human invasive carcinomas with increased vascularization and overexpression of vascular endothelial growth factor (VEGF). Our results demonstrate that Cyr61 is a tumor-promoting factor and a key regulator of breast cancer progression. This study provides evidence that Cyr61 is sufficient to induce E2-independence and anti-E2 resistance, and to promote invasiveness in vitro, and to induce tumorigenesis in vivo, all of which are characteristics of an aggressive breast cancer phenotype.
Date: January 16, 2002
Creator: Tsai, Miaw-Sheue; Bogart, Daphne F.; Castaneda, Jessica M.; Li, Patricia & Lupu, Ruth
Partner: UNT Libraries Government Documents Department

Analysis of Gene Targeting & Nonhomologous End-joining. Final Report

Description: Overall, we identified a number of new proteins that participate in nonhomologous end-joining and also in telomere addition to the ends of broken chromosomes. We showed that NHEJ is severely reduced in cells expressing both yeast mating-type genes and then went on to identify the NEJ1 gene that was under this control. We showed the epistasis relations among a set of mutations that impair telomere addition and we showed that there are in fact two pathways to repair broken chromosomes in the absence of telomerase. We characterized the DNA damage checkpoint pathway in response to a single broken chromosome and characterized especially the adaptation of cells arrested by an unrepaired DSB. We demonstrated that the DNA damage response is nuclear-limited. We showed adaptation defects for Tid1and Srs2 proteins and showed that Srs2 was also recovery-defective, even when DNA was repaired.
Date: November 30, 2002
Creator: Haber, J. E.
Partner: UNT Libraries Government Documents Department

Genetic Construction of Truncated and Chimeric Metalloproteins Derived from the Alpha Subunit of Acetyl-CoA Synthase from Clostridium thermoaceticum

Description: In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an alpha2beta2 tetrameric 310 kda bifunctional enzyme. ACS catalyzes the reversible reduction of CO2 to CO and the synthesis of acetyl-CoA from CO (or CO2 in the presence of low-potential reductants), CoA, and a methyl group bound to a corrinoid-iron sulfur protein (CoFeSP). ACS contains 7 metal-sulfur clusters of 4 different types called A, B, C, and D. The B, C, and D clusters are located in the 72 kda beta subunit while the A-cluster, a Ni-X-Fe4S4 cluster that serves as the active site for acetyl-CoA synthase activity, is located in the 82 kda alpha subunit. The extent to which the essential properties of the cluster, including catalytic, redox, spectroscopic, and substrate-binding properties, were retained as ACS was progressively truncated was determined. Acetyl-CoA synthase catalytic activity remained when the entire alpha subunit was removed, as long as CO, rather than CO2 and a low-potential reductant, was used as a substrate. Truncating an {approx} 30 kda region from the N-terminus of the alpha subunit yielded a 49 kda protein that lacked catalytic activity but exhibited A-cluster-like spectroscopic, redox, and CO binding properties. Further truncation afforded a 23 kda protein that lacked recognizable A-cluster properties except for UV-vis spectra typical of [Fe4S4]2+ clusters. Two chimeric proteins were constructed by fusing the gene encoding a ferredoxin from Chromatium vinosum to genes encoding the 49 kda and 82 kda fragments of the alpha subunit. The chimeric proteins exhibited EPR signals that were not the simple sum of the signals from the separate proteins, suggesting magnetic interactions between clusters. This study highlights the potential for using genetics to simplify the study of complex multi-centered metalloenzymes and to generate new complex metalloenzymes with interesting properties.
Date: June 28, 2002
Creator: Loke, Huay-Keng; Tan, Xiangshi & Lindahl, Paul A.
Partner: UNT Libraries Government Documents Department

Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem.

Description: This project was based on our discovery that sucrose acts as a signaling molecule that regulates the activity of a proton-sucrose symporter in sugar beet leaf tissue. A major objective here was determining how sucrose transporter activity is being regulated. When sucrose accumulates in the phloem sucrose transport activity drops dramatically. Western blots of plasma membrane proteins isolated from sucrose treated leaves showed that the loss of sucrose transport activity was proportional to a decline in symporter abundance, demonstrating that sucrose transport is regulated by changes in the amount of BvSUT1 protein. BvSUT1 transcript levels decreased in parallel with the loss of sucrose transport activity. Nuclear run-on experiments demonstrated that BvSUT1 gene transcription was repressed significantly in nuclei from leaves fed 100 mM exogenous sucrose, showing that sucrose-dependent modulation of BvSUT1 mRNA levels is mediated by changes in transcription. To identify which secondary messenger systems might be involved in regulating symporter activity, we used a variety of pharmacological agents to probe for a role of calcium or protein phosphorylation in sucrose signaling. In a detailed analysis, only okadaic acid altered sucrose transport activity. These results suggest a protein phosphatase is involved. We hypothesized that protein kinase inhibitors would have a neutral affect or increase symporter transcription. Transpirational feeding of the protein kinase inhibitor staurosporine had no impact on sucrose transport while calphostin C, an inhibitor of protein kinase C, caused a 60% increase. These data provided good evidence that protein phosphorylation plays a central role in regulating sucrose symporter expression and sucrose transport activity. To determine whether protein phosphorylation is involved in sucrose regulation of proton-sucrose symporter activity, we pre-fed leaves with staurosporine for 4 h and then fed the treated leaves water or 100 mM sucrose for an additional 20 h. Sucrose transport activity was higher than the water ...
Date: August 6, 2002
Creator: Vaughn, Matt; Harrington, Greg & Bush, Daniel R.
Partner: UNT Libraries Government Documents Department

Building beauty: the genetic control of floral patterning

Description: OAK-B135 Floral organ identity is controlled by combinatorial action of homeotic genes expressed in different territories within the emerging flower. This review discusses recent progress in our understanding of floral homeotic genes, with an emphasis on how their region-specific expression is regulated.
Date: February 1, 2002
Creator: Lohmann, J. U. & Weigel, D.
Partner: UNT Libraries Government Documents Department

Time-Resolved Sequence Analysis on High Density Fiberoptic DNA Probe

Description: A universal array format has been developed in which all possible n-mers of a particular oligonucleotide sequence can be represented. The ability to determine the sequence of the probes at every position in the array should enable unbiased gene expression as well as arrays for de novo sequencing.
Date: November 19, 2002
Creator: Walt, D. R. & Lee, K-H
Partner: UNT Libraries Government Documents Department

Vadose zone microbial community structure and activity in metal/radionuclide contaminated sediments. Final technical report

Description: This final technical report describes the research carried out during the final two months of the no-cost extension ending 11/14/01. The primary goals of the project were (1) to determine the potential for transformation of Cr(VI) (oxidized, mobile) to Cr(III) (reduced, immobile) under unsaturated conditions as a function of different levels and combinations of (a) chromium, (b) nitrate (co-disposed with Cr), and (c) molasses (inexpensive bioremediation substrate), and (2) to determine population structure and activity in experimental treatments by characterization of the microbial community by signature biomarker analysis and by RT-PCR and terminal restriction fragment length polymorphism (T-RFLP) and 16S ribosomal RNA genes. It was determined early in the one-year no-cost extension period that the T-RFLP approach was problematic in regard to providing information on the identities of microorganisms in the samples examined. As a result, it could not provide the detailed information on microbial community structure that was needed to assess the effects of treatments with chromium, nitrate, and/or molasses. Therefore, we decided to obtain the desired information by amplifying (using TR-PCR, with the same primers used for T-RFLP) and cloning 16S rRNA gene sequences from the same RNA extracts that were used for T-RFLP analysis. We also decided to use a restriction enzyme digest procedure (fingerprinting procedure) to place the clones into types. The primary focus of the research carried out during this report period was twofold: (a) to complete the sequencing of the clones, and (b) to analyze the clone sequences phylogenetically in order to determine the relatedness of the bacteria detected in the samples to each other and to previously described genera and species.
Date: August 17, 2002
Creator: Balkwill, David L.
Partner: UNT Libraries Government Documents Department

Development of Novel Polymeric Materials for Gene Therapy and pH-Sensitive Drug Delivery: Modeling, Synthesis, Characterization, and Analysis

Description: The underlying theme of this thesis is the use of polymeric materials in bioapplications. Chapters 2-5 either develop a fundamental understanding of current materials used for bioapplications or establish protocols and procedures used in characterizing and synthesizing novel materials. In chapters 6 and 7 these principles and procedures are applied to the development of materials to be used for gene therapy and drug delivery. Chapter one is an introduction to the ideas that will be necessary to understand the subsequent chapters, as well as a literature review of these topics. Chapter two is a paper that has been published in the ''Journal of Controlled Release'' that examines the mechanism of drug release from a polymer gel, as well as experimental design suggestions for the evaluation of water soluble drug delivery systems. Chapter three is a paper that has been published in the ''Journal of Pharmaceutical Sciences'' that discusses the effect ionic salts have on properties of the polymer systems examined in chapter two. Chapter four is a paper published in the Materials Research Society Fall 2000 Symposium Series dealing with the design and synthesis of a pH-sensitive polymeric drug delivery device. Chapter five is a paper that has been published in the journal ''Biomaterials'' proposing a novel polymer/metal composite for use as a biomaterial in hip arthroplasty surgery. Chapter six is a paper that will appear in an upcoming volume of the Journal ''Biomaterials'' dealing with the synthesis of a novel water soluble cationic polymer with possible applications in non-viral gene therapy. Chapter seven is a paper that has been submitted to ''Macromolecules'' discussing several novel block copolymers based on poly(ethylene glycol) and poly(diethylamino ethyl methacrylate) that possess both pH-sensitive and temperature sensitive properties. Chapter eight contains a summary of the research contained in chapters 2-7 and proposes future research ...
Date: August 27, 2002
Creator: Anderson, Brian Curtis
Partner: UNT Libraries Government Documents Department

Genetic polymorphism in three glutathione s-transferase genes and breast cancer risk

Description: The role of the glutathione S-transferase (GST) enzyme family is to detoxify environmental toxins and carcinogens and to protect organisms from their adverse effects, including cancer. The genes GSTM1, GSTP1, and GSTT1 code for three GSTs involved in the detoxification of carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and benzene. In humans, GSTM1 is deleted in about 50% of the population, GSTT1 is absent in about 20%, whereas the GSTP1 gene has a single base polymorphism resulting in an enzyme with reduced activity. Epidemiological studies indicate that GST polymorphisms increase the level of carcinogen-induced DNA damage and several studies have found a correlation of polymorphisms in one of the GST genes and an increased risk for certain cancers. We examined the role of polymorphisms in genes coding for these three GST enzymes in breast cancer. A breast tissue collection consisting of specimens of breast cancer patients and non-cancer controls was analyzed by polymerase chain reaction (PCR) for the presence or absence of the GSTM1 and GSTT1 genes and for GSTP1 single base polymorphism by PCR/RFLP. We found that GSTM1 and GSTT1 deletions occurred more frequently in cases than in controls, and GSTP1 polymorphism was more frequent in controls. The effective detoxifier (putative low-risk) genotype (defined as presence of both GSTM1 and GSTT1 genes and GSTP1 wild type) was less frequent in cases than controls (16% vs. 23%, respectively). The poor detoxifier (putative high-risk) genotype was more frequent in cases than controls. However, the sample size of this study was too small to provide conclusive results.
Date: April 1, 2002
Creator: Woldegiorgis, S.; Ahmed, R.C.; Zhen, Y.; Erdmann, C.A.; Russell, M.L. & Goth-Goldstein, R.
Partner: UNT Libraries Government Documents Department

rVISTA for Comparative Sequence-Based Discovery of Functional Transcription Factor Binding Sites

Description: Identifying transcriptional regulatory elements represents a significant challenge in annotating the genomes of higher vertebrates. We have developed a computational tool, rVISTA, for high-throughput discovery of cis-regulatory elements that combines transcription factor binding site prediction and the analysis of inter-species sequence conservation. Here, we illustrate the ability of rVISTA to identify true transcription factor binding sites through the analysis of AP-1 and NFAT binding sites in the 1 Mb well-annotated cytokine gene cluster1 (Hs5q31; Mm11). The exploitation of orthologous human-mouse data set resulted in the elimination of 95 percent of the 38,000 binding sites predicted upon analysis of the human sequence alone, while it identified 87 percent of the experimentally verified binding sites in this region.
Date: March 8, 2002
Creator: Loots, Gabriela G.; Ovcharenko, Ivan; Pachter, Lior; Dubchak, Inna & Rubin, Edward M.
Partner: UNT Libraries Government Documents Department

Interaction of LEAFY, AGAMOUS, and TERMINAL FLOWER1 in maintaining floral identity in Arabidopsis

Description: OAK-B135 The Arabidopsis transcription factor LEAFY (LFY) acts upstream of homeotic genes such as AGAMOUS (AG) to confer floral identity on meristems that arise after the transition to reproductive development. Compared to the genetic circuitry regulating the establishment of floral meristem identity, little is known about its maintenance. Previous experiments with fly heterozygous plants and ag mutants grown in conditions that reduce the floral inductive stimulus have shown that both genes are required to prevent reversion of floral to inflorescence meristems. Here, we present evidence that LFY maintains floral meristem identity independently of AG, and that the primary role of LFY is either direct repression of shoot identity genes or repression of an intermediate factor that activates shoot identity genes. The latter conclusions were deduced from the phenotypes conferred by a gain-of-function transgene, LFY:VP16, that appears to act as a dominant negative, or antimorphic, allele during maintenance of floral meristem identity. These observations contrast with previous findings that LFY acts as a direct activator of floral homeotic genes, supporting the hypothesis that the transcriptional activity of LFY is dependent on specific co-regulators.
Date: February 22, 2002
Creator: Parcy, F., Bomblies, K., and Weigel, D.
Partner: UNT Libraries Government Documents Department

Enzymatic Upgrading of Heavy Crudes via Partial Oxidation or Conversion of PAHs

Description: The objective of this program was to investigate new enzyme-based technologies for upgrading of heavy oils. Enzymes were selected for screening from those capable of conversion of polyaromatic hydrocarbons (PAHs) reported in the literature. Oxidative reactions of PAHs using hydrogen peroxide as an oxidant with conversion to partially oxidized products were used. The enzymes (lignin peroxidase, cytochrome c) were tested in various organic solvents and found to loose activity in pure organic solvents. A thermodynamic analysis revealed lack of effective interaction between the substrate and enzyme as the cause for low activity. The protein cytochrome c was modified to work in organic media by chemical hydrophobic group attachment. Two different modifications were made: attachment of polyethylene glycol (PEG) and alkyl groups. Alkyl groups, being small could be attached at interior locations within the core of the enzyme and possibly near the active site. Increase in the threshold solvent concentration where maximum enzyme activity occurred indicated potential of this strategy for effective enzyme-substrate interaction. Further improvements in enzyme activity called for other diverse methods due to the unavailability of sufficient chemical modification sites. Genetic techniques were therefore explored for further improvements. These experiments focused on cloning of a gene for the fungal enzyme lignin peroxidase (lip) into yeast Pichia pastoris, which would allow easy manipulation of the gene. However, differences in the fungal and yeast cellular machinery impeded significant expression of the fungal enzyme. Several strategies were explored to allow higher-level expression of the enzyme, which was required for enzyme improvement. The strategies used in this investigation are described in the report. Industrial in-kind support was available throughout the project period. review of the research results was carried out on a regular basis (bimonthly reports and annual meetings) followed by suggestions for improvement in ongoing work and direction for future work. ...
Date: July 1, 2002
Creator: Borole, A.P.; Davison, B.H. & Kuritz, T.
Partner: UNT Libraries Government Documents Department

Global Expression Studies of Yersinia Pestis Pathogenicity

Description: The aim of these studies continues to be the investigation into the molecular mechanisms that underlie the virulence process in Yersinia pestis. In particular, the focus of this work centers on the identification of novel genes and pathways responsible for the pathogenic properties of this organism. In spite of more than four decades of intense investigation in this field, the dilemma as to what makes Y. pestis such a virulent and lethal pathogen remains unanswered. The method being employed makes use microarray technology (DNA chip) that enables the examination of the global activities of the whole complement of genes in this pathogen. Two primary resources available to the investigators (one directly obtained from a separate CBNP-funded project) make these studies possible: (1) Whole genome comparisons of the genes in Y. pestis and its near neighbors with attenuated or non pathogenic characteristics, and (2) the ability to duplicate in vitro, conditions that mimic the infection process of this pathogen. This year we have extended our studies from the original work of characterizing the global transcriptional regulation in Y. pestis triggered during temperature transition from 26 C to 37 C (roughly conditions found in the flea vector and the mammalian host, respectively) to studies of regulation encountered during shift between growth from conditions of neutral pH to acidic pH (the latter conditions, those mimic the environment found inside macrophages, a likely environment found by these cells during infection.). For this work, DNA arrays containing some 5,000 genes (the entire genome of Y. pestis plus those genes found uniquely in the enteropathogen, and near neighbor, Y. pseudotuberculosis) are used to monitor the simultaneous expression levels of each gene of known and unknown function in Y. pestis. Those genes that are up-regulate under the experimental conditions represent genes potentially involved in the pathogenic process. ...
Date: October 15, 2002
Creator: Garcia, E; Motin, V; Brubaker, R & Fitch, P
Partner: UNT Libraries Government Documents Department