KVP40 is a T4-like bacteriophage whose dsDNA genome (244,835 bp) has been sequenced (21). It infects Vibrio parahaemolyticus, which can cause disease in fish and shellfish, and gastroenteritis in humans when consumed in raw or under-cooked seafood (i.e., oysters). The KVP40 genome carries bacterial-like genes that have the potential to encode a pyridine nucleotide scavenging system for synthesis of NAD+. This is the first pyridine nucleotide salvage pathway predicted from a phage or eukaryotic viral genome (21). nadV and natV are the two genes that encode the hypothetical two-reaction NAD+ scavenging pathway. NadV, a nicotinamide phosphoribosyltransferase, catalyzes the first reaction that converts nicotinamide to nicotinamide mononucleotide (NMN). The NatV NMN adenylyltransferase activity yields NAD+. Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor involved in fundamental processes in cell metabolism, and pathways for its synthesis are potential anti-microbial, therapeutic targets. The phage enzymes provide model targets for these studies. NatV, which catalyzes the second step of the pathway, also has a Nudix hydrolase domain in the C-terminal half. The purpose of this project was to characterize expression patterns of nadV and natV during KVP40 development in V. parahaemolyticus by using qRT-PCR; to characterize the enzymatic activity of NadV and NatV in a coupled-enzyme assay using alcohol dehydrogenase (ADH) to connect to fluorescent NADH; to identify possible substrates for the NatV Nudix hydrolase; and to use mass spectrometry to quantify product yields of the NatV reactions. qRT-PCR analysis showed that KVP40 nadV and natV were expressed early during infection relative to other phage genes used in this study. NadV-His6 and NatV-His6 enzymes were successfully purified by Ni2+ affinity HPLC and the levels of NADH produced were measured in a three step NadV-NatV-ADH reaction system. In the coupled assay, NatV-His6 converted NMN to 50 μmole of NAD+/sec/μg at 25°C. NatV NMNATase activity was also confirmed by mass spectrometry, in this assay, the rate was 350 μmole NAD+/sec/μg NatV-His6 at 25°C. NadV NAmPRTase activity was confirmed by producing 5.2, 4.9 and 5.0 μmole NMN/sec/μg NadV-His6 enzyme at 25, 30 and 37°C, respectively. Purified NadV used in the coupled enzyme also produced 2.7, 1.5, 1.3, 1.8 or 1.5 μmole NMN/sec/μg NadV-His6, when 10, 25, 40, 50 or 100 pmole NadV-His6 was respectively used in the reaction at 25°C. The âNudixâ activity of purified NatV was measured by a phosphate releasing assay and by mass spectrometry. Using the phosphate release Nudix hydrolase assay, ADP-ribose was identified as a preferred substrate for KVP40 NatV, followed by NAD+, NADH, and NADPH. In this assay, ADP-ribose as substrate yielded 0.6 μmole phosphate/sec/μg NatV-His6 at 37°C, when Mg2+ was supplied as the required metal ion. Mass spectrometry verified the NatV Nudix hydrolase preference for ADP-ribose as substrate, yielding the same rate of 0.6 μmole AMP/sec/μg NatV-His6 at 37°C in the presence of Mg2+. Together these data confirm the various enzymatic activities of key pyridine nucleotide scavenging enzymes encoded by phage KVP40. The time course of expression and in vivo activities suggest that pyridine nucleotide scavenging during KVP40 infection of V. parahaemolyticus is a functional and potential relevant pathway used by the phage.

Geminiviruses are a group of plant-infecting viruses that include the genera Begomovirus and Curtovirus. Although members of these two genera are similar in regards to their mechanisms of replication and encapsidation, they exhibit differences in movement as well as in transcriptional activation of late gene promoters. This study evaluated protein-protein interactions of the begomovirus AL2 protein and a curtovirus C2 protein using the yeast two-hybrid system. The AL2 proteins of TGMV, BGMV, and CabLCV were evaluated for their ability to self-interact, as well as to interact with heterologous AL2 proteins, and host proteins. The AL2 proteins tested were all shown to contain the ability to self-interact and to interact heterologously with one another. However, interactions between the AL2 proteins and the host proteins could not be assayed due to lack of expression of certain AL2 'bait' proteins. The BCTV C2 protein was also evaluated for self-interaction and interaction with the begomovirus AL2 proteins. Unlike AL2, C2 was not shown to contain the property of self-interaction and the preponderance of the evidence demonstrated that it also did not interact with AL2. However, the BCTV C2 protein was shown for the first time to possess transcriptional activation activity. In addition, self-interaction of TGMV AL2 was tested using a biochemical assay. However, technical problems prevented the detection of TGMV AL2 self-interaction using an immunocapture approach, although such self-interaction was observed using the yeast two-hybrid system.

Salmonella species cause a variety of infections in humans and domestic animals, ranging from mild food poisoning-gastroenteritis Salmonella typhimurium to severe systemic disease Salmonella typhi. Salmonella is used as a paradigm to understand how intracellular pathogens withstand the onslaught of the host innate immune system, namely the professional macrophage. SlyA is a global transcriptional regulator that is necessary for the virulence of Salmonella typhimurium. In a mouse model system, a slyA mutant is profoundly attenuated for virulence and is unable to survive in macrophages. To understand the evolutionary history of SlyA in Eubacteria, a phylogenetic analysis was performed. Several alignments of amino acid sequences were constructed with MarR and also with known SlyA homologues. Searches for other SlyA homologues were undertaken using databases of unfinished and finished microbial genomes and more than 50 putative homologues were found. SlyA has been classified as a MarR-like transcriptional regulator by homology. This classification may not be appropriate given the differences in function. Therefore it is suggested that a new class of SlyA-like regulators be formed incorporating all of the homologues found in this study. In order to determine the extent of the SlyA regulon, studies were conducted to analyze the DNA-binding properties of SlyA. The target promoters from this study included genes that were shown to be either activated or repressed by SlyA by utilizing a Salmonella genomic DNA microarray. SlyA was shown to bind specifically to the promoters of clyA, pagC, pagK and mig-14. The approximate Kd values appear to be similar between each of the promoters indicating a similar propensity for SlyA to bind.

HARRIS, VIRGINIA GAIL The Intracellular Targeting of cPLA2. (Under the direction of Scott M. Laster). Tumor necrosis factor-á (TNF) is an inflammatory cytokine that can induce apoptosis in virus-infected cells, susceptible tumor cells, and cells whose transcriptional or translational processes have been disrupted. The apoptotic pathway that is activated by TNF is dependent on the activity of the enzyme cytosolic phospholipase A2 (cPLA2). This enzyme cleaves arachidonic acid from membrane phospholipid, which in turn causes mitochondrial dysfunction leading to cell death. The goal of this research project was to identify the specific intracellular membrane to which cPLA2 binds during TNF-induced apoptosis. The membrane site for cPLA2 interaction during apoptosis would be a possible target for anti-apoptotic drug development. Three methods were tested in an attempt to identify the membrane location, including; immunofluorescence, biochemical fractionation, and GC/MS. Our results with immunofluorescence suggested that cPLA2 translocates to the nuclear membrane during apoptosis. The identity of the nuclear membrane was confirmed by staining with an antibody to the nuclear pore complex. In addition, cPLA2 staining was noted within the nucleus, perhaps indicating an interaction with chromatin, and small areas of punctate staining were noted in the perinuclear region. Biochemical fractionation indicated that the association of cPLA2 with the nuclear membrane was calcium-dependent since this association could not be stabilized in the absence of calcium. GC/MS, which was used in an attempt to find amputated phospholipids remaining in membranes as a result of cPLA2 activity, instead revealed higher levels of arachidonic acid suggesting that cells may increase the synthesis or repair of arachidonic acid containing membranes following the activation of cPLA2. Finally, our results revealed several novel, lower molecular weight forms of cPLA2 that were associated with nuclei in a calcium-independent fashion. Taken together these results suggest that it will be important to understand the mechanisms controlling the association of cPLA2 with nuclei.

This Master?s Thesis contains a description of the results of two research projects that were worked on over the course of two years. The first was an attempt to heterologously reconstitute the Bacillus subtilis RNase P RNA with the RNase P protein subunits of Methanothermobacter thermoautotrophicus, to identify a functional homology between one or more of the archaeal proteins with the Bacillus subtilis RNase P protein. Unfortunately, the reconstitution experiment could not be completed due to the poor quality of the pre-tRNAasp substrate synthesized. The second project dealt with comparative analysis of bacterial RNase P RNA. Eleven new RNase P RNA genes were amplified, sequenced, and their secondary structures derived by comparative analysis.

Reduction/oxidation (redox) balance is a critical component of cell viability. When oxidants reach levels that overcome the ability of antioxidants to eliminate them, it results in damage to cellular macromolecules. The damage to DNA, lipids and protein initiates a cascade of physiological events in response to the oxidative stress. Researchers have been studying these cellular responses by analyzing gene expression and protein activity for many years. Recently, new technologies have emerged that allow scientists to analyze the differential expression of extremely large sets of genes in response to oxidative stressors and other toxicants. Most experiments performed to date have involved a single dose of a chemical and a single timepoint for analysis. However, gene expression has proven to be a dynamic process with many transcriptional changes over a relatively short timecourse. In order to study the dynamic nature of gene expression and its effects on cellular physiology, experiments were performed to analyze the effects of oxidative stress on HepG2 cells over a 24 hour timecourse with a range of doses of the glutathione depletor, diethylmaleate (DEM). Using Clontech microarrays, TaqMan RT-PCR, and assays to measure reduced glutathione (GSH) concentrations and to determine cell cycle status, an overall picture of the effects of oxidative stress in relation to dose and time was created. DEM caused GSH depletion to the extent that cells treated with 1.25mM DEM for 4 hours contained less than 20% of the GSH levels in untreated cells. The redox imbalance caused the transcription of genes that initiate cell cycle arrest, DNA repair, and induction of stress proteins. The p53-independent induction of p21 initiated a cascade of events including the decreased transcription of cyclins that resulted in cell cycle arrest. Additionally, the transcription of stress induced genes such as HSP70 and heme oxygenase-1 exhibited significant time and dose-dependent increases in reponse to DEM. While the genes exhibiting differential expression remained generally the same between doses, it was the time taken for these gene changes to occur that varied greatly from the highest dose to the lowest dose of DEM. These experiments demonstrate the importance of analyzing an effective dose range over an extended time period when using differential gene expression to study the mechanisms of toxicity.

The hyperthermophilic archaeon, Pyrococcus furiosus depends on the element tungsten for growth since tungsten-containing enzymes such as aldehyde ferredoxin oxidoreductase (AOR) are key to its metabolism. Crystal structure analysis of the tungsten cofactor in AOR indicated that the tungsten cofactor exists as part of a tricyclic pterin moiety analogous to molybdopterin cofactor present in molybdoenzymes such as nitrate reductase. Molybdopterin cofactor synthesis has been well characterized in Escherichia coli with the identification of at least 14 genes that participate in this process. Analysis of the P. furiosus genome revealed that it has homologs to all cofactor synthesis genes except for modE, a transcriptional regulator of molybdopterin cofactor synthesis, and mogA, a putative molybdo-chelator. Two of the molybdenum cofactor biosynthesis genes, moeA and moeB, involved in activation of molybdenum and in donation of sulfur to the pterin ring structure, respectively, each have two homologs in P. furiosus (MoeA, 45%, MoeA2, 44%, MoeB, 50%, and MoeB2, 47% similar to E. coli MoeA and MoeB respectively). The MoeA and MoeB homologs were targeted for initial functional activity studies to determine if they participate in cofactor formation. The activity studies entailed complementing E. coli strains mutant in moeA or moeB with recombinant P. furiosus homologs in an in vitro system and assaying for restoration of the molydoenzyme nitrate reductase (NR) activity. Partial complementation of defects in E. coli moeA and moeB were observed for assays including P. furiosus MoeA2 and MoeB2 which supported 13.1 nmole NO2-?min-1?mg-1 (10µg MoeA2) and 19.6 nmole NO2-?min-1?mg-1 (100µg MoeB2) activity respectively. These specific activities represent 10.1% and 15.1% of wild type E.coli nitrate reductase activity (130 nmole NO2-?min-1?mg-1) Only negligible restoration of nitrate reductase activity was observed when P. furiosus MoeA or MoeB was included in the assay, with specific activities 0.16 nmole NO2-?min-1?mg-1 (1µg MoeA) and 0.74 NO2-?min-1?mg-1 (50µg MoeB). Partial complementation of the E. coli moeA mutant was also observed for in vitro trimethyl amine oxide (TMAO) reductase assays where 10 g MoeA2 supported a specific activity of 40.97 nmole TMAO reduced?min-1?mg-1 and 10 g MoeB2 supported a specific activity of 6.3 nmole TMAO reduced?min-1?mg-1 compared to 2,374 nmole TMAO reduced?min-1?mg-1 for the wild type E. coli. When TMAO assays were conducted in the presence of tungsten rather than molybdenum, the wild type E. coli had a specific activity of 1,297 nmole TMAO reduced-?min-1?mg-1. E. coli moeA mutant had an activity of 2.07 nmole TMAO reduced -?min-1?mg-1 when supplemented with 10 g MoeA2 and the E. coli moeB mutant supported 6.01 nmole TMAO reduced-?min-1?mg-1 when supplemented with 100 g MoeB2. The partial nature of the complementation seen in these studies is likely due in part to the use of sub optimal assay temperatures (37C), as required for E. coli NR, well below the optimum temperature of 95C seen for most P. furiosus enzymes. Nevertheless, these complementation assays demonstrate that P. furiosus MoeA2, and MoeB2 homologs likely function as MoeA and MoeB in tungsten cofactor synthesis in P. furiosus.

Bacillus licheniformis PWD-1 is a keratin-degrading, spore-forming bacterium isolated from a poultry waste digester. A mutant of B. licheniformis PWD-1, named B. licheniformis WBG-2, was developed that is deficient in sporulation. The mutation was created using the Splicing by Overlap-Extension PCR method (Gene SOE?ing) to create a 256bp deletion in the spoIIAC gene, which encodes an essential sporulation-specific sigma factor. In-vivo gene replacement was accomplished with the use of a temperature-sensitive plasmid that is able to integrate and excise from the B. licheniformis chromosome. PCR and DNA sequencing were used to confirm the deletion, while heat-treatment assays and electron microscopy verified the absence of spores. The mutant, while completely asporogenic, is able to expresses normal levels of keratinase as compared with B. licheniformis PWD-1.

<p>The RNase P RNAs from Methanococcus and Archaeoglobus lack secondary structural features essential for the recognition of pre-tRNA. In the absence of protein, these RNase P RNAs lack catalytic activity. How the Methanococcus and Archaeoglobus RNase P holoenzymes compensate for the absent RNA structural elements is not known, but we hypothesize that it is via additional proteins. The Methanococcus jannaschii RNase P holoenzyme has been purified for structural and functional characterization. This enzyme has a buoyant density in Cs2SO4 of 1.39 g/ml and an apparent molecular weight of greater than 400kDa. The holoenzyme has a Km of 68 nM, a kcat of 37nM min-1 (similar to that of other RNase P enzymes) and tolerates a wide range of ionic conditions. Six potential protein subunits have been identified on the basis of copurification with enzymatic activity. The molecular weight of three of these bands is consistent with apparent holomologs of RNase P proteins from Methanobacterium thermoautotrophicum and Saccharomyces cerevisiae. <P>

<p><p>     RNase P removes the 5´ leader from all transfer RNA precursors, and is thus a required factor for protein synthesis. RNase P consists of a catalytic RNA molecule and associated protein, the amount of which varies among phylogenetic domains. The ca. 120 kDa RNA component of RNase P from Bacteria is catalytic <p>     Previous characterizations of archaeal RNase P have presented conflicting and somewhat confusing results. RNase P from <p>     <P>