Replication of plus (+)-strand RNA viruses proceeds through minus (−)-strand intermediates. Satellite RNA C (satC), one of the nonessential subviral RNAs of Turnip crinkle virus (TCV), is dependent on the TCV-encoded RdRp for its replication. Earlier work showed that a stem-loop structure at the 3′ end of (+)-strand satC is required for synthesis of (−)-strands (Song and Simon, 1995a). Using an in vitro RdRp assay, I defined two separate cis-acting elements on satC (−)-strands that can promote complementary strand synthesis. One element comprises 11 bases and is located near the 3′ end (3′-proximal), and the other consists of 14 bases and is located 41 bases from the 5′ end (5′ -proximal). Both elements contain multiple consecutive C residues followed by multiple consecutive purines. In vivo mutagenesis and genetic selection (SELEX) studies were carried out to investigate the functional significance of the two elements as well as the satC (−)-strand 3′ terminus (3 ′ OH-CCCUAU), which contains the (−)-strand 3′ -end sequence 3′ OH-CC1–2(A/U)(A/U)(A/U) found in all carmovirus RNAs (named the carmovirus consensus sequence or CCS). My results indicate that the 3′-terminal CCS and the 5′-proximal element are highly conserved and required for satC (+)-strand synthesis. Although mutations introduced into the 3′-proximal element were tolerable, this element preferentially contains a sequence similar to the CCS and/or polypurines, suggesting that this element may also contribute to satC accumulation in vivo. All RNAs associated with TCV terminate with the motif CCUGCCC-3 ′ at the 3′ end. Transcripts of satC containing a deletion of the motif, or the 3′-terminal 6 bases, are nearly always repaired to wild-type in vivo by RdRp-mediated primer extension of oligoribonucleotides synthesized by abortive initiation and complementary to the 3′ end of TCV genomic RNA (Nagy et al., 1997). In this thesis, I provide evidence that two additional mechanisms are used by the TCV RdRp to repair shorter deletions of the 3 ′-end motif of satC. Deletion of the 3′-terminal CCC residues along with addition of 8 non-template bases is repaired in vivo mainly by homologous recombination between the similar 3 ′ ends of satC and TCV. Deletion of the 3′-terminal 4 or 5 bases, in the presence or absence of non-template bases, led to recovery of progeny containing a mixture of wild-type 3′ ends and non-wild-type 3′ ends that included base alterations, deletions and insertions. Assays using an in vitro RdRp transcription system indicate that the TCV RdRp is likely able to polymerize nucleotides in a template-independent, non-random fashion before initiating transcription of deletion-containing satC. The existence of 3 different repair mechanisms associated with a single virus suggests an intrinsic need for 3′ -end reconstruction in the cellular environment.

Biologically synthesized poly(α,L-glutamic acid) (PLGA) can be chemically modified to form monodisperse poly(γ-benzyl α,L-glutamate) (PBLG). This material shows rare smectic ordering where macromolecular rods organize into highly-ordered layers. Analysis of PBLG has been hindered by low level biosynthesis of PLGA. An unusual feature of PLGA expression is that its accumulation is inversely related to the levels of its mRNA. This phenomenon has been investigated with the objective of improving the bioproduction of PLGA. PLGA was expressed as a C-terminal fusion with dihydrofolate reductase (DHFR) in E. coli strain BL21, carrying an IPTG-inducible DHFR-PLGA gene fusion in the pQE15 plasmid. In exponentially growing cells, accumulation of DHFR-PLGA was optimal at 0.01 mM IPTG and decreased at higher concentrations of IPTG. However, maximal DHFR-PLGA accumulation occurred in cells grown to saturation with no IPTG induction. It appears, therefore, that the accumulation of DHFR-PLGA is optimal in nongrowing cells translating low levels of DHFR-PLGA mRNA over long periods of incubation. Overexpression of both E. coli tRNAGlu and the glutamyl-tRNA synthetase did not improve DHFR-PLGA production. In vivo incorporation of [35S]-methionine was inhibited >95% by induction of either translatable or untranslatable PLGA constructs, but induction of the corresponding anti-sense constructs was not inhibitory. Sucrose gradient centrifugation analysis showed that expression of PLGA RNA resulted in nearly complete depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient. These new complexes were enriched in 16S rRNA but also contained 23S rRNA, and unlike normal polysomes, they were resistant to breakdown in the presence of puromycin. These results support the conclusion that multiple internal ribosome binding sites in the PLGA coding sequence inhibit translation of both DHFR-PLGA and cellular proteins by sequestering ribosomal subunits in nonfunctional complexes on the PLGA mRNA.

The nematode parasite Brugia malayi is one of the two major causative agents of human lymphatic filariasis and elephantiasis. The parasite is transmitted by biting mosquitoes, mostly of the Mansonia genus and this disease affects more than 100 million people in tropical and subtropical regions around the world. Global eradication of the disease using current chemotherapeutic regimes and mosquito control has proven unsuccessful. Production of a vaccine that provides immunity or more effective drug treatments are prospects for the future but will be difficult tasks. A better understanding of differential gene expression during the Brugia life cycle is crucial. One of the critical life cycle stages for establishment of infection in the human host are the L3 infective stage (the first stage to enter the human host and exposed to the host immune system). Identification of L3 stage-specific genes and /or up-regulated genes will contribute to increased understanding of the infective capabilities and survival strategies of Brugia. The gene that encodes thioredoxin peroxidase-2 (Bm-tpx-2) was found to be highly expressed in the L3 stage of the parasite. Thioredoxin peroxidase-2 is an antioxidant enzyme important in the detoxification of H 2O2 produced as a result of aerobic metabolism and by host immune cells as an assault against the invading parasite. RT-PCR shows Bm-tpx-2 to be up-regulated in the vector derived L3, the L4 and the adult female stages. The protein was localized within the protective cuticle of the vector derived L3 stage and the L4 stage, as well as within the uterus of the adult female. These expression and localization patterns suggest a protective role for TPX-2 against the host immune system. ELISA studies using sera from individuals living in an endemic area for B. malayi showed total IgG production against Bm-TPX-2 in both symptomatic and asymptomatic patients. These ELISA data indicate that TPX-2 is exposed to the host immune system sometime during development and this protein is immunogenic. A vaccine trial using an animal model (jird) showed production of IgG against TPX-2 in the immunized animals, although these animals did not exhibit significant protection against subsequent challenge with L3 larvae.

Our lab studies oxidative regulation in defense against extracellular pathogens, especially African Trypanosomes. Trypanosomes are protozoan hemoflagellates. The organisms are transmitted by tsetse flies (Golssina spp) and cause fatal trypanosomiasis in people and domestic animals. Previous work in our lab showed that African Cape buffalo and eland plasma contain a trypanocidal protein which kills all species of African trypanosomes during short term incubation in vitro (Black, 1999). The protein was identified as xanthine oxidase (Muranjan, 1997). On-going work in our lab has shown that the level of xanthine oxidase is a species and breed characteristic. The main focus of this investigation was to explore the molecular basis underlying the intrinsic systemic XO activity differences between different mammal species, with a view toward elucidating strategies that might enhance resistance of domestic animals to trypanosomiasis. My work has shown that the systemic XO activity differences observed in different mammal species, including Cape buffalo, eland, cattle, human, rat and mouse, does not result form XO coding sequence variation. Neither does it result from differences in tissue expression or transcription efficiency. Xanthine oxidase was proposed as a cytosolic protein, lacking any known leader/targeting sequences. My work also showed that there was more intensive signal in the paranuclear region. Expression fragments of the XOR protein revealed unconventional targeting sequence residing in both the N-terminus and the C-terminus of the enzyme, and they all target the recombinant protein to the paranuclear region. Sequence analysis confirmed the presence of two nucleus targeting sequence, which seem to agree with the role of xanthine oxidase in oxidant-mediated signal transduction in ischemia/reperfusion injury. We also found that XOR was not secreted into the medium under standard tissue culture growth conditions, nor was it displayed on the cell surface. This strongly suggests that the XOR protein might not be transported into the circulatory system under normal physiological condition and the presence of XOR in plasma could simply result from its release during cell death as a result of normal cell turn-over.

Hedgehog (Hh) signaling is required in many developmental processes including overall growth, induction of various cell types and morphogenesis. One of the earliest discoveries regarding Hh signaling was its role as a morphogen in the spinal cord. Hh expression in the adjacent notochord and in the floor plate result in the induction of ventral cell types in a dose-dependent manner based on their distance from these sources of Hh. The downstream transcription factors, the Gli proteins, mediate the transcriptional response to Hh signaling. The functions of the three vertebrate Glis diverge somewhat among species and appear to be both temporally regulated and tissue specific. The first goal of my dissertation was to examine whether Hh was necessary and sufficient for proper pituitary development in zebrafish. I used a combination of genetic analysis, overexpression, and a pharmacological blocking agent and discovered that Hh signaling is not only required for pituitary development but is also capable of inducing ectopic secretory cells outside of the pituitary. I was also able to define precisely when Hh signaling is required for induction and patterning of the pituitary. The second goal of my dissertation was to illuminate the roles of the Gli proteins in transducing Hh signaling in pituitary development. Additional genetic analysis as well as temporally regulated knockdowns of each Gli protein have unveiled both overlapping and distinct functions for the Glis. During early development, both Gli1 and Gli2 have activator roles and are necessary for proper induction of the pituitary and for the Hh response in the pituitary. Later, Gli1 remains a Hh-dependent activator of the pituitary while the role of Gli2 changes to that of a repressor. Removal of the Gli2 repressor function results in transfating of pituitary cells such that normally anterior cell types differentiate in the posterior at the expense of existing posterior cell types. My analyses, therefore, have uncovered multiple roles for Hh signaling in pituitary development as well as both overlapping and divergent functions for the Glis in a manner that is temporally regulated.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants routinely found in human and animal tissues. Developmental exposure to PCBs is associated with neuropsychological deficits, which may be related to effects on thyroid hormone (TH) signaling in the developing brain. However, PCBs may interfere with TH signaling solely by reducing circulating levels of thyroid hormone, or they may exert direct effects on thyroid hormone receptors (TRs). Therefore, the goal of this work was to examine the effects of polychlorinated biphenyl (PCB) exposure on TH receptor (TR)-mediated gene expression in vivo, including both dams and their fetuses, as well as in vitro utilizing a cell culture system. The work described in this dissertation demonstrates that PCBs exert TH-like effects both in vivo and in vitro. Specifically, a commercial PCB mixture (Aroclor 1254; A1254) significantly reduced circulating levels of triiodothyronine (T3) and thyroxine (T4) in pregnant rats but increased the expression of several TH-responsive genes in the fetal cortex, including RC3/Neurogranin and Oct-1. These findings are consistent with a direct action of PCBs on TRs. However, I did not identify parent PCB congeners or metabolites that bound to rat TRs isolated from hepatic nuclei. Nonetheless, I showed that a mixture of 6 PCB congeners (PCB Mix 6), chosen based on their ortho substitution pattern, significantly reduced serum TH levels in pregnant rats on G16. Moreover, I found that exposure to this PCB Mix 6 up-regulated the expression malic enzyme (ME), a gene well-known to be positively regulated by TH in the adult liver, and significantly increased TH response element (TRE) driven luciferase reporter activity in vitro. Taken together, these studies suggest that PCBs can act as TH agonists. Finally, I demonstrated that PCB Mix 6 can exert agonistic actions on the TR by a two-step process. In the first step, dioxin-like PCB 126 activates AhR-regulated cytochrome P450 enzymes. In the second step, CYP1A1 metabolizes non-coplanar PCBs 105 and 118 converting them into TR agonists. The present findings clearly indicate that the effects of PCB exposure on circulating levels of TH can only partly explain developmental abnormalities observed in both humans and animals. Moreover, these studies are the first to demonstrate that PCBs increase the transcription of several different TH-responsive genes via a TR dependent mechanism by which one PCB up-regulates the molecular machinery necessary to metabolically activate other PCBs, which ultimately generates a putative TH agonist. The implication for these findings is important because PCBs are likely to exert very different tissue and cellular specific effects which may be attributable to the expression pattern of the molecular machinery involved in TH agonist production. Therefore, depending on the cellular make up of different tissues, PCBs may exert TH-like effects or the tissue response may be solely due to the PCB induced decrease in TH levels.

The secreted Hedgehog (Hh) molecule acts as a morphogen, mitogen, and cell survival factor during embryogenesis and during later growth and tissue homeostasis periods in vertebrates. Hh plays these diverse roles by regulating the transcription of a number of genes that promote cellular processes including differentiation, proliferation, and resistance to programmed cell death. At the cellular level, the response to Hh signaling appears to change depending on the period during development that the cells are exposed to Hh, potentially allowing the switch, particularly in the brain, from early morphogen to a late mitogen. Mutations that reduce Hh signaling lead to congenital disorders in humans (e.g. holoprosencephaly), while mutations that inappropriately activate Hh signaling can lead to cancer (e.g. medulloblastoma and basal cell carcinoma). One goal of my research was to identify the major transcriptional targets of this important signaling pathway, work that may identify new genes that are targets of mutation in human disease. A collaborative effort using gene profiling techniques on zebrafish embryos with either increased or decreased Hh signaling was undertaken to identify genes regulated by the Hh/Gli signaling cascade. Our microarray approach led us to both known and novel zebrafish genes that are regulated by Hh signaling, including several with putative Gli transcription factor binding sites in their regulatory regions. We have confirmed the regulation of some known and novel genes by Hh in embryos with increased and decreased Hh signaling. This now published work has identified a large number of direct and indirect targets of the Hh signaling pathway. The major goal of my research was to both phenotypically and genetically characterize the zebrafish umleitung (uml) (meaning "detour" in German) mutation. The umlty54 mutant was identified due to its aberrant retinal tectal axon projections, and here I show that these mutants also have defects in Hh-mediated ventral neural patterning. These defects are accompanied by reduced expression of Hh-regulated genes in the forebrain and spinal cord, similar to known mutations that affect the Hh signaling pathway. Consistent with impaired Hh signaling, uml mutants have fewer endocrine cell types in the developing anterior pituitary, a reduced number of differentiated slow-muscle fibers in the tail, and defects in development of the jaw. To further understand how uml affects Hh signaling, I isolated the gene disrupted in uml by positional cloning. I have now shown that the defects seen in uml are due to a loss of function mutation in the gene encoding an immunoglobulin (Ig) and fibronectin type III (FNIII) domain containing cell-surface Hh-receptor, Brother of Cdo (Boc). Mosaic analysis indicates that Boc exerts its affects on Hh signaling cell-autonomously and may have a very early role in establishing the dose dependent response of cells along the dorsal/ventral axis of the central nervous system (CNS) to Sonic Hedgehog (Shh). This study demonstrates a previously unknown necessity for Boc in multiple Hh-mediated vertebrate developmental processes including, forebrain patterning, endocrine cell type specification, and jaw development. My analysis also reveals intriguing new ways in which Hh signaling is regulated within the vertebrate embryo and further implicates boc as a candidate gene in the treatment of disease caused by aberrant Hh signaling.

The main goal of this dissertation has been to identify the molecular and cellular mechanisms that underlie dopamine signaling. To this end, we used genetic, behavioral and molecular approaches available in C. elegans, a free-living soil nematode with a simple nervous system comprised of just 302 neurons. Out of these 302 neurons, there are only eight neurons that synthesize and release neurotransmitter dopamine. These features, in combination with an abundance of dopamine-dependent behavioral assays and amenability to forward and reverse genetic approaches, makes C. elegans an ideal model organism to dissect the molecular and cellular mechanisms that regulate dopamine signaling. In C. elegans, dopamine is released from dopaminergic neurons in response to mechanical stimuli and it binds to G protein-coupled D1- (DOP-1) and D2-like (DOP-3) dopamine receptors to regulate locomotion behavior. This regulation occurs in C. elegans cholinergic and GABAergic ventral cord motor neurons that control locomotion by innervating body wall muscles. To identify novel signaling components that underlie dopamine signaling, we performed a large-scale RNAi (RNA interference) screen in C. elegans . This screen identified eat-16 and rsbp-1 , C. elegans ,homologs of mammalian R7 family RGS (regulator of G protein signaling) protein and R7BP (R7 RGS binding protein), respectively. Since RGS proteins and their binding partners regulate G protein signaling, we characterized the roles of EAT-16 and RSBP-1 in the regulation of G protein-coupled dopamine receptor signaling in C. elegans. To this end, we used a combination of mutant analyses and cell-specific transgenic rescue experiments to investigate the functional interaction between EAT-16 and RSBP-1 within single cell types and to examine their role in the modulation of dopamine receptor signaling. We found that EAT-16 and RSBP-1 function together to specifically regulate DOP-1 and not DOP-3 receptor signaling by acting in cholinergic motor neurons. All together, my PhD work has shed light on the importance of RGS proteins and their binding partners in the regulation of dopamine receptor signaling and demonstrated that genetic and molecular approaches in C. elegans can be used to understand the mechanisms that underlie dopamine signaling.

Notch signaling is an evolutionarily conserved pathway that mediates a variety of cell fate decisions. In the immune system, the Notch pathway plays an important role throughout the life of T cells. Currently, the role of Notch signaling in peripheral T cells is being actively investigated. The Notch pathway has already been shown to be important for proliferation, IL-2 production and helper T cell differentiation following peripheral T cell activation. In this study, we show that the E3 ubiquitin ligase Cbl-b regulates Notch protein levels by targeting it for proteosomal degradation. Notch and Cbl-b associate with each other in bulk splenocytes and when co-transfected in 293T cells. Cbl-b co-expression led to reduction in the activity of a Notch reporter construct and enhanced the ubiquitination of the Notch protein. We have also used various deletion mutants of Notch and Cbl-b to localize the interacting regions on both proteins. ^ T cell anergy is a state of T-cell hyporesponsiveness following incomplete activation. Cbl-b has been shown to be upregulated in anergic T cells and is crucial for anergy induction. As Notch is important for effector functions of peripheral T cells, our discovery that Cbl-b negatively regulates Notch signaling has led us to investigate the role of Notch in anergy induction. Using an ex-vivo system to induce anergy in CD4+ T cells we show that Notch expression is down regulated upon induction of anergy. Also, Notch and CSL bind to the IL-2 promoter and positively regulate transcription and Notch also augments NF-κB binding to this promoter. Finally, we show that Notch expression during anergy induction rescues cytokine production and proliferation of these cells upon restimulation. Our findings suggest a novel role for Notch signaling in opposing the establishment of anergy.^

Plasmid pSPNl is a 26.5kb cryptic plasmid, originally isolated from Streptomyces penemafaciens ATCC 31599. A 12.5kb BglII fragment of pSPNl was cloned into the vector pLR2, and this conferred on pLR2 which lacks a Streptomyces origin of replication, the ability to replicate in a number of Streptomyces species. A vector pBlue was constructed by inserting a streptomycin resistance gene from plasmid pIJ4642 into the ampicillin resistance gene of the vector Bluescript. The resistance gene was able to function in both E.coli and Streptomyces species and thus pBlue could serve as a vector for shortening and sequencing in E. coli as well as a origin-probe vector in Streptomyces. The origin-containing BglII fragment of pSPNl was cloned into pBlue to create pFull, which was able to be selected for and replicate in Streptomyces. The conditions affecting selection of pFull in Streptomyces were investigated and optimized. The copy number of pFull was found to be 0.2 per chromosome. Attempts were made to clone origin-containing fragments smaller than the 12.5kb BglII fragment. Initially a Sau3A partial library was made of the origin-containing fragment, this however did not produce any replicating plasmids. As an alternative approach, pFull was extensively mapped and a series of deletion derivatives were constructed. The derivatives were tested for the ability to replicate in Streptomyces. Judging from the deletions that were and were not able to replicate it is apparent that at least 5.5kb of DNA is required for pFull and hence for pSPNl to replicate.