Biochemistry, Biophysics, and Structural Biology Commons^™

Crystallization And Structure Determination Of The Catalytic Trimer Of Methanococcus Jannaschii Aspartate Transcarbamoylase, Jacqueline Vitali, Tatyana Vorobyova, Gordon Websterb, Evan R. Kantrowitza

Physics Faculty Publications

Aspartate transcarbamoylase (ATCase) catalyzes the first step in the pyrimidine biosynthetic pathway, the reaction between carbamoyl phosphate and L-aspartate to form N-carbamoyl-L-aspartate and phosphate. The structural analysis of the ATCase catalytic trimer from Methanococcus jannaschii, a unicellular thermophilic archaeabacterium, has been undertaken in order to gain insight into the structural features that are responsible for the thermostability of the enzyme. As a first step, the catalytic trimer was crystallized in space group R32, with unit-cell parameters a = b = 265.3, c = 195.5 Å and two trimers in the asymmetric unit. Its structure was determined using …

Intracellular Transport, Assembly, And Degradation Of Wild-Type And Disease-Linked Mutant Gap Junction Proteins, Judy K. Vanslyke, Suzanne M. Deschênes, Linda S. Musil

Biology Faculty Publications

More than 130 different mutations in the gap junction integral plasma membrane protein connexin32 (Cx32) have been linked to the human peripheral neuropathy X-linked Charcot–Marie–Tooth disease (CMTX). How these various mutants are processed by the cell and the mechanism(s) by which they cause CMTX are unknown. To address these issues, we have studied the intracellular transport, assembly, and degradation of three CMTX-linked Cx32 mutants stably expressed in PC12 cells. Each mutant had a distinct fate: E208K Cx32 appeared to be retained in the endoplasmic reticulum (ER), whereas both the E186K and R142W mutants were transported to perinuclear compartments from which …

Functional Characterization Of The Nonstructural Protein 2 Of Bluetongue Virus Serotype 17, Gary Christopher Fillmore

Biology

Bluetongue virus (BTV) is an arthropod-borne pathogen that causes bluetongue disease (BD) throughout the world, with economic losses primarily coming from infection of domestic livestock. Although BTV has been well studied at the molecular level since 1969, the mechanism of viral replication has not yet been fully determined. The virus is composed of seven structural and four nonstructural proteins, and the role of each protein has been investigated but has not been conclusively established within the viral life cycle. This study focused on the nonstructural protein NS2 in an attempt to examine its functions as part of the viral replication …

Electrically Mediated Plasmid Dna Delivery To Hepatocellular Carcinomas In Vivo, L. Heller, M. J. Jaroszeski, D. Coppola, C. Pottinger, R. Gilbert, Richard Heller

Bioelectrics Publications

Gene therapy by direct delivery of plasmid DNA has several advantages over viral gene transfer, but plasmid delivery is less efficient. In vivo electroporation has been used to enhance delivery of chemotherapeutic agents to tumors in both animal and human studies. Recently, this delivery technique has been extended to large molecules such as plasmid DNA. Here, the successful delivery of plasmids encoding reporter genes to rat hepatocellular carcinomas by in vivo electroporation is demonstrated.

Test Of Intron Predictions Reveals Novel Splice Sites, Alternatively Spliced Mrnas And New Introns In Meiotically Regulated Genes Of Yeast, Carrie Davis, Leslie Grate, Marc Spingola, Manuel Ares

Biology Department Faculty Works

Correct identification of all introns is necessary to discern the protein-coding potential of a eukaryotic genome. The existence of most of the spliceosomal introns predicted in the genome of Saccharomyces cerevisiae remains unsupported by molecular evidence. We tested the intron predictions for 87 introns predicted to be present in non-ribosomal protein genes, more than a third of all known or suspected introns in the yeast genome. Evidence supporting 61 of these predictions was obtained, 20 predicted intron sequences were not spliced and six predictions identified an intron-containing region but failed to specify the correct splice sites, yielding a successful prediction …

Molecular Evolution Of Insecticidal Spore-Forming Bacteria, John Pool

Honors Theses

Molecular methods are increasingly being used to determine the phylogeny of microorganisms. This research was intended to determine phylogenetic relationships for bacteria of the species Bacillus thuringiensis and other members of the Bacillus cereus group. Each strain was analyzed by its sasp-B gene sequence to determine its species classification and relation to other strains studied. Results of this study indicated that according to the sasp-B gene tree, the species Bacillus thuringiensis is a paraphyletic with respect to both Bacillus cereus and Bacillus anthracis. Some unexpected results and implications for species designations are also discussed.

Characterization Of Human T-Cell Leukemia Virus 1 Reverse Transcriptase, Pinky Gundayao Agbuya

Theses and Dissertations in Biomedical Sciences

Human T-cell leukemia virus 1 (HTLV-1) is a type C human retrovirus which has been the causative agent of Adult T-cell leukemia. Replication of the retrovirus; requires a reverse transcriptase which converts the retroviral RNA into DNA which is later incorporated into the host's genome. Very little is known about the reverse transcriptase of HTLV-1. Researchers have attempted to purify HTLV-1 RT by isolating the enzyme from human cell lines. Because large amounts of protein could not be produced by this isolation method, the reverse transcriptase cannot be fully characterized. In this research, a recombinant protein expressed in E. coli …

Multi-Pass Reflectron Time-Of-Flight Mass Spectrometer, Curtiss D. Hanson

Patents (University of Northern Iowa)

A novel design for a time-of-flight mass spectrometer capable of tandem mass spectrometry measurements with high resolution and high sensitivity using two variable reflectrons in a co-linear geometry. Variably switched reflectrons are oriented coaxially on opposing ends of the ion flight region allowing multiple passes of the ions along the flight region permitting high resolution, tandem mass spectrometry experiments to be performed. An electrostatic particle guide is incorporated to ensure high ion transmission efficiency in a multi-pass system. In addition to permitting the high transmission efficiency of ions, the EPG can be used in a bipolar pulsed mode to isolate …

Melanocortin-1-Receptor (Mcr-1) Gene Polymorphisms Associated With The Chicken E Locus Alleles, Andrew Ellett

Inquiry: The University of Arkansas Undergraduate Research Journal

The melanocortin 1-receptor (MCl-R) gene has been associated with E locus phenotypes in chickens. Variant alleles of the E locus are important for accurate down color sexing and also for the inhibition of unwanted tissue pigmentation in broilers. PCR (polymerase chain reaction) based tests for various replacement substitutions found in the published E allele sequences gave unexpected results when tested against known alleles of the E locus. To resolve these issues and gain a better understanding of how replacement substitutions in the MCl-R gene are affecting E locus phenotypes, a number of accessions of the e+, eb, wheaten ( ewh …

Eph Receptors And Ephrins, Masaru Nakamoto

Biology Faculty Publications

The Eph receptors are the largest known family of receptor tyrosine kinases. The Eph receptors and theirmembrane-attached ligands, ephrins, show diverse expression patterns during development. Recent studies havedemonstrated that Eph receptors and ephrins play important roles in many developmental processes, includingneuronal network formation, the patterning of the neural tube and the paraxial mesoderm, the guidance of cellmigration, and vascular formation. In the nervous system, Eph receptors and ephrins have been shown to act aspositional labels to establish topographic projections. They also play a key role in pathway ®nding by axons andneural crest cells. The crucial roles of Eph receptors and …

Absence Of Insect Juvenile Hormones In The American Dog Tick, Dermacentor Veriabilis (Say) (Acari: Ixodidae), And In Ornithodoros Parkeri Cooley (Acari: Argasidae), P. A. Neese, Daniel E. Sonenshine, V. L. Kallapur, C. S. Apperson, R. M. Roe

Biological Sciences Faculty Publications

Synganglia, salivary gland, midgut, ovary, fat body and muscle alone and in combination from the ixodid tick, Dermacentor variabilis (Say), or the argasid tick, Ornithodoros parkeri Cooley, were incubated in vitro in separate experiments with _L-[methyl-³H]methionine and farnesoic acid or with [1-¹⁴C]acetate. Life stages examined in D. variabilis were 3 and 72 h old (after ecdysis) unfed nymphs, partially fed nymphs (18 and 72 h after attachment to the host), fully engorged nymphs (2 d after detachment from host), 3 and 72 h old (after eclosion) unfed females, partially fed unmated females (12–168 …

Human Gonadotropin-Releasing Hormone (Hgnrh) Gene Expression And Hormone Regulation In Human Placental Jeg-3 Cells, Heming Zheng

Theses and Dissertations in Biomedical Sciences

Using the human placental choriocarcinoma JEG-3 cell line as an in vitro human placental model, I studied the mechanisms of the tissue-specific expression and steroid hormone regulation of the hGnRH gene in the human placenta. The results showed that all of the previously identified four elements are required for the full activity of the hGnRH upstream promoter in JEG-3 cells, while the element 4 (FP4, −987/−968) is the most important. Studies performed with 5′ end deletion of this region confirmed these observations. Further, supershift assay using Oct-1 antibody demonstrated the involvement of Oct-1 in the FP4 DNA-protein interaction in JEG-3 …

Structure And Function Analysis Of Lin-14, A Temporal Regulator Of Postembryonic Developmental Events In Caenorhabditis Elegans, Yang Hong, Rosalind C. Lee, Victor Ambros

Dartmouth Scholarship

During postembryonic development of Caenorhabditis elegans, the heterochronic gene lin-14 controls the timing of developmental events in diverse cell types. Three alternativelin-14 transcripts are predicted to encode isoforms of a novel nuclear protein that differ in their amino-terminal domains. In this paper, we report that the alternative amino-terminal domains of LIN-14 are dispensable and that a carboxy-terminal region within exons 9 to 13 is necessary and sufficient for in vivo LIN-14 function. A transgene capable of expressing only one of the three alternativelin-14 gene products rescues a lin-14 null mutation and is developmentally regulated by lin-4. …

A Defect In Interleukin 12-Induced Activation And Interferon Gamma Secretion Of Peripheral Natural Killer T Cells In Nonobese Diabetic Mice Suggests New Pathogenic Mechanisms For Insulin-Dependent Diabetes Mellitus., Marika Falcone, Brian Yeung, Lee Tucker, Enrique Rodriguez, Nora Sarvetnick

Journal Articles: Regenerative Medicine

The function of natural killer T (NKT) cells in the immune system has yet to be determined. There is some evidence that their defect is associated with autoimmunity, but it is still unclear how they play a role in regulating the pathogenesis of T cell-mediated autoimmune diseases. It was originally proposed that NKT cells could control autoimmunity by shifting the cytokine profile of autoimmune T cells toward a protective T helper 2 cell (Th2) type. However, it is now clear that the major function of NKT cells in the immune system is not related to their interleukin (IL)-4 secretion. In …

Tissue-Specific Expression And Steroid Hormone Regulation Of Human Gonadotropin -Releasing Hormone (Hgnrh) Gene In Placental Cells (Jeg -3 Cells), Zhengguang Chen

Theses and Dissertations in Biomedical Sciences

Using JEG-3 cells as an in vitro model, I investigated the mechanisms behind the tissue-specific expression and steroid hormone regulation of the hGnRH gene in the human placenta. The hGnRH upstream promoter was found to be functionally active in JEG-3 cells. The DNA sequence responsible for functioning of the upstream promoter in JEG-3 cells is narrowed to a region between –1048 bp and –730 bp. This DNA fragment contains four elements, which can bind with nuclear extract from JEG-3 cells (but not from GT1-7 cells).

Estradiol (E2) represses the hGnRH upstream promoter activity in JEG-3 cells. This inhibition is receptor-mediated, …

Production Of Copper-Complexing Ligands In Response To Elevated Concentrations Of Copper In Marine Synechococcus Spp., Arunsri Chatkaeomorakot Brown

Theses and Dissertations in Biomedical Sciences

Synechococcus spp. (marine cyanobacteria) are extremely sensitive to copper toxicity and can produce high-affinity ligands of unknown structure which form complexes with free cupric ion. These ligands may contribute to the biological control of the levels of free cupric ions in surface seawater. Synechococcus spp. are known to produce metallothioneins (MT) in response to cadmium and zinc stress. In the present study the hypothesis that marine Synechococcus produce MT in response to copper was tested. Three marine Synechococcus spp. i.e., PCC 7001, PCC 73109, and PCC 7003, were exposed to different concentrations of CUSO 4 for various time periods. Size …

Genome-Wide Bioinformatic And Molecular Analysis Of Introns In Saccharomyces Cerevisiae, Marc Spingola, Leslie Grate, David Haussler, Manuel Ares

Biology Department Faculty Works

Introns have typically been discovered in an ad hoc fashion: introns are found as a gene is characterized for other reasons. As complete eukaryotic genome sequences become available, better methods for predicting RNA processing signals in raw sequence will be necessary in order to discover genes and predict their expression. Here we present a catalog of 228 yeast introns, arrived at through a combination of bioinformatic and molecular analysis. Introns annotated in the Saccharomyces Genome Database (SGD) were evaluated, questionable introns were removed after failing a test for splicing in vivo, and known introns absent from the SGD annotation were …

Archaeal Aminoacyl-Trna Synthesis: Unique Determinants Of A Universal Genetic Code?, Michael Ibba, A. W. Curnow, J. Bono, P. A. Rosa, C. R. Woese, D. Söll

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

No abstract provided.

Transcriptional Regulation Of The Bmp2 Gene: Retinoic Acid Induction In F9 Embryonal Carcinoma Cells And Saccharomyces Cerevisiae, Loree C. Heller, Yong Li, Kevin L. Abrams, Melissa B. Rogers

Bioelectrics Publications

Bmp2, a highly conserved member of the transforming growth factor-beta gene family, is crucial for normal development. Retinoic acid, combined with cAMP analogs, sharply induces the Bmp2 mRNA during the differentiation of F9 embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA) also induces the Bmp2 gene in chick limb buds. Since normal Bmp2 expression may require an endogenous retinoid signal and aberrant Bmp2 expression may cause some aspects of RA-induced teratogenesis, we studied the mechanism underlying the induction of Bmp2. Measurements of the Bmp2 mRNA half-life and nuclear run-on assays …

Construction Of An Escherichia Coli Lac Repressor-Based System To Study Human Cytomegalovirus Gene Expression, Laura Fernanda Cageao-Luchetti

Theses and Dissertations in Biomedical Sciences

An Escherichia coli lac repressor-based system was developed to study the roles of human cytomegalovirus (HCMV) genes during viral replication. To this end, a recombinant HCMV expressing the lac repressor was generated (RVlac), and an HCMV-specific promoter was targeted for conditional expression by inserting the lac operator sequence. The promoter of a nonessential gene was chosen in order to be able to assess parameters of repression and derepression of the operator-containing promoter in the endogenous locus, without having virus growth dependent on the specific inducer isopropylthiogalactoside (IPTG). The feasibility of this approach to conditionally express an HCMV promoter …

Development Of 31p Nuclear Magnetic Resonance Methods For The Study Of Phosphate Metabolisms In E. Coli And B. Subtilis, Fabian M. Dayrit, Emilyn Q. Espiritu, Noreen Gonzalez, Nina Rosario L. Rojas, Jennifer T. Aguilan, Antonio M. Basilio, Edward T. Chainani, Enrico Cruz, Bernadette Matanguihan

Environmental Science Faculty Publications

31P NMR experiments were performed on Escherichia coli and Bacillus subtilis at various temperatures under aerobic and anaerobic conditions. The total soluble intracellular phosphate concentration was estimated to be 2 x 10^-17 mole/cell, while intracellular orthophosphate concentration was around 1 x 10^-17 mole/cell. Addition of glucose resulted in a general decrease in intracellular pH and was accompanied by the formation of sugar monophosphates. The concentrations of soluble intracellular phosphates, both inorganic and organic phosphates, were estimated by integration versus methylene diphosphonic acid (MDPA) standard. Although intracellular and extracellular orthophosphate could be observed, these appear to exchange rapidly on the NMR …

Carmoviruses (Tombusviridae), Feng Qu, Thomas Jack Morris

School of Biological Sciences: Faculty Publications

Carmovirus is one of two officially recognized genera of Tombusviridae. Members of Tombusviridae all have icosahedral virions of about 30 nm in diameter with T = 3 symmetry that consists of 180 coat protein (CP) subunits of about 38- 43 kDa and a single-stranded (ss) RNA genome ranging in size from 4.0 to 4.7 kb. Carmoviruses share recognizable sequence similarity in both the polymerase and structural genes with members of the other genus, Tombusvirus. Sequence comparisons and phylogenetic analysis of these genes support the view that the genera are distinct but closely related. In addition, the genome organization of the …

In Vitro Assessment Of The Toxicity Of Cocaine And Its Metabolites In The Human Umbilical Artery, Tessa L. Long

Electronic Theses and Dissertations

An in vitro model was used to assess the effect of cocaine and its metabolites on the umbilical artery. Objectives were to pharmacologically confirm the presence of adrenergic innervation using tyramine, evaluate the ability of cocaine, benzoylecgonine, norcocaine and cocaethylene to potentiate vasoconstriction by serotonin and norepinephrine, examine the ability of ketanserin to block the enhanced vasoconstriction produced by cocaine, and determine displacement of 3 H-ketanserin by serotonin, norepinephrine, tyramine and mianserin. The vasoconstrictive effect of tyramine (100 μM) was enhanced in the presence of cocaine by 257%. Vasoconstrictive effects of serotonin and norepinephrine were significantly enhanced by cocaine by …

Expression, Isolation And Purification Of Human Zona Pellucida Protein 3, Ting-Fung Chi

Theses and Dissertations in Biomedical Sciences

Mammalian fertilization involves interactions of sperm surface receptors with the ligands of the zona pellucida, an extracellular matrix surrounding the ovulated oocytes. In humans, the zona pellucida is composed of three major glycoproteins. One of them, ZP3, participates in the primary sperm binding and in the subsequent triggering of the spermatozoa's acrosome reaction. Studies on the role of this specific protein in the human fertilization process are hampered by the limited amount of available biologically functional proteins.

By use of a pcDNA 3.1(+) expression vector, a transfecting-vector was constructed containing a 1.3 kb histidine tagged hZP3 cDNA. This histidine tagged …

Characterization Of The Biological Functions Of Human Recombinant Zona Pellucida Protein 3, Yu Wen Juan

Theses and Dissertations in Biomedical Sciences

Recombinant human zona pellucida protein 3 (rhZP3), expressed. isolated and purified from PA-1 cells, is characterized for its biological activity and the role in the signal transduction pathway. Characterization of the biological activity of rhZP3 was detected by hemizona assay and immunofluorescence staining of acrosome reaction The results indicated that rhZP3 exhibited an inhibition in the binding assay (HZI 43.6 +/-3.3; n = 9; 30 ng/mL rhZP3) and induction of acrosome reaction (198.6% +/- 77.2% increase from baseline; n = 29; 30 ng/mL rhZP3). It was further confirmed by the transmission electron microscopy that there was no difference in morphology …

Leukemia Inhibitory Factor (Lif): Murine Preimplantation Embryo Development, Implantation Rates, And Skeletal Development, Michael Hayes Mitchell

Theses and Dissertations in Biomedical Sciences

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine which demonstrates perplexing physiological effects. It has been demonstrated that LIF is essential for implantation in mice. Little is known relating to the manner by which LIF effects pre-implantation and post-implantation development. The objectives of this project were to determine the effects LIF on pre-implantation development, to determine the effects that it may have on implantation rates, successful pregnancy rates, and resorption rates, and to determine the effects that LIF has on the skeletal development of mice. For the embryo transfer experiments, embryos were exposed to test compounds in the transfer medium …

Expression, Purification, And Characterization Of Nonstructural Protein 2 (Ns2) Of Bluetongue Virus Serotype 17, Hai Lin

Biology

The NS2 protein of bluetongue virus (BTV) serotype 17 (B17) was expressed in large scale as a Glutathione-S-Transferase (GST) fusion protein in E. coli cells. The size of the expressed NS2/GST fusion protein was about 68 kilodaltons (kDa) when analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This is equal to the sum of the predicted size from the deduced amino acid sequence of the NS2 protein (41 kDa) and the known mass of the GST protein (27 kDa). The NS2/GST fusion protein was purified by absorption to glutathione-conjugated agarose beads (G-beads), and then analyzed on western blots by two …

The Influence Of A Human Repetitive Dna On Genome Stability, Eugenia L. Posey

Electronic Theses and Dissertations

A uniquely human interspersed repetitive DNA sequence family, the L2Hs, are highly polymorphic in human genomes. Several features of interspersed repeated DNA may contribute to the instability observed. Certain motifs (direct repeats, palindromes, and inverted repeats) comprising L2Hs elements may adopt unusual secondary structures such as cruciforms or hairpins. These motifs have been associated with features of genome instability in recombination, insertions and deletions. The L2Hs elements also are AT-rich (76%) compared to the bulk of human DNA (52%). That their dynamic nature (i.e. polymorphisms) may arise from recombination, insertions and deletions has led to the hypothesis that the L2Hs …

Fission Yeast Mitotic Regulator Dsk1 Is An Sr Protein-Specific Kinase, Zhaohua Irene Tang, Mitsuhiro Yanagida, Ren-Jang Lin

WM Keck Science Faculty Papers

Intricate interplay may exist between pre-mRNA splicing and the cell division cycle, and fission yeast Dsk1 appears to play a role in such a connection. Previous genetic analyses have implicated Dsk1 in the regulation of chromosome segregation at the metaphase/anaphase transition. Yet, its protein sequence suggests that Dsk1 may function as a kinase specific for SR proteins, a family of pre-mRNA splicing factors containing arginine-serine repeats. Using an in vitro system with purified components, we showed that Dsk1 phosphorylated human and yeast SR proteins with high specificity. The Dsk1-phosphorylated SF2/ASF protein was recognized strongly by a monoclonal antibody (mAb104) known …

Land Use Patterns In Relation To Lake Water Quality In The Great Pond Watershed, Problems In Environmental Science Course (Biology 493), Colby College, Colby Environmental Assessment Team, Colby College

Colby College Watershed Study: Great Pond (2012, 2010, 1998)

Lakes are natural resources, which have many effects on the land surrounding them, They support adjacent communities by providing water and regulating temperatures, helping to define the surrounding ecosystem, and serving as sources of drinking water as well as recreation. The prolonged presence of human activity in a watershed can disturb the physical and chemical cycles of the lake and its surrounding ecosystems (Henderson-Sellers and Markland 1987). Over time, lakes undergo a process called eutrophication, a natural aging process during which the nutrient levels increase and dissolved oxygen levels decrease (Smith and Smith 1998). As the lake ages or becomes …