Molecular Biology Commons^™

Detection Of Insulin Receptor, Epidermal Growth Factor Receptor, And Interleukin-6 On Individual Mouse Embryos By Immuno-Polymerase Chain Reaction, Kun Xu

Electronic Theses and Dissertations

Three series of experiments were conducted to: 1) optimize the conditions for the production of pUC19 plasmid and of biotinylated pUC19 fragments; 2) optimize the conditions for the production of protein A-streptavidin chimera (chimeric protein); and 3) detect soluble antigens [bovine serum albumin (BSA) and interleukin-6 (IL-6)] and membrane-bound antigens [insulin receptor and epidermal growth factor receptor (EGFr)] of mouse embryos by immuno-polymerase chain reaction (I-PCR). The first experimental series, which included bacterial culture, chimeric protein purification, and chimeric protein functional experiments, was performed to investigate the effects of IPTG (isopropyl-β-D-thiogalactopyranoside) induction time and temperature, bacterial culture medium, and protein …

Cold-Temperature Adaptation Of Muscle Creatine Kinase From An Antartic Teleost (Chaenocephalus Aceratus), Paul Winnard Jr.

Electronic Theses and Dissertations

The white muscle of Chuenocephulus aceratus, an Antarctic teleost of the Channicthyidae family, has a compromised glycoiytic capacity and this fish cannot depend on glycolysis for rapid ATP generation For C. aceratus, creatine kinase (CK) and phosphocreatine (PCr) reserves comprise the metabolic pathway that may supplement and overcome this deficiency in energy transduction. Two conditions, low glycolytic capacity and evolution in a chronically cold habitat (-1.86°C), give us reason to believe that C. aceratus muscle CK (MMCK) has been subjected to strong selective pressure. Thus, the hypothesis of this thesis is that MMCK fiom C. aceratus white muscle …

Quinic Acid-Mediated Induction Of Hypovirulence And A Hypovirulence-Associated Double-Stranded Rna In Rhizoctonia Solani, Chunyu Liu

Electronic Theses and Dissertations

This study is a part of a project focused on the relationship between dsRNA and hypovirulence in R. solani. Hypovirulence refers to a condition in which a pathogen has a drastically reduced ability to cause disease. Rhizoctonia solani is a soil-borne pathogen causing diseases in numerous plants. Phenyl acetic acid (PAA), a metabolite of phenylalanine, causes Rhizoctonia disease symptoms on potato in the absence of the pathogen itself. The amount of PAA produced by a hypovirulent isolate is 10% of that produced by virulent isolates. A 3.6 kb dsRNA (M2) has been shown to be associated with hypovirulence in …

Sir3-Dependent Assembly Of Supramolecular Chromatin Structures In Vitro, Philippe T. Georgel, Madeleine A. Palacios Debeer, Gregory Pietz, Catherine A. Fox, Jeffrey C. Hansen

Biological Sciences Faculty Research

Baculovirus-expressed recombinant Sir3p (rSir3p) has been purified to near homogeneity, and its binding to naked DNA, mononucleosomes, and nucleosomal arrays has been characterized in vitro. At stoichiometric levels rSir3p interacts with intact nucleosomal arrays, mononucleosomes, and naked DNA, as evidenced by formation of supershifted species on native agarose gels. Proteolytic removal of the core histone tail domains inhibits but does not completely abolish rSir3p binding to nucleosomal arrays. The linker DNA in the supershifted complexes remains freely accessible to restriction endonuclease digestion, suggesting that both the tail domains and nucleosomal DNA contribute to rSir3p–chromatin interactions. Together these data indicate …

Positive Regulation Of Pka On Human Gonadotropin-Releasing Hormone (Hgnrh) Gene Expression In Human Placental Jeg-3 Cells, Zhaoyang Wen

Chemistry & Biochemistry Theses & Dissertations

Using the human placental choriocarcinoma JEG-3 cell line as an in vitro human placental model, we studied the mechanisms of the PKA positive regulation of the hGnRH gene expression in the human placenta. Studies in JEG3 cells showed that through the PKA catalytic subunit a, human GnRH upstream promoter activity was stimulated by PKA signaling pathway in a cAMP dependent mechanism. The sequence between —202 (Afl II) and —554 (BamH I) base pair in the human GnRH upstream promoter region appeared to be responsible for the PKA positive regulation of the gene expression. Furthermore, Western blot analysis demonstrated the involvement …

Interactions Among Murine Cytomegalovirus Us22 Family Gene Products That Influence Viral Pathogenesis, Zaruhi Karabekian

Theses and Dissertations in Biomedical Sciences

Cytomegalovirus (CMV) is a complex, ubiquitous herpesvirus that is characterized by acute, chronic, and latent infections. Monocytes-macrophages are the key target cell type involved in pathogenesis, which is most effectively studied using the murine model of CMV infection. Previously three murine CMV (MCMV) genes (M139, M140, and M141) were identified to regulate viral expression in cultured macrophages and in mice. These genes are members of the US22 gene family with respect to HCMV homology. There is no function assigned to the proteins encoded by these genes. However, deletion of M139, M140, and M141 significantly curtails growth of MCMV in macrophages …

Regulation Of The Hcmv Ul98 Promoter By Cooperation Of The Cyclic Amp Response Element And Gamma Interferon Response Element, Tabmitha Patricia Young Jervey

Theses and Dissertations in Biomedical Sciences

The HCMV UL98 early alkaline exonuclease gene promoter was examined to determine the DNA sequences as well as viral and/or cellular proteins functional in the regulation of this early gene. To assess promoter activation, UL98 promoter sequences were first cloned upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with expression plasmids which express the HCMV major immediate early (IE) proteins 1E72 and 1E86. To more specifically determine the importance of individual cis-acting elements in UL98 promoter activation, the promoter region underwent mutagenesis to delete or alter sequences. The variant promoters were again cloned into a reporter-CAT construct and …

Studies On The Formation Of Dna-Cationic Lipid Composite Films And Dna Hybridization In The Composites, Murali Sastry, Vidya Ramakrishnan, Mrunalini Pattarkine, Krishna N. Ganesh

Faculty Works

The formation of composite films of double-stranded DNA and cationic lipid molecules (octadecylamine, ODA) and the hybridization of complementary single-stranded DNA molecules in such composite films are demonstrated. The immobilization of DNA is accomplished by simple immersion of a thermally evaporated ODA film in the DNA solution at close to physiological pH. The entrapment of the DNA molecules in the cationic lipid film is dominated by attractive electrostatic interaction between the negatively charged phosphate backbone of the DNA molecules and the protonated amine molecules in the thermally evaporated film and has been quantified using quartz crystal microgravimetry (QCM). Fluorescence studies …

Detection Of Aneuploidy For Chromosomes 7 And 8 Using Fluorescence In Situ Hybridization In Patients With Aplastic Anemia And Sequencing Of The Mitotic Checkpoint Gene Hbub1, Laura Jane Aridgides

Theses and Dissertations in Biomedical Sciences

Aplastic anemia (AA) is characterized by complete bone marrow failure. Progression to myelodysplastic syndromes (MDS) and acute nonlymphocytic leukemia (ANLL) occurs frequently. At the time of transformation, cytogenetic abnormalities are common. Detection of cytogenetic abnormalities prior to leukemic transformation may indicate future disease progression. Karyotype analysis is the current method of choice to evaluate chromosome aberrations. However, fluorescence in situ hybridization (FISH) is more sensitive in detecting these abnormalities.

hBUB1, a mitotic spindle checkpoint gene, was shown to be mutated in two colorectal cancer cell lines with high levels of aneuploidy (Cahill, et al., 1998). Although theoretically possible, conclusive …

Nuclear Export Of 60s Ribosomal Subunits Depends On Xpo1p And Requires A Nuclear Export Sequence-Containing Factor, Nmd3p, That Associates With The Large Subunit Protein Rpl10p, Olivier Gadal, Daniela Strau, Jacques Kessl, Bernard Trumpower

Dartmouth Scholarship

Nuclear export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified. Using a large subunit reporter (Rpl25p-eGFP), we have isolated several temperature-sensitive ribosomal export (rix) mutants. One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits. We find that thermosensitive nmd3 mutants are impaired in large subunit export. Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is exported by the nuclear export receptor Xpo1p. Moreover, we show that export of 60S subunits is Xpo1p dependent. We …

Protein Targeting And Translocation, Barry D. Bruce, D.A. Phoenix

Faculty Publications and Other Works -- Biochemistry, Cellular and Molecular Biology

No abstract provided.

Phylogeographic Analysis Of The Threatened And Endangered Superconglutinate-Producing Mussels Of The Genus Lampsilis (Bivalvia: Unionidae), Kevin J. Roe, Charles Lydeard, Paul D. Hartfield

Kevin J. Roe

Several species of freshwater unionid mussels in the genus Lampsilis exhibit a remarkable reproductive strategy. Female mussels of these species enclose their larvae in a minnow-like lure, called a ‘superconglutinate’, to attract piscivorous fishes. When a fish attempts to ingest the superconglutinate the lure ruptures and the larvae are released to parasitize the fish. Of the four species of mussel which exhibit this strategy and are endemic to the Gulf Coast drainages of the southeastern United States, three are protected under the Endangered Species Act, and one is recognized as imperilled. Phylogenetic analysis of nucleotide sequences of the mitochondrial 16S …

Abnormal Expression Of P27kip1 Protein In Levator Ani Muscle Of Aging Women With Pelvic Floor Disorders – A Relationship To The Cellular Differentiation And Degeneration, Antonin Bukovsky, Pleas Copas, Michael R. Caudle, Maria Cekanova Ms, Rndr, Phd, Tamara Dassanayake, Bridgett Asbury, Stuart E. Van Meter, Robert F. Elder, Jeffrey B. Brown, Stephanie B. Cross

Maria Cekanova MS, RNDr, PhD

Background Pelvic floor disorders affect almost 50% of aging women. An important role in the pelvic floor support belongs to the levator ani muscle. The p27/kip1 (p27) protein, multifunctional cyclin-dependent kinase inhibitor, shows changing expression in differentiating skeletal muscle cells during development, and relatively high levels of p27 RNA were detected in the normal human skeletal muscles.

Methods Biopsy samples of levator ani muscle were obtained from 22 symptomatic patients with stress urinary incontinence, pelvic organ prolapse, and overlaps (age range 38–74), and nine asymptomatic women (age 31–49). Cryostat sections were investigated for p27 protein expression and type I (slow …

Protein Targeting And Translocation, Barry D. Bruce, D.A. Phoenix

Barry D. Bruce

No abstract provided.

Voltage Gating Of A Model Membrane Spanning Channel, Eric Miller

Inquiry: The University of Arkansas Undergraduate Research Journal

Channels in cell membranes are important for intercellular communication and especially for the function of the nervous system in higher vertebrates. These channels consist of proteins made from the 20 common amino acids. Channel proteins are embedded into the lipid bilayer membranes of living cells and function by allowing the specific passage of a positively charged material such as sodium or potassium ions across the membrane in response to an external signal. The external signaloeither a chemical signal or a voltageoregulates the opening and closing of channels. In an attempt to understand the voltage-dependent opening of channels (igatingi), we are …

Molecular Characterization Of Cultured Perkinsus Marinus Isolates, Gwynne D. Brown

Dissertations, Theses, and Masters Projects

Perkinsus marinus is the causative agent of the oyster disease Dermo in the eastern oyster, Crassostrea virginica. In vitro propagation of the parasite has led to the establishment of multiple isolates by several investigators. Little work, however, has been done to characterize different isolates. In this study multiple isolates were examined for genetic and biochemical diversity. at two loci, the ITS region and the ATAN region, there was as much intra-isolate genetic variation among DNA sequences of some isolates as there was inter-isolate variation. Variation was also observed at a third loci, a subtilisin-like serine protease gene. This is the …

Adrenal Expression Of Cyp17 And Hsd3b1 Mrna In Peromyscus Maniculatus Bairdii, Brian Thomas Lovitt

Dissertations, Theses, and Masters Projects

No abstract provided.

Cationic Surfactant Mediated Hybridization And Hydrophobization Of Dna Molecules At The Liquid/Liquid Interface And Their Phase Transfer, Murali Sastry, Ashavani Kumar, Mrunalini Pattarkine, Vidya Ramakrishnan, Krishna N. Ganesh

Faculty Works

Hybridization of complementary oligonucleotides mediated by a cationic surfactant at the water/hexane interface leads to hydrophobic, double-helical DNA which may be readily phase transferred to the organic phase and cast into thin films on solid substrates.

Characterization Of The Human Cytomegalovirus Ul75 Late Gene Promoter, Bernard J. P. Mcwatters

Theses and Dissertations in Biomedical Sciences

Gene expression during productive infection by the human cytomegalovirus (HCMV) occurs in an ordered and sequential manner, beginning with immediate early (IE), then early (E) and finally late (L) gene expression. Significant work has addressed the regulation of IE and E gene expression while relatively little work has addressed the control of late gene expression. In order to further address HCMV late gene expression, the promoter of the HCMV UL75 (glycoprotein H, gH) late gene was characterized. The data obtained in this study were combined with observations made in two other studies that have addressed HCMV late gene expression to …

Characterization Of The Biological Activities Of Recombinant Fusion Protein Green Fluorescent Protein/Human Zona Pellucida Protein 3 (Gfp/Hzp3), Zhiyong Lin

Theses and Dissertations in Biomedical Sciences

Despite numerous reports indicating the successful production of bioactive recombinant ZP3, no report has shown the rhZP3 having direct binding activity with human sperm. Recombinant ZP3 generated from our previous study displayed binding activity with human sperm through indirect evidence from hemizona assay (HZA).

This present study focused on the production of recombinant ZP3 with direct binding activity with human sperm. Through the application of a pEGFP expression vector, fusion protein GFP/ZP3 was successfully generated and expressed. The expression of GFP/ZP3 was evidenced by RT-PCR and western blot. The fusion protein was partially purified by Ni-NTA affinity column from cell …