Biochemistry, Biophysics, and Structural Biology Commons^™

Construction Of Rhas And Rhar Overexpression Plasmids And The Effects Of Crp Mutants On E.Coli L-Rhamnose Gene Expression, Amber Bromenshenkel, Susan Egan

Honors Capstones

Capstone submitted as a graduation requirement for the BSU Honors Program.

Production Of A Monoclonal Antibody Against Benzo[Α]Pyrene Diol Epoxide Dna Adducts, Brian Peden Austin

Chemistry & Biochemistry Theses & Dissertations

Benzo[α]pyrene is a ubiquitous pollutant produced from the incomplete combustion of organic material such as fossil fuels. It is found in the workplace, urban air, drinking water, and the food supply. Recently, it has been proposed that benzo[α]pyrene may be the causative agent in the formation of lung adenocarcinomas among some Taiwanese women exposed to cooking oil fumes without adequate ventilation. In this study, calf thymus DNA was modified in vitro with benzo[α]pyrene-diol epoxide (BPDE) to a level consistent with that found in biological samples. This DNA of low modification was used as an immunogen in the production of a …

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 …

Genomic Diversity Of Pseudomonas Aeruginosa In Cystic Fibrosis, Nathaniel Edwards Head

Theses, Dissertations and Capstones

Pseudomonas aeruginosa is a common environmental microorganism. However, it has the genetic capacity to cause diseases in patients with compromised host defense systems. Cystic fibrosis (CF) is one of the major hereditary diseases among Caucasian populations. CF patients are born with a defective chloride channel that is responsible for maintaining fluid and electrolyte balance across the lumen of the lung. This imbalance leads to the production of an abnormal dehydrated viscous mucous, prohibiting the patient from normally clearing the respiratory airway. Chronic pulmonary infections with P. aeruginosa are the major causes of high morbidity and mortality in CF. Two of …

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 …

Cbfa2 Is Required For The Formation Of Intra-Aortic Hematopoietic Clusters, Trista North, Ting-Lei Gu, Stacy Terryl, Qing Wang, Louisa Howard, Michael Binder, Miguel Marín-Padilla, Nancy A. Speck

Dartmouth Scholarship

Cbfa2 (AML1) encodes the DNA-binding subunit of a transcription factor in the small family of core-binding factors (CBFs). Cbfa2 is required for the differentiation of all definitive hematopoietic cells, but not for primitive erythropoiesis. Here we show that Cbfa2 is expressed in definitive hematopoietic progenitor cells, and in endothelial cells in sites from which these hematopoietic cells are thought to emerge. Endothelial cells expressing Cbfa2 are in the yolk sac, the vitelline and umbilical arteries, and in the ventral aspect of the dorsal aorta in the aorta/genital ridge/mesonephros (AGM) region. Endothelial cells lining the dorsal aspect of the aorta, and …

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 …

Sex Chromosome Ratios And Aneuploidy Rates In The Zona Pellucida Selected Spermatozoa From Normal And Subfertile Men, Qinuo Fan Van Dyk

Theses and Dissertations in Biomedical Sciences

The human hemizona has been demonstrated to select spermatozoa with good motility, normal morphology, and the capacity to undergo the zona-induced acrosomal reaction. The studies conducted here are directed at using the human hemizona to investigate two key questions: (1) whether human X-chromosome bearing spermatozoa (X-sperm) and Y-chromosome bearing spermatozoa (Y-sperm) differ in their functional survival time (assessed by their capacity to bind to the human zona pellucida) after prolonged in vitro incubation, and (2) whether hemizona-bound spermatozoa have a reduced aneuploidy rate compared to unbound (and therefore, unselected) spermatozoa?

In the functional survival study, donor spermatozoa were held in …

Ms2 Coat Protein Mutants Which Bind Qβ Rna, Marc Spingola, David Peabody

Biology Department Faculty Works

The coat proteins of the RNA phages MS2 and Qβ are structurally homologous, yet they specifically bind different RNA structures. In an effort to identify the basis of RNA binding specificity we sought to isolate mutants that convert MS2 coat protein to the RNA binding specificity of Qβ. A library of mutations was created which selectively substitutes amino acids within the RNA binding site. Genetic selection for the ability to repress translation from the Qβ translational operator led to the isolation of several MS2 mutants that acquired binding activity for Qβ RNA. Some of these also had reduced abilities to …

Fluorescence In Situ Hybridization Analysis Of Human Embryos Derived From In Vitro And In Vivo Matured Oocytes, Constance Descisciolo

Theses and Dissertations in Biomedical Sciences

Despite adequate hormonal stimulation, oocytes collected for the purpose of in vitro fertilization and embryo transfer display several levels of nuclear maturity. Preovulatory or mature oocytes, technically those that are Metaphase I or II, are inseminated shortly after aspiration and assessed for fertilization the following day. Prophase I oocytes, also called germinal vesicle-bearing or immature oocytes, require a 24-36 hour period in culture before being exposed to spermatozoa. During this time, the majority of Prophase I oocytes complete nuclear maturation in vitro, progressing from germinal vesicle breakdown through first polar body extrusion. If inseminated, many in vitro matured oocytes fertilize …

Oral Cancer Screening, Damon Omar Watson

Honors Theses

Cancers of the oral cavity and pharynx have increased throughout the decades. Since oral cancer represents a small percentage of the total cancers in the United States, it is often overlooked. Though this is the case, the results of this cancer can be devastating. To address this issue, an Oral Cancer Screening was organized on August 14, 1996 at the West Michigan Cancer Center in Kalamazoo. This was a joint collaboration between the author, the West Michigan Cancer Center, and the Kalamazoo Valley Dental Society. This event allowed research to take place concerning the data obtained. Confidential results and information …

Circadian Clock-Controlled Genes Isolated From Neurospora Crassa Are Late Night- To Early Morning-Specific, Deborah Bell-Pedersen, Mari L. Shinohara, Jennifer J. Loros, Jay C. Dunlap

Dartmouth Scholarship

An endogenous circadian biological clock controls the temporal aspects of life in most organisms, including rhythmic control of genes involved in clock output pathways. In the fungus Neurospora crassa, one pathway known to be under control of the clock is asexual spore (conidia) development. To understand more fully the processes that are regulated by the N. crassa circadian clock, systematic screens were carried out for genes that oscillate at the transcriptional level. Time-of-day-specific cDNA libraries were generated and used in differential screens to identify six new clock-controlled genes (ccgs). Transcripts specific for each of the ccgs …

Rosalind Elsie Franklin (1920-1958) Biologist, Margaret Sylvia

Faculty Publications

A biography of the biologist Rosalind Elsie Franklin whose work on x-ray crystallography contributed to Watson and Crick's elucidation of the structure of DNA.

Investigation Of The Substrate Recognition Characteristics And Kinetics Of Mammalian Mitochondrial Dna Topoisomerase I, Zeki Topcu

Theses and Dissertations in Biomedical Sciences

Topoisomerases are DNA-modifying enzymes found in prokaryotes, eukaryotes, viruses and organelles such as chloroplast and mitochondria. Information about these enzymes in eukaryotic systems is mostly limited to nuclear enzymes, although our laboratory has been characterizing the biochemical and biophysical properties of the mammalian mitochondrial topoisomerases. We have determined the polarity of the attachment of mitochondrial topoisomerase I to its substrate DNA. To study the substrate preference and kinetic parameters of mitochondrial topoisomerase I, selected regions of mammalian mitochondrial DNA (mtDNA) were inserted into pGEM plasmid vectors following a series of modification and optimization experiments of currently available methods for PCR-cloning. …

Identification And Characterization Of Mitochondrial Dna Variants In Alzheimer's Disease, Natasha Singh Hamblet

Theses and Dissertations in Biomedical Sciences

Alzheimer's Disease (AD) is a complex neurodegenerative disorder that affects a significant portion of the human population regardless of ethnicity or gender. A mitochondrial hypothesis of AD has been proposed based on a number of studies which establish altered oxidative phosphorylation (OXPHOS) and ATP synthesis in AD tissue. ATP demand is most prevalent in the brain; damage to OXPHOS could severely impair brain metabolism, thereby leading to a decline in cognitive function. Four out of five complexes in the OXPHOS pathway are partly encoded by mitochondrial DNA (mtDNA); thus, this may be a crucial site of lesions that alter brain …

Identification And Characterization Of Genes Associated With V-Jun Induced Cell Transformation, Martin Toralballa Hadman

Biological Sciences Theses & Dissertations

The v-jun oncogene was initially identified as the causative agent for fibrosarcomas in chickens. Studies show that overexpression of v-Jun proteins transforms chicken embryo fibroblasts (CEF) in vitro, and forms tumors in chickens in vivo. The mechanisms for this are not clearly defined. Conceivably, overexpression of an unregulated transcription factor would cause cell transfonnation by illicit regulation of its target genes. In support of this, we show that in vivo v-Jun complexes exhibit differential binding to in vitro generated AP-1 and 'AP-1 like' target sequences, suggesting that the pattern of target gene expression is altered during cell transformation. …

Pathogenicity Of Murine Cytomegalovirus Mutants, Victoria Jean Cavanaugh

Theses and Dissertations in Biomedical Sciences

The purpose of this study was to identified nonessential murine cytomegalovirus (MCMV) genes involved in pathogenesis in vivo. Our approach to identifyjng these genes consisted of constructing MCMV mutants, and then analyzing these mutants in vitro and in vivo. Recombinant viruses (RV) expressing the β-glucuronidase marker gene were constructed by site-directed insertion and deletion mutagenesis of the MCMV Hind III-J and -I regions of the viral genome. Mutations were targeted to this region of the MCMV genome because the corresponding region of the human CMV genome is nonessential and is involved in down-regulating major histocompatibility complex (MHC) class I expression …

An Analysis Of Mitochondrial Dna In Rett Syndrome And Other Neurodegenerative Disorders, Catherine Erickson Burgess

Theses and Dissertations in Biomedical Sciences

Mitochondrial dysfunction resulting from mutations on mitochondrial DNA (mtDNA) is being recognized in a growing spectrum of diseases. These diseases, resulting from single base mutations, large deletions, or insertions, have been largely neuromuscular in origin. However, as an understanding of the effects of mtDNA mutations progresses, attention is now focusing on neurodegenerative diseases. Rett Syndrome (RS), a progressive neurodegenerative disease with predominantly female cases, demonstrates morphologic mitochondrial changes, mitochondrial enzyme deficiencies and maternal inheritance (characteristic of mtDNA diseases). No investigation of mtDNA involvement has been previously conducted and, to date, no biological marker exists for this disorder.

Our preliminary studies …

Saccharomyces Cerevisiae Sec59 Cells Are Deficient In Dolichol Kinase Activity, Loree Heller, Peter Orlean, W. Lee Adair Jr.

Bioelectrics Publications

The temperature-sensitive Saccharomyces cerevisiae mutant sec59 accumulates inactive and incompletely glycosylated protein precursors in its endoplasmic reticulum at the restrictive temperature. O-mannosylation and glycosyl phosphatidylinositol membrane anchoring of protein are also abolished, consistent with a deficiency in dolichyl phosphate mannose. Membranes prepared from sec59 cells that had been shifted to the restrictive temperature, however, made normal amounts of dolichyl phosphate mannose when exogenous dolichyl phosphate was supplied, but dolichyl phosphate mannose synthesis was severely depressed in the absence of exogenous dolichyl phosphate. Quantitative measurements of dolichyl phosphate in sec59 cells showed that the levels were decreased to 48% of wild …

A Temperature-Sensitive Mutant Of Escherichia Coli Affected In The Alpha Subunit Of Rna Polymerase, Majid Mehrpouyan

Electronic Theses and Dissertations

A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase has been investigated. Gene mapping and complementation experiments placed the mutation to temperature-sensitivity within the alpha operon at 72 min on the bacterial chromosome. The rate of RNA synthesis in vivo and the accumulation of ribosomal RNA were significantly reduced in the mutant at 44$\sp\circ$C. The thermostability at 44$\sp\circ$C of the purified holoenzyme from mutant cells was about 20% of that of the normal enzyme. Assays with T7 DNA as a template showed that the fraction of active enzyme competent for transcription was reduced as a …

The Use Of Synthetic Oligodeoxyribonucleotides In Direct Analysis Of Single Nucleotide Variations And Inhibition Of Eukaryotic Gene Expression, Daniel Y. Wu

Loma Linda University Electronic Theses, Dissertations & Projects

The present thesis work contributes to two areas of molecular genetics, namely the development of methods for identifying single nucleotides and the understanding of the mechanism by which antisense oligonucleotides inhibit eukaryotic gene expression. Three single nucleotide detection methods are presented. The first method utilizes hybridization with synthetic oligonucleotide probes to detect single or multiple nucleotide differences between alleles in genomic DNA without electrophoretic separation. Total genomic DNA (either digested or undigested with restriction endonucleases) was immobilized in depressions in an agarose gel (in situ dots) and hybridized with radiolabeled, allele-specific oligonucleotide probes. We show that this method can be …

Variants And Polymorphisms Of Three Repetitive Dna Families In The Human Genome, Robert M. Roudabush

Electronic Theses and Dissertations

A novel 0.6 kb LINE family in human DNA, designated L2Hs, has been described (Musich and Dykes 1986). Studies employing clone N6.4, containing three 0.6 kb segments of this family, indicate that these sequences are interspersed and moderately repetitive. Two additional variant sequences of the L2Hs family, N6.1 and N6.3, have been identified. Restriction mapping of each cloned segment indicates similarities among N6.4, N6.3 and N6.1. When the cloned DNAs were cleaved with restriction enzymes and subjected to cross-hybridization, each cloned insert produced a pattern indicating that the sequences contained in N6.1 and N6.3 are represented in at least one …

The Characterization Of Ribosomal Rna Gene Chromatin From Physarum Polycephalum, Sally A. Amero, Vicky L. Montoya, Wendy L. Murdoch, Roy C. Ogle, John L. Keating, Robert M. Grainger

School of Medical Diagnostics & Translational Sciences Publications

We have isolated ribosomal RNA gene (rDNA) chromatin from Physarum polycephalum using a nucleolar isolation procedure that minimizes protein loss from chromatin and, subsequently, either agarose gel electrophoresis or metrizamide gradient centrifugation to purify this chromatin fraction (Amero, S. A., Ogle, R. C., Keating, J. L., Montoya, V. L., Murdoch, W. L., and Grainger, R. M. (1988) J. Biol. Chem. 263, 10725-10733). Metrizamide-purified rDNA chromatin obtained from nucleoli isolated according to the new procedure has a core histone/DNA ratio of 0.77:1. The major core histone classes comigrate electrophoretically with their nuclear counterparts on Triton-acid-urea/sodium dodecyl sulfate two-dimensional gels, although they …

The Purification Of Ribosomal Rna Gene Chromatin From Physarum Polycephalum, Sally A. Amero, Roy C. Ogle, John L. Keating, Vicky L. Montoya, Wendy L. Murdoch, Robert M. Grainger

School of Medical Diagnostics & Translational Sciences Publications

We have undertaken the purification of ribosomal RNA gene (rDNA) chromatin from the slime mold Physarum polycephalum, in order to study its chromatin structure. In this organism rDNA exists in nucleoli as highly repeated minichromosomes, and one can obtain crude chromatin fractions highly enriched in rDNA from isolated nucleoli. We first developed a nucleolar isolation method utilizing polyamines as stabilization agents that results in a chromatin fraction containing far more protein than is obtained by the more commonly used divalent cation isolation methods. The latter method appears to result in extensive histone loss during chromatin isolations. Two methods were then …

An Improved Method For Transferring Nucleic Acids To Nylon Membranes, Bruce D. Parker

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Methods currently in use for the separation and identification of specific segments of nucleic acids involve long transfer periods or elaborate apparatuses and result in the production of a single blot. Contamination by organisms or enzymes is always a factor to be dealt with. An improved method for transferring nucleic acids from acrylamide or agarose gels for use in hybridization has been developed. This method uses NaOH as the blotting medium to improve the rate and efficiency of transfer to nylon membranes. As many as six blots can be obtained within one hour using this method. This method is effective …

Photoreactivation Of Lethal Damage Induced In Hamster X Xenopus Hybrid Cells And Their Parentals By Uv Light, David Bohlender, Stuart Williams, Emir Cruz, H. Gaston Griggs

Journal of the Arkansas Academy of Science

A85 Xenopus cells that exhibited a high level of photoreactivation (PR) and V79B2 hamster cells that exhibited little PR were fused to produce the V79B2 x A85 cell line — a hybrid line which possessed a relatively stable karyotype, with most cells containing the entire V79B2 and A85 genomes. UV and UV plus PR fluence-survival relations were then determined and compared for the hybrid and parental lines in a first attempt to elucidate interactions of the parental PR mechanisms in the hybrid. It was anticipated that the A85 genome in the hybrid would produce PR enzyme in sufficient concentration and …