Biochemistry, Biophysics, and Structural Biology Commons^™

Interactions Involving The Human Rna Polymerase Ii Transcription/Nucleotide Excision Repair Complex Tfiih, The Nucleotide Excision Repair Protein Xpg, And Cockayne Syndrome Group B (Csb) Protein, Narayan Iyer, Michael S. Reagan, Kou-Juey Wu, Bertram Canagarajah, Errol C. Friedberg

Biology Faculty Publications

The human basal transcription factor TFIIH plays a central role in two distinct processes. TFIIH is an obligatory component of the RNA polymerase II (RNAP II) transcription initiation complex. Additionally, it is believed to be the core structure around which some if not all the components of the nucleotide excision repair (NER) machinery assemble to constitute a nucleotide excision repairosome. At least two of the subunits of TFIIH (XPB and XPD proteins) are implicated in the disease xeroderma pigmentosum (XP). We have exploited the availability of the cloned XPB, XPD, p62, p44, and p34 genes (all …

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 …

Molecular Cloning And Rare Cleavage Mapping Of Human 2p, 6q, 8q, 12q, And 18q Telomeres, Roberto A. Macina, Ken Morii, Xue-Lan Hu, Dimitri G. Negorev, Chrysanthe Spais, Lisa A. Ruthig, Harold C. Riethman

School of Medical Diagnostics & Translational Sciences Publications

Large terminal fragments of human chromosomes 2p, 6q, 8q, 12q, and 18q were cloned using yeast artificial chromosomes (YACs). RecA-assisted restriction endonuclease (RARE) cleavage analysis of genomic DNA samples from 11 unrelated individuals using YAC-derived probes confirmed the telomeric localizations of the half-YACs studied. The cloned Fragments provide telomeric closure of maps for the respective chromosome arms and will supply the reagents needed for analyzing and sequencing these distal subtelomeric regions.

A Study Of Euplotes Crassus Telomere Proteins And Related Genes, John Scott Perez

Department of Chemistry: Dissertations, Theses, and Student Research

A novel PCR technique used to amplify Euplotes crassus macronuclear chromosomes was developed to provide scientific proof that an Euplotes crassus 1.0 kb gene is not genetically related to an Oxytricha nova 1.8 kb β-telomere protein gene. Theories and experimental procedures associated with the development of this PCR technique were the product of research that will have a profound impact on future studies of telomere protein genes and telomeric DNA in a multitude of eukaryotic cells. The hypothesis that a β-telomere protein does not exist in Euplotes crassus was supported by the result of this study.

Expression of the recombinant …

Circadian Clock Locus Frequency: Protein Encoded By A Single Open Reading Frame Defines Period Length And Temperature Compensation., Benjamin D. Aronson, Keith A. Johnson, Jay C. Dunlap

Dartmouth Scholarship

The frequency (frq) locus encodes a key component, a state variable, in a cellular oscillator generating circadian rhythmicity. Two transcripts have been mapped to this region, and data presented here are consistent with the existence of a third transcript. Analysis of cDNA clones and clock mutants from this region focuses attention on one transcript encoding a protein. FRQ, which is a central clock component: (i) mutations in all of the semidominant frq alleles are the result of single amino acid substitutions and map to the open reading frame (ORF) encoding FRQ; (ii) deletion of this ORF, or a frameshift mutation …

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 …

Gal4 Disrupts A Repressing Nucleosome During Activation Of Gal1 Transcription In Vivo, Jeffrey D. Axelrod, Michael S. Reagan, John Majors

Biology Faculty Publications

Photofootprinting in vivo of GALl reveals an activation- dependent pattern between the UASG and the TATA box, in a sequence not required for transcriptional activation by GAL4. The pattern results from a nucleosome whose position depends on sequences within the UASG. In the wild-type gene, activation by GAL4 and derivatives disrupts this nucleosome. This activity is independent of interactions with DNA-bound core transcription factors and is proportional to the strength of the activator. Presence of the nucleosome correlates with low basal transcription levels under various conditions, suggesting a role in limiting basal expression. We propose a role for the GAL4 …

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 …

The Cγ Subunit Is A Unique Isozyme Of The Camp-Dependent Protein Kinase, Stephen J. Beebe, Paul Salomonsky, Tore Jahnsen, Yixin Li

Bioelectrics Publications

There are at least three isozymes (Cα, Cβ, and Cγ) of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase (PKA) (Beebe, S., Oyen, O., Sandberg, M., Froysa, A., Hansson, V., and Jahnsen, T. (1990) Mol. Endocrinol. 4, 465-475). To compare the Cγ and Cα isozymes, the respective cDNAs were expressed in permanently transformed Kin-8 PKA-deficient Y1 adrenal cells using the mouse metallothionein promoter. The recombinant C subunits were characterized as immunoreactive, zinc-inducible, cAMP-dependent kinase activities. In contrast to Cα, histone was a better substrate than Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) for Cγ. Furthermore, Cγ histone kinase activity was not inhibited by the …

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 …

Effects Of Chemical Aneuploidogens On Taxol Purified Drosophila And Mouse Brain Microtubules Polymerization And Depolymerization In Vitro, Anil Sehgal

Biological Sciences Theses & Dissertations

The effects of aneuploidogens (aneuploidy causing agents) on taxol-purified microtubules from Drosophila and mouse brain in vitro were studied by using a spectrophotometric assay and electron microscopy. Colchicine, acetonitrile, propionitrile, acrylonitrile, dimethyl sulfoxide (DMSO), griseofulvin and cadmium chloride inhibited microtubule polymerization whereas methoxyethyl acetate (MEA) and methyl mercuric chloride (MMC) did not. All aneuploidogens tested (at 50mM) resulted in reduced rate of elongation of mouse brain microtubules. MMC, cadmium chloride and DMSO resulted in increased rates of Drosophila microtubule elongation whereas the rest of the drugs resulted in decreases. The in vitro results from Drosophila correlate well with the previously …

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 …

Isolation And Partial Characterization Of Basic, Sperm-Specific, Nuclear Proteins In The Dog And Hampster, Richard William Hall

Biological Sciences Theses & Dissertations

The purpose was to determine whether dogs and hamsters contain the same sperm-specific protamine and testis specific proteins (TP) as other eutherian mammals previously studied. The proteins (protamine and TPs) were isolated from spermatids and mature spermatozoa, and were subjected to electrophoresis in an acid-urea, polyacrylamide gel system according to the procedure of Panyim and Chalkley (1962). The results indicated that, regardless of the anatomical location of sperm acquisition, acid precipitation technique, or denaturing agents used, epididymal and testicular samples from both the dog and hamster contained protamine in elongated spermatids and mature spermatozoa. The protamines from dogs and hamsters …

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 …

Solid Phase Extraction Of Mammalian Cell Mitochondrial Dna And Its Electrophoretic Separation In Agarose Gels, Theresa Brick-Miller

Biological Sciences Theses & Dissertations

An improved and efficient electrophoretic procedure for the isolation of mitochondrial DNA (mtDNA) is described. The solid phase extraction procedure for the isolation of mtDNA involves embedding as few as 200,000 Ehrlich ascites tumor cells into a block of agarose, digestion of cellular membranes by detergent action and subsequent electrophoretic separation of nucleic acids in agarose gels. Genomic DNA remains in the original block of agarose while the mtDNA migrates in the separation gel to a position equivalent to that obtained for mtDNA isolated by traditional procedures.

The efficacy of a large number of detergents, agarose concentrations, embedding conditions, embedding …

A Comparison Of The Acidic Extracellular Polysaccharides From Rhizobium Trifolii Mutants Affected In Trifolium Repens Root Hair Curling, Brian Andrew Hanley

Masters Theses

The compositions and sugar linkages of the acidic extracellular polysaccharide (EPS) from several Rhizobium trifolii mutants were compared. These mutants were derived from a wild type Rhizobium trifolii, ANU843, and are unable to cause normal root hair curling, Hac-, or nodulate clover, Nod-. Three of the mutants contain a Tn5 insertion in a 5.3 kb BglII fragment of the symbiotic plasmid (pSym). A fourth mutant is missing the pSym, which includes much of the 5.3 kb BglII fragment, both of these deletions result in Hac-Nod- phenotypes. When the 5.3 kb BglII fragment is cloned (pRT150) and reinserted into the …

The Association Of 5.8 S With 28s Ribosomal Rna, Nandita Banerjee

Chemistry & Biochemistry Theses & Dissertations

5.8S rRNA is a low molecular weight ribosomal RNA which is noncovalently bonded to the larger ribosomal subunit 28S rRNA; through its 3' end and through its 5' end. This interaction is an integral part of the ribosome, and plays an important role in the ribosome structure and function.

There is a high degree of homology between the 5.8S rRNA primary structures of rat, turtle and chicken. The base sequence of rat 5.8S rRNA differs only in one position from that of turtle and in three positions fr.om that of cl1.icken. Tl1ere is a single purine substitution at the 5' …