Biochemistry, Biophysics, and Structural Biology Commons^™

31p Nmr Of Backbone Conformation And Dynamics In Dna At Cre Binding Site In Terms Of Sequence Context, Kelly A. Garton

Scripps Senior Theses

The Cre sequence (ACGT) is a site responsible for the binding of specific transcription factors that determine the activation of genes. Due to its major role in gene transcription, it has become a subject of immense research. The binding of transcription factors to the Cre binding site has been determined to be dependent on DNA conformation. In this study, the effects of flanking sequence around the Cre binding site on the conformation and the dynamics of DNA were investigated. The Cre binding site was studied in its native form with differing flanking sequences to determine the BI/BII profile (conformation) and …

Cooperative Cluster Formation, Dna Bending And Base-Flipping By O6-Alkylguanine-Dna Alkyltransferase, Ingrid Tessmer, Manana Melikishvili, Michael G. Fried

Center for Structural Biology Faculty Publications

O⁶-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O⁶-alkylguanine and O⁴-alkylthymine adducts in DNA, protecting the genome and also contributing to the resistance of tumors to chemotherapeutic alkylating agents. AGT binds DNA cooperatively, and cooperative interactions are likely to be important in lesion search and repair. We examined morphologies of complexes on long, unmodified DNAs, using analytical ultracentrifugation and atomic force microscopy. AGT formed clusters of ≤11 proteins. Longer clusters, predicted by the McGhee–von Hippel model, were not seen even at high [protein]. Interestingly, torsional stress due to DNA unwinding has the potential to limit cluster size …

Lesion-Specific Dna-Binding And Repair Activities Of Human O⁶-Alkylguanine Dna Alkyltransferase, Manana Melikishvili, Michael G. Fried

Center for Structural Biology Faculty Publications

Binding experiments with alkyl-transfer-active and -inactive mutants of human O⁶-alkylguanine DNA alkyltransferase (AGT) show that it forms an O⁶-methylguanine (6mG)-specific complex on duplex DNA that is distinct from non-specific assemblies previously studied. Specific complexes with duplex DNA have a 2:1 stoichiometry that is formed without accumulation of a 1:1 intermediate. This establishes a role for cooperative interactions in lesion binding. Similar specific complexes could not be detected with single-stranded DNA. The small difference between specific and non-specific binding affinities strongly limits the roles that specific binding can play in the lesion search process. Alkyl-transfer kinetics with …

Artificial And Natural Nucleic Acid Self Assembling Systems, Marcus Wood

Wayne State University Dissertations

Nucleic acids are good candidates for nanomachine construction. They participate in all the processes of life, and so can function as structural building blocks and dynamic catalysts. However, to use nucleic acids as nanomachines, a better understanding of their material properties, how to design structures using them, and their dynamics is needed. We have tried to address these issues, in a small way, with nucleic acid force field development, an attempt at nanostructural design and synthesis using DNA, and a study of the RNA/protein regulatory dynamics of the tryptophan regulatory attenuation protein.

Meiotic Dna Re-Replication And The Recombination Checkpoint, Nicole Ann Najor

Wayne State University Dissertations

Progression through meiosis occurs through a strict sequence of events, so that one round of DNA replication precedes programmed recombination and two nuclear divisions. Cyclin dependent kinase 1 (Cdk1) is required for meiosis, and any disruption in its activity leads to meiotic defects. The Cdk1 inhibitor, Sic1, regulates the G1-S transition in the mitotic cell cycle and the analogous transition in meiosis. We have employed a form of Sic1, Sic1deltaP^HA, that is mutated at multiple phosphorylation sites and resistant to degradation. Meiosis specific expression of Sic1deltaP^HA disrupts Cdk1 activity and leads to significant accumulation of over replicated …

Electroporation-Mediated Delivery Of A Naked Dna Plasmid Expressing Vegf To The Porcine Heart Enhances Protein Expression, W. G. Marshall Jr., B. A. Boone, J. D. Burgos, S. I. Gografe, M. K. Baldwin, M. L. Danielson, M. J. Larson, D. R. Caretto, Y. Cruz, B. Ferraro, L. C. Heller, K. E. Ugen, M. J. Jaroszeski, R. Heller

Bioelectrics Publications

Gene therapy is an attractive method for the treatment of cardiovascular disease. However, using current strategies, induction of gene expression at therapeutic levels is often inefficient. In this study, we show a novel electroporation (EP) method to enhance the delivery of a plasmid expressing an angiogenic growth factor (vascular endothelial growth factor, VEGF), which is a molecule previously documented to stimulate revascularization in coronary artery disease. DNA expression plasmids were delivered in vivo to the porcine heart with or without coadministered EP to determine the potential effect of electrically mediated delivery. The results showed that plasmid delivery through EP significantly …

Due-B, A New Human Dna Replication Protein, Is The Functional Homolog Of S. Cerevisiae Sld3, Jianhong Yao

Browse all Theses and Dissertations

DNA unwinding elements (DUEs) are commonly found at DNA replication origins. The DUE binding protein (DUE-B) is crucial for the initiation of DNA replication in eukaryotes. The unique 59 amino acid C-terminal part of DUE-B shares nearly 50% similarity with yeast the C-terminus of Sld3. DUE-B plays a key role in eukaryotic DNA replication because it is required for the loading of Cdc45, the MCM helicase activator, on chromatin. Here we show that DUE-B, just like yeast Sld3, binds to Cdc45 and TopBP1 through its C-terminus in Sf9 cells and in vitro. We also show that DUE-B, Cdc45 and TopBP1 …

Generation Of A Reporter Library Of Eralpha Interacting Dna Sequences, Cormac Jennings

Masters

Oestrogens are a group of steroid hormones of which oestradiol is the major form. Oestrogens are present in men and women and some of their main physiological roles involve female and male reproduction, bone metabolism and homeostasis. Oestrogens carry out their actions by diffusing across the cell membrane and binding to their receptor called the oestrogen receptor (ER), of which two types exist, ERα and ERβ. ERs regulate gene expression through the binding of DNA, most notably the estrogen response element (ERE). Breast cancer is the most common malignancy in women in industrialised countries and 65 percent of breast cancers …

Atf4 Is An Oxidative Stress–Inducible, Prodeath Transcription Factor In Neurons In Vitro And In Vivo, Philipp Lange, Juan Chavez, John T. Pinto, Giovanni Coppola, Chiao-Wang Sun, Tim Townes, Rajiv Ratan

NYMC Faculty Publications

Oxidative stress is pathogenic in neurological diseases, including stroke. The identity of oxidative stress-inducible transcription factors and their role in propagating the death cascade are not well known. In an in vitro model of oxidative stress, the expression of the bZip transcription factor activating transcription factor 4 (ATF4) was induced by glutathione depletion and localized to the promoter of a putative death gene in neurons. Germline deletion of ATF4 resulted in a profound reduction in oxidative stress-induced gene expression and resistance to oxidative death. In neurons, ATF4 modulates an early, upstream event in the death pathway, as resistance to oxidative …

Intracellular Levels Of Centrin2, Xpc, & Hhr23b Increase After Dna Damage, Nicholas A. Rowland

Honors Capstones

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

Electrical Detection Of The Temperature Induced Melting Transition Of A Dna Hairpin Covalently Attached To Gold Interdigitated Microelectrodes, Greg P. Brewood, Yaswanth Rangineni, Daniel J. Fish, Ashwini Bhandiwad, David R. Evans, Raj Solanki, Albert S. Benight

Chemistry Faculty Publications and Presentations

The temperature induced melting transition of a self-complementary DNA strand covalently attached at the 5' end to the surface of a gold interdigitated microelectrode (GIME) was monitored in a novel, label-free, manner. The structural state of the hairpin was assessed by measuring four different electronic properties of the GIME (capacitance, impedance, dissipation factor and phase angle) as a function of temperature from 25 degrees C to 80 degrees C. Consistent changes in all four electronic properties of the GIME were observed over this temperature range, and attributed to the transition of the attached single-stranded DNA (ssDNA) from an intramolecular, folded …

Water. New Waters And New Life, Juan Enriquez

New England Journal of Public Policy

An excerpt from an article about life sciences taken from the proceedings of the Education for Public Inquiry and International Citizenship (EPIIC) Symposium held at Tufts University in Massachusetts in February 2005 is presented.

Dna-Templated Nanomaterials, Hector Alejandro Becerril-Garcia

Theses and Dissertations

Nanomaterials display interesting physical and chemical properties depending on their shape, size and composition. Self assembly is an intriguing route to producing nanomaterials with controllable compositions and morphologies. DNA has been used to guide the self assembly of materials, resulting in: (1) metal nanowires; (2) metal or semiconductor nanorods; (3) carbon nanotubes; and (4) semiconductor, metal or biological nanoparticles. My work expands the range of DNA templated nanomaterials and develops novel ways of using DNA to pattern nanostructures on surfaces. I have performed the first synthesis of silver nanorods on single stranded DNA, an attractive material for localizing DNA coupled …

A Model Of Dna Knotting And Linking, Erica Flapan, Dorothy Buck

Pomona Faculty Publications and Research

We present a model of how DNA knots and links are formed as a result of a single recombination event, or multiple rounds of (processive) recombination events, starting with an unknotted, unlinked, or a (2,m)-torus knot or link substrate. Given these substrates, according to our model all DNA products of a single recombination event or processive recombination fall into a single family of knots and links.

Beta-Lactamase: An Ideal Reporter System For Monitoring Gene Expression In Live Eukaryotic Cells, Sohail A. Qureshi

Department of Biological & Biomedical Sciences

To gain insightful information about the mechanisms through which genes are activated and repressed requires gene reporter systems that are sensitive, robust, and cost-effective. Although numerous reporter gene technologies are commercially available, none are as sophisticated and user-friendly as beta-lactamase (BLA) when it comes to studying gene expression in live cells. This article presents an overview of the BLA technology and describes how it can be exploited for studying rare events such as homologous recombination in somatic cells and be used to deliver any DNA sequence of choice anywhere within the genome.

Dna Damage Responses In Progeroid Syndromes Arise From Defective Maturation Of Prelamin A, Michael Sinensky, Y. Liu, A. Rusinol, Y. Wang, Y. Zou

Faculty Publications, Biological Sciences

The genetic diseases Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD) arise from accumulation of farnesylated prelamin A because of defects in the lamin A maturation pathway. Both of these diseases exhibit symptoms that can be viewed as accelerated aging. The mechanism by which accumulation of farnesylated prelamin A leads to these accelerated aging phenotypes is not understood. Here we present evidence that in HGPS and RD fibroblasts, DNA damage checkpoints are persistently activated because of the compromise in genomic integrity. Inactivation of checkpoint kinases Ataxia-telangiectasia-mutated (ATM) and ATR (ATM- and Rad3-related) in these patient cells can partially overcome their …

Atm-Dependent Erk Signaling In Response To Dna Double Strand Breaks, Ashraf Khalil

Theses and Dissertations

Ionizing radiation (IR) triggers many signaling pathways stemming from DNA damage, and, independently, from extra-nuclear events. To generate radio-mimetic DNA double-strand breaks (DSBs) without and minimizing the effects on extra-nuclear radiation targets, human (p53+) glioma and carcinoma cells containing bromodeoxyuridine (BrdU)- substituted DNA were treated with Hoechst 33258 followed by long wave-length UV (UV-A) (BrdU photolysis). BrdU photolysis resulted in well-controlled, dose-dependent generation of DSBs equivalent to 0.2 - 20 Gy of IR, as detected by pulse-field gel electrophoresis, accompanied by dose-dependent H2AX phosphorylation at ser-139 and ATM phosphorylation at ser-1981, indicating ATM activation. Furthermore, BrdU photolysis increased phosphorylation of …

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.

The Drosophila Melanogaster Rad54 Homolog, Dmrad54, Is Involved In The Repair Of Radiation Damage And Recombination, Rolf Kooistra, José B. M. Zonneveld, Anja De Jong, Jan C. J. Eeken, Chris J. Osgood, Jean-Marie Buerstedde, Paul H. M. Lohman, Albert Pastink

Biological Sciences Faculty Publications

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRAD54-deficient flies develop normally, but the females …

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.

Production Of Interleukin 10 By Islet Cells Accelerates Immune-Mediated Destruction Of Beta Cells In Nonobese Diabetic Mice., Lise Wogensen, Myung-Shik Lee, Nora Sarvetnick

Journal Articles: Regenerative Medicine

The T helper type 2 (Th2) cell product interleukin 10 (IL-10) inhibits the proliferation and function of Th1 lymphocytes and macrophages (M phi). The nonobese diabetic mouse strain (NOD/Shi) develops a M phi and T cell-dependent autoimmune diabetes that closely resembles human insulin-dependent diabetes mellitus (IDDM). The objective of the present study was to explore the consequences of localized production of IL-10 on diabetes development in NOD/Shi mice. Surprisingly, local production of IL-10 accelerated the onset and increased the prevalence of diabetes, since diabetes developed at 5-10 wk of age in 92% of IL-10 positive I-A beta g7/g7, I-E- mice …

Expression Of Prelamin A Confers Sensitivity Of Dna Biosynthesis To Lovastatin On F9 Teratocarcinoma Cells, Michael Sinensky, T. Mclain, K. Fantle

Faculty Publications, Biological Sciences

No abstract provided.

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 …

Unusual Structure Of A Human Middle Repetitive Dna, Duminda D. Ratnasinghe

Electronic Theses and Dissertations

The L2Hs sequences are a polymorphic, interspersed, middle repetitive DNA family unique to human genomes. Genomic fingerprinting indicates that these DNAs vary from one individual to another and between tissues of the same individual. Sequence analysis reveals that they are AT-rich (76%) and contain many unusual sequence arrangements (palindromes, inverted and direct repeats). These sequence properties confer on the L2Hs elements the potential to fold into non-B-form structures, a characteristic of recombination hot spots. To test this hypothesis carbodiimide, osmium tetroxide and S$\sb1$ nuclease were used as single-strand specific probes to study a recombinant plasmid, pN6.4.39, containing a single L2Hs …

The Role Of The Voltage Gradient In The Agarose Gel Electrophoresis Of Dna, David Wheeler

Theses and Dissertations in Biomedical Sciences

In Part I of this dissertation, empirical equations for predicting DNA mobility during agarose gel electrophoresis (AGE) from voltage gradient are developed from the data of McDonnel (36) for electrophoresis in a 1.6% agarose gel. These equations represented the data well for DNA between 2 and 10 kilobase pairs (KBp) in length. A computer program, called GELSIM, which incorporates these equations is described in Part II. GELSIM was designed to allow researchers to analyze electrophoresis data by predicting the effect on DNA migration of altering the voltage of electrophoresis. In this way, electrophoretic banding patterns produced using different voltages could …

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 …

Molecular Study Of The B19 (Human) Pathogenic Parvovirus, Jamshed Ayub

Theses and Dissertations in Biomedical Sciences

The B19 (human) parvovirus is a small single stranded DNA virus of 5.4 kilobases. B19 is specific for erythroid progenitor cells and has been propagated in vitro only with human erythroid bone marrow. Replication of viral DNA and the viral protein products of B19 appear similar to those of other animal parvoviruses. However, B19 differs from other parvoviruses in some important aspects, which include the initiation of all transcripts at a strong left side promoter (p6) and the absence of a functional internal promoter. B19 has an unusual transcription map which is described in this study.

The transcription map of …

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 …

Transforming Ability Of Bacillus Subtilis Dna Taken Up By Barley Embryos, Christine Tolman Ence

Theses and Dissertations

Purified Bacillus subtilis DNA taken up by one-day-old barley embryos and permanently fixed within the barley cell is shown to have maintained its biological activity in B. subtilis transformation. Cesuim chloride density gradient centrifugation of DNA isolated from the roots of barley embryos after the uptake and incorporation of radioactive, bromouracil-labelled B. subtilis DNA indicates the presence of the bacterial DNA in several discreet bands of well-defined density, whose density is changed by sonication. These preliminary findings concerning the fate of bacterial DNA taken up by barley roots are discussed in terms of the recombination model of Ledoux and Huart …