<p>Pyrosequencing is a DNA sequencing method based on thedetection of released pyrophosphate (PPi) during DNA synthesis.In a cascade of enzymatic reactions, visible light isgenerated, which is proportional to the number of nucleotidesincorporated into the DNA template. When dNTP(s) areincorporated into the DNA template, inorganic PPi is released.The released PPi is converted to ATP by ATP sulfurylase, whichprovides the energy to luciferase to oxidize luciferin andgenerate light. The excess of dNTP(s) and the ATP produced areremoved by the nucleotide degrading enzyme apyrase.</p><p>The commercially available enzymes, isolated from nativesources, show batch-tobatch variations in activity and quality,which decrease the efficiency of the Pyrosequencing reaction.Therefore, the aim of the research presented in this thesis wasto develop methods to recombinantly produce the enzymes used inthe Pyrosequencing method. Production of the nucleotidedegrading enzyme apyrase by Pichia pastoris expression system,both in small-scale and in an optimized large-scale bioreactor,is described. ATP sulfurylase, the second enzyme in thePyrosequencing reaction, was produced in<i>Escherichia coli</i>. The protein was purified and utilizedin the Pyrosequencing method. Problems associated with enzymecontamination (NDP kinase) and batch-to-batch variations wereeliminated by the use of the recombinant ATP sulfurylase.</p><p>As a first step towards sequencing on chip-format,SSB-(single-strand DNA binding protein)-luciferase and KlenowDNA polymerase-luciferase fusion proteins were generated inorder to immobilize the luciferase onto the DNA template.</p><p>The application field for the Pyrosequencing technology wasexpanded by introduction of a new method for clone checking anda new method for template preparation prior the Pyrosequencingreaction.</p><p><b>Keywords:</b>apyrase, Pyrosequencing technology, Z_basictag fusion, luciferase, ATP sulfurylase, dsDNAsequencing, clone checking, Klenow-luciferase, SSB-luciferase,<i>Pichia pastoris, Echerichia coli</i>.</p>

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains x, 169 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

The human mitochondrial DNA (mtDNA) genome must be faithfully maintained by the mitochondrial DNA replication machinery. Deficiencies in mtDNA maintenance result in the accumulation of mutations and deletions, which have been associated with a number of neuromuscular degenerative disorders including, mtDNA depletion syndrome, Alpers syndrome, progressive external opthalmoplegia (PEO), and sensory ataxic neuropathy, dysarthria, and opthalmoparesis (SANDO). The mtDNA replication machinery is comprised of a nuclearly-encoded DNA polymerase gamma (Pol γ), single-stranded DNA binding protein (mtSSB), and a hexameric mtDNA helicase. In this work, we employed quantitative pre-steady state kinetic techniques to establish the mechanisms responsible for the replication of the human mitochondrial DNA by Pol γ and explored the effects of point mutations that are observed in heritable diseases. With our biochemical characterization of mutants of Pol γ, we have shown unique characteristics that would lead to profound physiological consequences over time. Additionally, we have made significant progress towards reconstitution of the mitochondrial DNA replisome by monitoring DNA polymerization that is dependent on helicase unwinding of double stranded DNA. Overall, this work provides a better understanding of the mechanism of mtDNA replication and has important implications toward understanding the role of mitochondrial DNA replication in mitochondrial disease, ageing and cancer. In addition to the work on the mtDNA replisome, we have applied pre-steady state kinetic techniques to better understand the mechanism of RNA-dependent DNA polymerization by HIV reverse transcriptase (HIV-RT). This enzyme is responsible for the replication of the viral genome in HIV and is a common target for anti-HIV drugs. We have characterized the role of enzyme conformational changes in the kinetics of incorporation of correct nucleotide and the Nucleotide Reverse Transcriptase Inhibitor (NRTI) AZT by wild-type enzyme, as well as a mutant with clinical resistance to AZT. This work provides a better understanding of the complete mechanism of RNA-dependent DNA polymerization, the changes in the mechanism in the presence of inhibitor and the development of resistance to this nucleoside analog; and thereby this work contributes to the long-term goal of designing more effective drugs that can possibly deter resistance and be used successfully for treatment of HIV. / text

The human mitochondrial DNA polymerase (Pol γ) catalyzes mitochondrial DNA synthesis, and thus is essential for the integrity of the organelle. Mutations of Pol γ have been implicated in more than 150 human diseases. Reduced Pol γ activity caused by inhibition of anti-HIV drugs targeted to HIV reverse transcriptase confers major drug toxicity. To illustrate the structural basis for mtDNA replication and facilitate rational design of antiviral drugs, I have determined the crystal structure of human Pol γ holoenzyme. The structure reveals heterotrimer architecture of Pol γ holoenzyme with a monomeric catalytic subunit Pol γA, and a dimeric processivity factor Pol γB. While the polymerase and exonuclease domains in Pol γA present high structural homology with the other members of the DNA Pol I family, the spacer between the two functional domains shows a unique fold, and constitutes the subunit interface. The structure suggests a novel mechanism for Pol γ’s high processivity of DNA replication. Furthermore, the structure reveals dissimilarity in the active sites between Pol γ and HIV RT, thereby indicating an exploitable space for design of less toxic anti-HIV drugs. Interestingly, the structure shows an asymmetric subunit interaction, that is, one monomer of dimeric Pol γB primarily participates in interactions with Pol γA. To understand the roles of each Pol γB monomer, I generated a monomeric human Pol γB variant by disrupting the dimeric interface of the subunit. Comparative studies of this variant and dimeric wild-type Pol γB reveal that each monomer in the dimeric Pol γB makes a distinct contribution to processivity: one monomer (proximal to Pol γA) increases DNA binding affinity whereas the other monomer (distal to Pol γA) enhances the rate of polymerization. The pol γ holoenzyme structure also gives a rationale to establish the genotypic-phenotypic relationship of many disease-implicated mutations, especially for those located outside of the conserved pol or exo domains. Using the structure as a guide, I characterized a substitution of Pol γA residue R232 that is located at the subunit interface but far from either active sites. Kinetic analyses reveal that the mutation has no effect on intrinsic Pol γA activity, but shows functional defects in the holoenzyme, including decreased polymerase activity and increased exonuclease activity, as well as reduced discrimination between mismatched and corrected base pair. Results provide a molecular rationale for the Pol γA-R232 substitution mediated mitochondrial diseases. / text

Molecular biology has become an integral part of the diagnosis of infectious diseases. Recently, quantitative real-time PCR (QPCR) methods (often in the form of so-called TaqMan® systems) have been developed for the diagnosis of a wide range of infectious diseases; these techniques found valuable clinical application in the diagnosis and evaluation of progress and therapeutic success of viral diseases. The use of QPCR as a tool for diagnostic virological and viral research laboratories has greatly increased in recent years. It often replaces conventional PCR and amplicon detection systems which are more complex and laborious, with a higher risk of amplicon carry-over contamination. The new QPCR methods presented here utilize broadly targeted primers and probes for rational and sensitive detection and quantification of variable RNA viruses. They take advantage of the dual properties, both RNA and DNA dependent DNA polymerase activities, of the rTth thermostable polymerase, and thermolabile UNG with dUTP to protect against inadvertent contamination of samples with amplimers. In paper one, a novel QPCR approach to detect and quantify human enteroviral (EV) RNA in patients with neurological disorders such as aseptic meningitis is presented. In the second paper, the development of a novel serological technique, quantitative PCR enhanced immunoassay (QPIA), for serodiagnosis of EV infection, is described. In paper three the subject is the development of a touch-down QPCR (TD-QPCR) for detection and preliminary genogrouping of norovirus (NV), a group of Caliciviruses. In paper four a rational, broadly targeted, system for detection of diverse influenza viruses, yet being able to discriminate between influenza A, B and C, is designed and evaluated. In the last paper, another rational broadly targeted system, for detection of corona viruses in humans and animals, is described. The technologies described in this collection of papers have common features. They are a platform for further development of diagnostic tools for screening and detection of viruses in known viral diseases, maybe also for discovering new viruses.

No description available.

The nucleoside phosphonates cidofovir (CDV) and (S)-9-[3-hydroxy-(2-phosphonomethoxy)propyl]adenine [(S)-HPMPA] are analogs of dCMP and dAMP, respectively. Collectively these drugs are effective inhibitors of a wide range of DNA viruses, RNA viruses, and retroviruses. Because they are nucleotide analogs, the drugs are thought to target viral polymerases and inhibit viral genome replication. However, the precise mechanism by which these drugs block viral growth remains unclear. We have studied the mechanism of action of these antivirals against three viral polymerases, vaccinia virus DNA polymerase and the reverse transcriptases from human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (MMLV). In vitro experiments using the active intracellular metabolites of CDV and (S)-HPMPA, CDV diphosphate (CDVpp) and (S)-HPMPA diphosphate [(S)-HPMPApp], respectively, showed that the drugs are substrates for each enzyme and can be incorporated into DNA without causing chain termination, although the rate of DNA elongation catalyzed by the vaccinia virus and MMLV polymerases is slowed. We have also found that incorporation of CDV or (S)-HPMPA blocked the 3′-to-5′ proofreading exonuclease activity of the vaccinia virus DNA polymerase. In addition, we determined that when these drugs are incorporated into a template DNA strand, they inhibited replication across the drug lesion. These results indicate that although CDV and (S)-HPMPA can inhibit some enzymes when incorporated into the primer strand, the main effects of drug action occur when they are incorporated into the template strand. Our findings point to a new avenue of targeted drug design, one in which nucleoside or nucleotide analogues are efficient substrates for the viral nucleic acid polymerase, do not inhibit primer strand elongation, but exert their effects in subsequent rounds of nucleic acid synthesis. / Virology

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (Ph.D. in Biophysics & Genetics) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 165-185). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [114]-116).