Download or read book Vaccinia Virus DNA Polymerase and Ribonucleotide Reductase written by Donald Brad Gammon and published by . This book was released on 2010 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Vaccinia Virus Ribonucleotide Reductase written by Ralph Eugene Davis and published by . This book was released on 1992 with total page 210 pages. Available in PDF, EPUB and Kindle. Book excerpt: Vaccinia virus infected monkey kidney cells had been previously shown to have an increased ribonucleoside diphosphate reductase (RR) activity. DNA from mutant virus resistant to hydroxyurea were digested with restriction endonucleases and were shown to have substoichiometric amounts of the Hind III F fragment. Additional information from Southern blotting experiments localized the putative small subunit (R2) gene to the left end of the Hind III F fragment of the vaccinia virus genome. The entire open reading frame of the R2 gene and the flanking regions was sequenced and the translated sequence found to be 80% homologous to the mouse R2 polypeptide. A combination of in situ and in vitro experiments addressed the question of macromolecular interactions involving vaccinia ribonucleotide reductase (FIR). Replication of double stranded viral DNA occurs in very discrete loci in infected cells and these DNA factories can be isolated from gently lysed cell in sucrose gradients. RR was detected at low levels (less than 5% of the total R2) with the rapidly sedimenting DNA by using antibodies against FIR. In situ crosslinking experiments were attempted with no specific interaction determined at this time. Immunolocalization experiments have given evidence for localization of large subunit (R1) polypeptide to the viral inclusion bodies. The most conclusive results utilized anti-idiotypic antibodies against the antibodies to R2 protein. lmmunolocalization experiments have shown the putative R2 binding protein to be localized at the sites of viral DNA synthesis. lmmunoprecipitations show a single predominant viral polypeptide which also has proven to be a DNA binding (phospho)protein. Screening a lambda phale expression library of vaccinia with the anti-idiotypic antibody localized the binding site to the carboxy terminal 81 amino acids in open reading frame 1-3 of the vaccinia genome. The open reading frame was cloned into a pET11c expression vector and the partially purified recombinant protein was shown to have specificity for single-stranded DNA as well as stimulate vaccinia RR activity.
Download or read book Poxviruses written by Richard W. Moyer and published by Springer Science & Business Media. This book was released on 2012-12-06 with total page 220 pages. Available in PDF, EPUB and Kindle. Book excerpt: The purpose of this volume is to highlight some current areas of poxvirus research which are likely to be particularly fruitful in the upcoming few years. The first chapter, by Drs. Condit and Niles, discusses poxvirus genetics. Work in this area has provided mutants, produced practical procedures to simplify the manipulation of viral genes, and generated information about the molecular architecture and organization of genes characteristic of pox viruses. One of the most intensively studied regions of the viral genome is the HindIII D region of vaccinia, in which a combination of classical and molecular genetic analysis of the region has been particularly revealing. Within this region are open reading frames, some of which are expressed early and others late, organized in a fashion which is now known to be typical of these viruses. Other studies, related to temperature sensitive, drug resistant, and drug dependent mutants, are also discussed. Each of the other reviews included in this volume summarizes areas of research which have depended heavily on the genetics of the system. The intracellular site of a poxvirus infection is mostly, if not exclusively, limited to the cytoplasm which dictates several interesting biological ramifications. For example, poxvirus transcription must occur in the cytoplasm, rather than in the nucleus. The virus copes with this situation by incorporating into the virion the enzymatic machinery necessary to initiate transcription from input virus.
Download or read book Studies on the Vaccinia Virus DNA Polymerase written by William F. McDonald and published by . This book was released on 1993 with total page 286 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Vaccinia Virus and Poxvirology written by Stuart N. Isaacs and published by Springer Science & Business Media. This book was released on 2008-02-03 with total page 402 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Right Book at the Right Time The poxviruses comprise a family of complex DNA viruses that replicate in the cytoplasm of vertebrate or invertebrate cells. Of the eight recognized g- era of vertebrate poxviruses, those belonging to the orthopoxvirus genus have been most intensively studied. This group includes variola virus, the agent of smallpox, as well as cowpox virus and vaccinia virus. Jenner’s original sma- pox vaccine, described in 1798, consisted of live cowpox virus, but vaccinia virus later replaced it (1). There has been speculation as to the origin of v- cinia virus; the most likely idea is that it is a separate species, possibly ori- nally isolated from a horse, and is now extinct or rare in nature (2). Recent genome sequencing studies confirm the distinctness of variola virus, cowpox virus, and vaccinia virus and also their very close genetic relationship, which accounts for the cross protection of smallpox vaccines. The novelty of the smallpox vaccine can be readily appreciated by the time it took, about 80 years, before the next live vaccine against rabies was developed, and another 50 years for the yellow fever vaccine. Moreover, the eradication of smallpox in 1977 stands as a unique medical achievement. Because of its historical role, sma- pox vaccination contributed greatly to present concepts of infectious disease, immunity, and pathogenesis. Less well known, however, are the many other “firsts” for vaccinia virus.
Download or read book Viral Polymerases and Related Proteins written by Lawrence C. Kuo and published by Elsevier. This book was released on 1996 with total page 698 pages. Available in PDF, EPUB and Kindle. Book excerpt: The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. More than 270 volumes have been published (all of them still in print) and much of the material is relevant even today--truly an essential publication for researchers in all fields of life sciences. Key Features* Expression, purification, and characterization* Activity assays* Kinetic and screening* Design and analysis of substrates and inhibitors* Molecular and structural characterizations.
Download or read book Vaccinia Virus Ribonucleotide Reductase written by Meredith L. Howell and published by . This book was released on 1992 with total page 426 pages. Available in PDF, EPUB and Kindle. Book excerpt: Ribonucleotide reductase is a remarkable enzyme that catalyzes the rate-limiting step in the synthesis of the 2'-deoxynucleoside triphosphates. The intent of this project was to characterize the ribonucleotide reductase encoded by the orthopoxvirus, vaccinia. The first objective was to study the structural and functional features of the viral small subunit protein of ribonucleotide reductase. The viral reductase gene was engineered into an expression vector and expressed in Escherichia coli. The purified recombinant protein was then characterized and compared with other ribonucleotide reductase small subunits from different organisms. The physical characteristics of the vaccinia virus enzyme showed a strong similarity to the features of the mammalian counterpart. A second aim of this project was to establish the transcriptional and translational kinetics of ribonucleotide reductase gene expression during the time course of viral infection in cultured mammalian cells. In addition, the activity and stability of the enzyme in the viral system was measured and the accumulation of ribonucleotide reductase protein was quantitated. By also quantitating the accumulation of viral DNA synthesis, a direct comparison can be made between the the synthesis and utilization of deoxynucleotide precursors. A third objective of this work was to detail the mechanism by which hydroxyurea inactivates the vaccinia virus ribonucleotide reductase. Visible spectroscopy and electron paramagnetic resonance spectroscopy clearly demonstrated that the inhibitor destroys the free radical moiety in the viral small subunit protein. In addition, in vivo studies revealed that inhibition by hydroxyurea can be circumvented during viral infection. The exogenous addition of deoxyadenosine reversed the block to viral growth that was imposed by hydroxyurea, and stabilized hydroxyurea induced deoxynucleotide pool imbalances. These inhibition studies suggest that there may be a differential sensitivity of the enzyme towards hydroxyurea in the presence of various substrates.
Download or read book DNA Polymerase in Vaccinia Virus infected and Uninfected Animal Cells written by Catherine B. Lazier and published by . This book was released on 1967 with total page 212 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Studies on the DNA Polymerase Induced by Vaccinia Virus written by Mark D. Challberg and published by . This book was released on 1979 with total page 160 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Studies on DNA Precursor Metabolism in Vaccinia Virus infected Mamalian Cells written by Craig Spiro and published by . This book was released on 1987 with total page 254 pages. Available in PDF, EPUB and Kindle. Book excerpt: Vaccinia virus-infected animal cells have been used to study the interactions between the replication of deoxyribonucleic acid (DNA) and the biosynthesis of its nucleotide precursors. Some antimetabolites that inhibit DNA replication have as their targets enzymes of nucleotide biosynthesis. Furthermore, the disruption of nucleotide metabolism can alter the fidelity of DNA replication. The isolation of viral mutants resistant to the nucleoside analogue arabinosyl cytosine has shown that resistance to certain drugs is associated with altered fidelity of replication of DNA. Furthermore, single mutations can cause altered sensitivity to several compounds. On the other hand, selection of apparent revertants indicates that several sites can be involved in drug resistance and replication fidelity. Vaccinia virus has previously been shown to code for enzymes of DNA precursor metabolism. However, attempts to isolate mutants resistant to drugs that target thymidylate synthase and dihydrofolate reductase were not successful. In fact, stimulation of host cell DNA synthesis makes vaccinia virus extremely sensitive to the effects of the folate analogue methotrexate. Taken together these observations suggest that despite its cytoplasmic site of DNA replication and its large coding capacity, vaccinia virus depends, at least in part, on host cell enzymes to supply deoxynucleoside triphosphates for DNA replication.
Download or read book Genetic Characterization of the Vaccinia Virus DNA Polymerase written by John Anthony Taddie and published by . This book was released on 1992 with total page 468 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Large Subunit of Vaccinia Cirus Ribonucleotide Reductase written by Rainer K. Warth and published by . This book was released on 1993 with total page 170 pages. Available in PDF, EPUB and Kindle. Book excerpt: Ribonucleoside diphosphate reductase (RR) from vaccinia virus was recently cloned and overexpressed rn Escherichia coli. The amino acid sequence identities of the small and large subunits between the mouse and the vaccinia virus reductase are approximately 80 and 72 percent, respectively. In addition, vaccinia virus RR displays similar complex allosteric regulation to the mouse enzyme and other eukaryotic reductases. The overall activity of the enzyme, which has two subunits (Rl and R2), is regulated through binding to ATP, which activates the enzyme, and dATP which seryes as an inhibitor. Both nucleotides bind to the same allosteric site, called the activity site, on the large subunit of RR. The specificity of the enzyme towards the four ribonucleoside diphosphate substrates is regulated by the binding of ATP, dATP, dTTP and GTp. Each of these nucleotides affects the reduction of a specific nucleoside diphosphate. Although this enzyme's allosteric regulation is kinetically well understood it has not been possible so far to gain further structural information about the location of the activity site and specificity site. The use of deletion mutants and photoaffinity labeling of the large subunit to identify the location of the binding sites is the incentive for this thesis. With the introduction of 6xHis/Nickel Nitrilo-tri-acetic acid (Ni-NTA) chromatography, the purification of the large subunit was improved in the E. coli and vaccinia virus/T7 RNA polymerase hybrid system. The purification of several deletion mutant forms of the large subunit was also attempted, but it was not possible to purify any of them from either of the expression systems. The purified full-length large subunit obtained with the Ni-NTA-chromatography system was used for a photoaffinity labeling experiment with [32P]dATP and [32P]dTTP. The labeled proteins were proteolytically digested to find out about the specificity of the labeling experiment and also to map the binding site of the nucleotide. It was found that labeling of dATP yielded few discrete bands indicating specific binding, while a comparable experiment with dTTP indicated less specific binding, based on a larger number of labeled bands. In competition experiments with non-radioactive nucleotides, vaccinia virus R1 featured the same properties as the mouse and E. coli counterparts. This is consistent with data from kinetic experiments, which also establish the same kinetic properties between vaccinia virus RR with those of mouse and E. coli (RR). To identify the sequence of the fragments carrying the label the digests were subjected to mass spectrometric analysis. However, it was not possible to determine the sequence of the labeled fragment by mass spectrometry due to poor spectral resolution.
Download or read book Biochemical and Enzymatic Characterization of the Heterodimeric Processivity Factor for the Vaccinia Virus DNA Polymerase the Role of the Vaccinia Uracil DNA Glycosylase UDG Enzyme written by Eleni Stanitsa and published by . This book was released on 2006 with total page 195 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Vaccinia Virus DNA Replication written by Dennis M. Lambert and published by . This book was released on 1977 with total page 452 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Investigations of Vaccinia Virus DNA Replication The Roles of Three Viral Proteins A50 I3 and G5 written by Maciej W. Czarnecki and published by . This book was released on 2017 with total page 276 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Fine Structure Mapping Phenotypic and Molecular Biological Characterization of Mutants in Three Different Subunits of Vaccinia Virus DNA dependent RNA Polymerase written by Urvashi H. Dhingra and published by . This book was released on 1989 with total page 416 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book DNA Replication and Telomere Resolution in Vaccinia Virus written by and published by . This book was released on 1997 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
