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Author : Wing-hong Chin
Publisher :
ISBN 13 :
Total Pages : 194 pages
Book Rating : 4.:/5 (828 download)
Download or read book Polymerase Activity of Chimeric Polymerase written by Wing-hong Chin and published by . This book was released on 2012 with total page 194 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Author : Wing-Hong Chin
Publisher : Open Dissertation Press
ISBN 13 : 9781361005026
Total Pages : pages
Book Rating : 4.0/5 (5 download)
Download or read book Polymerase Activity of Chimeric Polymerase written by Wing-Hong Chin and published by Open Dissertation Press. This book was released on 2017-01-26 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: This dissertation, "Polymerase Activity of Chimeric Polymerase: a Determining Factor for an Influenza Virus to Be a Pandemic Strain" by Wing-hong, Chin, 錢永康, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: The influenza polymerase is a complex of three subunits, polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1) and polymerase acidic protein (PA). It associates with the viral RNA segment and nucleoprotein (NP) to form a viral ribonucleoprotein (vRNP) complex which is important for transcription and replication of the viral genome. Concurrently, the previous three influenza pandemics viruses contain reassorted vRNP of different origins. This leads to the aim of study to investigate the role of polymerase in the pandemic viruses. By reconstitution of vRNPs in human cells, it was demonstrated that vRNPs of H2N2 and H3N2 pandemic viruses had higher polymerase activity than the H2N2 seasonal viruses in-between them. The recombinant virus with H2N2 pandemic vRNP also showed faster growth kinetics in the early stage of viral replication and better adaptability to the selective environment with neuraminidase inhibitor than the recombinant virus with H2N2 seasonal vRNP, which had a lower polymerase activity. Reconstitution of chimeric vRNPs of H2N2 pandemic and seasonal viruses revealed that PB2, PB1 and PA were responsible for the difference in polymerase activity between them. Five residues, one in PB2, three in PB1 and one in PA were identified to be significant for the polymerase activity change. These polymerase subunits and residues may act as part of the determining factors for the H2N2 pandemic virus. Furthermore, PB2-627 has been shown to have stringent host specificity and affect polymerase activity and viral replication. Recombinant viruses in mammalian and avian cells with random mutation were generated at this position. It showed that the amino acids at this position are not restricted to those appear in the nature for generating viable viruses. It was also observed that the avian-derived viruses generally had lower polymerase activity and reduced growth kinetics in mammalian cells, while part of the mammalian-derived viruses had lower polymerase activity and reduced growth kinetics in avian cells. This consolidated the role of PB2-627 on host specificity and demonstrated the possibility of some novel amino acids for this position, which may play a role in the future influenza pandemic. The 2009 H1N1 pandemic virus contains a reassorted vRNP with subunits of avian, human and swine origins. This prompts me to compare the polymerase activity of all the 81 possible combinations of chimeric vRNPs of three different origins. The results were statistically analyzed and several single subunit factors and interactions between vRNP subunits were identified to significantly affect the polymerase activity. In order to reduce the effort and resources required, a fractional factorial design of 27 experimental runs was developed to substitute the 81-combination full factorial design for identifying the significant single subunit factors that affect the polymerase activity. Overall, this study identified some factors that may contribute to a pandemic virus and allows us to have better understanding of the role of polymerase in a pandemic virus. These findings may contribute to evaluating the pandemic potential of the novel virus that emerges or may emerge in the nature and enhances the preparedness towards the next pandemic influenza. DOI: 10.5353/th_b4979918 Subjects: RNA polymerases Influenza viruses Nucleoproteins
Author : Wing-hong Chin
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (828 download)
Download or read book Polymerase Activity of Chimeric Polymerase written by Wing-hong Chin and published by . This book was released on 2012 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Author : Lei Zhang
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (13 download)
Download or read book DNA-Directed DNA Polymerases Evolve From Reverse Transcriptase written by Lei Zhang and published by . This book was released on 2013 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Author : Hans Klenow
Publisher :
ISBN 13 :
Total Pages : 7 pages
Book Rating : 4.:/5 (115 download)
Download or read book Differential Effect of N- Carboxymethylisatoylation on the DNA Polymerase Activity, the 5′ → 3′-exonuclease Activity and the 3′ → 5′-exonuclease Activity of DNA Polymerase I of Escherichia Coli written by Hans Klenow and published by . This book was released on 1981 with total page 7 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Author : Michael G Mohsen
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (115 download)
Download or read book Chimeric Dinucleotides written by Michael G Mohsen and published by . This book was released on 2020 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Author : Zvi Kelman
Publisher : Frontiers Media SA
ISBN 13 : 2889194558
Total Pages : 147 pages
Book Rating : 4.8/5 (891 download)
Download or read book DNA polymerases in Biotechnology written by Zvi Kelman and published by Frontiers Media SA. This book was released on 2015-03-18 with total page 147 pages. Available in PDF, EPUB and Kindle. Book excerpt: DNA polymerases are core tools for molecular biology including PCR, whole genome amplification, DNA sequencing and genotyping. Research has focused on discovery of novel DNA polymerases, characterization of DNA polymerase biochemistry and development of new replication assays. These studies have accelerated DNA polymerase engineering for biotechnology. For example, DNA polymerases have been engineered for increased speed and fidelity in PCR while lowering amplification sequence bias. Inhibitor resistant DNA polymerase variants enable PCR directly from tissue (i.e. blood). Design of DNA polymerases that efficiently incorporate modified nucleotide have been critical for development of next generation DNA sequencing, synthetic biology and other labeling and detection technologies. The Frontiers in Microbiology Research Topic on DNA polymerases in Biotechnology aims to capture current research on DNA polymerases and their use in emerging technologies.
Author : Kary B. Mullis
Publisher : Birkhauser
ISBN 13 :
Total Pages : 490 pages
Book Rating : 4.3/5 (91 download)
Download or read book The Polymerase Chain Reaction written by Kary B. Mullis and published by Birkhauser. This book was released on 1994 with total page 490 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Author : Maria Giulia Battelli
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (31 download)
Download or read book DNA Polymerase Activity in Proliferating and Antigen-stimulated Tissues written by Maria Giulia Battelli and published by . This book was released on 1979 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Author : Joseph L. Stodola
Publisher :
ISBN 13 :
Total Pages : 178 pages
Book Rating : 4.:/5 (953 download)
Download or read book Functions of the DNA Polymerase Delta Replicase in Lagging Strand Replication written by Joseph L. Stodola and published by . This book was released on 2016 with total page 178 pages. Available in PDF, EPUB and Kindle. Book excerpt: The work described in this dissertation focuses on several aspects of DNA replication in the model organism Saccharomyces cerevisiae, with particular attention paid to the function of the replicative DNA polymerase [delta] (Pol [delta]), and its functions in Okazaki fragment synthesis and maturation. The first major theme of this dissertation is investigating the role that metal binding motifs play in the structure and function of Pol [delta] and other budding yeast polymerases. First, I discuss the role that two metal binding motifs within the catalytic subunit of Pol [delta] play in creating the multi-subunit polymerase complex and in promoting crucial interactions with the replication sliding clamp, proliferating cell nuclear antigen (PCNA). Next, I describe work defining the importance of similar metal binding motifs in the translesion DNA polymerase [zeta] (Pol [zeta]). This yielded the observation that the two accessory subunits of Pol [delta], Pol31 and Pol32, are also constitutive members of a four-subunit Pol [zeta] complex. Finally, I describe the creation of a chimeric DNA polymerase comprising the bacteriophage RB69 DNA polymerase fused to the metal binding domain of the Pol [delta] catalytic subunit. We show that this chimeric polymerase can form a multimeric complex containing the Pol [delta] accessory subunits, interact with PCNA, and support DNA replication in vivo. This data provided insight into the structural requirements of the lagging strand replication machinery. The second major theme is Pol [delta]'s crucial role in synthesizing Okazaki fragments and participating in the removal of initiator RNA, called Okazaki fragment maturation. I first describe my work developing a system to study the activity of Pol [delta] in higher kinetic detail than previous studies, using rapid-quench techniques. This work yielded insights into how Pol [delta] performs DNA synthesis and strand displacement synthesis, as well as accomplishes nick translation, requiring collaboration between Pol [delta] and the flap-endonuclease FEN1. The next chapter describes the production and characterization of engineered PCNA heterotrimers. These proteins were produced to test the 'toolbelt' model, which is the hypothesis that PCNA binds multiple enzymes simultaneously to increase the efficiency of DNA metabolism processes involving multiple enzymes. Finally, there has been a growing interest among those studying lagging strand synthesis into how potential impediments to the Okazaki fragment maturation machinery are resolved; we show that although the transcription factor Rap1 can block strand displacement synthesis by Pol [delta] when it is bound to DNA, this block can be resolved through the action of the helicase Pif1. In sum, these studies provide insight into how Pol [delta]'s structure dictates its function, as well as addresses larger mechanistic questions concerning how lagging strand DNA replication is accomplished.
Author : Ramon Kranaster
Publisher :
ISBN 13 :
Total Pages : 117 pages
Book Rating : 4.:/5 (731 download)
Download or read book DNA Polymerase Activity on Solid Support written by Ramon Kranaster and published by . This book was released on 2009 with total page 117 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Author : Stefan Lutz
Publisher : John Wiley & Sons
ISBN 13 : 3527666982
Total Pages : 723 pages
Book Rating : 4.5/5 (276 download)
Download or read book Protein Engineering Handbook, Volume 3 written by Stefan Lutz and published by John Wiley & Sons. This book was released on 2012-09-14 with total page 723 pages. Available in PDF, EPUB and Kindle. Book excerpt: This introduction collects 17 innovative approaches to engineer novel and improved proteins for diverse applications in biotechnology, chemistry, bioanalytics and medicine. As such, key developments covered in this reference and handbook include de novo enzyme design, cofactor design and metalloenzymes, extremophile proteins, and chemically resistant proteins for industrial processes. The editors integrate academic innovations and industrial applications so as to arrive at a balanced view of this multi-faceted topic. Throughout, the content is chosen to complement and extend the previously published two-volume handbook by the same editors, resulting in a superb overview of this burgeoning field.
Author : Nikunj Bhatt
Publisher :
ISBN 13 :
Total Pages : 96 pages
Book Rating : 4.:/5 (712 download)
Download or read book Characterization of Two Polymerases written by Nikunj Bhatt and published by . This book was released on 2006 with total page 96 pages. Available in PDF, EPUB and Kindle. Book excerpt: Abstract: Since 1999, the human genome project has led to the discovery of several novel DNA polymerases. Among these new polymerases were human DNA polymerase ?. (Pol?) and Sulfolobus solfataricus DNA polymerase IV (Dpo4). PoP, an X family polymerase, displays both 5'-2-deoxyribose-5-phosphate lyase (dRPase) activity and polymerase activity and efficiently incorporates nucleotides into short-gapped primer- primer/templates, the natural substrate for short-patch base excision repair (BER). Crystal studies of the ternary structure of truncated PoIA (tPolX) reveal that a 5'- phosphate of a downstream primer interacts with a positively-charged pocket in the dRPase domain. In this study, we constructed three substrates: (i) a 21-19/41-mer single nucleotide gapped DNA with a 5'-phosphate on the terminal end of the downstream 19- mer; (ii) a 21-19/41-mer single nucleotide gapped DNA with a 5'-OH on the downstream 19-mer; and (iii) a 21/41-mer with no downstream primer. Pre-steady state kinetics revealed that Pol[lamda]'s incorporation efficiency for the DNA substrate with the 5'-phosphate moiety on the downstream 1 9-mer primer is 11-fold more efficient at incorporating a correct nucleotide compared to the DNA substrate with the 5'-OH on the terminal end of the downstream 19-mer, and 160-fold more efficient than the DNA substrate with no downstream primer. Another aim of this thesis was to characterize Po1[lamda]' s preference for deoxynucleotides over ribonucleotides via pre-steady state kinetics. A previous study with polymerase u (Polu) revealed that the glycine433 residue mutated to tyrosine dramatically increased sugar selectivity for that enzyme. Sequence alignment of the M a-helix of Po1[lamda] and three other X family polymerases, Polu, terminal deoxynucleotidyl transferase (TDT), and polymerase [beta] (Polf[beta]), suggested that its tyrosine505 may be involved in determining sugar selectivity, while the crystal structure of the tPo[lamda]. ternary complex suggested that the backbone of the M a-helix blocks the 2'-OH of ribonucleotides forming a "stearic gate". In this thesis, the tyrosine505 mutated to glycine actually increased sugar selectivity indirectly supporting the stearic gate hypothesis. The second enzyme studied, Dpo4, a Y family polymerase that bypasses lesions and exhibits low fidelity, consists of an extra little finger domain in addition to the standard catalytic core, consisting of the finger, thumb, and palm domains. The little finger, which has been shown to be important to polymerase activity, is tethered to the thumb domain via 14 amino acid linker (P1 = 10), known as the little finger linker. Using 1, 4, and 6 glycine additions and 1, 4, and 6 deletions in the center of the little finger linker, a fluorescent titration assay revealed a significant decrease in binding affinity as the size of the little finger linker was increased and decreased. This suggests nature has optimized the length of the little finger linker. In addition, a kink in the little finger linker was removed by a proline236 substitution to alanine which did not significantly effect DNA binding to Dpo4. Finally, an optimized procedure for the cisplatination of an 1 8-mer DNA substrate was prepared and described in this thesis.
Author :
Publisher : Elsevier
ISBN 13 : 0080496830
Total Pages : 701 pages
Book Rating : 4.0/5 (84 download)
Download or read book Applications of Chimeric Genes and Hybrid Proteins, Part C: Protein-Protein Interactions and Genomics written by and published by Elsevier. This book was released on 2000-10-28 with total page 701 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. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant today--truly an essential publication for researchers in all fields of life sciences.
Author : Wolfram Saenger
Publisher :
ISBN 13 :
Total Pages : 230 pages
Book Rating : 4.F/5 ( download)
Download or read book Protein-nucleic Acid Interaction written by Wolfram Saenger and published by . This book was released on 1989 with total page 230 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume contains a series of essays which describe a range of problems in the field of nucleic-acid interactions, investigated by a variety of techniques. An introductory chapter on DNA-protein interactions in the regulation of gene expression is followed by papers on selected model systems.
Author : Erko Stackebrandt
Publisher :
ISBN 13 :
Total Pages : 370 pages
Book Rating : 4.3/5 (91 download)
Download or read book Nucleic Acid Techniques in Bacterial Systematics written by Erko Stackebrandt and published by . This book was released on 1991-04-19 with total page 370 pages. Available in PDF, EPUB and Kindle. Book excerpt: One of a series whose aim is to identify specialist areas in microbiology and to provide up-to-date methodological information for laboratory microbiologists, active researchers and graduate students. This volume addresses nucleic acid techniques in bacterial systematics.
Author : Gerrit J. Viljoen
Publisher : Springer Science & Business Media
ISBN 13 : 1402034040
Total Pages : 325 pages
Book Rating : 4.4/5 (2 download)
Download or read book Molecular Diagnostic PCR Handbook written by Gerrit J. Viljoen and published by Springer Science & Business Media. This book was released on 2005-12-06 with total page 325 pages. Available in PDF, EPUB and Kindle. Book excerpt: PREFACE The Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture is involved in agricultural research and development and assists Member States of FAO and IAEA in improving strategies to ensure food security through the use of nuclear techniques and related biotechnologies, where such techniques have a valuable and often unique role. In particular, molecular diagnostic methods have rapidly evolved in the past twenty years, since the advent of the Polymerase Chain Reaction (PCR). They are used in a wide range of agricultural areas such as, improving soil and water management; producing better crop varieties; diagnosing plant and animal diseases; controlling insect pests and improving food quality and safety. The uses of nucleic acid-directed methods have increased significantly in the past five years and have made important contributions to disease control country programmes for improving national and international trade. These developments include the more routine use of PCR as a diagnostic tool in veterinary diagnostic laboratories. However, there are many problems associated with the transfer and particularly, the application of this technology. These include lack of consideration of: the establishment of quality-assured procedures, the required set-up of the laboratory and the proper training of staff. This can lead to a situation where results are not assured. This book gives a comprehensive account of the practical aspects of PCR and strong consideration is given to ensure its optimal use in a laboratory environment. This includes the setting-up of a PCR laboratory; Good Laboratory Practice and standardised of PCR protocols.
