DNA polymerases are found in a diverse range of organisms, prokaryotes, eukaryotes, viruses and bacteriophage. T7 DNA polymerase is a replicative enzyme from E. coli bacteriophage T7. It relies on the thioredoxin binding domain (TBD) of phage gene 5 protein (gp5) and E. coli thioredoxin (Trx) for processive replication of phage DNA. Although T7 DNA polymerase is processive, it is also thermolabile. In order to design a thermostable and processive DNA polymerase, the structural stabilities of the TBD and Trx were studied in respect to their binding affinity and affect on enzyme processivity. An artificial operon was designed for coexpression of subunits of T7 DNA polymerase. By means of a 9??His-tag at the amino terminus of gp5, T7 DNA polymerase complex was purified by one-step nickel-agarose chromatography, with subunits gp5 and Trx co-eluting in a one to one molar ratio. Purified T7 DNA polymerase was assayed for polymerase activity, processivity and residual activity and compared to the commercial T7 DNA polymerase. The two enzymes were not identical with commercial T7 DNA polymerase being less processive at 37??C. Mass spectrometry of the two enzymes identified a mutation of Phe102 to Ser in the Trx subunit (TrxS102) of commercial T7 DNA polymerase. The Ser102 mutation, was found near the carboxyl terminal helix of Trx. TrxS102 was less stable than wild type Trx. In the study of the TBD structural stability, a hybrid polymerase was constructed by inserting the TBD motif into the homologous position in the Stoffel fragment of Taq DNA polymerase. The hybrid enzyme was coexpressed with Trx from an artificial operon; however, the TBD inserted retained a mesophilic binding affinity to Trx. The chimeric polymerase required 100 molar excess of Trx for processive polymerase activity at 60??C. TBD structural deformation at elevated temperatures was hypothesized to be the cause of the change in the subunit stoichiometry. Mutagenesis of TBD would be required to increase its thermostability. An efficient, rapid high throughput mutagenesis method (SLIM) was invented and would be appropriate for further studies.

DNA polymerases are found in a diverse range of organisms, prokaryotes, eukaryotes, viruses and bacteriophage. T7 DNA polymerase is a replicative enzyme from E. coli bacteriophage T7. It relies on the thioredoxin binding domain (TBD) of phage gene 5 protein (gp5) and E. coli thioredoxin (Trx) for processive replication of phage DNA. Although T7 DNA polymerase is processive, it is also thermolabile. In order to design a thermostable and processive DNA polymerase, the structural stabilities of the TBD and Trx were studied in respect to their binding affinity and affect on enzyme processivity. An artificial operon was designed for coexpression of subunits of T7 DNA polymerase. By means of a 9??His-tag at the amino terminus of gp5, T7 DNA polymerase complex was purified by one-step nickel-agarose chromatography, with subunits gp5 and Trx co-eluting in a one to one molar ratio. Purified T7 DNA polymerase was assayed for polymerase activity, processivity and residual activity and compared to the commercial T7 DNA polymerase. The two enzymes were not identical with commercial T7 DNA polymerase being less processive at 37??C. Mass spectrometry of the two enzymes identified a mutation of Phe102 to Ser in the Trx subunit (TrxS102) of commercial T7 DNA polymerase. The Ser102 mutation, was found near the carboxyl terminal helix of Trx. TrxS102 was less stable than wild type Trx. In the study of the TBD structural stability, a hybrid polymerase was constructed by inserting the TBD motif into the homologous position in the Stoffel fragment of Taq DNA polymerase. The hybrid enzyme was coexpressed with Trx from an artificial operon; however, the TBD inserted retained a mesophilic binding affinity to Trx. The chimeric polymerase required 100 molar excess of Trx for processive polymerase activity at 60??C. TBD structural deformation at elevated temperatures was hypothesized to be the cause of the change in the subunit stoichiometry. Mutagenesis of TBD would be required to increase its thermostability. An efficient, rapid high throughput mutagenesis method (SLIM) was invented and would be appropriate for further studies.

DNA polymerases are found in a diverse range of organisms, prokaryotes, eukaryotes, viruses and bacteriophage. T7 DNA polymerase is a replicative enzyme from E. coli bacteriophage T7. It relies on the thioredoxin binding domain (TBD) of phage gene 5 protein (gp5) and E. coli thioredoxin (Trx) for processive replication of phage DNA. Although T7 DNA polymerase is processive, it is also thermolabile. In order to design a thermostable and processive DNA polymerase, the structural stabilities of the TBD and Trx were studied in respect to their binding affinity and affect on enzyme processivity. An artificial operon was designed for coexpression of subunits of T7 DNA polymerase. By means of a 9??His-tag at the amino terminus of gp5, T7 DNA polymerase complex was purified by one-step nickel-agarose chromatography, with subunits gp5 and Trx co-eluting in a one to one molar ratio. Purified T7 DNA polymerase was assayed for polymerase activity, processivity and residual activity and compared to the commercial T7 DNA polymerase. The two enzymes were not identical with commercial T7 DNA polymerase being less processive at 37??C. Mass spectrometry of the two enzymes identified a mutation of Phe102 to Ser in the Trx subunit (TrxS102) of commercial T7 DNA polymerase. The Ser102 mutation, was found near the carboxyl terminal helix of Trx. TrxS102 was less stable than wild type Trx. In the study of the TBD structural stability, a hybrid polymerase was constructed by inserting the TBD motif into the homologous position in the Stoffel fragment of Taq DNA polymerase. The hybrid enzyme was coexpressed with Trx from an artificial operon; however, the TBD inserted retained a mesophilic binding affinity to Trx. The chimeric polymerase required 100 molar excess of Trx for processive polymerase activity at 60??C. TBD structural deformation at elevated temperatures was hypothesized to be the cause of the change in the subunit stoichiometry. Mutagenesis of TBD would be required to increase its thermostability. An efficient, rapid high throughput mutagenesis method (SLIM) was invented and would be appropriate for further studies.

The A segment of infectious pancreatic necrosis virus (IPNV) is expressed as a polyprotein encoding three primary gene products, VP2, NS and VP3, from a large open reading frame. The nucleotide sequence for the A segment of the Sp isolate of IPNV was determined. The NS protein is the putative autocatalytic proteinase responsible for the cleavage of the polyprotein. The functional boundaries of the NS proteinase were mapped by plasmid deletion analysis and examined in an La vitro, translation system. The NS proteolytic activity was determined to lie within the EcoRI and Nsil restriction sites. Characterization of the NS proteinase also was approached by use of proteinase inhibitors and site-directed mutagenesis of the putative catalytic and cleavage sites. Eight proteinase inhibitors, representative of all four proteinase classes, were tested and all failed to inhibit the NS enzyme. Mutagenesis of a putative aspartyl proteinase catalytic motif, DTG, to VTG did not affect proteolytic processing. Additionally, the mutagenesis of the predicted N-terminal cleavage site did not alter processing, however, altered processing was observed when the predicted C-terminal cleavage site was mutated. The major capsid protein, VP2, was mapped with polyclonal and monoclonal antisera. The VP2 gene was digested with Sau3A and subcloned into the pATH expression vector. The trpE-fusion proteins were characterized with polyclonal and monoclonal antisera. Two immunoreactive regions were identified with anti IPNV-Sp sera. A common immunoreactive region, B10, was reactive with antisera to three serotypes of IPNV as well as a neutralizing monoclonal antibody, AS-1. A serotype specific immunoreactive region, A43, also was identified, being recognized only by anti IPNV-Sp sera. The B segment of IPNV encodes the putative RNA-dependent RNA polymerase (RdRp), VP1. The nucleotide sequence for the B segment of the Sp isolate was determined and the deduced amino acid sequences were compared to other polymerases. Concensus sequences associated with GTP-binding proteins and RdRps were identified in the VP1 sequence. However, unlike RdRps associated with single-stranded RNA viruses, the IPNV VP1 proteins lack the Gly-Asp-Asp motif characteristic of this enzyme family. Additionally, the VP1 protein was expressed in a bacterial system and polyclonal antisera was raised against the protein. / Graduation date: 1993

Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 128-143). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains x, 200 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xiii, 108 p.; also includes graphics. Includes abstract and vita. Advisor: Paul K. Herman, Dept.of Molecular, Cellular, and Developmental Biology. Includes bibliographical references (p. 96-108).

Thesis (Ph. D.)--University of Oklahoma Health Sciences Center, 1998. / Includes bibliographical references (leaves 102-112).

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. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy