Dilantin (DPH) is a common anticonvulsant drug used to prevent seizures. It is known to be a human teratogen causing fetal hydantoin syndrome (FHS). FHS is characterized by multiple developmental and growth related abnormalities and mental retardation. Previous studies demonstrated that DPH slowed growth and division in preimplantation mouse embryos in vivo and in vitro. DHP exposure in utero decreased the crown to rump length and weight of 25-35% of embryos and reduced the rate of endochondral bone conversion from cartilage. In vitro preimplantation mouse embryos treated with DPH at 5, 10 and 20 μg/ml showed a reduction of 25-35% in their development, and block at 2-cell or 3-4-cell stages. These embryos also showed a prolonged DNA synthesis (S) phase during the second cell cycle. Nuclear localization and concentration levels of cyclin A , the S phase cyclin, were also altered in vivo in 2-cell DPH treated embryos compared with NaOH control embryos during G1, S phase and G2 of the first, second and third cell cycles. DPH altered patterns of expression of cyclin A were associated with cell cycle disregulation during preimplantation development. The purpose of the current study was to determine whether DPH also affects the concentration of DNA pol δ catalytic subunit in 2-cell preimplantation mouse embryos at G1 and S phases, thus delaying DNA synthesis and contributing to FHS. Immunofluorescence and confocal microscopy were used as tools to determine relative levels and distribution of DNA pol δ (for consistency with text) in the cytoplasm and the nuclei of DPH and NaOH treated 2-cell embryos at G1 and S phase of the second cell cycle. DPH decreased DNA pol δdelta total embryo and nuclear levels by 43% and 36%, respectively, in G1 compared with NaOH controls. Similarly, nuclear levels of DNA pol δ in DPH embryos in S phase near the G2 transition of the second cell cycle increased to 144% of NaOH control levels; there was not a statistically significant difference between total embryonic levels of late S phase DNA pol δ in DPH and NaOH treated control embryos. The results indicated that DPH affects the levels of DNA pol δduring G1 and S phase near the G2 transition of the second cell cycle in preimplantation mouse embryos. The significant alteration in the levels of DNA pol δ during S phase and its probable consequent altered polymerase activity could contribute to an explanation for the extension of S phase in preimplantation embryos observed by Blosser and Chatot. Even more, the alteration in the levels of DNA pol δ and potentially in its exonuclease activity could lead to an increase in the rate of mismatches and mutations suggesting a likely explanation for some features of FHS. / Department of Biology

Hepatitis C virus (HCV) replication is directed by NS5b, the viral RNA dependent RNA polymerase (RdRp). To date, our understanding of the HCV polymerase has come almost entirely from genotype 1. The aim of this study was to examine the influence of sequence variation in the polymerase region by characterising a polymerase derived from genotype 3a. The genotype 3a CB strain polymerase was cloned into the bacterial expression vector pTrcHis2C incorporating a hexahistidine tag to facilitate purification. An optimised process produced 2.5 mg of highly purified recombinant protein per litre of bacterial culture. The 3a preparation possessed an RdRp activity and could utilise both homopolymeric and heteropolymeric RNA templates. Optimal activity was seen at 30oC at pH 8 in reactions containing 160nM enzyme, 10??g/ml RNA template and 2.5mM MnCl2. Subsequently, three genotype 1b polymerases including the HCV-A, Con1 and JK1 strains were cloned for the comparison of activity under identical conditions. Steady state kinetic parameters for GMP incorporation revealed the 3a polymerase exhibited the highest activity, with an almost two fold higher catalytic efficiency (Kcat/Km) than HCVA-1b, primarily due to differences in Km for GTP (2.984??M vs 5.134??M). Furthermore, the 3a polymerase was 3.5 fold and 15 fold more active than JK1-1b and Con1-1b respectively. Improving our understanding of the influence of sequence difference on polymerase activity, particularly in the context of replication will be crucial to developing effective antiviral therapies.

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 80-87). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Biochemistry, 1981.

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 250-289). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 250-289). Also available on the Internet.

Thesis (Ph. D.)--University of Oregon, 2004. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 163-172). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Computational Biology and Molecular Biophysics." Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 182-210). Also available on the Internet.

Thesis (PH.D.)--Michigan State University. Cell and Molecular Biology, 2008. / Title from PDF t.p. (viewed on Aug. 11, 2009) Includes bibliographical references. Also issued in print.