The purpose of this research was to determine the effects of retinoic acid on bovine papillomavirus (BPV) transformed cells. Since BPV transformed cells are able to form colonies in agar and their untransformed counterparts are not, of particular interest was the effect of retinoic acid on this marker. Retinoic acid inhibited the growth of colony forming cells, but the extent of inhibition varied among several cloned cell lines. Retinoic acid inhibited the proliferation of BPV transformed cells and the extent of this inhibition also varied. The copy number of the virus was determined for each of three cell lines by liquid reassociation experiments. The copy number did not change when the cells were treated with RA for a prolonged period of time. Gel electrophoresis and Southern blotting of extrachromosomal DNA from the cell lines revealed the presence of unit length BPV DNA in all three transformed cell lines. The amount of BPV DNA per cell was decreased when the cells were treated with 10⁻⁵ M RA for three days.

Vaccinia virus (VV) is a large double-stranded DNA virus that is a prototypic member of the orthopoxvirus family. Previous works has showed that three of the major structural proteins found within the mature VV virion core 4a, 4b, and 25K are produced from higher molecular weight precursors at late times during infection and processed via a common morphogenic cleavage pathway that is intimately linked with virion assembly and maturation. The enzyme that carries out these cleavage reactions is unknown. A transient expression assay was used to demonstrate that the 17L gene product and its encoded cysteine proteinase activity is responsible for cleavage of each of the three major core protein precursors. Cleavage was demonstrated to occur at the authentic Ala-Gly-Xaa cleavage sites and require active enzyme. A truncated 17L protein lost the ability to cleave the core protein precursors. A conditional-lethal recombinant virus was constructed in which the expression of the 17L gene is under the control of the tetracycline operator/repressor system. In the absence of 17L expression, processing of the major VV core proteins is inhibited and electron microscopy revealed defects in virion morphogenesis prior to complete core condensation. Plasmid-borne 17L is capable of rescuing the growth of this virus. A structural model of 17L was developed and a unique chemical library was assayed for both cell toxicity and the ability to inhibit the growth of VV in tissue culture cells. A novel class of inhibitors was discovered that is capable of inhibiting VV. An in-vitro cleavage assay was developed to further characterize the activity of 17L. This assay is based on producing the major core protein precursors in a coupled transcription and translation assay and then mixing them with 17L enzyme extracts. Using this assay, 17L is shown to be capable of cleavage of each substrate. 17L is further characterized as a cysteine proteinase due to the inhibitory effects of known cysteine proteinase inhibitors such as NEM and iodoacetic acid, as well as through the use of specific small molecule inhibitors in this in-vitro assay. / Graduation date: 2005

1,10-phenanthroline and its non-chelating isomer, 1,7-phenanthroline were used to inhibit the replication of vaccinia virus (VV). Serial passage of VV in the presence of various concentrations of either 1,10-phenanthroline or 1,7-phenanthroline was carried out. No drug resistant mutants were isolated, suggesting that the observed inhibition was due to a cellular protease as opposed to the putative viral protease G1L. Cultures infected in the presence of the inhibitors, were radio labeled with �����S-methionine at various time points post infection, to determine which step of VV replication was inhibited. Infections in the presence of 1,10-phenanthroline proceeded only through early gene transcription, suggesting that the point of inhibition was uncoating. Finally, cells infected with VV with or without the inhibitors at time zero and eight hours post infection were used to generate transmission electron microscopic images. Taken together these results indicate that inhibition was occurring at the level of uncoating. / Graduation date: 1999

Thesis (Ph. D.)--University of Rhode Island, 2003. / Typescript. Includes bibliographical references (leaves 167-178).

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 25, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: David J. Pintel. Vita. Includes bibliographical references.

Bovine parvovirus DNA is associated with the nuclear matrix of infected bovine fetal lung cells as shown by Southern blot analysis of matrix DNA isolated by two procedures differing in the order of exposure of detergent-treated nuclei to high salt conditions and DNase I. Protein analysis of the two matrix types showed the polypeptide composition to be similar. Both procedures showed enrichment for BPV DNA with progressive DNase I digestion. Over the course of infection the amount of BPV DNA associated with the matrix increased, yet the amount of BPV DNA associated with matrix DNA as opposed to total DNA decreased from 21% at two hours to 7% at eight hours with a subsequent rise to 13% at sixteen hours. Restriction enzyme analysis of the matrix DNA indicated that no specific portion of the BPV genome was responsible for its attachment to the matrix at the selected times. In addition both the nonstructural BPV protein, NP-1, and the capsid proteins VP1, VP2, and VP3 were associated with the matrix at sixteen hours. The association of BPV DNA and proteins with the nuclear matrix implies structural if not functional significance for the matrix in BPV replication. / M.S.

We have developed crown-gall resistant transgenic plants capable of suppressing Agrobacterium tumefaciens T-DNA oncogenes. Crown gall tumors result from overproduction of auxin and cytokinin in plant cells transformed by A. tumefaciens. High phytohormone levels result from expression of two auxin biosynthetic genes, tryptophan monooxygenase (iaaM) and indole acetamide hydrolase (iaaH), and isopentenyl transferase (ipt), which mediates cytokinin synthesis. Inactivation of ipt and either one of the two auxin biosynthesis genes prevents crown gall formation. To suppress T-DNA oncogene expression, we created transgenic tobacco that produce the corresponding untranslatable sense-strand RNAs. This phenomenon, called cosuppression, frequently blocks expression of transgenes in plants. Often, expression of an untranslatable sense-strand transgene elicits sequence-specific destruction of both the mutant mRNA and the corresponding wild-type mRNA. Here we show that cosuppression can block expression of A. tumefaciens T-DNA oncogenes, resulting in plants that are resistant to gall induction by certain strains of A. tumefaciens. / Graduation date: 1999

Graduation date: 1995

Thesis (Ph. D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed Jan. 26, 2010). Additional advisors: Asim K. Bej, Gail E. Christie, Peter E. Prevelige, Jr., R. Douglas Watson. Includes bibliographical references.

by Jose Victor Lopez. / Typescript. / Thesis (M.S.)--Florida State University, 1988. / Bibliography: leaves 98-109.