The adenovirus replication origins reside in the inverted terminal repetition (ITR) sequences. Replication proceeds unidirectionally by a strand-displacement mechanism from either or both origins to produce duplex daughter DNA plus a displaced parental strand. The displaced strand can be duplicated by two different routes. The cis pathway involves intramolecular hybridization between the inverted terminal repeats to form a panhandle replicative intermediate, where synthesis of the complementary strand is initiated from the newly created duplex origin on the panhandle. Alternatively, complementary strands arising from replication on different molecules can directly hybridize intermolecularly to reform a duplex viral genome. This path is called trans replication. Using plasmid mini-chromosomes as model systems, the structural requirements for adenovirus DNA replication and the relationship between the cis and trans pathways for complementary strand replication was investigated. Plasmids containing single or dual adenovirus origins, with or without inverted repeats, were specially constructed to mimic the structures of the adenovirus genome. Linear plasmids which contain exposed adenovirus origins can engage in adenovirus helper-dependent strand-displacement replication. A special class of panhandle intermediates (foldback structures), arising from the replication of symmetrical dimers and multimers generated by end-to-end ligation during transfection, was identified by two-dimensional gel electrophoresis and physically characterized. Foldback molecules provide the first evidence for the existence of the adenovirus cis replication pathway. Comparing the yields of cis replication products from different plasmids, it is clear that the efficiency of cis replication increases with longer inverted repeats. In addition, work presented here demonstrates that the conversion of displaced strands into duplexes occurs simultaneously and independently by both the cis and the trans pathways. The ability of embedded replication origins to direct adenovirus DNA replication was investigated in transfected cells using the plasmid mini-chromosome model system. Plasmids with origins embedded in circular or linear templates gave rise to replication-proficient linear molecules. Inverted repeats were necessary and sufficient in order to rescue displaced strands from circular or linear templates by the cis pathway. In general, the efficiency of replication on linear templates with embedded origins was higher than on circular templates, but was lower when compared to linear templates with exposed origins. This suggests that the creation of a 3'-end, essential for the cis replication pathway, on displaced strands arising from cicluar molecules may be rate limiting. Alternatively, this might imply that the efficiency of initiation on linear templates is higher than on circular templates. A mutant adenovirus helper was used to investigate the role of homologous recombination in activating plasmids with wild-type adenovirus origin sequences. Recombination between the exposed origins on helper DNA and the embedded origins in linear plasmids might be one possible mechanism to activate embedded origins. However, recombination is clearly not necessary.' Other mechanisms must be involved in the replication of templates which contain embedded origins. The production of tandemly repeated multimers from circular templates suggests that randomly initiated displacement reactions might proceeed continuously around the circles. Subsequently, the displaced strands could be converted into replication-proficient molecules by the cis replication pathway. / Graduation date: 1990

Thesis (M.Phil.) - University of St Andrews, February 2008.

No description available.

Typescript. / Digitized by Kansas Correctional Industries

The work in this thesis has provided conclusive genetic evidence that "panhandle" intermediates form during adenovirus replication. Adenovirus chromosomes lacking 51 by from their left -hand termini are infectious and capable of regenerating the missing origin sequence. Yet if an entire inverted terminal repeat is removed, the adenovirus chromosome is no longer viable. This first suggested, but did not prove, that "panhandles" formed during adenovirus replication. Homologous recombination or postreplicative overlap recombination could generate the same outcome. Analysis of the segregation of markers in the inverted repeats of adenovirus minichromosomes shows that homologous recombination does not mediate end repair. A special case was also found where postreplicative overlap recombination failed to transfer sequences between the inverted repeats, but similar molecules could exchange sequence information during "panhandle" formation. The exchange of information between inverted repeats is referred to as sequence conversion. A number of length and/or orientation constraints on sequence conversion during adenovirus DNA replication were identified. A length- and orientation-dependent constraint was found for gap filling close to "panhandle" loops. Polymerization towards the loop could occur even when the gap was only 6 by away. In contrast, polymerization away from the "panhandle" loop at a gap at 6 bp, did not take place. This steric constraint could reflect an asymmetry in the action of adenovirus DNA polymerase. A similar length and/or orientation dependent constraint was found for the removal of bulges (3 by and 4 by mismatches). Incision in the bulge of the 5' inverted repeat caused a block to the completion of sequence conversion at that site. When the bulge was in the 3' inverted repeat, a length requirement for successful removal was demonstrated. When 6 by or 39 by separated the bulge from the "panhandle" loop, removal of the bulge was not detected. When the distance was 79 bp, 184 bp, or 217 bp, bulges were successfully removed. The molecular basis for this obstruction remains to be determined. Moreover, incision in bulges located in the 3' inverted repeat triggers directional coconversion. Finally, small loops placed close to the site of polymerization did not cause the same length and orientation dependent constraints as did the "panhandle" loop. / Graduation date: 1991

Fifteen adenoviruses from cattle, sheep, horses, and llamas were studied by virus neutralization to determine their degree of antigenic similarity. Prototype viruses included bovine adenoviruses species 1-8, ovine adenoviruses species 5 and 6, and equine adenovirus species 1. Unclassified viruses that were compared to the prototype viruses were isolated from different locations within Oregon and were represented by bovine isolate 32CN, ovine isolates 47F and 475N, and llama isolate 7649. Reciprocal virus neutralization tests were performed and the degree of antigenic similarity, i.e., species differentiation was determined by criteria established by the International Committee for the Nomenclature of Viruses. The study showed that many of the adenoviruses, both prototype and unclassified, shared minor antigenic components with each other. Prototype viruses possessed major antigenic differences and, as previously demonstrated by other investigators, should be classified as separate virus species. Bovine adenovirus isolate 32CN was shown to be of the same species as ovine adenovirus isolate 475N, but neither isolate was similar to any of the prototype virus species studied. Ovine adenovirus isolate 47F was shown to be of the same species as ovine adenovirus species 5 strain RTS 42. Llama adenovirus isolate 7649, while sharing minor antigens with different viruses from cattle and sheep, was shown to be a distinct species. This represents the first species of adenovirus from llama. / Graduation date: 1993

Thesis (Ph. D.)--Miami University, Dept. of Microbiology, 2003. / Title from first page of PDF document. Document formatted into pages; contains v, 78 p. : ill. Includes bibliographical references (p. 68-78).

Thesis (M.Phil.) - University of St Andrews, March 2007.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.