The requirement for identical inverted terminal repeats (ITRs) for viral viability and the role of internal viral sequences in the specification of the sequences of the termini were investigated. The viral strains used in this study were a variant Ad2 strain Ad2 (mac) and the wild type Ad5 strain which was very similar to the former one in sequence except at the extreme end of the terminal repeat. A hybrid virus (sub54), obtained by recombination between Ad2 (mac) and Ad5, derived the left 41-51% of its genome from Ad2 (mac) and the right 59-49% from Ad5. The identity of the termini was determined by Southern blotting analysis using 32p end labeled oligocleoxynucleotides. Analysis of the sub54 isolate indicated that both Ad2 (mac) and Ad5 ITRs were present. Plaque purification of sub54 demonstrated that viruses with non identical terminal sequences were viable and allowed their characterization. This analysis also indicated that Ad5 ITRs are converted to Ad2 (mac) ITRs possibly as a result of repair of the ends to yield viruses with identical termini. A model involving replication and emphasizing the importance of panhandle formation as a replicative intermediate is proposed. These results also indicated a possible role of the internal sequences of adenovirus in the selection and maintenance of serotype specific ITRs. The preference for Ad2 (mac) termini observed during repair of the ends of sub54 may be related to the origin of the genes coding for the adenoviral polymerase and/or the terminal protein both of which were derived from Ad2 (mac). Further investigation would be required to determine whether these replicative proteins are actually involved in ITR conversion. Transformation of Escherichia coli with a DNA preparation from sub54 infected rat embryo cells resulted in the isolation of the plasmid pFG154. This plasmid contained the entire adenovirus genome with an Ad2 (mac) ITR at the "left" terminus covalently linked to an Ad5 ITR at the "right". Analysis of the viral progeny generated upon transfection of mammalian cells with pFG154 indicated that the Ad2 (mac) ITRs were very efficiently converted to Ad5 termini. These results, although apparently contradictory to those initially obtained from the plaque purification of sub54, may be explained by an ITR repair model which is specific for infectious circles. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23573 |
| Date | 03 1900 |
| Creators | Lippe, Roger |
| Contributors | Graham, Frank, Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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