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UBC Theses and Dissertations

DNA polymerase sequence analysis of bacteriophage, podoviridae Reid, Karen E.

Abstract

Our oceans are biologically complex systems that incorporate a collection of participants. The most abundant players, which are the bacteria and viruses, are invisible to the naked eye. Seawater typically contains about a million bacteria and 10 million viruses per millilitre. Despite the abundance of viruses in seawater, little is known about their composition or diversity. This dissertation presents the design and application of a new set of PCR primers created to target a subset of bacteriophages. Bacteriophages are the viral fraction that infect and propagate through living bacterial cells. They vary both in terms of their morphology and their genetic constituents. My work focused on the family Podoviridae, one of 3 bacteriophage families that share similar morphological characteristics. The most notable characteristics include an isometric capsid about 60 nm in diameter, and a short, stubby tail. Moreover, although this family has been extensively reported in transmission electron microscopy scans, this study is the first to obtain Podoviridae genetic information from marine viral communities. To have a sense of the level of diversity that this Family constitutes, I designed a set of primers targeting the DNA polymerase gene (pol). The gene is approximately 2.0 kb in length and the primers target a 1.2-1.4 kb region for amplification. Following product verification and method optimization, I turned my attention to analysing environmental samples. Seven environmental samples from the Straight of Georgia, British Columbia, and one sample from the Gulf of Mexico, within the Mississippi River plume, were selected. The amplified products generated from each sample were cloned and 20 clones for each were randomly chosen for genetic comparison. Restriction fragment length polymorphism (RFLP) and sequencing were the methods adopted for analysis. RFLP analysis revealed 29 distinct restriction patterns of DNA pol genetic variation. These patterns indicate that certain samples contain greater genetic diversity than others and also that podovirus communities within a site are more similar than between sites. Sequencing these restriction patterns revealed that at the nucleotide level, 17 sequences were at least 5% different from each other. DNA pol genes from environmental podoviruses are divergent from coliphage T7 and T3, and are more similar to the marine isolates Roseophage SIO1 and Cyanophage P60. These latest environmental sequences clustered into four groups, two of which contain no cultured representatives. Moreover, the results indicate that the marine podoviruses share a common ancestor with the coliphage lineage. The work presented in this dissertation is the first study to target and examine the genetic diversity of environmental Podoviridae.

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