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Characterization of diversity-generating retroelement reverse transriptases from thermophilic bacteria

  • Author(s): Shaw, Kharissa Litín
  • et al.
Abstract

Diversity-generating retroelements (DGRs) are a class of genetic elements that create massive diversity in a target protein. DGRs are capable of creating at least 1020 unique amino acid sequences. The only other systems known to create such diversity in proteins are found in the adaptive immune systems of vertebrates. DGRs were initially discovered in the temperate bacteriophage BPP-1. This DGR was shown to be beneficial to the phage because it enables the phage to adapt to changes in its host. DGRs operate in a reverse transcriptase-mediated and template- dependent process. The DGR reverse transcriptase has the unique ability to create adenine-specific infidelity. DGR reverse transcriptases belong to a distinct clade of reverse transcriptases, one that little is known about. This thesis focuses on the characterization of such reverse transcriptases from two thermophilic bacteria. Thermus aquaticus was previously identified as encoding a DGR. In this thesis, I show that the T. aquaticus reverse transcriptase, when expressed recombinantly in Escherichia coli, likely forms soluble aggregates and is not amenable to purification. Additionally, I identify Caloramator australicus as a thermophilic bacterium with a DGR. I developed a means to express and purify a truncated version of the C. australicus reverse transcriptase, termed CalRT-33. I show that CalRT-33 is thermostable but does not form a stable complex with the accessory variable determinant, which is distinct from the case for the BPP-1 reverse transcriptase and its accessory variability determinant. Lastly, I identify future directions for the study of DGR reverse transcriptases from thermophilic organisms

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