Date of Award

8-2008

Degree Type

Thesis

Degree Name

MS Biology

Department

Biology

Advisor

Anne M. Pumfery

Committee Member

Allan Blake

Committee Member

Heping Zhou

Committee Member

Carroll Rawn

Committee Member

Carolyn Bentivegna

Keywords

Kaposi's sarcoma, Herpesvirus, Enzymes, dUTPase

Abstract

A dUTPase is a crucial enzyme that hydrolyzes dUTP to dUMP. This reaction prevents the mutagenic or lethal misincorporation of uracil into DNA. For that reason, the enzyme is required for efficient DNA replication. Previous studies have shown that has ORFl l dUTPase-like motifs and thus may be a dUTPase. Generally, gammaherpesviruses contain six characteristic dUTPase motifs. In particular ORFl 1 and contains motifs 1, 2, 4, 6. While the characteristic motifs of gamroaberpesviruses include motifs 1, 2, 3, 4, 5, and 6, the number of dUTPase-like motifs in ORFl 1 's protein sequence is substantial. Thus, ORFl 1 may be a dUTPase. To further investigate this hypothesis, ORFl 1 was cloned, expressed in E. coli, and the protein was examined in a dUTPase assay. ORFl 1 was cloned into pGEX-5x-3 through a sequential process starting with a polymerase chain reaction (PCR) and ending with the isolation of plasmid DNA containing ORFl 1. The positive clones were sequenced and confirmed. Subsequently, ORFl 1 was expressed as a GST fusion protein in E. coli. Verification of expression was done by purifying the GST proteins using glutathione beads. The purified proteins were evaluated by SOS-PAGE. The SOS-PAGE demonstrated purification of proteins with their expected sizes. Finally, the lack of dUTPase activity was demonstrated by a dUTPase assay. Bacterial extracts expressing ORFl 1 were incubated with dUTP and the separation of nucleotides was evaluated using thin layer chromatography. In summary, our results demonstrate that Kaposi's sarcoma-associated herpesvirus ORFl 1 does not code for a functional dUTPase. Further studies are needed to determine the function of the protein.

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