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A cDNA clone encoding the entire amino acid sequence of rabbit brown adipose tissue uncoupling protein has been isolated and sequenced. The coding region of this cDNA is 80.6% identical to that of the rat uncoupling protein cDNA. In contrast to rat uncoupling protein for which there are two mRNAs of 1500 and 2000 nucleotides there is only one rabbit uncoupling protein mRNA of 2000 nucleotides. Whereas the rat cDNA hybridizes more strongly to the shorter rat uncoupling protein mRNA the rabbit cDNA hybridizes more strongly to the longer rat uncoupling protein mRNA. Primer extension and Northern blot analysis were performed to try to account for the difference of 430 ± 75 nucleotides between the two rat uncoupling protein mRNAs. Northern blot analysis indicated the presence of 355 more nucleotides in the 3'-untranslated region of the 2000 nucleotide long rat uncoupling protein mRNA than in the 1500 nucleotide long rat uncoupling protein mRNA. The two rat uncoupling protein mRNAs could therefore arise by differential processing. Primer extension revealed that the two rat uncoupling protein mRNAs have a 5'-untranslated region of approximately 186 nucleotides. The deduced amino acid sequence of rabbit UCP is 86% identical with both the rat and hamster proteins. Several regions are conserved in all three uncoupling proteins. The two longest regions of conservation are residues 52 to 69 and 82 to 100 of the mature proteins and correspond to two of several basic regions of the protein that have been suggested as possible targeting sequences. These conserved regions fall within amino acids 52 to 104 of the mature rat protein, which has been shown by others to target a passenger protein to mitochondria. Helical wheel diagrams that correspond to residues 52 to 68 and residues 72 to 92 reveal possible amphiphilic α-helical formations that may be involved in targeting. Regions corresponding to those conserved in the three UCPs are also conserved in three mammalian ADP/ATP carriers and may indicate a common role for these regions, perhaps including targeting. There is almost complete conservation of lysine, arginine, and cysteine residues that are thought to be involved in nucleotide binding and proton transport in the three UCPs. There is a threonine to alanine change at the carboxyl-terminus of the rabbit protein compared to the rat protein. This amino acid difference may explain the differential reactivities of rabbit and rat UCP with an antibody preparation against rat UCP. / Thesis / Master of Science (MSc)

Cells infected with herpes simplex virus show a rapid cessation of protein synthesis and a dramatic decline in the levels of mRNA; a process known as host shutoff. This effect is attributed to a viral tegument protein called the virion host shutoff protein, or vhs. The mechanism by which vhs induces mRNA degradation is not yet understood. It is not known whether vhs possesses RNase activities or if it acts in combination with other cellular factors. To gain a better understanding of the function of vhs, I examined RNA degradation in detail by analyzing the RNA decay products generated in the presence of vhs. 𝘐𝘯 𝘷𝘪𝘷𝘰 experiments, performed by infecting murine erythroid leukemia cells with HSV, revealed that beta-globin mRNA is rapidly degraded, in the presence of vhs, without being converted to detectable decay intermediates. The half-life of this mRNA was 15 and 60 minutes for HSV-2 and HSV-1, respectively. Using vhs translated in a rabbit reticulocyte lysate system, I found that vhs induced rapid decay at the 5' end of a capped RNA molecule. The decay event was endonucleolytic and occurred at preferred sites downstream of the cap, generating capped oligonucleotides. Unlike influenza RNA polymerase, the cleavage event did not occur at a fixed distance from the cap since capped oligos of differing size were generated from different RNA substrates. My data indicate that vhs induced cleavage exhibits a strong, but not absolute preference for RNAs possessing an m⁷G cap, which may account for vhs' specificity for m RNAs 𝘪𝘯 𝘷𝘪𝘷𝘰. / Thesis / Master of Science (MSc)