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Characterization of promoter of a novel GTPase in T cell, TGTP

University of British Columbia

Our laboratory have previously described the cloning of a cDNA encoding a 50 kDa GTPase designated TGTP. TGTP s expression was induced rapidly after TCR crosslinking. However, subsequent studies showed that the induction was nevertheless mediated by IFN-y and that TGTP is inducible by IFN-y in cell lines of diverse lineages. More interestingly, studies in our laboratory showed that TGTP-transfected L cells displayed resistance to infection by the negative strand RNA virus, vesicular stomatitis virus. The main objective of my thesis project was to isolate, characterize and study the promoter of Tgtp, with the hope that this information may further elucidate the function of TGTP. The methologies employed in this study included: (1) identifying the genomic clones that contain the 5 end of the cDNA encoding TGTP; (2) mapping restriction sites and localizing the 5 end of cDNA in a genomic clone; (3) determining the orientation of the genomic DNA and, hence, the location of the promoter region; (4) assessing the promoter activity of a genomic DNA fragment that may carry the promoter region; (5) determining the sequence of the promoter region; and (6) analyzing the sequence of the promoter region to determine and locate different DNA motifs such as the GAS motif that is required for IFN-y induction. In this study I showed that the promoter of Tgtp contains a TATA box, a GAS sequence (TTCCAGGAA), a Myb binding site, two CF-2 binding sites, three c-Myc/Max binding sites and a SP-1 binding site. In vitro functional assay of the Tgtp promoter with a luciferase reporter gene showed that the Tgtp promoter is optimally activated by low concentrations (5.5 U/ml) of IFN-y and, to a lesser extent, by high concentrations (5,500 U/ml) of IFN-a. A unique sequence of eleven repeats of TCTCCCC was also found in the Tgtp promoter. However, the potential role of these repeats in transcriptional regulation of TGTP remains to be determined. These repeats of high GC content (71%) also presented a special challenge to my sequencing efforts. The sequencing difficulty was finally resolved by using PCR templates synthesized in presence of 75% or 100% 7- deaza dGTP.

Thesis/Dissertation

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2009-07-09

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http://hdl.handle.net/2429/10550

Microbiology and Immunology

University of British Columbia

UBCV

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