Title page for ETD etd-05162002-100901


Type of Document Master's Thesis
Author Roche, Rebecca I
URN etd-05162002-100901
Title Role of the Intron 13 Polypyrimidine Tract in Soluble Flt-1 Expression
Degree Master of Science
Department Veterinary Medical Sciences
Advisory Committee
Advisor Name Title
Huckle, William R. Committee Chair
Gillaspy, Glenda E. Committee Member
Robertson, John L. Committee Member
Keywords
  • Angiogenesis
  • Vascular Endothelial Growth Factor
  • RNA Splicing
  • Flt-1
  • Polypyrimidine Tract
  • sFlt-1
Date of Defense 2002-05-02
Availability unrestricted
Abstract
Angiogenesis is the formation of new blood vessels from existing vasculature. Vascular Endothelial Growth Factor (VEGF), a known angiogenic protein, stimulates endothelial cell proliferation and migration via interactions with its receptors, KDR and Flt-1. A secreted form of Flt-1 (sFlt-1), derived from alternatively-spliced RNA, can inhibit actions of VEGF in vivo. It has been suggested that alterations in sFlt-1 expression could significantly change the angiogenic VEGF activity. This project focuses on characterizing intronic elements that regulate Flt-1 mRNA splicing. A "wild-type" construct (pFIN13), containing the first 13 exons, intron 13 and exons 14-30 of mouse Flt-1, was shown to produce both forms of Flt-1 mRNA after transfection into HEK293 cells. To gauge the strength of the native splicing signals in intron 13 of Flt-1, a series of point mutations were made in the polypyrimidine tract using pFIN13. After transient transfection, the levels of Flt-1 and sFlt-1 protein and mRNA were compared using quantitative PCR, RNA hybridization analysis, and protein immunoblotting. Results from quantitative PCR showed that purine substitutions were associated with 120 to 350 fold decreases in Flt-1 mRNA (normalized against neor), consistent with less efficient splicing. These large decreases in Flt-1 mRNA were accompanied by increases in sFlt-1 mRNA. Modest (20 to 100%) increases in Flt-1 mRNA, reflecting improved splicing, resulted from increasing the uridine complement in the polypyrimidine tract. These results suggest that the wild-type polypyrimidine tract is of intermediate strength and may be a regulatory locus for modulating Flt-1: sFlt-1 ratios.
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