Title page for ETD etd-11012009-210522


Type of Document Dissertation
Author Ajlouni, Burouj Kayed
Author's Email Address bajlouni@vt.edu
URN etd-11012009-210522
Title Polymorphisms in the Flt1 gene and their relation to expression of the secreted Flt1 variant
Degree PhD
Department Veterinary Medical Sciences
Advisory Committee
Advisor Name Title
No Advisors Found
Keywords
  • RNA Processing
  • sFlt-1
  • SNPs
  • ESEs
  • Vascular Endothelial Growth Factor
  • Angiogenesis
  • Flt-1
Date of Defense 2009-09-07
Availability unrestricted
Abstract
Vascular endothelial growth factor (VEGF) is a potent angiogenic agent. VEGF activates its biologic responses through two cell-surface receptors, Flt1 and Flk1. In addition to the transmembrane form of Flt1, the Flt1 gene also encodes a secreted, truncated form of the receptor (sFlt1) translated from an mRNA in which a portion of intron 13 is preserved. sFlt1 retains high affinity for VEGF and thereby inhibits its

angiogenic activity. Intron 13 contains important cis elements involved in sFlt1 mRNA formation. Here, we test the hypothesis that polymorphisms in the human Flt1 gene, particularly SNPs at sites suspected to contain splicing or cleavage-polyadenylation

signals, influence Flt1 pre-mRNA processing and rates of Flt1 and sFlt1 expression. The NCBI SNP database contained 23 SNPs in the region of interest, one each in exons 13 and 14. An independent human SNP screen confirmed several of the reported SNPs. The web-based ESEfinder software predicted that the exon 13/14 SNPs had reduced potential for recruitment of splicing components. To test effects of exonic SNPs

on Flt1 pre-mRNA processing, wild type and mutant Flt1 minigene plasmids were transfected into NIH/3T3 cells. Both exonic SNPs were associated with ~40% decreases in Flt1:sFlt1 mRNA ratios determined by real-time PCR. To facilitate

exploration of ESEs in regulated RNA splicing, a PERL computer program, “EXONerator,” was written to silence predicted ESEs without altering polypeptide sequence. These results support the notion that small changes in exon composition can

have significant effects on splicing activity and underscore the utility of software tools for hypothesis generation.

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