Defense Date

2006

Document Type

Thesis

Degree Name

Master of Science

Department

Pharmacology & Toxicology

First Advisor

Dr. Richard G. Moran

Abstract

Folic acid is an essential vitamin utilized in the one-carbon metabolism pathway for the synthesis of purine and thymidine nucleotides, which are necessary for cell growth and proliferation. As a result, the enzymes that participate in the metabolism of folic acid have been good targets for cancer chemotherapy. Folylpoly-γ-glutamate synthetase (FPGS) is an enzyme in the folate metabolism pathway that catalyzes the addition of glutamic acid to the naturally occurring folates, thereby allowing the retention of folate cofactors in cells. Similarly, in the case of cancer chemotherapy, antifolates, such as Lometrexol and Tomudex are retained in cells through the activity of FPGS. Consequently, any single nucleotide polymorphisms (SNPs) that exist in the fpgs gene may decrease or increase the cytotoxicity of antifolates and, ultimately, the clinical response rate to antifolate therapy. The goal of this project is to define the position and frequency of single nucleotide polymorphisms (SNPs) in the mRNA made from the fpgs gene from peripheral blood of one hundred normal individuals. Six Polymerase Chain Reaction (PCR) primers were designed to amplify the gene as three overlapping pieces and four primers were designed for sequencing of the three PCR products. In this study, we found polymorphic sites at nucleotides 64, 123, 253, 423, 1334 and 1781. The majority of the samples (49/88) expressed rnRNA with point mutations on at least one allele at base 64, while 8 samples had a SNP at base 123. At nucleotide 123, 6 samples expressed the heterozygote GIA genotype, and one sample expressed the homozygote A/A allele at this site. At nucleotide 423, two samples expressed a G allele and also the common C allele. While the SNPs at nucleotide 64, 123, and 423 caused a silent or conservative mutation in the gene, in sample 82, a mutation C253T produced an amino acid change from an arginine to tryptophan, which may cause a functional change in the fpgs protein, due to the significant change in size and charge of the wild type amino acid. Similarly, sample 26 expessed a homozygote T/T allele at nucleotide 1334 instead of the common C/C allele expressed in the remaining samples. This point mutation caused a valine to alanine amino acid change. We also detected a SNP that is expressed after the stop codon in sample 40.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

June 2008

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