Adaku Iwueze
James Madison University
Subject Listing - Biology: Molecular Biology
Advisor: Dr. Judith Wubah
Thursday, Poster Session 1, Presentation Kiosk 7 B, Health & Fitness Center
THE USE OF GREEN FLUORESCENT PROTEIN TO CREATE EXPRESSION PLASMIDS FOR TWO SALVAGE PATHWAY ENZYMES
In the United States, 4 million babies are born each year and approximately 150,000 of these are affected by congenital malformations. It is not known whether the 4,000 neonates born each year that suffer from mitochondrial DNA defects are included in this number. The mitochondrion, which is the energy generator in the cell, functions in other metabolic processes. Deoxyguanosine kinase (dGK) and thymidine kinase2 (TK2) are nuclear genes, which encode the rate-limiting enzymes in the mitochondrial salvage pathway, a process for generating deoxynucleotide triphosphates. Mutations in dGK and TK2 lead to various forms of mtDNA depletion syndrome and early infantile deaths. A novel isoform of dGK, dGK3 and TK2 are being analyzed with a long-term goal to determine their possible role in birth defects. The objective of this study is to create expression plasmids of dGK3 and TK2 which will be used to determine the intracellular location of these two genes. Using total RNA from adult mouse liver, full-length dGK3 and TK2 complementary DNA (cDNA) were amplified which contained EcoR1 sites at both ends. The cDNA products were each cloned into pGEM-T vector and digested with EcoR1 enzyme to ensure incorporation of the sites. The digested products were ligated with a digested pEGFP vector. Screening of several clones has yielded an expression plasmid for dGK3 and three possible expression plasmids for TK2. The generated TK2-GFP expression plasmids will be sequenced for confirmation. In summary, GFP expression plasmids will be confirmed by sequencing and used in future transfection studies.
Funded by the Jefferres Memorial Trust Fund Grant # J699 and the NIH/Bridges Grant #R25GM063995.
Advisor: Dr. Judith Wubah, Assistant Professor, Biology, James Madison University, Harrisonburg, VA