Polymorphisms in the 3'-untranslated region of human and monkey dopamine transporter genes affect reporter gene expression.
Dopamine transporter (DAT) levels vary in normal subjects and deviate from the normal range in pathological states. We investigated mechanisms by which the DAT gene may influence DAT protein expression. As the 3'-untranslated region (3'-UTR) of the DAT gene varies with regard to length and single nucleotide polymorphisms (SNPs), we addressed whether the 3'-UTR of sequence-defined DAT alleles can differentially affect the level of reporter gene expression in vitro. We first established that within individual rhesus monkeys, two alleles of the DAT gene were expressed in the substantia nigra. We then transfected HEK-293 cells with HSV-TK- and SV40-driven luciferase expression vectors harboring downstream DAT 3'-UTR segments of alleles containing polymorphisms of length (human: 9 or 10 repeat units) or SNPs within alleles of fixed length (human: DraI-sensitive (DraI+) vs. DraI-insensitive (DraI-) 10-repeat alleles; rhesus monkey: Bst1107I-sensitive (Bst+) vs. Bst1107I-insensitive (Bst-) 12-repeat alleles). Vectors containing the 3'-UTR segment of a human DAT allele containing nine tandem repeat units resulted in significantly higher levels of luciferase production than analogous vectors containing 10 tandem repeat units. Depending on the promoter used, vectors containing the human or monkey 3'-UTR segments that differed on the basis of an SNP resulted in increases or decreases in luciferase gene expression. This report provides experimental evidence that variability in the length or the sequence of the 3'-UTR of the DAT gene may influence levels of DAT protein in the brain.
Miller
G M
GM
Harvard Medical School, Division of Neurochemistry, New England Regional Primate Research Center, One Pine Hill Drive, Southborough, MA 01772-9102, USA.
Madras
B K
BK
D020413:3' Untranslated Regions / Q000235:genetics*; D000483:Alleles; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D050483:Dopamine Plasma Membrane Transport Proteins; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008253:Macaca mulatta / Q000235:genetics*; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000235:genetics*; D009419:Nerve Tissue Proteins; D020641:Polymorphism, Single Nucleotide; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D013378:Substantia Nigra / Q000378:metabolism; D014162:Transfection
D020413:3' Untranslated Regions; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C493214:SLC6A3 protein, human; D008156:Luciferases