Miller G M, Madras B K
Harvard Medical School, Division of Neurochemistry, New England Regional Primate Research Center, One Pine Hill Drive, Southborough, MA 01772-9102, USA.
Mol Psychiatry. 2002;7(1):44-55. doi: 10.1038/sj.mp.4000921.
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.
多巴胺转运体（DAT）水平在正常受试者中有所不同，在病理状态下则偏离正常范围。我们研究了DAT基因可能影响DAT蛋白表达的机制。由于DAT基因的3'非翻译区（3'-UTR）在长度和单核苷酸多态性（SNP）方面存在差异，我们探讨了序列定义的DAT等位基因的3'-UTR是否能在体外差异影响报告基因的表达水平。我们首先确定，在恒河猴个体中，DAT基因的两个等位基因在黑质中表达。然后，我们用携带长度多态性（人类：9或10个重复单元）或固定长度等位基因内SNP（人类：Dral敏感（Dral+）与Dral不敏感（Dral-）10重复等位基因；恒河猴：Bst1107I敏感（Bst+）与Bst1107I不敏感（Bst-）12重复等位基因）的DAT等位基因下游3'-UTR片段的单纯疱疹病毒胸苷激酶（HSV-TK）和猴空泡病毒40（SV40）驱动的荧光素酶表达载体转染人胚肾293（HEK-293）细胞。含有9个串联重复单元的人类DAT等位基因3'-UTR片段的载体产生的荧光素酶水平明显高于含有10个串联重复单元的类似载体。根据所使用的启动子，含有基于SNP而不同的人类或猴3'-UTR片段的载体导致荧光素酶基因表达增加或减少。本报告提供了实验证据，表明DAT基因3'-UTR的长度或序列变异可能影响大脑中DAT蛋白的水平。
