Polymorphisms in the 3′-untranslated region of human and monkey dopamine transporter genes affect reporter gene expression
Polymorphisms in the 3′-untranslated region of human and monkey dopamine transporter genes affect reporter gene expression
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DOI：
10.1038/sj.mp.4000921
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发表时间：
2002
影响因子：
11
通讯作者：
Gregory M. Miller;B. Madras
中科院分区：
文献类型：
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作者：
Gregory M. Miller;B. Madras
多巴胺转运蛋白（DAT）水平在正常受试者中变化，在病理状态下偏离正常范围。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）方面不同，我们探讨了序列定义的DAT等位基因的3′-UTR是否可以在体外差异影响报告基因表达水平。我们首先确定，在个体恒河猴，DAT基因的两个等位基因在黑质中表达。然后，我们用HSV-TK-和SV 40驱动的荧光素酶表达载体转染HEK-293细胞，所述荧光素酶表达载体携带含有长度多态性的等位基因的下游DAT 3′-UTR片段。（人类：9或10个重复单位）或固定长度等位基因内的SNP（人类：DraI敏感（DraI+）vs DraI不敏感（DraI-）10-重复等位基因;恒河猴：Bst 1107 I敏感（Bst+）vs Bst 1107 I不敏感（Bst-）12-重复等位基因）。与含有10个串联重复单元的类似载体相比，含有含有9个串联重复单元的人DAT等位基因的3′-UTR区段的载体导致显著更高水平的荧光素酶产生。根据使用的启动子，含有基于SNP而不同的人或猴3′-UTR片段的载体导致荧光素酶基因表达的增加或减少。该报告提供了实验证据，表明DAT基因的3′-UTR的长度或序列的变化可能会影响大脑中DAT蛋白的水平。
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.