Elsevier

Gene

Volume 195, Issue 1, 11 August 1997, Pages 11-18
Gene

Structure and organization of the gene encoding human dopamine transporter

https://doi.org/10.1016/S0378-1119(97)00131-5Get rights and content

Abstract

The human dopamine transporter (hDAT) is a member of the subfamily of monoamine transporters which show a common topological structure and possess significant amino acid sequence homology. We isolated and characterized the hDAT gene including about 1 kb of 5′-flanking region. The hDAT gene spans over 64 kb, consisting of 15 exons separated by 14 introns. The intron-exon structure of the hDAT gene is most similar to that of the human noradrenaline transporter (hNAT) gene. Promoter sequence analysis demonstrated a `TATA'-less, `CAT'-less and G+C-rich structure. Two E box and several Sp-1-binding sites exist in the promoter region. These structural features are similar to that of the human D1A dopamine receptor gene and the human monoamine oxidase A gene. The transcription start site was determined by both 5′-RACE and RNase protection assay. We determined the 5′ end of the mRNA by identifying the 5′-terminal cap-G residue in 5′-RACE and RNase protection assay.

Introduction

Dopamine (DA) is the major neurotransmitter of the nigrostriatal system and is involved in the control of motor function, cognition and affect (Iversen, 1971). DA is released from presynaptic vesicle into the synaptic cleft and interacts with postsynaptic DA receptors. Synaptic transmission is terminated by the rapid reuptake of DA into the presynaptic nerve terminals via a specific DA transporter (DAT). Disruption of the mouse DA transporter gene using homologous recombination dramatically affected the system. DA clearance was prolonged and there was a significant downregulation of D1 and D2 dopamine receptor expression in the DAT knockout mice. The reduction of DA reuptake resulted in both a decreased content of DA and loss of tyrosine hydroxylase (TH) in the presynaptic terminals. As well as these biochemical deficits, the DAT knockout mice show spontaneous hyperactivity (Giros et al., 1996), consistent with prolonged activity of DA at postsynaptic receptors. Recent molecular cloning study has showed that the DA transporter is one of the family of Na+/Cl-dependent membrane transporter proteins and contains twelve transmembrane α-helical domains, cytoplasmic N and C termini and a large glycosylated extracellular loop. Psychostimulants, such as cocaine and amphetamine, are known to bind to the DA transporter and inhibit DA reuptake (Giros et al., 1992). The inhibition of dopamine transport is sufficient to account for addictive and psychomotor effects of cocaine. The neurotoxin 1-methyl-4-phenylpyridinium (MPP+), which causes parkinsonism, is accumulated into dopaminergic neurons via the DA transporter. The accumulation of MPP+ by the nigral dopamine cells resulted in selective cell death (German et al., 1988). Downregulation of hDAT mRNA has been observed in Parkinson disease, suggesting that a reduction of dopamine storage (Harrington et al., 1996) may contribute to the pathology of Parkinson disease.

The hDAT gene contains a variable number of tandem repeats (VNTR) at its 3′ non-coding region (Vandenbergh et al., 1992). Investigation of VNTR in various diseases has revealed genetic associations with cocaine-induced paranoia (Gelernter et al., 1994), alcoholism (Muramatsu and Higuchi, 1995) and attention-deficit hyperactivity disorder (Cook et al., 1996). In addition, mutagenesis of DA transporter produced dramatic alternations in dopamine/MPP+ uptake and cocaine binding (Kitayama et al., 1992, Kitayama et al., 1993). Therefore, some of these important diseases may be related to mutations of the hDAT gene. Thus the elucidation of the hDAT genomic structure will contribute considerably to our further understanding of the causes of the diseases mentioned above. To examine the physiological regulation of the expression of the hDAT gene, we have cloned the hDAT gene and clarified its intron-exon structure including approx. 1 kb of the 5′-flanking sequence.

Section snippets

Materials and methods

We used human substantia nigra cDNA (Clontech Laboratories, Palo Alto, CA) as a template for PCR. We synthesized degenerate oligonucleotides which were designed based on the nt sequence of the hDAT cDNA (Giros et al., 1992). PCR fragment was subcloned into pBluescript (Stratagene) and subjected to nt sequence analysis. Nucleotide sequence analysis was performed using the Sanger dideoxynucleotide chain-termination method and cycle sequencing (Sanger et al., 1977). We used the Sequenase enzyme

cDNA cloning and genomic library screening

The first PCR amplification was performed using two degenerate oligonucleotides: 5′-GCCAGGACTCGCGTGCAAAG-3′ and 5′-TTGTGGTTTCCTCCATTTCACCTCT-3′. This reaction resulted in a 2.3-kb band which was very thin. Therefore, we used 1/10 vol. of the first PCR product as a template for a second PCR. In this reaction hemi-`nested' PCR amplification was performed using an internal primer: 5′-GGCCAGACCAAGAGGGAAGAA-3′ and the same primer as used in the first PCR: 5′-TTGTGGTTTCCTCCATTTCACCTTCT-3′. After the

Acknowledgements

This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture and from the Ministry of Health and Welfare of Japan. Part of this work was carried out at the Medical Molecules Exploration Center (MMEC) at Hiroshima University. We thank Dr. P.C. Emson, the Babraham Institute, for thoughtful review of the manuscript.

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