To investigate the regulatory processes involved in the expression of the D2 dopamine receptor gene, a rat genomic clone was isolated using a 21-mer oligonucleotide probe made of exon 1 sequences. A 1.3-kb region including all of exon 1, its 5ʹ-flanking region, and part of intron 1 was sequenced. S1 nuclease analysis indicated three consecutive nucleotides as the main transcription start sites; several weaker sites were also noted between 321 and 363 nucleotides upstream from the 3ʹ end of exon 1. The promoter region lacks TATA and CAAT boxes and is rich in G+C content with several putative Spl binding sites. Transient expression assays using chimeric constructs of D2 promoter deletion mutants-chloramphenicol acetyltransferase gene in the neuroblastoma cell line NB41 A3 which expresses D2 binding sites indicated strong transcription enhancing activity between nucleotides -75 and -30 and silencing activity between nucleotides -217 and -76. DNase I footprinting studies using nuclear extract from NB41A3 suggested Spl binding to its consensus sequence at nucleotide -48 but inhibition of Spl binding at nucleotide -86 by the extract. The D2 promoter could not induce transcription of the heterologous CAT gene in C6 glioma, embryonal NIH 3T3, or hepatic Hep G2 cells. It is concluded that the rat D2 gene shares with the human DIA dopamine receptor gene several features typical of “housekeeping” genes but they are both tissue-specific, regulated genes. Unlike the D1A gene, however, the D2 gene has a strong preference for transcription initiation to three consecutive nucleotides. The homology of the D2 sequence between nucleotides -6 and +11 to the “initiator” sequence might be of functional significance in determining this preference.
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