Negative modulator of the rat D2 dopamine receptor gene.
The rat D2 dopamine receptor gene is transcribed from a TATA-less promoter that has an initiator-like sequence and several putative Sp1 binding sites. The main activator of this gene is between nucleotides -75 and -29, and a strong negative modulator is located between bases -217 and -76 (Minowa, T., Minowa, M. T., and Mouradian, M. M. (1992) Biochemistry 31, 8389-8396). In the present investigation, a small deletion series within this negative modulator fused with the reporter gene for chloramphenicol acetyltransferase was used to transfect the D2-expressing cells, NB41A3. Two cis-acting functional DNA sequences were identified: a 41-base pair segment between nucleotides -116 and -76 (D2Neg-B), which decreased transcription from the D2 promoter by about 45%, and a 26-base pair segment between nucleotides -160 and -135 (D2Neg-A), which, in the presence of the downstream negative modulator, reduced transcription down to the level of a promoterless vector. DNase I footprinting, gel mobility shift, and competitive cotransfection experiments suggested that D2Neg-A functions without trans-acting factors, whereas D2Neg-B interacts with nuclear factors at its Sp1 binding sequences. Gel supershift with anti-Sp1 antibody and UV cross-linking experiments revealed that a novel 130-kDa factor as well as Sp1 interact with D2Neg-B in NB41A3 cells. This novel protein recognizing Sp1 binding sequences in the D2 gene negative modulator is also found in nuclear extract from the rat striatum.
Minowa
T
T
Genetic Pharmacology Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
Minowa
M T
MT
Mouradian
M M
MM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D016384:Consensus Sequence; D003342:Corpus Striatum / Q000378:metabolism; D017931:DNA Primers; D015342:DNA Probes; D003850:Deoxyribonuclease I; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D051381:Rats / Q000235:genetics*; D017448:Receptors, Dopamine D2 / Q000096:biosynthesis* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D015342:DNA Probes; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I