Cloning of cDNA for DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein.
A cDNA clone for the mRNA of bovine DARPP-32 (dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein, Mr = 32,000) was isolated from a modified Okayama-Berg plasmid library. Transformed Escherichia coli colonies were screened by in situ colony hybridization with 2 different oligonucleotide probes corresponding to a region unusually rich in glutamate within the protein. Three positive clones were isolated and shown to encode DARPP-32 by an in situ immunoblot assay of their fusion protein products with beta-galactosidase. The results of the sequence analysis of the longest cDNA clone, pTKD7 (1771 nucleotides), revealed a 606-nucleotide-long coding region, in exact agreement with the bovine DARPP-32 amino acid sequence (Williams et al., 1986). Southern blot analysis of total bovine genomic DNA showed that there is a single gene coding for DARPP-32. Northern blot analysis of caudate nucleus RNA using antisense RNA derived from the clone pTKD7 demonstrated the existence of 2 abundant mRNA species, corresponding to 1.8 and 1.65 kilobase in length. The high concentration of DARPP-32 mRNAs in the caudate nucleus is in agreement with the known distribution of this protein.