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Cloning of cDNA for DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein
T Kurihara1, R M Lewis, J Eisler
1Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.
Abstract:
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.
Insights
Researchers isolated a bovine DARPP-32 (dopamine- and adenosine 3
Area of Science:
- Molecular Biology
- Neuroscience
- Genetics
Background:
- Dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein (DARPP-32) is a key signaling molecule in the brain.
- Understanding the genetic basis and expression of DARPP-32 is crucial for neuroscience research.
Purpose of the Study:
- To isolate and characterize the complementary DNA (cDNA) encoding bovine DARPP-32.
- To investigate the gene structure and mRNA expression patterns of bovine DARPP-32.
Main Methods:
- Construction and screening of a bovine cDNA library in *Escherichia coli* using colony hybridization.
- In situ immunoblot assay to confirm protein expression.
- DNA sequencing of the longest cDNA clone (pTKD7).
- Southern blot analysis of bovine genomic DNA.
- Northern blot analysis of caudate nucleus RNA.
Main Results:
- Isolation of three positive cDNA clones encoding bovine DARPP-32.
- Sequence analysis confirmed a coding region matching the known amino acid sequence.
- Southern blot indicated a single gene for DARPP-32 in the bovine genome.
- Northern blot revealed two abundant mRNA species (1.8 and 1.65 kb) in the caudate nucleus.
Conclusions:
- The study successfully isolated and sequenced the cDNA for bovine DARPP-32.
- Evidence suggests a single gene encodes DARPP-32, with specific mRNA expression in the caudate nucleus.
- These findings provide a molecular basis for studying DARPP-32 function in the brain.