The long range objectives of this research program are to understand the structure, function, evolution and regulation of the cyclic nucleotide phosphodiesterase (PDE) enzymes and the genes which encode them. These enzymes hydrolyze cyclic nucleotides and therefore serve to inactivate or modulate the biological signals produced by hormones, neurotransmitters or other agents which function by increasing intracellular Cyclic nucleotide levels. Studies of three mammalian genes, each homologous to the Drosophila dunce gene, and each coding for a cAMP PDE are proposed. Nucleic acid and antibody. probes specific for each will be used to reveal the populations of cells expressing the mRNAs and proteins in the rat brain. This will allow determination of which of the mammalian PDEs are concentrated in specific neuronal processes, like the dunce PDE in flies. To evaluate the physiological role(s) of one or more of the PDE genes, mutant mice will be obtained and analyzed phenotypically, especially with respect to potential neurological problems. This is particularly important, since one major phenotype associated with dunce mutation in flies is a pronounced memory deficit. Since existing cDNA clones and Northern blotting suggest that the PDE genes code for several proteins by alternative splicing of RNAs, additional mouse brain cDNAs will be isolated for a representative PDE gene to reveal the protein product diversity and to isolate full-length copies of the mRNAs. The cDNA clones will be expressed in yeast, to characterize the biochemical properties of the enzymes and to determine whether they are inhibited by the antidepressant rolipram, like the existing representatives. The cDNA clones, in turn, will be used to isolate genomic clones for the gene. The structure of the gene will be elucidated to compare its structure with the remarkably complex counterpart from Drosophila. The transcription start site(s) will be mapped and the promoter region(s) characterized to potentially understand the signals responsible for spatial expression patterns in the brain. We expect these experiments to contribute critical structural and functional information about these members of an important family of enzymes, the cyclic nucleotide PDEs.

R01GM047680

1991-09-01

1995-08-31