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Isolation and characterization of a mammalian gene encoding a high-affinity cAMP phosphodiesterase
J Colicelli1, C Birchmeier, T Michaeli
1Cold Spring Harbor Laboratory, NY 11724.
Abstract:
A rat brain cDNA library has been constructed in a Saccharomyces cerevisiae expression vector and used to isolate genes that can function in yeast to suppress the phenotypic effects of RAS2val19, a mutant form of the RAS2 gene analogous to an oncogenic mutant of the human HRAS gene. One cDNA, DPD, was cloned and its genetic and biochemical properties were characterized. A DPD product would share 80% amino acid sequence identity with the Drosophila melanogaster dunce-encoded protein over an extended region. We have shown that the DPD protein is a high-affinity cAMP-specific phosphodiesterase.
Insights
Researchers identified a rat brain gene, DPD, that suppresses a yeast RAS2 mutant. The DPD protein functions as a high-affinity, cAMP-specific phosphodiesterase, offering insights into cellular signaling pathways.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Neuroscience
Background:
- The RAS2 gene in yeast is crucial for cellular signaling pathways.
- Mutations in RAS genes, like RAS2val19, can lead to uncontrolled cell growth, similar to human oncogenes.
- Identifying suppressors of mutant RAS proteins can reveal new regulatory mechanisms.
Purpose of the Study:
- To isolate and characterize genes from a rat brain cDNA library that can suppress the phenotypic effects of a constitutively active RAS2 mutant (RAS2val19) in yeast.
- To determine the function of a novel cloned gene, DPD, identified as a suppressor.
Main Methods:
- Construction of a rat brain cDNA library in a Saccharomyces cerevisiae expression vector.
- Screening the library for clones that suppress the RAS2val19 phenotype in yeast.
- Genetic and biochemical characterization of the isolated DPD gene and its product.
Main Results:
- One cDNA clone, designated DPD, was isolated and shown to suppress the RAS2val19 phenotype.
- The DPD protein shares significant sequence identity (80%) with the Drosophila melanogaster dunce protein.
- Biochemical assays confirmed that the DPD product is a high-affinity, cAMP-specific phosphodiesterase.
Conclusions:
- The DPD gene encodes a cAMP-specific phosphodiesterase that can functionally suppress a mutant RAS2 protein in yeast.
- This finding suggests a conserved role for phosphodiesterases in regulating RAS signaling pathways across species.
- DPD represents a potential target for understanding and modulating cellular signaling in both yeast and potentially mammalian systems.