Identification of a protein kinase multigene family of Dictyostelium discoideum: molecular cloning and expression of a cDNA encoding a developmentally regulated protein kinase. |
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| Authors: | B Haribabu and R P Dottin |
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| Affiliation: | Department of Biological Sciences, Hunter College, City University of New York, NY 10021. |
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| Abstract: | We have identified protein kinase genes of Dictyostelium by using highly conserved amino acid sequence motifs to design the synthesis and amplification of DNA fragments by polymerase chain reactions (PCRs). Cloning and sequencing the PCR products have revealed five different members of the protein kinase multigene family. These five putative kinases showed varying degrees of amino acid sequence similarity (40-70%) to protein kinases in data bases and contained invariant amino acid residues characteristic of protein kinases. DNA from PCR was labeled and used to isolate several lambda gt11 cDNA clones, including one full-length one (Dd kinase-2). The nucleotide sequence of Dd kinase-2 contained a region identical to one of the cloned kinase fragments amplified by PCR, and based on the deduced amino acid sequence Dd kinase-2 encodes a protein of 479 amino acids. A 350-amino acid kinase domain at the C-terminal end shows high homology to the catalytic domains of protein kinase A, protein kinase C, S-6 kinase of Xenopus, and the suppressor of cdc25 of yeast. The N-terminal domain is highly basic and also contains alternating threonine/proline residues. The cDNA hybridized to a single copy gene but to two differentially regulated mRNAs--a 2.0-kilobase mRNA that is expressed in vegetative cells and a 2.2-kilobase mRNA that is expressed during development. The larger mRNA is induced by cAMP by using a cell-surface receptor-mediated signal transduction pathway. |
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