Homology cloning of protein kinase and phosphoprotein phosphatase sequences of Dictyostelium discoideum.
Reversible protein phosphorylation appears to be important at several stages in the signal transduction pathways in Dictyostelium discoideum. To elucidate its role, we have isolated sequences encoding putative protein kinases and phosphoprotein phosphatases by homology cloning using polymerase chain reactions (PCRs). Oligonucleotide primers were synthesized for use as forward and reverse primers with their nucleotide sequences deduced from the amino acid sequences of conserved domains of several protein kinases and phosphoprotein phosphatases. The fragments amplified by PCR were cloned, sequenced, and shown to encode parts of five different protein kinases and two phosphoprotein phosphatases. Several features such as the deduced amino acid sequence homology, location of invariant amino acids, GC content, and the codon usage confirmed that one set of clones encode parts of different protein kinases of Dictyostelium. Two clones derived from phosphoprotein phosphatase primers encode fragments of type 1 and type 2A phosphoprotein phosphatases. Amplified fragments were used to screen a lambda gt11 bank, and several cDNA clones for protein kinases were isolated. Some of these show differential expression during development or in response to exogenous cAMP.
Haribabu
B
B
Department of Biological Sciences, Hunter College, CUNY, NY 10021.
Dottin
R P
RP
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004271:DNA, Fungal; D004023:Dictyostelium / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction / Q000379:methods; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction
D004271:DNA, Fungal; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases