Gene targeting of the aggregation stage cAMP receptor cAR1 in Dictyostelium.
cAR1, a G-protein-linked surface cAMP receptor, plays a central role in the development of Dictyostelium. To investigate its role, we sought to target the cAR1 gene by homogolous recombination. Transformation of these amoebas with appropriately designed vectors results in integration into the cAR1 locus with high frequency. cAR1 "null" mutants, resulting from double crossover events, fail to bind or sense cAMP and arrest in early development. The null mutants can be rescued by constitutive expression of a wild-type cAR1 cDNA. Carboxy-terminal deletion mutants, derived from single crossover events, express a truncated form of cAR1 that binds and senses cAMP. These cells proceed through the developmental program, albeit with a delay. Constitutive expression of a similar truncated form of cAR1 also rescues the null mutant. These observations prove that cAR1-mediated signal transduction controls the development of Dictyostelium and allow structural/functional studies of a G-protein-linked receptor in its natural context.
Sun
T J
TJ
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
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D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002633:Chemotaxis; D002872:Chromosome Deletion; D000242:Cyclic AMP / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004023:Dictyostelium / Q000235:genetics / Q000502:physiology*; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D005810:Multigene Family; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011953:Receptors, Cyclic AMP / Q000235:genetics* / Q000378:metabolism
D004271:DNA, Fungal; D015345:Oligonucleotide Probes; D011953:Receptors, Cyclic AMP; C068125:cyclic AMP receptor cAR1; D000242:Cyclic AMP