RESUMO
The myosin II heavy chain (MHC)-specific protein kinase C (MHC-PKC) isolated from Dictyostelium discoideum has been implicated in the regulation of myosin II assembly in response to the chemoattractant, cAMP (Ravid, S., and Spudich, J. A. (1989) J. Biol. Chem. 264, 15144-15150). Here we report that elimination of MHC-PKC results in the abolishment of MHC phosphorylation in response to cAMP. Cells devoid of MHC-PKC exhibit substantial myosin II overassembly, as well as aberrant cell polarization, chemotaxis, and morphological differentiation. Cells overexpressing the MHC-PKC contain highly phosphorylated MHC and exhibit impaired myosin II localization and no apparent cell polarization and chemotaxis. The results presented here provide direct evidence that MHC-PKC phosphorylates MHC in response to cAMP and plays an important role in the regulation of myosin II localization during chemotaxis.
Assuntos
Quimiotaxia , Dictyostelium/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Proteína Quinase C/fisiologia , Animais , Polaridade Celular , AMP Cíclico/metabolismo , FosforilaçãoAssuntos
Genes de Protozoários , Sinais Direcionadores de Proteínas/genética , Splicing de RNA/genética , RNA de Protozoário/genética , Trypanosomatina/genética , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , Primers do DNA/química , DNA de Protozoário/análise , DNA de Protozoário/química , Densitometria , Biblioteca Gênica , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico , Ribonucleoproteínas/genéticaRESUMO
Transmission of potyviruses by aphids depends on the presence of a virus encoded helper-component protein (HC) that also exhibits protease activity. HC was expressed in E. coli from two types of clones: a full-length cDNA clone of PVY and two 5' end clones containing the first three cistrons (3.6-3.7 kbp). The clones derived from the 5' end of PVY expressed HC of the size of the mature component. Other proteins reacting with antibodies to HC were also observed, and their sizes corresponded with those of expected intermediates resulting from partial protease cleavage of the three-cistron polyprotein. On the other hand, the only detectable HC-related product of the full-length clone was a mature-size HC. The presence of a third PVY protease among the first three cistrons is therefore suggested.
Assuntos
Cisteína Endopeptidases/genética , Escherichia coli/genética , Vírus de Plantas/genética , Proteínas Virais/genética , Clonagem Molecular , DNA Viral/genética , Endopeptidases/genética , Expressão Gênica , Vírus de Plantas/enzimologiaRESUMO
A clone harboring the full-length cDNA of potato virus Y in a lambda-DASH vector under the control of a T7 promoter was introduced into Escherichia coli carrying the T7-RNA-polymerase gene on a plasmid. The viral coat protein was expressed and the product was of the same size as the corresponding mature protein in infected plants. Immunoelectronmicroscopy of transfected cell extracts revealed virus-like particles, indicating that the proteins involved in its processing and the viral coat protein retained their native activity.
Assuntos
Capsídeo/genética , Escherichia coli/genética , Regulação Viral da Expressão Gênica , Vírus de Plantas/genética , Capsídeo/química , Microscopia Imunoeletrônica , Solanum tuberosum/microbiologia , TransfecçãoRESUMO
Full-length cDNA of genomic RNA of potato virus Y (PVY) was cloned in one piece into a lambda vector. The order of the EcoRI and SalI fragments of the inserted cDNA was determined. This is the first report of the cloning of a long, expressible, potyvirus genome. The availability of such a clone is a prerequisite for any further study of the molecular biology of this group of viruses, as they are expressed into a self-processed primary polyprotein.
