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We placed a specific inhibitor of cyclic AMP-dependent protein kinase (PKA) under the control of a prestalk-specific promoter. Cells containing this construct form normally patterned slugs, but under environmental conditions that normally trigger immediate culmination, the slugs undergo prolonged migration. Slugs that eventually enter culmination do so normally but arrest as elongated, hairlike structures that contain neither stalk nor spore cells. Mutant cells do not migrate to the stalk entrance when codeveloped with wild-type cells and show greatly reduced inducibility by DIF, the stalk cell morphogen. These results suggest that the activity of PKA is necessary for the altered pattern of movement of prestalk cells at culmination and their differentiation into stalk cells. We propose a model whereby a protein repressor, under the control of PKA, inhibits precocious induction of stalk cell differentiation by DIF and so regulates the choice between slug migration and culmination.  相似文献
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During the aggregation of Dictyostelium discoideum extracellular cAMP is known to act as a chemotractant and as an inducer of cellular differentiation. However, its intracellular role as a second messenger remains obscure. We have constructed a fusion gene consisting of the cDNA encoding the regulatory subunit (R) of the cAMP-dependent protein kinase fused to the promoter and N-terminal-proximal sequences of a Dictyostelium actin gene. Stable transformants, containing multiple copies of this gene, overproduce the R subunit which accumulates prematurely relative to the endogenous protein. These transformants fail to aggregate. Detailed analysis has shown that they are blocked at interphase, the period prior to aggregation, and that they are severely defective in most responses to cAMP including the induction of gene expression. Our observations suggest that intracellular cAMP acts, presumably by activation of the catalytic subunit of the cAMP-dependent protein kinase, to facilitate early development.  相似文献
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Vectors for expression of truncated coding sequences in Escherichia coli   总被引:6,自引:0,他引:6  
M N Simon  R Mutzel  H Mutzel  M Véron 《Plasmid》1988,19(2):94-102
We describe the construction of vectors for expressing in Escherichia coli DNA fragments obtained by progressive deletions of DNA inserts in single-stranded sequencing vectors as M13 or pTZ according to the methode of Dale et al. (Plasmid 1985, 13, 31-40). These vectors, pIMS1, pIMS5, and pIMS6, harbor all of the elements required for the regulated expression of any open reading frame flanked by EcoRI restriction sites. The encoded peptides contain only a few vector-derived amino acids. A method is described for direct selection of recombinant clones by in situ RNA hybridization. The properties of the expression vector have been analyzed with a DNA deletion series obtained from the cDNA coding for the regulatory subunit of Dictyostelium discoideum cAMP-dependent protein kinase.  相似文献
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A Abrieu  D Fisher  M N Simon  M Dorée    A Picard 《The EMBO journal》1997,16(21):6407-6413
Down-regulation of MAP kinase (MAPK) is a universal consequence of fertilization in the animal kingdom, although its role is not known. Here we show that MAPK inactivation is essential for embryos, both vertebrate and invertebrate, to enter first mitosis. Suppressing down-regulation of MAPK at fertilization, for example by constitutively activating the upstream MAPK cascade, specifically suppresses cyclin B-cdc2 kinase activation and its consequence, entry into first mitosis. It thus appears that MAPK functions in meiotic maturation by preventing unfertilized eggs from proceeding into parthenogenetic development. The most general effect of artificially maintaining MAPK activity after fertilization is prevention of the G2 to M-phase transition in the first mitotic cell cycle, even though inappropriate reactivation of MAPK after fertilization may lead to metaphase arrest in vertebrates. Advancing the time of MAPK inactivation in fertilized eggs does not, however, speed up their entry into first mitosis. Thus, sustained activity of MAPK during part of the first mitotic cell cycle is not responsible for late entry of fertilized eggs into first mitosis.  相似文献
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The cAMP-dependent protein kinase (PKA) holoenzyme of Dictyostelium comprises a single regulatory (R) and catalytic (C) subunit, and both proteins increase in concentration during cellular aggregation. In order to determine the role of the kinase, we have constructed mutants of the R subunit that are defective in cAMP binding, in inhibition of the C subunit, or in both functions. Analysis of these mutants suggests that overexpression of the unmutated R subunit, which is known to block development, occurs by direct inactivation of the C subunit rather than by an effect on intracellular cAMP levels. Cells with an inactive C subunit (PKA- cells) are defective in cAMP relay, the production of cAMP in response to extracellular cAMP stimulation. This presumably accounts for their inability to undertake aggregation. When mixed with wild-type cells, PKA- cells migrate toward the signalling centre but remain confined to the periphery of the tight aggregate and are lost from the back of the migratory slug. This suggests that PKA may be required during the late, multicellular stages of development. Consistent with this, we find that a number of postaggregative genes are not expressed in PKA- cells, even when they are allowed to synergise with normal cells.  相似文献
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lambda gt11 phages harboring five different cDNA fragments for the regulatory (R) subunit of Dictyostelium discoideum cAMP-dependent protein kinase (CAK) directed the synthesis of this protein in Escherichia coli cells. Crude bacterial extracts were probed with an antiserum against the Dictyostelium R subunit. The presence of specific epitopes for the R subunit in a given extract was compared with high-affinity cAMP-binding activity and with the ability to inhibit the catalytic (C) subunit through protein-protein interaction. The expression and the biochemical properties of these proteins were correlated with their cDNA nucleotide sequence. The results show that the Dictyostelium R subunit can be functionally expressed in E. coli cells either as a fusion protein with beta-galactosidase or as a nonfusion protein. In both cases, the products of cDNA clones containing the entire coding sequence retained high-affinity cAMP-binding activity and the capacity to interact with the catalytic subunit. One of the fusions, lacking the 94 N-terminal residues, failed to inhibit catalytic activity, although it bound cAMP with an affinity similar to that of the native R protein from D. discoideum.  相似文献
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cAMP-dependent protein kinase from Dictyostelium discoideum   总被引:1,自引:0,他引:1  
The cAMP-dependent protein kinase (cAK) from Dictyostelium discoideum is an enzyme composed of one catalytic and one regulatory subunit. Upon binding of cAMP, the holoenzyme dissociates to liberate free active catalytic subunits. The cAK is developmentally regulated, ranging from very little activity in vegetative cells to maximal expression in postaggregative cells. Although there is no immunological cross-reaction between the subunits of cAKs from Dictyostelium and from other organisms, they share several biochemical properties. A complete cDNA for the regulatory subunit has been cloned and sequenced. Only one copy of the gene for the regulatory subunit is present per haploid genome. On the basis of the comparison of the structure of the cAK from Dictyostelium with its counterparts in yeast and higher eukaryotes, we propose a model for the evolution of cyclic-nucleotide-binding proteins.  相似文献
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