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A functional homolog of mammalian protein kinase C participates in the elicitor-induced defense response in potato. 总被引:6,自引:1,他引:5
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The elicitor-induced activation of the potato pathogenesis-related gene PR-10a is positively controlled by a protein kinase(s) that affects the binding of the nuclear factors PBF-1 (for PR-10a binding factor-1) and PBR-2 to an elicitor response element (ERE). In this study, we have identified a kinase that has properties similar to the conventional isoenzymes of the mammalian protein kinase C (PKC) family. the treatment of potato tuber discs with specific inhibitors of PKC abolished the elicitor-induced binding of the nuclear factor PBF-2 to the ERE. This correlated with a reduction in the accumulation of the PR-10a protein. In contrast, treatment of the tuber discs with 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, led to an increase in binding of PBF-2 to the ERE and the corresponding increase in the level of the PR-10a protein, mimicking the effect seen with the elicitor arachidonic acid. Biochemical characterization of proteins extracted from the particulate fraction of potato tubers demonstrated that a kinase belonging to the conventional isoforms of PKC is present. This was confirmed by immunoprecipitation with antibodies specific to the conventional isoforms of human PKC and in-gel kinase assays. The ability of the immunoprecipitates to phosphorylate the alpha-peptide (a PKC specific substrate) in the presence of the coactivators calcium, phosphatidylserine, and TPA strongly suggested that the immunoprecipitated kinase is similar to the kinase characterized biochemically. Finally, the similar effects of the various modulators of PKC activity on the elicitor-induced resistance against a compatible race of Phytophthora infestans implicate this kinase in the overall defense response in potato. 相似文献
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The Activation of the Potato PR-10a Gene Requires the Phosphorylation of the Nuclear Factor PBF-1 总被引:8,自引:3,他引:5
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The pathogenesis-related gene PR-10a (formerly STH[middot]2) is induced in various organs of potato after wounding, elicitor treatment, or infection by Phytophthora infestans. Deletion analysis of the promoter of the PR-10a gene enabled us to identify a 50-bp region, located between positions -155 and -105, necessary for the elicitor responsiveness of the [beta]-glucuronidase reporter gene in transgenic potato plants. Within this region, a 30-bp sequence, located between positions -135 and -105, was necessary for the activation of the promoter by the elicitor. However, strong promoter activity after elicitor treatment required the presence of a 20-bp sequence located between positions -155 and -135. The region between -135 and -105 was specifically recognized by two nuclear factors, PBF-1 (PR-10a Binding Factor 1) and PBF-2, and binding of PBF-1 was coordinated with the accumulation of the PR-10a mRNA. Gel shift assays using nuclear extracts pretreated with sodium deoxycholate or alkaline phosphatase suggested that PBF-1 is a multimeric factor in which at least one of the constituent proteins can be phosphorylated. Treatment with alkaline phosphatase also indicated that binding of PBF-1 is positively regulated by phosphorylation and that it is phosphorylated only in tissues in which PR-10a is expressed. The use of protein phosphatase and kinase inhibitors in vivo provided additional evidence that wounding and elicitor treatment induce the phosphorylation of PBF-1 and that this phosphorylation is associated with gene activation. 相似文献
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The structure of the nuclear factor-kappaB protein-DNA complex varies with DNA-binding site sequence 总被引:3,自引:0,他引:3
Menetski JP 《The Journal of biological chemistry》2000,275(11):7619-7625
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Jennifer H. Anolik Carolyn M. Klinge Colleen L. Brolly Robert A. Bambara Russell Hilf 《The Journal of steroid biochemistry and molecular biology》1996,59(5-6):413-429
To determine whether accessory proteins mediate the ligand- and DNA sequence-dependent specificity of estrogen receptor (ER) interaction with DNA, the binding of partly purified vs highly purified bovine ER to various estrogen response elements (EREs) was measured in the presence of different ER ligands. Partly purified estradiol-liganded ER (E2-ER) binds cooperatively to stereoaligned tandem EREs flanked by naturally occurring AT-rich sequences, with a stoichiometry of one E2-ER dimer per ERE. In contrast, highly purified E2-ER binds with a 10-fold lower affinity and non-cooperatively to EREs flanked by the AT-rich region. Moreover, the binding stoichiometry of highly purified E2-ER was 0.5 E2-ER dimer, or one monomer per ERE, independent of the ERE flanking sequence. Interestingly, the binding of ER liganded with the antiestrogen 4-hydroxytamoxifen (4-OHT-ER) was non-cooperative with an apparent stoichiometry of 0.5 4-OHT-ER dimer per ERE, regardless of ER purity or ERE flanking sequence. We recently showed that when 4-OHT-ER binds DNA, one molecule of 4-OHT dissociates from the dimeric 4-OHT-ER-ERE complex, accounting for the reduced apparent binding stoichiometry. In contrast, ER covalently bound by tamoxifen aziridine (TAz) gave an ERE binding stoichiometry of one TAz-ER dimer per ERE, and TAz-ER binds cooperatively to multiple AT-rich EREs, regardless of the purity of the receptor. We have obtained evidence that purification of ER removes an accessory protein(s) that interacts with ER in a sequence- and/or DNA conformational-dependent manner, resulting in stabilization of E2, but not 4-OHT, in the ligand binding domain when the receptor binds to DNA. We postulate that retention of ligand by ER maintains the receptor in a conformation necessary to achieve high-affinity, cooperative ERE binding. 相似文献
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Uterine estrogen receptor-DNA complexes: effects of different ERE sequences, ligands, and receptor forms 总被引:2,自引:0,他引:2
Previous studies used the gel retardation assay to examine the binding of the mouse estrogen receptor (ER) to the estrogen-responsive element (ERE) from the vitellogenin A2 gene (VitA2ERE). Multiple specific complexes were formed when the ER was bound to various estrogen agonists or antagonists, or in the absence of bound hormone. The ERE from the human PS2 gene, which varies from the consensus ERE by one base change in the right arm, was used in this study to determine the effect of DNA sequence on ER-ERE interaction with various ligand-receptor complexes. Partially purified ligand-free soluble ER showed a 3-fold lower affinity for the PS2ERE than for the VitA2ERE, suggesting a possible influence of the imperfect DNA sequence on certain binding interactions. However, multiple complexes of similar affinity were formed with the PS2 sequence by nuclear ER regardless of the agonist or antagonist bound. In gel retardation experiments, antagonist (LY117018) nuclear ER complexes bound to either PS2 or VitA2ERE migrated more slowly than agonist complexes, indicating that the slower migrating form of the complex was not due to the DNA sequence. Interestingly, soluble ER bound by LY 117018 did not produce this decreased mobility complex, suggesting that it was specific to the nuclear form of the ER antagonist complex. Receptor activation has been linked with exposure to increased temperature, resulting in an ER form that has an increased affinity for DNA. The binding of molybdate-stabilized nonactivated 8S ER to VitA2ERE was studied to determine the effect of temperature on ER binding.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Selection of novel, specific single-stranded DNA sequences by Flp, a duplex-specific DNA binding protein. 总被引:2,自引:0,他引:2
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Flp is a member of the integrase family of site-specific recombinases. Flp is known to be a double-stranded (ds)DNA binding protein that binds sequence specifically to the 13 bp binding elements in the FRT site (Flprecognitiontarget). We subjected a random pool of oligonucleotides to the in vitro binding site selection method and have unexpectedly recovered a series of single-stranded oligonucleotides to which Flp binds with high affinity. These single-stranded oligonucleotides differ in sequence from the duplex FRT site. The minimal length of the oligonucleotides which is active is 29 nt. This single strand-specific DNA binding activity is located in the same C-terminal 32 kDa domain of Flp in which the site-specific dsDNA binding activity resides. Competition studies suggest that the apparent affinity of Flp for single-stranded oligonucleotide is somewhat less than for a complete duplex FRT site but greater than for a single duplex 13 bp binding element. We have also shown that Cre, another member of the integrase family of site-specific recombinases, also exhibits single-stranded DNA binding similar to that of Flp. 相似文献
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The binding affinity of Ff gene 5 protein depends on the nearest-neighbor composition of the ssDNA substrate
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The Ff gene 5 protein (g5p) is considered to be a nonspecific single-stranded DNA binding protein, because it binds cooperatively to and saturates the Ff bacteriophage single-stranded DNA genome and other single-stranded polynucleotides. However, the binding affinity Komega (the intrinsic binding constant times a cooperativity factor) differs by over an order of magnitude for binding to single-stranded polynucleotides such as poly[d(A)] and poly[d(C)]. A polynucleotide that is more stacked, like poly[d(A)], binds more weakly than one that is less stacked, like poly[d(C)]. To test the hypothesis that DNA base stacking, a nearest-neighbor property, is involved in the binding affinity of the Ff g5p for different DNA sequences, Komega values were determined as a function of NaCl concentration for binding to six synthetic sequences 48 nucleotides in length: dA48, dC48, d(AAC)16, d(ACC)16, d(AACC)12, and d(AAACC)9A3. The binding affinities of the protein for these sequences were indeed found to be related to the nearest-neighbor compositions of the sequences, rather than to simple base compositions. That is, the g5p binding site, which is spanned by four nucleotides, discriminates among these sequences on the basis of the relative numbers of nearest neighbors (AA, CC, and AC plus CA) in the sequence. The results support the hypothesis that the extent of base stacking/unstacking of the free, nonbound ssDNA plays an important role in the binding affinity of the Ff gene 5 protein. 相似文献