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OREB1 is a rice ABRE binding factor characterized by the presence of multiple highly-conserved phosphorylation domains (C1, C2, C3, and C4) and two kinase recognition motifs, RXXS/T and S/TXXE/D, within different functional domains. An in vitro kinase assay showed that OREB1 is phosphorylated not only by the SnRK2 kinase, but also by other Ser/Thr protein kinases, such as CaMKII, CKII, and SnRK3. Furthermore, the N-terminal phosphorylation domain C1 was found to be differentially phosphorylated by the SnRK2/SnRK3 kinase and by hyperosmotic/cold stress, suggesting that the C1 domain may function in decoding different signals. The phosphorylation-mediated regulation of OREB1 activity was investigated through mutation of the SnRK2 recognition motif RXXS/T within each phosphorylation module. OREB1 contains a crucial nine-amino acid transactivation domain located near the phosphorylation module C1. Deletion of the C1 domain increased OREB1 activity, whereas mutation of Ser 44, Ser 45, and Ser 48 of the C1 domain to aspartates decreased OREB1 activity. In the C2 domain, a double mutation of Ser 118 and Ser 120 to alanines suppressed OREB1 activity. These findings strongly suggest that selective phosphorylation of the C1 or C2 modules may positively or negatively regulate OREB1 transactivation. In addition, mutation of Ser 385 of the C4 domain to alanines completely abolished the interaction between OREB1 and a rice 14-3-3 protein, GF14d, suggesting that SnRK2-mediated phosphorylation may regulate this interaction. These results indicate that phosphorylation domains of OREB1 are not functionally redundant and regulate at least three different functions, including transactivation activity, DNA binding, and protein interactions. The multisite phosphorylation of OREB1 is likely a key for the fine control of its activity and signal integration in the complex stress signaling network of plant cells. 相似文献
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The bZIP transcription factor OsABF1 is an ABA responsive element binding factor that enhances abiotic stress signaling in rice 总被引:5,自引:0,他引:5
Md. Amir Hossain Yongjoo Lee Jung-Il Cho Chul-Hyun Ahn Sang-Kyu Lee Jong-Seong Jeon Hun Kang Choon-Hwan Lee Gynheung An Phun Bum Park 《Plant molecular biology》2010,72(4-5):557-566
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We have used whole PCR in an attempt to isolate novel retinoic acid (RA) responsive genes. We cloned several small genomic fragments from total human DNA containing putative retinoic acid responsive elements (RAREs) selected by direct binding to the retinoic acid receptor alpha (RAR alpha). We report here that an oligonucleotide containing a sequence from one of the cloned human DNA fragments, and referred to as alpha 1, functions as an authentic RARE. It is shown that both RAR alpha and RAR beta produced in Cos cells as well as in vitro translated RAR alpha bind directly and sequence-specifically to the alpha 1RARE. By mutational analysis it is demonstrated that the alpha 1RARE consists of an imperfect direct repeat of the estrogen- and thyroid hormone-related AGGTCA half-site motif separated by a 5 bp spacer. The orientation and spacing of the half-site repeats are shown to play a critical role in RAR recognition. When cloned upstream of a TK-Luc reporter, the alpha 1RARE is shown to confer responsiveness to RA in an orientation-independent fashion in F9 and CV-1 cells. The magnitude of the RA response mediated by the alpha 1RARE differed in these cell lines. 相似文献
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Background
Many SCCmec elements of coagulase-negative staphylococci (CoNS) could not be typed using multiplex PCR. Such a ‘non-typable’ SCCmec was encountered in a Staphylococcus cohnii isolate.Methodology/Principal Findings
The SCCmec type of methicillin-resistant S. cohnii clinical isolate WC28 could not be assigned using multiplex PCR. Newly-designed primers were used to amplify ccrA and ccrB genes. The whole SCCmec was obtained by three overlapping long-range PCR, targeting regions from left-hand inverted repeat (IRL) to ccrA/B, from ccrA/B to mecA and from mecA to orfX. The region abutting IRL was identified using inverse PCR with self-ligated enzyme-restricted WC28 fragments as the template. WC28 SCCmec had a class A mec gene complex (mecI-mecR1-mecA). The ccrA and ccrB genes were closest (89.7% identity) to ccrA SHP of Staphylococcus haemolyticus strain H9 and to ccrB3 (90% identity) of Staphylococcus pseudintermedius strain KM241, respectively. Two new genes potentially encoding AAA-type ATPase were found in J1 region and a ψTn554 transposon was present in J2 region, while J3 region was the same as many SCCmec of Staphylococcus aureus. WC28 SCCmec abutted an incomplete SCC element with a novel allotype of ccrC, which was closest (82% identity) to ccrC1 allele 9 in Staphylococcus saprophyticus strain ATCC 15305. Only two direct target repeat sequences, one close to the 3′-end of orfX and the other abutting the left end of WC28 SCCmec, could be detected.Conclusions/Significance
A new 35-kb SCCmec was characterized in a S. cohnii isolate, carrying a class A mec gene complex, new variants of ccrA5 and ccrB3 and two novel genes in the J1 region. This element is flanked by 8-bp perfect inverted repeats and is similar to type III SCCmec in S. aureus and a SCCmec in S. pseudintermedius but with different J1 and J3 regions. WC28 SCCmec was arranged in tandem with an additional SCC element with ccrC, SCCWC28, but the two elements might have integrated independently rather than constituted a composite. This study adds new evidence of the diversity of SCCmec in CoNS and highlights the need for characterizing the ‘non-typable’ SCCmec to reveal the gene pool associated with mecA. 相似文献11.
Localization of an RNA binding element of the iron responsive element binding protein within a proteolytic fragment containing iron coordination ligands. 总被引:3,自引:1,他引:2
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The iron responsive element binding protein (IRE-BP) regulates iron storage and uptake in response to iron. This control results from the interaction of the IRE-BP with the iron responsive element (IRE), a conserved sequence/structure element located near the 5' end of all ferritin mRNAs and in the 3' UTR of transferrin receptor mRNAs. Proteolysis was used to probe for functional elements of the IRE-BP. Partial chymotrypsin digestion generates a simple digestion pattern yielding fragments of 68, 56, 41, and 30 kDa. The 68 and 30 kDa fragments are derived from a single cleavage at Trp623. Further cleavages of the 68 kDa polypeptide yield the 56 and 41 kDa peptides. A combination of UV-crosslinking and chymotrypsin digestion was used to localize an RNA binding element within the C-terminus of the 68 kDa fragment, between amino acid residues 480 and 623. This region includes cysteine residues 503 and 506 which have been shown to be required for iron-sulfur cluster assembly and for iron regulation of the IRE-BP. Proteolytic fragments of the IRE-BP that contain this RNA binding region can be crosslinked to the IRE but do not bind with high affinity, suggesting that elements within the IRE-BP, in addition to those located between residues 480 and 623, are required for high affinity binding to the IRE. 相似文献
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Efficient binding of glucocorticoid receptor to its responsive element requires a dimer and DNA flanking sequences 总被引:4,自引:0,他引:4
A combination of the gel retardation assay and interference by hydroxyl radical modification (missing nucleoside technique) was used to analyze the interaction of the glucocorticoid receptor (GR) with various glucocorticoid responsive elements (GRE). Short oligonucleotides containing the 15-bp GRE and 1 to 3 flanking base pairs on each side, are bound with very low affinity. The same GREs, when positioned in the center of a large DNA fragment (40-50 bp), show high affinity for the receptor. However, when the GRE is positioned at the border of a 54-bp fragment, the affinity of the GR for the GRE decreases markedly. The DNA binding affinity increases linearly with each added flanking base pair and optimal binding is observed with 8-10 flanking bp. Thus, the nonconserved DNA sequences flanking the GRE contribute significantly to the free energy of receptor binding to DNA. Using larger DNA fragments (greater than 100 bp) and a smaller form of the receptor (40 kD), two retarded complexes are found that correspond to monomeric and homodimeric receptor DNA complexes. The DNA-binding domain of the GR (20 kD), expressed in bacteria, binds to the GRE as a monomer as well as a dimer and can form heterodimers with the native 94-kD GR. Insertion or deletion of one single base pair between the two halves of the GRE reduces the affinity for the homodimeric form of the native GR, and inhibits the function of the GRE in gene transfer experiments, suggesting that a dimer of the GR is the functional entity that binds to the GRE. 相似文献
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We report the existence and partial purification of sporulation factor, which stimulates sporulation of Bacillus subtilis at low cell density. Proline or arginine is required for stimulation under the conditions of our assay. Sporulation factor is a small heat-stable substance produced by the cells during exponential growth phase. It is required in small amounts and is resistant to various proteolytic agents. Several spo mutants were tested for the ability to produce functional sporulation factor. All of these mutants produce factor and do not sporulate in the presence of factor from wild-type cells. Sporulation factor is not involved in the induction of alpha-amylase synthesis at the initiation of sporulation. 相似文献
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1. The data herein reveal the existence of cAMP-responsive element (CRE)-binding factors (CRF) in the nuclear extracts from cAMP-treated rat liver. 2. DNAase I and DMS footprinting analysis showed that the CRFs protected the CRE (-77 to -92) in the phosphoenolpyruvate carboxykinase (PEPCK) promoter and the TGACGTCA motif in a consensus oligodeoxynucleotide based on the sequence of the CRE's of 6 cAMP-regulated genes (C32mer). 3. Competition assays indicate that the CRF(s) is a CGTCA-specific, ATF/CREB-like factor(s). 4. Southwestern (SW) blot analysis detected 2 apparent CRFs which have molecular weights of about 30 and 32 kDa, respectively. 5. Based on the comparison of the size and binding specificity of the CRFs with the CREBs reported to date, the CRFs appear to be novel CRE-binding nuclear factors. 相似文献