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Purification and characterization of SopA and SopB proteins essential for F plasmid partitioning 总被引:24,自引:0,他引:24
H Mori Y Mori C Ichinose H Niki T Ogura A Kato S Hiraga 《The Journal of biological chemistry》1989,264(26):15535-15541
Mini-F plasmid has the trans-acting genes sopA and sopB and the cis-acting site sopC which are essential for accurate partitioning of plasmid DNA molecules into both daughter cells. In this study, we purified independently SopA and SopB proteins, analyzed the in vitro DNA-binding activity of these proteins by the gel retardation assay, and determined the precise binding sites of DNA by the footprinting method. SopA binds to four repeated sequences (CTTTGC) located in the promoter-operator region of the sopAB operon. The SopA binding activity is enhanced by the addition of SopB protein. SopB protein itself does not bind to this DNA region. These results suggest that the complex of SopA and SopB proteins autoregulate the expression of the sopA-sopB operon. On the other hand, SopB protein binds to the sopC region, in which 12 direct repeats of 43-base pairs nucleotides exist. SopB protein recognizes the inverted repeats of 7 base pairs in each direct repeats. SopA protein does not affect the SopB binding activity to the sopC DNA segment. 相似文献
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The SopA protein plays an essential, though so far undefined, role in partition of the mini-F plasmid but, when overproduced, it causes loss of mini-F from growing cells. Our investigation of this phenomenon has revealed that excess SopA protein reduces the linking number of mini-F. It appears to do so by disturbing the partition complex, in which SopB normally introduces local positive supercoiling upon binding to the sopC centromere, as it occurs only in plasmids carrying sopC and in the presence of SopB protein. SopA-induced reduction in linking number is not associated with altered sop promoter activity or levels of SopB protein and occurs in the absence of changes in overall supercoil density. SopA protein mutated in the ATPase nucleotide-binding site (K120Q) or lacking the presumed SopB interaction domain does not induce the reduction in linking number, suggesting that excess SopA disrupts the partition complex by interacting with SopB to remove positive supercoils in an ATP-dependent manner. Destabilization of mini-F also depends on sopC and SopB, but the K120Q mutant retains some capacity for destabilizing mini-F. SopA-induced destabilization thus appears to be complex and may involve more than one SopA activity. The results are interpreted in terms of a regulatory role for SopA in the oligomerization of SopB dimers bound to the centromere. 相似文献
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Low-copy number plasmids of bacteria rely on specific centromeres for regular partition into daughter cells. When also present on a second plasmid, the centromere can render the two plasmids incompatible, disrupting partition and causing plasmid loss. We have investigated the basis of incompatibility exerted by the F plasmid centromere, sopC, to probe the mechanism of partition. Measurements of the effects of sopC at various gene dosages on destabilization of mini-F, on repression of the sopAB operon and on occupancy of mini-F DNA by the centromere-binding protein, SopB, revealed that among mechanisms previously proposed, no single one fully explained incompatibility. sopC on multicopy plasmids depleted SopB by titration and by contributing to repression. The resulting SopB deficit is proposed to delay partition complex formation and facilitate pairing between mini-F and the centromere vector, thereby increasing randomization of segregation. Unexpectedly, sopC on mini-P1 exerted strong incompatibility if the P1 parABS locus was absent. A mutation preventing the P1 replication initiation protein from pairing (handcuffing) reduced this strong incompatibility to the level expected for random segregation. The results indicate the importance of kinetic considerations and suggest that mini-F handcuffing promotes pairing of SopB-sopC complexes that can subsequently segregate as intact aggregates. 相似文献
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Partitioning of the F plasmid: Overproduction of an essential protein for partition inhibits plasmid maintenance 总被引:2,自引:0,他引:2
Noriko Kusukawa Hirotada Mori Akihiro Kondo Sota Hiraga 《Molecular & general genetics : MGG》1987,208(3):365-372
Summary Multicopy plasmids carrying the sopB gene of the F plasmid inhibit stable inheritance of a coexisting mini-F plasmid. This incompatibility, termed IncG, is found to be caused by excess amounts of the SopB protein, which is essential for accuratepartitioning of plasmid DNA molecules into daughter cells. A sopB-carrying multicopy plasmid that shows the IncG+ phenotype was mutagenized in vitro and IncG negative mutant plasmids were isolated. Among these amber and missense mutants of sopB, mutants with a low plasmid copy number and a mutant in the Shine-Dalgarno sequence for translation of the SopB protein were obtained. These results demonstrate that the IncG phenotype is caused by the SopB protein, and that the incompatibility is expressed only when the protein is overproduced. This suggests that the protein must be kept at appropriate concentrations to ensure stable maintenance of the plasmid. 相似文献
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SopA, SopB proteins and the cis-acting sopC DNA region of F plasmid are essential for partitioning of the plasmid, ensuring proper subcellular positioning of the plasmid DNA molecules. We have analyzed by immunofluorescence microscopy the subcellular localization of SopA and SopB. The majority of SopB molecules formed foci, which localized frequently with F plasmid DNA molecules. The foci increased in number in proportion to the cell length. Interestingly, beside the foci formation, SopB formed a spiral structure that was dependent on SopA, which also formed a spiral structure, independent of the presence of SopB, and these two structures partially overlapped. On the basis of these results and previous biochemical studies together with our simulations, we propose a theoretical model named "the reaction-diffusion partitioning model", using reaction-diffusion equations that explain the dynamic subcellular localization of SopA and SopB proteins and the subcellular positioning of F plasmid. We hypothesized that sister copies of plasmid DNA compete with each other for sites at which SopB multimer is at the optimum concentration. The plasmid incompatibility mediated by the Sop system might be explained clearly by this hypothesis. 相似文献
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In the yeast Saccharomyces cerevisiae, mitochondria form a branched, tubular reticulum in the periphery of the cell. Mmm1p is required to maintain normal mitochondrial shape and in mmm1 mutants mitochondria form large, spherical organelles. To further explore Mmm1p function, we examined the localization of a Mmm1p-green fluorescent protein (GFP) fusion in living cells. We found that Mmm1p-GFP is located in small, punctate structures on the mitochondrial outer membrane, adjacent to a subset of matrix-localized mitochondrial DNA nucleoids. We also found that the temperature-sensitive mmm1-1 mutant was defective in transmission of mitochondrial DNA to daughter cells immediately after the shift to restrictive temperature. Normal mitochondrial nucleoid structure also collapsed at the nonpermissive temperature with similar kinetics. Moreover, we found that mitochondrial inner membrane structure is dramatically disorganized in mmm1 disruption strains. We propose that Mmm1p is part of a connection between the mitochondrial outer and inner membranes, anchoring mitochondrial DNA nucleoids in the matrix. 相似文献
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Partition of prokaryotic DNA requires formation of specific protein-centromere complexes, but an excess of the protein can disrupt segregation. The mechanisms underlying this destabilization are unknown. We have found that destabilization by the F plasmid partition protein, SopB, of plasmids carrying the F centromere, sopC, results from the capacity of the SopB-sopC partition complex to stimulate plasmid multimerization. Mutant SopBs unable to destabilize failed to increase multimerization. Stability of wild-type mini-F, whose ResD/rfsF site-specific recombination system enables it to resolve multimers to monomers, was barely affected by excess SopB. Destabilization of plasmids lacking the rfsF site was suppressed by recF, recO and recR, but not by recB, mutant alleles, indicating that multimerization is initiated from single-strand gaps. SopB did not alter the amounts or distribution of replication intermediates, implying that SopB-DNA complexes do not create single-strand gaps by blocking replication forks. Rather, the results are consistent with SopB-DNA complexes channelling gapped molecules into the RecFOR recombination pathway. We suggest that extended SopB-DNA complexes increase the likelihood of recombination between sibling plasmids by keeping them in close contact prior to SopA-mediated segregation. These results cast plasmid site-specific resolution in a new role - compensation for untoward consequences of partition complex formation. 相似文献
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In bacteria, mitotic stability of plasmids and many chromosomes depends on replicon-specific systems which comprise a centromere, a centromere-binding protein and an ATPase. Dynamic self-assembly of the ATPase appears to enable active partition of replicon copies into cell-halves, but for most ATPases (the Walker-box type) the mechanism is unknown. Also unknown is how the host cell contributes to partition. We have examined the effects of non-sequence-specific DNA on in vitro self-assembly of the SopA partition ATPase of plasmid F. SopA underwent polymerization provided ATP was present. DNA inhibited this polymerization and caused breakdown of pre-formed polymers. Centromere-binding protein SopB counteracted DNA-mediated inhibition by itself binding to and masking the DNA, as well as by stimulating polymerization directly. The results suggest that in vivo, SopB smothers DNA by spreading from sopC, allowing SopA-ATP polymerization which initiates plasmid displacement. We propose that SopB and nucleoid DNA regulate SopA polymerization and hence partition. 相似文献
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Active partition of the F plasmid to dividing daughter cells is assured by interactions between proteins SopA and SopB, and a centromere, sopC. A close homologue of the sop operon is present in the linear prophage N15 and, together with sopC-like sequences, it ensures stability of this replicon. We have exploited this sequence similarity to construct hybrid sop operons with the aim of locating specific interaction determinants within the SopA and SopB proteins that are needed for partition function and for autoregulation of sopAB expression. Centromere binding was found to be specified entirely by a central 25 residue region of SopB strongly predicted to form a helix-turn-helix structure. SopB protein also carries a species-specific SopA-interaction determinant within its N-terminal 45 amino acids, and, as shown by Escherichia coli two-hybrid analysis, a dimerization domain within its C-terminal 75 (F) or 97 (N15) residues. Promoter-operator binding specificity was located within an N-terminal 66 residue region of SopA, which is predicted to contain a helix-turn-helix motif. Two other regions of SopA protein, one next to the ATPase Walker A-box, the other C-terminal, specify interaction with SopB. Yeast two-hybrid analysis indicated that these regions contact SopB directly. Evidence for the involvement of the SopA N terminus in autoinhibition of SopA function was obtained, revealing a possible new aspect of the role of SopB in SopA activation. 相似文献
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以绿荧光蛋白(GFP)为标记,构建了一系列伪狂犬病毒VP22蛋白的C-端缺失突变体与GFP融合表达的真核表达质粒,脂质体介导转染Hela细胞,通过荧光显微镜观察分析各个缺失突变体的亚细胞定位,发现伪狂犬病毒VP22蛋白与核定位有关的结构域在第60个到第90个氨基酸残基之间,第111个到第159个氨基酸残基有可能与形成细胞核内的颗粒有关,与微管蛋白结合有关的结构域可能在第187到第241个氨基酸残基之间。上述研究结果为进一步深入研究伪狂犬病毒VP22蛋白的结构与功能奠定了基础。 相似文献
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Two isoforms, 1 and 2, of human DNA topoisomerase IIIbeta were expressed in HeLa cells as a fusion protein to the C-terminus of green fluorescent protein (GFP). The fusion protein of the isoform 1 was found to be localized to the nucleus, and to be associated with chromosomes during metaphase and anaphase. As yeast top3 mutants are known to exhibit phenotypes indicative of defective chromosome segregation, the result suggests that the isoform 1 of the human enzyme may also be involved in chromosome segregation. Two-hybrid screening for interaction partners of the isoform identified three candidate genes: CENP-F, a gene encoding a centromere protein and two genes of no known function, one of which was novel. The GFP fusion of the isoform 2 was found in the cytoplasm, indicating the nuclear localization signal sequence in the isoform 1 is in the C-terminal part that is different between the two isoforms. 相似文献
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We characterized the gene expression, subcellular localization, and in vivo functions of a Nicotiana benthamiana small GTPase belonging to the RabE family, designated NbRabE1. The NbRabE1 promoter drove strong β-glucuronidase reporter expression in young tissues containing actively dividing cells and in stomata guard cells. GFP fusion proteins of NbRabE1 and its dominant-negative and constitutively active mutants were all localized to the Golgi apparatus and the plasma membrane but showed different affinities for membrane attachment. Virus-induced gene silencing of NbRabE1 resulted in pleiotropic phenotypes, including growth arrest, premature senescence, and abnormal leaf development. At the cellular level, the leaves in which NbRabE1 was silenced contained abnormal stomata that lacked pores or contained incomplete ventral walls, suggesting that NbRabE1 deficiency leads to defective guard cell cytokinesis. Ectopic expression of the dominant-negative mutant of NbRabE1 in Arabidopsis thaliana resulted in retardation of shoot and root growth accompanied by defective root hair formation. These developmental defects are discussed in conjunction with proposed functions of RabE GTPases in polarized secretory vesicle trafficking. 相似文献
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A Begomovirus DNAbeta-encoded protein binds DNA, functions as a suppressor of RNA silencing, and targets the cell nucleus
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Our previous results demonstrated that the DNAbeta satellite (Y10beta) associated with Tomato yellow leaf curl China virus Y10 isolate (TYLCCNV-Y10) is essential for induction of leaf curl symptoms in plants and that transgenic expression of its betaC1 gene in Nicotiana plants induces virus-like symptoms. In the present study, in vitro DNA binding activity of the betaC1 proteins of Y10beta and DNAbeta (Y35beta) found in the Tobacco curly shoot virus Y35 isolate (TbCSV-Y35) were studied following their expression as six-His fusion proteins in Escherichia coli. Electrophoretic mobility shift assays and UV cross-linking experiments revealed that betaC1 proteins could bind both single-stranded and double-stranded DNA without size or sequence specificity. Suppression of green fluorescent protein (GFP) transgene silencing was observed with the new leaves of GFP-expressing Nicotiana benthamiana plants coinoculated by TYLCCNV-Y10 plus Y10beta or by TbCSV-Y35 plus Y35beta. In a patch agroinfiltration assay, the transiently expressed betaC1 gene of Y10beta or Y35beta was able to suppress host RNA silencing activities and permitted the accumulation of high levels of GFP mRNA in the infiltrated leaf patches of GFP transgenic N. benthamiana plants. The betaC1 protein of Y10beta accumulated primarily in the nuclei of plant and insect cells when fused with beta-glucuronidase or GFP and immunogold labeling showed that the betaC1 protein is present in the nuclei of infected N. benthamiana plants. A mutant version of Y10beta carrying the mutations within the putative nuclear localization sequence of the Y10 betaC1 protein failed to induce disease symptoms, suppress RNA silencing, or accumulate in the nucleus, suggesting that nuclear localization of the betaC1 protein is a key requirement for symptom induction and silencing suppression. 相似文献
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Identification and characterization of gyrB mutants of Escherichia coli that are defective in partitioning of mini-F plasmids.
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T Ogura H Niki H Mori M Morita M Hasegawa C Ichinose S Hiraga 《Journal of bacteriology》1990,172(3):1562-1568
hopA mutants, which have been suggested to be defective in mini-F plasmid partitioning (H. Niki, C. Ichinose, T. Ogura, H. Mori, M. Morita, M. Hasegawa, N. Kusukawa, and S. Hiraga, J. Bacteriol. 170:5272-5278, 1988), were found to carry mutations in the gyrB gene, coding for the B subunit of DNA gyrase. In gyrB(HopA) mutants, relaxation of the superhelicity of plasmids, increased IncG incompatibility, and increased SopB protein production were observed. It is suggested that altered expression of the sop genes, which is due to relaxation of the mini-F plasmid DNA, causes both defective partitioning of the mini-F plasmids and increased IncG incompatibility in gyrB(HopA) mutants. 相似文献
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利用PCR方法扩增FAM92A1-289全长,经BamH I和Xho I酶切后连接入pEGFP-N1真核表达载体,构建pEGFP-N1-FAM92A1-289重组表达质粒,转染Hela细胞,利用荧光显微镜观察FAM92A1-289在细胞中的定位。经双酶切和核酸序列分析证实重组质粒包含有正确编码的FAM92A1-289读码框。荧光显微镜观察到空质粒pEGFP-N1转染后,整个细胞内弥散绿色荧光,而转染pEGFP-N1-FAM92A1-289重组载体后,可见绿色荧光分布于Hela细胞核中,显示FAM92A1-289定位于细胞核。成功构建人FAM92A1-289真核表达载体,FAM92A1-289定位于哺乳细胞的细胞核中。 相似文献
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Asif Khan Anna Garbelli Serena Grossi Assa Florentin Giorgia Batelli Tania Acuna Gaston Zolla Yuval Kaye Laju K. Paul Jian‐Kang Zhu Giovanni Maga Gideon Grafi Simon Barak 《The Plant journal : for cell and molecular biology》2014,79(1):28-43
DEAD‐box RNA helicases are involved in many aspects of RNA metabolism and in diverse biological processes in plants. Arabidopsis thaliana mutants of two DEAD‐box RNA helicases, STRESS RESPONSE SUPPRESSOR1 (STRS1) and STRS2 were previously shown to exhibit tolerance to abiotic stresses and up‐regulated stress‐responsive gene expression. Here, we show that Arabidopsis STRS‐overexpressing lines displayed a less tolerant phenotype and reduced expression of stress‐induced genes confirming the STRSs as attenuators of Arabidopsis stress responses. GFP–STRS fusion proteins exhibited localization to the nucleolus, nucleoplasm and chromocenters and exhibited relocalization in response to abscisic acid (ABA) treatment and various stresses. This relocalization was reversed when stress treatments were removed. The STRS proteins displayed mis‐localization in specific gene‐silencing mutants and exhibited RNA‐dependent ATPase and RNA‐unwinding activities. In particular, STRS2 showed mis‐localization in three out of four mutants of the RNA‐directed DNA methylation (RdDM) pathway while STRS1 was mis‐localized in the hd2c mutant that is defective in histone deacetylase activity. Furthermore, heterochromatic RdDM target loci displayed reduced DNA methylation and increased expression in the strs mutants. Taken together, our findings suggest that the STRS proteins are involved in epigenetic silencing of gene expression to bring about suppression of the Arabidopsis stress response. 相似文献
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Ito M Ito R Huang Y Miura S Imamura A Suzuki Y Shimozawa N 《Biochimica et biophysica acta》2000,1496(2-3):232-242
We isolated and characterized CHO mutants deficient in peroxisome assembly using green fluorescent protein (GFP) and blue fluorescent protein (BFP) as the fluorescent probes to study the molecular mechanism of peroxisome biogenesis. We used stable transformants of CHO cells expressing GFP appending peroxisome targeting signal-1 (PTS1) and/or peroxisome targeting signal-2 (PTS2) as the parent strains for rapid isolation of the mutants. We have obtained six peroxisome-deficient mutants by visual screening of the mislocalizations of the peroxisomal GFPs. Mutual cell fusion experiments indicated that the six mutants isolated were divided into four complementation groups. Several of the mutants obtained possessed defective genes: the PEX2 gene was defective in SK24 and PT54; the PEX5 gene in SK32 and the PEX7 gene in PT13 and PT32. BE41, which belonged to the fourth complementation group, was not determined. When peroxisomal forms of BFP were transiently expressed in mutant cells, the peroxisomal BFPs appending both PTS1 and PTS2 appeared to bypass either the PTS1 or PTS2 pathway for localization in SK32. This observation suggested that other important machinery, in addition to the PTS1 or PTS2 pathway, could be involved in peroxisome biogenesis. Thus, our approach using peroxisomal fluorescent proteins could facilitate the isolation and analysis of peroxisome-deficient CHO mutants and benefit studies on the identification and role of the genes responsible for peroxisome biogenesis. 相似文献