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1.
小黑杨PsnWRKY70是能够响应盐胁迫的转录因子。为了进一步探究该转录因子在参与盐胁迫应答途径中与哪些蛋白质之间存在相互作用关系,本研究以140 mmol/L NaCl溶液处理过的小黑杨叶片为材料,利用热稳定核酸酶(DSN)构建了均一化的pGADT7-DEST酵母双杂交cDNA文库,将PsnWRKY70基因亚克隆至pGBKT7载体,形成BD-WRKY重组质粒,并以之为诱饵蛋白表达载体筛选小黑杨酵母双杂交cDNA文库。经过2轮酵母双杂交筛选以及回转验证试验,初步选出了5种与PsnWRKY70相互作用的蛋白质。对所筛选出的5种蛋白质进行保守结构域分析发现:有2种预测蛋白(HP1和HP2,其中HP1包含ClpP结构域),1种环化酶相关蛋白(CAP1),1种包含RNA识别基序的蛋白(RRM)以及1种泛素样修饰蛋白酶(Ulp1);对CAP1、HP1、RRM、HP2和Ulp1的毛果杨同源基因编码区上游2 000 bp范围内的顺式作用元件进行分析发现:CAP1、HP1、RRM、HP2和Ulp1的毛果杨同源基因上游启动子区均富含能够特异性结合WRKY转录因子的W-box。采用GST-pull down技术验证CAP1、HP1、RRM、HP2、Ulp1蛋白全长与PsnWRKY70转录因子之间是否存在直接的相互作用关系,将等量的His-X(X:CAP1、HP1、RRM、HP2或Ulp1)融合蛋白分别上样于正常谷胱甘肽琼脂糖树脂以及结合了GST或GST-WRKY蛋白的谷胱甘肽琼脂糖树脂中,SDS-PAGE电泳分离互作蛋白,最终Western blot结果显示:在体外状态下,HP1、RRM和Ulp1蛋白能够直接与PsnWRKY70相互作用,而CAP1和HP2则不能直接与PsnWRKY70相互作用。 相似文献
2.
High sequence selectivity in DNA-protein interactions was analyzed by measuring discrimination by Eco RI endonuclease between the recognition site GAATTC and systematically altered DNA sites. Base analogue substitutions that preserve the sequence-dependent conformational motif of the GAATTC site permit deletion of single sites of protein-base contact at a cost of +1 to +2 kcal/mol. However, the introduction of any one incorrect natural base pair costs +6 to +13 kcal/mol in transition state interaction energy, the resultant of the following interdependent factors: deletion of one or two hydrogen bonds between the protein and a purine base; unfavourable steric apposition between a group on the protein and an incorrectly placed functional group on a base; disruption of a pyrimidine contact with the protein; loss of some crucial interactions between protein and DNA phosphates; and an increased energetic cost of attaining the required DNA conformation in the transition state complex. Eco RI endonuclease thus achieves stringent discrimination by both "direct readout" (protein-base contracts) and "indirect readout" (protein-phosphate contacts and DNA conformation) of the DNA sequence. 相似文献
3.
Structure of complex of synthetic HIV-1 protease with a substrate-based inhibitor at 2.3 A resolution 总被引：22，自引：0，他引：22
M Miller J Schneider B K Sathyanarayana M V Toth G R Marshall L Clawson L Selk S B Kent A Wlodawer 《Science (New York, N.Y.)》1989,246(4934):1149-1152
The structure of a complex between a peptide inhibitor with the sequence N-acetyl-Thr-Ile-Nle-psi[CH2-NH]-Nle-Gln-Arg.amide (Nle, norleucine) with chemically synthesized HIV-1 (human immunodeficiency virus 1) protease was determined at 2.3 A resolution (R factor of 0.176). Despite the symmetric nature of the unliganded enzyme, the asymmetric inhibitor lies in a single orientation and makes extensive interactions at the interface between the two subunits of the homodimeric protein. Compared with the unliganded enzyme, the protein molecule underwent substantial changes, particularly in an extended region corresponding to the "flaps" (residues 35 to 57 in each chain), where backbone movements as large as 7 A are observed. 相似文献
4.
目的：观察大鼠脊髓Ｐ物质对细胞免疫水平的影响，方法：两组新生大鼠分别皮下注射辣椒素（ＣＡＰ）和Ｐ物质抗血清，成年大鼠则于脊髓蛛网膜下腔注射，用丝裂原激活的大鼠脾细胞方法测定了淋巴细胞的白细胞介素－２（ＩＬ－２）水平。结果：大鼠皮下及脊髓蛛网膜下腔各分别注射ＣＡＰ，Ｐ物质抗血清后，ＩＬ－２的水平较对照组显著升高（Ｐ〈０．０１）。结论：新生大鼠（皮下）与成年大鼠（蛛网膜下腔）注射ＣＡＰ或Ｐ物质抗血清垃 相似文献
5.
用人工合成的氯霉素-卵清蛋白(CAP-OVA)免疫BALB/c小鼠,通过杂交瘤技术筛选出1株能稳定传代并分泌抗氯霉素(CAP)单克隆抗体(McAb)的杂交瘤细胞4C9,并以此制备腹水单抗.经间接竞争ELISA(ciELISA)检测,该单抗亚类重链类型是IgG1,轻链为κ类型,单抗腹水效价为1∶1×106,与甲砜霉素、氟甲砜霉素以及其他常见抗生素的交叉反应率小于0.01%.用过碘酸钠氧化法合成CAP酶标记单抗,建立了CAP直接竞争ELISA(cdELISA)方法.此法检测的线性范围为0.1~100 ng·mL-1,半数抑制浓度(IC50)为5.81 ng?L-1.添加回收实验表明所建立的CAP cdELISA方法检测限达到0.1 ng·L-1,对比试验表明其检测灵敏度与商品试剂CAP ELISA基本相当. 相似文献
6.
Recent advances in far-field optical nanoscopy have enabled fluorescence imaging with a spatial resolution of 20 to 50 nanometers. Multicolor super-resolution imaging, however, remains a challenging task. Here, we introduce a family of photo-switchable fluorescent probes and demonstrate multicolor stochastic optical reconstruction microscopy (STORM). Each probe consists of a photo-switchable "reporter" fluorophore that can be cycled between fluorescent and dark states, and an "activator" that facilitates photo-activation of the reporter. Combinatorial pairing of reporters and activators allows the creation of probes with many distinct colors. Iterative, color-specific activation of sparse subsets of these probes allows their localization with nanometer accuracy, enabling the construction of a super-resolution STORM image. Using this approach, we demonstrate multicolor imaging of DNA model samples and mammalian cells with 20- to 30-nanometer resolution. This technique will facilitate direct visualization of molecular interactions at the nanometer scale. 相似文献
7.
Genome-wide mapping of in vivo protein-DNA interactions 总被引：5，自引：0，他引：5
8.
[目的]采用时间分辨荧光技术建立氯霉素（CAP）直接竞争免疫检测法（CAPTRFIA）。[方法]以羊抗鼠IgG二抗为固定相,游离CAP与生物素化的CAP人工抗原（CAP—BSA）共同竞争限量的CAP抗体;用铕标记的链霉亲和素示踪。[结果]该方法的灵敏度为0．016ng／ml。用于牛奶样品检测的添加回收率为89．6％,批内和批间变异系数分别为5．5％和10．1％,与甲砜霉素的交叉反应率〈0．01％。[结论]CAPTRFIA直接竞争法灵敏度高、特异性好、性能稳定,适于高通量筛查,有良好的应用前景。 相似文献
9.
Forces between surfaces in liquids 总被引：1，自引：0，他引：1
Recent developments in the direct measurements of forces between surfaces in liquids at the ?ngstrom resolution level are reviewed. The results reveal a rich variety of interactions and interaction potentials that depend on the nature of the surfaces and intervening liquids. These results also shed new insights into liquid structure adjacent to surfaces and the interactions occurrig in complex systems, with implications in many different areas of chemical physics, biology, and technology. The origin of some important fundamental interactions, such as repulsive "hydration" forces and attractive "hydrophobic" forces, are still not understood and offer a challenge for experimental and theoretical work in this area. 相似文献
10.
Methylation of cytosine residues in eukaryotic DNA is common, but poorly understood. Typically several percent of the cytosines are methylated; however, it is unclear what governs which sequences eventually become modified. Neurospora crassa DNA containing the "zeta-eta" (zeta-eta) region, which is a region of unusually heavy methylation, was tested for its ability to direct DNA methylation de novo. DNA stripped of its methylation by propagation in Escherichia coli was reintroduced into Neurospora crassa by transformation. The zeta-eta region reproducibly became "properly" methylated whether inserted at its native chromosomal position or at ectopic sites. Adjacent Neurospora and bacterial sequences in the transforming DNA rarely became methylated. A model is presented that accounts for position-independent faithful methylation as observed in the zeta-eta region, as well as position-dependent methylation, as occasionally observed, especially with sequences not native to Neurospora. 相似文献
11.
12.
对96孔板上氯霉素分子印迹膜的制备及其吸附性能进行了研究,并通过试验优化了该分子印迹膜的制备方法.以氯霉素为模板分子,四氢呋喃为致孔剂,偶氮二异丁腈为引发剂,采用紫外灯引发聚合在MaxiSorp 96孔板上合成氯霉素分子印迹聚合膜.结果表明,当模板分子、功能单体（甲基丙烯酸二乙基氨基乙酯）、交联剂（乙二醇二甲基丙烯酸酯）的摩尔比为1∶4∶1,模板分子物质的量为0.25 mmol,溶剂的量为5 mL时,合成的氯霉素分子印迹膜对氯霉素的吸附效果最佳,可在50 min内达到吸附平衡,当氯霉素的初始浓度为120 mg/L时,吸附量可达1.51μg/孔.吸附特异性试验结果表明,当氯霉素、甲砜霉素和氟甲砜霉素的初始浓度为50 mg/L时,该膜对氯霉素的吸附量为0.83μg/孔,而对甲砜霉素和氟甲砜霉素的吸附量分别仅为0.49、0.40μg/孔.研究表明,将该氯霉素分子印迹膜作为人工抗体代替氯霉素的生物抗体,采用直接竞争原理,应用化学发光免疫法检测氯霉素,具有可行性. 相似文献
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14.
Specific DNA probe for the diagnosis of Plasmodium falciparum malaria 总被引：14，自引：0，他引：14
R H Barker L Suebsaeng W Rooney G C Alecrim H V Dourado D F Wirth 《Science (New York, N.Y.)》1986,231(4744):1434-1436
Malaria can be diagnosed either by direct microscopic examination of blood smears, which is time consuming and requires expertise, or by immunological techniques, which are effective but do not distinguish between past and present infections. In this study, a simple procedure was developed for spotting lysed blood from infected patients directly onto nitrocellulose paper and identifying the malaria species on the basis of hybridization of parasite DNA with a species-specific probe. A genomic DNA library of Plasmodium falciparum was screened to detect clones containing DNA sequences that are highly repeated within the parasite genome. Several such clones were further analyzed to identify those that hybridize specifically with P. falciparum DNA but not with DNA from humans, P. vivax, or P. cynomolgi. This technique appears to be sensitive enough to detect 10 picograms of purified P. falciparum DNA (equivalent to 100 parasites) and in field studies is able to detect approximately 40 parasites per microliter of blood. 相似文献
15.
北疆棉花群体光合特性研究 总被引：1，自引：0，他引：1
北疆主要棉花品种光合性能的差异主要表现在棉花生育后期,吐絮期群体光合速率（CAP）与产量呈正相关。盛花期～吐絮期,冠层中的上、中、下层CAP变化具有一定的规律,吐絮期冠层上层作为棉株C的主要来源。棉花CAP与光密度通量（PFD）呈极显著正相关。 相似文献
16.
为了探讨抗生素对植物生长的影响,以3年生毛竹Phyllostachys edulis实生苗为材料,用Yaxin-1161型非调制式叶绿素荧光仪和JIP-test数据分析方法,研究了采用不同浓度氯霉素（CAP）对其叶片色素质量分数和叶绿素荧光特性的影响。结果表明:氯霉素处理后,毛竹幼苗叶片叶绿素a, 叶绿素b和总叶绿素质量分数显著降低（P＜0.05）;捕获的激子将电子传递到电子传递链中超过初级醌受体（QA）的其他电子受体的效率（ET0 /TR0）。用于电子传递的量子产额（ET0 /ABS）,最大光化学效率（P0）和吸收光能为基础的性能指数（PIABS）均显著下降（P＜0.05）,表明氯霉素具有抑制毛竹幼苗光合性能的作用。图1表3参29 相似文献
17.
S ED 《Science (New York, N.Y.)》1991,254(5031):502
In the Research News article "A ;mitey' theory for gene jumping" (6 Sept., p. 1093), Jean Marx erred when she wrote that the groups of Marilyn A. Houck and Margaret G. Kidwell (Reports, 6 Sept., p. 1125) focused on Proctolaelaps regalis as a possible carrier of P elements among Drosophila species by a "process of elimination." Viruses were not screened for P elements during the study, as implied, nor were any other organisms. Rather, the work was initiated by acarologist Houck (then on the faculty of the University of Arizona), who was aware of P element history from conversations with the Kidwell lab. Houck and Ken Peterson, a postdoc in the Kidwell lab, then initiated the Southern blot analysis that pointed to the presence of P element DNA in P. regalis, and Jonathan Clark, a postdoc at Arizona's Center for Insect Science, significantly extended the work through the application of polymerase chain reaction. Their data do not exclude the possibility of viral participation in the system, however. 相似文献
18.
本研究采用“大量注射直接导入法”(经检索国内外文献,在水稻上此法系初次使用)将农垦58S的外源DNA导入籼稻品种晚40中,考察其后代结果:D_1代部份单株性状发生变异,D_2代出现籼粳亚种间性状的重组类型,D_3代重组性状基本趋于稳定。证明直接在籼稻中导入粳稻外源DNA,可实现部分DNA片段与受体植株染色体重组。从而获得育种所需要的籼粳亚种间中间类型材料。此法在育种实践中具有较高的实用价值。 相似文献
19.
DNA-binding proteins 总被引：54，自引：0，他引：54
The structures of three proteins that regulate gene expression have been determined recently and suggest how these proteins may bind to their specific recognition sites on the DNA. One protein (Cro) is a repressor of gene expression, the second (CAP) usually stimulates gene expression, and the third (lambda repressor) can act as either a repressor or an activator. The three proteins contain a substructure consisting of two consecutive alpha helices that is virtually identical in each case. Structural and amino acid sequence comparisons suggest that this bihelical fold occurs in a number of proteins that regulate gene expression, and is an intrinsic part of the DNA-protein recognition event. The modes of repression and activation by Cro and lambda repressor are understood reasonably well, but the mode of action of CAP is still unclear. 相似文献
20.
Using single-molecule DNA nanomanipulation, we show that abortive initiation involves DNA "scrunching"--in which RNA polymerase (RNAP) remains stationary and unwinds and pulls downstream DNA into itself--and that scrunching requires RNA synthesis and depends on RNA length. We show further that promoter escape involves scrunching, and that scrunching occurs in most or all instances of promoter escape. Our results support the existence of an obligatory stressed intermediate, with approximately one turn of additional DNA unwinding, in escape and are consistent with the proposal that stress in this intermediate provides the driving force to break RNAP-promoter and RNAP-initiation-factor interactions in escape. 相似文献