Intrinsic coupling of lagging-strand synthesis to chromatin assembly

Fifty per cent of the genome is discontinuously replicated on the lagging strand as Okazaki fragments. Eukaryotic Okazaki fragments remain poorly characterized and, because nucleosomes are rapidly deposited on nascent DNA, Okazaki fragment processing and nucleosome assembly potentially affect one another. Here we show that ligation-competent Okazaki fragments in Saccharomyces cerevisiae are sized according to the nucleosome repeat. Using deep sequencing, we demonstrate that ligation junctions preferentially occur near nucleosome midpoints rather than in internucleosomal linker regions. Disrupting chromatin assembly or lagging-strand polymerase processivity affects both the size and the distribution of Okazaki fragments, suggesting a role for nascent chromatin, assembled immediately after the passage of the replication fork, in the termination of Okazaki fragment synthesis. Our studies represent the first high-resolution analysis--to our knowledge--of eukaryotic Okazaki fragments in vivo, and reveal the interconnection between lagging-strand synthesis and chromatin assembly.     

利用ARF综合ACF的方法

本文研究了有源 C 滤波器的基本组成规律,得出了综合这种滤波器的一般方法和公式,证明了从原有有源 R 滤波器电路可直接得到有相同传递函数的有源 C 滤波器。     

In vitro centromere and kinetochore assembly on defined chromatin templates

During cell division, chromosomes are segregated to nascent daughter cells by attaching to the microtubules of the mitotic spindle through the kinetochore. Kinetochores are assembled on a specialized chromatin domain called the centromere, which is characterized by the replacement of nucleosomal histone H3 with the histone H3 variant centromere protein A (CENP-A). CENP-A is essential for centromere and kinetochore formation in all eukaryotes but it is unknown how CENP-A chromatin directs centromere and kinetochore assembly. Here we generate synthetic CENP-A chromatin that recapitulates essential steps of centromere and kinetochore assembly in vitro. We show that reconstituted CENP-A chromatin when added to cell-free extracts is sufficient for the assembly of centromere and kinetochore proteins, microtubule binding and stabilization, and mitotic checkpoint function. Using chromatin assembled from histone H3/CENP-A chimaeras, we demonstrate that the conserved carboxy terminus of CENP-A is necessary and sufficient for centromere and kinetochore protein recruitment and function but that the CENP-A targeting domain--required for new CENP-A histone assembly--is not. These data show that two of the primary requirements for accurate chromosome segregation, the assembly of the kinetochore and the propagation of CENP-A chromatin, are specified by different elements in the CENP-A histone. Our unique cell-free system enables complete control and manipulation of the chromatin substrate and thus presents a powerful tool to study centromere and kinetochore assembly.     

自相关基频提取算法的MATLAB实现

在对自相关基音检测算法进行分析的基础上,对自相关基频提取算法进行了探讨.考虑到检测准确度和检测速率两方面的因素,结合带通滤波及三电平削波模块,设计了基于Matlab的估计方法,然后通过对一段具体的语音进行处理,得到了比较准确的浊音语音的基音周期.实验证明,该方法简单有效.     

The RCAF complex mediates chromatin assembly during DNA replication and repair

Chromatin assembly is a fundamental biological process that is essential for the replication and maintenance of the eukaryotic genome. In dividing cells, newly synthesized DNA is rapidly assembled into chromatin by the deposition of a tetramer of the histone proteins H3 and H4, followed by the deposition of two dimers of histones H2A and H2B to complete the nucleosome-the fundamental repeating unit of chromatin. Here we describe the identification, purification, cloning, and characterization of replication-coupling assembly factor (RCAF), a novel protein complex that facilitates the assembly of nucleosomes onto newly replicated DNA in vitro. RCAF comprises the Drosophila homologue of anti-silencing function 1 protein ASF1 and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones. Genetic analyses in Saccharomyces cerevisiae demonstrate that ASF1 is essential for normal cell cycle progression, and suggest that RCAF mediates chromatin assembly after DNA replication and the repair of double-strand DNA damage in vivo.     

ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication

Simian virus 40 (SV40) replicates in nuclei of human and monkey cells. One viral protein, large tumour (T) antigen, is required for the initiation of DNA replication. The development of in vitro replication systems which retain this property has facilitated the identification of the cellular components required for replication. T antigen recognizes the pentanucleotide 5'-GAGGC-3' which is present in four copies within the 64 base-pairs (bp) of the core origin. In the presence of ATP it binds with increased affinity forming a distinctive, bilobed structure visible in electron micrographs. As a helicase, it unwinds SV40 DNA bidirectionally from the origin. We report here that in vitro and in the presence of ATP, T antigen assembles a double hexamer, centred on the core origin and extending beyond it by 12 bp in each direction. The assembly of this dodecamer initiates an untwisting of the duplex by 2-3 turns. In the absence of ATP, a tetrameric structure is the largest found at the core origin. In the absence of DNA, but in the presence of ATP or its non-hydrolysable analogues, T antigen assembles into hexamers. This suggests that ATP effects an allosteric change in the monomer. The change alters protein-protein interactions and allows the assembly of a double hexamer, which initiates replication at the core origin.     

底部钻具组合动力学模型及涡动特性仿真

根据转子动力学理论,把底部钻具组合(BHA)简化成Jeffcott转子模型,同时考虑不同钻压形式对BHA的作用建立BHA的动力学模型,并对BHA的动力学特性进行数值仿真.结果表明:钻压的形式只影响恢复力项,对动力学模型的一般形式无影响;在不同钻压形式的作用下,BHA的动力学特性总体上保持一致;钻压为常数时BHA主要体现为经过一段时间的波动后转为规则向后涡动的特性,钻压为波动值时底部钻具组合也主要体现为向后涡动的特性,但BHA更容易贴着井壁运动.     

负载纳米TiO2的ACF滤料的空气净化性能

针对活性碳纤维（ACF）只能吸附多种有机污染物，并不分解污染物，而纳米光催化可以光降解有机污染物，但只在污染物浓度较高时有优异的光降解性能，而当污染物浓度较低时，光催化降解速率较慢，影响净化效果等问题，将纳米光催化与ACF吸附技术相组合，研究了二者组合后的吸附光催化机理及对ACF滤料的阻力等。     

用杂化粒子场分子动力学模拟嵌段共聚物在本体中的自组装

用杂化粒子场分子动力学模拟方法模拟大体系（约56 nm）长时间(100 μs)的嵌段共聚物在本体中的自组装, 以避免小体系的有限尺寸效应. 通过调控嵌段共聚物中的嵌段数目、 嵌段比例及嵌段间的相互作用参数, 获得两嵌段共聚物和三嵌段共聚物在本体中的自组装结构. 结果表明, 模拟结果与自洽场理论、 Monte Carlo方法、 耗散粒子动力学方法的模拟结果一致.     

纤维状催化剂ACF的织构和表面化学结构的研究

借助Ｘ－光电子能谱分析、红外光谱分析、元素分析、孔径及比表面积测定、Ｘ－射线衍射分析及电镜分析等手段,较全面地研究了自制的纤维状催化剂 ＡＣＦ的织构及表面化学结构,讨论了 ＡＣＦ吸附 ＳＯ_２的活性中心,并得出结论：ＡＣＦ比普通的活性炭 ＧＡＣ对 ＳＯ_２具有更强、更有效的吸附能力．     

ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin

Proteins contain thiol-bearing cysteine residues that are sensitive to oxidation, and this may interfere with biological function either as 'damage' or in the context of oxidant-dependent signal transduction. Cysteine thiols oxidized to sulphenic acid are generally unstable, either forming a disulphide with a nearby thiol or being further oxidized to a stable sulphinic acid. Cysteine-sulphenic acids and disulphides are known to be reduced by glutathione or thioredoxin in biological systems, but cysteine-sulphinic acid derivatives have been viewed as irreversible protein modifications. Here we identify a yeast protein of relative molecular mass M(r) = 13,000, which we have named sulphiredoxin (identified by the US spelling 'sulfiredoxin', in the Saccharomyces Genome Database), that is conserved in higher eukaryotes and reduces cysteine-sulphinic acid in the yeast peroxiredoxin Tsa1. Peroxiredoxins are ubiquitous thiol-containing antioxidants that reduce hydroperoxides and control hydroperoxide-mediated signalling in mammals. The reduction reaction catalysed by sulphiredoxin requires ATP hydrolysis and magnesium, involving a conserved active-site cysteine residue which forms a transient disulphide linkage with Tsa1. We propose that reduction of cysteine-sulphinic acids by sulphiredoxin involves activation by phosphorylation followed by a thiol-mediated reduction step. Sulphiredoxin is important for the antioxidant function of peroxiredoxins, and is likely to be involved in the repair of proteins containing cysteine-sulphinic acid modifications, and in signalling pathways involving protein oxidation.     

活性碳纤维的SO2和NO脱除特性

通过自行搭建实验系统进行动态活性碳纤维(ACF)脱除SO2和NO的观测实验,借助电镜、Xps等深入了解脱除过程的基本特性,在此基础上比较分析主要因素的影响.结果表明,ACF能够吸附、脱除SO2和NO,而且都是先吸附,后反应;脱除过程的化学机理不同:ACF催化SO2被氧化成SO3,催化NO被还原成N2.ACF表面上的官能团和表面势场能够降低反应所需的活化能,起到类似催化剂的作用,ACF表面官能团与吸附对象之间势能状态的不同导致脱除过程中发生氧化和还原两种截然不同的化学反应.反应温度,混合气体流速和水蒸汽含量等对吸附的影响进而影响脱除效率.     

The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus

In sexually reproducing animals, a crucial step in zygote formation is the decondensation of the fertilizing sperm nucleus into a DNA replication-competent male pronucleus. Genome-wide nucleosome assembly on paternal DNA implies the replacement of sperm chromosomal proteins, such as protamines, by maternally provided histones. This fundamental process is specifically impaired in sésame (ssm), a unique Drosophila maternal effect mutant that prevents male pronucleus formation. Here we show that ssm is a point mutation in the Hira gene, thus demonstrating that the histone chaperone protein HIRA is required for nucleosome assembly during sperm nucleus decondensation. In vertebrates, HIRA has recently been shown to be critical for a nucleosome assembly pathway independent of DNA synthesis that specifically involves the H3.3 histone variant. We also show that nucleosomes containing H3.3, and not H3, are specifically assembled in paternal Drosophila chromatin before the first round of DNA replication. The exclusive marking of paternal chromosomes with H3.3 represents a primary epigenetic distinction between parental genomes in the zygote, and underlines an important consequence of the critical and highly specialized function of HIRA at fertilization.     

基于纳米Fe/ACF电极的阴极E-Fenton氧化处理生活污水的研究

成功制备了活性碳纤维负载的纳米铁电极(Fe/ACF),并通过SEM、EDS进行了电极表征.某小区生活污水基于该电极的阴极E-Fenton氧化处理结果表明,在电压1.2 V、中性条件下处理2 h,初始COD由200 mg/L降低到70 mg/L,COD去除率达到65%,出水水质达到国家排放标准GB8978-2000中的一级标准.     

Control of transmembrane lipid asymmetry in chromaffin granules by an ATP-dependent protein

The Ca2+-dependent binding of annexin proteins to secretory granule membranes seems to be involved in the early stage of exocytosis. Binding studies have shown that these proteins have a specificity for phosphatidylserine (PtdS) interfaces. Furthermore, aminolipids are necessary for contact and fusion between lipid vesicles or between liposomes and chromaffin granules. Thus, PtdS must be present on the granule outer (cytoplasmic) monolayer. We report here that chromaffin granules possess a mechanism to maintain PtdS orientation, comparable to the ATP-dependent aminophospholipid translocase from human erythrocytes. The translocase, in granules, selectively transports PtdS from the luminal to the cytoplasmic monolayer, provided the incubation medium contains ATP. As this protein shares several properties with the granule vanadate-sensitive ATPase II, we infer that this ATPase, of relative molecular mass 115,000, is the protein responsible for aminophospholipid translocation. This is the first evidence for an ATP-dependent specific phospholipid 'flippase' in intracellular organelles.     

ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex.

A multiprotein complex that specifically recognizes cellular origins of DNA replication has been identified and purified from the yeast Saccharomyces cerevisiae. We observe a strong correlation between origin function and origin recognition by this activity. Interestingly, specific DNA binding by the origin recognition complex is dependent upon the addition of ATP. We propose that the origin recognition complex acts as the initiator protein for S. cerevisiae origins of DNA replication.     

Gene-specific control of inflammation by TLR-induced chromatin modifications

Toll-like receptors (TLRs) induce a multi-component inflammatory response that must be tightly regulated to avoid tissue damage. Most known regulatory mechanisms target TLR signalling pathways and thus broadly inhibit multiple aspects of the inflammatory response. Given the functional diversity of TLR-induced genes, we proposed that additional, gene-specific regulatory mechanisms exist to allow individual aspects of the TLR-induced response to be differentially regulated. Using an in vitro system of lipopolysaccharide tolerance in murine macrophages, we show that TLR-induced genes fall into two categories on the basis of their functions and regulatory requirements. We demonstrate that representatives from the two classes acquire distinct patterns of TLR-induced chromatin modifications. These gene-specific chromatin modifications are associated with transient silencing of one class of genes, which includes pro-inflammatory mediators, and priming of the second class, which includes antimicrobial effectors. These findings illustrate an adaptive response in macrophages and reveal component-specific regulation of inflammation.     

UV/H2O2/ACF对水中染料靛红的氧化脱色研究

采用紫外杀菌灯(UV)、双氧水(H2O2)和活性炭纤维(ACF)联合技术,对水中染料靛红的氧化脱色进行了研究。考察了溶液初始pH、H2O2用量以及外加Fe3 浓度对UV/H2O2/ACF光解氧化脱色过程的影响,并比较了UV、H2O2、ACF各自在氧化脱色过程的作用。结果表明,UV/H2O2/ACF是一种脱除水中靛红颜色的有效方法,在靛红初始质量浓度0ρ=50 mg/L,pH=6,ρ(H2O2)=200 mg/L条件下,经60 min的光解反应,脱色率达到98.7%。添加Fe3 离子,降低溶液初始pH,可提高颜色脱除效果。     

Modulation of spectrin-actin assembly by erythrocyte adducin

The spectrin-based membrane skeleton, an assembly of proteins tightly associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Spectrin, the most abundant component of this assembly, is an elongated and flexible molecule that, with potentiation by protein 4.1, is cross-linked at its ends by short actin filaments to form a lattice beneath the membrane. These and other proteins stabilize the plasma membrane, organize integral membrane proteins and maintain specialized regions of the cell surface. A membrane-skeleton-associated calmodulin-binding protein of erythrocytes is a major substrate for Ca2+- and phospholipid-dependent protein kinase C (ref. 5), and thus is a target for Ca2+ by two regulatory pathways. Here we demonstrate that this protein, called adducin: (1) binds tightly in vitro to spectrin-actin complexes but with much less affinity either to spectrin or to actin alone; (2) promotes assembly of additional spectrin molecules onto actin filaments; and (3) is inhibited in its ability to induce the binding of additional spectrin molecules to actin by micromolar concentrations of calmodulin and Ca2+. Adducin may be involved in the action of Ca2+ on erythrocyte membrane skeleton and in the assembly of spectrin-actin complexes.     

Microtubule assembly nucleated by isolated centrosomes

Microtubules are involved in the morphogenesis of most cells and are the structural basis of the mitotic spindle. We report here that purified centrosomes nucleate the assembly of microtubules with unusual dynamic properties. This may have important implications for the mechanism by which microtubule arrays are organized and stabilized in cells.