分离酶的作用及其调节

姐妹染色单体的分离是一精确时空调控事件,分离的紊乱会造成遗传物质传递的不稳定,从而可能引起严重的后果—细胞或个体的死亡或病态。在真核生物细胞中,一种比较保守的机制调控着姐妹染色单体的分离：随DNA复制过程建立由黏合素维持的姐妹染色单体的结合,在有丝分裂中期向后期转变过程中,随保全素的降解,分离酶发挥活性,裂解黏合素一个亚单位,促成黏合素蛋白质复合体的解离和姐妹染色单体的分离。Abstract: Sister chromatids separation is under precise regulation during cell cycle. Any turbulence happened in the separation process can cause instability in the transmission of inherited material ,and may cause: death or disease of cell or even individual. In eukaryotic cells, one conserved mechanism governs the separation of sister chromatids. Cohesion between sister chromatids is established during DNA replication and depends on a multiprotein complex called cohesin. At the metaphase to anaphase transition, separase is activated by proteolysis of securin. Separase can cleave one of cohesin's subunits, and then promote cohesin dissociation and sister chromatids separation.     

非诱变剂对姐妹染色单体交换的诱导效应

检测了１６种非诱变剂对大麦根端分生细胞姐妹染色单体交换（ＳＣＥ）的影响及其中９种成份对人外周血淋巴细胞ＳＣＥ的影响。在大麦细胞中,用高浓度的葡萄糖、氯化钠、维生素、氨基酸及脱氧核糖核苷三磷酸（ｄＮＴＰ）处理均能显著提高ＳＣＥ频率。维生素、氨基酸和ｄＮＴＰ对人外周血淋巴细胞中的ＳＣＥ频率也有显著影响,实验结果表明,不只是诱变剂能提高ＳＣＥ频率,非诱变剂在一定高剂量时也能提高ＳＣＥ频率。非诱变剂影响Ｓ     

姐妹染色单体交换(SCE)的检测原理及其分子机理

姐妹染色单体交换(sister-chromatid exchange,SCE)是当前生物学研究的热点之一。SCE检测技术已广泛地应用于环境科学、医学、生物学等研究领域,具有重要的意义。环境胁迫会造成细胞内DNA不同程度的损伤,并可能进一步导致染色体畸变,甚至引发癌变。SCE作为一种灵敏而有效的指标,能够反映DNA损伤程度和遗传不稳定性。本文主要介绍了有关SCE的由来,色差显示原理、分子机理、SCE与染色体畸变的关系以及SCE的诱导因子。文章最后还对今后有关SCE的研究及其应用提出一些新的看法。     

香烟烟雾水溶液中的活性氧及其对姊妹染色单体互换的影响

SCE频率分析是研究环境诱变剂和致癌剂引起人类遗传物质损伤的有效手段。以人类外周血林巴细胞姊妹染色单体交换（SCE）为指标,评价了香烟烟雾水溶液的遗传毒性。首先,定量测定了香烟烟雾水溶液中含有H2O2、O2-等活性氧,从而间接证明了其潜在的遗传毒性。然后,将一定量的香烟烟雾水溶液加入人外周血淋巴细胞培养液中,结果表明实验组SCE频率明显高于空白对照组,因此直接证明了香烟烟雾水溶液的遗传毒性。     

中国林蛙卵核糖核酸酶的分离纯化及其抗肿瘤作用

以中国林蛙卵为原料,采用丙酮分级沉淀、SP-Trisacryl阳离子交换色谱、Sephadex G-75凝胶过滤色谱、C8反相色谱等纯化方法,得到一种具有核糖核酸酶活性的蛋白质,采用SDS-PAGE电泳对该蛋白质进行了相对分子质量和纯度测定。结果表明：纯化的中国林蛙卵核糖核酸酶为相对分子质量13kDa的单一成分。该酶最适反应温度为65℃,最适反应pH为5.5~6.0,米氏常数为4.11μmol/L,最大反应速率为2.82 pmol/s。在体外细胞毒性实验中,对人三种肿瘤细胞HeLa、K562、MCF-7具有抑制作用,其IC50分别为0.6μmol/L、0.8μmol/L和4μmol/L,而对于正常人成纤维细胞在酶浓度达到8μmol/L时仍未见明显细胞毒性。这种从中国林蛙卵中分离纯化出的具有选择性细胞毒性的小分子量核糖核酸酶,为肿瘤的治疗提供了新的候选蛋白分子。     

白灵侧耳漆酶分离纯化及其酶学性质研究

为了探索白灵侧耳Pleurotus eryngii var.tuoliensis漆酶性质,以白灵侧耳菌株00485为试验材料,从发酵液中分离纯化得到胞外漆酶并对其酶学性质进行测定。纯化流程依次为DEAE-Cellulose阴离子交换层析,CM-Cellulose阳离子交换层析,SP-Sepharose强阳离子交换层析以及Superdex 75凝胶过滤层析,获得胞外白灵侧耳漆酶(Pn Lac)。SDS-PAGE检测结果表明Pn Lac为65k Da的单一蛋白。Pn Lac经过胰蛋白酶水解得到3种肽段,经过NBCI-BLAST后发现它们与糙皮侧耳、环柄韧伞、刺芹侧耳等的漆酶具有同源性。底物为2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)时该种漆酶的最适反应温度和p H分别为50℃和3.0,Ca2+和Hg2+能够抑制它的活性,相反地Cu2+和Mn2+能够提高它的活性,米氏常数Km和Vmax分别是0.17mmol/L和1.76OD/min/U。     

果胶裂解酶的分离,提纯及其特征

用等电聚焦层析方法从酶制剂Pectinex Ultra SP-L中分离获得制备标准的内切果胶裂解酶(PL),并测定了这个内切-PL的一些重要物理、化学及生物化学特性。用分析用等电聚焦电泳,生物化学反应及薄层层析证明所制备的PL为纯品。所制备的内切-PL的一些特性数据如下:pl∶pH=3.9,K_m=1.91 mg/ml,V_(max)=0.88单位/min。用苹果果胶(酯化度为71%)为底物时,PL的最适pH为6.0,最适温度是60℃。用H-果胶(酯化度为94%)为底物时,作用的最适pH为8.5,最适温度是65℃。所制备的PL是高温稳定和pH稳定的。PL的活性被Na~+激活。     

肝癌高发区霉变粮食致突变性的研究——SCE实验

从肝癌高发的扶绥县主粮中分离的常见污染真菌25株,分别接种于玉米-大米培养基培养,其培养物的甲醇-氯仿提取液经SCE试验,结果有10株为阳性。提示霉变粮食中存在着使人的体细胞突变的物质,这对肝癌的发生可能超重要作用。防止粮食霉变,不吃发霉的粮食可能会降低肝癌发病率。     

一株产卡拉胶酶细菌的分离鉴定及其酶学性质

【目的】从红树林土壤腐叶中分离出能够产生卡拉胶酶的菌株,对其进行鉴定,并研究其酶学性质。【方法】利用以卡拉胶为唯一碳源的培养基,分离出产卡拉胶酶的菌株;通过形态学观察、16S r DNA序列分析对其进行种属鉴定;对该菌株所产卡拉胶酶进行纯化并采用DNS测酶活的方法测定酶学性质。【结果】从红树林土壤腐叶中分离出1株高产κ-卡拉胶酶的菌株ASY5,经鉴定该菌株为假交替单胞菌属(Pseudoalteromonas sp.ASY5)。纯化得到的κ-卡拉胶酶的分子量约为30 k Da;酶学性质试验表明,其最适反应温度和p H分别为60℃和7.5,在50℃以下酶的稳定性较好,在p H 7.0-9.0范围内酶活力较稳定,对κ-卡拉胶具有良好的底物特异性,以κ-卡拉胶为底物时Km值和Vmax值分别为2.28 mg/m L和147.06μmol/(min·mg),Na+、K+、Ca2+、Mg2+、Al3+等对酶活有显著的促进作用,而Ag+、Zn2+、Cd2+及SDS对酶活有强烈的抑制作用。【结论】分离到的细菌假交替单胞菌Pseudoalteromonas sp.ASY5产生的κ-卡拉胶酶在较高的温度和碱性条件下均具有较高的酶活性,为利用卡拉胶水解酶产生卡拉寡糖的研究和应用奠定了基础。     

多酶复配合成海藻糖及其分离提取的研究

以大米淀粉为原料,多酶复配制备海藻糖。确定了实验室条件下多酶复配生产海藻糖的最佳条件:以15%(m/V)大米淀粉为底物,催化温度45℃、pH 6. 0、DE值16、α/β-CGTase加量为1. 4U/ml、催化28h后糖化处理12h,海藻糖转化率由双酶法催化的50%提高至73%。在底物浓度为25%(m/V)时,海藻糖产量最高达到182. 5g/L,随后对高浓度海藻糖进行分离提取,分别考察了活性炭脱色、离交分离、浓缩结晶等对海藻糖提取效率的响。     

Biology and insights into the role of cohesin protease separase in human malignancies

Separase, an enzyme that resolves sister chromatid cohesion during the metaphase‐to‐anaphase transition, plays a pivotal role in chromosomal segregation and cell division. Separase protein, encoded by the extra spindle pole bodies like 1 (ESPL1) gene, is overexpressed in numerous human cancers including breast, bone, brain, and prostate. Separase is oncogenic, and its overexpression is sufficient to induce mammary tumours in mice. Either acute or chronic overexpression of separase in mouse mammary glands leads to aneuploidy and tumorigenesis, and inhibition of separase enzymatic activity decreases the growth of human breast tumour xenografts in mice. This review focuses on the biology of and insights into the molecular mechanisms of separase as an oncogene, and its significance and implications for human cancers.     

Chromosome cohesion and segregation in mitosis and meiosis

The faithful segregation of the genetic material into daughter cells during cell division is crucial for the production of healthy progeny. Sister chromatid cohesion and separation are fundamental to this process. Progress has been made in our molecular understanding of cohesion and mechanisms for the dissolution of cohesion have been uncovered.     

Separase cleaves the kinetochore protein Meikin at the meiosis I/II transition

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Comparative analysis of chromosome segregation in human,yeasts and trypanosome

Chromosome segregation is a tightly regulated process through which duplicated genetic materials are equally partitioned into daughter cells. During the past decades, tremendous efforts have been made to understand the molecular mechanism of chromosome segregation using animals and yeasts as model systems. Recently, new insights into chromosome segregation have gradually emerged using trypanosome, an early branching parasitic protozoan, as a model organism. To uncover the unique aspects of chromosome segregation in trypanosome, which potentially could serve as new drug targets for anti-trypanosome chemotherapy, it is necessary to perform a comparative analysis of the chromosome segregation machinery between trypanosome and its human host. Here, we briefly review the current knowledge about chromosome segregation in human and Trypanosoma brucei, with a focus on the regulation of cohesin and securin degradation triggered by the activation of the anaphase promoting complex/cyclosome （APC/C）. We also include yeasts in our comparative analysis since some of the original discoveries were made using budding and fission yeasts as the model organisms and, therefore, these could provide hints about the evolution of the machinery. We highlight both common and unique features in these model systems and also provide perspectives for future research in trypanosome.     

The Sister Chromatid Cohesion Pathway Suppresses Multiple Chromosome Gain and Chromosome Amplification

Gain or loss of chromosomes resulting in aneuploidy can be important factors in cancer and adaptive evolution. Although chromosome gain is a frequent event in eukaryotes, there is limited information on its genetic control. Here we measured the rates of chromosome gain in wild-type yeast and sister chromatid cohesion (SCC) compromised strains. SCC tethers the newly replicated chromatids until anaphase via the cohesin complex. Chromosome gain was measured by selecting and characterizing copper-resistant colonies that emerged due to increased copies of the metallothionein gene CUP1. Although all defective SCC diploid strains exhibited increased rates of chromosome gain, there were 15-fold differences between them. Of all mutants examined, a hypomorphic mutation at the cohesin complex caused the highest rate of chromosome gain while disruption of WPL1, an important regulator of SCC and chromosome condensation, resulted in the smallest increase in chromosome gain. In addition to defects in SCC, yeast cell type contributed significantly to chromosome gain, with the greatest rates observed for homozygous mating-type diploids, followed by heterozygous mating type, and smallest in haploids. In fact, wpl1-deficient haploids did not show any difference in chromosome gain rates compared to wild-type haploids. Genomic analysis of copper-resistant colonies revealed that the “driver” chromosome for which selection was applied could be amplified to over five copies per diploid cell. In addition, an increase in the expected driver chromosome was often accompanied by a gain of a small number of other chromosomes. We suggest that while chromosome gain due to SCC malfunction can have negative effects through gene imbalance, it could also facilitate opportunities for adaptive changes. In multicellular organisms, both factors could lead to somatic diseases including cancer.     

C-terminus of Sororin interacts with SA2 and regulates sister chromatid cohesion

Sororin is a conserved protein required for accurate separation of sister chromatids in each cell cycle. Sororin is recruited to chromatin during DNA replication, protects sister chromatid cohesion in S and G2 phase, and regulates the resolution of sister chromatid cohesion in mitosis. Sororin binds to cohesin complex, but how Sororin and cohesin subunits interact remains unclear. Here we report that the C-terminus of Sororin, especially the last 12 amino acid (aa) residues, is important for Sororin to bind cohesin core subunit SA2. Deletion of the last 12aa residues not only inhibits the interactions between Sororin and SA2 but also causes precocious chromosome separation. Our data suggest that the C-terminus of Sororin functions as an anchor binding to SA2, which facilitates other conserved motifs on Sororin to interact with other proteins to regulate sister chromatid cohesion and separation.     

Sororin actively maintains sister chromatid cohesion

Cohesion between sister chromatids is established during DNA replication but needs to be maintained to enable proper chromosome–spindle attachments in mitosis or meiosis. Cohesion is mediated by cohesin, but also depends on cohesin acetylation and sororin. Sororin contributes to cohesion by stabilizing cohesin on DNA. Sororin achieves this by inhibiting WAPL, which otherwise releases cohesin from DNA and destroys cohesion. Here we describe mouse models which enable the controlled depletion of sororin by gene deletion or auxin‐induced degradation. We show that sororin is essential for embryonic development, cohesion maintenance, and proper chromosome segregation. We further show that the acetyltransferases ESCO1 and ESCO2 are essential for stabilizing cohesin on chromatin, that their only function in this process is to acetylate cohesin''s SMC3 subunit, and that DNA replication is also required for stable cohesin–chromatin interactions. Unexpectedly, we find that sororin interacts dynamically with the cohesin complexes it stabilizes. This implies that sororin recruitment to cohesin does not depend on the DNA replication machinery or process itself, but on a property that cohesin acquires during cohesion establishment.     

Rtt101‐Mms1‐Mms22 coordinates replication‐coupled sister chromatid cohesion and nucleosome assembly

Two sister chromatids must be held together by a cohesion process from their synthesis during S phase to segregation in anaphase. Despite its pivotal role in accurate chromosome segregation, how cohesion is established remains elusive. Here, we demonstrate that yeast Rtt101‐Mms1, Cul4 family E3 ubiquitin ligases are stronger dosage suppressors of loss‐of‐function eco1 mutants than PCNA. The essential cohesion reaction, Eco1‐catalyzed Smc3 acetylation is reduced in the absence of Rtt101‐Mms1. One of the adaptor subunits, Mms22, associates directly with Eco1. Point mutations (L61D/G63D) in Eco1 that abolish the interaction with Mms22 impair Smc3 acetylation. Importantly, an eco1LGpol30A251V double mutant displays additive Smc3ac reduction. Moreover, Smc3 acetylation and cohesion defects also occur in the mutants of other replication‐coupled nucleosome assembly (RCNA) factors upstream or downstream of Rtt101‐Mms1, indicating unanticipated cross talk between histone modifications and cohesin acetylation. These data suggest that fork‐associated Cul4‐Ddb1 E3s, together with PCNA, coordinate chromatin reassembly and cohesion establishment on the newly replicated sister chromatids, which are crucial for maintaining genome and chromosome stability.     

Disengaging the Smc3/kleisin interface releases cohesin from Drosophila chromosomes during interphase and mitosis

Cohesin's Smc1, Smc3, and kleisin subunits create a tripartite ring within which sister DNAs are entrapped. Evidence suggests that DNA enters through a gate created by transient dissociation of the Smc1/3 interface. Release at the onset of anaphase is triggered by proteolytic cleavage of kleisin. Less well understood is the mechanism of release at other stages of the cell cycle, in particular during prophase when most cohesin dissociates from chromosome arms in a process dependent on the regulatory subunit Wapl. We show here that Wapl‐dependent release from salivary gland polytene chromosomes during interphase and from neuroblast chromosome arms during prophase is blocked by translational fusion of Smc3's C‐terminus to kleisin's N‐terminus. Our findings imply that proteolysis‐independent release of cohesin from chromatin is mediated by Wapl‐dependent escape of DNAs through a gate created by transient dissociation of the Smc3/kleisin interface. Thus, cohesin's DNA entry and exit gates are distinct.