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Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins

MOLECULAR CELL 2000年第2期5卷 243-254页

作者： Ciosk, R Shirayama, M Shevchenko, A Tanaka, TU Toth, A Shevchenko, A Nasmyth, K Res Inst Mol Pathol A-1030 Vienna Austria European Mol Biol Lab Peptide & Prot Grp D-69012 Heidelberg Germany

Cohesion between sister chromatids depends on a multisubunit cohesin complex that binds to chromosomes around DNA replication and dissociates from them at the onset of anaphase. Scc2p, though not a cohesin subunit, is also required for sister chromatid cohesion. We show here that Scc2p forms a complex with a novel protein, Scc4p, which is also necessary for sister cohesion. In scc2 or scc4 mutants, cohesin complexes form normally but fail to bind both to centromeres and to chromosome arms. Our data suggest that a major role for the Scc2p/Scc4p complex is to facilitate the loading of cohesin complexes onto chromosomes.

关键词：细胞周期蛋白质类/遗传学细胞周期蛋白质类/分离和提纯细胞周期蛋白质类/代谢染色单体/代谢染色质/代谢染色体蛋白质类非组蛋白染色体分离染色体真菌/超微结构真菌蛋白质类核蛋白质类/代谢蛋白质结合酿酒酵母蛋白质类酵母菌

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Direct characterization of the physicochemical properties of fungal spores using functionalized AFM probes

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BIOPHYSICAL JOURNAL 2000年第6期78卷 3286-3291页

作者： Dufrêne, YF Univ Catholique Louvain Unite Chim Interfaces B-1348 Louvain Belgium

A new method is described for characterizing the physicochemical properties of native microbial cells by using atomic force microscopy (AFM) with chemically functionalized probes. Adhesion forces were measured, under deionized water, between probes and model substrata functionalized with alkanethiol self-assembled monolayers terminated with OH and CH3 groups. These were found to be 6 +/- 2 nN (n = 1024), 0.9 +/- 0.4 nN, and similar to 0 nN, for CH3/CH3, CH3/OH, and OH/OH surfaces, respectively, and were not significantly influenced by changes of ionic strength (0.1 M NaCl versus deionized water). This shows that functionalized probes are very sensitive to changes of surface hydrophobicity. Using OH- and CH3-terminated probes, patterns of rodlets, similar to 10 nm in diameter, were visualized, under physiological conditions, at the surface of spores of Phanerochaete chrysosporium. Multiple (1024) force-distance curves recorded over 500 x 500-nm areas at the spore surface, either in deionized water or in 0.1 M NaCl solutions, always showed no adhesion for both OH- and CH3-terminated probes. Control experiments indicated that the lack of adhesion is not due to transfer of cellular material onto the probe, but to the hydrophilic nature of the spore surface.

关键词：细胞黏附指示剂和试剂显微镜检查原子力/方法白腐真菌/生理学白腐真菌/超微结构可重复性结果孢子真菌/生理学孢子真菌/超微结构

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Effects of modifying topoisomerase II levels on cellular recovery from radiation damage

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RADIATION RESEARCH 2000年第4期154卷 461-466页

作者： Gaffney, DK Lundquist, M Warters, RL Rowley, R Univ Utah Med Ctr Dept Radiat Oncol Salt Lake City UT 84132 USA

Experiments were performed with the budding yeast, Saccharomyces cerevisiae, to test whether DNA topoisomerase II is involved in repair of DNA damage induced by ionizing radiation. Topoisomerase II was inactivated by use of a temperature-sensitive mutation. Enzyme inactivation increased cellular radiosensitivity, blocked the restitution of broken chromosomes, assayed by pulsed-held gel electrophoresis, and prolonged the induction of a DNA damage-inducible gene (RNR3). Overexpression of the topoisomerase II gene did not alter cellular radiosensitivity. The data support a role for topoisomerase II in the repair of DNA strand breaks. (C) 2000 by Radiation Research Society.

关键词：染色体真菌/辐射效应染色体真菌/超微结构 DNA损伤 DNA修复 DNA拓扑异构酶类 Ⅱ型/遗传学 DNA拓扑异构酶类 Ⅱ型/生理学 DNA 真菌/遗传学 DNA 真菌/代谢 DNA 真菌/辐射效应电泳凝胶脉冲场酶诱导酶抑制剂/药理学真菌蛋白质类/拮抗剂和抑制剂真菌蛋白质类/遗传学真菌蛋白质类/生理学基因表达调控真菌/辐射效应基因报告热温度新生霉素/药理学启动区遗传辐射耐受性/遗传学重组融合蛋白质类/生物合成重组融合蛋白质类/遗传学酿酒酵母菌/酶学酿酒酵母菌/遗传学酿酒酵母菌/辐射效应拓扑异构酶II抑制剂

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MAP kinase and protein kinase A-dependent mobilization of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea

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PLANT CELL 2000年第9期12卷 1703-1718页

作者： Thines, E Weber, RWS Talbot, NJ Univ Exeter Sch Biol Sci Washington Singer Labs Exeter EX4 4QG Devon England

Magnaporthe grisea produces an infection structure called an appressorium, which is used to breach the plant cuticle by mechanical force. Appressoria generate hydrostatic turgor by accumulating molar concentrations of glycerol. To investigate the genetic control and biochemical mechanism for turgor generation, we assayed glycerol biosynthetic enzymes during appressorium development, and the movement of storage reserves was monitored in developmental mutants. Enzymatic activities for glycerol generation from carbohydrate sources were present in appressoria but did not increase during development. In contrast, triacylglycerol lipase activity increased during appressorium maturation. Rapid glycogen degradation occurred during conidial germination, followed by accumulation in incipient appressoria and dissolution before turgor generation. Lipid droplets also moved to the incipient appressorium and coalesced into a central vacuole before degrading at the onset of turgor generation. Glycogen and lipid mobilization did not occur in a Delta pmk1 mutant, which lacked the mitogen-activated protein kinase (MAPK) required for appressorium differentiation, and was retarded markedly in a Delta cpkA mutant, which lacks the catalytic subunit of cAMP-dependent protein kinase A (PKA). Glycogen and lipid degradation were very rapid in a Delta mac1 sum1-99 mutant, which carries a mutation in the regulatory subunit of PKA, occurring before appressorium morphogenesis was complete. Mass transfer of storage carbohydrate and lipid reserves to the appressorium therefore occurs under control of the PMK1 MARK pathway. Turgor generation then proceeds by compartmentalization and rapid degradation of lipid and glycogen reserves under control of the CPKA/SUM1-encoded PKA holoenzyme.

关键词：生物转运环AMP依赖性蛋白激酶类/遗传学环AMP依赖性蛋白激酶类/生理学酶诱导甘油/代谢糖原/代谢脂肪酶/代谢稻温病菌/遗传学稻温病菌/代谢稻温病菌/致病力丝裂原激活蛋白激酶类/遗传学丝裂原激活蛋白激酶类/生理学突变容积渗克分子浓度渗透压孢子真菌/生长和发育孢子真菌/代谢孢子真菌/超微结构时间因素甘油三酯类/代谢毒力

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Biosynthesis and immunolocalization of Lewis a-containing N-glycans in the plant cell

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PLANT PHYSIOLOGY 1999年第2期121卷 333-343页

作者： Fitchette, AC Cabanes-Macheteau, M Marvin, L Martin, B Satiat-Jeunemaitre, B Gomord, V Crooks, K Lerouge, P Faye, L Hawes, C Univ Rouen Fac Sci Transports Intracellulaires Lab CNRSESA 6037European Inst Peptide ResIFRMP 23 F-76821 Mt St Aignan France Univ Rouen Fac Sci CNRSESA 6014 IFRMP 23 F-76821 Mt St Aignan France Oxford Brookes Univ Res Sch Biol & Mol Sci Oxford OX3 0BP England CNRS UPR 40 Inst Sci Vegetales F-91198 Gif Sur Yvette France

We recently demonstrated the presence of a new asparagine-linked complex glycan on plant glycoproteins that harbors the Lewis a (Le(a)), or Gal beta(1-3)[Fuc alpha(1-4)]GlcNAc, epitope, which in mammalian cells plays an important role in cell-to-cell recognition. Here we show that the monoclonal antibody JIM 84, which is widely used as a Golgi marker in light and electron microscopy of plant cells, is specific for the Le(a) antigen. This antigen is present on glycoproteins of a number of flowering and non-flowering plants, but is less apparent in the Cruciferae, the family that includes Arabidopsis. Le(a)-containing oligosaccharides are found in the Golgi apparatus, and our immunocytochemical experiments suggest that it is synthesized in the trans-most part of the Golgi apparatus. Le(a) epitopes are abundantly present on extracellular glycoproteins, either soluble or membrane bound, but are never observed on vacuolar glycoproteins. Double-labeling experiments suggest that vacuolar glycoproteins do not bypass the late Golgi compartments where Le(a) is built, and that the absence of the Lea epitope from vacuolar glycoproteins is probably the result of its degradation by glycosidases en route to or after arrival in the vacuole.

关键词：拟南芥属/超微结构碳水化合物序列表位/化学真菌/细胞学真菌/超微结构糖蛋白类/生物合成糖蛋白类/化学高尔基体/超微结构 Lewis血型系统/化学哺乳动物分子序列数据植物/细胞学植物/超微结构多糖类/分析多糖类/生物合成多糖类/化学种特异性动物

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Dependence of the regulation of telomere length on the type of subtelomeric repeat in the yeast Saccharomyces cerevisiae

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GENETICS 1999年第4期152卷 1531-1541页

作者： Craven, RJ Petes, TD Univ N Carolina Dept Biol Curriculum Genet & Mol Biol Chapel Hill NC 27599 USA

In the yeast Saccharomyces cerevisiae, chromosomes terminate with similar to 400 bp of a simple repeat poly(TG(1-3)). Based on the arrangement of subtelomeric X and Y' repeats, two types of yeast telomeres exist, those with both X and Y' (Y' telomeres) and those with only X (X telomeres). Mutations that result in abnormally short or abnormally long poly(TG(1-3)) tracts have been previously identified. In this study, we investigated telomere length in strains with two classes of mutations, one that resulted in short poly(TG(1-3)) tracts (tel1) and one that resulted in elongated tracts (pif1, rap1-17, rif1, or rif2). In the tel1 pif1 strain, Y' telomeres had about the same length as those in tell strains and X telomeres had lengths intermediate between those in tel1 and pif1 strains. Strains with either the tel1 rap1-17 or tel1 rif2 genotypes had short tracts for all chromosome ends examined, demonstrating that the telomere elongation characteristic of rap1-17 and rif2 strains is Tel1p-dependent. In strains of the tel1 rif1 or tel1 rif1 rif2 genotypes, telomeres with Y' repeats had short terminal tracts, whereas most of the X telomeres had long terminal tracts. These results demonstrate that the regulation of telomere length is different for X and Y' telomeres.

关键词：载体蛋白质类/遗传学载体蛋白质类/生理学染色体真菌/超微结构 DNA螺旋酶类/遗传学 DNA螺旋酶类/生理学 DNA 真菌/遗传学 DNA结合蛋白质类/遗传学 DNA结合蛋白质类/生理学上位性遗传真菌蛋白质类/遗传学真菌蛋白质类/生理学细胞内信号肽和蛋白质类蛋白质丝氨酸苏氨酸激酶重复序列核酸阻遏蛋白质类/遗传学阻遏蛋白质类/生理学酵母菌酿酒/遗传学酵母菌酿酒/超微结构酿酒酵母蛋白质类端粒/超微结构端粒结合蛋白质类

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Genetic control of telomere integrity in Schizosaccharomyces pombe:: rad3⁺ and tel1⁺ are parts of two regulatory networks independent of the downstream protein kinases chk1⁺ and cds1⁺

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GENETICS 1999年第4期152卷 1501-1512页

作者： Matsuura, A Naito, T Ishikawa, F Tokyo Inst Technol Grad Sch Biosci & Biotechnol Dept Life Sci Midori Ku Yokohama Kanagawa 2268501 Japan

The Schizosaccharomyces pombe checkpoint gene named rad3(+) encodes an ATM-homologous protein kinase that shares a highly conserved motif with proteins involved in DNA metabolism. Previous studies have shown that Rad3 fulfills its function via the regulation of the Chk1 and Cds1 protein kinases. Here we describe a novel role for Rad3 in the control of telomere integrity. Mutations in the rad3(+) gene alleviated telomeric silencing and produced shortened lengths in the telomere repeat tracts. Genetic analysis revealed that the other checkpoint rad mutations rad1, rad17, and rad26 belong to the same phenotypic class with rad3 with regard to control of the telomere length. Of these mutations, rad3 and rad26 have a drastic effect on telomere shortening, tel1(+), another ATM homologue in S. pombe, carries out its telomere maintenance function in parallel with the checkpoint rad genes. Furthermore, either a single or double disruption of cds1(+) and chk1(+) caused no obvious changes in the telomeric DNA structure. Our results demonstrate a novel role of the S. pombe ATM homologues that is independent of chk1(+) and cds1(+).

关键词：腺苷三磷酸酶类/遗传学腺苷三磷酸酶类/生理学细胞周期蛋白质类染色体真菌/超微结构 DNA螺旋酶类/遗传学 DNA螺旋酶类/生理学 DNA结合蛋白质类真菌蛋白质类/遗传学真菌蛋白质类/生理学乳清酸/类似物和衍生物乳清酸/药理学蛋白激酶类/生理学蛋白质丝氨酸苏氨酸激酶蛋白质类/生理学辐射耐受性/遗传学酿酒酵母蛋白质类裂殖酵母菌属/药物作用裂殖酵母菌属/遗传学裂殖酵母菌属/超微结构非洲粟酒人体酵母蛋白质类信号传导/生理学端粒/超微结构肿瘤抑制蛋白质类

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Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)-binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 1999年第7期96卷 3757-3762页

作者： Pietrasanta, LI Thrower, D Hsieh, W Rao, S Stemmann, O Lechner, J Carbon, J Hansma, H Univ Calif Santa Barbara Dept Mol Cellular & Dev Biol Santa Barbara CA 93106 USA Univ Calif Santa Barbara Dept Phys Santa Barbara CA 93106 USA Univ Regensburg Inst Biochem Genet & Mikrobiol D-93040 Regensburg Germany

Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3-CEN DNA complex by atomic force microscopy, Assembly of CBF3-CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is approximate to 9% shorter than the DNA alone, suggesting some winding of DNA within the complex, The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent approximate to 55 degrees at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms,,vith molecular volumes approximately three times the mean volumes of two-armed complexes, These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.

关键词：着丝粒/超微结构染色体图染色体真菌/超微结构 DNA 真菌/化学 DNA 真菌/代谢 DNA 真菌/超微结构 DNA结合蛋白质类/化学 DNA结合蛋白质类/代谢 DNA结合蛋白质类/超微结构真菌蛋白质类/化学真菌蛋白质类/代谢真菌蛋白质类/超微结构染色粒显微镜检查原子力核蛋白质类酿酒酵母菌/遗传学酿酒酵母菌/超微结构酿酒酵母蛋白质类

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The morphology of Ichthyophonus sp in their mugilid hosts (Pisces: Teleostei) and following cultivation in vitro.: A light and electron microscopy study

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PARASITOLOGY RESEARCH 1999年第7期85卷 562-575页

作者： Franco-Sierra, A Alvarez-Pellitero, P CSIC Inst Acuicultura Torre Sal E-12595 Ribera De Cabanes Castellon Spain

The morphology of Ichthyophonus sp., a parasite of Mugil capito and Liza saliens, was investigated by light and transmission electron microscopy. The most frequent stage found in the fish hosts was the multinucleate spore, though germinating stages, hyphae, and endospores were also found. Different development patterns were observed in the media assayed for in vitro culture. Optimal growth and development were obtained in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum at pH 7. Ultrastructural features of multinucleate spores, both in the fish host and in culture, were a fibrillar thick wall and an electron-lucent matrix, with large glycogen granules, some electron-dense bodies, large vacuoles, lipid inclusions, and endoplasmic reticulum mainly appearing among the nuclei. Mitochondria with scarce tubulovesicular cristae were observed in the different stages, mainly near the wall and the germinating sites. Condensed heterochromatin was rarely seen. A nucleus-associated organelle (NAO) was frequently observed, and dictyosome cisternae and vesicles appeared in its vicinity.

关键词：培养基鱼疾病/微生物学氢离子浓度显微镜检查电子鲈形目/寄生虫学孢子真菌/超微结构接合菌亚纲/生长和发育接合菌亚纲/分离和提纯接合菌亚纲/超微结构动物牛

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Identification of cohesin association sites at centromeres and along chromosome arms

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CELL 1999年第6期98卷 847-858页

作者： Tanaka, TU Cosma, MP Wirth, K Nasmyth, K Res Inst Mol Pathol A-1030 Vienna Austria

A multisubunit cohesin complex holds sister chromatids together after DNA replication. Using chromatin immunoprecipitation, we detected cohesin association with centromeres and with discrete sites along chromosome arms from S phase until metaphase in S. cerevisiae, Short DNA sequences (130-280 bp) are sufficient to confer cohesin association. Cohesin association with a centromere depends on Mif2p, the centromere binding factor CBF3, and a centromere-specific histone variant, Cse4p. Because only active centromeres confer cohesin association with centromeric DNA, we suggest that cohesin is recruited by the same chromatin structure that confers the attachment of microtubules. Propagation of this structure might be partly epigenetic. Finally, cohesion associated with "minimal" centromeres is insufficient to resist the splitting force exerted by microtubules and appears to be reinforced by cohesion provided by their flanking DNA sequences.

关键词：结合部位细胞周期蛋白质类/分离和提纯着丝粒/化学着丝粒/超微结构染色单体/化学染色单体/超微结构染色质/代谢染色质/超微结构染色体蛋白质类非组蛋白/分离和提纯染色体真菌/化学染色体真菌/超微结构 DNA复制 DNA 真菌/代谢 DNA结合蛋白质类/代谢真菌蛋白质类/分离和提纯真菌蛋白质类/代谢组蛋白类/代谢染色粒/化学染色粒/代谢核蛋白质类/分离和提纯磷蛋白类蛋白质结合蛋白硫酸软骨素类酵母菌酿酒/遗传学酵母菌酿酒/超微结构酿酒酵母蛋白质类

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