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鞭毛是衣藻在水中的运动器官.衣藻的鞭毛很纤细,不经染色,难以在普通显微镜下观察.观察衣藻的鞭毛,通常用鲁哥氏液染色,虽然这种方法很简便,但用此法染色后,经过脱水,鞭毛的颜色就会复失.为了制成永久装片长期观察,我们认为用以下方法制作手续比较简单,鞭毛的染色效果也较好.具体做法是: 相似文献
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单细胞光合生物莱茵衣藻光系统Ⅱ产生超氧阴离子自由基的ESR研究 总被引:1,自引:0,他引:1
强光辐照下高等植物光系统Ⅱ(PSⅡ)会产生超氧阴离子自由基(O-2),该过程中产生的O-2在高等植物光合作用活性氧自由基代谢中具有重要作用.莱茵衣藻光系统结构与高等植物非常相近.应用新型高特异性自旋捕获.ESR方法及四唑氮蓝还原法,首次在莱茵衣藻的类囊体膜和PSⅡ中检测到O-2的生成,通过与有关菠菜的ESR实验结果对比发现两者的O-2产生分子机制可能极为相似.相比高等植物,单细胞莱茵衣藻具有结构简单、遗传背景清晰与基因组测序工作完备等诸多特点.因而,针对莱茵衣藻的O-2分析可能为深入研究高等植物光合系统中的活性氧代谢机制提供新的契机. 相似文献
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纤毛/鞭毛是从细胞膜表面突出的真核细胞器,它能调节细胞运动及细胞周围液体流动,或者参与机体的感知功能,其异常会引发多种人类纤毛病。作为最早被发现的细胞器之一,纤毛一直是细胞生物学领域的重点研究对象,但是因结构大且复杂,其分子组装机制的揭示长期以来一直是个难题。近些年,随着冷冻电镜技术的发展,多个课题组先后报道了从衣藻到哺乳动物精子鞭毛的轴丝各部分高分辨率结构。该文综述了动纤毛的结构组成和最新的分子组装研究进展,重点描述了轴丝各个组成部分包括双联微管(DMT)、内外动力臂(IDA和ODA)、辐条结构(RS)、中央微管对(CP)和连接复合物(N-DRC)的蛋白组成和分布,为深入理解纤毛的组装过程和功能调节提供了科学依据。 相似文献
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报道了团藻目衣藻科拟衣藻属一新种。此种从中国科学院武汉植物研究所一浇园粪缸中采得,在分离室内单种培养过程中,对其形态学和生活史进行了研究。营养细胞具有多个盘状色素体,与拟衣藻属已知的种类有明显的差别。经培养观察,作者发现除细胞分裂、无性生殖外,还有同宗异配式有性生殖,在生活史各阶段该藻中绿体中均未见蛋白核,因此,拟衣藻作为分类单元是一个有效的属。 相似文献
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模式生物衣藻及其研究进展 总被引:5,自引:0,他引:5
单细胞衣藻(Chlamydomonas)由于其生活周期简单,培养方法简便,易于分离得到系列的突变体,并已建立了分子遗传学研究技术与遗传分析系统,成为植物光合作用、鞭毛组装与功能、细胞周期及节律、细胞信号传导与光感受、细胞识别等重要生物学过程研究的模式生物体。本文对模式生物衣藻及其相关生物学途径的研究进展作一综述。
Abstract:The unicellular alga Chlamydomonas offers a simple life cycle,easy culture and isolation of series of mutants,established the techniques and tool kit for molecular genetics and genetics analysis.It is now becoming the model organism for studies on photosynthesis in plant,flagellar assembly and function,cell cycle and circadian rhythms,signal transduction,light perception and cell recognition.It is summarized the progress of study on Chlamydomonas as a model organism in this paper. 相似文献
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目的:在莱茵衣藻细胞中构建并筛选鞭毛组装缺陷突变体,克隆缺陷基因,探索其对鞭毛组装的影响。方法:使用带有巴龙霉素(Paromomycin)抗性的基因片段随机插入衣藻细胞基因组中,通过性状筛选和基因序列分析获得与CrPP2C(Chlamydomonas reinhardtii type 2C protein phosphatase)基因相关的鞭毛异常突变体,根据突变体基本生物学性状和生化分析对CrPP2C基因的功能进行分析。结果:采用电转法成功获得衣藻细胞鞭毛缺陷相关突变体,部分细胞具有短鞭毛,部分细胞则不具有鞭毛;通过RESDA-PCR(restriction enzyme site-directed amplification PCR)对突变体基因序列分析,鞭毛缺陷性状由CrPP2C基因遭到破坏导致;把含有完整CrPP2C基因的重组质粒通过电转法导入突变体后,其鞭毛几乎恢复为野生型长度,并可检测到PP2C-HA融合蛋白的表达;观察鞭毛再生,突变体鞭毛只能再生为原有长度;使用药物处理使鞭毛缩短,突变体鞭毛能正常解聚;电镜检测突变体的鞭毛显微结构,发现过渡区的Y形结构缺陷。结论:CrPP2C基因的破坏导致鞭毛过渡区结构缺失,影响鞭毛组装过程,不组装鞭毛或组装短鞭毛。 相似文献
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真核细胞的纤毛(也称鞭毛)是一种突出于细胞表面的极性细胞器,纤毛不仅参与细胞运动,还参与信号传导等过程,其结构或功能异常引发的一系列人类疾病称为"纤毛相关性疾病"。纤毛相关性疾病巴德-毕德氏综合征(Bardet-Biedl syndrome,简称BBS)由BBS相关基因缺陷导致,为了研究致病基因BBS8的生理作用和功能,构建模式生物莱茵衣藻BBS8基因缺陷突变体,利用性状观测和生化分析检测突变体的表现型和生理功能。免疫荧光表明BBS8蛋白是一种鞭毛蛋白且在基体有特异性定位;bbs8突变体感光极性运动消失,并在解聚诱导实验中鞭毛解聚缓慢;鞭毛的银染和质谱结果表明突变体的鞭毛膜蛋白在鞭毛内异常积累。文中通过实验证据说明BBS8蛋白在参与鞭毛内膜蛋白运输中起到重要作用,并极可能通过介导膜蛋白反向运输发挥生理功能。 相似文献
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细菌鞭毛马达——一种卓越的分子机器 总被引:1,自引:0,他引:1
鞭毛马达(flagellar motor)是一种分子旋转马达,它在细菌鞭毛的结构与功能中起着中心作用.鞭毛马达的结构已基本清楚,主要由Mot A、Mot B、Fli G、Fli M和Fli N 5种蛋白组成定子(stator)和转子(rotor),其驱动力来自于跨膜的H+或Na+流.目前对鞭毛马达的旋转动力学及旋转力矩产生机制已有初步的了解.鞭毛马达可作为研究分子旋转马达的理想模型,对其深入研究将有助于认识生物能量转化利用及细胞运动的机制并具有广泛的生物学意义. 相似文献
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Cell body and flagellar agglutinins in Chlamydomonas reinhardtii: the cell body plasma membrane is a reservoir for agglutinins whose migration to the flagella is regulated by a functional barrier 总被引:4,自引:3,他引:1
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Fertilization in Chlamydomonas reinhardtii is initiated when gametes of opposite mating types adhere to each other via adhesion molecules (agglutinins) on their flagella. Adhesion leads to loss of active agglutinins from the flagella and recruitment of new agglutinins from a pool associated with the cell body. We have been interested in determining the precise cellular location of the pool and learning more about the relationship between agglutinins in the two domains. In the studies reported here we describe methods for purification of mt+ cell body agglutinins by use of ammonium sulfate precipitation, chromatography (molecular sieve, ion exchange, and hydrophobic interaction), and sucrose gradient centrifugation. About 90% of the total agglutinins were associated with the cell body and the remainder were on the flagella. Cell body agglutinins were indistinguishable from mt+ flagellar agglutinins by SDS-PAGE, elution properties on a hydrophobic interaction column, and in sedimentation properties on sucrose gradients. The nonadhesiveness of cell bodies suggested that the cell body agglutinins would be intracellular, but our results are not consistent with this interpretation. We have demonstrated that brief trypsin treatment of deflagellated gametes destroyed all of the cell body agglutinins and, in addition, we showed that the cell body agglutinins were accessible to surface iodination. These results indicated that C. reinhardtii agglutinins have a novel cellular disposition: active agglutinins, representing approximately 10% of the total cellular agglutinins, are found only on the flagella, whereas the remaining 90% of these molecules are on the external surface of the cell body plasma membrane in a nonfunctional form. This segregation of cell adhesion molecules into distinct membrane domains before gametic interactions has been demonstrated in sperm of multicellular organisms and may be a common mechanism for sequestering these critical molecules until gametes are activated for fusion. In experiments in which surface-iodinated cell bodies were permitted to regenerate new flagella, we found that the agglutinins (as well as the 350,000 Mr, major flagellar membrane protein) on the newly regenerated flagella were iodinated. These results indicate that proteins destined for the flagella can reside on the external surface of the cell body plasma membrane and are recruited onto newly forming flagella as well as onto preexisting flagella during fertilization. 相似文献
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Rapid and slow mechanisms for loss of cell adhesiveness during fertilization in Chlamydomonas 总被引:4,自引:0,他引:4
Although vegetative cells, gametes, and zygotes of the biflagellated alga Chlamydomonas bear flagella, only the flagella of mt+ and mt- gametes are adhesive. The molecules responsible for adhesiveness, mt+ and mt- agglutinins, are long rod-shaped glycoproteins displayed on the flagellar membrane. These flagellar agglutinins, which gametes use both as adhesion and signaling molecules during the early events of fertilization, are lost from the flagella during adhesion. Flagellar adhesiveness can be maintained, however, by recruitment and activation of preexisting, inactive agglutinins from the plasma membrane of the cell body (Hunnicutt et al, 1990, J. Cell Biol. 111, 1605-1616) unless the gametes of opposite mating types fuse to form zygotes. Upon cell fusion, flagellar adhesiveness is lost. In the studies presented here, we have employed an in vitro bioassay to measure agglutinins in both cell bodies and flagella at various times during gametogenesis, during fertilization, and after zygote-formation. By use of the bioassay, which can detect agglutinins that are functionally inactive in vivo, we found that vegetative cells are devoid of agglutinins. These adhesion molecules appear only after gametogenesis is underway with the cell body agglutinins appearing first and then the flagellar agglutinins. Surprisingly, 30 min after zygote formation, when the zygotes' flagella are no longer adhesive, the flagellar agglutinin activity detectable with the bioassay remains high. One interpretation of these results is that zygotes continue to recruit agglutinins from the cell body to the flagella, but cell fusion abrogates activation of the agglutinins. Within 45-90 min after fusion both the cell body and flagellar agglutinins are lost and can be detected in the medium. These mechanisms, which render the zygotes nonadhesive to other zygotes and unmated gametes, contribute to the Chlamydomonas equivalent of a block to polyspermy. 相似文献
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The ultrastructure of adhesion complexes between gametic flagellar membranes of Chlamydomonas reinhardtii Dangeard was analyzed using the quick-freeze deep-etch technique. The sexual agglutinin fibrils interact by forming hybrid fibers that frequently branch, forming extensively cross-bridged meshworks. This pattern of interaction mimics a prominent mode of cell wall formation in Chlamydomonas, supporting the notion that the agglutinins evolved from cell wall proteins and that sexual adhesion and cell wall assembly are homologous events. 相似文献
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Sexual agglutinins from the Chlamydomonas flagellar membrane. Partial purification and characterization 总被引:15,自引:0,他引:15
W S Adair B C Monk R Cohen C Hwang U W Goodenough 《The Journal of biological chemistry》1982,257(8):4593-4602
Chlamydomonas sexual agglutinins have been quantitatively extracted from isolated flagella in vitro using the dialyzable nonionic detergent octyl-D-glucopyranoside and from cells in vivo with 12.5 mM EDTA. Both preparations elicit normal sexual responses from gametes of complementary, but not like, mating types. Extracts of vegetative cells and several agglutination-deficient (imp) mutants are totally inactive. Agglutinin activity is sensitive to trypsin, mild periodate oxidation, and heating at 60 degrees C for 1 min. These findings, coupled with the size of the molecule (it is excluded from Sepharose 6B and sediments as a 12 S particle in sucrose gradients) lead us to propose that the Chlamydomonas sexual agglutinins are large glycoproteins or glycoprotein aggregates which associate with the flagellar membrane in an extrinsic fashion. Partial purification of in vivo 125I-surface labeled EDTA extracts rules out several surface polypeptides, including the bulk of material migrating in the region of the major membrane glycoprotein (Mr 350,000), as agglutinin candidates and indicates that the active molecule is a minor component of the flagellar membrane. In addition, in vitro assays suggest a mechanism for in vivo sexual agglutination whereby stable adhesion is achieved by the active redistribution of agglutinins to the flagellar tips. 相似文献
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Marieke R. Samson Frans M. Klis Wieger L. Homan Piet van Egmond Alan Musgrave Herman van den Ende 《Planta》1987,170(3):314-321
Sexual interaction between gametes of opposite mating type (mt) of the unicellular green alga Chlamydomonas eugametos starts with agglutination of the cells via particular glycoproteins on the flagellar surface. Purification of these socalled agglutinins was achieved by a three-step procedure consisting of, successively, gel filtration, anion-exchange chromatography, and high-performance gel filtration. The amino-acid and sugar compositions of both agglutinins showed a high degree of similarity; the most prominent amino acids were hydroxyproline, serine and glycine, and the main sugars were arabinose and galactose. The carbohydrate portions represented about half of the molecular mass of both agglutinins. Using high-performance gel filtration, a calibration curve was constructed for high-molecular-mass compounds from which the Stokes' radius of the sexual agglutinins could be estimated. The mt
+ agglutinin had a Stokes' radius of 39 nm and a sedimentation coefficient of 9.3 S. From these data its molecular mass was estimated to be 1.2·106. The corresponding data for the mt
- agglutinin were 38 nm, 9.7 S and 1.3·106, respectively. The biological activity of both agglutinins was destroyed by mild periodate treatment. Treatment with specific glycosidases had a differential effect on the biological activity of the agglutinins. These observations indicate that carbohydrate side-chains are needed for biological activity and perhaps are responsible for the specifity of the sexual agglutinins. A comparison of both agglutinins is given and their possible structure is discussed in relation to their amino-acid and sugar compositions.Abbreviations HP
high performance
-
mt
mating type
- SDS
sodium dodecyl sulfate 相似文献
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A. M. Tomson R. Demets E. A. van Spronsen G. J. Brakenhoff D. Stegwee H. van den Ende 《Protoplasma》1990,155(1-3):200-209
Summary During gamete-gamete adhesion in the unicellular green algaChlamydomonas eugametos, the sexual adhesion molecules or agglutinins that are located on the flagella are subject to tip-oriented migration and rapid inactivation. It is demonstrated that sexual adhesiveness is maintained by incorporation of additional agglutinins, recruited from a cellular pool. The location of this reservoir is unknown but, as indicated by its insensitivity to the chaotropic agent guanidine thiocyanate, it appears to be distinct from the large amount of agglutinins on the plasma membrane of the cell body. By viewing flagella of conjugating gametes in a confocal scanning laser microscope after immuno-labelling of the agglutinins, evidence was obtained for a linear arrangement of the agglutinins in two rows on the flagellar surface. This suggests that after insertion at the base of the flagellum, the agglutinins follow linear tracks to the tip and that the transport system is confined to two longitudinal domains. It is estimated that the half-life of flagellar agglutinins drops from 1–2 h in nonconjugating gametes to 1 min during conjugation, which suggests that after incorporation at the flagellar base, the agglutinins migrate to the tip with a velocity of 100 nm/s. Presumably after arrival at the tip, the molecules are inactivated. It is postulated that rapid turnover and transport of agglutinins are required for optimal signalling between partner gametes.Abbreviations BSA
bovine serum albumine
- CHI
cycloheximide
- CSLM
confocal scanning laser microscope
- GA
glutaraldehyde
- GTC
guanidine thiocyanate
- GAM-IgG
goat-anti-mouse immuno-globuline
- mAb
monoclonal antibody
- mt
mating type
- PBS
phosphate-buffered saline
- SDS
sodiumdodecyl sulphate
- TRIS
tris-(hydroxymethyl)-aminomethane 相似文献
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The effect of EDTA on the mating-type-specific agglutinins located on the flagellar surfaces of Chlamydomonas reinhardii gametes was investigated. The mating-type minus (mt-) gametes lost their agglutinability without apparent loss of motility soon after addition of EDTA at low concentrations (1-2 mM). At the same time, the cells released into the medium agglutinins which can elicit agglutinative responses of mating-type plus (mt+) gametes specifically. When EDTA was neutralized with Mg2+ or removed by centrifugation, the mt- cells quickly replaced agglutinins by protein synthesis: the recovery process was sensitive to cycloheximide, but not to tunicamycin. The EDTA-treated mt+ gametes lost their agglutinins much more slowly than the mt- gametes. The replacement of mt+ agglutinins was inhibited by both cycloheximide and tunicamycin. 相似文献
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Sexual agglutinins located on the flagellar membranes of Chlamydomonas gametes mediate a mating-type-specific adhesion reaction that brings complementary gametes together for zygotic cell fusion. We identify the mating-type plus agglutinin, using a combination of biochemical and genetic analysis, as a glycopolypeptide with an apparent molecular weight of >106 by SDS-polyacylamide gel electrophoresis. Its core polypeptide migrates as a ~480-kd species, and it is estimated to be present in ~30 copies per gametic flagellum. The agglutinin is present in the wild type, in a mutant that agglutinates but cannot fuse, and in a complementing diploid, whereas it is absent from four nonagglutinating mutants and from a noncomplementing diploid. Electron microscopy shows the purified agglutinin to be a highly asymmetric molecule, 220 × 4 nm. To our knowledge, this is the first reported purification and visualization of a membrane-associated cell-cell recognition protein. 相似文献
19.
Between-species analysis of short-repeat modules in cell wall and sex-related hydroxyproline-rich glycoproteins of Chlamydomonas
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Protein diversification is commonly driven by single amino acid changes at random positions followed by selection, but, in some cases, the structure of the gene itself favors the occurrence of particular kinds of mutations. Genes encoding hydroxyproline-rich glycoproteins (HRGPs) in green organisms, key protein constituents of the cell wall, carry short-repeat modules that are posited to specify proline hydroxylation and/or glycosylation events. We show here, in a comparison of two closely related Chlamydomonas species-Chlamydomonas reinhardtii (CC-621) and Chlamydomonas incerta (CC-1870/3871)-that these modules are prone to misalignment and hence to both insertion/deletion and endoduplication events, and that the dynamics of the rearrangements are constrained by purifying selection on the repeat patterns themselves, considered either as helical or as longitudinal face modules. We suggest that such dynamics may contribute to evolutionary diversification in cell wall architecture and physiology. Two of the HRGP genes analyzed (SAG1 and SAD1) encode the mating-type plus and minus sexual agglutinins, displayed only by gametes, and we document that these have undergone far more extensive divergence than two HRGP genes (GP1 and VSP3) that encode cell wall components-an example of the rapid evolution that characterizes sex-related proteins in numerous lineages. Strikingly, the central regions of the agglutinins of both mating types have diverged completely, by selective endoduplication of repeated motifs, since the two species last shared a common ancestor, suggesting that these events may have participated in the speciation process. 相似文献
20.
Marinus Versluis Frans Schuring Frans M. Klis Piet van Egmond Herman van den Ende 《FEMS microbiology letters》1992,97(1-2):101-105
Abstract The sexual mating reaction between gametes of the green alga Chlamydomonas eugametos starts by cell-cell contacts involving sex-specific cell-adhesion molecules (agglutinins) at the flagellar membrane. An in vitro adhesion assay is described using glutaraldehyde-fixed gametes. In vitro adhesion was fully comparable to in vivo adhesion, making it a reliable assay to study the initial recognition step of sexual adhesion in vivo. It was shown that both agglutinins are capable of inhibiting sexual adhesion at similar concentrations (1−2×10−10 M), indicating that mt+ and mt− agglutinins interact with each other during binding. This was confirmed by demonstrating that charcoal particles adsorbed with purified agglutinins of the opposite mating type aggregate with each other. 相似文献