包囊游仆虫休眠包囊的超微结构研究

包囊游仆虫休眠包囊中,各类纤毛器的纤毛基体上方的大部分纤毛杆退化,或仅保留毛基体,有时部分额腹棘毛的毛基体也瓦解消失。残留纤毛的纤毛杆周围微管和中央微管仍具有“9 2”结构特征,也有少数纤毛杆出现2套“9 2”微管共处于一层纤毛膜内的现象。毛基体中周围三联体微管的中央形成微管形结构聚合体,基体附属结构仅存在基体间连接及纤毛器托架的残余物;非纤毛区皮层表膜下未见微管层。纤毛区皮层含纤毛器腔周围微管层(相当于表膜下微管层)、纤毛器深部及附近的微管束和分散的微管群。并且,纤毛区皮层囊泡内含有呈不同形态的纤毛杆结构;大核核孔明显变大,核孔数目减少,核孔内膜附着染色质。     

Electron microscopy of negatively-stained lipids

Conclusions The present studies appear to confirm previous suggestions (Baker 1958) that some of the fixatives used for the study of membranes may not give an adequate preservation of the lipid components unless a sufficient concentration of calcium is present.These experiments are being continued, and we plan to investigate any changes that may occur during subsequent dehydration and embedding processes. It is hoped to preserve the helices so that they may be studied by thin-sectioning techniques, since a procedure that is satisfactory for these helices may also be of value in the examination of thin sections of biological membranes. Furthermore, as the helical structures appear to contain globular micelles of phospholipid, 40 to 50 Å in diameter (Lucy and Glauert 1964), continued investigation of these structures may also shed light on the formation, stability and function of globular micelles of lipid within natural membranes (Lucy 1964).     

Evidence for the role of the glomerulocyte in cellulose synthesis in the tunicate,Metandrocarpa uedai

Summary The tunicate,Metandrocarpa uedai, contains a large quantity of cellulose; however, it is not known how and where the cellulose is synthesized. Based on evidence from electron diffraction and conventional thin-sectioning for electron microscopy, this study shows that the glomerulocyte is involved in the synthesis of cellulose. The bundles of microfibrils in the glomerulocyte as well as the tunic were identified as cellulose I using selected area electron diffraction analysis. The diffraction pattern of cellulose in the glomerulocyte was similar to that from the tunic, suggesting that the crystallization of cellulose already is initiated in the glomerulocyte. The diameter of cellulose microfibrils, both in the glomerulocyte and the tunic was the same, about 16 nm. These results suggest that the glomerulocyte is the most probable site for the synthesis of cellulose in the tunic ofM. uedai. Using thin-sectioning techniques, a series of observations showed that individual microfibrils are primarily assembled in structures tentatively identified as vacuole-like structures, then they are bundled by a tapering region within the vacuole-like structures. These bundles of microfibrils are deposited in a continuously circular arrangement. The microtubules are oriented parallel to the bundles of microfibrils at the tapering vacuole-like structure, and they may be involved in the tapering of these structures (perhaps controlling the shape). This study also provides the first account for the involvement of a vacuole-like structure in the synthesis of cellulose microfibrils among living organisms.     

Cloning and analysis of 16 Rab genes from macronuclear DNA of Euplotes octocarinatus.

Rab proteins belong to the largest family of the Ras superfamily of small GTPase that play an important role in intracellular vesicular traffic. So far, almost 60 members of Rab family have been identified in mammalian cells. To further study the diversity and function of Rab protein in evolution, unicellular protozoa ciliates, Euplotes octocarinatus, were used in this study, Rab genes were screened by PCR method from macronuclear DNA of E. octocarinatus. Sixteen Rab genes were obtained. They share 87.6-99.5% identities. Highly conserved GTP-binding domains were found. There are some hot regions that diverse sharply in these genes as well.     

用非离子去垢剂抽提获得的小游仆虫皮层细胞骨架的构形

由扫描电镜术显示,应用非离子去垢剂抽提获得的小游仆虫(Euplotes grocilis)皮层细胞骨架是由非纤毛区皮层骨架、纤毛器骨架及其附属纤维等构成的三维结构网架。各类细胞骨架以纤维物质为基本成分组成纤维网、纤维层、纤维束和纤维薄片等不同形态单元。其中：非纤毛区皮层骨架以表面纤维网和表膜下纤维层为形态单元位于细胞的外周层;纤毛器骨架中的口围带骨架、口侧膜骨架、额腹横棘毛骨架按各自的分布图式在皮层内定位,成为主要的皮层骨架结构。尽管这些纤毛器骨架显示不同的形态,但却具有相同的建构特征,即都是由纤毛器的毛基体、纤毛器托架和骨架附属纤维相互联系镶嵌在一起形成的相对独立的结构单元。分析推测,游仆虫皮层表面纤维网使细胞表面形成区域化结构,它也可能与细胞表面各部分的联系及其细胞与环境的相互作用有关;纤毛器骨架中各个纤毛器的毛基体复合结构可能对纤毛器托架和骨架附属纤维等起到微管组织中心的作用。     

Electron Microscopy of the Longitudinal Fibrillar Bundle and the Contractile Fibrillar System in Spirostomum ambiguum

SYNOPSIS. A study of the 'longitudinal fibrillar bundle' (LFB) and the 'contractile fibrillar system' (CFS) of a large protozoan ciliate, Spirostomum ambiguum , has been performed by means of an electron microscope. A system of sub-pellicular fibrils has been newly found and its function is discussed. Each LFB runs parallel with a longitudinal row of ciliary bases. It seems to be identical with the so-called kinetodesma. It is composed of tubular fibrils arranged in layered sheets, each of which contains 13 to 35 fibrils with the same diameter as the intra-ciliary fibrils and has a close connection to each of the ciliary bases. The CFS lies on a transitional plane between ectoplasm and endoplasm of the organism and forms a cobweb-like system of myofibrils as a whole. It stands in an intimate relationship with a characteristic vacuolar system. In a peristomial field, the fibrous structures are interrupted and somewhat thickened. A sub-pellicular system is composed of minute fibrils 20 to 26 mμ in diameter. The fibrils run parallel with each other in an antero-posterior direction, immediately beneath the inner pellicular membrane.     

Subunit structure of junctional feet in triads of skeletal muscle: a freeze-drying, rotary-shadowing study

Isolated heavy sarcoplasmic reticulum vesicles retain junctional specializations (feet) on their outer surface. We have obtained en face three-dimensional views of the feet by shadowing and replicating the surfaces of freeze-dried isolated vesicles. Feet are clearly visible as large structures located on raised platforms. New details of foot structure include a four subunit structure and the fact that adjacent feet do not abut directly corner to corner but are offset by half a subunit. Feet aligned within rows were observed to be rotated at a slight angle off the long axis of the row creating a center-to-center spacing (32.5 nm) slightly less than the average diagonal of the feet (35.3 nm). Comparison with previous information from thin sections and freeze-fracture showed that this approach to the study of membranes faithfully preserves structure and allows better visualization of surface details than either thin-sectioning or negative-staining.     

Host-parasite interactions and trypanosome morphogenesis: a flagellar pocketful of goodies

Trypanosomes are characterised by the possession of a single flagellum and a subpellicular microtubule cytoskeleton. The flagellum is more than an organelle for motility; its position and polarity along with the sub-pellicular cytoskeleton enables the morphogenesis of a distinct flagellar pocket and the flagellar basal body is responsible for positioning and segregating the kinetoplast--the mitochondrial genome. Recent work has highlighted the molecules and morphogenesis of these cytoskeletal/flagellum structures and how dynamic events, occurring in the flagellar pocket and kinetoplast, are critical for host-parasite interactions.     

Comparing two methods for aging trees with suppressed,diffuse-porous rings (Betula pubescens ssp. czerepanovii)

The annual growth rings of diffuse porous species such as mountain birch are often difficult to distinguish when samples are collected from trees that grow at treeline or in other harsh environments. In this study we document the differences in seedling and sapling ring counts obtained from two methods of analysis: a traditional analysis based on reflected light and low-power microscopy and one based on transmitted light with higher power magnification that uses thin-sections of the samples. Rings are easier to resolve using the more labor-intensive transmitted light method. Small rings are often missed when using the reflected light method, resulting in an underestimation of tree age. The dates estimated by the standard method agreed with those determined using the thin-sectioning method in 9.6% of the cases. Most commonly, the standard method gave a younger age than did thin-sectioning (72.4% of the trees). In only 18.03% of the cases did the standard method result in a greater age than did thin-sectioning. The reflected light method produced age estimations that were on average 1.37 years younger than those determined using the transmitted light method. The difference between the two methods was positively correlated with age and negatively correlated with mean ring-width. Age-class histograms based on the two methods show little difference at coarser aggregation levels (decades and pentads), but annualized age-class histograms have less agreement between the two methods. Therefore, we suggest using the more labor-intensive thin-sectioning method when annualized age counts are necessary in suppressed seedlings and saplings, for example, comparing tree establishment with annual climate conditions at treeline.     

显示纤毛虫细胞微管骨架的两种荧光标记方法

应用直接荧光标记和免疫荧光标记显微术显示了几种原生动物纤毛虫(如尾草履虫、大尾柱虫、阔口游仆虫)的细胞微管骨架,并据结果提出了用所述方法制备标本时需注意的几个方面:对游仆虫,可以忽略某些步骤;对大尾柱虫各种药品的浓度以及处理时间以较小为宜。     

Sub-Pellicular Microtubules in Crithidia fasciculata

SYNOPSIS. Crithidia fasciculata sectioned transversally and studied with the electron microscope had gaps among an otherwise uniform row of sub-pellicular microtubules. Cultures were submitted to various treatments known to affect the structure of microtubules in other cell types. Only those very drastic treatments that killed the cells affected the sub-pellicular microtubules. Others, altho affecting the structure of the cell, left the sub-pellicular gaps and microtubules unchanged.     

Isolation and characterization of chromatin replication bands and macronuclei from Euplotes eurystomus

A method is described for isolating replication bands (RBs) from Euplotes eurystomus in quantities sufficient for biochemical analysis. The method involves the disruption of whole cells in a low ionic strength buffer that maintains RB integrity while dispersing macronuclear chromatin. The RBs are then stabilized with MgCl2 and spermidine phosphate and isolated by gradient centrifugation. Staining with silver nitrate and thiol-specific coumarin maleimide has been demonstrated in the RBs of Euplotes and other hypotrichs; both of these properties were maintained in isolated RBs. A method is also described in this study for isolating highly purified macronuclei. Examination of isolated macronuclei and RBs with electron microscopy (EM) indicates that the morphology of both structures remain essentially intact during purification. We also observe with EM an increase in the number of replicating molecules in RBs compared to macronuclei. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrates a consistent but minor enrichment of a 55 kilodalton protein in RBs when compared to macronuclear proteins.     

Euplotes crassus has genes encoding telomere-binding proteins and telomere-binding protein homologs.

We have identified two 1.6 kb macronuclear DNA molecules from Euplotes crassus that hybridize to the alpha subunit of the Oxytricha telomere protein. We have shown that one of these molecules encodes the 51 kDa Euplotes telomere protein while the other appears to encode a homolog of the telomere protein. Although this homolog clearly differs in sequence from the Euplotes telomere protein, the two proteins share extensive amino acid sequence identity with each other and with the alpha subunit of the Oxytricha telomere protein. In all three proteins 35-36% of the amino acids are identical, while 54-56% are similar. The most extended regions of sequence conservation map within the N-terminal section; this section has been shown to comprise the DNA-binding domain in the Euplotes telomere protein. Our findings suggest that some of the conserved amino acids may be involved in DNA recognition and binding. The gene encoding the telomere protein homolog contains two introns; one of these introns is only 24 bp in length. This is the smallest mRNA intron reported to date.     

原生动物八肋游仆虫cDNA文库的构建

细胞内蛋白质合成过程是一个由多种蛋白质相互作用参与调节的开放系统,形成了复杂的mRNA代谢和蛋白质翻译为核心的基因表达调控的网络和信号转导途径。【目的】为了获得更多参与调节蛋白质合成终止过程的蛋白质种类和功能信息,进一步了解其中的网络和信号转导途径,本研究构建了原生动物八肋游仆虫的cDNA文库。【方法】构建过程严格遵循Clontech公司的BD MatchmakerTM Library ConstructionScreening kit提供的方案进行文库构建和筛选.【结果】首次得到了可用于筛选功能基因的原生动物纤毛虫的cDNA文库,文库滴度为2.437×107cfu/mL。利用第二类肽链释放因子为诱饵,筛选得到了一些可能与之相互作用的蛋白质,其中包括一个可能编码RNA解旋酶的基因序列。该文库为进一步筛选和研究八肋游仆虫功能基因提供了便利的平台。     

Cold-adapted signal proteins: NMR structures of pheromones from the Antarctic ciliate Euplotes nobilii

Cell type-specific signal proteins, known as pheromones, are synthesized by ciliated protozoa in association with their self/nonself mating-type systems, and are utilized to control the vegetative growth and mating stages of their life cycle. In species of the most ubiquitous ciliate, Euplotes, these pheromones form families of structurally homologous molecules, which are constitutively secreted into the extracellular environment, from where they can be isolated in sufficient amounts for chemical characterization. This paper describes the NMR structures of En-1 and En-2, which are members of the cold-adapted pheromone family produced by Euplotes nobilii, a species inhabiting the freezing coastal waters of Antarctica. The structures were determined with the proteins from the natural source, using homonuclear (1)H NMR techniques in combination with automated NOESY peak picking and NOE assignment. En-1 and En-2 have highly homologous global folds, which consist of a central three-alpha-helix bundle with an up-down-up topology and a 3(10)-helical turn near the N-terminus. This fold is stabilized by four disulfide bonds and the helices are connected by bulging loops. Apparent structural specificity resides in the variable C-terminal regions of the pheromones. The NMR structures of En-1 and En-2 provide novel insights into the cold-adaptive modifications that distinguish the E. nobilii pheromone family from the closely related E. raikovi pheromone family isolated from temperate waters.     

Aplysia attractin: biophysical characterization and modeling of a water-borne pheromone.

Attractin, a 58-residue protein secreted by the mollusk Aplysia californica, stimulates sexually mature animals to approach egg cordons. Attractin from five different Aplysia species are approximately 40% identical in sequence. Recombinant attractin, expressed in insect cells and purified by reverse-phase high-performance liquid chromatography (RP-HPLC), is active in a bioassay using A. brasiliana; its circular dichroism (CD) spectrum indicates a predominantly alpha-helical structure. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) characterization of proteolytic fragments identified disulfide bonds between the six conserved cysteines (I-VI, II-V, III-IV, where the Roman numeral indicates the order of occurrence in the primary sequence). Attractin has no significant similarity to any other sequence in the database. The protozoan Euplotes pheromones were selected by fold recognition as possible templates. These diverse proteins have three alpha-helices, with six cysteine residues disulfide-bonded in a different pattern from attractin. Model structures with good stereochemical parameters were prepared using the EXDIS/DIAMOD/FANTOM program suite and constraints based on sequence alignments with the Euplotes templates and the attractin disulfide bonds. A potential receptor-binding site is suggested based on these data. Future structural characterization of attractin will be needed to confirm these models.     

艾美游仆虫大核基因组与转录组测序及结构特征

为获得艾美游仆虫(Euplotes amieti)大核基因组结构特征、分析基因功能及其表达调控方式,研究采用高通量测序技术对艾美游仆虫进行了大核基因组与转录组测序,结果显示基因组测序最终得到原始reads数据为10.92 Gb,过滤后得到50287条Contigs。GC含量较低,为31%;其中两端同时具有端粒的微染色体数量为27542条,占54.76%,只含有一端端粒的基因数量为6118条。Contigs进行基因结构分析, 96.5%的基因能够被预测出功能,最终得到27650条基因,平均外显子长度为311.69 bp;内含子较短,平均长度为150 bp。转录组测序结果为76219898条,拼接后获得38588条转录组Unigenes,其平均长度为1189 bp。将转录组的38588条Unigenes比对发现有2%—3%基因发生了编程性移码,其中,绝大多数为+1PRF;除此之外,艾美游仆虫的终止密码子还存在重配现象,其终止密码子为UAA和UAG,而UGA编码半胱氨酸或硒代半胱氨酸。这与游仆虫属的编程性移码及终止密码子重配的特点一致。将27650条基因组Contigs与38588条转录组U...     

Microtubules beneath the pellicles of two ciliate protozoa as seen with the SEM

Chemical procedures remove some of the outer 3 limiting membranes of 2 ciliate protozoa, Euplotes eurystomus and Tetrahymena pyriformis, and reveal sheets of microtubules in their ectoplasm for SEM study. This greatly enhances the analysis of the 3-dimensional geometry of these sheets, as is shown especially for E. eurystomus. In this organism, sheets of microtubules can readily be observed and described as they course through or around parts of th oral apparatus and other 3-dimensionally complex regions.     

滑动序列对游仆虫中识别+1位和+2位编程性核糖体移码具有关键作用

编程性核糖体移码(programmed ribosomal frameshifting,PRF)广泛存在于生命进化谱系的各个分支,是一种翻译水平上的基因表达调控方式。单细胞真核生物游仆虫(Euplotes)中不仅PRF基因比例高,而且移码类型有+1和+2位两种。本研究从基因组水平对八肋游仆虫(E.octocarinatus)中的+2 PRF基因进行了鉴定,比较分析+1及+2 PRF基因中可能的调控元件。为了探讨游仆虫中滑动序列对移码类型的影响,克隆了八肋游仆虫的+1 PRF基因——η微管蛋白基因,将其构建到含绿色荧光蛋白报告基因的游仆虫大核人工染色体中,转染游仆虫细胞,通过检测GFP的表达来确定不同滑动序列突变体对应的移码类型。结果表明,滑动序列的改变能使游仆虫+1 PRF转变为+2 PRF,且这种移码类型的改变与滑动序列第1个密码子编码何种氨基酸无关。本研究揭示了滑动序列对游仆虫中识别+1和+2位的编程性核糖体移码具有关键作用。