Structure of the Large Subunit rDNA from a Diatom, and Comparison Between Small and Large Subunit Ribosomal RNA for Studying Stramenopile Evolution

The aim of this study was to compare the usefulness of complete small and large subunit rRNA, and a combination of both molecules, for reconstructing stramenopile evolution. To this end, phylogenies from species of which both sequences are known Acre constructed with the neighbor-joining, maximum parsimony, and maximum likelihood methods. Also the use of structural features of the rRNAs was evaluated. The large subunit rRNA from the diatom Skeletonema pseudocostatum was sequenced in order to have a more complete taxon sampling, and a group I intron was identified. Our results indicated that heterokont algae are monophyletic, with diatoms diverging first. However, as the analysis was restricted to a particular data set containing merely six taxa, the outcome has limited value for elucidating stramenopile relationships. On the other hand, this approach permits comparison of the performance of both rRNA molecules without interference from other factors, such as a different species selection for each molecule. For the taxa used, the large subunit rRNA clearly contained more phylogenetic information than the small subunit rRNA. Although this result can definitely not be generalized and depends on the phvlogeny to be studied, in some cases determining complete large subunit rRNA sequences certainly seems worthwhile.     

Strategies for Amplification by Polymerase Chain Reaction of the Complete Sequence of the Gene Encoding Nuclear Large Subunit Ribosomal RNA in Corals

The nearly complete nuclear large subunit ribosomal RNA (LSU rRNA) gene in corals was amplified by primers designed from polymerase chain reaction (PCR) strategies. The motif of the putative 3′-terminus of the LSU rRNA gene was sequenced and identified from intergenic spacer (IGS) clones obtained by PCR using universal primers designed for corals. The 3′-end primer was constructed in tandem with the universal 5′-end primer for the LSU rRNA gene. PCR fragments of 3500 bp were amplified for octocorals and non-Acropora scleractinian corals. More than 80% of the Acropora LSU rRNA gene (3000 bp) was successfully amplified by modification of the 5′-end of the IGS primer. Analysis of the 5′-end of LSU rDNA sequences, including the D1 and D2 divergent domains, indicates that the evolutionary rate of the LSU rDNA differs among these taxonomic groups of corals. The genus Acropora showed the highest divergence pattern in the LSU rRNA gene, and the presence of a long branch of the Acropora clade from the other scleractinian corals in the phylogenetic tree indicates that the evolutionary rate of Acropora LSU rDNA might have accelerated after divergence from the common ancestor of scleractinian corals. Received February 17, 2000; accepted June 12, 2000.     

Methods for Cryo-EM Single Particle Reconstruction of Macromolecules Having Continuous Heterogeneity

Macromolecules change their shape (conformation) in the process of carrying out their functions. The imaging by cryo-electron microscopy of rapidly-frozen, individual copies of macromolecules (single particles) is a powerful and general approach to understanding the motions and energy landscapes of macromolecules. Widely-used computational methods already allow the recovery of a few distinct conformations from heterogeneous single-particle samples, but the treatment of complex forms of heterogeneity such as the continuum of possible transitory states and flexible regions remains largely an open problem. In recent years there has been a surge of new approaches for treating the more general problem of continuous heterogeneity. This paper surveys the current state of the art in this area.     

Cytoplasmic Solvent Structure of Single Barnacle Muscle Cells Studied by Electron Spin Resonance

A free radical probe was introduced into single barnacle muscle cells, and its freedom of motion inferred from the spin resonance spectra. The probe reported an average local viscosity of 5-10 cp compared with 1 cp for pure water. From a comparison of the temperature dependence of the probe's tumbling rate in model aqueous systems and in the muscle we concluded that in the muscle the probe was undergoing fast exchange between sites of different mobility. Thus 10 cp must be taken as an upper limit for the viscosity of most cell water.     

A New Version of the Insertion Particle Method for Determining the Chemical Potential by Monte Carlo Simulation

A new version of the test particle method for determining the chemical potential by Monte Carlo simulations is proposed. The method, applicable to any fluid at any density, combines the Widom's test particle insertion method with the ideas of the scaled particle theory, gradual insertion method and multistage sampling. Its applicability is exemplified by evaluating the chemical potential of the hard sphere fluid at a very high density in semi-grand-canonical and grand-canonical ensembles. A theory estimating the efficiency (i.e. statistical errors) of the method is proposed and the results are compared with the Widom's and gradual insertion methods, and the analytic results.     

The First Three-Dimensional Structure of Phosphofructokinase from Saccharomyces cerevisiae Determined by Electron Microscopy of Single Particles

Phosphofructokinaseis a key regulatory enzyme of the glycolytic pathway. We have determined the structure of this enzyme from Saccharomyces cerevisiae to a resolution of 2.0 nm. This is the first structure available for this family of enzymes in eukaryotic organisms. Phosphofructokinase is an octamer composed of 4α and 4β subunits arranged in a dihedral point group symmetry D₂. The enzyme has a very open and elongated structure, with dimensions of 24 nm in length and 17 nm in width. The final structure, calculated from 0° tilt projections of the molecule at random orientations using as reference the volume obtained by the random conical reconstruction technique in ice, has allowed us to discern the shapes of the subunits and their mutual arrangement in the octamer.     

Nuclear Pore Complex Number and Distribution throughout the Saccharomyces cerevisiae Cell Cycle by Three-Dimensional Reconstruction from Electron Micrographs of Nuclear Envelopes

The number of nuclear pore complexes (NPCs) in individual nuclei of the yeast Saccharomyces cerevisiae was determined by computer-aided reconstruction of entire nuclei from electron micrographs of serially sectioned cells. Nuclei of 32 haploid cells at various points in the cell cycle were modeled and found to contain between 65 and 182 NPCs. Morphological markers, such as cell shape and nuclear shape, were used to determine the cell cycle stage of the cell being examined. NPC number was correlated with cell cycle stage to reveal that the number of NPCs increases steadily, beginning in G₁-phase, suggesting that NPC assembly occurs continuously throughout the cell cycle. However, the accumulation of nuclear envelope observed during the cell cycle, indicated by nuclear surface area, is not continuous at the same rate, such that the density of NPCs per unit area of nuclear envelope peaks in apparent S-phase cells. Analysis of the nuclear envelope reconstructions also revealed no preferred NPC-to-NPC distance. However, NPCs were found in large clusters over regions of the nuclear envelope. Interestingly, clusters of NPCs were most pronounced in early mitotic nuclei and were found to be associated with the spindle pole bodies, but the functional significance of this association is unknown.     

测定λ原噬菌体诱导频率的新方法

本文报道两种测定λ原噬菌体紫外诱导频率的新方法 - 菌落计数法和平板诱导法.将溶源菌液经紫外诱导暗培养稀释后直接涂布在平板上培养,根据平板上菌落形成单位数计算λ噬菌体紫外诱导频率.另一种是将溶源菌与指示菌混合制备的平板用紫外线诱导,根据平板上噬菌体形成单位确定λ噬菌体紫外诱导频率.这两种方法不仅能准确测定噬菌体紫外诱导频率,而且操作简便,节省时间和用具,重复性好.本研究还将冬虫夏草浸出汁与溶源菌混合后进行紫外辐射,通过几种方法进行比较,结果证明建立的新方法确实可行,易操作;同时也表明冬虫夏草具有较强的抗紫外辐射作用.     

New Methods for Detecting Positive Selection at Single Amino Acid Sites

Inferring positive selection at single amino acid sites is of particular importance for studying evolutionary mechanisms of a protein. For this purpose, Suzuki and Gojobori (1999) developed a method (SG method) for comparing the rates of synonymous and nonsynonymous substitutions at each codon site in a protein-coding nucleotide sequence, using ancestral codons at interior nodes of the phylogenetic tree as inferred by the maximum parsimony method. In the SG method, however, selective neutrality of nucleotide substitutions cannot be tested at codon sites, where only termination codons are inferred at any interior node or the number of equally parsimonious inferences of ancestral codons at all interior nodes exceeds 10,000. Here I present a modified SG method which is free from these problems. Specifically, I use the distance-based Bayesian method for inferring the single most likely ancestral codon from 61 sense codons at each interior node. In the computer simulation and real data analysis, the modified SG method showed a higher overall efficiency of detecting positive selection than the original SG method, particularly at highly polymorphic codon sites. These results indicate that the modified SG method is useful for inferring positive selection at codon sites where neutrality cannot be tested by the original SG method. I also discuss that the p-distance is preferable to the number of synonymous substitutions for inferring the phylogenetic tree in the SG method, and present a maximum likelihood method for detecting positive selection at single amino acid sites, which produced reasonable results in the real data analysis.     

Exercise-Induced Neuroprotection in SMA Model Mice: A Means for Determining New Therapeutic Strategies

Due to the prevalence of neuromuscular disorders such as amyotrophic lateral sclerosis and spinal muscular atrophy in modern societies, defining new and efficient strategies for the treatment of these two neurodegenerative diseases has become a vital and still unfulfilled urge. Several lines of experimental evidence have emphasized the benefits of regular exercise training in mouse models for these affections in terms of life span increase and improvement of both motor capacities and motoneuron survival. Identifying molecules that could mimic the neuroprotective effects of exercise represents a promising way to find novel therapies. Some of the effects of exercise are caused by the overproduction of circulating neurotrophic factors, such as IGF-I, whereas others may be due to modifications of the intrinsic properties of the motoneurons within the spinal cord. The causal relationship that links these potential effects of exercise training and the improvement of motor capacity and life span expectancy is consequently discussed.     

New Evidence for Differential Roles of L10 Ribosomal Proteins from Arabidopsis

The RIBOSOMAL PROTEIN L10 (RPL10) is an integral component of the eukaryotic ribosome large subunit. Besides being a constituent of ribosomes and participating in protein translation, additional extraribosomal functions in the nucleus have been described for RPL10 in different organisms. Previously, we demonstrated that Arabidopsis (Arabidopsis thaliana) RPL10 genes are involved in development and translation under ultraviolet B (UV-B) stress. In this work, transgenic plants expressing Pro_RPL10:β-glucuronidase fusions show that, while AtRPL10A and AtRPL10B are expressed both in the female and male reproductive organs, AtRPL10C expression is restricted to pollen grains. Moreover, the characterization of double rpl10 mutants indicates that the three AtRPL10s differentially contribute to the total RPL10 activity in the male gametophyte. All three AtRPL10 proteins mainly accumulate in the cytosol but also in the nucleus, suggesting extraribosomal functions. After UV-B treatment, only AtRPL10B localization increases in the nuclei. We also here demonstrate that the three AtRPL10 genes can complement a yeast RPL10 mutant. Finally, the involvement of RPL10B and RPL10C in UV-B responses was analyzed by two-dimensional gels followed by mass spectrometry. Overall, our data provide new evidence about the nonredundant roles of RPL10 proteins in Arabidopsis.In eukaryotes, the cytosolic ribosomes consist of large 60S and small 40S subunits. In Arabidopsis (Arabidopsis thaliana), ribosomal protein genes exist as families composed of two to seven members that could be differentially incorporated into the cytosolic ribosome under specific situations (Schmid et al., 2005; Byrne, 2009). In this way, ribosomal heterogeneity would allow selective translation of specific mRNAs under particular cell conditions (Barakat et al., 2001; Szick-Miranda and Bailey-Serres, 2001; Giavalisco et al., 2005; Carroll et al., 2008; Carroll, 2013). Arabidopsis mutants in ribosomal proteins exhibit a large range of developmental phenotypes with extreme abnormalities, including embryonic lethality, suggesting that ribosomes also have specific functions regulating the expression of developmental genes (Van Lijsebettens et al., 1994; Degenhardt and Bonham-Smith, 2008; Byrne, 2009; Horiguchi et al., 2011, 2012; Szakonyi and Byrne, 2011). Furthermore, it has been recently demonstrated that ribosomal proteins control auxin-mediated developmental programs by translational regulation of auxin response factors (Rosado et al., 2012). In addition, the characterization of single, double, and, in certain cases, triple mutants as well as complementation by paralog genes have demonstrated full, partial, and no redundancy between members of ribosomal protein families (Briggs et al., 2006; Guo and Chen, 2008; Guo et al., 2011; Horiguchi et al., 2011; Stirnberg et al., 2012).RIBOSOMAL PROTEIN L10 (RPL10) was initially identified in humans as a putative suppressor of Wilms’ tumor (Dowdy et al., 1991). Since then, RPL10 has been studied in different organisms from archaea and bacteria to eukaryotes such as mammals, insects, yeast, and plants (Marty et al., 1993; Mills et al., 1999; Hwang et al., 2000; Zhang et al., 2004; Wen et al., 2005; Singh et al., 2009). A remarkable property of this protein is its high degree of amino acid conservation, suggesting fundamental and critical conserved functions of RPL10 in different organisms (Farmer et al., 1994; Eisinger et al., 1997; Hofer et al., 2007; Nishimura et al., 2008). Likewise, the crystallographic structural similarity observed among RPL10 orthologs in eukaryotes, bacteria, and archaea (called L16) established the conservation of this universal ribosomal protein family and provided evidence of the inalterability of the ribosome during evolution (Spahn et al., 2001; Nishimura et al., 2008). Nevertheless, besides being a constituent of ribosomes and participating in protein translation, additional extraribosomal functions have been described for RPL10 (Mills et al., 1999; Hwang et al., 2000; Chávez-Rios et al., 2003; Zhang et al., 2004; Singh et al., 2009). In yeast, RPL10 is essential for viability, organizes the union site of the aminoacyl-tRNA, and its incorporation into the 60S subunit is a prerequisite for subunit joining and the initiation of translation (West et al., 2005; Hofer et al., 2007). Extensive analysis of the in vivo assembly of ribosomes revealed that RPL10 is loaded to the ribosome in the cytosol with the assistance of its chaperone suppressor of QSR1 truncations (Hedges et al., 2005; West et al., 2005).Arabidopsis has three genes encoding RPL10 proteins, AtRPL10A, AtRPL10B, and, AtRPL10C. Recently, we demonstrated that Arabidopsis RPL10 genes are differentially regulated by UV-B radiation: RPL10B is down-regulated, RPL10C is up-regulated, while RPL10A is not UV-B regulated. Arabidopsis single mutants showed that RPL10 genes are not functionally equivalent. Heterozygous rpl10a mutant plants are translation deficient under UV-B conditions, knockout rpl10A mutants are not viable, and knockdown homozygous rpl10B mutants show abnormal growth. Conversely, knockout homozygous rpl10C mutants do not exhibit any visible phenotype. Overall, RPL10 genes are involved in development and translation under UV-B stress (Falcone Ferreyra et al., 2010b). Furthermore, coimmunoprecipitation studies showed an association of RPL10 with nuclear proteins, suggesting that at least one of the RPL10 isoforms could have an extraribosomal function in the nucleus (Falcone Ferreyra et al., 2010a).The aim of this work was to further investigate the contribution of each Arabidopsis RPL10 to plant development and UV-B responses. We examined the spatiotemporal expression of each AtRPL10 using transgenic plants expressing Pro_RPL10:GUS fusions. By AtRPL10-GFP fusions, we analyzed the subcellular localization of each RPL10, demonstrating that the three isoforms are mainly localized in the cytosol but also in the nucleus. In order to investigate the functional redundancy between AtRPL10 genes in more detail, we generated and characterized double rpl10 mutants. We also here demonstrate that the three AtRPL10 genes can complement a yeast RPL10 mutant. Finally, the involvement of RPL10B and RPL10C in UV-B responses was analyzed by two-dimensional (2D) gels followed by mass spectrometry. Overall, our data provide new insights into the role of each RPL10 in Arabidopsis.     

Pollen Wall Structure Using a New Stripping-Sputtering Device for Scanning Electron Microscopy

A new method has been developed to elucidate pollen wall architecture by the separation of wall layers and the use of scanning electron microscopy (SEM). Separation of wall layers at natural boundaries, breakage across the wall and metal-coating of the specimen have been achieved by controlled ramming of free scattered ions produced by a novel “ion separating and coating – model A” instrument. The stripping treatment reveals interfaces and cross profiles of pollen walls and the sputtering treatment results in metal coating for examination with SEM. An advantage of the method is that it provides intact interfaces that are not eroded or damaged. The application of the method is exemplified by SEM analyses of pollen grains of Gossypium hirsutum L., Zea mays L., Sesamum indicum L. and Brassica napus L. var. oleifera. Interfaces between the tectum, column, foot, nexine-2 and intine layers of the pollen wall were all portrayed in G. hirsutum and to a great part in the other species. In G. hirsutum, it was possible to document the attachment point of surface spines, the appearance of individual baculae and the irregular labrum-operculum but regular inner labrum-aperture structure. No tectum was found in S. indicum. In all four species it was not possible to separate the intine from the sporoplast. The numbers of apertures were 20, 1, 10–14 and 3 in G. hirsutum, Z. mays, S. indicum and B. napus, respectively. The dumbell-shaped arrangement of apertures in G. hirsutum, the gear-shaped oblate sporoplast of S. indicum and the abundance of micropores on the intine of B. napus are characteristic features.     

Forest Walk Methods for Localizing Body Joints from Single Depth Image

We present multiple random forest methods for human pose estimation from single depth images that can operate in very high frame rate. We introduce four algorithms: random forest walk, greedy forest walk, random forest jumps, and greedy forest jumps. The proposed approaches can accurately infer the 3D positions of body joints without additional information such as temporal prior. A regression forest is trained to estimate the probability distribution to the direction or offset toward the particular joint, relative to the adjacent position. During pose estimation, the new position is chosen from a set of representative directions or offsets. The distribution for next position is found from traversing the regression tree from new position. The continual position sampling through 3D space will eventually produce an expectation of sample positions, which we estimate as the joint position. The experiments show that the accuracy is higher than current state-of-the-art pose estimation methods with additional advantage in computation time.     

Three-Dimensional Structure of Macronuclei from Paramecium Determined by Scanning Electron Microscopy*

SYNOPSIS. Macronuclei of Paramecium primaurelia were isolated and examined by scanning electron microscopy. These nuclei consisted of a closely packed array of chromatin bodies measuring ～ 0.2 μm in diameter. We estimated there were ～ 30,000 such bodies/macronucleus, 20 times more than the number of unit genome equivalents. This suggests that a unit genome is physically shared by several chromatin bodies.     

蓖麻蚕核糖体大亚基RNA基因3‘—端序列分析及进化研究

测定了蓖麻蚕核糖体大亚基ＲＮＡ编码区３’－端ＤＮＡ序列,分析了其二级结构,并与昆虫伊蚊、果蝇;线虫;脊椎动物人、小鼠、爪蟾;低等脊索动物海鞘以及真菌酵母、毛霉相应的保守区段进行了同源比较。邻接法分析表明,昆虫核糖体大亚基ＲＮＡ在进化上与５ＳｒＲＮＡ相似,有加快的趋势。