H5N2亚型禽流感病毒血凝素裂解位点碱性氨基酸对应mRNA核苷酸的二级结构分析

以H5N2亚型禽流感病毒毒株血凝素蛋白裂解位点碱性氨基酸为研究对象,对其密码子偏好性和对应mRNA序列的折叠二级结构特点进行研究和分析.旨在探讨裂解位点氨基酸对应mRNA核苷酸片段的二级结构与病毒致病力的关系,希望能对禽流感病毒的研究提供一些基础性信息.将mRNA样本按照序列等步长递增的方法,用RNAstructure 4.1程序预测这些样本的动态延伸折叠二级结构.序列和结构的分析结果:裂解位点的碱性氨基酸对富含腺嘌呤的密码子有强烈偏好;与碱性氨基酸对应的mRNA片段上的核苷酸主要位于折叠二级结构的单链环区,少数位于配对螺旋区.结果表明:裂解位点氨基酸对应的mRNA核苷酸形成发夹端环的大小与其碱性氨基酸的多少具有正相关性.

Performance of thin film carbon materials and carbon nanotubes as cold cathodes

The field emissions from three different types of carbon films are studied using a Kiethly voltage-current source-measure unit under computer control. The three types of carbon films are : 1) a-C:H:N deposited using an inductively coupled rf PECVD process, where the N content in the films can be as high as 30 at %; 2) cathodic arc deposited tetrahedral amorphous carbon with embedded regions of carbon nanotube and anion structures and 3) unoriented carbon nanotube films on a porous substrate. The films are formed by filtering a solution of nanotubes dispersed in alcohol through the pores and drying.

THE ORIGIN AND GENETIC DIFFERENTIATION OF NATIVE BREEDS OF PIGS IN SOUTHWEST CHINA - AN APPROACH FROM MITOCHONDRIAL-DNA POLYMORPHISM

Mitochondrial DNA (mtDNA) of six breeds of native domestic pigs from Yunnan province, southwest China, and two wild boars obtained from Sichuan, China, and Vietnam was analyzed using 20 restriction endonucleases that recognize six nucleotides. Restriction maps were made by double-digestion methods and polymorphic sites were located on the map. According to their mtDNA restriction types, all the breeds were classified into six groups. Genetic distances among groups were calculated to define their phylogenetic relationships. The relationship between the Sichuan wild boar and domestic pigs is close, while the Vietnamese wild boar is relatively far from them, so the domestic pigs in southwest China are likely to have originated from a wild pig which distributed in west China. We compare our results with previous reports in literature and discuss the relationship among Chinese pigs, Japanese pigs, and European pigs. The mtDNA cleavage pattern of the Mingguang pig digested by EcoRV was identical to that of Duroc; mutations at the EcoRI site, detected in the mtDNA of two Dahe pigs, are the same as in the Vietnamese wild boar, suggesting that mutational hot spots exist in the mtDNA of pigs.

Secondary structure of yeast genomic downstream region and polyadenylation signals

Polyadenylation of 3 ' -forming in eukaryote concerns three elements located in precursor mRNA downstream region: efficiency element (EE), position element (PE) and the actual site for cleavage and polyadenylation. Several base sequences of EE and PE have

Regulation of zygotic gene activation in the preimplantation mouse embryo: Global activation and repression of gene expression

Superimposed on the activation of the embryonic genome in the preimplantation mouse embryo is the formation of a transcriptionally repressive state during the two-cell stage. This repression appears mediated at the level of chromatin structure, because it is reversed by inducing histone hyperacetylation or inhibiting the second round of DNA replication. We report that of more than 200 amplicons analyzed by mRNA differential display, about 45% of them are repressed between the two-cell and four-cell stages. This repression is scored as either a decrease in amplicon expression that occurs between the two-cell and four-cell stages or on the ability of either trichostatin A tan inhibitor of histone deacetylases) or aphidicolin tan inhibitor of replicative DNA polymerases) to increase the level of amplicon expression. Results of this study also indicate that about 16% of the amplicons analyzed likely are novel genes whose sequence doesn't correspond to sequences in the current databases, whereas about 20% of the sequences expressed during this transition likely are repetitive sequences. Lastly, inducing histone hyperacetylation in the two-cell embryos inhibits cleavage to the four-cell stage. These results suggest that genome activation is global and relatively promiscuous and that a function of the transcriptionally repressive state is to dictate the appropriate profile of gene expression that is compatible with further development.

Measurement of the elastic constants of nanometric films

Carbon coatings of thickness down to 2 nanometers are needed to increase the storage density in magnetic hard disks and reach the 100 Gbit/in2 target. Methods to measure the properties of these ultrathin hard films still have to be developed. We show that combining Surface Brillouin Scattering (SBS) andX-ray reflectivity measurements the elastic constants of such films are accessible. Tetrahedral amorphous carbofilms of thickness down to about 2 nm were deposited on Si by an S bend filtered cathodic vacuum arc, achieving a continuous coverage on large areas free of macroparticles. Film thickness and mass density are measured by X-ray reflectivity: densities above 3 g/cm3 are found, indicating a significant sp3 content. The dispersion relations of surface acoustic waves are measured by SBS. We show that for thicknesses above ∼4 nm these waves can be described by a continuum elastic model based on a single homogeneous equivalent film. The elastic constants can then be obtained by fitting the dispersion relations, computed for given film properties, to the measured dispersion relations. For thicknesses of 3 nm or less qualitative differences among films are well measurable, but quantitative results are less reliable. We have thus shown that we can grow and characterise nanometer size tetrahedral amorphous carbon film, which maintain their high density and peculiar mechanical properties down to around 4 nm thickness, satisfying the requirements set for the hard disk coating material.

Rapid quantification of total microcystins in cyanobacterial samples by periodate-permanganate oxidation and reversed-phase liquid chromatography

Microcystins (MCs) comprise a family of more than 80 related cyclic hepatotoxic heptapeptides. Oxidation of MCs causes cleavage of the chemically unique C-20 beta-amino acid (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) amino to form 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB), which has been exploited to enable analysis of the entire family. In the present study, the reaction conditions (e.g. concentration of the reactants. temperature and pH) used in the production of MMPB by oxidation of cyanobacterial samples with permanganate-periodate were optimized through a series of well-controlled batch experiments. The oxidation product (MMPB) was then directly analyzed by high-performance liquid chromatography with diode array detection. The results of this study provided insight into the influence of reaction conditions on the yield of MMPB. Specifically, the optimal conditions, including a high dose of permanganate (>= 50 mM) in saturated periodate solution at ambient temperature under alkaline conditions (pH similar to 9) over 1-4 h were proposed, as indicated by a MMPB yield of greater than 85%. The technique developed here was applied to determine the total concentration of MCs in cyanobacterial bloom samples, and indicated that the MMPB technique was a highly sensitive and accurate method of quantifying total MCs. Additionally, these results will aid in development of a highly effective analytical method for detection of MMPB as an oxidation product for evaluation of total MCs in a wide range of environmental sample matrices, including natural waters, soils (sediments) and animal tissues. (C) 2009 Elsevier B.V. All rights reserved.

Histone H2A Has a Novel Variant in Fish Oocytes

Histone variants and their modification have significant roles in many cellular processes. In this study, we identified and characterized the histone H2A variant h2af1o in fish and revealed its oocyte-specific expression pattern during oogenesis and embryogenesis. Moreover, posttranslational modification of H2af1o was observed that results from phosphorylation during oocyte maturation. To understand the binding dynamics of the novel core histone variant H2af1o in nucleosomes, we cloned ubiquitous gibel carp h2afx as a conventional histone control and investigated the dynamic exchange difference in chromatin by fluorescence recovery after photobleaching. H2af1o has significantly higher mobility in nucleosomes than ubiquitous H2afx. Compared with ubiquitous H2afx, H2af1o has a tightly binding C-terminal and a weakly binding N-terminal. These data indicate that fish oocytes have a novel H2A variant that destabilizes nucleosomes by protruding its N-terminal tail and stabilizes core particles by contracting its C-terminal tail. Our findings suggest that H2af1o may have intrinsic ability to modify chromatin properties during fish oogenesis, oocyte maturation, and early cleavage.

Origin and evolution of the RIG-I like RNA helicase gene family

Background: The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria. Results: The DExD/H containing RNA helicases including RIG-I, MDA5 and LGP2 were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. The gene synteny of MDA5 and LGP2 is well conserved among vertebrates whilst conservation of the gene synteny of RIG-I is less apparent. Invertebrate homologues had a closer phylogenetic relationship with the vertebrate RIG-Is than the MDA5/LGP2 molecules, suggesting the RIG-I homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. Our data suggest that the RIG-I like helicases possibly originated from three distinct genes coding for the core domains including the HELICc, CARD and ATP dependent DExD/H domains through gene fusion and gene/domain duplication. Furthermore, presence of domains similar to a prokaryotic DNA restriction enzyme III domain (Res III), and a zinc finger domain of transcription factor (TF) IIS have been detected by bioinformatic analysis. Conclusion: The RIG-I/MDA5 viral surveillance system is conserved in vertebrates. The RIG-I like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. Diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral PAMPs.

Kinetic study of the 2-methyl-3-methoxy-4-phenylbutanoic acid produced by oxidation of microcystin in aqueous solutions

Microcystins (MCs) are a family of related cyclic hepatotoxic heptapeptides, of which more than 70 types have been identified. The chemically unique nature of the C20 beta-amino acid, (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca4,6-dienoic acid (Adda), portion of the MCs has been exploited to develop a strategy to analyze the entirety. Oxidation of MCs causes the cleavage of MC Adda to form 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB). In the present study, we investigated the kinetics of MMPB produced by oxidation of the most-often-studied MC variant, MC-LR (L = leucine, R = arginine), with permanganate-periodate. This investigation allowed insight regarding the influence of the reaction conditions (concentration of the reactants, temperature, and pH) on the conversion rate. The results indicated that the reaction was second order overall and first order with respect to both permanganate and MC-LR. The second-order rate constant ranged from 0.66 to 1.35 M/s at temperatures from 10 to 30 degrees C, and the activation energy was 24.44 kJ/mol. The rates of MMPB production can be accelerated through increasing reaction temperature and oxidant concentration, and sufficient periodate is necessary for the formation of MMPB. The initial reaction rate under alkaline and neutral conditions is higher than that under acidic conditions, but the former decreases faster than the latter except under weakly acidic conditions. These results provided new insight concerning selection of the permanganate-periodate concentration, pH, and temperature needed for the oxidation of MCs with a high and stable yield of MMPB.

Isolation, characterization and genome sequence of a birnavirus strain from flounder Paralichthys olivaceus in China

A birnavirus strain, Paralichthys olivaceus birnavirus (POBV), was isolated and characterized from cultured flounder in China, and its complete genomic sequence was subsequently determined. The virus could induce cytopathic effects (CPE) in four of seven fish cell lines and was resistant to chloroform, 5-iodo-2'-deoxyuridine, acid and alkaline pH, and heat treatment. Purified virus particles had a typical icosahedral shape, with a diameter of approximately 55-60 nm. The genomic segments A and B of POBV were 3,091 and 2,780 bp in length and shared many of the features of the members of the family Birnaviridae. Segment A contained two partially overlapping ORFs encoding a polyprotein, pVP2-VP4-VP3, and a nonstructural protein, VP5, while segment B had only one ORF encoding for the VP1, a viral RNA-dependent RNA polymerase (RdRp). This is the first report about a birnavirus strain from a new non-salmonid host in China and its complete genome sequence.

Early ontogeny of Ancherythroculter nigrocauda and effects of delayed first feeding on larvae

Development of embryos and larvae in Ancherythroculter nigrocauda Yih et Woo (1964) and effects of delayed first feeding on larvae were observed after artificial fertilization. The fertilized eggs were incubated at an average temperature of 26.5 degrees C (range: 25.7-27) and the larvae reared at temperatures ranging from 21.8 to 28 degrees C. First cleavage was at 50 min, epiboly began at 7 h 5 min, heartbeat reached 72 per min at 24 h 40 min and hatching occurred at 43 h 15 min after insemination. Mean total length of newly hatched larvae was 4.04 +/- 0.03 mm (n = 15). A one-chambered gas bladder was observed at 70 h 50 min, two chambers occurred at 15 days, and scales appeared approximately 30 days after hatching. Larvae began to feed exogenously at day 4 post-hatch at an average temperature of 24 degrees C. Food deprivation resulted in a progressive atrophy of skeletal muscle fibres, deterioration of the larval digestive system and cessation of organ differentiation. Larval growth under food deprivation was significantly affected by the time of first exogenous feeding. Starved larvae began to shrink, with negative growth from day 6 post-hatch. The point of no return (PNR) was reached at day 11 after hatching. Mortality of starved larvae increased sharply from day 12 after hatching.

The functional divergence of two glgP homologues in Synechocystis sp PCC 6803

Glycogen phosphorylase (GlgP, EC 2.4.1.1) catalyzes the cleavage of glycogen into glucose-1-phosphate (Glc-1-P), the first step in glycogen catabolism. Two glgP homologues are found in the genome of Synechocystis sp. PCC 6803, a unicellular cyanobacterium: sll1356 and slr1367. We report on the different functions of these glgP homologues. sll1356, rather than slr1367, is essential for growth at high temperatures. On the other hand, when CO2-fixation and the supply of glucose are both limited, slr1367 is the key factor in glycogen metabolism. In cells growing autotrophically, sll1356 plays a more important role in glycogen digestion than slr1367. This functional divergence is also supported by a phylogenetic analysis of glgP homologues in cyanobacteria.

Two cathelicidin genes are present in both rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

Further to the previous finding of the rainbow trout rtCATH_1 gene, this paper describes three more cathelicidin genes found in salmonids: two in Atlantic salmon, named asCATH_1 and asCATH_2, and one in rainbow trout, named rtCATH_2. All the three new salmonid cathelicidin genes share the common characteristics of mammalian cathelicidin genes, such as consisting of four exons and possessing a highly conserved preproregion and four invariant cysteines clustered in the C-terminal region of the cathelin-like domain. The asCATH_1 gene is homologous to the rainbow trout rtCATH_1 gene, in that it possesses three repeat motifs of TGGGGGTGGC in exon IV and two cysteine residues in the predicted mature peptide, while the asCATH_2 gene and rtCATH_2 gene are homologues of each other, with 96% nucleotide identity. Salmonid cathelicidins possess the same elastase-sensitive residue, threonine, as hagfish cathelicidins and the rabbit CAP18 molecule. The cleavage site of the four salmonid cathelicidins is within a conserved amino acid motif of QKIRTRR, which is at the beginning of the sequence encoded by exon W. Two 36-residue peptides corresponding to the core part of rtCATH_1 and rtCATH_2 were chemically synthesized and shown to exhibit potent antimicrobial activity. rtCATH_2 was expressed constitutively in gill, head kidney, intestine, skin and spleen, while the expression of rtCATH_1 was inducible in gill, head kidney, and spleen after bacterial challenge. Four cathelicidin genes have now been characterized in salmonids and two were identified in hagfish, confirming that cathelicidin genes evolved early and are likely present in all vertebrates.

Identification of a Spindlin homolog in gibel carp (Carassius auratus gibelio)

Spindlin has been suggested to play an important role during the transition from oocyte maturation to embryo development in mouse, but its homolog similar to the mouse Spindlin in molecular and expression characterization has not been identified up to now in other vertebrates. In this study, a full length of cDNA sequence is cloned and sequenced from the gibel carp (Carassius auratus gibelio). It contains 1240 nucleotides with an open reading frame of 771 nt encoding 257 amino acids. Based on its amino acid sequence alignment and comparison analysis with the known Spin family proteins, the newly cloned Spin is named Carassius auratus gibelio Spindlin (CagSpin). Its product could be detected from mature eggs to blastula embryos, but its content decreased from the two-cell stage, and could not be detected after the gastrula stage. It suggests that the CagSpin should be a maternal protein that is expressed during oocyte maturation, and plays a crucial role in early cleavage of embryogenesis. CagSpin is the first homolog similar to mouse spindlin identified in fish, and also in other vertebrates. GST pull-down assay reveals the first biochemical evidence for the association of CagSpin and p-tubulin, the microtubule component. Therefore, CagSpin may play important functions by interacting with beta-tubulin and other spindle proteins during oocyte maturation and egg fertilization. (c) 2005 Elsevier Inc. All rights reserved.