Statistical correlation between protein secondary structure and messenger RNA stem-loop structure

Anew integrated sequence-structure database, called IADE (Integrated ASTRAL-DSSP-EMBL), incorporating matching mRNA sequence, amino acid sequence, and protein secondary structural data, is constructed. It includes 648 protein domains. Based on the IADE database, we studied the relation between RNA stem-loop frequencies and protein secondary structure. It was found that the alpha-helices and beta-strands on proteins tend to be preferably "coded" by mRNA stem region, while the coils on proteins tend to be preferably "coded" by mRNA loop region. These tendencies are more obvious if we observe the structural words (SWs). An SW is defined by a four-amino-acid-fragment that shows the pronounced secondary structural (alpha-helix or beta-strand) propensity. It is demonstrated that the deduced correlation between protein and mRNA structure can hardly be explained as the stochastic fluctuation effect. (C) 2003 Wiley Periodicals, Inc.

PURIFICATION AND CHARACTERIZATION OF A KINASE SPECIFIC FOR THE SERINE-RICH AND ARGININE-RICH PRE-MESSENGER-RNA SPLICING FACTORS

Members of the SR family of pre-mRNA splicing factors are phosphoproteins that share a phosphoepitope specifically recognized by monoclonal antibody (mAb) 104. Recent studies have indicated that phosphorylation may regulate the activity and the intracellular localization of these splicing factors. Here, we report the purification and kinetic properties of SR protein kinase 1 (SRPK1), a kinase specific for SR family members. We demonstrate that the kinase specifically recognizes the SR domain, which contains serine/arginine repeats. Previous studies have shown that dephosphorylated SR proteins did not react with mAb 104 and migrated faster in SDS gels than SR proteins from mammalian cells. We show that SRPK1 restores both mobility and mAB 104 reactivity to a SR protein SF2/ASF (splicing factor 2/alternative splicing factor) produced in bacteria, suggesting that SRPK1 is responsible for the generation of the mAb 104-specific phosphoepitope in vivo. Finally, we have correlated the effects of mutagenesis in the SR domain of SF2/ASF on splicing with those on phosphorylation of the protein by SRPK1, suggesting that phosphorylation of SR proteins is required for splicing.

运用减法杂交、差异筛选与信使RNA差别显示聚合酶链式反应克隆冬小麦春化相关基因

对于某些一年生或二年生高等植物，春化作用是诱导其成花的一个重要的环境因子。冬小麦春化进程中存在着一个核酸代谢的关键期，利用分子生物学技术分离特异表达的基因是研究春化诱导成花机理的一个突破口。 利用TRIzol试剂快速提取冬小麦燕大1817(Triticum aestivum L. cv Yanda 1817)未春化、春化4d、春化20d、5d脱春化的胚芽中的总RNA，去除污染的DNA后，将引物P_1(5'TTTTTTTTTTTCA3')、P_2(5'TTTTTTTTTTCC3')与10个碱基的随机引物OPF_1-OPF_(20)、OPG_1-OPG_(20)组成80个引物对，对不同来源的RNA进行差别显示，共显示了大约10,000种mRNA，结果发现了两个仅在春化20d这一关键期表达而在未春化、春化4d、5d脱春化时不表达的春化相关基因(VRG)VRG49与VRG54。Northern分析进一步表明这两个基因仅与春化20d的冬小麦RNA有杂交信号。将VRG49与VRG54亚克隆于pGEM-4Z载体上，利用T_7测序系统获得了VRG49和VRG54的DNA序列，它们的长度分别为307bp与169bp。 春化21d的冬小麦京冬1号(T. aestivum L. cv Jingdong No. 1)胚芽的mRNA在逆转酶作用下反转录成sscDNA杂交，将过量的未春化、脱春化的mRNA与sscDNA杂交，运用磁珠法分离出未杂交上的sscDNA，以特异的sscDNA为模板，用DNA聚合酷I合成了dscDNA。通过对dscDNA内部EcoRI位点的甲基化、末端补平、EcoRI接头的安装、连接进入λgt10载体的EcoRI位置，以及运用包装系统进行体外包装，建立了库容为4 * 10~6pfu的富集低温诱导的冬小麦cDNA噬菌体文库。用来源于未春化、春化21d、脱春化的冬小麦mRNA合成3种cDNA探针，对噬菌斑进行原位杂交，结果筛选出了3个春化相关基因(VRG)VRG79、VRG111和VRG231。Dot blotting与Northern分析表明VRG79仅在冬小麦春化关键期21d表达。运用PCR方法从λgt10DNA中扩增出VRG79片断并亚克隆于PUC18载体上，通过T_7测序系统获得了VGR79的序列，其包括349个碱基。 通过Internet将VRG49、VRG54、VRG79与GenBank、EMBL、DDBJ、PBD中的序列进行同源性分析，结果发现这些基因至少是在植物中新发现的基因，对这些基因推测的一些功能也进行了讨论。

气体信号分子一氧化氮和乙烯的生物学功能：一氧化氮在植物盐胁迫中的作用和乙烯激活钙离子通道

一氧化氮（NO）是重要的植物信号分子，参与许多植物生理过程。以拟南芥野生型和Atnoa1突变体为材料研究了NO在植物抗盐胁迫中的作用。 T-DNA插入AtNOA1基因的第一个外显子，使Atnoa1突变体中NOS活性大幅度下降，NO释放减少。用不同浓度的NaCl对拟南芥野生型和Atnoa1突变体进行盐胁迫处理后，Atnoa1突变体中Na+离子积累较野生型多，K+离子吸收较野生型少，从而使突变体中的Na+/K+比野生型高，对突变体造成了更大的伤害。Atnoa1突变体种子萌发和幼苗生长对盐胁迫更敏感。盐胁迫处理后，Atnoa1突变体的存活率比野生型低。无论是在正常生长条件下，还是盐胁迫条件下，Atnoa1突变体中的H2O2和TBARS含量都比野生型中高，说明Atnoa1突变体对盐胁迫和氧化胁迫都比野生型更敏感。用NOS抑制剂和NO清除剂处理拟南芥野生型，减少内源NO释放量，使其在盐胁迫条件下的Na+/K+比增高。盐胁迫处理降低了野生型体内的NOS活性，减少了NOA1蛋白的表达，DAF-2DA标记的NO荧光强度减弱。用NO供体SNP处理Atnoa1突变体，可以减少盐胁迫引起的Na+/K+比增加。以上研究结果证明NOS介导的NO合成在植物抗盐胁迫中起重要作用。 乙烯作为一种植物气体激素参与植物生长发育的许多生理生化过程。植物细胞自由钙离子（[Ca2+]c）是重要信号分子，在植物应答外界信号中起非常重要的作用。外界信号通过开启植物细胞质膜的钙离子通道，使得胞外钙离子进入细胞，导致瞬间[Ca2+]c的增加，激活钙依赖型的蛋白和蛋白激酶，从而改变生理生化过程。本研究利用膜片钳和激光共聚焦显微技术，研究了外源乙烯对烟草悬浮细胞质膜Ca2+离子通道和细胞中[Ca2+]c活性的影响。乙烯供体乙烯利和乙烯合成前体ACC能够迅速诱导内向型电流，表明这些处理能开启离子通道。通过离子替代实验和离子通道的药理学分析证实乙烯利和ACC激活了一种对Ba2+, Mg2+和Ca2+等阳离子具有通透性的离子通道，La3+、Gd3+和Al3+抑制该通道的活性。乙烯受体拮抗物（1-MPP）和ACC合成酶抑制剂，能够减弱乙烯利和ACC对这种通道的活化作用，说明乙烯利和ACC是通过乙烯活化此类Ca2+离子通道。用Ca2+敏感的荧光标记物Fluo-3标记，通过激光共聚焦显微观察，发现乙烯利能够诱导烟草悬浮细胞中[Ca2+]c离子浓度的增加，而且Gd3+和BAPTA显著抑制乙烯利诱导的细胞中[Ca2+]c离子的增加。说明外源Ca2+离子通过质膜上被激活的Ca2+离子通道进入细胞，使细胞中[Ca2+]c离子浓度增加。以上结果说明，乙烯活化质膜上的Ca2+离子通道，使细胞外Ca2+离子进入细胞，导致细胞中[Ca2+]c离子浓度增加，是乙烯信号转导途径的重要步骤。

Small but influential: the role of microRNAs on gene regulatory network and 3 ' UTR evolution

MicroRNAs (miRNAs) are endogenous similar to 22 nucleotide noncoding RNAs that regulate the expression of complementary messenger RNAs (mRNAs). Thousands of miRNA genes have been found in diverse species, and many of them are highly conserved. With the mi

Zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) mediates multiple intracellular signaling pathways

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-kappa B activity with and without lipopolysacharide (LPS) stimulation. (C) 2008 Elsevier Ltd. All rights reserved.

Functional domains and the antiviral effect of the double-stranded RNA-dependent protein kinase PKR from Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase PKR is thought to mediate a conserved antiviral pathway by inhibiting viral protein synthesis via the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2 alpha). However, little is known about the data related to the lower vertebrates, including fish. Recently, the identification of PKR-like, or PKZ, has addressed the question of whether there is an orthologous PKR in fish. Here, we identify the first fish PKR gene from the Japanese flounder Paralichthys olivaceus (PoPKR). PoPKR encodes a protein that shows a conserved structure that is characteristic of mammalian PKRs, having both the N-terminal region for dsRNA binding and the C-terminal region for the inhibition of protein translation. The catalytic activity of PoPKR is further evidence that it is required for protein translation inhibition in vitro. PoPKR is constitutively transcribed at low levels and is highly induced after virus infection. Strikingly, PoPKR overexpression increases eIF2 alpha phosphorylation and inhibits the replication of Scophthalmus maximus rhabdovirus (SMRV) in flounder embryonic cells, whereas phosphorylation and antiviral effects are impaired in transfected cells expressing the catalytically inactive PKR-K421R variant, indicating that PoPKR inhibits virus replication by phosphorylating substrate eIF2 alpha. The interaction between PoPKR and eIF2 alpha is demonstrated by coimmunoprecipitation assays, and the transfection of PoPKR-specific short interfering RNA further reveals that the enhanced eIF2 alpha phosphorylation is catalyzed by PoPKR during SMRV infection. The current data provide significant evidence for the existence of a PKR-mediated antiviral pathway in fish and reveal considerable conservation in the functional domains and the antiviral effect of PKR proteins between fish and mammals.

Mutations stabilize small subunit ribosomal RNA in desiccation-tolerant cyanobacteria Nostoc

The ribosomal RNA molecule is an ideal model for evaluating the stability of a gene product under desiccation stress. We isolated 8 Nostoc strains that had the capacity to withstand desiccation in habitats and sequenced their 16S rRNA genes. The stabilities of 16S rRNAs secondary structures, indicated by free energy change of folding, were compared among Nostoc and other related species. The results suggested that 163 rRNA secondary structures of the desiccation-tolerant Nostoc strains were more stable than that of planktonic Nostocaceae species. The stabilizing mutations were divided into two categories: (1) those causing GC to replace other types of base pairs in stems and (2) those causing extension of stems. By mapping stabilizing mutations onto the Nostoc phylogenetic tree based on 16S rRNA gene, it was shown that most of stabilizing mutations had evolved during adaptive radiation among Nostoc spp. The evolution of 16S rRNA along the Nostoc lineage is suggested to be selectively advantageous under desiccation stress.

Cyclin A2 is differentially expressed during oocyte maturation between gynogenetic silver crucian carp and gonochoristic color crucian carp

Silver crucian carp (Carassius auratus gibelio) is a unique gynogenetic fish. Because of its specific genetic background and reproduction mode, it is an intriguing model system for understanding regulatory mechanism of oocyte maturation division. It keeps its chromosomal integrity by inhibiting the first meiotic division (no extrusion of the first pole body). The spindle behavior during oocyte maturation is significantly different from that in gonochoristic fish. The chromosomes are first arranged in a tripolar spindle, and then they turn around and are reunited mutually to form a normal bipolar spindle. A new member of the fish A-type cyclin gene, cyclin A2, has been isolated by suppression of subtractive hybridization on the basis of its differential transcription in fully-grown oocytes between the gynogenetic silver crucian carp and gonochoristic color crucian carp. There are 18 differing amino acids in the total 428 residues of cyclin A2 between the two forms of crucian carps. In addition, cDNAs of cyclin A1 and cyclin B have also been cloned from them. Thus two members of A-type cyclins, cyclin A1 and cyclin A2, are demonstrated to exist in fish, just as in frog, humans, and mouse. Northern blotting reveals that cyclin A2 mRNA is more than 20-fold and cyclin A1 mRNA is about 2-fold in fully grown oocytes of gynogenetic silver crucian carp compared to gonochoristic color crucian carp. However, cyclin B does not show such a difference between them. Western blot analysis also shows that the cyclin A2 protein stockpiled in fully grown oocytes of gynogenetic crucian carp is much more abundant than in gonochoristic crucian carp. Moreover, two different cyclin A2 expression patterns during oocyte maturation have been revealed in the two closely related crucian carps. For color crucian carp, cyclin A2 protein is translated only after hormone stimulation. For silver crucian carp, cyclin A2 protein can be detected throughout the process of maturation division. The different expression of cyclin A2 may be a clue to understanding the special maturation division of gynogenetic silver crucian carp.

基于协议分析的IM阻断策略及算法分析

本文根据即时通讯（IM）的协议模型，在TCP层和即时通讯协议层分析了阻断即时通讯的可能性，分别提出了基于TCP层和即时通讯协议层的协议分析的阻断策略．在TCP层，通过插入FIN报文或RST报文，以关闭TCP连接（特别是与登录服务器的连接）；在即时通讯协议层，通过对相应字段的值进行设置，使客户端进入不活动状态．为使阻断策略能够有效地实施，本文也提出了一个消息驱动的实现算法．通过对Yahoo! Messenger的阻断实验，证明了本文提出的方法能够高效地完成阻断任务（平均数据包量不超过2．5个）．用统计分析的方法对它们的性能进行比较，结果表明各种方法的效率在不同的背景流量下没有显著的差异，通过消息驱动算法实现的基于协议分析的阻断策略，其工作效率不受背景流量的影响．另外，即时通讯数据的触发方式，使得发送的阻断包的数量与背景流量无关，也不对网络负载造成明显的影响．本文的工作为实施即时通讯的阻断提供了有效的解决方案．

Self-assembly of single-stranded RNA on carbon nanotube: Polyadenylic acid to form a duplex structure

All messenger-RNA (mRNA) molecules in eukaryotic cells have a polyadenylic acid [poly (rA)] tail at the 3'-end and human poly (rA) polymerase (PAP) has been considered as a tumor-specific target. A ligand that is capable of recognizing and binding to the poly(M) tail of mRNA might interfere with the full processing of mRNA by PAP and can be a potential therapeutic agent. We report here for the first time that single-walled carbon nanotubes (SWNTs) can cause single-stranded poly (M) to self-structure and form a duplex structure, which is studied by UV melting, atomic force microscopy, circular dichroism spectroscopy, and NMR spectrometry.

Preparation, Surface Properties and Biological Activity of the Composite of Poly (lactide-co-glycolide) and Bioglass Nanoparticles Surface Grafted with Poly(L-lactide)

SiO2-CaO-P2O5 gel bioglass (BG) nanoparticles with the diameter of 40 nm were synthesized by sol-gel approach. The surface of BG nanoparticles was grafted through the ring-open polymerization of the L-lactide to yield poly (L-lactide) (PLLA) grafted gel particle (PLLA-g-BG). The PLLA-g-BG was further blended with poly(lactide-co-glycolide) (PLGA) to prepare the nanocomposites of PLLA-g-BG/PLGA with the various blend ratios of two phases. PLLA-g-BG accounted 10%, 20% and 40% in the composite, respectively. TGA, ESEM and EDX were used to analyze the graft ratio of PLLA-g-BG, the dispersion of nano-particles and the surface elements of the composites respectively. The rabbit osteoblasts were seeded and cultured on the thin films of composites in vitro. The cell adhesion, spreading and growth of osteoblasts were analyzed with FITC staining, NIH Image J software and MTT assay. The change of cell cycle was monitored by flow cytometry (FCM). The results demonstrated that the Surface modification of BG with PLLA could significantly improve the dispersing of the particles in the matrix of PLGA. The nanocomposite with 20% PLLA-g-BG exhibited superior surface properties, including roughness and plenty of silicon, calcium and phosper, to enhance the adhesion, spreading and proliferation of osteoblasts.

A novel invariant and sequence comparison for DNA

We consider numerical characterization of DNA primary sequence based on the positions of bases (a, t, c, g) and the pairs of bases X, Y in DNA (X, Y=a, t, c, g). This leads to a representation of DNA by a numerical sequence. Then, we extract a novel invariant (molecular connectivity index) from the derived numerical sequences. The suitable invariant can offer a characterization of DNA primary sequence. Finally, we provide an illustration of its utility by making a comparison between ten DNA sequences belonging to beta-globin gene in different species. The evolutionary relationships of ten species we have revealed in this contribution accord with phylogenetic tree properly.

Progress in electrochemiluminescent analysis

The recent progress in electrochemiluminescent (ECL) assay is reviewed. This review begins with the fundamental researches in ECL, including the discovery of new ECL-active species, such as biochemical, organic and metallorganic materials, digital modeling of ECL process, the flow cells used in ECL assay, and electrochemiluminescent sensor. The application of ECL in environmental analysis, immunoassay, nucleotide acid hybridization sensor. The applications of ECL in environmental analysis, immunooassay, nucleic acid hybridization assay, and other aspects are reviewed with the latest references in detail. Finally, the main problems in the further investigation are outlooked, so are its prospects.