VitisMod:一个葡萄基因共表达数据库

葡萄转录组已在各种组织、发育阶段、生物胁迫、非生物胁迫和其他条件下被测定。目前,仍没有简单实用的网络工具来探索这些宝贵的数据。本文从美国国立生物技术信息中心的基因表达数据库(NCBI GEO)下载1019个基因表达芯片数据,进行权重基因共表达网络分析(WGCNA)。鉴定到41个基因共表达模块。功能富集分析发现这些模块具有不同的功能,并与实验/表型相关。通过模块内连接度筛选枢纽基因,这些基因可能具有重要功能。通过关联推定(Guilt-by-association)原理对模块内功能未知的基因进行功能预测。最后,构建了免费的网络工具VitisMod,为葡萄的基因功能研究提供新资源,网址为：http://bioinformatics.fafu.edu.cn/grape。     

Characterization of MicA interactions suggests a potential novel means of gene regulation by small non-coding RNAs

MicA is a small non-coding RNA that regulates ompA mRNA translation in Escherichia coli. MicA has an inhibitory function, base pairing to the translation initiation region of target mRNAs through short sequences of complementarity, blocking their ribosome-binding sites. The MicA structure contains two stem loops, which impede its interaction with target mRNAs, and it is thought that the RNA chaperone protein Hfq, known to be involved in MicA regulation of ompA, may structurally remodel MicA to reveal the ompA-binding site for cognate pairing. To further characterize these interactions, we undertook biochemical and biophysical studies using native MicA and a ‘stabilized’ version, modified to mimic the conformational state of MicA where the ompA-binding site is exposed. Our data corroborate two proposed roles for Hfq: first, to bring both MicA and ompA into close proximity, and second, to restructure MicA to allow exposure of the ompA-binding site for pairing, thereby demonstrating the RNA chaperone function of Hfq. Additionally, at accumulated MicA levels, we identified a Mg²⁺-dependent self-association that occludes the ompA-recognition region. We discuss the potential contribution of an Mg²⁺-mediated conformational switch of MicA for the regulation of MicA function.     

Characterization of a candidate Trypanosoma rangeli small nucleolar RNA gene and its application in a PCR-based parasite detection

In this study, we report the isolation and characterization of a candidate Trypanosoma rangeli small nucleolar RNA (snoRNA) gene, and the development of a PCR assay for detection of the parasite based on its nucleotide sequence. This gene, isolated from a T. rangeli genomic sub-library, was named snoRNA-cl1 and is encoded by a multi-copy gene of 801bp in length. Computer sequence analysis of snoRNA-cl1 showed the presence of two sequence motifs, box C and box D, as well as of two long stretches that perfectly complement the universal core region of the mature rRNA 28S, suggesting that cl1 encodes for a Box C/D snoRNA from the parasite. Hybridization analysis using cl1 as probe, showed a weak hybridization signal with Trypanosoma cruzi DNA, demonstrating the existence of differences in this locus between these two species. Two oligonucleotide primers from this gene, which specifically amplified a 620-bp fragment in KP1 (+) and KP1 (-) strains of T. rangeli, were used in a PCR assay. The amplification allowed the detection of 1pg of DNA in the presence of heterologous DNA and no amplification was observed with different T. cruzi strains (groups I and II). In addition, the PCR assay reported here is able to detect T. rangeli in the presence of T. cruzi DNA, and is useful for detection of the parasite in samples from infected vectors.     

Characterization of Escherichia coli O3 and O21 O antigen gene clusters and development of serogroup-specific PCR assays

Escherichia coli O3 and O21 are associated with enteroaggregative E. coli (EAEC). EAEC strains are often non-typable using the routine agglutination method due to their aggregative phenotype. Typing of E. coli O3 and O21 may also be impeded by cross-reactions with O152 or O83. In this study, the O antigen gene clusters of E. coli O3 and O21 were characterized, and PCR assays based on O antigen specific genes wzx (encoding O unit flippase) and wzy (encoding O unit polymerase) from each strain were developed. By screening against all 186 known E. coli O serotypes, the PCR assays were shown to be highly specific to O3 and O21 respectively. The sensitivity of the assays was determined to be 1 pg per mul of chromosomal DNA and 2 CFU per 10 g of water samples. The PCR assays were also applied to 658 clinical E. coli isolates, and 100% of detection accuracy was obtained. The PCR assays developed here are suitable for the detection and identification of E. coli O3 and O21 strains in environmental and clinical samples.     

Characterization of a novel rice kinesin O12 with a calponin homology domain

Genomic analysis predicted that the rice (Oryza sativa var. japonica) genome encodes at least 41 kinesin-like proteins including the novel kinesin O12, which is classified as a kinesin-14 family member. O12 has a calponin homology (CH) domain that is known as an actin-binding domain. In this study, we expressed the functional domains of O12 in Escherichia coli and determined its enzymatic characteristics compared with other kinesins. The microtubule-dependent ATPase activity of recombinant O12 containing the motor and CH domains was significantly reduced in the presence of actin. Interestingly, microtubule-dependent ATPase activity of the motor domain was also affected by actin in the absence of the CH domain. Our findings suggest that the motor activity of the rice plant-specific kinesin O12 may be regulated by actin.     

Genes co-expressed with CPSAR1 identified using ATTED-II

Identification of interacting proteins will help to investigate further the relationship between CPSAR1 and the vesicle transport system or the ribosomes. Thus, we adopted a bioinformatic approach, using the publicly available Arabidopsis thaliana trans-factor and cis-element prediction database, ATTED-II (http://atted.jp/), to identify putative protein interactors. The proteins directly linked to CPSAR1 were almost exclusively nucleus encoded and several were involved in protein synthesis of which three were thylakoid localized. The list of putative interacting proteins does not exclude any of the previous proposed actions of CPSAR1 but encourage more detailed examination of the role of CPSAR1.Key words: Arabidopsis, chloroplast, co-expression, protein cargo, vesicle transportChloroplast protein targeting has been intensively studied.^1–4 Transport of non-proteinaceous material, such as lipids, to the thylakoid has not been studied to the same extent, although several reports do exist.^5–8 Since thylakoids do not produce lipids themselves any lipids present must have been transported from the production site, i.e., the envelope.^9,10 Experimental data support the theory of a vesicular transport system and it has been predicted that several proteins resembling those important in the cytosolic vesicle transport system are chloroplast localized.¹¹ Recently we confirmed that one of these proteins, CPSAR1, is located in the chloroplast and has features similar to the cytosolic COPII-related Sar1 protein, i.e., it is found at the donor membrane and in the vesicle, but not at the acceptor membrane. In addition, other research groups^12,13 have also found that CPSAR1 is chloroplast localized but refer to it as atOBGL or atObgM, respectively, since CPSAR1 shares high sequence similarity with proteins belonging to the Obg-family. The roles of Obg proteins are very diverse, but a role closely connected to ribosomes has been suggested for CPSAR1.¹² CPSAR1 being part of a vesicle system does not exclude it from having a role in relation to ribosomes. The existence of a coiled-coil motif and a GTP binding domain, in combination with the suggested role as part of a vesicle transport system, makes it a highly attractive idea that CPSAR1 interacts with other proteins.     

Characterization of SLAC: a small laccase from Streptomyces coelicolor with unprecedented activity

Laccases and other four-copper oxidases are usually constructed of three domains: Domains one and three house the copper sites, and the second domain often helps form a substrate-binding cleft. In contrast to this arrangement, the genome of Streptomyces coelicolor was found to encode a small, four-copper oxidase that lacks the second domain. This protein is representative of a new family of enzymes--the two-domain laccases. Disruption of the corresponding gene abrogates laccase activity in the growth media. We have recombinantly expressed this enzyme, called SLAC, in Escherichia coli and characterized it. The enzyme binds four copper ions/monomer, and UV-visible absorption and EPR measurements confirm that the conserved type 1 copper site and trinuclear cluster are intact. We also report the first known paramagnetic NMR spectrum for the trinuclear copper cluster of a protein from the laccase family. The enzyme is highly stable, retaining activity as a dimer in denaturing gels after boiling and SDS treatment. The activity of the enzyme against 2,6-dimethoxyphenol (DMP) peaks at an unprecedentedly high pH (9.4), whereas the activity against ferrocyanide decreases with pH. SLAC binds negatively charged substrates more tightly than positively charged or uncharged molecules.