大鼠肾脏组织cDNA文库的制备和葡萄糖转运蛋白cDNA克隆的筛选

 大鼠的肾脏组织经匀浆后,提取纯化总RNA和mRNA合成cDNA,进行甲基化,加人工接头,最后连入载体(λgt11)和外壳蛋白包装步骤制成肾脏组织的cDNA文库。插入载体的cDNA长度为0.5kb到8kb之间。经反复稀释,测定出该文库的效价为1.5×10~7pfu/mL。     

水稻二化螟和三化螟基因组ddRADseq文库的构建

本文介绍了构建水稻二化螟和三化螟"双酶切限制性酶切位点关联DNA测序"(Double digest restrictionsite associated DNA sequencing,ddRADseq)文库的方法。利用安捷伦2100生物分析仪对4种单酶切及2种双酶切的酶切产物片段大小及分布范围进行分析,筛选出Mlu C I和Nla III两种限制性内切酶组合对螟虫基因组DNA进行酶切。酶切后的DNA片段两端连接上特定的P1、P2接头后,用Pippin Prep回收大小为285-435 bp的DNA片段。通过PCR扩增进行文库的富集并引入index序列。构建好的ddRADseq文库用琼脂糖凝胶电泳和生物分析仪进行质量检测。本方法所构建的文库DNA片段长度、分布和摩尔浓度能够达到Illumina平台测序的技术要求。本研究证实了利用Mlu C I和Nla III组合酶切构建水稻螟虫基因组ddRADseq文库的可行性,为在水稻螟虫中利用ddRADseq技术开展生物地理学、种群遗传学和系统发育重建等方面的研究奠定基础。     

A Novel Community of Acidophiles in an Acid Mine Drainage Sediment

A sediment sample (pH 2.5) was collected at an acid mine drainage site in Anhui, China. The present acidophilic microbial community in the sediment was studied with a 16S rRNA gene clone library. Small-subunit rRNA genes were PCR amplified, cloned and screened by amplified rDNA restriction analysis (ARDRA). Subsequently, 10 different clones were identified and they were affiliated with Acidobacteria, β/γ-Proteobacteria, δ-Proteobacteria, Nitrospira, Candidate division TM7, and Low G + C Gram-positives. Phylogenetic analysis of 16S rRNA gene sequences revealed a diversity of acidophiles in the sediment that were mostly novel. Unexpectedly, 16S rRNA gene sequences affiliated with δ-Proteobacteria were found to constitute more than 60% of clone library. To our knowledge, this is the first occasion that bacteria of δ-Proteobacteria have been found dominant in the acidic habitat. Anaerobic sulfate- or metal reduction is the predominant physiological trait of bacteria of this subdivision. The high sulfate, ferric iron and the presence of bioavailable carbon in the anaerobic microenvironment may result in the dominance of bacteria of δ-Proteobacteria.     

Structural analysis of Arabidopsis thaliana chromosome 5. IX. Sequence features of the regions of 1,011,550 bp covered by seventeen P1 and TAC clones.

In this series of projects sequencing the entire genome of Arabidopsis thaliana chromosome 5, non-redundant P1 and TAC clones have been sequenced according to the fine physical map, and as of May 7, 1999, the sequences of 16.2 Mb representing approximately 60% of chromosome 5 have been accumulated and released at our web site. In parallel, structural features of the sequenced regions have been analyzed by applying a variety of computer programs, and to date we have predicted a total of 2380 potential protein-coding genes in the 10,154,580 bp regions, which are covered by 142 P1 and TAC clones. In this paper, we newly analyzed the structural features of the 1,011,550 bp regions covered by additional 17 P1 and TAC clones, and predicted 298 protein-coding genes. The average density of the genes identified was 1 gene per 3394 bp. Introns were observed in 67% of the genes, and the average number per gene and the average length of the introns were 3.2 and 159 bp, respectively. The gene density became higher than the value estimated in the previously analyzed regions (1 gene per 4,267 bp), as the data in this paper were compiled based on a new standard of gene assignment including the computer-predicted hypothetical genes. The regions also contained 8 tRNA genes when searched by similarity to reported tRNA genes and the tRNA scan-SE program. The sequence data and information on the potential genes are available on the database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.     

OPUS-Dom: Applying the Folding-Based Method VECFOLD to Determine Protein Domain Boundaries

In this article, we present a de novo method for predicting protein domain boundaries, called OPUS-Dom. The core of the method is a novel coarse-grained folding method, VECFOLD, which constructs low-resolution structural models from a target sequence by folding a chain of vectors representing the predicted secondary-structure elements. OPUS-Dom generates a large ensemble of folded structure decoys by VECFOLD and labels the domain boundaries of each decoy by a domain parsing algorithm. Consensus domain boundaries are then derived from the statistical distribution of the putative boundaries and three empirical sequence-based domain profiles. OPUS-Dom generally outperformed several state-of-the-art domain prediction algorithms over various benchmark protein sets. Even though each VECFOLD-generated structure contains large errors, collectively these structures provide a more robust delineation of domain boundaries. The success of OPUS-Dom suggests that the arrangement of protein domains is more a consequence of limited coordination patterns per domain arising from tertiary packing of secondary-structure segments, rather than sequence-specific constraints.     

Transforming activity of purinergic receptor P2Y, G-protein coupled, 2 revealed by retroviral expression screening

Colorectal cancer (CRC) is one of the leading causes of cancer death in humans. In order to identify novel cancer-promoting genes in CRC, we here constructed a retroviral cDNA expression library from a CRC cell line RKO, and used it for a focus formation assay with mouse 3T3 fibroblasts, leading to the identification of 42 independent cDNAs. One of such cDNAs turned out to encode purinergic receptor P2Y, G-protein coupled, 2 (P2RY2). The oncogenic potential of P2RY2 was confirmed in vitro with the focus formation assay as well as soft agar-growth assay, and also in vivo with a tumorigenicity assay in nude mice. While our P2RY2 cDNA encodes a protein with two amino-acid substitutions compared to the reported one, we have confirmed that the wild-type P2RY2 has a strong transforming potential as well. These results indicate an unexpected role of P2RY2 in the carcinogenesis of human cancers.     

Direct targeting and rapid isolation of BAC clones spanning a defined chromosome region

To isolate genes of interest in plants, it is essential to construct bacterial artificial chromosome (BAC) libraries from specific genotypes. Construction and organisation of BAC libraries is laborious and costly, especially from organisms with large and complex genomes. In the present study, we developed the pooled BAC library strategy that allows rapid and low cost generation and screening of genomic libraries from any genotype of interest. The BAC library is constructed, directly organised into a few pools and screened for BAC clones of interest using PCR and hybridisation steps, without requiring organization into individual clones. As a proof of concept, a pooled BAC library of approximately 177,000 recombinant clones has been constructed from the barley cultivar Cebada Capa that carries the Rph7 leaf rust resistance gene. The library has an average insert size of 140 kb, a coverage of six barley genome equivalents and is organised in 138 pools of about 1,300 clones each. We rapidly established a single contig of six BAC clones spanning 230 kb at the Rph7 locus on chromosome 3HS. The described low-cost cloning strategy is fast and will greatly facilitate direct targeting of genes and large-scale intra- and inter-species comparative genome analysis.Edwige Isidore and Beatrice Scherrer contributed equally to the work.     

靶向miRNA前体不同类型sgRNA的丰度及特异性评估

CRISPR/Cas技术能高效进行基因组定点编辑,但不同细菌来源或人工改造的Cas9以及Cpf1等核酸酶识别的PAM (protospacer adjacent motif)有差异,因此不同的基因编辑核酸酶可能采用不同类型的sgRNAs(small guide RNAs)。MicroRNAs (miRNAs)是一类调控性的小分子非编码RNAs,为了研究miRNA前体中是否可能存在特异性高的sgRNAs靶点,本文利用本课题组前期开发的生物信息学软件CRISPR-offinder,对靶向28 645条miRNA前体的11种不同类型sgRNA的丰度及特异性进行了分析,并利用CRISPR/Cas9慢病毒技术构建了猪miR-302/367基因簇敲除细胞系,对构建的猪miRNA敲除细胞系的效率进行了检测。结果表明,每个miRNA前体中平均存在约8种不同类型sgRNA的靶点;通过评估靶向猪miRNA前体sgRNA的脱靶效应,发现其中特异性高的sgRNA仅占18.2%;通过CRISPR/Cas9慢病毒技术成功构建了猪miR-302/367基因簇敲除细胞系,发现通过该技术构建miRNA敲除细胞系的效率为40%。本研究为利用CRISPR/Cas技术靶向敲除miRNA提供了重要资源。     

应用噬菌体随机肽库技术筛选丙肝病毒NS3抗原模拟表位

以抗 HCVNS3的单克隆抗体作为固相筛选分子 ,对人工合成的噬菌体随机 12肽库进行 5轮“吸附 洗脱 扩增”的筛选过程 ,随机挑取 4 2个克隆 ,经噬菌体酶联免疫吸附法 (ELISA)鉴定并进行交叉反应实验以及竞争抑制性结合实验 ,最后对所选克隆进行DNA序列分析 ,以确定HCVNS3抗原的模拟表位。经噬菌体富集后 ,从随机筛选的 4 2个克隆中得到 11个阳性克隆 ,确定氨基酸序列XXIXXXXMSNXX为HCVNS3的模拟表位。我们用噬菌体12肽库成功筛选得到HCVNS3的模拟表位 ,为开展用HCV模拟表位探索HCV的防治研究创造了条件     

Growth and development of biofeedback: A bibliographic update

Computerized literature searching techniques were used to examine publication patterns in the worldwide biofeedback literature. Searches were completed in the United States and in Japan for the years 1985 through 1987. The results were used to update the results of an earlier study (Hatch & Riley, 1985) that covered the years from 1964 through 1984. Publication growth curves were generated for several media, including scientific journal articles, books, doctoral dissertations, and popular magazine articles. Although publication of information about biofeedback remains active worldwide, there has been a declining trend in effect for the past several years. The American database grossly underestimated the number of Japanese biofeedback articles, and it is likely that the literatures of other countries outside of North America were similarly underestimated. Possible explanations for the various trends noted are discussed.These results were presented in part at the Twentieth Annual Meeting of the Association for Applied Psychophysiology and Biofeedback. We gratefully acknowledge the assistance of Margaret Cyr-Provost in preparing the data for analysis.