野生一粒小麦BAC文库的构建和鉴定

以细菌人工染色体pECBAC1为载体 ,构建了野生一粒小麦 (TriticumboeoticumBoiss)的基因组BAC文库。该文库共包含约 17万个克隆 ,平均插入片段长度为 10 4kb ,按野生一粒小麦基因组为 5 6 0 0Mb计算 ,文库覆盖了约 3倍的该物种基因组。用大麦叶绿体psbA基因和玉米线粒体atp6基因作混合探针 ,检测发现该文库中含细胞器基因组同源序列的克隆数小于 1%。该文库的建成 ,为小麦基因的克隆及基因组学研究提供了技术平台     

野生一粒小麦BAC文库的构建和鉴定

以细菌人工染色体pECBAC1为载体,构建了野生一粒小麦(Triticum boeoticum B oiss)的基因组BAC文库.该文库共包含约17万个克隆,平均插入片段长度为104 kb,按野生一粒小麦基因组为5 600 Mb计算,文库覆盖了约3倍的该物种基因组.用大麦叶绿体psb A基因和玉米线粒体atp6基因作混合探针,检测发现该文库中含细胞器基因组同源序列的克隆数小于1% .该文库的建成,为小麦基因的克隆及基因组学研究提供了技术平台.     

外显子捕获法分离FRAXA位点的新表达序列

外显子捕获是近年发展起来的一种自基因组DNA分离表达序列的有效方法.我们采用以pSPL3为剪接载体的外显子捕获系统,从覆盖FRAXA位点的酵母人工染色体YAC209G4中分离到2个新外显子序列A91和D12.A91在人胎肝和骨骼肌文库中丰度较高,在肝、肾和骨髓文库中也有PCR扩增.而D12在所分析的8个cDNA文库中均未见PCR扩增产物.从骨骼肌文库中克隆到一段包含A91的315 bp的cDNA(AM4470).多种组织RNA印迹显示,AM4470与骨骼肌和心脏mRNA杂交,转录本长度均为2.8 kb.     

Library of diversely substituted 2-(quinolin-4-yl)imidazolines delivers novel non-cytotoxic antitubercular leads

A novel library based on quinolin-4-ylimidazoline core was designed to incorporate a general quinoline antimicrobial pharmacophore. A synthesis of the well-characterized library of 36 compounds was achieved using the Pd-catalyzed Buchwald–Hartwig-type imidazoline arylation chemistry developed earlier. Compounds were tested for biological activity and were found to possess no antimalarial activity. However, the library delivered two promising antitubercular leads, which are non-cytotoxic and can be further optimized with respect to antimycobacterial potency.     

Characterization of expressed sequence tags generated from skin cDNA clones of Equus caballus by single pass sequencing

A cDNA library was built using RNA extracted from the skin tissue of an adult horse. The library was primed with oligo (dT) and sequences were directionally inserted in order to produce an expression library. The library has 5.8X 10(5) plaque forming units with 99.6% recombinant phage. The average insert size is 1.3 Kbp. Three hundred and thirteen expressed sequence tags (ESTs) were generated from sequencing of the 5 prime end of randomly selected skin cDNA clones. The ESTs were sequenced on an ABI 377 using Big-Dye chemistry. A similarity search was performed on each EST using the NCBI non-redundant protein database and 206 ESTs were putatively identified. Twenty six percent of the identified ESTs were redundant. The ESTs were categorized by function. The most frequently identified functional class was translational proteins.     

中华蜜蜂幼虫膜蛋白酵母双杂交文库的构建

【目的】本研究旨在利用位点特异性重组技术(FullCoV)将中华蜜蜂 Apis cerana cerana 幼虫膜蛋白cDNA连接到pPR3-N载体上,构建中华蜜蜂幼虫膜蛋白酵母双杂交cDNA文库。【方法】提取2-3日龄中华蜜蜂工蜂幼虫总RNA;分离mRNA后,在反转录酶的作用下合成幼虫膜蛋白cDNA第1链,并合成双链cDNA。在双链cDNA的5′端加上带有重组序列的接头后,通过FullCoV技术与载体pPR3-N进行连接,然后将连接产物电转化到DH10B感受态细胞,构建中华蜜蜂幼虫膜蛋白酵母双杂交cDNA文库,并对该文库插入片段大小和文库滴度进行检测。【结果】通过FullCoV技术成功构建了中华蜜蜂幼虫膜蛋白酵母双杂交cDNA文库,经检测,中华蜜蜂幼虫膜蛋白酵母cDNA文库的总库容量为1.5×10^7 cfu,文库滴度为3×10^6 cfu/mL,重组率达到100%。【结论】本研究利用FullCoV技术成功构建了中华蜜蜂幼虫膜蛋白酵母cDNA文库,为进一步探究感染中华蜜蜂的病原微生物与宿主蛋白互作研究奠定了基础。     

甜菜夜蛾cDNA文库的构建

以甜菜夜蛾Spodoptera exigua(Hübner)幼虫为材料建立了cDNA表达文库,经检测文库的初始滴度为1.1×105pfu/mL,重组率为97%,扩增后文库的滴度为5.4×107pfu/mL。用设计的2对引物筛选该文库,得到468 bp的1个片段,分析后证实是几丁质合成酶基因I保守区域的1个片段。该cDNA文库为进一步筛选甜菜夜蛾功能基因奠定了基础。     

cDNA文库固相构建法

本文介绍一种cDNA文库的固相构建法。文库构建过程cDNA的合成和修饰全部在固相介质—磁珠上完成,简便、迅速、文库质量高,能满足不同目的的cDNA文库构建,是一种值得借鉴和应用的好方法。     

青杄均一化cDNA文库构建及EST序列分析

以青杄花粉和针叶为材料,将青杄全长cDNA与Gateway供体载体pDONR222重组,构建了其非剪切型全长cDNA原始文库,利用基因组DNA饱和杂交技术对原始cDNA文库进行均一化处理,构建青杄的均一化全长cDNA文库。文库的总库容量为1.1×106CFU/mL,平均插入片段长度大于1.0 kb,重组率大于95%。定量RT-PCR检测表明,青杄高丰度表达基因EF1-α在均一化cDNA文库中的表达量下降了约41倍。接着对文库中随机的5 144个克隆进行了测序,获得高质量的有效EST(expressedsequence tag)序列为5 144条,经拼接共获得单一基因(unigene)为2 717个,其中包括片段重叠群(contig)628个和单一EST序列(singlet)2 089个。NCBI同源比对分析表明,其中1 887个序列unigenes获得分子功能注释,这些EST涉及细胞生长、信号转导、转录、抗逆、能量代谢等功能。这些数据有助于对青杄的相关功能蛋白及分子机制开展进一步的研究。     

CHARACTERIZATION OF EXPRESSED SEQUENCE TAGS GENERATED FROM SKIN CDNA CLONES OF Equus caballus BY SINGLE PASS SEQUENCING

A cDNA library was built using RNA extracted from the skin tissue of an adult horse. The library was primed with oligo (dT) and sequences were directionally inserted in order to produce an expression library. The library has 5.8X 10⁵ plaque forming units with 99.6% recombinant phage. The average insert size is 1.3 Kbp. Three hundred and thirteen expressed sequence tags (ESTs) were generated from sequencing of the 5 prime end of randomly selected skin cDNA clones. The ESTs were sequenced on an ABI 377 using Big-Dye chemistry. A similarity search was performed on each EST using the NCBI non-redundant protein database and 206 ESTs were putatively identified. Twenty six percent of the identified ESTs were redundant. The ESTs were categorized by function. The most frequently identified functional class was translational proteins.     

Solid-phase synthesis and biological evaluation of a uridinyl branched peptide urea library

A 1000-member uridinyl branched peptide library was synthesized on PS-DES support using IRORI technology. High-throughput screening of this library for anti-tuberculosis activity identified several members with a MIC₉₀ value of 12.5 μg/mL.     

Construction of a BAC library of pearl millet, Pennisetum glaucum

A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from pearl millet (Pennisetum glaucum), and used as a resource for the isolation of microsatellite sequences. The library contains a total of 159,100 clones with an average insert size of 90 kb, and corresponds to 5.8 haploid genome equivalents. The BAC library was pooled for screening by the polymerase chain reaction (PCR) as well as robotically gridded on high-density filters. PCR-based screening of a subset of the library (4.7 haploid genome equivalents) using five sequence-tagged site (STS) and six microsatellite markers identified between 2 and 11 positives superpools (5.4 on average). The frequency of BAC clones carrying inserts of chloroplast DNA was estimated to be less than 1% by hybridisation with a rice chloroplast probe. Received: 30 January 2000 / Accepted: 16 October 2000     

Construction of a chromosome-enriched HpaII library from flow-sorted wheat chromosomes.

We report here the first successful generation of a chromosome-enriched library from flow sorted plant chromosomes. Chromosomes with a characteristic DNA content (a peak) were sorted from a synchronized cell culture (TaKB1, derived from Triticum aestivum). A HpaII library was constructed from the sorted chromosomes and half of the cloned DNA sequences analysed are unique or low copy. Approximately half of these sequences when used as probes detect sequences on wheat chromosome 4A. The generation and analysis of the chromosome library is described in detail and the prospects of using flow-sorted plant chromosomes discussed.     

Cloned genomic segments of Zea mays homologous to zein mRNAs

A maize genome library was constructed using maize W22 DNA from leaf tissue nuclei and bacteriophage λCh4 as the vector. cDNA clones of zein mRNA were used to identify homologous genomic sequences in the Ch4 maize library. Each of the genomic clones identified has homology to a family of mRNAs in the zein mRNA population. This paper reports on the construction of the library, the isolation of the genomic clones and their partial characterization.     

Isolation of novel catalytic antibody clones from combinatorial library displayed on yeast-cell surface

A combinatorial library of the Fab fragment of a catalytic antibody able to hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol was constructed on yeast-cell surface. Interesting clones were selected using fluorescence-activated cell sorting (FACS). When binding affinity to a transition-state analog was detected, evolution of the catalytic antibody was carried out in vitro on yeast-cell surface. A number of variants with enhanced catalytic activity and binding affinity were obtained. The results showed that the improvement of catalytic antibody, which can be performed easily on yeast-cell surface using the cell-surface engineering system, is a good example of the application of protein library construction.     

Gateway技术构建交链孢菌JH505 cDNA文库

Gateway(R)技术构建Cdna文库,利用λ噬菌体的位点特异性重组,避免使用限制性内酶切割Cdna,能够解决常规方法构建Cdna文库的技术缺陷.首次应用Gateway(R)技术构建交链孢菌Cdna文库,经检测Cdna入门文库的滴度达到1×107cfu/Ml,文库总容量为9×107cfu,平均插入片段为1510bp.通过LR重组把入门文库转换为表达文库,表达文库的滴度为1.58×106cfu/Ml,文库总容量为6.32×106cfu,平均插入片段大小为1680bp.表达文库的构建为进一步克隆植物激活蛋白基因打下了基础.     

A combined physical and genetic map of Pseudomonas aeruginosa PAO

A combined physical and genetic map of Pseudomonas aeruginosa PAO was constructed by pulsed-field gel electrophoresis and Southern hybridization using cosmid clones from a genomic library carrying known genes. A total of 37 SpeI restriction fragments have been mapped on the 5862 kb genome, and fragment contiguity demonstrated by hybridization with clones from a SpeI junction fragment library and fragments obtained by partial SpeI digestion, both derived from the P. aeruginosa PAO chromosome.     

SIRT1 top 40 hits: use of one-bead, one-compound acetyl-peptide libraries and quantum dots to probe deacetylase specificity

A novel, high-throughput method for determining deacetylase substrate specificity was developed using a one-bead, one-compound (OBOC) acetyl-peptide library with a quantum dot tagging strategy and automated bead-sorting. A 5-mer OBOC peptide library of 104,907 unique sequences was constructed around a central epsilon-amino acetylated lysine. The library was screened using the human NAD+-dependent deacetylase SIRT1 for the most efficiently deacetylated peptide sequences. Beads preferentially deacetylated by SIRT1 were biotinylated and labeled with streptavidin-coated quantum dots. After fluorescent bead-sorting, the top 39 brightest beads were sequenced by mass spectrometry. In-solution deacetylase assays on randomly chosen hit and nonhit sequences revealed that hits correlated with increased catalytic activity by as much as 20-fold. We found that SIRT1 can discriminate peptide substrates in a context-dependent fashion.     

从噬菌体展示肽库中筛选脂质A的结合肽

以脂质A为靶标,筛选噬菌体展示十二肽库,三轮后随机挑取14个噬菌体克隆进行结合活性鉴定,并对5个克隆进行序列分析。结果表明,14个克隆全部是阳性克隆,测序结果显示4个阳性克隆的序列完全一样。说明筛选到了一个脂质A的结合多肽。     

盐生杜氏藻(Dunaliella salina)cDNA文库构建及功能基因筛选

采用Qiagen公司的植物总RNA提取技术、Clontech公司的CreatorTM技术平台以及SMARTTM技术进行cDNA文库构建.从杜氏藻中提取出了高质量的总RNA,通过PowerScript反转录酶反转录杜氏藻的总RNA,采用LD-PCR、酶处理等方法对cDNA进行等比例扩增、纯化,同时使用CHROMA SPIN-400柱子将cDNA分段化,最后将长片段连入pDNR-LIB质粒,1.5 kV,25 μ F电转化大肠杆菌JM109,得到含1.5×106个克隆子的原始文库,滴度为1.5×106cfu ml-1.结合酶切和PCR,对该文库的质量进行了鉴定和统计,文库的平均片段插入长度为1.5kb.采用烯醇酶和UDP葡萄糖脱氢酶的EST作为同源探针,对文库中的功能基因进行筛选,并采用放射性原位杂交法,对扩增文库进行了初筛和复筛,得到了含这两条基因全编码序列的cDNA,烯醇酶为1.8kb,UDP葡萄糖脱氢酶为1.9kb,为今后对该种进行大规模功能基因组学研究奠定基础.