Screening of a synthetic pentapeptide library composed of d-amino acids against fructose-1,6-biphosphate aldolase

Summary A synthetic peptide library, theoretically composed of 537 824 d-amino acid pentapeptides anchored on polystyrene beads, was prepared with each bead bearing a single pentapeptide sequence. This library was screened for interaction with fructose-1,6-biphosphate aldolase of T. brucei labelled with biotin. Affinity beads that bound the enzyme were selected with streptavidin-coated magnetic beads. A total of 19 beads were isolated and individually subjected to Edman microsequence analysis. The corresponding peptide sequences were synthesized and evaluated for enzyme activity inhibition.     

Development of a herbicide biosensor using a peptide receptor screened from a combinatorial library

The purpose of this study was to screen for peptides that bind herbicides with a chlorinated aniline chemical structure. A tetrapeptide library was constructed using a solid phase split synthesis approach. Peptide beads were suspended in a buffer containing fluorescent-labeled dichloroaniline (DCA) as the bait. Eighteen fluorescent peptide beads were selected which bound to the bait after two rounds of staining screenings. The beads were then stained and suspended in a solution containing an excess of DCA and five quenched peptide beads were subsequently selected that recognized the DCA moiety. The screened peptides had many sequence similarities. The binding affinity of the screened peptides to herbicides was analyzed using surface plasmon resonance (SPR). N′-(3,4-dichlorophenyl)-N,N-dimethylurea [3-(3,4-dichlorophenyl)-1,1-dimethylurea] solution was injected over the peptide immobilized SPR chip. The SPR signal was found to increase in proportion to the DCMU concentration, whereas no signal was obtained from the negative control, 2-(2-methyl-4-chlorophenoxy) propionic acid (MCPP). From these results it is suggested that the screened peptide selectively recognizes the chemical structure of DCA.     

Construction and characterization of a sheep BAC library of three genome equivalents

A sheep BAC library of over three genome equivalents was constructed and arrayed in superpools and row, column, and plate pools. The library contains 90,000 clones distributed in 39 superpools. The average insert size was estimated at 123 kb. The library was screened by PCR with 77 primer pairs corresponding to ovine microsatellites distributed throughout the genome. The probability of finding a random sequence in the library could be estimated at 0.96. Received: 2 November 1998 / Accepted: 29 January 1999     

中国美利奴细毛羊BAC文库的三维PCR筛选

本研究利用中国美利奴细毛羊全基因组BAC文库,构建了可供快速筛选的两级水平的混合池,一级混合池和二级混合池(Primary pools and secondary pools).一级混合池基于每一384-well盘而构建,由盘、行,列三维混合池组成,二级混合池基于整个BAC文库而构建.设计了一种基于PCR技术的快速筛选方法,先筛选二级混合池m再根据结果筛选相应的一级混合池.利用此方法只需一步共66个PCR反应即可从BAC丈库中7.4万个克隆中筛选出1个阳性克隆,或三步100个以内的PCR反应筛选出多个阳性克隆.以绵羊基因组多态性分子标记BF94-1为引物,用一步共66个PCR反应成功筛选到1个阳性克隆373D13.     

SELEX技术筛选纤维蛋白适配子的研究

目的:用纤维蛋白作为靶物质对ss DNA随机序列文库进行筛选,旨在获得高亲和力的纤维蛋白适配子。方法:在体外人工合成长度为99个核苷酸的ss DNA随机序列文库,文库中间区域为63个核苷酸的随机序列,两端为18个核苷酸的固定的引物序列;然后以羧基磁珠为介质包被纤维蛋白,利用指数级富集的配体系统进化技术(SELEX)对ss DNA随机序列文库进行反复筛选,当结合率不再提高时对筛选出的适配子进行连接、转化及测序分析。结果:羧基磁珠成功地包被了纤维蛋白,包被效率为87.65%,经15轮逐步递增压力的筛选,获得了纤维蛋白适配子群,经测序分析比对发现适配子有很好的多样性。结论:应用SELEX技术初步筛选出了亲和力较高的纤维蛋白适配子群,为下一步的鉴定及功能研究奠定了良好基础。     

小麦—簇毛表6VS／6AL易位系可转化人工染色体（TAC）文库的构建

可转化人工染色体（Ｔｒａｎｓｆｏｒｍａｔｉｏｎ ｃｏｍｐｅｔｅｎｔ Ａｒｔｉｆｉｃｉａｌ Ｃｈｒｏｍｏｓｏｍｅ,ＴＡＣ）是具有克隆和转移大片段基因能力的新型载体,是植被基因克隆和转化的有效工具。为了克隆泪科抗白粉病基因和其它基因,本研究用ＴＣＡ载体ｐＹＬＴＡＣ１７构建了带有抗白粉病基因Ｐｍ２１的小麦＝簇毛麦６ＶＳ／６ＡＬ易位系的基因组ＤＮＡ文库。该文库包含２１０万个克隆平均插入征段３５ｌｂ,相当于     

Screening of a synthetic peptide library against glycosomal phosphoglycerate kinase of Trypanosoma brucei

Summary A synthetic peptide library composed of polystyrene beads was prepared employing the split-synthesis approach, using the 20 natural l-amino acids except for cysteine. This library was screened for interaction with glycosomal phosphoglycerate kinase (gPGK) of T. brucei, labelled with fluorescein or with biotin. Affinity beads were individually subjected to Edman microsequence analysis. The corresponding peptide sequences were synthesized as free peptide acids and evaluated for enzyme activity inhibition. The pentapeptide NWMMF was able to selectively inhibit gPGK with an IC₅₀ of 80 M.     

Construction and characterization of a bovine BAC library with four genome-equivalent coverage

A bovine artificial chromosome (BAC) library of 105 984 clones has been constructed in the vector pBeloBAC11 and organized in 3-dimension pools and high density membranes for screening by PCR and hybridization. The average insert size, determined after analysis of 388 clones, was estimated at 120 kb corresponding to a four genome coverage. Given the fact that a male was used to construct the library, the probability of finding any given autosomal and X or Y locus is respectively 0.98 and 0.86. The library was screened for 164 microsatellite markers and an average of 3.9 superpools was positive for each PCR system. None of the 50 or so BAC clones analysed by FISH was chimeric. This BAC library increases the international genome coverage for cattle to around 28 genome equivalents and extends the coverage of the ruminant genomes available at the Inra resource center to 15 genome equivalents.     

Construction of a Coix BAC library and isolation of the 22 kDa α-coixin gene cluster

Coix lacryma-jobi L. (Coix) is a close relative of maize and is considered a valuable genetic resource for crop improvement. Here we report the construction of the first Coix bacterial artificial chromosome (BAC) library using accession PI 324059. This BAC library contains about 230?400 clones with an average insert size of 113?kb, has low organellar DNA contamination, and provides 16.3-fold coverage of the genome. The library was stored in 12?× 96 pools that could be screened with a PCR protocol. Library screening was performed for the 22?kDa α-coixin gene family. A total of 57 positive pools were identified, and single clones were isolated from 19 of these pools. Based on DNA fingerprinting and Southern blot analysis, these 19 BAC clones form a single contig of about 340?kb in length, indicating that the 22 kDa α-coixin genes occur in a cluster. These results demonstrated the suitability of this BAC library for gene isolation and comparative genomics studies of the Coix genome.     

A novel manual pooling system for preparing three-dimensional pools of a deep coverage soybean bacterial artificial chromosome library

Compared with hybridization‐based techniques, polymerase chain reaction‐based screening of large insert libraries has been used widely as it is fast, easy and sensitive. However, various pooling strategies are needed to ensure efficient screening. It is time‐consuming and labourious to prepare three‐dimensional pools for a deep coverage bacterial artificial chromosome (BAC) library of soybean (1.12 × 10⁹ bp) in the absence of robotic facility. In the present study, we describe a novel manual pooling system for preparing three‐dimensional pools of a soybean BAC library. This simple technique enables a single researcher to construct three‐dimensional pools for a deep‐coverage (12 haploid genome equivalents) BAC library of soybean in less than 2 months without any robotic manipulation. When the prepared three‐dimensional pools were screened with 29 polymerase chain reaction‐based markers, an average of 9.2 clones per marker were identified. These identified clones will be useful either in quantitative trait loci gene isolation or in synteny study between soybean and other legumes including Lotus japonicus. This efficient pooling system could be applied to any other BAC libraries without the need for robotic manipulation.     

Identification of protein-binding peptides by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of peptide beads selected from the screening of one bead-one peptide combinatorial libraries

A fast and inexpensive strategy for the identification of peptide ligands by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of peptide beads screened from one bead-one peptide combinatorial libraries is herein described. Streptavidin was used as the model protein. A combinatorial library of 6561 peptides was synthesized on ChemMatrix resin by the divide-couple-recombine method. 4-Hydroxymethylbenzoic acid was used as the linker and five residues of Gly were incorporated at the C termini to increase the final peptide molecular weight. Positive control peptides with the HPQ motif and negative control peptides without the HPQ motif evidenced that the linker and the five residues of Gly have neither impaired the specific binding nor facilitated unspecific binding. After screening the library, positive beads were isolated and washed with 8M guanidine hydrochloride. The beads were sliced into two or four pieces, deposited onto the stainless steel MALDI sample plate, and treated with ammonia vapor to release the peptides. In addition, 26 beads picked at random from the library were subjected to the same treatment. All samples were analyzed by MALDI-TOF-MS and the peptides were unambiguously identified with very good reproducibility between the bead pieces, thus evidencing the good homogeneity of the bead. All sequences obtained from the screening contained HPQ.     

Screening one bead one compound libraries against serum using a flow cytometer: Determination of the minimum antibody concentration required for ligand discovery

One bead one compound (OBOC) libraries can be screened against serum samples to identify ligands to antibodies in this mixture. In this protocol, hit beads are identified by staining with a fluorescent labeled secondary antibody. When screens are conducted against two different sets of serum, antibodies, and ligands to them, can be discovered that distinguish the two populations. The application of DNA-encoding technology to OBOC libraries has allowed the use of 10?µm beads for library preparation and screening, which pass through a standard flow cytometer, allowing the fluorescent hit beads to be separated from beads displaying non-ligands easily. An important issue in using this approach for the discovery of antibody biomarkers is its analytical sensitivity. In other words, how abundant must an IgG be to allow it to be pulled out of serum in an unbiased screen using a flow cytometer? We report here a model study in which monoclonal antibodies with known ligands of varying affinities are doped into serum. We find that for antibody ligands typical of what one isolates from an unbiased combinatorial library, the target antibody must be present at 10–50?nM. True antigens, which bind with significantly higher affinity, can detect much less abundant serum antibodies.     

Construction of a BAC Library for a Defoliating Insect-Resistant Soybean and Identification of Candidate Clones Using a Novel Approach

Positional cloning of an insect-resistance quantitative trait locus (QTL) requires the construction of a large-insert genomic DNA library from insect-resistant genotypes. To facilitate cloning of a major defoliating insect-resistance QTL on linkage group M of the soybean genetic map, a bacterial artificial chromosome (BAC) library for PI 229358 was constructed and characterized. The HindIII BAC library contains 55,296 clones with an average insert size 131 kb. This library represents a 6-fold soybean haploid genome equivalents, allowing a 99.8% probability of recovering any specific sequence of interest in soybean. BAC filters were screened with a genomic DNA probe Sat_258sc2 obtained through genome walking from flanking sequences of a simple sequence repeat (SSR) marker, Sat_258, which links to the insect-resistance QTL. Thirteen BAC clones were identified positive for Sat_258sc2, and two of them were confirmed to carry Sat_258. The results suggest that this library is useful in positional cloning of the major insect-resistance QTL, and the approach presented here can be used to screen a BAC library for a SSR marker without requiring the creation of BAC pools.     

Matrix Gla protein is differentially expressed during the deposition of a calcified matrix by vascular pericytes

PCR-based subtractive hybridisation was used to identify genes up-regulated when pericytes undergo osteogenic differentiation and deposit a calcified matrix. cDNA pools were generated from confluent pericytes and from pericyte cultures containing calcified nodules. A pericyte cDNA library was screened with the product of the subtraction procedure (calcified minus confluent cDNA) and the majority of the positive clones were identified as matrix Gla protein (MGP). Northern analysis and immunohistochemistry demonstrated that MGP was only expressed by pericytes in calcified nodules. Antibodies to MGP inhibited the deposition of a calcified matrix by pericytes, suggesting that MGP regulates both cell differentiation and calcification.     

A strategy for rapid production and screening of yeast artificial chromosome libraries

We describe methods for rapid production and screening of yeast artificial chromosome (YAC) libraries. Utilizing complete restriction digests of mouse genomic DNA for ligations in agarose, a 32,000-clone library was produced and screened in seven weeks. Screening was accomplished by subdividing primary transformation plates into pools of approximately 100 clones which were transferred into a master glycerol stock. These master stocks were used to inoculate liquid cultures to produce culture pools, and ten pools of 100 clones were then combined to yield superpools of 1,000 clones. Both pool and superpool DNA was screened by polymerase chain reaction (PCR) and positive pools representing 100 clones were then plated on selective medium and screened by in situ hybridization. Screening by the two tiered PCR assay and by in situ hybridization was completed in 4–5 days. Utilizing this methodology we have isolated a 150 kb clone spanning the 1(I) collagen (Colla1) gene as well as 40 kb clones from the Hox-2 locus. To characterize the representation of the YAC library, the size distribution of genomic Sal I fragments was compared to that of clones picked at random from the library. The results demonstrate significant biasing of the cloned fragment distribution, resulting in a loss of representation for larger fragments.     

Generation of a high-quality P1 library of Arabidopsis suitable for chromosome walking

Using improved techniques, a representative P1 library of Arabidopsis was constructed and characterized. Megabase genomic DNA was prepared from nuclei and partially digested with Sau3AI. DNA fragments of 75–100 kb were selected by size fractionation in low melting agarose, concentrated by a spot-evaporation/dialysis method, and cloned in the pAd10sacBII P1 vector. The library contains 10 080 clones individually stored in microtiter plates. With an average insert size of about 80 kb, the library represents about eight haploid genomic equivalents of this plant. This library can be screened rapidly by dot hybridization of plate and well position pools. Characterization of the library by restriction analysis, screening with RFLP probes, RFLP mapping of insert end sequences, and chromosome walking shows that the library is of high quality with respect to insert site, completeness, and absence of chimeric artifacts. With this library a contig of about 600 kb has been constructed in the cer9 locus region. This P1 library is expected to be useful for genome mapping and gene cloning in Arabidopsis research programs.     

Rapid isolation of rare clones from highly complex DNA libraries by PCR analysis of liquid gel pools

A semiliquid medium was employed in an efficient method to screen highly complex DNA libraries for clones with known sequences. Unbiased clone pools are generated in 2 ml vials and screened by whole-cell PCR, and individual clones are obtained by few additional rounds of dilution and PCR screening. To demonstrate the utility of this approach, the single positive clone present in a 400,000 member metagenomic fosmid library was isolated.     

Construction and Characterization of a Human Chromosome 2-Specific BAC Library

We have constructed a human chromosome 2-specific bacterial artificial chromosome (BAC) library using DNA from the somatic cell hybrid GM10826. The average size of the clones is about 63 kb. The coverage and distribution of the library were estimated by screening with known polymorphic genetic markers and fluorescence in situ hybridization (FISH). Twentyone markers tested positive when DNA pools prepared from approximately one-sixth of the library were screened with 33 known markers. This is consistent with the theoretical calculation of 63% coverage at one genomic equivalent. This suggested that the coverage of the library is approximately 5-6×. FISH analysis with 54 BACs revealed single site hybridization to chromosome 2, and the clones were distributed randomly on the chromosome. We have also performed direct sequencing of the BAC insert ends to generate sequence-tagged sites suitable for mapping and chromosome walking. This is the first reported human chromosome 2-specific BAC library and should provide a resource for physical mapping and disease searching for this chromosome.     

Generation of a soybean BAC library, and identification of DNA sequences tightly linked to the Rps1-k disease resistance gene

 A soybean bacterial artificial chromosome (BAC) library, comprising approximately 45 000 clones, was constructed from high-molecular-weight nuclear DNA of cultivar Williams 82, which carries the Rps1-k gene for resistance against Phytophthora sojae. The library is stored in 130 pools with about 350 clones per pool. Completeness of the library was evaluated for 21 random sequences including four markers linked to the Rps1 locus and 16 cDNAs. We identified pools containing BACs for all sequences except for one cDNA. Additionally, when screened for possible contaminating BAC clones carrying chloroplast genes, no sequences homologous to two barley chloroplast genes were found. The estimated average insert size of the BAC clones was about 105 kb. The library comprises about four genome equivalents of soybean DNA. Therefore, this gives a probability of 0.98 of finding a specific sequence from this library. This library should be a useful resource for the positional cloning of Rps1-k, and other soybean genes. We have also evaluated the feasibility of an RFLP-based screening procedure for the isolation of BAC clones specific for markers that are members of repetitive sequence families, and are linked to the Rps1-k gene. We show that BAC clones isolated for two genetically linked marker loci, Tgmr and TC1-2, are physically linked. Application of this method in expediting the map-based cloning of a gene, especially from an organism, such as soybean, maize and wheat, with a complex genome is discussed. Received: 12 May 1998/Accepted: 24 August 1998     

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.