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41.
We constructed a chromosome 9 lambda DNA library from flow-sorted maize chromosomes. Approximately 3 million maize chromosome
9 were collected with high purity by flow cytometric sorting of chromosomes isolated from an oat-maize chromosome 9 addition
line based on the cytogram of fluorescent pulse area versus fluorescent pulse width. Chromosome 9 DNA was partially digested
withBamH I, dephosphorylated, and ligated with arms ofBamH I-digested lambda DASH vector (Stratagene). A total of 2.0×106 independent recombinants with an average insert size of 15 kb were obtained. For a 99% probability that every sequence of
chromosome 9 is represented in at least one chimeric phage, 5.6×104 cloned fragments are needed. This library covers the entire maize chromosome 9. Hybridizing cloned fragments with labeled
maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones.
This individual chromosome library is useful in plant genome mapping and gene isolation. 相似文献
42.
Jane C. Spetzler Vibeke Westphal Jakob R. Winther Morten Meldal 《Journal of peptide science》1998,4(2):128-137
Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluoroescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluoroescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA‒beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead‒linked library and a peptide containing the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluoroescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were also synthesized. © 1998 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
43.
银合欢基因文库的构建和种子贮藏蛋白基因的分离 总被引:1,自引:0,他引:1
潘志强 《Acta Botanica Sinica》1990,32(6):420-424
本文以λEMBL_3为载体,构建了银合欢(Leucaena leucocephala)的基因文库,所得重组子为3.5×10~6pfu。以大豆种子贮藏蛋白α′亚基基因为探针,从基因文库中分离得到了4个阳性克隆,并初步绘制了其中3个重组子的物理图谱。结果表明在这3个重组子的基因内部有一致的酶切位点。亚克隆的部分核苷酸序列与 GenBank 中的基因序列比较,表明与大豆种子贮藏蛋白α′亚基基因高度同源。 相似文献
44.
《基因组蛋白质组与生物信息学报(英文版)》2020,18(2):104-119
To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000. 相似文献
45.
46.
Yésica García‐Ramos Marta Paradís‐Bas Judit Tulla‐Puche Fernando Albericio 《Journal of peptide science》2010,16(12):675-678
CM resin is a totally PEG‐based resin, made exclusively from primary ether bonds and therefore highly chemically stable. Compared to other PEG resins, it exhibits good loading and is user friendly because of its free‐flowing form upon drying. It shows improved performance over PS resins for the preparation of hydrophobic, highly structured poly‐Arg peptides. In combination with ψPros, it allows the synthesis of small proteins such as the chemokine RANTES. Like other PEG‐based resins, CM resin swells well in biocompatible solvents such as water, thereby allowing on‐bead screening. Furthermore, the high loading of this resin permits the use of a tiny quarter of a bead as a microreactor for HPLC and MALDI‐TOF analysis, thus further extending its applications in the field of combinatorial chemistry. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
47.
Peterson RW Dutton PL Wand AJ 《Protein science : a publication of the Protein Society》2004,13(3):735-751
Accurate prediction of the placement and comformations of protein side chains given only the backbone trace has a wide range of uses in protein design, structure prediction, and functional analysis. Prediction has most often relied on discrete rotamer libraries so that rapid fitness of side-chain rotamers can be assessed against some scoring function. Scoring functions are generally based on experimental parameters from small-molecule studies or empirical parameters based on determined protein structures. Here, we describe the NCN algorithm for predicting the placement of side chains. A predominantly first-principles approach was taken to develop the potential energy function incorporating van der Waals and electrostatics based on the OPLS parameters, and a hydrogen bonding term. The only empirical knowledge used is the frequency of rotameric states from the PDB. The rotamer library includes nearly 50,000 rotamers, and is the most extensive discrete library used to date. Although the computational time tends to be longer than most other algorithms, the overall accuracy exceeds all algorithms in the literature when placing rotamers on an accurate backbone trace. Considering only the most buried residues, 80% of the total residues tested, the placement accuracy reaches 92% for chi(1), and 83% for chi(1 + 2), and an overall RMS deviation of 1 A. Additionally, we show that if information is available to restrict chi(1) to one rotamer well, then this algorithm can generate structures with an average RMS deviation of 1.0 A for all heavy side-chains atoms and a corresponding overall chi(1 + 2) accuracy of 85.0%. 相似文献
48.
49.
破色期番茄果实均一化cDNA沉默文库的构建和功能基因筛选模型的初步建立 总被引:2,自引:0,他引:2
番茄果实的成熟是由多基因精细调控的一个过程.利用破色期番茄果实,根据复性动力学原理在mRNA水平进行均一化操作使高丰度和低丰度的mRNA丰度接近.然后把均一化之后mRNA反转录得到cDNA,再与基因沉默载体pTRV重组,最后把构建好的载体通过电转化的方法转入到GV3101农杆菌中,从而建立起破色期番茄果实均一化cDNA沉默文库.通过番茄果实中病毒诱导基因沉默技术,对cDNA沉默文库进行初步筛选,从而确定功能基因筛选模型.在模型建立阶段,以番茄红素合成途径相关的PDS基因作为内标基因,在100个混合农杆菌样中,成功筛选到了PDS基因. 相似文献
50.