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At least 1,423 records · Page 79Linked to original sources
PubMed1996

Construction of a normalized directionally cloned cDNA library from adult heart and analysis of 3040 clones by partial sequencing.

Large-scale sequencing of clones from cDNA libraries derived from specific tissues is a rapid and efficient way of discovering novel genes expressed in those tissues. However, because the heart is continually contracting and relaxing, it strongly expresses muscle-contractile genes and/or mitochondrial genes, a bias that reduces the efficiency of this method. To improve the efficiency of identifying novel genes expressed in the heart, we constructed a normalized directionally cloned cDNA library from adult heart and partially sequenced 3040 clones. Comparisons of these sequence data with known DNA sequences in the database revealed that 57.1% of the clones matched human genes already known, 23.4% were identical or almost identical to human expressed sequence tags (ESTs), 14.2% bore no significant homology to any sequences in the database, and 1.2% represented repetitive sequences. The remaining 4.1% showed some homology with known genes, and Northern blot analysis of several clones in this category revealed that most of them were expressed mainly in the heart and skeletal muscle. After redundancy was excluded, the 3040 clones accounted for 1395 distinctive ESTs, 446 of which exhibited no match to any known sequence. Our results suggest that our normalized library is less redundant than standard libraries and is a useful resource for cataloging genes expressed in the heart.

Adult↗
PubMed1997

Characterization of peptides that bind the tumor-associated Thomsen-Friedenreich antigen selected from bacteriophage display libraries.

Peptides with high affinities and specificities for numerous proteins and nucleic acids have been previously identified from random peptide bacteriophage display libraries. Here, random peptide bacteriophage display libraries were used to identify sequences that bound the cancer-associated Thomsen-Friedenreich glycoantigen (T antigen). The T antigen, present on most malignant cells, contains an immunodominant Gal beta1 --> 3GalNAc alpha disaccharide unmasked on the surfaces of most carcinomas. This antigen has been postulated to be involved in tumor cell aggregation and metastasis. Two 15 amino acid random peptide bacteriophage display libraries were affinity selected with glycoproteins displaying T antigen on their surfaces. Sequence analysis revealed that many of the peptides shared homology with sugar recognition sites in several carbohydrate-binding proteins. A comparison of affinity selected sequences from both libraries yielded a common motif (W-Y-A-W/F-S-P) rich in aromatic amino acids. Four peptides, corresponding to the affinity selected sequences, were chemically synthesized and characterized for their carbohydrate recognition properties. The synthetic peptides exhibited high specificities and affinities to T antigen displayed on asialofetuin or conjugated to bovine serum albumin (Kd = 5 nM for MAP-P30 binding to asialofetuin) as well as free T-antigen disaccharide in solution (Kd = 10 microM for MAP-P30, 20 microM for P10). Two peptides, P30 and P10, demonstrated high affinities and specificities for both asialofetuin and T antigen in solution. Iodination of a lone tyrosine residue in each sequence dramatically reduced their abilities to bind T antigen, suggesting that the tyrosine residue plays an important role in carbohydrate recognition. That these peptides are of functional significance is evidenced by the ability of both P30 and P10 to inhibit asialofetuin-mediated melanoma cell aggregation in vitro and to compete with peanut lectin for binding to T antigen displayed on the surface of MDA-MB-435 breast carcinoma cells in situ.

Amino Acid Sequence↗
PubMed2000

Display of active subtilisin 309 on phage: analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors.

Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentus on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-l-Ala-l-Ala-l-P ro-Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-l-Lys-l-A la-l-Pro-Phe(P)-diphenylester allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.

Alkanesulfonic Acids↗
PubMed2000

Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides.

By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V(H) and seven V(L) (four Vkappa and three Vlambda) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs. Molecular modeling verified that all canonical classes were present. We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A first set of antibody phage display libraries totalling 2x10(9) members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question. Diversity was created by replacing the V(H) and V(L) CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences. The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 %. Selection experiments against many antigens yielded a diverse set of binders with high affinities. Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes.

Amino Acid Sequence↗
PubMed2000

Bacteriophage lambda display of complex cDNA libraries: a new approach to functional genomics.

We describe the construction and characterization of two lambda surface displayed cDNA expression libraries derived from human brain and mouse embryo. cDNA inserts were obtained by tagged random-priming elongation of commercially available cDNA libraries and cloned into a novel lambda vector at the 3' end of the D capsid protein gene, which produced highly complex repertoires (1x10(8) and 2x10(7) phage). These libraries were affinity selected with a monoclonal antibody against the neural specific factor GAP-43 and with polyclonal antibodies that recognize the EMX1 and EMX2 homeoproteins. In both cases rapid identification of specific clones was achieved, which demonstrates the great potential of the lambda display system for generating affinity selectable cDNA libraries from complex genomes.

Amino Acid Sequence↗
PubMed2000

Identification of natural ligands for SH2 domains from a phage display cDNA library.

The cytoplasmic domain of the Fc gamma receptor IIB (FcgammaRIIB) can be successfully displayed on the surface of filamentous phage, and after phosphorylation in vitro, can interact specifically with the SH2 domains of SHP-2, a cytoplasmic tyrosine phosphatase. When full-length FcgammaRIIB is expressed on phage, however, this interaction is greatly compromised, illustrating that characteristics of the full-length sequence are not well tolerated by the phage display system. Many associations in cell physiology are driven by similar interactions involving small modular binding domains or ligands, and so a fragmented cDNA library will facilitate display of such domains free of sequences which compromise their expression. A fragmented leukocyte cDNA display library of 10(8) clones was constructed. This library was phosphorylated in vitro with fyn kinase and was selected against the tandem SH2 domains of SHP-2 in the search for additional ligands. A depletion strategy to remove non-specific clones was employed, using SHP-2 Sepharose, prior to in vitro phosphorylation and selection. This permitted the emergence of clones encoding the cytoplasmic domain of PECAM-1, another natural ligand for SHP-2. The importance of dual phosphorylation of tyrosine residues at positions 663 and 686 was confirmed in competition ELISA experiments using phosphorylated phage and synthetic peptides. Thus, phage display of fragmented cDNA libraries permits the identification and characterisation of phosphorylated ligands of modular binding domains based on their functional interaction.

Amino Acid Sequence↗
PubMed2001

Assignment of homology to genome sequences using a library of hidden Markov models that represent all proteins of known structure.

Of the sequence comparison methods, profile-based methods perform with greater selectively than those that use pairwise comparisons. Of the profile methods, hidden Markov models (HMMs) are apparently the best. The first part of this paper describes calculations that (i) improve the performance of HMMs and (ii) determine a good procedure for creating HMMs for sequences of proteins of known structure. For a family of related proteins, more homologues are detected using multiple models built from diverse single seed sequences than from one model built from a good alignment of those sequences. A new procedure is described for detecting and correcting those errors that arise at the model-building stage of the procedure. These two improvements greatly increase selectivity and coverage. The second part of the paper describes the construction of a library of HMMs, called SUPERFAMILY, that represent essentially all proteins of known structure. The sequences of the domains in proteins of known structure, that have identities less than 95 %, are used as seeds to build the models. Using the current data, this gives a library with 4894 models. The third part of the paper describes the use of the SUPERFAMILY model library to annotate the sequences of over 50 genomes. The models match twice as many target sequences as are matched by pairwise sequence comparison methods. For each genome, close to half of the sequences are matched in all or in part and, overall, the matches cover 35 % of eukaryotic genomes and 45 % of bacterial genomes. On average roughly 15% of genome sequences are labelled as being hypothetical yet homologous to proteins of known structure. The annotations derived from these matches are available from a public web server at: http://stash.mrc-lmb.cam.ac.uk/SUPERFAMILY. This server also enables users to match their own sequences against the SUPERFAMILY model library.

Amino Acid Sequence↗
PubMed1999

Combinatorial chemistry: polymer supported synthesis of peptide and non-peptide libraries.

In recent years, combinatorial chemistry has emerged as a powerful tool for accelerating drug discovery. While industry is rapidly embracing the technology, researchers continue to develop novel library methods including resins, linkers, tagging and deconvolution techniques. Newer strategies involving computer-customized combinatorial libraries offer enormous potential for the design of more "focused" and "smart" chemical libraries with maximal diversity. In addition, miniaturized systems for synthesizing chemical libraries are also being developed, which has made it possible to carry out reactions at submicroliter volumes.

Animals↗
PubMed1992

Construction and characterisation of a yeast artificial chromosome library containing three haploid maize genome equivalents.

We have constructed a yeast artificial chromosome (YAC) library using high-molecular-weight DNA prepared from agarose-embedded leaf protoplasts of the maize inbred line UE95. This library contains 79,000 clones with an average insert size of 145 kb and should therefore represent approximately three haploid genome equivalents. The library is organised as an ordered array in duplicate microtitre plates. Forty-one pools of DNA from 1920 individual clones have been prepared for rapid screening of the library by the polymerase chain reaction (PCR). Using this approach, together with conventional colony hybridisation, we have been able to identify between one and eight positive clones for every probe used.

Chloroplasts↗
PubMed1994

A region-specific microdissection library for human chromosome 2p23-p25 and the analysis of an interstitial deletion of 2p23.3-p25.1.

A region-specific library for human chromosome 2p23-p25 was constructed using microdissection and polymerase chain reaction (PCR)-mediated microcloning techniques. This library is large, comprising 300,000 recombinant microclones. The insert sizes range between 50-600 base pairs (bp) with a mean of 200 bp. About 50%-60% of the clones contain unique or very low copy number sequence inserts as determined by their weak or no hybridization to total human DNA. A subset of 48 microclones that did not hybridize to total human DNA after colony hybridization was analyzed, and 26 (54%) clones were shown to contain single-copy inserts and hybridize to human chromosome 2 DNAs, indicating that they are human chromosome 2 specific. The human genomic fragments identified by these clones after cleavage with HindIII have also been characterized. The single-copy microclones were used to analyze an interstitial deletion in the 2p23.3-p25.1 region--46,XY, del(2) (pter-->p25.1::p23.3-->qter)--previously reported in a patient with severe growth and mental retardation and multiple anomalies. Of the 26 microclones analyzed, 14 clones were mapped to the deletion region. The availability of the 2p23-p25 region-specific library and the probes derived from the library should be valuable for fine structure physical mapping analysis and the cloning of disease-related genes localized to the region. These studies also demonstrate the efficiency with which useful probes can be quickly generated for genome studies and for positional cloning.

Animals↗
PubMed1991

Construction and characterization of a yeast artificial chromosome library of Arabidopsis which is suitable for chromosome walking.

A yeast artificial chromosome (YAC) genomic library of Arabidopsis thaliana was constructed in a derivative of the vector pYAC4 which was modified to facilitate the production of end-specific probes for chromosome walking. Experiments in which a subset of 2300 clones from the library were probed with 30 restriction fragment length polymorphism (RFLP) markers indicated that, on the average, the entire genome is represented once in each 800 YAC clones. Thus, the complete library of more than 20,000 YACs is expected to contain most or all of the Arabidopsis genome with a high probability. The YAC clones examined in the sample had an average insert size of approximately 150 kb +/- 10 and represented more than 5% of the Arabidopsis genome. Based on the properties of the library and the currently available RFLP maps for Arabidopsis, only one or two steps from flanking RFLPs should be sufficient to isolate an average gene in Arabidopsis by chromosome walking with the YACs. In order to facilitate chromosome walking, a method for the production of hybridization probes from the ends of the inserts was employed that is based on a combination of pre-amplification of the vector/insert junction sequences via the polymerase chain reaction and specific transcription from T3 or T7 RNA polymerase promoters flanking the cloning site in the YAC vector.

Base Sequence↗
PubMed1992

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 alpha 1(I) collagen (Col1a1) 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.

Base Sequence↗
PubMed1992

A mouse genomic library in the bacteriophage P1 cloning system: organization and characterization.

Using the bacteriophage P1 cloning system, we have constructed a two to three times coverage, high-molecular-weight (HMW) genomic library from mouse C127 fibroblast cells. The library consists of about 127,500 clones with an average insert size of about 70 kb that are organized into 300 primary pools containing approximately 425 clones per pool. For screening purposes the primary pools are combined into secondary pools (approximately 4250 clones each) and tertiary pools (approximately 21,250 clones each). Screening is performed by the polymerase chain reaction (PCR) with DNA isolated from the secondary and tertiary pools. We have screened the library for 13 different mouse sequences and have detected 11. Clones generated from two of the eleven positive screens were isolated from the library (those containing the c-fos and G alpha i2 genes) and were further characterized. Direct double-stranded sequencing of DNA from P1 clones with primers bordering the insert provided sequence information from each end of the cloned DNA.

Animals↗
PubMed1995

Construction of a porcine YAC library and mapping of the cardiac muscle ryanodine receptor gene to chromosome 14q22-q23.

Large-scale physical mapping of the porcine genome has been limited because up to now no suitable genomic libraries for this purpose have been available. Therefore, we have constructed a yeast artificial chromosome (YAC) library from porcine lymphocytes. The library was cloned in the amplifiable vector pCGS966. A total of 10080 YAC clones was obtained and has been ordered into 105 96-well microtiter plates. An average insert size of 300 kb was calculated from the analysis of 78 randomly selected clones, giving a one-fold coverage of the porcine genome. To analyze the complexity, we have screened the library for five different genes and isolated four different clones containing parts of three of these genes. One YAC clone harboring parts of the porcine cardiac muscle ryanodine receptor (RYR2) gene allowed us to assign this locus to Chromosome (Chr) 14q22-q23. The data were confirmed by PCR analysis of a rodent-porcine hybrid cell panel.

Animals↗
PubMed1994

Characterization of a cosmid library from flow-sorted chromosomes 11.

A cosmid library specific for human chromosome 11 has been constructed from flow-sorted chromosomes. The flow-purified chromosomes were prepared from the hamster/human hybrid line J1 which contains chromosome 11 as the only human chromosome. Individual clones were sampled in 187 microtitre plates, resulting in a total of 17,952 colonies. Hybridization analysis revealed that 83.7% of these clones were of human and 10.4% of hamster origin. The average insert size was estimated at 33.6 kb, and only 2.4% of insert fragments appear to be rearranged. This should result in 494,487 kb of cloned human DNA representing 3.5 chromosome 11 equivalents. We have prepared high-density nylon membranes of the arrayed library containing 1,536 single colonies per filter. We have demonstrated the usefulness of the library in the molecular genetic analysis of human chromosome 11 by testing for the presence of possibly polymorphic simple repeat motifs, by identifying cosmids that contain inserts from the telomeric ends of chromosome 11 and by assessing the potential of the library for rapid chromosome walking.

Animals↗
PubMed1996

Binding properties of SH3 peptide ligands identified from phage-displayed random peptide libraries.

Combinatorial libraries have yielded high-affinity ligands for SH3 domains of a number of different proteins. We have shown that synthetic peptides containing these SH3 ligand sequences serve as specific probes of SH3 domains. Direct binding of the N-terminal biotinylated peptide ligands was conveniently detected in ELISA, filter-blotting, and dot-blotting experiments with the use of streptavidin-conjugated enzymes. In some cases, detection of peptide-SH3 interactions required that the biotinylated peptides first were preconjugated with streptavidin to form a multivalent complex. Interestingly, these nominally tetravalent SH3 peptide ligands cross-react to varying degrees with different SH3 domains. We have used such complexes to screen lambda cDNA expression libraries and have isolated clones that encode both known and novel SH3-domain-containing proteins. Based on the success of this methodology, we propose a general strategy by which ligands of a modular domain-containing protein can be isolated from random peptide libraries and used to screen cDNA expression libraries systematically for novel modular domain-containing proteins.

Amino Acid Sequence↗
PubMed1996

Identification of peptide sequences that bind the Thomsen-Friedenreich cancer-associated glycoantigen from bacteriophage peptide display libraries.

The goal of this study was to determine if polypeptides that bind specifically to the carcinoma-associated Thomsen-Friedenreich (T) antigen could be isolated from a random peptide bacteriophage display library. T antigen is a carbohydrate antigen that is exposed and immunoreactive on the surfaces of most primary carcinomas and their metastases, while it is masked on normal cells. Tumor-specific surface carbohydrates are often used as markers of cell differentiation and play a role in cell aggregation, which is an important step in the metastatic process. Therefore, peptides that bind and mask T antigen may yield useful carbohydrate-specific probes and provide insight into carbohydrate-mediated tumor-cell aggregation. A 15-amino acid random peptide bacteriophage display library was screened for polypeptides that exhibited high specificity to two glycoproteins which display T antigen on their surfaces. The results suggest that synthetic peptides identified from the bacteriophage display library have high affinities (Kd approximately 1 microM) and specificities for proteins and human tumor cells which present T antigen. Thus, random bacteriophage peptide display libraries may be a rich source of sequences that bind to carbohydrate antigen structures.

Amino Acid Sequence↗
PubMed1996

Exploring antibody polyspecificity using synthetic combinatorial libraries.

Extensive mapping studies for seven antigen-antibody interactions have been carried out using both individual analogs and peptide libraries. With competitive ELISA, these studies have revealed that monoclonal antibodies exhibit a broad range of specificities, from antibodies that recognize only conservative substitutions for 1-2 positions of the antigenic determinant, to antibodies that recognize sequences that are completely unrelated to the parent antigen with comparable affinities. Synthetic combinatorial libraries, containing millions of peptide sequences, permit a more systematic and rapid evaluation of the extent of multiple-binding specificities of monoclonal antibodies than individual analogs. The peptide libraries used here comprise mixtures of compounds having specifically defined positions and mixture positions. The same diversity of sequences in different formats, which differ by the numbers of positions singularly defined and different locations defined within the sequence, can be examined. Comparison of the screening results, selection criteria of the most active mixtures, and different approaches used for the deconvolution of active individual compounds are discussed. Synthetic combinatorial libraries greatly facilitate the understanding of antigen-antibody interactions at the amino acid level and will assist in the development of improved immunodiagnostics.

Amino Acid Sequence↗