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PubMed2005

Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP).

cDNA cloning is a central technology in molecular biology. cDNA sequences are used to determine mRNA transcript structures, including splice junctions, open reading frames (ORFs) and 5'- and 3'-untranslated regions (UTRs). cDNA clones are valuable reagents for functional studies of genes and proteins. Expressed Sequence Tag (EST) sequencing is the method of choice for recovering cDNAs representing many of the transcripts encoded in a eukaryotic genome. However, EST sequencing samples a cDNA library at random, and it recovers transcripts with low expression levels inefficiently. We describe a PCR-based method for directed screening of plasmid cDNA libraries. We demonstrate its utility in a screen of libraries used in our Drosophila EST projects for 153 transcription factor genes that were not represented by full-length cDNA clones in our Drosophila Gene Collection. We recovered high-quality, full-length cDNAs for 72 genes and variously compromised clones for an additional 32 genes. The method can be used at any scale, from the isolation of cDNA clones for a particular gene of interest, to the improvement of large gene collections in model organisms and the human. Finally, we discuss the relative merits of directed cDNA library screening and RT-PCR approaches.

Animals↗

PubMed2006

Four-base codon mediated mRNA display to construct peptide libraries that contain multiple nonnatural amino acids.

In vitro selection and directed evolution of peptides from mRNA display are powerful strategies to find novel peptide ligands that bind to target biomolecules. In this study, we expanded the mRNA display method to include multiple nonnatural amino acids by introducing three different four-base codons at a randomly selected single position on the mRNA. Another nonnatural amino acid may be introduced by suppressing an amber codon that may appear from a (NNK)(n) nucleotide sequence on the mRNA. The mRNA display was expressed in an Escherichia coli in vitro translation system in the presence of three types of tRNAs carrying different four-base anticodons and a tRNA carrying an amber anticodon, the tRNAs being chemically aminoacylated with different nonnatural amino acids. The complexity of the starting mRNA-displayed peptide library was estimated to be 1.1 x 10(12) molecules. The effectiveness of the four-base codon mediated mRNA display method was demonstrated in the selection of biocytin-containing peptides on streptavidin-coated beads. Moreover, a novel streptavidin-binding nonnatural peptide containing benzoylphenylalanine was obtained from the nonnatural peptide library. The nonnatural peptide library from the four-base codon mediated mRNA display provides much wider functional and structural diversity than conventional peptide libraries that are constituted from 20 naturally occurring amino acids.

Amino Acids↗

PubMed2002

Alpha-helically constrained phage display library.

The library described here is a collection of phages with six degenerate codons in gene VIII, specifying amino acids 12, 13, 15-17 and 19 of the major coat protein. The randomized positions are surface exposed in the wild-type protein and thus might be expected to tolerate a great diversity of side chains without compromising phage viability. In agreement with this supposition, the new library showed great diversity of amino acids at the randomized positions and diversity did not diminish noticeably during repeated subculture. Despite their diversity, however, the randomized positions should be strongly constrained conformationally because they lie in an extended alpha-helical portion of the protein, stabilized by numerous inter- and intra-subunit contacts--a presupposition corroborated by circular dichroism spectroscopy of many library members. To reflect this conformational homogeneity and the fact that random amino acids subtend a major fraction of the surface 'landscape' of the particle, we call the new construct an alpha landscape library. It can be used as a source of alpha-helical ligands and substitute antibodies.

Capsid Proteins↗

PubMed2002

Efficient selection of genomic clones from a female chicken bacterial artificial chromosome library by four-dimensional polymerase chain reactions.

A bacterial artificial chromosome (BAC) library consisting of 49,152 genomic clones was constructed from partially HindIII-digested female chicken embryo genomic DNA using the pBAC-Lac vector and maintained in 512 96-well plates. The mean insert size was approximately 150 kb, and the total library was estimated to contain about 3.2 times coverage of the diploid genome. In order to screen this library by the PCR, 296 BAC clone DNA samples were prepared: one sample each from 8 superpools (64 plates per superpool) and 36 samples of four-dimensionally (4-D) mixed clones from each superpool. A BAC clone of interest was selected by two-step PCR. First, 8 DNA samples representing superpools were subjected to PCR with a set of primers to amplify a part of the genomic sequence of interest. Second, 36 4-D DNA samples from the superpool that contained BAC clone(s) of interest were subjected to PCR with the same set of primers. The second step identified a plate and a well containing the BAC clone of interest. Selection of target BAC clone(s) from the whole library with the above procedure can be achieved within 1 to 4 d without using a radioactive probe. This procedure was applied successfully in the selection of BAC clones for Wpkci, chPKCI/HINT, ZOV3, and 17beta-HSD genes.

Amino Acid Sequence↗

PubMed2002

Development and mapping of microsatellite markers derived from chicken chromosome-specific libraries.

Chromosome-specific painting probes and libraries were developed for chicken Macrochromosomes 1, 2, 3, and 4 by chromosome microisolation and microcloning. Fluorescent in situ hybridization results using the painting probes on normal chicken metaphase chromosomes indicated the purity and specificity of each probe. Chromosome-specific libraries for chicken Macrochromosomes 1, 2, 3, and 4 were prepared in a phage vector. Fifty-two additional unique microsatellite markers of the (AC)n type were developed from these chromosome-specific libraries. These markers were mapped on the East Lansing reference population to increase the marker density on the four macrochromosomes. Results of the current study suggest that development of markers from chromosome-specific libraries is very useful for constructing high-density linkage maps for chicken macrochromosomes.

Animals↗

PubMed2005

Full transcriptome analysis of rhabdomyosarcoma, normal, and fetal skeletal muscle: statistical comparison of multiple SAGE libraries.

Rhabdomyosarcoma (RMS) is the most frequent soft tissue sarcoma in children. Improved treatment strategies have increased overall survival, but the response of approximately one-third of the patients is still poor. To increase the knowledge of RMS pathogenesis, we performed the first full transcriptome analysis of RMS using serial analysis of gene expression (SAGE). With a G-test for the simultaneous comparison of subsets of SAGE libraries of normal skeletal muscle, embryonal (ERMS) and alveolar (ARMS) RMS, we identified 251 differentially expressed genes. A literature-mining procedure demonstrated that 158 of these genes have not previously been associated with RMS or normal muscle. Gene Ontology (GO) analysis assigned 198 of the 251 genes to muscle-specific classes, including those involved in normal myogenic development, as well as tumor-related classes. Prominent GO classes were those associated with proliferation and actin reorganization, which are processes that play roles during early muscle development, muscle function, and tumor progression. Using custom microarrays, we confirmed the (up- or down-) regulation of 80% of 98 differentially expressed genes. Another SAGE library of 19- to 22-week-old fetal skeletal muscle was compared with the RMS and normal muscle transcriptomes. Cluster analysis showed that the RMS and fetal muscle SAGE libraries formed one cluster distinct from normal muscle samples. Moreover, the expression profile of 86% of the differentially expressed genes between normal muscle and RMS was highly similar in fetal muscle and RMS. In conclusion, the G-test is a robust tool for analyzing groups of SAGE libraries and correctly identifies genes marking the difference between fully differentiated skeletal muscle and RMS. This study not only substantiates the close association between embryonic myogenesis and RMS development but also provides a rich source of candidate genes to further elucidate the etiology of RMS or to identify diagnostic and/or prognostic markers.

Cell Adhesion↗

PubMed2001

A bacterial artificial chromosome library for sequencing the complete human genome.

A 30-fold redundant human bacterial artificial chromosome (BAC) library with a large average insert size (178 kb) has been constructed to provide the intermediate substrate for the international genome sequencing effort. The DNA was obtained from a single anonymous volunteer, whose identity was protected through a double-blind donor selection protocol. DNA fragments were generated by partial digestion with EcoRI (library segments 1--4: 24-fold) and MboI (segment 5: sixfold) and cloned into the pBACe3.6 and pTARBAC1 vectors, respectively. The quality of the library was assessed by extensive analysis of 169 clones for rearrangements and artifacts. Eighteen BACs (11%) revealed minor insert rearrangements, and none was chimeric. This BAC library, designated as "RPCI-11," has been used widely as the central resource for insert-end sequencing, clone fingerprinting, high-throughput sequence analysis and as a source of mapped clones for diagnostic and functional studies.

Chromosomes, Artificial, Bacterial↗

PubMed2002

Protein-protein interactions between large proteins: two-hybrid screening using a functionally classified library composed of long cDNAs.

Large proteins have multiple domains that are potentially capable of binding many kinds of partners. It is conceivable, therefore, that such proteins could function as an intricate framework of assembly protein complexes. To comprehensively study protein-protein interactions between large KIAA proteins, we have constructed a library composed of 1087 KIAA cDNA clones based on prior functional classifications done in silico. We were guided by two principles that raise the success rate for detecting interactions per tested combination: we avoided testing low-probability combinations, and reduced the number of potential false negatives that arise from the fact that large proteins cannot reliably be expressed in yeast. The latter was addressed by constructing a cDNA library comprised of random fragments encoding large proteins. Cytoplasmic domains of KIAA transmembrane proteins (>1000 amino acids) were used as bait for yeast two-hybrid screening. Our analyses reveal that several KIAA proteins bearing a transmembrane region have the capability of binding to other KIAA proteins containing domains (e.g., PDZ, SH3, rhoGEF, and spectrin) known to be localized to highly specialized submembranous sites, indicating that they participate in cellular junction formation, receptor or channel clustering, and intracellular signaling events. Our representative library should be a very useful resource for detecting previously unidentified interactions because it complements conventional expression libraries, which seldom contain large cDNAs.

Brain Chemistry↗

PubMed2006

Large-scale production of SAGE libraries from microdissected tissues, flow-sorted cells, and cell lines.

We describe the details of a serial analysis of gene expression (SAGE) library construction and analysis platform that has enabled the generation of >298 high-quality SAGE libraries and >30 million SAGE tags primarily from sub-microgram amounts of total RNA purified from samples acquired by microdissection. Several RNA isolation methods were used to handle the diversity of samples processed, and various measures were applied to minimize ditag PCR carryover contamination. Modifications in the SAGE protocol resulted in improved cloning and DNA sequencing efficiencies. Bioinformatic measures to automatically assess DNA sequencing results were implemented to analyze the integrity of ditag structure, linker or cross-species ditag contamination, and yield of high-quality tags per sequence read. Our analysis of singleton tag errors resulted in a method for correcting such errors to statistically determine tag accuracy. From the libraries generated, we produced an essentially complete mapping of reliable 21-base-pair tags to the mouse reference genome sequence for a meta-library of approximately 5 million tags. Our analyses led us to reject the commonly held notion that duplicate ditags are artifacts. Rather than the usual practice of discarding such tags, we conclude that they should be retained to avoid introducing bias into the results and thereby maintain the quantitative nature of the data, which is a major theoretical advantage of SAGE as a tool for global transcriptional profiling.

Animals↗

PubMed2003

Amyloid-forming peptides selected proteolytically from phage display library.

We demonstrated that amyloid-forming peptides could be selected from phage-displayed library via proteolysis-based selection protocol. The library of 28-residue peptides based on a sequence of the second zinc finger domain of Zif268, and computationally designed betabetaalpha peptide, FSD-1, was presented monovalently on the surface of M13 phage. The library coupled the infectivity of phage particles to proteolytic stability of a peptide introduced into the coat protein III linker. It was designed to include variants with a strong potential to fold into betabetaalpha motif of zinc finger domains, as expected from secondary structure propensities, but with no structure stabilization via zinc ion coordination. As our primary goal was to find novel monomeric betabetaalpha peptides, the library was selected for stable domains with the assumption that folded proteins are resistant to proteolysis. After less than four rounds of proteolytic selection with trypsin, chymotrypsin, or proteinase K, we obtained a number of proteolysis-resistant phage clones containing several potential sites for proteolytic attack with the proteinases. Eight peptides showing the highest proteolysis resistance were expressed and purified in a phage-free form. When characterized, the peptides possessed proteolytic resistance largely exceeding that of the second zinc finger domain of Zif268 and FSD-1. Six of the characterized peptides formed fibrils when solubilized at high concentrations. Three of them assembled into amyloids as determined through CD measurements, Congo red and thioflavin T binding, and transmission electron microscopy.

Amino Acid Sequence↗

PubMed2002

Phylogenetic analysis of the human gut microbiota using 16S rDNA clone libraries and strictly anaerobic culture-based methods.

The human gut microbiota from three healthy subjects were compared by the use of a sequence analysis of 16S rDNA libraries and a culture-based method. Direct counts ranged from 1.9 X 10" to 4.0 X 10" cells/g (wet weight), and plate counts totaled 6.6 X 10(10) to 1.2 X 10(11) CFU/g (wet weight). Sixty to seventy percent of the bacteria in the human intestinal tract cannot be cultured with currently available methods. The 16S rDNA libraries from three subjects were generated from total community DNA in the intestinal tract with universal primer sets. Randomly selected clones were partially sequenced. All purified colonies detected from the surface of the agar plate were used for a partial sequencing of 16S rDNA. On the basis of sequence similarities, the clones and colonies were classified into several clusters corresponding to the major phylum of the domain Bacteria. Among a total of 744 clones obtained, approximately 25% of them belonged to 31 known species. About 75% of the remaining clones were novel "phylotypes" (at least 98% similarity of clone sequence). The predominant intestinal microbial community consisted of 130 species or phylotypes according to the sequence data in this study. The 16S rDNA libraries and colonies included the Bacteroides group, Streptococcus group, Bifidobacterium group, and Clostridium rRNA clusters IV, IX, XIVa, and XVIII. Moreover, several previously uncharacterized and uncultured microorganisms were recognized in clone libraries and colonies. Our results also showed marked individual differences in the composition of intestinal microbiota.

Adult↗

PubMed2006

Market orientation: a concept for health libraries.

OBJECTIVES: This paper is the result of exploratory research forming part of ongoing study into the value and relevance of market orientation as a strategic option for library managers. The aim of the study is to gain an understanding of the concept of market orientation relative to the health library sector. METHODOLOGY: A focus group was used to gather data from health librarians working at different levels in the sector. The data was coded and categorized by an expert panel and analysed using a taxonomic map developed during the study. RESULTS: Health library professionals define and understand market orientation in the same way as the concept is defined in the management literature. Their understanding of the concept is developing. A greater emphasis is given to some aspects of market orientation than others. CONCLUSIONS: There are implications for further research. Methods used to measure market orientation in other domains are likely to be relevant for libraries. Research should be extended to different sectors to explore any cross-sector differences. Fostering an organizational culture that supports market orientation has implications for service management and development.

Communication↗

PubMedDate not supplied

Construction of a normalized cDNA library for the Trypanosoma cruzi genome project.

Sequencing of the Trypanosoma cruzi genome is underway. Expressed sequence tags, obtained from cDNA libraries, facilitate mapping and gene discovery. The efficiency of large-scale generation of such tags is increased when using normalized cDNA libraries, where the frequency of individual clones is brought within a narrow range. Repetitive sequencing of abundant clones is therefore minimized. We constructed a normalized cDNA library from epimastigotes of clone CL Brener, and the efficiency of normalization of representative clones was assessed and shown to be adequate. The normalized cDNA library has been distributed to several groups and large-scale sequencing is currently in progress.

Animals↗

PubMed2002

Rapid selection of anti-hapten antibodies isolated from synthetic and semi-synthetic antibody phage display libraries expressed in Escherichia coli.

Antibody phage display libraries (Griffin and Tomlinson I) displaying antibody genes and maintained and amplified in Escherichia coli were used to isolate antibodies to the hapten target microcystin LR (1000 Da) conjugated to either bovine serum albumin or keyhole limpet haemocyanin. In competition enzyme-linked immunosorbent assay, bacterially expressed antibodies selected via the Griffin library showed at least 300 times greater sensitivity than those isolated from the Tomlinson library, for free microcystin. Bacterially expressed phage antibody libraries provide a rapid and relatively easy route for the selection of monoclonal antibodies specific for even the most difficult of antigenic targets such as free haptens.

Antibodies, Monoclonal↗

PubMed2006

Selection of novel nickel-binding peptides from flagella displayed secondary peptide library.

Nickel (Ni) performs its biological or toxic functions in nickel-protein coordination form. Novel Ni-binding peptides were isolated from a random dodecapeptide library displayed on the flagella of Escherichia coli against immobilized ions. On the basis of isolated sequences rich in histidine residues, two secondary libraries were constructed respectively. By consequent selection, more Ni-chelating peptides were identified and the consensus motif RHXHR (where X was always H) was deduced. The result suggested that not only histidine, but also arginine, play an important role in Ni-binding. Furthermore, two selected clones (1035 and 2022) were chosen for further identification. They exhibited similar relative binding affinity, which was about nine times that of the original library derived clones and statistically much more significant than the positive control with polyhistidine insert. Free nickel ions could almost completely inhibit the binding of the clones 1035 and 2022 to immobilized nickel, implicating that the peptides were able to chelate nickel ions. These studies reveal that bacterial surface displayed peptide libraries may have promising future potential for the development of metal bioadsorbents. Furthermore, novel Ni-binding peptides may provide lead molecules for Ni-chelation and applications thereof.

Amino Acid Sequence↗

PubMed2004

DNA-templated organic synthesis and selection of a library of macrocycles.

The translation of nucleic acid libraries into corresponding synthetic compounds would enable selection and amplification principles to be applied to man-made molecules. We used multistep DNA-templated organic synthesis to translate libraries of DNA sequences, each containing three "codons," into libraries of sequence-programmed synthetic small-molecule macrocycles. The resulting DNA-macrocycle conjugates were subjected to in vitro selections for protein affinity. The identity of a single macrocycle possessing known target protein affinity was inferred through the sequence of the amplified DNA template surviving the selection. This work represents the translation, selection, and amplification of libraries of nucleic acids encoding synthetic small molecules rather than biological macromolecules.

Amides↗

PubMed2000

Suitability of PCR fingerprinting, infrequent-restriction-site PCR, and pulsed-field gel electrophoresis, combined with computerized gel analysis, in library typing of Salmonella enterica serovar enteritidis.

Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D = 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance of Salmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.

Animals↗

PubMed2003

Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products.

To further explore possible avenues for accessing microbial biodiversity for drug discovery from natural products, we constructed and screened a 5,000-clone "shotgun" environmental DNA library by using an Escherichia coli-Streptomyces lividans shuttle cosmid vector and DNA inserts from microbes derived directly (without cultivation) from soil. The library was analyzed by several means to assess diversity, genetic content, and expression of heterologous genes in both expression hosts. We found that the phylogenetic content of the DNA library was extremely diverse, representing mostly microorganisms that have not been described previously. The library was screened by PCR for sequences similar to parts of type I polyketide synthase genes and tested for the expression of new molecules by screening of live colonies and cell extracts. The results revealed new polyketide synthase genes in at least eight clones. In addition, at least five additional clones were confirmed by high-pressure liquid chromatography analysis and/or biological activity to produce heterologous molecules. These data reinforce the idea that exploiting previously unknown or uncultivated microorganisms for the discovery of novel natural products has potential value and, most importantly, suggest a strategy for developing this technology into a realistic and effective drug discovery tool.

Amino Acid Sequence↗