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PubMed2001

Hospital patient libraries and information services in Australia.

In order to find out how many and what kind of Hospital Patient Libraries exist in Australia I placed a "Request for Information" on our national electronic discussion list entitled 'aliaHealth'. This discussion list has presently nearly 300 subscribers. Most of them are located in Australia, but they also come from Canada, Germany, Hong Kong, the United Kingdom and New Zealand. The e-mail address for 'aliaHealth' is: aliaHealth@alianet.alia.org.au. The result of my enquiry was rather poor: there are three Hospital Patient Libraries in Western Australia, one in Queensland and two in Victoria. The criterion was that the Library or Patient Information Service had to be located within the Hospital grounds. The six Libraries or Information Services are described in detail.

Australia↗
PubMed2002

A method for the generation of combinatorial antibody libraries using pIX phage display.

For more than a decade, phage displayed combinatorial antibody libraries have been used to generate and select a wide variety of antibodies. We previously reported that the phage coat proteins pVII and pIX could be used to display the heterodimeric structure of the antibody Fv region. Herein, aspects of this technology were invoked and extended to construct a large, human single-chain Fv (scFv) library of 4.5 x 10(9) members displayed on pIX of filamentous bacteriophage. Furthermore, the diversity, quality, and utility of the library were demonstrated by the selection of scFv clones against six different protein antigens. Notably, more than 90% of the selected clones showed positive binding for their respective antigens after as few as three rounds of panning. Analyzed scFvs were also found to be of high affinity. For example, kinetic analysis (BIAcore) revealed that scFvs against staphylococcal enterotoxin B and cholera toxin B subunit had a nanomolar and subnanomolar dissociation constant, respectively, affording affinities comparable to, or exceeding that, of mAbs obtained from immunization. High specificity was also attained, not only between very distinct proteins, but also in the case of the Ricinus communis ("ricin") agglutinins (RCA(60) and RCA(120)), despite >80% sequence homology between the two. The results suggested that the performance of pIX-display libraries can potentially exceed that of the pIII-display format and make it ideally suited for panning a wide variety of target antigens.

Bacteriophages↗
PubMed1990

Peptides on phage: a vast library of peptides for identifying ligands.

We have constructed a vast library of peptides for finding compounds that bind to antibodies and other receptors. Millions of different hexapeptides were expressed at the N terminus of the adsorption protein (pIII) of fd phage. The vector fAFF1, derived from the tetracycline resistance-transducing vector fd-tet, allows cloning of oligonucleotides in a variety of locations in the 5' region of gene III. A library of 3 x 10(8) recombinants was generated by cloning randomly synthesized oligonucleotides. The library was screened for high-avidity binding to a monoclonal antibody (3-E7) that is specific for the N terminus of beta-endorphin (Tyr-Gly-Gly-Phe). Fifty-one clones selected by three rounds of the affinity purification technique called panning were sequenced and found to differ from previously known ligands for this antibody. The striking finding is that all 51 contained tyrosine as the N-terminal residue and that 48 contained glycine as the second residue. The binding affinities of six chemically synthesized hexapeptides from this set range from 0.35 microM (Tyr-Gly-Phe-Trp-Gly-Met) to 8.3 microM (Tyr-Ala-Gly-Phe-Ala-Gln), compared with 7.1 nM for a known high-affinity ligand (Tyr-Gly-Gly-Phe-Leu). These results show that ligands can be identified with no prior information concerning antibody specificity. Peptide libraries are also likely to be useful in finding ligands that bind to other classes of receptors and in discovering pharmacologic agents.

Amino Acid Sequence↗
PubMed1990

Improved genetic selection for screening bacteriophage libraries by homologous recombination in vivo.

Three major difficulties have hindered the general application of in vivo recombination techniques to library screening: (i) the original selection could not be applied to libraries prepared in phage vectors lacking amber mutations, (ii) nonirradiated packaging extracts gave high backgrounds even when amber mutated vectors were used, and (iii) most red- vectors lacked rap, a recently discovered phage gene promoting phage-plasmid recombination. Here we describe a selection scheme for phage bearing suppressor tRNA plasmids, which relies upon an Escherichia coli host bearing an amber mutation in the dnaB gene. The selection is tight enough to allow library screening by recombination, is applicable to almost every phage vector in common use, and overcomes the background associated with nonirradiated packaging extracts. We also describe an ancillary plasmid that supplies the rap gene function in trans, permitting the recombination level to be raised fruitfully in phage libraries lacking endogenous rap. Finally, we demonstrate simple procedures for preparing and detecting phages that have lost integrated suppressor tRNA plasmids by homologous recombination.

Base Sequence↗
PubMed1992

Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem.

The properties of naiveté and large diversity are considered to be essential starting features for combinatorial antibody libraries that eschew immunization by evolution in vitro. We have prepared large libraries with such properties by using random oligonucleotide synthesis, which has the potential to create approximately 10(20) complementarity-determining regions for antibody heavy chains. When combined with light chains and expressed on phage surfaces, high-affinity antibodies could be selected from 5.0 x 10(7) Escherichia coli transformants. Remarkably, antibodies selected only for binding displayed both general structural features known to be important in nature's own antibodies and specific consensus sequences thought to be critical for interaction with the hapten against which the library was selected. Semisynthetic and ultimately totally synthetic combinatorial libraries when coupled with mutation and selection procedures should replace immunization for generation of reagent, therapeutic, and catalytic antibodies.

Amino Acid Sequence↗
PubMed1993

Recombination walking: genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination.

Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unordered) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases.

Animals↗
PubMed1993

Human monoclonal antibodies against a plethora of viral pathogens from single combinatorial libraries.

Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro.

Amino Acid Sequence↗
PubMed1994

Construction of libraries enriched for sequence repeats and jumping clones, and hybridization selection for region-specific markers.

We describe a simple and rapid method for constructing small-insert genomic libraries highly enriched for dimeric, trimeric, and tetrameric nucleotide repeat motifs. The approach involves use of DNA inserts recovered by PCR amplification of a small-insert sonicated genomic phage library or by a single-primer PCR amplification of Mbo I-digested and adaptor-ligated genomic DNA. The genomic DNA inserts are heat denatured and hybridized to a biotinylated oligonucleotide. The biotinylated hybrids are retained on a Vectrex-avidin matrix and eluted specifically. The eluate is PCR amplified and cloned. More than 90% of the clones in a library enriched for (CA)n microsatellites with this approach contained clones with inserts containing CA repeats. We have also used this protocol for enrichment of (CAG)n and (AGAT)n sequence repeats and for Not I jumping clones. We have used the enriched libraries with an adaptation of the cDNA selection method to enrich for repeat motifs encoded in yeast artificial chromosomes.

Base Sequence↗
PubMed1994

Preparation and screening of an arrayed human genomic library generated with the P1 cloning system.

We describe here the construction and initial characterization of a 3-fold coverage genomic library of the human haploid genome that was prepared using the bacteriophage P1 cloning system. The cloned DNA inserts were produced by size fractionation of a Sau3AI partial digest of high molecular weight genomic DNA isolated from primary cells of human foreskin fibroblasts. The inserts were cloned into the pAd10sacBII vector and packaged in vitro into P1 phage. These were used to generate recombinant bacterial clones, each of which was picked robotically from an agar plate into a well of a 96-well microtiter dish, grown overnight, and stored at -70 degrees C. The resulting library, designated DMPC-HFF#1 series A, consists of approximately 130,000-140,000 recombinant clones that were stored in 1500 microtiter dishes. To screen the library, clones were combined in a pooling strategy and specific loci were identified by PCR analysis. On average, the library contains two or three different clones for each locus screened. To date we have identified a total of 17 clones containing the hypoxanthine-guanine phosphoribosyltransferase, human serum albumin-human alpha-fetoprotein, p53, cyclooxygenase I, human apurinic endonuclease, beta-polymerase, and DNA ligase I genes. The cloned inserts average 80 kb in size and range from 70 to 95 kb, with one 49-kb insert and one 62-kb insert.

Bacteriophage P1↗
PubMed1998

Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library.

Cancer/testis (CT) antigens-immunogenic protein antigens that are expressed in testis and a proportion of diverse human cancer types-are promising targets for cancer vaccines. To identify new CT antigens, we constructed an expression cDNA library from a melanoma cell line that expresses a wide range of CT antigens and screened the library with an allogeneic melanoma patient serum known to contain antibodies against two CT antigens, MAGE-1 and NY-ESO-1. cDNA clones isolated from this library identified four CT antigen genes: MAGE-4a, NY-ESO-1, LAGE-1, and CT7. Of these four, only MAGE-4a and NY-ESO-1 proteins had been shown to be immunogenic. LAGE-1 is a member of the NY-ESO-1 gene family, and CT7 is a newly defined gene with partial sequence homology to the MAGE family at its carboxyl terminus. The predicted CT7 protein, however, contains a distinct repetitive sequence at the 5' end and is much larger than MAGE proteins. Our findings document the immunogenicity of LAGE-1 and CT7 and emphasize the power of serological analysis of cDNA expression libraries in identifying new human tumor antigens.

Amino Acid Sequence↗
PubMed2001

Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.

Amino Acid Sequence↗
PubMed2003

An oriented peptide array library (OPAL) strategy to study protein-protein interactions.

One of the major questions in signal transduction is how the specificities of protein-protein interactions determine the assembly of distinct signaling complexes in response to stimuli. Several peptide library methods have been developed and widely used to study protein-protein interactions. These approaches primarily rely on peptide or DNA sequencing to identify the peptide or consensus motif for binding and may prove too costly or difficult to accommodate high throughput applications. We report here an oriented peptide array library (OPAL) approach that should facilitate high throughput proteomic analysis of protein-protein interactions. OPAL integrates the principles of both the oriented peptide libraries and array technologies. Hundreds of pools of oriented peptide libraries are synthesized as amino acid scan arrays. We demonstrate that these arrays can be used to map the specificities of a variety of interactions, including antibodies, protein domains such Src homology 2 domains, and protein kinases.

Animals↗
PubMed2004

Identification of a network involved in thapsigargin-induced apoptosis using a library of small interfering RNA expression vectors.

We describe here the construction of a library of small interfering RNA expression vectors targeted to a few hundred apoptosis-related genes and the application of this library to an investigation of thapsigargin (TG)-induced apoptosis. Thapsigargin triggers endoplasmic reticulum stress, with subsequent apoptosis, but the molecular mechanisms underlying this process are incompletely understood. Using our library, we identified three anti-apoptotic genes, namely, NOXA, E2F1, and MAPK1, in addition to already characterized genes in the apoptotic pathway. In contrast to proposals by others, our data revealed (i) that TG-induced apoptosis is associated with Apaf1 in a caspase-3- and caspase-9-independent manner; (ii) that the E2F1-PUMA pathway might be involved; and (iii) that the ERK pathway, via MAP3K8 (mitogen-activated protein kinase kinase 8), is required for the induction by TG of apoptosis. Our study demonstrates clearly that unexpected and novel genes can be identified effectively by our method, and it provides evidence for the efficacy and utility of the comprehensive analysis of signaling networks and pathways using a library of small interfering RNA expression vectors.

Animals↗
PubMed2005

Analysis of double-stranded RNA-induced apoptosis pathways using interferon-response noninducible small interfering RNA expression vector library.

We have developed an original vector library that allowed us to exploit the phenomenon of RNA interference but also allowed us to avoid the confounding effects of the interferon response. In the present work, we used our library of small interfering RNA expression vectors to examine the genes involved in apoptosis that was induced by double-stranded RNA. To our surprise, screening of our library revealed two novel double-stranded RNA-induced apoptotic pathways, a JNK/SAPK-mediated mitochondrial pathway and an ERK2-related pathway, both of which appeared to be independent of the serine-threonine protein kinase-dependent caspase pathway. We also found that MST2 and protein kinase Calpha both activated the pro-apoptotic signal mediated by ERK2. The results of our screening analysis suggested the utility of large scale screenings with libraries of small interfering RNA expression vectors.

Apoptosis↗
PubMed2003

Enrichment of a single clone from a high diversity library of phage-displayed antibodies by panning with Anopheles gambiae (Diptera: Culicidae) midgut homogenate.

A high diversity library of recombinant human antibodies was selected on complex antigen mixtures from midguts of female Anopheles gambiae Giles. The library of phage-displayed single chain variable region fragment constructs, derived from beta-lymphocyte mRNA of naïve human donors, was repeatedly selected and reamplified on the insoluble fraction of midgut homogenates. Five rounds of panning yielded only one midgut-specific clone, which predominated the resulting antibody panel. In A. gambiae, the epitope was found throughout the tissues of females but was absent from the midgut of males. The cognate antigen proved to be detergent soluble but very sensitive to denaturation and could not be isolated or identified by Western blot of native electrophoresis gels or by immunoprecipitation. Nevertheless, immunohistology revealed that this sex-specific epitope is associated with the lumenal side of the midgut. Severe bottlenecking may limit the utility of phage display selection from naïve libraries for generating diverse panels of antibodies against complex mixtures of antigens from insect tissues. These results suggest that the selection of sufficiently diverse antibody panels, from which mosquitocidal or malaria transmission-blocking antibodies can be isolated, may require improved selection methods or specifically enriched pre-immunized libraries.

Animals↗
PubMed1991

The hospital library is crucial to quality healthcare.

In a 1988 report, an advisory committee of the Medical Library Association (MLA) wrote that "no one would argue against information as the foundation for efficient cost-effective business or against access to knowledge as a prerequisite for developing new knowledge." Yet the increasing number of threats to the hospital library--largely from within the industry--suggest that many hospitals do not value information in the same way their counterparts in other businesses do. In the first of two articles in this issue on hospital library and information services, the executive director of the MLA uses the MLA's experience and a variety of research findings to restate the case for the hospital library's vital role in quality care.

Attitude of Health Personnel↗
PubMed1999

Construction of murine phage antibody library and selection of ricin-specific single-chain antibodies.

A murine phage antibody library containing 1.7 x 10(8) independent clones was constructed and then screened by affinity panning of anti-ricin antibody fragments. First, mRNA was purified from the total RNA extracted from fresh spleens of nonimmunized mice of Kunming, and then the total antibody variable region cDNA was amplified via reverse transcription-polymerase chain reaction. Gene fragments encoding VH and VL were amplified and assembled into a single gene by using a DNA linker encoding a pentadecapeptide (Gly4Ser)3 through primary PCR. Finally the recombinant DNA fragments were cloned into the phagemid pCANTAB 5E vector and electroporated into Escherichia coli TG1. The recombinant phage particles displaying functional single-chain fragment variable regions (scFvs) were rescued by reinfection of helper phage M13KO7, thus constructing a murine antibody library. Two ricin-specific scFvs strains were selected from the phage antibody library by using affinity panning. The target antigen, ricin, was biotinylated with photobiotin and the biotin-labeled ricin interacted with the phage antibody library. Subsequent addition of streptavidincoated paramagnetic beads isolated the biotinylated ricin-binding phage particle complex. Washing, elution, and reinfection of the isolated complex then proceeded sequentially. After six rounds of panning, 2 strains of the 34 clones were verified to show greater specific affinity to ricin.

Animals↗
PubMed2003

Construction and characterization of a novel 13.34-fold chicken bacterial artificial chromosome library.

A chicken bacterial artificial chromosome (BAC) library consisting of 138,240 clones was constructed in vector pBeloBAC11 with genomic DNA isolated from female white-silk chicken. An average insert size of 118 kb was estimated from 452 randomly isolated clones, which indicate the library to be approximate 13.34-fold genome coverage. For the demonstration of the probability to pick out any unique genes or DNA markers from the library, 8 single-copy genes were screened out and the positive clones were yielded between 2 and 15 with an average of 11.125, in agreement with the estimated high genomic coverage of this library. Positive superpools were obtained for 40 microsatellite markers selected from different regions of chicken genome. The number of positive superpools for each marker varies from 1 to 15 with an average of 9.475.

Animals↗