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PubMed1995

The library of the future: an informatics institution.

As technology transforms how information is accessed, stored and disseminated, the concept of the library changes as well. In its plans for a new building to house both library and computing services, the University of Maryland at Baltimore (UMAB) will give physical form and organizational dimensions to this new concept of the library. UMAB's library of the future will be an 'informatics institution,' designed to accommodate new technologies that support problem-based learning in the health sciences curricula. This new library will embody the concept and the discipline of health informatics.

Baltimore↗
PubMed1995

Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes.

In chickens, single functional immunoglobulin variable and joining gene segments at each of the heavy and light chain loci undergo V(D)J rearrangement. Diversity is subsequently introduced by conversions templated by upstream pseudo V region genes in such a way that practically all V regions in mature B cells have identical ends. This greatly simplifies the representative amplification of V region genes. Furthermore, the entire naive repertoire of the adult chicken is produced in the bursa of Fabricius of the young bird. These special properties of the generation of immunoglobulin diversity in chickens have been exploited in the development of procedures to produce large libraries of diverse antibody combining sites derived from chicken Ig genes and expressed on filamentous bacteriophage. The utility of this library was assessed by selection of specifically binding phage using three solid phase-bound protein antigens, hen egg white lysozyme, bovine thyroglobulin and bovine serum albumin. The sequences of the V region genes thus isolated demonstrated that selection was specific and that the library contained useful diversity of binding sites. This library provides access to a repertoire whose diversity is based on a mechanism different from that underlying previously available libraries. The demonstrated feasibility of generating chicken phage antibodies may lead to the production of monoclonal reagents from immunised chickens, and the derivation of reagents for studying immunoglobulin mediated selection in avian B cell development.

Amino Acid Sequence↗
PubMed1996

Retrieval of human antibodies from phage-display libraries using enzymatic cleavage.

A combinatorial human IgG1, kappa gene library of 2 x 10(7) clones was constructed from a pericolic lymph node using the phagemid vector pComb3H. Fabs with binding activity against tetanus toxoid (TT) and keyhole limpet hemocyanin (KLH) were isolated from this library, and one such TT binding Fab was used to further evaluate a new phagemid vector for the display of recombinant antibody fragments (MCO1). This vector was designed to incorporate a cleavage site for the enzyme Genenase I, a myc peptide tag, and an amber codon between the heavy chain cloning site and the truncated M13 phage gene III. When MCO1 phage displaying an anti-TT Fab were bound to TT on a solid substrate, elution with Genenase I at concentrations of 5-10 micrograms/ml proved as effective as acid elution in releasing bound phage. Furthermore, enzymatic elution with Genenase I was comparable to acid elution in the enrichment of a TT binding Fab from the pericolic library subcloned into the vector MCO1. Importantly, the use of enzymatic or acid elutions resulted in the retrieval of different anti-TT Fabs from this same library. We conclude that panning of phage-displayed combinatorial antibody libraries can be successfully performed using enzymatic elution, and that this offers a useful alternative to currently available phage elution techniques.

Aged↗
PubMed1996

Directing antigen specificity towards botulinum neurotoxin with combinatorial phage display libraries.

The production of antibodies towards antigens with low immunogenicity is enhanced by the intrinsic efficiency of screening combinatorial libraries of immunoglobulins. The need to isolate clones with rare binding specificities has dictated a highly efficient method of screening and isolating antibody clones. The production of recombinant immunoglobulin libraries in bacteria allows for a more controlled selection of antibody specificity and can be used in circumstances where hybridoma fusions are unable to isolate rare clones with the desired epitope specificity. Botulinum neurotoxin (NT) with associated non neurotoxin proteins (non-NT) as a complex was used to immunize mice to obtain mRNA for the production of a recombinant antibody library with a repertoire of specificities. Initial screens of the combinatorial library revealed clones which recognized the non-neurotoxin proteins of the toxin complex similar to monoclonal antibodies produced by conventional hybridoma fusions. The combinatorial library was re-screened in order to isolate antibodies that specifically recognized the neurotoxin component of the toxin complex. The ability to alter the biopanning selection process affords the researcher a measure of control in the selection process not available with traditional hybridoma fusions.

Animals↗
PubMed1991

Use of high coverage reference libraries of Drosophila melanogaster for relational data analysis. A step towards mapping and sequencing of the genome.

Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been generated. Also, a jumping library has been created by a new method that takes advantage of methylation differences between genomic DNA and vector. Thirdly, two cDNA libraries have been picked. All these libraries have been arrayed on high-density in situ filters, each containing 9216 clones. As a reference system, such filters are distributed and identified clones are provided. Single-copy probes have identified on average 1.4 cosmids per genome equivalent. Together with cytogenetically mapped yeast artificial chromosomes, the libraries are also being used for physically mapping the genome, mainly by oligonucleotide fingerprinting and pool hybridizations. cDNA clones are further examined by a partial sequencing analysis by oligomer hybridization.

Animals↗
PubMed1991

By-passing immunization. Human antibodies from V-gene libraries displayed on phage.

We have mimicked features of immune selection to make human antibodies in bacteria. Diverse libraries of immunoglobulin heavy (VH) and light (V kappa and V lambda) chain variable (V) genes were prepared from peripheral blood lymphocytes (PBLs) of unimmunized donors by polymerase chain reaction (PCR) amplification. Genes encoding single chain Fv fragments were made by randomly combining heavy and light chain V-genes using PCR, and the combinatorial library (greater than 10(7) members) cloned for display on the surface of a phage. Rare phage with "antigen-binding" activities were selected by four rounds of growth and panning with "antigen" (turkey egg-white lysozyme (TEL) or bovine serum albumin) or "hapten" (2-phenyloxazol-5-one (phOx], and the encoding heavy and light chain genes were sequenced. The V-genes were human with some nearly identical to known germ-line V-genes, while others were more heavily mutated. Soluble antibody fragments were prepared and shown to bind specifically to antigen or hapten and with good affinities, Ka (TEL) = 10(7) M-1; Ka (phOx) = 2 x 10(6) M-1. Isolation of higher-affinity fragments may require the use of larger primary libraries or the construction of secondary libraries from the binders. Nevertheless, our results suggest that a single large phage display library can be used to isolate human antibodies against any antigen, by-passing both hybridoma technology and immunization.

Amino Acid Sequence↗
PubMed1990

An M13 vector library for cloning DNA with four nucleotide 3' overhangs.

A flexible M13 vector library incorporating the BstXI site has been developed. DNA cut by any currently commercially available restriction endonuclease that generates a 4-nucleotide (nt) 3' overhang can be ligated into a specific clone of the library. The BstXI enzyme recognizes a 6-bp bipartite palindromic sequence. The central nucleotides are not specified, and form a 4-base, 3' overhang when cut by BstXI. 5' CCANNNNN NTGG GGTN NNNNNACC 5' Since the 4-base overhang formed is not part of the BstXI recognition sequence, it is possible to generate a library of 256 different clones by introducing the BstXI site, 151 of the possible 256-member library have been isolated, including all 13 M13BF clones in which the overhang formed by BstXI digestion is complementary to those formed by currently available restriction endonucleases. Of these 13 vectors, BstXI digestion of six clones results in nonpalindromic cohesive ends and should facilitate in vitro tandem gene amplification. The BstXI site is adjacent to the four codons corresponding to the factor Xa recognition sequence. Hence the vector library could facilitate the expression of a fusion protein that could be proteolytically cleaved by factor Xa.

Base Sequence↗
PubMed1995

Generating a phage display antibody library against an identified neuron.

The generation of monoclonal antibodies by conventional hybridoma technology is limited by the diversity of the clones and the difficulties of screening the antibodies and of their large-scale production from isolated clones. As an alternative approach, we have investigated the suitability of phage display libraries for the production of recombinant antibodies against an identified neuron. Mice were immunized with isolated leech Retzius (R) neurons. The spleen poly A+ RNA was isolated and first-strand cDNA was prepared. The variable regions of light- and heavy-chain IgG molecules were amplified by the polymerase chain reaction (PCR) and separate libraries of each were constructed and combined in the pComb8 vector to yield a combinatorial library of approximately 10(7) transformants. Single R neurons that were plated in culture bound approximately 100 phages on a first screen and several thousand when the first batch was re-screened. Of these, 96 individual phage colonies were isolated and used for immunocytochemistry with leech CNS ganglia: 41 exhibited general staining, 20 showed no detectable staining and 30 stained selective subsets of neurons including (but not specific for) the R neuron. The phage display library approach thus simplifies the screening of large libraries with small numbers of (and even single) cells. However, the combinatorial antibodies with binding activity must still be tested individually by immunocytochemistry, which is further limited by their apparently low affinity.

Animals↗
PubMed1990

Construction and characterization of a DNA library from mouse chromosomes 19 purified by flow cytometry.

In spite of the constant development concerning physical mapping of eukaryotic genomes, the mouse chromosome 19 remains poorly characterized. In order to improve the possibilities for studying this chromosome, we have constructed a chromosome-specific EcoRI DNA fragment library from mouse chromosomes 19 sorted by flow cytometry. The resulting library contains about 3 X 10(4) recombinant clones. The identified inserts range in size from about 0.2-10 kb, with a 4 kb average size and with no observable redundancy. The purity of the library has been analyzed by flow-blot. For that purpose, chromosomes from 2 cell lines, 1 with a normal karyotype and 1 with translocated chromosome 19, were sorted on nylon filters and hybridized with 9 clones of the library. Results show that 5 clones out of the 9 clearly originate from sorted chromosomes 19 and 3 and are likely to be derived from its DNA, thus indicating that the library of chromosome 19 is of high purity.

Animals↗
PubMed1994

cDNA libraries from a few neural cells.

One of the most powerful approaches to the molecular analysis of differential gene expression is to construct cDNA libraries corresponding to different tissues or developmental stages, and then to enrich for genes expressed in a particular tissue or at a particular time by subtractive hybridisation. Our aim is to reduce the complexity of neuronal cDNA libraries by generating libraries from the mRNA of a single cell. The system chosen is the Retzius cell of the leech, a large neurone which can be unambiguously dissected out. A cDNA library was generated from one leech ganglion (containing about 400 neurons) by anchor 1-oligo dT priming, the addition of dG tails, second strand synthesis primed by an anchor 2-oligo dC primer, followed by PCR from the two anchor regions. XBaI and EcoRI sites were included in the respective anchor primers, between the dT or dC run and the PCR primer sequence, allowing high-efficiency directional cloning. Eight clones picked and sequenced at random gave five with some homology to a known protein and three novel genes. The average insert size in the library was 600 bp, 0.2% of the clones hybridised to repetitive DNA, and 20/30,000 clones gave signals with the Drosophila actin gene. This approach has now been extended to a few pooled Retzius cells.

Animals↗
PubMed1994

The 9-kDa calbindin gene of Rousettus aegyptiacus: its identification and isolation from a genomic library.

A genomic library of the fruit bat (Rousettus aegyptiacus) was constructed in lambda phage gt11. The titre of the library was determined to be 2 x 10(5) pfu/ml. The genomic library was amplified and the titre of the amplified library increased 300-fold to 7 x 10(7) pfu/ml. The library was screened by in situ hybridization techniques using a fragment of the mouse 9-kDa calbindin cDNA as a probe. Screening of 10(5) plaques yielded a positive clone. Three additional rounds of screening were performed to purify the positive. Lambda phage DNA was isolated from the positive clone and restriction digest analysis, followed by hybridization studies, was performed on these digests in order to determine the location of the bat 9-kDa calbindin gene in the insert of the lambda phage vector. Restriction maps so derived were interpreted from the published sequence for the rat 9-kDa calbindin gene and indicate the successful isolation of the 9-kDa calbindin gene of Rousettus aegyptiacus.

Animals↗
PubMed1989

A reliable method for random mutagenesis: the generation of mutant libraries using spiked oligodeoxyribonucleotide primers.

A new procedure for the production of a defined library of random mutants is described. Long spiked oligodeoxyribonucleotides (oligos), in which a predetermined level of the three 'wrong' phosphoramidites are used at each position, are made as primers for a standard oligo-directed mutagenesis protocol. Spiked oligo synthesis on a DNA synthesizer is achieved using an in-line mixing procedure that only requires five phosphoramidite reservoirs and which avoids contamination of any of the pure phosphoramidite reagents. Immutable positions (i.e., positions in the oligo for which pure reagents are used) can be specified, and a silent 'marker' base can be included that allows an early estimate of the mutagenesis efficiency. The randomness of the library in respect to the number, type, and position of the altered bases, is easily verified by DNA sequencing. This procedure has been used to generate a random mutant library of the gene encoding a sluggish triosephosphate isomerase. Among the transformants from this library, a number of second-site suppressor mutations have been found that increase the specific catalytic activity of the starting isomerase. This approach provides a more complete library than a method using chemical mutagenic reagents.

Base Sequence↗
PubMed1989

Phasmids as effective and simple tools for construction and analysis of gene libraries.

Phasmid lambda pMYF131, a hybrid of phage lambda vectors and plasmid pUC19, was constructed. The phasmid and its derivatives were shown to be efficient vectors for construction and analysis of gene libraries in Escherichia coli cells. The lambda pMYF131 DNA molecule contains all the genes and regions essential for phage lytic development. The plasmid cannot be packaged either in the monomeric or the oligomeric form due to its specific length. Elongation of the DNA molecule by ligation with fragments of foreign DNA can make it packageable and this is easily detected by plaque formation. Hence, the procedures used to construct genomic libraries can be simplified by selection of only recombinant DNA molecules just at the time and on the basis of their packaging in vitro. The output of recombinant clones per vector molecule was several times higher for vector lambda pMYF131, compared to phage vector lambda L47.1AB, and attained 3 x 10(6) clones per micrograms DNA. Vector and recombinant phasmids can be obtained in large quantities in plasmid form. lambda pMYF131 contains nine unique restriction sites which allow the cloning of DNA fragments with blunt ends and of fragments with various types of cohesive ends, obtained by digestion with 14 prototype restriction enzymes. The maximal size of the cloned DNA fragments is approx. 20 kb for lambda pMYF131. Phasmid vectors were used to construct libraries of bovine, pig and quail genomes, and genomic libraries of 17 species of bacteria. Application of suitable methods allowed the identification 13 individual genes within these libraries.

Bacteriophage lambda↗
PubMed1990

Yeast artificial chromosomes containing human Xq24-Xq28 DNA: library construction and representation of probe sequences.

A library of yeast artificial chromosomes (YACs) with human DNA inserts has been assembled from a human/hamster somatic cell hybrid containing Xq24-Xqter human DNA. Screening of the agar-embedded transformants for human DNA used a manifold of 3000 stainless-steel pins to transfer colonies onto the surface of media. This facilitated the recovery of the 1 in 300 clones that contained a human DNA insert (the remainder had hamster DNA and were discarded). The library described here consists of about two genomic equivalents (102 Mb) of human DNA in 467 clones: 167 were generated by EcoRI partial digestion and contain 25.5 Mb of human DNA; 252 used partial digestion with TaqI and cover 64.2 Mb; and 48 were from sheared DNA inserts and cover 11.7 Mb. Clones were screened by hybridization with 70 probes previously assigned to Xq24-Xq28. Eleven probes did not hybridize to any YACs in the library, and 16 probes hybridized to one YAC each, 23 to two, 13 to three, and 7 to four. Also, individual YACs large enough to detect features like the clustering of polymorphic sequences in subregions of Xq24-Xqter have been obtained. For example, XY58 contained five probe sequences previously independently isolated. The overall yield of YACs containing probe sequences was indistinguishable from Poisson statistical expectations for random cloning (P = 0.9). Thus, YAC libraries such as the one described here can include most, if not all, of the sequences in the source DNA from which the library is derived. These results support the possibility that YACs may provide a reliable bridge between linkage studies and conventional recombinant DNA analyses in mapping of the human genome.

Animals↗
PubMed1991

New strategy for mapping the human genome based on a novel procedure for construction of jumping libraries.

A novel procedure for construction of jumping libraries is described. The essential features of this procedure are as follows: (1) two diphasmid vectors (lambda SK17 and lambda SK22) are simultaneously used in the library construction to improve representativity, (2) a partial filling-in reaction is used to eliminate cloning of artifactual jumping clones and to obviate the need for a selectable marker. The procedure has been used to construct a representative human NotI jumping library (220,000 independent recombinant clones) from the lymphoblastoid cell line CBMI-Ral-STO, which features a low level of methylation of its resident EBV genomes. A human chromosome 3-specific NotI jumping library (500,000 independent recombinant clones) from the human chromosome 3 x mouse hybrid cell line MCH 903.1 has also been constructed. Of these recombinant clones 50-80% represent jumps to the neighboring cleavable NotI site. With our previously published method for construction of linking libraries this procedure makes a new genome mapping strategy feasible. This strategy includes the determination of tagging sequences adjacent to NotI sites in random linking and jumping clones. Special features of the lambda SK17 and lambda SK22 vectors facilitate such sequencing. The STS (sequence tagged site) information obtained can be assembled by computer into a map representing the linear order of the NotI sites for a chromosome or for the entire genome. The computerized mapping data can be used to retrieve clones near a region of interest. The corresponding clones can be obtained from the panel of original clones, or necessary probes can be made from genomic DNA by PCR.

Animals↗
PubMed1991

Differences in the probability of cloning specific DNA between primary and amplified libraries: theoretical considerations.

We present a theoretical study of the probability of isolating a particular clone from a DNA library. There are differences in this probability between primary and amplified libraries even if the desired clone represents the same fraction of both libraries. As the result, we must screen severalfold more phage or bacteria in an amplified library than in a primary library.

Cloning, Molecular↗
PubMed1995

An arrayed library enriched in hncDNA corresponding to transcribed sequences of human chromosome 19: preparation and analysis.

A simple technique for preparation of libraries of the human chromosome specific transcribed sequences is developed. It uses hnRNA from human-rodent hybrid cell lines containing particular human chromosomes or their fragments and includes three stages: (i) reverse transcription of the hnRNA with Alu-specific primers directing the transcription beyond the Alu-repeats to flanking non-repetitive sequences of the chromosome; (ii) nested primer PCR strategy with specifically designed primers; (iii) direct selective cloning of the second-stage nested primer PCR products. An arrayed hncDNA library was prepared from a hybrid cell line containing chromosome 19 and fragments of 22 and X chromosomes. The library contains around 98% of human-specific transcribed sequences. Sequences of 52 human-specific, according to PCR analysis, clones differed from each other and had no close analogs in the EMBL Data Bank. Of 17 clones assigned to certain human chromosomes, 9 belonged to chromosome 19, 5 to chromosome 22 and 3 to chromosome X. Some of the human specific clones contained repetitive elements scattered over different human chromosomes. Clones from hncDNA libraries are useful as STSs/ESTs, as probes for detecting full-size genes in genomic libraries, for RFLP analysis and for identification of chromosome specific cDNAs.

Animals↗
PubMed2005

Reducing mutational bias in random protein libraries.

The success of protein optimization through directed molecular evolution depends to a large extent on the size and quality of the displayed library. Current low-fidelity DNA polymerases that are commonly used during random mutagenesis and recombination in vitro display strong mutational preferences, favoring the substitution of certain nucleotides over others. The result is a biased and reduced functional diversity in the library under selection. In an effort to reduce mutational bias, we combined two different low-fidelity DNA polymerases, Taq and Mutazyme, which have opposite mutational spectra. As a first step, random mutants of the Bacillus thuringiensis cry9Ca1 gene were generated by separate error-prone polymerase chain reactions (PCRs) with each of the two polymerases. Subsequent shuffling by staggered extension process (StEP) of the PCR products resulted in intermediate numbers of AT and GC substitutions, compared to the Taq or Mutazyme error-prone PCR libraries. This strategy should allow generating unbiased libraries or libraries with a specific degree of mutational bias by applying optimal mutagenesis frequencies during error-prone PCR and controlling the concentration of template in the shuffling reaction while taking into account the GC content of the target gene.

Bacillus thuringiensis↗