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PubMed2002

A new approach to genome mapping and sequencing: slalom libraries.

We describe here an efficient strategy for simultaneous genome mapping and sequencing. The approach is based on physically oriented, overlapping restriction fragment libraries called slalom libraries. Slalom libraries combine features of general genomic, jumping and linking libraries. Slalom libraries can be adapted to different applications and two main types of slalom libraries are described in detail. This approach was used to map and sequence (with approximately 46% coverage) two human P1-derived artificial chromosome (PAC) clones, each of approximately 100 kb. This model experiment demonstrates the feasibility of the approach and shows that the efficiency (cost-effectiveness and speed) of existing mapping/sequencing methods could be improved at least 5-10-fold. Furthermore, since the efficiency of contig assembly in the slalom approach is virtually independent of length of sequence reads, even short sequences produced by rapid, high throughput sequencing techniques would suffice to complete a physical map and a sequence scan of a small genome.

Chromosomes, Artificial, Human↗
PubMed2004

Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution.

Directed molecular evolution and combinatorial methodologies are playing an increasingly important role in the field of protein engineering. The general approach of generating a library of partially randomized genes, expressing the gene library to generate the proteins the library encodes and then screening the proteins for improved or modified characteristics has successfully been applied in the areas of protein-ligand binding, improving protein stability and modifying enzyme selectivity. A wide range of techniques are now available for generating gene libraries with different characteristics. This review will discuss these different methodologies, their accessibility and applicability to non-expert laboratories and the characteristics of the libraries they produce. The aim is to provide an up to date resource to allow groups interested in using directed evolution to identify the most appropriate methods for their purposes and to guide those moving on from initial experiments to more ambitious targets in the selection of library construction techniques. References are provided to original methodology papers and other recent examples from the primary literature that provide details of experimental methods.

Base Sequence↗
PubMed2005

Selection of functional human antibodies from retroviral display libraries.

Antibody library technology represents a powerful tool for the discovery and design of antibodies with high affinity and specificity for their targets. To extend the technique to the expression and selection of antibody libraries in an eukaryotic environment, we provide here a proof of concept that retroviruses can be engineered for the display and selection of variable single-chain fragment (scFv) libraries. A retroviral library displaying the repertoire obtained after a single round of selection of a human synthetic scFv phage display library on laminin was generated. For selection, antigen-bound virus was efficiently recovered by an overlay with cells permissive for infection. This approach allowed more than 10(3)-fold enrichment of antigen binders in a single selection cycle. After three selection cycles, several scFvs were recovered showing similar laminin-binding activities but improved expression levels in mammalian cells as compared with a laminin-specific scFv selected by the conventional phage display approach. Thus, translational problems that occur when phage-selected antibodies have to be transferred onto mammalian expression systems to exert their therapeutic potential can be avoided by the use of retroviral display libraries.

Animals↗
PubMed1996

cDNA libraries from identified neurons.

One approach to studying the changes in gene expression which underlie differentiation is to construct cDNA libraries from different tissues or at different stages of development. However, generating representative cDNA libraries from heterogeneous tissues such as the nervous system is often a real problem. Here, we describe a reproducible method for the construction of large and complex cDNA libraries from a few leech Retzius or P neurons (equivalent to about 50 pg of mRNA) using polymerase chain reaction-based technology. The libraries contain about 10(6) independent recombinants and are remarkably free from contaminating rRNA or polymerase chain reaction artefacts. Sequence analysis of randomly picked clones shows that the libraries contain a high proportion (more than 90%) of cDNAs larger than 500 b.p. As expected, many of the clones are novel, but two (alpha-tubulin and cyclophilin-A) have been extensively characterized in other species. To our knowledge, this is the first report of a cDNA library from identified neurons.

Amino Acid Isomerases↗
PubMed2000

A gene-enriched BAC library for cloning large allele-specific fragments from maize: isolation of a 240-kb contig of the bronze region.

A generic bacterial artificial chromosome (BAC) library from a complex plant genome like maize may not be suitable for some types of genomic analysis, for example, for establishing correlations between the genetic and the physical organization of a given chromosome region. Previously, we carried out extensive genetic analysis of the bronze (Bz) region in Zea mays using a W22 inbred line carrying the Bz-McC allele; however, BAC libraries of that line are neither available nor under construction. Here, we report the isolation of large, adjacent BAC clones of this region from a partial BAC library of W22. We developed a BAC vector suitable for cloning NotI fragments and used it to clone size-fractionated genomic DNA that had been cut to completion with the methylation-sensitive, rare-cutting enzyme NotI. This strategy resulted in a very significant enrichment of large genic DNA. From a library of about 20,000 BACs, containing just two-thirds of a maize genome, we isolated 16 BAC clones of the 110-kb distal Bz fragment and 10 BAC clones of the 130-kb proximal Bz fragment. This recovery means that our strategy resulted in a 15- to 24-fold enrichment of specific sequences. The order of the BAC clones in the 240-kb contig, predetermined from an internal NotI site in the Bz-McC allele was confirmed by hybridization with sequences from sites previously mapped proximal and distal to Bz and by sequencing. To show the general utility of our approach and the value of our partial BAC library, we also isolated BAC clones of other sequences, such as tub4 and the complex R-r allele, contained in the same size fraction of DNA. This is the first report of the use of a BAC vector to clone allele-specific large DNA fragments from a plant with a large genome, circumventing the need to construct a complete BAC library.

Alleles↗
PubMed2001

An oligonucleotide fingerprint normalized and expressed sequence tag characterized zebrafish cDNA library.

The zebrafish is a powerful system for understanding the vertebrate genome, allowing the combination of genetic, molecular, and embryological analysis. Expressed sequence tags (ESTs) provide a rapid means of identifying an organism's genes for further analysis, but any EST project is limited by the availability of suitable libraries. Such cDNA libraries must be of high quality and provide a high rate of gene discovery. However, commonly used normalization and subtraction procedures tend to select for shorter, truncated, and internally primed inserts, seriously affecting library quality. An alternative procedure is to use oligonucleotide fingerprinting (OFP) to precluster clones before EST sequencing, thereby reducing the re-sequencing of common transcripts. Here, we describe the use of OFP to normalize and subtract 75,000 clones from two cDNA libraries, to a minimal set of 25,102 clones. We generated 25,788 ESTs (11,380 3' and 14,408 5') from over 16,000 of these clones. Clustering of 10,654 high-quality 3' ESTs from this set identified 7232 clusters (likely genes), corresponding to a 68% gene diversity rate, comparable to what has been reported for the best normalized human cDNA libraries, and indicating that the complete set of 25,102 clones contains as many as 17,000 genes. Yet, the library quality remains high. The complete set of 25,102 clones is available for researchers as glycerol stocks, filters sets, and as individual EST clones. These resources have been used for radiation hybrid, genetic, and physical mapping of the zebrafish genome, as well as positional cloning and candidate gene identification, molecular marker, and microarray development.

Animals↗
PubMed1993

Simplified construction of a subtracted cDNA library using asymmetric PCR.

A novel method for the direct construction of subtracted plasmid cDNA libraries in the plasmid pBluescript is presented. Two libraries in lambda-ZAP were compared starting with general phagemid excision from both libraries. Thereafter, single-stranded (ss) plasmids from one library were subtracted with biotinylated cDNA molecules generated by asymmetric PCR on ss plasmid templates from the other library. The nonsubtracted plasmids were used to transform Escherichia coli directly, thus making a subtracted plasmid library. Preliminary data suggest that the specificity of the method is around 25%. The method is sensitive enough to detect low-abundance mRNAs. In contrast to other subtractive methods based on lambda-ZAP, the bias introduced using PCR in this case only affects the method's specificity and not its sensitivity.

Animals↗
PubMed2000

The comparison of gene expression from multiple cDNA libraries.

We describe a method for comparing the abundance of gene transcripts in cDNA libraries. This method allows for the comparison of gene expression in any number of libraries, in a single statistical analysis, to identify differentially expressed genes. Such genes may be of potential biological or pharmaceutical relevance. The formula that we derive is essentially the entropy of a partitioning of genes among cDNA libraries. This work goes beyond previously published analyses, which can either compare only two libraries, or identify a single outlier in a group of libraries. This work also addresses the problem of false positives associated with repeating the test on many thousands of genes. A randomization procedure is described that provides a quantitative measure of the degree of belief in the results; the results are further verified by considering a theoretically derived large deviations rate for the test statistic. As an example, the analysis is applied to four prostate cancer libraries from the Cancer Genome Anatomy Project. The analysis identifies biologically relevant genes that are differentially expressed in the different tumor cell types.

Gene Expression Profiling↗
PubMed2003

Stably folded de novo proteins from a designed combinatorial library.

Binary patterning of polar and nonpolar amino acids has been used as the key design feature for constructing large combinatorial libraries of de novo proteins. Each position in a binary patterned sequence is designed explicitly to be either polar or nonpolar; however, the precise identities of these amino acids are varied extensively. The combinatorial underpinnings of the "binary code" strategy preclude explicit design of particular side chains at specified positions. Therefore, packing interactions cannot be specified a priori. To assess whether the binary code strategy can nonetheless produce well-folded de novo proteins, we constructed a second-generation library based upon a new structural scaffold designed to fold into 102-residue four-helix bundles. Characterization of five proteins chosen arbitrarily from this new library revealed that (1) all are alpha-helical and quite stable; (2) four of the five contain an abundance of tertiary interactions indicative of well-ordered structures; and (3) one protein forms a well-folded structure with native-like features. The proteins from this new 102-residue library are substantially more stable and dramatically more native-like than those from an earlier binary patterned library of 74-residue sequences. These findings demonstrate that chain length is a crucial determinant of structural order in libraries of de novo four-helix bundles. Moreover, these results show that the binary code strategy--if applied to an appropriately designed structural scaffold--can generate large collections of stably folded and/or native-like proteins.

Amino Acid Sequence↗
PubMed2006

Use of baculovirus MHC/peptide display libraries to characterize T-cell receptor ligands.

Peptide/protein display libraries are powerful tools for identifying and manipulating receptor/ligand pairs. While the large size of bacterial phage display libraries has made them the platform of choice in many applications, often considerable engineering has been required to achieve display of properly folded and active eukaryotic proteins, such as antibodies. This problem has been partially solved in several eukaryotic display systems, e.g. using yeast or retroviruses, but these systems have their own limitations. Recently, baculovirus has been developed as a display system using the virus itself or infected insect cells as the display platform. Here, we review the development and use of baculovirus-infected cells as a platform for display libraries of peptides bound to major histocompatibility complex (MHC) class I (MHCI) or class II (MHCII). We have used fluorescent multimeric soluble T-cell receptors (TCRs) to screen these libraries and to identify peptide antigen mimotopes. We also present some improvements to this system that allow very large libraries to be constructed and screened. We have used these libraries to examine the role of MHCII-bound peptides in the presentation of the staphylococcal enterotoxin A (SEA) and to manipulate an MHCI tumor-associated antigen.

Amino Acid Sequence↗
PubMed2004

Characterization of a new BAC library for rainbow trout: evidence for multi-locus duplication.

A 10X rainbow trout bacterial artificial chromosome (BAC) library was constructed to aid in the physical and genetic mapping efforts of the rainbow trout genome. The library was derived from the Swanson clonal line (YY male) and consists of 184,704 clones with an average insert size of 137,500 bp (PFGE) or 118,700 bp (DNA fingerprinting). The clones were gridded onto 10 large nylon membranes to produce high-density arrays for screening the library by hybridization. The library was probed with 11 cDNAs from the NCCCWA EST project chosen because of interest in their homology to known gene sequences, seven known genes, and a Y-specific sex marker. Putative positive clones identified by hybridization were re-arrayed and gridded for secondary confirmation. FPC analysis of HindIII and EcoRV DNA fingerprinting was used to estimate the level of redundancy in the library, to construct BAC contigs and to detect duplicated loci in the semi-duplicated rainbow trout genome. A good correlation (R2 = 0.7) was found between the number of hits per probe and the number of contigs that were assembled from the positive BACs. The average number of BACs per contig was 9.6, which is in good agreement with 10X genome coverage of the library. Two-thirds of the loci screened were predicted to be duplicated as the positive BACs for those genes were assembled into two or three different contigs, which suggests that most of the rainbow trout genome is duplicated.

Animals↗
PubMed2005

The information needs of perioperative staff: a preparatory study for a proposed specialist library for theatres (NeLH).

AIMS/OBJECTIVES: To undertake an information-needs analysis of operating theatre staff in order to inform the development of a specialist library of the National Electronic Library of Health (NeLH). SETTING: Three hospitals in an NHS Trust in a rural county in the north of England. METHODS: In-depth interviews, during which staff were questioned in regard to their job roles and information needs, information-seeking behaviour, access to computers, use and perceptions of the NeLH, and what they would like from a specialist library. RESULTS: Information needs were found to concern direct care, professional and academic development, medical devices knowledge and administration. Currency and speed of delivery were the key information requirements. Poor communication appeared to be a major problem for many staff and inhibited efficient information exchange. When using the Web, both clinical and non-clinical staff searched in a similar fashion to health consumers generally, despite their need for highly specialist information. Most respondents reacted warmly to the idea of the proposed National Electronic Library for Theatre Staff (NeLTS) and made several practical suggestions for suitable material. However, access was problematic for nurses and might prove to be an obstacle to take-up of the Specialist Library. CONCLUSIONS: Communication difficulties, the fragmentation of data in the field and the consequent difficulties of finding information point to the need for an NeLTS. However, for this library to embrace and succeed with all the various groups that constitute the operating theatre staff, issues of access and digital literacy need to be addressed.

England↗
PubMed2003

Artificial chromosome libraries of Streptomyces coelicolor A3(2) and Planobispora rosea.

Using an Escherichia coli-Streptomyces shuttle vector derived from a bacterial artificial chromosome (BAC), we developed methodologies for the construction of BAC libraries of filamentous actinomycetes. Libraries of Streptomyces coelicolor, the model actinomycete, and Planobispora rosea, a genetically intractable strain, were constructed. Both libraries have an average insert size of 60 kb, with maximal insert larger than 150 kb. The S. coelicolor library was evaluated by selected hybridisations to DraI fragments and by end sequencing of a few clones. Hybridisation of the P. rosea library to selected probes indicates a good representation of the P. rosea genome and that the library can be used to facilitate the genomic analysis of this actinomycete.

Chromosomes, Artificial, Bacterial↗
PubMed2003

Metagenomic profiling: microarray analysis of an environmental genomic library.

Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms. However, conventional library-screening techniques permit characterization of relatively few environmental clones. Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities. A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing approximately 1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls. COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species. The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe. Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes. Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture. Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition. Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.

Base Sequence↗
PubMed2006

Identification of eukaryotic open reading frames in metagenomic cDNA libraries made from environmental samples.

Here we describe the application of metagenomic technologies to construct cDNA libraries from RNA isolated from environmental samples. RNAlater (Ambion) was shown to stabilize RNA in environmental samples for periods of at least 3 months at -20 degrees C. Protocols for library construction were established on total RNA extracted from Acanthamoeba polyphaga trophozoites. The methodology was then used on algal mats from geothermal hot springs in Tengchong county, Yunnan Province, People's Republic of China, and activated sludge from a sewage treatment plant in Leicestershire, United Kingdom. The Tenchong libraries were dominated by RNA from prokaryotes, reflecting the mainly prokaryote microbial composition. The majority of these clones resulted from rRNA; only a few appeared to be derived from mRNA. In contrast, many clones from the activated sludge library had significant similarity to eukaryote mRNA-encoded protein sequences. A library was also made using polyadenylated RNA isolated from total RNA from activated sludge; many more clones in this library were related to eukaryotic mRNA sequences and proteins. Open reading frames (ORFs) up to 378 amino acids in size could be identified. Some resembled known proteins over their full length, e.g., 36% match to cystatin, 49% match to ribosomal protein L32, 63% match to ribosomal protein S16, 70% to CPC2 protein. The methodology described here permits the polyadenylated transcriptome to be isolated from environmental samples with no knowledge of the identity of the microorganisms in the sample or the necessity to culture them. It has many uses, including the identification of novel eukaryotic ORFs encoding proteins and enzymes.

Acanthamoeba↗
PubMed1994

A simple and efficient procedure for generating stable expression libraries by cDNA cloning in a retroviral vector.

cDNA expression cloning is a powerful method for the rescue and identification of genes that are able to confer a readily identifiable phenotype on specific cell types. Retroviral vectors provide several advantages over DNA-mediated gene transfer for the introduction of expression libraries into eukaryotic cells since they can be used to express genes in a wide range of cell types, including those that form important experimental systems such as the hemopoietic system. We describe here a straightforward and efficient method for generating expression libraries by using a murine retroviral vector. Essentially, the method involves the directional cloning of cDNA into the retroviral vector and the generation of pools of stable ecotropic virus producing cells from this DNA. The cells so derived constitute the library, and the virus they yield is used to infect appropriate target cells for subsequent functional screening. We have demonstrated the feasibility of this procedure by constructing several large retroviral libraries (10(5) to 10(6) individual clones) and then using one of these libraries to isolate cDNAs for interleukin-3 and granulocyte-macrophage colony-stimulating factor on the basis of the ability of these factors to confer autonomous growth on the factor-dependent hemopoietic cell line FDC-P1. Moreover, the frequency at which these factor-independent clones were isolated approximated the frequency at which they were represented in the original plasmid library. These results suggest that expression cloning with retroviruses is a practical and efficient procedure and should be a valuable method for the isolation of important regulatory genes.

Animals↗
PubMed2005

Construction of a hexaploid wheat (Triticum aestivum L.) bacterial artificial chromosome library for cloning genes for stripe rust resistance.

A hexaploid wheat (Triticum aestivum L.) bacterial artificial chromosome (BAC) library was constructed for cloning Yr5 and other genes conferring resistance to stripe rust (Puccinia striiformis f. sp. tritici). Intact nuclei from a Yr5 near-isogenic line were used to isolate high molecular weight DNA, which was partially cleaved with HindIII and cloned into pECBAC1 and pIndigoBAC-5 vectors. The wheat BAC library consisted of 422,400 clones arrayed in 1100 micro-titer plates (each plate with 384 wells). Random sampling of 300 BAC clones indicated an average insert size of 140 kb, with a size range from 25 to 365 kb. Ninety percent of the clones in the library had an insert size greater than 100 kb and fewer than 5% of the clones did not contain inserts. Based on an estimated genome size of 15,966 Mb for hexaploid wheat, the BAC library was estimated to have a total coverage of 3.58x wheat genome equivalents, giving approximately 96% probability of identifying a clone representing any given wheat DNA sequence. Twelve BAC clones containing an Yr5 locus-specific marker (Yr5STS7/8) were successfully selected by PCR screening of 3-dimensional BAC pools. The results demonstrated that the T. aestivum BAC library is a valuable genomic resource for positional cloning of Yr5. The library also should be useful in cloning other genes for stripe rust resistance and other traits of interest in hexaploid wheat.

Basidiomycota↗
PubMed2000

Construction of an equalized cDNA library from Colletotrichum lagenarium and its application to the isolation of differentially expressed genes.

To establish an efficient screening system for differentially expressed genes of a phytopathogenic fungus Colletotrichum lagenarium, we constructed an equalized (normalized) cDNA library from C. lagenarium and used this library for differential screening. For the isolation of genes involved in infection-related developments of conidia, conidia undergoing appressorium differentiation were selected as the source of materials for construction of the cDNA library. The equalization of cDNA was performed twice using a kinetic method, and the products were cloned into a plasmid vector. Colony hybridization with nine probes of different abundance showed a reduction in abundance variation from at least 276-fold in the original library to 10-fold in the equalized cDNA library, which demonstrated that the cDNA was successfully equalized. By differential hybridization of 1900 cDNA clones in the equalized cDNA library and RNA blot analysis of candidate clones, we identified 11 independent cDNA clones, designated CAD1 through CAD11, that were expressed in appressorium-differentiating conidia, but not in vegetative mycelia. The transcripts of CAD1 and CAD2 hardly accumulated in preincubated conidia, whereas those of CAD3 and CAD4 accumulated highly and slightly, respectively. The amount of the four CAD transcripts increased at the early stage of the appressorium formation process. Sequence analysis of CAD1 revealed that CAD1 would encode for 101 amino acid polypeptides, which showed homology to metallothioneins. Deduced amino acid sequence of CAD2 would encode 278 amino acid polypeptides, and showed high homology to genes in aflatoxin, and sterigmatocystin gene clusters of Aspergillus parasiticus and A. nidulans, respectively.

Amino Acid Sequence↗