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PubMed2001

Creation of genome-wide protein expression libraries using random activation of gene expression.

Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 x 10(6) individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE libraries created from multiple human cell lines, demonstrating that RAGE libraries are inherently normalized. Pools of RAGE clones were used to isolate 19,547 human gene clusters, approximately 53% of which were novel when tested against public databases of expressed sequence tag (EST) and complementary DNA (cDNA). Isolation of individual clones confirmed that the activated endogenous genes can be expressed at high levels to produce biologically active proteins. The properties of RAGE libraries and RAGE expression clones are well suited for a number of biotechnological applications including gene discovery, protein characterization, drug development, and protein manufacturing.

Cell Line↗

PubMed2003

An improved method for constructing a full-length enriched cDNA library using small amounts of total RNA as a starting material.

We have developed an improved method for constructing a full-length cDNA library using small quantity of material by modifying the original oligo-capping method. In our devised method, total RNAs are used in sequential oligo-capping steps directly without preliminary mRNA purification. Using this method, we constructed full- length cDNA libraries from 100 mg of total RNA. These libraries contained 8x10(5) to 8x10(6) independent clones with average insert sizes of 2.0 kb. Moreover, the number of full-length cDNAs containing the translation initiation codon ATG in the constructed libraries was estimated to 60-70%. In addition, 54% of the known cDNAs had a longer 5' end than the corresponding genes in the public database. Our results show that the method can be effectively used to construct full-length enriched cDNA libraries, especially, if starting material is limited.

Base Sequence↗

PubMed2005

Second-generation shRNA libraries covering the mouse and human genomes.

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference-induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.

Animals↗

PubMed2001

Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step.

We have developed technologies that simplify genomic library construction and screening, substantially reducing both the time and the cost associated with traditional library screening methods and facilitating the generation of gene-targeting constructs. By taking advantage of homologous recombination in Escherichia coli, we were able to use as little as 80 bp of total sequence homology to screen for a specific gene from a genomic library in plasmid or phage form. This method, called recombination cloning (REC), takes only a few days instead of the several weeks required for traditional plaque-lift methods. In addition, because every clone in the mouse genomic library we have constructed has a negative selection marker adjacent to the genomic insert, REC screening can generate gene-targeting vectors in one step, from library screening to finished construct. Conditional targeting constructs can be generated easily with minimal additional manipulation.

Animals↗

PubMed2004

Characterization of an Aedes aegypti bacterial artificial chromosome (BAC) library and chromosomal assignment of BAC clones for physical mapping quantitative trait loci that influence Plasmodium susceptibility.

Previous studies have confirmed a genetic basis for susceptibility of mosquitoes to Plasmodium parasites. Here we describe our efforts to characterize a bacterial artificial chromosome genomic library for the yellow fever mosquito, Aedes aegypti, and to identify BAC clones containing genetic markers that define quantitative trait loci (QTL) for Plasmodium gallinaceum susceptibility. This library (NDL) was prepared from the Ae. aegypti Liverpool strain and consists of 50 304 clones arrayed in 384-well microplates. We used PCR analysis with oligonucleotide primer pairs specific to 106 genetic markers (as sequence-tagged sites or STS) to screen the NDL library. Each STS identified between one and thirteen independent clones with an average of 3.3 clones. The average insert size was 122 kb and therefore the NDL library provides approximately 7.87-fold genome coverage. The availability of the NDL library should greatly facilitate physical mapping efforts, including positional cloning of QTL for traits of interest such as Plasmodium susceptibility and for whole genome sequence determination and assembly.

Aedes↗

PubMed1998

Construction of a bovine yeast artificial chromosome (YAC) library.

We have constructed a bovine yeast artificial chromosome (YAC) library to provide a common resource for bovine genome research. We used leukocytes of a Japanese black bull (Bos taurus) as the DNA source, AB1380 for the yeast host, and pYAC4 for the vector. The library consists of 24,576 clones arranged in 256 96-well microtiter plates. An average insert size estimated from the analysis of 251 randomly selected clones was 480 kb. The rate of chimeric YACs evaluated by fluorescence in situ hybridization (FISH) analysis of 44 randomly selected clones was 36.4%. To estimate the number of genome equivalents, PCR-based screening was performed with 48 primer pairs and isolated 3.2 clones on average. In order to provide broad access for the scientific community, this library has been incorporated into the Reference Library system which provides high density filters for colony hybridization screening and a common database of the library.

Animals↗

PubMed2000

Large-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA library.

BACKGROUND: Intracellular localization is an important part of the characterization of a gene product. In an attempt to search for genes based on the intracellular localization of their products, we constructed a green fluorescent protein (GFP)-fusion genomic DNA library of S. pombe. RESULTS: We constructed the S. pombe GFP-fusion genomic DNA library by fusing, in all three reading frames, random fragments of genomic DNA to the 5' end of the GFP gene in such a way that expression of potential GFP-fusion proteins would be under the control of the own promoters contained in the genomic DNA fragments. Fission yeast cells were transformed with this plasmid library, and microscopic screening of 49 845 transformants yielded 6954 transformants which exhibited GFP fluorescence, of which 728 transformants showed fluorescence localized to distinct intracellular structures such as the nucleus, the nuclear membrane, and cytoskeletal structures. Plasmids were isolated from 516 of these transformants, and a determination of their DNA sequences identified 250 independent genes. The intracellular localizations of the 250 GFP-fusion constructs was categorized as an image database; using this database, DNA sequences can be searched for based on the localizations of their products. CONCLUSIONS: A number of new intracellular structural components were found in this library. The library of GFP-fusion constructs also provides useful fluorescent markers for various intracellular structures and cellular activities, which can be readily used for microscopic observation in living cells.

Amino Acid Sequence↗

PubMed2003

LISTENing to healthcare students: the impact of new library facilities on the quality of services.

Following a low assessment of 'Learning resources' provision by the Quality Assurance Agency, the librarian of Homerton College, School of Health Studies commenced the LISTEN Project, a long-term study to monitor the effects of planned interventions on the quality of library provision. Surveys of entry-to-register student nurses & midwives were conducted in 1999 and 2001 by extensive questionnaires, inviting Likert-scaled and free text responses. Following a college relocation, students made greater than expected use of a new health studies library in Cambridge, and significantly less use of the local teaching hospital library. Using both a satisfaction index and a non-parametric test of mean scores, student evaluation of library services in Cambridge significantly improved following relocation. The physical accommodation and location of library services remain important to healthcare students. Identifiable improvements to the quality of services, however, will overcome initial resistance to change. Education providers must ensure the best mix of physical and electronic services for students who spend much of their time on clinical placement.

Attitude of Health Personnel↗

PubMed1998

A detailed analysis of the C5a anaphylatoxin effector domain: selection of C5a phage libraries on differentiated U937 cells.

We have used a phage-display-based system to investigate the effect of simultaneous substitutions within the C5a effector domain. Two different libraries were constructed. In library I, known binding positions 67, 68, 72 and 74 of human complement C5a (hC5a) and in library II, positions 69-73 of hC5a without C-terminal Arg74 (des-Arg74-C5a) were randomly mutated. In more than 80% (position 72) or 90% (positions 68 and 74) of all cases, the original residues of hC5a were selected from library I, demonstrating that the phage system can be used to define binding points within the C5a molecule. Surprisingly, in more than 90% of all clones, a Phe residue was enriched at position 67 instead of the original His residue which, however, did not affect the binding affinity or the signalling activity. In library II, Leu was preferentially selected at positions 70-72 and Tyr at position 73, while no enrichment of an individual amino acid was observed at position 69. Mutants with (a) Leu in positions 71 and 72 (b) Ser or Leu in position 70 and (c) Arg or Tyr in position 73, showed a 4-10-fold higher binding affinity as compared to des-Arg74-[Ala27, Phe67]C5a. The binding affinity was indistinguishable from that of hC5a. In consequence, not only position 72 but also positions 70, 71 and 73 are able to interact with the C5a receptor, whereas position 69 is not. Intriguingly, one mutant with a high binding affinity but without signalling activity was selected. Thus, random mutagenesis of phage-displayed C5a was proven to be a powerful strategy to define receptor-binding points and to select C5aR antagonists based on the structure of the natural ligand.

Acetylglucosaminidase↗

PubMed1990

Construction and characterization of a yeast artificial chromosome library containing seven haploid human genome equivalents.

Prior to constructing a library of yeast artificial chromosomes (YACs) containing very large human DNA fragments, we performed a series of preliminary experiments aimed at developing a suitable protocol. We found an inverse relationship between YAC insert size and transformation efficiency. Evidence of occasional rearrangement within YAC inserts was found resulting in clonally stable internal deletions or clonally unstable size variations. A protocol was developed for preparative electrophoretic enrichment of high molecular mass human DNA fragments from partial restriction digests and ligation with the YAC vector in agarose. A YAC library has been constructed from large fragments of DNA from an Epstein-Barr virus-transformed human lymphoblastoid cell line. The library presently contains 50,000 clones, 95% of which are greater than 250 kilobase pairs in size. The mean YAC size of the library, calculated from 132 randomly isolated clones, is 430 kilobase pairs. The library thus contains the equivalent of approximately seven haploid human genomes.

Cloning, Molecular↗

PubMed1990

Streamlined approach to creating yeast artificial chromosome libraries from specialized cell sources.

The study of tumor-specific chromosomal abnormalities has been severely impeded by an inability to link cytogenetic to molecular data. Restriction fragment length polymorphism mapping of any particular chromosomal rearrangement to the resolution limit of genetic methodology generates sets of probes that frequently are still too widely spaced to render the rearrangement breakpoints accessible to molecular isolation. The stable propagation of genomic fragments of up to one million base pairs in size as yeast artificial chromosomes (YACs) represents an important development in this regard. However, existing YAC libraries have been made from karyotypically normal sources making the localization and cloning of specific rearrangement breakpoints much more difficult. As a solution to this problem, we present an improved method for creating YAC libraries that can utilize specialized tumor-derived materials and that can be executed effectively in a small laboratory setting. Procedures that enabled more consistent DNA insert size selection and enhanced yeast transformation frequency were employed to generate a human YAC library from a neuroepithelioma cell line containing a characteristic t(11;22) chromosomal translocation. Approximately 40,000 colonies with an average insert size of 330 kilobase pairs were created. This library was screened with two single-copy probes that bracket the translocation breakpoint. YAC clones ranging from 370 to 550 kilobase pairs that were specific for each single-copy probe were identified. Specialized YAC libraries will make many more tumor-specific chromosomal abnormalities accessible to molecular isolation.

Cell Line↗

PubMed1992

In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library.

We have used a combinatorial immunoglobulin library approach to obtain monoclonal antibodies from nonimmune adult mice, thereby establishing the principles of (i) accessing naive combinatorial antibody libraries for predetermined specificities and (ii) increasing the affinity of the selected antibody binding sites by random mutagenesis. A combinatorial Fab library expressing immunoglobulin mu and kappa light-chain fragments on the surface of filamentous phage was prepared from bone marrow of nonimmunized, adult BALB/c mice with the multivalent display vector pComb8. Phage displaying low affinity Fabs (binding constants, 10(4)-10(5) M-1) binding to a progesterone-bovine serum albumin conjugate were isolated from the library. Random mutagenesis of the heavy- and light-chain variable regions expressed in the mono-valent phage display vector pComb3 was performed by error-prone PCR, and subsequently clones with improved affinity for the hapten conjugate were selected. We demonstrate that antibodies with desirable characteristics from a nonimmune source may be selected and affinity maturation may be achieved by using the twin vectors pComb8 and pComb3, thus opening the route to obtaining specific antibodies from a generic library and bypassing immunization.

Amino Acid Sequence↗

PubMed1994

Construction and characterization of a normalized cDNA library.

We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each circular template, melting and reannealing of the partial duplexes at relatively low C0t, and hydroxyapatite column chromatography, unreassociated circles were recovered from the flow through fraction and electroporated into bacteria, to propagate a normalized library without a requirement for subcloning steps. An evaluation of the extent of normalization has indicated that, from an extreme range of abundance of 4 orders of magnitude in the original library, the frequency of occurrence of any clone examined in the normalized library was brought within the narrow range of only 1 order of magnitude.

Base Sequence↗

PubMed1995

Systematic screening of an arrayed cDNA library by PCR.

We have developed a PCR-based method for rapid and effective screening of arrayed cDNA libraries. This strategy directly addresses the limitations of conventional hybridization-based schemes and provides a more rapid, cost-effective, and sensitive method compatible with large-scale and routine cDNA clone recovery. To prepare arrayed libraries, 1-2 x 10(6) cDNA clones were propagated as individual plaques on solid medium in 24-well culture dishes at approximately 250 plaque-forming units per well. Phage suspensions were prepared from each well and transferred to a 96-well format. To screen the library, pools were generated that correspond to each individual 96-well plate and to each row and column within "blocks" of six plates each. Library screening for specific cDNA clones was conducted in a systematic and hierarchical fashion beginning with the plate pools. Next, the row/column pools corresponding to each positive plate pool were screened. Finally, isolated clones from within each positive well were identified by hybridization. We have applied this approach to the screening of an arrayed human brain cDNA library resulting in the recovery of cDNAs corresponding to > 25 genes and expressed sequence tags.

Adult↗

PubMed1997

Generation of cDNA expression libraries enriched for in-frame sequences.

Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore developed and tested a library construction methodology using a novel vector, pKE-1, with which translation in the correct reading frame confers kanamycin resistance on the host. Following kanamycin selection, the cDNA libraries contained 60-80% open, in-frame clones. These, compared with unselected libraries, showed a 10-fold increase in the number of matches between the cDNA-encoded proteins made by the bacteria and database protein sequences. cDNA sequencing programs will benefit from the enrichment for correct coding sequences, and screening methods requiring protein expression will benefit from the enrichment for authentic translation products.

Base Sequence↗

PubMed1999

Toward functional genomics in bacteria: analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus.

As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes.

Bacillus cereus↗

PubMed1996

Elucidation of the core residues of an epitope using membrane-based combinatorial peptide libraries.

Combinatorial peptide libraries have proved to be a valuable tool for the study of the interaction of a functional protein with its ligand. Here, the epitope for a monoclonal antibody 201/9, raised against beta-factor XIIa, has been identified with a two-step approach using peptide libraries attached to a polymer (polyvinylidene difluoride) membrane. First, the octapeptide libraries with two amino acids defined at position 2 and 4, represented by the formula X-O2-X-O4-X-X-X-X, were synthesized on a sheet of polymer membrane in which X represents a mixture of all the natural -amino acids except cysteine, while O2 and O4 each represent a single amino acid. The libraries were probed with the antibody 201/9, and the bound antibody was detected with a sensitive chemiluminescent method. In the first cycle, the peptide mixtures X-Phe-X-Gln-X-X-X-X showed the strongest signal development. In the second cycle Phe and Gln were incorporated into new libraries consisting of sequences O1-Phe-X-Gln-X-X-X-X, X-Phe-O3-Gln-X-X-X-X, X-Phe-X-Gln-O5-X-X-X, X-Phe-X-Gln-X-O6-X-X, X-Phe-X-Gln-X-X-O7-X, and X-Phe-X-Gln-X-X-X-O8. After probing these new peptides, the residues representing the core sequence of the epitope for monoclonal antibody 201/9 were elucidated. The sequence Ser-Phe-Leu-Gln-Glu-Asn, identified as the immunodominant epitope, correlates well with the sequence Ser-Phe-Leu-Gln-Glu-Ala previously identified (Gao, B., and Esnouf, M. P. (1996) J. Immunol. 157, 183-188) in a scan of overlapping peptides based on the sequence of human beta-factor XIIa.

Amino Acid Sequence↗

PubMed1999

A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies.

We report the design, construction, and use of the first very large non-immunized phage antibody library in Fab format, which allows the rapid isolation and affinity analysis of antigen-specific human antibody fragments. Individually cloned heavy and light chain variable region libraries were combined in an efficient two-step cloning procedure, permitting the cloning of a total of 3.7 x 10(10) independent Fab clones. The performance of the library was determined by the successful selection of on average 14 different Fabs against 6 antigens tested. These include tetanus toxoid, the hapten phenyl-oxazolone, the breast cancer-associated MUC1 antigen, and three highly related glycoprotein hormones: human chorionic gonadotropin, human luteinizing hormone, and human follicle-stimulating hormone. In the latter category, a panel of either homone-specific or cross-reactive antibodies were identified. The design of the library permits the monitoring of selections with polyclonal phage preparations and to carry out large scale screening of antibody off-rates with unpurified Fab fragments on BIAcore. Antibodies with off-rates in the order of 10(-2) to 10(-4) s-1 and affinities up to 2.7 nM were recovered. The kinetics of these phage antibodies are of the same order of magnitude as antibodies associated with a secondary immune response. This new phage antibody library is set to become a valuable source of antibodies to many different targets, and to play a vital role in target discovery and validation in the area of functional genomics.

Amino Acid Sequence↗