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PubMed1991

Dutch library services to disadvantaged persons.

Hospital libraries in the Netherlands provide a variety of services. In this paper two well-equipped hospital libraries, staffed by professionally qualified librarians, are described. Some hospital libraries rely on volunteers, supervised by professional librarians, while in others volunteers work completely on their own. Special services for elderly people are focused on persons who cannot visit the library by themselves. These services vary from visiting house-bound people to placing a small library in residential homes. Of special interest is the library work for aphasics in the Netherlands. The Section of Library Work for the Elderly, Sick and Handicapped Persons, a section of the Dutch Centre for Public Libraries and Literature (NBLC) is described.

Aged↗
PubMed2006

An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants.

Random transposon insertion libraries have proven invaluable in studying bacterial genomes. Libraries that approach saturation must be large, with multiple insertions per gene, making comprehensive genome-wide scanning difficult. To facilitate genome-scale study of the opportunistic human pathogen Pseudomonas aeruginosa strain PA14, we constructed a nonredundant library of PA14 transposon mutants (the PA14NR Set) in which nonessential PA14 genes are represented by a single transposon insertion chosen from a comprehensive library of insertion mutants. The parental library of PA14 transposon insertion mutants was generated by using MAR2xT7, a transposon compatible with transposon-site hybridization and based on mariner. The transposon-site hybridization genetic footprinting feature broadens the utility of the library by allowing pooled MAR2xT7 mutants to be individually tracked under different experimental conditions. A public, internet-accessible database (the PA14 Transposon Insertion Mutant Database, http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi) was developed to facilitate construction, distribution, and use of the PA14NR Set. The usefulness of the PA14NR Set in genome-wide scanning for phenotypic mutants was validated in a screen for attachment to abiotic surfaces. Comparison of the genes disrupted in the PA14 transposon insertion library with an independently constructed insertion library in P. aeruginosa strain PAO1 provides an estimate of the number of P. aeruginosa essential genes.

DNA Transposable Elements↗
PubMed1992

Construction of small-insert genomic DNA libraries highly enriched for microsatellite repeat sequences.

We describe an efficient method for the construction of small-insert genomic libraries enriched for highly polymorphic, simple sequence repeats. With this approach, libraries in which 40-50% of the members contain (CA)n repeats are produced, representing an approximately 50-fold enrichment over conventional small-insert genomic DNA libraries. Briefly, a genomic library with an average insert size of less than 500 base pairs was constructed in a phagemid vector. Amplification of this library in a dut ung strain of Escherichia coli allowed the recovery of the library as closed circular single-stranded DNA with uracil frequently incorporated in place of thymine. This DNA was used as a template for second-strand DNA synthesis, primed with (CA)n or (TG)n oligonucleotides, at elevated temperatures by a thermostable DNA polymerase. Transformation of this mixture into wild-type E. coli strains resulted in the recovery of primer-extended products as a consequence of the strong genetic selection against single-stranded uracil-containing DNA molecules. In this manner, a library highly enriched for the targeted microsatellite-containing clones was recovered. This approach is widely applicable and can be used to generate marker-selected libraries bearing any simple sequence repeat from cDNAs, whole genomes, single chromosomes, or more restricted chromosomal regions of interest.

Animals↗
PubMed1999

Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface.

Polypeptide library screening technologies are critically dependent upon the characteristics of the expression system employed. A comparative analysis of the lpp-lac, tet and araBAD promoters was performed to determine the importance of tight regulation and expression level in library screening applications. The surface display of single-chain antibody (scFv) in Escherichia coli as an Lpp-OmpA' fusion was monitored using a fluorescently tagged antigen in conjunction with flow cytometry. In contrast to the lpp-lac promoter, both tet and araBAD promoters could be tightly repressed. Tight regulation was found to be essential for preventing rapid depletion of library clones expressing functional scFv and thus for maintaining the initial library diversity. Induction with subsaturating inducer concentrations yielded mixed populations of uninduced and fully induced cells for both the tet and araBAD expression systems. In contrast, homogeneous expression levels were obtained throughout the population using saturating inducer concentrations and could be adjusted by varying the induction time and plasmid copy number. Under optimal induction conditions for the araBAD system, protein expression did not compromise either cell viability or library diversity. This expression system was used to screen a library of random scFv mutants specific for digoxigenin for clones exhibiting improved hapten dissociation kinetics. Thus, an expression system has been developed which allows library diversity to be preserved and is generally applicable to the screening of E. coli surface displayed libraries.

Antibodies↗
PubMed2006

EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. II. Fungal cells sporulating on chitin and producing oosporein.

In the accompanying paper [Cho, E.-M., Liu, L., Farmerie, W. & Keyhani, N. O. (2006). Microbiology 152, 2843-2854], the analysis of expressed sequence tag (EST) libraries derived from homogeneous single-cell populations of aerial conidia, in vitro blastospores and submerged conidia of the entomopathogenic fungus Beauveria (Cordyceps) bassiana has been reported. Here an extended EST analysis is presented of complex cell mixtures derived from fungal cells sporulating on chitin or grown under culture conditions inducing the production of the B. bassiana secondary metabolite, oosporein. Fungal material used for the construction of the libraries included germinating conidia and blastospores, mycelia, as well as cells in various developmental stages. Approximately 2,500 5' end sequences were determined from random sequencing of clones from each library, and were clustered into 277 contigs with 1,069 singlets, and 306 contigs with 1,064 singlets, for the chitin and oosporein libraries, respectively. Almost half (45-50 %) of the sequences in each library displayed either no significant similarity (e value >10(-4)) or similarity to hypothetical proteins found in the NCBI database. Approximately 20-25 % of the sequences in each library could be annotated by gene ontology terms. A comparative analysis between the two libraries, as well as the libraries in the accompanying paper, is presented. A set of 4,360 clustered and unique sequences was characterized. The data are indicative of a highly plastic gene expression repertoire being available to B. bassiana for growth during different environmental and developmental conditions, and provides a dataset for gene discovery and genome annotation.

Benzoquinones↗
PubMed2006

Improving library services for graduate nurse students in New Zealand.

BACKGROUND: Graduate students are expected to have basic information literacy skills. Knowing how to access and select relevant information and then utilize that information is a necessary skill in today's knowledge and information-age world. In the health-care sector, the rise in evidence-based medicine has brought into focus the importance of information literacy skills for nurses. In the case described, information literacy skills are embedded in the curriculum and strengthened in collaboration with library staff. AIMS: To identify graduate student nurses' use of the library, and the library's response to findings. METHODS: A case-study approach was used with both quantitative and qualitative data. An anonymous questionnaire was distributed to all nurses enrolled in graduate courses in the second semester of 2002. Interviews were then undertaken with library staff. RESULTS: The university library services were not used by 43% of graduate nursing students and the library responded by developing a number of initiatives. CONCLUSIONS: Finding a deficit in use of the library by graduate nurses, this paper outlines the response by the library to improving awareness of services, access and providing education in a bid to improve literacy skills.

Computer Literacy↗
PubMed2004

Integration of microbial ecology and statistics: a test to compare gene libraries.

Libraries of 16S rRNA genes provide insight into the membership of microbial communities. Statistical methods help to determine whether differences in library composition are artifacts of sampling or are due to underlying differences in the communities from which they are derived. To contribute to a growing statistical framework for comparing 16S rRNA libraries, we present a computer program, integral -LIBSHUFF, which calculates the integral form of the Cramér-von Mises statistic. This implementation builds upon the LIBSHUFF program, which uses an approximation of the statistic and makes a number of modifications that improve precision and accuracy. Once integral -LIBSHUFF calculates the P values, when pairwise comparisons are tested at the 0.05 level, the probability of falsely identifying a significant P value is 0.098 for a study with two libraries, 0.265 for three libraries, and 0.460 for four libraries. The potential negative effects of making the multiple pairwise comparisons necessitate correcting for the increased likelihood that differences between treatments are due to chance and do not reflect biological differences. Using integral -LIBSHUFF, we found that previously published 16S rRNA gene libraries constructed from Scottish and Wisconsin soils contained different bacterial lineages. We also analyzed the published libraries constructed for the zebrafish gut microflora and found statistically significant changes in the community during development of the host. These analyses illustrate the power of integral -LIBSHUFF to detect differences between communities, providing the basis for ecological inference about the association of soil productivity or host gene expression and microbial community composition.

Aeromonas↗
PubMed1995

Construction of human embryonic cDNA libraries: HD, PKD1 and BRCA1 are transcribed widely during embryogenesis.

We used the polymerase chain reaction (PCR) to construct cDNA libraries from small amounts of tissue and to screen cDNA libraries efficiently for the presence of given sequences. To isolate genes expressed in early human development, we constructed both oligo dT-primed and random hexamer-primed cDNA libraries from ten different tissues of human embryos aged 53 to 78 days post conception. Given the small amount of RNA available, it was necessary to amplify the resultant cDNA using PCR to generate sufficient amounts of cDNA for library construction. As a result of using PCR followed by sizing to eliminate smaller synthesized fragments, the size of the synthesized product was > or = 650 base pairs and the average initial complexity of the given libraries was 10(6). We screened these cDNA libraries efficiently using PCR. Primers corresponding to a given gene were used to amplify DNA from phages encompassing a cDNA library. Successful amplification of the appropriate-sized fragment demonstrated that the DNA in question was transcribed in a given tissue. We demonstrated that HD (huntingtin, the protein transcribed from the Huntington disease locus), PKD1 (the most common gene responsible for familial polycystic kidney disease) and BRCA1 (a gene responsible for familial breast cancer) are synthesized nearly ubiquitously (including during embryogenesis). Thus, these human embryonic cDNA libraries constitute a unique resource to study early human development.

Base Sequence↗
PubMed2005

Extension of Lander-Waterman theory for sequencing filtered DNA libraries.

BACKGROUND: The degree to which conventional DNA sequencing techniques will be successful for highly repetitive genomes is unclear. Investigators are therefore considering various filtering methods to select against high-copy sequence in DNA clone libraries. The standard model for random sequencing, Lander-Waterman theory, does not account for two important issues in such libraries, discontinuities and position-based sampling biases (the so-called "edge effect"). We report an extension of the theory for analyzing such configurations. RESULTS: The edge effect cannot be neglected in most cases. Specifically, rates of coverage and gap reduction are appreciably lower than those for conventional libraries, as predicted by standard theory. Performance decreases as read length increases relative to island size. Although opposite of what happens in a conventional library, this apparent paradox is readily explained in terms of the edge effect. The model agrees well with prototype gene-tagging experiments for Zea mays and Sorghum bicolor. Moreover, the associated density function suggests well-defined probabilistic milestones for the number of reads necessary to capture a given fraction of the gene space. An exception for applying standard theory arises if sequence redundancy is less than about 1-fold. Here, evolution of the random quantities is independent of library gaps and edge effects. This observation effectively validates the practice of using standard theory to estimate the genic enrichment of a library based on light shotgun sequencing. CONCLUSION: Coverage performance using a filtered library is significantly lower than that for an equivalent-sized conventional library, suggesting that directed methods may be more critical for the former. The proposed model should be useful for analyzing future projects.

Chromosome Mapping↗
PubMed2003

Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli.

BACKGROUND: Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H), is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost) in the amplified library. RESULTS: Analysis of candidate genes as Y2H fusion constructs has shown that, while stable in E. coli and yeast for genetic studies, they are rapidly lost in growth conditions for genomic libraries. This includes the rapid loss of a fragment of the E. coli cell division gene ftsZ which encodes the binding site for ZipA and FtsA. Expression of this clone causes slower growth in E. coli. This clone is also rapidly lost in yeast, when expressed from a GAL1 promoter, relative to a vector control, but is stable when the promoter is repressed. We have demonstrated in this report that the construction of libraries for the E. coli and B. subtilis genomes without passaging through E. coli is practical, but the number of transformants is less than for libraries cloned using E. coli as a host. Analysis of several clones in the libraries that are strongly growth inhibitory in E. coli include genes for many essential cellular processes, such as transcription, translation, cell division, and transport. CONCLUSION: Expression of Y2H clones capable of interacting with E. coli and yeast targets are rapidly lost, causing a loss of complexity. The strategy for preparing Y2H libraries described here allows the retention of genes that are toxic when inappropriately expressed in E. coli, or yeast, including many genes that represent potential antibacterial targets. While these methods are generally applicable to the generation of Y2H libraries from any source, including mammalian and plant genomes, the potential of functional clones interacting with host proteins to inhibit growth would make this approach most relevant for the study of prokaryotic genomes.

Bacillus subtilis↗
PubMed2006

Quality enhancement of the non-immune phage scFv library to isolate effective antibodies.

The non-immune phage antibody library system is one of the most attractive technologies available to current therapeutic, diagnostic and basic scientific research. This system allows the rapid isolation of antibodies of interest that could subsequently be applied directly to drug delivery systems and antibody therapy. Previously, we reported the primer sets to encompass the antibody repertoire and thus improve library quality. However, a wide number of varying primer sets cause to decrease the amplification efficiency of antibody genes. In the present study, we re-generated the library primer sets newly and constructed an improved library from non-immune mice that was far superior in terms of variety and quality. This new library contained 2.4 billion independent clones. In addition, we optimized the selection step from this library to isolate high-affinity antibodies. The optimization of an affinity panning protocol by the incorporation of an automated Microfluidics instrument led to the successful isolation of three different monoclonal antibodies for human vascular endothelial growth factor receptor 2 (KDR). These antibodies were demonstrated to exhibit high specificity and were able to detect a mere 0.6 fmol of KDR by dot blot analysis. Previously reported antibodies for luciferase were also isolated successfully from this library. Our results clearly demonstrate the importance of the improved protocol for the library preparation of antibodies and the resulting isolation of antibodies for clinical and research applications.

Amino Acid Sequence↗
PubMed1999

Preparation of a cDNA library and preliminary assessment of 1400 genes from mouse growth cartilage.

Cartilage is an inconvenient tissue for the isolation of mRNA, and this has hampered studies of its component mRNAs conducted to date. Here, we describe the preparation of a good quality cDNA library from mouse growth cartilage (mGC). A total of 1.7 microg of poly(A)+ RNA was obtained from about 1200 pieces of the mGC zone of 60 young mice (BALB/c, 4 weeks old). Using this poly(A)+ RNA, we constructed a cDNA library using the pAP3neo vector by the linker-primer method. The complexity of the cDNA library was 2.6 x 106 colony-forming units (cfu), which signified that almost all of the mRNA components in the mGC were present in this cDNA library. From this library, 1401 clones were randomly selected and their insert sizes were examined. Of these clones, 166 (12%) had no inserts, 466 (33%) had inserts ranging in size from 0-0.9 kbp, 480 (34%) had inserts of 1. 0-1.9 kbp, 162 (12%) had inserts of 2.0-2.9 kbp, and 127 (9%) had sizes greater than 3.0 kbp. The average insert size was 1.45 kbp. The number of cfu and the insert size data qualified this library as of reasonably good quality. Clones with an insert size greater than 1 kbp (769 clones) were sequenced from their 5' ends. Among the 769 clones examined, 608 gave sequence data. Among these, 196 (32%) were unknown, 2 were only poly A, and 410 (67%) coded for known proteins. Of these, 55 clones coded for type II (pro)collagen, 54 for osteonectin, and 22 for other cartilage collagens (type IX, type X, and type XI). The rest included cartilage extracellular matrix genes, general cellular genes, and others. To judge further the quality of the library, 45 species coding for type II collagen chain were aligned based on their 5' end sequences. Three species (7%) contained almost the full-length insert, and the shortest one was 1. 5 kbp in length (full-length 5.6 kbp). These data show that this cDNA library is of reasonably good quality, making it likely that the large number of unknown inserts (32%) will provide a suitable pool for the identification and functional determination of new GC genes.

Animals↗
PubMed2005

A method for generation of arbitrary peptide libraries using genomic DNA.

Random peptide libraries can be constructed either by in vitro synthesis of random peptides, or through translation of DNA sequences from synthetic random oligonucleotides. Here we describe an alternative way of making arbitrary peptide libraries with high diversity that can be used in screening as random peptide libraries. Genomic DNA digested with a frequent-cutting restriction enzyme recognizing four nucleotides will theoretically consist of small DNA pieces with average length of 256 nucleotides, and on average around 107 fragments can be generated from a genome of 3 x 109 bases. A peptide library translated from these fragments will have sufficient diversity for some protein interaction screening experiments. Moreover, the same genome digested with a different four-cutter enzyme or ligated into different reading frames will result in different nonoverlapping libraries. A series of such libraries could be generated with genomic DNAs from different species. In this study, human genomic DNA was digested with four-cutter restriction enzymes DpnII and Tsp509I, respectively, and cloned into yeast expression vector pGADT7 to generate arbitrary peptide libraries. These libraries were used in yeast two-hybrid assays to screen for binding motifs of the PDZ domain containing protein synectin. Our results showed that in addition to various native carboxy-terminal tails, synectin could also bind to many artificial ones, some of which contained a consensus sequence--(S/T)XC-COOH.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B.

OBJECTIVE: To construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B. METHODS: The total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5' end of the RNA transcript (SMART) technique and CDS III/3' primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction (LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to lambdaTriplEx2 vector. Then lambda phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. RESULTS: The titers of unamplifed and amplified libraries were 1.94 x 10(6) pfu/ml and 1.49 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and 98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29%, and 0.5-1.0 kb in 35.71%. CONCLUSION: A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.

DNA, Complementary↗
PubMed2005

Efficient construction of a large collection of phage-displayed combinatorial peptide libraries.

Selections from phage-displayed combinatorial peptide libraries are an effective strategy for identifying peptide ligands to target proteins. Existing protocols for constructing phage-displayed libraries utilize either ligation into double-stranded phage DNA or Kunkel mutagenesis with single-stranded phagemid DNA. Although the Kunkel approach rapidly provides library sizes of up to 10(11), as many as 20% of the phagemids may be non-recombinant. With several modifications to current Kunkel protocols, we have generated peptide libraries with sizes of up to 10(11) clones and recombination frequencies approaching 100%. The production of phage libraries, as opposed to phagemid libraries, simplifies selection experiments by eliminating the need for helper phage. Our approach relies upon the presence of an amber stop codon in the coding region of gene III of bacteriophage M13. Oligonucleotides containing randomized stretches of DNA are annealed to the phage genome such that the randomized region forms a heteroduplex with the stop codon. The oligonucleotide is then enzymatically extended to generate covalently-closed, circular DNA, which is electroporated into a non-suppressor strain of Escherichia coli. If the amber stop codon is present in the DNA molecule, protein III is not synthesized and the phage cannot propagate itself. This method is customizable for the display of either random or focused peptide libraries. To date, we have constructed 22 different libraries ranging from 8-20 amino acids in length, utilizing complete or reduced codon sets.

Amino Acid Sequence↗
PubMed2005

[Construction and characterization of a cDNA library from human liver tissue of cirrhosis].

OBJECTIVE: To construct a cDNA library from human liver tissue of cirrhosis. METHODS: The total RNA from human liver tissue of cirrhosis was extracted using Trizol method, and the mRNA was purified using mRNA purification kit. SMART technique and CDSIII/3' primer were used for first-strand cDNA synthesis. Long distance PCR was then used to synthesize the double-strand cDNA that was then digested by proteinase K and Sfi I, and was fractionated by CHOMA SPIN-400 column. The cDNA fragments longer than 0.4 kb were collected and ligated to lambdaTripl Ex2 vector. Then lambda-phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries was strictly checked by conventional titer determination. Eleven plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. RESULTS: The titers of unamplifed and amplified libraries were 1.03 x 10(6) pfu/ml and 1.36 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 97.24 % in unamplified library and 99.02 % in amplified library. The lengths of the inserts were 1.02 kb in average (36.36 % 1 approximately equals 2 kb and 63.64 % 0.5 approximately equals 1.0 kb). CONCLUSION: A high quality cDNA library from human liver tissue of cirrhosis was constructed successfully, which can be used for screening and cloning new special genes associated with the occurrence of cirrhosis.

DNA, Complementary↗
PubMed1999

Affinity chromatographic screening of soluble combinatorial peptide libraries.

Affinity chromatography using immobilized S-protein was used for the screening of affinity peptide ligands from two soluble peptide libraries. Peptide library I consisted of octamers with glycine (G) at both termini of each peptide, i.e. GXXXXXXG. The six center positions were constructed using random sequences of six L-amino acids (Y, N, F, E, V, and L). Peptide library II also consisted of octamers but with glycine and valine (V) at both termini of each peptide (GVZZZZVG). The four variable center positions of peptide library II were random sequences of 18 L-amino acids. Peptides that were retained specifically on the immobilized S-protein column were eluted by 2% acetic acid. The peptides in the acid eluate were further separated using reversed-phase HPLC. Each separated peptide fraction was collected and the peptide sequences deconvoluted by mass spectrometry (MS/MS). The screenings of peptide libraries I and II resulted in 12 and 7 affinity peptides, respectively. Eight out of the twelve peptides from peptide library I contained the clear consensus sequence NFEV. Peptide library II resulted in affinity peptides with the sequences GVNFEVVG, GVNFTVVG and GVFFEL(I)VG. The advantages and limitations of affinity chromatography in peptide library screening are discussed.

Amino Acid Sequence↗
PubMed2001

Designing a library: everyone on the same page?

Excerpts are presented from an interview by the Bulletin of the Medical Library Association buildings projects editor with four academic health sciences library directors: one who had recently completed a major library building project and three who were involved in various stages of new building projects. They share their experiences planning for and implementing library-building programs. The interview explores driving forces leading to new library buildings, identifies who should be involved, recalls the most difficult and exciting moments of the building projects, relates what they wished they had known before starting the project, assesses the impact of new library facilities on clients and services, reviews what they would change, and describes forces impacting libraries today and attributes of the twenty-first century library.

Architecture↗