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Nucleotide sequencing double-stranded plasmids with primers selected from a nonamer library.

Nonamer primers, selected from a nonamer library, were tested by sequencing two plasmid subclones containing known insert sequences. These sequences were scanned (nonamer-mapped) against the 2391-member nonamer library to identify all members that share a 100% match at only one site. A total of 59 nonamers were tested using a slightly modified T7 polymerase sequencing procedure for double-stranded DNA. The success rate for nonamer primed reactions was about 60%, and single-stranded coverage was obtained for approximately 90% of each plasmid insert. The results presented demonstrate that a nonamer library, with as few as 2391 members, can greatly aid the completion of many sequencing projects by reducing the number of required custom primers. With the development of a technique for the rapid identification of all useful library primers for a particular sequencing project, one could envision a high-throughput shotgun-type sequencing procedure that would not require large numbers of subclones.

DNA Primers↗

A method based on PCR for the construction of cDNA libraries and probes from small amounts of tissue.

A PCR-based method is described for the production of cDNA libraries and total cDNA probes from a few milligrams of tissue. Using a model system, we show how a PCR library and PCR probes can be used to identify genes expressed at different levels in two tissues. Small amounts of tissue derived from two plants, one infected with arabis mosaic virus and the other uninfected, were used to make a library and probes. This library and the probes were used to identify viral genes expressed only in the infected plant.

Base Sequence↗

Construction of subtracted cDNA libraries enriched for cDNAs for genes expressed in the mesoderm of early Xenopus gastrulae.

We present a new method for the construction of subtracted cDNA libraries. It makes use of two directional cDNA libraries in phagemid vectors and has two advantages over traditional protocols: it can be used when a limiting amount of driver material is available and the cDNA inserts in the subtracted libraries are long enough to encode functional proteins. Using this method, we have constructed two subtracted libraries enriched for sequences expressed in the mesoderm of early Xenopus gastrulae.

Animals↗

A method for linking VL and VH region genes that allows bulk transfer between vectors for use in generating polyclonal IgG libraries.

Libraries of Ab fragments have been produced by others from light and heavy chain cDNAs derived from populations of B lymphocytes and expressed in bacteria. However, such libraries have not been transferred to eukaryotic expression vectors to generate polyclonal libraries of intact glycosylated Abs that can mediate effector functions. We present a method for transferring pairs of linked VL-VH region genes between circular prokaryotic and eukaryotic vectors. The key feature of the transfer is that the VL and VH region genes are linked head to head (<-->) in opposite transcriptional orientations. To illustrate this method, a pair of VL and VH region cDNAs derived from an existing hybridoma cell line were linked head to head by PCR, transferred as a unit between vectors, and expressed as an IgG Ab with Ag binding activity. Although we tested the transfer of a single VL-VH region gene pair, this system is expected to allow the bulk transfer of physically linked VL-VH region gene combinations between different circular vectors and the expression of the same library as either Ab fragments or intact Abs.

Animals↗

Library fungi at the University of São Paulo and their relationship with respiratory allergy.

In this study, we investigated the fungi most frequently found in the air and the books of 28 libraries at the University of São Paulo, Brazil. In a second phase, 314 librarians were questioned about the presence of asthmatic or rhinitic symptoms, and the relationship with the site of work. Forty-nine percent of them reported this type of symptoms and 80% related them to the place of work. In the third stage, librarians underwent intracutaneous tests against the 20 fungi most frequently isolated in libraries. Eighteen librarians presented positive tests, 12 of whom reported rhinitis and 6 of whom were asymptomatic. In 19.5% of symptomatic patients wheals > 15 mm were observed after 6 h, while 9% of the asymptomatic patients presented similar wheals. Fourteen librarians with positive tests underwent tests with each of the 20 fungi constituting the pool. The airborne fungi isolated in libraries are likely to be found anywhere in the city of São Paulo, but they are likely to be present in higher concentrations in libraries, subsequently producing respiratory allergies.

Adolescent↗

Selection of an active single chain Fv antibody from a protein linker library prepared by enzymatic inverse PCR.

Enzymatic inverse PCR mutagenesis was developed as a simple and reliable method for the construction of large libraries of site-directed mutants. Enzymatic inverse PCR library mutagenesis uses a single PCR fragment and is restriction-site independent. The usefulness of the technique was demonstrated by the design of a single chain linker for an antibody Fv fragment without computer modeling. The Fv fragment of an antibody specific for a metal chelate was expressed in active form in the periplasm of E. coli. The light and the heavy chains of the Fv are expressed as a bicistronic mRNA. Enzymatic inverse PCR mutagenesis was used to construct a library of 3 x 10(5) Fv mutants, in which the C-terminus of the light chain was connected to the N-terminus of the heavy chain by a 15-amino acid peptide linker of variable composition. After plating, active mutant colonies were identified by screening colony filter lifts with a radiolabeled hapten, N'-(2-hydroxyethyl)-p-thioureidobenzyl EDTA. About 0.2% of the mutants were positive, and a selected sFv clone was shown to have the same affinity as the Fv (9 x 10(9)) and was similar to the whole antibody (11 x 10(9)). This example compares favorably with both of the other approaches to constructing sFv's; namely, molecularly modeled linkers as well as universal linkers, which have often yielded significantly lower affinities than whole antibodies or Fabs. The enzymatic inverse PCR library mutagenesis approach is simple and reliable and can be used to obtain linkers for the great majority of antibodies for which no structural data are available. More generally, it can be used to modify DNA coding for any structural protein or regulatory element.

Base Sequence↗

Human lymphocyte cDNA ordered library analyzed by 2D gel electrophoresis. 1. Pooling strategy and matching of gel patterns.

We have analyzed an ordered library of 4,608 cDNA clones from the CEM human leukemic cell line. The aim was to facilitate gene retrieval, to enable immediate access to cDNA clones and to provide information on the protein expression of the individual clones in a 2D gel readout. The matrix array of 24 x 16 x 12, each position of which contained lambda jacII phage from one plaque, enabled us to establish pools of clones along the three axes (24 pools of complexity 192 cloned entities, 16 pools of complexity 288 and 12 pools of complexity 384). The total cDNA complexity is here reduced to such a level that spots which in more complex gels served as landmark spots are not present in each pool, and thus cannot serve as landmarks anymore. The image analysis of such gels and especially the matching of spots is not reliable under these circumstances. In order to achieve reliable matching, additional samples were created, such that pools were co-electrophoresed according to a special concatenation scheme; these samples then contained over-lapping elements (e.g., pools 1 + 2 + 3 and 3 + 4 + 5 have at least those spots in common, which originate from pool 3). This approach turned out to be feasible and we have completed the matching of one half of the ordered library. Already from the present stage of analysis we have obtained valuable information on the cDNA library and on the distribution of clones in this library.

DNA, Complementary↗

["Spoils of war"--losses of the Berlin Veterinary Medicine Library in 1945].

All the German veterinary libraries suffered heavy losses during World War II and its aftermath, but the Berlin Veterinary Faculty, whose library was known until 1945 as the biggest veterinary book collection of the world, was exceedingly affected. The reports on the damage during these years note losses of up to 35,000 or 43,000 of nearly 60,000 books. Actually, diverse papers did mention this fact but they concealed the underlying causes of the losses or simply referred to effects of war. However, new evidence allows to claim, that most of the losses have been caused by a very different measure: the confiscation of parts of the library by units of the Soviet Army in May and June 1945 as spoils of war. Eye witnesses and unpublished official as well as unofficial reports show that the choice as to what books should be seized was purposefully made according to special criteria: the most modern specialists' literature (journals and books) and the rare books of bibliophilic value. The library lost important parts of its stock: medical books, parts of the irreplaceable historical collection in veterinary medicine, the foreign language books, the biggest part of the natural science literature (physics, chemistry, biology with botany and zoology) and books on adjacent fields of interest (geography and travel literature, belles lettres, history, philosophy and so on). This contribution deals with the events during the weeks before and after VE day and the course of measures taken in selecting and expropriating the books mentioned above. It is intended to invite additional contemporary witnesses who might bei knowledgeable about these events to help in specifying them more precisely and clearing open questions, which still remain.

Animals↗

PLANET: a phage library analysis expert tool.

In recent years random peptide libraries displayed on filamentous phage have been widely used and new ideas and techniques are continuously developing in the field (1-5). Notwithstanding this growing interest in the technique and in its promising results, and the enormous increase in usage and scope, very little effort has been devoted to the implementation of software able to handle and analyze the growing number of phage library-derived sequences. In our laboratory, phage libraries are extensively used and peptide sequences are continuously produced, so that the need arose of creating a database (6) to collect all the experimental results in a format compatible with GCG sequence analysis packages (7). We present here the description of an XWindow-based software package named PLANET (Phage Library ANalysis Expert Tool) devoted to the maintenance and statistical analysis of the database.

Bacteriophages↗

Construction of a human heart cDNA library and identification of cardiovascular based genes (CVBest).

The availability of high quality cDNA libraries is often crucial to the successful identification and characterization of genes. The concepts and potential pitfalls of constructing cDNA libraries are presented. Various applications requiring high quality cDNA libraries are outlined, including large-scale single pass sequencing of cDNA clones to generate expressed sequence tags (ESTs) and differential screening of cDNA libraries. The usefulness of combining such approaches for the discovery of novel disease-related and cardiovascular-based ESTs (CVBest) is discussed.

Base Sequence↗

A peptidomimetic antagonist of TNF-alpha-mediated cytotoxicity identified from a phage-displayed random peptide library.

Phage-displayed peptide libraries represent a vast collection of peptide sequences that can be used to identify novel therapeutic molecules. In this report, a 15-mer phage-displayed peptide library was used to identify potential TNF-alpha antagonists. After direct interaction of recombinant human TNF-alpha with the library, four randomly selected phage clones were shown to inhibit in a dose-dependent fashion both mouse and human TNF-alpha-induced cytotoxicity in vitro. DNA sequencing of the positive clones revealed a common amino acid sequence that does not bear any structural similarity to the known primary structures of the extracellular domains of either 55-kDa or 75-kDa TNF receptors. This sequence was synthesized, and the peptidomimotope was shown i) to bind to the recombinant human TNF-alpha using surface plasmon resonance (biosensor) technology and ii) to inhibit both recombinant mouse and human TNF-alpha-induced cytotoxicity in vitro in a dose-dependent fashion. These findings highlight the potential of phage-displayed random peptide libraries for the identification of novel low molecular antagonistic molecules that can block the biologic activities of TNF-alpha.

Amino Acid Sequence↗

Establishing a psychiatric library.

The amount of information in the psychiatric field has increased to such an extent that a specialized library is an essential part of the mental health facility. Guidelines are presented for establishing a psychiatric library, including staffing, budgeting, physical layout, and selection and handling of books, reference materials, journals and annuals, microfilm and microfiche, and audiovisuals. The expertise of the librarian is the single most important factor in determining the quality of the collection and the services provided, the author believes. Because of escalating costs, developing a cooperative library network of small, specialized mental health collections may be the most productive course for the future.

Humans↗

VARIETIES OF INFORMATION REQUESTS IN A MEDICAL LIBRARY.

In an analysis of the information requests received and recorded in the Cleveland Medical Library over a two-year period, the requests were classified in four groups according to whether they were direct inquiries (factual), indirect (subject), biographical and directory, or requests for verification of literature citations. An attempt was made to discover some of the major characteristics and the volume of activity in each class. The results reflect the information services supplied by a single library with its own unique clientele and functions, rather than provide a means of generalizing about information services in all libraries, although they may give us some useful insights. Detailed analyses are made of direct and of biographical and directory questions. The largest number of inquiries received were those in which literature references on a specified subject were requested (indirect inquiries); these represented over 50 percent of the total. Most of the requests in this category called for limited coverage (five articles or less) in the recent literature (not over five years). Requests for verification of citations ranked next with 25 percent of the total, followed by direct (factual) inquiries (12 percent) and biographical and directory inquiries (9 percent), although it is recognized that these last two classes are probably underrepresented in the sample.

Bibliographies as Topic↗

Providing library services in a time of fiscal crisis: alternatives.

The nature of the fiscal crisis in health science libraries in the United States is in part due to the style of management in these libraries, in part due to the lack of user identification, in part due to the lack of economically valid fees for service, and in part due to the success of the Biomedical Communications Network. These issues are discussed in terms of how they might be approached. A pragmatic stance is advocated for practitioners, the MLA, the NLM, and library schools to jointly address the questions raised.

Economics↗

Perioperative nurses' guide to the library.

Nurses have a professional responsibility, both morally and ethically, to provide the most current and informed patient care possible. Nurses should maintain competence in nursing, exercise informed judgment, participate in activities that contribute to the ongoing development of their profession's body of knowledge, and participate in their profession's efforts to implement and improve standards of nursing care. Nurses should stay current with new information and share this information with coworkers to help disseminate new knowledge. Such practices help advance both the body of knowledge for perioperative nursing and the profession of nursing. Nurses are encouraged to make at least two visits each year to the library. During this time, they can take the opportunity to review current periodicals, recent publications, do a CINAHL search, and browse the Internet. Nurses need to be familiar with library resources, so go to the library. Share your findings with colleagues and students. Acquire the information-seeking skills that will best serve you and your patients in the twenty-first century.

Catalogs, Library↗

Virtual shelves in a digital library: a framework for access to networked information sources.

OBJECTIVE: Develop a framework for collections-based access to networked information sources that addresses the problem of location-dependent access to information sources. DESIGN: This framework uses a metaphor of a virtual shelf. A virtual shelf is a general-purpose server that is dedicated to a particular information subject class. The identifier of one of these servers identifies its subject class. Location-independent call numbers are assigned to information sources. Call numbers are based on standard vocabulary codes. The call numbers are first mapped to the location-independent identifiers of virtual shelves. When access to an information resource is required, a location directory provides a second mapping of these location-independent server identifiers to actual network locations. RESULTS: The framework has been implemented in two different systems. One system is based on the Open System Foundation/Distributed Computing Environment and the other is based on the World Wide Web. CONCLUSIONS: This framework applies in new ways traditional methods of library classification and cataloging. It is compatible with two traditional styles of selecting information searching and browsing. Traditional methods may be combined with new paradigms of information searching that will be able to take advantage of the special properties of digital information. Cooperation between the library-informational science community and the informatics community can provide a means for a continuing application of the knowledge and techniques of library science to the new problems of networked information sources.

Catalogs, Library↗

Implementing a personalized resource management program for a small medical library by using LinkOut.

End users want easy access to electronic full-text articles from their desktops. This small medical library has implemented an easy and cost-effective resource management program to meet this user need. This column will describe the implementation, use, and maintenance of the National Library of Medicine's LinkOut, a personalized resource management program that provides immediate access to the library's online journal full-text holdings via hyperlinks.

Access to Information↗