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At least 1,459 records · Page 81Linked to original sources
PubMed1998

Construction and characterization of the IGF Arabidopsis BAC library.

A bacterial artificial chromosome (BAC) library has been established for Arabidopsis thaliana (ecotype Col-0) covering about seven haploid nuclear genome equivalents. This library, called the Institut für Genbiologische Forschung (IGF) BAC library, consists of 10,752 recombinant clones carrying inserts (generated by partial EcoRI digestion) of an average size of about 100 kb in a modified BAC vector, pBeloBAC-Kan. Hybridization with organellar DNA and nuclear repetitive DNA elements revealed the presence of 1.1% clones with mitochondrial DNA, 0.2% clones with plastid DNA, 3.2% clones with the 180 bp paracentromeric repeat, 1.6% clones with 5S rDNA, and 10.8% clones with the 18S-25S rDNA repeat. With its extensive genome coverage, its rather uniformly sized inserts (80 kb < 85% < 120 kb) and low contamination with organellar DNA, this library provides an excellent resource for A. thaliana genomic mapping, map-based gene cloning, and genome sequencing.

Arabidopsis↗
PubMed2001

Physical mapping across an avirulence locus of Phytophthora infestans using a highly representative, large-insert bacterial artificial chromosome library.

The oomycete plant pathogen Phytophthora infestans is the causal agent of late blight, one of the most devastating diseases of potato worldwide. As part of efforts to clone avirulence (Avr) genes and pathogenicity factors from P. infestans, we have constructed a bacterial artificial chromosome (BAC) library from an isolate containing six Avr genes. The BAC library comprises clones with an average insert size of 98 kb and represents an estimated 10 genome equivalents. A three-dimensional pooling strategy was developed to screen the BAC library for amplified fragment length polymorphism (AFLP) markers, as this type of marker has been extensively used in construction of a P. infestans genetic map. Multiple positive clones were identified for each AFLP marker tested. The pools were used to construct a contig of 11 BAC clones in a region of the P. infestans genome containing a cluster of three avirulence genes. The BAC contig is predicted to encompass the Avr11 locus but mapping of the BAC ends will be required to determine if the Avr3 and Avr10 loci are also present in the BAC contig. These results are an important step towards the positional cloning of avirulence genes from P. infestans, and the BAC library represents a valuable resource for largescale studies of oomycete genome organisation and gene content.

Chromosomes, Artificial, Bacterial↗
PubMed2003

Construction and characterization of a vestibular-specific cDNA library using T7-based RNA amplification.

Using a very small amount of inner-ear tissue, we constructed a human vestibular cDNA library by means of T7-based amplification of RNA. This library should allow us to identify genes likely to be involved in auditory and vestibular functions. Here we first report the characterization of the human vestibular cDNA library. Among 506 cDNA clones randomly selected from the vestibular cDNA library, DNA sequences of 301 cDNA clones were identical to those of genes of known function. Twenty-two cDNA clones were considered to be novel because they did not match any cDNA sequences in the public database. The information in our study will provide a valuable resource for identifying several novel genes underlying deafness disorders and vestibular dysfunction.

Bacteriophage T7↗
PubMed2004

Suppression subtractive hybridization cDNA libraries to identify differentially expressed genes from contrasting fish habitats.

Suppression subtractive hybridization complementary DNA libraries identified differentially expressed genes in liver tissue of winter flounder collected from the highly impacted Raritan-Hudson estuary versus those from less industrialized estuaries farther south in New Jersey. Distinct transcript profiles emerged in the fish from these different habitats. A total of 251 clones from the forward (upregulated with anthropogenic impact) and reverse (downregulated with anthropogenic impact) subtracted libraries were sequenced. In the upregulated library immune response transcripts, including complement C-3, C-7, factor H, factor Bf/C2, differentially regulated trout protein 1, and the antimicrobial hepcidin, indicated the pollution-impacted fish were under a high viral or bacterial load. Transcripts for cytochrome P450 1A, P450 3A, and glutathione S-transferase, important components of phase I and II metabolism of xenobiotics, were found in the upregulated-with-pollution library. Vitellogenins I and II and egg envelope protein (zp) appeared to be downregulated. A homologue of the tumor suppressor p33(ING1) (down) and hepatocyte growth factor-like protein (up) may indicate liver damage or hepatocellular carcinoma or hepatoma. These expression patterns, confirmed by quantitative polymerase chain reaction, indicate that transcript analysis is a useful method for assessing the health of local habitats and the organisms therein.

Animals↗
PubMed2006

New resources for marine genomics: bacterial artificial chromosome libraries for the Eastern and Pacific oysters (Crassostrea virginica and C. gigas).

Large-insert genomic bacterial artificial chromosome (BAC) libraries of two culturally and economically important oyster species, Crassostrea virginica and C. gigas, have been developed as part of an international effort to develop tools and reagents that will advance our ability to conduct genetic and genomic research. A total of 73,728 C. gigas clones with an average insert size of 152 kb were picked and arrayed representing an 11.8-fold genome coverage. A total of 55,296 clones with an average insert size of 150 kb were picked and arrayed for C. virginica, also representing an 11.8-fold genome coverage. The C. gigas and C. virginica libraries were screened with probes derived from selected oyster genes using high-density BAC colony filter arrays. The probes identified 4 to 25 clones per gene for C. virginica and 5 to 50 clones per gene for C. gigas. We conducted a preliminary analysis of genetic polymorphism represented in the C. gigas library. The results suggest that the degree of divergence among similar sequences is highly variable and concentrated in intronic regions. Evidence supporting allelic polymorphism is reported for two genes and allelic and/or locus specific polymorphism for several others. Classical inheritance studies are needed to confirm the nature of these polymorphisms. The oyster BAC libraries are publicly available to the research community on a cost-recovery basis at (www.genome.clemson.edu).

Animals↗
PubMed1991

Construction of representative immunoglobulin variable region cDNA libraries from human peripheral blood lymphocytes without in vitro stimulation.

We have developed a sensitive and specific method for preparation of representative IgM and IgG cDNA libraries of peripheral blood lymphocytes without in vitro stimulation of B cells and without primer-based bias. The procedure involves preparation of double-stranded cDNA from initial C mu and C gamma constant region primers. Linkers are attached to the cDNA and nonselective polymerase chain reaction (PCR) amplification is performed with the linkers as primers. This product is ligated to M13 RF DNA, and selective PCR amplification is achieved with a nested constant region primer and a vector primer. The product is cloned into M13 phage. More than 85% of the white plaques are positive with a JH probe. Diversity of the library is confirmed by hybridization with VH gene probes and by DNA sequencing. A sample of 7-30 ml of blood from a normal human adult can yield both mu and gamma cDNA libraries. Analysis of the libraries yields a picture of Ig variable gene usage at the time of sampling.

Base Sequence↗
PubMed1995

Conversion of murine Fabs isolated from a combinatorial phage display library to full length immunoglobulins.

The use of combinatorial Ig libraries displayed on the surface of bacteriophage has advantages over traditional hybridoma techniques for the generation of mAbs but in many instances full length Igs may be more desirable than Fab fragments. Two murine Fabs reactive with the human complement component C5a, recovered from a combinatorial library, were converted to full length IgG2a mAbs. The VH and VL domains of these antibodies were removed from the bacterial expression vector used for the combinatorial library construction, and subcloned into individual mammalian expression vectors containing the corresponding Ig heavy and light chain constant regions. The subcloning relied on 5' restriction endonuclease sites encoded by the oligonucleotide primers originally used to amplify the Ig cDNAs and 3' sites conserved in CH1 and C kappa. These vectors were co-transfected into COS cells yielding full length IgG2a versions of the anti-C5a antibodies. The mAbs, purified from the culture supernatant, retained the full activity of the Fabs, binding specifically to and neutralizing human recombinant C5a. Refined versions of the mammalian expression vectors have been constructed for single step conversion of murine recombinant Fabs, recovered from combinatorial libraries, to IgG2a mAbs.

Animals↗
PubMed1996

Application of linker-ligation-PCR for construction of phage display epitope libraries.

An efficient method for construction of random epitope libraries using filamentous phage is described. Random DNA fragments generated by DNase I digestion were blunt ended by T4 DNA polymerase and ligated with a 12-mer linker, followed by PCR amplification using the same oligonucleotide linker as primers. The results showed that only the ligated product containing linker sequences on both ends was specifically amplified. When the linker ligated-PCR (LL-PCR) product was used for the construction of phage display epitope libraries, the total number of independent clones in the libraries was increased by 100 to 1000 fold in comparison to that obtained for libraries constructed using other methods. In addition, the LL-PCR strategy also increased the probability of isolating recombined DNA fragments which are derived by random in-frame ligation of two or more discontinuous peptide-coding sequences before being inserted into the display vector. Such randomly recombined DNA fragments might be useful in defining conformational epitopes.

African Swine Fever Virus↗
PubMed1994

Relational genome analysis using reference libraries and hybridisation fingerprinting.

The genomes of eukaryotic organisms are studied by an integrated approach based on hybridisation techniques. For this purpose, a reference library system has been set up, with a wide range of clone libraries made accessible to probe hybridisation as high density filter grids. Many different library types made from a variety of organisms can thus be analysed in a highly parallel process; hence, the amount of work per individual clone is minimised. In addition, information produced on one analysis level instantly assists in the characterisation process on another level. Genetic, physical and transcriptional mapping information and partial sequencing data are obtained for the individual library clones and are cross-referenced toward a comprehensive molecular understanding of genome structure and organisation, of encoded functions and their regulation. The order of genomic clones is established by hybridisation fingerprinting procedures. On these physical maps, the location of transcripts is determined. Complementary, partial sequence information is produced from corresponding cDNAs by hybridising short oligonucleotides, which will lead to the identification of regions of sequence conservation and the constitution of a gene inventory. The hybridisation analysis of the cDNA clones, and the genomic clones as well, could potentially be expanded toward a determination of (nearly) the complete sequence. The accumulated data set will provide the means to direct large-scale sequencing of the DNA, or might even make the sequence analysis of large genomic regions a redundant undertaking due to the already collected information.

Animals↗
PubMed1994

Tomorrow's library.

The buildings in which we house libraries are like other special purpose structures; the needs they fill are significantly influenced by technology. A prime function of the library building is to house collections (of people, material, and systems) as well as collections of collections (networks). Electronic formats for library material offer new approaches to information service delivery. An example, the information access station, typifies how traditional functions can be reconfigured with respect to space. Flexible design can help ensure that tomorrow's libraries meet the users' needs, but we need to question all our assumptions about building design including those driven by our understanding of the browsing process.

Facility Design and Construction↗
PubMed1994

Trends and recent experiments in library design.

An architect relates his experiences in designing new and renovated libraries, 'from the inside out'. He raises questions regarding the impact of technology and addresses concerns about collections, but places primary focus on users--why they come into the library and how they use information in and outside the library building. Design projects reviewed include libraries at Yale, Columbia, the University of Southern California, the Medical College of Wisconsin, Emory, George Mason, and Cornell.

Architecture↗
PubMed1994

Library resources for problem-based learning: the program perspective.

The impact of a problem based curriculum has been the subject of increasing interest, as evidenced by several recent articles on the subject [1-4]. McMaster was able to design its library to serve a problem-based curriculum, but since there had been no prior experience with such a curriculum, the library was designed to meet needs that could only be guessed at [5]. In addition, the library serves the needs of a research intensive faculty. How the curriculum and the library have interacted over the past 25 years may be helpful to other schools considering problem-based learning.

Curriculum↗
PubMed1994

Evaluating library renovation at the University of Rochester Medical Center.

In 1987, the Edward G. Miner Library of the University of Rochester Medical Center completed the second major renovation in its 62-year history. The goal of the renovation was to provide additional space for the library's 3 main constituencies: its readers, its collections, and its staff. A case study of the renovation is presented, outlining processes and issues that shaped the final outcome. The success of the renovation is assessed in terms of project goals, design, and functionality. Changes made since 1987 to accommodate automation are also summarized. The conclusion suggests that library renovation be viewed as a continuing process, and proposes that principles of interface design are appropriate guidelines for evaluating library design across time.

Facility Design and Construction↗
PubMed1992

Isolation and characterization of a library of cDNA clones that are preferentially expressed in the embryonic telencephalon.

In order to isolate genes involved in development of the mammalian telencephalon we employed an efficient cDNA library procedure. By subtracting an adult mouse telencephalic cDNA library from an embryonic day 15 (E15) mouse telencephalic cDNA library we generated two subtracted libraries (ES1 and ES2). We estimate that ES1 contains between 200 and 600 different cDNA clones, which approximates the number of genes that are preferentially expressed in the E15 telencephalon, compared to the adult telencephalon. Northern analysis of 20 different cDNA clones shows that 14 of these are expressed at least 5-fold more in the E15 telencephalon than the adult telencephalon. Limited sequencing of the 14 differentially expressed clones reveals that 10 have no significant identity to sequences in GenBank and EMBL databases, whereas the other 4 have significant sequence identity to vimentin, histone 3.3, topoisomerase I and the B2 repeat element. In situ hybridization using one of the differentially expressed cDNAs, TES-1, demonstrates that it is transiently expressed in the anlage of the basal ganglia. In situ hybridization with another differentially expressed cDNA clone, TES-4, shows that it is specifically expressed in differentiating cells of the neural axis with a distinctive rostral-caudal temporal pattern. These findings, and the methods that we have developed, provide a framework for future investigations of the genetic control of telencephalon development.

Animals↗
PubMed1990

Differential screening of murine ascites cDNA libraries by means of in vitro transcripts of cell-cycle-phase-specific cDNA and digital image processing.

Cell-cycle-phase-specific cDNA libraries were prepared in the lambda gt10 vector and in the in vitro transcription vector, pBluescript. Plaques of the cDNA libraries prepared in the lambda gt10 vector were differentially screened with (a) in vitro transcripts of the cell-cycle-phase-specific cDNAs cloned in the transcription vector and (b) with first-strand cDNA of mRNA from phase-synchronous cells. The results suggest that first-strand cDNA can be replaced, at least in prescreening experiments, by in vitro transcripts of representative cDNA libraries prepared in in vitro transcription vectors. The fractions of differential clones detected with in vitro transcripts (1.2%) and with first-strand cDNA (1%) were in the same order. Individual clones selected by differential hybridization with in vitro transcripts could be verified by differential hybridization with cell-cycle-phase-specific first-strand cDNA. This indicates that the pattern of stage-specific prevalences of cDNA clones is essentially retained during careful amplifications of large cDNA libraries. The application of in vitro transcripts of stage-specific cDNA for differential screening experiments is of interest in cases where the amount of biological material is either limited or difficult to prepare. It also allows standardization of the probes in repeated screening experiments. Three clones reflecting cell-cycle-phase-specific mRNA prevalences were chosen and analyzed on the sequence level. Two sequences with S-phase prevalences were identified. They code for elongation factor EF1 alpha and for glyceraldehyde-3-phosphate dehydrogenase, respectively. The third sequence reflects the first cDNA of a mRNA with significant prevalence in G2-phase cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Optimised cDNA size selection and cloning procedure for the construction of representative plasmid cDNA libraries.

Plasmid libraries are more versatile than phage libraries since they allow expression cloning in eukaryotic systems. However, high numbers of primary clones are sometimes difficult to obtain and more efficient transformation procedures are often required. In this paper, a detailed protocol is presented, for the construction of large plasmid libraries from ng quantities of cDNA, based on a highly efficient transformation step. Drop dialysis and electroporation are optimised: complete ligase removal and yeast tRNA addition before dialysis appear critical, while exclusive use of double-distilled water as well as rapid preparation of fresh cells provide excellent electroporation yields. A novel, simple and flexible cDNA size selection procedure is also presented, based on sucrose density gradient. Two libraries were constructed using an expression vector for mammalian cells (pCDM8) and its Escherichia coli host strain (MC1061[p3]). Numbers of 2 to 10 x 10(6) primary transformants were obtained from 1 microgram of poly(A)+ RNA. Up to 85% of the clones had inserts and half of the inserts were larger than 1.5 kb.

Cloning, Molecular↗
PubMed1992

Derivation of a mathematical expression useful for the construction of complete genomic libraries.

We present and derive a formula that is useful for the design and evaluation of gene cloning experiments in which a complete gene library of the entire genome of an organism is desired. The formula n = ln(1-phi f)/ln(1-f) (in which n is the number of recombinant clones required to ensure a probability, phi, of obtaining at least one of each of all possible gene sequences, and f is the fraction of the genome contained in an average-sized DNA fragment) applies to construction of libraries, in which at least one copy of all the genetic information of a genome is required. The use of this formula for quantitative evaluation of genomic libraries should give greater assurance that a given library would be complete.

Cloning, Molecular↗
PubMed1992

Construction and characterisation of a yeast artificial chromosome library containing two haploid Beta vulgaris L. genome equivalents.

We have constructed a yeast artificial chromosome (YAC) library using high-molecular-weight DNA prepared from agarose-embedded protoplasts of a sugar beet (Beta vulgaris L.) cell suspension line, Ar+. This library contains 15,000 clones with an average insert size of 140 kb. Based on a sugar beet haploid genome size of 1.1 x 10(3) Mb it should represent approximately two haploid genome equivalents. The library is organised as an ordered array in duplicate microtitre plates. High-density filters, each containing 864 YAC clones, have been screened in colony hybridisation experiments. In this way, sugar beet YAC clones have been identified containing chloroplast DNA, mitochondrial DNA and two satellite DNAs. Ten pools of DNA from 1500 individual YAC clones have also been prepared for rapid PCR screening of the library. Using this approach, in combination with colony hybridisation, we have been able to isolate sugar beet YAC clones containing a transcribed low-copy-number gene.

Base Sequence↗