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At least 199 records · Page 11Linked to original sources
PubMed1994

Novel tenascin variants with a distinctive pattern of expression in the avian embryo.

Previous studies have shown that several forms of the glycoprotein tenascin are present in the embryonic extracellular matrix. These forms are the result of alternative splicing, which generates tenascin variants with different numbers of fibronectin type III repeats. We have used degenerate primers and PCR to isolate a novel tenascin exon from an avian genomic library. Genomic clones contained a sequence encoding a fibronectin type III repeat that corresponds to repeat 'C' from the variable domain of human tenascin. To demonstrate that tenascin containing repeat 'C' is actually synthesized by avian cells, a monospecific antiserum was raised against a repeat 'C' fusion protein. This antiserum recognized a novel high-molecular-weight variant on immunoblots of tenascin isolated from chicken embryo fibroblast-conditioned medium, and stained tendons on frozen sections of chicken embryos. A cDNA probe specific for mRNA encoding repeat 'C' was used for in situ hybridization. This probe hybridized in a subset of the embryonic tissues labelled with a universal tenascin probe, including tendons, ligaments and mesenchyme at sites of epithelial-mesenchymal interactions. Finally, we provide evidence that additional fibronectin type III repeats, one corresponding to a recently discovered human repeat as well as one entirely novel sequence, also exists in chicken tenascin mRNA. These data indicate that tenascin is present in the embryonic matrix in a multitude of forms and that these forms have distinctive distributions that may reflect more than one function for tenascin in development.

Amino Acid Sequence↗
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↗
PubMed2000

The use of competitive PCR mimic to evaluate a Limulus lambda phage genomic DNA library.

1. A lambda phage genomic DNA library for Limulus (L.) polyphemus brain was constructed using the AGEM-12 vector and the host strain KW251. 2. The primary library contained approximately 1.275 x 10(6) independent clones, increasing upon amplfication to 6.66 x 10(9) pfu/ml in a total volume of 58 ml. 3. A total of 28 clones was randomly chosen for a determination of the average size of inserts in the library. All clones contained inserts and the average size was 14.9 kb, ranging from 11.7 to 28.0 kb. The library provides a 10-fold equivalent of the L. polyphemus genome. 4. A new approach for evaluating a genomic DNA library was developed, in which competitive PCR MIMIC was employed to determine the target gene copy number in both constructed library and brain genomic DNA. The putative protein kinase C epsilon (PKCepsilon) was selected as the target gene because its partial sequence of cDNA was recently cloned from L. polyphemus brain in our laboratory (Cao et al., 1998). A 419-bp fragment of nonhomologous sequence derived from putative PKCepsilon and a 306-bp fragment from plasmid pUC 18 were generated for use as target and competitor in PCR MIMIC, respectively. 5. Within the genomic library DNA, a 0.8 value was obtained for the copy number of the putative PKCepsilon gene that was detected in 0.1 amol of one equivalent L. polyphemus genome in terms of the average recombinant molecular weight. In the genomic DNA, a single copy of putative PKCepsilon was found in 0.1 amol of one coverage for the L. polyphemus genome. Thus, it was implied that nearly 80% genetic resource was incorporated into the library. This percentage was termed the incorporation rate. 6. Based on these findings, we suggest that the incorporation rate is an essential factor for evaluating genomic libraries, particularly, when using partial digestion with restriction enzymes for library construction.

Animals↗
PubMed2003

Gene enrichment in plant genomic shotgun libraries.

The Arabidopsis genome (about 130 Mbp) has been completely sequenced; whereas a draft sequence of the rice genome (about 430 Mbp) is now available and the sequencing of this genome will be completed in the near future. The much larger genomes of several important crop species, such as wheat (about 16,000 Mbp) or maize (about 2500 Mbp), may not be fully sequenced with current technology. Instead, sequencing-analysis strategies are being developed to obtain sequencing and mapping information selectively for the genic fraction (gene space) of complex plant genomes.

DNA Methylation↗
PubMed2003

Preparation of a whole genome phage library using fragmented Escherichia coli genome and its characterization of protein binding properties by surface plasmon resonance.

A novel phage library has been prepared using the Escherichia coli genome digested with three restriction enzymes. The resulting DNA fragments were ligated to the expression vector pCANTAB5 to obtain the library of recombinant M13 phages displaying relatively long exogenous peptides. The library was screened to isolate recombinant phages with high affinity to alkaline phosphatase (AP) from calf intestine. After four rounds of panning three phages (AP1, AP2 and AP3) were shown to have specific binding properties toward AP by enzyme-linked immunosorbent assay. The phages were further characterized by surface plasmon resonance (SPR). Among the three phages AP3 bound the AP-immobilized sensor chip most and caused the highest resonant angle shift. The sensor response decreased with the decrease of the concentration of AP3 added. Furthermore, displacement of AP3 from the AP-immobilized sensor chip was observed upon injection of AP solution to the SPR system, whereas injection of bovine serum albumin solution led to the great increase of the sensor response. This result indicates the specific binding of AP3 to AP.

Alkaline Phosphatase↗
PubMed1997

[Creation of genomic DNA libraries for Pseudomonas mallei and Pseudomonas pseudomallei].

The genomic libraries of P. mallei and P. pseudomallei species were constructed in Escherichia coli. The chromosomal DNA of P. pseudomallei C-141 strain has been cloned into the cosmid vector pHC79 and the broad host range plasmid vector pES154. The chromosomal DNA of P. mallei [symbol: see text]-5 strain has been cloned into the plasmid vector pSUP202. The recombinant clones of the genomic libraries were screened by the enzyme-linked immunoadsorbent assay (ELISA) to detect the production of Pseudomonas antigens: 28 clones were positive. Twelve recombinant strains demonstrated specific antigenic determinants of P. mallei and P. pseudomallei by immunoblotting. Cloned proteins of P. mallei and P. pseudomallei have molecular weights from 30 to 70 kD. A new method for introducing foreign genes into Pseudomonas genomes is offered. P. mallei strains with the chromosomally integrated plasmids pSM are universal recipients for ColEI-based cloning vectors.

Burkholderia pseudomallei↗
PubMed1998

Construction and characterisation of a gridded chicken cosmid library with four-fold genomic coverage.

Gridded genomic libraries are crucial for the positional candidate gene approach. For this purpose we constructed a gridded genomic library from a female chicken using the vector sCos 1. About 110,000 cosmid clones were grown and replicated in 384-well plates. An average insert size of 39 kb was calculated from the analysis of 68 randomly selected clones. No chimerism could be observed from 31 in situ hybridisations. One replica of the library (number 125) has been transferred to the Resource Centre/Primary Database (RZPD) of the German Human Genome Project (DHGP). The whole library was gridded onto four nylon filters at high density for efficient identification of cosmid clones by colony hybridisation. Twenty-two probes were used for screening the library and each of them gave at least one positive signal. This result is in good agreement with a four-fold coverage of the genome as estimated from the insert length and number of recombinant clones. This library provides a powerful tool for rapid physical mapping and complex analysis of the chicken genome.

Animals↗
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↗
PubMed2003

Functional selection of vaccine candidate peptides from Staphylococcus aureus whole-genome expression libraries in vitro.

An in vitro protein selection method, ribosome display, has been applied to comprehensively identify and map the immunologically relevant proteins of the human pathogen Staphylococcus aureus. A library built up from genomic fragments of the virulent S. aureus COL strain (methicillin-resistant S. aureus) allowed us to screen all possible encoded peptides for immunoreactivity. As selective agents, human sera exhibiting a high antibody titer and opsonic activity against S. aureus were used, since these antibodies indicate the in vivo expression and immunoreactivity of the corresponding proteins. Identified clones cluster in distinct regions of 75 genes, most of them classifiable as secreted or surface-localized proteins, including previously identified virulence factors. In addition, 14 putative novel short open reading frames were identified and their immunoreactivity and in vivo mRNA expression were confirmed, underscoring the annotation-independent, true genomic nature of our approach. Evidence is provided that a large fraction of the identified peptides cannot be expressed in an in vivo-based surface display system. Thus, in vitro protein selection, not biased by the context of living entities, allows screening of genomic expression libraries with a large number of different ligands simultaneously. It is a powerful approach for fingerprinting the repertoire of immune reactive proteins serving as target candidates for active and passive vaccination against pathogens.

Antigens, Bacterial↗
PubMed1985

Surface proteins of Mycoplasma hyopneumoniae identified from an Escherichia coli expression plasmid library.

A genomic library of Mycoplasma hyopneumoniae was constructed by cloning random DNA fragments approximately 300 base pairs long in a fusion expression plasmid, pEx29, containing the N terminus of the phage MS2 polymerase under the control of the PL promoter of phage lambda. Clones that produced fusion proteins carrying surface-specific antigenic determinants were identified by using antiserum raised in a pig by intranasal inoculation of viable mycoplasmas. Rabbit antisera produced against gel-purified fusion proteins synthesized in Escherichia coli were analyzed by Western blotting to identify antigenically related mycoplasma components. Distinct mycoplasma proteins termed P90, P68, P50, P30, and P26 were identified. Evidence for the surface location of P90, P68, and P50 was provided by their sensitivity to trypsin and their comigration with lactoperoxidase-catalyzed iodinated proteins of intact mycoplasmas. Immune electron microscopy, performed with antiserum against the hybrid MS2-mycoplasma protein produced in E. coli and corresponding to P90, also showed that its antigenic determinant is associated with the mycoplasma surface.

Bacterial Proteins↗
PubMed2002

Structure and level of genetic diversity in various bean types evidenced with microsatellite markers isolated from a genomic enriched library.

We have constructed a common bean genomic library enriched for microsatellite motifs (ATA), (CA), (CAC) and (GA). After screening, 60% of the clones selected from the library enriched for the (ATA) repeat contained microsatellites versus 21% of the clones from the library enriched for (GA) (CA) and (CAC) repeats. Fifteen primer pairs have been developed allowing for the amplification of SSR loci. We have evaluated the genetic diversity of these loci between 45 different bean lines belonging to nine various quality types. A total of 81 alleles were detected at the 15 microsatellite loci with an average of 5.3 alleles per locus. We have investigated the origin of allelic size polymorphism at the locus PvATA20 in which the number of repeats ranges from 24 to 85. We have related these large differences in repeat number to unequal crossing-over between repeated DNA regions. The diversity analysis revealed contrasted levels of variability according to the bean type. The lower level was evidenced for the very fine French bean, showing the effect of breeders intensive selection.

Journal Article↗
PubMed2005

Random sheared fosmid library as a new genomic tool to accelerate complete finishing of rice (Oryza sativa spp. Nipponbare) genome sequence: sequencing of gap-specific fosmid clones uncovers new euchromatic portions of the genome.

The International Rice Genome Sequencing Project has recently announced the high-quality finished sequence that covers nearly 95% of the japonica rice genome representing 370 Mbp. Nevertheless, the current physical map of japonica rice contains 62 physical gaps corresponding to approximately 5% of the genome, that have not been identified/represented in the comprehensive array of publicly available BAC, PAC and other genomic library resources. Without finishing these gaps, it is impossible to identify the complete complement of genes encoded by rice genome and will also leave us ignorant of some 5% of the genome and its unknown functions. In this article, we report the construction and characterization of a tenfold redundant, 40 kbp insert fosmid library generated by random mechanical shearing. We demonstrated its utility in refining the physical map of rice by identifying and in silico mapping 22 gap-specific fosmid clones with particular emphasis on chromosomes 1, 2, 6, 7, 8, 9 and 10. Further sequencing of 12 of the gap-specific fosmid clones uncovered unique rice genome sequence that was not previously reported in the finished IRGSP sequence and emphasizes the need to complete finishing of the rice genome.

Base Sequence↗
PubMed2005

A novel yeast genomic DNA library on a geneticin-resistance vector.

We have constructed a Saccharomyces cerevisiae genomic library utilizing a novel centromere-containing vector bearing the TRP1 and KANMX4 G418-resistance selectable markers. Random Sau3AI genomic DNA fragments with an average size of 6.4 kb were introduced into a unique BamHI site in the vector's polylinker. Six independent library pools were generated and the characteristics of these pools were determined. This library can be used to introduce low-copy genomic DNA fragments into any strain that is not G418-resistant, making it useful for low-copy overexpression analysis or complementation of mutant phenotypes in strains that lack a suitable auxotrophy marker or only exhibit phenotypes on rich, undefined, growth media.

Aldose-Ketose Isomerases↗
PubMed2000

Development of an efficient maintenance and screening system for large-insert genomic DNA libraries of hexaploid wheat in a transformation-competent artificial chromosome (TAC) vector.

Three large-insert genomic DNA libraries of common wheat, Triticum aestivum cv. Chinese Spring, were constructed in a newly developed transformation-competent artificial chromosome (TAC) vector, pYLTAC17, which accepts and maintains large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. The vector contains the cis sequence required for Agrobacterium-mediated gene transfer into grasses. The average insert sizes of the three genomic libraries were approximately 46, 65 and 120 kbp, covering three haploid genome equivalents. Genomic libraries were stored as frozen cultures in a 96-well format, each well containing approximately 300-600 colonies (12 plates for small library, four for medium-size library and four for large library). In each of the libraries, approximately 80% of the colonies harbored genomic DNA inserts of >50 kbp. TAC clones containing gene(s) of interest were identified by the pooled PCR technique. Once the target TAC clones were isolated, they could be immediately transferred into grass genomes with the Agrobacterium system. Five clones containing the thionin type I genes (single copy per genome), corresponding to each of the three genomes (A, B and D), were successfully selected by the pooled PCR method, in addition to an STS marker (aWG464; single copy per genome) and CAB (a multigene family). TAC libraries constructed as described here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning.

Base Sequence↗
PubMed2000

Sequence analysis of trinucleotide repeat microsatellites from an enrichment library of the equine genome.

Microsatellites are useful tools for the construction of a linkage map and parentage testing of equines, but only a limited number of equine microsatellites have been elucidated. Thus, we constructed the equine genomic library enriched for DNA fragments containing (CAG)n repeats. The enriched method includes hybridization-capture of repeat regions using biotin-conjugated oligonucleotides, nucleotide substrate-biased polymerase reaction with the oligonucleotides and subsequent PCR amplification, because these procedures are useful for the cloning of less abundant trinucleotide microsatellites. Microsatellites containing (CAG)n repeats were obtained at the ratio of one per 3-4 clones, indicating an enrichment value about 10(4)-fold, resulting in less time consumption and less cost for cloning. In this study, 66 different microsatellites, (CAG)n repeats, were identified. The number of complete simple CAG repeats in our clones ranged 4-33, with an average repeat length of 8.8 units. The microsatellites were useful as sequence-tagged site (STS) markers. In addition, some clones containing (CAG)n repeats showed homology to human (CAG)n-containing genes, which have been previously mapped. These results indicate that the clones might be a useful tool for chromosome comparison between equines and humans.

Animals↗
PubMed2006

SCALEs: multiscale analysis of library enrichment.

We report a genome-wide, multiscale approach to simultaneously measure the effect that the increased copy of each gene and/or operon has on a desired trait or phenotype. The method involves (i) growth selections on a mixture of several different plasmid-based genomic libraries of defined insert sizes or SCALEs, (ii) microarray studies of enriched plasmid DNA, and a (iii) mathematical multiscale analysis that precisely identifies the relevant genetic elements. This approach allows for identification of all single open reading frames and larger multigene fragments within a genomic library that alter the expression of a given phenotype. We have demonstrated this method in Escherichia coli by monitoring, in parallel, a population of >10(6) genomic library clones of different insert sizes, throughout continuous selections over a period of 100 generations.

DNA Fragmentation↗