Evidence for a prokaryotic insertion-sequence contamination in eukaryotic sequences registered in different databases

An insertion-sequence of prokaryotic origin was detected in a genomic clone obtained from a Phaseolus vulgaris bacterial artificial chromosome (BAC) library. This BAC clone, characterized as part of a contig constructed near a virus resistance gene, exhibited restriction fragment length polymorphism with an overlapping clone of the contig. Restriction analysis of DNA obtained from individual colonies of the stock culture indicated the presence of a mixed population of wild-type and insertional mutants. Sequence analysis of both members of the population revealed the presence of IS10R, an insertion-sequence from Escherichia coli. A BLAST search for IS10-like sequences detected unexpected homologies with a large number of eukaryotic sequences from Homo sapiens, Arabidopsis thaliana, Drosophila melanogaster and Caenorhabditis elegans. Southern analysis of a random sample of BAC clones failed to detect IS10 in the BAC DNA. However, prolonged sub-culturing of a set of 15 clones resulted in transposition into the BAC DNA. Eventually, all cultures acquired a 2.3-kb fragment that hybridized strongly with IS10. Sequence analysis revealed the presence of a preferred site for transposition in the BAC vector. These results indicate that a large number, if not all, of the BAC libraries from different organisms are contaminated with IS10R. The source of this element has been identified as the DH10B strain of E. coli used as the host for BAC libraries. Received: 5 December 2000 / Accepted: 25 April 2001     

Construction of a BAC Contig for a 3 cM Biologically Significant Region of Mouse Chromosome 1

One QTL and genes and phenotypes have been localized in the region between 92 cM and 95cM of mouse chromosome 1. The QTL locus contributes to approximately 40% of the variation of the peak bone density between C57BL/6J (B6) and CAST/EiJ (CAST) strains. Other loci located in this chromosomal region include a neural tube defect mutant loop-tail (Lp), a lymphocyte-stimulating determinant (Lsd), and the Transgelin 2 (Tagln 2). The human chromosome region homologous to this region is 1q21-23, which also contains a QTL locus for high bone mineral density (BMD). Furthermore, it has been reported that this region may have duplicated several times in the mouse genome. Therefore, genomic sequencing of this region will provide important information for mouse genome structure, for positional cloning of mouse genes, and for the study of human homologous genes. In order to provide a suitable template for genomic sequencing by the NIH-sponsored genomic centers, we have constructed a BAC contig of this region using the RPCI-23 library. We have also identified the currently available mouse genomic sequences localized in our BAC contig. Further analysis of these sequences and BAC clones indicated a high frequency of repetitive sequences within this chromosomal area. This region also contains L1 retrotransposon sequences, providing a potential mechanism for the repetitive sequences described in the literature.     

Rapid Screening of a Novel Arrayed Medaka (Oryzias latipes) Cosmid Library

Medaka (Oryzias latipes) has many advantages for genetic and developmental studies. With recent advances in the genome analyses of other species, rapid accumulation of resources for medaka genomics is expected. In this study, we generated an arrayed medaka cosmid library from the HNI inbred strain, carrying a 40-kb insert on average. The library consists of approximately 120,000 clones with a 6-fold genomic coverage. Cosmid clones can be screened within 2 days using standard polymerase chain reaction. Considering the advantage of the cosmid insert size and the compact genome size of the medaka, this library provides a powerful tool for future genome analyses.     

AFLP-derived SCARs facilitate construction of a 1.1 Mb sequence-ready map of a region that spans the Vf locus in the apple genome

The availability of high-density anchored markers is a prerequisite for reliable construction of a deep coverage BAC contig, which leads to creation of a sequence-ready map in the target chromosomal region. Unfortunately, such markers are not available for most plant species, including woody perennial plants. Here, we report on an efficient approach to build a megabase-size sequence-ready map in the apple genome for the Vf region containing apple scab resistance gene(s) by targeting AFLP-derived SCAR markers to this specific genomic region. A total of 11 AFLP-derived SCAR markers, previously tagged to the Vf locus, along with three other Vf-linked SCAR markers have been used to screen two apple genome BAC libraries. A single BAC contig which spans the Vf region at a physical distance of approximately 1,100 kb has been constructed by assembling the recovered BAC clones, followed by closure of inter-contig gaps. The contig is 4 ×deep, and provides a minimal tiling path of 16 contiguous and overlapping BAC clones, thus generating a sequence-ready map. Within the Vf region, duplication events have occurred frequently, and the Vf locus is restricted to the ca. 290 kb region covered by a minimum of three overlapping BAC clones.     

Monoclonal antibody purification by affinity chromatography with ligands derived from the screening of peptide combinatory libraries

The peptide, Ala-Pro-Ala-Arg (APAR), was selected from the screening of a tetrapeptide combinatorial synthetic library as the ligand for affinity purification of an anti-Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) monoclonal antibody (Mab) developed in mouse ascitis. The affinity chromatographic matrix obtained by attachment of APAR to agarose, having a peptide density of 0.5 mol ml^–1, showed a maximum capacity of 9.1 mg Mab ml^–1 and a dynamic capacity of 3.9 mg Mab ml^–1. A 95% yield of electrophoretically pure anti-GM-CSF was obtained in a single step.     

Cerebellar granule cell Cre recombinase expression

The cerebellum maintains balance and orientation, refines motor action, stores motor memories, and contributes to the timing aspects of cognition. We generated two mouse lines for making Cre recombinase-mediated gene disruptions largely confined to adult cerebellar granule cells. For this purpose we chose the GABA(A) receptor alpha6 subunit gene, whose expression marks this cell type. Here we describe mouse lines expressing Cre recombinase generated by 1) Cre knocked into the native alpha6 subunit gene by homologous recombination in embryonic stem cells; and 2) Cre recombined into an alpha6 subunit gene carried on a bacterial artificial chromosome (BAC) genomic clone. The fidelity of Cre expression was tested by crossing the mouse lines with the ROSA26 reporter mice. The particular alpha6BAC clone we identified will be valuable for delivering other gene products to cerebellar granule cells.     

Reexamination of the recognition preference of the specificity pocket of the Abl SH3 domain

Src homology-3 (SH3) domains mediate important protein-protein interactions in a variety of normal and pathological cellular processes, thus providing an attractive target for the selective interference of SH3-dependent signaling events that govern these processes. Most SH3 domains recognize proline-rich peptides with low affinity and poor selectivity, and the goal to design potent and specific ligands for various SH3 domains remains elusive. Better understanding of the molecular basis for SH3 domain recognition is needed in order to design such ligands with potency and specificity. In this report, we seek to define a clear recognition preference of the specificity pocket of the Abl SH3 domain using targeted synthetic peptide libraries. High-resolution affinity panning coupled with mass spectrometric readout allows for quick identification of Trp as the preferred fourth residue in the decapeptide ligand APTWSPPPPP, which binds to Abl SH3 four times stronger than does the decapeptide containing Tyr or Phe in the fourth position. This finding is in contrast to several reports that Tyr is the only residue selected from phage displayed peptide libraries that interacts with the specificity pocket of Abl SH3. This simple, unbiased approach can fine-tune the affinity and selectivity of both natural and unnatural SH3 ligands whose consensus binding sequence has been pre-defined by combinatorial library methods.     

Construction and characterization of BAC libraries for three fish species; rainbow trout, carp and tilapia

Bacterial artificial chromosome (BAC) libraries are important tools for genomic research. We have constructed seven genomic BAC libraries from three fish species, rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio) and tilapia (Oreochromis niloticus). The two rainbow trout BAC libraries have average insert sizes of 58 and 110 kb. The average size of inserts in the carp BAC library is 160 kb. The average insert sizes of the four tilapia BAC libraries are 65, 105, 145 and 194 kb, respectively. These libraries represent good coverage of each genome (2-64 x coverage). The libraries can be screened by conventional colony hybridization and provide a starting point for the construction of high-density filtres or polymerase chain reaction (PCR) screening approaches. These BAC libraries will facilitate the positional cloning of quantitative trait loci (QTLs) for a variety of economically important traits in these species.     

Construction and characterization of a gridded cattle BAC library

A bovine genomic large-insert bacterial artificial chromosome (BAC) library has been constructed from leukocytes of a Holstein-Friesian male. Size fractionated DpnII-digested genomic DNA was ligated to the dephosphorylated BamH1 ends of a pBACe3.6 vector. Approximately 8.3 x 10(4) individual BAC clones were picked into 384-well plates. Two-hundred and sixty-seven randomly chosen clones were characterized by pulsed-field gel electrophoresis (PFGE). The average insert size was 104 kb with a frequency of clones without inserts of 5.5%. Thirty-four BAC clones were mapped by fluorescence in situ hybridization (FISH) to cattle chromosomes. Three showed signals at more than one location, one of them on the centromeric regions of all autosomes, indicating that the clone contains centromeric repeats. A subset of these BAC clones was used for the development of sequence tagged sites. Both subcloning and direct sequencing of the BACs were used for generating sequence tagged site information. The clones from the library were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Membranes and superpools are available through the Resource Centre of the German Human Genome Project in Berlin (http:// www.rzpd.de).