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Cosmid Clones (cosmid + clone)

Distribution by Scientific Domains

Life Sciences	75%

Selected Abstracts

Chromosomal anomalies on 6p25 in iris hypoplasia and Axenfeld-Rieger syndrome patients defined on a purpose-built genomic microarray,

HUMAN MUTATION, Issue 1 2004
Rosemary Ekong
Abstract In many inherited diseases, the same phenotype can be produced both by single-base changes and by large deletions, or in some cases by duplications. Routine high-throughput sequencing can now detect small mutations relatively easily in a diagnostic setting, but deletions and duplications in the 50,500-kb region remain a more difficult problem. We have explored the application of array-CGH to the detection of such changes on a set of 20 samples consisting of patients with eye diseases associated with changes on chromosome 6p25 together with unaffected individuals, as well as two samples from tuberous sclerosis 2 (TSC2)-affected patients. We developed a microarray consisting of degenerate oligonucleotide primer (DOP)-PCR products from 260 human genomic clones, including BACs, PACs, and cosmids. In a masked study, chromosome changes in patients with iris hypoplasia (duplication) and Axenfeld-Rieger syndrome (deletion) were unequivocally distinguished from controls. Of the 20 6p25 samples analyzed, 19 were analyzed correctly (10 duplication cases, two deletions, and seven normals), while one individual failed to give a result because of poor hybridization. The extent of the duplication or deletion estimated was similar to that obtained by independent and much more time-consuming FISH experiments. On the other hand, deletions in the two TSC2 -affected samples, previously mapped by DNA molecular combing, were not detected on the array, possibly due to the repeat content of that region. Excluding the 16p13 cosmids, consistent results were obtained from all other cosmid clones; the potential for producing affordable disease-specific diagnostic microarray as an adjunct to diagnosis is discussed. Hum Mutat 24:76,85, 2004. © 2004 Wiley-Liss, Inc. [source]

The conserved Xanthomonas campestris pv. vesicatoria effector protein XopX is a virulence factor and suppresses host defense in Nicotiana benthamiana

THE PLANT JOURNAL, Issue 6 2005
Matthew Metz
Summary Nicotiana benthamiana leaves display a visible plant cell death response when infiltrated with a high titer inoculum of the non-host pathogen, Xanthomonas campestris pv. vesicatoria (Xcv). This visual phenotype was used to identify overlapping cosmid clones from a genomic cosmid library constructed from the Xcv strain, GM98-38. Individual cosmid clones from the Xcv library were conjugated into X. campestris pv. campestris (Xcc) and exconjugants were scored for an altered visual high titer inoculation response in N. benthamiana. The molecular characterization of the cosmid clones revealed that they contained a novel gene, xopX, that encodes a 74-kDa type III secretion system (TTSS) effector protein. Agrobacterium -mediated transient expression of XopX in N. benthamiana did not elicit the plant cell death response although detectable XopX protein was produced. Interestingly, the plant cell death response occurred when the xopX Agrobacterium -mediated transient expression construct was co-inoculated with strains of either Xcv,xopX or Xcc, both lacking xopX. The co-inoculation complementation of the plant cell death response also depends on whether the Xanthomonas strains contain an active TTSS. Transgenic 35S- xopX -expressing N. benthamiana plants also have the visible plant cell death response when inoculated with the non- xopX -expressing strains Xcv,xopX and Xcc. Unexpectedly, transgenic 35S- xopX N. benthamiana plants displayed enhanced susceptibility to bacterial growth of Xcc as well as other non- xopX -expressing Xanthomonas and Pseudomonas strains. This result is also consistent with the increase in bacterial growth on wild type N. benthamiana plants observed for Xcc when XopX is expressed in trans. Furthermore, XopX contributes to the virulence of Xcv on host pepper (Capsicum annuum) and tomato (Lycopersicum esculentum) plants. We propose that the XopX bacterial effector protein targets basic innate immunity in plants, resulting in enhanced plant disease susceptibility. [source]

Cloning large natural product gene clusters from the environment: Piecing environmental DNA gene clusters back together with TAR

BIOPOLYMERS, Issue 9 2010
Jeffrey H. Kim
Abstract A single gram of soil can contain thousands of unique bacterial species, of which only a small fraction is regularly cultured in the laboratory. Although the fermentation of cultured microorganisms has provided access to numerous bioactive secondary metabolites, with these same methods it is not possible to characterize the natural products encoded by the uncultured majority. The heterologous expression of biosynthetic gene clusters cloned from DNA extracted directly from environmental samples (eDNA) has the potential to provide access to the chemical diversity encoded in the genomes of uncultured bacteria. One of the challenges facing this approach has been that many natural product biosynthetic gene clusters are too large to be readily captured on a single fragment of cloned eDNA. The reassembly of large eDNA-derived natural product gene clusters from collections of smaller overlapping clones represents one potential solution to this problem. Unfortunately, traditional methods for the assembly of large DNA sequences from multiple overlapping clones can be technically challenging. Here we present a general experimental framework that permits the recovery of large natural product biosynthetic gene clusters on overlapping soil-derived eDNA cosmid clones and the reassembly of these large gene clusters using transformation-associated recombination (TAR) in Saccharomyces cerevisiae. The development of practical methods for the rapid assembly of biosynthetic gene clusters from collections of overlapping eDNA clones is an important step toward being able to functionally study larger natural product gene clusters from uncultured bacteria. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 833,844, 2010. [source]