Biomedical subjects

Jim Beynon

Publications and source records attributed to Jim Beynon.

7 recordsLinked to original sources

PubMed2005

Benchmarking the CATMA microarray. A novel tool for Arabidopsis transcriptome analysis.

Transcript profiling is crucial to study biological systems, and various platforms have been implemented to survey mRNAs at the genome scale. We have assessed the performance of the CATMA microarray designed for Arabidopsis (Arabidopsis thaliana) transcriptome analysis and compared it with the Agilent and Affymetrix commercial platforms. The CATMA array consists of gene-specific sequence tags of 150 to 500 bp, the Agilent (Arabidopsis 2) array of 60mer oligonucleotides, and the Affymetrix gene chip (ATH1) of 25mer oligonucleotide sets. We have matched each probe repertoire with the Arabidopsis genome annotation (The Institute for Genomic Research release 5.0) and determined the correspondence between them. Array performance was analyzed by hybridization with labeled targets derived from eight RNA samples made of shoot total RNA spiked with a calibrated series of 14 control transcripts. CATMA arrays showed the largest dynamic range extending over three to four logs. Agilent and Affymetrix arrays displayed a narrower range, presumably because signal saturation occurred for transcripts at concentrations beyond 1,000 copies per cell. Sensitivity was comparable for all three platforms. For Affymetrix GeneChip data, the RMA software package outperformed Microarray Suite 5.0 for all investigated criteria, confirming that the information provided by the mismatch oligonucleotides has no added value. In addition, taking advantage of replicates in our dataset, we conducted a robust statistical analysis of the platform propensity to yield false positive and false negative differentially expressed genes, and all gave satisfactory results. The results establish the CATMA array as a mature alternative to the Affymetrix and Agilent platforms.

Arabidopsis↗

PubMed2004

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.

Arabidopsis↗

PubMed2004

Two TIR:NB:LRR genes are required to specify resistance to Peronospora parasitica isolate Cala2 in Arabidopsis.

Resistance responses that plants deploy in defence against pathogens are often triggered following a recognition event mediated by resistance (R) genes. The encoded R proteins usually contain a nucleotide-binding site (NB) and a leucine-rich repeat (LRR) domain. They are further classified into those that contain an N-terminal coiled coil (CC) motif or a Toll interleukin receptor (TIR) domain. Such R genes, when transferred into a susceptible plant of the same or closely related species, usually impart full resistance capability. We have used map-based cloning and mutation analysis to study the recognition of Peronospora parasitica (RPP)2 (At) locus in Arabidopsis accession Columbia (Col-0), which is a determinant of specific recognition of P. parasitica (At) isolate Cala2. Genetic mapping located RPP2 to a 200-kb interval on chromosome 4, which contained four adjacent TIR:NB:LRR genes. Mutational analysis revealed three classes of genes involved in specifying resistance to Cala2. One class, which resulted in pleiotropic effects on resistance to other P. parasitica (At) isolates, was unlinked to the RPP2 locus; this class included AtSGT1b. The other two classes were mapped within the interval and were specific to Cala2 resistance. Representatives of each of these classes were sequenced, and mutations were found in one or the other of two (RPP2A and RPP2B) of the four TIR:NB:LRR genes. RPP2A and RPP2B complemented their specific mutations, but failed to impart resistance when present alone, and it is concluded that both genes are essential determinants for isolate-specific recognition of Cala2. RPP2A has an unusual structure with a short LRR domain at the C-terminus, preceded by two potential but incomplete TIR:NB domains. In addition, the RPP2A LRR domain lacks conserved motifs found in all but three other TIR:NB:LRR class proteins. In contrast, RPP2B has a complete TIR:NB:LRR structure. It is concluded that RPP2A and RPP2B cooperate to specify Cala2 resistance by providing recognition or signalling functions lacked by either partner protein.

Amino Acid Sequence↗

PubMed2003

CATMA: a complete Arabidopsis GST database.

The Complete Arabidopsis Transcriptome Micro Array (CATMA) database contains gene sequence tag (GST) and gene model sequences for over 70% of the predicted genes in the Arabidopsis thaliana genome as well as primer sequences for GST amplification and a wide range of supplementary information. All CATMA GST sequences are specific to the gene for which they were designed, and all gene models were predicted from a complete reannotation of the genome using uniform parameters. The database is searchable by sequence name, sequence homology or direct SQL query, and is available through the CATMA website at http://www.catma.org/.

Arabidopsis↗

PubMed2002

Resistance to Ralstonia solanacearum in Arabidopsis thaliana is conferred by the recessive RRS1-R gene, a member of a novel family of resistance genes.

The identification of two Arabidopsis thaliana genes involved in determining recessive resistance to several strains of the causal agent of bacterial wilt, Ralstonia solanacearum, is reported. Dominant (RRS1-S) and recessive (RRS1-R) alleles from susceptible and resistant accessions encode highly similar predicted proteins differing in length and which present a novel structure combining domains found in plant Toll-IL-1 receptor-nucleotide binding site-leucin-rich repeat resistance proteins and a WRKY motif characteristic of some plant transcriptional factors. Although genetically defined as a recessive allele, RRS1-R behaves as a dominant resistance gene in transgenic plants. Sequence analysis of the RRS1 genes present in two homozygous intragenic recombinant lines indicates that several domains of RRS1-R are essential for its resistance function. Additionally, RRS1-R-mediated resistance is partially salicylic acid- and NDR1-dependent, suggesting the existence of similar signaling pathways to those controlled by resistance genes in specific resistance.

Alleles↗

PubMed2002

GARNet, the Genomic Arabidopsis Resource Network.

GARNet, the Genomic Arabidopsis Resource Network, was created to establish UK-based facilities for functional genomic research on Arabidopsis thaliana. In addition, GARNet provides a platform for international Arabidopsis research and for research on other plant species. To use the GARNet facilities apply via the website: http://garnet.arabidopsis.org.uk. All GARNet services and resources are publicly available, and data created using the GARNet resources will be freely distributed via databases held at the Nottingham Arabidopsis Stock Centre and the John Innes Centre.

Arabidopsis↗

PubMed2002

STAIRS: a new genetic resource for functional genomic studies of Arabidopsis.

Many biologically and economically important traits in plants and animals are quantitative/multifactorial, being controlled by several quantitative trait loci (QTL). QTL are difficult to locate accurately by conventional methods using molecular markers in segregating populations, particularly for traits of low heritability or for QTL with small effects. In order to resolve this, large (often unrealistically large) populations are required. In this paper we present an alternative approach using a specially developed resource of lines that facilitate QTL location first to a particular chromosome, then to successively smaller regions within a chromosome (< or = 0.5 cM) by means of simple comparisons among a few lines. This resource consists of "Stepped Aligned Inbred Recombinant Strains" (STAIRS) plus single whole Chromosome Substitution Strains (CSSs). We explain the analytical power of STAIRS and illustrate their construction and use with Arabidopsis thaliana, although the principles could be applied to many organisms. We were able to locate flowering QTL at the top of chromosome 3 known to contain several potential candidate genes.

Arabidopsis↗