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Christopher D Town

Publications and source records attributed to Christopher D Town.

9 recordsLinked to original sources
PubMed2004

Development and evaluation of an Arabidopsis whole genome Affymetrix probe array.

We describe the development of a high-density Arabidopsis'whole genome' oligonucleotide probe array for expression analysis (the Affymetrix ATH1 GeneChip probe array) that contains approximately 22 750 probe sets. Precedence on the array was given to genes for which either expression evidence or a credible database match existed. The remaining space was filled with 'hypothetical' genes. The new ATH1 array represents approximately 23 750 genes of which 60% were detected in RNA from cultured seedlings. Sensitivity of the array, determined using spiking controls, was approximately one transcript per cell. The array demonstrated high technical reproducibility and concordance with real-time PCR results. Indole-3 acetic acid (IAA)-induced changes in gene expression were used for biological validation of the array. A total of 222 genes were significantly upregulated and 103 significantly downregulated by exposure to IAA. Of the genes whose products could be functionally classified, the largest specific classes of upregulated genes were transcriptional regulators and protein kinases, many fewer of which were represented among the downregulated genes. Over one-third of the auxin-regulated genes have no known function, although many belong to gene families with members that have previously been shown to be auxin regulated. For the 6714 genes represented both on this and the earlier Arabidopsis Genome (AG) array, both signal intensities and gene expression ratios were very similar. Mapping of the oligonucleotides on the ATH1 array to the latest (version 4.0) annotation showed that over 95% of the probe sets (based on version 2.0 annotation) still fully represented their original target genes.

Arabidopsis↗
PubMed2003

A unique set of 11,008 onion expressed sequence tags reveals expressed sequence and genomic differences between the monocot orders Asparagales and Poales.

Enormous genomic resources have been developed for plants in the monocot order Poales; however, it is not clear how representative the Poales are for the monocots as a whole. The Asparagales are a monophyletic order sister to the lineage carrying the Poales and possess economically important plants such as asparagus, garlic, and onion. To assess the genomic differences between the Asparagales and Poales, we generated 11,008 unique ESTs from a normalized cDNA library of onion. Sequence analyses of these ESTs revealed microsatellite markers, single nucleotide polymorphisms, and homologs of transposable elements. Mean nucleotide similarity between rice and the Asparagales was 78% across coding regions. Expressed sequence and genomic comparisons revealed strong differences between the Asparagales and Poales for codon usage and mean GC content, GC distribution, and relative GC content at each codon position, indicating that genomic characteristics are not uniform across the monocots. The Asparagales were more similar to eudicots than to the Poales for these genomic characteristics.

Cytosine↗
PubMed2003

Improving the Arabidopsis genome annotation using maximal transcript alignment assemblies.

The spliced alignment of expressed sequence data to genomic sequence has proven a key tool in the comprehensive annotation of genes in eukaryotic genomes. A novel algorithm was developed to assemble clusters of overlapping transcript alignments (ESTs and full-length cDNAs) into maximal alignment assemblies, thereby comprehensively incorporating all available transcript data and capturing subtle splicing variations. Complete and partial gene structures identified by this method were used to improve The Institute for Genomic Research Arabidopsis genome annotation (TIGR release v.4.0). The alignment assemblies permitted the automated modeling of several novel genes and >1000 alternative splicing variations as well as updates (including UTR annotations) to nearly half of the approximately 27 000 annotated protein coding genes. The algorithm of the Program to Assemble Spliced Alignments (PASA) tool is described, as well as the results of automated updates to Arabidopsis gene annotations.

Algorithms↗
PubMed2003

Gene expression analyses of Arabidopsis chromosome 2 using a genomic DNA amplicon microarray.

The gene predictions and accompanying functional assignments resulting from the sequencing and annotation of a genome represent hypotheses that can be tested and used to develop a more complete understanding of the organism and its biology. In the model plant Arabidopsis thaliana, we developed a novel approach to constructing whole-genome microarrays based on PCR amplification of the 3' ends of each predicted gene from genomic DNA, and constructed an array representing more than 94% of the predicted genes and pseudogenes on chromosome 2. With this array, we examined various tissues and physiological conditions, providing expression-based validation for 84% of the gene predictions and providing clues as to the functions of many predicted genes. Further, by examining the distribution of expression along the physical chromosome, we were able to identify a region of repressed transcription that may represent a previously undescribed heterochromatic region.

Arabidopsis↗
PubMed2003

Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of an arbuscular mycorrhizal symbiosis.

The formation of symbiotic associations with arbuscular mycorrhizal (AM) fungi is a phenomenon common to the majority of vascular flowering plants. Here, we used cDNA arrays to examine transcript profiles in Medicago truncatula roots during the development of an AM symbiosis with Glomus versiforme and during growth under differing phosphorus nutrient regimes. Three percent of the genes examined showed significant changes in transcript levels during the development of the symbiosis. Most genes showing increased transcript levels in mycorrhizal roots showed no changes in response to high phosphorus, suggesting that alterations in transcript levels during symbiosis were a consequence of the AM fungus rather than a secondary effect of improved phosphorus nutrition. Among the mycorrhiza-induced genes, two distinct temporal expression patterns were evident. Members of one group showed an increase in transcripts during the initial period of contact between the symbionts and a subsequent decrease as the symbiosis developed. Defense- and stress-response genes were a significant component of this group. Genes in the second group showed a sustained increase in transcript levels that correlated with the colonization of the root system. The latter group contained a significant proportion of new genes similar to components of signal transduction pathways, suggesting that novel signaling pathways are activated during the development of the symbiosis. Analysis of the spatial expression patterns of two mycorrhiza-induced genes revealed distinct expression patterns consistent with the hypothesis that gene expression in mycorrhizal roots is signaled by both cell-autonomous and cell-nonautonomous signals.

Expressed Sequence Tags↗
PubMed2002

Full-length messenger RNA sequences greatly improve genome annotation.

BACKGROUND: Annotation of eukaryotic genomes is a complex endeavor that requires the integration of evidence from multiple, often contradictory, sources. With the ever-increasing amount of genome sequence data now available, methods for accurate identification of large numbers of genes have become urgently needed. In an effort to create a set of very high-quality gene models, we used the sequence of 5,000 full-length gene transcripts from Arabidopsis to re-annotate its genome. We have mapped these transcripts to their exact chromosomal locations and, using alignment programs, have created gene models that provide a reference set for this organism. RESULTS: Approximately 35% of the transcripts indicated that previously annotated genes needed modification, and 5% of the transcripts represented newly discovered genes. We also discovered that multiple transcription initiation sites appear to be much more common than previously known, and we report numerous cases of alternative mRNA splicing. We include a comparison of different alignment software and an analysis of how the transcript data improved the previously published annotation. CONCLUSIONS: Our results demonstrate that sequencing of large numbers of full-length transcripts followed by computational mapping greatly improves identification of the complete exon structures of eukaryotic genes. In addition, we are able to find numerous introns in the untranslated regions of the genes.

Alternative Splicing↗
PubMed2002

Genome-wide identification of nodule-specific transcripts in the model legume Medicago truncatula.

The Medicago truncatula expressed sequence tag (EST) database (Gene Index) contains over 140,000 sequences from 30 cDNA libraries. This resource offers the possibility of identifying previously uncharacterized genes and assessing the frequency and tissue specificity of their expression in silico. Because M. truncatula forms symbiotic root nodules, unlike Arabidopsis, this is a particularly important approach in investigating genes specific to nodule development and function in legumes. Our analyses have revealed 340 putative gene products, or tentative consensus sequences (TCs), expressed solely in root nodules. These TCs were represented by two to 379 ESTs. Of these TCs, 3% appear to encode novel proteins, 57% encode proteins with a weak similarity to the GenBank accessions, and 40% encode proteins with strong similarity to the known proteins. Nodule-specific TCs were grouped into nine categories based on the predicted function of their protein products. Besides previously characterized nodulins, other examples of highly abundant nodule-specific transcripts include plantacyanin, agglutinin, embryo-specific protein, and purine permease. Six nodule-specific TCs encode calmodulin-like proteins that possess a unique cleavable transit sequence potentially targeting the protein into the peribacteroid space. Surprisingly, 114 nodule-specific TCs encode small Cys cluster proteins with a cleavable transit peptide. To determine the validity of the in silico analysis, expression of 91 putative nodule-specific TCs was analyzed by macroarray and RNA-blot hybridizations. Nodule-enhanced expression was confirmed experimentally for the TCs composed of five or more ESTs, whereas the results for those TCs containing fewer ESTs were variable.

Agglutinins↗
PubMed2002

Cloning and sequencing of cDNAs for hypothetical genes from chromosome 2 of Arabidopsis.

About 25% of the genes in the fully sequenced and annotated Arabidopsis genome have structures that are predicted solely by computer algorithms with no support from either nucleic acid or protein homologs from other species or expressed sequence matches from Arabidopsis. These are referred to as "hypothetical genes." On chromosome 2, sequenced by The Institute for Genomic Research, there are approximately 800 hypothetical genes among a total of approximately 4,100 genes. To test their expression under various growth conditions and in specific tissues, we used six cDNA populations prepared from cold-treated, heat-treated, and pathogen (Xanthomonas campestris pv campestris)-infected plants, callus, roots, and young seedlings. To date, 169 hypothetical genes were tested, and 138 of them are found to be expressed in one or more of the six cDNA populations. By sequencing multiple clones from each 5'- and 3'-rapid amplification of cDNA ends (RACE) product and assembling the sequences, we generated full-length sequences for 16 of these genes. For 14 genes, there was one full-length assembly that precisely supported the intron-exon boundaries of their gene predictions, adding only 5'- and 3'-untranslated region sequences. However, for three of these genes, the other assemblies represent additional exons and alternatively spliced or unspliced introns. For the remaining two genes, the cDNA sequences reveal major differences with predicted gene structures. In addition, a total of six genes displayed more than one polyadenylation site. These data will be used to update gene models in The Institute for Genomic Research annotation database ATH1.

Alternative Splicing↗