Biomedical subjects

W Richard McCombie

Publications and source records attributed to W Richard McCombie.

At least 19 recordsLinked to original sources

PubMed2026

Uncovering the early and conserved molecular mechanisms of root nitrogen foraging in model and crops.

BACKGROUND: Nitrogen (N) foraging, the ability of plants to promote preferential root growth in N-rich patches of soil, is fundamental to the competitiveness and wellbeing of plants. A unique “split-root” system, where a heterogenous N environment stimulates root foraging, provides a powerful experimental model to study the mechanisms underlying root foraging in model (Arabidopsis) and/or crop plants. RESULTS: We used the split-root set up to capture early molecular events involved in systemic N-signaling after exposure to a heterogeneous N signal, through time-course transcriptomic analysis across shoots and roots of Arabidopsis. We found that a histone methyltransferase, SET DOMAIN GROUP 8 (SDG8), is necessary for root N-foraging, suggesting a previously unknown role for chromatin regulation in mediating the preferential root growth response to colonize N-rich patches. To determine if the underlying molecular mechanism is conserved in evolution, we compared the root foraging behavior from model-to-crop (Arabidopsis, tomato and maize). Our analysis showed the model and crop species shared a root N-foraging growth response, with some variation among specific genotypes. Interestingly, we observed both shared and distinct transcriptional responses to heterogenous N environments among these three species. CONCLUSIONS: Our study has generated insights into the molecular basis of root N-foraging, with the potential to improve nutrient use efficiency in crop plants in a heterogeneous field environment.

Crops, Agricultural↗

PubMed2007

Curated genome annotation of Oryza sativa ssp. japonica and comparative genome analysis with Arabidopsis thaliana.

We present here the annotation of the complete genome of rice Oryza sativa L. ssp. japonica cultivar Nipponbare. All functional annotations for proteins and non-protein-coding RNA (npRNA) candidates were manually curated. Functions were identified or inferred in 19,969 (70%) of the proteins, and 131 possible npRNAs (including 58 antisense transcripts) were found. Almost 5000 annotated protein-coding genes were found to be disrupted in insertional mutant lines, which will accelerate future experimental validation of the annotations. The rice loci were determined by using cDNA sequences obtained from rice and other representative cereals. Our conservative estimate based on these loci and an extrapolation suggested that the gene number of rice is approximately 32,000, which is smaller than previous estimates. We conducted comparative analyses between rice and Arabidopsis thaliana and found that both genomes possessed several lineage-specific genes, which might account for the observed differences between these species, while they had similar sets of predicted functional domains among the protein sequences. A system to control translational efficiency seems to be conserved across large evolutionary distances. Moreover, the evolutionary process of protein-coding genes was examined. Our results suggest that natural selection may have played a role for duplicated genes in both species, so that duplication was suppressed or favored in a manner that depended on the function of a gene.

Arabidopsis↗

PubMed2006

Loss of p53 impedes the antileukemic response to BCR-ABL inhibition.

Targeted cancer therapies exploit the continued dependence of cancer cells on oncogenic mutations. Such agents can have remarkable activity against some cancers, although antitumor responses are often heterogeneous, and resistance remains a clinical problem. To gain insight into factors that influence the action of a prototypical targeted drug, we studied the action of imatinib (STI-571, Gleevec) against murine cells and leukemias expressing BCR-ABL, an imatinib target and the initiating oncogene for human chronic myelogenous leukemia (CML). We show that the tumor suppressor p53 is selectively activated by imatinib in BCR-ABL-expressing cells as a result of BCR-ABL kinase inhibition. Inactivation of p53, which can accompany disease progression in human CML, impedes the response to imatinib in vitro and in vivo without preventing BCR-ABL kinase inhibition. Concordantly, p53 mutations are associated with progression to imatinib resistance in some human CMLs. Our results identify p53 as a determinant of the response to oncogene inhibition and suggest one way in which resistance to targeted therapy can emerge during the course of tumor evolution.

Animals↗

PubMed2006

Comparative analysis of methylthioalkylmalate synthase (MAM) gene family and flanking DNA sequences in Brassica oleracea and Arabidopsis thaliana.

Gene BoGSL-PRO is associated with presence of 3-carbon side-chain glucosinolates (GSL). This gene is a member of the methylthioalkylmalate synthase (MAM) gene family. A BAC clone of Brassica oleracea, B21F5, containing this gene, was sequenced, annotated and compared to its corresponding region in Arabidopsis thaliana. Twelve protein-coding genes and 10 transposable elements were found in this clone. The corresponding region in A. thaliana chromosome I has 14 genes and no transposable elements. Analysis of MAM gene family in both species, which also include genes controlling 4-carbon side-chain GSL, separated the genes in two groups based on exon numbers and function. Phylogenetic analysis of the amino acid sequences encoded by these genes suggest that these two groups were produced by a duplication that must have occurred before the divergence of the Rosid and Asterid lineages of angiosperms. Comparison with putative orthologs from several prokaryotes further suggest that the members of the gene family with 10 exons, which encode proteins involved in 4-carbon side-chain GSL biosynthesis, were derived via truncation of the 3' end from ancestral genes more similar in length to those with 12 exons, which encode proteins involved in 3-carbon side-chain GSL biosynthesis. Lower gene density in B. oleracea compared to A. thaliana is due in part to presence of transposable elements (TE) mostly in inter-genic regions.

Arabidopsis↗

PubMed2005

Comparative analysis of a transposon-rich Brassica oleracea BAC clone with its corresponding sequence in A. thaliana.

We compared the sequence of a 96.7 Kb-long BAC clone (B 19 N 3) from Brassica oleracea (broccoli) with its corresponding regions in Arabidopsis thaliana. B 19 N 3 contains eight genes and 15 transposable elements (TEs). The first two genes in this clone, Bo 1 and Bo 2, have its corresponding region at the end of chromosome V of Arabidopsis (24 Mb). The third gene, Bo 3, corresponds to an ortholog at the opposite end (2.6 Mb) of the same chromosome. The other five genes, Bo 4 to Bo 8 also have a corresponding region on the same chromosome but at 7.7 Mb . These five genes are colinear with those found in the corresponding region of Arabidopsis, which contains, however, 15 genes. Therefore, a cluster of 10 genes is missing in B. oleracea clone (B 19 N 3). All five genes in common have the same order and orientation in the genomes of both species. Their 36 exons constituting the eight homologous genes have high conservation in size and sequence identity in both species. Among these, there is a major gene involved in aliphatic glucosinolate biosynthesis, Bo GSL-ELONG (Bo 4). Similar to A. thaliana, this gene, has a tandem duplicate, Bo 5. A contig for this region was constructed by primer walking and BAC-end-sequencing, revealing general gene colinearity between both species. During the 20 million years separating A. thaliana from B. oleracea from a common ancestor both genomes have diverged by chromosomal rearrangements and differential TE activity. These events, in addition to changes in chromosome number are responsible for the evolution of the genomes of both species. In spite of these changes, both species conserve general colinearity for their corresponding genes.

Arabidopsis↗

PubMed2005

Second-generation shRNA libraries covering the mouse and human genomes.

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference-induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.

Animals↗

PubMed2005

Sequence, annotation, and analysis of synteny between rice chromosome 3 and diverged grass species.

Rice (Oryza sativa L.) chromosome 3 is evolutionarily conserved across the cultivated cereals and shares large blocks of synteny with maize and sorghum, which diverged from rice more than 50 million years ago. To begin to completely understand this chromosome, we sequenced, finished, and annotated 36.1 Mb ( approximately 97%) from O. sativa subsp. japonica cv Nipponbare. Annotation features of the chromosome include 5915 genes, of which 913 are related to transposable elements. A putative function could be assigned to 3064 genes, with another 757 genes annotated as expressed, leaving 2094 that encode hypothetical proteins. Similarity searches against the proteome of Arabidopsis thaliana revealed putative homologs for 67% of the chromosome 3 proteins. Further searches of a nonredundant amino acid database, the Pfam domain database, plant Expressed Sequence Tags, and genomic assemblies from sorghum and maize revealed only 853 nontransposable element related proteins from chromosome 3 that lacked similarity to other known sequences. Interestingly, 426 of these have a paralog within the rice genome. A comparative physical map of the wild progenitor species, Oryza nivara, with japonica chromosome 3 revealed a high degree of sequence identity and synteny between these two species, which diverged approximately 10,000 years ago. Although no major rearrangements were detected, the deduced size of the O. nivara chromosome 3 was 21% smaller than that of japonica. Synteny between rice and other cereals using an integrated maize physical map and wheat genetic map was strikingly high, further supporting the use of rice and, in particular, chromosome 3, as a model for comparative studies among the cereals.

Arabidopsis↗

PubMed2005

Sorghum genome sequencing by methylation filtration.

Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF) technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis.

Arabidopsis↗

PubMed2005

Comparing low coverage random shotgun sequence data from Brassica oleracea and Oryza sativa genome sequence for their ability to add to the annotation of Arabidopsis thaliana.

Since the completion of the Arabidopsis thaliana genome sequence, there is an ongoing effort to annotate the genome as accurately as possible. Comparing genome sequences of related species complements the current annotation strategies by identifying genes and improving gene structure. A total of 595,321 Brassica oleracea shotgun reads were sequenced by TIGR (The Institute for Genome Research) and the collaboration of Washington University and Cold Spring Harbor. Vicogenta (a genome viewer based on GMOD and GBrowse) was created to view the current annotation and sequence alignments for Arabidopsis. Brassica reads were compared with the Arabidopsis genome and proteome databases using BLAST. Hypothetical genes and conserved unannotated regions on the short arm of chromosome 4 from Arabidopsis were experimentally verified using RT-PCR. We were able to improve the Arabidopsis annotation by identifying 25 genes that were missed, and confirming expression of 43 hypothetical genes in Arabidopsis. We were also able to detect conservation in genes whose transcription is normally suppressed due to methylation. We also examined how useful the O. sativa genome and ESTs from other species are, compared with Brassica, in improving the Arabidopsis annotation.

Amino Acid Sequence↗

PubMed2005

Differential methylation of genes and repeats in land plants.

The hypomethylated fraction of plant genomes is usually enriched in genes and can be selectively cloned using methylation filtration (MF). Therefore, MF has been used as a gene enrichment technology in sorghum and maize, where gene enrichment was proportional to genome size. Here we apply MF to a broad variety of plant species spanning a wide range of genome sizes. Differential methylation of genic and non-genic sequences was observed in all species tested, from non-vascular to vascular plants, but in some cases, such as wheat and pine, a lower than expected level of enrichment was observed. Remarkably, hexaploid wheat and pine show a dramatically large number of gene-like sequences relative to other plants. In hexaploid wheat, this apparent excess of genes may reflect an abundance of methylated pseudogenes, which may thus be more prevalent in recent polyploids.

DNA Methylation↗

PubMed2004

Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis.

Generation of complex libraries of defined nucleic acid sequences can greatly aid the functional analysis of protein and gene function. Previously, such studies relied either on individually synthesized oligonucleotides or on cellular nucleic acids as the starting material. As each method has disadvantages, we have developed a rapid and cost-effective alternative for construction of small-fragment DNA libraries of defined sequences. This approach uses in situ microarray DNA synthesis for generation of complex oligonucleotide populations. These populations can be recovered and either used directly or immortalized by cloning. From a single microarray, a library containing thousands of unique sequences can be generated. As an example of the potential applications of this technology, we have tested the approach for the production of plasmids encoding short hairpin RNAs (shRNAs) targeting numerous human and mouse genes. We achieved high-fidelity clone retrieval with a uniform representation of intended library sequences.

Animals↗

PubMed2004

Role of transposable elements in heterochromatin and epigenetic control.

Heterochromatin has been defined as deeply staining chromosomal material that remains condensed in interphase, whereas euchromatin undergoes de-condensation. Heterochromatin is found near centromeres and telomeres, but interstitial sites of heterochromatin (knobs) are common in plant genomes and were first described in maize. These regions are repetitive and late-replicating. In Drosophila, heterochromatin influences gene expression, a heterochromatin phenomenon called position effect variegation. Similarities between position effect variegation in Drosophila and gene silencing in maize mediated by "controlling elements" (that is, transposable elements) led in part to the proposal that heterochromatin is composed of transposable elements, and that such elements scattered throughout the genome might regulate development. Using microarray analysis, we show that heterochromatin in Arabidopsis is determined by transposable elements and related tandem repeats, under the control of the chromatin remodelling ATPase DDM1 (Decrease in DNA Methylation 1). Small interfering RNAs (siRNAs) correspond to these sequences, suggesting a role in guiding DDM1. We also show that transposable elements can regulate genes epigenetically, but only when inserted within or very close to them. This probably accounts for the regulation by DDM1 and the DNA methyltransferase MET1 of the euchromatic, imprinted gene FWA, as its promoter is provided by transposable-element-derived tandem repeats that are associated with siRNAs.

Arabidopsis↗

PubMed2004

Sequence analysis of the long arm of rice chromosome 11 for rice-wheat synteny.

The DNA sequence of 106 BAC/PAC clones in the minimum tiling path (MTP) of the long arm of rice chromosome 11, between map positions 57.3 and 116.2 cM, has been assembled to phase 2 or PLN level. This region has been sequenced to 10x redundancy by the Indian Initiative for Rice Genome Sequencing (IIRGS) and is now publicly available in GenBank. The region, excluding overlaps, has been predicted to contain 2,932 genes using different software. A gene-by-gene BLASTN search of the NCBI wheat EST database of over 420,000 cDNA sequences revealed that 1,143 of the predicted rice genes (38.9%) have significant homology to wheat ESTs (bit score >/= 100). Further BLASTN search of these 1,143 rice genes with the GrainGenes database of sequence contigs containing bin-mapped wheat ESTs allowed 113 of the genes to be placed in bins located on wheat chromosomes of different homoeologous groups. The largest number of genes, about one-third, mapped to the homoeologous group 4 chromosomes of wheat, suggesting a common evolutionary origin. The remaining genes were located on wheat chromosomes of different groups with significantly higher numbers for groups 3 and 5. Location of bin-mapped wheat contigs to chromosomes of all the seven homoeologous groups can be ascribed to movement of genes (transpositions) or chromosome segments (translocations) within rice or the hexaploid wheat genomes. Alternatively, it could be due to ancient duplications in the common ancestral genome of wheat and rice followed by selective elimination of genes in the wheat and rice genomes. While there exists definite conservation of gene sequences and the ancestral chromosomal identity between rice and wheat, there is no obvious conservation of the gene order at this level of resolution. Lack of extensive colinearity between rice and wheat genomes suggests that there have been many insertions, deletions, duplications and translocations that make the synteny comparisons much more complicated than earlier thought. However, enhanced resolution of comparative sequence analysis may reveal smaller conserved regions of colinearity, which will facilitate selection of markers for saturation mapping and sequencing of the gene-rich regions of the wheat genome.

Base Sequence↗

PubMed2004

A resource for large-scale RNA-interference-based screens in mammals.

Gene silencing by RNA interference (RNAi) in mammalian cells using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool. Here, we report the construction and application of a shRNA expression library targeting 9,610 human and 5,563 mouse genes. This library is presently composed of about 28,000 sequence-verified shRNA expression cassettes contained within multi-functional vectors, which permit shRNA cassettes to be packaged in retroviruses, tracked in mixed cell populations by means of DNA 'bar codes', and shuttled to customized vectors by bacterial mating. In order to validate the library, we used a genetic screen designed to report defects in human proteasome function. Our results suggest that our large-scale RNAi library can be used in specific, genetic applications in mammals, and will become a valuable resource for gene analysis and discovery.

Animals↗

PubMed2004

Sequencing the maize genome.

Sequencing of complex genomes can be accomplished by enriching shotgun libraries for genes. In maize, gene-enrichment by copy-number normalization (high C(0)t) and methylation filtration (MF) have been used to generate up to two-fold coverage of the gene-space with less than 1 million sequencing reads. Simulations using sequenced bacterial artificial chromosome (BAC) clones predict that 5x coverage of gene-rich regions, accompanied by less than 1x coverage of subclones from BAC contigs, will generate high-quality mapped sequence that meets the needs of geneticists while accommodating unusually high levels of structural polymorphism. By sequencing several inbred strains, we propose a strategy for capturing this polymorphism to investigate hybrid vigor or heterosis.

Chromosomes, Artificial, Bacterial↗

PubMed2004

The mouse genome: experimental examination of gene predictions and transcriptional start sites.

The completion of the mouse and other mammalian genome sequences will provide necessary, but not sufficient, knowledge for an understanding of much of mouse biology at the molecular level. As a requisite next step in this process, the genes in mouse and their structure must be elucidated. In particular, knowledge of the transcriptional start site of these genes will be necessary for further study of their regulatory regions. To assess the current state of mouse genome annotation to support this activity, we identified several hundred gene predictions in mouse with varying levels of supporting evidence and tested them using RACE-PCR. Modifications were made to the procedure allowing pooling of RNA samples, resulting in a scaleable procedure. The results illustrate potential errors or omissions in the current 5' end annotations in 58% of the genes detected. In testing experimentally unsupported gene predictions, we were able to identify 58 that are not usually annotated as genes but produced spliced transcripts (approximately 25% success rate). In addition, in many genes we were able to detect novel exons not predicted by any gene prediction algorithms. In 19.8% of the genes detected in this study, multiple transcript species were observed. These data show an urgent need to provide direct experimental validation of gene annotations. Moreover, these results show that direct validation using RACE-PCR can be an important component of genome-wide validation. This approach can be a useful tool in the ongoing efforts to increase the quality of gene annotations, especially transcriptional start sites, in complex genomes.

Animals↗

PubMed2004

Comparative analysis of a Brassica BAC clone containing several major aliphatic glucosinolate genes with its corresponding Arabidopsis sequence.

We compared the sequence of a 101-kb-long bacterial artificial chromosome (BAC) clone (B21H13) from Brassica oleracea with its homologous region in Arabidopsis thaliana. This clone contains a gene family involved in the synthesis of aliphatic glucosinolates. The A. thaliana homologs for this gene family are located on chromosome IV and correspond to three 2-oxoglutarate-dependent dioxygenase (AOP) genes. We found that B21H13 harbors 23 genes, whereas the equivalent region in Arabidopsis contains 37 genes. All 23 common genes have the same order and orientation in both Brassica and Arabidopsis. The 16 missing genes in the broccoli BAC clone were arranged in two major blocks of 5 and 7 contiguous genes, two singletons, and a twosome. The 118 exons comprising these 23 genes have high conservation between the two species. The arrangement of the AOP gene family in A. thaliana is as follows: AOP3 (GS-OHP) - AOP2 (GS-ALK) - pseudogene - AOP1. In contrast, in B. oleracea (broccoli and collard), two of the genes are duplicated and the third, AOP3, is missing. The remaining genes are arranged as follows: Bo-AOP2.1 (BoGSL-ALKa) - pseudogene - AOP2.2 (BoGSL-ALKb) - AOP1.1 - AOP1.2. When the survey was expanded to other Brassica accessions, we found variation in copy number and sequence for the Brassica AOP2 homologs. This study confirms that extensive rearrangements have taken place during the evolution of the Brassicacea at both gene and chromosomal levels.

Arabidopsis↗

PubMed2004

A workshop report on wheat genome sequencing: International Genome Research on Wheat Consortium.

Sponsored by the National Science Foundation and the U.S. Department of Agriculture, a wheat genome sequencing workshop was held November 10-11, 2003, in Washington, DC. It brought together 63 scientists of diverse research interests and institutions, including 45 from the United States and 18 from a dozen foreign countries (see list of participants at http://www.ksu.edu/igrow). The objectives of the workshop were to discuss the status of wheat genomics, obtain feedback from ongoing genome sequencing projects, and develop strategies for sequencing the wheat genome. The purpose of this report is to convey the information discussed at the workshop and provide the basis for an ongoing dialogue, bringing forth comments and suggestions from the genetics community.

Chromosome Mapping↗