Biomedical subjects

John Castle

Publications and source records attributed to John Castle.

7 recordsLinked to original sources

PubMed2005

An integrative genomics approach to infer causal associations between gene expression and disease.

A key goal of biomedical research is to elucidate the complex network of gene interactions underlying complex traits such as common human diseases. Here we detail a multistep procedure for identifying potential key drivers of complex traits that integrates DNA-variation and gene-expression data with other complex trait data in segregating mouse populations. Ordering gene expression traits relative to one another and relative to other complex traits is achieved by systematically testing whether variations in DNA that lead to variations in relative transcript abundances statistically support an independent, causative or reactive function relative to the complex traits under consideration. We show that this approach can predict transcriptional responses to single gene-perturbation experiments using gene-expression data in the context of a segregating mouse population. We also demonstrate the utility of this approach by identifying and experimentally validating the involvement of three new genes in susceptibility to obesity.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗

PubMed2005

Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.

MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression in plants and animals. To investigate the influence of miRNAs on transcript levels, we transfected miRNAs into human cells and used microarrays to examine changes in the messenger RNA profile. Here we show that delivering miR-124 causes the expression profile to shift towards that of brain, the organ in which miR-124 is preferentially expressed, whereas delivering miR-1 shifts the profile towards that of muscle, where miR-1 is preferentially expressed. In each case, about 100 messages were downregulated after 12 h. The 3' untranslated regions of these messages had a significant propensity to pair to the 5' region of the miRNA, as expected if many of these messages are the direct targets of the miRNAs. Our results suggest that metazoan miRNAs can reduce the levels of many of their target transcripts, not just the amount of protein deriving from these transcripts. Moreover, miR-1 and miR-124, and presumably other tissue-specific miRNAs, seem to downregulate a far greater number of targets than previously appreciated, thereby helping to define tissue-specific gene expression in humans.

3' Untranslated Regions↗

PubMed2004

A comprehensive transcript index of the human genome generated using microarrays and computational approaches.

BACKGROUND: Computational and microarray-based experimental approaches were used to generate a comprehensive transcript index for the human genome. Oligonucleotide probes designed from approximately 50,000 known and predicted transcript sequences from the human genome were used to survey transcription from a diverse set of 60 tissues and cell lines using ink-jet microarrays. Further, expression activity over at least six conditions was more generally assessed using genomic tiling arrays consisting of probes tiled through a repeat-masked version of the genomic sequence making up chromosomes 20 and 22. RESULTS: The combination of microarray data with extensive genome annotations resulted in a set of 28,456 experimentally supported transcripts. This set of high-confidence transcripts represents the first experimentally driven annotation of the human genome. In addition, the results from genomic tiling suggest that a large amount of transcription exists outside of annotated regions of the genome and serves as an example of how this activity could be measured on a genome-wide scale. CONCLUSIONS: These data represent one of the most comprehensive assessments of transcriptional activity in the human genome and provide an atlas of human gene expression over a unique set of gene predictions. Before the annotation of the human genome is considered complete, however, the previously unannotated transcriptional activity throughout the genome must be fully characterized.

Chromosomes, Human, Pair 20↗

PubMed2004

Expression of alternatively spliced sodium channel alpha-subunit genes. Unique splicing patterns are observed in dorsal root ganglia.

Molecular medicine requires the precise definition of drug targets, and tools are now in place to provide genome-wide information on the expression and alternative splicing patterns of any known gene. DNA microarrays were used to monitor transcript levels of the nine well-characterized alpha-subunit sodium channel genes across a broad range of tissues from cynomolgus monkey, a non-human primate model. Alternative splicing of human transcripts for a subset of the genes that are expressed in dorsal root ganglia, SCN8A (Na(v)1.6), SCN9A (Na(v)1.7), and SCN11A (Na(v)1.9) was characterized in detail. Genomic sequence analysis among gene family paralogs and between cross-species orthologs suggested specific alternative splicing events within transcripts of these genes, all of which were experimentally confirmed in human tissues. Quantitative PCR revealed that certain alternative splice events are uniquely expressed in dorsal root ganglia. In addition to characterization of human transcripts, alternatively spliced sodium channel transcripts were monitored in a rat model for neuropathic pain. Consistent down-regulation of all transcripts was observed, as well as significant changes in the splicing patterns of SCN8A and SCN9A.

Alternative Splicing↗

PubMed2004

Individual-specific variation of gene expression in peripheral blood leukocytes.

DNA microarray technology is used to determine gene expression profiles of various cell types, especially abnormal cells, such as cancer. By contrast, relatively little attention has been given to expression profiling of normal tissues. Here we describe studies of gene expression in peripheral blood leukocytes (PBL) from normal individuals sampled multiple times over periods ranging from several weeks up to 6 months. We demonstrate stable patterns of gene expression that differ between individuals. Among the genes whose expression varies by individual is a group of genes responsive to interferon stimulation. Certain individuals ( approximately 10-20% of those tested) showed higher baseline levels and lower inducibility of these genes in response to in vitro interferon stimulation. These studies demonstrate the feasibility of using DNA microarrays to measure the variations in gene expression of PBL from different individuals in response to environmental and genetic factors.

Gene Expression Profiling↗

PubMed2003

Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays.

Alternative pre-messenger RNA (pre-mRNA) splicing plays important roles in development, physiology, and disease, and more than half of human genes are alternatively spliced. To understand the biological roles and regulation of alternative splicing across different tissues and stages of development, systematic methods are needed. Here, we demonstrate the use of microarrays to monitor splicing at every exon-exon junction in more than 10,000 multi-exon human genes in 52 tissues and cell lines. These genome-wide data provide experimental evidence and tissue distributions for thousands of known and novel alternative splicing events. Adding to previous studies, the results indicate that at least 74% of human multi-exon genes are alternatively spliced.

Alternative Splicing↗

PubMed2003

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

Alternative Splicing↗