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Biomedical subjects

R Stoughton

Publications and source records attributed to R Stoughton.

9 recordsLinked to original sources

Experimental annotation of the human genome using microarray technology.

The most important product of the sequencing of a genome is a complete, accurate catalogue of genes and their products, primarily messenger RNA transcripts and their cognate proteins. Such a catalogue cannot be constructed by computational annotation alone; it requires experimental validation on a genome scale. Using 'exon' and 'tiling' arrays fabricated by ink-jet oligonucleotide synthesis, we devised an experimental approach to validate and refine computational gene predictions and define full-length transcripts on the basis of co-regulated expression of their exons. These methods can provide more accurate gene numbers and allow the detection of mRNA splice variants and identification of the tissue- and disease-specific conditions under which genes are expressed. We apply our technique to chromosome 22q under 69 experimental condition pairs, and to the entire human genome under two experimental conditions. We discuss implications for more comprehensive, consistent and reliable genome annotation, more efficient, full-length complementary DNA cloning strategies and application to complex diseases.

Algorithms↗

Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.

We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.

Cells, Cultured↗

Functional discovery via a compendium of expression profiles.

Ascertaining the impact of uncharacterized perturbations on the cell is a fundamental problem in biology. Here, we describe how a single assay can be used to monitor hundreds of different cellular functions simultaneously. We constructed a reference database or "compendium" of expression profiles corresponding to 300 diverse mutations and chemical treatments in S. cerevisiae, and we show that the cellular pathways affected can be determined by pattern matching, even among very subtle profiles. The utility of this approach is validated by examining profiles caused by deletions of uncharacterized genes: we identify and experimentally confirm that eight uncharacterized open reading frames encode proteins required for sterol metabolism, cell wall function, mitochondrial respiration, or protein synthesis. We also show that the compendium can be used to characterize pharmacological perturbations by identifying a novel target of the commonly used drug dyclonine.

Cell Wall↗

Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles.

Genome-wide transcript profiling was used to monitor signal transduction during yeast pheromone response. Genetic manipulations allowed analysis of changes in gene expression underlying pheromone signaling, cell cycle control, and polarized morphogenesis. A two-dimensional hierarchical clustered matrix, covering 383 of the most highly regulated genes, was constructed from 46 diverse experimental conditions. Diagnostic subsets of coexpressed genes reflected signaling activity, cross talk, and overlap of multiple mitogen-activated protein kinase (MAPK) pathways. Analysis of the profiles specified by two different MAPKs-Fus3p and Kss1p-revealed functional overlap of the filamentous growth and mating responses. Global transcript analysis reflects biological responses associated with the activation and perturbation of signal transduction pathways.

Cell Cycle Proteins↗

Drug target validation and identification of secondary drug target effects using DNA microarrays.

We describe here a method for drug target validation and identification of secondary drug target effects based on genome-wide gene expression patterns. The method is demonstrated by several experiments, including treatment of yeast mutant strains defective in calcineurin, immunophilins or other genes with the immunosuppressants cyclosporin A or FK506. Presence or absence of the characteristic drug 'signature' pattern of altered gene expression in drug-treated cells with a mutation in the gene encoding a putative target established whether that target was required to generate the drug signature. Drug dependent effects were seen in 'targetless' cells, showing that FK506 affects additional pathways independent of calcineurin and the immunophilins. The described method permits the direct confirmation of drug targets and recognition of drug-dependent changes in gene expression that are modulated through pathways distinct from the drug's intended target. Such a method may prove useful in improving the efficiency of drug development programs.

Calcineurin↗

Data-adaptive algorithms for calling alleles in repeat polymorphisms.

Data-adaptive algorithms are presented for separating overlapping signatures of heterozygotic allele pairs in electrophoresis data. Application is demonstrated for human microsatellite CA-repeat polymorphisms in LiCor 4000 and ABI 373 data. The algorithms allow overlapping alleles to be called correctly in almost every case where a trained observer could do so, and provide a fast automated objective alternative to human reading of the gels. The algorithm also supplies an indication of confidence level which can be used to flag marginal cases for verification by eye, or as input to later stages of statistical analysis.

Algorithms↗

Release of vasopressin in response to altered plasma volume and sodium concentrations following pinealectomy in the rat.

Pinealectomy has been shown to alter daily rhythms of neurohypophysial hormone release, with plasma hormone concentrations being elevated in the morning, as compared to intact rats. To determine whether pineal removal also altered the response to known stimuli of hormone release, vasopressin concentrations were measured in control, sham-operated, and pinealectomized animals during extracellular fluid hypertonicity produced by an intraperitoneal (i.p.) injection of hypertonic saline or hypovolaemia produced by an i.p. injections of polyethylene glycol. In the combined sham-operated and unoperated groups, injection of hypertonic saline produced a marked increase in plasma vasopressin concentrations from 2.18 +/- 0.28 to 7.2 +/- 1.24 pmol/liter, but the response was attenuated in pinealectomized animals, concentrations increasing to only 3.4 +/- 1.2 pmol/liter. Similarly, following infusion of hypertonic saline, the increase in plasma vasopressin per unit increase in plasma sodium was lower in pinealectomized animals than the pineal intact controls. The response to hypovolaemia was also attenuated, plasma hormone concentrations following reduction in blood volume of approximately 10% increasing to only 3.6 +/- 0.6 pmol/liter as compared to 7.3 +/- 2.2 pmol/liter in the control groups. There were no significant differences in pituitary vasopressin content in any of the groups studied. Thus, the pineal may influence the vasopressin response to physiological stimuli.

Animals↗

Long-term monitoring of mouse ultrasonic vocalizations.

The temporal pattern of ultrasonic vocalizations by mice in an undisturbed 'home' environment can now be assessed using a system based on amplitude discrimination. Within a chosen frequency band, vocalizations of sufficient intensity are detected by an amplitude discriminator. The output from a pulse generator is sent to a microcomputer which records the time of the incoming event. The system has been validated for monitoring ultrasonic vocalizations in the mouse.

Animals↗