Biomedical subjects

John Healy

Publications and source records attributed to John Healy.

9 recordsLinked to original sources

PubMed2006

Mouse genomic representational oligonucleotide microarray analysis: detection of copy number variations in normal and tumor specimens.

Genomic amplifications and deletions, the consequence of somatic variation, are a hallmark of human cancer. Such variation has also been observed between "normal" individuals, as well as in individuals with congenital disorders. Thus, copy number measurement is likely to be an important tool for the analysis of genetic variation, genetic disease, and cancer. We developed representational oligonucleotide microarray analysis, a high-resolution comparative genomic hybridization methodology, with this aim in mind, and reported its use in the study of humans. Here we report the development of a representational oligonucleotide microarray analysis microarray for the genomic analysis of the mouse, an important model system for many genetic diseases and cancer. This microarray was designed based on the sequence assembly MM3, and contains approximately 84,000 probes randomly distributed throughout the mouse genome. We demonstrate the use of this array to identify copy number changes in mouse cancers, as well to determine copy number variation between inbred strains of mice. Because restriction endonuclease digestion of genomic DNA is an integral component of our method, differences due to polymorphisms at the restriction enzyme cleavage sites are also observed between strains, and these can be useful to follow the inheritance of loci between crosses of different strains.

Animals↗

PubMed2005

Implications for repetitive application of acetylcholine in the determination of the mechanisms of endothelium-dependent relaxation.

The influence of repetitive vasodilator concentration response curve determination was investigated in rat mesenteric and femoral small arteries. Arteries were precontracted with noradrenaline and relaxed with acetylcholine (ACh) or K+. Mesenteric arteries exhibited attenuation of ACh-stimulated relaxations during repetitive precontraction/relaxation cycles that was not prevented by SQ29548. Apamin, but not charybdotoxin, prevented the attenuation of this relaxation response. Borderline (p=0.064) statistical differences in the relaxations of mesenteric arteries in response to ACh remained in the presence of the nitric oxide synthase inhibitor L-NNA. In contrast, femoral arterial relaxations increased with repeated acetylcholine challenges. The enhanced responsiveness was prevented with L-NNA or Ba2+. In one experimental series, Ba2+ appeared to be without influence upon K+-stimulated relaxation of femoral arteries but a significant inhibitory effect was revealed when appropriate time control experiments were considered. These experiments reveal that impairment of SK(Ca) function and, to a lesser extent, an impaired NO signalling account for the attenuation of the relaxation responses of rat mesenteric arteries. In contrast, alterations of nitric oxide signalling and inward rectifier K channel activity contribute to enhanced relaxation responses in rat femoral arteries. These experiments highlight the importance of appropriate time control experiments for the proper interpretation of results derived from pharmacological experiments directed toward the elucidation of mechanisms of arterial vasorelaxation.

Acetylcholine↗

PubMed2004

Large-scale copy number polymorphism in the human genome.

The extent to which large duplications and deletions contribute to human genetic variation and diversity is unknown. Here, we show that large-scale copy number polymorphisms (CNPs) (about 100 kilobases and greater) contribute substantially to genomic variation between normal humans. Representational oligonucleotide microarray analysis of 20 individuals revealed a total of 221 copy number differences representing 76 unique CNPs. On average, individuals differed by 11 CNPs, and the average length of a CNP interval was 465 kilobases. We observed copy number variation of 70 different genes within CNP intervals, including genes involved in neurological function, regulation of cell growth, regulation of metabolism, and several genes known to be associated with disease.

Alleles↗

PubMed2004

Distribution of short paired duplications in mammalian genomes.

Mammalian genomes are densely populated with long duplicated sequences. In this paper, we demonstrate the existence of doublets, short duplications between 25 and 100 bp, distinct from previously described repeats. Each doublet is a pair of exact matches, separated by some distance. The distribution of these intermatch distances is strikingly nonrandom. An unexpectedly high number of doublets have matches either within 100 bp (adjacent) or at distances tightly concentrated approximately 1,000 bp apart (nearby). We focus our study on these proximate doublets. First, they tend to have both matches on the same strand. By comparing nearby doublets shared in human and chimpanzee, we can also see that these doublets seem to arise by an insertion event that produces a copy without markedly affecting the surrounding sequence. Most doublets in humans are shared with chimpanzee, but many new pairs arose after the divergence of the species. Doublets found in human but not chimpanzee are most often composed of almost tandem matches, whereas older doublets (found in both species) are more likely to have matches spaced by approximately 1 kb, indicating that the nearly tandem doublets may be more dynamic. The spacing of doublets is highly conserved. So far, we have found clearly recognizable doublets in the following genomes: Homo sapiens, Mus musculus, Arabidopsis thaliana, and Caenorhabditis elegans, indicating that the mechanism generating these doublets is widespread. A mechanism that generates short local duplications while conserving polarity could have a profound impact on the evolution of regulatory and protein-coding sequences.

Animals↗

PubMed2004

Occupational exposure to dioxins at UK worksites.

Following a request from a Governmental Interdepartmental Group, the Health and Safety Executive undertook a polychlorinated dibenzo-p-dioxin (PCDD) and polychorinated dibenzofuran (PCDF) sampling exercise at several work sites in the UK. An initial survey suggested potential PCDD/F production at metal recycling sites, during cement manufacture, at municipal waste incinerators and landfill sites and during the use of thermal oxygen lances. PCDD/F sampling, using static and personal air samplers, revealed that the highest PCDD/F exposures were found at metal recycling sites, particularly aluminium recycling sites. The reasons for these results and the possible consequential intakes are discussed.

Air Pollutants, Occupational↗

PubMed2003

Representational oligonucleotide microarray analysis: a high-resolution method to detect genome copy number variation.

We have developed a methodology we call ROMA (representational oligonucleotide microarray analysis), for the detection of the genomic aberrations in cancer and normal humans. By arraying oligonucleotide probes designed from the human genome sequence, and hybridizing with "representations" from cancer and normal cells, we detect regions of the genome with altered "copy number." We achieve an average resolution of 30 kb throughout the genome, and resolutions as high as a probe every 15 kb are practical. We illustrate the characteristics of probes on the array and accuracy of measurements obtained using ROMA. Using this methodology, we identify variation between cancer and normal genomes, as well as between normal human genomes. In cancer genomes, we readily detect amplifications and large and small homozygous and hemizygous deletions. Between normal human genomes, we frequently detect large (100 kb to 1 Mb) deletions or duplications. Many of these changes encompass known genes. ROMA will assist in the discovery of genes and markers important in cancer, and the discovery of loci that may be important in inherited predispositions to disease.

Aneuploidy↗

PubMed2003

Annotating large genomes with exact word matches.

We have developed a tool for rapidly determining the number of exact matches of any word within large, internally repetitive genomes or sets of genomes. Thus we can readily annotate any sequence, including the entire human genome, with the counts of its constituent words. We create a Burrows-Wheeler transform of the genome, which together with auxiliary data structures facilitating counting, can reside in about one gigabyte of RAM. Our original interest was motivated by oligonucleotide probe design, and we describe a general protocol for defining unique hybridization probes. But our method also has applications for the analysis of genome structure and assembly. We demonstrate the identification of chromosome-specific repeats, and outline a general procedure for finding undiscovered repeats. We also illustrate the changing contents of the human genome assemblies by comparing the annotations built from different genome freezes.

Algorithms↗

PubMed2003

GapCoder automates the use of indel characters in phylogenetic analysis.

BACKGROUND: Several ways of incorporating indels into phylogenetic analysis have been suggested. Simple indel coding has two strengths: (1) biological realism and (2) efficiency of analysis. In the method, each indel with different start and/or end positions is considered to be a separate character. The presence/absence of these indel characters is then added to the data set. ALGORITHM: We have written a program, GapCoder to automate this procedure. The program can input PIR format aligned datasets, find the indels and add the indel-based characters. The output is a NEXUS format file, which includes a table showing what region each indel characters is based on. If regions are excluded from analysis, this table makes it easy to identify the corresponding indel characters for exclusion. DISCUSSION: Manual implementation of the simple indel coding method can be very time-consuming, especially in data sets where indels are numerous and/or overlapping. GapCoder automates this method and is therefore particularly useful during procedures where phylogenetic analyses need to be repeated many times, such as when different alignments are being explored or when various taxon or character sets are being explored. GapCoder is currently available for Windows from http://www.home.duq.edu/~youngnd/GapCoder.

Algorithms↗

PubMedDate not supplied

Validation of S. pombe sequence assembly by microarray hybridization.

We describe a method to make physical maps of genomes using correlative hybridization patterns of probes to random pools of BACs. We derive thereby an estimated distance between probes, and then use this estimated distance to order probes. To test the method, we used BAC libraries from Schizzosaccharomyces pombe. We compared our data to the known sequence assembly, in order to assess accuracy. We demonstrate a small number of significant discrepancies between our method and the map derived by sequence assembly. Some of these discrepancies may arise because genome order within a population is not stable; imposing a linear order on a population may not be biologically meaningful.

Algorithms↗