Biomedical subjects

Timothy P O'Brien

Publications and source records attributed to Timothy P O'Brien.

8 recordsLinked to original sources

PubMed2006

Folding and organization of a contiguous chromosome region according to the gene distribution pattern in primary genomic sequence.

Specific mammalian genes functionally and dynamically associate together within the nucleus. Yet, how an array of many genes along the chromosome sequence can be spatially organized and folded together is unknown. We investigated the 3D structure of a well-annotated, highly conserved 4.3-Mb region on mouse chromosome 14 that contains four clusters of genes separated by gene "deserts." In nuclei, this region forms multiple, nonrandom "higher order" structures. These structures are based on the gene distribution pattern in primary sequence and are marked by preferential associations among multiple gene clusters. Associating gene clusters represent expressed chromatin, but their aggregation is not simply dependent on ongoing transcription. In chromosomes with aggregated gene clusters, gene deserts preferentially align with the nuclear periphery, providing evidence for chromosomal region architecture by specific associations with functional nuclear domains. Together, these data suggest dynamic, probabilistic 3D folding states for a contiguous megabase-scale chromosomal region, supporting the diverse activities of multiple genes and their conserved primary sequence organization.

Animals↗

PubMed2004

Moving forward with chemical mutagenesis in the mouse.

The study of genetic variation in mice offers a powerful experimental platform for understanding gene function. Complex trait analysis, gene-targeting and gene-trapping technologies, as well as insertional and chemical mutagenesis approaches are becoming increasingly sophisticated and provide a variety of options for cataloguing gene activities and interactions. In this review we discuss fundamental and practical concepts related to chemical mutagenesis and we highlight the growing list of strategies for performing mutagenesis screens in mice. Gene-driven and diverse types of phenotype-driven screens provide several options for the recovery of the invaluable variety of alleles generated by chemical mutagenesis. The unique advantages offered using chemical mutagenesis compare favourably to and complement the spectrum of approaches available for functional annotation of the mammalian genome.

Animals↗

PubMed2004

Evidence for a conserved function in synapse formation reveals Phr1 as a candidate gene for respiratory failure in newborn mice.

Genetic studies using a set of overlapping deletions centered at the piebald locus on distal mouse chromosome 14 have defined a genomic region associated with respiratory distress and lethality at birth. We have isolated and characterized the candidate gene Phr1 that is located within the respiratory distress critical genomic interval. Phr1 is the ortholog of the human Protein Associated with Myc as well as Drosophila highwire and Caenorhabditis elegans regulator of presynaptic morphology 1. Phr1 is expressed in the embryonic and postnatal nervous system. In mice lacking Phr1, the phrenic nerve failed to completely innervate the diaphragm. In addition, nerve terminal morphology was severely disrupted, comparable with the synaptic defects seen in the Drosophila hiw and C. elegans rpm-1 mutants. Although intercostal muscles were completely innervated, they also showed dysmorphic nerve terminals. In addition, sensory neuron terminals in the diaphragm were abnormal. The neuromuscular junctions showed excessive sprouting of nerve terminals, consistent with inadequate presynaptic stimulation of the muscle. On the basis of the abnormal neuronal morphology seen in mice, Drosophila, and C. elegans, we propose that Phr1 plays a conserved role in synaptic development and is a candidate gene for respiratory distress and ventilatory disorders that arise from defective neuronal control of breathing.

Amino Acid Sequence↗

PubMed2003

Development of an enhanced GFP-based dual-color reporter to facilitate genetic screens for the recovery of mutations in mice.

Mutagenesis screens to isolate a variety of alleles leading to null and non-null phenotypes represent an important approach for the characterization of gene function. Genetic schemes that use visible markers permit the efficient recovery of chemically induced mutations. We have developed a universal reporter system to visibly mark chromosomes for genetic screens in the mouse. The dual-color reporter is based on a single vector that drives the ubiquitous coexpression of the enhanced GFP (EGFP) spectral variants yellow and cyan. We show that widespread expression of the dual-color reporter is readily detected in embryonic stem cells, mice, and throughout developmental stages. CRE-loxP- and FLPe-FRT-mediated deletion of each color cassette demonstrates the modular design of the marker system. Random integration followed by plasmid rescue and sequence-based mapping was used to introduce the marker to a defined genomic location. Thus, single-step placement will simplify the construction of a genomewide bank of marked chromosomes. The dual-color nature of the marker permits complete identification of genetic classes of progeny as embryos or mice in classic regionally directed screens. The design also allows for more efficient and novel schemes, such as marked suppressor screens, in the mouse. The result is a versatile reporter that can be used independently or in combination with the growing sets of deletion and inversion resources to enhance the design and application of a wide variety of genetic schemes for the functional dissection of the mammalian genome.

Animals↗

PubMed2003

Genome function and nuclear architecture: from gene expression to nanoscience.

Biophysical, chemical, and nanoscience approaches to the study of nuclear structure and activity have been developing recently and hold considerable promise. A selection of fundamental problems in genome organization and function are reviewed and discussed in the context of these new perspectives and approaches. Advancing these concepts will require coordinated networks of physicists, chemists, and materials scientists collaborating with cell, developmental, and genome biologists.

Animals↗

PubMed2003

Notch signaling regulates left-right asymmetry determination by inducing Nodal expression.

Generation of left-right asymmetry is an integral part of the establishment of the vertebrate body plan. Here we show that the Notch signaling pathway plays a primary role in the establishment of left-right asymmetry in mice by directly regulating expression of the Nodal gene. Embryos mutant for the Notch ligand Dll1 or doubly mutant for the Notch1 and Notch2 receptors exhibit multiple defects in left-right asymmetry. Analysis of the enhancer regulating node-specific Nodal expression revealed the presence of binding sites for the RBP-J protein, the primary transcriptional mediator of Notch signaling. Mutation of these sites destroyed the ability of this enhancer to direct node-specific gene expression in transgenic mice. Our results demonstrate that Dll1-mediated Notch signaling is essential for generation of left-right asymmetry, and that the Notch pathway acts upstream of Nodal expression during left-right asymmetry determination in mice.

Animals↗

PubMed2003

Spontaneous deletion of epilepsy gene orthologs in a mutant mouse with a low electroconvulsive threshold.

The electroconvulsive threshold (ECT) test has been used extensively to determine the protection conferred by antiepileptic drug candidates against induced seizures in rodents. Despite its clinical relevance, the potential of ECT to identify mouse epilepsy models in genetic studies has not been thoroughly assessed. We adopted the ECT test to screen the progeny of ethylnitrosourea treated male C57BL/6J mice. In a small-scale screen, several mutant lines conferring a low threshold to ECT minimal clonic seizures were mapped to the telomeric region of mouse chromosome 2 in independent founder families. This high incidence was suggestive of a single spontaneous event that pre-existed in the founders of mutagenized stock. Genetic and physical mapping led to the discovery that several lines shared a single mutation, Szt1 (seizure threshold-1), consisting of a 300 kb deletion of genomic DNA involving three known genes. Two of these genes, Kcnq2 and Chrna4, are known to be mutated in human epilepsy families. Szt1 homozygotes and heterozygotes display similar phenotypes to those found in the respective Kcnq2 knockout mutant mice, suggesting that Kcnq2 haploinsufficiency is at the root of the Szt1 seizure sensitivity. Our results provide a novel genetic model for epilepsy research and demonstrate that the approach of using ECT to study seizures in mice has the potential to lead to the identification of human epilepsy susceptibility genes.

Animals↗

PubMed2002

Functional and comparative genomic analysis of the piebald deletion region of mouse chromosome 14.

Several developmentally important genomic regions map within the piebald deletion complex on distal mouse chromosome 14. We have combined computational gene prediction and comparative sequence analysis to characterize an approximately 4.3-Mb segment of the piebald region to identify candidate genes for the phenotypes presented by homozygous deletion mice. As a result we have ordered 13 deletion breakpoints, integrated the sequence with markers from a bacterial artificial chromosome (BAC) physical map, and identified 16 known or predicted genes and >1500 conserved sequence elements (CSEs) across the region. The candidate genes identified include Phr1 (formerly Pam) and Spry2, which are mouse homologs of genes required for development in Drosophila melanogaster. Gene content, order, and position are highly conserved between mouse chromosome 14 and the orthologous region of human chromosome 13. Our studies combining computational gene prediction with genetic and comparative genomic analyses provide insight regarding the functional composition and organization of this defined chromosomal region.

Animals↗