Experimental Comparison and Evaluation of the Affymetrix Exon and U133Plus2 GeneChip Arrays

Affymetrix exon arrays offer scientists the only solution for exon-level expression profiling at the whole-genome scale on a single array. These arrays feature a new chip design with no mismatch probes and a radically new random primed protocol to generate sense DNA targets along the entire length of the transcript. In addition to these changes, a limited number of validating experiments and virtually no experimental data to rigorously address the comparability of all-exon arrays with conventional 3'-arrays result in a natural reluctance to replace conventional expression arrays with the new all-exon platform.Using commercially available Affymetrix arrays, we assess the performance of the Human Exon 1.0 ST (HuEx) and U133 Plus 2.0 (U133Plus2) platforms directly through a series of 'spike-in' hybridizations containing 25 transcripts in the presence of a fixed eukaryotic background. Specifically, we compare the measures of expression for HuEx and U133Plus2 arrays to evaluate the precision of these measures as well as the specificity and sensitivity of the measures' ability to detect differential expression.This study presents an experimental comparison and systematic cross-validation of Affymetrix exon arrays and establishes high comparability of expression changes and probe performance characteristics between Affymetrix conventional and exon arrays. In addition, this study offers a reliable benchmark data set for the comparison of competing exon expression measures, the selection of methods suitable for mapping exon array measures to the wealth of previously generated microarray data, as well as the development of more advanced methods for exon- and transcript-level expression summarization

RhoA Activates Purified Phospholipase C-ϵ by a Guanine Nucleotide-dependent Mechanism

Phospholipase C-epsilon (PLC-epsilon) is a recently identified PLC isoform activated by subunits of heterotrimeric G proteins (Galpha(12), Galpha(13), and Gbetagamma) as well as by the low molecular weight GTPases, Rho and Ras. To define the enzymatic activity and substrate specificity of PLC-epsilon as well as its potential direct activation by Rho family GTPases, a major fragment of PLC-epsilon encompassing the catalytic core (EF-hand repeats through the tandem Ras-associating domains; approximately 118 kDa) was purified to near homogeneity and assayed after reconstitution under various conditions. Similar to the enzymatic profiles of previously purified PLC-beta isozymes, the purified fragment of PLC-epsilon maximally hydrolyzed phosphatidylinositol 4-phosphate at a rate of approximately 10 mumol/mg of protein/min, exhibited phospholipase activity dependent on the concentration of free calcium, and favored phosphatidylinositol 4,5-bisphosphate as substrate relative to other phosphoinositides. Furthermore, in mixed detergent phospholipid micelles, RhoA stimulated the phospholipase activity of the PLC-epsilon fragment in both a concentration-dependent and guanosine 5'-O-(3-thiotriphosphate)-dependent manner. This activation was abolished by the deletion of a unique approximately 65 amino acid-insert within the catalytic core of PLC-epsilon. Although Rac1 activated purified PLC-beta2ina guanine nucleotide-dependent manner, Rac1 failed to promote guanine nucleotide-dependent activation of purified PLC-epsilon. These results indicate that PLC-epsilon is a direct downstream effector for RhoA and that RhoA-dependent activation of PLC-epsilon depends on a unique insert within the catalytic core of the phospholipase

Activation of Phospholipase C-ε by Heterotrimeric G Protein βγ-Subunits

PLC-epsilon was identified recently as a phosphoinositide-hydrolyzing phospholipase C (PLC) containing catalytic domains (X, Y, and C2) common to all PLC isozymes as well as unique CDC25- and Ras-associating domains. Novel regulation of this PLC isozyme by the Ras oncoprotein and alpha-subunits (Galpha(12)) of heterotrimeric G proteins was illustrated. Sequence analyses of PLC-epsilon revealed previously unrecognized PH and EF-hand domains in the amino terminus. The known interaction of Gbetagamma subunits with the PH domains of other proteins led us to examine the capacity of Gbetagamma to activate PLC-epsilon. Co-expression of Gbeta(1)gamma(2) with PLC-epsilon in COS-7 cells resulted in marked stimulation of phospholipase C activity. Gbeta(2) and Gbeta(4) in combination with Ggamma(1), Ggamma(2), Ggamma(3), or Ggamma(13) also activated PLC-epsilon to levels similar to those observed with Gbeta(1)-containing dimers of these Ggamma-subunits. Gbeta(3) in combination with the same Ggamma-subunits was less active, and Gbeta(5)-containing dimers were essentially inactive. Gbetagamma-promoted activation of PLC-epsilon was blocked by cotransfection with either of two Gbetagamma-interacting proteins, Galpha(i1) or the carboxyl terminus of G protein receptor kinase 2. Pharmacological inhibition of PI3-kinase-gamma had no effect on Gbeta(1)gamma(2)-promoted activation of PLC-epsilon. Similarly, activation of Ras in the action of Gbetagamma is unlikely, because a mutation in the second RA domain of PLC-epsilon that blocks Ras activation of PLC failed to alter the stimulatory activity of Gbeta(1)gamma(2). Taken together, these results reveal the presence of additional functional domains in PLC-epsilon and add a new level of complexity in the regulation of this novel enzyme by heterotrimeric G proteins

Genomic inversions and GOLGA core duplicons underlie disease instability at the 15q25 locus.

Human chromosome 15q25 is involved in several disease-associated structural rearrangements, including microdeletions and chromosomal markers with inverted duplications. Using comparative fluorescence in situ hybridization, strand-sequencing, single-molecule, real-time sequencing and Bionano optical mapping analyses, we investigated the organization of the 15q25 region in human and nonhuman primates. We found that two independent inversions occurred in this region after the fission event that gave rise to phylogenetic chromosomes XIV and XV in humans and great apes. One of these inversions is still polymorphic in the human population today and may confer differential susceptibility to 15q25 microdeletions and inverted duplications. The inversion breakpoints map within segmental duplications containing core duplicons of the GOLGA gene family and correspond to the site of an ancestral centromere, which became inactivated about 25 million years ago. The inactivation of this centromere likely released segmental duplications from recombination repression typical of centromeric regions. We hypothesize that this increased the frequency of ectopic recombination creating a hotspot of hominid inversions where dispersed GOLGA core elements now predispose this region to recurrent genomic rearrangements associated with disease

Gβ Association and Effector Interaction Selectivities of the Divergent Gγ Subunit Gγ 13

G gamma(13) is a divergent member of the G gamma subunit family considered to be a component of the gustducin G-protein heterotrimer involved in bitter and sweet taste reception in taste bud cells. G gamma(13) contains a C-terminal asparagine-proline-tryptophan (NPW) tripeptide, a hallmark of RGS protein G gamma-like (GGL) domains which dimerize exclusively with G beta(5) subunits. In this study, we investigated the functional range of G gamma(13) assembly with G beta subunits using multiple assays of G beta association and G beta gamma effector modulation. G gamma(13) was observed to associate with all five G beta subunits (G beta(1-5)) upon co-translation in vitro, as well as function with all five G beta subunits in the modulation of Kir3.1/3.4 (GIRK1/4) potassium and N-type (alpha(1B)) calcium channels. Multiple G beta/G gamma(13) pairings were also functional in cellular assays of phospholipase C (PLC) beta 2 activation and inhibition of G alpha(q)-stimulated PLC beta 1 activity. However, upon cellular co-expression of G gamma(13) with different G beta subunits, only G beta(1)/G gamma(13), G beta(3)/G gamma(13), and G beta(4)/G gamma(13) pairings were found to form stable dimers detectable by co-immunoprecipitation under high-detergent cell lysis conditions. Collectively, these data indicate that G gamma(13) forms functional G beta gamma dimers with a range of G beta subunits. Coupled with our detection of G gamma(13) mRNA in mouse and human brain and retina, these results imply that this divergent G gamma subunit can act in signal transduction pathways other than that dedicated to taste reception in sensory lingual tissue

A genome-wide association study for diabetic nephropathy genes in African Americans

A genome-wide association study was performed using the Affymetrix 6.0 chip to identify genes associated with diabetic nephropathy in African Americans. Association analysis was performed adjusting for admixture in 965 type 2 diabetic African American patients with end-stage renal disease (ESRD) and in 1029 African Americans without type 2 diabetes or kidney disease as controls. The top 724 single nucleotide polymorphisms (SNPs) with evidence of association to diabetic nephropathy were then genotyped in a replication sample of an additional 709 type 2 diabetes-ESRD patients and 690 controls. SNPs with evidence of association in both the original and replication studies were tested in additional African American cohorts consisting of 1246 patients with type 2 diabetes without kidney disease and 1216 with non-diabetic ESRD to differentiate candidate loci for type 2 diabetes-ESRD, type 2 diabetes, and/or all-cause ESRD. Twenty-five SNPs were significantly associated with type 2 diabetes-ESRD in the genome-wide association and initial replication. Although genome-wide significance with type 2 diabetes was not found for any of these 25 SNPs, several genes, including RPS12, LIMK2, and SFI1 are strong candidates for diabetic nephropathy. A combined analysis of all 2890 patients with ESRD showed significant association SNPs in LIMK2 and SFI1 suggesting that they also contribute to all-cause ESRD. Thus, our results suggest that multiple loci underlie susceptibility to kidney disease in African Americans with type 2 diabetes and some may also contribute to all-cause ESRD

OSSOS. IV. DISCOVERY OF A DWARF PLANET CANDIDATE IN THE 9 : 2 RESONANCE WITH NEPTUNE

We report the discovery and orbit of a new dwarf planet candidate, 2015 RR245, by the Outer Solar System Origins Survey (OSSOS). The orbit of 2015 RR245 is eccentric (e = 0.586), with a semimajor axis near 82 au, yielding a perihelion distance of 34 au. 2015 RR245 has g - r = 0.59 +/- 0.11 and absolute magnitude H-r = 3.6 +/- 0.1; for an assumed albedo of p(V) = 12%, the object has a diameter of similar to 670. km. Based on astrometric measurements from OSSOS and Pan-STARRS1, we find that 2015 RR245 is securely trapped on ten-megayear timescales in the 9: 2 mean-motion resonance with Neptune. It is the first trans-Neptunian object (TNO) identified in this resonance. On hundred-megayear. timescales, particles in 2015 RR245-like orbits depart and sometimes return to the resonance, indicating that 2015 RR245 likely forms part of the long-lived metastable population of distant TNOs that drift between resonance sticking and actively scattering via gravitational encounters with Neptune. The discovery of a 9: 2 TNO stresses the role of resonances in the long-term evolution of objects in the scattering disk. and reinforces the view that distant resonances are heavily populated in the current solar system. This object further motivates detailed modeling of the transient sticking population.Peer reviewe

OSSOS. VII. 800+Trans-Neptunian Objects-The Complete Data Release

The Outer Solar System Origins Survey (OSSOS), a wide-field imaging program in 2013-2017 with the Canada-France-Hawaii Telescope, surveyed 155 deg(2) of sky to depths of m(r) = 24.1-25.2. We present 838 outer solar system discoveries that are entirely free of ephemeris bias. This increases the inventory of trans-Neptunian objects (TNOs) with accurately known orbits by nearly 50%. Each minor planet has 20-60 Gaia/Pan-STARRS-calibrated astrometric measurements made over 2-5 oppositions, which allows accurate classification of their orbits within the trans-Neptunian dynamical populations. The populations orbiting in mean-motion resonance with Neptune are key to understanding Neptune's early migration. Our 313 resonant TNOs, including 132 plutinos, triple the available characterized sample and include new occupancy of distant resonances out to semimajor axis a similar to 130 au. OSSOS doubles the known population of the nonresonant Kuiper Belt, providing 436 TNOs in this region, all with exceptionally high-quality orbits of a uncertainty sigma(a)Peer reviewe

The Outer Solar System Origins Survey : I. Design and First-Quarter Discoveries

We report the discovery, tracking, and detection circumstances for 85 trans-Neptunian objects (TNOs) from the first 42 deg(2) of the Outer Solar System Origins Survey. This ongoing r-band solar system survey uses the 0.9 deg(2) field of view MegaPrime camera on the 3.6m Canada-France-Hawaii Telescope. Our orbital elements for these TNOs are precise to a fractional semimajor axis uncertaintyPeer reviewe