Genetics

Rapid and Robust Resampling-based Multiple Testing Correctionwith Application in Genome-wide eQTL Study.

Publication Date: 2012 Jan 31 PMID: 22298711
Authors: Zhang, X. - Huang, S. - Sun, W. - Wang, W.
Journal: Genetics

Genome-wide expression quantitative loci (eQTL) studies have emerged as a powerful tool to understand the genetic basis of gene expression and complex traits. In a typical eQTL study the huge number of genetic markers and expression traits and their complicated correlations present a challenging multiple testing correction problem. The resampling-based test using permutation or bootstrap procedures is a standard approach to address the multiple testing problem in eQTL studies. A brute force application of the resampling-based test to large-scale eQTL datasets is often computationally infeasible. Several computationally efficient methods have been proposed to calculate approximate resampling-based p-values. However, these methods rely on certain assumptions about the correlation structure of the genetic markers, which may not be valid for certain studies. We propose a novel algorithm, REM (Rapid and Exact Multiple testing correction by resampling), to address this challenge. REM calculates the exact resampling-based p-values in a computationally efficient manner. The computational advantage of REM lies in its strategy of pruning the search space by skipping genetic markers whose upper bounds on test statistics are small. REM does not rely on any assumption about the correlation structure of the genetic markers. It can be applied to a variety of resampling-based multiple testing correction methods including permutation and bootstrap methods. We evaluate REM on three eQTL datasets (yeast, inbred mouse, and human rare variants) and show that it achieves accurate resampling-based p-value estimation with much less computational cost than existing methods. The software is available at http://csbio.unc.edu/eQTL.

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Sensory Organ Remodeling in Caenorhabditis elegans Requires the Zinc-finger Protein ZTF-16.

Publication Date: 2012 Jan 31 PMID: 22298710
Authors: Procko, C. - Lu, Y. - Shaham, S.
Journal: Genetics

Neurons and glia display remarkable morphological plasticity, and remodeling of glia may facilitate neuronal shape changes. The molecular basis and control of glial shape changes is not well understood. In response to environmental stress, the nematode Caenorhabditis elegans enters an alternative developmental state, called dauer, in which glia and neurons of the amphid sensory organ remodel. Here, we describe a genetic screen aimed at identifying genes required for amphid glia remodeling. We previously demonstrated that remodeling requires the Otx-type transcription factor TTX-1 and its direct target, the receptor tyrosine kinase gene ver-1. We now find that the hunchback/Ikaros-like C2H2 zinc finger factor ztf-16 is also required. We show that ztf- 16 mutants exhibit pronounced remodeling defects, explained, at least in part, by defects in the expression of ver-1. Expression and cell-specific rescue studies suggest that ztf-16, like ttx-1, functions within glia; however, promoter deletion studies show that ztf-16 acts through a site on the ver-1 promoter that is independent of ttx-1. Our studies identify an important component of glia remodeling, and suggest that transcriptional changes may underlie glial morphological plasticity in the sensory organs of C. elegans.

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UNDERSTANDING AND PREDICTING THE FITNESS DECLINE OF SHRUNK POPULATIONS: Inbreeding, purging, mutation and standard selection.

Publication Date: 2012 Jan 31 PMID: 22298709
Authors: Garcia-Dorado, A.
Journal: Genetics

The joint consequences of inbreeding, natural selection, and deleterious mutation on mean fitness after population shrinkage, are of great importance in evolution and can be critical to the conservation of endangered populations. I present simple analytical equations that predict these consequences, improving and extending a previous heuristic treatment. Purge is defined as the "extra" selection induced by inbreeding, due to the "extra" fitness disadvantage (2d) of homozygotes for (partially) recessive deleterious alleles. Its effect is accounted for by using, instead of the classical inbreeding coefficient f, a purged inbreeding coefficient g that is weighed by the reduction of the frequency of deleterious alleles caused by purging. When the effective size of a large population is reduced to a smaller stable value N (with Nd>/=1), the purged inbreeding coefficient after t generations can be predicted as g(t) - [(1- 1/2N) g(t-1) + 1/2N)](1-2d f(t-1) ), showing how purging acts upon previously accumulated inbreeding and how its efficiency increases with N. This implies an early fitness decay, followed by some recovery. During this process, the inbreeding depression rate shifts from its ancestral value (delta) to that of the Mutation-Selection-Drift balance corresponding to N (delta*), and standard selection cancels out the inbreeding depression ascribed to delta*. Therefore, purge and inbreeding only operate upon the remaining delta -delta*. The method is applied to the conservation strategy in which family contributions to the breeding pool are equal, and is extended to make use of genealogical information. All these predictions are checked using computer simulation.

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Flowering time in Maize: Linkage and Epistasis at aMajor Effect Locus.

Publication Date: 2012 Jan 31 PMID: 22298708
Authors: Durand, E. - Bouchet, S. - Bertin, P. - Ressayre, A. - Jamin, P. - Charcosset, A. - Dillmann, C. - Tenaillon, M. I.
Journal: Genetics

In a previous study, we identified a candidate fragment length polymorphism associated with flowering time variation after 7 generations of selection for flowering time, starting from the maize inbred line F252. Here, we characterized the candidate region and identified underlying polymorphisms. Then, we combined QTL mapping, association mapping and developmental characterization to dissect the genetic mechanisms responsible for the phenotypic variation. The candidate region contained the Eukaryotic Initiation Factor (eIF-4A), and revealed a high level of sequence and structural variation beyond the 3'UTR of eIF 4A including several insertions of truncated transposable elements. Using a bi-allelic SNP (C/T) in the candidate region, we confirmed its association with flowering time variation in a panel of 317 maize inbred lines. However, while the T allele was correlated with late flowering time within the F252 genetic background, it was correlated with early flowering time in the association panel with pervasive interactions between allelic variation and the genetic background, pointing to underlying epistasis. We also detected pleiotropic effects of the candidate polymorphism on various traits including flowering time, plant height and leaf number. Finally, we were able to break down the correlation between flowering time and leaf number in the progeny of a heterozygote (C/T) within the F252 background consistent with causal loci in linkage disequilibrium. We therefore propose that both a cluster of tightly-linked genes and epistasis contribute to the phenotypic variation for flowering time.

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A General Population Genetic Framework for Antagonistic Selection that Accounts for Demography and Recurrent Mutation.

Publication Date: 2012 Jan 31 PMID: 22298707
Authors: Connallon, T. - Clark, A. G.
Journal: Genetics

Antagonistic selection-where alleles at a locus have opposing effects on male and female fitness ("sexual antagonism"), or between components of fitness ("antagonistic pleiotropy")-might play an important role in maintaining population genetic variation, and in driving phylogenetic and genomic patterns of sexual dimorphism and life-history evolution. While prior theory has thoroughly characterized the conditions necessary for antagonistic balancing selection to operate, we currently know little about the evolutionary interactions between antagonistic selection, recurrent mutation, and genetic drift, which should collectively shape empirical patterns of genetic variation. To fill this void, we developed and analyzed a series of population genetic models that simultaneously incorporate these processes. Our models identify two general properties of antagonistically selected loci. First, antagonistic selection inflates heterozygosity and fitness variance across a broad parameter range-a result that applies to alleles maintained by balancing selection and by recurrent mutation. Second, effective population size and genetic drift profoundly affect the statistical frequency distributions of antagonistically selected alleles. The "efficacy" of antagonistic selection (i.e., its tendency to dominate over genetic drift) is extremely weak relative to classical models, such as directional selection and overdominance. Alleles meeting traditional criteria for strong selection (N(e)s >> 1, where N(e) is the effective population size, and s is a selection coefficient for a given sex or fitness component) may nevertheless evolve as if neutral. The effects of mutation and demography may generate population differences in overall levels of antagonistic fitness variation, as well as molecular population genetic signatures of balancing selection.

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TORC2 Signaling is Antagonized by Protein Phosphatase 2A and the Far Complex in Saccharomyces cerevisiae.

Publication Date: 2012 Jan 31 PMID: 22298706
Authors: Pracheil, T. - Thornton, J. - Liu, Z.
Journal: Genetics

The target of rapamycin (TOR) kinase, a central regulator of eukaryotic cell growth, exists in two essential, yet distinct TOR kinase complexes in the budding yeast Saccharomyces cerevisiae: rapamycin-sensitive TORC1 and rapamycin-insensitive TORC2. Lst8, a component of both TOR complexes, is essential for cell viability. However, it is unclear whether the essential function of Lst8 is linked to TORC1, TORC2, or both. To that end, we carried out a genetic screen to isolate lst8 deletion suppressor mutants. Here we report that mutations in SAC7 and FAR11 suppress lethality of lst8Delta and TORC2-deficient (tor2-21) mutations but not TORC1 inactivation, suggesting that the essential function of Lst8 is linked only to TORC2. More importantly, characterization of lst8Delta bypass mutants reveals a role for Protein Phosphatase 2A (PP2A) in the regulation of TORC2 signaling. We show that Far11, a member of the Far3-7-8-9-10-11 complex involved in pheromone induced cell cycle arrest, interacts with Tpd3 and Pph21, conserved components of PP2A, and deletions of components of the Far3-7-8-9-10-11 complex and PP2A rescue growth defects in lst8Delta and tor2-21 mutants. Additionally, loss of the regulatory B' subunit of PP2A Rts1 or Far11 restores phosphorylation to the TORC2 substrate Slm1 in a tor2-21 mutant. Mammalian Far11 orthologs, FAM40A/B, exist in a complex with PP2A known as STRIPAK, suggesting conserved functional association of PP2A and Far11. Antagonism of TORC2 signaling by PP2A-Far11 represents a novel regulatory mechanism for controlling spatial cell growth of yeast.

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Genetic Analysis of Complex Interactions Amongst Components of the Mitochondrial Import Motor and Translocon in Saccharomyces cerevisiae.

Publication Date: 2012 Jan 31 PMID: 22298705
Authors: Schilke, B. A. - Hayashi, M. - Craig, E. A.
Journal: Genetics

A highly conserved, Hsp70-based, import motor, which is associated with the translocase on the matrix side of the inner mitochondrial membrane, is critical for protein translocation into the matrix. Hsp70 is tethered to the translocon via interaction with Tim44. The J-protein co-chaperone Pam18 and Pam16, a structurally related protein with which it forms a heterodimer, are also critical components of the motor. Their N-termini are important for the heterodimer's translocon association, with Pam18's and Pam16's N-termini interacting in the intermembrane space and the matrix, respectively. Here, using the model organism Saccharomyces cerevisiae, we report the identification of an N-terminal segment of Tim44 important for association of Pam16 with the translocon. We also report that higher amounts of Pam17, a nonessential motor component, are found associated with the translocon in both PAM16 and TIM44 mutants that affect their interaction with one another. These TIM44 and PAM16 mutations are also synthetically lethal with a deletion of PAM17. In contrast, a deletion of PAM17 has little, or no, genetic interaction with a PAM18 mutation that affects translocon association of the Pam16:Pam18 heterodimer, suggesting a second role for the Pam16:Tim44 interaction. A similar pattern of genetic interactions and enhanced Pam17 translocon association were observed in the absence of the C-terminus of Tim17, a core component of the translocon. We suggest the Pam16:Tim44 interaction may play two roles: (1) tethering the Pam16:Pam18 heterodimer to the translocon and (2) positioning the import motor for efficient engagement with the translocating polypeptide along with Tim17 and Pam17.

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Tissue Architecture in the Caenorhabditis elegans Gonad Depends on Interactions among Fibulin-1, Type IV Collagen and the ADAMTS Extracellular Protease.

Publication Date: 2012 Jan 31 PMID: 22298704
Authors: Kubota, Y. - Nagata, K. - Sugimoto, A. - Nishiwaki, K.
Journal: Genetics

Molecules in the extracellular matrix (ECM) regulate cellular behavior in both development and pathology. Fibulin-1 is a conserved ECM protein. The Caenorhabditis elegans ortholog, FBL-1, regulates gonad-arm elongation and expansion by acting antagonistically to GON-1, an ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family protease. The elongation of gonad arms is directed by gonadal distal tip cells (DTCs). Here we report that a dominant mutation in the EMB-9/type IV collagen alpha1 subunit can compensate for loss of FBL-1 activity in gonadogenesis. A specific amino acid substitution in the NC1 domain of EMB-9 suppressed the fbl-1 null mutant. FBL-1 was required to maintain wild-type EMB-9 in the basement membrane (BM), whereas mutant EMB-9 was retained in the absence of FBL-1. EMB-9 (either wild type or mutant) localization in the BM enhanced PAT-3/beta-integrin expression in DTCs. In addition, overexpression of PAT-3 partially rescued the DTC migration defects in fbl-1 mutants, suggesting that EMB-9 acts in part through PAT-3 to control DTC migration. In contrast to the suppression of fbl-1(tk45), mutant EMB-9 enhanced the gonadal defects of gon-1(e1254), suggesting that it gained a function similar to that of wild-type FBL-1, which promotes DTC migration by inhibiting GON- 3 1. We propose that FBL-1 and GON-1 control EMB-9 accumulation in the BM and promote PAT-3 expression to control DTC migration.

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Neural Maintenance Roles for the Matrix Receptor Dystroglycan and the Nuclear Anchorage Complex in Caenorhabditis elegans.

Publication Date: 2012 Jan 31 PMID: 22298703
Authors: Johnson, R. P. - Kramer, J. M.
Journal: Genetics

Recent studies in C. elegans have revealed specific neural maintenance mechanisms that protect soma and neurites against mispositioning due to displacement stresses, such as muscle contraction. We report that C. elegans dystroglycan DGN-1 functions to maintain the position of lumbar neurons during late embryonic and larval development. In the absence of DGN-1 the cell bodies of multiple lumbar neuron classes are frequently displaced anterior of their normal positions. Early but not later embryonic pan-neural expression of DGN-1 rescues positional maintenance, suggesting that dystroglycan is required for establishment of a critical maintenance pathway that persists throughout later developmental stages. Lumbar neural maintenance requires only a membrane-tethered N-terminal domain of DGN-1 and may involve a novel extracellular partner for dystroglycan. A genetic screen for similar lumbar maintenance mutants revealed a role for the nesprin/SYNE family protein ANC-1 as well as for the extracellular protein DIG-1, previously implicated in lumbar neuron maintenance. The involvement of ANC-1 reveals a previously unknown role for nucleus-cytoskeleton interactions in neural maintenance. Genetic analysis indicates that lumbar neuron position is maintained in late embryos by parallel DGN-1/DIG-1 and ANC-1 dependent pathways, and in larvae by separate DGN-1 and ANC-1 pathways. The effect of muscle paralysis on late embryonic or larval stage maintenance defects in mutants indicates that lumbar neurons are subject to both muscle contraction-dependent and contraction-independent displacement stresses, and that different maintenance pathways may protect against specific types of displacement stress.

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Roles for Receptors, Pheromones, G proteins and Mating Type Genes During Sexual Reproduction in Neurospora crassa.

Publication Date: 2012 Jan 31 PMID: 22298702
Authors: Kim, H. - Wright, S. J. - Park, G. - Ouyang, S. - Krystofova, S. - Borkovich, K. A.
Journal: Genetics

Here we characterize the relationship between the PRE-2 pheromone receptor and its ligand, CCG-4, and the general requirements for receptors and pheromones during fusion of opposite mating type cells and sexual sporulation in the multicellular fungus Neurospora crassa. PRE-2 is highly expressed in mat a cells and is localized in male and female reproductive structures. Deltapre-2 mat a females do not respond chemotropically to mat A males (conidia) or form mature fruiting bodies (perithecia) or meiotic progeny (ascospores). Strains with swapped identity due to heterologous expression of pre-2 or ccg-4 behave normally in crosses with opposite mating type strains. Co-expression of pre-2 and ccg-4 in the mat A background leads to self-attraction and development of barren perithecia without ascospores. Further perithecial development is achieved by inactivation of Sad-1, a gene required for meiotic gene silencing. Findings from studies involving forced heterokaryons of opposite mating type strains show that presence of one receptor and its compatible pheromone is necessary and sufficient for perithecial development and ascospore production. Taken together, the results demonstrate that although receptors and pheromones control sexual identity, the mating-type genes (mat A and mat a) must be in two different nuclei to allow meiosis and sexual sporulation to occur.

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A Test for Selection Employing Quantitative Trait Locus and Mutation Accumulation Data.

Publication Date: 2012 Jan 31 PMID: 22298701
Authors: Rice, D. P. - Townsend, J. P.
Journal: Genetics

Evolutionary biologists attribute much of phenotypic diversity observed in nature to the action of natural selection. However, for many phenotypic traits, and especially quantitative phenotypic traits, it has been challenging to test for the historical action of selection. An important challenge for biologists studying quantitative traits, therefore, is to distinguish between traits that have evolved under the influence of strong selection and those that have evolved neutrally. Most existing tests for selection employ molecular data, but selection also leaves a mark on the genetic architecture underlying a trait. In particular, the distribution of quantitative trait locus (QTL) effect sizes and the distribution of mutational effects together provide information regarding the history of selection. Despite the increasing availability of QTL and mutation accumulation data, such data have not yet been effectively exploited for this purpose. We present a model of the evolution of QTL and employ it to formulate a test for historical selection. To provide a baseline for neutral evolution of the trait, we estimate the distribution of mutational effects from mutation accumulation experiments. We then apply a maximum likelihood-based method of inference to estimate the range of selection strengths under which such a distribution of mutations could generate the observed QTL. Our test thus represents the first integration of population genetic theory and QTL data to measure the historical influence of selection.

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Inferring Coancestry in Population Samples in the Presence of Linkage Disequilibrium.

Publication Date: 2012 Jan 31 PMID: 22298700
Authors: Brown, M. D. - Glazner, C. G. - Zheng, C. - Thompson, E. A.
Journal: Genetics

In both pedigree linkage studies and in population-based association studies there has been much interest in the use of modern dense genetic marker data to infer segments of gene identity by descent (ibd) among individuals not known to be related, in order to increase power and resolution in localizing genes affecting complex traits. In this paper, we present a hidden Markov model (HMM) for ibd among a set of chromosomes, and describe methods and software for inference of ibd among the four chromosomes of pairs of individuals, using either phased (haplotypic) or unphased (genotypic) data. The model allows for missing data and typing error, but does not model linkage disequilibrium (LD), because fitting an accurate LD model requires large samples from well studied populations. However, LD remains a major confounding factor, since LD is itself a reflection of coancestry at the population level. To study the impact of LD, we have developed a novel simulation approach to generate realistic dense marker data for the same set of markers but at varying levels of LD. Using this approach, we present results of a study of the impact of LD on the sensitivity and specificity of our HMM model in estimating segments of ibd among sets of four chromosomes and between genotype pairs. We show that, despite not incorporating LD, our model has been quite successful in detecting segments as small as 10(6) base pairs (1 Mpb); we present also comparisons with fastIBD which uses an LD model in estimating ibd.

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Accuracy of Genomic Selection Methods in a Standard Dataset of Loblolly Pine (Pinus taeda L.).

Publication Date: 2012 Jan 23 PMID: 22271763
Authors: Resende, M. F. Jr - Munoz, P. - Resende, M. D. - Garrick, D. J. - Fernando, R. L. - Davis, J. M. - Jokela, E. J. - Martin, T. A. - Peter, G. F. - Kirst, M.
Journal: Genetics

Genomic selection can increase genetic gain per generation through early selection. Genomic selection is expected to be particularly valuable for traits that are costly to phenotype, and expressed late in the life-cycle of long-lived species. Alternative approaches to genomic selection prediction models may perform differently for traits with distinct genetic properties. Here the performance of four different original methods of genomic selection that differ with respect to assumptions regarding distribution of marker effects, including (i) Ridge Regression - Best Linear Unbiased Prediction (RR-BLUP), (ii) Bayes A, (iii) Bayes Cpi, and (iv) Bayesian LASSO are presented. In addition, a modified RR-BLUP (RR-BLUP B) that utilizes a selected subset of markers was evaluated. The accuracy of these methods was compared across 17 traits with distinct heritabilities and genetic architectures, including growth, development and disease-resistance properties, measured in a Pinus taeda (loblolly pine) training population of 951 individuals genotyped with 4,853 SNPs. The predictive ability of the methods was evaluated using a 10-fold, cross-validation approach, and differed only marginally for most method/trait combinations. Interestingly, for fusiform rust disease-resistance traits Bayes Cpi, Bayesian LASSO and RR-BLUB B had higher predictive ability than RR-BLUP and Bayes A. Fusiform rust is controlled by few genes of large effect. A limitation of RR-BLUP is the assumption of equal contribution of all markers to the observed variation. The genotypic and phenotypic data used in this study is publically available for comparative analysis of genomic selection prediction models.

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Surrogate Genetics and Metabolic Profiling for Characterization of Human Disease Alleles.

Publication Date: 2012 Jan 20 PMID: 22267502
Authors: Mayfield, J. A. - Davies, M. W. - Dimster-Denk, D. - Pleskac, N. - McCarthy, S. - Boydston, E. A. - Fink, L. - Lin, X. X. - Narain, A. S. - Meighan, M. - Rine, J.
Journal: Genetics

Cystathionine-beta-synthase (CBS) deficiency is a human genetic disease causing homocystinuria, thrombosis, mental retardation, and a suite of other devastating manifestations. Early detection coupled with dietary modification greatly reduces pathology, but the response to treatment differs with the allele of CBS. A better understanding of the relationship between allelic variants and protein function will improve both diagnosis and treatment. To this end, we tested the function of 84 CBS alleles previously sequenced from patients with homocystinuria by ortholog replacement in Saccharomyces cerevisiae. Within this clinically-associated set, 15% of variant alleles were indistinguishable from the predominant CBS allele in function, suggesting enzymatic activity was retained. An additional 37% of the alleles were partially functional or could be rescued by cofactor supplementation in the growth medium. This large class included alleles rescued by elevated levels of the cofactor vitamin B6, but also alleles rescued by elevated heme, a second CBS cofactor. Measurement of the metabolite levels in CBS-substituted yeast grown with different B6 levels using LC-MS revealed changes in metabolism that propagated beyond the substrate and product of CBS. Production of the critical antioxidant glutathione through the CBS pathway was greatly decreased when CBS function was restricted through genetic, cofactor, or substrate restriction, a metabolic consequence with implications for treatment.

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The Abundance of Deleterious Polymorphisms in Humans.

Publication Date: 2012 Jan 20 PMID: 22267501
Authors: Subarmanian, S.
Journal: Genetics

Here I show a gradual decline in the proportion of deleterious nonsynonymous SNPs (nSNPs) from tip to root of the human population tree. This study reveals that up to 48% of nSNPs specific to a single genome are deleterious in nature, which underscores the abundance of deleterious polymorphisms in humans.

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High-resolution Genome-wide Analysis of Irradiated (UV and gamma rays) Diploid Yeast Cells Reveals a High Frequency of Genomic Loss of Heterozygosity (LOH) Events.

Publication Date: 2012 Jan 20 PMID: 22267500
Authors: St Charles, J. - Hazkani-Covo, E. - Yin, Y. - Andersen, S. L. - Dietrich, F. S. - Greenwell, P. W. - Malc, E. - Mieczkowski, P. - Petes, T. D.
Journal: Genetics

In diploid eukaryotes, repair of double-stranded DNA breaks (DSBs) by homologous recombination often leads to loss of heterozygosity (LOH). Most previous studies of mitotic recombination in S. cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected. In this study, we used two techniques (single-nucleotide polymorphism [SNP] microarrays and high-throughput DNA sequencing [HTS]) to examine genome-wide LOH in a diploid yeast strain at a resolution averaging one kb. We examined both selected LOH events on chromosome V and unselected events throughout the genome in untreated cells, and cells treated with either gamma radiation or ultraviolet radiation (UV). Our analysis shows: 1) spontaneous and damageinduced mitotic gene conversion tracts are more than three times larger than meiotic conversion tracts, and conversion tracts associated with crossovers are usually longer and more complex than those unassociated with crossovers, 2) most of the crossovers and conversions reflect the repair of two sister chromatids broken at the same position, and 3) both UV and gamma radiation efficiently induce LOH at doses of radiation that cause no significant loss of viability. Using HTS, we also detected new mutations induced by gamma-rays and UV. To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote.

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Characterisation of ypa1 and ypa2, the Schizosaccharomyces pombe orthologues of the peptidyl proyl isomerases that activate PP2A, reveals a role for Ypa2p in the regulation of cytokinesis.

Publication Date: 2012 Jan 20 PMID: 22267499
Authors: Goyal, A. - Simanis, V.
Journal: Genetics

The Schizosaccharomyces pombe septation initiation network regulates cytokinesis. Cdc7p is the first kinase in the core septation initiation network; we have screened genetically for SIN regulators by isolating cold-sensitive suppressors of cdc7-24. Our screen yielded a mutant in SPAC1782.05, one of the two fission yeast orthologues of mammalian phosphotyrosyl phosphatase activator. We have characterised this gene and its orthologue SPAC4F10.04, which we have named ypa2 and ypa1, respectively. We find that Ypa2p is the major form of Protein Phosphatase Type 2A activator in S. pombe. A double ypa1-Delta ypa2-Delta null mutant is inviable, indicating that the two gene products have at least one essential overlapping function. Individually, the ypa1 and ypa2 genes are essential for survival only at low temperatures. The ypa2-Delta mutant divides at a reduced cell size, and displays aberrant cell morphology and cytokinesis. Genetic analysis implicates Ypa2p as an inhibitor of the septation initiation network. We also isolated a cold-sensitive allele of ppa2, the major Protein Phosphatase Type 2A catalytic subunit, implicating this enzyme as a regulator of the septation initiation network.

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Detecting Rare Variant Associations by Identity by DescentMapping in Case-control Studies.

Publication Date: 2012 Jan 20 PMID: 22267498
Authors: Browning, S. R. - Thompson, E. A.
Journal: Genetics

Identity by descent (IBD) mapping tests whether cases share more segments of IBD around a putative causal variant than do controls. These segments of IBD can be accurately detected from genome-wide SNP data. We investigate the power of IBD mapping relative to that of SNP association testing for genome-wide case-control SNP data. Our focus is particularly on rare variants, as these tend to be more recent and hence more likely to have recent shared ancestry. We simulate data from both large and small populations and find that the relative performance of IBD mapping and SNP association testing depends on population demographic history and the strength of selection against causal variants. We also present an IBD mapping analysis of a type 1 diabetes data set. In those data we find that we can detect association only with the HLA region using IBD mapping. Overall, our results suggest that IBD mapping may have higher power than association analysis of SNP data when multiple rare causal variants are clustered within a gene. However, for outbred populations, very large sample sizes may be required for genome-wide significance unless the causal variants have strong effects.

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Allelic Ratios and the Mutational Landscape Reveal Biologically Significant Heterozygous SNVs.

Publication Date: 2012 Jan 31 PMID: 22267497
Authors: Chu, J. S. - Johnsen, R. C. - Chua, S. Y. - Tu, D. - Dennison, M. - Marra, M. - Jones, S. J. - Baillie, D. L. - Rose, A. M.
Journal: Genetics

The issue of heterozygosity continues to be a challenge in the analysis of genome sequences. In this paper, we describe the use of allele ratios to distinguish biologically significant single nucleotide variants (SNVs) from background noise. An application of this approach is the identification of lethal mutations in C. elegans essential genes, which must be maintained by the presence of a wild-type allele on a balancer. The h448 allele of let-504 is rescued by the duplication balancer, sDp2. We readily identified the extent of the duplication when the percent read support for the lesion was between 70-80%. Examination of the EMS-induced changes throughout the genome revealed that these mutations exist in contiguous blocks. During early embryonic division in self-fertilizing C. elegans, alkylated guanines pair with thymines. As a result, EMS-induced changes become fixed as either G to A or C to T changes along the length of the chromosome. Thus, examination of the distribution of EMS-induced changes revealed the mutational and recombinational history of the chromosome, even generations later. We identified the mutational change responsible for the h448 mutation and sequenced PCR products for an additional four alleles, correlating let-504 with the DNA coding region for an ortholog of NFkappaB-activating protein, NKAP. Our results confirm that whole genome sequencing (WGS) is an efficient and inexpensive way of identifying nucleotide alterations responsible for lethal phenotypes and can be applied on a large scale to identify the molecular basis of essential genes.

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Crossover Distribution and Frequency are Regulated by him-5 in Caenorhabditis elegans.

Publication Date: 2012 Jan 20 PMID: 22267496
Authors: Meneely, P. M. - McGovern, O. L. - Heinis, F. I. - Yanowitz, J. L.
Journal: Genetics

Mutations in the him-5 gene in C. elegans strongly reduce the frequency of crossovers on the X chromosome, with lesser effects on the autosomes. him-5 mutants also show a change in crossover distribution on both the X and autosomes. These phenotypes are accompanied by a delayed entry into pachytene and premature desynapsis of the X chromosome. The nondisjunction, progression defects and desynapsis can be rescued by an exogenous source of double strand breaks (DSBs), indicating that the role of HIM-5 is to promote the formation of meiotic DSBs. Molecular cloning of the gene shows that the inferred HIM-5 product is a highly basic protein of 252 amino acids with no clear orthologs in other species, including other Caenorhabditis species. Although him-5 mutants are defective in segregation of the X chromosome, HIM-5 protein localizes preferentially to the autosomes. The mutant phenotypes and localization of him-5 are similar but not identical to the results seen with xnd-1, although unlike xnd-1, him-5 has no apparent effect on the acetylation of histone H2A on lysine 5 (H2A K5Ac). The localization of HIM-5 to the autosomes depends on the activities of both xnd-1 and him-17 allowing us to begin to establish pathways for the control of crossover distribution and frequency.

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An ORFan No More: The Bacteriophage T4 39.2 Gene Product, NwgI, Modulates GroEL Chaperone Function.

Publication Date: 2012 Jan 10 PMID: 22234860
Authors: Ang, D. - Georgopoulos, C. P.
Journal: Genetics

Bacteriophages are the most abundant biological entities in our biosphere, characterized by their hyperplasticity, mosaic composition, and the many unknown functions (ORFans) encoded by their immense genetic repertoire. These genes are potentially maintained by the bacteriophage to allow efficient propagation on hosts encountered in nature. To test this hypothesis, we devised a selection to identify bacteriophage-encoded gene(s) that modulate the host Escherichia coli GroEL/GroES chaperone machine, which is essential for the folding of certain host and bacteriophage proteins. As a result, we identified the bacteriophage RB69 gene 39.2, of previously unknown function, and showed that homologs of 39.2 in bacteriophages T4, RB43 and RB49 similarly modulate GroEL/GroES. Production of wild type bacteriophage T4 Gp39.2, a 58 amino acid protein, (a) enables diverse bacteriophages to plaque on the otherwise nonpermissive groES or groEL mutant hosts in an allele-specific manner, (b) suppresses the temperature-sensitive phenotype of both groES and groEL mutants, (c) suppresses the defective UV-induced PolV function (UmuCD) of the groEL44 mutant, and (d) is lethal to the host when overproduced. Finally, as proof of principle that Gp39.2 is essential for bacteriophage growth on certain bacterial hosts, we constructed a T4 39.2 deletion strain and showed that, unlike the isogenic wild type parent, it is incapable of propagating on certain groEL mutant hosts. We propose a model of how Gp39.2 modulates GroES/GroEL function.

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Membrane Phospholipid Asymmetry Counters the Adverse Effects of Sterol Overloading in the Golgi Membrane of Drosophila.

Publication Date: 2012 Jan 10 PMID: 22234859
Authors: Ma, Z. - Liu, Z. - Huang, X.
Journal: Genetics

Cholesterol and phospholipids serve as structural and functional components of cellular membranes in all eukaryotes. Heterogeneity in cholesterol and phospholipid content both within and between different organelles is an important characteristic of eukaryotic membranes. How this heterogeneity is achieved and orchestrated to maintain proper cellular physiology remains poorly understood. We previously found that overexpression of the Drosophila oxysterol binding protein, OSBP, leads to sterol accumulation in the Golgi apparatus. Here, we show that Osbp overexpression in a set of neuroendocrine neurons compromises the function of the Golgi apparatus. It impairs trafficking of the neuropeptide bursicon and results in post-eclosion behavior defects characterized by unexpanded wings. We performed a genetic screen to identify modifiers that suppress the unexpanded wing phenotype. A putative phospholipid flippase-encoding gene, CG33298, was validated, suggesting that a membrane-asymmetry-directed mechanism balances cholesterol chaos within the Golgi membranes. Since the functional connection between cholesterol metabolism and the activity of phospholipid flippase has been implicated in studies in yeast and worms, our findings here support an evolutionarily conserved causal link between cholesterol homeostasis and phospholipid asymmetry that maintains normal cellular physiology.

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Gene Genealogies Within a Fixed Pedigree, and the Robustness of Kingman's Coalescent.

Publication Date: 2012 Jan 10 PMID: 22234858
Authors: Wakeley, J. - King, L. - Low, B. S. - Ramachandran, S.
Journal: Genetics

We address a conceptual flaw in the backward-time approach to population genetics called coalescent theory as it is applied to diploid bi-parental organisms. Specifically, the way random models of reproduction are used in coalescent theory is not justified. Instead, the population pedigree for diploid organisms-that is, the set of all family relationships among members of the population-although unknown, should be treated as a fixed parameter, not as a random quantity. Gene genealogical models should describe the outcome of the percolation of genetic lineages through the population pedigree according to Mendelian inheritance. Using simulated pedigrees, some of which are based on family data from 19th century Sweden, we show that in many cases the (conceptually wrong) standard coalescent model is difficult to reject statistically, and in this sense may provide a surprisingly accurate description of gene genealogies on a fixed pedigree. We study the differences between the fixed-pedigree coalescent and the standard coalescent by analysis and simulations. Differences are apparent in recent past generations, roughly < log(2) (N) generations, but then disappear as genetics lineages are traced into the more distant past.

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A Novel Downstream Regulatory Element Cooperates With the Silencing Machinery to Repress EPA1 Expression in Candida glabrata.

Publication Date: 2012 Jan 10 PMID: 22234857
Authors: Gallegos-Garcia, V. - Pan, S. J. - Juarez-Cepeda, J. - Ramirez-Zavaleta, C. Y. - Briones-Martin-Del-Campo, M. - Martinez-Jimenez, V. - Castano, I. - Cormack, B. - De Las Penas, A.
Journal: Genetics

Candida glabrata, an opportunistic fungal pathogen, adheres to mammalian epithelial cells; adherence is mediated primarily by the Epa1 adhesin. EPA1 is a member of a large gene family of about 25 paralogues, which encode putative adhesins. In this study, we address how EPA1 transcription is regulated. Our data show that EPA1 expression is subject to two distinct negative regulatory mechanisms. EPA1 transcription is repressed by sub-telomeric silencing: the Sir Complex (Sir2, Sir3 and Sir4), Rap1, Rif1, yKu70 and yKu80 are required for full repression. Activation of EPA1 occurs immediately after dilution of stationary phase (SP) cells into fresh media; however, transcription is rapidly repressed again, limiting expression to lag phase, just as the cells exit stationary phase. This repression following lag phase requires a cis-acting regulatory negative element (NE) located in the EPA1 3' intergenic region, and is independent of telomere proximity. Bioinformatic analysis shows that there are 10 copies of the NE-like sequence in the Candida glabrata genome associated with other EPA genes as well as non-EPA genes.

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'Point' Centromeres of Saccharomyces Harbor Single CenH3 Nucleosomes.

Publication Date: 2012 Jan 10 PMID: 22234856
Authors: Henikoff, S. - Henikoff, J. G.
Journal: Genetics

The 'point' centromere of budding yeast is genetically defined by an ~125-bp sequence. Recent fluorescence measurements of kinetochore clusters have suggested that this sequence specifies multiple centromere-specific (CenH3) nucleosomes. However, high-resolution mapping demonstrates that there is only one CenH3 nucleosome per centromere, providing biochemical confirmation of the point centromere model.

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A Novel Approach for Directing Transgene Expression in Drosophila: T2A-Gal4 In-Frame Fusion.

Publication Date: 2012 Jan 10 PMID: 22209908
Authors: Diao, F. - White, B. H.
Journal: Genetics

In Drosophila, the Gal4-UAS system permits a transgene to be expressed in the same pattern as a gene of interest, by placing the Gal4 transcription factor under control of the gene's DNA regulatory elements. If these regulatory elements are not known, however, expression of Gal4 in the desired pattern may be difficult or impossible. To solve this problem, we have developed a method for co-expressing Gal4 with the endogenous gene by exploiting the "ribosomal skipping" mechanism of the viral T2A peptide. This method requires explicit knowledge only of the endogenous gene's open reading frame and not its regulatory elements.

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Estimating the Distribution of Selection Coefficientsfrom Phylogenetic Data Using Sitewise Mutation-Selection Models.

Publication Date: 2012 Jan 20 PMID: 22209901
Authors: Tamuri, A. U. - Dos Reis, M. - Goldstein, R. A.
Journal: Genetics

Estimation of the distribution of selection coef fi cients of mutations is a long-standing is- sue in molecular evolution. In addition to population-based methods, the distribution can be estimated from DNA sequence data by phylogenetic-based models. Previous models have generally found unimodal distributions where the probability mass is concentrated between mildly deleterious to nearly neutral mutations. Here we use a site-wise mutation-selection phylogenetic model to estimate the distribution of selection coef fi cients among novel and fi xed mutations (substitutions) in a data set of 244 mammalian mitochondrial genomes and a set of 401 PB2 proteins from influenza. We fi nd a bimodal distribution of selection coef fi cients for novel mutations in both the mitochondrial dataset and for the influenza protein evolving in its natural reservoir, birds. Most of the mutations are strongly deleterious with the rest of the probability mass concentrated around mildly deleterious to neutral mutations. The distribution of the coef fi cients among substitutions is unimodal and symmetrical around nearly neutral substitutions for both data sets at adaptive equilibrium. About 0.5% of the non-synonymous mutations and 14% of the non-synonymous substitutions in the mitochondrial proteins are advantageous, with 0.5% and 24% observed for the influenza protein. Following a host shift of influenza from birds to humans, however, we fi nd among novel mutations in PB2 a trimodal distribution with a small mode of advantageous mutations.

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Notch Signaling is Antagonized by SAO-1, a Novel GYF-domain Protein that Interacts with the E3 Ubiquitin Ligase SEL-10 in Caenorhabditis elegans.

Publication Date: 2012 Jan 10 PMID: 22209900
Authors: Hale, V. A. - Guiney, E. L. - Goldberg, L. Y. - Haduong, J. H. - Kwartler, C. S. - Scangos, K. W. - Goutte, C.
Journal: Genetics

Notch signaling pathways can be regulated through a variety of cellular mechanisms, and genetically compromised systems provide useful platforms from which to search for the responsible modulators. The Caenorhabditis elegans gene aph-1 encodes a component of gamma-secretase, which is essential for Notch signaling events throughout development. By looking for suppressors of the incompletely penetrant aph-1(zu147) mutation, we identify a new gene, sao-1 (suppressor of aph-one), that negatively regulates aph-1(zu147) activity in the early embryo. The sao-1 gene encodes a novel protein that contains a GYF protein-protein interaction domain and interacts specifically with SEL-10, an Fbw7 component of SCF E3 ubiquitin ligases. We demonstrate that the embryonic lethality of aph-1(zu147) mutants can be suppressed by removing sao-1 activity or by mutations that disrupt the SAO-1 - SEL-10 protein interaction. Decreased sao-1 activity also influences Notch signaling events when they are compromised at different molecular steps of the pathway, such as at the level of the Notch receptor GLP-1 or the downstream transcription factor LAG-1. Combined analysis of the SAO-1 - SEL-10 protein interaction and comparisons of sao-1 and sel-10 genetic interactions suggest a possible role for SAO-1 as an accessory protein that participates with SEL-10 in down regulation of Notch signaling. This work provides the first mutant analysis of a GYF-domain protein in either C. elegans or Drosophila, and introduces a new type of Fbw7-interacting protein that acts in a subset of Fbw7 functions.

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