Genetics

Extended Bayesian LASSO for Multiple Quantitative Trait Loci Mapping and Unobserved Phenotype Prediction.

Publication Date: 2010 Aug 30 PMID: 20805559
Authors: Mutshinda, C. M. - Sillanpaa, M. J.
Journal: Genetics

The Bayesian LASSO (BL) has been pointed out to be an effective approach to sparse model representation, and successfully applied to quantitative trait loci (QTL) mapping and genomic breeding value (GBV) estimation using genome-wide dense sets of markers. However, the BL relies on a single parameter known as the regularization parameter to simultaneously control the overall model sparsity and the shrinkage of individual covariate effects. This may be idealistic when dealing with a large number of predictors whose effect sizes may differ by orders of magnitude. Here we propose the extended Bayesian LASSO (EBL) for QTL mapping and GBV estimation, which introduces an additional level to the hierarchical specification of the BL to explicitly separate out these two model features. Compared to the adaptiveness of the BL, the EBL is "doubly adaptive" and thus, more robust to tuning. In simulations, the EBL outperformed the BL in regard to the accuracy of both effect size estimates and phenotypic value predictions, with comparable computational time. Moreover, the EBL proved to be less sensitive to tuning than the related Bayesian adaptive LASSO (BAL) which introduces locus-specific regularization parameters as well, but involves no mechanism for distinguishing between model sparsity and parameter shrinkage. Consequently, the EBL seems to point to a new direction for QTL mapping, phenotype prediction, and GBV estimation.

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Mapping Environment Specific Quantitative Trait Loci.

Publication Date: 2010 Aug 30 PMID: 20805558
Authors: Chen, X. - Zhao, F. - Xu, S.
Journal: Genetics

Environment specific quantitative trait loci (QTL) refer to QTL that express differently in different environments, a phenomenon called QTL by environment (QxE) interaction. QxE interaction is a difficult problem extended from traditional QTL mapping. The mixture model maximum likelihood method is commonly adopted for interval mapping of QTL, but the method is not optimal in handling QTL interacting with environments. We partitioned QTL effects into main and interaction effects. The main effects are represented by the means of QTL effects in all environments and the interaction effects are represented by the variances of the QTL effects across environments. We used the MCMC implemented Bayesian method to estimate both the main and interaction effects. The residual error covariance matrix was modeled using the factor analytic covariance structure. A simulation study showed that the factor analytic structure is robust and can handle other structures as special cases. The method was also applied to QxE interaction mapping for the yield trait of barley. Eight markers showed significant main effects and 18 markers showed significant QxE interaction. The 18 interacting markers were distributed across all seven chromosomes of the entire genome. Only one marker had both the main and the QxE interaction effects. Each of the other markers had either a main effect or a QxE interaction effect but not both.

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Genetic Mechanisms of Coffee Extract Protection in a Caenorhabditis elegans Model of {beta}-amyloid Peptide Toxicity.

Publication Date: 2010 Aug 30 PMID: 20805557
Authors: Dostal, V. - Roberts, C. M. - Link, C. D.
Journal: Genetics

Epidemiological studies have reported that coffee and/or caffeine consumption may reduce Alzheimer's disease risk. We found that coffee extracts can similarly protect against beta-amyloid peptide (Abeta) toxicity in a transgenic Caenorhabditis elegans Alzheimer's disease model. The primary protective component(s) in this model are not caffeine, although caffeine by itself can show some protection. Coffee exposure did not decrease Abeta transgene expression and did not need to be present during Abeta induction to convey protection, suggesting that coffee exposure protection might act by activating a protective pathway. By screening the effects of coffee on a series of transgenic C. elegans stress reporter strains, we identified activation of the skn-1 (Nrf2 in mammals) transcription factor as a potential mechanism of coffee extract protection. Inactivation of skn-1 genetically or by RNAi strongly blocked the protective effects of coffee extract, indicating activation of the skn-1 pathway was the primary mechanism of coffee protection. Coffee also protected against toxicity resulting from an aggregating form of Green Fluorescent Protein (GFP) in a skn-1-dependent manner. These results suggest that the reported protective effects of coffee in multiple neurodegenerative diseases may result from a general activation of the Nrf2 phase II detoxicification pathway.

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Genetics of Extracellular Matrix Remodelling During Organ Growth Using the Caenorhabditis elegans Pharynx Model.

Publication Date: 2010 Aug 30 PMID: 20805556
Authors: Jafari, G. - Burghoorn, J. - Kawano, T. - Mathew, M. - Morck, C. - Axang, C. - Ailion, M. - Thomas, J. H. - Culotti, J. G. - Swoboda, P. - Pilon, M.
Journal: Genetics

The organs of animal embryos are typically covered with an extracellular matrix (ECM) that must be carefully remodelled as these organs enlarge during post-embryonic growth, or else their shape and functions may be compromised. We previously described the twisting of the Caenorhabditis elegans pharynx (here called the Twp phenotype) as a quantitative mutant phenotype that worsens as that organ enlarges during growth. Mutations previously known to cause pharyngeal twist affect membrane proteins with large extracellular domains (DIG-1 and SAX-7), as well as a C. elegans septin (UNC-61). Here we show that two novel alleles of the C. elegans papilin gene, mig-6(et4) and mig-6(sa580), can also cause the Twp phenotype. We also show that overexpression of the ADAMTS protease gene mig-17 can suppress the pharyngeal twist in mig-6 mutants, and identified several alleles of other ECM-related genes that can cause or influence the Twp phenotype, including alleles of fibulin (fbl-1), perlecan (unc-52), collagens (cle-1, dpy-7), laminins (lam-1, lam-3), one ADAM protease (sup-17) and one ADAMTS protease (adt-1). The Twp phenotype in C. elegans is easily monitored using light microscopy, is quantitative via measurements of the torsion angle, and reveals that ECM components, metalloproteinases and ECM attachment molecules are important for this organ to retain its correct shape during post-embryonic growth. The Twp phenotype is therefore a promising experimental system to study ECM remodelling and diseases.

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Different Aneuploidies Arise From the Same Bridge-induced Cchromosomal Translocation Event in Saccharomyces cerevisiae.

Publication Date: 2010 Aug 30 PMID: 20805555
Authors: Rossi, B. - Noel, P. - Bruschi, C. V.
Journal: Genetics

Chromosome translocations are gross chromosomal rearrangements (GCRs) that have often been associated with cancer development in mammalian cells. The feasibility to drastically reshape the genome with a single translocation event also gives this molecular event a powerful capacity to drive evolution. Despite these implications and their role in genome instability, still very little is known about the molecular mechanisms that promote and accompany these events. Here, we describe at the molecular level, ten morphologically and physiologically different translocants ensuing from the induction of the same bridge-induced translocation (BIT) event in the budding yeast Saccharomyces cerevisiae. We have demonstrated that despite their common origin from the integration of the same linear DNA construct, all ten translocation mutant strains have different phenotypes, ability to sporulate, regulation of gene expression and morphology. We also provide insights on how heterogeneous phenotypic variations originate from the same initial genomic event. Here we show eight different ways in which yeast cells have dealt with a single initial event inducing translocation. Our results are in agreement with the formation of complex rearrangements and abnormal karyotypes described in many leukemia patients, thus confirming the modellistic value of the yeast BIT system for mammalian cells.

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Genetic Differentiation, Clinal Variation and Phenotypic Associations With Growth Cessation Across the Populus tremula Photoperiodic Pathway.

Publication Date: 2010 Aug 30 PMID: 20805554
Authors: Ma, X. F. - Hall, D. - St Onge, K. R. - Jansson, S. - Ingvarsson, P. K.
Journal: Genetics

Perennial plants monitor seasonal changes through changes in environmental conditions such as the quantity and quality of light. To ensure a correct initiation of critical developmental processes, such as the initiation and cessation of growth plants have adapted to spatially variable light regime and genes in the photoperiodic pathway have been implicated as likely sources for these adaptations. Here we examine genetic variation in genes from the photoperiodic pathway in Populus tremula (Salicaceae) for signatures diversifying selection in response to varying light regimes across a latitudinal gradient. We fail to identify any loci with unusually high levels of genetic differentiation among populations despite identifying four SNPs which show significant allele frequency clines with latitude. We do however observe large covariance in allelic effects across populations for growth cessation, a highly adaptive trait in P. tremula. High covariances in allelic effects is a signature compatible with diversifying selection along an environmental gradient. We also observe significantly higher heterogeneity in genetic differentiation among SNPs from the photoperiod genes than among SNPs from randomly chosen genes. This suggest that spatially variable selection could be affecting genes from the photoperiod pathway even if selection is not strong enough to cause individual loci to be identified as outliers. SNPs from three genes in the photoperiod pathway (PHYB2, LHY1 and LHY2) show significant associations with natural variation in growth cessation. Collectively these SNPs explain 10-15% of the phenotypic variation in growth cessation. Covariances in allelic effects across populations help explain an additional 5-7% of the phenotypic variation in growth cessation.

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Avoidance of Long Mononucleotide Repeats in Codon Pair Usage.

Publication Date: 2010 Aug 30 PMID: 20805553
Authors: Gu, T. - Tan, S. - Gou, X. - Araki, H. - Tian, D.
Journal: Genetics

Protein is an essential component for life, and its synthesis is mediated by codons in any organisms on earth. While some codons encode the same amino acid, their usage is often highly biased. There are many factors that can cause the bias, but a potential effect of mononucleotide repeats, which are known to be highly mutable, on codon usage and codon pair preference is largely unknown. In this study we performed a genomic survey on the relationship between mononucleotide repeats and codon pair bias in 53 bacteria, 68 archaea and 13 eukaryotes. By distinguishing the codon pair bias from the codon usage bias, four general patterns were revealed: strong avoidance of five or six mononucleotide repeats in codon pairs; lower observed/expected (o/e) ratio for codon pairs with C- or G-repeats (C/G pairs) than that with A- or T-repeats (A/T pairs); a negative correlation between genomic GC contents and the o/e ratios, particularly for C/G pairs; avoidance of C/G pairs in highly conserved genes. These results support natural selection against long mononucleotide repeats, which could induce frameshift mutations in coding sequences. The fact that these patterns are found in all kingdoms of life suggests that this is a general phenomenon in living organisms. Thus, long mononucleotide repeats may play an important role in base composition and genetic stability of a gene and gene functions.

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The Confounding Effects of Population Structure, Genetic Diversity and the Sampling Scheme on the Detection and Quantification of Population Size Changes.

Publication Date: 2010 Aug 25 PMID: 20739713
Authors: Chikhi, L. - Sousa, V. C. - Luisi, P. - Goossens, B. - Beaumont, M. A.
Journal: Genetics

The idea that molecular data should contain information on the recent evolutionary history of populations is rather old. However, much of the work carried out today owes to the work of the statisticians and theoreticians who demonstrated that it was possible to detect departures from equilibrium conditions (e.g. panmictic population/mutation-drift equilibrium) and interpret them in terms of deviations from neutrality or stationarity. During the last 20 years the detection of population size changes has usually been carried out under the assumption that samples were obtained from populations that can be approximated by a Wright-Fisher model (i.e. assuming panmixia, demographic stationarity, etc.). However, natural populations are usually part of spatial networks and are interconnected through gene flow. Here we simulated genetic data at mutation and migration-drift equilibrium under an n-island and a stepping-stone model. The simulated populations were thus stationary and not subject to any population size change. We varied the level of gene flow between populations and the scaled mutation rate. We also used several sampling schemes. We then analysed the simulated samples using the Bayesian method implemented in the MSVAR program to detect and quantify putative population size changes using microsatellite data. Our results show that all three factors (genetic differentiation/gene flow, genetic diversity and the sampling scheme) play a role in generating false bottleneck signals. We also suggest an ad hoc method to counter this effect. The confounding effect of population structure and of the sampling scheme has practical implications for many conservation studies. Indeed, if population structure is creating 'spurious' bottleneck signals, the interpretation of bottleneck signals from genetic data might be less straightforward than it would seem, and several studies may have overestimated or incorrectly detected bottlenecks in endangered species.

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A Genetic Survey of Fluoxetine Action on Synaptic Transmission in Caenorhabditis elegans.

Publication Date: 2010 Aug 25 PMID: 20739712
Authors: Kullyev, A. - Dempsey, C. M. - Miller, S. - Kuan, C. J. - Hapiak, V. M. - Komuniecki, R. W. - Griffin, C. T. - Sze, J. Y.
Journal: Genetics

Fluoxetine is one of the most commonly prescribed medications for many behavioral and neurological disorders. Fluoxetine primarily acts as an inhibitor of the 5-HT reuptake transporter (SERT) to block the removal of 5-HT from the synaptic cleft, thereby enhancing 5-HT signals. Although the effects of fluoxetine on behavior are firmly established, debate is ongoing whether inhibition of 5-HT reuptake is a sufficient explanation for its therapeutic action. In this study, we investigated the effects of fluoxetine on the serotonergic system and its inputs to neuronal functions, using C. elegans as a genetic model. We found that null mutation in the sole SERT gene mod-5 or fluoxetine treatment may eliminate 5-HT in specific neurons. These neurons cannot synthesize 5-HT but use SERT to absorb extrasynaptic 5-HT released from other neurons. Analyses of neuronal markers of the locomotory system revealed that fluoxetine and exogenous 5-HT cause similar changes in synaptic properties of ACh, GABA and glutamate, indicating that fluoxetine can influence neuronal targets regulated by 5-HT. However, we found that these effects of fluoxetine are independent of mod-5/SERT. We identified two G protein-coupled 5-HT receptors, SER-7 and SER-5, antagonistically regulating the effects of fluoxetine on locomotion. We demonstrate that fluoxetine binds SER-7. Epistatic analyses suggest that SER-7 and SER-5 act upstream of AMPA receptor GLR-1 signaling. Our work provides genetic evidence that fluoxetine may directly target 5-HT receptors to influence 5-HT downstream targets.

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Cooperation Between the Septins and the Actomyosin Ring and Role of a Cell-integrity Pathway During Cell Division in Fission Yeast.

Publication Date: 2010 Aug 25 PMID: 20739711
Authors: Wu, J. Q. - Ye, Y. - Wang, N. - Pollard, T. D. - Pringle, J. R.
Journal: Genetics

A major question about cytokinesis is the role of the septin proteins, which localize to the division site in all animal and fungal cells but are essential for cytokinesis only in some cell types. For example, in Schizosaccharomyces pombe, four septins localize to the division site, but deletion of the four genes produces only a modest delay in cell separation. To ask if the S. pombe septins function redundantly in cytokinesis, we conducted a synthetic-lethal screen in a septin-deficient strain and identified seven mutations. One affects Cdc4, a myosin light chain that is an essential component of the cytokinetic actomyosin ring. Five others cause frequent cell lysis during cell separation and map to two loci. These mutations and their dosage suppressors define a signaling pathway (including Rho1 and a novel arrestin) for repairing cell-wall damage. The seventh mutation affects the poorly understood RNA-binding protein Scw1 and severely delays cell separation when combined either with a septin mutation or with a mutation affecting the septin-interacting, anillin-like protein Mid2, suggesting that Scw1 functions in a pathway parallel to that of the septins. Taken together, our results suggest that the S. pombe septins participate redundantly in one or more pathways that cooperate with the actomyosin ring during cytokinesis, and that a septin defect causes septum defects that can be repaired effectively only when the cell-integrity pathway is intact.

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Novel Acid Phosphatase in Candida glabrata Suggests Selective Pressure and Niche Specialization in the Phosphate Signal Transduction Pathway.

Publication Date: 2010 Aug 25 PMID: 20739710
Authors: Orkwis, B. R. - Davies, D. L. - Kerwin, C. L. - Sanglard, D. - Wykoff, D. D.
Journal: Genetics

Evolution through natural selection suggests unnecessary genes are lost. We observed that the yeast Candida glabrata lost the gene encoding a phosphate-repressible acid phosphatase (PHO5) present in many yeasts including S. cerevisiae. However, C. glabrata still had phosphate starvation inducible phosphatase activity. Screening a C. glabrata genomic library, we identified CgPMU2, a member of a three gene family which contains a phosphomutase-like domain. This small scale gene duplication event could allow for sub- or neofunctionalization. Based on phylogenetic and biochemical characterizations, CgPMU2 has neofunctionalized to become a broad range, phosphate starvation-regulated acid phosphatase, which functionally replaces PHO5 in this pathogenic yeast. We determined that CgPmu2, unlike ScPho5, is not able to hydrolyze phytic acid (inositol hexakisphosphate). Phytic acid is present in fruits and seeds where S. cerevisiae grows, but is not abundant in mammalian tissues where C. glabrata grows. We demonstrated that C. glabrata is limited from an environment where phytic acid is the only source of phosphate. Our work suggests that during evolutionary time, the selection for the ancestral PHO5 was lost, and that C. glabrata neofunctionalized a weak phosphatase to replace PHO5. Convergent evolution of a phosphate-starvation inducible acid phosphatase in C. glabrata relative to most yeast species provides an example of how small changes in signal transduction pathways can mediate genetic isolation and uncovers a potential speciation gene.

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Functional Dissection of IME1 Transcription using Quantitative Promoter-reporter Screening.

Publication Date: 2010 Aug 25 PMID: 20739709
Authors: Kahana, S. - Pnueli, L. - Kainth, P. - Sassi, H. - Andrews, B. J. - Kassir, Y.
Journal: Genetics

Transcriptional regulation is a key mechanism that controls the fate and response of cells to diverse signals. Therefore, the identification of the DNA-binding proteins which mediate these signals is a crucial step in elucidating how cell fate is regulated. In this report, we applied both bioinformatics and functional genomic approaches to scrutinize the unusually large promoter of the IME1 gene in budding yeast. Using a recently described fluorescent protein based reporter screen (R-SGA), we assessed the effect of viable deletion mutants on transcription of various IME1 promoter-reporter genes. We discovered potential transcription factors, many of which have no perfect consensus site within the IME1 promoter. Moreover, most of the cis-regulatory sequences with perfect homology to known TF consensus were found to be non-functional in the R-SGA analysis. In addition, our results suggest that lack of conservation may not discriminate against a TF regulatory role at a specific promoter. We demonstrate that Sum1 and Sok2 which regulate IME1 , bind to non-perfect consensuses within non-conserved regions in the sensu stricto Saccharomyces strains. Our analysis supports the view that although comparative analysis can provide a useful guide, functional assays are required for accurate identification of TF-binding site interactions in complex promoters.

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Yin and Yang of Histone H2B Roles in Silencing and Longevity: A Tale of Two Arginines.

Publication Date: 2010 Aug 16 PMID: 20713692
Authors: Dai, J. - Hyland, E. M. - Norris, A. - Boeke, J. D.
Journal: Genetics

In budding yeast, silent chromatin is defined at the region of telomeres, rDNA loci and silent mating loci. Although the silent chromatin at different loci shows structural similarity, the underlying mechanism to establish, maintain and inherit these structures may be fundamentally different. In this study, we found two arginine residues within histone H2B, which are specifically required to maintain either the telomeric or the rDNA silenct chromatin. Arginine 95 (R95) plays a specific role at telomeres whereas Arginine 102 (R102) is required to maintain the silent chromatin at rDNA and to ensure the integrity of rDNA loci by suppressing recombination between rDNA repeats. R95 mutants show enhanced rDNA silencing but a paradoxically low Sir2 protein abundance. Furthermore weakened silencing at telomeres in R95 mutants can be suppressed by a specific SIR3 allele, SIR3-D205N, which increases the affinity of Sir proteins to telomeres, suggesting H2B-R95 may directly mediate telomeric Sir protein-nucleosome interactions. Double mutations of R95 and R102 lead to desilencing of both rDNA and telomeres, indicating both arginines are necessary to ensure integrity of silent chromatin at these loci. Furthermore, mutations of R102 cause accumulation of extrachromosomal rDNA circles and reduce lifespan, suggesting that histone H2B contributes to longevity.

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Placental Inactivity of an Imprinted Gene Underlies Midgestation Death of Mice With Paternal Duplication of Distal Chromosome 7.

Publication Date: 2010 Aug 16 PMID: 20713691
Authors: Rentsendorj, A. - Mohan, S. - Szabo, P. E. - Mann, J. R.
Journal: Genetics

Mammalian androgenones have two paternally or sperm derived genomes. In mouse (Mus musculus) they die at peri-implantation due to the misexpression of imprinted genes-these being genes expressed monoallelically according to parent-of-origin. The misexpressions involved are poorly defined. To gain further insight, we examined the causes of midgestation death of embryos with paternal duplication (PatDp) of distal chromosome 7 (dist7), a region replete with imprinted genes. PatDp(dist7) embryos have a similar phenotype to mice with a knockout of a maternally expressed imprinted gene, Ascl2 'achaete-scute complex homolog-like 2 (Drosophila)', and their death at midgestation could result from two inactive paternal copies of this gene. However, other dist7 misexpressions could duplicate this phenotype, and the potential epistatic load is undefined. We show that an Ascl2 transgene is able to promote the development of PatDp(dist7) embryos to term, providing strong evidence that Ascl2 is the only imprinted gene in the genome for which PatDp results in early embryonic death. While some of the defects in perinatal transgenic PatDp(dist7) fetuses were consistent with known misexpressions of dist7 imprinted genes, the overall phenotype indicates a role for additional undefined misexpressions of imprinted genes. This study provides implications for the human imprinting-related fetal overgrowth disorder, Beckwith-Wiedemann syndrome.

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Changes in the Nuclear Envelope Environment Affect Spindle Pole Body Duplication in Saccharomyces cerevisiae.

Publication Date: 2010 Aug 16 PMID: 20713690
Authors: Witkin, K. L. - Friederichs, J. M. - Cohen-Fix, O. - Jaspersen, S. L.
Journal: Genetics

The Saccharomyces cerevisiae nuclear membrane is part of a complex nuclear envelope environment also containing chromatin, integral and peripheral membrane proteins, and large structures such as nuclear pore complexes and the spindle pole body. In order to study how properties of the nuclear membrane affect nuclear envelope processes, we altered the nuclear membrane by deleting the SPO7 gene. We found that spo7 cells were sickened by mutation of genes coding for spindle pole body components, and that spo7 was synthetically lethal with mutations in the SUN domain gene MPS3. Mps3p is required for spindle pole body duplication and a variety of other nuclear envelope processes. In spo7 cells, the spindle pole body defect of mps3 mutants was exacerbated, suggesting that nuclear membrane composition affects spindle pole body function. The synthetic lethality between spo7 and mps3 mutants was suppressed by deletion of specific nucleoporin genes. In fact, these gene deletions bypassed the requirement for Mps3p entirely, suggesting that under certain conditions spindle pole body duplication can occur via an Mps3p-independent pathway. These data point to an antagonistic relationship between nuclear pore complexes and the spindle pole body. We propose a model whereby nuclear pore complexes either compete with the spindle pole body for insertion into the nuclear membrane, or affect spindle pole body duplication by altering the nuclear envelope environment.

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Multilevel Selection 4: Modeling the Relationship of Indirect Genetic Effects and Group Size.

Publication Date: 2010 Aug 16 PMID: 20713689
Authors: Bijma, P.
Journal: Genetics

Indirect Genetic Effects (IGE) occur when individual trait values depend on genes in others. With IGEs, heritable variance and response to selection depend on the relationship of IGEs and group size. Here I propose a simple and tractable model for this relationship, which can be implemented in standard ReML software.

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Estimating Indirect Genetic Effects: Precision of Estimates and Optimum Designs.

Publication Date: 2010 Aug 16 PMID: 20713688
Authors: Bijma, P.
Journal: Genetics

Social interactions among individuals are abundant both in natural and domestic populations. There is increasing evidence that such social interactions cause phenotypes of individuals to depend on genes carried by other individuals, a phenomenon known as Indirect Genetic Effects (IGE). Because IGEs may have drastic effects on the rate and direction of response to selection, knowledge of their magnitude and relationship to direct genetic effects (DGE) is indispensable for understanding response to selection. Very little is known, however, of the statistical power and optimum experimental designs for identifying IGEs. This work, therefore, presents expressions for the standard errors of the estimated (co)variances of DGEs and IGEs, and identifies optimum experimental designs for their estimation. It also provides a deterministic expression for optimum family size, and a numerical investigation of optimum group size. Designs with groups composed of two families were near optimal, and substantially better than designs with groups composed at random with respect to family. Results suggest that IGEs can be detected with ~1,000 to 2,000 individuals and/or ~250 to 500 groups when using optimum designs. Those values are within the feasible range for many species in agriculture and aquaculture, and for the smaller laboratory species. In summary, this work provides the tools to optimize and quantify the required size of experiments aiming to identify IGEs. An R-package SE.IGE is available, which predicts SE's and identifies optimum family and group sizes for each parameter.

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Refinement of Tools for Targeted Gene Expression in Drosophila.

Publication Date: 2010 Aug 9 PMID: 20697123
Authors: Pfeiffer, B. D. - Ngo, T. T. - Hibbard, K. L. - Murphy, C. - Jenett, A. - Truman, J. W. - Rubin, G. M.
Journal: Genetics

A wide variety of biological experiments rely on the ability to express an exogenous gene in a transgenic animal at a defined level and in a spatially and temporally controlled pattern. We describe major improvements of the methods available for achieving this objective in Drosophila melanogaster. We have systematically varied core promoters, UTRs, operator sequences, and transcriptional activating domains used to direct gene expression with the GAL4, LexA, and Split GAL4 transcription factors and the GAL80 transcriptional repressor. The use of site-specific integration allowed us to make quantitative comparisons between different constructs inserted at the same genomic location. We also characterized a set of PhiC31 integration sites for their ability to support transgene expression of both drivers and responders in the nervous system. The increased strength and reliability of these optimized reagents overcome many of the previous limitations of these methods and will facilitate genetic manipulations of greater complexity and sophistication.

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C-type CytochromeAssembly in Saccharomyces Cerevisiae: A Key Residue for Apocytochrome C1/Lyase Interaction.

Publication Date: 2010 Aug 10 PMID: 20697122
Authors: Corvest, V. - Murrey, D. A. - Bernard, D. G. - Knaff, D. B. - Guiard, B. - Hamel, P. P.
Journal: Genetics

The electron transport chains in the membranes of bacteria and organelles generate protonmotive force essential for ATP production. The c-type cytochromes, defined by the covalent attachment of heme to a CXXCH motif, are key electron carriers in these energy-transducing membranes. In mitochondria, cytochromes c and c1 are assembled by the cytochrome c heme lyases (CCHL and CC1HL) and Cyc2p, a putative redox protein. A cytochrome c1 mutant with a CAPCH heme- binding site instead of the wild-type CAACH is strictly dependent upon Cyc2p for assembly. In this context, we found that overexpression of CC1HL, as well as mutations of the proline in the CAPCH site to H, L, S or T residues, can by-pass the absence of Cyc2p. The P mutation was postulated to shift the CXXCH motif to an oxidized form, which must be reduced in a Cyc2p-dependent reaction before heme ligation. However, measurement of the redox midpoint potential of apocytochromes c1 indicates that neither the P nor the T residues impact the thermodynamic propensity of the CXXCH motif to occur in a disulfide versus dithiol form. We show instead that the identity of the second intervening residue in the CXXCH motif is key in determining the CCHL-dependent versus CC1HL-dependent assembly of holocytochrome c1. We also provide evidence that Cyc2p is dedicated to the CCHL pathway and is not required for the CC1HL- dependent assembly of cytochrome c1.

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Meiotic Chromosome Segregation in Triploid Strains of Saccharomyces cerevisiae.

Publication Date: 2010 Aug 10 PMID: 20697121
Authors: St Charles, J. - Hamilton, M. L. - Petes, T. D.
Journal: Genetics

Meiosis in triploids results in four highly aneuploid gametes because six copies of each homologue must be segregated into four meiotic products. Using DNA microarrays and other physical approaches, we examined meiotic chromosome segregation in triploid strains of Saccharomyces cerevisiae. In most tetrads with four viable spores, two of the spores had two copies of a given homologue and two spores had only one copy. Chromosomes segregated randomly into viable spores without preferences for generating near haploid or near diploid spores. Using single-nucleotide polymorphisms, we showed that, in most tetrads, all three pairs of homologues recombined. Strains derived from some of the aneuploid spore colonies had very high frequencies of mitotic chromosome loss, resulting in genetically diverse populations of cells.

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A Two-pathway Analysis of Meiotic Crossing Over and Gene Conversion in Saccharomyces cerevisiae.

Publication Date: 2010 Aug 16 PMID: 20679514
Authors: Stahl, F. W. - Foss, H. M.
Journal: Genetics

Several apparently paradoxical observations regarding meiotic crossing over and gene conversion are readily resolved in a framework that recognizes the existence of two recombination pathways that differ in mismatch repair, structures of intermediates, crossover interference, and in the generation of noncrossovers. One manifestation of these differences is that simultaneous gene conversion on both sides of a recombination-initiating DNA double-strand break ("two-sidedness") characterizes only one of the two pathways and is promoted by mismatch repair. Data from previous work are analyzed quantitatively within this framework, and a molecular model for meiotic double-strand-break repair based on the concept of sliding D-loops is offered as an efficient scheme for visualizing the salient results from studies of crossing over and gene conversion, the molecular structures of recombination intermediates, and the biochemical competencies of the proteins involved.

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RecG Protein and Single-strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity in Escherichia coli.

Publication Date: 2010 Aug 16 PMID: 20647503
Authors: Rudolph, C. J. - Mahdi, A. A. - Upton, A. L. - Lloyd, R. G.
Journal: Genetics

Replication of the Escherichia coli chromosome usually initiates at a single origin (oriC) under control of DnaA. Two forks are established and move away in opposite directions. Replication is completed when these meet in a broadly defined terminus area half way around the circular chromosome. RecG appears to consolidate this arrangement by unwinding D-loops and R-loops that PriA might otherwise exploit to initiate replication at other sites. It has been suggested that without RecG such replication generates 3' flaps as the additional forks collide and displace nascent leading strands, providing yet more potential targets for PriA. Here we show that to stay alive, cells must have either RecG or a 3' ssDNA exonuclease, which can be Exonuclease I, Exonuclease VII or SbcCD. Cells lacking all three nucleases are inviable without RecG. They also need RecA recombinase and a Holliday junction resolvase to survive rapid growth, but SOS induction, although elevated, is not required. Additional requirements for Rep and UvrD are identified and linked with defects in DNA mismatch repair and in the ability to cope with conflicts between replication and transcription, respectively. Eliminating PriA helicase activity removes the requirement for RecG. The data are consistent with RecG and ssDNA exonucleases acting to limit PriA-mediated re-replication of the chromosome and the consequent generation of linear DNA branches that provoke recombination and delay chromosome segregation.

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The Rapidly Evolving Centromere-specific Histone Has Stringent Functional Requirements in Arabidopsis thaliana.

Publication Date: 2010 Aug 16 PMID: 20628040
Authors: Ravi, M. - Kwong, P. N. - Menorca, R. M. - Valencia, J. T. - Ramahi, J. S. - Stewart, J. L. - Tran, R. K. - Sundaresan, V. - Comai, L. - Chan, S. W.
Journal: Genetics

Centromeres control chromosome inheritance in eukaryotes, yet their DNA structure and primary sequence are hypervariable. Most animals and plants have megabases of tandem repeats at their centromeres, unlike yeast with unique centromere sequences. Centromere function requires the centromere-specific histone CENH3 (CENP-A in human), which replaces histone H3 in centromeric nucleosomes. CENH3 evolves rapidly, particularly in its N-terminal tail domain. A portion of the CENH3 histone fold domain, the CENP-A targeting domain (CATD), has been previously shown to confer kinetochore localization and centromere function when swapped into human H3. Furthermore, CENP-A in human cells can be functionally replaced by CENH3 from distantly related organisms including S. cerevisiae. We have used cenh3-1 (a null mutant in Arabidopsis thaliana) to replace endogenous CENH3 with GFP-tagged variants. A H3.3 tail domain-CENH3 histone fold domain chimera rescued viability of cenh3-1, but CENH3s lacking a tail domain were non-functional. In contrast to human results, H3 containing the A. thaliana CATD cannot complement cenh3-1. GFP-CENH3 from the sister species Arabidopsis arenosa functionally replaces A. thaliana CENH3. GFP-CENH3 from the close relative Brassica rapa was targeted to centromeres, but did not complement cenh3-1, indicating that kinetochore localization and centromere function can be uncoupled. We conclude that CENH3 function in Arabidopsis thaliana, an organism with large tandem repeat centromeres, has stringent requirements for functional complementation in mitosis.

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A Pathogenic Relationship Between a Regulator of the Actin Cytoskeleton and Serum Response Factor.

Publication Date: 2010 Aug 9 PMID: 20610412
Authors: Verdoni, A. M. - Schuster, K. J. - Cole, B. S. - Ikeda, A. - Kao, W. W. - Ikeda, S.
Journal: Genetics

Cell hyperproliferation, inflammation, and angiogenesis are biological processes central to the pathogenesis of corneal disease, as well as other conditions including tumorigenesis and chronic inflammatory disorders. Due to the number of disease conditions that arise as a result of these abnormalities, identifying the molecular mechanisms underlying these processes is critical. The avascular and transparent cornea serves as a good in vivo model to study the pathogenesis of cell hyperproliferation, inflammation, and angiogenesis. Corneal disease 1 (Dstn(corn1)) mice are homozygous for a spontaneous null allele of the destrin (Dstn) gene, which is also known as actin depolymerizing factor (ADF). These mice exhibit abnormalities in the cornea including epithelial cell hyperproliferation, and stromal inflammation and neovascularization. We previously identified that the transcription factor, serum response factor (SRF), and a number of its target genes are upregulated in the cornea of these mice. In this study, we show that conditional ablation of Srf in the corneal epithelium of a diseased Dstn(corn1) cornea results in the rescue of the epithelial cell hyperproliferation, inflammation, and neovascularization phenotypes, delineating an epithelial cell specific role for SRF in the development of all of these abnormalities. Our study also demonstrates that Dstn is genetically upstream of Srf, and defines a new functional role for SRF as the master regulator of a hyperproliferative, inflammatory phenotype accompanied by neovascularization.

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Control of Arabidopsis Leaf Morphogenesis Through Regulation of the YABBY and KNOX Families of Transcription Factors.

Publication Date: 2010 Aug 9 PMID: 20610407
Authors: Ha, C. M. - Jun, J. H. - Fletcher, J. C.
Journal: Genetics

The patterning of initiating organs along specific axes of polarity is critical for the proper development of all higher organisms. Plant lateral organs, such as leaves, are derived from the shoot apical meristems located at the growing tips. After initiation, the leaf primordia of species such as Arabidopsis thaliana differentiate into a polarized structure consisting of a proximal petiole and a distal blade, but the molecular mechanisms that control proximal-distal pattern formation are poorly understood. The transcriptional activators BLADE-ON-PETIOLE1 (BOP1) and BOP2 are known to control Arabidopsis lateral organ differentiation by regulating gene expression along the adaxial-abaxial (dorsal-ventral) and proximal-distal polarity axes. Here, we demonstrate that the development of ectopic blade tissue along bop1 bop2 leaf petioles is strongly suppressed in a dosage-dependant manner by mutations in either of two closely related YABBY (YAB) genes, FILAMENTOUS FLOWER (FIL) and YAB3. Three KNOTTED-LIKE HOMEOBOX (KNOX1) genes also make lesser, and partially redundant, contributions to ectopic blade development in bop1 bop2 leaves. Mutation of these YAB and KNOX1 genes together cause nearly complete suppression of bop1 bop2 ectopic organ outgrowth at the morphological and cellular levels. Our data demonstrate that BOP1 and BOP2 regulate leaf patterning by controlling YAB and KNOX1 gene activity in the developing petiole.

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Reproductive Isolation in Hybrid Mice Due to Spermatogenesis Defects at Three Meiotic Stages.

Publication Date: 2010 Aug 9 PMID: 20610405
Authors: Oka, A. - Mita, A. - Takada, Y. - Koseki, H. - Shiroishi, T.
Journal: Genetics

Early in the process of speciation, reproductive failures occur in hybrid animals between genetically diverged populations. The sterile hybrid animals are often males in mammals and they exhibit spermatogenic disruptions, resulting in decreased number and/or malformation of mature sperms. Despite the generality of this phenomenon, comparative study of phenotypes in hybrid males from various crosses have not been done, and thereby comprehensive genetic basis of the disruption is still elusive. In this study, we characterized the spermatogenic phenotype especially during meiosis in four different cases of reproductive isolation: B6-ChrX(MSM), PGN-ChrX(MSM), (B6 x Mus musculus musculus-NJL/Ms) F1 and (B6 x Mus spretus) F1. The first two are consomic strains, both bearing the X chromosome of M. m. molossinus; in B6-ChrX(MSM), the genetic background is the laboratory strain C57BL/6J (predominantly M. m. domesticus), while in PGN-ChrX(MSM) the background is the PGN2/Ms strain purely derived from wild M. m. domesticus. The last two cases are F1 hybrids between mouse subspecies or species. Each of the hybrid males exhibited cell-cycle arrest and/or apoptosis at either one or two of three distinct meiotic stages: premeiotic stage, zygotene-to-pachytene stage of prophase I and metaphase I. This study shows that the sterility in hybrid males is caused by spermatogenic disruptions at multiple stages, suggesting that the responsible genes function in different cellular processes. Furthermore, the stages with disruptions are not correlated with the genetic distance between the respective parental strains.

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Behavioral Responses to Hypoxia in Drosophila Larvae are Mediated by Atypical Soluble Guanylyl Cyclases.

Publication Date: 2010 Aug 9 PMID: 20592263
Authors: Vermehren-Schmaedick, A. - Ainsley, J. A. - Johnson, W. A. - Davies, S. A. - Morton, D. B.
Journal: Genetics

The three Drosophila atypical soluble guanylyl cyclases, Gyc-89Da, Gyc-89Db and Gyc-88E, have been proposed to act as oxygen detectors mediating behavioral responses to hypoxia. Drosophila larvae mutant in any of these subunits were defective in their hypoxia escape response - a rapid cessation of feeding and withdrawal from their food. This response required cGMP and the cyclic nucleotide-gated ion channel, cng, but did not appear to be dependent on either of the cGMP dependent protein kinases, dg1 and dg2. Specific activation of the Gyc-89Da neurons using channel rhodopsin showed that activation of these neurons was sufficient to trigger the escape behavior. The hypoxia escape response was restored by reintroducing either Gyc-89Da or Gyc-89Db into either Gyc-89Da or Gyc-89Db neurons in either mutation. This suggests that neurons that co-express both Gyc-89Da and Gyc-89Db subunits are primarily responsible for activating this behavior. These include sensory neurons that innervate the terminal sensory cones. Although the roles of Gyc-89Da and Gyc-89Db in the hypoxia escape behavior appeared to be identical, we also showed that changes in larval crawling behavior in response to either hypoxia or hyperoxia differed in their requirements for these two atypical sGCs, with responses to 15% oxygen requiring Gyc-89Da and responses to 19% and 25% requiring Gyc-89Db. For this behavior, the identity of the neurons appeared to be critical in determining the ability to respond appropriately.

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Interaction Between Eye Pigment Genes and Tau-induced Neurodegeneration in Drosophila melanogaster.

Publication Date: 2010 Aug 9 PMID: 20592261
Authors: Ambegaokar, S. S. - Jackson, G. R.
Journal: Genetics

Null mutations in the genes white and brown, but not scarlet, enhance a rough eye phenotype in a Drosophila melanogaster model of tauopathy; however, adding rosy mutations suppresses these effects. Interaction with nucleotide-derived pigments or increased lysosomal dysregulation are potential mechanisms. Finally, tau toxicity correlates with increased GSK-3beta activity, but not with tau phosphorylation at Ser202/Thr205.

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The Effective Size of Populations Infected With Cytoplasmic Sex-ratio Distorters.

Publication Date: 2010 Aug 16 PMID: 20592260
Authors: Engelstadter, J.
Journal: Genetics

Many arthropod species are infected with maternally inherited endosymbionts that induce a shift in the sex-ratio of their hosts by feminizing or killing males ('cytoplasmic sex-ratio distorters', or SRDs). These endosymbionts can have profound impacts on evolutionary processes of their hosts. Here, I derive analytical expressions for the coalescent effective size Ne of populations that are infected with SRDs. Irrespective of the type of SRD, Ne for mitochondrial genes is given by the number of infected females. For nuclear genes, the effective population size generally decreases with increasing prevalence of the SRD and can be considerably lower than the actual size of the population. For example, with male-killing bacteria that have near perfect maternal transmission, Ne is reduced by a factor that is given to a good approximation by the proportion of uninfected individuals in the population. The formulae derived here also yield the effective size of populations infected with mutualistic endosymbionts or maternally inherited bacteria that induce cytoplasmic incompatibility, although in these cases, the reduction in Ne is expected to be less severe than for cytoplasmic SRDs.

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Using Student-Generated UV-Induced Escherichia coli Mutants in a Directed Inquiry Undergraduate Genetics Laboratory.

Publication Date: 2010 Aug 16 PMID: 20592259
Authors: Healy, F. G. - Livingstone, K. D.
Journal: Genetics

We report a thematic sequence of directed inquiry-based labs taking students from bacterial mutagenesis and phenotypic identification of their own self-created mutant, through identification of mutated genes by biochemical testing, to verification of mutant alleles by complementation, and lastly to mutant allele characterization by DNA sequence analysis. The lab utilizes UV mutagenesis with wild type Escherichia coli and a UV-sensitive isogenic derivative optimized for undergraduate use. The labs take advantage of the simplicity of E. coli in a realistic genetic investigation using safe UV irradiation methods for creation and characterization of novel mutants. Assessment data collected over three offerings of the course suggest that the labs, which combine original investigation in a scientifically realistic intellectual environment with learned techniques and concepts, were instrumental in improving students' learning in a number of areas. These include the development of critical thinking skills and understanding of concepts and methods. Student responses also suggest the labs were helpful in improving students' understanding of the scientific process as a rational series of experimental investigations, each building upon interpretation of previously acquired results. Survey data also suggest the labs are helpful in improving student awareness of the interdisciplinary nature of scientific inquiry.

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The Genetic Architecture of Grain Yield and Related Traits in Zea maize L. Revealed by Comparing Intermated and Conventional Populations.

Publication Date: 2010 Aug 9 PMID: 20592258
Authors: Huang, Y. F. - Madur, D. - Combes, V. - Ky, C. L. - Coubriche, D. - Jamin, P. - Jouanne, S. - Dumas, F. - Bouty, E. - Bertin, P. - Charcosset, A. - Moreau, L.
Journal: Genetics

Using advanced intermated populations has been proposed as a way to increase the accuracy of mapping experiments. An F3 population of 300 lines and an advanced intermated F3 population of 322 lines, both derived from the same parental maize inbred lines, were jointly evaluated for dry grain yield, grain moisture and silking date. Genetic variance for dry grain yield was significantly lower in the intermated population compared to the F3 population. The confidence interval around a QTL was on average 2.31 times smaller in the intermated population compared to the F3 population. One controversy surrounding QTL mapping is whether QTL identified in fact represent single loci. This study identifies two distinct loci for dry grain yield in the intermated population, in coupling phase, while the F3 identifies only a single locus. Surprisingly, fewer QTLs were detected in the intermated population than the F3 (21 versus 30) and less than 50% of the detected QTLs were shared among the two populations. Cross-validation showed that selection bias was more important in the intermated population than in the F3 and that each detected QTL explained a lower percentage of the variance. This finding supports the hypothesis that QTLs detected in conventional populations correspond mainly to clusters of linked QTLs. The actual number of QTLs involved in the genetic architecture of complex traits may be substantially larger, with effect sizes substantially smaller than in conventional populations.

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Natural Diversity in Flowering Responses of Arabidopsis thaliana Caused by Variation in a Tandem Gene Array.

Publication Date: 2010 Aug 9 PMID: 20551443
Authors: Rosloski, S. M. - Jali, S. S. - Balasubramanian, S. - Weigel, D. - Grbic, V.
Journal: Genetics

Tandemly arrayed genes that belong to gene families characterize genomes of many organisms. Gene duplication and subsequent relaxation of selection can lead to the establishment of paralogous cluster members that may evolve along different trajectories. Here, we report on the structural variation in MADS AFFECTING FLOWERING 2 (MAF2) gene, one member of the tandemly duplicated cluster of MADS-box containing transcription factors in Arabidopsis thaliana. The altered gene structure at the MAF2 locus is present as a moderate-frequency polymorphism in Arabidopsis and leads to the extensive diversity in transcript patterns due to alternative splicing. Rearrangements at the MAF2 locus are associated with an early flowering phenotype in BC5 lines. The lack of suppression of flowering time in a MAF2-insertion line expressing the MAF2-specific artificial miRNA suggests that these MAF2 variants are behaving as loss-of-function alleles. The variation in gene architecture is also associated with segregation distortion, which may have facilitated the spread and the establishment of the corresponding alleles throughout the Eurasian range of the Arabidopsis thaliana population.

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The Transcriptional Landscape of Cross-specific Hybrids and its Possible Link with Growth in Brook Charr (Salvelinus fontinalis Mitchill).

Publication Date: 2010 Aug 9 PMID: 20551437
Authors: Bougas, B. - Granier, S. - Audet, C. - Bernatchez, L.
Journal: Genetics

The genetic mechanisms underlying hybridization are poorly understood despite their potentially important roles in speciation processes, adaptative evolution, and agronomical innovation. In this study, transcription profiles were compared among three populations of brook charr and their hybrids using microarrays to assess the influence of hybrid origin on modes of transcription regulation inheritance and on the mechanisms underlying growth. We found that twice as many transcripts were differently expressed between the domestic population and the two wild populations (Rupert and Laval) than between wild ones, despite their deeper genetic distance. This could reflect the consequence of artificial selection during domestication. We detected that hybrids exhibited strikingly different patterns of mode of transcription regulation, being mostly additive (94%) for domestic x Rupert, and non-additive for Laval x domestic (45.7%) and Rupert x Laval hybrids (37.5%). Both heterosis and outbreeding depression for growth were observed among the crosses. Our results indicated that prevalence of dominance in transcription regulation seems related to growth heterosis, while prevalence of transgressive transcription regulation may be more related to outbreeding depression. Our study clearly shows, for the first time in vertebrates, that the consequences of hybridization on both the transcriptome level and the phenotype are highly dependent on the specific genetic architectures of crossed populations and therefore hardly predictable.

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