Genetics

The waved with open eyelids (woe) Locus is a Hypomorphic Mouse Mutation in Adam17.

Publication Date: 2010 Mar 1 PMID: 20194968
Authors: Hassemer, E. L. - Le Gall, S. M. - Liegel, R. - McNally, M. - Chang, B. - Zeiss, C. J. - Dubielzig, R. D. - Horiuchi, K. - Kimura, T. - Okada, Y. - Blobel, C. P. - Sidjanin, D. J.
Journal: Genetics

The waved with open eyes (woe) locus is a spontaneous recessive mouse mutation that exhibits wavy fur, eyelids open at birth, and enlarged heart and esophagus. In this study, we confirmed the previously identified woe phenotypes and additionally identified anterior eye segment defects, absence of the meibomian glands, and defects in the semilunar cardiac valves. Positional cloning identified a C794T substitution in the Adam17 gene that ablates a putative exonic splicing enhancer (ESE) sequence in exon 7 resulting in aberrant Adam17 splicing. The predominant woe transcript Adam17(Deltaexon7) lacks exon 7 resulting in an in-frame 90 bp deletion and a putative Adam17(Delta252-281) lacking residues 252-281 from the metalloprotease domain. Western blot analysis identified in woe only the precursor form of Adam17(Delta252-281). In the absence of cleavage of the prodomain Adam17(Delta252-281) is most likely functionally inactive, however, constitutive and stimulated shedding of Adam17-substrates was detected in woe, albeit at significantly reduced levels. This residual Adam17 shedding activity in woe most likely originates from full-length Adam17(T265M) encoded by the Adam17(C794T) transcript identified expressed at severely reduced levels. These results show that even small amounts of functional Adam17 allow woe mice to survive into adulthood. In contrast to Adam17(-/-) mice that die at birth, the viability of woe mice provides an excellent opportunity for studying the role of Adam17 throughout postnatal development and homeostasis.

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SIAMESE Cooperates with the CDH1-like Protein CCS52A1 to Establish Endoreplication in Arabidopsis thaliana Trichomes.

Publication Date: 2010 Mar 1 PMID: 20194967
Authors: Kasili, R. - Walker, J. D. - Simmons, L. A. - Zhou, J. - De Veylder, L. - Larkin, J. C.
Journal: Genetics

Endoreplication, also known as endoreduplication, is a phyogenetically wide-spread modified version of the cell cycle in which DNA replication is not followed by cell division. The SIAMESE (SIM) gene of Arabidopsis thaliana encodes the founding member of a novel class of plant-specific cyclin-dependent kinase (CDK) inhibitors and is a key regulator of endoreplication during the development of trichomes (shoot epidermal hairs). Here, we have identified mutations in the CCS52A1 gene as genetic modifiers of the multicellular trichome phenotype of sim mutants. Loss-of-function ccs52A1 mutations dramatically enhance the multicellularity of sim mutants trichomes in double mutants, whereas over-expression of CCS52A1 completely suppresses the sim mutant phenotype. CCS52A1 encodes a CDH1/FZR-like protein, a class of proteins that function as activators of the anaphase-promoting complex. Unicellular ccs52A1 trichomes become multicellular upon overexpression of B-type cyclin, consistent with repression of the accumulation of mitotic cyclins in the developing trichome by CCS52A1. As these M-phase-specific cyclins are known to accumulate in sim mutant trichomes, our data suggest that CCS52A1 and SIM cooperate in repressing accumulation of mitotic cyclins to establish the trichome endocycle. Comparison with endoreplication pathways in Drosophila and mammals indicates that while these organisms all use similar components to initiate endoreplication, the components are deployed differently in each organism.

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The Influence of Horizontal Gene Transfer on the Mean Fitness of Unicellular Populations in Static Environments.

Publication Date: 2010 Mar 1 PMID: 20194966
Authors: Raz, Y. - Tannenbaum, E. D.
Journal: Genetics

Horizontal Gene Transfer (HGT) is believed to be a major source of genetic variation, particularly for prokaryotes. It is believed that Horizontal Gene Transfer plays a major role in shaping bacterial genomes, and is also believed to be responsible for the relatively rapid dissemination and acquisition of new, adaptive traits across bacterial strains. Despite the importance of Horizontal Gene Transfer as a major source of genetic variation, the bulk of research on theoretical evolutionary dynamics and population genetics has focused on point-mutations (sometimes coupled with gene duplication events) as the main engine of genomic change. Here, we seek to specifically model HGT processes in bacterial cells, by developing a mathematical model describing the influence that conjugation-mediated HGT has on the mutation-selection balance in an asexually reproducing population of unicellular, prokaryotic organisms. It is assumed that mutation-selection balance is reached in the presence of a fixed background concentration of antibiotic, to which the population must become resistant in order to survive. We find that HGT has a non-trivial affect on the mean fitness of the population. However, one of the central results that emerge from our analysis is that, at mutation-selection balance, conjugation-mediated HGT has a slightly deleterious effect on the mean fitness of a population. Therefore, we conclude that HGT does not confer a selection advantage in static environments. Rather, its advantage must lie in its ability to promote faster adaptation in dynamic environments, an interpretation that is consistent with the observation that HGT can be promoted by environmental stresses on a population.

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The Nucleosome Remodeling Factor ISWI Functionally Interacts with an Evolutionarily Conserved Network of Cellular Factors.

Publication Date: 2010 Mar 1 PMID: 20194965
Authors: Arancio, W. - Onorati, M. C. - Burgio, G. - Collesano, M. - Ingrassia, A. M. - Genovese, S. I. - Fanto, M. - Corona, D. F.
Journal: Genetics

ISWI is an evolutionarily conserved ATP-dependent chromatin remodeling factor playing central roles in DNA replication, RNA transcription and chromosome organization. The variety of biological functions dependent on ISWI suggests that its activity could be highly regulated. Our group has previously isolated and characterized new cellular activities that positively regulate ISWI in D.melanogaster. In order to identify factors that antagonize ISWI activity we developed a novel in vivo eye-based assay to screen for genetic suppressors of ISWI. Our screen revealed that ISWI interacts with an evolutionarily conserved network of cellular and nuclear factors that escaped previous genetic and biochemical analyses.

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Quantitative Trait Locus Mapping of Genes Under Selection Across Multiple Years and Sites in Avena barbata: Epistasis, Pleiotropy, and Genotype by Environment Interactions.

Publication Date: 2010 Mar 1 PMID: 20194964
Authors: Latta, R. G. - Gardner, K. M. - Staples, D. A.
Journal: Genetics

The genetic architecture of variation in evolutionary fitness determines the trajectory of adaptive change. We identified Quantitative Trait Loci (QTL) affecting fitness in a mapping population of Recombinant Inbred Lines (RILs) derived from a cross between moist- and dry- associated ecotypes of Avena barbata. We estimated fitness in 179 RILs in each of two natural environments in each of four years. Two loci account for over half of the variation in geometric mean fitness across environments. These loci are associated in repulsion phase in the wild ecotypes suggesting the potential for strong transgressive segregation, but also show significant epistasis giving hybrid breakdown. This epistasis is the result of sharply lower fitness in only one of the recombinant genotypes suggesting that the loci may contain synergistically acting mutations. Within each trial, (year/site combination) we can explain less of the variation than for geometric mean fitness, but the two major loci are associated with variation in fitness in most environments. Tests for pleiotropic effects of QTL on fitness in different environments reveal that the same loci are under selection in all trials. Genotype by Environment interactions are significant for some loci, but this reflects variation in the strength, not the direction of selection.

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TRRAP-mediated Regulation of Wee1.

Publication Date: 2010 Mar 1 PMID: 20194963
Authors: Calonge, T. M. - Eshaghi, M. - Liu, J. - Ronai, Z. - O'Connell, M. J.
Journal: Genetics

The G2 DNA damage checkpoint inhibits Cdc2 and mitotic entry through the dual regulation of Wee1 and Cdc25 by the Chk1 effector kinase. Up-regulation of Chk1 by mutation or overexpression bypasses the requirement for up-stream regulators or DNA damage to promote a G2 cell cycle arrest. We screened in fission yeast for mutations that rendered cells resistant to overexpressed chk1(+). We identified a mutation in tra1, which encodes one of two homologs of TRRAP, an ATM/R-related pseudokinase that scaffolds several histone acetyltransferase (HAT) complexes. Inhibition of histone deacetylases reverts the resistance to overexpressed chk1(+), suggesting this phenotype is due to a HAT activity, though expression of checkpoint and cell cycle genes is not greatly affected. Cells with mutant or deleted tra1 activate Chk1 normally and are checkpoint proficient. However, these cells are semi-wee even when overexpressing chk1(+), and accumulate inactive Wee1 protein. The Cdr (changed division response) kinases Cdr1 and Cdr2 are negative regulators of Wee1, and we show that they are required for the Tra1-dependent alterations to Wee1 function. This identifies Tra1 as another component controlling the timing of entry into mitosis via Cdc2 activation.

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Fast Transcriptional Responses to Domestication in the Brook Charr Salvelinus fontinalis.

Publication Date: 2010 Mar 1 PMID: 20194962
Authors: Sauvage, C. - Derome, N. - Normandeau, E. - St-Cyr, J. - Audet, C. - Bernatchez, L.
Journal: Genetics

Domestication has been practiced for centuries yet directed towards relatively few terrestrial crops and animals. While phenotypic and quantitative genetic changes associated with domestication have been amply documented, little is known about the molecular changes underlying the phenotypic evolution during the process. Here, we have investigated the brook charr (Salvelinus fontinalis) responses to artificial selection by means of transcriptional analysis of ~ 32 000 cDNA features performed in both a selected and control populations reared under identical environmental conditions during four generations. Our results indicate that selective breeding led to significant changes in the transcription of genes at the juvenile stage, where we observed 4.16% (156/3750) of differentially expressed genes between the two lines. No significant genes were revealed at the earlier life stage. Moreover, when comparing our results to those of previous studies on Atlantic salmon that compared lines that were selected for 5-7 generations for similar traits (e.g. growth), genes with similar biological functions were found to be under selection in both studies. These observations indicate that (1) four generations of selection caused substantial changes in regulation of gene transcription between selected and control populations and (2) selective breeding for improving the same phenotypic traits (e.g. rapid growth) in brook charr and Atlantic salmon tended to select for the same changes in transcription profiles as the expression of a small and similar set of genes were affected by selection.

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Condensins Promote Co-orientation of Sister Chromatids During Meiosis I in Budding Yeast.

Publication Date: 2010 Mar 1 PMID: 20194961
Authors: Brito, I. L. - Yu, H. G. - Amon, A.
Journal: Genetics

The condensin complex is a key determinant of higher-ordered chromosome structure. We show here that the complex is also important for the correct alignment of chromosomes on the meiosis I spindle. Unlike during mitosis and meiosis II, when sister chromatids attach to microtubules emanating from opposite spindle poles (bi-orientation), accurate meiosis I chromosome segregation requires that sister chromatids attach to microtubules emanating from the same spindle pole (co-orientation). The monopolin complex, consisting of Lrs4, Csm1 and the meiosis-specific component Mam1, brings about meiosis I co-orientation. We find that in the absence of functional condensin complexes, a fraction of sister kinetochores bi-orient on the meiosis I spindle and association of the monopolin complex subunit Mam1 with kinetochores is decreased. Our studies uncover a new locus-specific effect of the condensin complex.

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Identification of a Maize Locus that Modulates the Hypersensitive Defense Response, Using Mutant-Assisted Gene Identification and Characterization.

Publication Date: 2010 Feb 22 PMID: 20176981
Authors: Chintamanani, S. - Hulbert, S. H. - Johal, G. S. - Balint-Kurti, P. J.
Journal: Genetics

Potentially useful naturally occurring genetic variation is often difficult to identify as the effects of individual genes are subtle and difficult to observe. In this study, a novel genetic technique called Mutant-Assisted Gene Identification and Characterization is used to identify naturally-occurring loci modulating the hypersensitive defence response (HR) in maize. Mutant-Assisted Gene Identification and Characterization facilitates the identification of naturally occurring alleles underlying phenotypic variation from diverse germplasm, using a mutant phenotype as a "reporter". In this study the reporter phenotype was caused by a partially-dominant autoactive disease resistance gene, Rp1-D21 ,which caused HR lesions to form spontaneously all over the plant. Here it is demonstrated that the Rp1-D21 phenotype is profoundly affected by genetic background. By crossing the Rp1-D21 gene into the IBM mapping population, it was possible to map and identify Hrml1 on chromosome 10, a locus responsible for modulating the HR phenotype conferred by Rp1-D21. Other loci with smaller effects were identified on chromosomes 1 and 9. These results demonstrate that Mutant-Assisted Gene Identification and Characterization is a viable approach for identifying naturally-occurring useful genetic variation.

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Fission Yeast Hsk1 (Cdc7) Kinase is Required After Replication Initiation for Induced Mutagenesis and Proper Response to DNA Alkylation Damage.

Publication Date: 2010 Feb 22 PMID: 20176980
Authors: Dolan, W. P. - Le, A. H. - Schmidt, H. - Yuan, J. P. - Green, M. - Forsburg, S. L.
Journal: Genetics

Genome stability in fission yeast requires the conserved S phase kinase Hsk1 (Cdc7) and its partner Dfp1 (Dbf4). In addition to their established function in the initiation of DNA replication, we show these proteins are important to maintain genome integrity later in S phase and G2. hsk1 cells suffer increased rates of mitotic recombination and require recombination proteins for survival. Both hsk1 and dfp1 mutants are acutely sensitive to alkylation damage yet defective in induced mutagenesis. Hsk1 and Dfp1 are associated with the chromatin even after S phase, and normal response to MMS damage correlates with the maintenance of intact Dfp1 on chromatin. A screen for MMS sensitive mutants identified a novel truncation allele, rad35 (dfp1-(1-519)), as well as alleles of other damage-associated genes. Although Hsk1-Dfp1 function with the Swi1-Swi3 fork protection complex, it also acts independently of the FPC to promote DNA repair. We conclude that Hsk1-Dfp1 kinase functions post-initiation to maintain replication fork stability, an activity potentially mediated by the C-terminus of Dfp1.

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Unified Framework to Evaluate Panmixia and Migration Direction Among Multiple Sampling Locations.

Publication Date: 2010 Feb 22 PMID: 20176979
Authors: Beerli, P. - Palczewski, M.
Journal: Genetics

For many biological investigations, groups of individuals are genetically sampled from several geographic locations. These sampling locations often do not reflect the genetical population structure. We describe a framework using marginal likelihoods to compare and order structured population models, such as testing whether the sampling locations belong to the same randomly mating population or comparing unidirectional and multidirectional gene flow models. In the context of inferences employing Markov chain Monte Carlo methods, the accuracy of the marginal likelihoods depends heavily on the approximation method used to calculate the marginal likelihood. Two methods, modified thermodynamic integration and a stabilized harmonic mean estimator, are compared. With finite Markov chain Monte Carlo run lengths, the harmonic mean estimator may not be consistent. Thermodynamic integration, in contrast, delivers considerably better estimates of the marginal likelihood. The choice of prior distributions does not influence the order and choice of the better models when the marginal likelihood is estimated using thermodynamic integration, whereas with the harmonic mean estimator the influence of the prior is pronounced and the order of the models changes. The approximation of marginal likelihood using thermodynamic integration in MIGRATE allows the evaluation of complex population genetic models; not only of whether sampling locations belong to a single panmictic population, but also of competing complex structured population models.

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An auxiliary silencer and a boundary element maintain high levels of silencing proteins at HMR in Saccharomyces cerevisiae.

Publication Date: 2010 Feb 22 PMID: 20176978
Authors: Lynch, P. J. - Rusche, L. N.
Journal: Genetics

Heterochromatin is notable for its capacity to propagate along a chromosome. The prevailing model for this spreading process postulates that silencing proteins are first recruited to silencer sequences and then spread from these sites independently of the silencers. However, we found that in Saccharomyces cerevisiae silencers also influence the extent of silenced chromatin domains. We compared the abilities of two different silencers, HMR-E and a telomeric repeat, to promote silencing and found that the HMR-E silencer contributed to an increased steady-state association of Sir proteins over a region of several kilobase-pairs compared to the telomeric repeat, even though both silencers recruited similar levels of Sir proteins. We also discovered that, although the HMR-E silencer alone was sufficient to block transcription of the HMR locus, a secondary silencer, HMR-I, boosted the level of Sir proteins at HMR, apparently beyond the level necessary to repress transcription. Finally, we discovered that a tRNA(Thr) gene near HMR-I helped maintain silenced chromatin and transcriptional repression under conditions of reduced deacetylase activity. This study highlights the importance of auxiliary elements, such as HMR-I and the tRNA(Thr) gene, in enhancing the association of Sir silencing proteins with appropriate genomic locations, thereby buffering the capacity of silenced chromatin to assemble under suboptimal conditions.

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Amplification of the Gene for isoleucyl-tRNA Synthetase Facilitates Adaptation to the Fitness Cost of Mupirocin Resistance in Salmonella enterica.

Publication Date: 2010 Feb 22 PMID: 20176977
Authors: Paulander, W. - Andersson, D. I. - Maisnier-Patin, S. E.
Journal: Genetics

Mutations that cause resistance to antibiotics in bacteria often reduce growth rate by impairing some essential cellular function. This growth impairment is expected to counter-select resistant organisms from natural populations following discontinuation of antibiotic therapy. Unfortunately (for disease control) bacteria adapt and improve their growth rate, often without losing antibiotic resistance. This adaptation process was studied in mupirocin-resistant (Mup(R)) strains of Salmonella enterica. Mupirocin (Mup) is an isoleucyl-adenylate analogue that inhibits the essential enzyme, isoleucyl-tRNA synthetase (IleRS). Mutations causing Mup(R) alter IleRS and reduce growth rate. Fitness is restored by any of 23 secondary IleS amino acid substitutions, sixty percent of which leave resistance unaffected. Evidence is presented that increased expression of the original mutant ileS gene (Mup(R)) also improves fitness while maintaining resistance. Expression can be increased by amplification of the ileS gene (more copies) or mutations that improve the ileS promoter (more transcription). Some adapted strains show both ileS amplification and an improved promoter. This suggests a process of adaptation initiated by common amplifications and followed by later acquisition of rare point mutations. Finally, a point mutation in one copy relaxes selection and allows loss of defective ileS copies. This sequence of events is demonstrated experimentally. A better understanding of adaptation can explain why antibiotic resistance persists in bacterial populations and may help identify drugs that are least subject to this problem.

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Regulation of Septum Formation by the Bud3-Rho4 GTPase Module in Aspergillus nidulans.

Publication Date: 2010 Feb 23 PMID: 20176976
Authors: Si, H. - Justa-Schuch, D. - Seiler, S. - Harris, S. D.
Journal: Genetics

The ability of fungi to generate polarized cells with a variety of shapes likely reflects precise temporal and spatial control over the formation of polarity axes. The bud site selection system of Saccharomyces cerevisiae represents the best-understood example of such a morphogenetic regulatory system. However, the extent to which this system is conserved in the highly polarized filamentous fungi remains unknown. Here, we describe the functional characterization and localization of the Aspergillus nidulans homologue of the axial bud site marker Bud3. Our results show that AnBud3 is not required for polarized hyphal growth per se, but is involved in septum formation. In particular, our genetic and biochemical evidence implicates AnBud3 as a guanine nucleotide exchange factor for the GTPase Rho4. Additional results suggest that the AnBud3-Rho4 module acts downstream of the septation initiation network to mediate recruitment of the formin SepA to the site of contractile actin ring assembly. Our observations provide new insight into the signaling pathways that regulate septum formation in filamentous fungi.

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Activation of SREBP in the Absence of Scap in Drosophila melanogaster.

Publication Date: 2010 Feb 22 PMID: 20176975
Authors: Matthews, K. A. - Ozdemir, C. - Rawson, R. B.
Journal: Genetics

The escort factor Scap is essential in mammalian cells for the regulated activation of sterol regulatory element binding proteins (SREBPs), which are membrane-bound transcription factors. Cells lacking Scap cannot activate SREBP and are therefore deficient in the transcription of numerous genes involved in lipid synthesis and uptake; they cannot survive in the absence of exogenous lipid. Here we report that, conversely, Drosophila completely lacking dscap are viable. Flies lacking dscap emerge at about 70 % of the expected rate and readily survive as homozygous stocks. These animals continue to cleave dSREBP in some tissues. Transcription of dSREBP target genes in dscap mutant larvae is reduced compared to wild type but is greater than in mutants lacking dSREBP and remains responsive to dietary lipids in dscap mutants. In contrast to flies lacking ds2p, a gene encoding a protease that releases the transcription factor domain of dSREBP from the membrane, dscap mutants do not require the caspase Drice to activate dSREBP. Larvae doubly mutant for dscap and ds2p exhibit phenotypes similar to those of ds2p single mutants. Thus, dScap and dS2P, essential components of the SREBP activation machinery in mammalian cells, are dispensable in Drosophila owing to different compensatory mechanisms.

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Caenorhabditis elegans TRPV Channels Function in a Modality-Specific Pathway to Regulate Response to Aberrant Sensory Signaling.

Publication Date: 2010 Feb 22 PMID: 20176974
Authors: Ezak, M. J. - Hong, E. - Chaparro-Garcia, A. - Ferkey, D. M.
Journal: Genetics

Olfaction and some forms of taste (including bitter) are mediated by G protein-coupled signal transduction pathways. Olfactory and gustatory ligands bind to chemosensory G protein-coupled receptors (GPCRs) in specialized sensory cells to activate intracellular signal transduction cascades. G protein-coupled receptor kinases (GRKs) are negative regulators of signaling that specifically phosphorylate activated GPCRs to terminate signaling. Although loss of GRK function usually results in enhanced cellular signaling, Caenorhabditis elegans lacking GRK-2 function are not hypersensitive to chemosensory stimuli. Instead, grk-2 mutant animals do not chemotax towards attractive olfactory stimuli or avoid aversive tastes and smells. We show here that loss-of-function mutations in the transient receptor potential vanilloid (TRPV) channels OSM-9 and OCR-2 selectively restore grk-2 behavioral avoidance of bitter tastants, revealing modality-specific mechanisms for TRPV channel function in the regulation of C. elegans chemosensation. Additionally, a single amino acid point mutation in OCR-2 that disrupts TRPV channel-mediated gene expression, but does not decrease channel function in chemosensory primary signal transduction, also restores grk-2 bitter taste avoidance. Thus, loss of GRK-2 function may lead to changes in gene expression, via OSM-9/OCR-2, to selectively alter the levels of signaling components that transduce or regulate bitter taste responses. Our results suggest a novel mechanism and multiple modality-specific pathways that sensory cells employ in response to aberrant signal transduction.

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Structure Prediction-driven Genetics in Saccharomyces cerevisiae Identifies an Interface Between the t-RPA Proteins Stn1 and Ten1.

Publication Date: 2010 Feb 18 PMID: 20157006
Authors: Paschini, M. - Mandell, E. K. - Lundblad, V.
Journal: Genetics

In Saccharomyces cerevisiae, Cdc13, Stn1 and Ten1 are essential for both chromosome capping and telomere length homeostasis. These three proteins have been proposed to perform their roles at chromosome termini as a telomere-dedicated t-RPA complex, based on several parallels with the conventional RPA complex. In this study, we have used several approaches to test whether a predicted alpha-helix in the N-terminal domain of the S. cerevisiae Stn1 protein is required for formation of the proposed t-RPA complex, in a manner analogous to the comparable helix in Rpa2. Analysis of a panel of Rpa2-OB(Stn1) chimeras indicates that whether a chimeric protein contains the Rpa2 or Stn1 version of this alpha-helix dictates its ability to function in place of Rpa2 or Stn1, respectively. In addition, mutations introduced into a hydrophobic surface of the predicted Stn1 alpha-helix eliminated association with Ten1. Strikingly, allele-specific suppression of a stn1 mutation in this helix (stn1-L164D) by a ten1 mutation (ten1-D138Y) resulted in a restored Stn1-Ten1 interaction, supporting the identification of a Stn1-Ten1 interface. We conclude that Stn1 interacts with Ten1 through an alpha-helix, in a manner analogous to the interaction between the comparable subunits of the RPA complex.

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Fitness Epistasis and Constraints on Adaptation in a Human Immunodeficiency Virus Type 1 Protein Region.

Publication Date: 2010 Feb 15 PMID: 20157005
Authors: da Silva, J. - Coetzer, M. - Nedellec, R. - Pastore, C. - Mosier, D. E.
Journal: Genetics

Fitness epistasis, the interaction among alleles at different loci in their effects on fitness, has potentially important consequences for adaptive evolution. We investigated fitness epistasis among amino acids of a functionally important region of the human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein (gp120). Seven mutations putatively involved in the adaptation of the second conserved to third variable protein region (C2-V3) to the use of an alternative host-cell chemokine coreceptor (CXCR4) for cell entry were engineered singly and in combinations on the wild type genetic background and their effects on viral infectivity were measured. Epistasis was found to be common and complex, involving not only pairwise interactions, but also higher-order interactions. Interactions could also be surprisingly strong, changing fitness by more than nine orders of magnitude, which is explained by some single mutations being practically lethal. A consequence of the observed epistasis is that many of the minimum-length mutational trajectories between the wild type and the mutant with highest fitness on cells expressing the alternative coreceptor are selectively inaccessible. These results may help explain the difficulty of evolving viruses that use the alternative coreceptor in culture and the delayed evolution of this phenotype in natural infection. Knowledge of common, complex and strong fitness interactions among amino acids is necessary for a full understanding of protein evolution.

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A Tetrad Analysis of the Basidiomycete Fungus Cryptococcus neoformans.

Publication Date: 2010 Feb 15 PMID: 20157004
Authors: Idnurm, A.
Journal: Genetics

Cryptococcus neoformans is a basidiomycete fungus that is found worldwide and causes disease in humans and animal species. The fungus grows asexually as a budding yeast. Under laboratory conditions it is capable of sexual reproduction between two mating types. After cell fusion a dikaryotic filament develops, at the tip of which a basidium gives rise to four chains of basidiospores. Because the chains comprise each 10-30 spores, rather than single spores, the analysis of individual meiotic events has not been attempted in C. neoformans in the style of tetrad analyses performed in other fungal species. Here, the basidiospores from more than 100 basidia were micromanipulated and the resultant >2,500 progeny analyzed for three genetic markers to understand the sexual process in this fungus, leading to four observations. (i) Marker segregation provides genetic evidence for a single meiotic event within the basidium followed by multiple rounds of mitosis. (ii) Using each basidium as an unordered tetrad, the ADE2 and URA5 genes are linked to their centromeres, consistent with adjacent genomic regions rich in repetitive elements predicted to comprise Cryptococcus centromeres. (iii) Lack of germination of basidiospores is attributed to aneuploidy, rather than dormancy. (iv) Analysis of basidiospores derived from single chains demonstrates that each chain can contain different genotypes. This mechanism of sexual spore production would benefit the species with a high rate of dispersal and at the same time aid in simultaneous dissemination of both mating types to new locations in the environment.

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Genome-wide Multiple Loci Mapping in Experimental Crosses by the Iterative Adaptive Penalized Regression.

Publication Date: 2010 Feb 15 PMID: 20157003
Authors: Sun, W. - Ibriham, J. G. - Zou, F.
Journal: Genetics

Genome-wide multiple loci mapping can be viewed as a variable selection problem where the major objective is to select genetic markers related with a trait of interest. This is a challenging variable selection problem because the number of genetic markers is large (often much larger than the sample size) and there are often strong linkage or linkage disequilibrium between markers. In this paper, we developed two methods for genome-wide multiple loci mapping: the Bayesian adaptive Lasso and the iterative adaptive Lasso. Compared to the existing methods, the advantages of our methods come from the assignment of adaptive weights to different genetic makers and the iterative updating of these adaptive weights. We evaluate these two methods as well as several existing methods in the application of genome-wide multiple loci mapping in experimental cross. Both large-scale simulation and real data analyses show that the proposed methods have improved variable selection performance. The iterative adaptive Lasso is also computationally much more efficient than the commonly used marginal regression and step-wise regression methods. Although our methods are motivated by multiple loci mapping, they are general enough to be applied to other variable selection problems.

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Promoting and Avoiding Recombination: Contrasting Activities of the Escherichia coli RuvABC Holliday Junction Resolvase and RecG DNA Translocase.

Publication Date: 2010 Feb 15 PMID: 20157002
Authors: Zhang, J. - Mahdi, A. A. - Briggs, G. S. - Lloyd, R. G.
Journal: Genetics

RuvABC and RecG are thought to provide alternative pathways for the late stages of recombination in Escherichia coli. Inactivation of both blocks the recovery of recombinants in genetic crosses. RuvABC resolves Holliday junctions, with RuvAB driving branch migration and RuvC catalysing junction cleavage. RecG also drives branch migration, but no nuclease has been identified that might act with RecG to cleave junctions, apart from RusA, which is not normally expressed. We searched for an alternative nuclease using a synthetic lethality assay to screen for mutations causing inviability in the absence of RuvC, on the premise that a strain without any ability to cut junctions might be inviable. All the mutations identified mapped to polA, dam or uvrD. None of these genes encodes a nuclease that cleaves Holliday junctions. Probing the reason for the inviability using the RusA Holliday junction resolvase provided strong evidence in each case that the RecG pathway is very ineffective at removing junctions, and indicated that a nuclease component most probably does not exist. It also revealed new suppressors of recG, which were located to the ssb gene. Taken together with the results from the synthetic lethality assays, the properties of the mutant SSB proteins provide evidence that, rather than promoting recombination, a major function of RecG is to curb pathological replication initiated via PriA protein at sites remote from oriC.

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A Hierarchical Bayesian Model for a Novel Sparse Partial Diallel Crossing Design.

Publication Date: 2010 Feb 15 PMID: 20157001
Authors: Greenberg, A. J. - Hackett, S. R. - Harshman, L. G. - Clark, A. G.
Journal: Genetics

Partial diallel crossing designs are in common use among evolutionary geneticists, as well as plant and animal breeders. When the goal is to make statements about populations represented by a given set of lines it is desirable to maximize the number of lines sampled given a set number of crosses among them. We propose an augmented round-robin design that accomplishes this. We develop a hierarchical Bayesian model to estimate quantitative genetic parameters from our scheme. For example, we show how to partition genetic effects into specific and general combining abilities, and the method provides estimates of heritability, dominance and genetic correlations in the face of complex and unbalanced designs. We test our approach with simulated and real data. We show that although the models slightly overestimate genetic variances, main effects are assessed accurately and precisely. We also illustrate how our approach allows the construction of posterior distributions of combinations of parameters by calculating narrow-sense heritability and a genetic correlation between activities of two enzymes.

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The Effect of Recombination on the Reconstruction of Ancestral Sequences.

Publication Date: 2010 Feb 22 PMID: 20124027
Authors: Arenas, M. - Posada, D.
Journal: Genetics

While a variety of methods exist to reconstruct ancestral sequences, all of them assume that single phylogeny underlies all the positions in the alignment, and therefore that recombination has not taken place. Using computer simulations we show that recombination can severely bias ancestral sequence reconstruction (ASR), and quantify this effect. If recombination is ignored, the ancestral sequences recovered can be quite distinct from the grand most recent common ancestor of the sample (GMRCA) and better resemble the concatenate of partial most recent common ancestors (MRCAs) at each recombination fragment. When independent phylogenetic trees are assumed for the different recombinant segments, the estimation of the fragment MRCAs improves significantly. Importantly, we show that recombination can change the biological predictions derived from ASRs carried out with real data. Given that recombination is widespread on nuclear genes and in particular in RNA viruses and some bacteria, the reconstruction of ancestral sequences in these cases should consider the potential impact of recombination and ideally be carried out using approaches that accommodate recombination.

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