Deregulated Ras signaling compromises DNA damage checkpoint recovery in S. cerevisiae

The DNA damage checkpoint maintains genome stability by arresting the cell cycle and promoting DNA repair under genotoxic stress. Cells must downregulate the checkpoint signaling pathways in order to resume cell division after completing DNA repair. While the mechanisms of checkpoint activation have been well-characterized, the process of checkpoint recovery, and the signals regulating it, has only recently been investigated. We have identified a new role for the Ras signaling pathway as a regulator of DNA damage checkpoint recovery. Here we report that in budding yeast, deletion of the IRA1 and IRA2 genes encoding negative regulators of Ras prevents cellular recovery from a DNA damage induced arrest. the checkpoint kinase Rad53 is dephosphorylated in an IRA-deficient strain, indicating that recovery failure is not caused by constitutive checkpoint pathway activation. the ira1Δ ira2Δ recovery defect requires the checkpoint kinase Chk1 and the cAMP-dependent protein kinase (PKA) catalytic subunit Tpk2. Furthermore, PKA phosphorylation sites on the anaphase promoting complex specificity factor Cdc20 are required for the recovery defect, indicating a link between the recovery defect and PKA regulation of mitosis. This work identifies a new signaling pathway that can regulate DNA damage checkpoint recovery and implicates the Ras signaling pathway as an important regulator of mitotic events.Key words: DNA damage checkpoint, Ras signaling, budding yeast, cAMP-dependent protein kinase, anaphase promoting complex, neurofibromatosis type 1     

Rhizosphere Selection of Highly Motile Phenotypic Variants of Pseudomonas fluorescens with Enhanced Competitive Colonization Ability

Phenotypic variants of Pseudomonas fluorescens F113 showing a translucent and diffuse colony morphology show enhanced colonization of the alfalfa rhizosphere. We have previously shown that in the biocontrol agent P. fluorescens F113, phenotypic variation is mediated by the activity of two site-specific recombinases, Sss and XerD. By overexpressing the genes encoding either of the recombinases, we have now generated a large number of variants (mutants) after selection either by prolonged laboratory cultivation or by rhizosphere passage. All the isolated variants were more motile than the wild-type strain and appear to contain mutations in the gacA and/or gacS gene. By disrupting these genes and complementation analysis, we have observed that the Gac system regulates swimming motility by a repression pathway. Variants isolated after selection by prolonged cultivation formed a single population with a swimming motility that was equal to the motility of gac mutants, being 150% more motile than the wild type. The motility phenotype of these variants was complemented by the cloned gac genes. Variants isolated after rhizosphere selection belonged to two different populations: one identical to the population isolated after prolonged cultivation and the other comprising variants that besides a gac mutation harbored additional mutations conferring higher motility. Our results show that gac mutations are selected both in the stationary phase and during rhizosphere colonization. The enhanced motility phenotype is in turn selected during rhizosphere colonization. Several of these highly motile variants were more competitive than the wild-type strain, displacing it from the root tip within 2 weeks.     

Effects of parental care on the accumulation and release of cryptic genetic variation: <Emphasis Type="Italic">review of mechanisms and a case study of dung beetles</Emphasis>

Cryptic genetic variation plays an important role in the emergence of disease and evolutionary responses to environmental change. Focusing on parental care behavior, we discuss three mechanisms by which behavior can affect the accumulation and release of cryptic genetic variation. We illustrate how these hypotheses might be tested with preliminary data from Onthophagus dung beetles, which provide indirect parental care by provisioning their offspring with dung and sheltering them underground. The environmental stress hypothesis states that parental care reduces selection intensity on novel mutations when increased parental care results in a less stressful offspring environment. A review of recent literature, coupled with an irradiation experiment in beetles, suggests this mechanism may operate in some situations, but depends on the types of mutations under consideration. The relaxed selection hypothesis states that genes expressed in low care environments should be under weakened selection because their phenotypic manifestations are exposed to selection less frequently, and thus are prone to mutation accumulation. If parental care is reduced, for instance due to population-wide environmental changes, such cryptic variation may exert phenotypic effects, becoming exposed to selection. There is substantial theory in support of this hypothesis, and comparisons between beetle populations that differ in parental care behavior further support this idea. Finally, the compensation hypothesis states that organisms with direct parental care may be able to respond to cues or signals from offspring and compensate for genetic variants. We highlight the extensive discussion of this hypothesis with respect to medical care and genetic load in humans and explore invertebrate systems that may constitute powerful models for further inquiry. In summary, several mechanisms exist by which care behavior may shape the accumulation and release of cryptic genetic variation, thereby affecting the potential emergence of diseases and the rate and direction of evolutionary responses to novel environments.     

Compensatory Evolution and the Origins of Innovations

Cryptic genetic sequences have attenuated effects on phenotypes. In the classic view, relaxed selection allows cryptic genetic diversity to build up across individuals in a population, providing alleles that may later contribute to adaptation when co-opted—e.g., following a mutation increasing expression from a low, attenuated baseline. This view is described, for example, by the metaphor of the spread of a population across a neutral network in genotype space. As an alternative view, consider the fact that most phenotypic traits are affected by multiple sequences, including cryptic ones. Even in a strictly clonal population, the co-option of cryptic sequences at different loci may have different phenotypic effects and offer the population multiple adaptive possibilities. Here, we model the evolution of quantitative phenotypic characters encoded by cryptic sequences and compare the relative contributions of genetic diversity and of variation across sites to the phenotypic potential of a population. We show that most of the phenotypic variation accessible through co-option would exist even in populations with no polymorphism. This is made possible by a history of compensatory evolution, whereby the phenotypic effect of a cryptic mutation at one site was balanced by mutations elsewhere in the genome, leading to a diversity of cryptic effect sizes across sites rather than across individuals. Cryptic sequences might accelerate adaptation and facilitate large phenotypic changes even in the absence of genetic diversity, as traditionally defined in terms of alternative alleles.     

Genetic Origins of Lactase Persistence and the Spread of Pastoralism in Africa

In humans, the ability to digest lactose, the sugar in milk, declines after weaning because of decreasing levels of the enzyme lactase-phlorizin hydrolase, encoded by LCT. However, some individuals maintain high enzyme amounts and are able to digest lactose into adulthood (i.e., they have the lactase-persistence [LP] trait). It is thought that selection has played a major role in maintaining this genetically determined phenotypic trait in different human populations that practice pastoralism. To identify variants associated with the LP trait and to study its evolutionary history in Africa, we sequenced MCM6 introns 9 and 13 and ∼2 kb of the LCT promoter region in 819 individuals from 63 African populations and in 154 non-Africans from nine populations. We also genotyped four microsatellites in an ∼198 kb region in a subset of 252 individuals to reconstruct the origin and spread of LP-associated variants in Africa. Additionally, we examined the association between LP and genetic variability at candidate regulatory regions in 513 individuals from eastern Africa. Our analyses confirmed the association between the LP trait and three common variants in intron 13 (C-14010, G-13907, and G-13915). Furthermore, we identified two additional LP-associated SNPs in intron 13 and the promoter region (G-12962 and T-956, respectively). Using neutrality tests based on the allele frequency spectrum and long-range linkage disequilibrium, we detected strong signatures of recent positive selection in eastern African populations and the Fulani from central Africa. In addition, haplotype analysis supported an eastern African origin of the C-14010 LP-associated mutation in southern Africa.     

Morphotypic Conversion in Listeria monocytogenes Biofilm Formation: Biological Significance of Rough Colony Isolates

Adherence to a stainless steel surface selected isolates of Listeria monocytogenes with enhanced surface colonization abilities and a change in phenotype from the common smooth colony morphology to a succession of rough colony morphotypes. Growth in broth culture of the best-adapted, surface-colonizing rough colony morphotype gave a smooth colony revertant. Comparative analysis revealed that the smooth and rough variants had similar phenotypic and biochemical characteristics (e.g., identical growth rates and tolerances to antibiotics and environmental stressors). Rough colony isolates, however, failed to coordinate motility or induce autolysis. The defect in autolysis of rough colony isolates, which involved impaired cellular localization of several peptidoglycan-degrading enzymes, including cell wall hydrolase A (CwhA), suggested a link to a secretory pathway defect. The genetic basis for the impairment was studied at the level of the accessory secretory pathway component SecA2. DNA sequencing of the secA2 gene in smooth and rough colony isolates found no mutations in the coding or promoter regions. Analysis of SecA2 expression with an integrated secA2-FLAG tag construct found the protein to be upregulated in the rough and revertant backgrounds compared to the parental smooth colony isolate. A compensatory mechanism involving the SecA2 secretion pathway components is postulated to control smooth to rough interconversion of L. monocytogenes. Such phenotypic variation may enhance the ability of this opportunistic pathogen to colonize environments as diverse as processing surfaces, food products, and animal hosts.     

Sporulation Genes Associated with Sporulation Efficiency in Natural Isolates of Yeast

Yeast sporulation efficiency is a quantitative trait and is known to vary among experimental populations and natural isolates. Some studies have uncovered the genetic basis of this variation and have identified the role of sporulation genes (IME1, RME1) and sporulation-associated genes (FKH2, PMS1, RAS2, RSF1, SWS2), as well as non-sporulation pathway genes (MKT1, TAO3) in maintaining this variation. However, these studies have been done mostly in experimental populations. Sporulation is a response to nutrient deprivation. Unlike laboratory strains, natural isolates have likely undergone multiple selections for quick adaptation to varying nutrient conditions. As a result, sporulation efficiency in natural isolates may have different genetic factors contributing to phenotypic variation. Using Saccharomyces cerevisiae strains in the genetically and environmentally diverse SGRP collection, we have identified genetic loci associated with sporulation efficiency variation in a set of sporulation and sporulation-associated genes. Using two independent methods for association mapping and correcting for population structure biases, our analysis identified two linked clusters containing 4 non-synonymous mutations in genes – HOS4, MCK1, SET3, and SPO74. Five regulatory polymorphisms in five genes such as MLS1 and CDC10 were also identified as putative candidates. Our results provide candidate genes contributing to phenotypic variation in the sporulation efficiency of natural isolates of yeast.     

Bias and Evolution of the Mutationally Accessible Phenotypic Space in a Developmental System

Genetic and developmental architecture may bias the mutationally available phenotypic spectrum. Although such asymmetries in the introduction of variation may influence possible evolutionary trajectories, we lack quantitative characterization of biases in mutationally inducible phenotypic variation, their genotype-dependence, and their underlying molecular and developmental causes. Here we quantify the mutationally accessible phenotypic spectrum of the vulval developmental system using mutation accumulation (MA) lines derived from four wild isolates of the nematodes Caenorhabditis elegans and C. briggsae. The results confirm that on average, spontaneous mutations degrade developmental precision, with MA lines showing a low, yet consistently increased, proportion of developmental defects and variants. This result indicates strong purifying selection acting to maintain an invariant vulval phenotype. Both developmental system and genotype significantly bias the spectrum of mutationally inducible phenotypic variants. First, irrespective of genotype, there is a developmental bias, such that certain phenotypic variants are commonly induced by MA, while others are very rarely or never induced. Second, we found that both the degree and spectrum of mutationally accessible phenotypic variation are genotype-dependent. Overall, C. briggsae MA lines exhibited a two-fold higher decline in precision than the C. elegans MA lines. Moreover, the propensity to generate specific developmental variants depended on the genetic background. We show that such genotype-specific developmental biases are likely due to cryptic quantitative variation in activities of underlying molecular cascades. This analysis allowed us to identify the mutationally most sensitive elements of the vulval developmental system, which may indicate axes of potential evolutionary variation. Consistent with this scenario, we found that evolutionary trends in the vulval system concern the phenotypic characters that are most easily affected by mutation. This study provides an empirical assessment of developmental bias and the evolution of mutationally accessible phenotypes and supports the notion that such bias may influence the directions of evolutionary change.     

A naturally occurring variant of Hsp90 that is associated with decanalization

The heat shock protein Hsp90 has been the focus of many studies since it was suggested that it acts to mediate the buffering of phenotypic variation. Hsp90-mediated buffering may result in the accumulation of cryptic genetic variation that, when released either as a consequence of environmental or genetic stress, increases the evolvability of a population. Recent studies using laboratory-induced mutations of Hsp90 and/or chemical inhibition to disrupt Hsp90 function confirm that Hsp90 can buffer cryptic genetic variation. We have previously identified a naturally occurring variant in the charged linker region of the Hsp90 gene, and now examine whether this variant is associated with altered levels of trait variability. The variant is associated with the release of cryptic genetic variation for canalized morphological (bristle) traits, but not for uncanalized morphological (wing and bristle) traits, and the effect on canalized traits depends on culture temperature. This suggests that natural genetic variation in Hsp90 may mediate the evolution of canalized morphological traits even if it does not influence the expression of variation for uncanalized traits.     

Linked genetic variants on chromosome 10 control ear morphology and body mass among dog breeds

Background

The domestic dog is a rich resource for mapping the genetic components of phenotypic variation due to its unique population history involving strong artificial selection. Genome-wide association studies have revealed a number of chromosomal regions where genetic variation associates with morphological characters that typify dog breeds. A region on chromosome 10 is among those with the highest levels of genetic differentiation between dog breeds and is associated with body mass and ear morphology, a common motif of animal domestication. We characterised variation in this region to uncover haplotype structure and identify candidate functional variants.

Results

We first identified SNPs that strongly associate with body mass and ear type by comparing sequence variation in a 3 Mb region between 19 breeds with a variety of phenotypes. We next genotyped a subset of 123 candidate SNPs in 288 samples from 46 breeds to identify the variants most highly associated with phenotype and infer haplotype structure. A cluster of SNPs that associate strongly with the drop ear phenotype is located within a narrow interval downstream of the gene MSRB3, which is involved in human hearing. These SNPs are in strong genetic linkage with another set of variants that correlate with body mass within the gene HMGA2, which affects human height. In addition we find evidence that this region has been under selection during dog domestication, and identify a cluster of SNPs within MSRB3 that are highly differentiated between dogs and wolves.

Conclusions

We characterise genetically linked variants that potentially influence ear type and body mass in dog breeds, both key traits that have been modified by selective breeding that may also be important for domestication. The finding that variants on long haplotypes have effects on more than one trait suggests that genetic linkage can be an important determinant of the phenotypic response to selection in domestic animals.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1702-2) contains supplementary material, which is available to authorized users.     

Complex genetic diversity patterns of cryptic,sympatric brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>) populations in tiny mountain lakes

Intraspecific genetic variation can have similar effects as species diversity on ecosystem function; understanding such variation is important, particularly for ecological key species. The brown trout plays central roles in many northern freshwater ecosystems, and several cases of sympatric brown trout populations have been detected in freshwater lakes based on apparent morphological differences. In some rare cases, sympatric, genetically distinct populations lacking visible phenotypic differences have been detected based on genetic data alone. Detecting such “cryptic” sympatric populations without prior grouping of individuals based on phenotypic characteristics is more difficult statistically, though. The aim of the present study is to delineate the spatial connectivity of two cryptic, sympatric genetic clusters of brown trout discovered in two interconnected, tiny subarctic Swedish lakes. The structures were detected using allozyme markers, and have been monitored over time. Here, we confirm their existence for almost three decades and report that these cryptic, sympatric populations exhibit very different connectivity patterns to brown trout of nearby lakes. One of the clusters is relatively isolated while the other one shows high genetic similarity to downstream populations. There are indications of different spawning sites as reflected in genetic structuring among parr from different creeks. We used >3000 SNPs on a subsample and find that the SNPs largely confirm the allozyme pattern but give considerably lower F _ST values, and potentially indicate further structuring within populations. This type of complex genetic substructuring over microgeographical scales might be more common than anticipated and needs to be considered in conservation management.     

Molecular Population Genetics and Phenotypic Diversification of Two Populations of the Thermophilic Cyanobacterium Mastigocladus laminosus

We investigated the distributions of genetic and phenotypic variation for two Yellowstone National Park populations of the heterocyst-forming cyanobacterium Mastigocladus (Fischerella) laminosus that exhibit dramatic phenotypic differences as a result of environmental differences in nitrogen availability. One population develops heterocysts and fixes nitrogen in situ in response to a deficiency of combined nitrogen in its environment, whereas the other population does neither due to the availability of a preferred nitrogen source. Slowly evolving molecular markers, including the 16S rRNA gene and the downstream internal transcribed spacer, are identical among all laboratory isolates from both populations but belie considerable genetic and phenotypic diversity. The total nucleotide diversity at six nitrogen metabolism loci was roughly three times greater than that observed for the human global population. The two populations are genetically differentiated, although variation in performance on different nitrogen sources among genotypes could not be explained by local adaptation to available nitrogen in the respective environments. Population genetic models suggest that local adaptation is mutation limited but also that the populations are expected to continue to diverge due to low migratory gene flow.     

Gene-by-Temperature Interactions and Candidate Plasticity Genes for Morphological Traits in Drosophila melanogaster

Understanding the genetic architecture of any quantitative trait requires identifying the genes involved in its expression in different environmental conditions. This goal can be achieved by mutagenesis screens in genetically tractable model organisms such as Drosophila melanogaster. Temperature during ontogenesis is an important environmental factor affecting development and phenotypic variation in holometabolous insects. In spite of the importance of phenotypic plasticity and genotype by environment interaction (GEI) for fitness related traits, its genetic basis has remained elusive. In this context, we analyzed five different adult morphological traits (face width, head width, thorax length, wing size and wing shape) in 42 co-isogenic single P-element insertional lines of Drosophila melanogaster raised at 17°C and 25°C. Our analyses showed that all lines differed from the control for at least one trait in males or females at either temperature. However, no line showed those differences for all traits in both sexes and temperatures simultaneously. In this sense, the most pleiotropic candidate genes were CG34460, Lsd-2 and Spn. Our analyses also revealed extensive genetic variation for all the characters mostly indicated by strong GEIs. Further, our results indicate that GEIs were predominantly explained by changes in ranking order in all cases suggesting that a moderate number of genes are involved in the expression of each character at both temperatures. Most lines displayed a plastic response for at least one trait in either sex. In this regard, P-element insertions affecting plasticity of a large number of traits were associated to the candidate genes Btk29A, CG43340, Drak and jim. Further studies will help to elucidate the relevance of these genes on the morphogenesis of different body structures in natural populations of D. melanogaster.     

Guanyl nucleotide exchange factor Sql2 and Ras2 regulate filamentous growth in Ustilago maydis

The cyclic AMP (cAMP)-signaling pathway regulates cell morphology and plays a crucial role during pathogenic development of the plant-pathogenic fungus Ustilago maydis. Strains lacking components of this signaling pathway, such as the Gα-subunit Gpa3 or the adenylyl cyclase Uac1, are nonpathogenic and grow filamentously. On the other hand, strains exhibiting an activated cAMP pathway due to a dominant-active allele of gpa3 display a glossy colony phenotype and are unable to proliferate in plant tumors. Here we present the identification of sql2 as a suppressor of the glossy colony phenotype of a gpa3_Q206L strain. sql2 encodes a protein with similarity to CDC25-like guanine nucleotide exchange factors, which are known to act on Ras proteins. Overexpression of sql2 leads to filamentous growth that cannot be suppressed by exogenous cAMP, suggesting that Sql2 does not act upstream of Uac1. To gain more insight in signaling processes regulated by Sql2, we isolated two genes encoding Ras proteins. Expression of dominant active alleles of ras1 and ras2 showed that Ras2 induces filamentous growth while Ras1 does not affect cell morphology but elevates pheromone gene expression. These results indicate that Ras1 and Ras2 fulfill different functions in U. maydis. Moreover, observed similarities between the filaments induced by sql2 and ras2 suggest that Sql2 is an activator of Ras2. Interestingly, sql2 deletion mutants are affected in pathogenic development but not in mating, indicating a specific function of sql2 during pathogenesis.     

An Evaluation of High-Throughput Approaches to QTL Mapping in Saccharomyces cerevisiae

Dissecting the molecular basis of quantitative traits is a significant challenge and is essential for understanding complex diseases. Even in model organisms, precisely determining causative genes and their interactions has remained elusive, due in part to difficulty in narrowing intervals to single genes and in detecting epistasis or linked quantitative trait loci. These difficulties are exacerbated by limitations in experimental design, such as low numbers of analyzed individuals or of polymorphisms between parental genomes. We address these challenges by applying three independent high-throughput approaches for QTL mapping to map the genetic variants underlying 11 phenotypes in two genetically distant Saccharomyces cerevisiae strains, namely (1) individual analysis of >700 meiotic segregants, (2) bulk segregant analysis, and (3) reciprocal hemizygosity scanning, a new genome-wide method that we developed. We reveal differences in the performance of each approach and, by combining them, identify eight polymorphic genes that affect eight different phenotypes: colony shape, flocculation, growth on two nonfermentable carbon sources, and resistance to two drugs, salt, and high temperature. Our results demonstrate the power of individual segregant analysis to dissect QTL and address the underestimated contribution of interactions between variants. We also reveal confounding factors like mutations and aneuploidy in pooled approaches, providing valuable lessons for future designs of complex trait mapping studies.