Phenotypic Consequences of Aneuploidy in Arabidopsis thaliana

Aneuploid cells are characterized by incomplete chromosome sets. The resulting imbalance in gene dosage has phenotypic consequences that are specific to each karyotype. Even in the case of Down syndrome, the most viable and studied form of human aneuploidy, the mechanisms underlying the connected phenotypes remain mostly unclear. Because of their tolerance to aneuploidy, plants provide a powerful system for a genome-wide investigation of aneuploid syndromes, an approach that is not feasible in animal systems. Indeed, in many plant species, populations of aneuploid individuals can be easily obtained from triploid individuals. We phenotyped a population of Arabidopsis thaliana aneuploid individuals containing 25 different karyotypes. Even in this highly heterogeneous population, we demonstrate that certain traits are strongly associated with the dosage of specific chromosome types and that chromosomal effects can be additive. Further, we identified subtle developmental phenotypes expressed in the diploid progeny of aneuploid parent(s) but not in euploid controls from diploid lineages. These results indicate long-term phenotypic consequences of aneuploidy that can persist after chromosomal balance has been restored. We verified the diploid nature of these individuals by whole-genome sequencing and discuss the possibility that trans-generational phenotypic effects stem from epigenetic modifications passed from aneuploid parents to their diploid progeny.THE genome of aneuploid individuals contains incomplete chromosome sets. The balance between chromosome types, and the genes they encode, is compromised, resulting in altered expression of many genes, including genes with dosage-sensitive effects on phenotypes. In humans, only a few types of aneuploid karyotypes are viable (Hassold and Hunt 2001), highlighting the deleterious effect of chromosome imbalance. The most commonly known viable form of aneuploidy in humans is Down syndrome, which results from a trisomy of chromosome 21 in an otherwise diploid background. Down syndrome patients exhibit many specific phenotypes, sometimes visible only in a subset of patients (Antonarakis et al. 2004). For phenotypes found in all Down syndrome patients, the penetrance of each phenotype varies between patients (Antonarakis et al. 2004). Despite the increasing amount of information available about the human genome and the availability of a mouse model for Down syndrome (O''Doherty et al. 2005), the genes responsible for most of the phenotypes associated with Down syndrome are still unknown (Patterson 2007; Korbel et al. 2009; Patterson 2009). Recently, detailed phenotypic analyses of as many as 30 aneuploid patients have allowed the identification of susceptibility regions for several specific phenotypes (Patterson 2007, 2009; Korbel et al. 2009; Lyle et al. 2009), but the specific genes remain to be identified. Understanding the physiology of aneuploidy is not only relevant to those individuals with aneuploid genomes but also to understanding cancer since most cancerous cells are aneuploid (Matzke et al. 2003; Pihan and Doxsey 2003; Storchova and Pellman 2004; Holland and Cleveland 2009; Williams and Amon 2009) or the consequences of copy number variation and dosage sensitivity (Dear 2009; Henrichsen et al. 2009).Plants are more tolerant of aneuploidy than animals (Matzke et al. 2003) for reasons that remain unclear. Since the discovery of the Datura trisomic “chromosome mutants” by Blakeslee (1921, 1922), viable trisomics of each chromosome type have been described in numerous species. Trisomics exhibit phenotypes specific to the identity of the triplicated chromosome (Blakeslee 1922; Khush 1973; Koornneef and Van der Veen 1983; Singh 2003). More complex aneuploids, i.e., individuals carrying more than one additional chromosome, can be viable as well and have been observed in many plants species, especially among the progeny of triploid individuals (McClintock 1929; Levan 1942; Johnsson 1945; Khush 1973). Some species appear to be more tolerant of complex aneuploidies than others, suggesting a genetic basis for aneuploidy tolerance (Satina and Blakeslee 1938; Khush 1973; Ramsey and Schemske 2002; Henry et al. 2009). Aneuploid individuals frequently appear spontaneously within polyploid plant populations, presumably due to a failure to equally partition the multiple chromosome sets at meiosis (Randolph 1935; Doyle 1986). These aneuploids exhibit few or subtle phenotypic abnormalities and can often compete with their euploid progenitors (Ramsey and Schemske 1998). Plants therefore provide an excellent opportunity for a genome-wide investigation of aneuploid syndromes: sample size is not limited, phenotypes can be described and assessed in detail, and plant aneuploid populations provide a complex mixture of viable karyotypes.In this article, we report our investigation of the relationship between phenotype and karyotype in populations of aneuploid Arabidopsis thaliana plants. All simple trisomics of A. thaliana have been previously isolated and phenotypically characterized (Steinitz-Sears 1962; Lee-Chen and Steinitz-Sears 1967; Steinitz-Sears and Lee-Chen 1970; Koornneef and Van der Veen 1983), demonstrating that they are tolerated in A. thaliana. We previously reported that aneuploid swarms—populations of aneuploid individuals of varying aneuploid karyotypes—could be obtained from the progeny of triploid A. thaliana individuals (Henry et al. 2005, 2009). Using a combination of a quantitative PCR-based method and flow cytometry, we were able to derive the full aneuploid karyotype of each of these individuals (Henry et al. 2006). We further crossed triploid A. thaliana to diploid or tetraploid individuals and demonstrated that at least 44 of the 60 possible aneuploid karyotypes that could result from these crosses (aneuploid individuals carrying between 11 and 19 chromosomes) were viable and successfully produced adult plants. Taken together, these populations and methods make it possible to explore the basis of aneuploid syndromes in A. thaliana. In this study, we were able to phenotypically characterize at least one individual from 25 different aneuploid karyotypes falling between diploidy and tetraploidy. We demonstrated that specific phenotypes are affected by the dosage of specific chromosome types. The effect of the dosage of specific chromosome types on traits was additive and could be used to predict the observed phenotype. The availability of multiple generations of aneuploid and euploid individuals allowed us to investigate potential long-term effects of aneuploidy as well as parent-of-origin effects on aneuploid phenotypes.     

Exponential Fitness Gains of RNA Virus Populations Are Limited by Bottleneck Effects

Fitness is a parameter that quantitatively measures adaptation of a virus to a given environment. We have previously reported exponential fitness gains of large populations of vesicular stomatitis virus replicating in a constant environment (I. S. Novella et al., Proc. Natl. Acad. Sci. USA 92:5841–5844, 1995). In this paper, we report that during long-term passage of such large viral populations, fitness values reached a high-fitness plateau during which stochastic fitness variations were observed. This effect appears likely to be due to bottleneck effects on very high fitness populations.     

不同启动子驱动下acdS基因转化烟草及耐盐性研究

acdS基因编码产生ACC脱氨酶,该酶属于脱巯基家族,可以降低逆境乙烯的合成量。利用农杆菌介导的叶盘法将CaMV35S-2启动子和rolD启动子驱动下的acdS基因转入烟草NC89叶片中。在含有卡那霉素的MS培养基上筛选得到Kanr转化烟草。通过PCR、Southern blotting对得到的Kanr转基因烟草进行分析,结果表明,acdS基因已经整合到了烟草的基因组中。对转基因烟草的RT-PCR及cDNA进行测序分析表明,acdS基因能够正确转录。对转基因烟草进行耐盐性测定,结果显示,与对照相比,两种启动子驱动下的转基因烟草耐盐性均有增强。但是rolD启动子驱动下的转基因植株的耐盐性最强。     

Surprising Fitness Consequences of GC-Biased Gene Conversion: I. Mutation Load and Inbreeding Depression

GC-biased gene conversion (gBGC) is a recombination-associated process mimicking selection in favor of G and C alleles. It is increasingly recognized as a widespread force in shaping the genomic nucleotide landscape. In recombination hotspots, gBGC can lead to bursts of fixation of GC nucleotides and to accelerated nucleotide substitution rates. It was recently shown that these episodes of strong gBGC could give spurious signatures of adaptation and/or relaxed selection. There is also evidence that gBGC could drive the fixation of deleterious amino acid mutations in some primate genes. This raises the question of the potential fitness effects of gBGC. While gBGC has been metaphorically termed the “Achilles'' heel” of our genome, we do not know whether interference between gBGC and selection merely has practical consequences for the analysis of sequence data or whether it has broader fundamental implications for individuals and populations. I developed a population genetics model to predict the consequences of gBGC on the mutation load and inbreeding depression. I also used estimates available for humans to quantitatively evaluate the fitness impact of gBGC. Surprising features emerged from this model: (i) Contrary to classical mutation load models, gBGC generates a fixation load independent of population size and could contribute to a significant part of the load; (ii) gBGC can maintain recessive deleterious mutations for a long time at intermediate frequency, in a similar way to overdominance, and these mutations generate high inbreeding depression, even if they are slightly deleterious; (iii) since mating systems affect both the selection efficacy and gBGC intensity, gBGC challenges classical predictions concerning the interaction between mating systems and deleterious mutations, and gBGC could constitute an additional cost of outcrossing; and (iv) if mutations are biased toward A and T alleles, very low gBGC levels can reduce the load. A robust prediction is that the gBGC level minimizing the load depends only on the mutational bias and population size. These surprising results suggest that gBGC may have nonnegligible fitness consequences and could play a significant role in the evolution of genetic systems. They also shed light on the evolution of gBGC itself.GC-BIASED gene conversion (gBGC) is increasingly recognized as a widespread force in shaping genome evolution. In different species, gene conversion occurring during double-strand break recombination repair is thought to be biased toward G and C alleles. In heterozygotes, GC alleles undergo a kind of molecular meiotic drive that mimics selection (reviewed in Marais 2003). This process can rapidly increase the GC content, especially around recombination hotspots (Spencer et al. 2006), and, more broadly, can affect genome-wide nucleotide landscapes (Duret and Galtier 2009a). For instance, it is thought to play a role in shaping isochore structure evolution in mammals (Galtier et al. 2001; Meunier and Duret 2004; Duret et al. 2006) and birds (Webster et al. 2006). Direct experimental evidence of gBGC mainly comes from studies in yeast (Birdsell 2002; Mancera et al. 2008; but see Marsolier-Kergoat and Yeramian 2009) and humans (Brown and Jiricny 1987). However, associations between recombination and the nucleotide landscape and frequency spectra biased toward GC alleles provide indirect evidence in very diverse organisms (

Gene Mutations and Aneuploidy: The Instability That Causes Cancer

Cancer cells have been characterized with activated mutant oncogenes and inactivated or deleted tumor suppressor genes. Cancer cells are also aneuploid, displaying a jumble of chromosomal anomalies including gain or loss of whole chromosomes or transposed chromosomal fragments. Whether mutation of specific genes or aneuploidy is more critical for tumorigenesis is very much a contentious issue. We recently showed that activated oncogenes induce oxidative damage that is exacerbated by conventional cell culture conditions. This “culture shock” or a loss of p53 function creates a precarious environment that permits oncogenes to induce rapid chromosomal instability and transformation. We found that mutant genes and aneuploidy were prerequisites and collaborators for neoplastic transformation.     

Fitness Consequences of Maternal Effects in Streblospio benedicti (Annelida: Polychaeta)

SYNOPSIS. The degree to which a female partitions resourcesbetween fecundity and per offspring investment is a centralquestion in life-history theory. Maternal effects may influencethe nature of this tradeoff through their effect on per offspringinvestment and subsequent offspring fitness. The purpose ofthis study was to determine the effect of female age and sizeon brood size (number of offspring), per offspring investment,and fitness in the polychaete Streblospio benedicti. Early stageembryos were collected from brooding females of known age andsize over a period of 100 days; these embryos were counted andanalyzed for their C and N content. Female size had a positiveeffect on brood size; larger females produced larger broods.However, brood size decreased with female age (females did notincrease in size after reaching sexual maturity). Brood sizedeclined 20–46% between 60 and 160 days of age. Duringthis same age period per offspring investment, measured in termsof C and N, increased by 25%. Offspring survivorship and sizeat two weeks post-release from the female were used as measuresof offspring fitness. Offspring survivorship increased 28% between60 and 160 days of age. Increased growth in offspring from olderfemales resulted in a 23% increase in offspring size at twoweeks. Including the maternal age effect in two population modelsfor S. benedicti increased population growth rate (). Populationgrowth was increased to a greater degree when the maternal effectwas modeled by enhancing offspring survival compared to whenfecundity was increased by the same proportional amount. Thissuggests that the maternal effect may be adaptive, particularlywhen conditions for offspring survival and growth are poor.     

Acquisition of Aneuploidy Provides Increased Fitness during the Evolution of Antifungal Drug Resistance

The evolution of drug resistance is an important process that affects clinical outcomes. Resistance to fluconazole, the most widely used antifungal, is often associated with acquired aneuploidy. Here we provide a longitudinal study of the prevalence and dynamics of gross chromosomal rearrangements, including aneuploidy, in the presence and absence of fluconazole during a well-controlled in vitro evolution experiment using Candida albicans, the most prevalent human fungal pathogen. While no aneuploidy was detected in any of the no-drug control populations, in all fluconazole-treated populations analyzed an isochromosome 5L [i(5L)] appeared soon after drug exposure. This isochromosome was associated with increased fitness in the presence of drug and, over time, became fixed in independent populations. In two separate cases, larger supernumerary chromosomes composed of i(5L) attached to an intact chromosome or chromosome fragment formed during exposure to the drug. Other aneuploidies, particularly trisomies of the smaller chromosomes (Chr3–7), appeared throughout the evolution experiment, and the accumulation of multiple aneuploid chromosomes per cell coincided with the highest resistance to fluconazole. Unlike the case in many other organisms, some isolates carrying i(5L) exhibited improved fitness in the presence, as well as in the absence, of fluconazole. The early appearance of aneuploidy is consistent with a model in which C. albicans becomes more permissive of chromosome rearrangements and segregation defects in the presence of fluconazole.     

Elevated Tolerance to Aneuploidy in Cancer Cells: Estimating the Fitness Effects of Chromosome Number Alterations by In Silico Modelling of Somatic Genome Evolution

An unbalanced chromosome number (aneuploidy) is present in most malignant tumours and has been attributed to mitotic mis-segregation of chromosomes. However, recent studies have shown a relatively high rate of chromosomal mis-segregation also in non-neoplastic human cells, while the frequency of aneuploid cells remains low throughout life in most normal tissues. This implies that newly formed aneuploid cells are subject to negative selection in healthy tissues and that attenuation of this selection could contribute to aneuploidy in cancer. To test this, we modelled cellular growth as discrete time branching processes, during which chromosome gains and losses were generated and their host cells subjected to selection pressures of various magnitudes. We then assessed experimentally the frequency of chromosomal mis-segregation as well as the prevalence of aneuploid cells in human non-neoplastic cells and in cancer cells. Integrating these data into our models allowed estimation of the fitness reduction resulting from a single chromosome copy number change to an average of ≈30% in normal cells. In comparison, cancer cells showed an average fitness reduction of only 6% (p = 0.0008), indicative of aneuploidy tolerance. Simulations based on the combined presence of chromosomal mis-segregation and aneuploidy tolerance reproduced distributions of chromosome aberrations in >400 cancer cases with higher fidelity than models based on chromosomal mis-segregation alone. Reverse engineering of aneuploid cancer cell development in silico predicted that aneuploidy intolerance is a stronger limiting factor for clonal expansion of aneuploid cells than chromosomal mis-segregation rate. In conclusion, our findings indicate that not only an elevated chromosomal mis-segregation rate, but also a generalised tolerance to novel chromosomal imbalances contribute to the genomic landscape of human tumours.     

Fitness Consequences of Timing of Migration and Breeding in Cormorants

In most bird species timing of breeding affects reproductive success whereby early breeding is favoured. In migratory species migration time, especially arrival at the breeding grounds, and breeding time are expected to be correlated. Consequently, migration time should also have fitness consequences. However, in contrast to breeding time, evidence for fitness consequences of migration time is much more limited. Climate change has been shown to negatively affect the synchrony between trophic levels thereby leading to directional selection on timing but again direct evidence in avian migration time is scarce. We here analysed fitness consequences of migration and breeding time in great cormorants and tested whether climate change has led to increased selection on timing using a long-term data set from a breeding colony on the island of Vorsø (Denmark). Reproductive success, measured as number of fledglings, correlated with breeding time and arrival time at the colony and declined during the season. This seasonal decline became steeper during the study period for both migration and breeding time and was positively correlated to winter/spring climate, i.e. selection was stronger after warmer winters/springs. However, the increasing selection pressure on timing seems to be unrelated to climate change as the climatic variables that were related to selection strength did not increase during the study period. There is indirect evidence that phenology or abundances of preferred prey species have changed which could have altered selection on timing of migration and breeding.     

Vector-Dependent Gene Expression Driven by Insulated P-Element Reporter Vectors

The Pelican and Stinger series of P-element transformation vectors are a popular choice for reporter gene expression in transgenic flies. We report here as a cautionary note that these vectors on their own can drive reporter gene expression in the larval and pupal salivary gland.     

A Mechanism of Gene Amplification Driven by Small DNA Fragments

DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s). Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA) occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB) external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation in nature.     

The Evolutionarily Stable Distribution of Fitness Effects

The distribution of fitness effects (DFE) of new mutations is a key parameter in determining the course of evolution. This fact has motivated extensive efforts to measure the DFE or to predict it from first principles. However, just as the DFE determines the course of evolution, the evolutionary process itself constrains the DFE. Here, we analyze a simple model of genome evolution in a constant environment in which natural selection drives the population toward a dynamic steady state where beneficial and deleterious substitutions balance. The distribution of fitness effects at this steady state is stable under further evolution and provides a natural null expectation for the DFE in a population that has evolved in a constant environment for a long time. We calculate how the shape of the evolutionarily stable DFE depends on the underlying population genetic parameters. We show that, in the absence of epistasis, the ratio of beneficial to deleterious mutations of a given fitness effect obeys a simple relationship independent of population genetic details. Finally, we analyze how the stable DFE changes in the presence of a simple form of diminishing-returns epistasis.     

Induced Fungal Resistance to Insect Grazing: Reciprocal Fitness Consequences and Fungal Gene Expression in the Drosophila-Aspergillus Model System

Background

Fungi are key dietary resources for many animals. Fungi, in consequence, have evolved sophisticated physical and chemical defences for repelling and impairing fungivores. Expression of such defences may entail costs, requiring diversion of energy and nutrients away from fungal growth and reproduction. Inducible resistance that is mounted after attack by fungivores may allow fungi to circumvent the potential costs of defence when not needed. However, no information exists on whether fungi display inducible resistance. We combined organism and fungal gene expression approaches to investigate whether fungivory induces resistance in fungi.

Methodology/Principal Findings

Here we show that grazing by larval fruit flies, Drosophila melanogaster, induces resistance in the filamentous mould, Aspergillus nidulans, to subsequent feeding by larvae of the same insect. Larval grazing triggered the expression of various putative fungal resistance genes, including the secondary metabolite master regulator gene laeA. Compared to the severe pathological effects of wild type A. nidulans, which led to 100% insect mortality, larval feeding on a laeA loss-of-function mutant resulted in normal insect development. Whereas the wild type fungus recovered from larval grazing, larvae eradicated the chemically deficient mutant. In contrast, mutualistic dietary yeast, Saccharomyces cerevisiae, reached higher population densities when exposed to Drosophila larval feeding.

Conclusions/Significance

Our study presents novel evidence that insect grazing is capable of inducing resistance to further grazing in a filamentous fungus. This phenotypic shift in resistance to fungivory is accompanied by changes in the expression of genes involved in signal transduction, epigenetic regulation and secondary metabolite biosynthesis pathways. Depending on reciprocal insect-fungus fitness consequences, fungi may be selected for inducible resistance to maintain high fitness in fungivore-rich habitats. Induced fungal defence responses thus need to be included if we wish to have a complete conception of animal-fungus co-evolution, fungal gene regulation, and multitrophic interactions.