Purifying selection maintains highly conserved noncoding sequences in Drosophila

The majority of metazoan genomes consist of nonprotein-coding regions, although the functional significance of most noncoding DNA sequences remains unknown. Highly conserved noncoding sequences (CNSs) have proven to be reliable indicators of functionally constrained sequences such as cis-regulatory elements and noncoding RNA genes. However, CNSs may arise from nonselective evolutionary processes such as genomic regions with extremely low mutation rates known as mutation "cold spots." Here we combine comparative genomic data from recently completed insect genome projects with population genetic data in Drosophila melanogaster to test predictions of the mutational cold spot model of CNS evolution in the genus Drosophila. We find that point mutations in intronic and intergenic CNSs exhibit a significant reduction in levels of divergence relative to levels of polymorphism, as well as a significant excess of rare derived alleles, compared with either the nonconserved spacer regions between CNSs or with 4-fold silent sites in coding regions. Controlling for the effects of purifying selection, we find no evidence of positive selection acting on Drosophila CNSs, although we do find evidence for the action of recurrent positive selection in the spacer regions between CNSs. We estimate that approximately 85% of sites in Drosophila CNSs are under constraint with selection coefficients (N(e)s) on the order of 10-100, and thus, the estimated strength and number of sites under purifying selection is greater for Drosophila CNSs relative to those in the human genome. These patterns of nonneutral molecular evolution are incompatible with the mutational cold spot hypothesis to explain the existence of CNSs in Drosophila and, coupled with similar findings in mammals, argue against the general likelihood that CNSs are generated by mutational cold spots in any metazoan genome.     

The mutational hazard hypothesis of organelle genome evolution: 10 years on

Why is there such a large variation in size and noncoding DNA content among organelle genomes? One explanation is that this genomic variation results from differences in the rates of organelle mutation and random genetic drift, as opposed to being the direct product of natural selection. Along these lines, the mutational hazard hypothesis (MHH) holds that ‘excess’ DNA is a mutational liability (because it increases the potential for harmful mutations) and, thus, has a greater tendency to accumulate in an organelle system with a low mutation rate as opposed to one with a high rate of mutation. Various studies have explored this hypothesis and, more generally, the relationship between organelle genome architecture and the mode and efficiency of organelle DNA repair. Although some of these investigations are in agreement with the MHH, others have contradicted it; nevertheless, they support a central role of mutation, DNA maintenance pathways and random genetic drift in fashioning organelle chromosomes. Arguably, one of the most important contributions of the MHH is that it has sparked crucial, widespread discussions about the importance of nonadaptive processes in genome evolution.     

Identification and characterization of new long conserved noncoding sequences in vertebrates

Comparative sequence analyses have identified highly conserved genomic DNA sequences, including noncoding sequences, between humans and other species. By performing whole-genome comparisons of human and mouse, we have identified 611 conserved noncoding sequences longer than 500 bp, with more than 95% identity between the species. These long conserved noncoding sequences (LCNS) include 473 new sequences that do not overlap with previously reported ultraconserved elements (UCE), which are defined as aligned sequences longer than 200 bp with 100% identity in human, mouse, and rat. The LCNS were distributed throughout the genome except for the Y chromosome and often occurred in clusters within regions with a low density of coding genes. Many of the LCNS were also highly conserved in other mammals, chickens, frogs, and fish; however, we were unable to find orthologous sequences in the genomes of invertebrate species. In order to examine whether these conserved sequences are functionally important or merely mutational cold spots, we directly measured the frequencies of ENU-induced germline mutations in the LCNS of the mouse. By screening about 40.7 Mb, we found 35 mutations, including mutations at nucleotides that were conserved between human and fish. The mutation frequencies were equivalent to those found in other genomic regions, including coding sequences and introns, suggesting that the LCNS are not mutational cold spots at all. Taken together, these results suggest that mutations occur with equal frequency in LCNS but are eliminated by natural selection during the course of evolution.

     

Identification and characterization of new long conserved noncoding sequences in vertebrates

Comparative sequence analyses have identified highly conserved genomic DNA sequences, including noncoding sequences, between humans and other species. By performing whole-genome comparisons of human and mouse, we have identified 611 conserved noncoding sequences longer than 500 bp, with more than 95% identity between the species. These long conserved noncoding sequences (LCNS) include 473 new sequences that do not overlap with previously reported ultraconserved elements (UCE), which are defined as aligned sequences longer than 200 bp with 100% identity in human, mouse, and rat. The LCNS were distributed throughout the genome except for the Y chromosome and often occurred in clusters within regions with a low density of coding genes. Many of the LCNS were also highly conserved in other mammals, chickens, frogs, and fish; however, we were unable to find orthologous sequences in the genomes of invertebrate species. In order to examine whether these conserved sequences are functionally important or merely mutational cold spots, we directly measured the frequencies of ENU-induced germline mutations in the LCNS of the mouse. By screening about 40.7 Mb, we found 35 mutations, including mutations at nucleotides that were conserved between human and fish. The mutation frequencies were equivalent to those found in other genomic regions, including coding sequences and introns, suggesting that the LCNS are not mutational cold spots at all. Taken together, these results suggest that mutations occur with equal frequency in LCNS but are eliminated by natural selection during the course of evolution.     

Genetic drift and mutational hazard in the evolution of salamander genomic gigantism

Salamanders have the largest nuclear genomes among tetrapods and, excepting lungfishes, among vertebrates as a whole. Lynch and Conery (2003) have proposed the mutational‐hazard hypothesis to explain variation in genome size and complexity. Under this hypothesis, noncoding DNA imposes a selective cost by increasing the target for degenerative mutations (i.e., the mutational hazard). Expansion of noncoding DNA, and thus genome size, is driven by increased levels of genetic drift and/or decreased mutation rates; the former determines the efficiency with which purifying selection can remove excess DNA, whereas the latter determines the level of mutational hazard. Here, we test the hypothesis that salamanders have experienced stronger long‐term, persistent genetic drift than frogs, a related clade with more typically sized vertebrate genomes. To test this hypothesis, we compared dN/dS and Kr/Kc values of protein‐coding genes between these clades. Our results do not support this hypothesis; we find that salamanders have not experienced stronger genetic drift than frogs. Additionally, we find evidence consistent with a lower nucleotide substitution rate in salamanders. This result, along with previous work showing lower rates of small deletion and ectopic recombination in salamanders, suggests that a lower mutational hazard may contribute to genomic gigantism in this clade.     

Low Genetic Quality Alters Key Dimensions of the Mutational Spectrum

Mutations affect individual health, population persistence, adaptation, diversification, and genome evolution. There is evidence that the mutation rate varies among genotypes, but the causes of this variation are poorly understood. Here, we link differences in genetic quality with variation in spontaneous mutation in a Drosophila mutation accumulation experiment. We find that chromosomes maintained in low-quality genetic backgrounds experience a higher rate of indel mutation and a lower rate of gene conversion in a manner consistent with condition-based differences in the mechanisms used to repair DNA double strand breaks. These aspects of the mutational spectrum were also associated with body mass, suggesting that the effect of genetic quality on DNA repair was mediated by overall condition, and providing a mechanistic explanation for the differences in mutational fitness decline among these genotypes. The rate and spectrum of substitutions was unaffected by genetic quality, but we find variation in the probability of substitutions and indels with respect to several aspects of local sequence context, particularly GC content, with implications for models of molecular evolution and genome scans for signs of selection. Our finding that the chances of mutation depend on genetic context and overall condition has important implications for how sequences evolve, the risk of extinction, and human health.     

Evolution of genome size: multilevel selection, mutation bias or dynamical chaos?

In the past two years, new data on conceptual aspects of the evolution of eukaryotic genome size have appeared, including the adaptivity of genome enlargement, the mechanisms of genome size change and the relation of genome size to organismal complexity. New data on the hypotheses of "selfish DNA" and "mutational equilibrium" have been recently obtained. A relationship is emerging between the intragenomic distribution of noncoding DNA and differential gene expression, which suggests that noncoding DNA is involved in epigenetic organization of the genome and organismal complexity. The standpoint of dynamical chaos, which integrates multilevel selection and mutation biases, may provide a framework for studying the evolution of genome size.     

RIP: the evolutionary cost of genome defense

Repeat-induced point mutation (RIP) is a homology-based process that mutates repetitive DNA and frequently leads to epigenetic silencing of the mutated sequences through DNA methylation. Consistent with the hypothesis that RIP serves to control selfish DNA, an analysis of the Neurospora crassa genome sequence reveals a complete absence of intact mobile elements. As in most eukaryotes, the centromeric regions of N. crassa are rich in sequences that are related to transposable elements; however, in N crassa these sequences have been heavily mutated. The analysis of the N. crassa genome sequence also reveals that RIP has impacted genome evolution significantly through gene duplication, which is considered to be crucial for the evolution of new functions. Most if not all paralogs in N. crassa duplicated and diverged before the emergence of RIP. Thus, RIP illustrates the extraordinary extent to which genomes will go to defend themselves against mobile genetic elements.     

The impact of transposable elements on eukaryotic genomes: From genome size increase to genetic adaptation to stressful environments

Transposable elements (TEs) are present in roughly all genomes. These mobile DNA sequences are able to invade genomes and their impact on genome evolution is substantial. The mobility of TEs can induce the appearance of deleterious mutations, gene disruption and chromosome rearrangements, but transposition activity also has positive aspects and the mutational activities of TEs contribute to the genetic diversity of organisms. This short review aims to give a brief overview of the impact TEs may have on animal and plant genome structure and expression, and the relationship between TEs and the stress response of organisms, including insecticide resistance.     

Evidence for widespread degradation of gene control regions in hominid genomes

Although sequences containing regulatory elements located close to protein-coding genes are often only weakly conserved during evolution, comparisons of rodent genomes have implied that these sequences are subject to some selective constraints. Evolutionary conservation is particularly apparent upstream of coding sequences and in first introns, regions that are enriched for regulatory elements. By comparing the human and chimpanzee genomes, we show here that there is almost no evidence for conservation in these regions in hominids. Furthermore, we show that gene expression is diverging more rapidly in hominids than in murids per unit of neutral sequence divergence. By combining data on polymorphism levels in human noncoding DNA and the corresponding human–chimpanzee divergence, we show that the proportion of adaptive substitutions in these regions in hominids is very low. It therefore seems likely that the lack of conservation and increased rate of gene expression divergence are caused by a reduction in the effectiveness of natural selection against deleterious mutations because of the low effective population sizes of hominids. This has resulted in the accumulation of a large number of deleterious mutations in sequences containing gene control elements and hence a widespread degradation of the genome during the evolution of humans and chimpanzees.     

Mutational Dynamics of Microsatellites

Microsatellites are a ubiquitous class of simple repetitive DNA sequences, which are widespread in both eukaryotic and prokaryotic genomes. The use of microsatellites as polymorphic DNA markers has considerably increased both in the number of studies and in the number of organisms, primarily for genetic mapping, studying genomic instability in cancer, population genetics, forensics, conservation biology, molecular anthropology and in the studies of human evolutionary history. Although simple sequence repeats have been extensively used in studies encompassing varied areas of genetics, the mutation dynamics of these genome regions is still not well understood. The present review focuses on the mutational dynamics of microsatellite DNA with special reference to mutational mechanisms and their role in microsatellite evolution.     

High divergence rate of sequences located on different DNA strands in closely related bacterial genomes

One of the common features of bacterial genomes is a strong compositional asymmetry between differently replicating DNA strands (leading and lagging). The main cause of the observed bias is the mutational pressure associated with replication. This suggests that genes translocated between differently replicating DNA strands are subjected to a higher mutational pressure, which may influence their composition and divergence rate. Analyses of groups of completely sequenced bacterial genomes have revealed that the highest divergence rate is observed for the DNA sequences that in closely related genomes are located on different DNA strands in respect to their role in replication. Paradoxically, for this group of sequences the absolute values of divergence rate are higher for closely related species than for more diverged ones. Since this effect concerns only the specific group of orthologs, there must be a specific mechanism introducing bias into the structure of chromosome by enriching the set of homologs in trans position in newly diverged species in relatively highly diverged sequences. These highly diverged sequences may be of varied nature: (1) paralogs or other fast-evolving genes under weak selection; or (2) pseudogenes that will probably be eliminated from the genome during further evolution; or (3) genes whose history after divergence is longer than the history of the genomes in which they are found. The use of these highly diverged sequences for phylogenetic analyses may influence the topology and branch length of phylogenetic trees. The changing mutational pressure may contribute to arising of genes with new functions as well.     

Sequencing and comparative analysis of a conserved syntenic segment in the Solanaceae

Comparative genomics is a powerful tool for gaining insight into genomic function and evolution. However, in plants, sequence data that would enable detailed comparisons of both coding and noncoding regions have been limited in availability. Here we report the generation and analysis of sequences for an unduplicated conserved syntenic segment (CSS) in the genomes of five members of the agriculturally important plant family Solanaceae. This CSS includes a 105-kb region of tomato chromosome 2 and orthologous regions of the potato, eggplant, pepper, and petunia genomes. With a total neutral divergence of 0.73-0.78 substitutions/site, these sequences are similar enough that most noncoding regions can be aligned, yet divergent enough to be informative about evolutionary dynamics and selective pressures. The CSS contains 17 distinct genes with generally conserved order and orientation, but with numerous small-scale differences between species. Our analysis indicates that the last common ancestor of these species lived approximately 27-36 million years ago, that more than one-third of short genomic segments (5-15 bp) are under selection, and that more than two-thirds of selected bases fall in noncoding regions. In addition, we identify genes under positive selection and analyze hundreds of conserved noncoding elements. This analysis provides a window into 30 million years of plant evolution in the absence of polyploidization.     

What controls the length of noncoding DNA?

Several recent studies of genome evolution indicate that the rate of DNA loss exceeds that of DNA gain, leading to an underlying mutational pressure towards collapsing the length of noncoding DNA. That such a collapse is not observed suggests opposing mechanisms favoring longer noncoding regions. The presence of transposable elements alone also does not explain observed features of noncoding DNA. At present, a multidisciplinary approach--using population genetics techniques, large-scale genomic analyses, and in silico evolution--is beginning to provide new and valuable insights into the forces that shape the length of noncoding DNA and, ultimately, genome size. Recombination, in a broad sense, might be the missing key parameter for understanding the observed variation in length of noncoding DNA in eukaryotes.     

Ten years of bacterial genome sequencing: comparative-genomics-based discoveries

It has been more than 10 years since the first bacterial genome sequence was published. Hundreds of bacterial genome sequences are now available for comparative genomics, and searching a given protein against more than a thousand genomes will soon be possible. The subject of this review will address a relatively straightforward question: “What have we learned from this vast amount of new genomic data?” Perhaps one of the most important lessons has been that genetic diversity, at the level of large-scale variation amongst even genomes of the same species, is far greater than was thought. The classical textbook view of evolution relying on the relatively slow accumulation of mutational events at the level of individual bases scattered throughout the genome has changed. One of the most obvious conclusions from examining the sequences from several hundred bacterial genomes is the enormous amount of diversity—even in different genomes from the same bacterial species. This diversity is generated by a variety of mechanisms, including mobile genetic elements and bacteriophages. An examination of the 20 Escherichia coli genomes sequenced so far dramatically illustrates this, with the genome size ranging from 4.6 to 5.5 Mbp; much of the variation appears to be of phage origin. This review also addresses mobile genetic elements, including pathogenicity islands and the structure of transposable elements. There are at least 20 different methods available to compare bacterial genomes. Metagenomics offers the chance to study genomic sequences found in ecosystems, including genomes of species that are difficult to culture. It has become clear that a genome sequence represents more than just a collection of gene sequences for an organism and that information concerning the environment and growth conditions for the organism are important for interpretation of the genomic data. The newly proposed Minimal Information about a Genome Sequence standard has been developed to obtain this information.     

What transposable elements tell us about genome organization and evolution: the case of Drosophila

Transposable elements (TEs) have been identified in every organism in which they have been looked for. The sequencing of large genomes, such as the human genome and those of Drosophila, Arabidopsis, Caenorhabditis, has also shown that they are a major constituent of these genomes, accounting for 15% of the genome of Drosophila, 45% of the human genome, and more than 70% in some plants and amphibians. Compared with the 1% of genomic DNA dedicated to protein-coding sequences in the human genome, this has prompted various researchers to suggest that the TEs and the other repetitive sequences that constitute the so-called "noncoding DNA", are where the most stimulating discoveries will be made in the future (Bromham, 2002). We are therefore getting further and further from the original idea that this DNA was simply "junk DNA", that owed its presence in the genome entirely to its capacity for selfish transposition. Our understanding of the structures of TEs, their distribution along the genomes, their sequence and insertion polymorphisms within genomes, and within and between populations and species, their impact on genes and on the regulatory mechanisms of genetic expression, their effects on exon shuffling and other phenomena that reshape the genome, and their impact on genome size has increased dramatically in recent years. This leads to a more general picture of the impact of TEs on genomes, though many copies are still mainly selfish or junk DNA. In this review we focus mainly on discoveries made in Drosophila, but we also use information about other genomes when this helps to elucidate the general processes involved in the organization, plasticity, and evolution of genomes.     

Evolution of genome architecture

Charles Darwin believed that all traits of organisms have been honed to near perfection by natural selection. The empirical basis underlying Darwin's conclusions consisted of numerous observations made by him and other naturalists on the exquisite adaptations of animals and plants to their natural habitats and on the impressive results of artificial selection. Darwin fully appreciated the importance of heredity but was unaware of the nature and, in fact, the very existence of genomes. A century and a half after the publication of the "Origin", we have the opportunity to draw conclusions from the comparisons of hundreds of genome sequences from all walks of life. These comparisons suggest that the dominant mode of genome evolution is quite different from that of the phenotypic evolution. The genomes of vertebrates, those purported paragons of biological perfection, turned out to be veritable junkyards of selfish genetic elements where only a small fraction of the genetic material is dedicated to encoding biologically relevant information. In sharp contrast, genomes of microbes and viruses are incomparably more compact, with most of the genetic material assigned to distinct biological functions. However, even in these genomes, the specific genome organization (gene order) is poorly conserved. The results of comparative genomics lead to the conclusion that the genome architecture is not a straightforward result of continuous adaptation but rather is determined by the balance between the selection pressure, that is itself dependent on the effective population size and mutation rate, the level of recombination, and the activity of selfish elements. Although genes and, in many cases, multigene regions of genomes possess elaborate architectures that ensure regulation of expression, these arrangements are evolutionarily volatile and typically change substantially even on short evolutionary scales when gene sequences diverge minimally. Thus, the observed genome architectures are, mostly, products of neutral processes or epiphenomena of more general selective processes, such as selection for genome streamlining in successful lineages with large populations. Selection for specific gene arrangements (elements of genome architecture) seems only to modulate the results of these processes.     

The evolving fungal genome

Fungal genomes vary considerably in size and organization. The genome of Microsporidium contains less than 3 Mb while the genomes of several Basidiomycetes and Ascomycetes greatly exceed 100 Mb. Likewise chromosome numbers and ploidy levels can differ even between closely related species. The differences in genome architecture among fungi reflect the interplay of different mutational processes as well as the population biology of the different species. Comparative genome studies have elucidated the underlying mechanisms of genome evolution in different groups of fungi and have provided insight into species-specific genomic traits. Mobile genetic elements have been instrumental in shaping the genome architecture and gene content in many fungal species. In many pathogenic fungi the mobile genetic elements even play a crucial role in rapid adaptive evolution by mediating high rates of sequence mutations, chromosomal rearrangements, and ploidy changes. But in many species mobile elements are efficiently restricted by defense mechanisms, which have evolved to suppress and regulate parasitic elements. Different rates of genome dynamic and adaptive evolution may reflect varying effective population sizes through which genetic drift and natural selection have differentially affected genome architecture in fungi over time.     

Evolution of mutational robustness in an RNA virus

Mutational (genetic) robustness is phenotypic constancy in the face of mutational changes to the genome. Robustness is critical to the understanding of evolution because phenotypically expressed genetic variation is the fuel of natural selection. Nonetheless, the evidence for adaptive evolution of mutational robustness in biological populations is controversial. Robustness should be selectively favored when mutation rates are high, a common feature of RNA viruses. However, selection for robustness may be relaxed under virus co-infection because complementation between virus genotypes can buffer mutational effects. We therefore hypothesized that selection for genetic robustness in viruses will be weakened with increasing frequency of co-infection. To test this idea, we used populations of RNA phage φ6 that were experimentally evolved at low and high levels of co-infection and subjected lineages of these viruses to mutation accumulation through population bottlenecking. The data demonstrate that viruses evolved under high co-infection show relatively greater mean magnitude and variance in the fitness changes generated by addition of random mutations, confirming our hypothesis that they experience weakened selection for robustness. Our study further suggests that co-infection of host cells may be advantageous to RNA viruses only in the short term. In addition, we observed higher mutation frequencies in the more robust viruses, indicating that evolution of robustness might foster less-accurate genome replication in RNA viruses.