Estimating the Fitness Effects of New Mutations in the Wild Yeast Saccharomyces paradoxus.

The nature of selection acting on a population is in large measure determined by the distribution of fitness effects of new mutations. In this study, we use DNA sequences from four closely related clades of Saccharomyces paradoxus and Saccharomyces cerevisiae to identify and polarize new mutations and estimate their fitness effects. By progressively restricting the analyses to narrower categories of sites, we further seek to characterize sites with predictable mutational effects, that is, unconditionally deleterious, neutral or beneficial. Consistent with previous studies on S. paradoxus, we have failed to find evidence for mutations with beneficial effects, even in regions that were divergent in two outgroup clades, perhaps a consequence of the relatively unchallenged, predominantly asexual and highly inbred lifestyle of this species. On the other hand, there is abundant evidence of deleterious mutations, varying in severity of effect from strongly deleterious to very mild, particularly in regions conserved in the outgroup taxa, indicating a history of persistent purifying selection. Narrowing the analysis down to individual amino acids reduces further the range of effects: for example, mutations changing cysteine are predicted to be nearly always strongly deleterious, whereas those changing arginine, serine, and tyrosine are expected to be nearly neutral. The proportion of mutations with deleterious effects for a particular amino acid is correlated with long-term stasis of that amino acid among highly divergent sequences from a variety of organisms, showing that functionality of sites tends to persist through the diversification of clades and that our findings are also relevant to longer evolutionary times and other taxa.

Transmissible [corrected] dog cancer genome reveals the origin and history of an ancient cell lineage.

Canine transmissible venereal tumor (CTVT) is the oldest known somatic cell lineage. It is a transmissible cancer that propagates naturally in dogs. We sequenced the genomes of two CTVT tumors and found that CTVT has acquired 1.9 million somatic substitution mutations and bears evidence of exposure to ultraviolet light. CTVT is remarkably stable and lacks subclonal heterogeneity despite thousands of rearrangements, copy-number changes, and retrotransposon insertions. More than 10,000 genes carry nonsynonymous variants, and 646 genes have been lost. CTVT first arose in a dog with low genomic heterozygosity that may have lived about 11,000 years ago. The cancer spawned by this individual dispersed across continents about 500 years ago. Our results provide a genetic identikit of an ancient dog and demonstrate the robustness of mammalian somatic cells to survive for millennia despite a massive mutation burden.

Mitochondrial capture by a transmissible cancer.

Canine transmissible venereal tumor (CTVT) is an infectious cell line circulating in many feral dog populations. It originated once, about 10,000 years ago. Phylogenetic analyses of mitochondrial sequences from dogs, wolves, and a geographically diverse collection of CTVT samples indicate that the cancer has periodically acquired mitochondria from its host. We suggest that this may be because the cancer's own mitochondria have a tendency to degenerate, due to high mutation rates and relaxed selection, resulting in host mitochondria being more fit.

Extensive conservation of genomic imbalances in canine transmissible venereal tumors (CTVT) detected by microarray-based CGH analysis.

Canine transmissible venereal tumor (CTVT) is an intriguing cancer that is transmitted naturally as an allograft by transplantation of viable tumor cells from affected to susceptible dogs. At least initially, the tumor is able to evade the host's immune response; thus, CTVT has potential to provide novel insights into tumor immunobiology. The nature of CTVT as a "contagious" cancer, originating from a common ancestral source of infection, has been demonstrated previously by a series of studies comparing geographically distinct tumors at the molecular level. While these studies have revealed that apparently unrelated tumors share a striking degree of karyotypic conservation, technological restraints have limited the ability to investigate the chromosome composition of CTVTs in any detail. We present characterization of a strategically selected panel of CTVT cases using microarray-based comparative genomic hybridization analysis at ~one-megabase resolution. These data show for the first time that the tumor presents with an extensive range of non-random chromosome copy number aberrations that are distributed widely throughout the dog genome. The majority of abnormalities detected were imbalances of small subchromosomal regions, often involving centromeric and telomeric sequences. All cases also showed the sex chromosome complement XO. There was remarkable conservation in the cytogenetic profiles of the tumors analyzed, with only minor variation observed between different cases. These data suggest that the CTVT genome demonstrates a vast degree of both structural and numerical reorganization that is maintained during transmission among the domestic dog population.

Origins and evolution of a transmissible cancer.

Canine transmissible venereal tumor (CTVT) is an infectious disease of dogs. Remarkably, the infectious agent is the cancerous cell itself. To investigate its origin and spread, we collected 37 tumor samples from four continents and determined their evolutionary relationships using microsatellite length differences and microarray-based comparative genomic hybridization (aCGH). The different tumors show very little microsatellite variation, and the pattern of variation that does exist is consistent with a purely asexual mode of transmission. Approximately one quarter of the loci scored by aCGH show copy number variation relative to normal dogs, again with little variation among different tumor samples. Sequence analysis of the RPPH1 gene indicates an origin from either dogs or wolves, and microsatellite analysis indicates that the tumor is more than 6000 years old, and perhaps originated when dogs were first domesticated. By contrast, the common ancestor of extant tumors lived within the last few hundred years, long after the first tumor. The genetic and genomic patterns we observe are typical of those expected of asexual pathogens, and the extended time since first origin may explain the many remarkable adaptations that have enabled this mammalian cell lineage to live as a unicellular pathogen.

Degeneration and domestication of a selfish gene in yeast: molecular evolution versus site-directed mutagenesis.

VDE is a homing endonuclease gene in yeasts with an unusual evolutionary history including horizontal transmission, degeneration, and domestication into the mating-type switching locus HO. We investigate here the effects of these features on its molecular evolution. In addition, we correlate rates of evolution with results from site-directed mutagenesis studies. Functional elements have lower rates of evolution than degenerate ones and higher conservation at functionally important sites. However, functionally important and unimportant sites are equally likely to have been involved in the evolution of new function during the domestication of VDE into HO. The domestication event also indicates that VDE has been lost in some species and that VDE has been present in yeasts for more than 50 Myr.

High copy number in human endogenous retrovirus families is associated with copying mechanisms in addition to reinfection.

There are at least 31 families of human endogenous retroviruses (HERVs), each derived from an independent infection by an exogenous virus. Using evidence of purifying selection on HERV genes, we have shown previously that reinfection by replication-competent elements was the predominant mechanism of copying in some families. Here we analyze the evolution of 17 HERV families using d(N)/d(S) ratios and find a positive relationship between copy number and the use of additional copying mechanisms. All families with more than 200 elements have also used one or more of the following mechanisms: (1) complementation in trans (elements copied by other elements of the same family; HERV-H and ERV-9), (2) retrotransposition in cis (elements copying themselves) within germ-line cells (HERV-K(HML3)), and (3) being copied by non-HERV machinery (HERV-W). We discuss why these other mechanisms are rare in most families and suggest why complementation in trans is significant only in the larger families.

Evolution of divergent DNA recognition specificities in VDE homing endonucleases from two yeast species.

Homing endonuclease genes (HEGs) are mobile DNA elements that are thought to confer no benefit to their host. They encode site-specific DNA endonucleases that perpetuate the element within a species population by homing and disseminate it between species by horizontal transfer. Several yeast species contain the VMA1 HEG that encodes the intein-associated VMA1-derived endonuclease (VDE). The evolutionary state of VDEs from 12 species was assessed by assaying their endonuclease activities. Only two enzymes are active, PI-ZbaI from Zygosaccharomyces bailii and PI-ScaI from Saccharomyces cariocanus. PI-ZbaI cleaves the Z.bailii recognition sequence significantly faster than the Saccharomyces cerevisiae site, which differs at six nucleotide positions. A mutational analysis indicates that PI-ZbaI cleaves the S.cerevisiae substrate poorly due to the absence of a contact that is analogous to one made in PI-SceI between Gln-55 and nucleotides +9/+10. PI-ZbaI cleaves the Z.bailii substrate primarily due to a single base-pair substitution (A/T+5 --> T/A+5). Structural modeling of the PI-ZbaI/DNA complex suggests that Arg-331, which is absent in PI-SceI, contacts T/A+5, and the reduced activity observed in a PI-ZbaI R331A mutant provides evidence for this interaction. These data illustrate that homing endonucleases evolve altered specificity as they adapt to recognize alternative target sites.

Cancer selection.

Cancers are often thought to be selectively neutral. This is because most of the individuals that they kill are post-reproductive. Some cancers, however, kill the young and so select for anticancer adaptations that reduce the chance of death. These adaptations could reduce the somatic mutation rate or the selective value of a mutant clone of cells, or increase the number of stages required for neoplasia. New theory predicts that cancer selection--selection to prevent or postpone deaths due to cancer--should be especially important as animals evolve new morphologies or larger, longer-lived bodies, and might account for some of the differences in the causes of cancer between mice and men.

Adaptation for horizontal transfer in a homing endonuclease.

Selfish genes of no function other than self-propagation are susceptible to degeneration if they become fixed in a population, and regular transfer to new species may be the only means for their long-term persistence. To test this idea we surveyed 24 species of yeast for VDE, a nuclear, intein-associated homing endonuclease gene (HEG) originally discovered in Saccharomyces cerevisiae. Phylogenetic analyses show that horizontal transmission has been a regular occurrence in its evolutionary history. Moreover, VDE appears to be specifically adapted for horizontal transmission. Its 31-bp recognition sequence is an unusually well-conserved region in an unusually well-conserved gene. In addition, the nine nucleotide sites most critical for homing are also unusually well conserved. Such adaptation for horizontal transmission presumably arose as a consequence of selection, both among HEGs at different locations in the genome and among variants at the same location. The frequency of horizontal transmission must therefore be a key feature constraining the distribution and abundance of these genes.