Thomas G. Doak

RNA-mediated epigenetic programming of a genome-rearrangement pathway

Genome-wide DNA rearrangements occur in many eukaryotes but are most exaggerated in ciliates, making them ideal model systems for epigenetic phenomena. During development of the somatic macronucleus, Oxytricha trifallax destroys 95% of its germ line, severely fragmenting its chromosomes, and then unscrambles hundreds of thousands of remaining fragments by permutation or inversion. Here we demonstrate that DNA or RNA templates can orchestrate these genome rearrangements in Oxytricha, supporting an epigenetic model for sequence-dependent comparison between germline and somatic genomes. A complete RNA cache of the maternal somatic genome may be available at a specific stage during development to provide a template for correct and precise DNA rearrangement. We show the existence of maternal RNA templates that could guide DNA assembly, and that disruption of specific RNA molecules disables rearrangement of the corresponding gene. Injection of artificial templates reprogrammes the DNA rearrangement pathway, suggesting that RNA molecules guide genome rearrangement.

Drift-barrier hypothesis and mutation-rate evolution.

Mutation dictates the tempo and mode of evolution, and like all traits, the mutation rate is subject to evolutionary modification. Here, we report refined estimates of the mutation rate for a prokaryote with an exceptionally small genome and for a unicellular eukaryote with a large genome. Combined with prior results, these estimates provide the basis for a potentially unifying explanation for the wide range in mutation rates that exists among organisms. Natural selection appears to reduce the mutation rate of a species to a level that scales negatively with both the effective population size (Ne), which imposes a drift barrier to the evolution of molecular refinements, and the genomic content of coding DNA, which is proportional to the target size for deleterious mutations. As a consequence of an expansion in genome size, some microbial eukaryotes with large Ne appear to have evolved mutation rates that are lower than those known to occur in prokaryotes, but multicellular eukaryotes have experienced elevations in the genome-wide deleterious mutation rate because of substantial reductions in Ne.

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

With more chromosomes than any other sequenced genome, the macronuclear genome of Oxytricha trifallax has a unique and complex architecture, including alternative fragmentation and predominantly single-gene chromosomes.

A Parsimony Approach to Biological Pathway Reconstruction/Inference for Genomes and Metagenomes

A common biological pathway reconstruction approach -- as implemented by many automatic biological pathway services (such as the KAAS and RAST servers) and the functional annotation of metagenomic sequences -- starts with the identification of protein functions or families (e.g., KO families for the KEGG database and the FIG families for the SEED database) in the query sequences, followed by a direct mapping of the identified protein families onto pathways. Given a predicted patchwork of individual biochemical steps, some metric must be applied in deciding what pathways actually exist in the genome or metagenome represented by the sequences. Commonly, and straightforwardly, a complete biological pathway can be identified in a dataset if at least one of the steps associated with the pathway is found. We report, however, that this naïve mapping approach leads to an inflated estimate of biological pathways, and thus overestimates the functional diversity of the sample from which the DNA sequences are derived. We developed a parsimony approach, called MinPath (Minimal set of Pathways), for biological pathway reconstructions using protein family predictions, which yields a more conservative, yet more faithful, estimation of the biological pathways for a query dataset. MinPath identified far fewer pathways for the genomes collected in the KEGG database -- as compared to the naïve mapping approach -- eliminating some obviously spurious pathway annotations. Results from applying MinPath to several metagenomes indicate that the common methods used for metagenome annotation may significantly overestimate the biological pathways encoded by microbial communities.

A Functional Role for Transposases in a Large Eukaryotic Genome

Editing the Genome The ciliate Oxytricha trifallax has an unusual genome with the coding regions of genes (the exons) scattered through the genome. The exons are then somehow knitted together following transcription prior to their translation into proteins. As part of this process Oxytricha eliminates all transposable elements, stripping the genome down to 5% of the original germline DNA during development. Nowacki et al. (p. 935, published online 16 April) show that germline-limited transposases appear to be important for these large-scale DNA rearrangements. The ciliate Oxytricha expresses transposase genes to influence thousands of DNA rearrangements required for proper development. Despite comprising much of the eukaryotic genome, few transposons are active, and they usually confer no benefit to the host. Through an exaggerated process of genome rearrangement, Oxytricha trifallax destroys 95% of its germline genome during development. This includes the elimination of all transposon DNA. We show that germline-limited transposase genes play key roles in this process of genome-wide DNA excision, which suggests that transposases function in large eukaryotic genomes containing thousands of active transposons. We show that transposase gene expression occurs during germline-soma differentiation and that silencing of transposase by RNA interference leads to abnormal DNA rearrangement in the offspring. This study suggests a new important role in Oxytricha for this large portion of genomic DNA that was previously thought of as junk.

Diverse CRISPRs Evolving in Human Microbiomes

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, together with cas (CRISPR–associated) genes, form the CRISPR/Cas adaptive immune system, a primary defense strategy that eubacteria and archaea mobilize against foreign nucleic acids, including phages and conjugative plasmids. Short spacer sequences separated by the repeats are derived from foreign DNA and direct interference to future infections. The availability of hundreds of shotgun metagenomic datasets from the Human Microbiome Project (HMP) enables us to explore the distribution and diversity of known CRISPRs in human-associated microbial communities and to discover new CRISPRs. We propose a targeted assembly strategy to reconstruct CRISPR arrays, which whole-metagenome assemblies fail to identify. For each known CRISPR type (identified from reference genomes), we use its direct repeat consensus sequence to recruit reads from each HMP dataset and then assemble the recruited reads into CRISPR loci; the unique spacer sequences can then be extracted for analysis. We also identified novel CRISPRs or new CRISPR variants in contigs from whole-metagenome assemblies and used targeted assembly to more comprehensively identify these CRISPRs across samples. We observed that the distributions of CRISPRs (including 64 known and 86 novel ones) are largely body-site specific. We provide detailed analysis of several CRISPR loci, including novel CRISPRs. For example, known streptococcal CRISPRs were identified in most oral microbiomes, totaling ∼8,000 unique spacers: samples resampled from the same individual and oral site shared the most spacers; different oral sites from the same individual shared significantly fewer, while different individuals had almost no common spacers, indicating the impact of subtle niche differences on the evolution of CRISPR defenses. We further demonstrate potential applications of CRISPRs to the tracing of rare species and the virus exposure of individuals. This work indicates the importance of effective identification and characterization of CRISPR loci to the study of the dynamic ecology of microbiomes.

The Architecture of a Scrambled Genome Reveals Massive Levels of Genomic Rearrangement during Development

Programmed DNA rearrangements in the single-celled eukaryote Oxytricha trifallax completely rewire its germline into a somatic nucleus during development. This elaborate, RNA-mediated pathway eliminates noncoding DNA sequences that interrupt gene loci and reorganizes the remaining fragments by inversions and permutations to produce functional genes. Here, we report the Oxytricha germline genome and compare it to the somatic genome to present a global view of its massive scale of genome rearrangements. The remarkably encrypted genome architecture contains >3,500 scrambled genes, as well as >800 predicted germline-limited genes expressed, and some posttranslationally modified, during genome rearrangements. Gene segments for different somatic loci often interweave with each other. Single gene segments can contribute to multiple, distinct somatic loci. Terminal precursor segments from neighboring somatic loci map extremely close to each other, often overlapping. This genome assembly provides a draft of a scrambled genome and a powerful model for studies of genome rearrangement.

Bats Use Magnetite to Detect the Earth's Magnetic Field

While the role of magnetic cues for compass orientation has been confirmed in numerous animals, the mechanism of detection is still debated. Two hypotheses have been proposed, one based on a light dependent mechanism, apparently used by birds and another based on a “compass organelle” containing the iron oxide particles magnetite (Fe3O4). Bats have recently been shown to use magnetic cues for compass orientation but the method by which they detect the Earths magnetic field remains unknown. Here we use the classic “Kalmijn-Blakemore” pulse re-magnetization experiment, whereby the polarity of cellular magnetite is reversed. The results demonstrate that the big brown bat Eptesicus fuscus uses single domain magnetite to detect the Earths magnetic field and the response indicates a polarity based receptor. Polarity detection is a prerequisite for the use of magnetite as a compass and suggests that big brown bats use magnetite to detect the magnetic field as a compass. Our results indicate the possibility that sensory cells in bats contain freely rotating magnetite particles, which appears not to be the case in birds. It is crucial that the ultrastructure of the magnetite containing magnetoreceptors is described for our understanding of magnetoreception in animals.

Peptidergic signaling in Calanus finmarchicus (Crustacea, Copepoda): in silico identification of putative peptide hormones and their receptors using a de novo assembled transcriptome.

The copepod Calanus finmarchicus is the most abundant zooplankton species in the North Atlantic. While the life history of this crustacean is well studied, little is known about its peptidergic signaling systems despite the fact that these pathways are undoubtedly important components of its physiological/behavioral control systems. Here we have generated and used a de novo assembled transcriptome for C. finmarchicus (206,041 sequences in total) to identify peptide precursor proteins and receptors. Using known protein queries, 34 transcripts encoding peptide preprohormones and 18 encoding peptide receptors were identified. Using a combination of online software programs and homology to known arthropod isoforms, 148 mature peptides were predicted from the deduced precursors, including members of the allatostatin-A, allatostatin-B, allatostatin-C, bursicon, crustacean cardioactive peptide (CCAP), crustacean hyperglycemic hormone, diuretic hormone 31 (DH31), diuretic hormone 44 (DH44), FMRFamide-like peptide (myosuppressin, neuropeptide F [NPF] and extended FL/IRFamide subfamilies), leucokinin, neuroparsin, orcokinin, orcomyotropin, periviscerokinin, RYamide and tachykinin-related peptide (TRP) families. The identified receptors included ones for allatostatin-A, allatostatin-C, bursicon, CCAP, DH31, DH44, ecdysis-triggering hormone, NPF, short NPF, FMRFamide, insulin-like peptide, leucokinin, periviscerokinin, pigment dispersing hormone, and TRP. Developmental profiling of the identified transcripts in embryos, early nauplii, late nauplii, early copepodites, late copepodites, and adult females was also undertaken, with all showing the highest expression levels in the naupliar and copepodite stages. Collectively, these data radically expand the catalog of known C. finmarchicus peptidergic signaling proteins and provide a foundation for experiments directed at understanding the physiological roles served by them in this species.

Extraordinary genome stability in the ciliate Paramecium tetraurelia

Mutation plays a central role in all evolutionary processes and is also the basis of genetic disorders. Established base-substitution mutation rates in eukaryotes range between ∼5 × 10−10 and 5 × 10−8 per site per generation, but here we report a genome-wide estimate for Paramecium tetraurelia that is more than an order of magnitude lower than any previous eukaryotic estimate. Nevertheless, when the mutation rate per cell division is extrapolated to the length of the sexual cycle for this protist, the measure obtained is comparable to that for multicellular species with similar genome sizes. Because Paramecium has a transcriptionally silent germ-line nucleus, these results are consistent with the hypothesis that natural selection operates on the cumulative germ-line replication fidelity per episode of somatic gene expression, with the germ-line mutation rate per cell division evolving downward to the lower barrier imposed by random genetic drift. We observe ciliate-specific modifications of widely conserved amino acid sites in DNA polymerases as one potential explanation for unusually high levels of replication fidelity.