The Genome Sequence of Caenorhabditis briggsae: A Platform for Comparative Genomics

The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.     

fog-2 and the Evolution of Self-Fertile Hermaphroditism in Caenorhabditis

Somatic and germline sex determination pathways have diverged significantly in animals, making comparisons between taxa difficult. To overcome this difficulty, we compared the genes in the germline sex determination pathways of Caenorhabditis elegans and C. briggsae, two Caenorhabditis species with similar reproductive systems and sequenced genomes. We demonstrate that C. briggsae has orthologs of all known C. elegans sex determination genes with one exception: fog-2. Hermaphroditic nematodes are essentially females that produce sperm early in life, which they use for self fertilization. In C. elegans, this brief period of spermatogenesis requires FOG-2 and the RNA-binding protein GLD-1, which together repress translation of the tra-2 mRNA. FOG-2 is part of a large C. elegans FOG-2-related protein family defined by the presence of an F-box and Duf38/FOG-2 homogy domain. A fog-2-related gene family is also present in C. briggsae, however, the branch containing fog-2 appears to have arisen relatively recently in C. elegans, post-speciation. The C-terminus of FOG-2 is rapidly evolving, is required for GLD-1 interaction, and is likely critical for the role of FOG-2 in sex determination. In addition, C. briggsae gld-1 appears to play the opposite role in sex determination (promoting the female fate) while maintaining conserved roles in meiotic progression during oogenesis. Our data indicate that the regulation of the hermaphrodite germline sex determination pathway at the level of FOG-2/GLD-1/tra-2 mRNA is fundamentally different between C. elegans and C. briggsae, providing functional evidence in support of the independent evolution of self-fertile hermaphroditism. We speculate on the convergent evolution of hermaphroditism in Caenorhabditis based on the plasticity of the C. elegans germline sex determination cascade, in which multiple mutant paths yield self fertility.     

The Obligate Human Pathogen, Neisseria gonorrhoeae, Is Polyploid

We show using several methodologies that the Gram-negative, diplococcal-bacterium Neisseria gonorrhoeae has more than one complete genome copy per cell. Gene dosage measurements demonstrated that only a single replication initiation event per chromosome occurs per round of cell division, and that there is a single origin of replication. The region containing the origin does not encode any genes previously associated with bacterial origins of replication. Quantitative PCR results showed that there are on average three genome copies per coccal cell unit. These findings allow a model for gonococcal DNA replication and cell division to be proposed, in which a minimum of two chromosomal copies exist per coccal unit within a monococcal or diplococcal cell, and these chromosomes replicate in unison to produce four chromosomal copies during cell division. Immune evasion via antigenic variation is an important mechanism that allows these organisms to continually infect a high risk population of people. We propose that polyploidy may be necessary for the high frequency gene conversion system that mediates pilin antigenic variation and the propagation of N. gonorrhoeae within its human hosts.     

The Complete Mitochondrial Genome of the Entomopathogenic Nematode Steinernema carpocapsae: Insights into Nematode Mitochondrial DNA Evolution and Phylogeny

We determined the complete sequence of the mitochondrial DNA of the entomopathogenic nematode Steinernema carpocapsae and analyzed its structure and composition as well as the secondary structures predicted for its tRNAs and rRNAs. Almost the complete genome has been amplified in one fragment with long PCR and sequenced using a shotgun strategy. The 13,925-bp genome contains genes for 2 rRNAs, 22 tRNAs, and 12 proteins and lacks an ORF encoding ATPase subunit 8. Four initiation codons were inferred, TTT, TTA, ATA, and ATT, most of the genes ended with TAA or TAG, and only two had a T as an incomplete stop codon. All predicted tRNAs showed the nonconventional secondary structure typical of Secernentea. Although we were able to fold the sequences of trnN, trnD, and trnC into more conventional cloverleaf structures after adding adjacent nucleotides, northern blot experiments showed that the nonstandard tRNAs are actually expressed. Phylogenetic and comparative analyses showed that the mitochondrial genome of S. carpocapsae is more closely related to the genomes of A. suum and C. elegans than to that of Strongyloides stercoralis. This finding does not support the phylogeny based on nuclear small subunit ribosomal DNA sequences previously published. This discrepancy may result from differential reproductive strategies and/or differential selective pressure acting on nuclear and mitochondrial genes. The distinctive characteristics observed among mitochondrial genomes of Secernentea may have arisen to counteract the deleterious effects of Muller’s ratchet, which is probably enhanced by the reproductive strategies and selective pressures referred to above. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zardoya]     

The Evolution of Senescence and Post-Reproductive Lifespan in Guppies (Poecilia reticulata)

The study of post-reproductive lifespan has been of interest primarily with regard to the extended post-menopausal lifespan seen in humans. This unusual feature of human demography has been hypothesized to have evolved because of the “grandmother” effect, or the contributions that post-reproductive females make to the fitness of their children and grandchildren. While some correlative analyses of human populations support this hypothesis, few formal, experimental studies have addressed the evolution of post-reproductive lifespan. As part of an ongoing study of life history evolution in guppies, we compared lifespans of individual guppies derived from populations that differ in their extrinsic mortality rates. Some of these populations co-occur with predators that increase mortality rate, whereas other nearby populations above barrier waterfalls are relatively free from predation. Theory predicts that such differences in extrinsic mortality will select for differences in the age at maturity, allocation of resources to reproduction, and patterns of senescence, including reproductive declines. As part of our evaluation of these predictions, we quantified differences among populations in post-reproductive lifespan. We present here the first formal, comparative study of the evolution of post-reproductive lifespan as a component of the evolution of the entire life history. Guppies that evolved with predators and that experienced high extrinsic mortality mature at an earlier age but also have longer lifespans. We divided the lifespan into three non-overlapping components: birth to age at first reproduction, age at first reproduction to age at last reproduction (reproductive lifespan), and age at last reproduction to age at death (post-reproductive lifespan). Guppies from high-predation environments live longer because they have a longer reproductive lifespan, which is the component of the life history that can make a direct contribution to individual fitness. We found no differences among populations in post-reproductive lifespan, which is as predicted since there can be no contribution of this segment of the life history to an individual's fitness. Prior work on the evolution of post-reproductive lifespan has been dominated by speculation and correlative analyses. We show here that this component of the life history is accessible to formal study as part of experiments that quantify the different segments of an individual's life history. Populations of guppies subject to different mortality pressures from predation evolved differences in total lifespan, but not in post-reproductive lifespan. Rather than showing the direct effects of selection characterizing other life-history traits, post-reproductive lifespan in these fish appears to be a random add-on at the end of the life history. These findings support the hypothesis that differences in lifespan evolving in response to selection are confined to the reproductive lifespan, or those segments of the life history that make a direct contribution to fitness. We also show, for the first time, that fish can have reproductive senescence and extended post-reproductive lifespans despite the general observation that they are capable of producing new primary oocytes throughout their lives.     

Genome Announcement: The Draft Genome of the Carrot Cyst Nematode Heterodera Carotae

Heterodera carotae, the carrot cyst nematode, is a significant pest affecting carrot globally. Here we present the draft genome of H. carotae, which was generated from short read libraries from Illumina HiSeq technology, and the corresponding genome annotation.     

GAD2 on Chromosome 10p12 Is a Candidate Gene for Human Obesity

The gene GAD2 encoding the glutamic acid decarboxylase enzyme (GAD65) is a positional candidate gene for obesity on Chromosome 10p11–12, a susceptibility locus for morbid obesity in four independent ethnic populations. GAD65 catalyzes the formation of γ-aminobutyric acid (GABA), which interacts with neuropeptide Y in the paraventricular nucleus to contribute to stimulate food intake. A case-control study (575 morbidly obese and 646 control subjects) analyzing GAD2 variants identified both a protective haplotype, including the most frequent alleles of single nucleotide polymorphisms (SNPs) +61450 C>A and +83897 T>A (OR = 0.81, 95% CI [0.681–0.972], p = 0.0049) and an at-risk SNP (−243 A>G) for morbid obesity (OR = 1.3, 95% CI [1.053–1.585], p = 0.014). Furthermore, familial-based analyses confirmed the association with the obesity of SNP +61450 C>A and +83897 T>A haplotype (χ² = 7.637, p = 0.02). In the murine insulinoma cell line βTC3, the G at-risk allele of SNP −243 A>G increased six times GAD2 promoter activity (p < 0.0001) and induced a 6-fold higher affinity for nuclear extracts. The −243 A>G SNP was associated with higher hunger scores (p = 0.007) and disinhibition scores (p = 0.028), as assessed by the Stunkard Three-Factor Eating Questionnaire. As GAD2 is highly expressed in pancreatic β cells, we analyzed GAD65 antibody level as a marker of β-cell activity and of insulin secretion. In the control group, −243 A>G, +61450 C>A, and +83897 T>A SNPs were associated with lower GAD65 autoantibody levels (p values of 0.003, 0.047, and 0.006, respectively). SNP +83897 T>A was associated with lower fasting insulin and insulin secretion, as assessed by the HOMA-B% homeostasis model of β-cell function (p = 0.009 and 0.01, respectively). These data support the hypothesis of the orexigenic effect of GABA in humans and of a contribution of genes involved in GABA metabolism in the modulation of food intake and in the development of morbid obesity.     

Survey Sequencing and Comparative Analysis of the Elephant Shark (Callorhinchus milii) Genome

Owing to their phylogenetic position, cartilaginous fishes (sharks, rays, skates, and chimaeras) provide a critical reference for our understanding of vertebrate genome evolution. The relatively small genome of the elephant shark, Callorhinchus milii, a chimaera, makes it an attractive model cartilaginous fish genome for whole-genome sequencing and comparative analysis. Here, the authors describe survey sequencing (1.4× coverage) and comparative analysis of the elephant shark genome, one of the first cartilaginous fish genomes to be sequenced to this depth. Repetitive sequences, represented mainly by a novel family of short interspersed element–like and long interspersed element–like sequences, account for about 28% of the elephant shark genome. Fragments of approximately 15,000 elephant shark genes reveal specific examples of genes that have been lost differentially during the evolution of tetrapod and teleost fish lineages. Interestingly, the degree of conserved synteny and conserved sequences between the human and elephant shark genomes are higher than that between human and teleost fish genomes. Elephant shark contains putative four Hox clusters indicating that, unlike teleost fish genomes, the elephant shark genome has not experienced an additional whole-genome duplication. These findings underscore the importance of the elephant shark as a critical reference vertebrate genome for comparative analysis of the human and other vertebrate genomes. This study also demonstrates that a survey-sequencing approach can be applied productively for comparative analysis of distantly related vertebrate genomes.     

Accelerated Evolution of the ASPM Gene Controlling Brain Size Begins Prior to Human Brain Expansion

Primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by global reduction in cerebral cortical volume. The microcephalic brain has a volume comparable to that of early hominids, raising the possibility that some MCPH genes may have been evolutionary targets in the expansion of the cerebral cortex in mammals and especially primates. Mutations in ASPM, which encodes the human homologue of a fly protein essential for spindle function, are the most common known cause of MCPH. Here we have isolated large genomic clones containing the complete ASPM gene, including promoter regions and introns, from chimpanzee, gorilla, orangutan, and rhesus macaque by transformation-associated recombination cloning in yeast. We have sequenced these clones and show that whereas much of the sequence of ASPM is substantially conserved among primates, specific segments are subject to high Ka/Ks ratios (nonsynonymous/synonymous DNA changes) consistent with strong positive selection for evolutionary change. The ASPM gene sequence shows accelerated evolution in the African hominoid clade, and this precedes hominid brain expansion by several million years. Gorilla and human lineages show particularly accelerated evolution in the IQ domain of ASPM. Moreover, ASPM regions under positive selection in primates are also the most highly diverged regions between primates and nonprimate mammals. We report the first direct application of TAR cloning technology to the study of human evolution. Our data suggest that evolutionary selection of specific segments of the ASPM sequence strongly relates to differences in cerebral cortical size.     

The Wolbachia genome of Brugia malayi: endosymbiont evolution within a human pathogenic nematode

Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.     

The Streamlined Genome of Phytomonas spp. Relative to Human Pathogenic Kinetoplastids Reveals a Parasite Tailored for Plants

Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.     

Genome wide computational analysis of Brugia malayi helicases: a comparison with human host

The availability of Brugia malayi genome sequence has paved ways for the search of homologues for a variety of genes. Helicases are ubiquitous enzymes involved in all the nucleic acid metabolic pathways and are essential for the development and growth. The genome wide analysis of B. malayi for different helicases showed the presence of a number of DEAD box helicases, 7 DEAH box helicases, RecQ helicases, repair helicases, super killer helicases, MCM2-7 complex, Rad54 and two subunits of Ku helicase. The comparison of protein sequence of each helicase with its human counterpart indicated characteristic differences in filarial helicases. There are noticeable differences in some of the filarial helicases such as DHX35, RecQL1 and Ku. Further characterization of these helicases will help in understanding physiological significance of these helicases in filarial parasites, which in future can be utilized for chemotherapy of parasitic infection.     

Resistance Evolution to Bt Crops: Predispersal Mating of European Corn Borers

Over the past decade, the high-dose refuge (HDR) strategy, aimed at delaying the evolution of pest resistance to Bacillus thuringiensis (Bt) toxins produced by transgenic crops, became mandatory in the United States and is being discussed for Europe. However, precopulatory dispersal and the mating rate between resident and immigrant individuals, two features influencing the efficiency of this strategy, have seldom been quantified in pests targeted by these toxins. We combined mark-recapture and biogeochemical marking over three breeding seasons to quantify these features directly in natural populations of Ostrinia nubilalis, a major lepidopteran corn pest. At the local scale, resident females mated regardless of males having dispersed beforehand or not, as assumed in the HDR strategy. Accordingly, 0–67% of resident females mating before dispersal did so with resident males, this percentage depending on the local proportion of resident males (0% to 67.2%). However, resident males rarely mated with immigrant females (which mostly arrived mated), the fraction of females mating before dispersal was variable and sometimes substantial (4.8% to 56.8%), and there was no evidence for male premating dispersal being higher. Hence, O. nubilalis probably mates at a more restricted spatial scale than previously assumed, a feature that may decrease the efficiency of the HDR strategy under certain circumstances, depending for example on crop rotation practices.     

Mutation Exposed: A Neutral Explanation for Extreme Base Composition of an Endosymbiont Genome

The influence of neutral mutation pressure versus selection on base composition evolution is a subject of considerable controversy. Yet the present study represents the first explicit population genetic analysis of this issue in prokaryotes, the group in which base composition variation is most dramatic. Here, we explore the impact of mutation and selection on the dynamics of synonymous changes in Buchnera aphidicola, the AT-rich bacterial endosymbiont of aphids. Specifically, we evaluated three forms of evidence. (i) We compared the frequencies of directional base changes (ATGC vs. GCAT) at synonymous sites within and between Buchnera species, to test for selective preference versus effective neutrality of these mutational categories. Reconstructed mutational changes across a robust intraspecific phylogeny showed a nearly 1:1 ATGC:GCAT ratio. Likewise, stationarity of base composition among Buchnera species indicated equal rates of ATGC and GCAT substitutions. The similarity of these patterns within and between species supported the neutral model. (ii) We observed an equivalence of relative per-site AT mutation rate and current AT content at synonymous sites, indicating that base composition is at mutational equilibrium. (iii) We demonstrated statistically greater equality in the frequency of mutational categories in Buchnera than in parallel mammalian studies that documented selection on synonymous sites. Our results indicate that effectively neutral mutational pressure, rather than selection, represents the major force driving base composition evolution in Buchnera. Thus they further corroborate recent evidence for the critical role of reduced N_e in the molecular evolution of bacterial endosymbionts.Reviewing Editor: Dr. J. William Ballard     

The Molecular Biology of a Bacterial Endosymbiont1

ABSTRACT. The marine ciliate Parauronema acutum harbors a group of symbiotic bacteria, termed xenosomes, which reside exclusively in the cytoplasm where they grow and divide in a remarkable synchronism with the host. When released into the ambient culture medium by gentle rupture of the protozoans, the symbionts can infect homologous as well as heterologous Parauronema stocks and, oddly, Miamiensis avidus, a distantly related marine ciliate. Xenosomes from certain Parauronema stocks can kill other marine ciliates, particularly those of the genus Uronema. Our principal aims with this host-symbiont system have been to study, at the molecular level, the nature of the interaction of the xenosome with the host, infection and the killer effect. Our most recent investigations have been directed toward establishing the phylogenetic origins of the symbionts using molecular approaches. This paper summarizes our previous work and updates our more recent studies on the association of a bacterial symbiont with its protozoan host. The reader is referred to previous reviews for more details on the subject [4, 5].     

Spliceosomal genes in the D. discoideum genome: a comparison with those in H. sapiens, D. melanogaster, A. thaliana and S. cerevisiae

Little is known about pre-mRNA splicing in Dictyostelium discoideum although its genome has been completely sequenced. Our analysis suggests that pre-mRNA splicing plays an important role in D. discoideum gene expression as two thirds of its genes contain at least one intron. Ongoing curation of the genome to date has revealed 40 genes in D. discoideum with clear evidence of alternative splicing, supporting the existence of alternative splicing in this unicellular organism. We identified 160 candidate U2-type spliceosomal proteins and related factors in D. discoideum based on 264 known human genes involved in splicing. Spliceosomal small ribonucleoproteins (snRNPs), PRP19 complex proteins and late-acting proteins are highly conserved in D. discoideum and throughout the metazoa. In non-snRNP and hnRNP families, D. discoideum orthologs are closer to those in A. thaliana, D. melanogaster and H. sapiens than to their counterparts in S. cerevisiae. Several splicing regulators, including SR proteins and CUG-binding proteins, were found in D. discoideum, but not in yeast. Our comprehensive catalog of spliceosomal proteins provides useful information for future studies of splicing in D.βdiscoideum where the efficient genetic and biochemical manipulation will also further our general understanding of pre-mRNA splicing.