Loss of sexual reproduction is considered an evolutionary dead end for metazoans, but bdelloid rotifers challenge this view as they appear to have persisted asexually for millions of years 1 . Neither male sex organs nor meiosis have ever been observed in these microscopic animals: oocytes are formed through mitotic divisions, with no reduction of chromosome number and no indication of chromosome pairing 2 . However, current evidence does not exclude that they may engage in sex on rare, cryptic occasions. Here we report the genome of a bdelloid rotifer, Adineta vaga (Davis, 1873) 3 , and show that its structure is incompatible with conventional meiosis. At gene scale, the genome of A. vaga is tetraploid and comprises both anciently duplicated segments and less divergent allelic regions. However, in contrast to sexual species, the allelic regions are rearranged and sometimes even found on the same chromosome. Such structure does not allow meiotic pairing; instead, we find abundant evidence of gene conversion, which may limit the accumulation of deleterious mutations in the absence of meiosis. Gene families involved in resistance to oxidation, carbohydrate metabolism and defence against transposons are significantly expanded, which may explain why transposable elements cover only 3% of the assembled sequence. Furthermore, 8% of the genes are likely to be of non-metazoan origin and were probably acquired horizontally. This apparent convergence between bdelloids and prokaryotes sheds new light on the evolutionary significance of sex.With more than 460 described species 4 , bdelloid rotifers ( Fig. 1) represent the highest metazoan taxonomic rank in which males, hermaphrodites and meiosis are unknown. Such persistence and diversification of an ameiotic clade of animals are in contradiction with the supposed long-term disadvantages of asexuality, making bdelloids an 'evolutionary scandal' 5 . Another unusual feature of bdelloid rotifers is their extreme resistance to desiccation at any stage of their life cycle 6 , enabling these microscopic animals to dwell in ephemeral freshwater habitats such as mosses, lichens and forest litter; this ability is presumably the source of their extreme resistance to ionizing radiation 7 .We assembled the genome of a clonal A. vaga lineage into separate haplotypes with a N 50 of 260 kilobases (kb) (that is, half of the assembly was composed of fragments longer than 260 kb). Assembly size was 218 megabases (Mb) but 26 Mb of the sequence had twice the average sequencing coverage, suggesting that some nearly identical regions were not resolved during assembly ( Supplementary Fig. 3); hence, the total genome size is likely to be 244 Mb, which corresponds to the estimate obtained independently using fluorometry (Supplementary Note C2). Annotation of the complete assembly (including all haplotypes) yielded 49,300 genes. Intragenomic sequence comparisons revealed numerous homologous blocks with conserved gene order (colinear regions). For each such block we computed the per-site synonymous d...

Horizontal gene transfer in metazoans has been documented in only a few species and is usually associated with endosymbiosis or parasitism. By contrast, in bdelloid rotifers we found many genes that appear to have originated in bacteria, fungi, and plants, concentrated in telomeric regions along with diverse mobile genetic elements. Bdelloid proximal gene-rich regions, however, appeared to lack foreign genes, thereby resembling those of model metazoan organisms. Some of the foreign genes were defective, whereas others were intact and transcribed; some of the latter contained functional spliceosomal introns. One such gene, apparently of bacterial origin, was overexpressed in Escherichia coli and yielded an active enzyme. The capture and functional assimilation of exogenous genes may represent an important force in bdelloid evolution.

Sexual reproduction allows deleterious transposable elements to proliferate in populations, whereas the loss of sex, by preventing their spread, has been predicted eventually to result in a population free of such elements [Hickey, D. A. (1982) Genetics 101, 519 -531]. We tested this expectation by screening representatives of a majority of animal phyla for LINE-like and gypsy-like reverse transcriptases and mariner͞Tc1-like transposases. All species tested positive for reverse transcriptases except rotifers of the class Bdelloidea, the largest eukaryotic taxon in which males, hermaphrodites, and meiosis are unknown and for which ancient asexuality is supported by molecular genetic evidence. Mariner-like transposases are distributed sporadically among species and are present in bdelloid rotifers. The remarkable lack of LINE-like and gypsy-like retrotransposons in bdelloids and their ubiquitous presence in other taxa support the view that eukaryotic retrotransposons are sexually transmitted nuclear parasites and that bdelloid rotifers evolved asexually.T ransposons are commonly thought to be of universal occurrence in eukaryotes, even though only a few major phyla have actually been examined. We tested representatives of the majority of animal phyla for the most prominent superfamilies of the two classes of eukaryotic transposons: (i) retrotransposons, which transpose via an RNA intermediate copied into DNA by an element-encoded reverse transcriptase (RTase), and (ii) DNA transposons, which transpose as DNA by a cut-and-paste mechanism using an element-encoded transposase (1, 2). Amino acid sequences of RTases or transposases exhibit more similarity within the same superfamily, even in distantly related host species, than between superfamilies, even within the same host. This similarity allows the design of PCR screens capable of detecting transposons belonging to a given superfamily in diverse taxa. Even within relatively conserved domains, however, sequence diversity necessitates the use of highly degenerate primer pools, in most cases giving no transposon-specific bands when only a single pair of domains is targeted. To achieve the necessary specificity, we developed a two-step PCR procedure that takes advantage of the presence of more than two conserved domains in each enzyme. This procedure was used to screen for RTases of LINE-like and gypsy-like superfamilies and DNA transposases of the mariner͞Tc1-like superfamily. Melton (Harvard University), (r) M. Evgen'ev (Institute of Molecular Biology, Moscow), (s) S. Palumbi (Harvard University), and (t) Sigma. Letter designations correspond to superscripts in Table 1. Materials and MethodsPCR, Cloning, and Sequencing. RTase sequences were first amplified with primers GAYITIINNNVNGSNTWY (A) and ANI-NINAINCCNARRWM (E) for 5 min at 95°C, 10 ϫ (1 min at 94°C, 1 min at 47°C, 1 min at 72°C), 50 ϫ (20 sec at 94°C, 45 sec at 52°C, 45 sec at 72°C), 10 min at 72°C. Second-step primers were INGGNIBNCSNCARGG (B-LINE) and RNNRNRT-CRTCNGCRWA (C-LINE) or HIIDBNNTNCCNTTYGG (Bgyp)...

The evolutionary origin of telomerases, enzymes that maintain the ends of linear chromosomes in most eukaryotes, is a subject of debate. Penelope-like elements (PLEs) are a recently described class of eukaryotic retroelements characterized by a GIY-YIG endonuclease domain and by a reverse transcriptase domain with similarity to telomerases and group II introns. Here we report that a subset of PLEs found in bdelloid rotifers, basidiomycete fungi, stramenopiles, and plants, representing four different eukaryotic kingdoms, lack the endonuclease domain and are located at telomeres. The 5 truncated ends of these elements are telomereoriented and typically capped by species-specific telomeric repeats. Most of them also carry several shorter stretches of telomeric repeats at or near their 3 ends, which could facilitate utilization of the telomeric G-rich 3 overhangs to prime reverse transcription. Many of these telomere-associated PLEs occupy a basal phylogenetic position close to the point of divergence from the telomerase-PLE common ancestor and may descend from the missing link between early eukaryotic retroelements and present-day telomerases.reverse transcriptase ͉ telomerase ͉ transposable elements G enomic DNA in many eukaryotes is composed, to a large extent, of transposable elements (TEs), especially retrotransposons, which multiply via an RNA intermediate copied into DNA by reverse transcriptase (RT) and inserted into new sites by an endonuclease (EN)/integrase. Although RT creates new copies, DNA cleavage is essential for TE proliferation, i.e., insertion into previously unoccupied sites. Integrases of retrovirus-like (LTR) retrotransposons insert dsDNA into chromosomes, whereas EN of non-LTR retrotransposons generate the 3ЈOH-end that primes cDNA synthesis directly onto the chromosome (target-primed reverse transcription). The only known eukaryotic RT-containing genes lacking EN domains are telomerase RTs (TERTs), which are not TEs but specialized ribonucleoprotein enzymes maintaining telomeres by repeated copying of a short segment of an unlinked template RNA, primed by the 3ЈOH end of a linear chromosome (see refs. 1-5 for review).Penelope-like elements (PLEs) are a widespread but not very extensively studied class of eukaryotic TEs characterized by a single ORF coding for RT and an unusual GIY-YIG EN domain also found in bacterial group I introns, and by the presence of spliceosomal introns in several members (4, 6). They occupy a special place in retroelement phylogeny by sharing a common ancestor with TERTs (4). PLEs insert relatively randomly throughout the genome, preferring AT-rich targets (6). Indeed, the element-encoded EN, in which the conserved residues are essential for transposition, exhibits some sequence preferences but no pronounced sequence specificity (7).Rotifers of the class Bdelloidea, a large taxon of multicellular freshwater invertebrates considered to be anciently asexual (8,9), contain a distinct group of PLEs, called Athena (4), carrying spliceosomal introns within highly conserve...

We report that two structurally similar transposable elements containing reverse transcriptase (RT), Penelope in Drosophila virilis and Athena in bdelloid rotifers, have proliferated as copies containing introns. The ability of Penelope-like elements (PLEs) to retain introns, their separate phylogenetic placement and their peculiar structural features make them a novel class of eukaryotic retroelements.

The genomes of virtually all sexually reproducing species contain transposable elements. Although active elements generally transpose more rapidly than they are inactivated by mutation or excision, their number can be kept in check by purifying selection if its effectiveness becomes disproportionately greater as their copy number increases. In sexually reproducing species, such synergistic selection can result from ectopic crossing-over or from homologous recombination under negative epistasis. In addition, there may be controls on transposon activity that are associated with meiosis. Because a sexual lineage that abandons sex must lack such mechanisms, it may be driven to extinction by the unchecked proliferation of deleterious transposons inherited from its sexual progenitor. An important component of the evolutionary advantage of sex over asex may therefore lie in the ability of sex, despite facilitating the spread of deleterious elements within interbreeding populations, also to restrain their intragenomic proliferation.

HeT-A elements are non-long terminal repeat (non-LTR) retrotransposons found in head-to-tail arrays on Drosophila chromosome ends, where they form telomeres. We report that HeT-A promoter activity is located in the 3' end of the element, unlike the 5' location seen for other non-LTR retrotransposons. In HeT-A arrays the 3' sequence of one element directs transcription of its downstream neighbor. Because the upstream promoter has the same sequence as the 3' end of the transcribed element, the HeT-A promoter is effectively equivalent to a 5' LTR in both structure and function. Retroviruses and LTR retrotransposons have their promoters and transcription initiation sites in their 5' LTRs. Thus HeT-A appears to have the structure of an evolutionary intermediate between non-LTR and LTR retrotransposons.

Reverse transcriptases (RTs) polymerize DNA on RNA templates. They fall into several structurally related but distinct classes and form an assemblage of RT-like enzymes that, in addition to RTs, also includes certain viral RNA-dependent RNA polymerases (RdRP) synthesizing RNA on RNA templates. It is generally believed that most RT-like enzymes originate from retrotransposons or viruses and have no specific function in the host cell, with telomerases being the only notable exception. Here we report on the discovery and properties of a unique class of RT-related cellular genes collectively named rvt. We present evidence that rvts are not components of retrotransposons or viruses, but single-copy genes with a characteristic domain structure that may contain introns in evolutionarily conserved positions, occur in syntenic regions, and evolve under purifying selection. These genes can be found in all major taxonomic groups including protists, fungi, animals, plants, and even bacteria, although they exhibit patchy phylogenetic distribution in each kingdom. We also show that the RVT protein purified from one of its natural hosts, Neurospora crassa, exists in a multimeric form and has the ability to polymerize NTPs as well as dNTPs in vitro, with a strong preference for NTPs, using Mn 2+ as a cofactor. The existence of a previously unknown class of single-copy RT-related genes calls for reevaluation of the current views on evolution and functional roles of RNA-dependent polymerases in living cells.D NA-dependent polymerases are essential for cellular function, as they mediate the flow of genetic information from DNA to RNA to proteins (1). In contrast, RNA-dependent polymerases have long been associated with replication of selfish and parasitic genetic elements, such as viruses or transposons. Although the discovery of reverse transcriptase (RT) challenged the concept of unidirectionality of the flow of genetic information, this reverse direction has been reserved for retroviruses, pararetroviruses (hepadna-and caulimoviruses), and other RT-containing multicopy entities such as non-LTR and LTR retrotransposons, group II introns, retrons, and retroplasmids, as well as occasional retro (pseudo)genes (2-4). Similarly, viral RNA-dependent RNA polymerases (RdRPs), enzymes structurally related to RTs, serve to replicate the genomes of viruses that use RNA as genetic material (5). These and certain other polymerases are unified by the architecture known as "right hand," composed of the three subdomains called fingers, palm, and thumb (6). Like all polymerases, they use two-metal-ion catalysis for phosphoryl transfer reactions resulting in nucleotide addition.In 1997, this diverse superfamily of enzymes was joined by the telomerase reverse transcriptase (TERT), a specialized RT that maintains the ends of eukaryotic linear chromosomes by addition of short G-rich repeated DNA sequences that are copied multiple times via reverse transcription of a specific region of the associated RNA template constituting part of the holoenzyme (...