Not so bad after all: retroviruses and long terminal repeat retrotransposons as a source of new genes in vertebrates.

Viruses and transposable elements, once considered as purely junk and selfish sequences, have repeatedly been used as a source of novel protein-coding genes during the evolution of most eukaryotic lineages, a phenomenon called 'molecular domestication'. This is exemplified perfectly in mammals and other vertebrates, where many genes derived from long terminal repeat (LTR) retroelements (retroviruses and LTR retrotransposons) have been identified through comparative genomics and functional analyses. In particular, genes derived from gag structural protein and envelope (env) genes, as well as from the integrase-coding and protease-coding sequences, have been identified in humans and other vertebrates. Retroelement-derived genes are involved in many important biological processes including placenta formation, cognitive functions in the brain and immunity against retroelements, as well as in cell proliferation, apoptosis and cancer. These observations support an important role of retroelement-derived genes in the evolution and diversification of the vertebrate lineage.

Sex differentiation in an all-female (XX) rainbow trout population with a genetically governed masculinization phenotype.

Sex determination is known to be male heterogametic in the rainbow trout, Oncorhynchus mykiss; however, scattered observations that deviate from this rather strict genetic control have been reported. Here, we provide a detailed morphological and histological characterization of the gonadal differentiation and development (from 43 days postfertilization to 11 months of age) in an all-female (XX) population with a genetically governed masculinization phenotype. In comparison with control males and females, the gonadal differentiation in these animals was characterized by many perturbations, including significantly fewer germ cells. This decrease in germ cells was confirmed by the significantly decreased expression of 2 germ cell maker genes (vasa and sycp3) in the masculinized XX populations as compared with the control females and control males. Although only a proportion of the total adult population was partially or fully masculinized, this early differentiating phenotype affected nearly all the sampled animals. This suggests that the adult masculinization phenotype is the consequence of an early functional imbalance in ovarian differentiation in the entire population. We hypothesize that the lower number of germ cells that we observed in this population could be one cause of their masculinization.

Sex determination diversity and sex chromosome evolution in poeciliid fish.

Poeciliids, a family of live-bearing freshwater fish, including among others platyfish, swordtails and guppies, fully illustrate the diversity of genetic sex determination mechanisms observed in teleosts. Besides unisexuality, a variety of sex-determining systems has been described in this group of fish, including male and female heterogamety with or without autosomal influence, as well as more complicated situations such as multichromosomal and polyfactorial sex determination. Due to the presence of different mechanisms in closely related species or even between populations within a same species, poeciliids are a very attractive model to study the evolutionary dynamics of sex determination. For one species, the Southern platyfish Xiphophorus maculatus, positional cloning of the master sex-determining gene has been initiated through the construction and sequencing of bacterial artificial chromosome contigs covering the region differentiating the X from the Y chromosome. Initial analysis revealed a high plasticity of the sex-determining region and the absence of synteny with other fish and vertebrate sex chromosomes, indicating an independent evolutionary origin.

Modern genomes with retro-look: retrotransposed elements, retroposition and the origin of new genes.

A fascinating evolutionary facet of retroposition is its ability to generate a dynamic reservoir of sequences for the formation of new genes within genomes. Retroelement genes, such as gag from retrotransposons or envelope genes from endogenous retroviruses, have been repeatedly exapted and domesticated during evolution. Such genes fulfill now useful novel functions in diverse aspects of host biology, for example placenta formation in mammals. New protein-coding genes can also be generated through the reverse transcription of mRNA from 'classical' genes by the enzymatic machinery of autonomous retroelements. Many of these retrogenes, which generally show a modified expression pattern compared to their molecular progenitor, have a testis-biased expression and a potential role in spermatogenesis in different animals. New non-protein-coding RNA genes have also been repeatedly generated through retroposition during evolution. A striking evolutionary parallel has been observed between two such RNA genes, the rodent BC1 and the primate BC200 genes. Although both genes are derived from different types of sequences (tRNA and Alu short interspersed element, respectively), they are both expressed almost specifically in neurons, transported into the dendrites and included in ribonucleoprotein complexes containing the poly(A)-binding protein PABP. Both BC1 and BC200 RNA are able to inhibit translation in vitro and are progenitors of new families of short interspersed elements. These genes, which might play a role in animal behavior, provide an astonishing example of evolutionary convergence in two distinct mammalian lineages, which is also observed for placenta genes derived from endogenous retroviruses. Finally, there are indications that genes for small nucleolar RNAs (snoRNAs) and possibly microRNAs (miRNAs) can also be duplicated via retroposition. Taken together, these observations definitely demonstrate the major role of retroposition as mediator of genomic plasticity and contributor to gene novelties. Therefore, the 'retro-look' of genomes is in fact indicative of their modernity.

Governing sex determination in fish: regulatory putsches and ephemeral dictators.

In contrast to the rather stable regulatory regimes established over more that 100 million years in birds and mammals, sex determination in fish might frequently undergo evolutionary changes bringing the sex-determining cascade under new master sex regulators. This phenomenon, possibly associated with the emergence of new sex chromosomes from autosomes, would explain the frequent switching between sex determination systems observed in fish. In the medaka Oryzias latipes, the Y-specific master sex-determining gene dmrt1bY has been formed through duplication of the autosomal gene dmrt1 onto another autosome, thus generating a new Y chromosome. Dmrt1bY emerged about 10 million years ago and is restricted to several Oryzias species, indicating that the Y chromosome of the medaka is evolutionarily much younger than mammalian and bird sex chromosomes. Fertile males without dmrt1bY have been detected in some medaka populations, and this gene might even have been inactivated in one Oryzias species, indicating the existence of sexual regulators already able to supplant dmrt1bY. Studies on other models have confirmed that fish sex chromosomes are generally young and occurred independently in different fish lineages. The identification of new sex-determining genes in these species will shed new light on the exceptional evolutionary instability governing sex determination in fish.

Synaptonemal complex protein SYCP3 of the rat: evolutionarily conserved domains and the assembly of higher order structures.

SYCP3 is a major structural protein component of vertebrate synaptonemal complexes as well as an important determinant of male fertility, at least in mammals. The elucidation of SYCP3 polymerization properties would provide important information towards our understanding as to how synaptonemal complexes are assembled and disassembled during meiotic prophase. To this end we have investigated the possible contribution of different SYCP3 domains to the assembly of higher order structures. We observed that the evolutionarily conserved domains of the molecule (i.e. the alpha-helix together with the two flanking motifs CM1 and CM2) are not only necessary but also sufficient for SYCP3 polymerization. The relevance of these results for reproduction biology is underscored by recent studies showing that the deletion of the very end of the alpha-helix and CM2 leads to meiosis disruption and infertility in humans.

A family of neofunctionalized Ty3/gypsy retrotransposon genes in mammalian genomes.

A family of at least eleven genes called Mar related to long terminal repeat retrotransposons from the Ty3/gypsy group, including two genes previously identified as such, is present in human and mouse genomes. Single orthologous copies were identified for most Mar genes in different mammals. All of them have lost essential structural features necessary for autonomous retrotransposition before divergence between mouse and human. Three Mar genes also have introns at identical positions in human and mouse. Hence, Mar genes do not correspond to functional retrotransposons. Mar genes evolved under purifying selection, strongly suggesting that they are not pseudogenic relics but rather neofunctionalized retrotransposon genes. All putative Mar proteins display sequence similarity to the capsid-like domain of the Gag protein of Tf1/Sushi retrotransposons. In addition, three Mar proteins have conserved the Gag CCHC zinc finger motif, suggesting a role in nucleic acid binding. Some Mar genes have also retained from their retrotransposon origin a -1 ribosomal frameshifting between the gag-related open reading frame and a region encoding a putative aspartyl protease domain. EST analysis revealed that the majority of Mar genes are expressed in brain as well as in other tissues and organs. Some Mar proteins might function as transcription factors or be involved in the control of cell proliferation and apoptosis. Strikingly, as many as eight Mar genes are located on the X chromosome in human, mouse and other mammals, and at least two of the autosomal genes are subject to imprinting. We suggest that retrotransposons might be a source for epigenetically regulated genes. Epigenetic regulation of these neogenes might be derived from the cellular defense mechanisms having controlled their retrotransposon ancestor.

Diversity and clustered distribution of retrotransposable elements in the compact genome of the pufferfish Tetraodon nigroviridis.

We report the characterization and chromosomal distribution of retroelements in the compact genome of the pufferfish Tetraodon nigroviridis. We have reconstructed partial/complete retroelement sequences, established their phylogenetic relationship to other known eukaryotic retrotransposons, and performed double-color FISH analyses to gain new insights into their patterns of chromosomal distribution. We could identify 43 different reverse transcriptase retrotransposons belonging to the three major known subclasses (14 non-LTR retrotransposons from seven clades, 25 LTR retrotransposons representing the five major known groups, and four Penelope-like elements), and well as two SINEs (non-autonomous retroelements). Such a diversity of retrotransposable elements, which seems to be relatively common in fish but not in mammals, is astonishing in such a compact genome. The total number of retroelements was approximately 3000, roughly representing only 2.6% of the genome of T. nigroviridis. This is much less than in other vertebrate genomes, reflecting the compact nature of the genome of this pufferfish. Major differences in copy number were observed between different clades, indicating differential success in invading and persisting in the genome. Some retroelements displayed evidence of recent activity. Finally, FISH analysis showed that retrotransposable elements preferentially accumulate in specific heterochromatic regions of the genome of T. nigroviridis, revealing a degree of genomic compartmentalization not observed in the human genome.

Genome evolution and biodiversity in teleost fish.

Teleost fish, which roughly make up half of the extant vertebrate species, exhibit an amazing level of biodiversity affecting their morphology, ecology and behaviour as well as many other aspects of their biology. This huge variability makes fish extremely attractive for the study of many biological questions, particularly of those related to evolution. New insights gained from different teleost species and sequencing projects have recently revealed several peculiar features of fish genomes that might have played a role in fish evolution and speciation. There is now substantial evidence that a round of tetraploidization/rediploidization has taken place during the early evolution of the ray-finned fish lineage, and that hundreds of duplicate pairs generated by this event have been maintained over hundreds of millions of years of evolution. Differential loss or subfunction partitioning of such gene duplicates might have been involved in the generation of fish variability. In contrast to mammalian genomes, teleost genomes also contain multiple families of active transposable elements, which might have played a role in speciation by affecting hybrid sterility and viability. Finally, the amazing diversity of sex determination systems and the plasticity of sex chromosomes observed in teleost might have been involved in both pre- and postmating reproductive isolation. Comparison of data generated by current and future genome projects as well as complementary studies in other species will allow one to approach the molecular and evolutionary mechanisms underlying genome diversity in fish, and will certainly significantly contribute to our understanding of gene evolution and function in humans and other vertebrates.

The teleost fish medaka (Oryzias latipes) as genetic model to study gravity dependent bone homeostasis in vivo.

Long-term space flight and microgravity result in bone loss that can be explained by reduced activity of bone-forming cells (osteoblasts) and/or an increase in activity of bone resorbing cells (osteoclasts). Osteoprotegerin (OPG) has been shown to regulate the balance between osteoblast and osteoclast cell numbers and is involved in maintaining constant bone mass under normal gravitational conditions. The small bony fish medaka (Oryzias latipes) has attracted increasing attention as a genetic model system to study normal embryonic developmental and pathological processes. To analyze the molecular mechanisms of bone formation in this small vertebrate, we have isolated two opg genes, opgl and opg2, from medaka. Our phylogenetic analysis reveals that both genes originated from a common ancestor by fish-specific gene duplication and represent the orthologs of the mammalian opg gene. Both opg genes are differentially expressed during embryonic and larval development, in adult tissues and in cultured primary osteoblast-like cells. Furthermore, we have characterized the opg2 promoter region and identified consensus binding sites for the transcription factor core-binding-factor-1A (CBFA1). In mammals, CBFA1 has been shown to be a regulator of opg expression and to be essential for several steps during osteoblast differentiation. Here we show that sequence and expression domains of opg, cbfal and a member of the dlx gene family are highly conserved between medaka and higher vertebrates. This suggests that not only single genes but entire genetic networks for bone formation are conserved between teleosts and mammals. These findings will open medaka fish as a genetic model to monitor bone formation under different gravity conditions in a living whole animal allowing the identification of novel factors involved in bone homeostasis.

Sex determination and sex chromosome evolution in the medaka, Oryzias latipes, and the platyfish, Xiphophorus maculatus.

The first vertebrate master sex-determining gene different from Sry has been very recently discovered in a small aquarium fish, the medaka (Oryzias latipes). In this fish, the X and Y chromosomes apparently differ only by a 250-kb Y-specific region containing dmrt1bY (also called DMY and dmrt1Y), a male-specific copy of the autosomal gene dmrt1. Dmrt1 is a putative transcription factor probably involved in testis formation in different vertebrate lineages. Dmrt1bY is the only gene having escaped the drastic process of degeneration that devastated the small Y-specific region of the medaka. Mutations leading to truncation or lower expression of dmrt1bY result in XY male-to-female sex reversal. Hence, both genetic and functional evidences converge in making dmrt1bY an outstanding candidate for the function of a master sex-determining gene in fish. Nevertheless, dmrt1bY could not be detected in certain other Oryzias species or in more divergent fishes. Phylogenetic analysis revealed that the duplication of the autosomal dmrt1 that formed dmrt1bY is young in evolutionary terms. Hence, dmrt1bY is not the universal master sex-determining gene in fish. Because the classical fish models, such as zebrafish and pufferfish, are not very adequate to study the basis of genetic sex determination, alternative models, such as the platyfish (Xiphophorus maculatus), are re-emerging. In this fish, which is a well-suited laboratory organism, gene loci involved in pigmentation, melanoma formation, and sexual maturity have been mapped close to the master sex-determining gene. Interestingly, the platyfish can harbor three different sex chromosomes (W, X, and Y) in certain natural populations. Bacterial artificial chromosome contigs covering the sex-determining region of the platyfish are already available, and the positional cloning of the master sex-determining gene(s) should provide new insights into sex determination and sex chromosome evolution in fish and other vertebrates.

Fish retroposons related to the Penelope element of Drosophila virilis define a new group of retrotransposable elements.

Poseidon and Neptune are two ancient lineages of retroposons related to the Penelope element from Drosophila virilis. They have been identified in various teleost fish species, including the medakafish (Oryzias latipes), and the pufferfishes Fugu rubripes and Tetraodon nigroviridis, whose genomes are currently being sequenced. Some of these elements are highly reiterated in fish genomes. Penelope-related elements were also identified in blood fluke, shrimp, sea urchin, cichlid fish and frog, showing that they are widespread in animals. Penelope-related retroposons were not detected among sequences from the Drosophila melanogaster and human genome projects, suggesting that they have been lost from certain animal lineages. A sequence encoding a putative Uri (also called GIY-YIG) endonuclease domain was detected downstream from the gene for reverse transcriptase. To the best of our knowledge, this type of endonuclease sequence has previously been identified in group I introns and in genes for prokaryotic excinucleases but not in retrotransposable elements. Penelope-related elements are frequently truncated at their 5' ends and can also be flanked by long terminal repeat-like structures. Phylogenetic analysis of the reverse transcriptase domain failed to assign Penelope-related retroposons to one of the major groups of retroelements. Overall, therefore, the evidence strongly suggests that these sequences represent a new group of retrotransposable elements.

Non-LTR retrotransposons encoding a restriction enzyme-like endonuclease in vertebrates.

All autonomous non-long terminal repeat (non-LTR) retrotransposons reported to date in vertebrates encode an apurinic/apyrimidinic endonuclease-like enzyme necessary for target sequence cleavage and subsequent target-primed reverse transcription. We describe here vertebrate non-LTR retrotransposons encoding another type of endonuclease more related to type IIS restriction enzymes. Such retrotransposons have been detected until now only in trypanosomes, nematodes, and arthropods. The retrotransposon Rex6 was identified in the genome of several teleost fish including Xiphophorus maculatus (platyfish), Oryzias latipes (medakafish), Oreochromis niloticus (Nile tilapia), and Fugu rubripes (Japanese pufferfish). Rex6 encodes a reverse transcriptase and a putative restriction enzyme-like endonuclease and is a member of the R4 family of non-LTR retrotransposons containing the Dong and R4 elements found in nematodes and insects. Rex6 was active in many species during teleost evolution and underwent several bursts of retrotransposition (some of them being relatively recent) leading to a high copy number of Rex6 in the genome of numerous fish. Extremely truncated Rex6-related sequences were detected by database screening in reptiles, including the snake Trimeresus flavoviridis and the lizard Anolis carolinensis, but not in sequences from the human genome project, suggesting that this element might have been lost from certain vertebrate lineages.

Jule from the fish Xiphophorus is the first complete vertebrate Ty3/Gypsy retrotransposon from the Mag family.

Jule is the second complete long-terminal-repeat (LTR) Ty3/Gypsy retrotransposon identified to date in vertebrates. Jule, first isolated from the poeciliid fish Xiphophorus maculatus, is 4.8 kb in length, is flanked by two 202-bp LTRs, and encodes Gag (structural core protein) and Pol (protease, reverse transcriptase, RNase H, and integrase, in that order) but no envelope. There are three to four copies of Jule per haploid genome in X. maculatus. Two of them are located in a subtelomeric region of the sex chromosomes, where they are associated with the Xmrk receptor tyrosine kinase genes, of which oncogenic versions are responsible for the formation of hereditary melanoma in Xiphophorus. One almost intact copy of Jule was found in the first intron of the X-chromosomal allele of the Xmrk proto-oncogene, and a second, more corrupted copy is present only 56 nt downstream of the polyadenylation signal of the Xmrk oncogene. Jule-related elements were detected by Southern blot hybridization with less than 10 copies per haploid genome in numerous other poeciliids, as well as in more divergent fishes, including the medakafish Oryzias latipes and the tilapia Oreochromis niloticus. Database searches also identified Jule-related sequences in the zebrafish Danio rerio and in both genome project pufferfishes, Fugu rubripes and Tetraodon nigroviridis. Phylogenetic analysis revealed that Jule is the first member of the Mag family of Ty3/Gypsy retrotransposons described to date in vertebrates. This family includes the silkworm Mag and sea urchin SURL retrotransposons, as well as sequences from the nematode Caenorhabditis elegans. Additional related elements were identified in the genomes of the malaria mosquito Anopheles gambiae and the nematode Ascaris lumbricoides. Phylogeny of Mag-related elements suggested that the Mag family of retrotransposons is polyphyletic and is constituted of several ancient lineages that diverged before their host genomes more than 600 MYA.

Genomic plasticity and melanoma formation in the fish Xiphophorus.

Melanoma formation in certain interspecific hybrids of the genus Xiphophorus (Teleostei: Poeciliidae) is associated with the overexpression of the Xmrk receptor tyrosine kinase oncogene. The Xmrk oncogene arose by duplication of the pre-existing Xmrk protooncogene in a highly unstable subtelomeric region of the X and Y sex chromosomes undergoing frequent rearrangements including duplications, deletions, amplifications, and transpositions. Some of these rearrangements are likely to be responsible for the overexpression of the Xmrk oncogene in melanoma. The oncogene itself is very unstable in Xiphophorus and is frequently removed by deletion or disrupted by transposable elements. The Xmrk oncogene region displays a high concentration of retroelements not observed in the corresponding Xmrk protooncogene region. Particularly, a retrovirus long terminal repeat-like sequence was amplified in the proximity of the Xmrk oncogene. Additional genes, some of them also duplicated copies, were detected in this region and might be involved in modulating the melanoma's phenotype.

Transformation-associated recombination (TAR) cloning of tumor-inducing Xmrk2 gene from Xiphophorus maculatus.

We modified the TAR methodology of YAC clone construction for application to fish genomic DNA isolated from Xiphophorus maculatus. YAC libraries were developed using the XIR1 repeat sequence as the recombinational hook. Construction of these libraries demonstrates that Xiphophorus DNA sequences can function as hooks in the yeast recombination system and that X. maculatus genomic DNA contains sequences that provide origin of replication function in yeast. By screening a subset of Xiphophorus YAC clones, we isolated a clone harboring the Xmrk2 locus that is associated with spontaneous and induced melanomagenesis. Modifications of the TAR technique allowed the targeted cloning of specific genes from genomic regions ranging in size from cDNAs to several hundred kilobases. Specific genomic regions can be isolated in a directional manner from fixed map locations to saturate these areas with physical markers. We discuss the applications of these and other yeast recombinational processes to fish genetics.

Variability of genetic sex determination in poeciliid fishes.

Poeciliids are one of the best-studied groups of fishes with respect to sex determination. They present an amazing variety of mechanisms, which span from simple XX-XY or ZZ-ZW systems to polyfactorial sex determination. The gonosomes of poeciliids generally are homomorphic, but very early stages of sex chromosome differentiation have been occasionally detected in some species. In the platyfish Xiphophorus maculatus, gene loci involved in melanoma formation, in different pigmentation patterns and in sexual maturity are closely linked to the sex-determining locus in the subtelomeric region of the X- and Y- chromosomes. The majority of traits encoded by these loci are highly polymorphic. This phenomenon might be explained by the high level of genomic plasticity apparently affecting the sex-determining region, where frequent rearrangements such as duplications, deletions, amplifications, and transpositions frequently occur. We propose that the high plasticity of the sex-determining region might explain the variability of sex determination in Xiphophorus and other poeciliids.

Multiple lineages of the non-LTR retrotransposon Rex1 with varying success in invading fish genomes.

Rex1, together with the related BABAR: elements, represents a new family of non-long-terminal-repeat (non-LTR) retrotransposons from fish, which might be related to the CR1 clade of LINE elements. Rex1/BABAR: retrotransposons encode a reverse transcriptase and an apurinic/apyrimidinic endonuclease, which is very frequently removed by incomplete reverse transcription. Different Rex1 elements show a conserved terminal 3' untranslated region followed by oligonucleotide tandem repeats of variable size and sequence. Phylogenetic analysis revealed that Rex1 retrotransposons were frequently active during fish evolution. They formed multiple ancient lineages, which underwent several independent and recent bursts of retrotransposition and invaded fish genomes with varying success (from <5 to 500 copies per haploid genome). At least three of these ancient Rex1 lineages were detected within the genome of poeciliids. One lineage is absent from some poeciliids but underwent successive rounds of retrotransposition in others, thereby increasing its copy number from <10 to about 200. At least three ancient Rex1 lineages were also detected in the genome project fish Fugu rubripes. Rex1 distribution within one of its major lineages is discontinuous: Rex1 was found in all Acanthopterygii (common ancestor in the main teleost lineage approximately 90 MYA) and in both European and Japanese eels (divergence from the main teleost lineage about 180 MYA) but not in trout, pike, carp, and zebrafish (divergence 100-120 MYA). This might either result from frequent loss or rapid divergence of Rex1 elements specifically in some fish lineages or represent one of the very rare examples of horizontal transfer of non-LTR retrotransposons. This analysis highlights the dynamics and complexity of retrotransposon evolution and the variability of the impact of retrotransposons on vertebrate genomes.

Amplification of a long terminal repeat-like element on the Y chromosome of the platyfish, Xiphophorus maculatus.

The platyfish (Xiphophorus maculatus), in which sex chromosomes are evident from stable and predictable inheritance of sex, is one of the best-studied lower vertebrates with respect to sex determination. In order to identify the structural equivalent for this in the karyotype, which does not contain heteromorphic pairs of chromosomes, two sex-linked molecular probes were used for fluorescent in situ hybridization analysis. One probe, derived from the melanoma oncogene locus ONC-Xmrk, stained both the X and the Y chromosome. This cytogenetic analysis mapped the sex-determining locus to the subtelomeric region of a medium-sized telocentric chromosome. Another probe, a repetitive element (XIR), specifically labeled the Y chromosome in metaphase spreads and in interphase nuclei. The sex chromosomes of X. maculatus can be considered to be at an early stage of evolution of gonosomes. Expansion of the XIR repeat is obviously one of the earliest of the molecular events that lead to divergence of the Y chromosome and recombinational isolation of the sex-determining locus.