Bov-B LINE Research Articles

Geography-Dependent Horizontal Gene Transfer from Vertebrate Predators to Their Prey

Horizontal transfer (HT) of genes between multicellular animals, once thought to be extremely rare, is being more commonly detected, but its global geographic trend and transfer mechanism have not been investigated. We discovered a unique HT pattern of Bovine-B (BovB) LINE retrotransposons in vertebrates, with a bizarre transfer direction from predators (snakes) to their prey (frogs). At least 54 instances of BovB HT were detected, which we estimate to have occurred across time between 85 and 1.3 Ma. Using comprehensive transcontinental sampling, our study demonstrates that BovB HT is highly prevalent in one geographical region, Madagascar, suggesting important regional differences in the occurrence of HTs. We discovered parasite vectors that may plausibly transmit BovB and found that the proportion of BovB-positive parasites is also high in Madagascar where BovB thus might be physically transported by parasites to diverse vertebrates, potentially including humans. Remarkably, in two frog lineages, BovB HT occurred after migration from a non-HT area (Africa) to the HT hotspot (Madagascar). These results provide a novel perspective on how the prevalence of parasites influences the occurrence of HT in a region, similar to pathogens and their vectors in some endemic diseases.

Existence of Bov-B LINE Retrotransposons in Snake Lineages Reveals Recent Multiple Horizontal Gene Transfers with Copy Number Variation.

Transposable elements (TEs) are dynamic elements present in all eukaryotic genomes. They can “jump” and amplify within the genome and promote segmental genome rearrangements on both autosomes and sex chromosomes by disruption of gene structures. The Bovine-B long interspersed nuclear element (Bov-B LINE) is among the most abundant TE-retrotransposon families in vertebrates due to horizontal transfer (HT) among vertebrate lineages. Recent studies have shown multiple HTs or the presence of diverse Bov-B LINE groups in the snake lineage. It is hypothesized that Bov-B LINEs are highly dynamic and that the diversity reflects multiple HTs in snake lineages. Partial sequences of Bov-B LINE from 23 snake species were characterized. Phylogenetic analysis resolved at least two Bov-B LINE groups that might correspond to henophidian and caenophidian snakes; however, the tree topology differed from that based on functional nuclear and mitochondrial gene sequences. Several Bov-B LINEs of snakes showed greater than 80% similarity to sequences obtained from insects, whereas the two Bov-B LINE groups as well as sequences from the same snake species classified in different Bov-B LINE groups showed sequence similarities of less than 80%. Calculation of estimated divergence time and pairwise divergence between all individual Bov-B LINE copies suggest invasion times ranging from 79.19 to 98.8 million years ago in snakes. Accumulation of elements in a lineage-specific fashion ranged from 9 × 10−6% to 5.63 × 10−2% per genome. The genomic proportion of Bov-B LINEs varied among snake species but was not directly associated with genome size or invasion time. No differentiation in Bov-B LINE copy number between males and females was observed in any of the snake species examined. Incongruence in tree topology between Bov-B LINEs and other snake phylogenies may reflect past HT events. Sequence divergence of Bov-B LINEs between copies suggests that recent multiple HTs occurred within the same evolutionary timeframe in the snake lineage. The proportion of Bov-B LINEs varies among species, reflecting species specificity in TE invasion. The rapid speciation of snakes, coinciding with Bov-B LINE invasion in snake genomes, leads us to better understand the effect of Bov-B LINEs on snake genome evolution.

STRUCTURE AND DISTRIBUTION OF THE RETROTRANSPOSON BOV-B LINE

The classification of mobile elements was discussed. Special attention was devoted to the retroelement of the LINE group: retrotransposon Bov-B LINE. The history of its origin and distribution in the nature was considered. The results of the phenomenon of horizontal transition of the retrotransposon Bov-B LINE between evolutionally distant classes were discussed.

Characterization of retrotransposon Bov-B LINE reverse transcriptase gene sequences in parthenogenetic lizards Darevskia unisexualis and bisexual species D. nairensis and D. valentini

Cloning and sequencing of the partial reverse transcriptase gene (750 bp) of the Bov-B LINE retrotransposon have been held in parthenogenetic lizards Darevskia unisexualis and its assumed parental bisexual species D. nairensis and D.valentini. It was shown that the percentage of transcriptionally active copies of this gene, which does not contain a stop codon, is almost the same in the three species and is about 75%. The intragenomic divergence level of these sequences is low and was found to be 2.6% in D. unisexualis, 1.9% in D. nairensis, and 1.6% in D. valentini. The phylogenetic analysis shows the distribution of copies of D. unisexualis in each of the two clusters of RT sequences characteristic of D. nairensis and D. valentini. This result supports the view of the hybrid origin of D. unisexualis and does not exclude intraspecific hybridization between D. nairensis and D. valentini.

Finding of Bov-B LINE retroelement in parthenogenetic and bisexual lizard species of the genus Darevskia (Lacertidae)

The Bov-B LINE retrotransposon was first discovered in Ruminantia and was long considered to be specific for this order. Later, this mobile element was described in snakes and some lizard species. Analysis of phylogenetic relationships of Bov-B LINE elements from different ruminants, snakes, and lizard species led to the suggestion on horizontal transfer of this retrotransposon from Squamata to Ruminantia. In the Squamata group, Bov-B LINE element was found in all snakes and some lizard species examined. The element was not detected in the genomes of some species of the genera Lacerta and Podarcis. In the present study, using PCR amplification and sequencing of PCR products, Bov-B LINE element was identified in the genomes of parthenogenetic and bisexual species of the genus Darevskia (Lacertidae), as well as in such species as Lacerta agilis and Zootoca vivipara, where this retrotransposon had not been not detected before.

Sequences and structural organization of phospholipase A2 genes from Vipera aspis aspis, V. aspis zinnikeri and Vipera berus berus venom

We used a PCR-based method to determine the genomic DNA sequences encoding phospholipases A2 (PLA2s) from the venoms of Vipera aspis aspis (V. a. aspis), Vipera aspis zinnikeri (V. a. zinnikeri), Vipera berus berus (V. b. berus) and a neurotoxic V. a. aspis snake (neurotoxic V. a. aspis) from a population responsible for unusual neurotoxic envenomations in south-east France. We sequenced five groups of genes, each corresponding to a different PLA2. The genes encoding the A and B chains of vaspin from the neurotoxic V. a. aspis, PLA2-I from V. a. zinnikeri, and the anticoagulant PLA2 from V. b. berus are described here. Single nucleotide differences leading to amino-acid substitutions were observed both between genes encoding the same PLA2 and between genes encoding different PLA2s. These differences were clustered in exons 3 and 5, potentially altering the biological activities of PLA2. The distribution and characteristics of the PLA2 genes differed according to the species or subspecies. We characterized for the first time genes encoding neurotoxins from the V. a. aspis and V. b. berus snakes of central France. Genes encoding ammodytins I1 and I2, described previously in Vipera ammodytes ammodytes (V. am. ammodytes), were also present in V. a. aspis and V. b. berus. Three different ammodytin I1 gene sequences were characterized: one from V. b. berus, the second from V. a. aspis, V. a. zinnikeri and the neurotoxic V. a. aspis, and the third from the neurotoxic V. a. aspis. This third sequence was identical with the reported sequence of the V. am. ammodytes ammodytin I1 gene. Genes encoding monomeric neurotoxins of V. am. ammodytes venom, ammodytoxins A, B and C, and the Bov-B LINE retroposon, a phylogenetic marker found in V. am. ammodytes genome, were identified in the genome of the neurotoxic V. a. aspis. These results suggest that the population of neurotoxic V. a. aspis snakes from south-east France may have resulted from interbreeding between V. a. aspis and V. am. ammodytes.

Evolutionary dynamics and evolutionary history in the RTE clade of non-LTR retrotransposons.

This study examined the evolutionary dynamics of Bov-B LINEs in vertebrates and the evolution of the RTE clade of non-LTR retrotransposons. The first full-length reptilian Bov-B LINE element is described; it is 3.2 kb in length, with a structural organization typical of the RTE clade of non-LTR retrotransposons. The long-term evolution of Bov-B LINEs was studied in 10 species of Squamata by analysis of a PCR-amplified 1.8-kb fragment encoding part of apurinic/apyrimidinic endonuclease, the intervening domain, and the palm/fingers subdomain of reverse transcriptase. A very high level of conservation in Squamata Bov-B long interspersed nuclear elements has been found, reaching 86% identity in the nearly 600 amino acids of ORF2. The same level of conservation exists between the ancestral snake lineage and Ruminantia. Such a high level is exceptional when compared with the level of conservation observed in nuclear and mitochondrial proteins and in other transposable elements. The RTE clade has been found to be much more widely distributed than previously thought, and novel representatives have been discovered in plants, brown algae, annelids, crustaceans, mollusks, echinoderms, and teleost fishes. Evolutionary relationships in the RTE clade were deduced at the amino acid level from three separate regions of ORF2. By using different independent methods, including the divergence-versus-age analysis, several examples of horizontal transfer in the RTE clade were recognized, with important implications for the existence of HT in non-LTR retrotransposons.

Gene organization of bovine BCNT that contains a portion corresponding to an endonuclease domain derived from an RTE-1 (Bov-B LINE), non-LTR retrotransposable element: duplication of an intramolecular repeat unit downstream of the truncated RTE-1

BCNT (a protein named after Bucentaur or craniofacial development protein 1) has a unique structure in Ruminantia. Bovine BCNT contains a region of the endonuclease domain derived from a truncated RTE-1 (previously called Bov-B LINE), a non-LTR retrotransposable repetitive element, and two repeat units (intramolecular repeat, IR) each with 40 amino acids in the C-terminal region. In contrast the human and mouse BCNT proteins contain one repeat unit and lack the RTE-1-derived portion. The 3′ UTR of bovine bcnt cDNA also contains an approximately 300-bp portion homologous to the 3′-part of RTE-1. We examined the bovine bcnt genomic DNA sequence to understand how the bovine bcnt gene has been organized. The sequence of 3′ UTR homologous portion was found to more closely resemble the Art2 element than the bovine RTE-1. By PCR screening a bovine/hamster hybrid somatic cell panel, the bovine bcnt gene was mapped to chromosome 18, syntenic human chromosome 16q on which human BCNT is located. The bcnt genomic DNA sequence corresponding to the cDNA downstream of a RTE-1 derived portion reveals that each IR unit is flanked by both 5′-side and 3′-side introns and that 3′-UTR consists of one exon. The alignment of the above sequence with a bovine RTE-1 did not show any significant homology downstream of the endonuclease domain. On the other hand, the alignment of the intron sequences with each other revealed that the six sequential homologous segments ranging in size from 40 to 453 bp existed over a 1 kb long sequence between both the 5′- and 3′-side introns flanking each bovine IR unit. In addition, both the 174-bp of 5′-side intron and 80-bp of 3′-side intron neighboring each 120-bp IR exon are significantly homologous among the two bovine IRs, human IR and mouse IR. These results suggest that a truncated bovine RTE-1 was inserted into the intron upstream of an IR unit of an ancestor bcnt gene and that a duplication of a relatively long region that includes IR occurred in the bovine genome.

Molecular evolution of Bov-B LINEs in vertebrates.

Since their discovery in family Bovidae (bovids), Bov-B LINEs, believed to be order-specific SINEs, have been found in all ruminants and recently also in Viperidae snakes. The distribution and the evolutionary relationships of Bov-B LINEs provide an indication of their origin and evolutionary dynamics in different species. The evolutionary origin of Bov-B LINE elements has been shown unequivocally to be in Squamata (squamates). The horizontal transfer of Bov-B LINE elements in vertebrates has been confirmed by their discontinuous phylogenetic distribution in Squamata (Serpentes and two lizard infra-orders) as well as in Ruminantia, by the high level of nucleotide identity, and by their phylogenetic relationships. The direction of horizontal transfer from Squamata to the ancestor of Ruminantia is evident from the genetic distances and discontinuous phylogenetic distribution of Bov-B LINE elements. The ancestor of Colubroidea snakes has been recognized as a possible donor of Bov-B LINE elements to Ruminantia. The timing of horizontal transfer has been estimated from the distribution of Bov-B LINE elements in Ruminantia and the fossil data of Ruminantia to be 40-50 My ago. The phylogenetic relationships of Bov-B LINE elements from the various Squamata species agrees with that of the species phylogeny, suggesting that Bov-B LINE elements have been stably maintained by vertical transmission since the origin of Squamata in the Mesozoic era.

Horizontal transfer of non-LTR retrotransposons in vertebrates.

Since their discovery in family Bovidae (bovids), Bov-B LINEs, believed to be order-specific SINEs, have been found in all ruminants and recently also in Viperidae snakes. The distribution and the evolutionary relationships of Bov-B LINEs provide an indication of their origin and evolutionary dynamics in different species. The evolutionary origin of Bov-B LINE elements has been shown unequivocally to be in Squamata (squamates). The horizontal transfer of Bov-B LINE elements in vertebrates has been confirmed by their discontinuous phylogenetic distribution in Squamata (Serpentes and two lizard infra-orders) as well as in Ruminantia, by the high level of nucleotide identity, and by their phylogenetic relationships. The direction of horizontal transfer from Squamata to the ancestor of Ruminantia is evident from the genetic distances and discontinuous phylogenetic distribution of Bov-B LINE elements. The ancestral snake lineage (Boidae) has been recognized as a possible donor of Bov-B LINE elements to Ruminantia. The timing of horizontal transfer has been estimated from the distribution of Bov-B LINE elements in Ruminantia and the fossil data of Ruminantia to be 40-50 mya. The phylogenetic relationships of Bov-B LINE elements from the various Squamata species agrees with that of the species phylogeny, suggesting that Bov-B LINE elements have been stably maintained by vertical transmission since the origin of Squamata in the Mesozoic era.

The Bov-B LINEs found in Vipera ammodytes toxic PLA 2 genes are widespread in snake genomes

In the fourth intron of two toxic Vipera ammodytes PLA 2 genes a Ruminantia specific 5′-truncated Bov-B LINE element was identified. Southern blot analysis of Bov-B LINE distribution in vertebrates shows that, apart from the Ruminantia, it is limited to Viperidae snakes ( V. ammodytes, Vipera palaestinae, Echis coloratus, Bothrops alternatus, Trimeresurus flavoviridis and Trimeresurus gramineus). The copy number of the 3′ end of Bov-B LINE in the V. ammodytes genome is between 62 000 and 75 000. At orthologous positions in other snake PLA 2 genes the Bov-B LINE element is absent, indicating that its retrotransposition in the V. ammodytes PLA 2 gene locus has occurred quite recently, about 5 Myr ago. The amplification of Bov-B LINEs in snakes may have occurred before the divergence of the Viperinae and Crotalinae subfamilies. Due to its wide distribution in Viperidae snakes it should be a valuable phylogenetic marker. The neighbour-joining phylogenetic tree shows two clusters of truncated Bov-B LINE, a Bovidae and a snake cluster, indicating an early horizontal transfer of this transposable element.

Existence of a bovine LINE repetitive insert that appears in the cDNA of bovine protein BCNT in ruminant, but not in human, genomes.

A novel protein, BCNT, originally isolated from bovine brain and named after Bucentaur, contains an internal portion that is translated from part of bovine LINE repetitive sequence (Bov-B LINE). Human cDNA highly homologous to the bovine bcnt (bbcnt) cDNA has been isolated but does not contain a sequence similar to the Bov-B LINE insert (Nobukuni, T., Kobayashi, M., Omori, A., Ichinose, S., Iwanaga, T., Takahashi, I., Hashimoto, K., Hattori, S., Kaibuchi, K., Miyata, Y., Masui, T., Iwashita, S., 1997. An Alu-linked repetitive sequence corresponding to 280 amino acids is expressed in a novel bovine protein, but not in its human homologue. J. Biol. Chem. 272, 2801-2807). In this study, we conducted a polymerase chain reaction analysis to investigate whether such a Bov-B LINE insert is present in bcnt orthologs in other animals and in the genomic sequence of the human BCNT (hBCNT) gene. The results indicate that the Bov-B LINE insert is present in the genomic sequences of bcnt orthologs from sheep, goats, axis deer, and mouse deer (chevrotain), that is in Ruminantia, but not in pigs or human. Analysis of the bbcnt genomic sequence around the Bov-B LINE insert revealed a large part of the inserted Bov-B LINE sequence to be included in an exon; this is followed by a 54-nucleotide sequence that is highly homologous to Bov-B LINE in the 3'-side intron. The hBCNT gene was isolated and found to consist of seven exons and six introns, among which the intron corresponding to the Bov-B LINE insertion site in the bbcnt genome is 16.5kb in length with no sequence similar to Bov-B LINE. Based on these results, it seems likely that the Bov-B LINE insert is derived from a long Bov-B LINE repetitive sequence transposed to an ancestral bcnt gene in Ruminantia and reformed as a new exon through new splicing sites in the transposed sequence.

Bov-B long interspersed repeated DNA (LINE) sequences are present in Vipera ammodytes phospholipase A2 genes and in genomes of Viperidae snakes.

Ammodytin L is a myotoxic Ser49 phospholipase A2 (PLA2) homologue, which is tissue-specifically expressed in the venom glands of Vipera ammodytes. The complete DNA sequence of the gene and its 5' and 3' flanking regions has been determined. The gene consists of five exons separated by four introns. Comparative analysis of the ammodytin L and ammodytoxin C genes shows that all intron and flanking sequences are considerably more conserved (93-97%) than the mature protein-coding exons. The pattern of nucleotide substitutions in protein-coding exons is not random but occurs preferentially on the first and the second positions of codons, which suggests positive Darwinian evolution for a new function. An Ruminantia specific ART-2 retroposon, recently recognised as a 5'-truncated Bov-B long interspersed repeated DNA (LINE) sequence, was identified in the fourth intron of both genes. This result suggests that ammodytin L and ammodytoxin C genes are derived by duplication of a common ancestral gene. The phylogenetic distribution of Bov-B LINE among vertebrate classes shows that, besides the Ruminantia, it is limited to Viperidae snakes (Vipera ammodytes, Vipera palaestinae, Echis coloratus, Bothrops alternatus, Trimeresurus flavoviridis and Trimeresurus gramineus). The copy number of the 3' end of Bov-B LINE in the Vipera ammodytes genome is between 62,000 and 75,000. The absence of Bov-B LINE at orthologous positions in other snake PLA2 genes indicates that its retrotransposition in the V. ammodytes PLA2 gene locus has occurred quite recently, about 5 My ago. The amplification of Bov-B LINEs in snakes may have occurred before the divergence of the Viperinae and Crotalinae subfamilies. Due to its wide distribution in Viperidae snakes it may be a valuable phylogenetic marker. The neighbor-joining phylogenetic tree shows two clusters of truncated Bov-B LINE, a Bovidae and a snake cluster, indicating an early horizontal transfer of this transposable element.