The non-LTR retrotransposons in Ciona intestinalis: new insights into the evolution of chordate genomes

Background: Non-long terminal repeat (non-LTR) retrotransposons have contributed to shaping the structure and function of genomes. In silico and experimental approaches have been used to identify the non-LTR elements of the urochordate Ciona intestinalis. Knowledge of the types and abundance of non-LTR elements in urochordates is a key step in understanding their contribution to the structure and function of vertebrate genomes. Results: Consensus elements phylogenetically related to the I, LINE1, LINE2, LOA and R2 elements of the 14 eukaryotic non-LTR clades are described from C. intestinalis. The ascidian elements showed conservation of both the reverse transcriptase coding sequence and the overall structural organization seen in each clade. The apurinic/apyrimidinic endonuclease and nucleic-acid-binding domains encoded upstream of the reverse transcriptase, and the RNase H and the restriction enzyme-like endonuclease motifs encoded downstream of the reverse transcriptase were identified in the corresponding Ciona families. Conclusions: The genome of C. intestinalis harbors representatives of at least five clades of non-LTR retrotransposons. The copy number per haploid genome of each element is low, less than 100, far below the values reported for vertebrate counterparts but within the range for protostomes. Genomic and sequence analysis shows that the ascidian non-LTR elements are unmethylated and flanked by genomic segments with a gene density lower than average for the genome. The analysis provides valuable data for understanding the evolution of early chordate genomes and enlarges the view on the distribution of the non-LTR retrotransposons in eukaryotes.

Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Transcriptionally active LTR retrotransposons in Eucalyptus genus are differentially expressed and insertionally polymorphic

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns

Background: Sugarcane is an important crop worldwide for sugar production and increasingly, as a renewable energy source. Modern cultivars have polyploid, large complex genomes, with highly unequal contributions from ancestral genomes. Long Terminal Repeat retrotransposons (LTR-RTs) are the single largest components of most plant genomes and can substantially impact the genome in many ways. It is therefore crucial to understand their contribution to the genome and transcriptome, however a detailed study of LTR-RTs in sugarcane has not been previously carried out. Results: Sixty complete LTR-RT elements were classified into 35 families within four Copia and three Gypsy lineages. Structurally, within lineages elements were similar, between lineages there were large size differences. FISH analysis resulted in the expected pattern of Gypsy/heterochromatin, Copia/euchromatin, but in two lineages there was localized clustering on some chromosomes. Analysis of related ESTs and RT-PCR showed transcriptional variation between tissues and families. Four distinct patterns were observed in sRNA mapping, the most unusual of which was that of Ale1, with very large numbers of 24nt sRNAs in the coding region. The results presented support the conclusion that distinct small RNA-regulated pathways in sugarcane target the lineages of LTR-RT elements. Conclusions: Individual LTR-RT sugarcane families have distinct structures, and transcriptional and regulatory signatures. Our results indicate that in sugarcane individual LTR-RT families have distinct behaviors and can potentially impact the genome in diverse ways. For instance, these transposable elements may affect nearby genes by generating a diverse set of small RNA's that trigger gene silencing mechanisms. There is also some evidence that ancestral genomes contribute significantly different element numbers from particular LTR-RT lineages to the modern sugarcane cultivar genome.

Terminal-repeat retrotransposons with GAG domain in plant genomes: a new testimony on the complex world of transposable elements

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Bursts of transposition from non-long terminal repeat retrotransposon families of the RTE clade in Schistosoma mansoni

The genus Schistosoma is composed of blood flukes that infect vertebrates, from which three species are major causative agents of human schistosomiasis, a tropical disease that affects more than 200 million people. Current models of the recent evolution of Schistosoma indicate multiple events of migration and speciation from an Asian ancestral species. Transposable elements are important drivers of genome evolution and have been hypothesised to have an important role in speciation. In this work, we describe a comprehensive inventory of Schistosoma mansoni and Schistosoma japonicum retrotransposons, based on their recently published genomic data. We find a considerable difference in retrotransposon representation between the two species (22% and 13%, respectively). A large part of this difference can be attributed to higher representation of two previously described families of S. mansoni retrotransposons (SR2 and Perere-3/SR3), compared with the representation of their closest relative families in S. japonicum. A more detailed analysis suggests that these two S. mansoni families were the subject of recent bursts of transposition that were not paralleled by their S. japonicum counterparts. We hypothesise that these bursts could be a consequence of the evolutionary pressure resulting from migration of Schistosoma from Asia to Africa and their establishment in this new environment, helping both speciation and adaptation. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Gulliver, a long terminal repeat retrotransposon from the genome of the oriental blood fluke Schistosoma japonicum

We characterized the consensus sequence and structure of a long terminal repeat (LTR) retrotransposon from the genome of the human blood fluke, Schistosoma japonicum, and have earned this element, Gulliver. The full length, consensus Gulliver LTR retrotransposon was 4788 bp, and it was flanked at its 5'- and 3'-ends by LTRs of 259 bp. Each LTR included RNA polymerase II promoter sequences, a CAAT signal and a TATA box, Gulliver exhibited features characteristic of a functional LTR retrotransposon including two read through (termination) ORFs encoding retroviral gag and pol proteins of 312 and 1071 amino acid residues, respectively. The gag ORF encoded motifs conserved in nucleic acid binding proteins, while the pol ORF encoded conserved domains of aspartic protease, reverse transcriptase (RT), RNaseH and integrase, in that order, a pol pattern conserved in the gypsy lineage of LTR retrotransposons. Whereas the sequence and structure of Gulliver was similar to that of gypsy, phylogenetic analysis revealed that Gulliver did not group particularly closely with the gypsy family. Rather, its closest relatives were a LTR retrotransposon from Caenorhabditis elegans, mag from Bombyx mori and, to a lesser extent, easel from the salmon Oncorhynchus keta. Dot blot hybridizations indicated that Gulliver was present at between 100 and several thousand copies in the S. japonicum genome, and Southern hybridization analysis suggested its probable presence in the genome of Schistosoma mansoni. Transcripts encoding the RT domain of Gulliver were detected by RT-PCR in larval and adult stages of S. japonicum, indicating that (at least) the RT domain of Gulliver is transcribed. This is the first report of the sequence and structure of an LTR retrotransposon from any schistosome or indeed from any species belonging to the phylum Platyhelminthes. (C) 2001 Elsevier Science B.V. All rights reserved.

pido, a non-long terminal repeat retrotransposon of the chicken repeat 1 family from the genome of the Oriental blood fluke, Schistosoma japonicum

A newly described non-long terminal repeat (non-LTR) retrotransposon element was isolated from the genome of the Oriental schistosome, Schistosoma japonicum. At least 1000 partial copies of the element, which was named pido, were dispersed throughout the genome of S. japonicum. As is usual with non-LTR retrotransposons, it is expected that many pido elements will be 5'-truncated. A consensus sequence of 3564 bp of the truncated pido element was assembled from several genomic fragments that contained pido-hybridizing sequences. The sequence encoded part of the first open reading frame (ORF), the entire second ORF and, at its 3'-terminus, a tandemly repetitive, A-rich (TA(6)TA(5)TA(8)) tail, The ORF1 of pido encoded a nucleic acid binding protein and ORF2 encoded a retroviral-like polyprotein that included apurinic/apyrimidinic endonuclease (EN) and reverse transcriptase (RT) domains, in that order. Based on its sequence and structure, and phylogenetic analyses of both the RT and EN domains, pido belongs to the chicken repeat 1 (CR1)-like lineage of elements known from the chicken, turtle, puffer fish, mosquitoes and other taxa. pido shared equal similarity with CRI from chicken, an uncharacterized retrotransposon from Caenorhabditis elegans and SR1 (a non-LTR retrotransposon) from the related blood fluke Schistosoma mansoni; the level of similarity between pido and SR1 indicated that these two schistosome retrotransposons were related but not orthologous. The findings indicate that schistosomes have been colonized by at least two discrete CRI-like elements. Whereas pido did not appear to have a tight target site specificity, at least one copy of pido has inserted into the 3'-untranslated region of a protein-encoding gene (GeriBank AW736757) of as yet unknown identity. mRNA encoding the RT of pido was detected by reverse transcription-polymerase chain reaction in the egg, miracidium. and adult developmental stages of S. japonicum, indicating that the RT domain was transcribed and suggesting that pido was replicating actively and mobile within the S. japonicum genome. (C) 2002 Elsevier Science B.V. All rights reserved.

Reverse transcriptase activity and untranslated region sharing of a new RTE-like, non-long terminal repeat retrotransposon from the human blood fluke, Schistosoma japonicum

A new RTE-like, non-long terminal repeat retrotransposon, termed SjR2, from the human blood fluke, Schistosoma japonicum, is described. SjR2 is similar to3.9 kb in length and is constituted of a single open reading frame encoding a polyprotein with apurinic/apyrimidinic endonuclease and reverse transcriptase domains. The open reading frame is bounded by 5'- and 3'-terininal untranslated regions and, at its 3-terminus, SjR2 bears a short (TGAC)(3) repeat. Phylogenetic analyses based on conserved domains of reverse transcriptase or endonuclease revealed that SjR2 belonged to the RTE clade of non-long terminal repeat retrotransposons. Further, SjR2 was homologous, but probably not orthologous, to SR2 front the African blood fluke, Schistosoma mansoni; this RTE-like family of non-long terminal repeat retrotransposons appears to have arisen before the divergence of the extant schistosome species. Hybridisation analyses indicated that similar to 10,000 copies of SjR2 were dispersed throughout the S. japonicum chromosomes, accounting for up to 14% of the nuclear genome. Messenger RNAs encoding the reverse transcriptase and endonuclease domains of SjR2 were detected in several developmental stages of the schistosome, indicating that the retrotransposon was actively replicating within the genome of the parasite. Exploration of the coding and non-coding regions of SjR2 revealed two notable characteristics. First, the recombinant reverse transcriptase domain of SjR2 expressed in insect cells primed reverse transcription of SjR2 mRNA in vitro. By contrast, recombinant SjR2-endonuclease did not appear to cleave schistosome or plasmid DNA. Second, the 5'-untranslated region of SjR2 was >80% identical to the 3-untranslated region of a schistosome heat shock protein-70 gene (hsp-70) in the antisense orientation, indicating that SjR2-like elements were probably inserted into the non-coding regions of ancestral S. japonicum HSP-70, probably after the species diverged from S. mansoni. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

EGASP: the human ENCODE Genome Annotation Assessment Project

Background: Non-long terminal repeat (non-LTR) retrotransposons have contributed to shaping the structure and function of genomes. In silico and experimental approaches have been used to identify the non-LTR elements of the urochordate Ciona intestinalis. Knowledge of the types and abundance of non-LTR elements in urochordates is a key step in understanding their contribution to the structure and function of vertebrate genomes. Results: Consensus elements phylogenetically related to the I, LINE1, LINE2, LOA and R2 elements of the 14 eukaryotic non-LTR clades are described from C. intestinalis. The ascidian elements showed conservation of both the reverse transcriptase coding sequence and the overall structural organization seen in each clade. The apurinic/apyrimidinic endonuclease and nucleic-acid-binding domains encoded upstream of the reverse transcriptase, and the RNase H and the restriction enzyme-like endonuclease motifs encoded downstream of the reverse transcriptase were identified in the corresponding Ciona families. Conclusions: The genome of C. intestinalis harbors representatives of at least five clades of non-LTR retrotransposons. The copy number per haploid genome of each element is low, less than 100, far below the values reported for vertebrate counterparts but within the range for protostomes. Genomic and sequence analysis shows that the ascidian non-LTR elements are unmethylated and flanked by genomic segments with a gene density lower than average for the genome. The analysis provides valuable data for understanding the evolution of early chordate genomes and enlarges the view on the distribution of the non-LTR retrotransposons in eukaryotes.

PCR amplification and sequence analyses of reverse transcriptase-like genes in Crinipellis perniciosa isolates

Reverse transcriptase (RT) sequence analysis is an important technique used to detect the presence of transposable elements in a genome. Putative RT sequences were analyzed in the genome of the pathogenic fungus C. perniciosa, the causal agent of witches' broom disease of cocoa. A 394 bp fragment was amplified from genomic DNA of different isolates of C. perniciosa belonging to C-, L-, and S-biotypes and collected from various geographical areas. The cleavage of PCR products with restriction enzymes and the sequencing of various RT fragments indicated the presence of several sequences showing transition events (G:C to A:T). Southern blot analysis revealed high copy numbers of RT signals, forming different patterns among C-, S-, and L-biotype isolates. Sequence comparisons of the predicted RT peptide indicate a close relationship with the RT protein from thegypsy family of LTR-retrotransposons. The possible role of these retrotransposons in generating genetic variability in the homothallic C. perniciosa is discussed.

Ty-1 copia retrotransposon-based SSAP marker development in Cashew

The most popular retrotransposon-based molecular marker system in use at the present time is the sequence-specific amplification polymorphism (SSAP) system . This system exploits the insertional polymorphism of long terminal repeat (LTR) retrotransposons around the genome. Because the LTR sequence is used to design primers for this method, its successful application requires sequence information from the terminal region of the mobile elements . In this study, two LTR sequences were isolated from the cashew genome and used successfully to develop SSAP marker systems. These were shown to have higher levels of polymorphism than amplified fragment length polymorphic markers for this species.

Copia Retrotransposon in the Zaprionus Genus: Another Case of Transposable Element Sharing with the Drosophila melanogaster Subgroup

Copia is a retrotransposon that appears to be distributed widely among the Drosophilidae subfamily. Evolutionary analyses of regulatory regions have indicated that the Copia retrotransposon evolved through both positive and purifying selection, and that horizontal transfer (HT) could also explain its patchy distribution of the among the subfamilies of the melanogaster subgroup. Additionally, Copia elements could also have transferred between melanogaster subgroup and other species of Drosophilidae-D. willistoni and Z. tuberculatus. In this study, we surveyed seven species of the Zaprionus genus by sequencing the LTR-ULR and reverse transcriptase regions, and by using RT-PCR in order to understand the distribution and evolutionary history of Copia in the Zaprionus genus. The Copia element was detected, and was transcriptionally active, in all species investigated. Structural and selection analysis revealed Zaprionus elements to be closely related to the most ancient subfamily of the melanogaster subgroup, and they seem to be evolving mainly under relaxed purifying selection. Taken together, these results allowed us to classify the Zaprionus sequences as a new subfamily-ZapCopia, a member of the Copia retrotransposon family of the melanogaster subgroup. These findings indicate that the Copia retrotransposon is an ancient component of the genomes of the Zaprionus species and broaden our understanding of the diversity of retrotransposons in the Zaprionus genus.

High homologous gene conservation despite extreme autopolyploid redundancy in sugarcane

P>Modern sugarcane (Saccharum spp.) is the leading sugar crop and a primary energy crop. It has the highest level of `vertical` redundancy (2n = 12x = 120) of all polyploid plants studied to date. It was produced about a century ago through hybridization between two autopolyploid species, namely S. officinarum and S. spontaneum. In order to investigate the genome dynamics in this highly polyploid context, we sequenced and compared seven hom(oe)ologous haplotypes (bacterial artificial chromosome clones). Our analysis revealed a high level of gene retention and colinearity, as well as high gene structure and sequence conservation, with an average sequence divergence of 4% for exons. Remarkably, all of the hom(oe)ologous genes were predicted as being functional (except for one gene fragment) and showed signs of evolving under purifying selection, with the exception of genes within segmental duplications. By contrast, transposable elements displayed a general absence of colinearity among hom(oe)ologous haplotypes and appeared to have undergone dynamic expansion in Saccharum, compared with sorghum, its close relative in the Andropogonea tribe. These results reinforce the general trend emerging from recent studies indicating the diverse and nuanced effect of polyploidy on genome dynamics.

The Tnt1 family member Retrosol copy number and structure disclose retrotransposon diversification in different Solanum species

Eukaryotic genome expansion/retraction caused by LTR-retrotransposon activity is dependent on the expression of full length copies to trigger efficient transposition and recombination-driven events. The Tnt1 family of retrotransposons has served as a model to evaluate the diversity among closely related elements within Solanaceae species and found that members of the family vary mainly in their U3 region of the long terminal repeats (LTRs). Recovery of a full length genomic copy of Retrosol was performed through a PCR-based approach from wild potato, Solanum oplocense. Further characterization focusing on both LTR sequences of the amplified copy allowed estimating an approximate insertion time at 2 million years ago thus supporting the occurrence of transposition cycles after genus divergence. Copy number of Tnt1-like elements in Solanum species were determined through genomic quantitative PCR whereby results sustain that Retrosol in Solanum species is a low copy number retrotransposon (1-4 copies) while Retrolyc1 has an intermediate copy number (38 copies) in S. peruvianum. Comparative analysis of retrotransposon content revealed no correlation between genome size or ploidy level and Retrosol copy number. The tetraploid cultivated potato with a cellular genome size of 1,715 Mbp harbours similar copy number per monoploid genome than other diploid Solanum species (613-884 Mbp). Conversely, S. peruvianum genome (1,125 Mbp) has a higher copy number. These results point towards a lineage specific dynamic flux regarding the history of amplification/activity of Tnt1-like elements in the genome of Solanum species.