Looking for ltr very specific

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Kenji K. Ribosomal RNA genes are abundant repetitive sequences in most eukaryotes. R2 is a large family sharing the same insertion specificity and classified into 4 clades R2-A, -B, -C, and -D based on the N-terminal domain structure and the phylogeny.

There is no observation of horizontal transfer of R2; therefore, the origin of R2 dates back to before the split between protostomes and deuterostomes. Here, we in silico identified 1 R2 element from the sea anemone Nematostella vectensis and 2 R2-like retrotransposons from the hydrozoan Hydra magnipapillata.

R2 from N. We deated the Hydra R2—like elements R8. There is no obvious sequence similarity between targets of R2 and R8, probably because they recognize long DNA sequences. Domain structure and phylogeny indicate that R2 from N. These suggest that R2 had been generated before the split between cnidarians and bilaterians and that R8 is a retrotransposon family that changed its target from the 28S rDNA to the 18S rDNA. Most of the insertions belong to group I self-splicing introns Haugen et al.

Group I introns have an ability to splice themselves from rRNA, which makes them neutral to the host. In contrast, non-LTR retrotransposons do not have the ability of self-splicing. R1, R6, RT, and R7 are related to one another both structurally and phylogenetically and all classified into the R1 clade Kojima and Fujiwara They are considered to share the common sequence-specific ancestor Kojima and Fujiwara Thus, the acquisition of sequence specificity of R2, R4, and R5 can be older than that of the R1 clade elements.

Their insertion sites are very close fig. R4 is closely related to the TAA repeat—specific retrotransposon and elements with no obvious specificity Van Dellen et al. R5 is related to the spliced leader exon—specific retrotransposon Burke et al. R2 constitutes a large group that includes many retrotransposons with the same sequence specificity. Ribosomal DNA sequences are represented by that of Anopheles albimanus Land the insertion site of each retrotransposon is shown Looking for ltr very specific parentheses. The cleavage site of R2 is represented by that of R2Bm.

R2 is distributed over at least 4 animal phyla—Arthropoda, Chordata, Echinodermata, and Platyhelminthes Burke et al. Because obvious horizontal transfer of R2 has not been observed, R2 could date back to before the split between protostomes and deuterostomes. At present, R2 is the sequence-specific non-LTR retrotransposon family, whose origin can be traced back the earliest.

The R2-A, -C, and -D clades have 3, 2, and 1 zinc-finger motif srespectively. The N-terminal structure of the R2-B clade is undetermined. The members of the R2-A and the R2-D clades have been found from diverse phyla of bilaterian animals.

One of the questions about the evolution of R2 is when R2 appeared. The answer could be helpful to understand the ancestral state of non-LTR retrotransposons. We tried to search for R2 from organisms belonging to Cnidaria as an approach to answer this question. In this report, we describe new non-LTR retrotransposons from 2 cnidarian species, Nematostella vectensis and Hydra magnipapillata. We found that an element from Nematostella has the same specificity as R2.

So it is an authentic R2 element and belongs to the R2-D clade. In contrast, 2 elements from Hydra show novel sequence specificity and are integrated Looking for ltr very specific the 18S rDNA. We Looking for ltr very specific them R8. The sequence around the insertion site of R8 is not similar to that of R2. Although R8 has distinct sequence specificity from R2, the phylogenetic analyses revealed that R8 belongs to the R2-A clade.

R8 is likely to be a retrotransposon family that changed its target from the 28S to the 18S rDNA in the past. Identification of R2 and R8 from Cnidaria suggests that the origin of R2 antedates the appearance of triploblasts. Genomic sequence traces of the starlet sea anemone N. We used approximately 2. The hit sequence traces of N. The hit sequence traces of H. BlastN was performed using one representative sequence trace per group as a query in order to search the sequence traces that overlap with the query.

BlastN was done iteratively using the assembled sequence as a query. The longest assembled sequence both ends of which ad to ribosomal RNA genes was defined as the full-length retrotransposon. If the assembled retrotransposon sequence contained inframe stop codons or frameshifts compared with known R2 elements, it was corrected using other Looking for ltr very specific traces as references.

The sequence alignments used for the phylogenetic analyses are available as Supplementary Material online. Maximum likelihood trees were constructed using Treefinder Jobb et al. Modelgenerator Keane et al. Nonparametric bootstrap analyses for the maximum likelihood and the Neighbor-ing trees were performed with 1, replicates. Bayesian phylogenetic inference trees were constructed using MrBayes 3. Markov chain Monte Carlo chain length was 1, generations with trees sampled every 10 generations; the first 10, trees were discarded as burn-in.

We identified one non-LTR retrotransposon from the genomic sequence traces of the starlet sea anemone N. We deated it R2Nvec-A. R2Nvec-A is 3, bp long. The insertion site of R2Nvec-A is a bit different from other R2 elements. Insertion of R2Nvec-A makes neither target-site duplications nor deletions.

Therefore, the cleavage of R2Nvec-A endonuclease is considered to make a blunt end fig. The boundary of R2Nvec-A can be explained in another way. Equally, it is possible that the endonuclease of R2Nvec-A cleaves the bottom strand at the same site as other R2 fig. Experimental evidences are needed to determine the cleavage sites of endonuclease.

Insertion sites of R2Nvec-A. A Boundaries of the full-length R2Nvec-A. Nucleotides identical to the 28S rRNA gene are shaded. s of sequence Looking for ltr very specific are shown at the right. The horizontal solid line shows the region used to make the phylogenetic tree in figure 5whereas the broken line shows that in figure 6. B Putative cleavage sites of R2 endonucleases.

Vertical lines represent the cleavage sites of bottom and top strands. Only the cleavage site of R2Bm has been characterized experimentally Luan et al. We identified 2 closely related non-LTR retrotransposons from the sequence traces of the hydrozoan H. As their sequences have high similarity to those of R2, we checked the boundaries of these retrotransposons. Beyond our expectation that they share the target sequence with R2, these retrotransposons are not inserted into the 28S rDNA.

They are not the authentic R2 elements but constitute a novel sequence-specific non-LTR retrotransposon family. Because R2 not only is a phylogenetic group but also constitutes a group with the same sequence specificity, we propose the new family R8 including R8Hm-A and R8Hm-B. R8Hm-A follows the position and is followed by the position Most of them are flanked with several bases upstream compared with the complete copies. Putative target-site duplication sequences are boxed. Nucleotides identical to the 18S rRNA gene are shaded. Abbreviations are the same as figure 2. A Boundaries of the full-length R8Hm-A.

B Boundaries of the full-length R8Hm-B. Because there is no conclusive evidence to suggest which possibility is true, we here represent the insertion site of R8 by that of R8Hm-A. There is no sequence similarity between R2 target site and R8 target site. R8 target site is similar to neither R4 nor R5 target sites. The putative protein is 1, amino acids long. The protein of R8Hm-A encodes 3 zinc-finger motifs at the N terminus figs. The protein of R8Hm-B also encodes 3 zinc-finger motifs, but the first motif is defective figs.

They also have a reverse transcriptase domain, a C-terminal zinc finger, and an RLE. N-terminal zinc-finger motifs of R2 and R8. Conserved residues are shaded. The spacing between zinc-finger motifs is shown in parenthesis. The first zinc-finger motif of R8Hm-B is defective. We roughly estimated the copy of each retrotransposon.

Because the genome size of H. To confirm the estimation, we similarly estimated the copy of single-copy genes. We used 10 genes that had been characterized as single copy in H. The average of estimated copy was 0. Thus, our estimated copy seems close to the actual one.

In order to infer the phylogenetic relationships of 4 rDNA-specific retrotransposons, R2, R4, R5, and R8, we constructed the phylogenetic trees based on the amino acid sequences of the reverse transcriptase domain. The length of the reverse transcriptase domain is approximately amino acids. We used 3 methods for inferring the phylogeny—maximum likelihood method, Neighbor-ing method, and Bayesian phylogenetic inference. Because the phylogenies based on the 3 methods were nearly the same, only the consensus maximum likelihood tree is shown fig.

R2Nvec-A is located inside of the R2 superclade as Looking for ltr very specific. R8Hm-A and R8Hm-B are closely related with each other and are the most closely related to the R2-A clade, which is consistent with the N-terminal domain structure fig. R8 is closely related to neither R4 nor R5. The phylogenetic tree was constructed using the amino Looking for ltr very specific sequences of the reverse transcriptase domains figs. The root was determined using 5 sequences of the same group II introns as the study Malik et al.

Elements newly identified in this study are in boldface. Elements specific for rDNA are italicized. The left at each branch represents the percentage of maximum likelihood bootstrap value. The middle and the right s indicate the percentages of Neighbor-ing bootstrap value and Bayesian posterior probability, respectively, for the corresponding cluster. Clade names are shown at the right. Further, to resolve the detailed relationships of R2 and R8, we made other phylogenetic trees using the C-terminal half of putative proteins.

Because there is more sequence information of the C-terminal half of R2 protein than that of the full reverse transcriptase domain, we can include 40 R2 and 2 R8 elements in figure 6although we can use only 24 R2 and 2 R8 elements in figure 5.

Looking for ltr very specific

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