Long-read sequencing identifies an SVA_D retrotransposon insertion deep within the intron of ATP7A as a novel cause of occipital horn syndrome.

BACKGROUND: SINE-VNTR-Alu (SVA) retrotransposons move from one genomic location to another in a 'copy-and-paste' manner. They continue to move actively and cause monogenic diseases through various mechanisms. Currently, disease-causing SVA retrotransposons are classified into human-specific young SVA_E or SVA_F subfamilies. In this study, we identified an evolutionarily old SVA_D retrotransposon as a novel cause of occipital horn syndrome (OHS). OHS is an X-linked, copper metabolism disorder caused by dysfunction of the copper transporter, ATP7A. METHODS: We investigated a 16-year-old boy with OHS whose pathogenic variant could not be detected via routine molecular genetic analyses. RESULTS: A 2.8 kb insertion was detected deep within the intron of the patient's ATP7A gene. This insertion caused aberrant mRNA splicing activated by a new donor splice site located within it. Long-read circular consensus sequencing enabled us to accurately read the entire insertion sequence, which contained highly repetitive and GC-rich segments. Consequently, the insertion was identified as an SVA_D retrotransposon. Antisense oligonucleotides (AOs) targeting the new splice site restored the expression of normal transcripts and functional ATP7A proteins. AO treatment alleviated excessive accumulation of copper in patient fibroblasts in a dose-dependent manner. Pedigree analysis revealed that the retrotransposon had moved into the OHS-causing position two generations ago. CONCLUSION: This is the first report of a human monogenic disease caused by the SVA_D retrotransposon. The fact that the evolutionarily old SVA_D is still actively transposed, leading to increased copy numbers may make a notable impact on rare genetic disease research.

Daidara: A gigantic Gypsy LTR retrotransposon lineage in the springtail Allacma fusca genome.

Long terminal repeat (LTR) retrotransposons are the major contributor to genome size expansion, as in the cases of the maize genome or the axolotl genome. Despite their impact on the genome size, the length of each retrotransposon is limited, compared to DNA transposons, which sometimes exceed over 100 kb. The longest LTR retrotransposon known to date is Burro-1 from the planarian Schmidtea medierranea, which is around 35.7 kb long. Here through bioinformatics analysis, a new lineage of gigantic LTR retrotransposons, designated Daidara, is reported from the springtail Allacma fusca genome. Their entire length (25-33 kb) rivals Burro families, while their LTRs are shorter than 1.5 kb, in contrast to other gigantic LTR retrotransposon lineages Burro and Ogre, whose LTRs are around 5 kb long. Daidara encodes three core proteins corresponding to gag, pol, and an additional protein of unknown function. The phylogenetic analysis supports the independent gigantification of Daidara from Burro or Ogre.

Population Evolution of Helicobacter pylori through Diversification in DNA Methylation and Interstrain Sequence Homogenization.

Decoding of closely related genomes is now revealing the process of population evolution. In bacteria, population divergence appears associated with a unique set of sequence-specific epigenetic DNA methylation systems, often within restriction-modification (RM) systems. They might define a unique gene expression pattern and limit genetic flux between lineages in population divergence. We addressed the contribution of methylation systems to population diversification in panmictic bacterial species, Helicobacter pylori, which shows an interconnected population structure through frequent mutual recombination. We analyzed complete genome sequences of 28 strains collected in Fukui, Japan. Their nucleotide sequences are closely related although fine-scale analyses revealed two subgroups likely reflecting human subpopulations. Their sequences are tightly connected by homologous recombination. Our extensive analysis of RM systems revealed an extreme variability in DNA methyltransferases, especially in their target recognition domains. Their diversity was, however, not immediately related to the genome sequence diversity, except for very closely related strains. An interesting exception is a hybrid strain, which likely has conserved the methylation gene repertoire from one parent but diversified in sequence by massive acquisition of fragmentary DNA sequences from the other parent. Our results demonstrate how a bacterial population can be extremely divergent in epigenetics and yet homogenized in sequence.

Transmission of the PabI family of restriction DNA glycosylase genes: mobility and long-term inheritance.

BACKGROUND: R.PabI is an exceptional restriction enzyme that functions as a DNA glycosylase. The enzyme excises an unmethylated base from its recognition sequence to generate apurinic/apyrimidinic (AP) sites, and also displays AP lyase activity, cleaving the DNA backbone at the AP site to generate the 3'-phospho alpha, beta-unsaturated aldehyde end in addition to the 5'-phosphate end. The resulting ends are difficult to religate with DNA ligase. The enzyme was originally isolated in Pyrococcus, a hyperthermophilic archaeon, and additional homologs subsequently identified in the epsilon class of the Gram-negative bacterial phylum Proteobacteria, such as Helicobacter pylori. RESULTS: Systematic analysis of R.PabI homologs and their neighboring genes in sequenced genomes revealed co-occurrence of R.PabI with M.PabI homolog methyltransferase genes. R.PabI and M.PabI homolog genes are occasionally found at corresponding (orthologous) loci in different species, such as Helicobacter pylori, Helicobacter acinonychis and Helicobacter cetorum, indicating long-term maintenance of the gene pair. One R.PabI and M.PabI homolog gene pair is observed immediately after the GMP synthase gene in both Campylobacter and Helicobacter, representing orthologs beyond genera. The mobility of the PabI family of restriction-modification (RM) system between genomes is evident upon comparison of genomes of sibling strains/species. Analysis of R.PabI and M.PabI homologs in H. pylori revealed an insertion of integrative and conjugative elements (ICE), and replacement with a gene of unknown function that may specify a membrane-associated toxin (hrgC). In view of the similarity of HrgC with toxins in type I toxin-antitoxin systems, we addressed the biological significance of this substitution. Our data indicate that replacement with hrgC occurred in the common ancestor of hspAmerind and hspEAsia. Subsequently, H. pylori with and without hrgC were intermixed at this locus, leading to complex distribution of hrgC in East Asia and the Americas. In Malaysia, hrgC was horizontally transferred from hspEAsia to hpAsia2 strains. CONCLUSIONS: The PabI family of RM system behaves as a mobile, selfish genetic element, similar to the other families of Type II RM systems. Our analysis additionally revealed some cases of long-term inheritance. The distribution of the hrgC gene replacing the PabI family in the subpopulations of H. pylori, hspAmerind, hspEAsia and hpAsia2, corresponds to the two human migration events, one from East Asia to Americas and the other from China to Malaysia.

Evolution and gene capture in ancient endogenous retroviruses - insights from the crocodilian genomes.

BACKGROUND: Crocodilians are thought to be hosts to a diverse and divergent complement of endogenous retroviruses (ERVs) but a comprehensive investigation is yet to be performed. The recent sequencing of three crocodilian genomes provides an opportunity for a more detailed and accurate representation of the ERV diversity that is present in these species. Here we investigate the diversity, distribution and evolution of ERVs from the genomes of three key crocodilian species, and outline the key processes driving crocodilian ERV proliferation and evolution. RESULTS: ERVs and ERV related sequences make up less than 2% of crocodilian genomes. We recovered and described 45 ERV groups within the three crocodilian genomes, many of which are species specific. We have also revealed a new class of ERV, ERV4, which appears to be common to crocodilians and turtles, and currently has no characterised exogenous counterpart. For the first time, we formally describe the characteristics of this ERV class and its classification relative to other recognised ERV and retroviral classes. This class shares some sequence similarity and sequence characteristics with ERV3, although it is phylogenetically distinct from the other ERV classes. We have also identified two instances of gene capture by crocodilian ERVs, one of which, the capture of a host KIT-ligand mRNA has occurred without the loss of an ERV domain. CONCLUSIONS: This study indicates that crocodilian ERVs comprise a wide variety of lineages, many of which appear to reflect ancient infections. In particular, ERV4 appears to have a limited host range, with current data suggesting that it is confined to crocodilians and some lineages of turtles. Also of interest are two ERV groups that demonstrate evidence of host gene capture. This study provides a framework to facilitate further studies into non-mammalian vertebrates and highlights the need for further studies into such species.

Helenus and Ajax, Two Groups of Non-Autonomous LTR Retrotransposons, Represent a New Type of Small RNA Gene-Derived Mobile Elements.

Terminal repeat retrotransposons in miniature (TRIMs) are short non-autonomous long terminal repeat (LTR) retrotransposons found from various eukaryotes. Cassandra is a unique TRIM lineage which contains a 5S rRNA-derived sequence in its LTRs. Here, two new groups of TRIMs, designated Helenus and Ajax, are reported based on bioinformatics analysis and the usage of Repbase. Helenus is found from fungi, animals, and plants, and its LTRs contain a tRNA-like sequence. It includes two LTRs and between them, a primer-binding site (PBS) and polypurine tract (PPT) exist. Fungal and plant Helenus generate 5 bp target site duplications (TSDs) upon integration, while animal Helenus generates 4 bp TSDs. Ajax includes a 5S rRNA-derived sequence in its LTR and is found from two nemertean genomes. Ajax generates 5 bp TSDs upon integration. These results suggest that despite their unique promoters, Helenus and Ajax are TRIMs whose transposition is dependent on autonomous LTR retrotransposon. These TRIMs can originate through an insertion of SINE in an LTR of TRIM. The discovery of Helenus and Ajax suggests the presence of TRIMs with a promoter for RNA polymerase III derived from a small RNA gene, which is here collectively termed TRIMp3.

IS481EU Shows a New Connection between Eukaryotic and Prokaryotic DNA Transposons.

DDD/E transposase gene is the most abundant gene in nature and many DNA transposons in all three domains of life use it for their transposition. A substantial number of eukaryotic DNA transposons show similarity to prokaryotic insertion sequences (ISs). The presence of IS481-like DNA transposons was indicated in the genome of Trichomonas vaginalis. Here, we surveyed IS481-like eukaryotic sequences using a bioinformatics approach and report a group of eukaryotic IS481-like DNA transposons, designated IS481EU, from parabasalids including T. vaginalis. The lengths of target site duplications (TSDs) of IS481EU are around 4 bps, around 15 bps, or around 25 bps, and strikingly, these discrete lengths of TSDs can be observed even in a single IS481EU family. Phylogenetic analysis indicated the close relationships of IS481EU with some of the prokaryotic IS481 family members. IS481EU was not well separated from IS3EU/GingerRoot in the phylogenetic analysis, but was distinct from other eukaryotic DNA transposons including Ginger1 and Ginger2. The unique characteristics of IS481EU in protein sequences and the distribution of TSD lengths support its placement as a new superfamily of eukaryotic DNA transposons.

Base-excision restriction enzymes: expanding the world of epigenetic immune systems.

The restriction enzymes examined so far are phosphodiesterases, which cleave DNA strands by hydrolysing phosphodiester bonds. Based on the mobility of restriction-modification systems, recent studies have identified a family of restriction enzymes that excise a base in their recognition sequence to generate an abasic (AP) site unless the base is properly methylated. These restriction glycosylases also show intrinsic but uncoupled AP lyase activity at the AP site, generating an atypical strand break. Action of an AP endonuclease at the AP site may generate another atypical break, rejoining/repairing of which is difficult. This PabI family of restriction enzymes contain a novel fold (HALFPIPE) and show unusual properties, such as non-requirement of divalent cations for cleavage. These enzymes are present in Helicobacteraceae/Campylobacteraceae and in few hyperthermophilic archaeal species. In Helicobacter genomes, their recognition sites are strongly avoided, and the encoding genes are often inactivated by mutations or replacement, indicating that their expression is toxic for the cells. The discovery of restriction glycosylases generalizes the concept of restriction-modification systems to epigenetic immune systems, which may use any mode of damage to DNA that are considered 'non-self' based on epigenetic modifications. This concept will add to our understanding of immunity and epigenetics.

Alu monomer revisited: recent generation of Alu monomers.

Alu is a predominant short interspersed element (SINE) family in the human genome and consists of two monomer units connected by an A-rich linker. At present, dimeric Alu elements are active in humans, but Alu monomers are present as fossilized sequences. A comparative genome analysis of human and chimpanzee genomes revealed eight recent insertions of Alu monomers. One of them was a retroposed product of another Alu monomer with 3' transduction. Further analysis of 1,404 loci of the Alu monomer in the human genome revealed that some Alu monomers were recently generated by recombination between the internal and 3' A-rich tracts inside of dimeric Alu elements. The data show that Alu monomers were generated by 1) retroposition of other Alu monomers and 2) recombination between two A-rich tracts.

Recent expansion of a new Ingi-related clade of Vingi non-LTR retrotransposons in hedgehogs.

Autonomous non-long terminal repeat (non-LTR) retrotransposons and their repetitive remnants are ubiquitous components of mammalian genomes. Recently, we identified non-LTR retrotransposon families, Ingi-1_AAl and Ingi-1_EE, in two hedgehog genomes. Here we rename them to Vingi-1_AAl and Vingi-1_EE and report a new clade "Vingi," which is a sister clade of Ingi that lacks the ribonuclease H domain. In the European hedgehog genome, there are 11 non-autonomous families of elements derived from Vingi-1_EE by internal deletions. No retrotransposons related to Vingi elements were found in any of the remaining 33 mammalian genomes nearly completely sequenced to date, but we identified several new families of Vingi and Ingi retrotransposons outside mammals. Our data suggest the horizontal transfer of Vingi elements to hedgehog, although the vertical transfer cannot be ruled out. The compact structure and trans-mobilization of nonautonomous derivatives of Vingi can make them useful for in vivo retrotransposition assay system.

Diversity and Evolution of DNA Transposons Targeting Multicopy Small RNA Genes from Actinopterygian Fish.

Dada is a unique superfamily of DNA transposons, inserted specifically in multicopy RNA genes. The zebrafish genome harbors five families of Dada transposons, whose targets are U6 and U1 snRNA genes, and tRNA-Ala and tRNA-Leu genes. Dada-U6, which is inserted specifically in U6 snRNA genes, is found in four animal phyla, but other target-specific lineages have been reported only from one or two species. Here, vertebrate genomes and transcriptomes were surveyed to characterize Dada families with new target specificities, and over 120 Dada families were characterized from the genomes of actinopterygian fish. They were classified into 12 groups with confirmed target specificities. Newly characterized Dada families target tRNA genes for Asp, Asn, Arg, Gly, Lys, Ser, Tyr, and Val, and 5S rRNA genes. Targeted positions inside of tRNA genes are concentrated in two regions: around the anticodon and the A box of RNA polymerase III promoter. Phylogenetic analysis revealed the relationships among actinopterygian Dada families, and one domestication event in the common ancestor of carps and minnows belonging to Cyprinoidei, Cypriniformes. Sequences targeted by phylogenetically related Dada families show sequence similarities, indicating that the target specificity of Dada is accomplished through the recognition of primary nucleotide sequences.

A unique eukaryotic lineage of composite-like DNA transposons encoding a DDD/E transposase and a His-Me finger homing endonuclease.

BACKGROUND: DNA transposons are ubiquitous components of eukaryotic genomes. A major group of them encode a DDD/E transposase and contain terminal inverted repeats (TIRs) of varying lengths. The Kolobok superfamily of DNA transposons has been found in a wide spectrum of organisms. RESULTS: Here we report a new Kolobok lineage, designated KolobokP. They were identified in 7 animal phyla (Mollusca, Phoronida, Annelida, Nemertea, Bryozoa, Chordata, and Echinodermata), and are especially rich in bivalves. Unlike other Kolobok families, KolobokP adopts a composite-like architecture: an internal region (INT) flanked by two long terminal direct repeats (LTDRs), which exhibit their own short terminal inverted repeats ranging up to 18 bps. The excision of LTDRs was strongly suggested. The LTDR lengths seem to be constrained to be either around 450-bp or around 660-bp. The internal region encodes a DDD/E transposase and a small His-Me finger nuclease, which likely originated from the homing endonuclease encoded by a group I intron from a eukaryotic species. The architecture of KolobokP resembles composite DNA transposons, usually observed in bacterial genomes, and long terminal repeat (LTR) retrotransposons. In addition to this monomeric LTDR-INT-LTDR structure, plenty of solo LTDRs and multimers represented as (LTDR-INT)n-LTDR are also observed. Our structural and phylogenetic analysis supported the birth of KolobokP in the late stage of the Kolobok evolution. We propose KolobokP families propagate themselves in two ways: the canonical transposition catalyzed by their transposase and the sequence-specific cleavage by their endonuclease followed by the multimerization through the unequal crossover. CONCLUSIONS: The presence of homing endonuclease and long terminal direct repeats of KolobokP families suggest their unique dual replication mechanisms: transposition and induced unequal crossover.

Whole chromosome elimination and chromosome terminus elimination both contribute to somatic differentiation in Taiwanese hagfish Paramyxine sheni.

Chromosome elimination is a process in which some chromatins are discarded from the presumptive somatic cells during early embryogenesis. Eliminated chromatins in hagfish generally consist of repetitive sequences, and they are highly heterochromatinized in germ cells. In this study, we characterized four novel eliminated DNA families, EEPs1-4, from the Taiwanese hagfish Paramyxine sheni. Sequences of these four elements occupied 20-27% of eliminated DNA in total, and each family was arranged mainly in tandem in the germline genome with high copy numbers. Although most of these elements were eliminated, a minor fraction remained in somatic cells. Some eliminated DNA families are shared as eliminated sequences between Eptatretidae and Myxinidae. Fluorescence in situ hybridization (FISH) of these elements showed that not only heterochromatic chromosomes but also both ends of euchromatic chromosomes in germ cells are absent in somatic cells of P. sheni. It strongly suggests that chromosome terminus elimination, in addition to whole chromosome elimination, contributes to somatic chromosome differentiation. Telomere-FISH further showed that chromosome fragmentation and the subsequent de novo addition of telomeric repeats are the likely mechanisms underlying chromosome terminus elimination. These characteristics make it indispensable to study the evolution and mechanisms underlying chromosome elimination in hagfish.

mRNA retrotransposition coupled with 5' inversion as a possible source of new genes.

Human long interspersed nuclear element-1 (L1) occupies one-sixth of our genome and has contributed to genome evolution in various ways. Approximately 10% of human L1 copies are composed of two L1 segments; the 5' segment and 3' segment are in head-to-head (i.e., 5'-inverted) orientation. Besides mediating their own retrotransposition, L1 has the ability to mobilize mRNA "in trans," and the number of retrotransposed mRNA sequences (retrocopies) is estimated to be >6,000. In this study, we identified 48 human-specific retrocopies and 95 chimpanzee-specific retrocopies by comparing the human and chimpanzee genomes. Among these retrocopies, 12 were 5'-inverted. The characteristics of these 5'-inverted retrocopies were similar to those of 5'-inverted L1 copies, indicating that the 5' inversion is generated by the same mechanism. With these findings, we examined the possibility that 5' inversion of the retrocopy generates a new gene that codes for a peptide with a different N terminus. We identified several potential 5'-inverted retrogenes, including those of thymopoietin beta (TMPO) and eukaryotic translation initiation factor 3 subunit 5 (EIF3F). The most interesting candidate was the 5'-inverted retrocopy of small nuclear ribonucleoprotein polypeptide N (SNRPN). This retrocopy was transcribed in the reverse orientation in several organs, had multiple transcript variants, and encoded a protein containing a peptide fragment derived from the N-terminal portion of SNRPN. Our results suggest that mRNA retrotransposition coupled with 5' inversion may be a mechanism to generate new genes distinct from parental genes.

Structural and sequence diversity of eukaryotic transposable elements.

The majority of eukaryotic genomes contain a large fraction of repetitive sequences that primarily originate from transpositional bursts of transposable elements (TEs). Repbase serves as a database for eukaryotic repetitive sequences and has now become the largest collection of eukaryotic TEs. During the development of Repbase, many new superfamilies/lineages of TEs, which include Helitron, Polinton, Ginger and SINEU, were reported. The unique composition of protein domains and DNA motifs in TEs sometimes indicates novel mechanisms of transposition, replication, anti-suppression or proliferation. In this review, our current understanding regarding the diversity of eukaryotic TEs in sequence, protein domain composition and structural hallmarks is introduced and summarized, based on the classification system implemented in Repbase. Autonomous eukaryotic TEs can be divided into two groups: Class I TEs, also called retrotransposons, and Class II TEs, or DNA transposons. Long terminal repeat (LTR) retrotransposons, including endogenous retroviruses, non-LTR retrotransposons, tyrosine recombinase retrotransposons and Penelope-like elements, are well accepted groups of autonomous retrotransposons. They share reverse transcriptase for replication but are distinct in the catalytic components responsible for integration into the host genome. Similarly, at least three transposition machineries have been reported in eukaryotic DNA transposons: DDD/E transposase, tyrosine recombinase and HUH endonuclease combined with helicase. Among these, TEs with DDD/E transposase are dominant and are classified into 21 superfamilies in Repbase. Non-autonomous TEs are either simple derivatives generated by internal deletion, or are composed of several units that originated independently.

AcademH, a lineage of Academ DNA transposons encoding helicase found in animals and fungi.

BACKGROUND: DNA transposons are ubiquitous components of eukaryotic genomes. Academ superfamily of DNA transposons is one of the least characterized DNA transposon superfamilies in eukaryotes. DNA transposons belonging to the Academ superfamily have been reported from various animals, one red algal species Chondrus crispus, and one fungal species Puccinia graminis. Six Academ families from P. graminis encode a helicase in addition to putative transposase, while some other families encode a single protein which contains a putative transposase and an XPG nuclease. RESULTS: Systematic searches on Repbase and BLAST searches against publicly available genome sequences revealed that several species of fungi and animals contain multiple Academ transposon families encoding a helicase. These AcademH families generate 9 or 10-bp target site duplications (TSDs) while Academ families lacking helicase generate 3 or 4-bp TSDs. Phylogenetic analysis clearly shows two lineages inside of Academ, designated here as AcademH and AcademX for encoding helicase or XPG nuclease, respectively. One sublineage of AcademH in animals encodes plant homeodomain (PHD) finger in its transposase, and its remnants are found in several fish genomes. CONCLUSIONS: The AcademH lineage of TEs is widely distributed in animals and fungi, and originated early in the evolution of Academ DNA transposons. This analysis highlights the structural diversity in one less studied superfamily of eukaryotic DNA transposons.

Hagfish genome reveals parallel evolution of 7SL RNA-derived SINEs.

BACKGROUND: Short interspersed elements (SINEs) are ubiquitous components of eukaryotic genomes. SINEs are composite transposable elements that are mobilized by non-long terminal repeat (non-LTR) retrotransposons, also called long interspersed elements (LINEs). The 3' part of SINEs usually originated from that of counterpart non-LTR retrotransposons. The 5' part of SINEs mostly originated from small RNA genes. SINE1 is a group of SINEs whose 5' part originated from 7SL RNA, and is represented by primate Alu and murine B1. Well-defined SINE1 has been found only from Euarchontoglires, a group of mammals, in contrast to the wide distribution of SINE2, which has a tRNA-derived sequence, from animals to plants to protists. Both Alu and B1 are mobilized by L1-type non-LTR retrotransposons, which are the only lineage of autonomous non-LTR retrotransposons active in these mammalian lineages. RESULTS: Here a new lineage of SINE1 is characterized from the seashore hagfish Eptatretus burgeri genome. This SINE1 family, designated SINE1-1_EBu, is young, and is transposed by RTE-type non-LTR retrotransposon, not L1-type. Comparison with other SINE families from hagfish indicated the birth of SINE1-1_EBu through chimera formation of a 7SL RNA-derived sequence and an older tRNA-derived SINE family. It reveals parallel evolution of SINE1 in two vertebrate lineages with different autonomous non-LTR retrotransposon partners. The comparison between two SINE1 lineages supports that the RNA secondary structure of the Alu domain of 7SL RNA is required for the efficient retrotransposition. CONCLUSIONS: The hagfish SINE1 is the first evident SINE1 family found outside of Euarchontoglires. Independent evolution of SINE1 with similar RNA secondary structure originated in 7SL RNA indicates the functional importance of 7SL RNA-derived sequence in the proliferation of SINEs.

Bioinformatic and Molecular Analysis of Satellite Repeat Diversity in Vaccinium Genomes.

Bioinformatic and molecular characterization of satellite repeats was performed to understand the impact of their diversification on Vaccinium genome evolution. Satellite repeat diversity was evaluated in four cultivated and wild species, including the diploid species Vaccinium myrtillus and Vaccinium uliginosum, as well as the tetraploid species Vaccinium corymbosum and Vaccinium arctostaphylos. We comparatively characterized six satellite repeat families using in total 76 clones with 180 monomers. We observed that the monomer units of VaccSat1, VaccSat2, VaccSat5, and VaccSat6 showed a higher order repeat (HOR) structure, likely originating from the organization of two adjacent subunits with differing similarity, length and size. Moreover, VaccSat1, VaccSat3, VaccSat6, and VaccSat7 were found to have sequence similarity to parts of transposable elements. We detected satellite-typical tandem organization for VaccSat1 and VaccSat2 in long arrays, while VaccSat5 and VaccSat6 distributed in multiple sites over all chromosomes of tetraploid V. corymbosum, presumably in long arrays. In contrast, very short arrays of VaccSat3 and VaccSat7 are dispersedly distributed over all chromosomes in the same species, likely as internal parts of transposable elements. We provide a comprehensive overview on satellite species specificity in Vaccinium, which are potentially useful as molecular markers to address the taxonomic complexity of the genus, and provide information for genome studies of this genus.

Systematic survey for novel types of prokaryotic retroelements based on gene neighborhood and protein architecture.

Retroelements, elements encoding reverse transcriptase (RT), are ubiquitous in eukaryotes and have a great influence on the evolution of our genome. Detailed information is available on eukaryotic retroelements; however, prokaryotic retroelements are poorly understood. Recently, new types of eukaryotic retroelements were characterized on the basis of their gene composition and their phylogenetic positions. Here we performed a systematic survey to identify novel types of prokaryotic retroelements by analyzing gene neighborhood and protein architecture. We found novel types of gene combination and examined whether they represent actual retroelements. Five monophyletic groups were identified that were distinct from characterized prokaryotic retroelements, showed specific gene combination, were distributed patchily, and included at least 1 example of recent integration. These results strongly indicated the frequent horizontal transfer of these elements. One group encoded DNA polymerase A. A possible function of DNA polymerase A in the life cycle of retroelements is catalyzing second-strand cDNA synthesis, which is DNA polymerization performed using a DNA template not an RNA template. Another group encoded both bacterial primase and carbon-nitrogen hydrolase. Primase is likely to synthesize primers to initiate reverse transcription. Two other groups also encoded carbon-nitrogen hydrolase as a fusion protein with RT. It is difficult to speculate on the function of hydrolase in the life cycle of retroelements. The last group encoded dual RT proteins, which are likely to form heterodimers during replication. The protein sets of these 5 groups of prokaryotic retroelements were completely different from those of eukaryotic retroelements, indicating that the survival constraints of prokaryotic elements were distinct from those of eukaryotic elements. It is likely that these prokaryotic retroelements are maintained as extrachromosomal DNA or RNA or are accidentally integrated into genomes. Our findings presented the possibility that many types of extrachromosomal prokaryotic retroelements remain to be characterized. In addition, we found 8 RT genes were associated with clustered regularly interspaced short palindrome repeats (CRISPRs) of the CRISPR-Cas system. These RT genes are likely to work in immunity against RNA phages via cDNA synthesis.

Eukaryotic translational coupling in UAAUG stop-start codons for the bicistronic RNA translation of the non-long terminal repeat retrotransposon SART1.

Most eukaryotic cellular mRNAs are monocistronic; however, many retroviruses and long terminal repeat (LTR) retrotransposons encode multiple proteins on a single RNA transcript using ribosomal frameshifting. Non-long terminal repeat (non-LTR) retrotransposons are considered the ancestor of LTR retrotransposons and retroviruses, but their translational mechanism of bicistronic RNA remains unknown. We used a baculovirus expression system to produce a large amount of the bicistronic RNA of SART1, a non-LTR retrotransposon of the silkworm, and were able to detect the second open reading frame protein (ORF2) by Western blotting. The ORF2 protein was translated as an independent protein, not as an ORF1-ORF2 fusion protein. We revealed by mutagenesis that the UAAUG overlapping stop-start codon and the downstream RNA secondary structure are necessary for efficient ORF2 translation. Increasing the distance between the ORF1 stop codon and the ORF2 start codon decreased translation efficiency. These results are different from the eukaryotic translation reinitiation mechanism represented by the yeast GCN4 gene, in which the probability of reinitiation increases as the distance between the two ORFs increases. The translational mechanism of SART1 ORF2 is analogous to translational coupling observed in prokaryotes and viruses. Our results indicate that translational coupling is a general mechanism for bicistronic RNA translation.