A single amino acid switch converts the Sleeping Beauty transposase into an efficient unidirectional excisionase with utility in stem cell reprogramming.

The Sleeping Beauty (SB) transposon is an advanced tool for genetic engineering and a useful model to investigate cut-and-paste DNA transposition in vertebrate cells. Here, we identify novel SB transposase mutants that display efficient and canonical excision but practically unmeasurable genomic re-integration. Based on phylogenetic analyses, we establish compensating amino acid replacements that fully rescue the integration defect of these mutants, suggesting epistasis between these amino acid residues. We further show that the transposons excised by the exc+/int- transposase mutants form extrachromosomal circles that cannot undergo a further round of transposition, thereby representing dead-end products of the excision reaction. Finally, we demonstrate the utility of the exc+/int- transposase in cassette removal for the generation of reprogramming factor-free induced pluripotent stem cells. Lack of genomic integration and formation of transposon circles following excision is reminiscent of signal sequence removal during V(D)J recombination, and implies that cut-and-paste DNA transposition can be converted to a unidirectional process by a single amino acid change.

The ctenophore genome and the evolutionary origins of neural systems.

The origins of neural systems remain unresolved. In contrast to other basal metazoans, ctenophores (comb jellies) have both complex nervous and mesoderm-derived muscular systems. These holoplanktonic predators also have sophisticated ciliated locomotion, behaviour and distinct development. Here we present the draft genome of Pleurobrachia bachei, Pacific sea gooseberry, together with ten other ctenophore transcriptomes, and show that they are remarkably distinct from other animal genomes in their content of neurogenic, immune and developmental genes. Our integrative analyses place Ctenophora as the earliest lineage within Metazoa. This hypothesis is supported by comparative analysis of multiple gene families, including the apparent absence of HOX genes, canonical microRNA machinery, and reduced immune complement in ctenophores. Although two distinct nervous systems are well recognized in ctenophores, many bilaterian neuron-specific genes and genes of 'classical' neurotransmitter pathways either are absent or, if present, are not expressed in neurons. Our metabolomic and physiological data are consistent with the hypothesis that ctenophore neural systems, and possibly muscle specification, evolved independently from those in other animals.

A novel group of diverse Polinton-like viruses discovered by metagenome analysis.

BACKGROUND: The rapidly growing metagenomic databases provide increasing opportunities for computational discovery of new groups of organisms. Identification of new viruses is particularly straightforward given the comparatively small size of viral genomes, although fast evolution of viruses complicates the analysis of novel sequences. Here we report the metagenomic discovery of a distinct group of diverse viruses that are distantly related to the eukaryotic virus-like transposons of the Polinton superfamily. RESULTS: The sequence of the putative major capsid protein (MCP) of the unusual linear virophage associated with Phaeocystis globosa virus (PgVV) was used as a bait to identify potential related viruses in metagenomic databases. Assembly of the contigs encoding the PgVV MCP homologs followed by comprehensive sequence analysis of the proteins encoded in these contigs resulted in the identification of a large group of Polinton-like viruses (PLV) that resemble Polintons (polintoviruses) and virophages in genome size, and share with them a conserved minimal morphogenetic module that consists of major and minor capsid proteins and the packaging ATPase. With a single exception, the PLV lack the retrovirus-type integrase that is encoded in the genomes of all Polintons and the Mavirus group of virophages. However, some PLV encode a newly identified tyrosine recombinase-integrase that is common in bacteria and bacteriophages and is also found in the Organic Lake virophage group. Although several PLV genomes and individual genes are integrated into algal genomes, it appears likely that most of the PLV are viruses. Given the absence of protease and retrovirus-type integrase, the PLV could resemble the ancestral polintoviruses that evolved from bacterial tectiviruses. Apart from the conserved minimal morphogenetic module, the PLV widely differ in their genome complements but share a gene network with Polintons and virophages, suggestive of multiple gene exchanges within a shared gene pool. CONCLUSIONS: The discovery of PLV substantially expands the emerging class of eukaryotic viruses and transposons that also includes Polintons and virophages. This class of selfish elements is extremely widespread and might have been a hotbed of eukaryotic virus, transposon and plasmid evolution. New families of these elements are expected to be discovered.

ISC, a Novel Group of Bacterial and Archaeal DNA Transposons That Encode Cas9 Homologs.

UNLABELLED: Bacterial genomes encode numerous homologs of Cas9, the effector protein of the type II CRISPR-Cas systems. The homology region includes the arginine-rich helix and the HNH nuclease domain that is inserted into the RuvC-like nuclease domain. These genes, however, are not linked to cas genes or CRISPR. Here, we show that Cas9 homologs represent a distinct group of nonautonomous transposons, which we denote ISC (insertion sequences Cas9-like). We identify many diverse families of full-length ISC transposons and demonstrate that their terminal sequences (particularly 3' termini) are similar to those of IS605 superfamily transposons that are mobilized by the Y1 tyrosine transposase encoded by the TnpA gene and often also encode the TnpB protein containing the RuvC-like endonuclease domain. The terminal regions of the ISC and IS605 transposons contain palindromic structures that are likely recognized by the Y1 transposase. The transposons from these two groups are inserted either exactly in the middle or upstream of specific 4-bp target sites, without target site duplication. We also identify autonomous ISC transposons that encode TnpA-like Y1 transposases. Thus, the nonautonomous ISC transposons could be mobilized in trans either by Y1 transposases of other, autonomous ISC transposons or by Y1 transposases of the more abundant IS605 transposons. These findings imply an evolutionary scenario in which the ISC transposons evolved from IS605 family transposons, possibly via insertion of a mobile group II intron encoding the HNH domain, and Cas9 subsequently evolved via immobilization of an ISC transposon. IMPORTANCE: Cas9 endonucleases, the effectors of type II CRISPR-Cas systems, represent the new generation of genome-engineering tools. Here, we describe in detail a novel family of transposable elements that encode the likely ancestors of Cas9 and outline the evolutionary scenario connecting different varieties of these transposons and Cas9.

The amphioxus genome and the evolution of the chordate karyotype.

Lancelets ('amphioxus') are the modern survivors of an ancient chordate lineage, with a fossil record dating back to the Cambrian period. Here we describe the structure and gene content of the highly polymorphic approximately 520-megabase genome of the Florida lancelet Branchiostoma floridae, and analyse it in the context of chordate evolution. Whole-genome comparisons illuminate the murky relationships among the three chordate groups (tunicates, lancelets and vertebrates), and allow not only reconstruction of the gene complement of the last common chordate ancestor but also partial reconstruction of its genomic organization, as well as a description of two genome-wide duplications and subsequent reorganizations in the vertebrate lineage. These genome-scale events shaped the vertebrate genome and provided additional genetic variation for exploitation during vertebrate evolution.

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.

Modeling the asymmetric evolution of a mouse and rat-specific microRNA gene cluster intron 10 of the Sfmbt2 gene.

BACKGROUND: The total number of miRNA genes in a genome, expression of which is responsible for the miRNA repertoire of an organism, is not precisely known. Moreover, the question of how new miRNA genes arise during evolution is incompletely understood. Recent data in humans and opossum indicate that retrotranspons of the class of short interspersed nuclear elements have contributed to the growth of microRNA gene clusters. METHOD: We studied a large miRNA gene cluster in intron 10 of the mouse Sfmbt2 gene using bioinformatic tools. RESULTS: Mice and rats are unique to harbor a 55-65 Kb DNA sequence in intron 10 of the Sfmbt2 gene. This intronic region is rich in regularly repeated B1 retrotransposons together with inverted self-complementary CA/TG microsatellites. The smallest repeats unit, called MSHORT1 in the mouse, was duplicated 9 times in a tandem head-to-tail array to form 2.5 Kb MLONG1 units. The center of the mouse miRNA gene cluster consists of 13 copies of MLONG1. BLAST analysis of MSHORT1 in the mouse shows that the repeat unit is unique for intron 10 of the Sfmbt2 gene and suggest a dual phase model for growth of the miRNA gene cluster:arrangement [corrected] of 10 MSHORT1 units into MLONG1 and further duplication of 13 head-to-tail MLONG1 units in the center of the miRNA gene cluster. Rats have a similar arrangement [corrected] of repeat units in intron 10 of the Sfmbt2 gene. The discrepancy between 65 miRNA genes in the mouse cluster as compared to only 1 miRNA gene in the corresponding rat repeat cluster is ascribed to sequence differences between MSHORT1 and RSHORT1 that result in lateral-shifted, less-stable miRNA precursor hairpins for RSHORT1. CONCLUSION: Our data provides new evidence for the emerging concept that lineage-specific retroposons have played an important role in the birth of new miRNA genes during evolution. The large difference in the number of miRNA genes in two closely related species (65 versus 1, mice versus rats) indicates that this species-specific evolution can be a rapid process.

Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae.

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.

Transposition of a reconstructed Harbinger element in human cells and functional homology with two transposon-derived cellular genes.

Ancient, inactive copies of transposable elements of the PIF/Harbinger superfamily have been described in vertebrates. We reconstructed components of the Harbinger3_DR transposon in zebrafish, including a transposase and a second, transposon-encoded protein that has a Myb-like trihelix domain. The reconstructed Harbinger transposon shows efficient cut-and-paste transposition in human cells and preferentially inserts into a 15-bp consensus target sequence. The Myb-like protein is required for transposition and physically interacts with the N-terminal region of the transposase via its C-terminal domain. The Myb-like protein enables transposition in part by promoting nuclear import of the transposase, by directly binding to subterminal regions of the transposon, and by recruiting the transposase to the transposon ends. We investigated the functions of two transposon-derived human proteins: HARBI1, a domesticated transposase-derived protein, and NAIF1, which contains a trihelix motif similar to that described in the Myb-like protein. Physical interaction, subcellular localization, and DNA-binding activities of HARBI1 and NAIF1 suggest strong functional homologies between the Harbinger3_DR system and their related, host-encoded counterparts. The Harbinger transposon will serve as a useful experimental system for transposon biology and for investigating the enzymatic functions of domesticated, transposon-derived cellular genes.

Population analysis of retrotransposons in giraffe genomes supports RTE decline and widespread LINE1 activity in Giraffidae.

BACKGROUND: The majority of structural variation in genomes is caused by insertions of transposable elements (TEs). In mammalian genomes, the main TE fraction is made up of autonomous and non-autonomous non-LTR retrotransposons commonly known as LINEs and SINEs (Long and Short Interspersed Nuclear Elements). Here we present one of the first population-level analysis of TE insertions in a non-model organism, the giraffe. Giraffes are ruminant artiodactyls, one of the few mammalian groups with genomes that are colonized by putatively active LINEs of two different clades of non-LTR retrotransposons, namely the LINE1 and RTE/BovB LINEs as well as their associated SINEs. We analyzed TE insertions of both types, and their associated SINEs in three giraffe genome assemblies, as well as across a population level sampling of 48 individuals covering all extant giraffe species. RESULTS: The comparative genome screen identified 139,525 recent LINE1 and RTE insertions in the sampled giraffe population. The analysis revealed a drastically reduced RTE activity in giraffes, whereas LINE1 is still actively propagating in the genomes of extant (sub)-species. In concert with the extremely low activity of the giraffe RTE, we also found that RTE-dependent SINEs, namely Bov-tA and Bov-A2, have been virtually immobile in the last 2 million years. Despite the high current activity of the giraffe LINE1, we did not find evidence for the presence of currently active LINE1-dependent SINEs. TE insertion heterozygosity rates differ among the different (sub)-species, likely due to divergent population histories. CONCLUSIONS: The horizontally transferred RTE/BovB and its derived SINEs appear to be close to inactivation and subsequent extinction in the genomes of extant giraffe species. This is the first time that the decline of a TE family has been meticulously analyzed from a population genetics perspective. Our study shows how detailed information about past and present TE activity can be obtained by analyzing large-scale population-level genomic data sets.

New superfamilies of eukaryotic DNA transposons and their internal divisions.

Despite their enormous diversity and abundance, all currently known eukaryotic DNA transposons belong to only 15 superfamilies. Here, we report two new superfamilies of DNA transposons, named Sola and Zator. Sola transposons encode DDD-transposases (transposase, TPase) and are flanked by 4-bp target site duplications (TSD). Elements from the Sola superfamily are distributed in a variety of species including bacteria, protists, plants, and metazoans. They can be divided into three distinct groups of elements named Sola1, Sola2, and Sola3. The elements from each group have extremely low sequence identity to each other, different termini, and different target site preferences. However, all three groups belong to a single superfamily based on significant PSI-Blast identities between their TPases. The DDD TPase sequences encoded by Sola transposons are not similar to any known TPases. The second superfamily named Zator is characterized by 3-bp TSD. The Zator superfamily is relatively rare in eukaryotic species, and it evolved from a bacterial transposon encoding a TPase belonging to the "transposase 36" family (Pfam07592). These transposons are named TP36 elements (abbreviated from transposase 36).

Helitrons on a roll: eukaryotic rolling-circle transposons.

Rolling-circle eukaryotic transposons, known as Helitron transposons, were first discovered in plants (Arabidopsis thaliana and Oryza sativa) and in the nematode Caenorhabditis elegans. To date, Helitrons have been identified in a diverse range of species, from protists to mammals. They represent a major class of eukaryotic transposons and are fundamentally different from classical transposons in terms of their structure and mechanism of transposition. Helitrons seem to have a major role in the evolution of host genomes. They frequently capture diverse host genes, some of which can evolve into novel host genes or become essential for helitron transposition.

Fungal literature records database of the Northern West Siberia (Russia).

BACKGROUND: Mycological research in the Northern part of West Siberia has now become sufficient for review and digitisation as over 460 scientific works have been completed mainly since the beginning of the 20th century. The history of research in the region started from isolated studies at the beginning of the 20th century, but regular and systematic research started from the 1970s. Over the following decades, several dozens of researchers have worked in the area, but the reported occurrences were scattered amongst a broad variety of publications, mainly hardly available. The great need in digitisation and accumulation of fungal records reported in published literature in a standardised regional database has now become evident. The «Fungal records database of the Northern West Siberia¼ (FuNWS) was initiated in 2016 according to contemporary biodiversity data standards (Darwin Core), to be compatible and accessible by the broad research community. The database has been supplemented ever since by the collective effort of specialists working in the area. According to the database summary report, there are 3358 fungal and fungus-like species revealed in the Northern West Siberia at present. The richest in species number classes are Agaricomycetes (60%) and Lecanoromycetes (33%) with a total of 25 classes represented. The FuNWS database was uploaded to Global Biodiversity Information Facility (GBIF) (Ygra State University Biological Collection publisher) on 11 November 2017 (earlier titled «Fungal Records Database of Yugra, FReDY¼) to provide open access to the data and its reusability (Filippova et al. 2020). NEW INFORMATION: This publication summarises the results of the digitisation of literature-based occurrence records of fungi and fungus-like organisms initiated in the Northern part of West Siberia for the first time in the history of mycological research. The bibliography of regional mycological publications was created to include about 460 published works (Suppl. material 2). In total, about 140 literature sources were digitised and about 22000 occurrence records were integrated into the FuNWS database (Filippova et al. 2020).

Profiles of Little-Known Medicinal Polypores: Haploporus odorus (Agaricomycetes).

The purpose of this study was to compile a comprehensive characterization of little-known polypores, which have recently been found to possess anticancer activity and thus can also be used in cancer target therapy. Haploporus odorus is a polypore of Holarctic distribution and has been found by harvesters working in taiga floodlands and broadleaf forests of the Northern Hemisphere. A substance known as haploporic acid A was determined in methylene chloride extract from the dried basidiomata of H. odorus. This substance can be used in cancer therapy; more details of its health benefits could be used in mycotherapy.

RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons.

The V(D)J recombination reaction in jawed vertebrates is catalyzed by the RAG1 and RAG2 proteins, which are believed to have emerged approximately 500 million years ago from transposon-encoded proteins. Yet no transposase sequence similar to RAG1 or RAG2 has been found. Here we show that the approximately 600-amino acid "core" region of RAG1 required for its catalytic activity is significantly similar to the transposase encoded by DNA transposons that belong to the Transib superfamily. This superfamily was discovered recently based on computational analysis of the fruit fly and African malaria mosquito genomes. Transib transposons also are present in the genomes of sea urchin, yellow fever mosquito, silkworm, dog hookworm, hydra, and soybean rust. We demonstrate that recombination signal sequences (RSSs) were derived from terminal inverted repeats of an ancient Transib transposon. Furthermore, the critical DDE catalytic triad of RAG1 is shared with the Transib transposase as part of conserved motifs. We also studied several divergent proteins encoded by the sea urchin and lancelet genomes that are 25%-30% identical to the RAG1 N-terminal domain and the RAG1 core. Our results provide the first direct evidence linking RAG1 and RSSs to a specific superfamily of DNA transposons and indicate that the V(D)J machinery evolved from transposons. We propose that only the RAG1 core was derived from the Transib transposase, whereas the N-terminal domain was assembled from separate proteins of unknown function that may still be active in sea urchin, lancelet, hydra, and starlet sea anemone. We also suggest that the RAG2 protein was not encoded by ancient Transib transposons but emerged in jawed vertebrates as a counterpart of RAG1 necessary for the V(D)J recombination reaction.

High power CW (16W) and pulse (145W) laser diodes based on quantum well heterostructures.

We suggested and experimentally confirmed the effective method of internal optical loss reduction by high order mode suppression in a separate confinement quantum well laser heterostructure with asymmetric ultra thick waveguide. Manufacturing of InGaAs/GaAs/AlGaAs laser heterostructure with a 1.7 microm-thick asymmetric waveguide allowed attaining super low value of internal optical loss alphai=0.34 cm-1 preserving high efficiency and fundamental transverse mode operation. Record-high 16 W continuous wave (CW) and 145 W pulse room temperature front facet output optical power and 74% wallplug efficiency were attained in 100-microm-aperture 1.06-microm-emitting laser diodes with 3 mm cavity length.

HERVd: the Human Endogenous RetroViruses Database: update.

An elaboration of HERVd (http://herv.img.cas.cz) is being carried out in two directions. One of them is the integration and better classification of families that diverge considerably from typical retroviral genomes. This leads to a more precise identification of members with individual families. The second improvement is better accessibility of the database and connection with human genome annotation.

A Helitron transposon reconstructed from bats reveals a novel mechanism of genome shuffling in eukaryotes.

Helitron transposons capture and mobilize gene fragments in eukaryotes, but experimental evidence for their transposition is lacking in the absence of an isolated active element. Here we reconstruct Helraiser, an ancient element from the bat genome, and use this transposon as an experimental tool to unravel the mechanism of Helitron transposition. A hairpin close to the 3'-end of the transposon functions as a transposition terminator. However, the 3'-end can be bypassed by the transposase, resulting in transduction of flanking sequences to new genomic locations. Helraiser transposition generates covalently closed circular intermediates, suggestive of a replicative transposition mechanism, which provides a powerful means to disseminate captured transcriptional regulatory signals across the genome. Indeed, we document the generation of novel transcripts by Helitron promoter capture both experimentally and by transcriptome analysis in bats. Our results provide mechanistic insight into Helitron transposition, and its impact on diversification of gene function by genome shuffling.

Evolution of the RAG1-RAG2 locus: both proteins came from the same transposon.

The RAG1 and RAG2 proteins are essential subunits of the V(D)J recombinase that is required for the generation of the enormous variability of antibodies and T-cell receptors in jawed vertebrates. It was demonstrated previously that the 600-aa catalytic core of RAG1 evolved from the transposase of the Transib superfamily transposons. However, although homologs of RAG1 and RAG2 genes are adjacent in the purple sea urchin genome, a transposon encoding both proteins so far has not been reported. Here we describe such transposons in the genomes of green sea urchin, a starfish and an oyster. Comparison of the domain architectures of the RAG1 homologs in these transposons, denoted TransibSU, and other Transib superfamily transposases provides for reconstruction of the structure of the hypothetical TransibVDJ transposon that gave rise to the VDJ recombinases at the onset of vertebrate evolution some 500 million years ago.