Mobilizing the genome of Lepidoptera through novel sequence gains and end creation by non-autonomous Lep1 Helitrons

Transposable elements (TEs) can affect the structure of genomes through their acquisition and transposition of novel DNA sequences. The 134-bp repetitive elements, Lep1, are conserved non-autonomous Helitrons in lepidopteran genomes that have characteristic 5'-CT and 3'-CTAY nucleotide termini, a 3'-terminal hairpin structure, a 5'- and 3'-subterminal inverted repeat (SIR), and integrations that occur between AT or TT nucleotides. Lep1 Helitrons have acquired and propagated sequences downstream of their 3'-CTAY termini that are 57-344-bp in length and have termini composed of a 3'-CTRR preceded by a 3'-hairpin structure and a region complementary to the 5'-SIR (3'-SIRb). Features of both the Lep1 Helitron and multiple acquired sequences indicate that secondary structures at the 3'-terminus may have a role in rolling circle replication or genome integration mechanisms, and are a prerequisite for novel end creation by Helitron-like TEs. The preferential integration of Lep1 Helitrons in proximity to gene-coding regions results in the creation of genetic novelty that is shown to impact gene structure and function through the introduction of novel exon sequence (exon shuffling). These findings are important in understanding the structural requirements of genomic DNA sequences that are acquired and transposed by Helitron-like TEs.     

Landscape and evolutionary dynamics of Helitron transposons in plant genomes as well as construction of online database HelDB

Helitron transposons play an important role in host genome evolution due to their ability to capture genes and regulatory elements. In this study, we developed a pipeline to identify and annotate Helitrons systematically from 358 plant and 178 animal high-quality genomes. All these data were organized into HelDB, a database where Helitrons can be explored with a user-friendly Web interface and related software. Based on these data, further analysis showed that the number or the cumulative length of Helitrons is positively correlated with genome size. Helitrons had experienced two expansion periods in plants, with the first occurring 20–30 Ma and peaking at approximately 24 Ma. The second expansion occurred in the last 4 million years. The expansions might be due to stimulation of paleogeographic environment. Detailed investigation of gene capture by Helitrons in Brassicaceae and Solanaceae plants showed that the captured genes showed diverse functions. Interestingly, metal ion binding function was enriched in these captured genes in most species. This phenomenon might be due to the need for binding of divalent metal ions to the Rep domain required for Helitron transposition. This study improves our knowledge of the landscape and evolution of Helitron transposons in plants and paves a way for further functional studies of this kind of transposable element.     

The maize genome contains a helitron insertion

The maize mutation sh2-7527 was isolated in a conventional maize breeding program in the 1970s. Although the mutant contains foreign sequences within the gene, the mutation is not attributable to an interchromosomal exchange or to a chromosomal inversion. Hence, the mutation was caused by an insertion. Sequences at the two Sh2 borders have not been scrambled or mutated, suggesting that the insertion is not caused by a catastrophic reshuffling of the maize genome. The insertion is large, at least 12 kb, and is highly repetitive in maize. As judged by hybridization, sorghum contains only one or a few copies of the element, whereas no hybridization was seen to the Arabidopsis genome. The insertion acts from a distance to alter the splicing of the sh2 pre-mRNA. Three distinct intron-bearing maize genes were found in the insertion. Of most significance, the insertion bears striking similarity to the recently described DNA helicase-bearing transposable elements termed HELITRONS: Like Helitrons, the inserted sequence of sh2-7527 is large, lacks terminal repeats, does not duplicate host sequences, and was inserted between a host dinucleotide AT. Like Helitrons, the maize element contains 5' TC and 3' CTRR termini as well as two short palindromic sequences near the 3' terminus that potentially can form a 20-bp hairpin. Although the maize element lacks sequence information for a DNA helicase, it does contain four exons with similarity to a plant DEAD box RNA helicase. A second Helitron insertion was found in the maize genomic database. These data strongly suggest an active Helitron in the present-day maize genome.     

<Emphasis Type="Italic">Helitron</Emphasis> mediated amplification of cytochrome P450 monooxygenase gene in maize

The mass movement of gene sequences by Helitrons has significantly contributed to the lack of gene collinearity reported between different maize inbred lines. However, Helitron captured-genes reported to date represent truncated versions of their progenitor genes. In this report, we provide evidence that maize CYP72A27-Zm gene represents a cytochrome P450 monooxygenase (P450) gene recently captured by a Helitron and transposed into an Opie-2 retroposon. The four exons of the CYP72A27 gene contained within the element contain a putative open reading frame (ORF) for 428 amino acid residues. We provide evidence that Helitron captured CYP72A27-Zm is transcribed. To identify the progenitor gene and the evolutionary time of capture, we searched the plant genome database and discovered other closely related CYP72A27-Zm genes in maize and grasses. Our analysis indicates that CYP72A27-Zm represents an almost complete copy of maize CYP72A26-Zm gene captured by a Helitron about 3.1 million years ago (mya). The Helitron-captured gene then duplicated twice, approximately 1.5-1.6 mya giving rise to CYP72A36-Zm and CYP72A37-Zm. These data provide evidence that Helitrons can capture and mobilize intact genes that are transcribed and potentially encode biologically relevant proteins.     

Population and evolutionary dynamics of Helitron transposable elements in Arabidopsis thaliana

Helitrons, a recently discovered superfamily of DNA transposons that capture host gene fragments, constitute up to 2% of the Arabidopsis thaliana genome. In this study, we identified 565 insertions of a family of nonautonomous Helitrons, known as Basho elements. We aligned subsets of these elements, estimated their phylogenetic relationships, and used branch lengths to yield insight into the age of each Basho insertion. The age distribution suggests that 87% of Bashos inserted within 5 Myr, subsequent to the divergence between A. thaliana and its sister species Arabidopsis lyrata. We screened 278 of these insertions for their presence or absence in a sample of 47 A. thaliana accessions. With both phylogenetic and population frequency data, we investigated the effects of gene density, recombination rate, and element length on Basho persistence. Our analyses suggested that longer Basho copies are less likely to persist in the genome, consistent with selection against the deleterious effects of ectopic recombination between Basho elements. Furthermore, we determined that 39% of Basho elements contain fragments of expressed protein-coding genes, but all of these fragments were explained by only 5 gene-capture events. Overall, the picture of A. thaliana Helitron evolution is one of rapid expansion, relatively few gene-capture events, and weak selection correlated with element length.     

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.     

The limited distribution of Helitrons to vesper bats supports horizontal transfer

Transposable elements (TEs) have the unique ability to move and replicate within the genome and therefore engender dramatic changes to genome architecture. Among different types of TEs, rolling-circle transposons (Helitrons) are well known for their ability to capture and amplify host gene fragments. Bioinformatic analysis revealed that Helitrons constitute ~3% of the Myotis lucifugus, (little brown bat) genome, while no Helitrons were found in any of the other 44+ sequenced mammalian genomes. Recently horizontal transfer has been implicated for some of the M. lucifugus Helitrons, in part explaining this disparate distribution among mammals. The purpose of this work is to determine both the distribution of Helitrons among bats and to estimate the number of independent invasions. We employed a combination of in silico, PCR and hybridization based techniques to identify Helitrons from diverse bat species belonging to ten different families. This work reveals that Helitrons invaded the vesper bat lineage, at least once. Indeed, Helitrons were not identified in the sister taxa 'Miniopterus', which suggests that the amplification of Helibat occurred (30-36 mya) only in the vesper bat lineage. The estimated age of amplification of the Helibats and the rapid radiation of vesper bats are roughly coincidental and suggest that the invasion and amplification of these elements might have influenced their evolutionary trajectory potentially contributing to phenotypic and genotypic diversity.     

The origin and evolution of geminivirus-related DNA sequences in Nicotiana

A horizontal transmission of a geminiviral DNA sequence, into the germ line of an ancestral Nicotiana, gave rise to multiple repeats of geminivirus-related DNA, GRD, in the genome. We follow GRD evolution in Nicotiana tabacum (tobacco), an allotetraploid, and its diploid relatives, and show GRDs are derived from begomoviruses. GRDs occur in two families: the GRD5 family's ancestor integrated into the common ancestor of three diploid species, Nicotiana kawakamii, Nicotiana tomentosa and Nicotiana tomentosiformis, on homeologous group 4 chromosomes. The GRD3 family was acquired more recently on chromosome 2 in a lineage of N. tomentosiformis, the paternal ancestor of tobacco. Both GRD families include individual members that are methylated and diverged. Using relative rates of synonymous and nonsynonymous nucleotide substitutions, we tested for evidence of selection on GRD units and found none within the GRD3 and GRD5 families. However, the substitutions between GRD3 and GRD5 do show a significant excess of synonymous changes, suggesting purifying selection and hence a period of autonomous evolution between GRD3 and GRD5 integration. We observe in the GRD3 family, features of Helitrons, a major new class of putative rolling-circle replicating eukaryotic transposon, not found in the GRD5 family or geminiviruses. We speculate that the second integration event, resulting in the GRD3 family, involved a free-living geminivirus, a Helitron and perhaps also GRD5. Thus our data point towards recurrent dynamic interplay between geminivirus and plant DNA in evolution.     

New Intraclass Helitrons Classification Using DNA-Image Sequences and Machine Learning Approaches

Helitrons, eukaryotic transposable elements (TEs) transposed by rolling-circle mechanism, have been found in various species with highly variable copy numbers and sometimes with a large portion of their genomes. The impact of helitrons sequences in the genome is to frequently capture host genes during their transposition. Since their discovery, 18 years ago, by computational analysis of whole genome sequences of Arabidopsis thaliana plant and Caenorhabditis elegans (C. elegans) nematode, the identification and classification of these mobile genetic elements remain a challenge due to the fact that the wide majority of their families are non-autonomous. In C. elegans genome, DNA helitrons sequences possess great variability in terms of length that varies between 11 and 8965 base pairs (bps) from one sequence to another. In this work, we develop a new method to predict helitrons DNA-sequences, which is particularly based on Frequency Chaos Game Representation (FCGR) DNA-images. Thus, we introduce an automatic system in order to classify helitrons families in C. elegans genome, based on a combination between machine learning approaches and features extracted from DNA-sequences. Consequently, the new set of helitrons features (the FCGR images and K-mers) are extracted from DNA sequences. These helitrons features consist of the frequency apparition number of K nucleotides pairs (Tandem Repeat) in the DNA sequences. Indeed, three different classifiers are used for the classification of all existing helitrons families. The results have shown potential global score equal to 72.7% due to FCGR images which constitute helitrons features and the pre-trained neural network as a classifier. The two other classifiers demonstrate that their efficiency reaches 68.7% for Support Vector Machine (SVM) and 91.45% for Random Forest (RF) algorithms using the K-mers features corresponding to the genomic sequences.     

Invertrons, a class of structurally and functionally related genetic elements that includes linear DNA plasmids, transposable elements, and genomes of adeno-type viruses.

Invertrons are genetic elements composed of DNA with inverted terminal repeats at both ends, covalently bonded to terminal proteins involved in the initiation of DNA replication at both their 5' termini when they exist in the cytoplasm of their host in free form. They function as viruses, linear DNA plasmids, transposable elements, and sometimes combinations of two of these properties. They differ from retroviruses and related retro-type transposons which have direct repeats on both their genomic ends and exploit RNA intermediates for replication of their DNA. A model for replication and integration of invertrons is presented, as well as a model for transposition of transposable elements.     

Interaction of related Tn916-like transposons: analysis of excision events promoted by Tn916 and Tn5386 integrases

In recent work, we described the excision of a large genomic region from Enterococcus faecium D344R in which the sequence from "joint" regions suggested that excision resulted from the interaction of conjugative transposon Tn916 and the related mobile element Tn5386. In the present study, we examined the ability of integrases and integrase-excisase combinations from Tn916 and Tn5386 to promote the excision of constructs consisting of the termini of Tn916, Tn5386, and the VanB mobile element Tn5382. Integrases alone from either Tn916 or Tn5386 promoted the circularization of constructs from the three different transposons, even when the different termini used in the constructs were discordant in their transposon of origin. The termini of Tn916 and Tn5382 found in all joints were consistent with previously identified Tn916 and Tn5382 termini. Substantial variation was seen in the integrase terminus of Tn5386 used to form joints, regardless of the integrase that was responsible for circularization. Variability was observed in joints formed from Tn5386 constructs, in contrast to joints observed with the termini of Tn916 or Tn5382. The coexpression of excisase yielded some variability in the joint regions observed. These data confirm that integrases from some Tn916-like elements can promote circularization with termini derived from heterologous transposons and, as such, could promote excision of large genomic regions flanked by such elements. These findings also raise interesting questions about the sequence specificities of the C terminals of Tn916-like integrases, which bind to the ends and facilitate strand exchange.     

Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases

The clinical success of multitargeted kinase inhibitors has stimulated efforts to identify promiscuous drugs with optimal selectivity profiles. It remains unclear to what extent such drugs can be rationally designed, particularly for combinations of targets that are structurally divergent. Here we report the systematic discovery of molecules that potently inhibit both tyrosine kinases and phosphatidylinositol-3-OH kinases, two protein families that are among the most intensely pursued cancer drug targets. Through iterative chemical synthesis, X-ray crystallography and kinome-level biochemical profiling, we identified compounds that inhibit a spectrum of new target combinations in these two families. Crystal structures revealed that the dual selectivity of these molecules is controlled by a hydrophobic pocket conserved in both enzyme classes and accessible through a rotatable bond in the drug skeleton. We show that one compound, PP121, blocks the proliferation of tumor cells by direct inhibition of oncogenic tyrosine kinases and phosphatidylinositol-3-OH kinases. These molecules demonstrate the feasibility of accessing a chemical space that intersects two families of oncogenes.     

Expansion and evolution of the X-linked testis specific multigene families in the melanogaster species subgroup

The testis specific X-linked genes whose evolution is traced here in the melanogaster species subgroup are thought to undergo fast rate of diversification. The CK2?tes and NACβtes gene families encode the diverged regulatory β-subunits of protein kinase CK2 and the homologs of β-subunit of nascent peptide associated complex, respectively. We annotated the CK2βtes-like genes related to CK2?tes family in the D. simulans and D. sechellia genomes. The ancestor CK2βtes-like genes preserved in D. simulans and D. sechellia are considered to be intermediates in the emergence of the D. melanogaster specific Stellate genes related to the CK2?tes family. The CK2?tes-like genes are more similar to the unique autosomal CK2?tes gene than to Stellates, taking into account their peculiarities of polymorphism. The formation of a variant the CK2?tes gene Stellate in D. melanogaster as a result of illegitimate recombination between a NAC?tes promoter and a distinct polymorphic variant of CK2?tes-like ancestor copy was traced. We found a close nonrandom proximity between the dispersed defective copies of DINE-1 transposons, the members of Helitron family, and the CK2βtes and NACβtes genes, suggesting an involvement of DINE-1 elements in duplication and amplification of these genes.     

Identification of an additional interaction domain in transmembrane domains 11 and 12 that supports oligomer formation in the human serotonin transporter

Na+/Cl--dependent neurotransmitter transporters form constitutive oligomers. The topological arrangement is not known, but a leucine heptad repeat in transmembrane domain (TM) 2 and a glycophorin-like motif in TM6 have been proposed to stabilize the oligomer. To determine the topology, we generated versions of the human serotonin transporter (hSERT) that carried cyan or yellow fluorescent proteins at their amino and/or carboxyl terminus. Appropriate pairs were coexpressed to measure fluorescence resonance energy transfer (FRET). Donor photobleaching FRET microscopy was employed to deduce the following arrangement: within the monomer, the amino and carboxyl termini are in close vicinity. In addition, in the oligomer, the carboxyl termini are closer to each other than the amino termini. Hence, a separate interaction domain (i.e. distinct from TM2 and TM6) must reside in the carboxyl-terminal half of hSERT. This was confirmed by expressing the amino- and carboxyl-terminal halves of hSERT. These were retained intracellularly; they also retained the coexpressed full-length transporter by forming export-deficient oligomers and, when cotransfected in all possible combinations, supported FRET. Hence, both the carboxyl and amino termini contain elements that drive oligomerization. By employing fragments comprising two neighboring TM helices, we unequivocally identified TM11/12 as a new contact site by donor photobleaching FRET and beta-lactamase protein fragment complementation assay. TM1/2 was also found to self-associate. Thus, oligomerization of hSERT involves at least two discontinuous interfaces. The currently identified interaction sites drive homophilic interactions. This is consistent with assembly of SERT oligomers in an array-like structure containing multimers of dimers.     

Novel strand exchanges in V(D)J recombination

We describe novel products of V(D)J recombination in which signal sequences become joined to coding elements, in contrast to the standard reaction whose products are junctions of two signal sequences or two coding elements. In this variant reaction, the recombination machinery evidently recognizes signal sequences and introduces strand breaks at the normal positions, but then connects the elements in unusual combinations. The lack of fixed directionality indicates that recombination sites are not uniquely aligned when strand exchange occurs. The discovery of these variant junctions suggests a model for the evolution of the antigen receptor loci.