In this work, we sequenced a 12 kbp-fragment of the mt genome of the katablepharid Leucocryptos marina. Among nine protein-coding genes included in the mt genome fragment, the genes encoding cytochrome b and cytochrome c oxidase subunit I (cob and cox1) were interrupted by group I introns. We further identified that the cob and cox1 introns host open reading frames for homing endonucleases (HEs) belonging to distantly related superfamilies. Phylogenetic analyses recovered an affinity between the HE in the Leucocryptos cob intron and two green algal HEs, and that between the HE in the Leucocryptos cox1 intron and a fungal HE, suggesting that the Leucocryptos cob and cox1 introns possess distinct evolutionary origins. Although the current intron (and intronic HE) data are insufficient to infer how the homologous introns were distributed to distantly related mt genomes, the results presented here successfully expanded the evolutionary dynamism of group I introns in mt genomes.Mitochondria are descendants of the endosymbiotic α-proteobacterium most likely engulfed by the ancestral eukaryotic cells, and the proto-mitochondrial genome should have been severely streamlined in terms of both genome size and gene repertoire. In addition, mitochondrial (mt) sequence data indicated that frequent intron gain/loss events contributed to shaping the modern mt genome organizations, resulting in the homologous introns being shared between two distantly related mt genomes. Unfortunately, the bulk of mt sequence data currently available are of phylogenetically restricted lineages,  Group I (gI) introns are a major class of introns found in bacterial genomes, mitochondrial and plastid genomes, and eukaryotic nuclear genomes http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome), as of January 2012. Thus, we can shed light on novel aspects in the evolution of gI introns in mt genomes by investigating the intron data from the lineages of which mt gene sequences have not been accumulated.Mitochondrial (mt) gene/genome data are potentially informative for inspecting the evolution of gI introns hosting HEs, as a number of gI introns has been identified in mt genomes Leucocryptos marina mt genome, and explored the evolutionary histories of these introns by combining their putative secondary structures, the intron positions, and the phylogenetic affinities of the intronic HEs.Katablepharida is a group of heterotrophic unicellular eukaryotes whose members widely distribute in aquatic environments Leucocryptos mt genome including NADH dehydrogenase subunit 11 (nad11), NADH dehydrogenase subunit 1 (nad1), NADH dehydrogenase subunit 6 (nad6), ATP synthase F0 subunit 6 (atp6), NADH dehydrogenase subunit 7 (nad7), cytochrome c oxidase subunit 2 (cox2), cytochrome c oxidase subunit 3 (cox3), cytochrome b (cob), and cytochrome c oxidase subunit 1 (cox1) genes in this order (cox1 and the 5′ terminus of nad11 are not available in this mt genome fragment). The intergenic spacer regions are short, ranging from 4–65 bp in length. Neither transfer RNA nor ribosomal RNA gene was found in the mt genome fragment determined in the current study. By the comparison between the cDNA and genomic sequences, two introns in this region, one in the cob gene and the other in the cox1 gene, were detected  with an E-value = 10−13]. On the other hand, the putative core region of the cox1 intron appeared to share sequence similarity to those of group IA1 introns . The two gI introns are also distinguishable from one another by the two following points: (i) The cox1 intron have two extra double helical domains, P7.1 and P9.1  An ORF occupies the loop region between P1 and P10 in the cob intron, while an ORF places in the loop region between P1 and P2 in the cox1 intron  residue-long and 267 aa residue-long polypeptides, respectively. The two intronic ORFs likely encode LAGLIDADG-type HEs, but no significant similarity was detected between their putative aa sequences by a BlastP search (bl2seq) with the default settings. Henceforth here, we designate the HE hosted in the cob intron as HELm-cob, while that hosted in the cox1 intron as HELm-cox1. HELm-cob and HELm-cox1 appeared to belong to distant superfamilies, LAGLIDADG_2 (pfam031611) and LAGLIDADG_1 (pfam00961), respectively.The ORFs hosted in the Lm-cob, and subjected to the maximum-likelihood (ML) and Bayesian phylogenetic analyses. In the unrooted phylogeny of this ‘LAGLIDADG_2’ alignment, HELm-cob and two HEs encoded in the cob introns of two green algae, Nephroselmis olivacea and Chlorokybus atmophyticus, grouped together with a BP of 98% and a Bayesian posterior probability (BPP) of 1.00  lied at phase-0 position of the codon corresponding to Gln138 in the Saccharomyces cerevisiae cob gene (GenBank accession number NC_001224), and (ii) hosted a HE belonging to LAGLIDADG_2 superfamily in the loop region between P1 and P2 as shown in Leucocryptos cob intron by intron positions, as the HEs hosted by the introns lying in the homologous positions were sporadically distributed in the LAGLIDADG_2 phylogeny . Thus, Leucocryptos cox1 intron and the forth intron in Rhizophydium cox1 gene likely derived from a single ancestral intron, which lied at phase-0 position of the codon corresponding to Thr93 in the S. cerevisiae cox1 gene, and hosted a LAGLIDADG_1-type HE in the loop region between P1 and P10 , being in close proximity to but apparently distinct from the homing position of the Leucocryptos and Rhizophydium introns (see above). One possibility is that HELm-cox1 and the Rhizophydium HE, and the Podospora and Dictyostelium HEs have evolved from a single ancestral HE and still recognize the identical nucleotide sequence (or very similar sequences), but the cleavage position altered after the separation of the two HE pairs. In any case, the evolutionary link between the cox1 introns in Leucocryptos and Rhizophydium, and those in Podospora and Dictyostelium can be assessed only after the enzymatic properties of the HEs hosted in the four cox1 introns are characterized.The clan of HEcob and cox1 genes discussed above have been vertically inherited from the common ancestor of katablepharids, green algae and fungi, but (ii) secondary intron loss occurred in other descendent lineages, as the HE sequences considered here unlikely represent the true diversity of LAGLIDADG_2-type or LAGLIDADG_1-type HE superfamily. The origins of the two gI introns found in Leucocryptos mt genome should be revisited after in-depth surveying introns and intronic HEs in the mt genomes of phylogenetically broad eukaryotic lineages, particularly those of close relatives of katablepharids, such as goniomonads and cryptomonads.Introns in organellar genomes are generally regarded as mobile genetic elements powered by intronic HEs, as ‘trans-genomic’ intron invasion have been accumulated in the literature Leucocryptos marina NIES-1335 and the haptophyte Chrysochromulina sp. NIES-1333 were purchased from the National Institute for Environmental Study (NIES). Leucocryptos was maintained in f/2 medium (http://mcc.nies.go.jp/02medium.html#f2) with Chrysochromulina as a prey at 20°C under 14∶10 hours of light∶dark cycles.The katablepharid Leucocryptos cells containing a small amount of the Chrysochromulina (prey) cells, and this sample was then subjected to DNA and RNA extractions by using Plant DNA Isolation Reagent (TaKaRa) and RNeasy Plant Mini Kit (QIAGEN), respectively. Total RNA was used for random hexamer-primed cDNA synthesis by Superscript II reverse transcriptase (Invitrogen). The experiments mentioned above were conducted by following the manufactures' instructions. The cDNA and total DNA were used as the templates for polymerase chain reactions (PCR) aiming at the amplification of gene transcripts and genome fragments, respectively.We harvested the cob, cox1, cox3, nad1, nad7, and nad11. PCR products were cloned into pGEM-TEasy vector (Promega). For each gene transcript, we completely sequenced eight clones and confirmed no sequence heterogeneity among clones, except the cob and cox3 transcripts. The cob and cox3 samples appeared to consist of two distinctive types of amplicons, one with and the other without in-frame TGA codons . We regarded the amplicons with in-frame TGA codons as the mt gene transcripts from the haptophyte (Chrysochromulina) prey cells for two reasons: Firstly, our preliminary phylogenetic analyses indicated that the two amplicons were distantly related to each other, and only the one with in-frame TGA codons displayed an intimate affinity to the haptophyte homologues  with the primer sets shown in mologues  & B. Secnad11 and nad1, nad1 and nad7, nad7 and cox3, cox3 and cob, and cob and cox1 with outwarded exact match primers designed based on the six mt gene transcripts initially determined (see above) as performed in previous works nad6, atp6, and cox2) were additionally found. Cloning and sequencing were performed as described above. The partial mt genome sequence was deposited to DNA Data Bank of Japan (GenBank/EMBL/DDBJ accession no. AB693966).We then amplified the mt genome fragments corresponding to the six transcripts with no in-frame TGA codon with exact-match primers (not shown). We also amplified the intergenic spacer regions between cob and cox1 genes in the Leucocryptos mt genome appeared to possess a single gI intron with a HE (see Results and Discussion). Both 5′ and 3′ splice sites were determined by comparing the cDNA and genomic sequences. Intron secondary structures were predicted using MFOLD Each of Leucocryptos cob intron (HELm-cob) was aligned with 29 HEs belonging to the LAGLIDADG_2 superfamily, which showed significant similarity to HELm-cob in TBlastN search against the GenBank nr database . We carefully assessed the alignments from the Blast search, and excluded redundant sequences and the sequences which produced very short alignments with HELm-cob. After manual refinement followed by the exclusion of ambiguously aligned positions, 183 aa positions were remained in the final ‘LAGLIDADG_2’ alignment. Pairwise aa identities and similarities ranged from 30 to 98%, and from 48 to 98%, respectively (Chlorokybus atmophyticus cob intron showed the highest aa identity (47%) to HELm-cob, while the ones hosted in the Millerozyma farinose and Chlorogonium elongatum cob genes showed the lowest aa identity (34%) to HELm-cob (see the upper triangular in Chlorokybus atmophyticus cob intron showed the highest aa similarity (65%) to the HELm-cob, while the one hosted in the Chlamydomonas incerta cob genes showed the lowest aa similarity (49%) to HELm-cob (see the lower triangular in The HE encoded in the ectively . The HE Leucocryptos cox1 intron (HELm-cox1) and 24 HEs belonging to the LAGLIDADG_1 superfamily, which can form global alignments with HELm-cox1 with TBlastN E-values<10−10. The final LAGLIDADG_1 alignment contains 191 unambiguously-aligned aa positions. Pairwise aa identities and similarities ranged from 14 to 95%, and from 34 to 97%, respectively (Rhizophydium sp. cox1 intron showed the highest aa identity (44%) to HELm-cox1, while the one hosted in the Flammulina velutipes cox1 gene showed the lowest aa identity (20%) to HELm-cox1 (see the upper triangular in Podospora anserina cox1 intron showed the highest aa similarity (62%) to HELm-cox1, while the ones hosted in the Blastomyces dermatitidis cox1 gene and Allomyces macrogynus cob gene showed the lowest aa similarity (41%) to HELm-cob . B. Schematic structures of Leucocryptos and Rhizophydium cox1 introns. Both introns harbor LAGLIDADG_1-type homing endonucleases in the region between P1 and P10 (shown as closed boxes).(TIF)Click here for additional data file.Figure S2Maximum-likelihood (ML) analyses of the COB and COX3 amino acid (aa) alignments.A. The ML phylogeny inferred from the COB alignment comprises 31 taxa with 368 unambiguously aligned aa positions B. The ML phylogeny inferred from the COX3 alignment comprising 26 taxa with 218 unambiguously aligned aa positions. Leucocryptos marina and Chrysochromulina sp. are highlighted by arrowheads. The haptophyte clade is shaded. Only ML bootstrap values equal to or greater than 50% are shown. Methods: The two aa alignments were separately analyzed with the ML method with the LG+Γ+F model by using RAxML ver. 7.2.1. The details of the ML and ML bootstrap analyses were same as described in Materials and Methods/Phylogenetic analyses of intronic HEs.(TIF)Click here for additional data file.Figure S3Amino acid (aa) sequence homology.A. Pairwise aa identity matrix of the 30 endonuclease (HE) sequences in the LAGLIDADG_2 alignment. We also recoded 20 aa characters in the HE sequences to six Dayhoff classes, and then made the identity matrix presented below diagonal. B. Pairwise aa identity matrix of the 25 HE sequences in the LAGLIDADG_1 alignment. We also provide the pairwise ‘Dayhoff-class’ identity matrix below diagonal. For each sequence, the GenBank accession no. is shown in brackets.(TIF)Click here for additional data file.


In this article, a new method for doping graphene using the conjugated organic molecule, tetracyanoquinodimethane (TCNQ), is described. Notably, TCNQ is well known as a powerful electron accepter and is expected to favor electron transfer from graphene into TCNQ molecules, thereby leading to p-type doping of graphene films. Small amounts of TCNQ drastically improved the resistivity without degradation of optical transparency. Our carrier doping method based on charge transfer has a huge potential for graphene-based TCFs.Transparent conductive films (TCFs) are critical components of a myriad of technologies including flat panel displays, light-emitting diodes, and solar cells. Graphene-based TCFs have attracted a lot of attention because of their high electrical conductivity, transparency, and low cost. Carrier doping of graphene would potentially improve the properties of graphene-based TCFs for practical industrial applications. However, controlling the carrier type and concentration of dopants in graphene films is challenging, especially for the synthesis of  Transparent conductive films (TCFs) are a class of extremely important components of modern technology for applications such as optical devices and solar energy utilization . Indium Meanwhile, graphene, a single atomic layer of carbon, has attracted greater attention as an alternative material of TCFs because of its high electrical conductivity and transparency . In addi2 [p-type films. With a view to improving the electrical properties of graphene-based TCFs, we propose a novel carrier doping method based on charge transfer from conjugated organic molecules. It is anticipated that liquid phase chemical interaction between graphene and conjugated organic molecules induces a high doping efficiency.The problem of high resistivity of chemically derived graphene-based TCFs  still re2 , and cha2 ,12. Howep-type doping of graphene films. Figure Tetracyanoquinodimethane (TCNQ) is well known as a powerful electron accepter and is expected to favor electron transfer from graphene into TCNQ molecules, thereby leading to 2SO4 and K2S2O8, and oxidized in KMnO4 and H2SO4. After centrifugation, the resulting graphite oxide was exfoliated into graphene oxide (GO) by ultra-sonication . Then, a GO aqueous dispersion was produced by centrifugation and dialysis to neutralize a pH. The morphology of GO synthesized by this procedure was characterized by Raman spectroscopy (excited by 532-nm Ne laser) [Chemically derived graphene was synthesized by the modified Hummer's method , a well-e laser) , optical2H4, a strong reductant, with NH3 to adjust pH [N,N-dimethylformamide (DMF) using an evaporator. RGO can be dispersed well in many kinds of organic solvents including DMF, while it is easily aggregated in aqueous solution because of its low electrostatic repulsion force. A RGO sample deposited on Au (10 nm)/SiO2 (90 nm)/Si substrate was prepared for the evaluation of the reduction state by x-ray photoelectron spectroscopy (monochrome Al Kα X-ray).A reduction step of GO into graphene plays an essential role to determine the electrical properties of the resulting graphene films. GO was reduced as follows: GO was dispersed in aqueous solution containing Ndjust pH . This waOur graphene films were deposited on glass substrates (Corning7059) by a spray-coat method at a substrate temperature of 200°C in an atmosphere containing the solvent vapor. The thickness of the films was controlled by varying the spray amounts. The optical transmittance was measured in the wavelength range from 250 to 2500 nm, and the sheet resistance was measured by van der Pauw method.Doping graphene via charge transfer by TCNQ molecules was carried out as follows. First, 0.01 g of TCNQ powder  was dissolved into 5 ml of DMF solvent. It is expected that TCNQ molecules in DMF are radicalized . Then, 52) were present in the GO aqueous dispersion as shown in Figure -1, referred to as the 2D band, which indicates about 25% of GO flakes were single layer of carbon as demonstrated in our previous article [Large GO flakes (over 30 × 30 μm article .The carbon 1s core level XPS spectra of GO, RGO, and graphite samples were shown in Figure 6 Ω/square with a transparency of 88% at 550 nm. Such a sheet resistance was the lowest obtained compared with previously reported chemically derived graphene films as deposited [Figure eposited . Post-aneposited  obtainedThe SEM images of individual doped graphene flakes indicate RGO flakes maintaining 2D structures after interaction with TCNQ molecules in liquid phase as shown in Figure 10 and 1.17 × 1012 cm-2 for the undoped and doped graphenes, respectively. These estimated values are similar to the reported values by Coletti et al. [Figure i et al. . They moThe authors developed a new and inexpensive fabrication method of chemically derived graphene-based TCFs and demonstrated a huge potential of doping effect via charge transfer by conjugated organic molecules. All of the fabrication steps including the reduction of GO and carrier doping were carried out in liquid phase. Therefore, this novel method proposed in this study does not require any vacuum system and is suitable for quantity synthesis. Furthermore, chemically derived graphene combined with the above doping technique could be a potential alternative to conventional transparent conductive materials.N,N-dimethylformamide; GO: graphene oxide; ITO: indium tin oxide; RGO: reduced graphene oxide; TCFs: transparent conductive films; TCNQ: tetracyanoquinodimethane.DMF: The authors declare that they have no competing interests.RI designed and conducted all experiments and characterisation and drafted the manuscript. MB helped in technical support for experiments and drafting the manuscript. Both YM and AS have read and approved the final manuscript.


In the course of sequencing the complete mitochondrial genome of the prasinophyte picoplanktonic green alga Prasinoderma coloniale CCMP 1220 , we uncovered two additional cases of trans-spliced group I introns. Here, we describe these introns and compare the 54,546 bp-long mitochondrial genome of Prasinoderma with those of four other prasinophytes . This comparison underscores the highly variable mitochondrial genome architecture in these ancient chlorophyte lineages. Both Prasinoderma trans-spliced introns reside within the large subunit rRNA gene (rnl) at positions where cis-spliced relatives, often containing homing endonuclease genes, have been found in other organelles. In contrast, all previously reported trans-spliced group I introns occur in different mitochondrial genes (rns or coxI). Each Prasinoderma intron is fragmented into two pieces, forming at the RNA level a secondary structure that resembles those of its cis-spliced counterparts. As observed for other trans-spliced group I introns, the breakpoint of the first intron maps to the variable loop L8, whereas that of the second is uniquely located downstream of P9.1. The breakpoint In each Prasinoderma intron corresponds to the same region where the open reading frame (ORF) occurs when present in cis-spliced orthologs. This correlation between the intron breakpoint and the ORF location in cis-spliced orthologs also holds for other trans-spliced introns; we discuss the possible implications of this interesting observation for trans-splicing of group I introns.Organelle genes are often interrupted by group I and or group II introns. Splicing of these mobile genetic occurs at the RNA level via serial transesterification steps catalyzed by the introns'own tertiary structures and, sometimes, with the help of external factors. These catalytic ribozymes can be found in  These intron pieces flanked by exon sequences must interact at the RNA level to produce a functional intron structure that allows splicing to take place; the separate primary transcripts derived from the individual exon sequences are joined and ligated after assembly and splicing of the flanking intron sequences. While trans-spliced group II introns have been known for decades trans-spliced group I introns were reported in 2009 Trans-spliced group I introns are widely distributed across lineages and have been found within fungi trans-spliced group I introns are restricted to the cox1 and rns genes in mitochondria and examples of trans-spliced introns in the plastid have yet to be identified. All reported trans-spliced group I introns display bipartite RNA structures, with one instance hypothesized to use an helper RNA fragment to guide splicing trans-spliced introns whose secondary structures have been predicted, the junction between the 5′ and 3′ fragments, i.e. the breakpoint, is usually located in the loop subtending the base-paired region P8 (L8) cox1 introns found in the lycophytes Isoetes engelmannii/Selaginella moellendorffii are uniquely split in the L9 loop Group I and group II introns can be found in Prasinoderma coloniale CCMP 1220, a marine green alga belonging to the prasinophyte clade VI , and report the characteristics of its trans-spliced group I introns. The Prasinoderma mitochondrial introns are the first trans-spliced introns reported within the rnl gene. One of these introns also provides the first example of a trans-spliced group I intron split in the region downstream of P9.1. We show that the breakpoint in the bipartite RNA structure of each Prasinoderma intron corresponds to the same region that contains an ORF in cis-spliced relatives at the same cognate site. This correlation between the intron breakpoint and the ORF location in cis-spliced orthologs also holds for other trans-spliced introns.In the course of investigating the mitochondrial genome of Prasinoderma coloniale CCMP1220 belongs to one of the deepest prasinophyte lineages. The prasinophytes are paraphyletic, forming at least seven lineages  at the base of the Chlorophyta Prasinoderma represents the Prasinococcales (clade VI), a lineage that had not been previously sampled for organelle genome studies. The complete mitochondrial genome sequence of Prasinoderma was compared with those of four other prasinophytes representing three distinct lineages: Ostreococcus tauri (clades II) Micromonas pusilla (clade II) Nephroselmis olivacea (clade III) Pycnococcus provasolii III (clade V) Nephroselmis, are picoplanktonic prasinophytes, with Ostreococcus being the smallest free-living eukaryote described to date.Prasinoderma mitochondrial genome [GenBank:KF387569] maps as a 54,546 bp-long single circular molecule and features two copies of a large inverted repeat  encompassing 53% of the genome  is slightly higher than that found in genes (52.3%).The e genome . While iclade II . With annophytes  and alsoPrasinoderma mitochondrial genome encodes a set of 55 unique genes, 20 of which (including both rRNA genes) are present in the large inverted repeat. This gene repertoire is intermediate between those of the gene-rich genomes of Ostreococcus tauri, Micromonas pusilla and Nephroselmis olivacea (clades II and III) and that of the gene-poor genome of Pycnococcus provasolii (clade V)  . Unlike nococcus . These dPrasinoderma genome; this is the least densely packed mtDNA among the prasinophyte mitochondrial genomes sequenced so far -3′, 5′-trnR(ucg)-trnI(gau)-3′, 5′-trnS(gcu)-trnA(ugc)-trnT(ggu)-3′ and 5′-rps11-rps13-rpl6-rps8-rps14-rpl5-rpl14-rpl16-rps3-rps19-3′ clusters have been eroded only in the Pycnococcus lineage. The genes comprised in the latter ribosomal protein cluster are missing entirely from the Pycnococcus mtDNA In terms of synteny, the Prasinoderma and Nephroselmis harbor these elements, with two and four group I introns found in these mtDNAs, respectively. Both Prasinoderma group I introns and three of the Nephroselmis introns reside in the large subunit rRNA gene (rnl), whereas the remaining Nephroselmis intron is located in cob. Unlike their Nephroselmis counterparts, the two Prasinoderma mitochondrial introns, named hereafter Pr.rnl.1 and Pr.rnl.2, are discontinuous. Each of these introns is fragmented into two non-adjacent pieces, thus accounting for the three distinct coding regions observed for the rnl gene. The rnl a and rnl b exons are separated from one another by rns, a gene encoded on the same DNA strand as the latter exons; in contrast, the third piece of rnl lies on the opposite strand between cob and rps12  had revealed important variations at the level of mitochondrial genome size, gene content, gene density, and overall genome structure among lineages Prasinoderma, a representative of clade VI, also differs substantially from its counterparts . The Prasinoderma genome has retained more genes than its Pycnococcus homolog but has lost many compared to the Ostreococcus, Micromonas and Nephroselmis mtDNAs, yet its genome size is the largest known among prasinophytes. Moreover, contrary to the genomes of the other three picoplanktonic prasinophytes, which are very tightly packed with genes and lack introns, that of Prasinoderma is much less compact than the Nephroselmis genome and like the latter contains introns. The comparative data reported here thus highlight differences in the types and extent of mtDNA changes that accompanied cell reduction in clades I, V and VI although these three lineages all display a reduced gene content.Sequencing of the terparts , includiPrasinoderma mtDNA was not surprising given the low gene density of this genome; however, the discovery that both are trans-spliced group I introns in the rnl gene was very unexpected. To our knowledge, these introns are the first trans-spliced group I introns reported in the rnl gene. In green algae/land plants, trans-splicing of group I introns had previously been reported only for the mtDNAs of the parasitic trebouxiophyte HelicosporidiumIsoetes and Selaginellatrans-spliced group I introns, cox1 was the gene interrupted.The finding of two introns in the trans-spliced group I introns are still fairly new molecular oddities, we expect that these catalytic ribozymes will be encountered more often in the future. This is due to the dramatically improved DNA sequencing capabilities that allow sampling both in depth and coverage of previously uninvestigated lineages at an unprecedented pace. It is perhaps not surprising that all of the reported examples of trans-spliced group I introns are located in the well-known cox1 and rns and, in this study, in the rnl gene. The main reason is that these genes, in particular cox1 and rnl, are often rich in introns and contain numerous potential intron insertion sites. Moreover, because the products of these genes are well conserved and essential for mitochondrion function, their partial or total absence from annotations is looked upon with suspicion. In contrast, divergent genes are often hard to analyze and thus annotation errors are more likely to be left unnoticed. Finding introns in such genes either in cis or trans configuration can be far from trivial, and when these introns are further split into distinct pieces jumbled across a whole genome, the complexity of this task is compounded.While cis to a trans configuration is rather straightforward and implies one or more recombination events in a segment of the intron that is malleable enough to accommodate the disruption. Therefore, variable loops containing expanded stretches of DNA between conserved pairings are the most obvious targets for recombination. Accordingly, all of the trans-spliced group I introns reported so far that have cis-spliced orthologs are broken at the same variable region as the one featuring the ORF in their cis-spliced relatives  acting as a de facto maturase might be required to yield the productive structure necessary for splicing. Reliance on many nuclear-encoded splicing factors (at least 14) has been demonstrated for the tripartite trans-spliced group II intron found in the chloroplast of the green alga Chlamydomonas reinhardtiiPrasinoderma Pa.rnl.1 intron, it is intriguing that the two fragments linked to the flanking exons cannot form the typical secondary structure expected for a group IB intron .Aside from the DNA rearrangements discussed above, at least two major conditions must be met for successful events of B intron . Indeed,Prasinoderma has several unique characteristics, including the presence of two trans-spliced group I introns in the rnl gene. Sampling of other prasinophyte lineages should provide further insights into the range of mtDNA variations seen in these basal chloroplast lineages and could also help deepen our understanding of how trans-spliced introns arise.The comparative genome analysis presented here underscores the high variability in mtDNA architecture among prasinophyte lineages. The newly sequenced mitochondrial genome of the picoplanktonic green alga Prasinoderma coloniale strain CCMP 1220 was obtained from the Provasoli-Guillard National Center for Marine Algae and Microbiota . Prasinoderma cells were cultured in K medium Prasinoderma A+T-rich organellar DNA (700 bp fragments) was constructed using the GS-FLX Titanium Rapid Library Preparation Kit from Roche 454 Life Sciences . Construction of this library as well as 454 GS-FLX DNA Titanium pyrosequencing (one eight of a run) were carried out by the Plate-forme d′Analyses Génomiques . The resulting reads were assembled with gsAssembler 2.5 from the Roche GS Data Analysis Software package . Contigs were visualized, linked, edited and polished using the CONSED 22 package Prasinoderma mtDNA, repeats ≥30 bp were retrieved using REPFIND of the REPuter 2.74 program http://www.repeatmasker.org/) running under the Crossmatch search engine (http://www.phrap.org/).A shotgun library of Prasinoderma was extracted from cells ground in liquid nitrogen with the Qiagen RNeasy Midi kit  as described in Turmel et al rnl transcripts undergo trans-splicing and also to confirm the insertion positions of the trans-spliced introns, RT-PCR reactions were performed on the DNA-free RNA using the Qiagen One-Step RT-PCR kit with the following primers: 1) 5′-ACCAAACTGTCTTACGACGTTC-3′, 2) 5′-ATACTGAACCGGAGTTTCCTTG-3′, 3) 5′-CTTCAATTTCACCGAGTCCATG-3′, 4) 5′-ACAGGTCTCTGCAAAGTCGAAG-3′, 5) 5′-GTGAAGTCGCAGAAAATTGTGG-3′ (for their genomic locations, consult Total RNA from


TAR DNA binding protein 43 KD (TDP-43) is an essential gene that regulates gene transcription, mRNA splicing and stability. In amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal neurodegenerative diseases, TDP-43 is fragmented, generating multiple fragments that include the C-terminal fragment of ∼25 KD. The role of these fragments in the pathogenesis of ALS and FTD is not clear. Here we investigated the aggregation propensity in various polypeptide regions of TDP-43 in mammalian cells and the effect of these fragments on cultured neurons. By expressing the full length and various TDP-43 fragments in motor neuron-derived NSC-34 cells and primary neurons, we found that both N- and C-terminal fragments of TDP-43 are prone to aggregate and the C-terminal end of RRM2 region is required, though not sufficient, for aggregation. The aggregation of the TDP-43 fragments can drive co-aggregation with the full-length TDP-43, consequently reducing the nuclear TDP-43. In addition, the TDP-43 fragments can impair neurite growth during neuronal differentiation. Importantly, overexpression of the full-length TDP-43 rescues the neurite growth phenotype whereas knockdown of the endogenous TDP-43 reproduces this phenotype. These results suggest that TDP-43 fragments, particularly the pathologically relevant C-terminal fragments, can impair neuronal differentiation by dominant-negatively interfering with the function of the full length TDP-43, thus playing a role in pathogenesis in ALS and FTD. TAR DNA binding protein-43 (TDP-43) has been identified as a major component of the ubiquitin-positive protein inclusions in neurons and glia in ALS, FTD as well as several other neurodegenerative disorders In both ALS and FTD, TDP-43 is fragmented, resulting in lower molecular weight C-terminal fragments of ∼35 to ∼25 KD To further understand the aggregation propensity of TDP-43 and the role of TDP-43 fragments in pathogenesis, we engineered a series of TDP-43 truncation fragments and expressed these fragments in cultured neurons. We found that both the C-terminal and the N-terminal fragments of TDP-43 can form aggregates as long as they contain the C-terminal end of the RRM2, which is necessary but by itself is insufficient for aggregation. The aggregation of the TDP-43 fragments can drive co-aggregate with the full-length TDP-43, thereby reducing nuclear TDP-43. Furthermore, the aggregation-prone fragments can impair neurite growth during neuronal differentiation. Importantly, this impairment can be rescued by an overexpression of full-length TDP-43 and can be reproduced by a knockdown of the endogenous TDP-43 expression. These results suggest a model where the C-terminal fragments of TDP-43, found in ALS and FTD patients, harm neurons by a dominant-negative mechanism.To understand the properties of fragmented TDP-43, we made a series of constructs that synthesize the N-terminal and C-terminal truncated TDP-43 fragments . We taggIn general, cells transfected with the C-terminal fragments 2, 3 and 4 had a high percentage of aggregate-containing cells than the cells transfected with the N-terminal fragments 6, 7 and 8 . In cellTo investigate the functional consequence of TDP-43 aggregation, we examined the cell death/viability. By several assays, including trypan blue staining, MTS assay, counting EGFP-fluorescent cells at 1, 2 and 3 days after transfection, TUNEL, activated caspase 3 staining and LDH release, we did not find a significant difference among the cells transfected with the constructs expressing EGFP and various TDP-43 fragments -expression construct that silences TDP-43 expression. This construct employed the CAG promoter to drive expression of a primary miRNA, which encode a red fluorescent protein (dsRed2) and a pre-miRNA targeting mouse TDP-43  [35]. ThTo confirm the neurite phenotype observed in NSC-34 cells, we transfected the constructs into the rat forebrain neural precursors and then differentiated them into neurons. The neurons transfected with full-length TDP-43-EGFP, the N-terminal fragments (construct 6-10) and the RRM2 fragment (construct 11) grew normal neurites that were not different from the cells transfected with EGFP alone . In contTo determine whether TDP-43 is required for neurite growth in primary neurons, we also conducted the knockdown experiments. Because the transfection efficiency was low, we first tested whether our two miRNAs (which were designed to silence mouse TDP-43) could knockdown rat TDP-43 by transfecting our constructs into rat-2 cells, which can be transfected with above 90% efficiency. Although both miR-TDP43a and miR-TDP43b matched rat TDP-43 mRNA sequence well, miR-TDP-43a did not knockdown rat TDP-43 (data not shown), probably due to the sequence differences in the regions flanking the miRNA target between the rat and mouse TDP-43 mRNA. miR-TDP43b knocked down rat TDP-43 efficiently . TherefoThe above data are consistent with the hypothesis that TDP-43 fragments that contain RRM2 are prone to aggregation and impairing neurite growth by dominant-negatively interfering with the normal TDP-43 function. The functional impairment could be mediated through the interaction between the TDP-43 fragments and the full-length TDP-43. To test this possibility, we cotransfected the EGFP-tagged TDP-43 fragments with a full-length TDP-43 that was tagged with a red fluorescent protein tdTomato (RFP). Unlike the EGFP-tagged TDP-43, this construct expressed the full-length fusion protein and unfused tdTomato was detected . When coTo determine whether the aggregation reduces the endogenous nuclear TDP-43, we transfected the cells with the EGFP-tagged fragments. To avoid the interference from the transfected TDP-43 fragments, we observed the endogenous TDP-43 using antibodies against the N- or C-terminal peptide. In cells transfected with wild type TDP-43, there was an increase in the nuclear TDP-43 staining compared with the untransfected cells , indicatTo rule out the possibility that the above observations were caused by the tags, we constructed untagged constructs and transfected them into NSC-34 cells. Like the tagged constructs, constructs 1 (the full-length TDP-43) and 11 did not form aggregates whereas the aggregation-prone constructs  formed aggregates . FurtherA well-known function of TDP-43 is modulation of mRNA splicing. A best characterized example is that it promotes exon 9 skipping during the CFTR mRNA splicing Our data show that both the C-terminal and the N-terminal fragments of TDP-43 can form aggregates in mammalian cells as long as they contain the C-terminal end of the RRM2 . The C-tConsistent with this model, in FTD and ALS patients, the C-terminal fragments are generated Our result that the RRM2 is necessary for TDP-43 aggregation agrees with the recent literature, where all fragments capable of forming aggregates in cells contain the RRM2 region The C-terminal half of TDP-43 is important for pathogenesis. In addition to the presence of the C-terminal fragments in the CNS tissues from the patients, most ALS-associated mutations are located in this region Our data indicate that the neurite growth can be an in vitro readout for the toxicity of TDP-43 fragments. Observations from other groups support the relevance and the reliability of this assay. In Drosophila, a loss of TDP-43 function leads to a decrease in dendritic branching during the development of larva nervous system While the data from our experiments and the literature are consistent with a dominant-negative effect of the C-terminal fragments in neurodegeneration, this does not preclude a role from other properties of TDP-43 in the pathogenesis. For example, numerous studies have shown that the full-length TDP-43 can drive aggregation and toxicity when TDP-43 is overexpressed in vitro and in vivo In summary, our results suggest a model where the C-terminal fragments cause neuronal toxicity by a dominant-negative mechanism. This model does not preclude a role of toxicity from the other properties of TDP-43 protein and is consistent with the data in the current literature. Many details in this model remain hypothetical and will require data from experiments to confirm. Nevertheless, this model may be a useful guide for designing future experiments.5′-AAGGTGTTTGTTGGACGTTGT-3′ for miR-TDP43a and 5′-ATGCTGAACCTAAGCATAA-3′ for miR-TDP43b. The sense and antisense strands of DNA encoding the miRNAs were synthesized, annealed and digested with KpnI and XhoI. They were then cloned into an intron in the 3′ untranslated region of the dsRed2 gene To construct the full-length and truncated TDP-43 tagged with EGFP, the open reading frame (ORF) of human TDP-43 was amplified by PCR using the forward primers with XhoI restriction site at the 5′ end and the reverse primers with BamHI restriction site at the 5′ end. The PCR product was digested with XhoI and BamHI and inserted into the pEGFP-N1 Vector (BD Biosciences Clontech) that was digested using the same restriction enzymes. To construct the full-length TDP-43 tagged with tdTomato (kindly provided by Dr. Roger Tsien 3 cells/cm2 in the medium consisting of 1∶1 DMEM and Ham's F12 (Gibco BRL) supplemented with 1% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM glutamine. After 72 hrs, the cells were fixed using 4% paraformaldehyde and 0.1% glutaraldehyde in PBS.NSC-34 cells 6 cells per 100-mm dishes precoated with 15 µg/mL poly-L-ornithine (Sigma) and 1 µg/mL fibronectin , and treated daily with 10 ng/mL of basic fibroblast growth factor 2 (FGF2) (R&D Systems). At 5 d in vitro, cells were passaged and plated out at 0.8–1.0×106 cells per precoated plate and expanded as above for an additional 3–4 d . Cells were passaged again and plated out at 1.2–1.4×106 per precoated plate. The cells were then transfected with constructs using Lipofectamine 2000 (Invitrogen). Two days after transfection, FGF2 was removed to induce neuronal differentiation. After another 2 days, cells were washed once with PBS and fixed.For primary neuronal cultures, neural precursors were prepared from forebrain of embryonic day 14.5 (E14.5) SASCO SD rat embryos . Cells were plated out at 1.2–1.4×10HeLa cells (ATCC) were grown at 37°C in DMEM/10% FBS. The cells were then transfected with constructs using Lipofectamine 2000 (Invitrogen), cultured for 48 hours and then fixed.NSC34 cells were transfected with different constructs and 72 hours after transfection, cell viability/death assays were performed. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed using the In Situ Cell Death Detection kit, TMR red  according to the manufacturer's instructions. The 3--5-(3-caboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based cell proliferation assay (MTS assay) was carried out using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay , according to the manufacturer's instructions. Absorbance at 490 nm was measured in a microplate reader . After 72 hours transfection, the medium was collected for NSC34 cells and a LDH assay (Promega) was used to measure LDH levels as an indicator of toxicity. Cells were then fixed and immunofluorescent staining was done using an antibody against activated caspase-3  and an Alexa Fluor®568-conjugated goat anti-rabbit IgG .After fixation for 15 min at room temperature, cells were permeabilized by using 0.5% Triton in PBS for 5 min. Nuclei were stained with 4-, 6-diamidino-2-phenylindole (DAPI). To stain for TDP43, the fixed cells were incubated in the blocking buffer  for 15 min at room temperature and then incubated with a rabbit polyclonal antibody  at 1∶100 dilution or N- or C-terminal rabbit polyclonal antibody (Kind gifts from Dr. Virginia Lee) at 1∶1000 dilution in the blocking buffer at 4°C. After overnight incubation, cells were washed and incubated with Alexa Fluor®568-conjugated goat anti-rabbit IgG (invitrogen) at room temperature for 1 hour. To stain Tubulin, a mouse monoclonal anti-α-tubulin (1∶100) and Alexa Fluor®488-conjugated goat anti-mouse IgG (invitrogen) were used.The cells were visualized and photographed using a Nikon Eclipse Ti fluorescence microscope. For observation of nuclear aggregates, a series of optical sections were taken along the z-axis and processed by deconvolution  to ensure that the aggregates are within the bounds of the nucleus. For quantification of neurites, the cells with at least one neurite longer than 10 µm in NSC-34 cells or longer than 30 µm in primary neurons were counted as having neurites. Otherwise, the cells were counted as having no neurites. For illustration, some panels were adjusted for brightness and contrast as a whole.The endogenous nuclear and cytoplasmic TDP-43 was estimated by fluorescence density measurement. When the cells were transfected with the full-length or the C-terminal fragments, the antibody against the N-terminus of TDP-43 was used for staining. When the cells were transfected with the N-terminal fragments, the antibody against the C-terminus of TDP-43 was used. After staining the cells were visualized and photographed. For each cell, average density of TDP43 staining was measured in the nucleus and the cytoplasm by Nikon NIS-Elements software. The density ratio of the nucleus to the cytoplasm was calculated and then normalized to the average ratio of the EGFP control.Cells were collected by a hard plastic cell scraper from the plates and centrifugation at 400 g. The pellets were resuspended in RIPA buffer  plus protease inhibitors (Pierce). After clearing the lysate by centrifugation, protein concentration of cell lysates or tissue homogenates was measured using BCA assay . The samples were heated in Laemmli's buffer, and equal amounts of protein were loaded and resolved by SDS-PAGE. After transfer to nitrocellulose membranes, blots were blocked with 5% nonfat dry milk in PBST  for 1 h, and then incubated with primary antibodies overnight. After washing 3 times in PBST, the membrane was incubated with anti-rabbit IgG  or mouse IgG  conjugated to horseradish peroxidase for 1 hour. Membranes were washed three times and proteins were visualized after ECL (Pierce) treatment and detected using the LAS-3000 imaging system. The primary antibodies used were rabbit polyclonal anti-EGFP  and anti-TDP-43 ; and mouse monoclonal anti-α-tubulin  and anti-HA .5′-CAACTTCAAGCTCCTAAGCCACTGC-3′) and reverse Bra2 (5′-TAGGATCCGGTCACCAGGAAGTTGGTTAAATCA-3′) primers. PCR was performed at 95°C for 5 min, followed by 30 cycles of denaturing at 95°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 60 s. PCR products were visualized on agarose gels.The experiment was conducted based on a previously published method


In particular, genotoxic agents , PPARα agonists (bezafibrate and fenofibrate), and non-steroidal anti-inflammatory drugs  induced L1 promoter activity. To examine their effects on L1 retrotransposition, we developed a high-throughput real-time retrotransposition assay using a novel secreted Gaussia luciferase reporter cassette. Three compounds  produced a significant enhancement in L1 retrotransposition. This is the first study to report the effects of a wide variety of compounds on L1 transcription and retrotransposition. These results suggest that certain chemical- and drug-induced stresses might have the potential to cause genomic mutations by inducing L1 mobilization. Thus, the risk of induced L1 transcription and retrotransposition should be considered during drug safety evaluation and environmental risk assessments of chemicals.The Long interspersed element 1 (LINE1 or L1) retrotransposon constitutes 17% of the human genome. There are currently 80–100 human L1 elements that are thought to be active in any diploid human genome. These elements can mobilize into new locations of the genome, resulting in changes in genomic information. Active L1s are thus considered to be a type of endogenous mutagen, and L1 insertions can cause disease. Certain stresses, such as gamma radiation, oxidative stress, and treatment with some agents, can induce transcription and/or mobilization of retrotransposons. In this study, we used a reporter gene assay in HepG2 cells to screen compounds for the potential to enhance the transcription of human L1. We assessed 95 compounds including genotoxic agents, substances that induce cellular stress, and commercially available drugs. Treatment with 15 compounds increased the L1 promoter activity by >1.5-fold ( Long interspersed element 1 (LINE1 or L1) is a non-long terminal repeat retrotransposon. L1 constitutes 17% of the human genome 2O2L1 transcription and/or mobilization is upregulated by certain types of stress: for example, genotoxic stress, which is induced by benzo[a]pyrene, UV light Chemical- or drug-induced retrotransposition of L1 into a functional gene in germ cells or somatic cells might cause teratogenicity or disease, including cancer. However, it is still unclear what kinds of compounds affect L1 retrotransposition, and the risk of inducing retrotransposition by compounds has not been considered during drug or chemical safety assessment.In this study, we used a reporter gene assay in HepG2 cells to investigate compounds for their potential to enhance the transcription of human L1. We examined 95 compounds, each of which was categorized as a genotoxic agent, cellular stress agent, or drug. Of these compounds, 54 are now available commercially. Among the 95 compounds, 15 compounds  increased the L1 promoter activity. To examine their effect on L1 retrotransposition, we designed a retrotransposition assay using a novel secreted Gaussia luciferase reporter cassette. Importantly, we found that salicylamide not only increased L1 promoter activity but also slightly enhanced L1 retrotransposition in HeLa cells. Because this drug is widely used as an anti-pyretic analgesic, the data presented here suggest a new paradigm for drug assessment.5′-ATGCCTCGAGGGGGGAGGAGCCAAGATGGCCG-3′) that contains the XhoI restriction site and a reverse primer (5′-GCATAAGCTTCTTTGTGGTTTTATCTACTTTTGGTCTTTG-3′) that contains the HindIII restriction site. The PCR product was cloned into XhoI- and HindIII-digested linearized pGL4.11 (Promega). pCEP4/L1.3mneoI400/ColE1 pGL4.11-L1.3 5′ UTR contains the 5′ UTR of L1.3 upstream of a Firefly luciferase (FLuc) gene. The 5′ UTR was amplified by PCR using a forward primer  signal of the construct pORF5-Fcy::Fur (InvivoGen) was replaced with a thymidine kinase (TK) poly(A) signal. Next, an EcoICRI restriction site was introduced into the GLuc gene NcoI and NheI sites) of pORF5-Fcy::Fur, immediately downstream of the EF-1α/eIFg hybrid promoter. The chimeric mini-intron of the plasmid psiCHECK-2 (Promega) was isolated and cloned in the EcoICRI site of GLuc in the antisense orientation. The entire reporter cassette was then extracted with flanking PacI and AsiSI restriction enzyme sites, blunted, and cloned in a BstZ17I site in the 3′ UTR of the active L1RP contained within the vector 99-PUR 99-PUR-RPS-mGLucI is the JM111 mutant of 99-PUR-RPS-mGLucI. The JM111 mutant contains two missense mutations in L1RP ORF1 that abolish retrotransposition 99-PUR-JM111-pSV40-CLuc (New England BioLabs), which contains an SV40 promoter and Cypridina luciferase (CLuc) Ultimate ORF cDNA clones (Invitrogen) were V5-tagged on their N-termini by shuttling them from pENTR221 vector into pcDNA3.1/nV5-DEST using Gateway Technology (Invitrogen) as previously described l-glutamine, and 10% fetal bovine serum . Human adenocarcinoma HeLa cells (ATCC) and HEK293T cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum . Cells were grown at 37°C in a humidified 5% CO2 atmosphere. Cells were grown in a T75 or T225 flask to ∼80% confluence and passaged using a 0.25% trypsin/0.02% EDTA solution (Sigma-Aldrich).Human hepatocellular carcinoma HepG2 cells (ATCC) were grown in minimum essential medium supplemented with 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2 mM Most of the compounds were purchased from Sigma-Aldrich, Wako Pure Chemical Industries, Ltd., or EMD Millipore. All test compounds were solubilized in dimethyl sulfoxide (DMSO) and then stored at −20°C until use. They were diluted with DMSO to different concentrations and added to the culture medium at a 1/1000 (vol/vol) dilution just before use.4 cells/100 µl/well) or HeLa (0.5×104 cells/100 µl/well) cells were seeded on 96-well plates. After a 24-hour incubation, cells were cotransfected with 30 ng pGL4.11-L1.3 5′ UTR and 30 ng pGL4.74 with 0.18 µl FuGENE6 reagent (Roche) per well. At 24 hours post-transfection, cells were exposed to the various concentrations of test compounds. After 6 or 24 hours of treatment, FLuc and RLuc luminescence were measured by Multilabel Counter 1420 ARVO (PerkinElmer) using the Dual-Glo Luciferase Assay System (Promega).HepG2  control. If the addition of the compound reduced the RLuc activity to <50% of the DMSO control, the data at those concentrations were not used for analysis.-mGLucI or 99-PUR- JM111-mGLucI was cotransfected with 0.5 µg of empty vector (pcDNA3) or V5-tagged Ultimate ORF cDNA construct (four replicate wells each). At 24 hours post-transfection, 3 mls of media were replaced, and at subsequent time-points 50 µl of media was sampled from each well for time-course determination of luminescence. All readings for a single experiment were made at the same time using the BioLux Gaussia luciferase assay kit (New England BioLabs). Readings were adjusted for background luminescence of media alone.Initially, Gaussia luciferase L1 reporter constructs were tested for efficacy in HEK293T cells and 6-well plates. One µg of 99-PUR-RPS4 cells/100 µl/well) were seeded on a 96-well plate. After a 24-hour incubation, cells were cotransfected with 100 ng 99-PUR-RPS-mGLucI or 99-PUR-JM111-mGLucI and 2 ng pSV40-CLuc with 0.3 µl FuGENE6 reagent per well. At 24 hours post-transfection, the medium was replaced with fresh medium containing 1 µg/ml puromycin and various concentrations of test compounds. Untreated cells were included in each plate for normalization between plates and experiments. After a 2-day culture, cells were exposed to fresh medium containing only the test compounds. After an additional 3-day incubation, two 20-µl aliquots of the medium were sampled to measure GLuc and CLuc. Luminescence was measured with the Multilabel Counter 1420 ARVO using the BioLux Gaussia luciferase assay kit (New England BioLabs) and BioLux Cypridina luciferase assay kit (New England BioLabs).To screen chemical compounds for effects on retrotransposition, HeLa cells  control was calculated. After luminescence in the medium was measured for the L1 retrotransposition assay, cell viability was determined (see below). The concentration at which cell viability was >80% was used to evaluate L1 retrotransposition activity.mGLucI-tagged L1s, HeLa cells were harvested at four days post-transfection and their DNA were extracted with a DNeasy Tissue Kit (Qiagen). PCR was performed using primers that flank the intron: sc-F4 (CTTTCCGGGCATTGGCTTCC) and sc-R4 (CAAGCCCACCGAGAACAACG). Digesting the genomic DNA prior to PCR with restriction enzymes that cut within the intron (BanI or BspT107I) increased the amount of 149-base pair (bp) spliced product and reduced the amount of 282-bp unspliced product.To confirm removal of the mini-intron, and therefore genomic insertion of Immunofluorescence techniques have been described Cell viability was determined by measuring the amount of ATP present using the Cell Titer-Glo Luminescent Cell Viability Assay (Promega). Luminescence was measured with the Multilabel Counter 1420 ARVO. The percentage of luminescence in treated wells relative to that in control wells was used to determine cell viability. Cell viability was evaluated for four or six independent experiments.t-test between the control and treated wells. When two or more concentrations were used, Dunnett’s multiple comparison test was applied. Statistical analyses were performed with SAS 9.1.3 software (SAS Institute Inc.), with p-values <0.05 considered significant.Statistical analyses were performed with Student’s We wished to determine the effects of a wide selection of environmental, genotoxic, and therapeutic compounds, including many of common human exposure, on the activity of the LINE1 retrotransposon, an insertional mutagen linked to the etiology of human disease and possibly cancers. To begin, we cloned the 5′ UTR of L1.3, one of the most active human L1s p<0.05) after exposure for 6 hours and/or 24 hours (p<0.05) in L1 promoter activity listed in Using the system described above, we screened 95 compounds for effects on L1 promoter activity. Among these 95 compounds, 21 genotoxic agents with various mechanisms of action, such as DNA alkylation, DNA crosslinking, and DNA topoisomerase inhibition, were evaluated . Five ge24 hours . Among 9Benzo[a]pyrene, a DNA intercalator, increased the L1 promoter activity in a dose-dependent manner by >1.5-fold at 24 hours. This is consistent with a previous report, which showed that Benzo[a]pyrene activates mouse L1 transcription p<0.05) after exposure for 6 and/or 24 hours  stress . Four ce24 hours .Among the cellular stresses, oxidative stress is induced by diethyl malate, iodoacetamide, and phorone, all of which increased L1 promoter activity 1.2- to 2-fold. Cyclosporin A, which induced L1 promoter activity at 24 hours, inhibits mitochondrial membrane permeabilization p<0.05) after exposure for 6 and/or 24 hours (p<0.05) after exposure for 6 and/or 24 hours , isolated from the marine copepod RP in the opposite orientation. L1RP is one of the most active human L1s RP containing the GLuc retrotransposition cassette into an episomally replicating mammalian expression vector that expresses a puromycin resistance gene. The resultant plasmid was named 99-PUR-RPS-mGLucI.The secreted GLuc retrotransposition cassette consists of the GLuc gene interrupted by an antisense mini-intron, the EF-1α/eIF4g hybrid promoter (which is stronger than the CMV promoter), and the TK poly(A) signal . The GLu-mGLucI with empty vector or cDNA constructs whose protein products were previously determined to alter EGFP-reporter retrotransposition in HEK293T cell culture . Heterogeneous nuclear ribonucleoprotein K (hnRNPK) increased retrotransposition as previously reported All evidence indicates that Gaussia luciferase secreted into the media serves as an effective read-out of accumulated retrotransposition events. GLuc is expressed only when the intron is spliced from the chimeric L1-reporter RNA, the RNA is reverse transcribed, and its cDNA is inserted in the genome by retrotransposition. Testing initially for efficacy in 6-well plates, we cotransfected 99-PUR-RPS culture . At 24 hKroutter et al. noted that earliest L1 retrotransposition events are detected only at 32 hours post-transfection mGLucI  inhibits reverse transcription by becoming integrated into the elongating cDNA sequence. AZT inhibits L1 retrotransposition as measured with a neomycin cassette p<0.0001; p<0.0001; p<0.05; p<0.0001; We evaluated the effects of 26 compounds on L1 retrotransposition with the dual secreted luciferase assay. Twenty one of the test compounds were chosen from among the agents that enhanced L1 promoter activity in the L1 reporter gene assay. The remaining five compounds were randomly chosen from among those that did not enhance L1 promoter activity. Among the 21 compounds that elevated L1 promoter activity, etomoxir elevated L1 retrotransposition by 40.6% as compared with the control at 50 µM  significantly increased L1 promoter activity by >1.5-fold at 6 and/or 24 hours. We then assessed the effects of compounds on L1 retrotransposition. The results revealed that 3 compounds significantly increased L1 retrotransposition activity by 20–40.6% . The datin vitro, there is a possibility that the effects of these compounds on retrotransposition in vivo might be larger than those observed in vitro. Several factors suggest that this might be the case. First, benzo[a]pyrene, which is a DNA intercalator and an inducer of genotoxicity, induces both transcription Although our retrotransposition experiments were conducted in vitro versus those in vivo has to do with the L1. Here we used L1RP, which has very high retrotransposition activity RP, we may not have been able to detect enhanced retrotransposition activity, even if it was substantial. Thus, compounds that only slightly elevated L1 retrotransposition might induce retrotransposition more in vivo than would have been expected based on experiments in vitro. Moreover, it is estimated that 80 to 100 L1s remain potentially active for retrotransposition in the average diploid human genome A second factor that may lead to a difference in the effects seen in vivo and in vitro assays. In the case of the PPARα agonists, bezafibrate and fenofibrate enhanced L1 transcription and bezafibrate and WY-14643 slightly increased L1 retrotransposition. Long-term exposure of mice to WY-14643 decreases the DNA methylation in the liver of LINE1s, LINE2s, and intracisternal A particles (IAPs); the latter is an active retrotransposon in mice in vivo. A stable assay system that allows us to assess retrotransposition in vivo, including effects that result from epigenetics, will be useful to evaluate the effects of compounds on L1 retrotransposition.The third factor concerns the difference in conditions between neoRT reporter cassette, containing a neomycin phosphotransferase (neo) gene and an artificial intron placed between the promoter and neo coding sequence Cell culture assays for retrotransposition are the most important tools for studying LINE1 elements in humans and mice. These assays report the final step of retrotransposition, the number of cells in a total population of cells that receive an insertion event. The first assay to measure retrotransposition in cells was developed over 25 years ago neoRTmneoI) included neo interrupted by antisense γ-globin intron 2. Moran et al. inserted mneoI in opposite transcriptional orientation into the 3′ untranslated regions (UTRs) of L1.2 and LRE2 RPFreeman et al. redesigned mneoI assay requires 3 weeks for antibiotic selection. A blasticidin S-deaminase gene (bsr) reporter cassette cloned in L1RP and a vector that constitutively expresses puromycin cut antibiotic selection time in half Limitations exist for each of the L1 cell culture assays in widespread use. The None of the current assays permit easy study of retrotransposition kinetics and the performance of multiple assays in a single well. Different wells may be sampled sequentially to establish a time-course series mGLucI) containing a modified Gaussia luciferase gene. The JM111 mutant, which exhibits almost no retrotransposition activity because of the introduction of two missense mutations in ORF1 Cognizant of assay limitations, we designed a “fourth generation” cell culture retrotransposition assay that is easy to execute, sensitive, amenable to high-throughput analyses, and permissive for time-course sampling from a single transfected well. We constructed a novel retrotransposition cassette reporter . However, the risk of retrotransposition has not yet been considered as part of drug safety assessment, even though there are several reports that chemical-induced stress activates L1 transcription and retrotransposition and that the integration of retrotransposition into genes causes disease  and Renilla luciferase (RLuc) expression in the L1 reporter gene assay in HepG2 cells. Relative FLuc luminescence, relative RLuc luminescence, and relative normalized luciferase luminescence at 6 hours (A) and 24 hours (B) after treatment with vehicle control (DMSO) in the L1 reporter gene assay using HepG2 cells. FLuc and RLuc indicate L1 promoter activity and expression of the internal control, respectively. Data are the mean ± SD from four independent experiments. TK pro-R: pGL4.74; no pro-F: pGL4.11; L1 5′ UTR-F: pGL4.11-L1.3 5′ UTR.(TIF)Click here for additional data file.Figure S2FLuc and RLuc expression in L1 reporter gene assay in HeLa cells. Relative FLuc luminescence, relative RLuc luminescence, and relative normalized luciferase luminescence at 6 hours (A) and 24 hours (B) after treatment with the vehicle control (DMSO) in the L1 reporter gene assay using HeLa cells. FLuc and RLuc indicate L1 promoter activity and an internal control, respectively. Data are the mean ± SD from four independent experiments. TK pro-R: pGL4.74; no pro-F: pGL4.11; L1 5′ UTR-F: pGL4.11-L1.3 5′ UTR.(TIF)Click here for additional data file.Figure S3Data for the earliest sampling time-points of the time-course GLuc retrotransposition assay shown in . See legend for that figure.(TIF)Click here for additional data file.Figure S4GLuc and CLuc expression in the novel dual secreted luciferase L1 retrotransposition assay. GLuc and CLuc luciferase luminescence in the novel L1 retrotransposition assay 6 days post-transfection. Either 99-PUR-RPS-mGLucI or 99-PUR -JM111-mGLucI containing two missense mutations in L1RP ORF1 and pSV40-CLuc were co-transfected. GLuc can be expressed only when retrotransposition occurs. CLuc was used as an internal control. Data are the mean ± SD from six independent experiments. RLU: Relative Light Units.(TIF)Click here for additional data file.Dataset S1Results of L1 reporter gene assay in HepG2 cells. L1 promoter activity 6 and 24 hours after treatment was evaluated by luciferase reporter gene assay. L1 promoter activities are represented as a percentage relative to that of the vehicle control. Mean and SD from at least two independent experiments  are shown.(XLS)Click here for additional data file.Dataset S2Results of L1 reporter gene assay in HeLa cells. L1 promoter activity 6 and 24 hours after treatment was evaluated by the luciferase reporter gene assay. L1 promoter activities are represented as a percentage relative to that of the vehicle control. Mean and SD from two independent experiments  are shown.(XLS)Click here for additional data file.Dataset S3Results of L1 retrotransposition assay and cell viability. L1 retrotransposition activity and cell viability after treatment are represented as a percentage relative to that of the vehicle control. Mean and SD from four independent experiments are shown.(XLS)Click here for additional data file.


Graphene's unique optoelectronic properties are promising to realize photodetectors with ultrafast photoresponse over a wide spectral range from far-infrared to ultraviolet radiation. The underlying mechanism of the photoresponse has been a particular focus of recent work and was found to be either photoelectric or photo-thermoelectric in nature and enhanced by hot carrier effects. Graphene supported by a substrate was found to be dominated by the photo-thermoelectric effect, which is known to be an order of magnitude slower than the photoelectric effect. Here we demonstrate fully-suspended chemical vapor deposition grown graphene microribbon arrays that are dominated by the faster photoelectric effect. Substrate removal was found to enhance the photoresponse by four-fold compared to substrate-supported microribbons. Furthermore, we show that the light-current input/output curves give valuable information about the underlying photophysical process responsible for the generated photocurrent. These findings are promising towards wafer-scale fabrication of graphene photodetectors approaching THz cut-off frequencies. Graphene's high carrier mobility combined with its universal optical absorption from terahertz to the ultraviolet wavelength regime, as well as its large nonlinear optical coefficients, are promising towards novel applications in photodetection123478910111234512131415161718192021123915161718181920222324Practical applications require scalable approaches such as chemical vapor deposition (CVD) grown graphene photodetectors. The photophysics of CVD-grown graphene devices is however less understood, and it is a priori unclear to what extent inherent grain boundaries and other defects affect the mechanism of photocurrent generation. A recent time-resolved study of the photoresponse in CVD-grown graphene embedded in a coplanar stripeline circuit reveals that the time constant of the photo-thermoelectric effect is, at 130 ps, more than an order of magnitude slower than the photoelectric effect, at 4 ps12912151620Here we demonstrate fully-suspended CVD-grown graphene microribbon arrays that are dominated by the faster photoelectric effect. Removal of the substrate was found to enhance the photoresponse by four-fold compared to substrate-supported microribbons. We also show that the slope of the light-current input/output curves (L-I curves) gives valuable information about the underlying photophysical process responsible for time-integrated photocurrents. These findings are promising towards wafer-scale fabrication of graphene photodetectors approaching THz cut-off frequencies.2 and patterned using electron-beam lithography . The graphene quality was evaluated by Raman spectroscopy as well as optical transmission measurements and found to be monolayer graphene . Ultimately, IPC saturates at the highest pump power with values for suspended graphene being approximately 6-times smaller than those for supported graphene, as indicated by the arrow labeled “A” in PC ∝ (Popt)α. For supported graphene we studied five different devices and found an average value of α = 0.62 ± 0.05 at zero gate voltage. In stark contrast, the power exponent of the fully suspended device is with 0.9, near unity. The pronounced change in slope of the L-I input-output curve indicates that the underlying physical mechanism has changed.While demonstrating four-fold enhanced photocurrents in large-scale CVD-grown graphene photodetectors is of great technological importance, one would also like to understand the underlying photophysical mechanism to be able to optimize the detector device performance. The two main competing mechanisms for photocurrent generation in graphene are the photo-thermoelectric effect and the photoelectric effect. One way to tell these two effects apart is to characterize their time constant in optical pump-probe experiments which differ by at least an order of magnitudeTE) and the optical pump power Popt can be estimated and yields ITE ∝ (Popt)2/3, i.e. α = 0.66 (see 131415161718192021e underneath the laser excitation spot located in the center of the microribbon. These photoexcited carriers interact strongly with the surface polar phonons (SPP) of the substrate2223242533e in carrier temperatures. This ΔTe is the drive for the Seebeck effect and thus a strong photo-thermoelectric contribution  effect still partly contributing to the photocurrent in this device. In addition, Quantitatively, the photoelectric contribution depends linearly on photogenerated carrier density which is directly proportional to the rate of absorbed photons and thus the incident excitation power. Therefore, the photoelectric contribution Ie, driving the Seebeck effect before reaching contacts. It is thus evident that novel graphene photodetectors can be engineered in which either the photo-thermoelectric or the photoelectric effect dominates the photoresponse. Since the time constant of the photoelectric effect is, at 4 ps, over an order of magnitude faster than the time constant of the photo-thermoelectric effect, at 130 psIn contrast to our findings, a recent study of partially supported graphene shows that such a sample design yields a dominant photo-thermoelectric effectIn conclusion, we have demonstrated four-fold enhancement of the photoresponsitivity of fully suspended CVD-grown graphene photodetector devices over substrate-supported graphene. In addition, we have shown that the light-current input/output curves give valuable information on the underlying photophysical process responsible for the generated photocurrent. Finally, we have found that fully suspended CVD-grown graphene devices are dominated by the photoelectric effect, which is promising towards CVD-grown graphene photodetectors approaching THz cut-off frequencies.2 substrate confirms good quality monolayer graphene film . The devices were then wire bonded using 25 μm thick Au wires on a chip carrier for electrical measurements. For suspending graphene, we etched the 300 nm thick underlying layer of SiO2 using (6:1) buffered oxide etchant (BOE) followed by critical point drying (CPD) to avoid damage to the suspended graphene ribbon.Large area graphene was grown on high purity copper foil using a low pressure CVD furnace at 1000°C in a hydrogen, argon and methane environment. Raman spectrum of the graphene transferred onto 300 nm SiO−6 mbar. The photoresponse of the graphene ribbons was measured using a lock-in amplifier by positioning a 532 nm wavelength laser spot onto the ribbon. A spot of approximately 1 μm diameter (FWHM) was focused using a 50 × objective with working distance of 13 mm. The position of the laser spot was manipulated by using piezoelectric motor controlled scanning mirrors with nanometer precision in X and Y directions. The incident laser power, which was measured to be 20 mW at maximum, was controlled by using neutral density filters to sweep between 1 nW and 2.8 mW. The beam was modulated with a chopper wheel at 2 kHz. All current and voltage values were supplied by two Keithly 2400 source meters, while output was read from the lock-in amplifier. A moderate source-drain bias of 0.1 V was used for photocurrent measurements. Back-gate sweeps were performed with 1 mV with voltage ranges selected to center the Dirac point.All the measurements were performed at room temperature in a chamber with vacuum level of 10V.P., S.S. and E.H.Y. conceived and designed the experiments. V.P fabricated the suspended CVD-graphene microribbons. V.P. carried out the electro-optical experiments and analyzed the data. V.P. and A.C. carried out the Raman experiments. V.P., S.S. and E.H.Y. co-wrote the manuscript. E.H.Y. supervised the device fabrication and S.S. supervised the electro-optical measurements. All authors contributed to scientific discussions.SI


The mechanical behavior of graphene under various indentation depths, velocities, and temperatures is studied using molecular dynamics analysis. The results show that the load, elastic and plastic energies, and relaxation force increased with increasing indentation depth and velocity. Nanoindentation induced pile ups and corrugations of the graphene. Resistance to deformation decreased at higher temperature. Strong adhesion caused topological defects and vacancies during the unloading process. Graphene has received a lot of attention due to its good mechanical and electromagnetic properties -3, incluNovoselov and Geim  used graMany studies -25 have In this study, the effects of nanoindentation depth and velocity on the mechanical properties and contact behavior of graphene at various temperatures are investigated using molecular dynamics (MD) simulations. Adhesion, relaxation, defects, and deformation are discussed.Figure The Lennard-Jones potential function is employed to describe the interaction between the diamond tip and the graphene atoms. The Tersoff empirical potential energy function  is generFigure The tip then indented the graphene. The absorptive force gradually turns into a repulsive force. As the indentation depth increased, the stress wave spread out farther from the center, inducing ripples and corrugations. Figure The effect of indentation depth was examined. With all other conditions fixed, indentation depths of 0, 0.2, 0.4, and 0.6 nm were investigated. Figure 2) defects, which are composed of one octagonal ring and two pentagonal rings, during the adhesion pulling process. Double vacancies are referred to as 5-8-5 defects [2 defects [Figure  defects . Our sim defects , who inv defects .Figure Figure Indentation velocities of 25, 50, 75, and 100 m/s were tested by fixing the temperature at 300 K and the packing time at 15 ps. Figure Figure The indentation behavior of graphene was studied using molecular dynamics simulations. The following conclusions were obtained:1. The affected area, load, elastic and plastic energies, and relaxation force increased with increasing indentation depth.2. Nanoindentation-induced pile ups and corrugations of graphene were observed. Strong adhesion causes topological defects and vacancies.3. The average contact stiffnesses of the graphene at temperatures of 0, 200, 300, and 400 K are 58.7, 58.1, 49.48, and 36.6 N/m, respectively.4. At higher temperature, the kinetic energy among atoms increases, which weakens covalent bonds and thus decreases resistance to deformation. The load, elastic and plastic energies, and relaxation force decrease with increasing temperature.5. With a fast indentation, the graphene has insufficient time to respond, which leads to a high load, elastic and plastic energies, and relaxation force.The authors declare that they have no competing interests.The work presented here was carried out in collaboration between all authors. THF, THW and JCY defined the research theme. THF and JCY designed methods and analyzed the data, interpreted the results and wrote the paper. YJH co-worked on associated data collection, their interpretation, and presentation. All authors have contributed to, seen and approved the final manuscript.


Group I introns are intervening sequences that have invaded tRNA, rRNA and protein coding genes in bacteria and their phages. The ability of group I introns to self-splice from their host transcripts, by acting as ribozymes, potentially renders their insertion into genes phenotypically neutral. Some group I introns are mobile genetic elements due to encoded homing endonuclease genes that function in DNA-based mobility pathways to promote spread to intronless alleles. Group I introns have a limited distribution among bacteria and the current assumption is that they are benign selfish elements, although some introns and homing endonucleases are a source of genetic novelty as they have been co-opted by host genomes to provide regulatory functions. Questions regarding the origin and maintenance of group I introns among the bacteria and phages are also addressed. Tetrahymena thermophila   , and theas.edu/] -33. Whenas.edu/] . In caseas.edu/] ,36.Group I introns are removed from precursor RNA by an autocatalytic RNA splicing event that is mediated by the intron’s RNA tertiary structure. Base-pairing interactions between the 5′-end of the intron and flanking exon sequences define the location of the 5′ and 3′ splice sites. The Internal Guide Sequence (IGS), which is a short intronic sequence near the 5′-end that pairs with sequences of the upstream exon to form P1, determines the 5′ splice site Figure . The 3′ Azoarcus sp. BH72 pre-tRNAIle intron-exon complexes ,6710,67]Currently, there are six families of HEases, classified primarily on the basis of conserved amino acids that correspond to structural or active site residues; the LAGLIDADG, H-N-H, His-Cys box, GIY-YIG, PD-(D/E)xK, and EDxHD families -73. The trans-acting HEases have been described in T4 and related phages that can promote the homing of either group I introns lacking ORFs or group I introns that encode defunct (degenerated) HEases  to avoid loss. Alternatively, it is thought that group I introns can ‘escape’ to a new population of intronless alleles by transposition to new sites (ectopic integration) by reverse splicing. Reverse splicing is the reverse of the forward splicing reaction, and theoretically allows a group I intron RNA to insert into a RNA molecule with four to six complementary bases to the P1 stem of the intron RNA ,85. ThisAnother mechanism for ectopic integration or transposition relates to the relaxed specificity of many intron-encoded HEases. For instance, cleavage at a site similar to a HEase’s native target site may promote intron mobility, and it has been shown that the cleavage specificity of the I-TevI HEase can be influenced by oxidative stress . HoweverClostridium difficile as a feature of a two-component riboswitch that involves c-di-GMP as an allosteric activator [There are a few instances where group I introns or their components may have been domesticated by their host genomes, or by other types of mobile genetic elements. The bacterial DUF199/WhiA protein is a transcription factor and its N-terminal region contains the same protein fold as found in monomeric LAGLIDADG HEases encoded within group I introns ,90. Thisctivator . Here, ictivator . In esseC. difficile in the tcdA locus. The composite element, termed an IStron, is composed of a splicing-competent group I intron (IA2 subgroup) that has an insertion element  embedded within its 3′-end and encoding two transposases [A unique composite element has been described in some enterotoxin producing strains of sposases ,94. One sposases . TnpA casposases ,97. Thissposases . Althougsposases ,100.Bacillus species [recA and nrdE genes in various Bacillus species [nrdE genes [Within bacteria, group I introns are predominately inserted within structural RNA genes such as tRNA and rRNA genes ,101-107. species ,112, rec species ,113, anddE genes ,110. ThidE genes ,114-117.in vivo splicing rates, and that self-splicing RNA introns became extinct in the archaeal lineage [Interestingly, group I introns have so far not been discovered in archaeal genomes, although group I intron derived HEase sequences are sometimes associated with archaeal introns -122. TheBacillus cereus group suggested that some of the genomic recA, nrdE, nrdF introns are similar to phage introns, indicating that phage infection could serve as a vector system for the lateral movement of introns among different genomes [Tetrahymena into the E. coli 23S gene resulted in the reduction of the growth rate which was correlated with poor splicing of the Tetrahymena intron. Moreover, the intron RNA was shown to associate with the 50 S ribosomal subunit and possibly interfere with translation. Clearly, there are barriers to intron spread in bacteria [The persistence and spread of group I introns in prokaryotic genomes is dependent on a number of factors including (1) the phenotypic cost associated with the insertion of a group I intron, (2) the availability of intronless alleles for endonuclease-mediated homing, (3) the presence of efficient homology-based DSB repair systems, (4) the availability of DNA or RNA transfer mechanisms such as DNA uptake by natural transformation, conjugation and plasmid transfer, and phages. Interestingly, recent work on the  genomes . However genomes  showed tbacteria  that areet al. [sunY gene of the T4 phage the intron sequences flanking the HEase ORF (I-TevII) were similar to the exon junction sequences that comprise the I-TevII target sequence. Importantly, they were able to demonstrate that a synthetic construct that included the fused sequence composed of the up- and down-stream sequences that flank the I-TevII ORF was indeed cleaved by I-TevII. This result provided strong circumstantial evidence for the ‘endonuclease-gene invasion’ hypothesis whereby a free-standing HEase cut an intron sequence that fortuitously contained a similar HEase target site. During the recombination-based repair process, the endonuclease gene sequence was inserted into the cleaved intron sequence, thus generating a composite potentially mobile intron.One of the most intriguing questions about mobile group I introns concerns their evolutionary origin. The current consensus is that HEases and group I introns had distinct evolutionary origins, and that HEases have on multiple independent occasions invaded an endonuclease-free intron. The alternative scenario, that group I introns always possessed an endonuclease gene is problematic for a number of reasons, including the fact that many group I introns do not contain ORFs, and the notion that group I introns were direct descents of catalytic RNAs from the RNA world. Moreover, the finding that HEases can exist outside of the protective confines of introns, as so-called free-standing homing endonucleases, lent credibility to the hypothesis that these free-standing enzymes could be a potential source of the ‘invading’ endonuclease. Two mechanisms that would lead to the formation of such a composite mobile intron have been proposed. Loizos et al.  noted thRecent studies ,76 proviRegardless of the origin of mobile group I introns, one would assume that endonuclease invasion would have a deleterious effect on intron splicing. In this respect, it is interesting to note that many endonuclease ORFs are inserted in loops that presumably do not interfere with folding and splicing. It is also possible that the intron-encoded endonucleases and/or host factors were able to compensate by stabilizing the intron tertiary RNA structure or discouraging misfolding of the intron RNAs -132. ThiThis returns us to the enigma of why group I introns and their associated HEases have been successful in spreading among the organellar genomes of plants, protozoans, and fungi but have very limited representation among bacterial and phage genomes. Koonin  proposedThe mechanisms that promote and prevent group I introns from proliferating among bacterial genomes are poorly understood, as is the long-term impact of introns on organismal viability. When present, it is assumed that introns are phenotypically neutral, yet the co-opting of intron functions by a riboswitch or the domestication of intron-encoded homing endonuclease as a regulatory protein (WhiA) indicates that introns can be a source of genetic novelty. Future research efforts directed at understanding the effect of group I introns on host gene expression, mechanisms of mobility to ectopic sites and their spread among bacterial genomes and phages will lead to valuable insights regarding the dynamics and evolution of group I introns.bp: base pair; c-di-GMP: cyclic diguanylate; DSB: double-strand break; GTP: guanosine-5′-triphosphate; HEase: homing endonuclease; HEG: homing endonuclease gene; HUH: endonuclease motif; IGS: Internal Guide Sequence; IS: insertion element; ORF: open-reading frame; rRNA: ribosomal RNA tRNA, transfer RNA; SDSA: synthesis-dependent strand annealing.The authors declare that they have no competing interests.GH: conception and design, figure preparation, manuscript writing and final approval of manuscript. MH: conception and design, compilation of data for Figures