A hybrid structure (HS) made of one-dimensional ZnO nanorods (NRs) and a two-dimensional synthesized graphene sheet was successfully constructed in this study. The uniform ZnO NRs were obtained by hydrothermal method and grown on a graphene surface that had been transferred to a polyethylene terephthalate substrate. The HS exhibited high transmittance (approximately 75%) over the visible wavelength range, even after cyclic bending with a small radius of curvature. Raman spectroscopy and Hall measurement were carried out to verify the chemical composition and electrical properties of the structure. Stable electrical conductance of the ZnO NR/graphene HS was achieved, and increase in carrier mobility decreased the resistance of the ZnO-with-graphene sheet in comparison with bare ZnO NRs. For ent years -6. Becaus [2/Vs) , a strets [2/Vs) . Moreoves [2/Vs) -13. Manys [2/Vs) , vapor–ls [2/Vs) , and puls [2/Vs) . Howevers [2/Vs) , light-es [2/Vs) , solar cs [2/Vs) , and pies [2/Vs) . In addis [2/Vs) ,22. To oIn this work, 1D ZnO NRs were synthesized by hydrothermal method onto a 2D graphene sheet to form an HS. High transmittance over the visible light region was obtained after synthesizing the ZnO NRs, and the sample displayed excellent mechanical properties after bending with a small radius. Notably, we essayed a Hall measurement of the HS, which consisted of ZnO NRs/graphene on a polyethylene terephthalate (PET) substrate.3/HCl solution for etching to strip the Cu foil. After etching, we retrieved the sample from the FeCl3/HCl solution, transferred it onto the PET substrate, and cleaned it with deionized water. Finally, the PMMA/graphene was soaked in an acetone solution to remove the PMMA and then washed with isopropyl alcohol [Each graphene sheet was prepared on a Cu foil by CVD and then spin-coated with a protective layer of poly(methyl methacrylate) (PMMA). The PMMA/graphene/Cu foil sample was immersed into an FeCl alcohol . A 100-nTo investigate the 3D hybrid nanostructure formed by combining 1D ZnO NRs with2D graphene, the ZnO seed layer was coated onto the graphene surface and annealed at a suitable temperature for the growth of ZnO NRs through hydrothermal method. The ZnO NRs presented here were obtained with a solution-based chemical synthesis. In a solution containing zinc nitrate hexahydrate and HMTA, hydroxyl ions were released through the thermal decomposition of the HMTA and reacted with zinc ions to form ZnO. The synthesis can be summarized in the following reactions:2− terminated surface, onto which the precursor molecules in the vicinity tend to be adsorbed [2+ cations usually form hydroxyl complexes as the precursors of ZnO.To observe the growth of the ZnO NRs on the graphene sheet, FE-SEM images were taken, as shown in Figure adsorbed . SimultaA concerning feature of the hybrid structure is that, although ZnO and graphene exhibit good optical transmittance in the visible spectral range, the scattering of light by ZnO NRs is suspected to lead to a decrease in transmittance to a certain extent. The optical transmittance of the ZnO NR/graphene hybrid structure was estimated by fabricating the structures on PET substrates. Figure −1 corresponds to the E2 mode of the ZnO hexagonal wurtzite structure [−1 is attributed to the E2g phonon of C sp2 atoms, and the D peak at approximately 1,350 cm−1 is accredited to local defects and disorder, such as the edges of graphene and graphite platelets [−1 has also been found that may be related to the formation and number of layers of the graphene [−1, the D peak at 1,353 cm−1, the G peak at 1,586 cm−1, and the 2D peak at 2,690 cm−1. The formation of few-layer graphene was verified by the intensity ratio of the G peak to that of the 2D peak, which was approximately 1 to 1.5, and by the position of the 2D peak [Raman spectroscopy is a promising method for inspecting the ordered/disordered crystal structures of carbonaceous materials and the different layer characteristics of graphene. It was also used to prove that ZnO nanostructures were grown on graphene surface. The usual peak at 437 to 439 cmtructure . The G platelets ,28. Moregraphene . Figure  2D peak ,31. In a2/Vs, 18 times higher than that of the ZnO film. It has been reported that there is an important relationship between mobility and sheet resistance because the carriers can be easily scattered by lattice defects [The device structure was fabricated as shown in Figure  defects . AccordiUniform ZnO NRs were obtained by hydrothermal method and grown on a graphene surface that had been transferred to a PET substrate. The ZnO NR/graphene HS exhibited high transmittance (approximately 75%) over the visible wavelength range, even after cyclic bending with a small radius of curvature. Stable electrical conductance of the ZnO NR/graphene HS was achieved, and the improvement of the ZnO sheet resistance by the incorporation of the graphene sheet can be attributed to the resultant increase in carrier mobility.The authors declare that they have no competing interests.RJC was the principal investigator and is also the corresponding author of this paper. ZCL and PKY were in charge of material preparation and characterization. KYL contributed to data analysis. SFJ and PWC contributed to graphene synthesis. All authors collaborated to complete this research and to compile this manuscript. All authors read and approved the final manuscript.


Myocardial differentiation is associated with the activation and expression of an array of cardiac specific genes. However, the transcriptional networks that control cardiac gene expression are not completely understood. Myocardin is a cardiac and smooth muscle-specific expressed transcriptional coactivator of Serum Response Factor (SRF) and is able to potently activate cardiac and smooth muscle gene expression during development. We hypothesize that myocardin discriminates between cardiac and smooth muscle specific genes by associating with distinct co-factors. Here, we show that myocardin directly interacts with Tbx5, a member of the T-box family of transcription factors involved in the Holt-Oram syndrome. Tbx5 synergizes with myocardin to activate expression of the cardiac specific genes atrial natriuretic factor (ANF) and alpha myosin heavy chain (α-MHC), but not that of smooth muscle specific genes SM22 or smooth muscle myosin heavy chain (SM-MHC). We found that this synergistic activation of shared target genes is dependent on the binding sites for Tbx5, T-box factor-Binding Elements (TBEs). Myocardin and Tbx5 physically interact and their interaction domains were mapped to the basic domain and the coil domain of myocardin and Tbx5, respectively. Our analysis demonstrates that the Tbx5G80R mutation, which leads to the Holt-Oram syndrome in humans, failed to synergize with myocardin to activate cardiac gene expression. These data uncover a key role for Tbx5 and myocardin in establishing the transcriptional foundation for cardiac gene activation and suggest that the interaction of myocardin and Tbx5 maybe involved in cardiac development and diseases. Heart formation is one of the earliest morphogenic events during animal development. The development of the heart is under the control of a complex transcriptional network, and dys-regulation of the expression or genetic mutation of cardiac genes often leads to abnormality in the morphology and the function of the heart Whereas haploinsufficiency of Tbx5 in transgenic mice mimics the HOS, homologous deletion of the Tbx5 gene led to early embryonic lethality Tbx5 cooperates with other transcriptional factors to regulate the expression of its target genes 6GG or CArG box Myocardin is a potent transcription cofactor for serum response factor (SRF) and is expressed in smooth and cardiac muscles Myocardin potently activates both cardiac and smooth muscle gene expression in a CArG-dependent manner To test whether Tbx5 and myocardin could synergistically activate the ANF promoter in cardiomyocytes, we transfected both myocardin and Tbx5 expression plasmids, together with the ANF luciferase reporter, into primarily cultured neonatal rat cardiomyocytes. As shown in The promoter/enhancer of the ANF gene contains multiple cis-elements for several transcription factors, including that of Nkx2.5-binding site (NKE), MEF2-binding site (MEF), GATA-element (GATA), T-box factor-Binding Element (TBE) as well as SRF-binding site (CArG) Next, we mutated the TBE site from the ANF115 minimal promoter and tested the activities of myocardin and Tbx5 to activate the reporter gene. Interestingly, we found that the TBE mutation abolished the synergy between myocardin and Tbx5 to activate this reporter . ConversTo test whether Tbx5 and myocardin interact directly, co-immunoprecipitation (Co-IP) assays were performed with cell extracts in which Myc- or Flag-tagged myocardin and Tbx5 were overexpressed in COS-7 cells. When Flag-Tbx5 was precipitated using anti-Flag antibodies, Tbx5-associated Myc-myocardin was readily detected by Western blotting . No suchWe used an alternative approach to confirm the interaction between myocardin and Tbx5. The full-length sequence of the myocardin gene was fused with the DNA-binding domain of the Gal4 gene to create a Gal4-myocardin fusion protein . The Gal4-myocardin was then transfected with the pL8G5-luciferase reporter in which the luciferase reporter is under the control of the Gal4 binding element UAS. Gal4-myocardin, but not pcDNA-Tbx5 or pcDNA-myocardin, potently activated the reporter gene . AdditioTo map the domains of myocardin that are responsible for its interaction with Tbx5, truncated myocardin mutants were generated and their potential interaction with Tbx5 was examined by co-immunoprecipitation (Co-IP) assays. The truncation mutants from the N-terminus up to the basic domain of myocardin can still interact with Tbx5, while further deletion past the basic domain abolished this capability . These rNext, we attempted to map the domains of the Tbx5 protein that mediate its interaction with myocardin. Series of Tbx5 deletion mutants were generated to test their interaction with myocardin using co-immunoprecipitation (Co-IP) assays. Our results showed that the N-terminal region (aa 1-100) was sufficient to interact with myocardin . SimilarMyocardin associates with the CArG boxes via the formation of a CArG/SRF/myocardin ternary complex Next, we performed EMSAs to test whether Tbx5 could affect the formation of myocardin/SRF/CArG ternary complex. When SRF was incubated with the CArG box probe, they form CArG/SRF complex lane 2, . InclusiTbx5 has been identified as the Holt-Oram syndrome (HOS) causative gene. Multiple missense mutations of the human Tbx5 gene have been identified from individuals with HOS In this study we demonstrated that Tbx5 and myocardin synergistically activate cardiac gene expression. This activity is specific to cardiac tissue as these two proteins failed to similarly regulate smooth muscle genes. Our results therefore reveal a transcriptional regulatory network for cardiac gene expression.How do Tbx5 and myocardin cooperate to activate cardiac gene expression? We found that myocardin and Tbx5 interact directly and we mapped their interaction domains to the N-termini of these two proteins. It was known that myocardin does not bind to DNA directly, but instead associates with SRF to form a myocardin/SRF/CArG ternary complex on the promoters/enhancers of its target genes Myocardin is expressed in both cardiac and smooth muscle cells and is capable of activating both cardiac and smooth muscle-specific gene expression. How is the specificity defined? We observed that the interaction between Tbx5 and myocardin helps mediate distinct smooth muscle and cardiac gene expression profiles. In differentiating smooth muscle cells, Tbx5 staining showed that it was present only in the cytoplasm, instead of the nucleus Tbx5 belongs to the family of T-box containing transcription factors. Several members of this family of transcription factors are also expressed in the heart. Interestingly, Tbx2 was shown to repress the expression of the ANF gene, in part, by impairing the recruitment of Nkx2.5 to the TBE or the NKE Multiple missense mutations of the human Tbx5 gene have been associated with the HOS. Interestingly, these mutations do not uniformly impair the function of Tbx5 in the same manner. For example, Tbx5 mutations G80R and R237Q have different effects on its synergistic transactivation of the cardiac-specific ANF gene, which will likely translate as subtle differences in the phenotype and severity of the HOS Myocardin and SRF-expression plasmids were described previously COS-7 cells were maintained in DMEM with 10% FBS as described Neonatal rat cardiomyocytes were prepared as described Co-IP and Western blot analyses were performed as described EMSAs were performed as described previously


Furthermore, L1-KD neurons showed longer and more undulated leading processes during translocation through the primitive cortical zone. The curvature index, a quantitative index for curvilinearity, as well as the length of the leading process, were increased, whereas the somal movement was decreased in L1-KD neurons during terminal translocation in the PCZ. These results suggest that L1 has a role in radial migration of cortical neurons.L1cam (L1) is a cell adhesion molecule associated with a spectrum of human neurological diseases, the most well-known being X-linked hydrocephalus. Although we recently demonstrated that L1 plays an important role in neuronal migration during cortical histogenesis, the mechanisms of delayed migration have still not been clarified. In this study, we found that cell locomotion in the intermediate zone and terminal translocation in the primitive cortical zone (PCZ) were affected by L1-knockdown (L1-KD). Time-lapse analyses revealed that L1-KD neurons produced by  The neural cell adhesion molecule L1cam (L1) is one of the adhesion molecules expressed in the developing central and peripheral nervous system in vitro was perturbed by the addition of anti-L1 antibody However, very few reports have supported the concept of neuronal migration in utero electroporation, we showed that in vivo L1-knockdown (KD) perturbed neuronal radial migration, accompanied with alterations in the expression of some transcription factors in the cortical plate Previously, using a shRNA strategy combined with All of the animal experiments conducted in this study were approved by the Institutional Review Board for Biomedical Research using Animals at Kyoto Prefectural University of Medicine, and the animals were handled according to the Institutional Guidelines and Regulations. Pregnant C57BL/6J mice were purchased from CLEA Co. Ltd.  or SHIMIZU Laboratory Supplies Co. Ltd., Japan SLC. . The day a vaginal plug was detected was designated as embryonic day 0.5 (E0.5).scrambled sequence) were cloned in linearized pGeneClipTM hMGFP Vector (Promega).We prepared shRNA expression plasmid as previously described The shRNA sequence is as follows.anti-L1 shRNA (shRNA2 and shRNA5):AGCCTTACCAGAAGGGAAAGT (location 3406–3426)shRNA2; GTGCTTCAGGATGAACGATTT (location 1652–1672)shRNA5; GGAATCTCATTCGATGCATACscramble shRNA (shNC); in utero electroporation . Pregnant mice were anesthetized by intraperitoneal injection of Ketamine , Xylazine  and saline cocktail (5/3/40 v/v/v 9.75 ml/kg bodyweight). After disinfection with 70% ethanol, a 2-cm midline laparotomy was performed, and the uterine horns were resected. In order to conduct the plasmid DNA microinjection, 0.85-mm internal diameter glass capillary tubes  were employed, using a micropipette puller PB-7 . After the glass capillary tubes were pulled out, the pipettes were broken at approximately 70–80 μm on the external diameter (at ca.1.0 cm from the shoulder of the pipette) by pinching with forceps, and then ground to make a 30° bevel. One μl of plasmid DNA solution (1.0 mg/ml) in endotoxin free TE buffer, with 0.005% trypan blue added to aid targeting, was injected into the lateral ventricles using a mouth-controlled pipette system  Embryos were harvested after 1 or 2 days, i.e., at E14.5 or E15.5. The brains were removed and transferred into a solution of 1.2% agarose  in phosphate-buffered saline  at 37°C and the agar was subsequently hardened on ice for 5 minutes. Each brain was coronally cut at 200 μm with a Vibratome .2 incubator at 37°C for 10 minutes, and kept in Neurobasal medium supplemented with B27 , 0.5 mM L-glutamine, and antibiotics.The following procedures were similar to those reported previously Z-section images were acquired, and all focal planes were merged using EZ-C1 software. The migrating neurons were traced and analyzed using NIH ImageJ software and the Manual Tracking plugin.The slices were observed with a laser confocal scanning microscope with epifluorescence, as well as differential interference contrast (DIC) optics . Fluorescent microscopic images were obtained sequentially at 10-minute intervals for 24–72 hours. Approximately 20 x and y values for each cell. The distances between the starting point  and the end point  were derived using the Pythagorean Theorem; the square root of the quantity of (x2 – x1)2 + (y2 – y1)2. The velocity was defined as the distance between starting and end points divided by time over the observation period The images acquired were analyzed to trace the neuronal migration and the velocity of the migration was measured with EZ-C1 software and the NIH ImageJ plugin . The position of the center of the soma was plotted in each image, and displacement of the soma was defined by the difference in location between the starting point and the end point. The locations were expressed as the Z-section images, were merged.The curvature index was defined as “the curvilinear length” in micrometers of a leading process divided by “the linear distance” between the ends of the leading process in utero electroporation, and then reached a point underneath the CP at E15.5. In contrast, L1-KD neurons mostly stayed in IZ at E15.5. Thus, in order to analyze the dynamic radial migration in IZ, we observed cultured slices from E14.5 to E17.5 (t-test)   shRNA:shNC, migrated into the IZ at E14.5, one day after the to E17.5 . Almost (t-test) . Interes t-test) movie S2in utero electroporation, when most of the electroporated cells were just migrating toward the pial surface in the PCZ .The L1-KD neurons in the upper CP showed an unusual morphology, although there were no significant differences in the morphology or migration speed in the deeper CP, compared with the control neurons (data not shown). In order to assess this phenomenon, cultured slices were prepared at E15.5, two days after the  the PCZ . Althoug the PCZ , the lea the PCZ . The meaP = 0.0431, unpaired Student's t-test). These observations indicate that the leading process of a L1-KD neuron tends to be longer and more undulated than that of a control neuron. It is likely, therefore, that the process of terminal translocation is affected by L1-KD.In order to analyze the aberrant morphology of the leading processes of the L1-KD neurons, we compared the extent of undulation by the curvature index. The mean curvature index of the leading processes in the L1-KD neurons was 1.073±0.026 (n = 11), whereas that of the leading processes in the control neurons was 1.019±0.006 (n = 13), showing a significantly higher curvature index in the L1-KD neurons  were cultured under time-lapse microscopy for up to 24 hours. The time-lapse observations showed that most of the control neurons demonstrated continuous translocation of the cell body toward the pial surface. The leading processes of the control neurons were straight and shortened in a smooth process along with somal translocation . HoweverP = 0.7870, paired Student's t-test) , and compared those leading processes to neurons 1 h after contact (1 h). In  t-test) . L1-KD n t-test) . In each t-test) , suggestx-axis value was the distance of each somal movement, while the y-axis value was the shortened length (Δ Leading process length) of its leading process, as shown in x-y distributions for a sample population of control and L1-KD neurons in the scatter plots were illustrated in We further evaluated the distance of the somal translocation in the PCZ. The in utero electroporation of L1-shRNA at E13.5.Recently, we demonstrated that L1cam-downregulation perturbed neuronal migration in developing murine cerebral cortex trans and perhaps in the cis, as well as heterophilic binding of L1 to its ligands would be lost in L1-KD neurons, because full-length L1 is deficient in the cell membrane of those neurons. It is thought that these homophilic and heterophilic interactions play critical roles in the developing brain by inducing sustained activation of ERK1/2, leading to an acceleration of cell migration and L1-dependent gene expression Our time-lapse analyses revealed that the locomotion of the L1-KD neurons was significantly decreased in the IZ, which would underlie our previous findings that significantly fewer L1-KD neurons migrated into the cortical plate at E16.5, compared with the control neurons The leading processes of the L1-KD neurons were significantly longer and undulated in the upper CP. It has been reported that integrins, particularly integrin alpha3-beta1 or alpha5-beta1, seemed to mediate the interaction between the tip of the leading process and the substrate, at least in the cerebral cortex in vivo, although the detailed molecular mechanisms remain unknown. We showed that L1-KD neurons can be classified into three groups according to the data on the curvature index and the process length. It is anticipated that these differences might reflect the extent of L1-KD in each cell, and/or the paracrine cue from heterophilic L1-integrin interactions.During the initial phase of terminal translocation, control neurons moved quickly into the PCZ associated with the smooth retraction of the leading process. However, in L1-KD neurons, the soma remained unmoved with a longer leading process. As previously reported Since studies have shown that knock-in mice in which the sixth Ig domain of L1 was deleted (L1-6D) The mechanism whereby abnormal radial migration results in hydrocephalus when L1cam is deficient is still not clear. In L1-6D mice, cerebral cortex showed a decreased number of neurons and a reduced amount of subcortical white matter associated with hydrocephalus. It is tempting to speculate that affected radial migration might subsequently induce cell loss in the cerebral cortex, which lost appropriate connections with subcortical or cortical projections due to the time lag between the migrating L1-KD neurons and the arrival of the incoming fibers. Further studies are required to evaluate this hypothesis.Finally, we propose the schema of a novel role of L1 in cortical migration during development . L1-KD iin utero electroporation of shRNA targeting L1 in murine corticogenesis. Time-lapse analyses revealed that L1-KD neurons showed decreased locomotion velocity and affected terminal translocation with aberrant leading processes and somal movement. Although the underlying molecular mechanisms remain unsolved, L1 plays an important role not only in axonal guidance, but also in neuronal migration during neocortical development.We analyzed the effect of L1-knockdown using Figure S1Neuro2a at 24h post-electroporation. Both shRNA2 and shRNA5 efficiently downregulated L1cam mRNA.(TIF)Click here for additional data file.Figure S2Both shRNAs downregulated L1cam at the protein level in Neuro2a. Western blot analyses revealed that both shRNA2 and shRNA5 efficiently downregulated L1cam protein in Neuro2a.(TIF)Click here for additional data file.Figure S3Cell surface L1cam was efficiently downregulated in Neuro2a. The expression of L1cam was significantly reduced on the cell membrane of Neuro2a cells transfected by shRNA2 or shRNA5.(TIF)Click here for additional data file.Figure S4Radial migration of cortical neurons was disrupted by in utero electroporation of shRNA5 as well as shRNA2.(TIF)Click here for additional data file.Text S1Materials and Methods of supporting experiments and Figure Legends of supporting figures and movies are described.(DOCX)Click here for additional data file.Movie S1This movie shows the time-lapse transition of migrating neurons expressing control-shRNA (shNC) from the intermediate zone (IZ) into the cortical plate (CP). The neurons with a single leading process migrate quickly in the IZ.(AVI)Click here for additional data file.Movie S2This movie shows the time-lapse transition of migrating neurons expressing L1cam-shRNA (shL1) from the intermediate zone (IZ) into the cortical plate (CP). The neurons migrate slowly and the somas often stay in the IZ.(AVI)Click here for additional data file.Movie S3This movie shows the time-lapse transition of migrating neurons expressing control-shRNA (shNC) in the cortical plate (CP). The leading processes of the control neurons are straight and shortened in a smooth process along with somal translocation.(AVI)Click here for additional data file.Movie S4This movie shows the time-lapse transition of migrating neurons expressing L1cam-shRNA (shL1) in the cortical plate (CP). The L1-KD neurons migrate with undulated long leading processes and some of the neurons transiently cease migration.(AVI)Click here for additional data file.


Terrestrial microbial decomposer communities thrive on a wide range of organic matter types that rarely ever meet their elemental demands. In this review we synthesize the current state-of-the-art of microbial adaptations to resource stoichiometry, in order to gain a deeper understanding of the interactions between heterotrophic microbial communities and their chemical environment. The stoichiometric imbalance between microbial communities and their organic substrates generally decreases from wood to leaf litter and further to topsoil and subsoil organic matter. Microbial communities can respond to these imbalances in four ways: first, they adapt their biomass composition toward their resource in a non-homeostatic behavior. Such changes are, however, only moderate, and occur mainly because of changes in microbial community structure and less so due to cellular storage of elements in excess. Second, microbial communities can mobilize resources that meet their elemental demand by producing specific extracellular enzymes, which, in turn, is restricted by the C and N requirement for enzyme production itself. Third, microbes can regulate their element use efficiencies , such that they release elements in excess depending on their demand . Fourth, diazotrophic bacteria and saprotrophic fungi may trigger the input of external N and P to decomposer communities. Theoretical considerations show that adjustments in element use efficiencies may be the most important mechanism by which microbes regulate their biomass stoichiometry. This review summarizes different views on how microbes cope with imbalanced supply of C, N and P, thereby providing a framework for integrating and linking microbial adaptation to resource imbalances to ecosystem scale fluxes across scales and ecosystems. Soil microbial communities are key players in global biogeochemical cycles, regulating core ecosystem processes such as organic matter decomposition, soil C sequestration and nutrient recycling. Microbial decomposers release extracellular enzymes (EEs), which deconstruct plant macromolecules and ultimately liberate soluble substrates for microbial uptake . In turnMicroorganisms can be linked to these ecosystem processes through the theory of ecological stoichiometry, which has emerged as a powerful tool for studying the functioning of both aquatic and terrestrial ecosystems . Soil isOn a global scale, the stoichiometry of soil microbial biomass has been shown to be more constrained in range and variance compared to its resource, which implies that microbes are largely homeostatic in terms of their biomass C:N:P . HomeostFigure 1), including (i) plasticity of microbial biomass C:N:P, (ii) production of EEs as C and nutrient acquisition strategy, (iii) adjustments in microbial element use efficiencies, and (iv) input of external nutrients by N-fixing prokaryotes or by saprotrophic fungi.The key questions regarding stoichiometric imbalances, i.e., how soil microbes regulate their C:N:P homeostasis and how this in turn affects the processing of organic matter, are still insufficiently understood. The aim of this review is to synthesize the current knowledge on the mechanisms that allow terrestrial microbial communities to thrive in a stoichiometrically imbalanced world. First, we review the spatial and temporal variability of resource stoichiometry and its effect on microbial biomass stoichiometry. Second, we present a mechanistic framework of how soil microbial communities cope with resource imbalances , exudates and soil organic matter (SOM). Decomposable plant products  exhibit much wider and more variable C:N:P ratios than microbes [see data compiled in le 1 and ; all C:Nle 1 and , or 3,05le 1 and . Leaf lile 1 and , and evele 1 and . Therefole 1 and . Neverthle 1 and . Woody dTable 1; . Wood ofTable 1; , and troAdding to this complexity, the availability and type of plant necromass varies seasonally e.g., . For insTable 2, dataset of Table 2). Significant differences in soil C:N:P along the SOM decomposition continuum therefore become evident, despite soils being stoichiometrically more constrained than plant detritus. As a result of the spatio-temporal variability in the availability and type of plant detritus and SOM, terrestrial microbial communities must cope with large spatio-temporal variability in resource C:N:P ratios.Soil organic matter, which is the result of microbial decomposition of plant necromass, is another major resource available for heterotrophic microorganisms. Microbial residues play an important role in the formation of SOM, as plant resources are converted to microbial biomass during decomposition, comprising a large proportion of SOM e.g., . ConsequThe range of biomass C, N, and P concentrations in soil microbial communities spans several orders of magnitude, although the linear and isometric relationships between these elements suggest a fixed, or at least a highly constrained, microbial C:N:P ratio . A meta-Table 2, shows that microbial biomass C:N and C:P, but not N:P, are positively correlated with SOC content. This points to indirect control of SOC content on microbial stoichiometry, i.e., soil C:N and C:P ratios increase with SOC content, and microbial C:N and C:P as well. Moreover, this pattern indicates that in soil with high SOC content and high C:N (C:P), N or P may become limiting while C availability is high, thereby increasing microbial biomass C:N:P. On a global scale, microbial C:N:P ratios were not (Table 2), indicating a large degree of stoichiometric homeostasis of soil microbial communities. Table 2), though the relationship between resource C:N and microbial C:N is strongly dampened in relation to the variability in resource elemental ratios. For example, a resource C:N ratio of 10 would predict a microbial biomass C:N of 8.1, but a 10-fold higher resource C:N of 100 would only lead to a 1.3-fold increase in microbial biomass C:N to 10.9.Biome- and ecosystem-level differences in microbial C:N:P ratios may be explained by co-variation with resource stoichiometry, or by soil type, soil pH, soil C content and/or latitude . were not , or onlywere not ; Table 2Table 1). We estimated the average global C:N imbalance – calculated as resource C:N divided by microbial biomass C:N – ranging from 2 (SOM) to values between 10 and 14 , and up to 50 (wood). The C:P imbalances are higher than the C:N imbalances, ranging from 7 (SOM), to 73 (litter), 100 (root litter) and 336 (wood). Smallest imbalances are found for N:P, ranging from 3 in SOM to 7 in plant detritus . These patterns in resource stoichiometry raise the question of how soil microbial communities cope with such large elemental imbalances.As a consequence of the relatively low variation in microbial biomass C:N:P, large C:N:P imbalances arise between different types of decomposable organic matter and decomposer communities : First, microbes can adjust their biomass C:N:P ratios to meet the elemental composition of their substrates; second, they change the elemental composition of their immediate substrates by producing EEs that preferentially deconstruct polymers that meet their demand for C and nutrients; third, after substrate uptake, they mineralize and excrete the elements in excess of their demand by regulating their element use efficiencies; and fourth, diazotrophic bacteria and saprotrophic fungi may trigger the input of external N and P to decomposer communities. Below, we review these four mechanisms in more detail:There are four main mechanisms for microbial decomposers to cope with elemental imbalances and thrive on substrates that do not meet their elemental demand for growth ((1)Non-homeostatic behavior of microbes reduces the stoichiometric imbalance to their resource. As demonstrated above, small adjustments of microbial biomass C:N:P ratios to resource C:N:P do indeed occur, and may help as an adaptive mechanism of microbial communities to stoichiometric imbalances. Adjustment of microbial biomass C:N:P to resource C:N:P ratios can occur due to two main mechanisms: first, microbial storage of elements in excess, leading to a convergence between the biomass and resource stoichiometries, and second, shifts in microbial community structure and concomitant shifts in biomass stoichiometry. Whereas the first mechanism would be a physiological adjustment of the stoichiometry of microorganisms, requiring that microbes are non-homeostatic, the latter is not a true adjustment, as a community change may have very different reasons.Despite the lack of information regarding changes in microbial C, N, or P storage, analyses of cultured organisms have shown that P storage in polyphosphates by bacteria and fungi is related to increased P availability , and C sin situ measurements of single-cell C:N:P ratios in soils by techniques such as X-ray microanalysis or NanoSIMS coupled with phylogenetic classification at broad scale  or fine scale Microbes adjust their EE production, to maximize the mobilization of substrates rich in the limiting element. Microbial regulation of EE production is complex, including constitutive secretion of low levels of EE (most likely to detect suitable substrates), induction of EE synthesis in response to increasing availability of complex substrates, and feedback inhibition of EE activity by their products  . In contIf microbes indeed reduce the elemental imbalance between bulk substrate (soil or litter) and their biomass composition through the release of EE, we would expect that the C:N:P ratio of dissolved organic matter (DOM) is closer to the stoichiometry of microbial biomass than the bulk resource. However, C:N ratios of DOM (DOC:DON) in soil water and leachates are often higher than C:N ratios of SOM and are only weakly, or not significantly, related across sites . Soil miin situ by soil microbes is to infer microbial C:N:P uptake from measured C:N:P ratios of mineralization fluxes, microbial biomass and bulk soil. However, this approach relies strongly on knowledge about microbial element use efficiencies Microbes excrete elements that are present in excess in their resource compared to their biomass composition by adjusting their element use efficiencies. If microbes cannot change the elemental composition of their immediate substrates or adjust their biomass composition accordingly, they could take up whatever substrate is available and release elements in excess of their requirement, while keeping those in short supply for growth. Microbes are able to achieve this balance by regulating their element use efficiencies, such as the carbon use efficiency . Microbial CUE is defined as the ratio of C invested in growth (new biomass production) over total C taken up . Progressively lower C:N and C:P ratios from litter to topsoil, and further to subsoil, correspond also to a decreasing C availability in relation to N and P (increasing C limitation). In order to cope with such differences in resource elemental composition, microbes are expected to adjust their element use efficiencies accordingly. For example, assuming strict homeostasis and negligible adjustment of EE, when microbes with a biomass C:N of 7  decompose plant litter with a relatively high C:N ratio of 70, they must release 63 units of C as CO2 per unit of N invested in growth, yielding a low CUE of about 0.1. By contrast, if SOM with a C:N close to that of the microbial biomass would be decomposed , CUE would converge toward the theoretical maximum of about 0.6 (The considerable differences in C:N:P stoichiometry between plant detritus (litter) and SOM result in distinct elemental limitations for the respective microbial communities , with broad implications for soil C sequestration and N losses from terrestrial ecosystems. This means that microbial homeostasis achieved through adaptations in microbial element use efficiencies is expected to cause a strong positive relationship between resource C:N:P and mineralization flux C:N:P , suggestive of increasing microbial NUE and PUE at high resource C:N and C:P ratios while microbial communities were homeostatic with respect to these element ratios . Moreove(4) Nitrogen-fixing prokaryotes and saprotrophic fungi increase the N and P availability by inputs from external sources. There are two major pathways for input of N or P to the decomposing material from external sources, (i) N fixation by prokaryotes and (ii) fungal transfer of N or P from nutrient-rich patches. Prokaryotic N fixers (diazotrophs) convert atmospheric N2 to ammonia, a highly energy demanding process that is under strict physiological control : (i) plasticity of microbial biomass C:N:P, (ii) compensatory regulation of EE production as C and nutrient acquisition strategy, (iii) adjustments in microbial element use efficiencies and (iv) input of external nutrients by diazotrophic bacteria or saprotrophic fungi. Although the four described mechanisms clearly operate in parallel, their contributions on spatial, temporal and ecosystem scales remain underexplored. The wealth of measurements of microbial biomass C:N:P ratios sets limits to the non-homeostatic behavior of soil microbial communities. Growth of terrestrial decomposer communities on many types of resources with either very wide C:nutrient ratios  or very low C:nutrient ratios  cannot be achieved solely through non-homeostatic behavior. Therefore, adaptation of microbial element use efficiencies and compensatory regulation of EE production are expected to contribute significantly to the adaptation of microorganisms to chemically diverse environments, together with external inputs of nutrients mediated by subgroups of the microbial community.In this review, we demonstrate that terrestrial microbial communities have to cope with a large spatio-temporal variability in resource stoichiometry, which can result in strong imbalances between resource composition and elemental demands by soil microbes. We highlight four major mechanisms that allow microbial communities to adapt to these environmental constraints (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Between 6 and 30% of human and mouse transcripts are initiated from transposable elements. However, the promoters driving such transcriptional activity are mostly unknown. We experimentally characterized an antisense (AS) promoter in mouse L1 retrotransposons for the first time, oriented antiparallel to the coding strand of L1 open reading frame-1. We found that AS transcription is mediated by RNA polymerase II. Rapid amplification of cDNA ends cloning mapped transcription start sites adjacent to the AS promoter. We identified >100 novel fusion transcripts, of which many were conserved across divergent mouse lineages, suggesting conservation of potential functions. To evaluate whether AS L1 transcription could regulate L1 retrotransposition, we replaced portions of native open reading frame-1 in donor elements by synonymously recoded sequences. The resulting L1 elements lacked AS promoter activity and retrotransposed more frequently than endogenous L1s. Overexpression of AS L1 transcripts also reduced L1 retrotransposition. This suppression of retrotransposition was largely independent of Dicer. Our experiments shed new light on how AS fusion transcripts are initiated from endogenous L1 elements across the mouse genome. Such AS transcription can contribute substantially both to natural transcriptional variation and to endogenous regulation of L1 retrotransposition. F, GF and A) reside in the mouse genome  are a major class of mammalian retrotransposons, comprising ∼19 and 21% of the mouse and human genomes, respectively s are a m. Ongoinge genome ,14,15. TMet proto-oncogene  in human and mouse appear to depend on their genomic context, their sequence structure and other factors . Endogenoncogene  and otheoncogene .Like other mammalian TEs, L1s are constrained by various cellular defenses including DNA methylation, histone modifications, Dicer-mediated RNA interference (RNAi) and other small RNA-mediated effects . BidirecDicer ex5 knockout cells were provided by Dr Bert Vogelstein (Johns Hopkins).Mice were maintained and euthanized according to approved Institutional Animal Care and Use Committee protocols . Mouse strains and purified genomic DNA were purchased from the Jackson Laboratory . A mouse spermatocyte cell line (CRL2196) was purchased from the American Type Culture Collection. HeLa cells were provided by Dr John V. Moran (University of Michigan). HCT116 Genomic DNA and pooled total RNAs were isolated from CRL2196 cells and from various tissues, ages and lineages of mice as indicated, using standard methods and Trizol (Invitrogen), respectively.Supplementary Table S1.Primer sequences and annotations are listed in F GenBank accession number AF016099; GF, AC068252; A, AY053456 and FIII, AC002406) and a synthetic L1 element smL1 . Amplicons included fragments of L1 TF (represented by L1spa in pTN201), sense promoter (primers DES1212 and DES1213), AS promoter  and AS ORF2 (DES1459 × DES1460). Promoter candidates were cloned directionally upstream of TEM1, a β-lactamase reporter gene, in pBLAK-b, which lacks a promoter (Invitrogen) . They were confirmed by sequencing. One microgram of BglII-digested (linearized) plasmid DNA was transiently transfected into CRL2196 or HeLa cells using FuGENE 6 (Roche). As positive and negative controls, plasmids with and without the SV40 promoter upstream of TEM1 were used .Genomic DNA fragments representing four mouse L1 subfamilies  (TF GenBent smL1  were amp2 well. Cells were incubated in the dark at room temperature (RT) for 1 h with gentle shaking, washed with HBSS and visualized using an Axiovert 200 M inverted fluorescence microscope (Zeiss) with blue/aqua and β-lactamase ratio filter sets (Chroma Technology Corp.) and ORCA-ER high resolution digital camera (Hamamatsu Photonics) using Openlab software . Flow cytometric analysis was performed using a BD LSR II flow cytometer with a 405 nm violet laser, 440/40 nm (blue) and 530/30 nm (green) filters, and FACSDiva software (BD Biosciences). Ratios of blue to green intensities were collected as a linear parameter. Each flow cytometry session included positive and negative controls to normalize output.To quantify β-lactamase protein expression, cells were stained with CCF2/AM substrate (Invitrogen) ,49 by reTEM1 expression also was quantified by quantitative reverse transcriptase-mediated PCR (qRT-PCR). Promoter candidates were linearized by BglII digestion and transfected into HeLa cells using FuGENE 6. Total RNAs were isolated ∼48 h after transfection using RNeasy kit (Qiagen). Standard curves were based on serial dilutions of control plasmids. First strand cDNAs were synthesized using oligo-d(T) (DES2633) primer and the SuperScript double-stranded cDNA synthesis kit (Invitrogen). As further controls, RNAs were treated with and without reverse transcriptase. qRT-PCR was performed on an iCycler (Bio-Rad) or Step One Plus (Applied Biosystems) instrument, using SYBR Green Supermix (Bio-Rad). TEM1 transcript concentrations were calculated by interpolation, after subtracting for input plasmid DNA contamination. Beta-actin transcript levels were calculated for each sample. Each sample was measured in triplicate. Results are presented for each sample as the normalized ratio of TEM1 to beta-actin transcript levels.Anti-mouse RNA polymerase III subunit RPC39 mouse monoclonal antibody was purchased from Santa Cruz . Anti-mouse RNA polymerase II mouse monoclonal antibody (Cat. 39097) was from Active Motif. For chromatin immunoprecipitation (ChIP), the Magna ChIP G Tissue kit (Millipore) was used following the manufacturer’s instructions.Supplementary Table S1) for first strand cDNA synthesis.We performed 5′ RACE cloning using a second-generation 5′/3′ RACE kit (Roche) with an AS L1 ORF1-specific primer , a representative full-length Ttemplate . BacteriSupplementary Table S1). Resulting PCR products were gel purified and radiolabeled by random nonamer priming. Commercial bacteriophage cDNA libraries from mouse testis (Clontech) and thymus (Stratagene) were plated at ∼50 000 plaques per dish, transferred to Hybond-N filters (Amersham) and hybridized with the ORF2 probe. Filters were washed at 65°C in 0.1 × SSC and 0.1% SDS and autoradiographed. This procedure was repeated with the ORF1 probe to identify ORF1+ORF2− clones, which were purified upon additional rounds of hybridization. Phage plaques were converted to plasmids and sequenced using BigDye v. 3.1 (Applied Biosystems) on a 96 capillary sequencer (Transgenomic Spectrumedix) with primers DES886 and DES837  and standard M13R and M13F oligonucleotides . Additional sequences for full-length cDNA sequencing by primer walking are available on request.The primer pairs used to amplify fragments from L1spa ORF2 and ORF1 were DES1165 × DES1166 and DES 1167 × DES1168, respectively  from DNase-treated total RNAs, using the M13F-anchored primer DES1141 paired with DES1256 for mouse L1spa ORF1, AS nucleotides 2011–1991. PCR products (1–3 kb) were isolated using gel purification columns (Qiagen), and cloned for sequencing. Tissue-specific expression patterns of AS L1 RIFTs and corresponding cognate genes were analyzed by RT-PCR using a commercial multiple-tissue mouse cDNA panel (Clontech).F subfamily) ORF1 as query, i.e. AS nucleotides 2225–1838 . Resulting AS RNA was purified; a second round of first strand cDNA synthesis was performed with reverse transcriptase, dUTP and random primers; cRNA was hydrolyzed using RNaseH, and resulting sense strand DNA was purified. Products were fragmented with uracil DNA glycosylase and apurinic/apyrimidinic (AP)-endonuclease I. Terminal labeling was performed with terminal deoxynucleotidyl transferase (TdT), and resulting labeled fragments were hybridized to the exon microarray.To develop a novel assay to identify L1 RIFTs, we modified the manufacturer’s protocol for the Affymetrix GeneChip mouse exon microarray. First strand cDNA synthesis was performed on total RNA isolated from various tissues and lineages, using a primer including both T7 promoter and oligo-d(T) sequences and SuperScript II reverse transcriptase (Invitrogen). Polyadenylated cDNAs containing AS L1 sequences were amplified using a primer for a particular L1 ORF1 AS template sequence paired with the T7 promoter primer. Resulting double-stranded RIFT cDNAs containing T7 sequences were used as templates for 2 intensity >7) as high expression probes and counted the number of consecutive high expression probes per annotated gene. On alignment with the reference mouse genome, candidate fusion transcripts were scored positive if a neighboring AS L1 could be identified within 30 kb of an overlapping RefSeq gene and/or within 100 kb of the upregulated probe(s). We also required five consecutive high expression probe intensities (corresponding to adjacent exons in a given gene) in exon microarray data; length of predicted initiating genomic L1 integrant had to be >5 kb; and its subtype had to be L1 TF, A, GF or F as per RepeatMasker (www.repeatmasker.org).Resulting raw data from an Affymetrix microarray chip reader were analyzed for transcript expression, using Partek Genomics Suite software. CEL files (MoEx-1_0-st-v1) were imported using RMA background correction and quantile normalization. Probe intensities were transformed to log base 2. We defined signals with intensity > mean + one standard deviation (i.e. ∼logNotI-XhoI fragment from pTN201 (L1spa)  (NotI-HindIII fragment back into the NotI-HindIII backbone (5.4 kb) from pMK20, resulting in ∼11-kb pMK27, i.e. a marked, full-length L1 donor element. This plasmid was Sanger sequenced , revealing a missense mutation in ORF2, i.e. Ala756Ser, along with two noncoding mutations present in pTN201. The missense mutation was repaired by replacement of a ∼1.4-kb EcoRI fragment in pMK27 with the corresponding fragment from pTN201. The 8.1-kb NotI-XhoI fragment of repaired pMK27 was ligated with a ∼11.7-kb NotI-XhoI pTN201 backbone fragment, yielding the desired final plasmid, i.e. pMK28 or L1spa::smL1-ORF1.To compare retrotransposition frequencies of various L1 donors, we started by replacing native L1 ORF1 sequences with a synonymous fragment of smL1 , by movi (L1spa) , into pBnd ORF1) , resultiSupplementary Figure S2). This fragment (Blue Heron Bio), which also included 50-nt flanking arms on both ends for recombineering, was cloned into pUC MinusMCS, resulting in plasmid pJL2. The recoded L1 ORF1 fragment from pJL2 was amplified by PCR using DES3353 × DES3354 and Platinum Taq DNA polymerase High Fidelity (Invitrogen), gel purified (Qiagen), mixed with PstI-linearized pMK20 and co-transformed into electrocompetent DY380 bacteria, bearing the lambda red recombination system for recombineering , using primer pairs DES2880 × DES2881 (L1spa nucleotides 2150–1286); DES2880 × DES2882 (nucleotides 2150–1636) and DES2879 × DES2881 (nucleotides 2823–1286). Resulting PCR products were digested with NotI and BamHI, electrophoresed on agarose gels, purified and ligated to linearized pCEP4 backbone. Fragments from AS synthetic L1 elements similarly were generated using primers DES3818 × DES3820 , DES3819 × DES3820 (nt 2150–1801), DES3818 × DES3821 (nt 2823–1121) and DES3819 × DES3821 (nt 2823–1801). Products were digested with NheI and BamHI and ligated to similarly linearized pCEP4.Various AS L1 transcript overexpression plasmids were engineered from L1 TR) cells then were grown without selection for several days, prior to selection for NeoR L1 integrants in 0.4 mg/ml G418 (Invitrogen) for 2 weeks. In the transient assay , fixed in 2% formaldehyde/0.2% glutaraldehyde in 1 x PBS, washed and stained using 0.4% Giemsa (Sigma) at RT overnight and then counted.HeLa cells were cultured in DMEM with 10% heat-inactivated fetal bovine serum and 2% penicillin/streptomycin (Gibco). Cells at ∼75% confluence in six-well plates or T25 flasks were transfected with plasmid DNA mixed with FuGENE HD (Roche) at a ratio of 1 µg to 3 µl FuGENE. To quantify transfection efficiency, GFP expression was assessed by fluorescence microscopy in cells transfected with pEGFP-N1 (Clontech). Both stable and transient transfection assays were performed. In the former ,23, cellnt assay , NeoR L1in trans, we co-transfected HeLa cells with smL1 donor plasmid together with AS smL1 fragment-expressing constructs. One µg of pCEP4/smL1/Neo donor plasmid DNA was mixed with 1 µg of various smL1 AS fragment-expressing constructs or empty vector pCEP4, respectively, in FuGENE 6. Two µg of vector pCEP4 was used as another negative control. A transient assay (Supplementary Information).To assess effects of AS L1 transcripts overexpressed nt assay  was perfTEM1 reporter constructs, we conducted qRT-PCR. Plasmids were transfected into HeLa cells, and total RNAs were isolated and treated with DNase (Ambion). First strand cDNAs were synthesized using an oligo-d(T) primer. As a control, samples were treated with no reverse transcriptase. L1 expression was measured by SYBR green PCR master mix (Applied Biosystems) using ORF2 primers DES2784 × DES2790 for pTN201/TEM1, pJL3/TEM1 and pMK28/TEM1; and DES1847 × DES1848 for pCEP4/smL1/TEM1. As a control for transfection, Hygromycin gene expression was measured by qRT-PCR using DES1249 × DES1250.In an independent assay of expression levels from various L1-R   reporter cassette and a portion of pCEP4 backbone were excised from pDES46 (which contains human L1.3) by digestion with NotI and BstZ17I. The resulting ∼13-kb backbone fragment was gel-purified. Native, hybrid or fully synthetic mouse L1 constructs in pTN201, pJL3, pMK28 and pCEP4/smL1 were digested by BamHI, ends were filled in by Klenow and digested by NotI. Each of the resulting ∼6.5-kb L1 fragments was ligated with the NotI - BstZ17I fragment. Positive candidates were confirmed by conventional Sanger sequencing. Resulting L1 donor plasmids, marked by the TEM1-AI reporter, included pTN201/TEM1, pJL3/TEM1, pMK28/TEM1 and pCEP4/smL1/TEM1.To assay L1 retrotransposition in Dicer ex5 −/− HCT116 human colorectal cells, which are constitutively NeoR , we usedron (AI) . The TEMTEM1-AI reporter in HCT116 wild-type versus Dicer −/− cell lines, transfectants were selected for 10 d on 400 mcg/ml hygromycin. Expression levels of spliced TEM1 transcripts were assayed by qRT-PCR using DES3062 × DES3063.To compare retrotransposition with native or hybrid L1 donors marked with TEM1 β-lactamase reporter gene otherwise lacking a promoter . The candidates were derived from mouse L1 subfamilies TF, GF, A and FIII; fully synthetic synonymously recoded smL1  .Previously, we and others identified mouse AS L1 RIFTs ,45. Basepromoter . To assa ORFeus) ; and a nF AS nucleotides 2823–2125, mapped as per L1spa coordinates  >> GF ∼ A (∼10% of control) > F (∼5% of control). For these functional promoter assays, we chose particular elements to represent the subfamilies, i.e. L1spa for L1 TF subfamily; L1 GF62 for the GF subfamily and L1Md_A2 for the A subfamily . Within ORF1, these individual surrogates were 99.8, 99.7 and 99.9% identical to the consensus subfamily sequences, respectively . Differences between the subfamily consensus sequences and the individual surrogates were predicted at 1944A>G and 2261G>C ; 1963C>T, 2687T>A, 2716T>C and 2857A>C (L1_GF>L1 GF62); and 2857G>A (L1_A>L1Md_A2).The highest level of AS promoter activity was found in L1Trdinates A. VariouF AS promoter activity was robust, i.e. again, approximately half that of the L1 5′ UTR sense promoter . By contrast, virtually no promoter activity was detected in various fragments derived from the sense (coding) orientation of ORF1, AS ORF2, L1 3′ UTR, smL1 or a novel recoded ORF1 sequence which we designed to contain A/T content comparable with natural L1 sequences  (A qRT-PCR assay for reporter transcript expression confirmed that L1 Tpromoter . Low butgure S1) .Supplementary Figure S2). Comparison of representative L1 subfamily amino acid sequences encoded by ORF1 indicated that the particular portions comprising the AS promoter were more conserved, but still distinct, between subfamilies, compared with the flanking, proximal and distal portions of ORF1 . By contrast, the L1 subfamily sequences within ORF2, which do not contain this AS promoter, were nearly identical (not shown). These results suggested that ORF2 and the AS promoter segment within ORF1 may have undergone strong purifying selection . A recent analysis of the evolution of mouse and human L1s confirmed that the mouse ORF1 coiled-coil domain has undergone much less adaptive change than that of human elements  II and III, either of which plausibly could bind to and initiate fusion transcription from various endogenous TE sequences. As shown in To confirm the role for RNA pol II in transcribing AS L1 RIFTs, we treated a mouse spermatocyte cell line, CRL2196, with α-amanitin B, a poteF subfamily probe for ORF2 . Sequential hybridization with an L1 TF ORF1 probe  were ORF1+.To find mouse transcripts that included sequences from genomic L1 templates, we screened full-length transcripts represented in bacteriophage libraries. Although 6–30% of all mouse and human transcripts recently were reported to be initiated from TEs including L1s , we obseF1 probe  revealed+ORF2- transcripts would include AS L1 RIFTs. This possibility was prompted by our previous identification of fusion transcripts in adult mouse tissues, mapping to L1 elements  contained AS L1 ORF1 sequences spliced with other exons in the sense orientation, forming AS L1 RIFTs. Additionally, 2 of 13 thymus cDNAs  also were spliced AS L1 RIFTs. Other ORF1+ORF2- cDNAs either were unspliced AS RIFTs, reading antiparallel to ORF1 through the 5′ UTR into flanking genomic sequences , or were prematurely polyadenylated, sense-strand transcripts   . Some RI+ORF2- transcripts initiated across the genome in a variety of chromosomal and tissue contexts, as illustrated by schematics of their genomic templates including the initiating L1 elements  (Supplementary Table S2) expressed in cultured mouse spermatocyte cells or adult testes. Twelve (29%) aligned to genomic regions lacking a previously annotated gene, and two (5%) were initiated from polymorphic L1s absent from B6 mice (Supplementary Table S2). Fifty-seven EST clones contained AS L1 sequences in their 5′ ends. Of these, 22 were spliced, but no splicing was observed within L1 sequences per se. Many of the AS L1 RIFTs identified by bioinformatics analysis were found in testis and embryonic cells at certain developmental stages, again suggesting a high level of tissue specificity. This search identified >80 EST clones with AS alignment ≥300 nt and >90% identity with L1 at their 5′ ends, of which 15 were full-length RIKEN cDNAs. In some cases, 3′ paired ends of other EST clones were identified from the EMBL/EBI database using 5′ clone IDs; 57 clones were sequenced from both ends.We identified diverse spliced ORF1gure S3) . To dete and two % were in B6 mice , reveali B6 mice  as splicSupplementary Figure S4). As expected, almost all of these RIFTs were confirmed in testis. Relatively few were expressed in other tissues assayed, but we did recover clones 1ASII1, additionally expressed in 11-day embryos; L1-5AS1-1, additionally expressed in brain; and CRL2196C10, widely expressed in most tissues assayed. We also assayed for overlapping spliced transcripts from cognate genes. Although AS L1 RIFTs that were spliced to downstream exons of Erbb2ip, Usp29 and Arhgap15 each were expressed in testis, the corresponding conventional transcripts of these genes (i.e. lacking sequences from L1s) were not detectable there.To compare RIFT expression levels in different tissues, we re-assayed 17 RIFTs identified initially in adult testis or from a spermatocyte cell line (Supplementary Table S2), of which many were also identified in phage cDNA libraries (Supplementary Table S2). Thus, many transcripts were corroborated by independent methods.To identify genes whose expression levels may be affected by AS L1 RIFTs, we probed Affymetrix mouse exon microarrays conventionally with total RNAs. As commercial exon microarrays typically exclude probes for repetitive elements such as L1 retrotransposons, we also developed a novel, unconventional assay using the arrays to screen specifically for AS L1 RIFTs that include downstream exons. In this assay, hereafter called the RIFT assay, we prepared cDNAs from several tissues and mouse lineages by RT-PCR, using an AS L1-specific primer paired with an oligo-d(T) primer. At least 130 unique spliced AS L1 RIFTs were identified in adult testis . In addition, the 5′ ends of 24 RIKEN cDNA clones, most of which were reported previously  analysis on fusion transcripts expressed in testis, kidney and brain. A primer specific for the L1 Tarby TSS . A largeeviously , mapped eviously .Figure F ORF1 , but it is likely too distant from the RIFTs’ 5′ ends, identified by RACE  of 65 RIFTs, was GATGgtgag , whereas the highest levels were seen for full-length smL1 . Intermediate levels were seen for the novel hybrid element containing recoded ORF1, harboring no AS promoter activity and neutral changes in A/T content, engineered upstream of native L1spa (TF) ORF2. Somewhat higher expression was seen for the second hybrid L1 element, i.e. smL1/L1spa in pMK28, which has lower A/T content in ORF1 . Th. Th13). trans, first we engineered AS smL1 fragments to overexpress them in the desired orientation. Four AS fragments from smL1, corresponding to AS L1spa coordinates 2119–1120, 2800–1120, 2119–1812 and 2800–1812, each were cloned downstream of the CMV promoter and were co-transfected with marked smL1 in a transient retrotransposition assay . As a positive control, where smL1 could mobilize in the absence of overexpressed AS L1 transcripts in trans, empty pCEP4 was co-transfected with smL1. A negative control consisted of cells transfected with no smL1 donor and pCEP4 alone. The overexpression of AS smL1 transcripts in trans significantly suppressed smL1 retrotransposition, i.e. by ∼50–75%  were overexpressed . These AS L1 transcripts overlapped in part with endogenous AS L1 RIFTs .To determine whether overexpressed AS L1 transcripts could inhibit retrotransposition in on assay  . Thus, Dicer played a modest role in suppressing native L1 retrotransposition, mediated by AS L1 transcription; most of the suppression by AS L1 transcripts occurred independent of Dicer. Previous experiments showed a similar ∼2-fold level of suppression of human L1 retrotransposition on knockdown of Dicer in cultured cells. That result was interpreted as showing the role for Dicer-dependent RNA interference in regulating human retrotransposition  RNAs. In turn, these dsRNAs could trigger formation of short interfering RNAs or microRNAs through a Dicer-dependent pathway ,61, therR , we engil intron . Either sertions . The retposition .Supplementary Figure S3 and Supplementary Table S3) (Supplementary Table S2) .A recent analysis of human and mouse transcriptomes suggested that 6–30% of all transcripts are initiated from repetitive elements . Here, wable S3) ,45. AS LThe co-existence of a protein-coding sequence together with an antiparallel promoter activity in opposite overlapping orientations is unusual, but is not unprecedented, in mammalian genomes .i.e. TF, GF and A elements . Several putative transcription factor binding sites in the AS promoter sequence of L1spa . Although members of each subfamily retrotransposed recently , consistent with a complete lack of AS promoter activity observed in those elements . Thus, we concluded that AS L1 promoter activities ranked as L1 TF >> GF ∼ A > F. Notably, the latter subfamilies possessed modest, but detectable, AS promoter activities.The various AS promoter activities associated with each L1 subfamily  were rouF elements range from 0.25 to 1.23 million years old , in this study we have not tested whether sense and AS L1 promoters may be active simultaneously in single cells. If they are not, the resulting unbalanced expression of sense versus AS L1 transcripts in distinct cells or tissues could allow particular L1 elements to evade this putative defense mechanism. Moreover, individual mouse and human L1 elements can mobilize over a wide range of frequencies, despite similar ORF sequences shared by ‘hot’ versus ‘cool’ elements . These included screens of phage cDNA libraries, RT-PCR followed by cloning and sequencing, bioinformatics surveys of transcript sequence databases, Northern blots .The presence of a particular full-length L1 element was necessary, but not sufficient, to initiate a locus-specific AS L1 RIFT. We found that only 13 (19%) of 68 polymorphic full-length L1s initiated AS L1 RIFTs in testis, as assayed by RT-PCR. Moreover, some RIFTs only were expressed in embryonic, newborn or adult mouse testis, whereas smaller numbers were expressed in other organs such as brain and kidney . A few Ain situ   may play several distinct biological roles including possible protein translation. In some cases, the predicted protein-coding ORF sequences of AS L1 RIFTs match the cognate ORF in transcripts from the associated native genes, suggesting that although they may encode identical proteins, their expression patterns may be added to, or modified by, the AS L1 promoter. Other AS L1 RIFTs may modify or replace cognate protein structures or expression, generate novel proteins or long noncoding RNAs  and mobilization on synonymous recoding of the AS L1 promoter in ORF1 in cis and upon overexpression of AS L1 RIFTs in trans . Hybrid L1s, containing either a recoded synonymous ORF1 segment from smL1 with decreased A/T content , the only elements capable of triggering dsRNAs that were tested here. By contrast, Dicer appeared to be a crucial component in RNAi-mediated regulation of L1 expression and mobilization in mouse ES cells . For this reason, we speculate that the RNAi pathway is not likely to be the predominant suppressive mechanism of mouse L1 elements, and that other suppressive mechanisms are involved, at least in the differentiated somatic cells tested here . Thus, we conclude that AS L1 transcripts act mostly independent of Dicer in decreasing L1 expression and retrotransposition.We showed that Dicer played a modest <2-fold role in suppression of endogenous mouse L1 elements ; federal funds from the National Cancer Institute, NIH to SAIC-Frederick [contract N01-CO-12400]; and Ohio State University Comprehensive Cancer Center. Funding for open access charge: Ohio State University Comprehensive Cancer Center.Funded by the Conflict of interest statement. None declared.


ARPES and XAS data of the H2O (NH3)/graphene/Ni(111) system give an information regarding the kind of interaction between the adsorbed molecules and the graphene on Ni(111). The presented experimental data are compared with the results obtained in the framework of the DFT approach.In this article, an angle-resolved photoelectron spectroscopy (ARPES), X-ray absorption spectroscopy (XAS), and density-functional theory (DFT) investigations of water and ammonia adsorption on graphene/Ni(111) are presented. The results of adsorption on graphene/Ni(111) obtained in this study reveal the existence of interface states, originating from the strong hybridization of the graphene π and spin-polarized Ni 3 These bands have a filled shell and, hence, form a deep valence band. The unaffected 2z porbital, which is perpendicular to the planar structure of the graphene layer, can bind covalently with neighboring carbon atoms, leading to the formation of a π band. Since each 2z porbital has one extra electron, the π band is half filled. The π and π* bands touch in a single point at the Fermi energy (EF) at the corner of the hexagonal graphene's Brillouin zone, and close to this so-called Dirac point, the bands display a linear dispersion and form perfect Dirac cones. Thus, undoped graphene is a semimetal ("zero-gap semiconductor"). The linear dispersion of the bands results in quasi-particles with zero mass, namely, the so-called Dirac fermions.Graphene is a single layer of carbon atoms arranged in a honeycomb lattice with two crystallographically equivalent atoms C1 and C2) in its primitive unit cell , and theoretical methods [density-functional theory (DFT) calculations]. XAS and ARPES studies of graphene/Ni(111) reveal the existence of the interface states, originating from the strong hybridization of the graphene π and Ni 3d valence band states with partial charge transfer of the spin-polarized electrons on the graphene π* unoccupied states. This leads to the appearance of induced magnetism in the carbon atoms of the graphene layer as confirmed by X-ray magnetic circular dischroism (XMCD). ARPES and XAS data of the H2O-NH3/graphene/Ni(111) systems enable us to discriminate between different strengths of interactions (physisorption or chemisorption), which appear between the adsorbed molecules and graphene on Ni(111). DFT calculations were used to model different geometries of the adsorbed molecules on top of graphene/Ni(111), and electronic structure calculations were performed for them. The results thus obtained and those of the previous theoretical studies are compared with the present experimental results.In this article, an attempt to modify the electronic structure of graphene via contact of this material with metal (ferromagnetic Ni substrate) and via adsorption of polar molecules (H3H6) according to the procedure described elsewhere , as well as intensity profiles as a function of water deposition time (t) taken at particular binding energies  are shown in the upper and right panels, respectively. The behavior of the water-related photoemission features, I and II, allows us to conclude that island-type growth of water on graphene/Ni(111) takes place: (i) These features start to grow simultaneously at t = 130 s, but slopes of the intensities growth are different; (ii) After t = 170 s, the intensity of feature I decreases via the exponential law, and there is a small plateau for the feature II (first ML is complete); (iii) At t = 230 s, when the thickness of deposited water is more than 2ML, probably, the structural phase transition takes place--formation of ice. Since ice is an insulator, the rapid decrease of the photoemission intensities of the Ni-related features and the shift of some states to higher binding energies can be explained by the formation of an insulating thin film of ice on top of the graphene/Ni(111) system. The delay in starting of the growth of the water-related photoemission features is somewhat puzzling , but this delay could be because some clustering centers on the graphene/Ni(111) surface are necessary to allow water growth process to start. As soon as sufficient numbers of such centers are formed, the process of growth is accelerated. The general trend in the observation of the ammonia-related photoemission features in the similar experiments is the same. In subsequent XAS and ARPES experiments, the thicknesses of water and ammonia layers were chosen to be 1/3-1/2 of the ML (as discussed above with regard to the structure).In order to study the growth modes of water or ammonia, the time sequences of the photoemission maps around the Γ point of the Brillouin zone  were recorded. The extracted photoemission intensity map showing the modification of the valence band at the Γ point of the graphene/Ni(111) system upon adsorption of water molecules (K-edge XAS spectra of (a) graphene/Ni(111) and this system after adsorption of 1/2 of the ML of (b) H2O and (c) NH3, respectively.The effects of the possible orbital mixing of the valence band states of the graphene layer on Ni(111) and orbitals of water and ammonia molecules were studied by XAS Figure . This fis core level to the interface state above the Fermi level , which originates from the C z p- Ni 3d hybridization and corresponds to the antibonding orbital between a carbon atom C-top and an interface Ni atom. The second peak in the XAS spectrum at 287.1 eV of photon energy is due to the dipole transition of an electron from the C 1s core level to the interface state above the Fermi level  which originates from C z p- Ni xp,yp,3d hybridization and corresponds to a bonding orbital between C-top and C-fcc atoms, involving a Ni interface atom. As was found in the experiment, the observed hybridization leads to the orbital mixing of the valence band states of graphene and Ni and to the appearance of the effective magnetic moment of carbon atoms in the graphene layer. This moment was detected in the recent XMCD measurements of this system [B per atom.The XAS spectra of the clean graphene/Ni(111) system Figure  were anas system , which a2O/graphene/Ni(111) and the NH3/graphene/Ni(111) systems measured at the C K absorption threshold are shown in Figure s → π* transitions; the photon energies in the region of 290-320 eV correspond to the C 1s → σ* transitions). In this context, it is worth emphasizing that the presented XAS measurements were recorded at the C K absorption edge and that they reflect (to some extent) the partial density of states of the carbon atoms in the system [2O and NH3 molecules on graphene/Ni(111).The XAS spectra of the He system , and thecenter or in the on-bond position on graphene/Ni(111)  with the lone-pair toward carbon atoms and N-H bonds along C-C bond of the graphene layer.The interpretation of the XAS spectra measured after water or ammonia adsorption can be performed on the basis of the peak-assignment, which has been presented above. For the water adsorbate, the new structure in the XAS spectra appears at the photon energy range corresponding to the hybrid state in the electronic structure of graphene/Ni111) involving both carbon atoms in the unit cell of graphene and interface Ni atom. This leads to the assumption that water molecules are adsorbed either in the 11 involvhν = 75 eV along the Γ-K direction of the Brillouin zone for the graphene/Ni(111), H2O/graphene/Ni(111), and NH3/graphene/Ni(111) systems. In all the series, one can clearly discriminate the dispersions of graphene π- and σ-derived states in the region below 2 eV of the binding energy as well as Ni 3d-derived states near EF. The binding energy difference of ≈2.4 eV for the π states and ≈1 eV for the σ states in the center of the Brillouin zone (in the Γ point) between graphite and graphene on Ni(111) is in good agreement with previously reported experimental and theoretical values [d states. The effect of hybridization between Ni 3d and graphene π states can be clearly demonstrated in the region around the K point of the Brillouin zone: (i) one of the Ni 3d bands at 1.50 eV changes its binding energy by ≈150 meV to larger binding energies when approaching the K point; (ii) a hybridization shoulder is visible in photoemission spectra which disperses from approximately 1.6 eV to the binding energy of the graphene π states at the K point. The full analysis of the electronic band structure and magnetic properties of the graphene/Ni(111) system were performed in Ref. [Figure l values , and it  in Ref. .2O-3a1 and NH3-1e states, respectively. As can be clearly seen from the photoemission spectra, the adsorption of H2O or NH3 on graphene/Ni(111) leaves the electronic structure of graphene π- and Ni 3d-states almost intact. This observation can be taken as an indication of the inertness of the graphene layer on Ni(111) as was earlier demonstrated in Ref. [K point, there is a shift of this band to the higher binding energies of about 50 and 70 meV for the water and ammonia adsorptions, respectively.The adsorption of 1/2 of ML of water and ammonia molecules on graphene/Ni(111) leads to the appearance of the additional photoemission signal in the spectra at 6.5 and 7.3 eV, respectively Figure . In thes in Ref. . There a6R-6 potentials  system as physisorption. From a theoretical point of view, physisorption can be considered as weak interaction arising due to two types of forces, namely, dispersion forces and/or classical electrostatic ones. The dispersion interactions are long-range electron correlation effects, which are not captured in DFT because of the local character of common functionals. Consequently, DFT often fails to describe physisorption correctly. For a correct and consistent treatment of physisorption interaction, it is necessary to use high-level wave-function-based post-Hartree-Fock methods like the Møller-Plesset perturbation theory 1 system . One prog., Ref. ).2O molecule adsorbed on freestanding graphene were performed by O. Leenaerts et al. [Eint) are listed in Table d0).  supercell yield similar values). One can observe very low interaction energies and no energetic preference regarding the adsorption site or orientation of the adsorbate. We have repeated the calculations taking into account the dispersion correction as proposed by Grimme [2O and graphene are significantly shorter. In addition, DOWN orientation is clearly more preferred in this case as compared to the opposite one . It can be noted that the obtained results are in reasonable agreement with the recent CCSD(T) data evaluated for the H2O/graphene system. Thus, for further consideration of the systems of interest, the PBE-D2 approximation will be used.Recently, studies based on first principles for single Hs et al. . For comy Grimme  (DFT-D2 R30° overstructures of adsorbed molecules on graphene/Ni(111) are presented in Table on-top positions have to be considered in these cases. The difference between adsorption behaviors of water on graphene and graphene/Ni(111) indicates the effect of the substrate underneath of the graphene layer, and can be explained by the fact, that in the latter case, the electron charge density is shifted to the interface between the graphene layer and the Ni(111) support. At the same time, similar to the case when free-standing graphene is used as a substrate, for the H2O/graphene/Ni(111) system, DOWN orientation is the energetically most favorable one and the preferable adsorption site is the center of the carbon ring. This theoretical observation confirms our prediction based on the interpretation of XAS spectra. It can be noted that during these calculations, structural optimization of the system was not preformed, and only the distance between graphene and adsorbate is relaxed. Full optimization of H2O geometry in the case of C_DOWN configuration leads to d0 = 2.51 Å, that is a deviation of 2% with respect to the non-relaxed value. The corresponding interaction energy is lower by 4%, than Eint given in Table The results obtained for the (√3 × √3)2O on top of graphene/Ni(111), the band structure calculations were performed. One finds the H2O-related states at the following binding energies: 3.97, 5.96, and 9.85 eV, which satisfactorily match the APRPES data.For the most stable arrangement of H2O/graphene/Ni(111) system, which is also in good agreement with the experimental results, where the modification of the XAS C K spectra was observed. In this context, the UP orientation is preferable for any adsorption position. Although on-top (T_C1) adsorption yields the highest interaction energy, one has to be aware that the present calculations cannot give exact answer regarding the energetically most favorable adsorption position since the obtained interaction energies are very close to each other (within 3%). Geometry optimization can make this difference more pronounced, especially when taking into account stronger interaction between ammonia and the considered substrate.One can see, when looking at data listed in Table 2O and NH3 on top of graphene/Ni(111) have to be considered. Optimization of molecular geometry as well as relaxation of interlayer distances within the substrate has to be performed. Furthermore, parameter-free way of accounting for dispersion corrections is preferable. The latter is possible via van der Waals density functional, developed by Dion et al. [Overall, from a theoretical side, one can see good agreement between the experimental data and the ones obtained by means of DFT calculations. However, further investigations are required before making the final conclusion regarding the position and orientation of the adsorbate with respect to the substrate under study. First, all possible arrangements of Hn et al. .K-edge spectra indicating the orbital mixing of the valence band states of graphene and adsorbates. For the occupied states, the small shifts of the graphene π states were detected in both cases with overall shift of the graphene π states to the lower binding energies reflecting the effect of p-doping  after adsorption of water and ammonia on graphene/Ni(111). Analysis of experimental results leads us to the idea of the site-selective adsorption: water is adsorbed either in the center of carbon ring or on the bond between two carbon atoms; ammonia molecules are adsorbed on the carbon atom, which is located above the Ni interface atom. This assumption is supported by the results obtained via DFT calculations.In conclusion, the authors have studied the modification of the electronic structure of the graphene/Ni(111) system upon adsorption of water and ammonia molecules at low temperature. Adsorption of both types of adsorbates leads to the modifications of the XAS C Ab Initio Simulation Package; XAS: X-ray absorption spectroscopy; XMCD: X-ray magnetic circular dischroism.ARPES: angle-resolved photoelectron spectroscopy; DFT: density-functional theory; PBE: Perdew-Burke-Ernzerhof; VASP: Vienna The authors declare that they have no competing interests.SB, MW and YSD carried out the experiment and perform treatment of experimental data. SB and ENV performed the calculations. YSD conceived of the study, and participated in its design and coordination. KH participated in design and coordination of the experimental part of this study. BP coordinated the theoretical part of this study. YSD and ENV prepared the manuscript initially. All authors read and approved the final manuscript.


Group I and II introns can be considered as molecular parasites that interrupt protein-coding and structural RNA genes in all domains of life. They function as self-splicing ribozymes and thereby limit the phenotypic costs associated with disruption of a host gene while they act as mobile DNA elements to promote their spread within and between genomes. Once considered purely selfish DNA elements, they now seem, in the light of recent work on the molecular mechanisms regulating bacterial and phage group I and II intron dynamics, to show evidence of co-evolution with their hosts. These previously underappreciated relationships serve the co-evolving entities particularly well in times of environmental stress. One of the most intricate relationships in biology is that between a host and a parasite. Almost all organisms studied so far harbor mobile genetic elements and/or their derivatives. At the genomic level, the traditional view of mobile elements is that they provide seemingly little or no benefit to the host while parasitizing the host's cellular machinery to promote element mobility through complex molecular pathways ,2. The hOne group of mobile genetic elements comprises the group I and II introns. These sequences interrupt protein-coding and structural RNA genes in all domains of life and can be considered as molecular parasites. When the gene is transcribed into RNA, the intron sequence acts as a ribozyme (an RNA with enzymatic activity), which removes the intron sequence from the primary RNA transcript, thus limiting the phenotypic cost associated with insertion of the element into a host gene and promoting their maintenance in the genome. In the case of group I and II introns, the host-parasite relationship is enriched by the fact that the introns themselves have been invaded by smaller parasitic elements - genes that encode mobility-promoting activities that enable the DNA element to move within and between genomes . Thus, aGroup I introns commonly inhabit bacterial, organellar, bacteriophage and viral genomes, and the ribosomal RNA genes (rDNA) of eukaryotes, and produce a self-splicing RNA . Group Iin trans to promote splicing of other group I introns in the same genome [ND5 gene of the mushroom coral Discosoma that encodes 15 mitochondrial genes in the P8 loop of the intron [cox1 gene, which is interrupted by another self-splicing group I intron.Many group I introns move by a DNA-based transposition mechanism known as 'homing'. Such introns harbor genes encoding so-called homing endonucleases, site-specific but sequence-tolerant DNA endonucleases that introduce double-strand breaks (DSBs) in cognate alleles that lack the intron, initiating intron mobility via a DSB-repair process  Figure . The oute genome ,18. Intee genome ,20. Groue genome ,22, as ee intron ,24. InteClostridium that contain an IS605-like element inserted at the 3' end of the intron [IS605 machinery. Intriguingly, another unusual clostridial group I intron arrangement was recently found by a bioinformatic search for riboswitches [Some bacterial group I introns have been invaded by mobile elements other than those that encode homing endonucleases. Notable among these are the chimeric intron/insertion sequence (IS) elements (IStrons) of e intron . It is nswitches , RNA strtd intron, the 3' end of an ORF called I-TevI  contributes 20 nucleotides that form part of the P6a, P6.0 and P7 structures that are essential for splicing of the intron [While many ORFs embedded within group I introns are entirely located in loop regions, a surprising number of ORFs extend beyond peripheral loops to contribute nucleotides to more distant regions of the intron that form key structural elements needed for splicing . The exte intron  . In the crystal structure of the I-TevI DNA-binding domain bound to DNA representing either its homing target site or its target operator site, only one of the carboxy-terminal residues (Tyr242) makes a hydrogen bond to the phosphate backbone of the DNA substrate, clearly not providing any specificity to the interaction of I-TevI with its DNA substrate [Importantly, this hypothesis gives rise to a number of testable predictions. First, the length of the 5' or 3' extension should be variable for each independent case of endonuclease invasion, and the position of the initiation or termination codon should be influenced by the GC content of the intron because termination and initiation codons are slightly more AT rich than GC rich and the GC content of the intron will therefore influence the probability of mutation of a sense codon into a nonsense (stop) codon. The second prediction is that the sequence at the 5' or 3' ends of an endonuclease ORF that extends into the intron may not be essential for endonuclease function. In the case of I-TevI, the 20 nucleotides that extend into the ubstrate . BioinfoThe successful spread and retention of mobile introns depends on expression of the mobility-promoting protein from within the intron, and on accurate splicing-out of the introns from the flanking exon sequences. For most group I introns, mobility and splicing are independent of each other, whereas for group II introns, and some organellar group I introns, these processes are not mutually exclusive. In these cases, translation of the IEP from the pre-splicing intron transcript is necessary for splicing because the IEP acts as an RNA maturase, in addition to facilitating mobility (reviewed in ). FurtheOne potential barrier to efficient intron splicing in bacterial and organellar genomes is the coupled nature of transcription and translation, which raises the possibility that ribosomes translating the RNA transcript could encounter the 5' exon-intron junction before the 3' splice site of the intron is transcribed, thus preventing the folding of critical intron structures and recognition of the correct splice sites by the intron. Ironically, a number of studies with bacterial group I introns have shown that translation of the exon upstream of the 5' splice site is necessary for efficient splicing, probably because a ribosome at this position acts as a 'chaperone' to prevent nonproductive interactions between exon and intron sequences that would disrupt the intron-folding pathway -51. MostLactococcus lactis [More complicated types of regulation are implied by numerous examples of ribosome-binding sites in introns that are sequestered by RNA secondary structure -58. Mutas lactis ,61. LtrDidymium [Structured group I introns interrupt the nuclear rDNA of many eukaryotes, in which coupled transcription and translation is not an issue, but they nonetheless face a different set of problems connected with intron-encoded ORFs. The well-studied group I introns in rDNA genes in the slime mold Didymium  contain Didymium . I-DirI Didymium , and hasDidymium . This isAs noted earlier, many proteins encoded within introns in organellar genomes are initially translated as fusions with upstream exon sequences, requiring subsequent proteolytic processing to provide an active protein with an amino terminus in domain IV ,41,66. L2+ is sufficient for efficient folding and splicing. The requirement for host-encoded proteins is especially evident for many organellar group II introns: at least 14 nuclear gene products promote efficient splicing of the two group II introns in the chloroplast-encoded psaA gene of Chlamydomonas reinhardtii [2+ transporter Mrs2p, and the chaperone activity of three DEAD-box proteins, Mss116p, Ded1p and Cyt19p, to promote group II intron splicing in the mitochondria of fungi [Host-encoded proteins function to stimulate the splicing of group I and II introns. In the case of group II introns, host-function-assisted splicing is also crucial for mobility, as the spliced intron RNA is an active intermediate in the mobility pathway . Detailenhardtii ,69. Anotof fungi -74.Escherichia coli where host factors, including the major replicative polymerase Pol III, repair polymerases Pol II, Pol IV and Pol V, the endonuclease RNase H1, and DNA ligase, all function to complete a retromobility event [In terms of mobility, the primary response of a host genome to the presence of mobile elements is repressive, as unregulated mobile element activity will lead to an unbearable mutational load. In recent years, a number of studies have uncovered host proteins that downregulate the activities of mobile introns, many of which (not unexpectedly) are involved in aspects of RNA processing. These proteins include RNase E and RNase I, which negatively regulate group II intron mobility by reducing the steady-state level of intron RNA -77. At tty event . Thus, tUp to this point we have considered the dynamic interplay between introns, their intramolecular inhabitants and their hosts, without considering evolutionary and environmental factors that might influence these partnerships. One traditional view of introns is that they are purely selfish DNA elements, imparting neither benefit nor burden to the host genome in which they reside. Recent evidence, however, has forced a re-evaluation of this viewpoint, particularly in the light of experimental data showing that introns can mobilize in response to stress-induced conditions ,83, as hFor instance, the group I intron endonuclease I-TevI (described in Figure E. coli is not only regulated by RNase E [Group II introns respond to metabolic stress with a burst of retrotransposition to new sites by a mechanism different from that used by group I introns. Retrotransposition of the lactococcal Ll.LtrB group II intron in  RNase E , but is  RNase E . These gStaphylococcus aureus, the ORF orf182 is interrupted by three similar group I introns, and analysis of spliced products revealed that some transcripts lack one exon, suggestive of programmed exon skipping [trans-splicing between highly similar group II introns in organellar genomes also has the potential to generate novel transcripts [Clearly, the mechanisms whereby these variant introns respond to oxidative and nutritional stresses in order to disseminate are different, but with similar outcomes - the 'abandoning of ship' for more hospitable genomic environs. For the group I intron, the mobility machinery itself, the intron endonuclease I-TevI, transduces the signal . Whereasskipping . Similarnscripts . It is tRecent results have challenged our perceptions regarding self-splicing introns, from the notion that they represent simple genomic parasites imparting neither cost nor benefit to the host genome, to that of sophisticated mobile DNA elements fully integrated into host-cell metabolism in ways that could be viewed as molecular mutualism. Host organisms devote considerable resources, whether by design or accident, to both positively and negatively influence intron behavior, and elucidating the molecular basis of host-factor involvement in the regulation of intron splicing and mobility is one area ripe for future investigation. In particular, the mechanism underlying the processing of intron ORFs that are initially translated as fusion proteins with upstream exons in organellar introns represents an obvious gap in our knowledge, but this is a technically daunting problem to address. However, it is questions of an evolutionary slant that will challenge intronologists for years to come. Foremost among these is the possibility that introns could provide some benefit to hosts by generating genetic diversity as a consequence of transposition events brought on by cellular stress.