Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses.Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2′-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Å 2 activation surface area in nsp10, and the resulting complex exhibits RNA cap (nucleoside-2′-O)-methyltransferase activity. We have performed mutational and functional assays to identify key residues involved in catalysis and/or in RNA binding, and in the association of nsp10 to nsp16. We present two additional crystal structures, that of the known inhibitor Sinefungin bound in the SAM binding pocket, and that of a tighter complex made of the mutant nsp10(Y96F) bound to nsp16. Our study provides a basis for antiviral drug design as well as the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses.A novel coronavirus emerged in 2003 and was identified as the etiological agent of the deadly disease called Severe Acute Respiratory Syndrome. This coronavirus replicates and transcribes its giant genome using sixteen non-structural proteins (nsp1-16). Viral RNAs are capped to ensure stability, efficient translation, and evading the innate immunity system of the host cell. The nsp16 protein is a RNA cap modifying enzyme only active in the presence of its activating partner nsp10. We have crystallized the nsp10/16 complex and report its crystal structure at atomic resolution. Nsp10 binds to nsp16 through a ∼930 Å In the eukaryotic cell, the cap is added co-transcriptionally in the nucleus by three sequential enzymatic reactionsMost eukaryotic cellular and viral mRNAs are modified by the addition of a polyadenine tail at the 3′- terminal and a cap structure at the 5′-terminal. The RNA cap protects mRNA from degradation by 5′ exoribonucleases, ensures efficient mRNA translation, and prevents recognition of viral RNA 7MeGMP to the nascent viral 5′-diphosphate mRNAThe viral RNA capping machinery is structurally and mechanistically diverse, and RNA viruses often deviate from the paradigmic eukaryotic mRNA capping scheme. For example, alphaviruses methylate GTP onto the N7-guanine before the presumed attachment of In 2003, a novel coronavirus named Severe Acute Respiratory Syndrome coronavirus SARS-CoV was respSAM-dependent MTases belong to a large class of enzymes present in all life forms. These enzymes catalyze the transfer of the SAM methyl group to a wide spectrum of methyl acceptors, indicating that a common chemical reaction is used on a variable active-site environment able to activate the methyl acceptor atom. Although SAM-dependent MTases share little sequence identity, 2′O-MTases exhibit a KDKE catalytic tetrad and a very conserved folding made of a seven-stranded β-sheet surrounded by one to three helices on each sidePoxviridae, Reoviridae, and Flaviviridae. The Vaccinia virus VP39 crystal structure was the first to be elucidated in 1996Reoviridae provided the first RNA cap MTase structures at 3.6 Å resolution as forming part of the reovirus coreStructural and functional studies of viral MTases involved in RNA capping is an expanding research area, since these enzymes show unexpected diversity relative to their cellular counterparts, and thus constitute attractive antiviral targets. Crystal structures of viral RNA cap MTases exist for only three viral families, namely Regarding (+)RNA viruses, MTase structural information at the atomic level is only available for a single genus. The flavivirus N-terminus domain (residues 1–265) of the NS5 RNA-dependent RNA polymerase harbors an RrmJ fold with an N-terminus extension able to accommodate RNA cap structuresIn 2003, the identification of the 2′-O-MTase signature sequence in the SARS-CoV genome added nsp16 to the list of putative targets for antiviral drugsWe observed that purified nsp16 was unstable in solution, impeding crystallogenesis. Yeast double-hybrid and co-immunoprecipitation experiments on purified SARS-CoV nsp10 and nsp16 have uncovered the reciprocal interaction of these two proteins2+ ions are not directly involved in the nsp10/nsp16 interface  conformation. All the residues involved in SAM/SAH binding are absolutely conserved in coronavirus np16s (Electron density corresponding to one molecule of S-adenosylhomocysteine (SAH), the co-product of the methylation reaction, was identified in the putative SAM-binding site  and 3A. us np16s . Bindingus np16s . The ribus np16s . BindingSoaking the crystals into a Sinefungin-containing buffer captured this MTase inhibitor in the SAH binding site almost perfectly superimposable on SAH . Binding2+. Although the crystallization buffer contains Mg2+, we were unable to locate any such cation in the nsp16 active site. In enzyme activity assays, the Mg2+ ion can be substituted by Mn2+ or Ca2+, but not Zn2+  could neither be co-crystallized with nsp10/nsp16 nor soaked and bound onto preformed nsp10/nsp16 crystals. However, the atomic coordinates of the N7-methyl guanine RNA oligomer in complex with VP39The nsp10/nsp16 complex absolutely requires an N7-methyl guanine capped RNA substrate to exhibit MTase activity2, with nsp10 contributing to 930 Å2 and nsp16 to 890 Å2.All nsp10 secondary structure elements but helices 2 and 5 contact nsp16 . The nspin vitro pull-down assays  abrogates interaction whereas a phenylalanine (Y96F) increases both interaction and MTase activityMutation analysis was also conducted on nsp16 residues presumably involved in the interface and interfacial activation  and S2. 2) able to activate the RNA 2′-hydroxyl at neutral pH. In VP39, K175 has been identified as the general base catalystin line attack through a SN2-like mechanism onto the electrophilic SAM methyl group. The methyl group would pass through a pentavalent intermediate with the 2′-O and sulfur at apical positions. D130 is positioned to stabilize the transient positive charge on the donated methyl atom of SAM before the sulfur recovers a neutral electric charge during SAH generation -MTase. The complex is assembled through a ∼890 Åbona fide viral RNA cap is key in evading the host cell innate immunityOur mutagenesis analysis shows that the formation of an nsp10/nsp16 complex is a pre-requisite for MTase activity  indicatii.e., involving mainly helices αA, αZ and strands β2 and β3 as depicted in MTase activation through dimerisation of two viral protein partners has already been reported in the case of the vaccinia virus D1/D12 N7-guanine MTaseWe have previously shown that the nsp10/nsp16 is only active as N7-guanine methylated capped RNA, implying that RNA cap methylation obeys to an ordered sequence of events where nsp14-mediated N7-guanine methylation precedes nsp10/nsp16 RNA 2′-O methylation50 of 0.74 µM, 16-fold lower than that of SAH as reported by Bouvet et al.Viral MTases are increasingly evaluated as potential drug design targetsIn conclusion, the crystal structures presented here extend our general understanding of the mechanism and regulation of viral RNA cap MTases in (+)RNA viruses, and point to both the nsp10/nsp16 interface and the substrate binding sites as putative antiviral targets.2 hexahydrate. Crystals  contain one nsp10/nsp16 complex per asymmetric unit, with a solvent content of 70 % and Vm of 4.17 Å3/Da. Crystals of nsp10(Y96F)/nsp16 were grown in 67 mM CHES pH 8.5, 0.99 M MgCl2 hexahydrate, 33 mM Tris-HCl, 8.3 % PEG 8000. Both crystallization conditions yielded crystals diffracting to 1.9 Å when exposed to synchroton radiation at the ID14-1 beamline of the European Synchrotron Radiation Facility, Grenoble, France. Crystals were cryo-cooled in the same buffer supplemented with 15 % glycerol. Crystal soaking was performed in the same buffer supplemented with 5 mM SAH or Sinefungin during 24 h.Both nsp10 and nsp16 were expressed from the same dual expression vector pmCOX The position of the nsp10 protein was unambiguously determined by molecular replacement using the program PHASER2. Conformational differences were analyzed using the DynDom server (http://www.cmp.uea.ac.uk/dyndom/main.jsp). Figures were created using PYMOL (http://www.pymol.org). The coordinates of the wild-type/SAH, mutant, and wild-type/Sinefungin structures have been deposited at the Protein Data bank under PDB codes 2XYQ, 2XYV, and 2XYR, respectively.Overall, the chain traces are unambiguous, with clear electron density including for a single SAH residue bound to the nsp16 protein. Solvent accessible surfaces were calculated using program AREAIMOL bona fide modeling. In order to fit the RNA molecule in the cavity, an alternative conformation was sought for this side chain. The second most common conformation for the tyrosine side chain was selected. Due to the biochemistry data and surface electrostatic analysis, it is not possible to describe with certainty the final position of the cap, thus the cap was removed and replaced by arrows symbolizing possible positions. No other modification was performed on the RNA, the Sinefungin molecule or the nsp16 structure.The modeling of the RNA cap structure in the nsp10/nsp16 complex structure is derived from the analysis of the structure of the vaccinia virus methyltransferase VP39 crystallized in complex with a capped RNA and a S-AdenosylhomocysteineThe SARS-CoV nsp10 and nsp16-coding sequences were amplified by RT-PCR from the genome of SARS-CoV Frankfurt-1 (accession number AY291315) as previously described7MeGpppA were purchased from New England BioLabs, the[3H]-AdoMet was purchased from Perkin Elmer and Sinefungin (adenosylornithine) from Sigma-Aldrich.AdoMet and cap analogs GpppA and E. coli C41 (DE3) cells , containing the pLysS plasmid (Novagen), were transformed with nsp10 or nsp16 cloned in pDest14, or nsp10/nsp16 cloned in pmCox, and grown in 2YT medium supplemented with appropriate antibiotics. The expression of strep-tagged nsp10 or 6His-tagged nsp16 mutants was induced (DO600 = 0.6) by adding 50 µM IPTG, and the expression of the nsp10/nsp16 complex by adding 50 µM IPTG and 200 µg/L of anhydrotetracycline. After an incubation for a 16 h at 24°C, the cell were pellets, frozen and resuspended in lysis buffer  supplemented with 1 mM PMSF, 40 mM imidazole, 10 µg/ml DNase I, and 0.5% Triton X-100. After sonication and clarification, proteins were purified either by IMAC  chromatography2, 2 µM 7MeGpppAC5 or GpppAC5, 10 µM AdoMet, and 0.03 µCi/µl [3H]AdoMet . Short capped RNAs  as previously described3H-CH3 transferred onto 7MeGpppAC5 substrates was determined by filter binding assay as previously describedMTase activity assays were performed in 40 mM Tris-HCl, pH 8.0, 5 mM DTT, 1 mM MgClFigure S1http://www.ebi.ac.uk/Tools/msa/muscle/), and the resulting alignment converted using the ESPript program, (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Residues that are conserved in all or >70% sequences are boxed in red and yellow, respectively. National Center for Biotechnology Information (NCBI) accession numbers for replicase polyprotein sequences that include nsp16 are as follows: Severe acute respiratory syndrome virus SARS-CoV Frankfurt isolate (SARF), Severe acute respiratory syndrome virus SARS-CoV Tor2 isolate (SART), NP_828873.2; Turkey coronavirus (Turk), YP_001941189; Infectious Bronchitis Virus (IBV), NP_066134; Feline Coronavirus (Feli), YP_239426;Porcine Transmissible Gastroenteritis Coronavirus (PTGC), P18457; Transmissible Gastroenteritis coronavirus (TGEV), NP_840013; Porcine epidemic diarrhea virus CV777 (PEDV), NP_839969; Bat coronavirus 512/2005 (BATC), YP_001351683; Human coronavirus NL63 (NL63), AAS58176; Human coronavirus 229E (229E), NP_073549; Murine hepatitis virus strain JHM (MurJ), YP_209243; Mouse Hepatitis virus strain A59 (MA59), NP_740613; Human coronavirus HKU1 genotype A (HKU1), YP_460023; Human enteric coronavirus 4408 (4408), ACJ35483; Bovine coronavirus (BCoV), NP_742142; Human coronavirus OC43 (OC43), AAT84359. Numbering was made using SARF as a reference.Sequence alignment and amino acid conservation in coronavirus nsp16. The alignment of coronavirus nsp16 sequences was generated with Muscle program ((TIF)Click here for additional data file.Figure S2www.ebi.ac.uk/thornton-srv/software/LIGPLOT/) of the SAH ligand molecule interacting with the nsp16 binding site. Ligand bonds are in purple, neighbor residue (non-ligand) bonds are in light brown, hydrogen bonds are green dashed lines. Ligand atoms surrounded by a yellow circle are highly accessible. Non-ligand residues in hydrophobic contacts with the ligand are presented by red semi-circles with radiating spokes. B) Superimposition of SAH and Sinefungin molecules. The nsp16 residues are green sticks, the SAH is represented in red sticks, and Sinefungin and water molecules are in sticks and spheres as in Binding determinants in the SAM/SAH/Sinfungin binding site. A) LIGPLOT diagram ((TIF)Click here for additional data file.Figure S3Primary and secondary structure elements of nsp16. Helices and sheets are colored according to rainbow colors from N- to C-terminus as in (TIF)Click here for additional data file.Figure S4Nsp10 interface comparison between the nsp10/nsp16 complex and nsp10 homo multimer. A) For the corresponding nsp10/nsp16 interface nsp10 (cyan) interacts with 2 monomers (green and orange). Left: Cartoon representation of 3 nsp10 monomer part of the dodecamer structure (PDB: 2G9T). Right: Cartoon representation of the nsp10(cyan)-nsp16(dark red) complex. Nsp10 molecules in cyan are in the same orientation. B) Nsp10 sequence is presented with above the corresponding secondary structure elements. Below dark red colored dots indicate residues involved in the interaction between nsp10 and nsp16 while green and orange dots indicate residues involved in the homomultimer in the dodecamer complex.(TIF)Click here for additional data file.Table S1http://www.ebi.ac.uk/msd-srv/prot_int/pi_link.html). Bold on grey background: strictly conserved residues amongst coronaviruses; Bold on white background: conserved a.a. (>70%) amongst coronaviruses Click here for additional data file.Table S2http://www.ebi.ac.uk/msd-srv/prot_int/pi_link.html). Same legend as Effect of alanine mutations in nsp16 interface residues. Residues were identified using PISA ((DOC)Click here for additional data file.


To further characterize this phenomenon, low speed extracts (LSE) from axolotl and Xenopus UFE were prepared and tested in an RNA polymerization assay. The major conclusions are: i) the amphibian extracts catalyze the incorporation of radioactive ribonucleotide in RNase but not DNase sensitive products showing that these products correspond to RNA; ii) the phenomenon is resistant to α-amanitin, an inhibitor of RNA polymerases II and III and to cordycepin (3′dAMP), but sensitive to cordycepin 5′-triphosphate, an RNA elongation inhibitor, which supports the existence of an RNA polymerase activity different from polymerases II and III; the detection of radiolabelled RNA comigrating at the same length as the exogenous transcript added to the extracts allowed us to show that iii) the RNA polymerization is not a 3′ end labelling and that iv) the radiolabelled RNA is single rather than double stranded. In vitro cell-free systems derived from amphibian UFE therefore validate our previous in vivo results hypothesizing the existence of an evolutionary conserved enzymatic activity with the properties of an RNA dependent RNA polymerase (RdRp).We have previously reported a post-transcriptional RNA amplification observed  Neurospora crassain vitro and biochemical and genetic experiments have established that RdRp plays a critical role in amplifying the RNAi effect Drosophila and vertebrates. However, post-transcriptional suppression of gene expression by siRNA was established in oocytes and preimplantation embryos of mice Xenopus embryos Drosophila RNAi where repeated cycles of dsRNA synthesis and concomitant siRNA/primer production result in mRNA degradation DrosophilaPost-transcriptional regulations modulating mRNA stabilities represent important mechanistic steps in the regulation of eukaryotic gene activity. Indeed, it is clearly assumed that half of all changes in the amounts of mRNA is attributed to altered rates of decay in vivo heterologous system to study RNA stability at the post-transcriptional level, we previously reported a coupling between RNA amplification and accelerated degradation of in vitro synthesized Xenopus transcripts injected into axolotl oocytes, fertilized or unfertilized (UFE) eggs in vivoin vivo after injection of axolotl RNA into Xenopus fertilized eggs, suggesting a phylogenetically conserved mechanism.Using an in vivo post-transcriptional process, we intended to use cell-free extracts. In the present study, we develop for the first time low speed extracts (LSE) from axolotl UFE, according to a protocol that minimizes dilution as previously published for Xenopus laevisXenopus UFE have provided the first non viral cell-free system able to faithfully reproduce nuclear synthesis events, from the replication of DNA to the assembly and reconstitution of a functional nucleus Xenopus extracts in an RNA polymerization assay. We show that the amphibian cell-free systems incorporate radioactive ribonucleotide precursors into discrete RNA polymers, indicating that axolotl and Xenopus extracts contain all the components necessary to polymerize RNA. When large amounts of an exogenous RNA are added to the extracts, the labelling of a single RNA band similar in size to that of the exogenous RNA is detected. This radioactive labelling does not depend on the free 3′OH extremity of the added RNA and RNA polymerization is not due to terminal labelling of the 3′ end of the RNA. Analysis of the radiolabelled RNA sensitivity to RNase A shows that the RNA produced is single stranded. These results support the hypothesis of an RNA dependent RNA neosynthesis in the axolotl and Xenopus mitotic extracts.In order to further investigate the molecular mechanism of this The protocol of animal handling and treatment was performed in accordance with the guidelines of the animal ethics committee of the Ministère de l′Agriculture of France.4, 0.5 mM Na2HPO4, 2 mM NaHCO3) at room temperature. An axolotl female lays between 800 to 1000 eggs (2.3 mm in diameter). Laid eggs were collected, quickly rinsed with 0.2X HSB brought to pH 8 by addition of NaOH 10N and completely dejellied by gently swirling at room temperature in 3% L-cysteine-hydrochloride, 0.1% papain in 0.2X HSB pH 8. Dejellied eggs were rinsed at least 5 times in 100 mL 0.2X HSB, EGTA 1 mM pH 7.8 per mL of eggs (EGTA : Ethylene Glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), sorted under a dissecting microscope to remove all damaged or abnormal eggs and eggs were immediately used for preparation of cell-free extracts. Xenopus UFE were obtained as previously described Axolotl UFE were obtained from females maintained at 18°C in our laboratory, stimulated 24 h earlier by subcutaneous injection (700 UI) of human chorionic gonadotrophin  and kept overnight in 1X HSB . At this point, Cytochalasin B was added (1 µL at 10 mg/mL per mL of supernatant). After gentle mixing, the clear extracts obtained were stored at −80°C in 50 µL aliquots rapidly frozen in liquid nitrogen. One milliliter of extract was prepared from 500 axolotl UFE. Xenopus low speed extracts (LSE) were prepared as previously described Xenopus LSE, the final concentration of non-yolk proteins was determined by Bradford dosage according to Maniatis and Sambrook Xenopus UFE. These values (25 µg) are in perfect accordance with those found for non yolk proteins in mature Xenopus stade VI oocytes 260) and estimated to respectively 1 and 1.5 µg per one equivalent UFE in axolotl and Xenopus extract.Axolotl eggs were first incubated during 5 min in 25 mL cold 1X CSF, 5 mM EGTA buffer extract  then transferred 5 min in 25 mL of 1X CSF, 5 mM EGTA buffer extract containing 10 µg/mL of each protease inhibitors . UFE were then transferred in 2.2 mL eppendorfs previously cooled on ice. Excess buffer was removed, leaving only interstitial buffer between packed eggs. After centrifugation at 20 000 g during 50 min (16°C), the cytosolic supernatant was collected, excluding the lipid overlayer, the yolk pellet and the particulate grey interface. The supernatant was collected and Cytochalasin B  was added to a final concentration of 10 µg/mL. All subsequent manipulations were carried out at 4°C. Cytosolic supernatant was cleared by centrifugation at 20 000 g during 50 min and the soluble phase was collected by puncturing the side of the tube with a 20-gauge needle inserted into a 1 to 5 mL syringue then stored on ice. Protease inhibitors at final concentration of 10 mg/mL each, Energy Mix (1/20 volume) and 5% glycerol were added . The Xenopus pGbORFA65 construct was used to synthesize globin RNA −  and poly(A)+  sense RNA were obtained by linearization of the plasmid respectively with BamHI or EcoRI (Biolabs) then transcription from T7 promoter of the Bluescript vector. C-myc Xenopus RNA was produced in vitro using T7 RNA polymerase and the template pXLmyc 260). The ≪standard≫ reaction for addition of cordycepin (3′-deoxy AMP) at the 3′OH ends of in vitro transcripts was done according to the poly(A) tailing kit procedure from Ambion (Ref : AM1350) except that cordycepin 5′-triphosphate was used instead of ATP. Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor , 5X poly(A) polymerase buffer, 2.5 mM MnCl2, 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C. To check the inability of these 3′OH-modified RNAs to be elongated by any radiolabelled ribonucleotide, 100 µg of in vitro Xenopus globin or c-myc transcripts modified at their 3′OH-end (globin 3′H or myc 3′H) or not (globin 3′OH or myc 3′OH) were incubated in a standard tailing reaction (Ambion) containing 1 mM ATP, RNase Inhibitor (30 U), [α–32P] rATP  and E. Coli poly(A) polymerase (12 U) in a final 200 µL reaction during 30 min at 22°C. The size and quality of the different DNA or RNA templates were respectively controlled on a native agarose gel (DNA) or on a formaldehyde gel (RNA) then by electrophoretic migration. RNase A (DNase and protease free) was purchased from Boehringer Mannheim, DNase RQ1 from Promega, α-amanitin, cordycepin and cordycepin 5′-triphosphate from Sigma and RiboRuler RNA ladders from Fermentas.All the plasmids used as templates in DNA polymerization experiments (single-stranded or double-stranded (7246 base pairs) M13mp18 circular plasmid and double-stranded (2464 base pairs) circular pSP73 plasmid DNA) were purchased at Biolabs as RNase-free grade products and resuspended in the manufacturer's buffer Tris-HCl 10 mM pH 7.5, EDTA 1 mM. Preparation of in vitro reaction for a DNA polymerization assay was as follows. Aliquots of frozen extracts were thawed at room temperature immediately before use. To 44 µL of egg extract, 2.5 µL of the 20X ATP-regenerating system (Energy mix) were added to give a final concentration (1X) of 10 mM phosphocreatine (Sigma), 10 mg/mL creatine phosphokinase (Sigma), 1 mM ATP and 1 mM MgCl2. Samples were then supplemented with 1 µL of DNA  and 0.5 µL of cycloheximide  or water to obtain a final volume of 50 µL. For a ≪standard≫ in vitro RNA polymerization reaction, 2.5 µL of the 20X ATP-regenerating Energy mix system were added to 42 µL of egg extract supplemented with 1 µL RNasin inhibitor , 2 µL of RNA in vitro template (500 ng), 1 µL  of [α–32P] CTP or [α–32P] UTP and 2 µL containing drugs at the desired concentrations  to obtain a final volume of 50 µL. For all the reactions, immediately after gentle homogenization, the addition of the radiolabelled precursor defined the t = 0 of the kinetics performed at 20°C, unless otherwise mentionned. At t = 0, five microlitters  was added to 45 µL of ≪Stop mix≫ solution  then spotted on a Whatmann GF/C glass filter to determine the total radioactivity of the extract (input). At each time of the kinetics, 1/10 of the reaction was withdrawn and added to 45 µL of ≪Stop mix≫ solution. At the end of the kinetics, each ≪Stop mix≫ solution containing the samples was incubated with 2 µL of Proteinase K  during 2 hr at 55°C, then the totality of the reaction was directly spotted on a Whatmann GF/C glass filter. Then filters were TCA (Trichloroacetic Acid) precipitated first at least 3 hr in two batches of cold 5% TCA, 2% PPNA solution (PPNA : tetrasodium diphosphate) (20 mL per filter) and three times two hours in cold 5% TCA then washed twice in ethanol 95%, dried and counting of the incorporated TCA precipitable radioactivity was done in 5 mL of Liquifluor (Amersham). For RNA polymerization assays, an RNase A treatment (Fermentas) was added to the standard procedure : 5 µL of the reaction were incubated with 1 µL of RNase A (10 µg) and water (14 µL) during 1 hr at 37°C.A ≪standard≫ + membrane , the filters were washed twice in 2X saline sodium citrate (SSC), pre-hybridized at 65°C in Church buffer For DNA agarose gel analyses, after the polymerization assays using incubations with the amphibian extracts, some samples were treated during 1 hr at 55°C with Proteinase K (10 U/assay), then phenol/chloroform and chloroform (V/V) treated before ethanol precipitation and totally loaded in an 1% agarose native gel before electrophoresis in TEA 1X buffer as described by Maniatis and Sambrook in vitro transcripts (5 or 10 µg each) were heat-denatured 10 min at 85°C in 80% deionized formamide, 40 mM PIPES pH 6.7, 0.4 M NaCl, 1 mM EDTA . Rehybridization was performed overnight at 65°C. Then the RNA solutions as well as 10X RNase A digestion buffer  were diluted 10 fold in a common mix to proceed to RNase A (Fermentas) digestion for 1 hr at 37°C . Digestions were stopped by incubation with proteinase K (2 mg mL−1), 8% SDS during 15 min at 37°C. After phenol/chloroform extraction, RNAs were ethanol precipitated in the presence of glycogen  and dissolved in 80% deionized formamide, 50 mM Tris-HCl pH 8.3, 1 mM EDTA, 1% bromophenol blue, 0.1% xylene cyanol. RNAs (corresponding to respectively 1/8 of the RNA polymerization sample or 1/5 of the RNA combinations) were electrophorezed on 8 M-urea 4% polyacrylamide gel for 75 or 30 min at 150 Volts in TBE buffer .Either total RNA extracted from an RNA polymerization sample using axolotl LSE (48 µl) and an exogenous synthetic transcript (24 µg) or two combinations (10 µg) of in vitro system according to procedures used for Xenopus LSE Xenopus sperm nuclei containing 100 ng DNA were incubated with axolotl or Xenopus extracts in reactions using [α–32P] dCTP as well as Ca++ addition or not. As expected, [32P] dCMP incorporation into TCA precipitable material was Ca++ dependent in Xenopus LSE . The use of the same extract in 7 independent experiments . These patterns of discrete bands detected in independent experiments performed from different axolotl and Xenopus extracts were not always as clearly observed as in i.e. RNA molecules, and indicate that the RNA polymerization is catalyzed by an RNA polymerase activity different from RNA pol II and pol III.Aliquots of a standard RNA polymerisation reaction without any exogenous RNA were processed at t = 0, t = 1 hr and t = 4 hr by LiCl method then analyzed by agarose gel electrophoresis and autoradiography. As shown in a representative experiment , low sigin vitro synthesized Xenopus 3′UTR poly(A)− globin RNA (555 bases). This amount of exogenous RNA was equivalent to 1/4 of the total RNA amount present in an aliquot of 44 µL axolotl extract (35–40 µg). Following a 4 hr incubation, the reaction sample was extracted by LiCl method, then analyzed by ethidium bromide staining/UV illumination and autoradiography. As shown in Interestingly, our preceeding experiment showed that the radiolabelled products were clearly resolved into few discrete bands. One of them corresponding to the 1.8–1.9 kb band comigrates with the 18S rRNA which represents an important endogenous amount of RNA in the extract. This observation led us to study the impact of the addition of a large amount of an exogenous RNA during RNA polymerization with the axolotl extract. Since no radiolabelled bands were detectable with sizes between 2 and 2.7 kb or smaller than 0.8 kb, we choose to design a 0.55 kb and 0.62 kb  as well Xenopus globin 3′UTR transcript containing a 65 adenosines poly(A) tail at its 3′ end (620 bases) was added either to the Xenopus  an RNA elongation inhibitor, but remained detectable after co-incubations of the transcript with cordycepin  or α-amanitin used at an RNA pol II and pol III inhibiting concentration . This supports the involvement of cellular enzymes other than RNA pol II and III activities catalyzing the ribonucleotide triphosphate dependent step during precursor incorporation into the radiolabelled RNA polymers.Similar results were obtained when 10 µg of the In summary, addition of large amount of exogenous RNA allows to observe a radiolabelled RNA with the same size as the added RNA. This RNA synthesis appears to occur to the detriment of the RNAs synthesized when no exogenous synthetic transcript is added.in vitro transcript and we checked that the resulting RNA with a 3′desoxy modified end (3′H end) was unable to incorporate [α–32P] ATP in a classical in vitro polyadenylation reaction. Synthetic globin and c-myc RNA, with 3′OH or 3′H ends, were incubated in vitro in the presence of [α–32P] ATP and poly(A) polymerase and further analyzed by electrophoretic migration and autoradiography. As shown in The preceeding observations were compatible with two hypothesis : elongation of preexisting RNAs from their 3′ ends or neosynthesis of RNA. Since the elongation hypothesis depends on the presence of RNA exhibiting a free 3′OH extremity and since the RNA labelling observed could be provoked by the addition of exogenous RNA, we decided to use exogenous RNA but lacking a free 3′OH extremity. Therefore, a 3′deoxyribonucleotide (cordycepin 5′-triphosphate) was added to the 3′OH extremity of the Xenopus synthetic globin (0.62 kb) and c-myc (2.2 kb) RNA would result in identical labelling intensities of the 0.62 kb and 2.2 kb radiolabelled signals (ratio = 1) whatever the 3′ ends of the transcripts. However, since an equal amount of each Xenopus synthetic RNA corresponds to about 4 fold more globin 0.62 kb transcript as compared to the c-myc 2.2 kb RNA (i.e. 4 fold more 3′ globin extremities as compared to 3′ myc extremities), the elongation hypothesis would result in a radiolabelling intensity of the 0.62 kb radioactive signal 4 fold higher than that of the 2.2 kb signal (ratio = 4) if both transcripts harbored 3′OH ends. We first tested the co-incubation of 13 µg of each unmodified Xenopus RNA in the axolotl extract in the presence of the radioactive precursor (CTP). As shown in Xenopus extract . Therefore, these results confirm that the RNA synthesis is not due to a template independent terminal labelling at the 3′OH-end of the exogenous RNA.We then compared the intensity of the radioactive labelling detected in axolotl polymerization reactions containing different combinations of exogenous transcripts with 3′OH or 3′H ends. We reasonned that in the neosynthesis hypothesis, an equal amount of each To further investigate the nature of radiolabelled RNAs, we wished to know if the detected RNAs could find a complementary strand in axolotl LSE. We used an experimental approach inspired by classical RNase protection protocols to allow RNA duplex hybridization within the total RNA pool extracted following a 3 hr RNA polymerization reaction. Moreover, we chose to use a large amount of an exogenous synthetic RNA during the RNA polymerization reaction since this favors the synthesis of a radiolabelled RNA at the same size as the exogenous RNA see . To overXenopus LSE as controls. Axolotl LSE is unable to replicate DNA from demembranated Xenopus sperm nuclei whether Ca++ was added to the incubation mix or not. In a Xenopus LSE, incubation of demembranated sperm nuclei leads to the loading of prereplication complexes on DNA and Ca++ addition allows the Cdk (cell-cycle dependent kinase) dependent initiation of DNA replication through activation of the licensing factor ++ addition : both the levels of the phosphorylated forms of MAP kinases and Cdk1 (on tyrosine 15) that accumulate in axolotl UFE ++ addition (data not shown), although they are modulated when intact axolotl UFE are activated It is the first time that low speed extracts (LSE) from unfertilized eggs are performed in the urodele axolotl. To characterize the replicating properties of these extracts, classical DNA templates were incubated in polymerization reactions using axolotl LSE and parallel incubations with Considering the molecular requirements for DNA dependent DNA polymerization, it was anticipated that SS M13 DNA would allow DNA polymerization driven by DNA primase and DNA polymerase activities of axolotl LSE. When 150 ng of SS M13 DNA are added to the axolotl extract, the SS M13 DNA is fully converted in DS DNA . These rXenopus extracts are intrinsically able to synthesize RNA since addition of a radiolabelled RNA precursor to the extracts allow registration of TCA precipitable cpm that i) are sensitive to 5′-triphosphate cordycepin, an RNA chain elongation inhibitor and that ii) totally disappear following RNase treatment. Previous works showing RNA synthesis in amphibian extracts indeed study transcription and its regulation, i.e. DNA dependent RNA synthesis in vitro assay.Our results show for the first time that axolotl and Xenopus extracts are well known to promote transcriptional repression due to the large store of histones and the resulting assembly of DNA into chromatin If The detection of radiolabelled RNA comigrating with synthetic or endogenous transcripts present at high concentrations in the amphibian extracts raised the possibility that the RNA synthesis corresponded to the template independent labelling of primer-RNAs at their 3′-ends. This could be due to various enzymatic activities : the first poly(A) polymerase activity identified in vertebrate cells Overall our results therefore suggest that the RNA synthesis does not depend neither on DNA-dependent RNA polymerization activities nor on template-independent 3′OH-end long-sized-RNA extension activities. The RNA labelling could therefore correspond to an RNA template dependent RNA synthesis.S. pombe or C. elegans RNA dependent RNA polymerases (RdRp)  of a standard reaction (50 µL) using [α−32P] CTP were withdrawn throughout kinetics ranging from t = 0 to t = 180 minutes (min), processed for [α−32P] CMP incorporation into acid-insoluble material by TCA precipitation and the [α−32P] CMP incorporated was expressed as counts per minute (cpm) for 106 cpm used in the reaction . B. Incorporation of [α−32P] CMP in four independent experiments using the same Xenopus mitotic LSE. Results obtained using the same LSE during 4 hr incubation independent experiments (1 to 4) are shown as histograms (white bars) where [α−32P] CMP incorporated into TCA precipitable material was expressed as counts per minute (cpm) for 106 cpm used in the reaction . Hatched bars correspond to samples incubated without exogenous RNA and processed in parallel of the standard incubation 2. White bars surrounded by a bold line (m) represent the mean of the different values with the corresponding standard deviation obtained at t = 0 h and t = 4 h in incubations with exogenous RNA. C. [α−32P] CMP is incorporated into RNA. Using a Xenopus LSE in a reaction (50 µL) with [α−32P] CTP, aliquots (10 µL) were collected at t = 0 and t = 4 h. [α−32P] CMP incorporated into TCA precipitable material (cpmTCA) was determined using a standard protocol (white bars) or a modified one including a further incubation at 37°C with RNase (black bar) or with RQ1 DNase (grey bar) prior to TCA precipitation. Similar results were obtained using another Xenopus LSE.Xenopus extracts incorporate [α−(0.40 MB TIF)Click here for additional data file.


Penicillium digitatum is a fungal necrotroph causing a common citrus postharvest disease known as green mold. In order to gain insight into the genetic bases of its virulence mechanisms and its high degree of host-specificity, the genomes of two P. digitatum strains that differ in their antifungal resistance traits have been sequenced and compared with those of 28 other Pezizomycotina.P. chrysogenum reveals a much smaller gene content in P. digitatum, consistent with a more specialized lifestyle. We show that large regions of the P. chrysogenum genome, including entire supercontigs, are absent from P. digitatum, and that this is the result of large gene family expansions rather than acquisition through horizontal gene transfer. Our analysis of the P. digitatum genome is indicative of heterothallic sexual reproduction and reveals the molecular basis for the inability of this species to assimilate nitrate or produce the metabolites patulin and penicillin. Finally, we identify the predicted secretome, which provides a first approximation to the protein repertoire used during invasive growth.The two sequenced genomes are highly similar, but important differences between them include the presence of a unique gene cluster in the resistant strain, and mutations previously shown to confer fungicide resistance. The two strains, which were isolated in Spain, and another isolated in China have identical mitochondrial genome sequences suggesting a recent worldwide expansion of the species. Comparison with the closely-related but non-phytopathogenic P. digitatum, the first of a phytopathogenic Penicillium species, is a valuable tool for understanding the virulence mechanisms and host-specificity of this economically important pest.The complete genome of  Penicillium digitatum can account for up to 90% of the total losses, especially in arid and sub-tropical climates [P. digitatum is a necrotrophic wound pathogen that requires pre-existing injured fruit peel to penetrate the plant tissue, and it colonizes mostly through the deployment of maceration enzymes. Remarkably, and despite this rather unspecific infection mechanism, P. digitatum exhibits a high degree of host specificity and has not been described as ‘naturally-occurring’ in other pathosystems outside citrus fruits [P. digitatum infection and host specificity remain largely unknown. We have previously analyzed the fruit’s transcriptomic response to P. digitatum infection and also to elicitors that trigger induced resistance [P. digitatum strains that differ in their resistance to common fungicides and a comprehensive comparison across 28 other sequenced Pezizomycotina. This resource will enable progress to be made towards understanding the physiology and virulence mechanisms of this economically important phytopathogen.Postharvest losses can have a significant impact on crops, ranging from 15 to as high as 50% of total production [climates . Nowadayclimates . P. digis fruits . Despitesistance . Here, wP. digitatum strains isolated from infected orange (PHI26) and grapefruit (Pd1) in Valencia (Spain) [P. chrysogenum, though there were differences in the distributions of the various types of elements. Using a combined approach that included the use of RNA-Seq data, we predicted approximately 9,000 protein-coding genes in each of the two strains. Predicted genes include 100% of a set of 69 core genes found to be ubiquitous across all fungal clades [P. digitatum physiology and pathogenicity. In the current study we explore the newly obtained genomes to gain insight into some of the main aspects of P. digitatum physiology.Two previously described  (Spain) ,8 were sefruit Pd in Valen (Spain)  identifiefruit Pd in Valenl clades ,12. The P. digitatum. We nevertheless found a conserved alpha-box mating-type protein (MAT1) locus in both strains , a nitrate reductase (NiaD) and a nitrite reductase (NiiA) [P. digitatum genome for the presence of homologs of the five above-mentioned A. nidulans proteins and found homologs to AreA, NirA, NiaD and NiiA, the latter two being encoded by contiguous genes. However, P. digitatum lacks a homolog for the nitrate transporter CrnA  mapping to the P. chrysogenum crnA locus at low stringency. The cluster formed by niaD-niiA-crnA and the surrounding genes is highly conserved among Aspergillus species. However, P. chrysogenum contains three putative open reading frames (ORFs) between niiA and crnA. P. digitatum shares the same basic genome organization as P. chrysogenum with the exception that it lacks crnA and the three ORFs that interrupt the nitrate assimilation cluster. This indicates that this region has suffered important re-arrangements in the Penicillium lineage, including the loss of the nitrate transporter in P. digitatum, which would explain the inability of this species to use nitrate. A comparison of the rates of non-synonymous vs synonymous codon substitutions (dN/dS) between P. chrysogenum and P. digitatum in the nitrate gene cluster shows values compatible with purifying selection (0.36 for niiA and 0.48 for niaD), and similar to the average value for orthologous genes in these two species (0.47). This finding suggests the recent loss of crnA or, alternatively, that niiA and niaD may still be functional in the absence of crnA.n source . This pae (NiiA) , the ence (NiiA) . We searA Figure . This isP. digitatum is able to synthesize siderophores. Similar to other Aspergillus and Penicillium species it contains the genes necessary to synthesize the extracellular siderophore fusarinine C, namely the homologs to sidA, sidF and sidD . This similarity extends to the chromosomal organization between the two strains, with 99.8% of neighboring gene pairs being conserved. We could only detect two putative chromosomal re-arrangements .Few single nucleotide polymorphisms (SNPs) were noted between the two strains. In total 1,441 high confidence SNPs were found, of which 553 affected coding sequences. Remarkably, there is an excess of non-synonymous (358) vs synonymous (195) SNPs, suggesting the possibility that part of the differences are the result of positive selection. Among the 22 genes with more than one non-synonymous SNPs . This enabled us to derive a complete catalogue of orthology and paralogy relationships among the species considered based on phylogenetic evidence [http://www.phylomedb.org, [P. digitatum phylome. The closest species related to P. digitatum with a completely sequenced genome is P. chrysogenum [P. chrysogenum had an ortholog in P. digitatum. Conversely, 7,107 (86%) P. digitatum genes have an ortholog in P. chrysogenum. Interestingly, and despite the large differences in the total number of genes between P. digitatum and P. chrysogenum, both genomes have similar representations of the different functional classes. Indeed, only small differences in the representation of COG functional categories were found  of the protein-coding genes of the latter seem to have emerged from expansions of gene families that are present in other chromosomes . In addysogenum . DiffereP. digitatum proteins lack homologs in the closely-related but non-pathogenic species P. chrysogenum. Of these, 97 have homologs exclusively in other plant pathogenic species thus highlighting them as good candidates for possible roles in virulence . Previous analyses [Pezizomycotina species are particularly enriched for genes acquired from prokaryotes. We have applied the same strategy to find genes of putatively prokaryotic origin in P. digitatum. Four genes fulfilled our criteria  that has been associated with virulence in maize infections and the synthesis of T-toxin in Cochliobolus heterostrophus [P. digitatum correspond to genes that have been described as HGT in P. chrysogenum, among which the penicillin gene cluster is included (see below). Another relevant HGT described in some fungi [P. digitatum, is arsenate reductase (arsC) which in P. chrysogenum [Saccharomyces cerevisiae (Acr2p) or in other distantly-related fungi [P. digitatum may be vulnerable to arsenic, or alternatively, that this species may be using another detoxification system not previously described in fungi. We next focused on five particular aspects of P. digitatum genomic content that are likely to provide hints for understanding this organism’s pathogenic lifestyle and constitute interesting targets for future research: namely i) secondary metabolism gene clusters, ii) the prediction of putative small cysteine-rich peptides (CRPs), iii) homologs of known effector proteins iv) carbohydrate active enzymes (CAZy), and finally iv) the repertoire of secreted proteins among which we expect to find those that potentially interact with the host and are involved in invasive growth of the fungus.Based on the phylome analysis we found that 843 analyses  have foustrophus . None ofme fungi , but absysogenum  forms paed fungi . This woPenicillium species are sources of compounds or enzymatic activities that have important clinical or biotechnological applications [P. chrysogenum and the antifungal and potential cancer chemotherapeutic agent griseofulvin produced by P. griseofulvum, as well as the capacity shown by diverse Penicillium species for the degradation of several xenobiotics [P. digitatum PHI26 containing, among others, 31 backbone genes, of which 13 were nonribosomal peptide synthases (NRPSs), 14 polyketide synthases (PKS), one prenyltransferase (DMATSs) and three NRPS/PKS hybrids , the difference is less when the relative sizes of the two genomes are taken into consideration , six of which are clustered  are located in a different scaffold separated by three genes. The presence of a truncated patI pseudogene, the loss of the other components and the absence of backbone genes around the cluster, suggest that these genes do not function in the patulin biosynthetic pathway. Consistent with this interpretation, we found ratios of non-synonymous versus synonymous substitutions (dN/dS) close to 1 in most of the genes in the cluster, which is consistent with relaxation of the selective pressure  or the clavatus ) which i expansum, anotherhiopicum  and abseAspergillus and Penicillium[P. chrysogenum [P. digitatum we found a CRP (PDIG_68840) showing 91% amino acid identity with the Pc12g08290 gene from P. chrysogenum which corresponds to one of the previously reported proteins [P. digitatum as it is located in the Pc24 supercontig which is virtually devoid of P. digitatum orthologs. We identified two additional putative CRPs in P. digitatum (PDIG_31210 and PDIG_70730) by performing profile-searches with CRP motifs identified in plants [Several fungi are known to secrete small cysteine-rich proteins (CRPs) that exhibit diverse biological functions including adherence , virulennicillium. These cnicillium. Two disysogenum ,46. Amonn plants . Due to P. digitatum proteins homologous to effector proteins from other pathogenic fungi [P. digitatum encodes a smaller number of homologs of known effector proteins compared to P. chrysogenum, but abundances relative to the total proteome are comparable , and single homologs to Npp1 , Gip1 and Gip2 (Phytophtora infestans). Additionally, five proteins in P. digitatum contain the LysM domain  across all the pezyzomycotina genomes analyzed regardless of their phytopathogenicity. This result illustrates the limitations of using mere homology to predict the functional roles of proteins.We next searched the pathogen-host interaction database  for P. dic fungi . These ie Figure . We founP. chrysogenum. Among P. digitatum CAZymes, 49 have clear assignments to either glycoside hydrolases (GH), carbohydrate esterases (CE) or polysaccharide lyases (PL) involved in fungal cell wall degradation, a number close to the 52 found in P. chrysogenum. In contrast, a larger difference was observed in the number of enzymes involved in plant cell wall degradation: 49 in P. digitatum as compared to 72 in P. chrysogenum. Most of this difference can be attributed to GH families 10, 31, 43 and 61, which account for 40 enzymes in P. chrysogenum and only 14 in P. digitatum. Enzymes in these families are mostly related to the degradation of celullose (endoglucanases) and hemicellulose . Interestingly, two CAZy families involved in pectin degradation are enriched in P. digitatum compared to P. chrysogenum. P. digitatum contains eight polygalacturonases belonging to familiy GH28, and three pectinesterases included in family CE8, whereas P. chrysogenum has five and two proteins in these two families, respectively. Given that P. digitatum is a phytopathogen, whereas P. chrysogenum is not, these differences could provide hints to help identify the CAZymes relevant for citrus fruit infection in P. digitatum.Plant cell wall carbohydrate components form a complex network of cellulose, hemicellulose and pectin. These polysaccharides, together with others not derived from plants, are targets of the carbohydrate-active enzymes  that degrade them into simple monomers that can be used as nutrients . A searcin silico pipeline (Methods) we predicted 552 secreted proteins, of which 133 (24%) code for various hydrolases such as proteases, lipases or nucleases , which in relative terms is somewhat smaller than the secretome of P. chrysogenum  and the average 8% found in Aspergillus species, and much lower than the 14% of secreted proteins found in the Magnaporthe species  Sacc. PHI26 and Pd1 strains have been used in this study. PHI26 was isolated from an infected orange [d orange  and Pd1 d orange , both inP. digitatum spores collected after 7 days of growth in potato dextrose agar, and three libraries from P. digitatum-infected oranges at 12, 24, and 48 h post inoculation. Fruit inoculation and RNA extraction were conducted following procedures previously described [RNA-Seq data were generated from four different libraries: a cDNA library synthesized from escribed . cDNA liP. digitatum PHI26 and Pd1 (5 × 105 conidia/mL) were grown in 500 mL Potato Dextrose Broth media  on a shaker at 180 rpm at 24°C for 4 days. Fungal mass was collected, dried and frozen in liquid nitrogen. DNA was extracted from 1 g of frozen mycelium essentially as previously described [escribed . The fin′-adenylation was performed by incubation with dATP and 3′-5′-exo− Klenow fragment (New England Biolabs). DNA was purified using MinElute spin columns (Qiagen) and double-stranded Illumina paired-end adapters were ligated to the DNA using rapid T4 DNA ligase (New England Biolabs). After another purification step, adapter-ligated fragments were enriched, and adapters were extended by selective amplification in an 18-cycle PCR reaction using Phusion DNA polymerase (Finnzymes). Libraries were quantified and loaded into Illumina flow-cells at concentrations of 7–20 pM , or 1.4–1.75 pM . Cluster generation was performed in an Illumina cluster station (GA) or in a cBOT (HS). Sequence runs of 2 × 50 cycles were performed on the sequencing instruments. Base calling was performed using Illumina pipeline software. In case of multiplexed libraries, we used 4 bp internal indices (5′ indexed sequences). De-convolution was performed using the CASAVA software (Illumina). A total of 3.4 Gb of pair-end (350 insert size) and 2.6 Gb of mate-pairs (5 kb inserts), were produced. Reads were trimmed at the base with a Phred quality score lower than 10, or at the first undetermined base. Reads shorter than 31 bases (and their corresponding pairs) were removed. Reads were assembled using SOAPdenovo [Genome sequencing was performed at the CRG ultrasequencing facility (PHI26) and at LifeSquencing (Pd1) using Illumina HiSeq 2000 and Roche 454 GS FLX Titanium technologies, respectively. For the Illumina sequencing (PHI26) DNA was fragmented by nebulization to a size ~400-600 bp. After shearing, the ends of DNA fragments were blunted with T4 DNA polymerase and Klenow fragment (New England Biolabs). DNA was purified with a QIAquick PCR purification kit (Qiagen). Thereafter, 3APdenovo . The besAPdenovo . We explAPdenovo  to creatP. digitatum Pd1 genomic DNA employing the GS FLX Titanium General Library Preparation Kit following the manufacturer’s recommendations . Briefly, DNA was randomly sheared via nebulization and double stranded DNA adaptors were blunt-ligated to fragment ends following post-electrophoresis agarose gel excision of the 500–800 bp fraction. The final single-stranded DNA library was isolated via streptavidin bead binding to biotinylated adaptors followed by alkaline treatment. The library was quantitated via fluorometry using Quant-iT RiboGreen reagent  prior to emulsion PCR amplification. Subsequently, a paired end library was constructed. Briefly, 30 μg of double stranded genomic DNA was randomly fragmented via hydrodynamic shearing to an average size of 15,000 or 20,000 bp using the HydroShear apparatus . Fragment ends were blunt ended via T4 DNA polymerase and T4 polynucleotide kinase  treatment, and a double-stranded DNA adaptor  was blunt-ligated to DNA ends. Library fragments in the desired size range were isolated from excised gel fragments via electroelution following overnight 0.5% agarose gel electrophoresis. Nicks present at the 3′-junctions of each of the adaptors and the library fragment were filled in by the strand-displacement activity of the Bst DNA polymerase . Linear library molecules were circularized via Cre recombinase  excision. These molecules along with 1 μg carrier DNA were randomly fragmented via nebulization to an average size of approximately 450–500 bp. Fragment ends were blunted and desired library fragments  were selected via the biotinylated linker, using 1 Dynal M-270 magnetic streptavidin beads . Double-stranded DNA adaptors were ligated to blunt fragment ends. The resulting library was amplified using 20 cycles of PCR and purified with AMPure beads  to remove small DNA fragments and amplification primers. Single stranded DNA library was isolated via Dynal M-270 bead binding followed by alkaline treatment and quantitation using Quant-iT RiboGreen  prior to emulsion PCR amplification. Genomic shotgun library molecules were clonally amplified via emulsion PCR following manufacturer’s recommendations employing the GS FLX Titanium LV emPCR Kit . Paired end library was handled similarly, with the exception that 30 μL of amplification primer was used per Large Volume Emulsion cup. Following amplification, emPCR reactions were collected, and emulsions broken according to the manufacturer’s protocols. Beads containing sufficient copies of clonally amplified library fragments were selected via the specified enrichment procedure and counted with a Multisizer 3 Coulter Counter  prior to sequencing. Following emulsion PCR enrichment, beads were deposited into the wells of a Titanium Series PicoTiterPlate device and 454 Sequencing was performed using the GS FLX instrument according to the manufacturer’s recommendations . Image analysis, signal processing and base calling were performed using supplied system software. Standard Flowgram Format (sff) files output from base calling were employed in subsequent genome assembly. The assembly of 454 reads was performed with Newbler (454-Roche).For the Roche 454 sequencing (Pd1), a random “shotgun” genomic library was generated via fragmentation of 10 μg of P. chrysogenum genome [A. nidulans files. RNA-Seq data from P. digitatum Pd1 in different growth conditions were incorporated in the predictions as hints. ii) a second prediction was done using JIGSAW [P. chrysogenum or A. nidulans, depending on availability; d) a custom python script was used to detect Open Reading Frames (ORFs) using the universal genetic code, e) homology data obtained from Blast searches for each protein encoded in the three sequenced Penicillium species  and several other Pezizomycotina species  run against the P. digitatum genomes; f) GMAP [P. chrysogenum to the newly sequenced P. digitatum genomes. iii) A third prediction was run using GeneWise [P. chrysogenum, P. marneffei, T. stipitatus, A. nidulans, Fusarium oxysporum, A. flavus, N. crassa, G. zeae, H. capsulatum) and the set of revised proteins found in Uniprot (as of June 2011). An initial Blast was performed to locate the contig in which a protein was potentially present, then GeneWise was run to establish its most likely position within the contig. Hits with a bit score over 50 were revised and extended in case of lack of starting or stop codon. If no internal stop codons were found in the final gene, this was added to the annotation. The final annotation was build using the first prediction as a starting point. Proteins detected in the second and third annotations where added when there was no overlapping with the initial set. The final predicted gene set included 9,153 and 8,969 genes for PHI26 and Pd1, respectively. Pfam domains [We used a genome annotation pipeline similar to that used for the closely-related m genome . In brieg JIGSAW  in orderg JIGSAW   g JIGSAW , which w f) GMAP  was usedGeneWise  to searc domains  were sea domains  pathwaysThe subcellular location of each protein in the 29 genomes used in this study was predicted using PSORT . AdditioP. digitatum (both strains), P. chrysogenum, A. clavatus, A. fumigatus, A. flavus, Aspergillus niger. Subsequently, HMM-derived profiles of 516 plant CRP motives described [Prediction of CRPs was based according to their expected sequence characteristics . Known pescribed  were useescribed . Hits weP. digitatum (strain PHI26) and of P. chrysogenum. The phylome was reconstructed using 28 other Pezizomycotina species. Considering the high degree of similarity between the two P. digitatum strains only the PHI26 strain was used in the phylomes. The phylome was reconstructed using an automated pipeline previously described in [http://phylomedb.org), with the phylome IDs 150 (P. digitatum) and 179 (P. chrysogenum). Trees were scanned automatically using ETE v2 [The phylome, meaning the complete collection of phylogenetic trees for each gene in a genome, was reconstructed for the genomes of ribed in , MAFFT vribed in , and DIAribed in . Alignmeribed in ), and thribed in . This coribed in ,  were computed on the protein sequences using MUSCLE v3.7 [Ratios of non-synonymous over synonymous substitutions (dN/dS) were calculated as follows. Alignments of the relevant one-to-one orthologs (CLE v3.7 . The amiCLE v3.7 . dN/dS wAgrobacterium tumefaciens-mediated transformation with plasmid pRFD519. This plasmid derives from plasmid pRFHU2 [′- GGTCTTAAUGGGGAGAGCATAGGTGGAAT-3′) and PD519O2 (5′-GGCATTAAUCTTGGATGGAGAGGGAACAA-3′) were used to amplify a fragment of 1.76 kb region upstream of the genes and primers PD519A3 (5′- GGACTTAAUCGTGACATAAGGGCCAAACT-3′) and PD519A4 (5′- GGGTTTAAUCCCCTTTGGTTACCCTCATT-3′) were used to amplify a 1.85 kb DNA fragment downstream of the genes. The two flanking fragments were introduced into pRFHU2 following the USER protocol described by [E. coli DH5α chemical competent cells. Proper fusions were checked by DNA sequencing and then the plasmid was transferred to A. tumefaciens AGL1 electrocompetent cells. Agrobacterium-mediated transformation of P. digitatum Pd1 was conducted basically as described in [5 con/mL was mixed with A. tumefaciens AGL1 cells that had been grown in liquid induction medium (IM) supplemented with 200 μM acetosyringone until they reached an A600 of ~0.75. Aliquots of the mixture were spread onto 0.45 μm nitrocellulose membranes (Abet) that were layered on solid IM plates. After three days of incubation at 24°C the membranes were transferred to Petri plates containing PDA medium supplemented with 100 μg/mL hygromycin B (Invitrogen), for selection of fungal transformants, and 200 μg/mL cefotaxim , for killing the bacterial cells. Putative transformants appeared after four days of incubation at 24°C. They were transferred to fresh plates containing PDA supplemented with hygromycin B and monosporic isolates were obtained. Conidia from monosporic isolates were inoculated in PDB medium containing 100 μg/mL hygromicin B and incubated at 24°C for two days with shaking. DNA was extracted from fungal mycelia following the procedure described in [′-GCTTTCCCGCTTTAGTGTGG-3′) and PD519R (5′-ACAGCCGCAGCCTGTTATCT-3′). In the wild type strain these two primers amplify a fragment of 2.3 kb, whereas in gene knockout transformants this fragment is replaced by a 3.0 kb band. Ectopic transformants contain both bands.To investigate the possible role in the differential susceptibility to fungicides of the two contiguous genes that were only present in the Pd1 strain, we transformed this strain via d pRFHU2  and contribed by  and the ribed in  with minribed in  and resu5 con/mL in PDB medium supplemented with fungicide. The fungicides azoxystrobin , imazalil , myclobutanil , prochloraz  and trifloxystrobin  were assayed at final concentrations of active ingredient of 0, 0.16, 0.31, 0.63, 1.25, 2.5, 5.0, and 10.0 μg/mL. Thiabendazole  was assayed at concentrations of 0, 0.63, 1.25, 2.5, 5.0, 10.0, 20.0 and 40.0 μg/mL. There were three replicate wells for each fungicide concentration. Plates were incubated at 24°C and growth, measured as A600, was followed for up to seven days. Minimum inhibitory concentration (MIC) values were considered as those in which no fungal growth was observed by seven days of incubation.Fungicide resistance assays were conducted in 96-well microtiter plates as described in . Wells cThe authors declare that they have no competing interests.ARB, BF, EH, and LGC performed experiments. MMH and ARB conducted bioinformatic analyses. JFM, TG and LGC conceived the study. TG and LGC coordinated the work. TG, LGC, MMH, ARB and JFM analyzed the data and drafted the manuscript. All authors read and approved the final manuscript.Supplementary materials.Click here for file


Also implicated in encapsidation are cis determinants in the R, U5, and PBS (primer binding site) from the 5' untranslated region (UTR). Although conventionally associated with nuclear export of HIV-1 RNA, there is a burgeoning role for the Rev/RRE in the encapsidation process. Pleiotropic effects exhibited by these cis and trans viral components may confound the ability to examine their independent, and combined, impact on encapsidation of RNA into HIV-1 viral particles in their innate viral context. We systematically reconstructed the HIV-1 packaging system in the context of a heterologous murine leukemia virus (MLV) vector RNA to elucidate a mechanism in which the Rev/RRE system is central to achieving efficient and specific encapsidation into HIV-1 viral particles.The process of HIV-1 genomic RNA (gRNA) encapsidation is governed by a number of viral encoded components, most notably the Gag protein and gRNA cis elements from the 5' UTR . Incorporation of all the 5' UTR cis elements did not enhance RNA encapsidation in the absence of the Rev/RRE system. In fact, we demonstrate that the Rev/RRE system is required for specific and efficient encapsidation commonly associated with the canonical packaging signal. The mechanism of Rev/RRE-mediated encapsidation is not a general phenomenon, since the combination of the Rev/RRE system and 5' UTR cis elements did not enhance encapsidation into MLV-derived viral particles. Lastly, we show that heterologous MLV RNAs conform to transduction properties commonly associated with HIV-1 viral particles, including in vivo transduction of non-dividing cells (i.e. mouse neurons); however, the cDNA forms are episomes predominantly in the 1-LTR circle form.We show for the first time that the Rev/RRE system can augment RNA encapsidation independent of all cis elements, Gag, and the Rev/RRE system, in which the Rev/RRE system is required to make the RNA amenable to the ensuing interaction between Gag and the canonical packaging signal for subsequent encapsidation.Premised on encapsidation of a heterologous RNA into HIV-1 viral particles, our findings define a functional HIV-1 packaging system as comprising the 5' UTR  Cis packaging signals in the viral RNA confer specific selection among the milieu of host cell RNAs through interactions with trans factors encoded by the virus, and host cell. The conventional canonical cis packaging signal (ψ) is a ~120 bp fragment comprised of four stem-loop structures located in the HIV-1 5' untranslated region (UTR), and extending into the 5' end of the HIV-1 Gag coding sequence , 6 μg/ml oligo dT16, 25 mM DTT, 0.06% Triton X-100, and 0.25 mCi/ml 3H-TTP [MP Biomedicals]). After incubation for 1 hour at 37°C the entire volume was spotted onto DE81 anion exchange chromatography paper (Whatman), and placed into 5% Na2PO4 for 5 minutes at room temperature. The samples were washed at room temperature five times, for 5 minutes each, with 5% Na2PO4 two times with water, and one time with 95% ethanol. The samples were dried, placed in scintillation fluid, and radioactivity was measured on a Beckman LS 6500 scintillation counter. Values were recorded as CPM. Data used for titer normalization, and determining amount of viral particles for RNA isolation, are a mean of replicate samples.MLV reverse transcriptase activity was executed on vector particles (5 μl) harvested from the media by mixing with 25 μl of RT activity reagent . Results are expressed as relative light units (RLU)/mg protein. Protein concentrations were assayed according to manufacturer's instructions for Pierce BCA Protein Assay kit.Luciferase lysates were prepared by pelleting transfected 293T cells at the time of vector collection, and resuspending the pellet in 1X passive lysis solution (Promega). After freeze-thawing cell debris was pelleted by centrifugation for 15 minutes at 14,000 rpm and 4°C. Equivalent volumes of supernatant were assayed for firefly luciferase expression using 100 μl luciferin reagent (Promega). Luminescence was measured with a VictorN-morpholino)ethanesulfonic acid) buffered 0.1% Triton X-100 solution  + protease inhibitor minicocktail (Roche) + 200 units/ml RNase Inhibitor (Fermentas). Pellets were incubated 2 minutes on ice, and nuclei were pelleted at 1500 rpm and 4°C. The cytoplasmic supernatant fraction was collected and separated for RNA isolation and protein analysis. Cytoplasmic fraction was monitored by western blot analysis for the absence of nucleolin protein, compared to total cellular proteins. RNA was purified from the cytoplasmic supernatant using the PARIS protein and RNA isolation kit (Ambion). Manufacturer's instructions were followed starting with addition of the 2X lysis/binding solution to an equal volume of cytoplasmic supernatant. Purified RNA was treated with DNase I Turbo (Ambion) for 1.5 hours at 37°C, and inactivated according to manufacturer's instructions. RNA integrity and concentration were regularly assessed by agarose gel electrophoresis. Cytoplasmic RNA was then utilized for analysis in qRT-PCR and northern blot.At 48-60 hours post-transfection vectors were harvested from the media, and cells were divided for luciferase assay, total protein, and cytoplasmic RNA/protein fractionation. Cells were removed from the plate by trypsinizing, and pelleted. The cytoplasmic fraction was separated by treating the pelleted cells with a MES (2-(6 293T cells were concomitantly added to the RLT lysis solution in preparation for column purification of the RNA with the RNeasy Plus Mini kit (Qiagen). Spiking the vector particles with 293T cells at time of purification has multiple advantages: i) 293T cell RNA is a carrier during RNA purification; ii) RNA can be easily quantified after purification; and iii) quantity and integrity of purified RNA can be monitored by agarose gel electrophoresis. For purposes of homogenization, the lysed particles/293T cell RNA were passed through a QIAshredder column (Qiagen) and subsequently through a genomic DNA eliminator column provided with the RNeasy plus mini kit. Lysate was then purified through the RNA isolation column provided with the RNeasy plus kit. Purified RNA was treated with DNase I Turbo (Ambion) as described for cytoplasmic RNA. Vector particle RNA was then utilized for analysis in qRT-PCR and northern blot. RNA was isolated from MLV viral particles in a similar fashion, except that viral particles were normalized for equivalent amounts of MLV reverse transcriptase. The reverse transcriptase assay is described above.Isolation of RNA from vector particles was carefully executed so that all vectors packaged into HIV-1 viral particles were normalized for equivalent amounts of p24 prior to isolation. For each sample at the time of RNA isolation p24 equivalents of vector particles and 5 × 10Purified cytoplasmic and vector RNA were heated to 65°C for 5 minutes prior to preparing each reverse transcription (RT) reaction. Each RT reaction was prepared at final concentrations of 1X RT Buffer (Qiagen OmniScript kit), 0.5 mM each dNTP, 10 μM random prime nonamer, 10 units RNase Inhibitor (Fermentas), 500 ng RNA, and 4 units OmniScript reverse transcriptase (Qiagen). As controls for the presence of contaminating DNA each reaction was also performed in the absence of reverse transcriptase. Reactions were executed for 1 cycle on a BioRad MyCycler at 37°C for 1 hour. Equivalent amounts of cDNA were used in the subsequent quantitative PCR reaction. Each qPCR reaction was prepared at final concentrations of 1X ABI Taqman mix , 0.9 μM forward primer, 0.9 μM reverse primer, and 0.1 μM probe. Reactions were performed at 1 cycle of 50°C/2 minutes, 1 cycle of 95°C/10 minutes, and 40 cycles of 95°C/15 seconds + 60°C/30 seconds on a 7300 real time PCR system (Applied Biosystems). Vector RNAs were detected with a primer/probe set to the luciferase gene: For Luc 5'-aggtcttcccgacgatga-3', Rev Luc 5'-gtctttccgtgctccaaaac-3', and probe #70 . All quantitative PCR reactions were normalized to an endogenous control reaction for TATA Binding Protein (TBP) using the following primer/probe set in independent reactions: For TBP 5'-gaaccacggcactgattttc-3', Rev TBP 5'-tgccagtctggactgttcttc-3', and probe #92 .t) for luciferase to that obtained for TBP, from each reaction. The calculation was as follows: 2-ΔCt, where the ΔCt is (Luc Ct - TBP Ct). Independent reactions were performed for each VRNA and CRNA sample. All isolated VRNA could be normalized to TBP since each sample was copurified with 293T cell RNA, as described above. Calculation of the encapsidation efficiency, where VRNA relative to CRNA is expressed as AU, was determined for each independent sample. The calculation was as follow: 2-ΔΔCt, where the ΔΔCt is (VRNA Luc Ct - VRNA TBP Ct) - (CRNA Luc Ct - CRNA TBP Ct). All relative encapsidation efficiencies are expressed as an average of at least three independent experiments with corresponding standard deviations (S.D.).The levels of vector RNA (VRNA) and cytoplasmic RNA (CRNA) derived from the qRT-PCR data are expressed as arbitrary units (AU). The values were determined by normalizing the cycle threshold  at 37°C for 1 hour. Probes were either a BstEII fragment from pTK1440 generated to the 5' end of the vector RNA, or to the GFP gene located in the 3' end of the RNA. Images were obtained on BioMax MR film (Kodak), or by phosphorimager (Molecular Dynamics Storm System).Cytoplasmic RNA was isolated as described, and vector RNA was isolated from concentrated vector particles. Vector RNA was prepared from equivalent levels of p24, and at the time of isolation the samples were copurified with 293T cells as described above. Prior to resolving RNA on a denaturing formaldehyde agarose gel, RNA was denatured at 65°C for 10 minutes. Equivalent amounts of cytoplasmic RNA, and vector RNAs, were resolved on the gel. Northern blot analysis was performed under standard conditions. Probes were random prime labeled with αCytoplasmic and total cell lysates were resolved by standard denaturing SDS-PAGE analysis on 10% gels and blotted to Hybond P membrane. Membranes were probed with rabbit anti-nucleolin  and rabbit anti-GAPDH . Detection was achieved by probing blots with goat anti-rabbit IgG peroxidase . All antibody incubations and washes were done in 1X PBST. Signals were detected using ECL .293T cells were transduced with MLV/HIV RRE + RU5PS vector packaged into HIV-1 or MLV viral particles, at a MOI of 5. Total cellular DNA was isolated at 5 days post-transduction , and after five passages (P5) of the transduced cells. To collect total DNA transduced cells were lysed in a proteinase K solution  for 48 hours at 55°C. Total DNA was extracted with v/v phenol (Invitrogen) and v/v phenol/chloroform/isoamyl alcohol (Invitrogen) and then treated with RNase A  at 37°C for 2 hours. A second round of extractions was performed and total DNA was precipitated using standard ethanol precipitation.Isolated gDNA (10 μg) was digested with BsrGI and DpnI for ~24 hours at 37°C. DpnI was included to eliminate putative plasmid DNA carry-over from the transfections during vector production in 293T cells. Equivalent amounts of gDNA were resolved on 1% agarose gels, transferred to Zetaprobe membrane, and probed with a BstEII fragment from pTK1440 that recognizes the 5' end of the vector enabling a size distinction between linear, 1-LTR, 2-LTR and backbone/integrated forms. Images were captured on BioMax MR Film (Kodak), or by phosphorimager (Molecular Dynamics Storm System).Total cellular DNA was isolated as described for Southern blot analysis and qPCR for vector and β-globin were described previously . Brieflyad libitum. All procedures were in accordance with the Guide for the Care and Use of Laboratory Animals  85-23), and all procedures received prior approval by the NIH Institutional Animal Care and Usage Committee. All procedures were performed according to animal protocol number, 396-LNS-2014.Female C57Bl/6 mice (n = 10), 3 months old (Jackson Labs) were housed in standard conditions with 3-4 mice per cage with food and water available Mice were anesthetized , and MLV retrovirus (n = 5 mice), or HIV lentivirus (n = 5 mice) was injected stereotactically (2 μl using a 5 μl Hamilton microliter syringe) into the right striatum . Studies were executed with the MLV/HIV RRE vector Figure  packagedDouble labeling for the neuronal marker NeuN and GFP was done on a 1:6 series of 40-μm free-floating horizontal sections as described previously [The authors declare that they have no competing interests.ASC, HvP and TK designed research, analyzed data and wrote the paper; ASC, HvP and NS performed research; HM contributed reagents and necessary materials. All authors read and approved the final manuscript.Figure S1. Cytoplasmic separation and transfection assessment. A. At the time of harvesting vectors total and cytoplasmic protein fractions were routinely collected from the producer 293T cells to monitor separation of the cytoplasmic fraction. Equivalent amounts of total (TP) and cytoplasmic (CP) protein lysates were analyzed for nucleolin (a nuclear specific protein), and GAPDH, by western blot analysis. Three independent experiments are shown for the MLV/HIV RRE + RU5PS vector packaged into HIV-1 particles in the presence of Rev. B. Cytoplasmic RNAs isolated from transfected producer 293T cells at the time of vector harvest. Four independent experiments (lanes 1-4) were analyzed by denaturing northern blot analysis. The MLV/HIV RRE + RU5PS vector RNA is shown in the absence of Rev  and in the presence of Rev . Dilutions of vector RNA in lane 4 were also resolved at 50% (lane 5), and 20% (lane 6). Actin was probed as a positive control. The band densities were quantitated by phosphorimager for vector (C) and actin (D). Numbers below each bar correlate with lanes on northern blots.Click here for file


Since then, paramutation and paramutation-like interactions have been discovered in other plants and animals, suggesting that they may underlie important mechanisms for gene expression. The molecular bases of these phenomena are unknown. However in some cases, the event of paramutation has been correlated with changes in DNA methylation, chromatin structure and recently several studies suggest that RNA could play a fundamental role. This last consideration is greatly supported by genetic screening for mutants inhibiting paramutation, which allowed the identification of genes involved in RNA-directed transcriptional silencing, although it is possible that proteins are also required for paramutation.Gene silencing is associated with heritable changes in gene expression which occur without changes in DNA sequence. In eukaryotes these phenomena are common and control important processes, such as development, imprinting, viral and transposon sequence silencing, as well as transgene silencing. Among the epigenetic events, paramutation occurs when a silenced allele (named paramutagenic) is able to silence another allele (paramutable) in trans and this change is heritable. The silenced paramutable allele acquires paramutagenic capacity in the next generations. In the 1950s, Alexander Brink described for the first time the phenomenon of paramutation, occurring in maize at the The meaning of paramutation in the life cycle and in evolution remains to be determined even though we might conjecture that this phenomenon could be involved in a fast heritability of favourable epigenetic states across generations in a non-Mendelian way. Cell specialization in complex organisms is possible by fine tuning of genome expression because all the cells of a multicellular organism carry the same DNA information but only a small sub-set of the genes must be active at a certain point of development and growth . FurtherSince Mendel’s laws were rediscovered a huge amount of work has been done confirming the universality of these findings: nevertheless some exceptions were identified from several studies done by earlier geneticists. In the 1940s Barbara McClintock was one of the first scientists to find exceptions to Mendel’s laws while working on anthocyanin pigments in maize: this work led to the discovery of transposons and to the Nobel prize in 1983 , 3.In particular the epigenetic phenomena defined by Riggs and colleagues as “the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence” have disclosed a new level of gene regulation .These phenomena seem as if they may exist in all phyla, and control a number of gene regulation processes ranging from embryo development to human diseases by DNA methylation, chromatin modification (histone methylation and nucleosome position) and noncoding RNA -7.1). Differently from a typical mutation, in which the change of the DNA sequence usually causes a switch off of the gene activity, paramutation generates different epialleles silenced with variable phenotypes due to modulation of gene expression, leading to a change in the conception of gene expression from a digital system  to an analog system  alter-ego[b1 loci : the repgenicity , 57. Thepl1 gene, repeated sequences have not yet been identified, although a complex allele named pl-bol3 containing multiple pl1 gene copies has been isolated from a Bolivian maize population [sulf locus [hygromycin phosphotransferase (HPT) transgene locus [In the case of the pulation  and it slf locus  and in Ane locus .B’ or Pl’ systems) using ethyl methanesulfonate have permitted the isolation of at least ten loci belonging to the first class named “mediator of paramutation” and to the second class named “required to maintain repression”. Out of the mutations isolated, all the genes cloned so far are involved in the RNA-directed transcriptional silencing: mediator of paramutation1 (mop1) encoding for RNA-dependent RNA polymerase [mediator of paramutation2 (mop2) gene encoding for a second-largest subunit of plant-specific RNA polymerases IV and V [required to maintain repression1 (rmr1) gene encoding for an SNF2-like ATPase, a chromatin-remodeling enzyme [required to maintain repression6 (rmr6) encoding the largest subunit of the plant specific DNA-dependent RNA polymerase [mop1 is involved in the biogenesis of 24 nt siRNA and synthesis of dsRNA [RDR2 Arabidopis gene [kit paramutation system in mouse an involvement of siRNA has been demonstrated, in fact microinjecting RNA extracted from sperm or brain of mice with the white tail tips phenotype  into fertilized wild mice eggs has been demonstrated to induce paramutation at the wild kit locus [C. elegans which led to the discovery of RNA interference (RNAi) [Drosophila melanogaster [b1, experimental data showed it was likely that the tandem repeats are not directly involved in the genesis of siRNAs but instead they are required as cis-signaling in the paramutation [A potent tool to dissect the paramutation phenomenon consists in the isolation and study of the mutations that perturb the paramutation process: they can be subdivided roughly into two classes: (1) modifying the establishment of paramutation and (2) modifying the epigenetic memory . Geneticlymerase , 35, medIV and V , requireg enzyme  and requlymerase . In partof dsRNA  as in thpis gene . Also init locus , resemble (RNAi)  and may nogaster . Taken tnogaster . It is wmutation .b1 in which a CXC-domain protein CBBP (CXC domain b1-repeat binding protein) has been isolated sharing homology with some transposases able to bind in vivo and in vitro specifically a sequence within the tandem repeats of 853 bp inducing repressive chromatin states [b1 locus and this change was hereditable and the silent epiallele obtained (in the absence of a transgenic construct) was paramutagenic although with a reduced strength in comparison with B’. Furthermore CBBP forms multimers binding the b1 tandem repeats suggesting a correlation between the strength of paramutation and the number of b1 repeats and a possible trans interaction between chromosomes as observed in Drosophila in the case of transvection [B-I and B’ whilst the CBBP protein is only detectable in the B’, suggesting that a posttranscriptional control of CBBP is involved in the establishment of the B’ state [With the aim to isolate proteins involved in paramutation processes, the yeast one-hybrid technology has been used to identify the proteins binding to the repeated sequences present in most paramutagenic alleles. This strategy has been used successfully in the case of n states . To confsvection . It is nB’ state .b1 locus it is possible to speculate regarding a paramutation model: an increase of CBBP protein level  causes the onset of B’ from BI, this state is maintained in the next generation by RNA and/or proteins signals associated with the b1 repeats during mitosis and meiosis; in some way a pair trans interaction between B’ and B-I repeats establishes the paramutation Fig. (4). Interestingly, another phenomenon involving RNAi-mediated heterochromatin in yeast and arabidopsis does not show paramutation capacity [So far the relationship between RNAi machinery and CBBP is not clear but CBBP might be involved in same way in the chromatin modification complex as hypothesized for Drosophila CXC domain proteins . Hence Ccapacity , 74 stre. Furthermore in the r1 paramutation system it has been shown that environmental stimuli such as temperature and light can modify in an hereditable way the r1 expression states, suggesting a Lamarckian-like behavior of this trait [Paramutation may represent a rare “dull” deregulation of the system involved in the establishment and maintaining of chromatin state in a particular genome region defining the epigenetic state. Otherwise the biological systems where paramutation has been discovered have in common two characteristics: first the genes involved determined a phenotype easy to score by a simple visual inspection such as pigment , 28, 41 is trait .B1 paramutation phenomenon [Paramutation is associated in some way to siRNA biogenesis and in most cases to repeated sequences closely linked to the gene undergoing paramutation. Although it has been hypothesized for many years that repeated sequences were involved in the transcription of the aberrant RNA triggering an RNA-directed transcriptional silencing, a recent paper regarding the enomenon  suggests


Although a large set of full-length transcripts was recently assembled in catfish, annotation of large gene families, especially those with duplications, is still a great challenge. Most often, complexities in annotation cause mis-identification and thereby much confusion in the scientific literature. As such, detailed phylogenetic analysis and/or orthology analysis are required for annotation of genes involved in gene families. The ATP-binding cassette (ABC) transporter gene superfamily is a large gene family that encodes membrane proteins that transport a diverse set of substrates across membranes, playing important roles in protecting organisms from diverse environment.In this work, we identified a set of 50 ABC transporters in catfish genome. Phylogenetic analysis allowed their identification and annotation into seven subfamilies, including 9 ABCA genes, 12 ABCB genes, 12 ABCC genes, 5 ABCD genes, 2 ABCE genes, 4 ABCF genes and 6 ABCG genes. Most ABC transporters are conserved among vertebrates, though cases of recent gene duplications and gene losses do exist. Gene duplications in catfish were found for ABCA1, ABCB3, ABCB6, ABCC5, ABCD3, ABCE1, ABCF2 and ABCG2.The whole set of catfish ABC transporters provide the essential genomic resources for future biochemical, toxicological and physiological studies of ABC drug efflux transporters. The establishment of orthologies should allow functional inferences with the information from model species, though the function of lineage-specific genes can be distinct because of specific living environment with different selection pressure. Fish are exposed to numerous toxicants present in the aquatic environment. Catfish, as a bottom-dwelling fish species, is highly adaptable to its associated aquatic environment such as low dissolved oxygen, high level of toxins  and various xenobiotics. Therefore, catfish can serve as a good research model for toxicological studies. Numerous genomic resources have been developed to study detoxification-related genes in catfish, including a large number of ESTs The ATP-binding cassette (ABC) transporters are one of the largest protein families and are present in all organisms from bacteria to human Eukaryotic ABC transporters are either full transporters combining all required domains in one polypeptide (2 NBDs and 2 TMDs), or half transporters consisting of one NBD and one TMD that need to form homo- or heterodimer to generate a functional pump The ABC transporter family from animals was first characterized in the human with a total of 48 members A total of 50 ABC transporter genes were identified in catfish genome. Their transcripts, coding sequences, domain structures and accession numbers are summarized in Nine ABCA genes were identified in the catfish genome including ABCA1a, ABCA1b, ABCA1-like, ABCA2, ABCA3, ABCA4, ABCA5, ABCA7, and ABCA12. Full-length coding sequences were obtained for six of the nine catfish ABCA transporters except ABCA4, ABCA7 and ABCA12. All catfish ABCA proteins are full transporters, even though some of the sequences are partial .Xenopus , but two duplicates were identified with ABCB3 and ABCB6 . Of the All the catfish ABCB transporters were placed into distinct clades well supported by phylogenetic analysis with the exception of ABCB1 and ABCB5 . As showSimilarly, phylogenetic analysis did not provide a concrete support for the annotation of ABCB5. On the one hand, the catfish ABCB5 fell into the same clade containing ABCB5 of zebrafish, Xenopus, chicken, and lizard, but on the other hand, these ABCB5s did not fall into the same clade containing the mammalian ABCB5s as well as two additional ABCB5s from Xenopus . We therThe ABCB1, ABCB4 and ABCB5 are closely related, which share a common ancestor in the history of chordates To determine the age of duplication for ABCB4, we adopted a molecular clock test with the protein sequences of ABCB1 and ABCB4 using the human ABCB11 as an outgroup. The branch-length test indicated that the sequences have evolved at similar rates , therefore, a linearized tree was constructed . The treTwelve ABCC genes were identified in the catfish genome including ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC5-like, ABCC6, ABCC7, ABCC8, ABCC9, ABCC10, and ABCC12 . In geneThe ABCC subfamily consists of transporters known as multidrug resistance-associated proteins (MRPs) which transport diverse substrates including drugs, endogenous compounds and xenobiotics. In addition to MRPs, the ABCC subfamily includes the chloride channel CFTR and the sulfonylurea receptors (SUR1 and SUR2) The ABCD transporters are located to the peroxisome and are involved in the transport of fatty acids and/or fatty acyl-CoAs into peroxisome All genes in the ABCD subfamily are highly conserved. As revealed by phylogenetic analysis, four major clades of ABCDs were formed with one catfish member in each clade except ABCD3 that included two catfish ABCD3s  and S2. The ABCE and ABCF subfamilies consist of genes that possess two NBDs but no TMDs, making them non-functional as transporters. The ABCE subfamily contains a single gene in human, but two ABCE genes were identified in catfish genome. There are three ABCF genes in human: ABCF1, ABCF2 and ABCF3. All ABCF homologs were identified in catfish genome. Phylogenetic analysis well supported the annotation of these genes. However, the catfish ABCF2 is duplicated in the catfish genome as is in the zebrafish genome, whereas the catfish ABCE1 is uniquely duplicated .The human ABCG subfamily contains five members: ABCG1, ABCG2, ABCG4, ABCG5 and ABCG8. All ABCG transporters in metazoans are half transporters. In contrast to other half transporters, ABCGs show distinct domain structure with TMDs being located at the C-terminus of the NBDs. We identified all the ABCG homologs in the catfish genome. The phylogenetic analysis as shown in Teleost fish constitute over half of all vertebrate species and have adapted to a variety of marine and freshwater habitats Several members of ABC transporters in catfish have undergone gene duplications. These genes include ABCA1, ABCB3, ABCB6, ABCC5, ABCD3, ABCE1, ABCF2 and ABCG2 . In addiXenopus in this study  Eleven ABC transporters have not been identified from any fish genomes characterized to date: ABCA6, 8, 9, 10 13, 14, 15, 17, ABCB4, ABCC11, and ABCG3, suggesting their absence from the teleost genomes; 2) In contrast, seven ABC transporters are duplicated in the teleost genomes. They include ABCA1, ABCA4, ABCB6, ABCC4, ABCC6, ABCG2, and ABCG4; and 3) A couple of ABC transporters were found only in zebrafish among teleost genomes characterized to date, and they include ABCC13 and ABCH1 .The high level of conservation of ABC proteins involved in fundamental physiological processes suggests an evolutionary ancestral origin of these proteins. Clear orthologous relationships were established for the majority of ABC genes, enabling the possibility for functional inference of the catfish transporters. However, specific gene duplications were observed in catfish as well as in other fish species. Further research is warranted to unravel the philological significance of gene duplications in adaptation to diverse aquatic environments in fish. The whole set of ABC transporters provide the essential genomic resources for future biochemical, toxicological and physiological studies in catfish.Homo sapiens) and zebrafish (Danio rerio) retrieved from GenBank (NCBI) were used as queries to search against the RNA-Seq transcriptome assembly database by standalone TBLASTN (available from NCBI). Same searches were also conducted against the catfish whole genome assembly which was also generated from a doubled haploid channel catfish (unpublished data) to confirm sequence accuracy. The coding sequences from retrieved RNA-Seq transcripts were predicted using getorf from the EMBOSS tools, and validated by BLASTP against NCBI non-redundant protein sequence database (nr). The retrieved genome sequences were subjected to ab initio gene prediction by GENSCAN The ABC transporter genes in catfish were mainly identified through mining a RNA-Seq assembly from our previous study Tetraodon, stickleback, tilapia, cod, coelacanth, lamprey, chicken/turkey, Xenopus, lizard, mouse and human Click here for additional data file.Figure S2Phylogenetic trees of catfish ABC transporters with reference ABC transporters from all other species.(PDF)Click here for additional data file.Table S1Gene names and accessions of reference ABC transporters used in this study.(XLSX)Click here for additional data file.