The intestinal mucosa is exposed to large amounts of foreign antigen (Ag) derived from commensal bacteria, dietary Ags, and intestinal pathogens. Dendritic cells (DCs) are believed to be involved in the induction of tolerance to harmless Ags and in mounting protective immune responses to pathogens and, as such, to play key roles in regulating intestinal immune homeostasis. The characterization of classical DCs (cDCs) in the intestinal lamina propria has been under intense investigation in recent years but the use of markers , which are also expressed by intestinal MΦs, has led to some controversy regarding their definition. Here we review recent studies that help to distinguish cDCs subsets from monocyte-derived cells in the intestinal mucosa. We address the phenotype and ontogeny of these cDC subsets and highlight recent findings indicating that these subsets play distinct roles in the regulation of mucosal immune responses in vivo. Dendritic cells (DCs) are present throughout most tissues of the body where their main function is to sample self and foreign antigens (Ags), migrate to draining lymph nodes (LNs), and present antigenic peptides to naïve T cells, inducing their proliferation and differentiation. In so doing, DCs provide a critical link between the innate and adaptive immune systems. The intestinal lumen contains huge quantities of foreign Ag derived from commensal bacteria, dietary components, and occasional pathogenic microbes. Intestinal DCs are believed to play a central role in intestinal immune homeostasis, by inducing tolerance to harmless Ags, initiating protective immunity against intestinal pathogens, and contributing to intestinal diseases including celiac disease and the inflammatory bowel diseases .Classical DCs (cDCs) are found throughout the intestinal lamina propria (LP), in gut-associated lymphoid tissue (GALT) including Peyer's patches (PPs) and isolated lymphoid follicles (ILFs), and in intestinal draining LNs such as the mesenteric LNs (MLNs). In the MLNs, cDCs can be broadly classified into lymphoid tissue resident cDCs, which are thought to derive from circulating precursors that enter directly from the blood, and migratory cDCs that arrive in organized lymphoid tissues via afferent lymph after acquiring Ag in the intestinal LP. While lymphoid tissue cDCs have been studied extensively, the functions of the intestinal cDCs that migrate to MLNs are less well understood.The characterization of cDCs in the intestinal LP has been the subject of intense investigation and controversy, in part because the markers initially used to define them ) are also expressed by MΦs. In this review we discuss recent findings that have started to unravel the complexity of migratory intestinal cDCs, including assessment of the precursor-product relationships of transferred monocytes and cDC precursors, the identification of novel markers that appear to distinguish cDCs and monocyte-derived MΦs and new models that shed light on their in vivo functionality.+ MPs do not express the integrin αE chain CD103 and include all CX3CR1high cells that we, and others, have shown to be tissue resident MΦs + population also contains a subset of CX3CR1int cells that co-express F4/80 and can be generated from adoptively transferred Ly6Chigh monocytes. These cells represent intermediaries in the local differentiation of resident MΦs +MHCII+CD64− MPs are F4/80− and the majority of these cells express CD103. These CD103+ DCs have been held to be the prototypic migratory cDCs in the LP and can be divided into two main populations, CD103+CD11b+ and CD103+CD11b− cDCs  such as monocytes and monocyte-derived MΦs in steady-state mouse LP can all express MHCII and CD11c, cDCs can be distinguished by their lack of expression of the high-affinity IgG receptor FcγR1, CD64 +CD11b+ and CD103+CD11b− cDCs in the intestinal LP −CD11c+α4β7+B220+CCR9− cells termed premucosal DC progenitors was recently identified, which appeared, upon adoptive transfer, to home preferentially to the intestinal mucosa and give rise to both CD103+ cDC subsets + cDCs) and, in contrast to pre-cDCs, they also maintained the potential to develop into plasmacytoid DCs + cDC compartment in the steady state remains unclear. It is also unknown whether there are distinct precursors for CD103+CD11b+ and CD103+CD11b− cDCs within these putative progenitor populations, or whether the final differentiation state of the precursors is determined by signals they receive within the intestinal environment. In this regard it is of interest to note that the relative proportion of CD103+CD11b+ and CD103+CD11b− cDCs changes throughout the intestine, with CD103+CD11b+ cDCs comprising ∼70% of total CD103+ cDCs in the duodenum and CD103+CD11b− cDCs comprising ∼75% of total CD103+ cDCs in the colon +CD11b− cDCs dominate over CD103+CD11b+ cDCs in GALT +CD11b− cDCs are present in normal numbers in the intestine of RORγt−/− mice suggesting that they do not require GALT for their final differentiation and development According to current models, cDCs derive from committed cDC progenitors that develop in the bone marrow (BM) and travel via the blood to lymphoid organs and nonlymphoid tissues, where they differentiate into mature cDCs −CD103−CD11b+ MPs remains unclear, as most studies that have assessed precursor differentiation in the intestine to date have not used CD64 or other appropriate markers to distinguish DCs from CD11b+ MΦs. As a result, it was frequently concluded that many or all CD103−CD11b+ MPs were derived from monocytes high monocytes gave rise exclusively to F4/80+CD64+ MΦs and not CD64−CD11b+ MPs in the steady state, strongly indicating that the latter cells do not derive from monocytes int cells was observed in the LP following transfer of pre-cDCs intLy6Clow cells expressing the DC-specific transcription factor Zbtb46 in DSS colitis The identity of the precursors of CD64+CD11b− and CD103+CD11b+ cDC subsets is dependent on distinct transcription factors. Peripheral tissue CD103+CD11b− cDCs, including those located in the intestinal LP and PPs, are dependent on basic leucine zipper transcription factor ATF-like 3 (Batf3), inhibitor of DNA-binding 2 (Id2), and IFN-regulatory factor 8 (IRF8) +CD11b− cDCs therefore appear developmentally linked to IRF8/Batf3-dependent lymphoid tissue resident CD8α+ cDCs +CD11b− cDCs in LP express CD8α + lymphoid cDCs, including the chemokine receptor XCR1 +CD11b+ intestinal cDCs develop independently of IRF8, Id2, and Batf3 +CD4+ DCs fl/fl mice, whose DCs lack the transcription factor IRF4, referred to here as ΔIRF4 DC mice, have significantly reduced numbers of intestinal CD103+CD11b+ cDCs + DCs +CD11b+ cDC numbers in intestinal draining MLNs +CD11b+ cDCs isolated from ΔIRF4 DC mice died more rapidly in vitro compared with WT CD103+CD11b+ cDCs, and the few CD103+CD11b+ cDCs that remained in the MLNs of ΔIRF4 DC mice expressed Annexin V. Together these results indicate a key role for IRF4 in intestinal CD103+CD11b+ cDC survival , IRF2, and Rel-B-deficient mice +CD11b+ cDC homeostasis remains to be explored. The transcription factor requirements for CD103− cDCs remain to be determined.It is now clear that the development of the intestinal CD103survival , Fig. 2.−/− mice) have a severe reduction in both CD103+CD11b− and CD103+CD11b+ intestinal LP cDC numbers + cDCs, it has a greater impact on intestinal CD103+CD11b− cDCs +CD11b− cDCs + cDC homeostasis. The role of CSF-2 in intestinal cDC development is less clear. Although utilized for generating human and murine DCs from blood monocytes and BM, respectively, in vitro −/− and Csf2rb−/− × Csf2rb2−/−), CSF-2 KO mice (CSF2−/−) and in WT mice following administration of exogenous CSF-2 have suggested that the homeostasis of intestinal CD103+CD11b+ DCs depends upon CSF-2 −/− × Csf2rb2−/− mice show a defect in CD103 upregulation cDC development and homeostasis are regulated by the growth factors FMS-like tyrosine kinase 3 ligand (Flt3L) and granulocyte MΦ colony-stimulating factor (GM-CSF/CSF-2) − cDCs is currently unclear. Administration of exogenous Flt3L to mice results in a significant expansion of both CD103−CD11b− and CD103−CD11b+ cDCs in vivo − DCs are virtually absent from the small intestine (SI) of Flt3L−/− mice −/− mixed BM chimeras, the numbers of CD103−CD11b+ LP cDCs that derived from Flt3−/− BM were only partially reduced compared with WT BM-derived CD103−CD11b+ LP cDCs Whether CSF-2 or Flt3L is required for the development of the intestinal CD103+ cells could extend transepithelial dendrites (TEDs) through tight junctions in the epithelium to capture luminal bacteria + cells was observed ex vivo in the terminal ileum of CX3CR1+/gfp mice and this appeared to be CX3CR1-dependent SalmonellaSalmonella usually gain entry to the intestine primarily via microfold cells in PPs and ILFs Salmonella nor noninvasive Aspergillus spores + cells in the intestine are tissue resident MΦs, rather than cDCs In their role as professional antigen presenting cells, intestinal DCs survey the content of the intestine and transport Ags to the MLNs where they initiate adaptive immune responses. Exactly how intestinal cDCs gain access to luminal Ag and whether intestinal cDC subsets sample soluble and particulate luminal Ags by similar routes is currently unclear. Initial studies suggested that CD11c+/yfp x CX3CR1+/gfp mice to allow distinction between CX3CR1+CD11c+ MΦs and CX3CR1−CD11c+ cDCs. McDole et al. −YFPhi cells. This route is unlikely to be involved in uptake of particulate Ag, such as luminal bacteria, as beads >0.02 μm diameter could not transit through GAPs. Similarly, Farache et al. + DCs within the epithelium itself, whose numbers increased in a CD103-and CCR6-independent manner following luminal administration of Salmonella. These intraepithelial CD103+ cDCs could be visualized extending intraepithelial dendrites into the lumen sampling Salmonella+ cDCs are present in the epithelium at steady state, but increase during inflammation high MΦs + DCs were poor at capturing soluble Ag, compared with CX3CR1high MΦs, which formed occasional TEDs. While the reason for the discrepant findings between the two studies regarding uptake of soluble Ag is not immediately apparent, Farache et al. −YFPhigh cells whereas Farache et al. −YFPint cells and provided data to suggest that YFPhigh cells were in fact CX3CR1high MΦs. Clearly, further studies are required to assess the mechanisms by which intestinal cDCs acquire soluble and particulate derived luminal Ag, and whether the different LP cDC subsets perform overlapping or distinct roles in these processes.Two recent multiphoton microscopy studies used refined methods for identifying hematopoietic Ag sampling cells in the intestine + cDCs derive from the intestinal LP + DCs as the major migratory cDC population in intestinal lymph. More recently, by cannulating thoracic duct lymph from mesenteric lymphadenectomized mice, Cerovic et al. +MHCII+ cells in steady-state intestinal lymph are CD103+ cDCs. CD103+CD11b+ and CD103+CD11b− cDCs were present in intestinal steady-state lymph at a ratio of ∼1.5–1, approximating that found in the MHCIIhigh migratory cDC population in MLNs. The remaining CD11c+MHCII+ cells were CCR7-expressing CD103− cDCs, the majority of which were CD11b+After acquiring Ag, intestinal cDCs migrate to draining MLNs in a CCR7-dependent manner high MΦs were not found in intestinal draining afferent lymph in the studies by Schulz et al. −CX3CR1high cells in intestinal lymph in the steady state. While the reason for these discrepant findings is currently unclear, it is unlikely to be due to differences in the microbiota, as similar numbers of putative CX3CR1high cells were observed in steady-state lymph following treatment of mice with broad spectrum antibiotics. One possible explanation is that the CX3CR1high cells detected by Diehl et al. int cells and represent the CD103− CD11b+ cDC subset of bona fide cDCs identified by Cerovic et al. Consistent with their lack of CCR7 expression and their absence from MLNs, CX3CR1+ cDCs isolated from the MLNs shortly after oral administration of soluble Ag induce Ag-specific proliferation of CD4+ and CD8+ T cells ex vivo + T cells in vitro + DCs and CD103+CD8α+ DCs in other peripheral tissues +CD11b− LP DCs appear superior to CD103+CD11b+ DCs at cross-presenting Ag to CD8+ T cells in vitro + and CD8+ T cells in MLN in vivo is currently unclear, but could potentially be explored utilizing mice whose DCs are deficient in the distinct transcription factors described above.Migratory intestinal LP cDCs carry soluble and particulate Ags to the MLNs + cDCs display an enhanced ability to induce CCR9 and α4β7 on responding T cells in vitro, compared with DCs isolated from other tissues + cDCs were shown to have an enhanced ability to generate the vitamin A metabolite, retinoic acid (RA) − cDCs in intestinal lymph metabolize vitamin A and induce gut-homing receptors on responding T cells in vitro, a finding that is consistent with the idea that migratory cDCs are imprinted with the ability to metabolize Vitamin A from signals they receive in the SI mucosa , respectively (for reviews see + regulatory T (iTreg) cells + Treg cells, and MLNs are all required for the induction of tolerance to soluble luminal Ag + cDCs display an enhanced capacity to generate iTreg cells from naïve precursors in vitro compared with other cDC subsets, in part through their ability to generate RA . Despite these findings, Batf3−/− mice, which lack CD103+CD11b− cDCs, have normal numbers of intestinal FoxP3+ Treg cells +CD11b+ cDCs have normal numbers of FoxP3+ Treg cells in the SI and colon + and neuropilin+ Treg cells when compared with WT mice The MLN is a site that favors the differentiation of inducible FoxP3erate RA . Mechani+ T cells with specificities against enteric microbial Ags and other luminal content + T cells include Th1 cells and the largest population of Th17 cells in the body. While it was thought initially that intestinally derived CD103+ cDCs preferentially induced Treg-cell responses +CD11b+ and CD103+CD11b− cDCs can drive Th1 polarization +CD11b− SI DCs are effective at driving IFN-γ production by CD8+ T cells  and MyD88 signaling has been suggested +CD11b+ and CD103+CD11b− cDCs, which derive from committed cDC precursors; and a more recently described CD103−CD11b+ population whose functions and origins remain to be characterized fully. The recent development of specific knock out mouse models with selective reduction in individual intestinal cDC populations has also allowed researchers to start addressing the functions of individual intestinal cDC subsets in the regulation of adaptive immune responses in vivo. Initial studies with these mice have demonstrated that the previous assertions that CD103+ cDCs were inherently tolerogenic are incorrect, and that, for example intestinal CD103+CD11b+ cDCs play an important role in driving Th17 differentiation in the MLNs + IRF4-expressing cDCs that we speculate are equivalent to murine intestinal CD103+CD11b+ DCs, and a minor population of CD141+ cDCs that are likely equivalents of CD103+CD11b− DCs − cDCs exists in the human intestine. Determining whether these human intestinal cDC subsets function in a similar way to their murine counterparts will be important if these cells are to be exploited for the treatment and/or prevention of human inflammatory bowel diseases and in the development of mucosal vaccines.Intestinal DCs are a crucial link between the innate and adaptive immune responses and thus provide an attractive target for therapeutic manipulation of the intestinal immune response. Although similar levels of CD11c and MHCII expression by intestinal DCs and MΦs have impeded reliable characterization of DCs in the past, recent advances in phenotyping now allow these cells to be more precisely discriminated. As a result, it is now believed that there are at least three populations of bona fide DCs in the murine intestinal LP: two CD103-expressing cDC subsets, CD103


T7 RNA polymerase synthesizes, in addition to run-off products of precise length, transcripts with an additional non-base-paired nucleotide at the 3′-terminus (N + 1 product). This contaminating product is extremely difficult to remove. We recently characterized the single-subunit RNA polymerase from marine cyanophage Syn5 and identified its promoter sequence. This marine enzyme catalyses RNA synthesis over a wider range of temperature and salinity than does T7 RNA polymerase. Its processivity is >30 000 nt without significant intermediate products. The requirement for the initiating nucleotide at the promoter is less stringent for Syn5 RNA polymerase as compared to T7 RNA polymerase. A major difference is the precise run-off transcripts with homogeneous 3′-termini synthesized by Syn5 RNA polymerase. Therefore, the enzyme is advantageous for the production of RNAs that require precise 3′-termini, such as tRNAs and RNA fragments that are used for subsequent assembly.The enzyme predominantly used for  RNA transcripts synthesized by RNA polymerase The RNA polymerase encoded by bacteriophage T7 is used widely to synthesize RNA molecules . These rSynechococcus and Prochlorococcus are estimated at 1030 particles in the oceans  were from New England Biolabs. Radiolabeled nucleotides were from Perkin Elmer. RNA Clean & Concentrator™-5 kit was from ZYMO Research. RNaseOUT™ Recombinant Ribonuclease Inhibitor was from Invitrogen. Yeast inorganic pyrophosphatase was from Sigma-Aldrich. Nucleoside-5′-triphosphates were from USB except for 5-methylcytidine-5′-triphosphate and pseudouridine-5′-triphosphate that were from Trilink. Escherichia coli BL21(DE3) cells over-producing the protein were cultured in 2 L LB medium containing 50 µg/ml kanamycin at 37°C until they reached an OD600 of 1.2. The gene for Syn5 RNA polymerase was induced by the addition of 0.5 mM IPTG at 16°C and incubation was continued for 3 h. The cells were harvested, resuspended in 50 mM sodium phosphate, pH 8.0, 100 mM NaCl and lysed by three cycles of freeze-thaw in the presence of 0.5 mg/ml lysozyme. NaCl at a final concentration of 2 M was then added to the lysed cells and the cleared lysate was collected by centrifugation. Ni-NTA agarose (2 ml) was added to the clear lysate and gently mixed at 4°C overnight. The resin was then washed with 30 ml of Wash Buffer . Syn5 RNA polymerase was eluted from the column using a 60 ml gradient (20–250 mM imidazole) in Wash Buffer. The majority of His-tagged Syn5 RNA Polymerase eluted at 20–100 mM imidazole. Fractions were analysed on SDS-PAGE gels and those in which Syn5 RNA polymerase was at least ∼80% of the total protein (40–100 mM imidazole elution) were collected and concentrated with Amicon Ultra-15 Centrifugal Filter Units (Millipore) to 1 ml. The concentrated sample was directly loaded onto a 200 ml preparative Superdex S200 column. The gel filtration buffer contained 20 mM Tris-HCl pH 7.5, 2 M NaCl, 0.5 mM DTT and 0.5 mM EDTA. Fractions were analysed on SDS-PAGE gels and those fractions that contained homogenous Syn5 RNAP were pooled. The pooled fractions were concentrated by Amicon Ultra-15 Centrifugal Filter followed by dialysis against Final Dialysis Buffer  and stored at –20°C. Dilutions for enzyme assays were made using the Final Dialysis Buffer. In this buffer the protein remains soluble at concentrations up to 400 µM and we did not try to concentrate the protein further. The yield of His-tagged Syn5 RNA polymerase following this procedure was 5 mg protein per gram of wet cells. His-tagged T7 RNA polymerase was purified from E. coli BL21(DE3) harboring plasmid pET28a with the T7 RNA polymerase gene inserted between the NdeI and HindIII sites, using Ni-NTA and DEAE ion-exchange chromatography.His-tagged Syn5 RNA polymerase was produced from a pET24 vector harboring the His-tagged Syn5 RNA polymerase gene between the NdeI and NotI sites  and puriATTGGGCACCCGTAAGGG-3′ or T7 5′-TAATACGACTCACTATAGGG-3′) was inserted between the BamHI and XbaI sites of plasmid pUC19 to form pUC19-S5P or pUC19-T7P. The templates for run-off tRNAArg synthesis were formed by annealing complementary synthesized oligonucleotides for each of the two sequences: S5P-tDNAR: 5′-GCCATTGGGCACCCGTAAGCATCCGTAGTTCAGCTGGATAGAGTACTCGGCTACGAACCGAGCGGTCGGAGGTTCGAATCCTCCCGGATGCACCA-3′ and T7P-tDNAR: 5′-GCCTAATACGACTCACTATAGCATCCGTAGTTCAGCTGGATAGAGTACTCGGCTACGAACCGAGCGGTCGGAGGTTCGAATCCTCCCGGATGCACCA-3′. These two templates are identical except for their promoter region. The plasmid pET24 harboring the Syn5 RNA polymerase gene (pET24-S5RNAP) containing a single Syn5 promoter in the RNA polymerase gene and a single T7 promoter preceding the gene was used for the filter binding assay shown in ATTGGGCACCCGTAAG+1(or A+1 or C+1 or T+1) G+2(or A+2 or C+2 or T+2) AGAACCTTAAGGTTTAACTTTAAGACCCTTAAGTG-3′.DNA templates for transcription assays were either plasmids or dsDNA fragments constructed using synthesized oligonucleotides. To test the processivity of Syn5 and T7 RNAP, a single Syn5 or T7 promoter containing three guanosine residues downstream of the promoter (Syn5 5′-3H-ATP (20 cpm/pmole), 1.5 U/µl RNaseOUTTM, 50 nM Syn5 or T7 RNA polymerase and 4 nM plasmid pET24-S5RNAP were incubated at 24°C. After various times the reactions were stopped by the addition of 20 mM EDTA. The mixtures (4 µl) were then loaded onto Whatman DE81 filter paper disks and the disks were washed to remove unincorporated 3H-ATP. The amount of bound 3H-AMP, corresponding to nucleotides incorporated into newly synthesized RNA, was measured using a scintillation counter. The data were analysed using Prism software.To compare the specific activities of purified RNA polymerases B, reacti2, 2 mM spermidine, 10 mM DTT, 600 µM ATP, GTP, CTP and UTP , 1.5 U/µl RNaseOUTTM recombinant ribonuclease inhibitor, 0.04 U/µl yeast inorganic pyrophosphatase, 10 nM pUC19-Syn5P3G or pUC19-T7P3G, 10 nM Syn5 or T7 RNA polymerase and varying amounts of KCl as indicated in the figure and described in the figure legend. After incubations for 1 h at 24°C, one unit of DNase I was added to each reaction mixture and incubated for an additional 15 min at 37°C to remove the DNA templates. For Reaction mixtures (10 µl) for the comparison of apparent processivity of Syn5 and T7 polymerases  containe2, 2 mM spermidine, 10 mM DTT, 200 µM ATP, GTP and UTP, 10 µM [α-32P]CTP, 1.5 U/µl RNaseOUTTM recombinant ribonuclease inhibitor, 0.5 µM DNA templates S5P-tDNAR or T7P-tDNAR, 100 nM Syn5 or T7 RNA polymerase, and varying amounts of KCl as noted in the figure were carried out at either 16, 24 or 37°C for 30 min before adding of one unit of DNase I to each reaction mixture, and then incubated for an additional 15 min at 37°C. Reactions were then terminated by the addition of 4 µl of loading dye containing 95% formamide and 40 mM EDTA. Samples were then heated at 90°C for 1 min and 4 µl of each sample was loaded onto a 15% TBE-Urea denaturing polyacrylamide gel. After electrophoresis, gels were dried and the radioactivity was analysed using a Fuji BAS 1000 Bioimaging Analyzer.In order to compare tRNA synthesis by Syn5 and T7 RNA polymerases , reactio2, 2 mM spermidine, 10 mM DTT, 200 µM ATP, GTP and UTP, 10 µM [α-32P]CTP, 1.5 U/µl RNaseOUTTM recombinant ribonuclease inhibitor, 0.5 µM of DNA templates S5P-tDNAR or T7P-tDNAR, and varying amounts of Syn5 or T7 RNA polymerase were incubated at 37°C for 30 min before adding of one unit of DNase I to each reaction mixture, and then incubated for an additional 15 min at 37°C. Reactions were then terminated by the addition of 4 µl of loading dye containing 95% formamide and 40 mM EDTA. Samples were then heated at 90°C for 1 min and 4 µl of each sample was loaded onto a 6% TBE-Urea denaturing polyacrylamide gel. After electrophoresis, gels were dried and analysed using a Fuji BAS 1000 Bioimaging Analyzer. For the results shown in 2, 2 mM spermidine, 10 mM DTT, 600 µM ATP, GTP CTP and UTP, 1.5 U/µl RNaseOUTTM recombinant ribonuclease inhibitor, 0.04 U/µl yeast inorganic pyrophosphatase, 0.5 µM of DNA templates S5P-tDNAR or T7P-tDNAR, and 100 nM Syn5 or T7 RNA polymerase. Reaction mixtures were incubated at 37°C for 2 h, followed by the addition of 10 unit of DNase I to each reaction mixture and an additional incubation of 20 min at 37°C. RNA products were then purified with RNA Clean & Concentrator™-5 kit or traditional phenol-chloroform extraction/ethanol precipitation with resuspension into 30 µl H2O. RNA was quantified by measurement of OD260 using NANOPHOTOMETER (IMPLEN). Syn5 RNA polymerase reaction mixture (5 µl) and T7 RNA polymerase reaction mixture (1 µl) were mixed with denaturing dye, heated at 90°C for 1 min and loaded onto a 15% TBE-Urea denaturing polyacrylamide gel. RNAs were visualized by staining with ethidium bromide. Native tRNAArg purified from E. coli  and UTP (or pseudo-UTP), 1.5 U/µl RNaseOUTTM recombinant ribonuclease inhibitor, 0.04 U/µl yeast inorganic pyrophosphatase, 10 nM linearized (NotI treated) pET24-Syn5RNAP, plus or minus 160 mM KCl, and 10 nM Syn5 or T7 RNA polymerase. Reactions with Syn5 RNAP were carried out at 24°C and reactions with T7 RNAP were carried out at 37°C. RNA products were then purified with RNA Clean & Concentrator™-5 kit using a final solution of 10 µl, and 5 µl of each sample was mixed with 5 µl denaturing RNA loading dye. Samples were then heated at 65°C for 5 min and loaded onto a 1.6% TAE agarose gel. RNA products were visualized by staining with ethidium bromide. For 2, 2 mM spermidine, 10 mM DTT, 200 µM ATP, GTP and UTP (or pseudo-UTP), 10 µM [α-32P]CTP, 1.5 U/µl RNaseOUTTM recombinant ribonuclease inhibitor, 0.5 µM of DNA templates S5P-tDNAR or T7P-tDNAR, and varying amounts of Syn5 or T7 RNAP. Reaction mixtures were incubated at 24°C (for Syn5 RNAP) or 37°C (for T7 RNA polymerase) for 30 min before adding one unit of DNase I to each reaction mixture and incubation for an additional 15 min at 37°C. Reactions were then terminated by the addition of 4 µl of loading dye containing 95% formamide and 40 mM EDTA. Samples were then heated at 90°C for 1 min and 4 µl of each sample was loaded onto a 6% TBE-Urea denaturing polyacrylamide gel. After electrophoresis, gels were dried and analysed using a Fuji BAS 1000 Bioimaging Analyzer.In order to examine the incorporation of modified nucleotides by Syn5 and T7 RNA polymerases , reactio2, 2 mM spermidine, 10 mM DTT, 200 µM GTP, CTP and UTP, 10 µM [α-32P]ATP, 1.5 U/µl RNaseOUTTM recombinant ribonuclease inhibitor, 1 µM DNA templates  was then added to the mixture. Samples were heated at 90°C for 1 min and were loaded onto a 15% TBE-Urea denaturing polyacrylamide gel. RNA products were visualized by staining with ethidium bromide.To examine the stability of Syn5 RNA polymerase over long incubation times , reactio2, 0.5 mM DTT, 4 mM ATP, 0.2 µM RNA (purified native E. coli tRNAArg (Arg transcripts from Syn5 or T7 RNA polymerase reaction using RNA Clean & Concentrator™-5 kit), 15 µM [3H] arginine (15 Ci/mmol) and 4 µM E. coli total aminoacyl-tRNA synthetases were incubated at 37°C for the indicated times. Each reaction mixture (5 µl) was spotted onto Whatman DE(81) filters and the reactions were terminated by washing the filters in 10% TCA solution containing 0.5 mM arginine. The filters were washed twice with 10% TCA and once with ethanol, and then dried. The amount of amino acid charged to tRNA was determined by measuring the radioactivity remaining bound to the filters.tRNA aminoacylation assay was performed as described previously . Reactio tRNAArg  or purifE. coli cells yielded active enzyme suitable for preliminary biochemical characterization and identification of its promoter. However, the low solubility and yield of protein, the tedious purification procedure and the contamination of nucleases led us to improve the purification procedure. Based on the properties of other marine viral proteins we have been characterizing we have modified and improved the purification procedure for Syn5 RNA polymerase. We improved the yield of protein more than 10-fold by using an unusually high concentration of NaCl (2 M) during chromatography . Additional benefits of this procedure are that the time required for purification is reduced by a factor of two and the specific activity of the enzyme is increased 2-fold  and pseudouridine (Ps-U) are introduced into mRNA to improve its function in vivo . We examin vivo . When CT present A. T7 RNA present B. Using T7 RNA polymerase has a stringent requirement for GTP as the initiating nucleotide and a strong preference for GG as the initiating sequence ,23. We fLong incubation times are often required when T7 RNA polymerase is used to obtain larger amounts of transcripts. Over a 4-h incubation at 37°C, Syn5 RNA polymerase exhibits greater stability than T7 RNA polymerase during the synthesis of run-off transcripts as judged by the amount of transcripts at 4 h .


Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in −/−Setx revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the  Setx (coding for senataxin) is part of a subgroup of autosomal recessive ataxias characterized by defects in genes responsible for the recognition and/or repair of damage in DNA. Cells from these patients are characterized by oxidative stress and are defective in RNA processing and termination of transcription. Recent data suggest that senataxin is involved in coordinating events between DNA replication forks and ongoing transcription. To further understand the role of senataxin, we disrupted the Setx gene in mice and demonstrated its essential role in spermatogenesis during meiotic recombination and in meiotic sex chromosome inactivation (MSCI). In the absence of senataxin, DNA double-strand breaks persist, RNA:DNA hybrids (R-loops) accumulate, and homologous recombination is disrupted. Senataxin localised to the XY chromosomes during pachytene. This was dependent on Brca1, which functions early in MSCI to recruit DNA damage response proteins to the XY body. In the absence of senataxin, there was incomplete accumulation of DNA damage response proteins on the XY chromosomes and no MDC1-dependent diffusion of ATR to the broader XY chromatin. The end result was a defect in MSCI, apoptosis, and a failure to complete meiosis.Ataxia with oculomotor apraxia type 2 (AOA2) caused by a defect in the gene  SETX, is also associated with amyotrophic lateral sclerosis 4 (ALS4), an autosomal dominant juvenile-onset form of ALS Saccharomyces cerevisiae splicing endonuclease 1 protein (Sen1p) which possesses helicase activity and is involved in RNA processing, transcription and transcription-coupled DNA repair 2O2, camptothecin and mitomycin C and have elevated levels of oxidative DNA damage Ataxia oculomotor apraxia type 2 (AOA2), a severe form of autosomal recessive cerebellar ataxia (ARCA) is characterised by progressive cerebellar atrophy and peripheral neuropathy, oculomotor apraxia and elevated α-fetoprotein Senataxin also plays a role in transcription regulation by its ability to modulate RNA Polymerase II (Pol II) binding to chromatin and through its interaction with proteins involved in transcription Setx knockout mouse model to investigate further the role of senataxin. Our data revealed that this protein is essential for male meiosis, acting at the interface of transcription and meiotic recombination, and also in the process of meiotic sex chromosome inactivation (MSCI).We generated a −/−Setx mice were produced using a Cre-LoxP system to delete exon 4 as outlined in Setx heterozygotes produced all 3 genotypes  as expected (Setx gene was confirmed by RT-PCR and the absence of Setx mRNA in the knockout mouse as compared to the wild type (+/+Setx mice but senataxin was not immunoprecipitated from −/−Setx extracts (−/−Setx mice (data not shown). Using a simple phenotypic scoring system that has been employed to evaluate mouse models of cerebellar ataxia +/+Setx and −/−Setx animals  revealed no overt phenotypic difference from their wild type littermates, with normal structure and presence of follicles at all stages and an ability to ovulate  embryos in order to compare their viability. A greater than 3.5-fold reduction in the yield of 0.5 dpc for −/−Setx was observed compared to wildtype animals (10–20 0.5 dpc embryos for −/−Setx compared to 50–70 for wild types). In addition, only 23% of viable embryos were obtained at 0.5 dpc for −/−Setx and most of the viable ones did not survive in culture, indicating that −/−Setx females have a reduced fertility.Multiple attempts to breed  ovulate . However−/−Setx with those from wild type mice. −/−Setx testes were smaller in size (50–60% reduction in size) than wild-type littermates  −/−Setx, indicating a block at the pachytene-diplotene transition  at pachytene stage in −/−Setx was also observed. To investigate the cause of the meiotic defect in more detail, we also determined the expression of spermatogenesis stage-specific markers  and throughout meiosis  up to the round spermatid stage showed only a small reduction in expression in −/−Setx as compared to +/+Setx. Markers for haploid mature germ cells Prm1, Prm2 and Tnp1 showed markedly reduced expression in −/−Setx compared to +/+Setx  catalysed by a type II topoisomerase-like protein Spo11 Setx−/− . At pachne stage . Staininabelling .+/+Setx with few foci labelling pachytene chromosomes  and involves the participation of various DNA repair factors including RPA, Dmc1 and Rad51 omosomes . In cont antibody −/−Setx, indicating heterogeneity in R-loop accumulation , Fthl17 (1.4 fold), Tktl1 (1.36 fold) and Ube1x (1.65 fold) genes was observed for −/−Setx mice compared to +/+Setx mice. This was also true for several Y-linked genes that include Ube1y (2 fold) and Rbmy (2.14 fold) indicating that MSCI is defective in −/−Setx. Normal expression for autosomal genes Actinb, Dazl, and Gapdh was also observed, confirming the specific nature of MSCI  lined the axes of these chromosomes. We did not detect a direct endogenous interaction between senataxin and Brca1, suggesting that the Brca1-dependent localisation of senataxin to the XY chromosome may be indirect and mediated by other DNA damage response proteins involved in MSCI. On the other hand, Brca1 still localised to the sex chromosomes in −/−Setx mutant mice. However, this was incomplete since it was excluded from part of the XY structure in pachytene. This is consistent with a recent report that the X and Y chromosomes have different patterns of incorporation and release of recombination/repair and MSCI-related factors during different stages of meiosis −/−Setx spermatocytes due to a block earlier in pachytene.Senataxin specifically localises to the XY body in pachytene stage partially co-localising with Brca1, MDC1 and ATR suggesting that it might have a role in MSCI. While Brca1 lines the unsynapsed axes of the XY chromosomes, senataxin is associated with these chromosomes but also has a more diffused distribution on chromatin. Prior to MSCI initiation, Brca1 is targeted to the unsynapsed axial elements of the X and Y chromosomes where it remains  XY body . It seem−/−Setx suggests that meiosis only proceeds from early to mid pachytene in these mice and that ATR re-localisation is dependent on senataxin . These data suggest that the major clinical neurodegenerative phenotype seen in AOA2 patients is more likely to be due to a more general defect in RNA processing leading to reduced transcription fidelity rather than a failure to resolve R-loops. Altogether, these findings reveal a complex and coordinated network between transcription, RNA processing, and DNA repair pathways  were subsequently micro-injected into C57BL6/129Sv mice blastocysts to generate chimeras. Excision of the Neo cassette was obtained by crossing the chimeras with a Cre deleter stain to generate −/−Setx mice containing only a LoxP site.To disrupt the 5′-TTTAAGGAACAGTGCTGC-3′, In3R: 5′-ATGAAGCAGGTAGGATT-3′ and LoxPR: 5′-CGAAGTTATATTAAGGGT-3′. PCR Cycling conditions were as follows: 35 cycles, denaturation at 95°C for 30 sec, annealing at 49°C for 30 sec, extension at 72°C for 1 min, with a final cycle and extension of 7 min at 72°C. Two PCR products were generated, a wild-type PCR product of 600 bp, and the targeted PCR product of 339 bp. PCR products were electrophoresed at 100 V for 30 min on 2% TAE Agarose  stained with Ethidium bromide and visualised with UV transillumination using a GelDoc XR .The mice were weaned at 21 days post-partum and ear clipped for identification. Genotyping was carried out by PCR on genomic DNA isolated from tail tips. Tail tips were lysed in directPCR Lysis eagent  as recommended by the manufacturer. The primers used were In3F: Testes from adult (35-day-old), 4 months, 8 months and 12 month-old mice were collected and fixed in PBS buffered 10% formalin, embedded in paraffin block and sectioned at 4 µm. Sections were stained with Hematoxylin and Eosin (H&E) and Toluidine blue. Slides were examined under light microscope and then scanned using Scanscope CS system . Images corresponding to ×10 and ×20 magnification were captured and assembled into Adobe Photoshop 7 .2 , 20 µM dNTPs, 1 µM of each primer, and 5 µl of AmpliTaq Gold DNA Polymerase . The primer pairs used for gene expression analysis are described in Total RNA was isolated from 35-day-old wild type and knockout mice testes using the RNeasy mini kit  according to the manufacturer's protocol. RNA concentrations were determined by UV spectrophotometry using a Nanodrop ND-1000 . cDNA was made from 5 µg of purified RNA. Briefly, RNA was mixed with 1 µl of random hexamer primers (Bio-Rad Laboratories Inc. USA), 1 µl of 10 mM dNTP mix and DEPC-treated water up to a 14 µl volume. The mixture was heat-denatured at 65°C for 5 min. 4 µl of First Strand buffer , 1 µl of 1 mM DTT, 1 µl of RNAaseIN , and 1 µl of SuperScriptIII reverse transcriptase enzyme  was added to the mixture, and incubated for 10 min at 25°C, then 60 min at 50°C, 15 min at 70°C, and chilled on ice. 1 µl of RNAse H was subsequently added to each tube and incubated for 20 min at 37°C, followed by heat inactivation for 20 min at 65°C. The resulting cDNA were stored at −20°C prior to use. Gene expression analysis was performed by PCR in a 2720 Thermal Cycler . Reactions (25 µl) contained 14.5 µl of sterile water, 50 ng of cDNA template, 1× PCR Buffer II , 2.5 mM MgClTestes from 35 day-old mice were collected and ground with a pestle to disrupt their structure and lysed for 1 h at 4°C on a rotating wheel with lysis buffer . Cellular debris were pelleted by centrifugation at 16,100×g at 4°C for 10 min, and protein concentration was determined using Lowry Assay . 2 mg of total cell extract were pre-cleared with 50 µl of a mixture of 1∶30 protein G+A beads  for 3 hours at 4°C on a rotating wheel. Extract were centrifuged for 5 min at 2000×g, beads were removed, and 20 µg of anti-human senataxin antibody (Ab1/Ab-3) was added to the extract. Extracts and antibody were incubated overnight at 4°C on a rotating wheel to allow binding of the antibody to mouse senataxin. The next day, 50 µl of protein G+A beads were added to the extract and incubated for 1 h at 4°C on a rotating wheel. The immunoprecipitate was subsequently washed 3 times with lysis buffer and the beads were resuspended in gel loading buffer and separated on 5% SDS-PAGE at constant current (20 mA per gel) for 1.5 h. Once separated, proteins were transferred onto a nitrocellulose membrane  for 1 h at 4°C with constant voltage (100 Volts). Immunoblotting with anti-senataxin (Ab-1) antibody was performed using standard procedure as previously described All spreads were made from testes collected from adult 35-day-old mice or at day 16, 20 and 22 post partum. Briefly, testes were decapsulated, finely chopped and rinsed in GIBCO DME medium . Large clumps were removed by centrifugation at 6780×g for 5 min at room temperature. The remaining supernatant was centrifuged to pellet the cell suspension and mixed with 0.1M sucrose and spread onto glass slides pre-wetted with 1% paraformaldehyde and 0.1% Triton X-100 in PBS. Cells were fixed on the glass slides for 2 h at room temperature. The slides were subsequently washed with PBS and air-dried in the presence of a wetting agent, 1∶250 Kodak Photo-Flo 200 . Once dried, spreads were stored at −80°C. For immunostaining, slides, were rehydrated in dH20, and blocked in blocking buffer  for 30 min at room temperature. Spreads were incubated with primary antibodies overnight at 4°C in a humidified chamber. Primary antibodies used included anti-SCP3 , anti-SCP1-DyeLight conjugated , anti-γH2AX , anti-Rad51 , anti-Dmc1 , anti-Mlh1 , anti-ATR , anti-senataxin  following the manufacturer's instructions. Slides were visualised under a fluorescent microscope and images were captured as previously described. The objective employed was a Zeiss Plan Neofluar ×10/0.30 (×10 magnification). For double staining, TUNEL was carried out first followed by immunostaining as described below.Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids. TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signaling cascades. The assay relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase (TdT), an enzyme that will catalyze the addition of Fluorescein-labeled dUTP. Paraffin sections were dewaxed and rehydrated with Shandon Varistain Gemini ES . TUNEL assay was performed using the Fluorescence −/−Setx testes sections with RNAse H .Slides with tissue sections were dewaxed and enzymatic antigen retrieval was performed by incubating the sections with 1∶10 Trypsin dilution in PBS for 20 min at 37°C. Slides were washed 3 times for 5 min with PBS at room temperature for 5 min each. Tissues sections were blocked in  for 1 h at room temperature. Slides were incubated with anti-R-loop  in situ Proximity Ligation Assay (PLA)  on wild type (+/+Setx) spermatocytes spreads. PLA allows the monitoring of protein interactions and modifications with high specificity and sensitivity. Protein targets can be readily detected and localized with single molecule resolution and objectively quantified in unmodified cells and tissues. Utilizing only a few cells, sub-cellular events, such as transient or weak interactions are revealed in situ. Two primary antibodies raised in different species recognize the target antigens of interest. Species-specific secondary antibodies, called PLA probes, each with a unique short DNA strand attached to it, bind to the primary antibodies. When the PLA probes are in close proximity, the DNA strands can interact through a subsequent addition of two other circle-forming DNA oligonucleotides. After joining of the two added oligonucleotides by enzymatic ligation, they are amplified via rolling circle amplification using a polymerase. After the amplification reaction, several-hundredfold replication of the DNA circle has occurred, labeled complementary oligonucleotide probes highlight the product. The resulting high concentration of fluorescence in each single-molecule amplification product is visible as a distinct bright spot when viewed with a fluorescence microscope. The assay was performed according to the manufacturer's protocol using rabbit anti-mouse Brca1 , sheep anti-senataxin  and goat anti-ATR antibody  antibodies and the corresponding anti-goat PLA Probe MINUS and anti-rabbit PLA probe PLUS. Identification of pachytene stage spermatocytes was determined by counterstaining with SCP3 antibody. PLA was also carried out on −/−Setx spermatocytes spreads as a negative control. Slide mounting and imaging was performed as described above.To investigate a possible interaction between Brca1, ATR and senataxin we employed Figure S1−/−Setx mice. Neurological phenotype and ataxia were examined according to the phenotypic scoring system developed by Guyenet et al. 2010 +/+Setx and −/−Setx at each age was calculated.Absence of neurological phenotype and ataxia in (TIF)Click here for additional data file.Figure S2−/−Setx mice. We examined the histology of −/−Setx ovaries at 8 months of age to determine whether they exhibited similar signs as in the human patients −/−Setx mouse ovaries. Pre-antral and antral follicles (F) are present in all sections and many follicles contain oocytes (asterisks). All sections also contain corpora lutea (CL), suggesting ovulation has occurred and the hypothalamic-pituitary-gonadal endocrine axis is intact in these mice.  Scale bar, 200 µm.Normal ovary structure in (TIF)Click here for additional data file.Figure S3Setx-deficient mice. A. Testis of a 35-day old +/+Setx mouse showing normal testis histology. Scale bar, 200 µm. B. Testis of a 35-day-old +/+Setx mouse. Scale bar, 100 µm. Interstitial (Leydig) cells lie between the seminiferous tubules (LD). C. Seminiferous tubule from a +/+Setx mouse at higher magnification. The tubule contains primary spermatocytes undergoing mitosis (short arrow), and elongated spermatids (asterisks) towards the lumen of the tubule, in which few sperm tails (T) can be seen. LD cells lie between the tubules. Sertoli cell nuclei (SCN) are present on the basement membrane of the tubule. D. Testis of a 35-day-old −/−Setx mouse showing seminiferous tubules with smaller diameter and disrupted spermatogenesis. Scale bar, 200 µm. E. Testis of a 35-day-old −/−Setx mouse, showing tubules at different stages of the seminiferous epithelial cycle. In some stages, spermatocytes (asterisks) are abundant. In other stages of the seminiferous cycle spermatogonia and some spermatocytes (Sp) line an almost empty tubule. Scale bar, 100 µm F. Seminiferous tubule of a 35-day-old −/−Setx mouse testis, showing spermatogonia (Spn) and Sertoli cell nuclei (SCN) lining the basement membrane of the tubule. The lumen appears to contain remnants of Sertoli cell cytoplasm. Elongated spermatids and spermatozoa are completely absent. G–J. Male +/−Setx mice exhibit signs of reduced fertility at 8 months of age. G. Seminiferous tubules contain few mature spermatozoa and large inclusions (arrowheads), suggesting increased apoptosis of spermatogenic cells. The seminiferous epithelium contains post-meiotic round and elongating spermatids. Scale bar, 200 µm. H. Seminiferous tubule from an 8-month +/−Setx mouse testis; Scale bar, 100 µm. The seminiferous epithelium contains all stages of spermatogenesis, including round (R) and elongating spermatids (asterisks) and there are sperm tails (T) in the lumen. I. Seminiferous tubule from an 8-month +/−Setx mouse testis. Scale bar, 100 µm. The seminiferous epithelium appears disrupted in places (arrow), with few round or elongated spermatids and debris in the lumen. J. In this seminiferous tubule from 8-month +/−Setx mouse testis there are few round (R) or elongated spermatids (asterisks) present and the lumen contains cell debris (arrow). Scale bar, 100 µm.Testis histology of (TIF)Click here for additional data file.Figure S4−/−Setx mice. A. Schematic representation of the various stages of spermatogenesis and the temporal expression of stage-specific makers as previously reported +/+Setx and −/−Setx testes. Similar levels of expression for mitosis and meiosis-specific markers  were observed in both +/+Setx and −/−Setx testes. A reduction of Pgk2 is noticable in −/−Setx. A marked reduction in expression for post-meiotic germ cells  is observed in −/−Setx testes in agreement the absence of these cells in −/−Setx seminiferous tubules as shown in −/−Setx spermatocytes do not proceed past meiosis. Gapdh was used as an internal standard. Cal, Calmegin.Abortion of meiosis following a block at pachytene stage in (TIF)Click here for additional data file.Figure S5−/−Setx testes sections. γH2AX staining of adult testes histological sections highlight the extensive amount of DNA DSB breaks in −/−Setx seminiferous tubules.Elevated levels of DSBs in adult (TIF)Click here for additional data file.Figure S6−/−Setx and Δ11/Δ11p53+/− spermatocytesBrca1. A. Persistence of DSB repair intermediates in pachytene cells of −/−Setx mice. Normal Rad51 foci formation occurred at leptotene and zygotene stage in +/+Setx and −/−Setx mice, there was persistence of Rad51 foci at pachytene stage in −/−Setx spermatocytes indicating the presence of unrepaired DSBs. Scale bar, 20 µm. B. Persistence of DSB repair intermediates in pachytene cells of −/−Setx mice. Normal Dmc1 foci formation occurred at leptotene and zygotene stage in +/+Setx and −/−Setx mice, there was persistence of Dmc1 foci at pachytene stage in −/−Setx spermatocytes indicating the presence of unrepaired DSBs (compare 1 and 2). Scale bar, 20 µm. C. Quantitation of Dmc1 foci at pachytene stage in +/+Setx and −/−Setx mice. A 10-fold increase of Dmc1 foci was observed in −/−Setx.  * indicates p<0.05. D. Rad51 foci in +/+p53+/−Brca1 and Δ11/Δ11p53+/−Brca1. Normal Rad51 foci formation occurred at leptotene and zygotene stage in +/+Setx and −/−Setx mice, there was persistence of Rad51 foci at pachytene stage in Δ11/Δ11p53+/−Brca1 spermatocytes indicating the presence of unrepaired DSBs. Scale bar, 20 µm. E. Quantitation Rad51 foci at pachytene stage in +/+p53+/−Brca1 and Δ11/Δ11p53+/−Brca1 spermatocytes. A 2.5-fold increase in the numbers of Rad51 foci was observed in Δ11/Δ11p53+/−Brca1 . * indicates p<0.05.Persistance of Rad51 and Dmc1 foci at pachytene stage in (TIF)Click here for additional data file.Figure S7Rad51 expression revealed that the increased number of Rad51 foci in −/−Setx is not due to an increased expression of Rad51 gene since similar levels of Rad511 mRNA levels were detected in +/+Setx and −/−Setx testes. B. Immunoblotting of +/+Setx and −/−Setx testes protein extracts with anti-Rad51 antibody shows reduced levels of Rad51 protein in −/−Setx. Anti-GAPDH was used as a loading control. C. RT-PCR analysis of Dmc1 expression revealed that the increased number of Dmc1 foci in −/−Setx is not due to an increased expression of Dmc1 gene since similar levels of Dmc1 mRNA levels were detected in +/+Setx and −/−Setx testes. D. RT-PCR analysis of Mlh1 expression revealed that the absence of Mlh1 foci in −/−Setx is not due to a lack of expression of Mlh1 gene since similar levels of Mlh1 mRNA levels were detected in +/+Setx and −/−Setx testes. E. Immunoblotting of +/+Setx and −/−Setx testes protein extracts with anti-Mlh1 antibody shows similar levels of Mlh1 protein. Anti-GAPDH was used as a loading control.Expression levels of recombination factors. A. RT-PCR analysis of (TIF)Click here for additional data file.Figure S8−/−Setx pachytene spermatocytes. ATR foci formation occurred at leptotene and zygotene stages in +/+Setx and −/−Setx spermatocytes, however, ATR failed to spread to XY chromatin domain in −/−Setx at pachytene stage. Scale bar, 20 µm.Abnormal localisation of ATR in (TIF)Click here for additional data file.Table S1Sequences of primers used for the gene expression analysis. Sequences of the primers used for the spermatogenesis stage specific markers and X- and Y-linked gene expression analysis.(PDF)Click here for additional data file.


Artificial reefs are increasingly used worldwide as a method for managing recreational diving since they have the potential to satisfy both conservation goals and economic interests. In order to help maximize their utility, further information is needed to drive the design of stimulating resources for scuba divers. We used a questionnaire survey to explore divers’ perceptions of artificial reefs in Barbados. In addition, we examined reef resource substitution behaviour among scuba divers. Divers expressed a clear preference for large shipwrecks or sunken vessels that provided a themed diving experience. Motives for diving on artificial reefs were varied, but were dominated by the chance of viewing concentrated marine life, increased photographic opportunities, and the guarantee of a ‘good dive’. Satisfaction with artificial reef diving was high amongst novices and declined with increasing experience. Experienced divers had an overwhelming preference for natural reefs. As a management strategy, our results emphasize the capacity of well designed artificial reefs to contribute towards the management of coral reef diving sites and highlight a number of important areas for future research. Suggested work should validate the present findings in different marine tourism settings and ascertain support of artificial reefs in relationship to level of diver specialization. Scuba diving is a burgeoning global activity with coral reefs being a major attraction to divers. As a niche market, recreational diving is widely acknowledged as being one of the tourism industry’s fastest growing markets Although there are negative impacts associated with mass diving tourism, scuba diving has the potential to generate substantial revenues Whilst artificial reefs are not viewed as ‘perfect’ substitutes for natural coral reefs The aim of this study was to explore the perceptions of diving on artificial reefs from a user perspective. Information was sought to characterize both resident and visitor scuba divers, to acquire an understanding of why individuals dive on artificial reefs, and the factors that inform their choice of dive site. We report on divers use, opinions, and preferences related to artificial reefs, including the environmental attributes and motivational factors that contribute to diver enjoyment. We also explore if reef habitat preference is influenced by diving experience. Our results are discussed within the context of scuba diving management where reef conservation is important.All divers completed the survey themselves and gave their permission to use the results. Individuals were not identifiable from the data provided. The work described in this paper was reviewed and approved by the Centre for Environmental and Marine Science departmental ethics committee. Verbal assurance was provided by a representative of the Barbadian Coastal Zone Management Unit that no permit is required to conduct questionnaire based research on the island.2), its current population of 276,300 www.reefballs.org) have been gradually deployed along the south-west coast , West Indies , betweenst coast . Barbadon = 10) aided by a survey assessment sheet that resulted in minor modifications to several of the questions.Data was collected from a 36 question, self-administered survey (in English only), using a combination of open-ended and closed questions. The questionnaire was designed to profile the diving clientele of Barbados, report on their artificial reef awareness and use, their satisfaction of artificial reef diving, and their habitat preferences. A series of Likert scales (5–point) and checklists were included in the survey design alongside 8 free-response questions that gave individuals an opportunity to express their own thoughts and feelings to a prior response. Specific questions included in the survey reflect previous works that have studied diver perceptions of artificial reefs a priori requirement of ≥10 logged dives and knowledge of local artificial reef diving. The rationale of the study was made clear to all participants prior to completion of the questionnaire. Two hundred questionnaires were fully completed within the study period.Sampling was conducted with the assistance of five of the diving companies situated along the south-west coast of Barbados . A twelvThe Statistical Package for the Social Sciences  software was used to analyze relevant questionnaire data. For this study and consistent with the methodology of Fitzsimmons Of the 200 divers surveyed, the sample included more men (60.5%) than women. Collectively respondents averaged 43 years of age (±13.4 s.d.), ranging from 12 to 71 years. Fifty percent of those surveyed were British, 24.5% American, 15.5% Canadian, and 6.5% resided in Barbados. The remaining 3.5% of respondents were represented by three countries; Germany, Australia, and Bulgaria. The higher numbers of visitors from the United Kingdom and the United States are consistent with figures reported in a study conducted on Barbados The diving experience of respondents was highly variable. A break down of diving qualifications held revealed that 66.5% possessed Open Water certification , followed by 27% of divers with Sport or Dive Master qualifications (CMAS***). The remaining participants were either Instructors (5.5%) or trainee divers (1%). To further assess each respondent’s level of diving experience, individuals were questioned on the number of dives they had logged. Respondents had logged an average of 190 (±264 s.d.) dives to date. Moreover, the study revealed novices, i.e. <100 logged dives, accounted for 52% of the sample , compared to 48% being experienced divers , with ≥100 dives logged. Five percent of participants  had logged 1000 dives or more in their diving history. An assessment of commitment to diving indicated a mean career length of 10.75 (±9.6 s.d.) years. One individual had been diving for 45 years on both reef types.a priori knowledge of artificial reefs. Most divers (96%) had heard of the term ‘artificial reef’. As a reflection of this, 95% of respondents reported having previously dived on what they considered artificial habitat, whilst all 200 respondents had dived on artificial reefs in Barbados at some point. Divers were questioned on whether their decision to visit the island was influenced by established artificial reefs such as the group of six wrecks situated within Carlisle Bay. Twenty percent of individuals were found to be influenced by these reefs, and as such had chosen to visit the Caribbean island. When participants were asked if they had dived on these wrecks, 76% had done so. Respondents were also asked to state their most favoured type of artificial reef structure. From a list of 9 structures; 76.5% selected shipwrecks and 15.5% sunken vessels as their most preferred type. A number of exploratory questions were presented to participants to assess their awareness, use, and Analysis of responses to rate level of satisfaction  to the experience of artificial reef diving in Barbados, revealed 90% of divers being either very satisfied (54%) or satisfied (36%) with the experience, while none reported being ‘very dissatisfied’. An exploratory analysis was conducted to assess any relationship between diver experience and level of satisfaction according to the number of dives respondents had logged .x2 (3) = 23.90, p≤0.001) (x2 (1) = 5.38, p≤0.020), and ‘neither satisfied nor dissatisfied’ responses (x2 (1) = 6.67, p≤0.001), between novice and experienced divers. It appears therefore with increasing diving experience, level of satisfaction with artificial reefs as diving sites, decreases. Conversely, novice divers experience greater satisfaction with artificial reef diving. Analysis conducted to assess differences in diver satisfaction suggested no significant differences between males and females (x2 (3) = 5.99, p≥0.112, phi = 0.174).A Kruskal-Wallis Test indicated a high level of association between diver experience and level of satisfaction relating to artificial reef diving (p≤0.001) . This inp≤0.004 and p≤0.001, respectively). However, closer inspection of mean scores highlighted differences in ‘groups of attributes’ between levels of diver experience. For example, experienced divers placed greater importance on biological attributes including coral cover, reef colours, and reef complexity; whereas novices derived greater satisfaction from environmental attributes such as reef depth, location and access of reef, and sea visibility. Whist these latter results are not of statistical significance, further research to examine specific artificial reef attributes and their levels of importance to novice divers and experienced divers would be worthwhile.The next set of questions explored the level of importance of 13 artificial reef attributes that divers considered would enhance their diving enjoyment and satisfaction. Mean scores and overall ranking of reef attributes are presented in Respondents were presented with 8 attitude statements relating to artificial reefs that broadly addressed a number of ecological based themes. x2 (1) = 103.68, p≤0.001. When divers were asked if they agreed or disagreed with the question; ‘if there were aspects of diving on artificial reefs which are more satisfying when compared to diving on natural reefs’ 58% agreed; x2 (1) = 5.12, p≤0.024. The relationships of responses to questions between diver experience categories and between genders were then examined.For the final stage of the questionnaire, opinions and preferences relating to artificial reefs were sought in comparison to diving on natural reefs. Respondents were questioned on ‘whether they perceived artificial reef diving to be a nature-based experience, or not’. A high level of agreement (86%) was given in support of this question, which was found to be highly significant; x2 (1) = 0.706, p≥0.401, phi = 0.074). However, the second question asking ‘if there were aspects of diving on artificial reefs which are more satisfying’ indicated a significant difference in attitude between novices and experienced divers, with novices tending to agree more (x2 (1) = 4.24, p≤0.039, phi = 0.156). Moreover, there was a significant association between gender and responses to the latter two questions (x2 (1) = 3.43, p≤0.044, phi = 0.151; x2 (1) = 11.01, p≤0.001, phi = 0.258, respectively), with males being much more enthusiastic about artificial reefs in each case.Both novice (88%) and experienced (83%) divers agreed strongly that ‘artificial reef diving is a nature based experience’ (x2 (1) = 18.00, p≤0.001) of divers chose natural reefs (65%). Differences between habitat preference and diver experience were explored. U = 2267, z = −5.848, p≤0.001, r = 0.41). It is clear from x2 (1) = 3.85, p≥0.062); whist experienced divers had a strong preference for natural reefs as diving sites that revealed a highly significant result (x2 (1) = 66.66, p≤0.001). However, no significant gender based association between these categorical variables was indicated (x2 (1) = 0.913, p≥0.339, phi = 0.078), despite females being less likely (30%) than males (38%) to choose artificial reefs for diving.Respondents were also requested to state their preference for diving either on an artificial reef or on a natural reef in Barbados. Analysis revealed that a significant proportion (x2 (1) = 24.48, p≤0.001). In relation to diver experience; experienced divers performed significantly more  of these dives, in comparison to dives recorded  for novices (x2 (1) = 20.76, p≤0.001). Experienced divers dived more on natural reefs  than novice divers  (x2 (1) = 55.97, p≤0.001). In contrast, no apparent differences could be detected between experienced divers  and novice divers’  use of artificial reef habitat (x2 (1) = 2.62, p≥0.105).During the study period, divers interviewed were found to have performed a total of 1,280 dives. Of these dives, 57% (n = 729 dives) were conducted on natural reefs and 43% (n = 551 dives) on artificial reefs, revealing a highly significant difference between habitat use  individuals surveyed had previously heard of the term artificial reef, and when invited to give an accurate description of this habitat, did so with accuracy. For example: ‘a structure placed on the seabed  that attracts corals and associated marine species that over time appears natural’ (Participant 15), ‘structures or objects deliberately placed in an accessible location for reef growth to be viewed by divers and snorkellers in the future’ (Participant 121). It was clear that embedded within a majority of the definitions was the perception that artificial reefs provide habitat for marine fauna and flora. In addition, the word shipwreck was used frequently in the descriptions provided by divers.Divers’ Shipwrecks and purposefully sunken vessels were identified as the most favoured artificial reefs to dive on by respondents. Consistent with our findings, divers studied by Ditton et al. In general, divers were found to derive a high level of satisfaction from artificial reef diving in Barbados, with few individuals being dissatisfied or ambivalent . Stolk eDespite such strong satisfaction reported for artificial reef diving, it was clear that as respondents experience increased, their level of satisfaction decreased . DeardenWhen participants were asked to rate various reef attributes considered important to the enjoyment and satisfaction of diving on artificial reef habitat, the most valued characteristics for both novice and experienced divers were; fish abundance, sea visibility, safety, and coral cover. These results broadly reflect previous findings of attributes To develop a greater understanding of why divers choose artificial reefs as diving attractions, respondents’ personal experiences of artificial reef diving were sought relative to natural reef diving. Qualitative work by Stolk et al. Previous studies Traditional practices aimed at controlling diver impacts on reefs have largely embraced the concept of ecological carrying capacity of divers e.g. . HoweverConsiderable economic and ecological benefits can be achieved by developing diving destinations through the provision of artificial reefs for diving. In the first instance, they provide divers with a more diverse range of diving opportunities and environmental settings, essential factors in maintaining diving market interest and facilitating a competitive market edge RMS Rhone as being 5,270 dives per year, representing four times as many dives undertaken in comparison to the busiest local natural reef.In the absence of formal statistics for diving in Barbados, Schuhmann et al. In order to enhance current dive management practices using artificial reefs, the following points are recommended for consideration by marine resource managers and policy makers. Where artificial reefs are present: (1) transfer all introductory courses and in-training dives to artificial reef sites, (2) reinforce the environmental education of divers through the provision of educational materials positioned on artificial reefs, and (3) use more ‘in-depth’ conservation education dive briefings that have been shown This study of artificial reef divers in Barbados contributes to the current body of knowledge (e.g. 32,33,40–43] and is useful to reef planners and marine tourism managers. Motives for diving on artificial reefs were dominated by the reliability of the diving experience and associated biodiversity viewing and wildlife photographic opportunities. Divers expressed a clear preference for themed diving experiences associated with large shipwrecks or sunken vessels.Our findings show however, that our sample of divers is not homogenous - they differ in their satisfaction of artificial reef diving and reef habitat preference. Novice divers derive greater enjoyment and show a greater preference for artificial reef diving sites than their experienced diving counterparts. These findings therefore suggest that novice divers are more likely to accept reef habitat substitution more readily than experienced divers. To our knowledge, this study is the first to reveal recreation specialization in scuba divers relative to resource substitution behavior, and these results could have significant implications for the way reef based tourism is managed.Further studies need to establish to what extent divers would support a reef substitution policy, as well as additional research to validate our present findings in different locations. Limited work exists in the field of diver specialization


Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA–seq and differential 5′-end RNA–seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions . This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA–based regulation of gene expression in this emergent enteropathogen. Clostridium difficile is a major cause of nosocomial diarrhea associated with antibiotic therapy. During the last few years, severe forms of C. difficile infections became more frequent due to the emergence of hypervirulent isolates. Despite intensive studies, many questions regarding the mechanisms controlling C. difficile virulence remain unanswered. We hypothesized that C. difficile, a member of an ancient group of bacteria, might widely use ancestral RNA–based mechanisms to control its gene expression for better adaptation to host conditions. Indeed, using next-generation sequencing technology, we identified a great number and a large diversity of potential RNA regulators in this pathogen. We obtained experimental evidence for regulatory roles of a particular class of regulatory RNAs responding to c-di-GMP, a universal bacterial signaling molecule regulating motility, biofilm formation, and virulence. We also detected abundant small RNA products of recently discovered adaptive prokaryotic immunity CRISPR-Cas systems that might be important for C. difficile survival in gut communities. Our findings suggest that small RNA molecules may play a major role in regulatory processes during C. difficile infection cycle and as such are promising targets of new therapeutic strategies.The emergent human pathogen  Upon interaction with ligands , riboswitches undergo a conformational change leading to positive or negative effects on transcription termination or translation cis-encoded RNAs, which are transcribed from a DNA strand opposite to the one from which mRNA to be regulated is transcribed and are thus fully complementary to their targets trans-encoded RNAs, which are transcribed from separate loci and are only partially complementary to target mRNAs trans-encoded sRNA-mediated control In recent years, the importance of regulatory mechanisms based on the action of RNA molecules became widely appreciated. In bacteria, regulatory RNAs play a critical role in adaptive responses and in various metabolic, physiological, and pathogenic processes. In particular, small noncoding RNAs (sRNAs) have been recently identified in many pathogenic bacteria Clostridium difficile has been reported. However, recent genome-wide in silico searches for sRNAs within intergenic regions (IGRs) C. difficile and could be involved in the control of metabolism and virulence. In Clostridium perfringens, four sRNAs controlling expression of toxin-encoding genes were experimentally identified Clostridium acetobutylicum, we have recently described a novel mechanism of control of a sulfur metabolic operon by an antisense RNA With the exception of recent identification of cyclic-di-guanosyl-5′monophosphate (c-di-GMP) riboswitches C. difficile is an anaerobic spore-forming bacterium that is a major cause of nosocomial infections associated with antibiotic therapy. This enteropathogen can lead to antibiotic-associated diarrhea and pseudomembranous colitis, a potentially lethal disease. Transmission of C. difficile is mediated by contamination of the gut by its spores. The disruption of colonic microflora by antimicrobial therapy precipitates colonization of the intestinal tract by C. difficile and ultimately leads to infection C. difficile infection cycle, including the identification of additional virulence and colonization factors and the determination of molecular mechanisms controlling their production in response to environmental signals, still remain poorly understood C. difficile, we here used a robust in silico approach that has been successfully applied for the identification of sRNAs in Escherichia coli and Streptococcus agalactiaeC. difficile genome. RNA-seq and differential 5′-end RNA-seq approaches were then used to validate these predictions and to globally identify C. difficile sRNAs and their transcriptional start sites. In this way, the expression of numerous sRNAs located in IGRs and representing potential trans riboregulators, cis-antisense RNAs, riboswitches, and CRISPR RNAs was detected. The expression of several sRNAs was independently confirmed by gene-specific approaches, and potential involvement of some of them in growth-stage specific control in C. difficile was revealed. This study thus constitutes a basis for the future detailed functional characterization of RNA-based regulatory mechanisms in C. difficile.To systematically search for regulatory RNAs in C. difficile Rho-independent terminator (RIT) sites, which often terminate sRNA gene transcription, were used as a starting point for an in silico sRNA search according to a method previously described for S. agalactiaeC. difficile strain 630, 2644 putative RIT sites were found. To identify the transcription termination signals associated with sRNA genes, we filtered out the RIT sites associated with protein coding genes ) as well as the RIT sites with calculated free Gibbs energy ΔG037>−4 kcal/mol. sRNA gene candidates located antisense to coding sequences (CDSs) were filtered out if ΔG°37 of their RIT sites was more than −8 kcal/mol. The conservation of remaining sequences and the presence of covariations in predicted RNA secondary structure elements of RIT was next analysed using comparison with available clostridial sequences in GenBank database. sRNA candidates were then scored on the basis of their RIT score, which was weighted by the number of covariation pairs found. Threshold ΔG037 values of −4 kcal/mol or −8 kcal/mol and a requirement for at least two covariations, including one in the RIT stem left 511 sRNA candidates (257 sRNAs in IGRs and 254 antisense RNAs) that were classified according to their position relative to adjacent CDSs. These predicted sRNAs are summarized in C. difficile 630 strain. The comparison of our data with the previous study revealed 95 IGRs containing common potential sRNAs  and differential 5′-end sequencing ). The RNA-seq analysis was performed with RNAs extracted from C. difficile 630Δerm strain after 6 h of growth . For 5′-end sequencing, we mixed three different RNA samples extracted from cells harvested in exponential growth phase (4 h of growth), at the onset of stationary phase (10 h of growth), and under nutrient starvation conditions. For RNA-seq, a non-orientated whole-transcript library was generated from late exponential phase RNA sample depleted for sRNAs. For 5′-end RNA-seq, two strand-specific cDNA libraries were generated from mixed RNA sample depleted for rRNAs. The first library was constructed from RNA sample treated with Tobacco Acid Pyrophosphatase (TAP+) allowing inclusion of reads associated with the TSS. A second library was constructed from the same RNA sample without TAP treatment (TAP−). The libraries were subjected to Illumina sequencing yielding a total of 37 million of whole-transcript reads (RNA-seq) and a total of 75.5 million and 38 million of 5′-end reads for TAP+ and TAP− libraries (5′-end RNA-seq), respectively. After removal of reads mapping to rRNA genes, about 6 to 10% of total reads were mapped to the genome of C. difficile strain 630  and Northern blot analysis. Expression of selected sRNAs was monitored under different growth conditions of C. difficile 630Δerm cultures to reveal the cases of growth phase-dependent expression. To determine if selected sRNAs are also present in another C. difficile strain, we extracted RNA from a PCR-ribotype 027 hypervirulent strain (R20291) during late exponential growth phase and also subjected it to analysis.We confirmed several selected sRNAs predicted in silico predicted sRNA candidates were transcribed in strain 630Δerm during the exponential growth phase and/or at the onset of stationary phase. A representative example of experimental validation of five sRNA candidates expression by RT-PCR is presented in in silico predicted sRNA candidates in strain 630Δerm and R20291, respectively, was detected by Northern blotting. in silico and detected by RNA-seq in both strains. As can be seen, Northern blot analysis with probes specific for SQ1002 candidate sRNA revealed an abundant ∼200-nt transcript, which agrees well with the 236-nt in silico predicted length and the 151-nt length deduced from RNA-seq data. For SQ1985 (predicted length of 233-nt), a much shorter, ∼70-nt low-abundance transcript was detected in accordance with the 84-nt length revealed from RNA-seq data. The discrepancy in lengths may be explained by in silico prediction criteria (maximum size limit fixed at 250 nt) as well as the fact that predictions did not take in account the positions of promoters. No growth phase dependence was observed for the expression of sRNA candidates tested. However, examples of growth phase-dependent regulation of C. difficile sRNAs will be discussed later in the text.RT-PCR analysis revealed that 28 out of 30 C. difficile 630Δerm sRNAs identified by deep sequencing, 35 out of 40 candidates assayed were detected by Northern blotting of 630Δerm strain RNA samples, while 23 were detected in RNA prepared from the R20291 strain samples. Transcript lengths agreed well with the sizes deduced from RNA-seq approach , a cis-antisense , and a riboswitch (Cdi2_4). The RCd1 sRNA is transcribed from IGR between CD1894 and CD1895 genes encoding conserved hypothetical proteins. An abundant transcript of about 100-nt was stably detected under all tested conditions by Northern blotting  sRNA matches the housekeeping 6S RNA and CD630_n00840 corresponds to selenocysteine tRNA. A total of 24 annotated tRNAs were also detected by deep sequencing. 12 other highly expressed sRNAs originated from C. difficile CRISPR cassettes (see below). Most of identified regulatory RNAs for which a functional class can be assigned correspond to riboswitches. Thus, in addition to 16 c-di-GMP-dependent riboswitches carrying either GEMM RNA or c-di-GMP II motif C. difficile RNA regulatory elements detected in our RNA-seq analysis corresponded to T-box riboswitches. We also detected that all of the 5 predicted SAM-riboswitches and all of the 4 predicted L-box riboswitches are located upstream of genes involved in methionine biosynthesis or uptake We next used the Rfam database (a collection of noncoding RNA families) to search for C. difficile 630Δerm sRNAs detected by deep sequencing were also present in the hypervirulent R20291 strain, which has about 80% genome sequence similarity with the 630 strain C. difficile species, we used BLAST to analyse the distribution of sRNAs among available C. difficile genomes. The results revealed that most sRNAs are conserved, with 69 IGR sRNAs being present in all 14 sequenced C. difficile strains  revealed that only few of  species . Two IGRC. difficile sRNAs belonging to different functional classes of riboregulators including IGR sRNAs, cis-antisense RNAs, riboswitches, and CRISPR RNAs.Below, we present a more detailed analysis of several C. difficile expressed one abundant not previously annotated sRNA  corresponding to the widely distributed housekeeping 6S RNA. The TSS identified by our 5′-end RNA-seq analysis is preceded by consensus −35 and −10 promoter elements of the sigma A RNA polymerase holoenzyme , respectively, and SQ1498 located in IGR between CD2112 and CD2113 genes encoding a conserved hypothetical protein and a two-component sensor histidine kinase, respectively . Thus, the mechanisms of regulation of these six growth-phase dependent C. difficile sRNAs remain to be determined.For four out of six growth phase-regulated sRNAs, the sigma A-dependent promoters were identified upstream of the TSS determined by 5′-end RNA-seq and confirmed by 5′RACE or 5′/3′RACE , while oC. difficile sRNAs (in trans by base-pairing with 5′UTRs of target genes mRNAs or coding regions. Three available softwares (see C. difficile strains carrying sRNA regions in sense or antisense orientation under the control of inducible tetP promoter of the pRPF185 vector derivative (C. difficile growth were observed (data not shown). One possibility would be that RCd6 and SQ1498 might affect the expression of their target genes at the translational initiation level as commonly found for known trans riboregulators in silico predictions may have failed to identify true RCd6 and SQ1498 targets. So, additional experiments will be required to assess the function of these sRNAs and their mechanisms of action.With the exception of a c-di-GMP-dependent riboswitch, the search with Rfam database did not allow us to assign a function for the growth phase-regulated le sRNAs . Since fares see  section ares see . To testrivative . In the CD0182 and CD0183 and overlapped the 3′-end of CD0182 (a gene of unknown function) and the 5′-end of CD0183  , 630/pRCd2 (overexpresses RCd2 from the tetP inducible promoter of pRPF185), and 630/pAS-RCd2 (overexpresses RCd2 in antisense orientation) (erm or 630/p strains (The RCd2 sRNA is down-regulated at the onset of stationary phase. It is transcribed in an antisense orientation to two adjacent genes drolase) . Northerdrolase) . Identiff CD0182 . Overexpf CD0182 , promptintation)  grew equntation) . Similar strains .CD0182 or CD0183. While the CD0182 mRNA was indetectable at conditions used, we observed a band of about 1000-nt with the CD0183-specific probe corresponding to the expected mRNA length (CD0183 transcript was further confirmed by qRT-PCR (>9-fold increase in 630/pRCd2 compared to 630/p). One hypothesis would be that the possible base-pairing between the 5′-parts of RCd2 RNA and CD0183 mRNA . Indeed, CD0183 overexpression leads to growth defect in E. coli and C. difficile (data not shown). Thus, RCd2 sRNA may represent an example of RNA-dependent control of autolysin function in C. difficile.Interestingly, the C. difficile encodes a large number of c-di-GMP turnover enzymes (up to 37) C. difficileAnother class of riboregulators detected by our analysis includes riboswitches responding to c-di-GMP. C-di-GMP is a signaling molecule with important functions in microbiological systems controlling lifestyle switches from free-living motile state to biofilm communities and virulence in Gram-negative bacteria C. difficile c-di-GMP riboswitches (CD2830 (encodes a precursor of exported protein) and CD2831 (encodes a precursor of a collagen-binding protein) are controlled by c-di-GMP-I and c-di-GMP-II riboswitches (Cdi1_12 and Cdi2_3), respectively. Two c-di-GMP-I riboswitches (Cdi1_4 and Cdi1_5) are located in distant loci within the C. difficile 630 genome in highly similar prophage regions (CD0904-979 and CD2889-2952) corresponding to host cell lysis module of bacteriophage PhiCD119. Therefore, c-di-GMP may control two homologous prophage genes CD2889 and CD0977.1. Four other regions carrying c-di-GMP-I riboswitches  are also highly homologous to each other (from 88 to 93% nucleic acid sequence identity) and must have arisen through multiple duplication events. They are located upstream of genes encoding conserved hypothetical proteins of unknown function .RNA-seq detected the expression of all switches . The anaflgB operon involved in the synthesis of basal body, hook and motor components of flagella and flagella specific sigma factor, SigD flgB was predicted in silico (SQ173 RNA) and experimentally confirmed by RNA-seq . However, by gene-specific RT-PCR we have clearly demonstrated the co-transcription of Cdi1_3 with flgB (flgB deserves further investigation.One c-di-GMP-I riboswitch (Cdi1_3) is found upstream of the large  RNA-seq . The prey 5′RACE . In accoB operon . It is iith flgB . The rolC. difficile, we cloned the CD1420-dccA gene encoding a c-di-GMP synthetase tetP promoter of plasmid pRPF185  or 170 nt (Cdi1_12) smaller bands with higher intensities that may represent the products of ribonucleolytic cleavage of the terminated transcript initiated in the presence of high c-di-GMP concentrations. Indeed, specific ribonucleases including RNase Y, RNase P, RNases J1 and J2 have been shown to cleave riboswitches or T-box motifs to initiate their ligand-dependent turnover in several bacteria C. difficile genome and may be also involved in riboswitch processing. Finally, a more general qRT-PCR analysis revealed the c-di-GMP-dependent negative control for all but one of the 12 c-di-GMP type I riboswitches in C. difficile  CRISPR-Cas system cas genes might be processed by Cas proteins encoded by the operons associated with CRISPR 12 and/or 17. Our RNA-seq analysis revealed the expression of all 12 CRISPR loci in C. difficile 630Δerm. 5′-end RNA-seq analysis allowed precise identification of the TSS for all CRISPR cassettes and most of them were associated with potential sigma A-dependent promoters  in TY C. difficile cultures. E. coli strains were grown in Luria-Bertani (LB) broth, and when indicated, ampicillin (100 µg/ml) or chloramphenicol (15 µg/ml) was added to the culture medium. The non-antibiotic analog anhydrotetracycline  was used for induction of the tetP promoter of pRPF185 vector derivatives in the C. difficileMotility was tested on semi-solid BHI medium (0.17% agar) containing 8 µg/ml Tm and 500 ng/ml ATc. Strains were inoculated into semi-solid medium tubes and incubated for at least 24 h at 37°C. Biofilm formation was assayed as previously described C. difficile genes, we used the pRPF185 vector expression system carrying a gusA gene between an ATc-inducible tetP promoter and a transcriptional terminator CD1420 gene  were amplified by PCR. To investigate the effects of down-regulation of expression of SQ1498, RCd6 and RCd2, antisense fragment to these sRNA genes was also cloned under the control of the inducible tetP promoter. We used primers containing a 5′-BamHI or a 3′-SacI site for sense and 5′-SacI or a 3′-BamHI site for antisense cloning orientation. These fragments were inserted between the BamHI and SacI sites of pRPF185 vector. For sRNA overexpression the pRPF185 vector was modified by reverse PCR approach with OS618 and OS620 primers to allow cloning of sRNA region directly between tetP promoter and vector-borne terminator. The CD0183 gene was cloned into StuI and BamHI sites of pRPF185ΔgusA derivative carrying SacI, StuI, XhoI and BamHI cloning sites instead of gusA region. DNA sequencing was performed to verify plasmid constructs using pRPF185 specific primers IMV507, OS621 and IMV508. The resulting derivative pRPF185 plasmids were transformed into the E. coli HB101 (RP4) and subsequently mated with C. difficile 630ΔermC. difficile transconjugants were selected by sub-culturing on BHI agar containing Tm (15 µg/ml) and Cfx (25 µg/ml).All routine plasmid constructions were carried out using standard procedures C. difficile 630Δerm strain grown in TY medium either after 4 h, 6 h or 10 h of growth and from R20291 strain during late exponential growth phase (6 h) as previously described Total RNA was isolated from + membrane (Amersham) by electroblotting using the Trans-blot cell from Bio-Rad in 1× TBE buffer . Following UV-cross-linking of the samples to the membrane, prehybridization was carried out for 2 h at 42°C in 7 mL of prehybridization buffer ULTRAHyb (Ambion). Hybridization was performed overnight at 42°C in the same buffer in the presence of a [gamma-32P]-labeled DNA oligonucleotide probe. Alternatively, the probe was synthesized using PCR with 5′end-labeled primer complementary to RNA sequence. After hybridization, membranes were washed twice for 5 min in 50 mL 2× SSC (300 mM sodium chloride and 30 mM sodium citrate) 0.1% sodium dodecyl sulphate (SDS) buffer and twice for 15 min in 50 mL 0.1× SSC 0.1% SDS buffer. Radioactive signal was detected with a Typhoon system (Amersham). The size of the transcripts was estimated by comparison with RNA molecular weight standards (Invitrogen).For Northern blot analysis, 5 µg of total RNA was separated on a denaturing 6% or 8% polyacrylamide gel containing 8 M urea, and transferred to Hybond-NC. difficile 630Δerm strain using a 5′RACE System kit (Invitrogen) as previously described 5′RACE (Rapid amplification of cDNA ends) assays were performed on total RNA extracted from C. difficile 630Δerm strain during late exponential growth phase (6 h) as previously described Non-orientated library for whole transcript analysis by RNA-seq was realized on the RNA sample extracted from C. difficile genome using Bowtie software (Options –sam -q -k 2000) The Illumina reads were first scanned for adaptor removal. Remaining sequencing reads were mapped to the in silico screening of the genome of C. difficile 630 strain was done as previously described for S. agalactiaeC. difficile 630 strain was done against all available sequences of clostridial strains in the GenBank database (March 2010). The prediction of secondary structure of each sRNA candidate was performed with Mfold 3.4 software The C. difficile genome E-value of 0.05 or less) and CDSs sized greater than 15 amino acids with no homologues were classified as possible novel proteins or peptides.To analyse possible coding capacities of sRNAs their sequences were scanned for the presence of protein coding sequences (CDSs) starting with ATG, TTG or GTG codons representing almost the totality of start codons identified in C. difficile, 14 other clostridial genomes and 5 additional bacterial genomes outside Clostridia. We used BLAST with standard parameters and selected matches corresponding to the alignments covering more than 80% of sRNA length . We performed a search for riboswitch conservation in the upstream regulatory regions of the orthologous genes from C. difficile strains  and other clostridial species  and selected matches corresponding to BLAST alignments covering more than 80% of riboswitch length for C. difficile strains and >30% for the other clostridia. The ORF conservation within C. difficile strains was analyzed by TBLASTX with standard parameters.The conservation of sRNAs detected in this study was determined by comparing them with sequence homologues in 13 The Rfam database was used to search for known sRNAs http://mmonot.eu/COV2HTML/visualisation.php?str_id=-13.Deep sequencing data are available at Figure S1in silico predicted sRNAs. RT-PCR was performed using gene-specific primers   or to the entry to stationary phase (10 h of growth) . Negative control reactions without reverse transcriptase performed using the same gene-specific primers and RNA samples are shown in lanes 11–20.Experimental validation of  primers  for SQ10(PDF)Click here for additional data file.Figure S2Detection of abundant 6S RNA by deep sequencing. The TAP−/TAP+ profile comparison for 5′-end RNA-seq data is aligned with RNA-seq data for RCd3 (6S RNA) genomic region. The TSS identified by 5′-end sequencing are indicated by red broken arrows in accordance with the positions of 5′-transcript ends shown by vertical green lines on the sequence read graphs corresponding either to TSS (broken arrows) or to processing sites . TSS corresponds to position with significantly greater number of reads in TAP+ sample, potential cleavage site corresponds to position with large number of reads in both TAP− and TAP+ samples. 5′-end sequencing data show 51-bp reads matching to the 5′-transcript ends, while RNA-seq data show reads covering whole transcript. Coding sequences are indicated by blue arrows and the 6S RNA is indicated by a grey arrow.(PDF)Click here for additional data file.Figure S3Expression analysis by deep sequencing of growth phase-regulated sRNAs. The results are given at the left for RCd6 sRNA and at the right for SQ1498. The TAP−/TAP+ profile comparison for 5′-end RNA-seq data is aligned with RNA-seq data for corresponding genomic region. The TSS identified by 5′-end sequencing are indicated by red broken arrows in accordance with the positions of 5′-transcript ends shown by vertical green lines on the sequence read graphs corresponding either to TSS (broken arrows) or to processing sites. TSS corresponds to position with significantly greater number of reads in TAP+ sample. 5′-end sequencing data show 51-bp reads matching to the 5′-transcript ends, while RNA-seq data show reads covering whole transcript. Coding sequences are indicated by blue arrows and new sRNA candidates identified in this study are indicated by grey arrows.(PDF)Click here for additional data file.Figure S4flgB operon. TAP−/TAP+ (5′-end RNA-seq) profile comparison and RNA-seq data are given for SQ173 located upstream of the flgB flagella operon. The positions of the primers used for RT-PCR are indicated at the bottom of the flgB region diagram with the estimated length of corresponding fragments. RT-PCR was performed using gene-specific primers   or to the entry to stationary phase (10 h of growth) . Negative control reactions without reverse transcriptase performed using the same gene-specific primers and RNA samples are shown in lanes 7–12.Co-transcription of SQ173 and  primers  for SQ17(PDF)Click here for additional data file.Figure S5Expression analysis of CRISPR 16 cassette by deep sequencing. The TAP−/TAP+ profile comparison for 5′-end RNA-seq is aligned with RNA-seq data for corresponding genomic region. The TSS identified by 5′-end sequencing are indicated by red broken arrows in accordance with the positions of 5′-transcript ends shown by vertical green lines on the sequence read graphs corresponding either to TSS (broken arrows) or to processing sites. TSS corresponds to position with significantly greater number of reads in TAP+ sample, potential cleavage site corresponds to position with large number of reads in both TAP− and TAP+ samples. 5′-end sequencing data show 51-bp reads matching to the 5′-transcript ends, while RNA-seq data show reads covering whole transcript.(PDF)Click here for additional data file.Table S1Complete list of predicted sRNAs.(PDF)Click here for additional data file.Table S2C. difficile.Summary of sequenced 51-bp reads from NGS of (PDF)Click here for additional data file.Table S3Complete list of sRNA detected by deep sequencing.(PDF)Click here for additional data file.Table S4sRNA extremity identification by 5′RACE and 5′/3′RACE.(PDF)Click here for additional data file.Table S5Conservation of regulatory RNAs in bacterial strains.(PDF)Click here for additional data file.Table S6Strains and plasmids used in this study.(PDF)Click here for additional data file.Table S7C-di-GMP riboswitches.(PDF)Click here for additional data file.Table S8CRISPR arrays.(PDF)Click here for additional data file.Table S9Oligonucleotides used in this study.(PDF)Click here for additional data file.