Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1's chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1's dominance in promoting strand exchange between homologs involves repression of Rad51's strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation in response to defective homolog interactions. Thus, robust crossover homeostasis is conferred by integrated regulation at initiation, strand-exchange and maturation steps of meiotic recombination.During meiosis, repair of programmed DNA double-strand breaks (DSBs) by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1's strand exchange activity, while its own exchange activity is inhibited. However, in a  Meiosis is the specialized cell division that produces gametes by precisely reducing the chromosome copy number from two to one. Accurate segregation of homologous chromosome pairs requires they be connected by crossing-over, the precise breakage and exchange of chromosome arms that is carried out by a process called recombination. Recombination is regulated so each pair of homologous chromosomes becomes connected by at least one crossover. We studied the roles of two recombination proteins, Rad51 and Dmc1, which can act directly to join homologous DNA molecules. Our evidence supports the idea that Dmc1 is the dominant joining activity, while Rad51 acts indirectly with other proteins to support and regulate Dmc1. Furthermore, Hed1, an inhibitor of Rad51's DNA joining activity, is also shown to enhance the efficiency of crossing-over. Cells in which Rad51 is activated to promote DNA joining in place of Dmc1 have unregulated and inefficient crossing-over that often leaves chromosome pairs without the requisite crossover. Despite these defects, most cells that use Rad51 in place of Dmc1 complete meiosis and produce high levels of crossovers. Our results indicate that compensatory processes ensure that meiotic cells accumulate high levels of crossover intermediates before progressing to the first round of chromosome segregation. During meiosis, haploid gametes are formed from diploid precursor cells via two successive rounds of chromosome segregation. By a program of events unique to meiosis, parental chromosomes (homologs) associate into homologous pairs and then disjoin from one another at the first division of meiosis (MI). In most organisms, the process of homologous recombination mediates both the pairing and disjunction of homologs The cell-to-cell variation in crossover numbers is much lower than the variation seen for DSB numbers In this study, specific relationships between Rad51, Dmc1, and their accessory factors are defined. Regulation of the strand-exchange step of meiotic recombination, especially template choice, has been a major focus of studies of meiotic recombination hed1 mutation also provides a means to test the inference that Rad51 can effectively substitute for Dmc1 during meiosis dmc1 hed1 cells, where Rad51 catalyzes DNA strand exchange. We show that efficient crossover assurance requires homolog template bias and that even a mild reduction in interhomolog bias causes defective SC morphogenesis and renders cells sensitive to modest reductions in DSB numbers.The Remarkably, and despite the profound primary defects associated with Rad51-catalyzed meiotic recombination, crossing-over occurs at nearly wild-type frequencies. We show that most cells are able to compensate for defective interhomolog bias by biasing the outcome of recombination to favor crossing-over; and by a second feedback process that we propose involves additional DSB formation at sites that normally experience relatively low levels of recombination.dmc1 mutants arrest in meiotic prophase with unrepaired DSBs and profound defects in homolog pairing and SC formation dmc1 mutants to progress through meiosis and form viable spores In the budding yeast SK1 strain background, HIS4::LEU2 DSB hotspot in cell cultures undergoing synchronous meiosis  chromosomes allow parental, DSB and crossover molecules to be resolved and monitored by Southern analysis . Increased levels of mcJMs provide more specific support for defective coordination between DSB ends. This defect could result from enhanced strand-invasion activity of the “second” end when Dmc1 is absent, or a defect in the helicase-dependent mechanism that normally acts to resolve mcJMs dmc1 hed1 cells comes from the observation that non-allelic crossing-over between HIS4::LEU2 and the native LEU2 locus dmc1 hed1; non-allelic recombination bands are indicated by an asterisk in dmc1 hed1 cells. Most notably, strand-exchange now occurs primarily between sister-chromatids instead of homologs.Additional changes in JM processing can also be discerned in te times . AccumulHIS4::LEU2, an artificial hotspot, were representative, we also examined dHJ intermediates and crossover products at ERG1 (YGR175c), the site of a natural DSB hotspot on chromosome VII ERG1 hotspot, with a IH/IS ratio of 4.5, similar to that seen at HIS4::LEU2 .Unexpectedly, the IH-dHJ/IS-dHJ ratio was also reduced in the e mutant . In hed1dmc1 single mutants, mnd1 mutants arrest during meiotic prophase with unrepaired DSBs  is not observed in mnd1 mutants (HIS4::LEU2 are not detected in mnd1 cells while they reach 3% (∼15% of wild-type levels) in the dmc1 mutant , were present in up to 40% of hed1 nuclei , a method of light microscopy that can achieve 50 nm resolution compared to the 200 nm resolution provided by standard widefield epifluorescence microscopy hed1 single mutants than in wild type. Indeed, the increased density of Red1 puncta resulted in regions where the parallel configuration of the underlying lateral elements was evident (as observed in the Red1+Rec8 mixed staining experiment) again demonstrating that Red1 is a component of axial/lateral elements  and the crossover-associated protein, Zip3  in map distances were detected in three of eight intervals, but overall map distance of chromosome III was reduced by only 11%, from 131.6 cM (2.6 crossovers per meiosis) in wild type to 116.8 cM (2.3 crossovers per meiosis) in dmc1 hed1  compared to 1.5% of dmc1 hed1 tetrads (45/3052).Spore viability of e spores . Allelicdmc1 hed1 mutants may be caused by defective crossover assurance, we determined the frequency of tetrads in which chromosome III failed to undergo any crossing-over (Reasoning that the reduced spore viability of ing-over . If crosdmc1 hed1 cells is very similar to wild type (2.3 versus 2.6 crossovers per chromosome III per meiosis), 15% (210/1376) of tetrads lack a crossover between chromosome III (dmc1 hed1 cells. The frequency of non-exchange tetrads seen in hed1 single mutants (2%) may by slightly higher than that of wild type (0.9%), but the difference is not significant in this data set.Remarkably, even though the average crossover frequency in some III . Not onldmc1 hed1 cells results from defective homolog pairing .Consistent with the possibility that crossover failures in  pairing , gene coA second aspect of crossover control, interference, was analyzed by a new approach that took advantage of the ability to purge the data of tetrads in which chromosome III did not undergo a crossover event. This approach avoids the diminution of the genetic signature of interference that is associated with defective crossover assurance dmc1 hed1 tetrads showed a general increase relative to wild type and significant interference was detected in only one of the seven intervals indicating substantially weaker crossover interference  tetrads. NPDs arise from a double crossover event within a single genetic interval that involves all four chromatids. The expected number of NPD tetrads is calculated assuming the absence of crossover interference rference . Mutatiodmc1 hed1 cells was confirmed by analysis of coincident crossovers in adjacent genetic intervals AdjCO/cMAdjPD in AdjCO/cMAdjPD<1) was detected for 5 of 6 intervals in wild type, hed1, and dmc1 hed1 indicating that significant interference can occur in the absence of both hed1 and dmc1. However, dmc1 hed1 shows significant decreases in the strength of interference in 4 of the 6 intervals tested . This suggests that enhancement of the crossover versus non-crossover outcome is not the only mechanism that compensates for the homolog bias defect of dmc1 hed1 , or spo11-HA, or heterozygous for spo11-HA/spo11-yf; these combinations of SPO11 alleles result in 100%, ∼80%, and ∼30% of normal DSB levels, respectively + HED1+DMC1 control strains, the fraction of recombination events that result in crossing-over increases with decreasing DSB levels +, spo11-HASPO11, and spo11-HA/spo11-yf strains are 1.3 (56% crossovers), 1.6 (61%), and 1.7 (63%), respectively . This ratio may increase slightly in the spo11-HA background , but no increase was seen for the most severe spo11 allele combination, spo11-HA/spo11-yf .We constructed sets of ectively . Consisthotspots , the crodmc1 hed1 cells and therefore are unable to further compensate for reduced DSB levels. Consistent with this interpretation, in the dmc1 hed1 background, spo11-HA caused a 2-fold drop in spore viability  and the spo11-HA/spo11-yf combination resulted in a 4.3-fold decrease  in hed1 SPO11 and increased to 1.8 (64% crossovers) in hed1 spo11-HA and 2.1 (68% crossovers) in hed1 spo11-HA/spo11-yf  were reduced 2.7-fold from 17.3% in wild-type cells to 6.5% in dmc1 hed1 cells  was reduced 2.2-fold, from 31.0 cM to 13.8 cM , is physically just over ten times larger than interval 2, but shows a very similar crossover rate (27.7 cM in wild type). Strikingly, crossing-over in interval 3 is not reduced in dmc1 hed1 cells (dmc1 hed1).Although chromosome-wide crossover levels in ild type , crossind1 cells . By tetr 13.8 cM . At 130 d1 cells . In factdmc1 hed1 cells, interhomolog interactions at DSB hotspots appear to be diminished to the point where homeostatic mechanisms are unable to maintain local crossover levels. However, crossover levels measured over larger distances remain similar to wild type. These data suggest that a second homeostatic mechanism operates over large chromosomal regions to help maintain crossover levels in dmc1 hed1 cells (discussed below).Thus, in dmc1 cells and results in near normal levels of crossing-over and high spore viability Previous work showed that activation of Rad51's strand-exchange alleviates the meiotic arrest of in vivo remains a key objective for understanding meiotic recombination.The precise spatial arrangement of Rad51 and Dmc1 on DSB-ends remains unclear. Cytological experiments argue against mixed filaments of Rad51 and Dmc1 in vivo. Moreover, Hop2-Mnd1 is required even when Dmc1 catalyzes strand exchange between sister chromatids Consonant with the fact that Hop2-Mnd1 is absent from the same evolutionary lineages that lack Dmc1 In vitro and in vivo studies provide clear evidence that Hed1 inhibits Rad51's strand exchange activity, which blocks meiotic DSB-repair when Dmc1 is absent hed1 mutation also renders cells unable to compensate for modest reductions in DSB levels, indicating that Hed1-dependent interhomolog bias is especially important in cells with suboptimal DSB levels. These data also raise the possibility that the ∼5-fold interhomolog bias seen in wild-type cells reflects a physiologically important “set point” that enables individual cells to compensate for stochastic variation in DSB numbers. Finally, we note that the phenotypes conferred by hed1 mutation are similar to those of cells lacking the AAA+ ATPase, Pch2, and may reflect a common defect in enforcing interhomolog template choice A Together with previous studies, our data indicate that the concerted action of at least four components promote interhomolog bias catalyzed by Dmc1-dependent DNA strand exchange . Early arising recombination events may not interfere with late events (and vice versa) resulting in dilution of the overall intensity of crossover interference. In support of this idea, delayed, asynchronous, and inefficient chromosome pairing can perturb interference in C. elegansReduced interference could arise from changes caused by defective interhomolog bias in dmc1 hed1 cells, 5-fold defects in template choice are observed at the HIS4::LEU2 and ERG1 hotspots ; and second, little or no additional increase in the crossover/non-crossover ratio is observed when global DSB levels are reduced . The NPD ratio for interference analysis is calculated as the fraction of NPDs observed/fraction of NPDs expected. NPD expected is calculated under the assumption where there is no interference using the Papazian equation: NPD expected = 0.5[(1-fT)−(1-(3fT/2))2/3], where fT denotes the observed frequency of tetratypes Haploid strains were mated for 4–24 hrs on YPD plates and sporulated on plates containing 1% potassium acetate and 0.02% raffinose at 30°C for 2 days. Alternatively, asci were taken from meiotic time course after 48 hrs. Asci were digested with zymolyase and dissected on YPD plates supplemented with adenine, uracil, methionine, lysine, and threonine. Only tetrads producing four viable spores and showing Mendelian segregation of markers were used to calculate genetic map distance. Map distances were determined using the Perkins equation: (100(6NPD+TT))/(2(PD+NPD+TT)) ERG1 was engineered by a series of two-step gene replacements. On one copy of chromosome VII, SacII restriction sites were engineered into intragenic regions at Saccharomyces Genome Database (SGD) (S288c genome assembly) coordinate 844276 (YGR173w) and coordinate 854464 (YGR179c); and a SalI site was engineered between ERG1 and YGR177c at coordinate 848724. For the SacII site at coordinate 844276 the region between coordinates 843173 and 845291 was PCR amplified (5′ and 3′ primers contain HindIII restriction sites), digested with HindIII, and cloned into pRS306. The SacII site was introduced by Quickchange Site-Directed Mutagenesis (Invitrogen) with the primers 5′-GTTGTTGCCACAGCAAGGACCGCGGATCTAGTATTAATGG and 5′-CCATTAATACTAGATCCGCGGTCCTTGCTGTGGCAACAAC (A→G). This plasmid was subsequently linearized using a unique MscI restriction site. For the SacII site at coordinate 854464, the region between coordinates 853473 and 855252 was PCR amplified (5′ and 3′ primers contain HindIII restriction sites), digested with HindIII, and cloned into pRS306. The SacII site was introduced by site-directed mutagenesis with the primers 5′-GCTTTAATTCATAATTCCGCGGCAACCTTTCTCTATACTCAGC and 5′-GCTGAGTATAGAGAAAGGTTGCCGCGGAATTATGAATTAAAGC (G→C). This plasmid was linearized using a unique EcoNI restriction site. For the SalI restriction site at coordinate 848724, the region between coordinates 847679 and 849776 was PCR amplified (5′ and 3′ primers contain HindIII restriction sites), digested with HindIII, and cloned into pRS306. The SalI site was introduced by site-directed mutagenesis with the primers 5′-GCAGCCACGGCATGCGTCGACTACGAGCGTATTGTG and 5′-CACAATACGCTCGTAGTCGACGCATGCCGTGGCTGC (A→G). This plasmid was linearized using a unique AgeI restriction site.The diploid strain used for JM analysis at VII, SacII restriction sites were engineered at SGD coordinate 845470 (intergenic) and coordinate 852145 (YGR178c) and a SpeI site was engineered between ERG1 and YGR177c at coordinate 848683. For the SacII restriction site at coordinate 845470, the region between coordinates 844520 and 846322 was PCR amplified (5′ and 3′ primers contain HindIII restriction sites), digested with HindIII, and cloned into pRS306. The SacII site was introduced by site-directed mutagenesis with the primers 5′-GGTTTAGATCCAAGATTCCGCGGTTCCACCATTTAATATG and 5′-CATATTAAATGGTGGAACCGCGGAATCTTGGATCTAAACC (C→G). This plasmid was linearized using a unique NruI restriction site. For the SacII restriction site at coordinate 852145, the region between coordinates 851037 and 853268 was PCR amplified , digested with SalI, and cloned into pRS306. The SacII site was introduced by site-directed mutagenesis with the primers 5′-CCTCTGGCGCACCTGCTGCCGCGGGAGTAGAGGTATCCG and 5′-CGGATACCTCTACTCCCGCGGCAGCAGGTGCGCCAGAGG (T→C). This plasmid was linearized using a unique BglII restriction site. For the SpeI restriction site at coordinate 848683, the region between coordinates 847679 and 849776 was PCR amplified (5′ and 3′ primers contain HindIII restriction sites), digested with HindIII, and cloned into pRS306. The SpeI site was introduced by site-directed mutagenesis with the primers 5′-CATGCGAGGTAAGACTAGTGTCTGAGACTTATACCCGACC and 5′-GGTCGGGTATAAGTCTCAGACACTAGTCTTACCTCGCATG (T→G). This plasmid was linearized using a unique AgeI restriction site.On the other copy of chromosome HIS4::LEU2 and ERG1 loci were similarly monitored by gel electrophoresis. The HIS4::LEU2 assay system contains XhoI restriction site polymorphisms between parental homologs producing fragments diagnostic for parental and recombinant chromosomes. A BamHI/NgoMIV polymorphism immediately at the DSB site allows detection of non-crossover products. Non-crossovers were analyzed by double digestion of genomic DNA with XhoI and NgoMIV and separation on one-dimensional gels. The ERG1 locus contains SacII restriction site polymorphisms that produce fragments diagnostic for JMs and recombinant products. To analyze relative amounts of crossover and non-crossover products at ERG1, genomic DNA was doubly digested with SacII and SalI and analyzed by one-dimensional gel and Southern hybridization. The polymorphic SalI site located at the DSB site is diagnostic for non-crossover products. Samples were treated with psoralen and UV to crosslink DNA and stabilize JM intermediates using previously described methods Recombination events at Yeast cells were fixed and spread as described previously ARG+ colonies were then tested for growth on plates lacking uracil and threonine.Random spore analysis was performed as described 2), and pelleted as before. Next, cells were resuspended in 1 ml MES and spun for 1 min at 200 rpm, followed by aspiration of the supernatant. To release nuclei from the spheroplasts, 5 µl of the pellet was placed into 50 µl MEM/protease inhibitor  with freshly added 0.1 M PMSF (10 µl of PMSF stock to 1 ml MEM), then gently resuspended by pipetting. To fix the released nuclei, 50 µl of PFA was added and mixed well. 50 µl of the final mixture was spread across microscope slide precoated with polystyrene plastic and incubated at room temperature in a humid chamber. After 5 min, an additional 400 µl of PFA was pipetted onto the slide. After 5 min more, preparations were rinsed with 4 ml of 0.4% Photoflo (Kodak) and allowed to dry completely before proceeding to silver staining.Nuclei were first fixed and spread followed by silver staining to visualize the synaptonemal complex 2O and allowed to dry. Images were taken at 3,500×magnification using a Hitachi H-7600 electron microscope housed in the Oklahoma Medical Research Foundation Core Facility for Imaging.200 µl of colloidal developer and 200 µl silver nitrate were mixed on a 24×50 coverslip and the mixture was put on top of a slide that had fixed nuclei. The preparation was placed on a 60°C hotplate for 2–5 min, then gently rinsed with water to remove the coverslip and stop the reaction. Once dry the polystyrene membrane was floated off on a water surface and clean EM grids were placed on the membrane. The membrane and grids were removed from HFigure S1hed1, dmc1, and dmc1 hed1 strains. A. Representative Southern images of JMs resolved by 2D gels. B. Timing and efficiency of meiotic divisions. C. Quantification of JMs over time. D. IH/IS dHJ ratios over time. E. Images of 1D Southern analysis of crossing-over at the HIS4::LEU2 locus. F. Quantification of final crossover levels at the HIS4::LEU2. Averages of four independent time courses are shown. Error bars show standard error.Independent analysis of JM formation and crossing-over in (TIFF)Click here for additional data file.Figure S2ndt80 background. A. 2D Southern images showing accumulated JMs in ndt80, hed1 ndt80 and dmc1 hed1 ndt80 strains. B. IH/IS dHJ ratios at 7 and 8 hrs after induction of meiosis. C. Quantification of various JM species in ndt80 strains. *Note that the SEI-like species are distinct from the SEIs that form at early times, which form more discrete signals and have a defined strand composition Analysis of JMs in the (TIFF)Click here for additional data file.Figure S3rad51 is epistatic to hed1. A. Representative Southern images showing 1D gel analysis of crossing over at HIS4::LEU2 in rad51 and rad51 hed1 time course experiments. B. Quantitation of crossovers at 12 and 24 hours in rad51 and rad51 hed1 cells. Each strain was analyzed in triplicate. C. Representative Southern images of 2D gels showing JM analysis at HIS4::LEU2 in wild-type, rad51 and rad51 hed1 strains. Lower panels show blowups of the JM regions. D. Quantification of IH-dHJs and IS-dHJs at HIS4::LEU2 in wild-type, rad51 and rad51 hed1 strains. E. Southern images showing 1D gel analysis of DSB formation and crossing over at HIS4::LEU2 in wild-type, dmc1 rad51 and dmc1 rad51 hed1 time course experiments. F. Quantification of the Southerns shown in E. The apparent reduction of DSB signals at late times in dmc1 rad51 and dmc1 rad51 hed1 strains results from excessive resection of DSBs past the diagnostic XhoI restriction site. Residual crossover products detected in rad51 dmc1 double mutants  have been noted previously bond fide reciprocal crossovers. The aberrant pathway responsible for these products has not been defined, but JMs are not detected in the rad51 dmc1 double mutant (TIFF)Click here for additional data file.Figure S4mek1-as strains. A. Representative Southern images of 2D gels showing JM analysis at HIS4::LEU2 in wild-type, mek1-as, hed1, and mek1-as hed1 cells. Lower panels show blowups of the JM regions. B. Timing and efficiency of meiotic divisions in wild-type, hed1, mek1-as, and mek1-as hed1 cells. C. Quantification of IH-dHJs, IS-dHJs and the IH/IS dHJ ratio in wild-type, mek1-as, hed1, and mek1-as hed1 cells.JM analysis in (TIFF)Click here for additional data file.Figure S5Additional images of Red1 immunostaining analyzed by STED microscopy. Red1 (red) is imaged via STED and Zip1 by confocal microscopy.(TIFF)Click here for additional data file.Figure S6dmc1 hed1 mutant are SCs. A. Representative immunostained nuclei showing Zip1 (green), Zip3 (red) and merged channels. Note that Zip3 foci colocalize with elongated ZIp1 structures. B. Average numbers of Zip3 foci per nucleus for wild-type, hed1, dmc1 hed1, and dmc1 cells. At lest 50 nuclei were analyzed for each strain. C. Electron micrographs of SCs stained with silver. Both wild-type nuclei and one of the dmc1 hed1 nuclei have fully formed tripratite SCs.The hed1 dmc1 nucleus to the left displays regions of tripartite SC as well as unsynapsed regions.Immunostaining and EM analysis of spread meiotic nuclei shows that elongated Zip1 structures formed in the (TIFF)Click here for additional data file.Figure S7dmc1 hed1 dataset that included non-exchange tetrads. In the graph on the right, non-exchange tetrads were excluded from the dmc1 hed1 dataset. The asterisks indicate intervals in dmc1 hed1 dataset that show a significant change from wild type. B. Analysis of interference using the “adjacent intervals” approach of Malkova et al. CO/AdjPD for wild-type, hed1, and dmc1 hed1 are shown. The reported ratios are averages of the two ratios for each interval pair. Solid lines represent significant interference and dashed lines represent non-significant deviations from a ratio of one. The panels on the left shows the analysis for dmc1 hed1 with non-exchange tetrads included and the panel on the right shows the analysis for dmc1 hed1 with non-exchange tetrads excluded. The asterisks indicate intervals with a significant change from wild type.Analysis of crossover interference along chromosome III with and without non-exchange tetrads. A. NPD ratios. In the graph on the left, NPD ratio were calculated for the (TIFF)Click here for additional data file.Figure S8CO/AdjPD for wild type, dmc1 hed1 including non-exchange tetrads, and dmc1 hed1 excluding non-exchange tetrad. The number is the average of the two AdjCO/AdjPD ratios obtained by taking each interval as reference. Solid lines represent significant interference and dotted lines represent non-significant interference. dmc1 hed1 shows significant negative interference when non-exchange tetrads are included in the analysis.Analysis of crossover interference for distant intervals. Analysis of interference between distant intervals using the nearest neighbor approach of Malkova et al. (2004). Ratios of map distances Adj(TIFF)Click here for additional data file.Table S1Quantitation of Red1, Zip1, and Zip3 localization.(PDF)Click here for additional data file.Table S2Quantitation and comparison of Red1 staining by STED microscopy.(PDF)Click here for additional data file.Table S3Analysis of crossover interference using non-parental ditype ratios.(PDF)Click here for additional data file.Table S4Analysis of crossover interference using adjacent intervals.(PDF)Click here for additional data file.Table S5Analysis of crossover interference for distant intervals.(PDF)Click here for additional data file.Table S6Yeast strains used in this study.(PDF)Click here for additional data file.Text S1Supplementary discussion.(PDF)Click here for additional data file.


We previously reported the existence of six alternative open reading frame proteins in WNV genome, one of which entitled WARF4 is exclusively restricted to the lineage I of the virus. WARF4 is able to elicit antibodies in WNV infected horses; however, there was no direct experimental proof of the existence of this novel protein. The purpose of this study was to demonstrate the We produced a monoclonal antibody against WARF4 protein (MAb 3A12) which detected the novel protein in WNV lineage I-infected, cultured VERO cells while it did not react with WNV lineage II infected cells. MAb 3A12 specificity to WARF4 protein was confirmed by its reactivity to only one peptide among four analyzed that cover the full WARF4 amino acids sequence. In addition, WARF4 protein was expressed in the late phase of WNV lineage I infection. Western blotting and bioinformatics analyses strongly suggest that the protein could be translated by programmed −1 ribosomal frameshifting process. Since WARF4 is embedded in the NS4B gene, we rename this novel protein N-NS4B/WARF4. Furthermore, serological analysis shows that N-NS4B/WARF4 is able to elicit antibodies in WNV infected individuals.N-NS4B/WARF4 is the second Alternative Reading Frame (ARF) protein that has been demonstrated to be produced following WNV infection and might represent a novel tool for a better characterization of immune response in WNV infected individuals. Further serological as well as functional studies are required to characterize the function of the N-NS4B/WARF4 protein. Since the virus might actually make an extensive use of ARFs, it appears important to investigate the novel six ARF putative proteins of WNV. West Nile virus (WNV) is an arthropod-borne virus maintained in a bird-mosquito transmission cycle. Birds are the natural reservoir hosts while humans and other mammals are dead-end hosts occasionally infected through mosquito bite . The virFlaviviriade family, genus Flavivirus and WNV lineage II  were propagated by infecting VERO E6 cells monolayers in MEM with 2% FCS and titrated according to the Reed and Muench formula.http://www.enivd.de/ENIVD_P.HTM) during the External Quality Assurance (EQA) for molecular detection of West Nile virus. The nucleic acids were extracted by NucleoSpin RNA Virus kit according to manufacturer’s instructions .Additional WNV genomic RNA  was obtained from European Network for Diagnostics of Imported Viral Disease (http://www.5PRIME.com). The His-tagged recombinant proteins were purified by Ni-NTA affinity chromatography kit according to manufacturer’s instructions . All the oligonucleotides were synthesized by Eurofins MWG Operon (http://www.eurofinsdna.com). Since the previously described recombinant His-WARF4 protein  does notFour week-old BALB/mice were immunized twice by intraperitonal injection with 25 μg of purified His-WARF4 protein emulsified in RIBI adjuvant . Mice were then given a booster immunization intravenously with 10 μg of the immunogen, and immune splenocytes were removed 3 days later. Somatic cell hybrids were prepared with NS-1 mouse non secreting myeloma cells as previously described -39. HybrFour synthetic peptides named SP1, SP2, SP3 and SP4, which cover the full N-NS4B/WARF4 COOH-terminal amino acid sequence and which have amino acids overlapping sequences were designed . Five hundred ng of peptide as well as Env and BSA proteins, 50 ng of His-WARF4 and 200 ng of His-NS4B were spotted in replicates on nitrocellulose membranes. Membranes were blocked for 4 h at RT in 5% non-fat dry milk-PBS-0.1% Tween-20 and incubated 1 hour at RT with MAb 3A12, anti-NS4B antibody  and MAb anti-Histidine (Invitrogen). The membranes were washed four times and then incubated with goat anti-mouse or rabbit IgG peroxidase-conjugated antibodies  and developed by a chemiluminescent kit  as previously described [SP1 (H-GNALSPAADSGWNHEKRCSGWHRGHGRPRIRAHHTHHAEESWANHA), SP2 (H-THHAEESWANHADLGVSSCSSSEPVCEDSARSRNSDHGSSGDTLGEW), SP3 (H-SDHGSSGDTLGEWSKLCLECNNCHRTLPHHAWGLVVMLIHNMDTHKE) and SP4 (HVVMLIHNMDTHKEHGKTRTKKRWGKGTHLGRGLERKTQPDDKRRVH) were synthesized by PEPSCAN  were used to infect VERO cells monolayers (40-50% confluent). WNV was subsequently allowed to adsorb for 1 hour at 37°C. MEM medium with 2% FCS, was then added to the infected cells monolayer. After 36 hours, the cells monolayer was washed 2 times in PBS 1X and fixed with 4% paraformaldehyde for 20 min at room temperature, followed by treatment with 0.1 M glycine for 20 min at 25°C and with 0.1% Triton X-100 for an additional 5 min at 25°C to allow permeabilization. Cells were incubated for 30 min at room temperature with MAb 3A12 or the antibody MOPC-21 used as negative control as previously described [The VERO E6 cell line was grown in eight wells Chamber slides™ . 200 μl of a viral suspension  of 0.1 for 24 to 72 hours or for 96 hours at 37°C in 5% CO2. Cells were scraped and harvested by centrifugation at 1,500 X g at 4°C and rinsed in ice-cold phosphate-buffered saline (PBS). Cell lysates were prepared in Staph A buffer  containing 1 mM PMSF ,47 and aHuman serum samples, obtained from convalescent patients suffering from neuro-invasive WNV infection, testing positive for IgGs anti-West Nile by IFA and confirmed by Micro-Neutralization Test Assay - MNTA  were kinhttp://bibiserv.techfak.uni-bielefeld.de/knotinframe/) to predict ribosomal −1 frameshift sites with a pseudoknot strucuture.384 full genomes of WNV were retrived from gene bank and aligned with BioNumerics software package . Genomes were further analized by KnotInFrame software . The protocol was approved under the regional plan of surveillance and control for west nile disease in Emilia Romagna .http://www.sta.uniroma2.it/) of the University of Rome “Tor Vergata” on 06/12/2010.Mice were sacrificed by cervical dislocation and later the spleens were removed by surgical resection. The procedures were carried out in the presence of a veterinarian who monitored proper compliance with the decree law 116/92. The protocol was approved by the ethics committee of the Station Technology for Animal (STA) (WARF4: West Nile Alternative open Reading Frame 4; WNV: West Nile Virus; ARF: Alternative Reading Frame.The authors declare that they have no competing interests.GF, RB, , and FL participated in the design of the study, carried out the experiments and wrote the manuscript. AP, RDS, LM, AC and LM carried out the experiments and wrote the manuscript. GR, FM, RL, ADG and VS have critically revised the manuscript and the experimental design. All authors read and approved the final manuscript.


To investigate the effect of 25(OH)D depletion on neuronal susceptibility to toxic insult, we induced a state of 25(OH)D deficiency in mice and then challenged them with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We found there was no significant difference between control and 25(OH)D-deficient animals in striatal dopamine levels or dopamine transporter and tyrosine hydroxylase expression after lesioning with MPTP. Additionally, we found no difference in tyrosine hydroxylase expression in the substantia nigra pars compacta. Our data suggest that reducing 25(OH)D serum levels in mice has no effect on the vulnerability of nigral dopaminergic neurons  Multiple factors, including increased oxidative stresses, varying genetic susceptibility, mitochondrial dysfunction, proteasomal dysfunction, inflammation and premature apoptosis contribute to the pathophysiology of Parkinson’s disease (PD) 2D)). Both vitamin D2 and vitamin D3 are rapidly hydroxylated to 25(OH)D, the body’s primary storage form for “vitamin D” and the best indicator of vitamin D status. This 25(OHD) metabolite has much higher circulating concentrations in serum and has a much longer half-life than the serum 1,25OH2D, which is considered the hormonal form of vitamin D. The hormonal form of vitamin D, 1,25OH2D is dependent on the substrate 25-hydroxyvitamin D. Therefore, circulating 1,25OH2D concentrations generally correlate with 25(OH)D concentrations. However, concentrations of the biologically active vitamin D hormone  are tightly regulated and in our neurology clinical settings it is rare to find patients who have normal renal function and abnormally low 1,25OH2D concentrations, even when 25(OH)D concentrations suggest severe vitamin D deficiency.The term “vitamin D” frequently refers to the family of compounds that include the nutritional supplements  and ergocalciferol (vitamin D2)), 25-hydroxyvitamin D (25(OH)D), and the biologically active vitamin D hormone, 1,25-dihydroxyvitamin D  has been shown in multiple injury models relevant to stroke, multiple sclerosis, and Alzheimer’s disease to differentially protect both neuronal populations and glia via upregulation of L-type calcium channels, immune modulation, increased antioxidant function, improved neurotrophin expression 2D appears to support homeostasis of the dopaminergic neuronal populations affected in PD. Both vitamin D hormone receptors and 1-alpha hydroxylase enzyme (the activating enzyme for vitamin D hormone) are expressed throughout the human brain, but are particularly enriched in the large (likely dopaminergic) neurons of the substantia nigra 2D) protects dopaminergic neurons from 6-hydroxydopamine and N-methyl-4-phenylpyridinium (MPP+) toxicity increasing vitamin D hormone appears to be neuroprotective. Alternatively, as Newmark and Newmark suggest and as seen in traumatic brain injury models, functional vitamin D deficiency (from reduced 25(OH)D, 1,25OH2D or vitamin D receptor dysfunction) may leave nigrostriatal neurons more vulnerable to insult reducing 25(OH)D concentrations (as seen in patients with PD) or vitamin D hormone levels on the vulnerability of the nigrostriatal system to insult has not been tested in vivo. To explore this possibility, we determined if 25(OH)D deficiency in mice renders the nigrostriatal system of those mice more vulnerable to insult by the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).Laboratory evidence also suggests plausibility that the vitamin D physiology may play a role in the PD. The hormone  male C57Bl/6J mice were purchased from Jackson Laboratory. Vesicular monoamine transporter 2 hypomorph (VMAT2 LO) and wild type mice have been previously described ad libitum. C57Bl/6J mice were randomly assigned to either the control or depleted group. Depleted mice were fed rodent chow devoid of vitamin D  while control mice were fed a chow containing identical components plus vitamin D . These diets contain 0.47% calcium, 0.3% phosphorus, 16.4% protein, 59.2% carbohydrate, and 10% fat by weight with 3.9 Kcal/g. Therefore, under these conditions, diet  was the only source of vitamin D; depleted animals received no dietary vitamin D and could not make vitamin D from UVB radiation. There was no difference between starting body mass in the control and vitamin D depletion animal groups prior to changing their chow . Both groups of mice gained body mass on the new chow formulation and no differences were observed between control-fed and vitamin D depletion chow-fed groups at the time of depletion .All animal procedures were conducted in accordance with the National Institutes of Health C57Bl/6J mice were subjected to the control or vitamin D-deficient diet for 6 weeks prior to MPTP challenge. Before MPTP challenge, blood was collected into a BD Microtainer® Serum Separation Tube from the lateral tail vein without restraint. Blood was held on ice for 10 minutes and then centrifuged at 10,000 x g. Serum 25(OH)D levels were assayed by 25(OH)D Direct EIA kit  per manufacturer’s protocol to confirm depletion. Additionally, tail vein blood was collected from VMAT2 LO and wild type (WT) mice for 25(OH)D measures.A diagram of the study design is shown . For theForepaw stride length was measured as described previously th day, forepaw stride lengths were collected for pretreatment baseline measures. Mice then received daily subcutaneous injections of either phosphate buffered saline (PBS) or 15 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine  in PBS for 4 days. Mice were allowed to recover for 7 days. On the 6th day of recovery, mice received an additional day of reinforcement training on the forepaw stride length task. On the 7th day, forepaw stride length measures were taken.After 25(OH)D depletion was confirmed, mice were trained daily on the task for 5 days. On the 6Tissues were analyzed by western blotting technique as previously g for 3 minutes to pellet precipitated proteins. 10 µL of each sample solution was injected and separated using a Thermo Scientific C18 Aquasil column . Mobile phase consisted of 50 mM citric acid, 1.8 mM sodium heptane sulfonate, 0.2% trifluoroacetic acid (v/v),pH 3.0 (A) and acetonitrile (B). Gradient conditions were as follows: 3% B at 0 min, 3% B at 8 min, 18% B at 19 min, 3% B at 20 min and 3% B at 35 min using a flow rate of 250 µL/min. Dopamine, DOPAC and HVA were detected using electrodes set at potentials of −150 and +300 mV. Calibration curves were generated using standards to convert peak area to concentration units.An Agilent 1100 Series Capillary HPLC system coupled with an ESA Coulochem III electrochemical detector was used for separation and quantification of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Briefly, the striatal region of each mouse was weighed and homogenized using a sucrose media  at 10% w/v. A 100 µL aliquot was taken and proteins were precipitated by addition of 5 µL perchloric acid. Samples were then spun at 10,000 x + levels, striata were sonicated  in 5 volumes of 5% TCA and centrifuged for 10 min at 14,000 g. MPP+ levels were determined by analysis of supernatant using an Agilent Model 1200 HPLC equipped diode array detector operated at a wavelength of 290 nm. Separation was achieved on a reverse phase Altima C18 column  with a mobile phase consisting of 89% 50 mM KH2PO4 and 11% acetonitrile. MPP+ was identified by comparison of retention time with known standards and concentrations were calculated from a 6-point standard curve of known concentrations of MPP+. Protein concentrations were determined by Bradford Assay.MPP+ levels were measured similarly as described previously Perfused brains were kept at 4°C for 12 hours in 4% PFA. Brains were then transferred to 30% sucrose and stored at 4°C. Forty µm sections were prepared (Microm HM450) and transferred to Tris-buffered saline and stored at 4°C until use. For immunohistochemical staiining, thin sections were washed six times in 1× TBS to remove all traces of PFA. Thin sections were incubated with a rat monoclonal anti-dopamine transporter (DAT) , or a rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody  overnight at 4°C and then incubated in a biotinylated-goat anti-rat or goat anti-rabbit secondary antibody  for 1 hour at room temperature. Visualization was performed using 0.03% 3,3′-diaminobenzidine  at room temperature.All statistical analyses were performed using Graph Pad Prism 5.0. Student t-tests were performed for initial two group comparisons with significant values reported as p<0.05. Two-way AVOVA analyses were performed on all multi-group comparisons with Bonferroni post-hoc tests to determine significant interactions. Significant values are reported as p<0.05.2D with a much shorter serum half-life (∼15 hours) In order to test the effect of 25(OH)D deficiency on the vulnerability of nigrostriatal neurons, we first depleted mice of serum levels of 25(OH)D. Since a laboratory animal’s sole source of vitamin D is dietary, mice were transferred from standard rodent chow (Purina) to a vitamin D deficient diet to specifically deplete serum 25(OH)D levels. Serum 25(OH) D levels are widely regarded as the most reliable predictor of vitamin D status due to a longer serum half-life (∼15 days) versus 1,25(OH)Next, we confirmed that 25(OH)D deficiency had no effect on the mice’s ability to learn or perform a behavioral index of nigrostriatal damage. Previously, gestational 25(OH)D deficiency has been shown to affect dopamine-dependent locomotor behaviors in mice We hypothesized that 25(OH)D depletion would not affect the dopamine system alone, rather render the system more vulnerable to future insult. To test this, we challenged 25(OH)D-depleted mice with the dopaminergic neurotoxin MPTP (4 x 15 mg/kg) to produce a mild lesion resulting in approximately 50–60% reduction of striatal dopamine. This dose was chosen so that exacerbation or protection could be detected in the vitamin D-depleted animals. MPTP lesion was confirmed by measuring changes in dopamine and markers of dopamine neuron. Striatal dopamine  was signIn order to determine whether 25(OH)D deficiency exacerbates MPTP-induced behavioral changes, forepaw stride length measures were performed. MPTP treatment did not significantly affect forepaw stride length measured at the end of the study  . InteresSince changes in markers of dopamine neuron integrity can be seen in the absence of any behavioral deficit and are often changed when dopamine levels are altered, we next investigated the effect of 25(OH)D depletion of expression of markers of dopamine neurons in the nigrostriatal system. A significant 46% and 49% reduction in striatal tyrosine hydroxylase (the rate limiting step in dopamine synthesis) and dopamine transporter protein levels were observed with MPTP lesion, respectively ; howeverIt is possible that 25(OH)D deficiency could affect the metabolism of the MPTP neurotoxin obscuring any effect that 25(OH)D depletion has on the vulnerability of the nigrostriatal system. Therefore, we tested the ability of 25(OH)D deficiency to interfere with MPTP metabolism. In a separate cohort of mice, we found that MPP+ levels were similar in mice challenged with a single dose of MPTP after 25(OH)D depletion . TherefoBecause 25(OH)D levels are reportedly lower in patients with more advanced PD, we hypothesized that perhaps acute or chronic dopamine loss drives the lower levels of serum 25(OH)D observed in patients with PD. To test this possibility, we also determined whether dopaminergic neuron damage acutely affected 25(OH)D serum levels. In mice on the vitamin D complete chow, there were no differences in 25(OH)D serum levels between mice treated with MPTP and those treated with saline after ten days of lesion . HoweverTherefore to test the effect of chronic dopamine deficiency as seen in PD on 25(OH)D serum levels, we used a mouse model that more closely matches the chronic state of dopamine deficiency, the vesicular monoamine transporter 2 hypomorph (VMAT2 LO) mouse model. These mice have severe impairment of their ability to package dopamine into synaptic vesicles for later release resulting in enhanced turnover of dopamine with a chronic state of hypodopminerigia similar to that seen in patients with PD hormone levels), future studies should include longer duration of 25(OH)D depletion in animals.With exception of bone and tooth health, the role that vitamin D supplements, 25OHD and vitamin D hormone play in the pathogenesis of disease has been understudied and underappreciated. Because epidemiological data have suggested a link between low 25(OH)D status and PD, we sought to investigate the effects of 25(OH)D depletion in the MPTP mouse model- of parkinsonism. We found that while we are able to effectively reduce serum 25(OH)D levels and lesion animals with MPTP, there was no relationship between serum 25(OH)D levels and the magnitude of MPTP injury as measured by striatal and nigral dopamine neuronal markers, striatal dopamine, and gait analysis. Also, we found no difference in the status of dopaminergic nigrostriatal neurons as measured above with serum 25(OH)D deficiency alone. This suggests that in animals, low serum 25(OH)D levels may not predict dopamine neuronal health. It is unlikely that 25(OH)D deficiency in PD patients is a surrogate marker for some other metabolic changes such as in calcium or parathyroid hormone levels as these levels are rarely altered in our clinical population with 25(OH)D insufficiency (data not shown). It is also unlikely based on previous studies that calcium levels are affected in our model system MPTP is a reliable toxicant used to selectively lesion dopamine neurons and is the only toxicant model available for animal studies that also undoubtedly causes a parkinsonian syndrome in humans Previously, we have demonstrated that changes in environmental factors during brain development can predispose the nigrostriatal system to damage later in life 2D  confers resistance of dopaminergic neurons to MPP+ (the active form of the toxin) 2D partially restored TH expression in the nigra of 6-hydroxydopamine lesioned rats 2D could be protective by several distinct mechanisms, including stimulating the action of growth factors, acting as an antioxidant, or increasing glutathione production In humans, midlife 25(OH)D insufficiency is associated with higher PD risk while higher serum levels are associated with reduced risk of PD Perhaps studies of other alteration of vitamin D status and models of PD (such as the transgenic A53T α-synuclein mouse or the VMAT2 LO mouse) in vivo and in vitro models of dopaminergic neuronal vulnerability to toxins. These large future studies are necessary to establish the role, if any, that vitamin D supplements, vitamin D status or the active 1,25OH2D (hormone) play PD pathophysiology.Our data suggest that short term 25(OH)D depletion over the course of a few weeks does not render dopamine neurons more susceptible to the neurotoxin, MPTP as we initially hypothesized. However, additional studies are warranted to explore whether more long-standing deficiency of 25(OH)D or gestational depletion have a role in the laboratory


Pollicipes species in an evolutionary context.The 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units that consist of a transcribing region (5S) and a variable nontranscribed spacer (NTS), in higher eukaryotes. Until recently the 5S rDNA was thought to be subject to concerted evolution, however, in several taxa, sequence divergence levels between the 5S and the NTS were found higher than expected under this model. So, many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA. In analyses of 5S rDNA evolution is found several 5S rDNA types in the genome, with low levels of nucleotide variation in the 5S and a spacer region highly divergent. Molecular organization and nucleotide sequence of the 5S ribosomal DNA multigene family (5S rDNA) were investigated in three Pollicipes genomes. They are clustered in up to seven different types based on differences in their nontranscribed spacers (NTS). Five different units of 5S rDNA were characterized in P. pollicipes and two different units in P. elegans and P. polymerus. Analysis of these sequences showed that identical types were shared among species and that two pseudogenes were present. We predicted the secondary structure and characterized the upstream and downstream conserved elements. Phylogenetic analysis showed an among-species clustering pattern of 5S rDNA types.The nucleotide sequence variation revealed that several 5S rDNA variants occur in Pollicipes 5S rDNA is driven by birth-and-death processes with strong purifying selection.These results suggest that the evolution of  In higher eukaryotes, nuclear ribosomal DNA (rDNA) genes are usually organized in two multigene families, each composed of hundreds to thousands of copies. A major family encodes for 28S, 5.8S, and 18S rRNA, and a minor family contains only 5S rRNA genes. The 5S rDNA consists of a conserved transcribing region of 120 bp (hereafter 5S) with a variable intergenic spacer usually referred to as the nontranscribed spacer (NTS). The 5S region is highly conserved in length and sequence even among unrelated species, although there is a high rate of heterogeneity within the NTS region among closely related species. This variation of NTS is due to insertions, deletions, mini-repeats, base-substitutions and pseudogenes and has been used for evolutionary studies and as a source of species-specific or population specific markers .The evolution of ribosomal gene families recently became controversial after it was analyzed in several taxa. The evolution of 5S rDNA units has classically been explained by the concerted evolution model, which suggests that molecular mechanisms such as gene conversion and unequal crossing-over play an important role in the homogenization of repeated units. These mechanisms maintain a high sequence similarity between copies and prevent the independent evolution of each member of a multigenic family . HoweverPollicipes consists of four species: P. pollicipes, P. elegans, P. polymerus and P. caboverdensis. These stalked barnacles are sessile pedunculate cirripede occurring in dense aggregations exposed to heavy swell on rocky intertidal sites. Distribution of P. pollicipes (Gmelin 1789) is on the northeastern Atlantic coast, from Dakar in Senegal to the north coast of Brittany in France   and 5S-Poll-R (5'-ACC GGT GTT TTC AAC GTG AT-3'), and 5S-Poll-F and 5S-Poll-R2 (5'-ACT GGT GTT TTC AAC GTG GT-3'). These designed primers have opposite orientations, are separated by 5 bp and anneal at positions 13-32 y 38-57 of the 5S transcribing region. They were designed for the amplification of one unit of any tandemly arranged 5S rDNA in the genus Pollicipes.The NucleoSpin Tissue kit (Macherey-Nagel and Co.) was used to extract genomic DNA from foot tissue. The 5S rDNA sequences from each genomic DNA were amplified by PCR. Three pairs of primers were used for this analysis. The first one pair was 5S-Univ-F and 5S-Univ-R [2. Thermocycling conditions were 5 min at 95°C; 35 cycles of 30 s at 95°C, 30 s at 50°C, and 30 s at 72°C; and a final 5 min extension at 72°C. A negative control was also included to test for any contamination. The PCR products were resolved in 1.5% agarose gel, and visualized after ethidium bromide staining via ultraviolet trans-illumination.PCRs were carried out in a BIORAD My Cycler tm thermocycler using a reaction volume of 25 μl containing ~ 25 ng genomic DNA, 200 μM each dNTP (Roche Diagnostics), 0.5 μM each primer, 0.625 U Taq DNA polymerase (Roche Diagnostics), the buffer recommended by the polymerase supplier and 2.5 mM MgClE. coli competent cells. A subset of transformant colonies from each cloning reaction was analyzed by PCR in order to check the insert size. A QiaPrep Spin Miniprep Kit (Qiagen) was used to purify the plasmids. Sequencing reactions were carried out using both M13 Forward and M13 Reverse primers in a capillary DNA sequencer .The PCR-generated 5S rDNA fragments were cloned in the pSC-A-amp/kan PCR Cloning Vector (StrataGene), and used to transform http://compbio.dfci.harvard.edu/tgi/software/). We also performed a statistical evaluation of the local similarities among spacer types or assembled clusters in a local BLAST in BioEdit 7.0.9.0. [The quality of the electropherograms was checked in BioEdit 7.0.9.0. . The BLA7.0.9.0. . The BLAThe number of polymorphic sites, the number of haplotypes and the nucleotide diversity were calculated from DnaSP 5.00.04 . All nucDrosophila Eukaryotic Promoter (EPD) and JASPAR database, and others were manually compared with published regulatory elements. The 5S sequences were folded into the RNA alifold web server [In order to search for putative regulatory conserved elements, we analysed sequences upstream and downstream of the 5S region. Searches were performed within the first 78-120 nt upstream and downstream of the DNA transcribing region. Some putative 5S rDNA transcriptional regulatory motifs were identified by the TOUCAN workbench  establisb server  applyingPollicipes 5S rDNA sequences were subjected to a neighbor-net analysis [analysis  implemenanalysis  using GTanalysis . A maximanalysis , selectianalysis . The relanalysis  with 100analysis  was emplAP obtained the sequence data, carried out the genetic analyses and drafted the manuscript. DS and FRF helped in the DNA cloning and sequencing. AMGT and AML coordinated the study and helped to draft the manuscript. All authors read and approved the final version of manuscript.Figure S1: Different 5S rDNA tandem arrangements. Drawings are done to scale. Scheme is as follows: a) last portion of the 5S (88 bp), NTS, 5S (120 pb), NTS, 5S (120 pb), NTS, and the first portion of the contiguous 5S (32 pb) for the trimer; b-g) last portion of the 5S (88 bp), NTS, 5S (120 pb), NTS, and the first portion of the contiguous 5S (32 pb) for dimers. Different colors show different types of NTS. Dotted arrows indicate putative pseudogenes. 5S, 5S rDNA gene; NTS, nontranscribed spacer.Click here for fileTable S1: Accession numbers and details of barnacle sequences used in this study.Click here for fileFigure S2: Alignments of different types. F and G types are aligned together. Putative pseudogenes (red) are aligned with C type sequences. Table S2: Values of similarity among NTS types.Click here for filePollicipes species reconstructed by means of a maximun likelihood treesFigure S3: Phylogenetic relationships of 5S rDNA for the three main variants in . Numbers on nodes represent bootstrap values based on 100 replicates. (a) Phylogeny of A type reconstruted by K81uf + I + G model. (b) Phylogeny of E type reconstruted by SYM + I + G model. (c) Phylogeny of F and G types reconstruted by HKY + I + G model. In (b) and (c), asterisks indicate P. pollicipes sequences. Figure S4: Identified regions  of 78 nucleotides upstream the transcriptional start site of 5S ribosomal DNA. Three conserved regions were identified. Nucleotides shaded in blue share the motif CGGCCACCGGC, those shaded in red correspond to an AT rich region that it was located about -25 bp, and the TTC sequence (shared with Bombyx mori silkworms) is shaded in green color. Figure S5: 5S ribosomal RNA predicted secondary structures of barnacles. Structures correspond to the b and c types sequences that excluding the primer-annealing regions. (a-h) Sequences used in the predicted consensus secondary structures type I Pollicipes and (i) type II Pollicipes. ((a) El04Tie09b;(b) El03Afu02b; (c) Py02Oly04b; (d) El04Afu04b, El03Afu19b, El05Afu04b, El03Afu02c, El01Tie02b, El01Tie01b; (e) El01Tie03b; (f) Py08Oly03b, Py03Oly01b; (g) El03Afu07b, El04Tie06b; (h) El03Afu16b; (i) Po06Bal01b, Po06Bal02b. Figure S6: 5S alignment. Upper line is the consensus 5S rDNA gene. White boxes represent the internal control regions (ICRs) involved in the transcription of the D. melanogaster 5S rDNA, and grey shading areas correspond to the three sequence elements that regulate transcription activity of X. laevis 5S rDNA . The b and c types sequences represent the second and third unit of the array.Click here for fileConstraints applied in RNA alifold.Click here for file


Nucleolus is a dynamic structure that controls biogenesis of ribosomal RNA and senses cellular stresses. Nucleolus contains a number of proteins including ribosomal proteins that conduct cellular stresses to downstream signaling such as p53 pathway. Recently, it has been reported that modification by a ubiquitin-like molecule, Nedd8, regulates subnuclear localization of ribosomal protein L11. Most of L11 is normally localized and neddylated in nucleolus. However, cellular stress triggers deneddylation and redistribution of L11, and subsequent activation of p53. Although Nedd8 modification is thought to be important for L11 localization, the mechanism of how neddylation of L11 is regulated remains largely unknown. Here, we show that Myeloma overexpressed 2 (Myeov2) controls L11 localization through down-regulation of Nedd8 modification. Expression of Myeov2 reduced neddylation of proteins including L11. We also found that Myeov2 associates with L11 and withholds L11 in nucleoplasm. Although Myeov2 interacted with a Nedd8 deconjugation enzyme COP9 signalosome, L11 deneddylation was mediated by another deneddylase Nedp1, independently of Myeov2. Finally, p53 transcriptional activity is upregulated by Myeov2 expression. These data demonstrate that Myeov2 hampers L11 neddylation through their interactions and confines L11 to nucleoplasm to modulate nucleolar integrity. Our findings provide a novel link between oncogenic stress and p53 pathway and may shed light on the protective mechanism against cancer. Nucleolus is a non-membrane organelle composed of ribosomal DNA and proteins. The major functions of nucleolus are synthesis of ribosomal RNA (rRNA) and ribosomal subunit assembly. In addition, nucleolus acts as a sensor of cellular stress response and transmits signals to stress response pathways It has been demonstrated that several nucleolar stress-related proteins including L11 are modified by a ubiquitin-like protein, Nedd8. A number of ribosomal proteins are usually neddylated in vivo www.ensembl.org). Myeov2 is evolutionarily conserved from plant to human, and thought to have similar functions among different species. Given that Myeov2 is expressed abnormally in myeloma cells, expression level should be tightly regulated to maintain normal state and it is likely that Myeov2 expression is associated with the onset of multiple myeloma. However, it is largely unknown how Meyov2 is regulated.Multiple myeloma is a plasma cell cancer. Plasma cells differentiate from B cells in bone marrow, and produce a huge number of specific antibodies In this study, we found that Myeov2 regulates L11 subnucleolar localization and consequently Nedd8 modification. Overexpression of Myeov2 led to a decrease of neddylated L11 and high proportion of nucleoplasmic L11. Intriguingly, Myeov2 interacted with COP9 signalosome, a Nedd8 deconjugation enzyme, although the interaction was not essential for Myeov2-mediated L11 deneddylation. Because expression level of Myeov2 is associated with multiple myeloma, our findings suggest that Myeov2 is a primary mediator that regulates nucleolus integrity and stress response pathway.Myeov2 is overexpressed in myeloma cells and appears to be associated with the plasmacytes proliferation and transformation. Although expression level of Myeov2 is considered to be important for the tumorigenesis of plasma cells, little is known about the function of Myeov2. To investigate this, we searched for target proteins that interact with Myeov2 in HEK293 cells. We identified 18 proteins that associate with Myeov2 at least three times of four experiments (score≥3). Surprisingly, all subunits of COP9 signalosome were co-precipitated with Flag-Myeov2 . COP9 siCOP9 signalosome mediates the deneddylation of Cullin family proteins and regulate CRLs activity; however, the mechanisms of how COP9 signalosome is activated are not well understood. Because our data imply that Myeov2 could be another component of COP9 signalosome, we asked whether Myeov2 mediates deneddylating activity. Unexpectedly, expression of Flag-Myeov2 did not change the neddylation status of Cul1, a cullin family protein . Thus, tWe next investigated the molecular mechanisms of how expression of Myeov2 causes a decrease of neddylation status of L11. To explore this question, we focused on another deneddylating enzyme, Nedp1, which possesses a deneddylation activity for a broad range of proteins in cells per se is not sufficient to disrupt nucleolus. However, treatment with 10 nM ActD for 1 hour induced EGFP-NPM relocation in Myeov2-expressing cells, but not control cells , Talon metal affinity resin (Clontech), MG132 (Peptide Institute), Cyclohexamide (WAKO), and Hoechst 33342 (Life Technologies) were purchased.Antibodies to Flag , Myc (9E10 Santa Cruz), HA , HA , CSN5 , Cul1 , Tubulin , His , p53 , and Mdm2  were used for immunoblot analyses. Antibodies to Myc , HA , GFP , and DYKDDDDK-tag  were used for immunocytochemistry and Anti-Flag M2-agarose bead (SIGMA) for immunoprecipitation. Antibody to Cul3 has been described previously HEK293 5′-AAAAGGATCCACATGTGGCGCGCGCCGGAAGC-3′; and reverse, 5′-AAAAAACTCGAGCTACCTCACATCTATGCATG-3′. Myeov2ΔC forward, 5′-AAAAGGATCCACATGTGGCGCGCGCCGGAAGC-3′; and reverse, 5′-AAAAAACTCGAGCTAATGTCATCATCATCAAAAAGATCTTCAAAA-3′.pCAGEN-His-Ub, pCAGEN-His-Nedd8, pCAGEN-Bla-HA-Nedp1, PG13-luc, pcDNA3-Flag-Mdm2 constructs were provided by Y. Gotoh  5′-GGATCCACATGGTTCCCGCACTGCAGCGAGGGCAGTCG-3′; and reverse, 5′-AAAAAACTCGAGCTACCTCACATCTATGCATG-3′. Myeov2ΔFD forward, 5′-AAAGGATCCACATGTGGCGCGCGCCGGAAGC; Myeov2ΔFD reverse 5′-ATGGACTTGGCAGCCAGCTCAGGACTGCC-3′.Myeov2ΔN forward, 5′-CACAGGACGCTTAAGAAACA-3′, #2 5′-GGACGCTTAAGAAACAGTTTA) were subcloned into pMXII vector.For the shMyeov2 contructs, the oligonucleotides corresponding to the target sequence for Myeov2 , 150 mM NaCl, 1 mM, EDTA, 1 mM DTT) and centrifuged at 14,000 rpm for 5 minutes. The cleared lysates were separated by SDS-PAGE, transferred to PVDF membrane, probed with primary antibodies, and detected with HRP-conjugated secondary antibodies and chemiluminescence reagent .The cell lysates  was rotated with anti-Flag agarose beads (SIGMA) or anti-HA agarose beads (SIGMA) for 4 hours at 4°C. The immunoprecipitants were washed and subjected to immunoblot analyses with indicated antibodies.Cells were transfected with the indicated constructs and lysed in extraction buffer , 300 mM NaCl and 5 mM imidazole). Cell lysates were sonicated briefly and were then incubated with Talon metal affinity resin (Clontech) for 4 hours at 4 C°. The precipitants were washed by buffer (50 mM sodium phosphate buffer (pH 8.0), 300 mM NaCl and 5 mM imidazole) and were then subjected to immunoblot anaylsis.HeLa cells plated on 15 mm coverslips and grown in 12-well plates were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 minutes at room temperature. The coverslips were washed in PBS, blocked with 5% bovine serum albumin (BSA), and 0.4% Triton X-100 in PBS, then incubated with the indicated primary antibodies for one hour at room temperature or overnight at 4°C. Following PBS washes, samples were incubated with secondary antibodies (Alexa Fluor 594 anti-mouse IgG (1∶500), Alexa Fluor 488 anti-rat IgG (1∶500) and Alexa Fluor 488 anti-chicken IgG (1∶500)) for 30 minutes at room temperature in blocking solution. Cells were imaged using a fluorescence microscope . Fluorescence images were analyzed using Image J.HCT116 cells were transfected with the PG13-Luc reporter plasmid, which contains the firefly luciferase gene driven by p53 promoter, and a renilla luciferase expression plasmid as an internal control. Cell lysates were subsequently assayed for both firefly and renilla luciferase activities with Dual-Luciferase Reporter Assay System (Promega), and the former activity was normalized on the basis of the latter.Figure S1Characterization of anti-Nedd8 antiserum. (A and B) Immunoprecipitation of neddylated proteins. Neddylated proteins were immunprecipiated from HEK293 cells using anti-Nedd8 antiserum and analyzed with anti-Nedd8 antiserum (A) and anti-Ub antibody .(TIF)Click here for additional data file.Figure S2Knockdown of Myeov2 has little effect on L11 neddylation. (A) HEK293 cells were transfected with expression vectors encoding Flag-Myeov2 and Myeov2 shRNA. Cell lysates were subjected to immunoblot anayses with antibodies to Flag, GFP and Tubulin. The GFP loading control is expressed from the same plasmid as the shRNA. Asterisk indicates non-specific bands. (B) HEK293T cells were transfected with indicated plasmids. Neddylated proteins were pulled down from denatured cell lysates using Talon metal affinity beads, and analyzed by immunoblot analyses using antibodies to His and Myc.(TIF)Click here for additional data file.Figure S3Myeov2 does not interact with Nedp1. (A and B) Co-immunoprecipitation experiment of HA-Nedp1 and Flag-Myeov2. HEK293T cells were co-transfected with HA-Nedp1 and either Flag-Myeov2 or Flag-Myeov2DFD. Cell lysates were immunprecipiated with either anti-HA or anti-Flag antibodies and analyzed using Flag and HA antibodies.(TIF)Click here for additional data file.Figure S4Myeov2 does not alter Mdm2-mediated p53 ubiquitination. (A) HEK293T cells were transfected with indicated plasmids. Ubiquitinated proteins were pulled down from denatured cell lysates using Talon metal affinity beads, and analyzed by immunoblot analyses using antibodies to His, HA, p53 and Mdm2.(TIF)Click here for additional data file.


Penaeus monodon based on the amplification of nine prostanoid biosynthesis genes: cytosolic phospholipase A2, hematopoietic prostaglandin D synthase, glutathione-dependent prostaglandin D synthase, prostaglandin E synthase 1, prostaglandin E synthase 2, prostaglandin E synthase 3, prostaglandin F synthase, thromboxane A synthase and cyclooxygenase. TBLASTX analysis confirmed the identities of these genes with 51-99% sequence identities to their closest homologs. In addition, prostaglandin F2α (PGF2α), which is a product of the prostaglandin F synthase enzyme, was detected for the first time in P. monodon ovaries along with the previously identified PUFAs and prostaglandin E2 (PGE2) using RP-HPLC and mass-spectrometry. The prostaglandin synthase activity was also observed in shrimp ovary homogenates using in vitro activity assay. When prostaglandin biosynthesis was examined in different stages of shrimp ovaries, we found that the amounts of prostaglandin F synthase gene transcripts and PGF2α decreased as the ovaries matured. These findings not only indicate the presence of a functional prostanoid pathway in penaeid shrimp, but also suggest a possible role of the PGF2α biosynthesis in shrimp ovarian development. The prostanoid pathway converts polyunsaturated fatty acids (PUFAs) into bioactive lipid mediators, including prostaglandins, thromboxanes and prostacyclins, all of which play vital roles in the immune and reproductive systems in most animal phyla. In crustaceans, PUFAs and prostaglandins have been detected and often associated with female reproductive maturation. However, the presence of prostanoid biosynthesis genes remained in question in these species. In this study, we outlined the prostanoid pathway in the black tiger shrimp  Theseectively ,5. Downsesponses .Penaeus chinensis [Litopenaeus vannamei [Penaeus semisulcatus [Marsupenaeus japonicus [Penaeus merguiensis [Penaeus monodon [Carcinus maenas [Callinectes sapidus [Procambarus clarkii [2 (PGE2) and prostaglandin F2α (PGF2α) have been identified in M. japonicus [Procambarus paeninsulanus [2 (PGD2), PGE2 and PGF2α have been detected in the fresh water field crab Oziotelphusa senex senex [2, thromboxane B2 (TXB2) and 6-keto-PGF1α have been reported in C. maenas [2 has been identified in hemolymph, muscle and ovary of domesticated P. monodon [2α has not been reported in this species.The presence of PUFAs and prostaglandins in crustaceans has long been the focus of aquaculture research. All three prostanoid precursors  have been identified in the Chinese prawn hinensis , the Pacvannamei , the gresulcatus , the kuraponicus ,11, the guiensis  and Pena monodon ,14. In as maenas  and the  sapidus , while D clarkii . On the aponicus  and the nsulanus ,19. Prosex senex , while P. maenas . In addi monodon , but the2 and PGF2α is positively correlated with ovarian maturation in P. paeninsulanus [O. senex senex [2 and PGF2α into ovaries of O. senex senex significantly increased the number and the diameter of the oocytes in a dose-dependent manner [P. monodon, the amounts of PGE2 in ovaries and haemolymph increased along with developing ovary stages [2 and PGF2α were highest in ovaries stage I and continued to decrease until stage IV in M. japonicus [In crustaceans, one of the more prominent roles of prostanoids is the regulation of female reproductive maturation. For instance, the production of PGEnsulanus  and O. sex senex . Furthert manner . In domey stages . Howeveraponicus . NeverthDaphnia pulex, whose annotated genome sequence revealed nine prostanoid biosynthesis genes: cytosolic phospholipase A2 (cPLA2), COX, prostaglandin D2 synthase A, prostaglandin D2 synthase B, prostaglandin E2 synthase (PGES), carbonyl reductase 1, thromboxane A and thromboxane B, and two prostanoid receptors prostanoid receptor EP4 isoform A and B [COX genes have been identified in Gammarus spp. and Caprella spp. [PGES genes have been characterized in L. vannamei [Homarus americanus (Accession: MGID155886 from the Marine Genomic Project) [Lepeophtheirus salmonis and Caligus rogercresseyi [Although the production of prostanoids and their precursors is well-established in most crustaceans, prostanoid biosynthesis genes in these species are poorly characterized. Thus far, the only crustacean with a fully constructed prostanoid pathway is the fresh water flea  A and B . In marilla spp. , while Pvannamei , the AmeProject)  and the cresseyi .P. monodon prostanoid pathway based on the identification of eight P. monodon prostanoid biosynthesis genes, the detection of lipid precursors and prostaglandins, and the detection of prostaglandin synthase activity. The correlations observed among gene transcription, prostaglandin production, and ovarian maturation also suggest that prostaglandin biosynthesis may be involved in the regulation of the P. monodon female reproductive system.Due to the roles of prostanoids in the reproductive system in most crustaceans, the characterization of the prostanoid pathway in economically valuable organisms, such as penaeid shrimp, is essential for both scientific gain and potential applications in aquaculture practice. In this study, we propose a scheme for the P. monodon prostanoid biosynthesis genes were amplified from shrimp ovary cDNA. RACE-PCR was used to obtain full-length gene sequences, resulting in the identification of nine putative P. monodon prostanoid biosynthesis genes: cytosolic phospholipase A2 (PmcPLA2), hematopoietic prostaglandin D synthase (PmhPGDS), glutathione-dependent prostaglandin D synthase (PmgPGDS), prostaglandin E synthase 1 (PmPGES1), prostaglandin E synthase 2 (PmPGES2), prostaglandin E synthase 3 (PmPGES3), prostaglandin F synthase (PmPGFS), thromboxane A synthase (PmTBXAS), and cyclooxygenase (PmCOX). These gene sequences were then analyzed by TBLASTX, revealing 51-99% sequence identities of the predicted P. monodon enzymes when compared with their closest homologs  for protein domain prediction, revealing that the P. monodon proteins contain the same domain types and positions as the prostanoid enzymes found in other species  [PGES1 requires glutathione for its enzymatic function, which involves converting PGH2 to PGE2 [2 (R108 and T112 - 64ERXXXAXXNXX75 E required for oxygenation product formation (In  (MAPEG) ,28. PGES to PGE2 . Multipl to PGE2 . In addiPGES2 or membrane-associated prostaglandin E synthase 2 is a Golgi membrane-associated pre-protein that requires spontaneous cleavage of the N-terminal hydrophobic domain to become a mature cytosolic enzyme [66C-X-X- 69C  is a 23 kDa GSH-requiring enzyme that was originally termed p23 based on its initial characterization as a co-chaperone of heat shock protein 90 [PmPGES3 gene.otein 90 ,36. Unliotein 90 . In humaotein 90 . CDART pPGFS encodes a bifunctional enzyme in the aldo-keto reductase (AKR) superfamily that converts PGD2 and PGH2 to -(15S)-9α,11β,15-trihydroxyprosta-5,13-dien-l-oic acid  and PGF2α, respectively [+ cofactor binding site  were collected from the Andaman Sea, Thailand. The weight and length of broodstock were measured and recorded prior to dissection. Shrimp ovaries were dissected, weighed and either subjected to metabolite extraction or flash frozen in liquid nitrogen and stored at -80 °C for RNA extraction. The gonadosomatic index (GSI) of each shrimp was calculated using the following equation: ovarian weight/body weight x 100. Ovarian developmental stages were assigned according to GSI, separating the broodstock into stage I (GSI < 1.5), stage II (GSI = 2-4), stage III (GSI > 4-6) and stage IV (GSI > 6) [Wild female GSI > 6) .TM, Vertical Chromatography, Co., Ltd., Thailand), which was previously washed with 10 ml methanol and 10 ml water. Columns were then washed with 10 ml water, 4 ml hexane, and again with 10 ml water, before being eluted with 10 ml ethyl acetate. The eluate was then evaporated and dissolved in ethanol for subsequent HPLC analysis.Tissue homogenates underwent extraction twice with an equal volume of ethyl acetate. The solvent phases were then pooled together and dried under vacuum. Afterward, the dried crude extract was dissolved in 5% methanol. The solution was filtered to remove insoluble material before being loaded onto a 6-ml C18 SPE cartridge  and a gradient mobile system consisting of acetonitrile (ACN): water: acetic acid (30:70:0.01) to 100% ACN in 35 min, with the flow rate of 0.8 ml/min. Resulting peaks were detected using the DIONEX Ultimate 3000 diode-array detector (DIONEX Ltd.). For RP-HPLC/MS analysis, samples were analyzed using the Agilent 1200 series LC system  coupled with the micrOTOF mass spectrometer and operated with HyStar version 3.2 .Shrimp tissue extracts were separated by RP-HPLC using an AcclaimTM First Strand cDNA Synthesis Kit with oligo (dT)18 primer  according to the manufacturer’s instructions.Shrimp organs were homogenized and subjected to total RNA extraction using the Trizol reagent . mRNA was purified from total RNA using the Oligotex mRNA Mini Kit . First strand cDNA was synthesized using the RevertAidhttp://pmonodon.biotec.or.th [http://www.marinegenomics.org [TM 2 PCR kit  and the SmartTM RACE cDNA amplification kit (Clontech). All PCR products were cloned into the pTZ57R/T vector (Fermentas), transformed into DH5α E. coli, and submitted for DNA sequencing . Identities of the obtained cDNA sequences were verified by TBLASTX analysis [Initial PCR fragments were obtained using primers based on short gene sequences from The Black Tiger Shrimp EST Project at ec.or.th  and the mics.org . 5′- andanalysis . Multiplanalysis . Rooted analysis . Proteinanalysis .TM EvaGreen® Supermix  according to the manufacturer’s instructions. Amounts of prostanoid biosynthesis gene transcripts relative to that of the house-keeping gene elongation factor 1α (EF1α) were obtained using the standard curve method [Quantitative real-time PCR analysis was performed using the SsoFaste method . The spein vitro PGE2 synthesis assay, ovary homogenates were incubated in a rotary shaker with 25 µM AA at 28 °C, 200 rpm and collected at 0, 30, 60, 120, 240, and 360 minutes post-incubation. The homogenates were centrifuged at 12,000 x g for 2 min at 4 °C and the amounts of PGE2 and PGF2α in the supernatant were estimated using the prostaglandin E2 EIA kit – Monoclonal and prostaglandin F2α EIA kit .Stage IV shrimp ovaries were harvested and homogenized in Hank’s Balanced Salt Solution (HBSS) with an osmolarity of 720 mmol/kg . For the P<0.05).Statistical significant was assessed in this study using the T-test with two samples assuming equal variances Click here for additional data file.Figure S2HPLC analysis and mass spectra of prostanoid precursors in shrimp ovary extract.Ovaries from 5 wild broodstock were homogenized in HBSS, pooled together, and incubated at 28 °C, 200 rpm for 1 h. The homogenate was extracted as described in materials and methods. The extract was then subjected to analysis by RP-HPLC and mass spectrometry. RP-HPLC elution profiles of commercially available prostanoid standards (A) and prostanoid precursors found in ovary homogenate (B) was obtained at 200 nm wavelength. Subsequent mass spectrometry analysis of prostanoid precursors in ovary homogenate revealed the mass spectra of EPA (C), DHA (D) and AA (E).(TIF)Click here for additional data file.Figure S3PmPGES1 and related sequences from vertebrates and invertebrates.Phylogenetic trees constructed from Predicted amino acid sequences from various organisms were obtained from GenBank and the Marine Genomics Project. Sequences were aligned using CLUSTALW multiple sequence alignment program and the rooted phylogenetic tree with branch length (UPGMA) were constructed.(TIF)Click here for additional data file.