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A high density of ancient spliceosomal introns in oxymonad excavates Slamovits, Claudio H; Keeling, Patrick J Apr 25, 2006

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ralssBioMed CentBMC Evolutionary BiologyOpen AcceResearch articleA high density of ancient spliceosomal introns in oxymonad excavatesClaudio H Slamovits and Patrick J Keeling*Address: Canadian Institute for Advanced Research, Botany Department, University of British Columbia, 3529-6270 University Boulevard, Vancouver, BC, V6T 1Z4, CanadaEmail: Claudio H Slamovits - claudio@mail.botany.ubc.ca; Patrick J Keeling* - pkeeling@interchange.ubc.ca* Corresponding author    AbstractBackground: Certain eukaryotic genomes, such as those of the amitochondriate parasites Giardiaand Trichomonas, have very low intron densities, so low that canonical spliceosomal introns haveonly recently been discovered through genome sequencing. These organisms were formerlythought to be ancient eukaryotes that diverged before introns originated, or at least becamecommon. Now however, they are thought to be members of a supergroup known as excavates,whose members generally appear to have low densities of canonical introns. Here we have usedenvironmental expressed sequence tag (EST) sequencing to identify 17 genes from the uncultivableoxymonad Streblomastix strix, to survey intron densities in this most poorly studied excavate group.Results: We find that Streblomastix genes contain an unexpectedly high intron density of about 1.1introns per gene. Moreover, over 50% of these are at positions shared between a broad spectrumof eukaryotes, suggesting theyare very ancient introns, potentially present in the last commonancestor of eukaryotes.Conclusion: The Streblomastix data show that the genome of the ancestor of excavates likelycontained many introns and the subsequent evolution of introns has proceeded very differently indifferent excavate lineages: in Streblomastix there has been much stasis while in Trichomonas andGiardia most introns have been lost.BackgroundOne of the prominent features that distinguishes eukary-otic genomes from those of prokaryotes is the presence ofspliceosomal introns. Introns are intervening sequencesthat are removed from expressed RNAs, in the case of spli-ceosomal introns through a series of transesterficationsmediated by a large riboprotein complex called the spli-ceosome [1]. Spliceosomal introns are only known fromeukaryotic nuclear genomes, and were the subject oflate debate [2-4]. One of the interesting features of intronevolution that came to light during this debate was thelarge range in intron density. At one extreme, intronsappeared to be lacking in several protist lineages thatwere, at the time, thought to be the earliest-branchingeukaryotes. These lineages included diplomonads (e.g.,Giardia) and parabasalia (e.g. Trichomonas).The early-branching status of these organisms has sincePublished: 25 April 2006BMC Evolutionary Biology 2006, 6:34 doi:10.1186/1471-2148-6-34Received: 26 January 2006Accepted: 25 April 2006This article is available from: http://www.biomedcentral.com/1471-2148/6/34© 2006 Slamovits and Keeling; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 8(page number not for citation purposes)intense controversy over their potential role in early geneorigins and evolution, the so-called introns early versusbeen undermined by a variety of data, and now diplo-monads and parabasalia are thought to be part of a largeBMC Evolutionary Biology 2006, 6:34 http://www.biomedcentral.com/1471-2148/6/34assemblage of protists called excavates, which alsoincludes trypanosomes, euglenids, and a number of para-sitic and free living flagellate or amoeboflagellate lineages[5]. However, despite the accumulation of a considerablequantity of molecular data from both Giardia and Tri-chomonas, as well as the identification of proteins involv-ing splicing in Trichomonas [6], evidence for introns intheir genomes remained intriguingly elusive. Indeed, onlyrecently were introns finally characterized in these organ-isms [7-9], and remain extremely rare. Only three intronshave been found in G. intestinalis among thousands ofknown genes [8,9] and forty-one introns were identifiedin the T. vaginalis genome after exhaustive searches [7].Information from excavates other than Trichomonas andGiardia is scarce, but overall there seems to be a generallylow density of introns (with the possible exception ofJakobid flagellates based on one family of genes[10]).Moreover, other instances of non-canonical introns andsplicing are known in excavates [11-13], as are systemswhere splicing machinery is put to a slightly different usesuch as trans-splicing [14-16].One of the excavate groups about which we know very lit-tle are the oxymonads. Oxymonads are anaerobic flagel-lates found almost exclusively in association withanimals, many in the guts of termites and wood-eatingroaches [17]. This is the only group of amitochondriatesfor which secondary loss of mitochondria has not beenyet demonstrated, but they are closely related to the flag-ellate Trimastix, which has a vestigial organelle, so a pri-mary lack of mitochondria in oxymonads is unlikely.Mostoxymonads are not available in culture because theylive in complex communities with other protists andprokaryotes. As a result, there are few molecular dataavailable from any oxymonad, and no introns have beenidentified [18]. The oxymonad Streblomastix strix is asym-biont of the dampwood termite Zootermopsis angusticollisfrom North American Pacific coastal region. This specieshas a number of unusual morphological characters,including a peculiar long slender cell shape with deep lon-gitudinal vanes which is apparently maintained by inti-mate association with epibiotic bacteria [19], So far, manycopies of four genes (alpha-tubulin, beta-tubulin, HSP90,and elongation factor-1 alpha) have been characterizedfrom S. strix [18], and the complete absence of intronsfrom all sequences (a total of 19,888 bp) suggests the oxy-monads might share low intron densities apparently com-mon to excavates. Here, we have used the recentdocumentation of a rare non-canonical genetic code inStreblomastix [18] to identify 17 oxymonad genes from anenvironmental expressed sequence tag (EST) pool fromthe hindgut of Zootermopsis. The genomic DNA sequencefor each mRNA was determined and we found that, inhigh density of canonical spliceosomal introns. Moreover,a large proportion of these introns are shared in positionwith other distantly related eukaryotes, suggesting thatthey are ancient intron positions retained in oxymonadsbut lost in other excavates such as Giardia and Tri-chomonas.Results and discussionIdentification of oxymonad sequences from ESTsA total of 5,337 ESTs from a Z. angusticollis termite hind-gut cDNAlibrary were sequenced and found to form 2,595clusters of unique sequences. Overall, the sample wasdominated by sequences of parabasalian origin (tran-scripts encoding parabasalian actin and actin-related pro-teins alone represented 32% of all ESTs). Moreover, thereare few oxymonad sequences known outside this sample,so Streblomastix cDNAs could not be identified based onsimilarity to known genes (only 2 ESTs, corresponding toknown Streblomastix alpha- and beta-tubulin sequences,were identified by BLASTX searches). Accordingly, weused the presence of a rare non-canonical genetic code inStreblomastix as a filter to identify at least those geneswhere non-canonical codons were sampled. In Streblomas-tix, TAA and TAG encode glutamine (Q) rather than stopas in the universal code [18], so all clusters were comparedto public databases using BLASTX and examined individ-ually for in frame stop codons, in particular at positionsnormally encoding glutamine. No other protist known toexist in Z. angusticollis has been shown to possess a non-canonical genetic code. The other prominent protists inthis insect are parabasalia, which are not known to deviatefrom the universal genetic code and whose sequences arealso easy to identify with BLASTX searches given their highsimilarity with T. vaginalis genomic sequences.Using the non-canonical code as a filter, we were able toidentify 17 protein-coding genes (Table 1), representing amajor increase in the available sequence data from oxy-monads. Formerly partial sequences of 4 protein codinggenes were known from Streblomastix, and a handful ofcDNAs were known from other species [18,20,21]. Fromthis sample we recovered 8 complete protein-codinggenes, an additional 5 genes missing only 1 to 30 codonsat the N-terminus, and another three lacking from 100 to160 codons at the N-terminus. In addition, a short frag-ment encoding 258 codons of the large protein UPF1 wasseverely truncated, but we failed to obtain more sequence.Complete or near-complete sequences included five ribos-omal proteins (RPS7 and 9, RPL4, 18 and 21), alpha- andbeta-tubulin, the nuclear transporter Ntf2, cyclophilin, apeptidyl-isomerase involved in assisting protein folding,and NAD-dependent glutamate dehydrogenase. Also, twoversions of the cystein-protease Cathepsin B werePage 2 of 8(page number not for citation purposes)contrast to other amitochondriate protists and the limiteddata previously available for Streblomastix, a relativelyobtained. Although related, these sequences exhibitedseveral differences at the amino acid level, so they areBMC Evolutionary Biology 2006, 6:34 http://www.biomedcentral.com/1471-2148/6/34likely to represent multiples copies of the gene. We alsoidentified two copies of the carbon metabolism enzymepyruvate phosphate dikinase (PPDK), the functional andevolutionary significance of which are discussed else-where [22]. One conserved hypothetical protein was alsofound to use the Streblomastix genetic code. This proteinhas homologues in diverse eukaryotes (e.g. Arabidopsisthaliana AAM67532), buthas no assigned function. UPF1is a key member of nonsense-mediated decay (NMD).This protein may be of interest in Streblomastix because itis involved in a mechanism of mRNA surveillance devotedto eliminating defective transcripts, such as those carryingpremature stop codons [23]. NMD has been describedand studied in animals and yeasts, but not yet found inprotists [24]. The presence of UPF1 in Streblomastix sug-gests NMD is used by oxymonads, and in organismswhere stop codons are reassigned to encode amino acids.Finally, UAP56 is a member of the DEAD box family ofRNA helicases that is associated with the spliceosome andintervenes in early steps of pre-mRNA splicing in mam-mals and yeasts, but is also linked to mRNA export[25,26]. Even in the absence of introns, the presence ofUAP56 indicates the likely presence of the spliceosome inoxymonads, and therefore by extension introns as well.Introns in Streblomastix genesGenomic DNA sequences were obtained for all Streblomas-tix coding regions identified from the cDNA library.Despite the fact that many alleles and loci representingtranscripts were interrupted by introns. In total, we found21 introns in our sample of 17 genes with genes having asmany as 5 introns (Table 1). Including previously knownintronless EF-1 alpha and HSP90 genes (alpha and betatubulin are included in our sample) [18], the overall den-sity is 1.1 introns per gene. However this is likely to be anunderestimation since some of our sequences are trun-cated and could contain further introns, and there is a biasfavouring genes that are more often intronless (e.g.HSP90). This density is less than that observed in the rel-atively intron-rich mammals and plants, but comparableto many other eukaryotic genomes, and certainly muchhigher than Giardia and Trichomonas where only 3 and 41introns have been detected despite very large quantities ofgenomic data [7,9].Overall, the Streblomastix introns were found to exhibitcharacteristics typical of eukaryotic spliceosomal introns.Introns ranged from 46 to 229 bases (Table 2), but mostwere between 60 and 100 bases long, and the AT contentwas markedly higher than that of the coding sequence(Table 2). Spliceosomal introns are flanked by GT and AGdinucleotides in the vast majority of known introns, whileabout 0.1% are U12 AT-AC introns[27] and a very smallproportion of known introns use other non-canonicalsplice boundaries. Interestingly, however, the first of onlythree introns from G. intestinalis to be discovered has CU-AG boundaries [8]. Of the twenty-one introns from Stre-blomastix, 20 featured canonical GT-AG boundaries, butTable 1: S. strix genes identified in this study. Streblomastix genes recovered from the Z. angusticollis hindgut RNA sample. For incomplete sequences, the number of missing amino acids were estimated from homologues from Giardia and/or Trichomonas. UPF1 shows extensive size variation among eukaryotic lineages (between 800 and 1600 amino acids, approximately), so it is difficult to determine how much sequence this fragment is lacking. ND: not determined.Protien name Length(AA) IntronsRPS7 189 0RPS9 188 3RPL4 445 NDRPL18 184 1RPL21 146 2Alpha-tubulin 450 0Beta-tubulin 447 0Cyclophillin 167 0Cathespin B (1) 312 1Cathespin B (2) 283 3NAD-dependent glutamate dehydrogenase 446 1Pyruvate phosphate dikinase (1) 779 1Pyruvate phosphate dikinase (2) 784 0UPF1 258 NDUAP56/BAT1 272 2Nuclear transport factor 2 123 2Conserved hypothetical protein 203 5Page 3 of 8(page number not for citation purposes)four proteins were previously found to contain nointrons, we found that most of the genes encoding theseone intron in rps9 was flanked by AC-AG splice sites.However, the Streblomastix intron is located very close toBMC Evolutionary Biology 2006, 6:34 http://www.biomedcentral.com/1471-2148/6/34the start of the transcript, so we cannot exclude the possi-bility that this intron sequence is incomplete and a canon-ical boundary lies upstream.We also inspected intron sequences to look for conservedfeatures that may correspond to functional motifs.Although signals important for intron recognition andremoval are not very well understood, some have beenstudied in certain detail, especially in mammals andyeasts. The branch-point is a sequence element requiredfor lariat formation during splicing [28]. The mammalianbranch point consensus sequence has been determined tobe CURAY, where the A corresponds to the actual branch-ing point. In yeast, the branch point sequence is morestrictly defined as UACUAAC [29]. The plant branch pointappears to be similar to that of mammals [30]. In all cases,the branch point is located near the 3' splice site, but theexact location varies. In contrast, the putative branchpoint found in the three introns of Giardia (ACURAC) islocated directly adjacent to the 3' splice site [9]. Likewise,the potential branch points in Trichomonas are invariablyACUAAC and are also adjacent to the 3' splice site [7]. Theapparently strict requirement for proximity between thebranch point and 3' splice site is rare in metazoa andyeast, but common to Trichomonas and Giardia. This led tothe suggestion that the branch point and 3' splice site rec-introns (Figure 1) reveals highly conserved A, U and G res-idues at positions +3 to +5, respectively. This is in goodagreement with the first 5 positions of the yeast 5' splicesite (typically GUAUGU), suggesting that interaction withU1 snRNA is conserved. At the 3' splice site no branchpoint motifs like those of Giardia or Trichomonas wereobserved, although the -1 position (adjacent to the AGdinucleotide) was invariably a pyrimidine and the regionis T-rich. Overall, branch point specification in Streblomas-tix introns is probably different from that of Giardia or Tri-chomonas. Under the assumption that these lineages arerelated it is possible that the peculiar features observed inGiardia and Trichomonas may be a consequence of second-ary implification in their spliceosomal apparatus.Conservation of intron positions in oxymonad genesStreblomastix intron-containing genes were compared tohomologues from other eukaryotes, and surprisinglymore than half of the Streblomastix intron positions wereshared with members of at least two different eukaryoticsupergroups, unikonts and plants (where the best sam-pling of intron-containing genes exists), and in one casealso with a chromalveolate (for six examples, see Figure2). This suggests these are relatively ancient introns andperhaps date back to the last common ancestor of alleukaryotes. This degree of conservation is high, takingTable 2: Basic features of the S. strix introns. Characteristics of 21 introns found in 17 Streblomastix genes analysed. Size and base composition are shown. GC% mRNA shows base composition of the coding sequence (excluding introns).Protien name Intron Size(bp) %GC(Intron) %GC(coding)RPS9 1 57 0.29 0.462 88 0.383 57 0.3RPL18 1 60 0.25 0.49RPL21 1 46 0.11 0.372 66 0.09Cathespin B (1) 1 122 0.16 0.35Cathespin B (2) 1 63 0.22 0.472 66 0.183 91 0.15Glutamate Dehydrogenase 1 100 0.36 0.38Pyruvate phosphate dikinase (1)1 229 0.21 0.44UAP56/BAT1 1 105 0.22 0.372 168 0.15Nuclear transport factor 2 1 56 0.27 0.412 58 0.2Conserved hypothetical 1 64 0.27 0.442 61 0.313 102 0.234 54 0.35 69 0.22Average 85 0.23 0.42Page 4 of 8(page number not for citation purposes)ognition could be combined in these species [7]. Aligningthe regions around the 5' splice site of all Streblomastixinto account data such as those of Rogozin et al., who cal-culated that approximately 20% of the introns in Plasmo-BMC Evolutionary Biology 2006, 6:34 http://www.biomedcentral.com/1471-2148/6/34Page 5 of 8(page number not for citation purposes)Examples of conserved intron positions between Streblomastix and other eukaryotesFigure 1Examples of conserved intron positions between Streblomastix and other eukaryotes. In each case a section of the gene is shown aligned at the amino acid level, and the position of the intron found in all aligned sequences is indicated above by a trian-gle with a number indicating the phase (0, 1, or 2). Aligned sequences are from three unikont groups, animals (H. sapiens and P. troglodytes), fungi (S. pombe, U. maydis and A. fumigatus), and slime molds (D. discoideum), from one chromalveolate group, the ciliate (P. tetraurelia), and from three plantae groups, land plants (A. thaliana), green algae (Bigelowiella natans nucleomorph), and red algae (Guillardia theta nucleomorph).H. sapiensS. pombe0TDPYRQLLHKLYQYLAHRIGNQDIYLRLLVKLYRFLARRTNSDDVYLKLTVKLYRFLVRRTNSQDPYLLLLVKLYRFLARRTDSS. strixH. sapiensA. thalianaU. maydisS. strixA. thalianaD. discoideumG. theta (nm)B. natans (nm)1IHHARLMITQGHIRIGKQIVTVPSYMVIHHARVLIRQRHIRVRKQVVNIPSFIVIHHSRVLIRQRHIRVGKQLVNIPSFMVIHEARILIMHKHIQVKNQIVNKPSFLVIHHARTLIRQRHFRVGKRLVNSPSFLVS. strixP. troglodyteA. thalianaB. natans (nm)P. tetraurelia2FIIDECDKVLEKN-------DMRGDVQRIFVSCFILDECDKMLEQL-------DMRRDVQEIFRMTFILDECDKMLESL-------DMRRDVQEIFKMTFVLDECDKMLDQIGKQAQIAHMRRDVQEIFRATS. strixH. sapiensA. thalianaA. fumigatus1EQEFPSISIHGDLPQDQRLKRYQEFKDFQSRIEQNFPAIAIHRGMPQEERLSRYQQFKDFQRRIECNFPSICIHSGMSQEERLTRYKSFKEGHKRIECNFPSIAVHSGVSQEERIKRYKEFKEFNKRIS. strixH. sapiensA. thalianaA. fumigatus2FDSDRSQLSSLYR----EESMLSFEGFDNDRTQLGAIYI----DASCLTWEGLDGNRDLLAPLYLGTPSQTSHMTMEGS. strix0MSRNYRKVFKTPRHPFERERIDSELRYYRNYGKTFKGPRRPYEKERLDSELKMSSNYSKTSHTPRRPFEKERIDAELKNYRNFSKTSKTPRRPYEKERLDYELKNYTNYSKIWNRPKNPYEKLRLCNEIRA. NTF2  (intron 1)B. rpl18C. rps9 (intron 1)D. rps9 (intron 3)E. UAP56/BAT1 (intron 1)F. UAP56/BAT1 (intron 2)BMC Evolutionary Biology 2006, 6:34 http://www.biomedcentral.com/1471-2148/6/34dium are shared by at least one of the other genomesanalysed (Human, Anopheles, Drosophila, Caenorhabditis,Arabidopsis, Schizosaccharomyces and Saccharomyces), andthat 25% of the human introns are shared by Arabidopsis[31]. It is also possible that shared intron positions aredue to independent gains, but it is very unlikely that theobserved level of shared positions (about 50%) resultedfrom parallel gains, in particular in the many cases wherethe intron is found in several of the major lineages ofeukaryotes. Whether intron gains or losses predominatein eukaryotic evolution is still a subject of controversy.Recently, several studies using different analyticapproaches and datasets addressed this question with var-ied results, but in all cases, they show that ancestral con-servation accounts for the large majority of sharedpositions [32-36]. The degree of conservation observed inStreblomastix intron positions suggests two things. First, itsuggests that the ancestor of excavates was relativelyintron rich and retained a large number of ancient introns,somes, Giardia and Trichomonas. This assumes the rela-tionship between oxymonads and other hypothesizedexcavates is correct, but this is not certain and oxymonadslack the morphological trait used to define excavates (theventral groove). However, other ultrastructural characters[37] as well as molecular phylogenies have shown a closeaffiliation between oxymonads and Trimastix [38], a freeliving flagellate that does have excavate characteristics[5,39]. Multi-gene phylogenies also lend additional sup-port for a common origin of the lineages leading to oxy-monads, diplomonads and parabasalia [21]. The secondimplication of this data is that intron gain and loss havetaken place very slowly in the lineage leading up to Stre-blomastix: if intron turnover were rapid, then we wouldexpect a low proportion of ancient introns to remainunless ancient intron positions were under some selectionto be retained. While this is probably true in a few individ-ual cases where introns have acquired some function inthe control of gene expression, there is presently no evi-Sequence logos showing conservation at intron bordersFig re 2Sequence logos showing conservation at intron borders. Top: 5’ splice site (position 1) and surrounding sequence. Bottom: 3’ splice site (-1) and surrounding sequence. Logos were made using Weblogo (http://weblogo.berkeley.edu).012bits5‘ -19T-18AT-17GCAT-16GAT-15GCAT-14GCAT-13GCAT-12CAT-11GCAT-10AGCT-9TA-8GAT-7ACT-6-5GCAT-4GCTA-3CT-2A-1G1 2CTGA3CT4TAG53‘012bits5‘ -6CT-5-4CTA-3GAC-2CTGA-1TCAGG1T2GTA3CT4TAG5TCA6 7TA8AT93‘Page 6 of 8(page number not for citation purposes)many of which were subsequently lost in the genomeswhere we have the most information, such as trypano-dence either for or against this as a common feature ofancient introns. None of these shared introns are knownBMC Evolutionary Biology 2006, 6:34 http://www.biomedcentral.com/1471-2148/6/34from either Giardia or Trichomonas, so any potential func-tion is clearly dispensable, although it is interesting tonote that the rps9 intron 1 has been retained by the G.theta nucleomorph, which is very intron poor, having kepta total of only 17 introns [40].ConclusionThe present sampling of protein-coding gene sequencesfrom Streblomastix suggests that oxymonad genomes con-tain a relatively large number of canonical splicesomalintrons, many of which are at ancient conserved positions.This is in contrast to the better studied excavate genomessuch as those of kinetoplastids, Giardia and Trichomonaswhere canonical spliceosomal introns are either rare orhave been co-opted in specific ways, such as the splicedleaders in euglenozoa. The fact that many Streblomastixintrons are ancient shows that the genome of the ancestorof these organisms, and indeed probably all extanteukaryotes, contained many introns and that the intron-poor state found in Giardia and Trichomonas is more likelyindependently derived.MethodscDNA library construction and EST sequencingTermites were collected from a rotten log in Point Grey,Vancouver, Canada. The whole hindgut content of about60 individuals of Zootermopsis angusticollis from a singlecolony was collected and total RNA was extracted usingTRIZOL (Invitrogen). A directionally cloned cDNA librarywas constructed (Amplicon Express) and 5,337 cloneswere sequenced from the 5' end. ESTs were trimmed forvector and quality, and assembled into clusters by PEPdbhttp://amoebidia.bcm.umontreal.ca/public/pepdb/agrm.php.Identification and genomic characterisation of Streblomastix genesStreblomastix sequences were recovered from EST data byidentifying protein coding sequences containing in-frameTAA and TAG stop codons. Putatively stop-coding con-taining mRNAs were re-sequenced in both strands. Incases where cDNA clones were truncated, the sequenceswere extended by means of 3' and 5' RACE (Ambion)using total termite hindgut RNA. The genomic sequencefor each mRNA was amplified using specific primers cor-responding to the ends of each complete or partial cDNAand PCR-amplified using genomic DNA purified from thetermite hindgut content. All PCR products were clonedusing TOPO and sequenced both strands. Accession num-bers for new sequences are [genbankDQ363664,genbankDQ363665, genbankDQ363666,genbankDQ363667, genbankDQ363668,genbankDQ363669, genbankDQ363670,genbankDQ363675, genbankDQ363676,genbankDQ363677, genbankDQ363678,genbankDQ363679].Authors' contributionsCHS analysed the EST data, performed PCR and sequenc-ing, and examined conservation of intron positions inother organisms. PJK collected the termites and purifiedRNA for library construction. Both authors participated inthe writing and editing of the manuscript. All authors readand approved the final manuscript.AcknowledgementsThis work was supported by a grant from the Natural Sciences and Engi-neering Research Council of Canada, and EST sequencing was supported by the Protist EST Program through Genome Canada/Genome Atlantic. We thank A. de Koning for help isolating termite gut RNA, and N. Fast for crit-ical reading of the manuscript. 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