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Draft genome sequence of the Daphnia pathogen Octosporea bayeri: insights into the gene content of a… Corradi, Nicolas; Haag, Karen L; Pombert, Jean-François; Ebert, Dieter; Keeling, Patrick J Oct 6, 2009

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Open Access2009Corradiet al.V lume 10, Issue 10, Article R106ResearchDraft genome sequence of the Daphnia pathogen Octosporea bayeri: insights into the gene content of a large microsporidian genome and a model for host-parasite interactionsNicolas Corradi*, Karen L Haag†‡, Jean-François Pombert*, Dieter Ebert¤† and Patrick J Keeling¤*Addresses: *Canadian Institute for Advanced Research, The Biodiversity Research Centre, University of British Columbia, University Boulevard, Vancouver, BC, V6T 1Z4, Canada. †Universität Basel, Zoologisches Institut, Evolutionsbiologie, Vesalgasse, CH-4051 Basel, Switzerland. ‡Department of Genetics, UFRGS, Porto Alegre, RS 91501-970, Brazil. ¤ These authors contributed equally to this work.Correspondence: Patrick J Keeling. Email: pkeeling@interchange.ubc.ca© 2009 Corradi et al.; 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.The Octosporea bayeri  genome sequence<p>The draft genome sequenc of Octosporea bayeri,  a microsporidian pathogen of Daphnia, provides insights into the content and evo-lution of a la ge microsporidian g nome</p>AbstractBackground: The highly compacted 2.9-Mb genome of Encephalitozoon cuniculi placed themicrosporidia in the spotlight, encoding a mere 2,000 proteins and a highly reduced suite ofbiochemical pathways. This extreme level of reduction is not universal across the microsporidia,with genomes known to vary up to sixfold in size, suggesting that some genomes may harbor a genecontent that is not as reduced as that of Enc. cuniculi. In this study, we present an in-depth surveyof the large genome of Octosporea bayeri, a pathogen of Daphnia magna, with an estimated genomesize of 24 Mb, in order to shed light on the organization and content of a large microsporidiangenome.Results: Using Illumina sequencing, 898 Mb of O. bayeri genome sequence was generated, resultingin 13.3 Mb of unique sequence. We annotated a total of 2,174 genes, of which 893 encodes proteinswith assigned function. The gene density of the O. bayeri genome is very low on average, but alsohighly uneven, so gene-dense regions also occur. The data presented here suggest that the O. bayeriproteome is well represented in this analysis and is more complex that that of Enc. cuniculi.Functional annotation of O. bayeri proteins suggests that this species might be less biochemicallydependent on its host for its metabolism than its more reduced relatives.Conclusions: The combination of the data presented here, together with the imminent annotatedgenome of Daphnia magna, will provide a wealth of genetic and genomic tools to study host-parasiteinteractions in an interesting model for pathogenesis.Published: 6 October 2009Genome Biology 2009, 10:R106 (doi:10.1186/gb-2009-10-10-r106)Received: 9 July 2009Revised: 2 September 2009Accepted: 6 October 2009The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2009/10/10/R106Genome Biology 2009, 10:R106106.2http://genomebiology.com/2009/10/10/R106 Genome Biology 2009,     Volume 10, Issue 10, Article R106       Corradi et al. RBackgroundMicrosporidia are extremely successful, highly adapted obli-gate intracellular parasites known to infect a wide range ofanimals, such as arthropods, fish, and mammals, includinghumans [1,2]. These parasites are characterized by the pres-ence of a highly specialized host invasion apparatus called thepolar tube (or polar filament), which is used to penetrate andinfect new host cells. Microsporidian cells significantly differfrom other eukaryotes, as they lack conventional mitochon-dria and Golgi apparatus and harbor 70S instead of 80Sribosomes [3-5]. These features were once taken to suggestthat microsporidia represent a very ancient eukaryotic line-age [6-11], but recent advances in cell biology, genomesequencing, and phylogenetic reconstruction have all shownthat all these apparently primitive features instead reflect anextreme state of reduction, perhaps a result of their obligateintracellular parasitic lifestyle. Instead, it is now widelyacknowledged that microsporidia are, in fact, related to fungi,and have relict mitochondria (called mitosomes) [12], degen-erated eukaryote-like ribosomal RNA subunits [13], andreduced genes and genomes [14-24].The extremely reduced nature of microsporidian genomeshas attracted attention since they were first noted at the endof the 1990s [13], culminating in 2001 with the completion ofthe first microsporidian genome from the mammalian para-site Encephalitozoon cuniculi [25]. The Enc. cuniculi genomeis extremely small, at only 2.9 Mb, and the 2,000 genes itencodes provided the first compelling evidence for a strongcorrelation between obligate intracellular parasitism and theloss of metabolically important genes in eukaryotes. Meta-bolic capabilities are indeed significantly reduced in Enc.cuniculi, and genes required for de novo biosynthesis ofpurine and pyrimidine nucleotides or those involved in thetricarboxylic acid cycle, fatty acid beta-oxidation, respiratoryelectron-transport chain and the F0F1-ATPase complex arecompletely absent from its genome. The reduction of severalmetabolic pathways in Enc. cuniculi implied that these para-sites might be extremely dependent on their host for obtain-ing most of their metabolites and energy. For example, it hasbeen indeed recently demonstrated that this parasite and itsmitosomes both import ATP from its host via specific trans-porters [26,27].In addition to a significant reduction in its metabolic capabil-ities, the genome of Enc. cuniculi is also very compact. Itsgenes are reduced in size and separated by remarkably shortintergenic regions. This extreme compaction has impactedthe process of transcription so that in the microsporidia Enc.cuniculi and Antonospora locustae a significant part of theirmRNA transcripts has been found to overlap between adja-cent genes [28-30]. Genome reduction has also apparentlyaffected the rate of gene rearrangement, as conservation ofSince the completion of the Enc. cuniculi genome, newgenomic data from other microsporidian parasites have beenlimited to two in-depth genome surveys from Enterocytozoonbieneusi and Nosema ceranae [33,34], a smaller survey fromA. locustae [32] and some very small surveys from variousother species [35-38]. The deeper-sampled genomes of Ent.bieneusi and A. locustae show many similarities with that ofEnc. cuniculi - all three genomes are compact and containroughly the same number of genes and pathways - but this isperhaps not surprising because all three genomes are also rel-atively small (ranging from 2.9 to 6 Mb) and might not, there-fore, represent all microsporidian genomes adequately.So how do larger microsporidian genomes compare withsmaller ones? Does their large size reflect the presence ofmore genes and pathways or do they harbor the same genesbut separated by much larger intergenic regions? These ques-tions have been partly addressed with genome surveys fromSpraguea lophii [35], Vittaforma cornea [36], Edhazardiaaedis, and Brachiola algerae [37,38], but because of theirvery low sequence coverage no conclusion can be drawnabout their overall gene content and evolution. In the presentstudy, we provide a 37× sequence coverage of the largegenome of the microsporidian Octosporea bayeri. O. bayeriis a parasite of the freshwater planktonic crusteacean Daph-nia magna [39]. Other Daphnia species have never beenfound to be infected. The parasite is both horizontally andvertically transmitted [40]. Vertical transmission occurs with100% efficiency to the asexual (parthenogenetic) eggs of thehost and with somewhat reduced efficiency to the sexual eggs.Horizontal transmission occurs after the host cadaver decom-poses and environmental spores are released. Infection fol-lows ingestion of spores by the filter feeding host. Theparasite reduces host survival and fecundity. Its geographicdistribution is limited to rock pool D. magna populationsalong the baltic Sea in Finland and Sweden [39] and a singlereport from the Czech Republic.From our sequence survey, over 13 Mb of unique O. bayerisequence data have been assembled and 2,174 ORFs havebeen identified, providing an excellent framework to charac-terize the overall gene content and structure of a large micro-sporidian genome, to compare it with its more reducedrelatives and to increase the availability of genetic markersfrom this latter species. Consistent with small surveys frommicrosporidia with large genomes, the gene density of the O.bayeri genome is generally low but also highly variable. Mostof the genes known in the Enc. cuniculi genome are also foundin O. bayeri, but a number of other genes are also found thatare apparently absent in other microsporidia. The functionaldistribution of the proteins significantly differed between O.bayeri and its more reduced relatives, suggesting the meta-bolic capacity and host dependency within the group is alsoGenome Biology 2009, 10:R106gene order is strikingly high among microsporidia comparedto what has been reported for other eukaryotes [31,32].variable. The wealth of genomic data from this parasite cou-pled with the annotation of the Daphnia genome should fur-106.3http://genomebiology.com/2009/10/10/R106 Genome Biology 2009,     Volume 10, Issue 10, Article R106       Corradi et al. Rther increase the interest for this model of host-parasiteinteractions [41].ResultsGene content of the O. bayeri genomeApproximately 898 Mb of DNA sequence was obtained fromshotgun and paired-end 35-bp reads with the IlluminaGenome Analyzer™, resulting in an estimated 34.2 to 37.2×coverage of the O. bayeri genome, which has been estimatedto 24 Mb based on total number of bases sequenced dividedby the average coverage. This calculation does not take intoaccount the fact that some assembled contigs might representseveral identical regions in the reference genome, and thatunassembled reads might represent DNA sequences fromother sources (that is, contaminants). Reads were assembledinto 41,804 contigs representing a total of 13.3 Mb ofsequence data (26% G+C), with only 20 contigs displayingevidence of contamination. The length of contigs averaged320 bp (100 bp to a maximum of 8 kb). The small size of mostcontigs resulted in the incompleteness of most ORFs identi-fied in this study and, on average, incomplete ORFs werefound to encode 60% of the amino acids of their respectiveeukaryotic homologs. This explains why the complete (oralmost complete) O. bayeri proteome has been identifiedwithin an assembly that is almost half the size of the esti-mated genome.A total of four rRNA genes, 37 tRNAs and 2,174 predicted pro-tein-coding ORFs were identified (Table 1). Of the O. bayeriORFs, 1,405 were found to have homologs in the Enc. cuniculigenome, representing about 70% of its annotated genes [25](Additional data file 1). Over 93% of Enc. cuniculi proteinswith assigned functions and 53% of its hypothetical proteinshad clear homologs in the O. bayeri genome [25,33]. Over25% of Enc. cuniculi homologs identified are full length, whileothers were slightly truncated in the carboxy-terminal oramino-terminal regions, or both. Another 80 ORFs wereidentified that were found to have homologs in other organ-isms, but not Enc. cuniculi, 72 of which could be assigned to afunctional category (Additional data file 2), the majority ofwhich have highest similarities with fungal homologs, sug-gesting that they are ancestral within the lineage and notrecently introduced into the O. bayeri genome. The remain-ing 689 O. bayeri putative ORFs (of at least 200 amino acids)returned no significant hits in BLAST homology searchesagainst the National Center for Biotechnology Information(NCBI) non-redundant database. However, 25 of theseshowed significant similarities with hypothetical proteinsfrom the A. locustae database, indicating that O. bayeri andA. locustae share a number of hypothetical proteins that areTable 1General characteristics of O. bayeri and other microsporidian genomesGeneral characteristics O. bayeri Enc. cuniculi Ent. BieneusiNumber of chromosomes NA 11 6Genome size (Mb) 24.2* 2.9 6Assembled Mb 13.3 2.5 3.86Genome coverage (%) 55† 86 64G+C content (%) 26 47 25Gene density 1 per 4,593 bases‡ 1 per 1,025 bases 1 per 1,148 basesMean intergenic region (bp) 429§ 129 127Presence of overlapping genes No Yes YesNumber of SSU-LSU rRNA genes 2¶ 22 Unkown¥Number of 5S rRNA genes 2¶ 3 Unkown¥Number of tRNAs 37 46 46Number of tRNA synthetases 21 21 21Number of tRNA introns (size in bp) 1 (50) 2 (16, 42) 2 (13, 30)Number of splicesomal introns (size in bp) 6 (24-33) 13 (23-52) 19 (36-306)Number of predicted ORFs 2,174# 1,997 3,804**Number of ORFs assigned to functional categories 894 (41%) 884 (44%) 669 (39%)Mean size of CDS (bp) 1,056†† 1,017†† 1,002††*The genome size has been estimated using total number of bases sequenced divided by the average coverage. †Based on the 24.2-Mb estimated genome size. ‡Based on the 200 largest contigs. §Based on contigs (n = 23) in which two or more ORFs of at least 100 amino acids have been identified. ¶Only two contigs harboring an SSU-5.8S-LSU gene array have been identified in the O. bayeri genome survey. ¥Based on [33]. #Includes ORFs with assigned functions, homologs of Enc. cuniculi hypothetical proteins, and hypothetical proteins of at least 200 amino acids identified in the Genome Biology 2009, 10:R106O. bayeri genome. **The Ent. bieneusi genome has been subjected to several segmental duplications and the number of ORFs identified in that study includes a very large number of duplicates [33]. This number should, therefore, not be taken into account to determine the haploid coding capacity of this species. ††Based on 95 and 63 complete Enc. cuniculi and Ent. bieneusi orthologs, respectively. CDS, coding sequence.106.4http://genomebiology.com/2009/10/10/R106 Genome Biology 2009,     Volume 10, Issue 10, Article R106       Corradi et al. Rabsent in Enc. cuniculi and Ent. bieneusi. It is also importantto note that a large proportion of microsporidian hypotheticalproteins have been found to be smaller than 200 amino acids[25,31-33], so the actual number of ORFs could be over 25%higher than what we report here, perhaps in the range of, orhigher than, what has been recently reported for N. ceranae[34].Functional categories represented in O. bayeriAll identified O. bayeri ORFs were assigned to the 11 func-tional categories listed in [25,33] (Figure 1; Additional datafile 3). Such comparison is currently unavailable for N. cera-nae [34]. O. bayeri ORFs are well distributed among the func-tional categories, yet display differences when compared toEnc. cuniculi and Ent. bieneusi. Specifically, five categories(metabolism, energy production, cell growth and DNA syn-thesis, transcription and protein destination) are more repre-sented in O. bayeri than in Enc. cuniculi and Ent. bieneusi,whereas four other categories (transport facilitation, intracel-lular transport, cellular organization - biogenesis, and cellrescue) are reduced in number in O. bayeri. Within eachfunctional category, several pathways stood out as being par-ticularly different among the three species. For instance,genes involved in lipid and fatty acid metabolism and glyco-sylation were better represented in O. bayeri (37 and 12 pro-teins, respectively) than either Enc. cuniculi (29 and 7proteins) or Ent. bieneusi (8 and 5 proteins), while proteinsinvolved in the translocation of various substrates acrossmembranes are underrepresented in O. bayeri (Figure 2).Finally, in contrast to what has been reported for other spe-cies with smaller genomes [33,34], no evidence for gene orsegmental genome duplication events has been identified inthe present survey.Phylogeny of O. bayeri and evolution of the ATP transporters in the microsporidiaO. bayeri was put into a phylogenetic context by comparingthe amino acid sequences from its newly identified alpha- andbeta-tubulins with those of other microsporidia (Figure 3a).Our tree is consistent with the most recently reported usingthe same amino acid sequences [42]. Specifically, Nosemaand Encephalitozoon are sisters to one another, as are Anton-ospora and Brachiola. The remaining species all branch moredeeply, and O. bayeri is in this tree basal to all other micro-sporidian species from which large genome sequence data arepresently available. Only a single ATP transporter protein wasidentified in O. bayeri, and phylogenetic analyses of all pres-ently known microsporidian members of this family show theO. bayeri protein clustering with strong support at the base ofa clade including Antonospora and Brachiola homologues,all of which are sister to the Encephalitozoon/Enterocyto-zoon/Nosema clade (Figure 3b). This is not consistent withthe rRNA tree, and might represent a mis-rooting of eithertree, or ancient paralogy of the ATP transporters.O. bayeri intronsOnly 13 introns have been annotated in the Enc. cuniculigenome at present, and we identified a total of 6 introns in theDistribution of O. bayeri (blue), Enc. cuniculi (yellow) and Ent. bieneusi (red) proteins among functional categoriesFigure 1Distribution of O. bayeri (blue), Enc. cuniculi (yellow) and Ent. bieneusi (red) proteins among functional categories. The ordinate represents the number of ORFs assigned to the corresponding category. Each of the O. bayeri proteins was assigned to only one of eleven functional categories listed in [25,33]. The corresponding gene list is presented in the online version of this manuscript (Additional data file 3). *Based on a 4× sequence coverage O. bayeriEnc. cuniculiEnt. bieneusi050100150200EnergyMetabolism Cell growth, division and DNA synthesisTranscription Protein synthesisProtein destinationTransport facilitationIntracellular TransportCellular organisation -   BiogenesisCommuni-cation - Signal transduc-tionCell rescue, defense, death and aging*Genome Biology 2009, 10:R106[33].106.5http://genomebiology.com/2009/10/10/R106 Genome Biology 2009,     Volume 10, Issue 10, Article R106       Corradi et al. Rpresent survey, all of which are homologous to intronsreported in Enc. cuniculi ribosomal protein genes (L19, L27a,L37a, L37, L39, S26) [25]. All the O. bayeri introns identifiedhere are located within or close to the start codon, which isconsistent with the introns in Enc. cuniculi [25], Saccharo-myces cerevisiae [43] and cryptomonad nucleomorphs [44].The retention of the majority of these introns leads to frame-shifts and termination codons, while their removal leads to acomplete ORF that is highly conserved with homologs fromother eukaryotes. The intron sequences are available with theonline version of this paper (Additional data file 4).O. bayeri-specific large amino acid insertionsA number of large insertions ranging from 15 to 57 aminoacids were identified in 14 conserved proteins in O. bayeri (O-sialoglycoprotein endopeptidase, 3-hydroxy-3-methylglu-taryl CoA reductase, 3-ketoacyl CoA thiolase,  -trehalaseprecursor, choline phosphate cytidyltranferase, transcriptionfactor of the E2F/DP family, tubulin -chain, kinesin-like pro-tein, pyruvate dehydrogenase E1 component subunit , repli-cation factor C, T complex protein 1  subunit, threonyl tRNAsynthetase, and translation elongation factor 2). These inser-tions are all in-frame and in most cases are surrounded byhighly conserved amino acid motifs, although they are notceosomal introns (data not shown). Similar insertions havebeen previously reported in the parasites Plasmodiumberghei and Toxoplasma gondii [45,46].Length of O. bayeri proteinsThe majority of O. bayeri proteins were found to be largerthan homologs from Enc. cuniculi (69%) and Ent. bieneusi(65%) (Figure 4). However, the opposite trend was identifiedwhen O. bayeri genes were compared with other fungal line-ages, in which case the majority of O. bayeri proteins (75% onaverage) were found to be smaller than homologs from theother fungal lineages, even when the fungal species comparedhad a smaller genome than O. bayeri. The difference in thenumber of amino acids was found to be significantly largerbetween O. bayeri and other fungal lineages (14% smaller onaverage) than between O. bayeri and other microsporidia (3%larger on average) (Figure 4).Gene density and syntenyGene density and synteny in O. bayeri were examined byannotating all ORFs of at least 100 amino acids on the 200largest contigs (average length of 2,795 bp). In more than halfof these contigs, no putative ORF could be identified. Onecontig was found to harbor three putative ORFs, whereas 72and 22 contigs harbored one or two recognizable ORFs,respectively. No correlation between the length of the contigsand the number of ORFs could be identified (Figure 5a).Based on these contigs, gene density was calculated to be 1gene every 4,593 bases. However, when two or more ORFswere identified on the same contig the average intergenicregion was calculated to be only 429 bp, suggesting the genedensity is highly variable across the genome. Conservation ingene order could be identified in only two cases, representing8% of all the gene pairs identified (Figure 5b).Repeated elementsThe large amount of small, non-coding DNA sequences iden-tified in this study could reflect the presence of highlyrepeated sequences in the O. bayeri genome. This possibilitywas investigated by measuring the sequence coverage of eachcontig and identifying a possible correlation with their length.As suspected, the contigs with highest coverage are also thesmallest. Specifically, all contigs with a coverage over 200×are smaller than 300 bp, suggesting these are highly repeti-tive (Additional data file 6).The presence of repeated elements was also investigatedamong all contigs. A total of 74 O. bayeri contigs harbor DNAsegments homologous to known fungal repeated elements(Additional data file 7). The Mariner, Gypsy and Copia classesof repeated elements are the most frequently observed in O.bayeri. The O. bayeri contigs also display DNA strings thatare repeated in tandem, with strings repeated at least twiceExamples of sub-functional categories showing sharp differences in distribution between O. b yeri (blue), Enc. cu iculi (yellow) and Ent. bieneusi (red) roteinsFigure 2Examples of sub-functional categories showing sharp differences in distribution between O. bayeri (blue), Enc. cuniculi (yellow) and Ent. bieneusi (red) proteins. (a) Functional sub-categories more highly represented in O. bayeri than in Enc. cuniculi and Ent. bieneusi. (b) Functional sub-categories less represented in O. bayeri than in Enc. cuniculi and Ent. bieneusi. *Based on a 4× sequence coverage [33] (that is, almost 10 times lower than the present genome draft), suggesting a number of these transporters may yet be identified in the Ent. bieneusi genome survey.0510152025303540Glycosylation Lipid fatty BiosynthesisADP/ATPTransportersABCTransporters(a) (b)O. bayeriEnc. cuniculiEnt. bieneusi*Genome Biology 2009, 10:R106generally located within functionally important domains(Additional data file 5). RT-PCR confirmed that none of theseinserts are removed from mRNA and so do not represent spli-identified in 1,345 contigs (data not shown). However, thesetandem repeats are usually short and rarely exceed ten con-secutive repeated strings. Putative stem-loop structures with106.6http://genomebiology.com/2009/10/10/R106 Genome Biology 2009,     Volume 10, Issue 10, Article R106       Corradi et al. RAT-rich palindromic stems have been identified in a numberof contigs, although the primary sequences of these potentialstructures, aside from their biased nucleotide composition,do not appear to be repeated per se.DiscussionArchitecture of a large microsporidian genomeThe currently available microsporidian genomes best repre-mediate in size, but our knowledge of microsporidiangenomes is still strongly biased, which might hinder the elu-cidation of the evolution of this poorly understood group. Ourpresent survey of the O. bayeri genome is the first deep sur-vey of a larger microsporidian genome, and estimates fromsequence coverage suggest it may even be the largest knownmicrosporidian genome (at 24 MB). What accounts for thisvariation in genome size and which features of microsporid-ian genomes have to be reconsidered after adding a genomePhylogenetic relationships of microsporidia and their ATP transportersFigure 3Phylogenetic relationships of microsporidia and their ATP transporters. (a) Phylogenetic reconstruction of the microsporidian phylogeny based on available - and -tubulin amino acid sequences and gains of ATP and ABC transporters. Known genome sizes and number of transporters are shown. Ent. bieneusi tubulins cluster as a sister group to the clade including Encephalitozoon and Nosema species; this position is represented by a black square. (b) Evolution of the ATP transporter family based on available amino acid sequences from a range of microsporidian parasites. 1, Putative ancestral duplication of ATP transporters within the microsporidia following lateral gene transfer from prokaryotes. 2, A putative secondary gene duplication occurred in the more diverged genera, Nosema, Enterocytozoon and Encephalitozoon. 3, Supported lineage including all three diverged genera. 4, Species-specific duplication of an ATP transporter. *Data from NC, JFP et al., unpublished.0.052.5Mb2.3Mb2.9Mb 4 1312841≥3*<10Mb15Mb5.4Mb<20Mb6.2Mb24Mb19.5Mb?? ??4*4*????????????00?????20Mb?~8.9 (?)Concatenated α- and β-tubulin microsporidian phylogeny Reported genome size# of ADP/ATP Transporters# of Proteins with "ABC" motifs Nosema bombycis Nosema ceranae Encephalitozoon cuniculi Encephalitozoon intestinalis Encephalitozoon hellem Antonospora locustae Brachiola algerae Microsporidia sp. AMVB Edhazardia aedis Glugea plecoglossi Trachipleistophora hominis Spraguea lophii Octosporea bayeri Conidiobolus coronatus Entomophaga maimaiga10010010010010098 -99100100Large set of transportersReduced set of transporters(a) Chlamydophila pneumoniae Chlamydophila abortus0.2 Nosema ceranae Nosema ceranae Nosema ceranae Nosema ceranae Antonospora locustae Antonospora locustae Antonospora locustae Octosporea bayeri Enterocytozoon bieneusi Paranosema grylli1001009910097100961001007981 7682100 Enterocytozoon bieneusi Enterocytozoon bieneusi Enterocytozoon bieneusi Encephalitozoon cuniculi Encephalitozoon cuniculi Encephalitozoon cuniculi Encephalitozoon cuniculiATP transporters Clade IIDiverged ATP transporters?ATP transporters Clade IAncestral ATP transporters?1274 3(b)Genome Biology 2009, 10:R106sent the lower limits in the spectrum of genome sizes, not onlyfor Eukaryotes as a whole, but also microsporidia. The singleexception to this is N. ceranea, whose genome is more inter-from the other end of the genome size spectrum? There areseveral answers to these questions.106.7http://genomebiology.com/2009/10/10/R106 Genome Biology 2009,     Volume 10, Issue 10, Article R106       Corradi et al. RGenomes might be larger due to the presence of more genes,which could be due to whole or partial genome duplications,repetitive sequences, expansion of gene families, or the reten-tion of a greater diversity of genes in general. They might alsohave about the same complement of genes but have largerintergenic regions, more or larger introns, more transposableelements, and so on. Previous small-scale surveys of micro-sporidia with larger genomes have demonstrated a higherproportion of non-coding DNA, but reveal nothing about theoverall organization of the genome because the fragmentssampled were small and only a tiny fraction of the genomewas characterized in any one case [35,36,38]. The data pre-sented here provide additional evidence that large micro-sporidian genomes have a very low gene density, in this caseup to a fivefold decrease compared to species with smallergenomes, but also provide information on the organizationand structure of a large genome in this group. [25,32-34].only 45 bp, which is even shorter than most intergenic regionsfound in Enc. cuniculi, Ent. bieneusi and A. locustae. Second,it now seems obvious that gene density alone accounts formost of the variation in genome size between different micro-sporidian species, although we did find numerous genes in O.bayeri that are absent in Enc. cuniculi (see below).Smaller microsporidian genomes have also been noted assharing a high conservation of gene order across distantlyrelated species, which has been attributed to compaction[31,32,34]. Despite the overall low gene density, we found 8%of all annotated gene pairs (equating to 2 out of 24 gene pairs)were conserved in order between O. bayeri and Enc. cuniculi.This is not very different to what is found in other micro-sporidia [31,32], and close to the expectation for closelyrelated fungi [47]. It is interesting that both cases describedhere involve pairs of genes that are unusually close to oneanother (423 and 15 bp apart). This may reflect the role ofDifferences in gene length among microsporidia and their fungal relativesFigu  4Differences in gene length among microsporidia and their fungal relatives. (a) Comparison of the length (in amino acids) of O. bayeri proteins to orthologs from Enc. cuniculi, Ent. bieneusi, S. cerevisiae, U. maydis, B. dendrobatidis and R. oryzae. In general, O. bayeri proteins are longer than microsporidian orthologues, but shorter than fungal orthologues. Vertical arrows indicate the average reduction or increase in protein size compared to O. bayeri. (b) Specific examples of length variation between orthologs from O. bayeri, Enc. cuniculi, Ent. bieneusi and S. cerevisiae.SameLargerSmaller020040060080010001200O. bayeriE. cuniculiE. bieneusiS. cerevisiaeProtein length (in amino acids)26Sprotea-some beta subunitRPL11 Thymidylate kinasePeptide chain release factor subunit 1Ribo-nucleoside diphos-phate reductase small chain Tubulin gamma chainGamma glutamyl transpep-tidaseDNA-directed RNA poly-merase III subunit 2 (130kDa)Zinc protein(ECU02_0310)(a)(b)S. cerevisiaen = 50  ~ 15%U. maydisn = 49  ~ 21%B. dendrobatidisn = 52   ~ 10%R. oryzaen = 51  ~ 11%E. cuniculin = 95   ~ 2%E. bieneusin = 63  ~ 4%Variation in gene density across the O. bayeri genomeFigure 5Variation in gene density across the O. bayeri genome. (a) Identification and distribution of ORFs (of at least 100 amino acids) among the largest O. bayeri contigs. Only the 100 largest contigs are shown here for convenience. Yellow dots represent contigs in which no ORF could be annotated. Blue and red arrows and dots represent contigs harboring two or one ORF, respectively. (b) Two cases of gene order conservation between O. bayeri and Enc. cuniculi.O. bayeriE. cuniculiEcu02_1420 Ecu02_1430(570bp) (813bp)71bpEcu02_1420 Ecu02_1430(791bp)(incomplete on 5’)423bpEcu06_0350 Ecu06_0360(743bp) (824bp)62bpEcu06_0360Ecu06_0350(incomplete on 5’)(399bp)45bp(b)(a)0 20 40 60 80 100 12010002000300040005000600070008000Length (in bp)ContigChromosome 2Contig 6939Chromosome 6Contig 4605Genome Biology 2009, 10:R106First, gene density is not homogeneous across the genome,but is instead a sum of long stretches (5.5 kb) of non-codingsequences, as well as regions where genes are separated bycompaction in conservation of gene order, but it might also bea sampling bias since closely spaced genes are more likely to106.8http://genomebiology.com/2009/10/10/R106 Genome Biology 2009,     Volume 10, Issue 10, Article R106       Corradi et al. Rbe found on the same contig in our survey, which is based oncontigs, rather than a complete genome.The large size of the O. bayeri genome does not reflect exten-sive and segmental gene duplication. However, numerousnon-coding and small genomic repetitions could have playeda role in its expansion. The origin of these repetitive regionsis difficult to assess without a better genome assembly.Because these do not encode known functional proteins, norharbor potential ORFs, however, it is possible that these rep-resent telomeric and sub-telomeric regions of the O. bayerigenome. If this is the case, genome size variation in micro-sporidia could also be a consequence of variation in the size oftelomeres. This prediction is supported by the recent acquisi-tion in our laboratory of genome data from other, muchsmaller genomes, showing that the vast majority of unassem-bled Illumina™ reads belong indeed to telomeric regions(NC, JFP and PJK, unpublished).Length of microsporidian genes and size of the protein networkMicrosporidian proteins are known to be shorter in generalthan orthologs in other organisms, a characteristic that hasbeen attributed to the reduction in gene content and, byextension, protein networks in these cells [25,48]. In keepingwith this, the majority of the O. bayeri proteins are shorterthan orthologs from S. cerevisiae (approximately 5,570genes, size 12 Mb), Ustilago maydis (approximately 6,500genes, 20 Mb genome), Batrachytridium dendrobatidis(approximately 8,700 genes, 24 Mb genome) and Rhizopusoryzae (approximately 17,459 genes, 35 Mb genome). Inter-estingly, however, O. bayeri proteins are also larger thanorthologues found in Enc. cuniculi and Ent. bieneusi. Conse-quently, the O. bayeri genome provides additional evidencethat microsporidian proteins are shorter than their homologsfrom other fungal phyla, but also that their size correlates bet-ter with the coding capacity rather than the size of the genomein which they are found.Evidence for the progressive loss of ancestral genes throughout the evolution of the microsporidian lineagePrior to this study, the vast majority of genes with predictedfunctions found in diverse microsporidia were also found inEnc. cuniculi [32-36,38]. Three exceptions were found in A.locustae [49-51] and a single one was found in Ent. bieneusi[33]. This suggested that all members of this group share acommon core set of genes that have been retained after mas-sive gene losses occurred in their ancestor, resulting in only asmall degree of variability in gene content. This predictionwas based, however, on a very low coverage for two largemicrosporidian genomes [38]. The O. bayeri genome and itsevolutionary position within the group suggest that perhapsearly microsporidians possessed many more genes with pre-by lineage-specific reductions and expansions in somebranches of the microsporidian tree. The total number ofORFs identified in O. bayeri also suggests an overall codingcapacity that is at least 10% larger than that of Enc. cuniculi.This is a conservative estimate based on the annotation of O.bayeri hypothetical proteins of at least 200 amino acids.Since it is known that Enc. cuniculi proteins shorter than 200amino acids make up over a quarter of its total coding capac-ity [25], the overall coding capacity of O. bayeri is almost cer-tainly greater still. It has been suggested that both N. ceranaeand Enc. bieneusi genomes contain genes that are absent inEnc. cuniculi; however, the novel sequences in these genomesare apparently all hypothetical ORFs or transposable-like ele-ments, and not genes with predicted functions. In these caseswe cannot rule out that these are rapidly evolving genes withunrecognized homologues in other microsporidian genomes,or in some cases are not functional genes at all. In contrast,the genome of O. bayeri contains at least 80 genes with pre-dicted functions and recognizable homologues in otherorganisms, but which are absent in Enc. cuniculi. This con-firms that the proteome complexity of the ancestral micro-sporidian was greater than that seen in Enc. cuniculi (andother current taxa for which genome level surveys have beenconducted to date), and suggests that further genomesequencing, especially of putatively deep-branching taxa,should reveal still more genes previously unseen in micro-sporidia. It is also formally possible that many genes wereacquired relatively recently in the lineage leading to O. bayeriby lateral gene transfer, which has indeed been observed inother microsporidia [49]. However, this does not seem likelyfor all these genes given the rarity of transferred genes inother microsporidia, and especially given that the most highlyconserved cases are all notably similar to homologs in fungi,suggesting they are more likely ancestral to the micro-sporidia. This implies that much more proteome diversityawaits discovery as more microsporidian genomes are char-acterized.Functional importance of O. bayeri proteins absent in other microsporidiaPerhaps the most intriguing finding of the present study is theidentification of 80 O. bayeri proteins sharing homology witheukaryotes but not with Enc. cuniculi. Not surprisingly, theseinclude eight transposable elements, some of which showed ahigh similarity to those reported from Nosema bombycis[52]. Transposable elements are absent in the most reducedmicrosporidian genomes [25,33], but are commonly reportedin the ones that are larger and less compact [34,37,38,52], soin this case our study simply corroborates previous findings.The remainder of these eukaryotic proteins stood out forbeing involved in important functional processes. In total, 14are involved in transcriptional processes, including RNAGenome Biology 2009, 10:R106dicted functions than previously thought. It now seems likelythat there was a large reduction in the ancestral proteome fol-lowing the origin of microsporidia, but this was also followedpolymerases or proteins involved in the transcription oftRNAs, while 19 are part of different metabolic pathways suchas the metabolism of fatty acids and lipids and nucleotide106.9http://genomebiology.com/2009/10/10/R106 Genome Biology 2009,     Volume 10, Issue 10, Article R106       Corradi et al. Rmetabolism. A whole set of proteins involved in the modifica-tion of proteins and three cation transporters are also presentin O. bayeri but absent in Enc. cuniculi. The identification ofthese eukaryotic proteins is important as it shows that the O.bayeri proteome is more complex than that of Enc. cuniculior Ent. bieneusi. Moreover, most of these proteins have high-est similarities with homolgs from fungal lineages, suggestingthey arose through common descent rather than by theirrecent incorporation into the genome by lateral gene transfer.Do O. bayeri protein categories reflect a lesser host dependency?Aside from the set of O. bayeri proteins that are absent inEnc. cuniculi, the overall number of proteins with assignedfunctions is generally similar in the two genomes. This doesnot imply that both species encode the same set of identifiableeukaryotic homologs and, indeed, we observed several differ-ences in the functional distribution of their proteins. Forinstance, genes involved in lipid and fatty acid metabolismare at least 25% more common in O. bayeri than in Enc.cuniculi or Ent. bieneusi. Similarly, O. bayeri harbors twoadditional genes involved in energy production compared toEnc. cuniculi, a trehalose synthase and an alternative oxi-dase.O. bayeri also harbors almost twice the number of pro-teins involved in glycosylation compared to Enc. cuniculi,suggesting a greater capacity to modify proteins, and perhapsthe presence of a less simplified endoplasmic reticulum andGolgi apparatus compared to other microsporidia [3].The presence of a larger number of genes for metabolic andenergy generating proteins in O. bayeri does not by itself nec-essarily mean that this species is less dependent on its host forenergy than are other microsporidia; however, we alsoobserved a marked underrepresentation of proteins involvedin stealing metabolites from the host. At the extreme, onlyone-quarter of the ATP transporters present in other micro-sporidia and around half of the Enc. cuniculi homologs ofamino acid and sugar transporters were found in O. bayeri.Octosporea also appears to harbor a reduced set of ABCtransporters compared to both Enc. cuniculi and Ent.bieneusi. Taken together, this implies that O. bayeri has abroader metabolic repertoire than other microsporidia whileat the same time a reduced capability to derive metabolicproducts and energy from its host, both of which suggest it isless host-dependent than other microsporidia with smallergenomes.The phylogenetic placement of O. bayeri is also consistentwith the idea that host dependency evolved hand in hand withreduction in genome size and hyper-adaptation for intracellu-lar parasitism. Indeed, O. bayeri clusters at a basal position inthe microsporidian phylogeny, in the proximity of other spe-cies characterized by large genomes, and the only ATP trans-sporidia, then the ancestral genome of microsporidia wasalmost certainly large, complex, and encoded few transport-ers. Certainly, genome surveys of other basal representativesof the group such as Glugea plecoglossi or Trachipleisto-phora hominis would provide decisive evidence in support oragainst the evolution of reduced microsporidian genomesfrom larger and complex relatives. This certainly warrantsfurther need for investigating the genomics of these highlyadapted and successful parasites.ConclusionsNot all microsporida are characterized by small and highlyreduced genomes. Here we demonstrate that the proteomecomplexity can vary greatly across the different species of thegroup, and that a larger genome size could be a good predictorof increased genomic complexity and reduced host depend-ency in microsporidia.Since a microsporidian genome has now been surveyed with454™ (N. ceranae [34]) and Illumina™ sequencing technol-ogy (this study), it might be interesting to compare theresults. The 454™ de novo genome assembly of N. ceranae[34] resulted in lower overall sequence coverage, but anassembly of larger contigs, on average, due to the longersequence reads. However, the Illumina™ methodology usedto survey O. bayeri required substantially less high molecularweight DNA - in our case only 100 ng of sheared DNA. Thedownside of very short reads (35 bp) was mostly offset by thedeep sequence coverage, allowing a detailed analysis of thecoding capacity of the O. bayeri genome, but not of its struc-ture (for example, conservation of gene order). Moreover, thesmall quantity of DNA required opens the door to genomicanalyses from a broad range of uncultivatable organismsfrom which only a handful of contaminant-free DNA can beextracted.Finally, an important goal of the present study was to gathera large amount of genome sequence information from O. bay-eri so that it may complement the soon-to-be annotatedgenome of its exclusive host, the crustacean D. magna. Thesetwo species represent an excellent and well-recognized modelto study host-parasite interactions [41]. The complementarynature of both genomic datasets will therefore form a greatstudy system and provide a unique opportunity to furtherexpand this specific field of evolutionary ecology into thepost-genomic eraMaterials and methodsDNA and RNA extraction and DNA sequencingTotal RNA and genomic DNA from O. bayeri (isolate OER 3-3 from the Island Oeren in the Tvärminne archipelago, south-Genome Biology 2009, 10:R106porter identified from this species was also found to be a basalrepresentative of the gene family. If both phylogenies depictthe correct evolutionary relationships within the micro-western Finland) were obtained from purified spores isolatedfrom a laboratory culture of infected D. magna hosts (Univer-sity of Basel, Switzerland). A total of 100 ng of genomic DNA106.10http://genomebiology.com/2009/10/10/R106 Genome Biology 2009,     Volume 10, Issue 10, Article R106       Corradi et al. Rwas sequenced with single and paired-end 35-bp reads on theIllumina™ Genome Analyzer from Solexa (San Diego, CA,USA) by FASTERIS SA (Geneva, Switzerland). Reads wereassembled using EDENA version 2.1.1, Velvet version 0.6.03and ELAND version GAPipeline-1.0rc4 programs. This wholegenome shotgun project has been deposited at GenBankunder project accession [GenBank:ACSZ00000000]. Theversion described in this paper is the first version [Gen-bank:ACSZ01000000].Identification of O. bayeri homologs present in the Enc. cuniculi genomeThe O. bayeri homologs that are present in the Enc. cuniculigenome were identified by BLAST homology searches [53]against the complete Enc. cuniculi genome using the NCBIBLASTALL suite. First, TBLASTX searches were performedunder a cutoff E-value (E  1E-10) against our local Enc.cuniculi database, then the Enc. cuniculi genes that were notfound in O. bayeri were searched for using TBLASTX againstthe O. bayeri contigs. The O. bayeri tRNAs and tRNA intronsidentified using tRNAscan-SE and default parameters [54]were searched for in the Enc. cuniculi genome manually.Identification of O. bayeri eukaryotic homologs that are absent in Enc. cuniculiThe contigs sharing no similarities in TBLASTX searches (E >1E-3) with the Enc. cuniculi genome have been annotated forpotential ORFs using the program GETORF from theEMBOSS package [55]. Eukaryotic homologs were identifiedby BLASTP searches (E  1E-10) against a local copy of theNCBI non-redundant database using the NCBI BLASTALLsuite. Following the BLASTP procedure, TBLASTX searcheson contigs harboring ORFs that retrieved significant BLASTPhits were performed for further validation. The resultingORFs were assigned to functional categories using the KyotoEncyclopedia of Genes and Genomes (KEGG) [56], Pfam [57],and UniProt [58] databases (Additional data file 2).Identification of putative O. bayeri-specific hypothetical proteinsORFs of at least 200 amino acids that did not retrieve signifi-cant homology in BLAST searches against the Enc. cuniculigenome or the NCBI non-redundant database were queriedagainst the genome survey of A. locustae [59] using TBLASTPsearches (E  1E-10) to identify potential hypothetical pro-teins of microsporidian origin. Potential functions for theseORFs were also searched for using the KEGG [56], Pfam [57],and UniProt [58] databases. ORFs of at least 200 amino acidsthat showed no homology in any of these searches were con-sidered O. bayeri-specific putative proteins.Phylogenetic reconstructionA total of 13 - and -tubulin amino acid sequences have beenthe group and have been successfully used in the past for sim-ilar purposes [42]. Ent. bieneusi tubulins have been discardedfrom the present phylogeny because of their extreme aminoacid divergence, resulting in its unsupported positioningwithin the tree and in an overall reduction in the statisticalsupport for all other phylogenetic clades. Two zygomyceteshave been used as outgroups as this phylum has been pro-posed to represent the most recent fungal common ancestorof microsporidia [20,22]. The - and -tubulin amino acidsequences were aligned using Muscle v3.7 [60] and the mostconserved regions selected using Gblocks 0.91b [61]. Themicrosporidia phylogeny was reconstructed using concate-nated - and -tubulin amino acid sequences and MrBayes v3.1.2 [62] with six General Time Reversible (GTR) types ofsubstitutions, Dayoff acid substitution model and invariableplus gamma rate variations across sites. The Markov chainMonte Carlo search was run for 10,000 generations, samplingthe Markov chain every 10 generations, and 250 were dis-carded as 'burn-in'. The relationships among microsporidiaATP transporters were studied in parallel using amino acidsequences retrieved from public databases and the parame-ters explained above.Introns, gene density, and gene lengthThe O. bayeri ORFs with assigned functions were screenedfor potential frameshit mutations caused by the potentialpresence of introns, with introns previously reported in Enc.cuniculi [25] searched for manually. Gene density in the O.bayeri genome was determined by annotating ORFs of atleast 100 amino acids along the 200 largest contigs used inthis study. A number of complete O. bayeri proteins havebeen compared against orthologs from Enc. cuniculi, Ent.bieneusi, S. cerevisiae, Neurospora crassa, U. maydis, B.dendrobatidis and R. oryzae to identify the presence of sig-nificant differences in gene length. O. bayeri-specific insertsin otherwise highly conserved proteins were screened for byvisual inspection of BLAST search results, compared withorthologs using MEGA 4 [63], and their presence in mRNAsconfirmed by RT-PCR. Locations of the O. bayeri-specificinserts on the corresponding protein three-dimensionalstructures were determined using SwissPDB-viewer andQuickPDB from the RSCB Protein Data Bank for availablestructures.Repeated elementsDNA regions in the O. bayeri contigs showing homology withfungal repeated elements were identified with CENSOR [64]from the Genetic Information Research Institute webserver.Repeated elements arrayed in tandem in the O. bayeri contigswere determined with Tandem Repeat Finder 4.03 [65] usinga match/mismatch/indel ratio of 2/7/7 and a minimum scoreof 50. Putative stem-loop structures in the O. bayeri contigswere screened for with PALINDROME from the EMBOSSGenome Biology 2009, 10:R106identified from a range of microsporidian species and used toreconstruct their phylogenetic relationships, as they repre-sent the most conserved and widely sampled proteins withinpackage using a minimum stem length of 10 and a maximumloop length of 4.106.11http://genomebiology.com/2009/10/10/R106 Genome Biology 2009,     Volume 10, Issue 10, Article R106       Corradi et al. RAbbreviationsGTR: General Time Reversible; KEGG: Kyoto Encyclopedia ofGenes and Genomes; NCBI: National Center for Biotechnol-ogy Information; ORF: open reading frame.Authors' contributionsNC conceived the study, performed molecular and bioinfor-matics analyses, contributed major scientific ideas anddrafted the manuscript. KLH cultured O. bayeri strains andprovided DNA and RNA samples required for sequencing.JFP performed bioinformatics analyses and drafted the man-uscript. DE provided the raw sequence data on which all pre-sented analyses have been performed and drafted themanuscript. PJK contributed to scientific ideas presentedhere and in conceiving the study, and drafted the manuscript.Additional data filesThe following additional data are available with the onlineversion of this paper: a table listing Enc. cuniculi predictedgenes and the putative counterparts we identified in O. bayeri(Additional data file 1); a table listing the 80 O. bayeri pro-teins with assigned functions and motifs that are absent inEnc. cuniculi (Additional data file 2); a table listing O. bayeriORFs and their assignment to functional categories (accord-ing to [25]) (Additional data file 3); the sequences of the sixintrons identified in O. bayeri (Additional data file 4); a figureshowing three examples of large gene inserts we identified inotherwise conserved eukaryotic proteins (Additional data file5); a graphical representation of the number of contigs usedin this study and their respective sequence coverage (Addi-tional data file 6); list of a number of repetitive elements weidentified in the O. bayeri genome (Additional data file 7).Additional data file 1Enc. cu iculi predicted genes and the putative counterparts identi-fie  in O. baye i.Cli k here for fil 2The 80 O. bayeri proteins with assigned functions and motifs that are abs nt in Enc. uniculi.3O. b y  ORFs and their as ig ment to al categorie(ac ordi g to [25]). 4S quences f h  s x intro  identifi d in O. bayeri.5re  ex mpl  of larg  gene inserts identified i  otherwise con-served uk r oti  prot ns w identifi d in othe wis  cons rved euk yotic pr teins.6n mber o  c igs us d n thi  tudy d th ir r s ec ive coverage. 7R p tit v em nts w d t fie in he O. bayeri geno e.AcknowledgementsThis work was supported by Canadian Institute of Health Research (CIHR)operating MOP (MOP-42517) to PJK and the Swiss National Foundation toDE. PJK is a Fellow of the Canadian Institute for Advanced Research(CIFAR) and a Senior Scholar of the Michael Smith Foundation for HealthResearch (MSFHR). NC is a Scholar of the Canadian Institute for AdvancedResearch (CIFAR) and a senior postdoctoral fellow of the Swiss NationalScience Foundation (PA00P3_124166). JFP is the recipient of the FondsQuébécois de la Recherche sur la Nature et les Technologies (FQRNT)/Génome Québec Louis-Berlinguet Postdoctoral Fellowship. KLH's work inBasel was supported by a Brazilian fellowship from CNPq, process#201401/2007-0. We thank Hilary Morrison and acknowledge the Jose-phine Bay Paul Center for Comparative Molecular Biology and Evolutionfor the use of data included in the Antonospora locustae Genome Projectfunded by NSF award number 0135272. We would like to thank Erick Jamesand two anonymous reviewers for their important comments on previousversions of the manuscript, Renny Lee for his help in identifying O. bayeriintrons, Sylvia Doan for her help in annotating O. bayeri full length ORFs andLaurent Farinelli (FASTERIS SA, Switzerland) for sequencing.References1999, 38:161-197.3. Vávra J, Larsson JIR: Structure of the microsporidia.  In The Micro-sporidia and Microsporidiosis Edited by: Wittner M, Weiss LM. Wash-ington, DC: ASM Press; 1999:7-84. 4. Ishihara R, Hayashi YJ: Some properties of ribosomes from thesporoplasm of Nosema bombycis.  Invert Pathol 1968, 11:377-385.5. Curgy JJ, Vávra J, Vivarès CP: Presence of ribosomal RNAs withprokaryotic properties in Microsporidia, eukaryotic organ-isms.  Biol Cell 1980, 38:49-51.6. Brown JR, Doolittle WF: Root of the universal tree of life basedon ancient aminoacyl-tRNA synthetase gene duplications.Proc Natl Acad Sci USA 1995, 92:2441-2445.7. 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