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ESTs from the microsporidian Edhazardia aedis Gill, Erin E; Becnel, James J; Fast, Naomi M Jun 20, 2008

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ralssBioMed CentBMC GenomicsOpen AcceResearch articleESTs from the microsporidian Edhazardia aedisErin E Gill1, James J Becnel2 and Naomi M Fast*1Address: 1Department of Botany, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada and 2Center for Medical, Agricultural and Veterinary Entomology, USDA/ARS, Gainesville, FL 32608, USAEmail: Erin E Gill - egill@interchange.ubc.ca; James J Becnel - james.becnel@ars.usda.gov; Naomi M Fast* - nfast@interchange.ubc.ca* Corresponding author    AbstractBackground: Microsporidia are a group of parasites related to fungi that infect a wide variety ofanimals and have gained recognition from the medical community in the past 20 years due to theirability to infect immuno-compromised humans. Microsporidian genomes range in size from 2.3 to19.5 Mbp, but almost all of our knowledge comes from species that have small genomes (primarilyfrom the human parasite Encephalitozoon cuniculi and the locust parasite Antonospora locustae). Wehave conducted an EST survey of the mosquito parasite Edhazardia aedis, which has an estimatedgenome size several times that of more well-studied species. The only other microsporidian ESTproject is from A. locustae, and serves as a basis for comparison with E. aedis.Results: The spore transcriptomes of A. locustae and E. aedis were compared and the numbers ofunique transcripts that belong to each COG (Clusters of Orthologous Groups of proteins)category differ by at most 5%. The transcripts themselves have widely varying start sites and encodea number of proteins that have not been found in other microsporidia examined to date. However,E. aedis seems to lack the multi-gene transcripts present in A. locustae and E. cuniculi. We alsopresent the first documented case of transcription of a transposable element in microsporidia.Conclusion: Although E. aedis and A. locustae are distantly related, have very disparate life cyclesand contain genomes estimated to be vastly different sizes, their patterns of transcription aresimilar. The architecture of the ancestral microsporidian genome is unknown, but the presence ofgenes in E. aedis that have not been found in other microsporidia suggests that extreme genomereduction and compaction is lineage specific and not typical of all microsporidia.BackgroundMicrosporidia are single-celled eukaryotic intracellularparasites that are related to fungi. Currently, over 1200species have been identified, infecting animals fromnearly every phylum, including commercially importantspecies such as honeybees and fish, as well as humans [1].Inside host cells, microsporidia proliferate as vegetativepossess a unique host cell invasion apparatus called thepolar filament, which is forcefully everted upon germina-tion to form a tube and can pierce a nearby host cell [1].The tube then acts as a conduit allowing the contents ofthe spore to be injected into the host cell's cytoplasm,where the parasite undergoes vegetative replication.Published: 20 June 2008BMC Genomics 2008, 9:296 doi:10.1186/1471-2164-9-296Received: 12 December 2007Accepted: 20 June 2008This article is available from: http://www.biomedcentral.com/1471-2164/9/296© 2008 Gill 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.Page 1 of 12(page number not for citation purposes)stages (meronts, schizonts) which eventually producespores that are released when the host cell lyses. SporesMicrosporidia are a diverse group of organisms, and varygreatly in the complexity of their life cycles. For instance,BMC Genomics 2008, 9:296 http://www.biomedcentral.com/1471-2164/9/296Encephalitozoon cuniculi and Antonospora locustae produceonly one type of spore (uninucleate in the former andbinucleate in the latter), and complete their entire lifecycles inside one host individual, while Amblyospora cali-fornica requires two host groups (mosquitoes and micro-crustacea) and produces three morphologically andfunctionally discrete spore types [1].Microsporida possess some of the smallest primarynuclear genomes known (as tiny as 2.3 Mbp). The onlymicrosporidian whose genome has been completelysequenced is the human parasite, E. cuniculi. At a meager2.9 Mbp, E. cuniculi's genome is extremely compact, withonly 2000 genes [2]. A small genome sequence survey(GSS) project has been conducted on A. locustae, a locustparasite that has been approved as a biological controlagent in the United States [3]. A. locustae's genome isroughly 5.4 Mbp in size [4], or about twice the size of E.cuniculi's genome. Despite the genome size difference,both genomes appear to be structured in much the sameway. Genes are closely packed (nearly one gene per kilo-base), are small in size compared to homologues in ani-mals and fungi, and are intron-poor. There is also a muchgreater degree of synteny between these two organismsthan would be expected given their phylogenetic relation-ship, which implies that although microsporidian genesare fast-evolving, genomic rearrangements occur onlyrarely [3] (See Fig. 1).However, we have very little information on microsporid-ian genomes of larger sizes. Edhazardia aedis is a micro-sporidian that infects Aedes aegypti, the mosquito vector ofthe Dengue hemorrhagic and yellow fever viruses. E. aedishas been intensively studied as a viable biological controlagent for A. aegypti [5] and has a genome estimated to beto be many times larger than that of E. cuniculi. There areseveral possible explanations for this difference: E. aedismay have more genes that control its complex life cycle.Genes may also be longer, more widely spaced, and con-tain more introns than E. cuniculi [2].Morphological studies conducted on E. aedis haverevealed at least four different types of spores – two uni-nucleate and two binucleate [6,7]. The two types of uninu-cleate spore types differ morphologically but possesssimilar pyriform shapes. However, the cell division eventsfrom which they arise differ. Spores produced via mitosisare roughly 8.5 μm in length, whereas spores produced viameiosis (meiospores) are about 7.5 μm. Small binucleatespores (~6.5 μm in length) that have short polar filamentsare formed first, followed by the production of largerbinucleate spores (~9 μm in length) that are ovoid inshape. Meiospore formation is usually abortive and rarelyE. aedis' life cycle is moderately complex and involves twogenerations of the mosquito host. It begins when a uninu-cleate spore is ingested by a mosquito larva from the envi-ronment. Once in the gut, the spore germinates andbegins to multiply in the host tissue. Within 48 hours,small binucleate spores are formed that are responsiblefor spread to other tissues. Orally infected larvae generallyexhibit reduced growth, and may die before reachingmaturity if the parasite load is high, thus releasing morespores into the environment. However, if the infectionload is sufficiently small, the larva will mature into anadult mosquito and survive to reproduce [6]. If the adultmosquito is female, large binucleate spores will developin her ovaries and will infect oocytes, thus passing theinfection on to the next generation where the majority ofmortality occurs in larvae. Little is known about the fac-tors that modulate the transition from one phase in thelife cycle to the next, or about the changes in gene expres-sion that occur during these transitions.It is also possible that the difference in genome sizebetween E. aedis and E. cuniculi or A. locustae may have lessto do with the number of genes, and more to do withgenome architecture. E. aedis genes could be longer, morewidely spaced, and contain more introns than E. cuniculi[2]. In an effort to learn more about E. aedis' genome, aGSS of >200 kbp was conducted [8]. This study concludedthat E. aedis' genome structure is very different from thoseof E. cuniculi and A. locustae. A large portion of the genomeis occupied by non-coding DNA and genes are not closelypacked together, although the existence of local areas ofcompaction could not be ruled out.Previous examinations of ESTs from microsporidia haveonly been conducted on microsporidia with smallgenomes. These transcripts possessed unusual featuresthat are atypical in eukaryotes. Examinations of ESTs fromA. locustae [9] and transcripts from E. cuniculi revealednumerous multi-gene transcripts. These transcripts are dif-ferent from prokaryotic operons, as the proteins encodedby the transcript do not have related functions and areoften not encoded on the same DNA strand. Many tran-scripts encode only a portion of one gene, while the otheris present in its entirety [9,10]. The reason for this phe-nomenon is not known, but it has been suggested thattranscriptional control elements have been lost (or movedinto adjacent genes) during the process of genome com-paction [10].As E. aedis' genome and life cycle are very different from E.cuniculi and A. locustae, it is reasonable to assume that thetranscript structure and number of genes present may dif-fer as well. In this study, we describe the first survey ofPage 2 of 12(page number not for citation purposes)produces normal spores [6]. ESTs from a microsporidian with a much larger genomesize and complex life cycle. In sequencing over 1300 tran-BMC Genomics 2008, 9:296 http://www.biomedcentral.com/1471-2164/9/296scripts, we have elucidated more of E. aedis' genome con-tent, and have gained a profile of its transcript structureand composition. Surprisingly, the E. aedis uninucleateResultsOverviewSequences were deposited into the Genbank EST databaseThe phylogenetic relationships between several microsporidiaFigure 1The phylogenetic relationships between several microsporidia. Species that house transposable elements belonging to the Ty3/gypsy family are highlighted in blue, while species containing LTR transposons are highlighted in yellow. Genome sizes are indicated to the right of each species. (Adapted from Slamovits et al., 2004.)%DHAZARDIAAEDIS!MBLYOSPORA!NTONOSPORALOCUSTAE"RACHIOLAALGERAE'URLEYA3PRAGUEALOPHII'LUGEAATHERINAE4RACHIPLEISTOPHORA6AVRAIAONCOPERAE6ITTAFORMACORNEAE%NTEROCYTOZOON%NCEPHALITOZOONCUNICULI.OSEMABOMBYCIS Page 3 of 12(page number not for citation purposes)spore transcriptome is remarkably similar to that of A.locustae.and have the accession numbers FG063843 to FG065106.From the 1307 clones sequenced, 133 unique genes wereBMC Genomics 2008, 9:296 http://www.biomedcentral.com/1471-2164/9/296Table 1: Gene Name Species Genbank Accession Number16S rRNA GENE Brachiola algerae AM4229051-ACYL-SN-GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE Encephalitozoon cuniculi NP_58614626S PROTEASOME REGULATORY SUBUNIT 4 Encephalitozoon cuniculi NP_58609126S PROTEASOME REGULATORY SUBUNIT 6 Encephalitozoon cuniculi NP_58612826S PROTEASOME REGULATORY SUBUNIT 8 Encephalitozoon cuniculi XP_95573840S RIBOSOMAL PROTEIN S2 Leishmania infantum XP_00146653740S RIBOSOMAL PROTEIN S3 Encephalitozoon cuniculi XP_95567640S RIBOSOMAL PROTEIN S4 Mycetophagus quadripustulatus CAJ1716840S RIBOSOMAL PROTEIN SA or P40 Encephalitozoon cuniculi NP_58472860S RIBOSOMAL PROTEIN L3 Encephalitozoon cuniculi NP_59763060S RIBOSOMAL PROTEIN L4 Encephalitozoon cuniculi NP_59721360S RIBOSOMAL PROTEIN L5 Encephalitozoon cuniculi NP_5858466-PHOSPHOFRUCTOKINASE Encephalitozoon cuniculi NP_597579ABC TRANSPORTER (MITOCHONDRIAL TYPE) #1 Encephalitozoon cuniculi NP_586426ABC TRANSPORTER (MITOCHONDRIAL TYPE) #2 Encephalitozoon cuniculi NP_586426ACTIN Blakeslea trispora AAW32475ARGININE/SERINE RICH PRE-mRNA SPLICING FACTOR Encephalitozoon cuniculi NP_597487ASSOCIATED WITH RAN (NUCLEAR IMPORT/EXPORT) FUNCTION FAMILY MEMBERCaenorhabditis elegans NP_499369ATP SYNTHASE Encephalitozoon cuniculi XP_955732BELONGS TO THE ABC TRANSPORTER SUPERFAMILY Encephalitozoon cuniculi NP_597462cAMP-DEPENDENT PROTEIN KINASE TYPE 1 REGULATORY CHAIN Encephalitozoon cuniculi NP_597223CASEIN KINASE 1 HOMOLOG (INVOLVED IN DNA REPAIR Encephalitozoon cuniculi NP_597600CATION-TRANSPORTING ATPase Encephalitozoon cuniculi NP_586078CHOLINE PHOSPHATE CYTIDYLYLTRANSFERASE Encephalitozoon cuniculi NP_586276DNA REPLICATION LICENSING FACTOR MCM2 Encephalitozoon cuniculi NP_584768DNA REPLICATION LICENSING FACTOR OF THE MCM FAMILY MCM6 Encephalitozoon cuniculi NP_597420DNA REPLICATION LICENSING FACTOR OF THE MCM FAMILY MCM7 Encephalitozoon cuniculi NP_585977DNAJ PROTEIN HOMOLOG 2 Encephalitozoon cuniculi NP_586004DNAK-LIKE PROTEIN Encephalitozoon cuniculi NP_586489EUKARYOTIC TRANSLATION INITIATION FACTOR 4A Encephalitozoon cuniculi XP_955671FIBRILLARIN (34kDa NUCLEOLAR PROTEIN) Encephalitozoon cuniculi NP_586197GENERAL TRANSCRIPTION FACTOR Encephalitozoon cuniculi NP_597292GLUCOSAMINE FRUCTOSE-6-PHOSPHATE AMINOTRANSFERASE Encephalitozoon cuniculi NP_586057GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE Encephalitozoon cuniculi NP_586008GUANINE NUCLEOTIDE BINDING PROTEIN BETA SUBUNIT Encephalitozoon cuniculi NP_597241HEAT SHOCK RELATED 70 kDa PROTEIN Encephalitozoon cuniculi NP_597563HEAT-SHOCK PROTEIN HSP90 HOMOLOG Encephalitozoon cuniculi NP_584635HISTIDYL tRNA SYNTHETASE Antonospora locustae AAT12372HISTONE ACETYLTRANSFERASE TYPE B SUBUNIT 2 Encephalitozoon cuniculi NP_586003HISTONE DEACETYLASE 1 Encephalitozoon cuniculi NP_597645HISTONE DEACETYLASE Encephalitozoon cuniculi XP_955621HISTONE H3 Mus musculus JQ1983HSP 101 RELATED PROTEIN Encephalitozoon cuniculi NP_586448HYPOTHETICAL PROTEIN ECU02_0840 Encephalitozoon cuniculi NP_584609HYPOTHETICAL PROTEIN ECU02_0950 Encephalitozoon cuniculi NP_584620HYPOTHETICAL PROTEIN ECU06_0450 Encephalitozoon cuniculi NP_585801HYPOTHETICAL PROTEIN ECU06_1280 Encephalitozoon cuniculi NP_585884HYPOTHETICAL PROTEIN ECU07_0530 Encephalitozoon cuniculi NP_585981HYPOTHETICAL PROTEIN ECU08_1500 Encephalitozoon cuniculi NP_597278HYPOTHETICAL PROTEIN ECU09_0740 Encephalitozoon cuniculi XP_955628HYPOTHETICAL PROTEIN ECU09_1700 Encephalitozoon cuniculi XP_955723HYPOTHETICAL PROTEIN ECU11_1720 Encephalitozoon cuniculi NP_586478LIM DOMAIN-CONTAINING PROTEIN Encephalitozoon cuniculi NP_586340LONG CHAIN FATTY ACID CoA LIGASE Encephalitozoon cuniculi NP_586206METHIONINE AMINOPEPTIDASE TYPE 2 Encephalitozoon cuniculi NP_586190METHIONINE PERMEASE Encephalitozoon cuniculi NP_585905NIFS-LIKE PROTEIN (CYSTEINE DESULFURASE) INVOLVED IN IRON- Encephalitozoon cuniculi NP_586483Page 4 of 12(page number not for citation purposes)SULFUR CLUSTER SYNTHESISBMC Genomics 2008, 9:296 http://www.biomedcentral.com/1471-2164/9/296found; 55 were represented by a single transcript, whilethe remaining 78 were represented by two or more. 97 ofthe 133 unique genes are present in other microsporidia(See Table 1), while 10 are present in other (non-micro-sporidian) organisms (See Table 2), 18 are putatively E.aedis-specific and 8 have no apparent open readingframes. Coding sequences contained 43% G+C while 5'and 3' untranslated regions possessed 27% and 26%,respectively.Approximately a quarter of the transcripts analyzed codedin E. cuniculi (NP_597563). Single nucleotide variationexists between sequences, usually as 3rd position synony-mous substitutions. Where non-synonymous substitu-tions exist, they are always a single nucleotide and thereare no indels between sequences. Mitochondrial-type andDNAK-like Hsp70s were also represented.Genes were assigned to COG categories to allow for com-parison with A. locustae. Figure 2 illustrates the percent-ages of total E. aedis transcripts that are dedicated to eachCOG category. Total A. locustae transcripts are providedNUCLEAR SER/THR PROTEIN PHOSPHATASE PP1-1 GAMMA CATALYTIC SUBUNITEncephalitozoon cuniculi NP_597385P68-LIKE PROTEIN (DEAD BOX FAMILY OF RNA HELICASES) Encephalitozoon cuniculi NP_597238PEPTIDE CHAIN RELEASE FACTOR SUBUNIT 1 Encephalitozoon cuniculi NP_597376PEPTIDE ELONGATION FACTOR 2 Glugea plecoglossi BAA11470PHOSPHATIDYLINOSITOL TRANSFER PROTEIN, ALPHA Danio rerio NP_957229PHOSPHOMANNOMUTASE Encephalitozoon cuniculi NP_597365POLYADENYLATE-BINDING PROTEIN 2 Encephalitozoon cuniculi NP_586226POLYPROTEIN Sorghum bicolor AAD27571PRE-mRNA SPLICING FACTOR Encephalitozoon cuniculi NP_586183PROTEIN KINASE B-LIKE PROTEIN Plasmodium falciparum AAT06260PROTEIN TRANSPORT PROTEIN SEC23 HOMOLOG (COPII COAT) Encephalitozoon cuniculi NP_586385PUTATIVE HYDROLASE-LIKE PROTEIN Antonospora locustae AAU11090PUTATIVE ZINC FINGER PROTEIN Encephalitozoon cuniculi NP_597297SER/THR PROTEIN PHOSPHATASE 2-A Encephalitozoon cuniculi NP_584753SER/THR PROTEIN PHOSPHATASE PP2-A REGULATORY SUBUNIT B Encephalitozoon cuniculi NP_597423SERINE/THREONINE PROTEIN KINASE (REQUIRED FOR ACTIN RING AND SEPTATION)Encephalitozoon cuniculi XP_965898SIMILAR TO DNAJ-LIKE PROTEIN Nasonia vitripennis XP_001602403SIMILARITY TO 14-3-3 PROTEIN 1 Encephalitozoon cuniculi NP_597610SIMILARITY TO ADP/ATP CARRIER PROTEIN Paranosema grylli CAI30461SIMILARITY TO CDC20 (WD-REPEAT PROTEIN) Encephalitozoon cuniculi NP_597660SIMILARITY TO Hsp70-RELATED PROTEIN Encephalitozoon cuniculi NP_584537SIMILARITY TO HYPOTHETICAL INTEGRAL MEMBRANE PROTEIN YQ55_CAEELEncephalitozoon cuniculi NP_597662SIMILARITY TO HYPOTHETICAL PROTEIN YAAT_BACSU Encephalitozoon cuniculi NP_597532SIMILARITY TO HYPOTHETICAL PROTEIN YB36_METJA Encephalitozoon cuniculi NP_597239SIMILARITY TO PUTATIVE AMINOACID TRANSPORTER YEU9_yeast Encephalitozoon cuniculi NP_584803SIMILARITY TO SKT5 PROTEIN Encephalitozoon cuniculi NP_586349SIMILARITY TO TRANSCRIPTION INITIATION FACTOR TFIIA Encephalitozoon cuniculi NP_597616STE12 TRANSCRIPTION FACTOR Encephalitozoon cuniculi NP_586509STRUCTURE-SPECIFIC RECOGNITION PROTEIN Encephalitozoon cuniculi NP_586030T COMPLEX PROTEIN 1 SUBUNIT BETA Encephalitozoon cuniculi XP_955601THREONYL tRNA SYNTHETASE #1 Encephalitozoon cuniculi NP_586084THREONYL tRNA SYNTHETASE #2 Encephalitozoon cuniculi NP_586084TRANSLATION ELONGATION FACTOR 1 ALPHA Glugea plecoglossi BAA12288TRIOSE PHOSPHATE ISOMERASE Encephalitozoon cuniculi NP_586329TUBULIN BETA CHAIN Encephalitozoon cuniculi NP_597591U5 ASSOCIATED snRNP Encephalitozoon cuniculi NP_586393UNNAMED PROTEIN PRODUCT (Hsp70) Candida glabrata XP_445544VACUOLAR ATP SYNTHASE CATALYTIC SUBUNIT A Encephalitozoon cuniculi NP_586434VACUOLAR ATP SYNTHASE SUBUNIT B Encephalitozoon cuniculi NP_586219ZINC FINGER PROTEIN Encephalitozoon cuniculi NP_584833The 97 unique E. aedis transcripts which are homologous to genes present in other microsporidia are listed above. Species names and Genbank accession numbers of top BLASTX hits are indicated. Bold text in the "Gene Name" column indicates instances where two different transcripts both had the same top BLASTX hit. Underlining indicates a copy of Hsp70 that is most similar to a protein that remains unnamed in Genbank.Table 1:  (Continued)for Hsp70. Almost all of the Hsp70 sequences were most for comparative purposes. As the randomness of thePage 5 of 12(page number not for citation purposes)similar to the "heat shock related 70 kDa protein" found library is uncertain, it is possible that some transcripts areBMC Genomics 2008, 9:296 http://www.biomedcentral.com/1471-2164/9/296artificially overrepresented. It is therefore more informa-tive to examine unique transcripts (ie. counting multipletranscripts for the same gene only once) rather than totaltranscripts. Figure 3 displays the percentages of unique E.aedis and A. locustae transcripts dedicated to each category.Surprisingly, the values are similar and sometimes identi-cal (maximum difference between E. aedis and A. locustaecategories is 5%).Notable transcripts include a retrotransposon that is sim-ilar to LTR retrotransposons present in Sorghum bicolor(AAD27571) and Nosema bombycis (ABE26655). Allbelong to the Ty3/Gypsy family of retrotransposons. E.aedis also possesses a methionine aminopeptidase 2 gene(MetAP-2), which is present in E. cuniculi. There were sev-eral transcripts present that appear homologous to pro-teins found in various eukaryotes, but are absent in othermicrosporidia examined to date. These include hypothet-ical or unknown proteins found in Oryza, Danio and Plas-modium, as well as genes encoding proteins with identifiedfunctions, such as an adenosine kinase, a lysine-tRNAligase and an L-asparaginase (See Table 2). In addition, E.aedis encodes a putative hydrolase-like protein that ispresent in A. locustae, but absent in E. cuniculi.E. cuniculi and A. locustae both contain a small number ofintrons in their genomes and consequently, they haveretained a minimal set of splicing machinery. These twoorganisms are not closely related (See Fig. 1), but they doshare a few conserved introns [11]. Therefore, there is rea-son to suspect that some of these introns may also bepresent in E. aedis. Fortunately, seven transcripts of thegene encoding ribosomal protein L5 (which contains anintron in E. cuniculi) were recovered from the E. aedislibrary. These sequences were used to design primers toamplify the L5 gene from genomic DNA. It was found thatthe E. aedis L5 gene does not contain an intron.Transcript structureAs E. aedis is an intracellular parasite and therefore cannotbe easily cultured, RNA was limited and the library couldnot be constructed in a 5' cap-dependent manner. There-fore, nearly all of the inserts encoding the same gene wereof different lengths, and most were 5' truncated. However,some of E. aedis' transcripts appear to have very long 5'untranslated regions (UTRs) of several hundred basepairs. To further assess transcript structure, cap-dependent5' RACE (rapid amplification of cDNA ends) was con-ducted on transcripts from a moderately represented gene,glucosamine fructose-6-phosphate aminotransferase. 5'RACE confirmed that transcript lengths for this gene dovary, with 5' UTRs ranging from 255 to 348 bp (See Fig.4).Contrary to the variable start sites of the transcripts, nearlyall appear to have identical end sites. The notable excep-tions are the heat shock related 70 kDa protein transcripts,which have somewhat variable 3' polyadenylation sites.There were frequently single nucleotide differencesbetween sequences in contigs, but these differences wereusually restricted to silent third position substitutions. Ininstances where the substitutions are not silent, they areconservative amino acid substitutions. These differencescould represent different copies of the same gene or differ-ent alleles within the population (UTRs were not availablein most cases to determine which).DiscussionComparing microsporidian transcriptomesThis is the second microsporidian EST project to be con-ducted and the first from a microsporidian possessing alarge genome, allowing for a meaningful comparison ofmicrosporidian spore transcriptomes. Despite the vast dif-ferences in genome size and life cycle complexity betweenTable 2: Gene Name Species Name Genbank Accession Number60S RIBOSOMAL PROTEIN L2 Babesia bovis XP_001612300ADENOSINE KINASE Homo sapiens AAA97893HYPOTHETICAL PROTEIN Candida albicans XP_717148HYPOTHETICAL PROTEIN PY5484 Plasmodium yoelii yoelii XP_725949L-ASPARIGINASE Dirofilaria immitis Q9U518LYSINE tRNA LIGASE Saccharomyces cerevisiae CAA39699PUTATIVE VESICULAR TRANSPORT FACTOR USO1P Candida albicans XP_710120SEC63 DOMAIN CONTAINING PROTEIN Trichomonas vaginalis XP_001580151PROTEIN PHOSPHATASE 2B Cryptosporidium hominis XP_666159WD-40 REPEAT FAMILY PROTEIN Arabidopsis thaliana NP_201533The 10 genes which are present in E. aedis but absent from other microsporidia are listed above. The species names and Genbank accession numbers of the top BLASTX hit for each gene are also listed.Page 6 of 12(page number not for citation purposes)E. aedis and A. locustae, their transcriptomes are highlysimilar in their compositions. The proportions of uniqueBMC Genomics 2008, 9:296 http://www.biomedcentral.com/1471-2164/9/296transcripts encoding proteins devoted to the "protein des-tination" COG category in both E. aedis and A. locustae arerelatively large (19% and 16%, respectively) (See Fig. 3).It is interesting to note that proteomic work correlatesa large percentage of the total proteins present (~28%)that have known functions [12].When the total number of unique genes found in E. aedisTotal E aedis transcripts represented by COG (Clusters of Orthologous Groups of proteins) category with and without Hsp70Figure 2Total E. aedis transcripts represented by COG (Clusters of Orthologous Groups of proteins) category with and without Hsp70. Total A. locustae transcripts are provided for comparison. (A. locustae data adapted from Williams et al., 2005.)4OTAL!NTONOSPORA%344RANSCRIPTS4OTAL%DHAZARDIA%344RANSCRIPTS 4OTAL%DHAZARDIA%344RANSCRIPTS NO(SP	0HWDEROLVP(QHUJ\&HOO*URZWK&HOO'LYLVLRQDQG'1$6\QWKHVLV7UDQVFULSWLRQ3URWHLQ6\QWKHVLV3URWHLQ'HVWLQDWLRQ7UDQVSRUW)DFLOLWDWLRQ,QWUDFHOOXODU7DQVSRUW&HOOXODU2UJDQL]DWLRQDQG%LRJHQHVLV&HOOXODU&RPPXQLFDWLRQ6LJQDO7UDQVGXFWLRQ&HOOUHVFXH'HIHQFH&HOO'HDWKDQG$JHLQJ7UDQVSRVDEOH(OHPHQWV3URWHLQ6\QWKHVLV3URWHLQ'HVWLQDWLRQ7UDQVSRUW)DFLOLWDWLRQ,QWUDFHOOXODU7DQVSRUW&HOOXODU&RPPXQLFDWLRQ6LJQDO7UDQVGXFWLRQ&HOOXODU2UJDQL]DWLRQDQG%LRJHQHVLV0HWDEROLVP7UDQVSRVDEOH(OHPHQWV(QHUJ\&HOOUHVFXH'HIHQFH&HOO'HDWKDQG$JHLQJ&HOO*URZWK&HOO'LYLVLRQDQG'1$6\QWKHVLV7UDQVFULSWLRQ0HWDEROLVP(QHUJ\&HOO*URZWK&HOO'LYLVLRQDQG'1$6\QWKHVLV7UDQVFULSWLRQ3URWHLQ6\QWKHVLV3URWHLQ'HVWLQDWLRQ7UDQVSRUW)DFLOLWDWLRQ,QWUDFHOOXODU7DQVSRUW&HOOXODU2UJDQL]DWLRQDQG%LRJHQHVLV&HOOXODU&RPPXQLFDWLRQ6LJQDO7UDQVGXFWLRQ7UDQVSRVDEOH(OHPHQWV&HOOUHVFXH'HIHQFH&HOO'HDWKDQG$JHLQJPage 7 of 12(page number not for citation purposes)with these results, as the number of proteins in E. cuniculidevoted to the "protein destination" COG category formand A. locustae are compared based on COG category clas-sification, the percentages in each category are close toBMC Genomics 2008, 9:296 http://www.biomedcentral.com/1471-2164/9/296identical (See Fig. 3). The largest differences lie in the cat-egories of cellular organization and biogenesis, cellularcommunication and signal transduction and cell rescue,defense, cell death and aging. One notable differencebetween the two spore transcriptomes is that no transpos-able elements were recovered in the A. locustae ESTs,whereas E. aedis transcribes a retrotransposon of the Ty3/gypsy family. Transposable elements have been previ-ously reported to exist in the genomes of Nosema bombycis[13], Spraguea lophii [14], Brachiola algerae and E. aedis [8](See below). To the best of our knowledge, this is the firstinstance of documented transposable element transcrip-tion in microsporidia, and could indicate active transposi-tion.Nearly 8% of the unique transcripts from E. aedis encodegenes that are present in various eukaryotes, but areabsent from other microsporidia. The existence of thesegenes has several possible explanations. Sequence datafrom microsporidia is scarce, and the only completelysequenced genome is that of E. cuniculi. Therefore, it iscurrently impossible to assert that these genes are absentin any microsporidia other than E. cuniculi. The possibilityexists that they were present in the genome of the micro-sporidian ancestor, and were lost during genome reduc-tion/compaction events in E. cuniculi. These genes couldalso have arisen from lateral transfer events or they couldhave come to resemble genes in other organisms bysuggest that the ancestor of microsporidia was not,indeed, compact to the extent of E. cuniculi.The MetAP-2 protein is a target for drug therapy in E.cuniculi [15]. The E. aedis copy of the MetAP-2 gene is verysimilar to that present in E. cuniculi, and contains theamino acid residues that bind the drug fumagillin as wellas those believed to coordinate metals. Like E. cuniculi, E.aedis lacks a polylysine tract at the N-terminus of theMetAP-2 protein that is present in animals, other fungiand plants. This tract plays a role in hindering the phos-phorylation of eukaryotic initiation factor 2α (eIF2α),and its absence indicates that the microsporidian proteinslikely lack this function [15].Although our work indicates that the E. aedis L5 gene doesnot contain an intron like its E. cuniculi homologue (seeResults, above), there is reason to believe that there areintrons elsewhere in the genome. There are several tran-scripts encoding proteins that act in pre-mRNA splicing:an arginine/serine rich pre-mRNA splicing factor(NP_597487 in E. cuniculi), a pre-mRNA splicing factor(NP_586183 in E. cuniculi) and a U5 associated snRNP(NP_586393 in E. cuniculi). These genes comprise 2.2% ofthe total unique genes found.Hsp70Roughly 28% of total E. aedis transcripts encoded someUnique E. aedis transcripts represented by COG categoryFigur 3Unique E. aedis transcripts represented by COG category. Unique A. locustae transcripts are provided for comparison. (A. locustae data adapted from Williams et al., 2005.)5NIQUE!NTONOSPORA%344RANSCRIPTS 5NIQUE%DHAZARDIA%344RANSCRIPTS0HWDEROLVP(QHUJ\&HOO*URZWK&HOO'LYLVLRQDQG'1$6\QWKHVLV7UDQVFULSWLRQ3URWHLQ6\QWKHVLV3URWHLQ'HVWLQDWLRQ7UDQVSRUW)DFLOLWDWLRQ,QWUDFHOOXODU7DQVSRUW&HOOXODU&RPPXQLFDWLRQ6LJQDO7UDQVGXFWLRQ&HOOUHVFXH'HIHQFH&HOO'HDWKDQG$JHLQJ7UDQVSRVDEOH(OHPHQWV&HOOXODU2UJDQL]DWLRQDQG%LRJHQHVLV(QHUJ\&HOO*URZWK&HOO'LYLVLRQDQG'1$6\QWKHVLV7UDQVFULSWLRQ3URWHLQ6\QWKHVLV3URWHLQ'HVWLQDWLRQ7UDQVSRUW)DFLOLWDWLRQ&HOOXODU&RPPXQLFDWLRQ6LJQDO7UDQVGXFWLRQ&HOOUHVFXH'HIHQFH&HOO'HDWKDQG$JHLQJ 0HWDEROLVP&HOOXODU2UJDQL]DWLRQDQG%LRJHQHVLV7UDQVSRVDEOH(OHPHQWV,QWUDFHOOXODU7DQVSRUWPage 8 of 12(page number not for citation purposes)chance or by convergence. Parsimoniously, the first expla-nation seems most likely, therefore, these data seem toform of Hsp70, a heat shock protein that assists in thefolding of other proteins. Hsp70 helps prevent proteinsBMC Genomics 2008, 9:296 http://www.biomedcentral.com/1471-2164/9/296from becoming insoluble and also plays a role in variousother intracellular processes, such as apoptosis [16]. Theaction of Hsp70 allows mutant proteins to continue func-tioning by being refolded instead of being degraded,which necessitates the costly synthesis of more protein.The number of Hsp70 transcripts in the E. aedis ESTs is anorder of magnitude higher than was found in A. locustae(2%) [17]. We are cautious in this interpretation as wehave not quantitatively assessed the transcription level ofHsp70 in E. aedis, and it is likely that transcripts of thisprotein are somewhat overrepresented in the library.Although no E. cuniculi ESTs have been published, Bros-son et al. [12] investigated the proteins present in spores.Hsp70 constitutes a moderate amount of all proteinpresent. Brosson and his colleagues classified all proteinsbased on their COG categories, and found that all "pro-tein destination" proteins together comprise 21% of E.cuniculi's proteome. Intriguingly, Brosson et al.'s [12]experiments indicate that of the four copies of Hsp70 in E.cuniculi, the predominately expressed copy of Hsp70 in E.cuniculi is homologous to the highly represented tran-script in E. aedis. In A. locustae, the most highly transcribedcopy was most similar to the abundantly transcribed copyin E. aedis as well [17]. Therefore, it is likely that micro-sporidia employ similar primary mechanisms to ensureIn other parasites and endosymbionts, such as Buchneraaphidicola, Hsp70 is also highly expressed [18] and mayconstitute up to 10% of the protein contained in the cellat any one time. In species that lead parasitic or endosym-biotic lifestyles, genetic drift and relaxed selection pres-sure frequently lead to an increased mutation rate. Theneed for Hsp70 in order for proteins to fold correctlyseems to increase with both the size and number of muta-tions in the protein [16]. Although microsporidiangenomes appear to have had little rearrangement, thenucleotide mutation rate seems to be high in this group oforganisms [19,20]. Microsporidia could, therefore, con-tain elevated levels of Hsp70 in order to allow folding ofmutant proteins.Transposable elementsOne of the E. aedis ESTs closely matches the integrasedomain of the Ty3/gypsy family of retrotransposons. Sev-eral of these elements were identified in a GSS of E. aedis[8] and a few other microsporidian species, but to the bestof our knowledge, this is the first instance in which tran-scripts of any microsporidian retrotransposon have beenfound. Transcripts could be indicative of active transposi-tion occurring in E. aedis' genome.Ty3/gypsy retrotransposons exist in many organisms rang-5' RACE conducted on a moderately represented transcript in E. aedis reveals multiple transcription start sitesFigure 45' RACE conducted on a moderately represented transcript in E. aedis reveals multiple transcription start sites. ESTs are depicted in green and RACE products in purple. The predicted translational start codon is indicated by the orange arrow. As indicated, the E. cuniculi homologue of this gene contains 128 amino acids at N-terminus that appear to be absent in E. aedis.      !!!!!!!!!!!!!!!!!!!!'LUCOSAMINE&RUCTOSE0HOSPHATE!MINOTRANSFERASE/2&BP%CUNICULI'ENE	Page 9 of 12(page number not for citation purposes)proper folding of proteins. ing from the microsporidia Spraguea lophii [14], Brachiolaalgerae [8], and Nosema bombycis [13] to Saccharomyces,BMC Genomics 2008, 9:296 http://www.biomedcentral.com/1471-2164/9/296Drosophila and Sorghum. Ty3 elements have been wellcharacterized in budding yeast, and exist in 1–4 copies pergenome, where they are transcribed by RNA polymeraseIII. Transcription typically occurs only in haploid cells inthe presence of mating pheromones [21]. The N. bombycisgenome contains at least 8 different retrotransposons inthe Ty3/gypsy family, but unlike yeast, they are not exclu-sively located upstream of tRNAs [13]. Nearly all N. bom-bycis retrotransposons encode a polyprotein containing 5domains, which exist in a defined order: Gag, protease,reverse transcriptase, RnaseH and integrase. As many ofthe sequences in the E. aedis library appear to be 5' trun-cated, it is possible that the other domains upstream ofthe integrase in the polyprotein are also present ingenomic DNA. Indeed, the GSS project revealedsequences matching the reverse transcriptase domain [8].Although the microsporidian Vittaforma corneae is alsoknown to possess at least one transposable element [22],it belongs to a different family than those present in E.aedis - the L1 family present in humans.The only completely sequenced microsporidian genome,that of E. cuniculi [2], is completely devoid of transposableelements. The existence of similar transposable elements(of the Ty3/gypsy family) in the distantly related S. lophii,N. bombycis, B. algerae and E. aedis (See Fig. 1) implies thatthis element may have been present in the genome of theancestor of microsporidia. Therefore, the process ofgenome compaction that gave rise to the E. cuniculigenome likely involved purging transposable elements.It has been suggested that transposable elements may actto reorganize genes within the genome. Xu et al. [13] com-pared regions of synteny between N. bombycis and E.cuniculi chromosomes, as selection appears to be acting toretain gene synteny among microsporidia, even if they areonly distantly related [3]. In N. bombycis, transposable ele-ments flank these syntenic regions [13]. If E. aedis' largegenome is partially a product of transposable elementproliferation, one would expect much less syntenybetween this species and other microsporidia. Perhapsfuture research will elucidate other roles that transposableelements have played in shaping microsporidiangenomes, especially since the minute genome of E.cuniculi seems to lack them, while they are present inlarger genomes.The functions that these transposable elements perform ina given genome are cryptic at best, but evidence is emerg-ing that they may be more than just simply parasitic DNA.Peaston et al. [23] recently discovered that a class ofmouse retrotransposons appears to regulate gene expres-sion in embryos.Transcript structureTranscripts in A. locustae typically contain more than onegene. These transcripts do not necessarily contain com-plete open reading frames for all genes and the genes arefrequently in opposite orientations [9]. It is not knownhow many proteins are made from each transcript orwhether this situation is typical for microsporidia, butrecent work by Corradi et al. [10] suggests that E. cuniculialso possesses multi-gene transcripts.Unlike A. locustae and E. cuniculi, E. aedis appears to tran-scribe very few multi-gene transcripts, if any at all. This isnot unexpected, given that E. aedis genes appear to be sep-arated by large intergenic spaces [8]. The E. aedis GSScould not rule out the possibility that local areas of com-pacted genes might exist [8]. Given the lack of multi-genetranscripts identified, this seems increasingly unlikely.Also contrary to what is found in A. locustae, nearly all ofE. aedis' transcripts encode proteins in a positive frame(<1% are in a negative frame, compared to 17% in A.locustae) [9]. Although antisense transcripts are used inmany organisms (possibly also A. locustae) to suppresstranslation, it appears unlikely that this type of regulationoccurs in E. aedis. Conversely, the large number of anti-sense transcripts in A. locustae may be due to a lack of tran-scriptional regulation resulting from genome compaction.E. aedis' transcripts seem to start at multiple locationsupstream of the start codon (5' UTR length is 180 bp onaverage) but terminate at the same position with a rela-tively short 3' UTR (51 bp on average) (See, for example,Figure 4). This is more in line with transcription in E.cuniculi and contrasts with the situation in A. locustae,where transcripts start directly upstream of the translationinitiation site, but often terminate much farther down-stream in the adjacent gene [10]. For comparison, theyeast S. cerevisiae contains much shorter 5' UTRs than 3'UTRs (15–75 and ~144 bp, respectively [24,25]), a com-mon trend seen in other fungi, plants and animals. Thereason for this reversal is unknown, since 3' UTRs areubiquitously used as translation regulators. It is likely thatE. aedis lacks some of the translational control mecha-nisms present in other fungi, plants and animals [26].ConclusionThis is the first examination of ESTs from a microsporid-ian containing a large genome. The extent of genomecompaction in the microsporidian ancestor is not known,but the presence of genes in E. aedis that have not beenfound in other microsporidia suggests that extreme reduc-tion and compaction occurred only in specific lineages.Surprisingly, E. aedis has a predicted uninucleate sporePage 10 of 12(page number not for citation purposes)transcriptome that is highly similar to that of the distantlyBMC Genomics 2008, 9:296 http://www.biomedcentral.com/1471-2164/9/296related A. locustae, although the two species have diverselife cycles and genome sizes.MethodsUninucleate E. aedis spores were grown and harvestedfrom A. aegypti larvae as described previously [27].E. aedis spores were lysed in Ambion's plant RNA isolationaid and lysis/binding solution from an Ambion RNAque-ous kit using a bead beater operating at 2500 rpm for 6minutes with glass beads. RNA was extracted from theresulting supernatant using the RNAqueous kit. A micro-quantity cDNA library was constructed by Marligen, usingthe pExpress-1 vector. 1307 clones with an average insertsize of 1.5 kb were uni-directionally sequenced using anautomated capillary sequencer. Sequences were manuallyedited and analyzed using Sequencher 4.2 software. Pro-teins encoded by the transcripts were identified viaBLASTX [28] searches performed on the NCBI website(Genbank). Transcripts were identified as encoding a par-ticular protein when BLASTX hits to Genbank proteinshad e-values of 10-4 or lower. Transcripts were scored as"present in other microsporidia" when the best BLASTXhit was a gene present in other microsporidia or when thebest hit was a gene that has a microsporidian homologue,and the homologue was identified in other microsporidiaby BLASTing the E. aedis transcript against availablemicrosporidian data. Putative E. aedis-specific genes aretranscripts that contain open reading frames at least 100base pairs in size and do not have any BLASTX hits with e-values lower than 10-3. In order to facilitate access to theEST sequences, they were uploaded and annotated by thedbEST website [17].Authors' contributionsEEG extracted RNA from the E. aedis spores, performedand interpreted the sequence analyses and drafted themanuscript. JJB cultivated insect larvae and harvested E.aedis spores. NMF conceived of this study, contributed tothe interpretation of the results and helped draft the man-uscript.AcknowledgementsEEG's work is supported by an NSERC Postgraduate Scholarship and research in the Fast lab is funded by an NSERC Discovery Grant to NMF.References1. Wittner M, Weiss LM: The Microsporidia and Microsporidosis Washing-ton, D. C.: ASM Press; 1999. 2. Katinka MD, Duprat S, Cornillot E, Metenier G, Thomarat F, PrensierG, Barbe V, Peyretaillade E, Brottier P, Wincker P, Delbac F, El AlaouiH, Peyret P, Saurin W, Gouy M, Weissenbach J, Vivares C: Genomesequence and gene compaction of the eukaryote parasiteEncephalitozoon cuniculi.  Nature 2001, 414:450-453.3. Slamovits CH, Fast NM, Law JS, Keeling PJ: Genome compactionand stability in microsporidian intracellular parasites.  Curr4. Streett DA: Analysis of Nosema locustae (microsporidia:Nose-matidae) chromosomal DNA with pulsed-field gel electro-phoresis.  J Invertebr Pathol 1994, 63:301-303.5. Becnel JJ: Edhazardia aedis (microsporidia: Amblyosporidae)as a biological control agent of Aedes aegypti (diptera: Culici-dae).  Proc Vth Int Colloq Invertebr Pathol Microb Control: Adelaide, Aus-tralia 1990:56-60.6. Becnel JJ, Sprague V, Fukuda T, Hazard EI: Development of Edhaz-ardia aedis (kudo, 1930 N. G., N. comb. (microsporidia:Amblyosporidae) in the mosquito Aedes aegypti (L.) (DIp-tera: Culicidae).  J Protozool 1989, 36:119-130.7. Johnson MA, Becnel JJ, Undeen AH: A new sporulation sequencein Edhazardia aedis (microsporidia: Culicosporidae), a para-site of the mosquito Aedes aegypti (diptera: Culicidae).  J Inver-tebr Pathol 1997, 70:69-75.8. Williams BAP, Lee RCH, Becnel JJ, Weiss LM, Fast NM, Keeling PJ:Genome sequence surveys of Brachiola algerae and Edhaz-ardia aedis reveal microsporidia with low gene densities.  BMCGenomics  in press.9. Williams BA, Slamovits CH, Patron NJ, Fast NM, Keeling PJ: A highfrequency of overlapping gene expression in compactedeukaryotic genomes.  Proc Natl Acad Sci USA 2005,102:10936-10941.10. Corradi N, Gangaeva A, Keeling PJ: Comparative profiling ofoverlapping transcription in the compacted genomes ofmicrosporidia Antonospora locustae and Encephalitozooncuniculi.  Genomics  in press.11. Limpright VO, Fast NM: personal communication.  .12. Brosson D, Kuhn L, Delbac F, Garin J, Vivares CP, Texier C: Pro-teomic analysis of the eukaryotic parasite Encephalitozooncuniculi (microsporidia): A reference map for proteinsexpressed in late sporogonial stages.  Proteomics 2006,6:3625-3635.13. Xu J, Pan G, Fang L, Li J, Tian X, Li T, Zhou Z, Xiang Z: The varyingmicrosporidian genome: Existence of long-terminal repeatretrotransposon in domesticated silkworm parasite Nosemabombycis.  Int J Parasitol 2006, 36:.14. Hinkle G, Morrison HG, Sogin ML: Genes coding for reversetranscriptase, DNA-directed RNA polymerase, and chitinsynthetase from the microsporidian Spraguea lophii.  Biol Bull1997, 193:250-251.15. Pandrea I, Mittleider D, Brindley PJ, Didier ES, Robertson DL: Phyl-ogenetic relationships of methionine aminopeptidase 2among Encephalitozoon species and genotypes of micro-sporidia.  Mol Biochem Parasit 2005, 140:141-152.16. Mayer MP, Bukau B: Hsp70 chaperones: Cellular functions andmolecular mechanism.  Cell Mol Life Sci 2005, 62:670-684.17. O'Brien E, Koski L, Zhang Y, Yang L, Wang E, Gray MW, Burger G,Lang BF: TBestDB: A taxinomically broad database ofexpressed sequence tags (ESTs).  Nucleic Acids Res 2007,35:D445-451.18. Wilcox J, Dunbar HE, Wolfinger RD, Moran NA: Consequences ofreductive evolution for gene expression in an obligate endo-symbiont.  Mol Microbiol 2003, 48:1491-1500.19. Thomarat F, Vivares CP, Gouy M: Phylogenetic analysis of thecomplete genome sequence of Encephalitozoon cuniculi sup-ports the fungal origin of microsporidia and reveals a highfrequency of fast-evolving genes.  J Mol Evol 2004, 59:780-791.20. Peer Y Van de, Ben Ali A, Meyer A: Microsporidia: Accumulatingmolecular evidence that a group of amitochondriate and sus-pectedly primitive eukaryotes are just curious fungi.  Gene2000, 246:1-8.21. Kinsey PT, Sandmeyer SB: Ty3 transposes in mating populationsof yeast: A novel transposition assay for Ty3.  Genetics 1995,139:81-94.22. Mittleider D, Green LC, Mann VH, Michael SF, Didier ES, Brindley PJ:Sequence survey of the genome of the opportunistic micro-sporidian pathogen, Vittaforma corneae.  J Eukaryot Microbiol2002, 49:393-401.23. Peaston AE, Evsikov AV, Graber JH, de Vries WN, Holbrook AE, Sol-ter D, Knowles BB: Retrotransposons regulate host genes inmouse oocytes and preimplantation embryos.  Dev Cell 2004,7:597-606.24. Zhang Z, Dietrich FS: Mapping of transcription start sites inPage 11 of 12(page number not for citation purposes)Biol 2004, 14:891-896. Saccharomyces cerevisiae using 5' SAGE.  Nucleic Acids Res 2005,33:2838-2851.Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central BMC Genomics 2008, 9:296 http://www.biomedcentral.com/1471-2164/9/29625. Graber JH, Cantor CR, Mohr SC, Smith TF: Genomic detection ofnew yeast pre-mRNA 3'-end-processing signals.  Nucleic AcidsRes 1999, 3:888-894.26. Mazumder B, Seshadri V, Fox PL: Translational control by the 3'-UTR: The ends specify the means.  Trends Biochem Sci 2003,2:91-98.27. Becnel JJ, Garcia JJ, Johnson MA: Edhazardia aedis (microspora:Culicosporidae) effects on the reproductive capacity ofAedes aegypti (diptera: Culicidae).  J Med Entomol 1995,32:549-553.28. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lip-man DJ: Gapped BLAST and PSI-BLAST: A new generation ofprotein database search programs.  Nucleic Acids Res 1997,25:3389-3402.yours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralPage 12 of 12(page number not for citation purposes)

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