DNA RESEARCH 4, 231-240 (1997)

Evaluation of cDNA Libraries from Different DevelopmentalStages of Schistosoma mansoni for Production of ExpressedSequence Tags (ESTs)

Gloria R. FRANCO,1 Elida M. L. RABELO,2 Vasco AZEVEDO,3 Heloisa B. PENA,1 J. Miguel ORTEGA,1

Tiilio M. SANTOS,1 Wendell S. F. MEIRA,1 Neuza A. RODRIGUES,1 Carlos M. M. DIAS,2 Richard HARROP,5

Alan WILSON,5 Mohamed SABER,6 Hannan ABDEL-HAMID,6 Michelyne S. C. FARIA,7

Maria Elizabeth B. MARGUTTI,4 Jugara C. PARRA,7 and Sergio D. J. PENA1'*

Departamento de Bioquimica e Imunologia,1 Departamento de Parasitologia,2 Departamento de BiologiaGeral 3 and Departamento de Microbiologia, Universidade Federal de Minas Gerais, Belo Horizonte,Brazil 31270-010,4 Department of Biology, University of York, York, Y015DD, UK,5 Theodore BilharzResearch Institute, Cairo, 12411, Egypt6 and Centro de Pesquisas Rene Rachou, Belo Horizonte,Brazil 30190-0027

(Received 7 April 1997)

Abstract

A comparative study of the gene expression profile in different developmental stages of Schistosomamansoni has been initiated based on the expressed sequence tag (EST) approach. A total of 1401 ESTswere generated from seven different cDNA libraries constructed from four distinct stages of the parasite lifecycle. The libraries were first evaluated for their quality for a large-scale cDNA sequencing program. Mostof them were shown to have less than 20% useless clones and more than 50% new genes. The redundancyof each library was also analyzed, showing that one adult worm cDNA library was composed of a smallnumber of highly frequent genes. When comparing ESTs from distinct libraries, we could detect that mostgenes were present only in a single library, but others were expressed in more than one developmental stageand may represent housekeeping genes in the parasite. When considering only once the genes present inmore than one library, a total of 466 unique genes were obtained, corresponding to 427 new S. mansonigenes. From the total of unique genes, 20.2% were identified based on homology with genes from otherorganisms, 8.3% matched S. mansoni characterized genes and 71.5% represent unknown genes.Key words: Key Words: Schistosoma mansoni; developmental stages; cDNA sequencing analysis; ex-pressed sequence tags

1. Introduction

Schistosoma mansoni (Sm) is a digenetic trematodeworm responsible for schistosomiasis, a parasitic diseasethat is estimated to affect at least 300 million people intropical and subtropical areas of the world (WHO, 1985).

Communicated by Kenichi Matsubara* To whom correspondence should be addressed. Departamento

de Bioquimica e Imunologia, ICB/UFMG. Av. Antonio Carlos,6627, Belo Horizonte, MG 31270-010, Brazil. Tel. +5531-227-3496, Fax. +5531-227-3792, E-mail: spena@dcc.ufmg.br

f EST sequences were deposited in dbEST and Gen-Bank with the following accession numbers: Adult 1 li-brary (T14340^T14651; T18616->T18626; T24126->T24150;W06712-»W06824); Adult 2 library (AA185747^AA185837),Adult 3 library (AA218448->AA218524); Adult 4 li-brary (AA125663^AA169943), Egg library (AA140558^AA140638);Cercariae library (AA143808^AA143896); Lungstage schistosomula library (AA125668—>AA125734).

Despite intense efforts dedicated to eradicating schisto-somiasis through sanitary measures, suppression of theintermediate host and drug treatment, the prevalence ofthe disease has not decreased. No vaccine is yet avail-able and control of the disease is primarily by chemother-apy. However, reinfection of patients is common and weneed new approaches to treatment and prevention, since5. mansoni is becoming increasingly resistant to drugtherapy. It is hoped that detailed information about thegenome of 5. mansoni might uncover key gene productsthat may constitute new targets for drug and vaccine de-velopment.

Accordingly, in 1992 we started a systematic gene dis-covery program study in S. mansoni using the strat-egy of partial sequencing of cDNA ends to generate ex-pressed sequence tags (EST).1 Initially, we utilized anadult worm cDNA library, from which 607 ESTs were

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232 ESTs from S. mansoni cDNA Libraries [Vol. 4,

obtained, corresponding to 169 different genes, 15 pre-viously known in 5. mansoni and 154 new genes.2 Thisincreased considerably the number of genes identified inthe parasite. However, we felt that studying only adultworms was insufficient. S. mansoni has a complex life cy-cle with several morphologically very diverse stages (ova,miracidia, cercariae, schistosomula and adult worms),during which different sets of genes are expressed. Ob-viously, if one considers the acquisition of informationabout the worm gene expression in the perspective ofdesigning new drugs and vaccines, the young stages cannot be overlooked. Actually, the schistosomula stage isincreasingly recognized as one of the main targets for thehost immune system.3

With this in mind, we planned to extend our EST pro-gram to the other life stages of S. mansoni. For that,stage-specific cDNA libraries were needed, some of which,unfortunately, are very difficult to construct because ofdifficulties in obtaining the necessary amounts of puremRNA. Thus, before embarking on large-scale studies,we decided to evaluate the libraries that were already inexistence, comparing them with our original adult wormlibrary. We here report our results with seven differentcDNA libraries constructed from four distinct stages ofthe parasite life cycle, from which a total of 1401 ESTswere generated, totaling 466 different genes, 427 of whichare newly describe in S. mansoni. From the total ofidentified genes, we can start to outline a pattern of geneexpression, with some genes expressed in a stage-specificmanner and others, housekeeping ones, in all develop-mental stages.

2. Methodology

2.1. Construction of cDNA libraries and sequencingThe following seven cDNA libraries were used in this

study: four libraries (Adult 1-4) from adult worms andone library each from ova (Egg), cercariae and lung stageschistosomula (Lung stage). The construction of theAdult 1 cDNA library, plasmidial DNA preparation andsequencing of clones from this library have been previ-ously described.2 The other six libraries were constructedin AZapII (Stratagene), according to the manufacturer'sinstructions. Total RNA was isolated from distinct5. mansoni developmental stages by the guanidiniumthiocyanate-phenol-chloroform method4 and poly(A)+RNA was obtained by chromatography on an oligo(dT) column.5 Double-stranded cDNA was cloned intoEcoRI/Xhol restriction sites of AZapII. pBluescript SK+phagemids were obtained by "en masse" in vivo exci-sion of AZap clones,6 by co-infecting Escherichia coliXL-1 Blue cells with the ExAssist helper phage (Strata-gene). The excised phagemids were used to infectE. coli SOLR™ cells (Stratagene) for production of

double-stranded DNA (dsDNA) templates. Transfor-mants were plated onto LB agar containing ampicillin, X-gal and isopropyl-/3-D(-)-thiogalactopyranoside (IPTG).White colonies were selected and grown for 16 hr in3 ml of Luria broth (LB) supplemented with ampicillin.Aliquots of the cultures (200 /xl) were mixed with thesame volume of 30% glycerol in LB and frozen at — 70° Cin 96-well plates. The rest of the cultures were used forplasmidial DNA preparation using the Wizard Plus MiniPrep DNA Purification System (Promega). dsDNA wassequenced by dideoxy chain-termination sequencing7 us-ing the Thermo-Sequenase Cycle Sequencing kit (Amer-sham) and M13 Reverse or M13-40 fluorescent-labeledprimers (Pharmacia). Single-pass runs of the sequencingreactions were performed on an A.L.F. automated DNAsequencer (Pharmacia).

2.2. Data analysisSequences were manually edited to eliminate vector re-

gions, poly(A) tails and lower quality data at the endof the sequence. ESTs containing less than 150 bp andmore than 4% ambiguity were rejected. ESTs were com-pared to DNA and protein sequences deposited in non-redundant databases using the Basic Local AlignmentSearch Tool (BLAST) programs8 at the National Centerfor Biotechnology Information (NCBI). Alignments scor-ing more than 200 for BLASTN and 100 for BLASTXwere selected and after meticulous visual inspection onthe biological significance of the alignment, ESTs werenamed as putative identification for the gene. ESTs withno significant database matches or showing only partialhomology with database sequences were grouped as non-identified genes.

2.3. Clustering analysis:Sequences sharing local similarities were clustered with

the ICATOOLS set of programs9 (freely available atftp.ebi.ac.uk). Initially, each library was independentlyanalyzed. The module ICAass was used to create an in-dex of clustered sequences (threshold and ktup set to 25and 8, respectively). One singular sequence was added tothe cluster with ICAass and used to run the module ICA-tool, under the same threshold and ktup settings. Thiswas followed by the run of ICAtool with all sequences inthe library. ICAprint was used to generate the outputfile, that was manually inspected since some clones hadbeen sequenced in both orientations and/or led to thesame identification when submitted to homology search.A second round of analysis was conducted with all li-braries concomitantly in order to join the clusters thathad been previously formed, but for this purpose onlyICAass followed by ICAtool with a singular sequence wasexecuted.

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Table 1. Information about the sequencing of different S. mansoni cDNA libraries.

NumberNumberNumberNumber

of ESTsof sequenced clonesof usable ESTsa>of usable clones'1'

Egg106107

80

80

Cercariae110107

98

98

S.Lung staj

107107

67

67

mansoni

;e Adult812617

657

504

cDNA libraries1 Adult 2

9494

91

91

Adult 3101101

78

78

Adult471715252

Total140112041123970

a ' ESTs/clones analyzed by ICATOOLS. These numbers correspond to the total number of ESTs/clones after removing sequences ofvector, mitochondrial DNA, rRNA and contaminating sequences from other organisms.

3. Results and Discussion

3.1. Quality control of the cDNA librariesSince the start of S. mansoni genome project, one of

our main focuses has been the large-scale sequencing ofcDNA to produce ESTs, in an attempt to identify newgenes of this organism. Initially, we used an adult wormcDNA library, from which we generated 607 ESTs cor-responding to 154 new S. mansoni genes.2 The goodquality of this library was attested by the diversity ofgenes that were isolated, even after the discovery of asignificant degree of redundancy (65% of the sequencedclones corresponded to 49 redundant genes).2 The suc-cess of this approach prompted us to extend the sequenc-ing program to include other libraries. We started witheight libraries from distinct developmental stages, all ofthem constructed using the AZap system (Stratagene):one egg. two cercariae (the human-infecting larvae), threeadult worms, one 7-day schistosomula (the lung stage)and one from 25-day old worms. All libraries were ex-cised "en masse" and at least 30 colonies from each li-brary were selected to evaluate the average size of the in-serts by polymerase chain reaction (PCR). Most of themhad an average insert size greater than 500 bp, exceptfor one cercariae and the 25-day worm libraries. Thus,we decided to use all three adult worm cDNA librariesand the Egg. one cercariae and the 7-day schistosomulalibraries in this study.

Table 1 summarizes data obtained from the sequenc-ing of the distinct libraries. A total of 1401 ESTs wereproduced from one or both ends of 1204 clones. Thedata from the Adult 1 library are cumulative since thebeginning of the program and includes ESTs publishedby Franco et al., 1995.2 In the Egg library, the numberof clones exceeds the number of ESTs and this is dueto the sequencing of a chimeric clone from which twoESTs were generated. Both ESTs were eliminated fromsubsequent analysis. After homology searches in non-redundant databases using BLAST programs8 and elim-ination of ESTs corresponding to useless sequences (vec-tor, mitochondrial DNA, rRNA and contaminating se-quences from other organisms), 1123 ESTs derived from970 clones were submitted to clustering analysis, usingICATOOLS program,9 resulting in a list of distinct genes.

Adams et al.10 proposed criteria to evaluate the qual-ity of the libraries used in large-scale EST analysis. Theystate that the sequencing of 100-200 clones from a li-brary is sufficient to assess the quality of this libraryand to detect problems that might have occurred dur-ing library construction. A useful library should con-tain no more than 20% useless sequences, at least 50%new genes and a broad variety of transcripts. We usedtheir criteria to evaluate the seven cDNA libraries usedin this study (Fig. 1). The first five parameters are ameasure of the proportion of useless clones. In general,the libraries were of good quality with respect to theseparameters, except for the Lung stage, Egg and Adult 3libraries. The Egg library contains 20% clones withoutan insert, even though a previous blue/white selectionof clones had been performed. The Adult 3 library isenriched in clones corresponding to mitochondrial DNAsequences. Most of them correspond to a polymorphicminisatellite sequence of 620 bp,11 that contains part ofan S. mansoni nuclear transcript denominated SM750.12

This transcript is composed of a invariable region that isfollowed by five copies of a 62-bp polymorphic repeat el-ement (PRE). Interestingly, five or more copies of the62-bp PRE were seen solely or as part of the mitochon-drial minisatellite in all libraries analyzed except the Egglibrary. This fact implies that PRE is a very frequent el-ement in the genome of the parasite and that it couldbe part of a nuclear sequence that was incorporated intothe mitochondrial genome.11 None of the libraries con-tains excessive number of sequences derived from riboso-mal RNA. The Lung stage library contains almost 20%contaminating sequences from other organisms. Thesecontaminating sequences are derived either from E. colior other bacteria, probably due to the contamination ofthe worm samples during the 7-day period of in vitrocultivation necessary to mature to lung stage schistoso-mula.

The quality of the construction of each library was alsoanalyzed. All of them were shown to be unidirectional(most ESTs had matches to database sequences on theexpected strand), composed of a high proportion of in-serts longer than 500 bp, composed of inserts with shortpoly(A) tails and containing no chimeric clones. The onlyexception was the Egg library, where we found a single

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12- % distinct unknown p w »

11-% distinct non-Sm match

10- % distinct Sm match f

9- % unknown *

Jj 8- % non-Sm match

jg 7- % Sm match

o 6- % useless clones

U

"3 5- % chimaeric clonesO

4- % contaminants

3- % rRNA

2- % mtDNA

1- % no insert

10 20 30 40 50 60 70 80 90 100

Percentage of total

Figure 1. Evaluation of the cDNA libraries according to the criteria of Adams et al.10 Parameters 1 to 5 indicate the percentage ofthe total of clones in each library that produced useless ESTs and this set of data is totaled in parameter 6. The percentage of thetotal of clones that are identified either by homology with previously reported S. mansoni genes (Sm match), putatively identifiedby homology with genes from other organisms (non-Sm match), or with partial homology with genes from other organisms andnon-database match sequences (unknown) is also shown (parameters 7 to 9). The percentage of useful clones that are distinct foreach category of genes was determined by clustering analysis and is shown in parameters 10 to 12.

chimeric clone (parameter 5). The sixth parameter is thesum of the first five parameters and totals the frequencyof useless clones in each library. Three out of the seven li-braries exceed 20% non-useful clones: Lung stage (37%).Egg (22%) and Adult 3 (21%), and this is mainly due tothe reasons discussed above. However, when analyzingthe gene content in each of these three libraries, we veri-fied that they have a high percentage of distinct genes anda low proportion of redundant genes (see below). This

fact justifies the continuation of using of these librariesin the EST sequencing program, but with the inclusionof a previous selection step to eliminate abundant uselessclones.

Parameters 7 to 9 of Fig. 1 concern to the analy-sis of the composition of the libraries after EST ho-mology searches in non-redundant databases. Most li-braries showed a low proportion of cDNA clones withexact match, to previously described S. mansoni genes

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Table 2. Gene content of the cDNA libraries after random-sampling of clones.

Distinct genesNew genes% of distinct genes% of new genes per

per total of sequenced clones*'total of sequenced clones'1'

Egg736768.262.6

Cercariae655860.754.2

5. mansoni cDNA librariesLung stage

625457.950.5

Adult 119817332.128.0

Adult 2191820.219.1

Adult 3574856.447.5

Adult 4484067.656.3

Total52245843.438.0

for the number of sequenced clones see Table 1.

(less than 20%), except for the Adult 1 library (parame-ter 7). This can be explained by the fact that this libraryis enriched in clones corresponding to the S. mansoniglycolytic enzyme glyceraldehyde 3-phosphate dehydro-genase (GAPDH),13 the most redundant gene found inthis library. Moreover, as the Adult 1 library was themost sequenced library in this program, it is possible thatit better represents the profile of genes expressed in adultworms. Remarkably, all adult worm libraries had, ingeneral, more cDNA matching S. mansoni known genesthan the libraries constructed from other developmentalstages. This is particularly interesting, since it reflectsthe sort of S. mansoni genes that have been deposited inpublic databases. Most of them are isolated from adultworms. However, the Cercariae library attained the sameproportion of clones matching 5. mansoni genes as theadult libraries. This can be explained by the presence ofa very abundant transcript in this category, the calcium-binding protein (CaBP),14 that corresponds to 10% ofthe total of useful clones. Most probably this protein isvery important for the cercariae metabolism and may beinvolved in movement. Few clones in all libraries could beputatively identified by significant homology with genesfrom other organisms (parameter 8) and the great major-ity of clones in each library (>35%) could not be identi-fied (parameter 9). These last ones correspond to cDNAthat had only partial matches to sequences from otherorganisms or non-database match cDNA.

Parameters 10 to 12 consist of the number of distinctgenes divided by the number of useful clones in each cat-egory and measure the diversity of transcripts. To obtainthe number of distinct genes, each library was submittedto clustering analysis, using the program ICATOOLS.The program grouped together as a single cluster cloneswith a high degree of identity; each cluster was treated asan independent gene. The veracity of such clusters wasattested by the correct grouping of clones that sharedthe same homology to 5. mansoni or other organismsdatabase sequences. Considering that one goal of theEST sequencing program is the discovery of new genes,the diversity in the non-Sra match and in the unknowncategories are particularly relevant. In this respect, inill libraries with exception of the Adult 1 and Adult 2libraries, more than 70% of the transcripts are distinct in;hese two categories. This fact counterbalances the low

efficiency in obtaining useful clones from the Egg, Lungstage and Adult 3 libraries. An intermediate degree ofdiversity is observed for the Adult 1 library, while a verylow diversity of transcripts is seen in the Adult 2 library.A tendency of decreasing the variety of transcripts inthe Sm match category is also observed, which can beexplained by the presence of very abundant transcriptsalready characterized in 5. mansoni. That is the casefor the Cercariae, Adult 1 and Adult 2 libraries due tothe enrichment of CaBP-, GAPDH- and eggshell protein-encoding15 transcripts, respectively.

3.2. Gene content and redundancy analysisThe strategy of random-sampling of cDNA libraries al-

ways produces a series of clones corresponding to a singletranscript; either because abundant mRNA will be morerepresented in the library, or because each library has aninherent bias that was introduced during its construction.Thus, clones obtained from a such library will reflect itscDNA composition. For this reason, we decided to an-alyze each library according to its gene content and toevaluate its quality based on the extent of redundancy.This was only possible after performing clustering anal-ysis by ICATOOLS.

Table 2 shows the number of distinct genes, as wellas the number of new genes obtained from each library.This last class includes genes homologous to genes fromother organisms (non-Sm match category) and genes ei-ther partially homologous to genes from other organismsor non-database match genes (unknown category). A to-tal of 522 distinct genes were obtained from the sevenlibraries, 458 of which (88%) were newly identified inS. mansoni. This corresponds to three times the numberof new genes obtained in the beginning of the sequencingprogram.2

Considering the effort to get distinct or new genes fromrandom selection of clones in each library, it is impor-tant to consider the percentage of genes in the total ofsequenced clones. This is a measure of the library qual-ity regarding both its redundancy and content of uselessclones. It can be seen that, in all libraries with the excep-tion of the Adult 1 and Adult 2 libraries, more than 50%of the sequenced clones were found to be distinct genes.It is important to note that the Adult 1 library was se-

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236 ESTs from S. mansoni cDNA Libraries [Vol. 4,

i of

Gel

100 V

7 5 -

5 0 -

2 5 -

1 Egg

i

W-& 7W \ f \ \ \ \ \ \ /

100-1 1CK) —ILung stage

Frequency Frequency Frequency

Frequency

Frequency Frequency Frequency

Figure 2. Redundancy in EST sequencing of the S. mansoni cDNA libraries. On the abcissa we show the number of times that eachgene was sampled and on the ordinate we depict the fraction of genes sharing a given sampling frequency.

quenced close to six times more than the other libraries(Table 1), and this might explain the rate of 32% of newgenes. The same tendency was seen for the ratio of newgenes per total of sequenced clones. Again, the Adult1 and Adult 2 libraries provided the lowest efficiencies.Rates of 50% in acquirement of new genes as observedfor the S. mansoni libraries met the criteria establishedfor the human EST program.10

A direct representation of the extent of redundancy ineach library is seen in Fig. 2, that shows the percentageof genes that appear in the library under a given fre-quency. As random sampling of a cDNA library shouldfollow a Poisson distribution for rare events, the unex-

pected presence of genes under classes of high frequencyof isolation reveals a bias in the library. This is evidentfor the Adult 2 library, where the profile of frequencydistribution clearly escapes a typical Poisson distribu-tion, which strongly supports our decision not to use thislibrary for large-scale EST production. The high pro-portion of redundant genes in this library might haveresulted from errors introduced during library construc-tion and amplification, "en masse" excision or clone sam-pling for EST generation. The occurrence of genes underclasses of high frequency of isolation is also seen in theCercariae and Adult 1 libraries. Nevertheless, it would bepossible to eliminate the most redundant genes (8 genes

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Table 3. Putatively identified genes homologous to 5. mansonigenes.a'

Gene Library EST accessionb )

EnzymeAspartic proteinaseCarbonyl reductaseCathepsin BCyclophilin BEnolaseER-luminal cysteine protease (ER-60)Fructose-l,6-bisphosphate aldolaseGlutathione peroxidaseGlutathione S-transferaseGlyceraldehyde-3-phosphate

dehydrogenaseHemoglobinase (Sm32)HexokinaseTriose phosphate isomeraseCytoskeletal/structural proteinActinAlpha-tubulinEggshell proteinFemale-specific polypeptideMyosin heavy chainP48 eggshell proteinSm23 integral membrane proteinTropomyosin (GB:SCMTPM)Tropomyosin (GB:SCMTROPO)AntigenAntigen 10-3Antigen Sm21.7Major egg Antigen (P40)Sml3 tegumental antigenTransport/storage proteinCalcium binding protein (CABP)Calcium-calmodulin binding proteinCalreticulin

Fatty-acid binding protein (Sml4)FerritinGlucose transporterOtherBreast basic conserved protein/

ribosomal protein L13Calnexin homolog SmlrVlElongation factor 1 alphaHeat shock protein 86S. mansoni mRNA for tandem repeatS. mansoni (Liberia)

zinc finger proteinY-box-binding protein

Adult 4Egg

CercariaeLung stage

Adult 1Adult 4

Lung stageCercariaeAdult 1Adult 1

Adult 1Adult 1

Egg

CercariaeEgg

Adult 4Adult 3

Lung satgeAdult 3Adult 1Adult 1Adult 1

Adult 1Adult 3

EggAdult 4

CercariaeCercariaeAdult 1Adult 1Adult 3Adult 1

Adult 1Adult 3

Lung stageAdult 1

EggLung stage

AA169900AA140589AA143823AA125705

T14396AA169915AA125670AA143892

T14549T14434

T14348T14603

AA140583

AA143846AA 140633AA169905AA218489AA125688AA218479

T14382W06805W06761

T14386AA218508AA140559AA169901

AA143886AA143883W06720T14374

AA218482T14364

T14585AA218511AA125724

T14407AA140585AA125700

Adult 1 T14571a' Genes putatively identified by homology with S. mansoni databasesequences. Only one representative EST matching the respective geneis shown, together with the name of the library it was isolated from.

' EST accession corresponds to the GenBank accession number.

for the Adult 1 library and 3 genes for the Cercariae li-brary) from these libraries by filter screening, using theabundant transcripts as probes, and this should result ina profile compatible with a Poisson distribution.

Although some libraries presented problems detectedby quality analysis, they all contributed to the list of pu-tatively identified genes, as well as 333 distinct unknowngenes (see below). Genes identified by homology withpreviously described S. mansoni genes are distributedamongst various classes, such as genes coding for en-zymes, structural proteins, antigens, proteins involved intransport and storage, etc (Table 3). Two genes in thislist have been the subject of a more extensive study andwere characterized in detail in our laboratory. They are

the 5. mansoni homologues of the Y-box-binding protein(Franco et al., submitted) and the breast basic conservedprotein, or the 60S ribosomal protein L13 (Franco et al.,submitted). Table 4 lists distinct genes putatively identi-fied by homology with genes from other organisms. Theycode for enzymes of different metabolic pathways, a greatvariety of ribosomal proteins, several constituents of tran-scriptional/translational machinery, and regulatory cyto-plasmic and membrane proteins, among others. Three ofthese genes were selected for further studies. One is thehomologue of mago nashi gene from Drosophila. Thisgene is necessary for proper germ plasm assembly andmutations in it result in sterility of Fl progeny.16 The5. mansoni purine nucleoside phosphorylase was selectedfor presenting a high similarity with the human counter-part, the 3D structure of which has already been resolvedand deposited in the Protein Data Bank. Modeling stud-ies with this protein have led to the identification of pow-erful inhibitors of this enzyme, whose activity is crucialin T cell guanosine metabolism.17 The third gene is thehomologue of the human HLA-DR-associated protein I,a protein which may be involved in signal transductionin B cells.18 We are interested in the selection of proteinsthat can interact with it, which may help to define itsbiological function in the parasite.

3.3. Gene expression profile in S. mansoniTo obtain an initial profile of gene diversity in the

parasite and a preliminary pattern of gene expressionin distinct stages of the development of S. mansoni,we performed a clustering analysis, joining sequencesfrom all libraries. This resulted in a total of 466 uniquegenes (considering only once the genes present in morethan one library), corresponding to 427 new S. mansonigenes. From the total of unique genes, 39 (8.3%) matchedpreviously characterized S. mansoni genes, 94 (20.2%)matched genes from other organisms and 333 (71.5%)represent unknown genes. From the clustering analysis,most genes (433 of 466) were present only in a singlelibrary (e.g. CaBP was found only in the Cercariae li-brary). Other genes were expressed in more than onedevelopmental stage and are listed in Table 5. They mayrepresent housekeeping genes in the parasite and, curi-ously, ten of them were unknown. The antigenic poten-tial of such genes should be investigated, since they mightbe specific to this parasite.

At this point of the sequencing program, only threegenes were found to be expressed in all developmentalstages analyzed: the cytochrome oxidase chain I, thefructose-1,6-bisphosphate aldolase and unknown gene 10.Somewhat unexpectedly, actin and GAPDH, the mostfrequent genes in the collection, were not isolated fromall stages, perhaps because the number of transcripts se-quenced in each library was not very large. Five genes

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Table 4. Identified genes homologous to non-5, mansoni genes.a)

Gene Library EST accessionb)

EnzymesAlcohol dehydrogenase class IIIAldehyde dehydrogenaseAldose reductaseATP synthase, vacuolarCytochrome Oxidase chain ICytocrome oxidase IIDaktl serine/threonine protein kinaseDihydrolipoamide acetyltransferaseEnoyl-CoA HydrataseGlutamine SynthetaseGlycerol 3-phosphate dehydrogenaseH+-transporting ATP synthase

alpha-chainLactate dehydrogenaseOligosaccharyl transferase 48 KDOrnithine aminotransferasePhosphoenolpyruvate CarboxykinasePhosphoglycerate kinasePhosphoglycerate mutase20S proteasoma subunit RC7-I=PREl

homologProteasome zeta chainPurine nucleoside phosphorylasePyruvato kinaseRibonuclease- phosphate 3-epimerase

(pentose-5-phosphate 3-epimerase)Vacuolar ATP synthase subunit BTranscriptional/

Translational Machinery40S ribosomal protein S340S ribosomal protein S440S ribosomal protein S740S ribosomal protein S l l40S ribosomal protein S1240S ribosomal protein S1440S ribosomal protein S1740S ribosomal protein S2040S ribosomal protein S2140S ribosomal protein S2660S ribosomal protein L560S ribosomal prote:

Table 4. Continued.

Gene Library EST accessionb )

m L7in L7an LlOan L25n L30

60S ribosomal prote;60S ribosomal prote:60S ribosomal prote:60S ribosomal proteiAsp-tRNA synthetaseElongation factor 1 gammaHomo sapiens 9G8 splicing factorJun-binding proteinLys-tRNA synthetasePolyadenylate binding proteinPutative transcriptional regulatorReverse transcriptaseRho-GDP dissociation inhibitorRNA poiymerase II subunitRNA-binding protein X-16Small nuclear ribonucleoprotein

Adult 3Adult 1Adult 1Adult 1

EggAdult 3Adult 4Adult 1

Lung stageAdult 1

CercariaeAdult 1

Adult 1Adult 3Adult 1

EggAdult 1Adult 1

Lung stage

Adult 1Adult 1Adult 1Adult 3

Adult 1

Adult 3Adult 1

EggAdult 4

Lung stageAdult 1

Lung stageEggEgg

Lung stageLung stage

EggAdult 1

EggLung stage

Adult 3Adult 1Adult 1Adult 1Adult 4Adult 1Adult 1

Lung stageEgg

Adult 1Lung stage

Adult 1Adult 1

AA218449T24129W06782T24142

AA140564AA218486AA169931

W06795AA125690W06821

AA143842W06794

W06744AA218463

T14588AA140576

T14620W06743

AA125733

T14568W06714T24140

AA218494

W06824

AA218471W06814

AA140626AA169892AA125707

T14564AA125727AA140581AA140582AA125695AA125687AA140600

T14431AA140612AA125723AA218468W06768T14422W06725

AA125664T14484W06727

AA125694AA140605W06723

AA125704T14358T14459

were present in two or more adult libraries, but absentin other stages. This is the case for the eggshell protein,that is recognized to be expressed in mature females, andalso unknown gene 2.

Clustering analysis also included formation of contigsof sequences. As an example, the cDNA sequence of anunknown gene, that is abundant in the Adult 1 library,was obtained after assembling ESTs from both cDNAends that were clustered together by ICATOOLS. Thisgene is currently being characterized in more detail. We

Membrane /cy top lasmADP/ATP carrier proteinAnnexin familyBeta-1 tubulinChaperonin-like proteinCytochrome cDNAJ homologGTP-binding proteinHeat shock protein 108HLA-DR associated protein IPolyubiquitinPossible membrane proteinProtein kinase C inhibitor proteinNonerythroid alpha-spectrinUDP-galactose translocadorOther52k active chromatin boundary proteinAlpha-collagenApoptosis-inducibleArginine-rich geneC. elegans hypothetical 272 KD protein

C50C3.6 in chromosome IIIC. elegans clone C16C10.10Coded for by C. elegans cDNACysteine-rich intestinal proteinE. coli hypothetical 53.1 KD protein

in LYSU-CADA intergenic regionFibrillin 2GATA-3 geneGolden Syrian Hamster repetitive DNAHistone H3.3H. sapiens mRNA for Sm protein FHuman Alu subfamilyHypothetical protein - D. melanogasterHypothetical protein 5 Xanthobacter spHypothetical 30.5 KD protein of C. elegansLiver regeneration factor augmenterMago nashi proteinMER5 ProteinNIFS-like 54.5 KD proteinProliferation-associated proteinRetrovirus-related GAG polyproteinSynaptophysinYeast hypothetical 103.7 KD ProteinValosin-containing protein homologue

Adult 1Adult 1

EggAdult 1

CercariaeAdult 1Adult 3Adult 1Adult 3

EggLung stage

Adult 1Adult 1Adult 3

Adult 1Adult 1

CercariaeAdult 1Adult 3

Adult 1Adult 3Adult 3

Lung stage

CercariaeEggEgg

CercariaeLung stage

Adult 1CercariaeAdult 1

EggAdult 2

Lung stageAdult 1Adult 1

Lung stageEgg

Adult 1Adult 1Adult 1

T14447T14511

A A140634T14632

AA143872W06722

AA218450T18621

AA218460A A140632AA125728

T14595T14622

AA218519

W06740T14493

AA143880T14555

AA218465

W06746AA218495AA218481AA125683

AA143891AA140598AA140590AA143814AA125673W06750

AA143820W06771

AA140628AA185826AA125719

T14649W06757

AA125729AA140602W06818W06819T14640

a ' Genes putatively identified by homology with genes from other or-ganisms. Only one representative EST matching the respective geneis shown, together with the name of the library it was isolated from.

' EST accession corresponds to GenBank accession number.

expect that, with the advance of the sequencing program,a higher number of partial cDNA sequences will be as-sembled as full-length contigs, increasing the ability toidentify unknown genes and more precisely define the realnumber of distinct genes in each library and in each de-velopmental stage.

Acknowledgments: The authors thank KatiaBarroso for carrying out automated DNA sequenc-ing. This investigation received financial support fromthe following sources: PADCT, CNPq, UNDP/WORLD BANK/WHO Special Program for Researchand Training in Tropical Diseases (TDR N°: 940325 and940751), USAID/HOH (N° 264.01.01.04), FAPEMIG,PAPES/ FIOCRUZ.

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No. 3] G. R. Franco et al. 239

Table 5. Frequence of genes present in multiple S. mansoni cDNA libraries.a

Genes

1- Actin2- Alpha tubulin3- ATP synthase4- Beta tubulin5- Carbonyl reductase6- Cathepsin7- Cyclophilin B8- Cysteine-rich intestinal protein9- Cytochrome oxidase chain I

10- EFlalpha11- Eggshell protein12- Enolase13- ER-luminal cysteine protease14- Fibrillin15- Fructose-l,6-BP aldolase16- GAPDH17- Major egg Antigen (P40)18- Myosin heavy chain19- Oligosaccharyl transferase 48 KD20- Triose phosphate isomerase21- Ubiquitin22- 60S ribosomal protein L523- 60S ribosomal protein L3024- Gene lb)25- Gene 226- Gene 327- Gene 428- Gene 529- Gene 630- Gene 731- Gene 832- Gene 933- Gene 10

Egg

—2.5—1.31.3———1.3—————1.3—1.3——1.31.3——————————1.31.3

Cercariae

1.0————1.0—1.02.0————1.01.02.0———————2.0———1.01.0——2.05.1

Lung stage

—1.51.5——1.51.5—1.51.5————1.54.5—1.5———1.51.5——1.51.5——1.51.5—1.5

Adult 1

6.90.80.20.40.20.4——0.43.00.20.80.40.63.27.30.20.40.20.41.01.0——0.4—0.20.20.2—0.20.21.6

Adult 2 Adult 3

— 3.9— 2.6— —— —— —— —— 2.6— 1.38.8 —— —

19.8 —— —— —— —— —— 1.3— —— —— 1.3— —— —— —— 1.3— 3.82.2 5.1— 1.3— —— —— —— —— —4.4 6.4— 1.3

Adult 4

1.9—————————1.91.91.9—3.8——————————————1.9—1.91.9

Total

4.10.90.20.30.20.40.30.21.41.62.10.50.30.42.24.40.20.30.20.30.60.60.20.50.80.20.20.20.20.20.21.41.8

a ' Percentage of clones matching the corresponding gene in the total of usable clones analyzed by ICATOOLS. For the total of usableclones see Table 1. b ' unknown genes are numbered 1-10.

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