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. Author manuscript; available in PMC: 2008 Nov 1.
Published in final edited form as: Exp Parasitol. 2007 Jun 12;117(3):229–235. doi: 10.1016/j.exppara.2007.06.001

The Schistosoma mansoni transcriptome: an update

Guilherme Oliveira 1,2,*
PMCID: PMC2140242  NIHMSID: NIHMS34271  PMID: 17624328

Abstract

Large scale EST sequencing projects have been carried out for S. mansoni and S. japonicum. This update will briefly review the most recent accomplishments in the area and discuss the use of EST data for the purposes of gene discovery, gene model development, genome annotation and SNP analysis. In addition, the use of ESTs for studying other features of the transcriptome such as splice site and transcription initiation variants will be discussed as well as approaches to assigning function to unknown transcripts. Although EST sequencing has contributed much for schistosome research, other data mining possibilities exist, including the identification of putative drug and vaccine targets.

Keywords: Trematode, schistosomiasis, expressed sequence tags, transcriptome

The transcriptome can be defined as a collection of the genes transcribed in an organism, tissue, or cell. Transcriptomic information has been extremely useful for gene discovery, the elaboration of gene models, training of gene finding algorithms (once a genome is available) the design of microarrays and has tremendously facilitated the cloning of genes of interest by the availability of cDNA clones. However, the mRNA transcribed is not expressed in equal amounts even in a single cell and they also differ in length. Therefore, experimental efforts towards obtaining the transcriptome of an organism generally will provide partial mRNA sequences and will be enriched for genes that are more highly transcribed. The partial cDNA sequences generated are called expressed sequence tags, ESTs. Despite some technical difficulties for producing full length cDNAs and incomplete views of the transcriptome provided, the approach has proven to be a powerful tool for the study of genes and their expression pattern.

The original and main motivation for the study of transcriptomes was the possibility of discovering the gene content of an organism and generating STSs that pointed to a gene, without the need to obtain the complete genome sequence (Adams et al., 1991). Although the unfinished genome sequences of S. mansoni and S. japonicum have been produced (El-Sayed et al., 2004;McManus et al., 2004), and are currently in the annotation stage, the transcriptome of both species have been studied on a large scale and have contributed to the discovery of genes and genome annotation, among other uses that will be further discussed in this update (genome sequencing will be discussed in this issue). Currently, GenBank contains information for over 650 species with at least 1,000 ESTs (Table 1).

Table 1.

Online resources related to Schistosoma ESTs available.

Resource Web site Reference
ORESTES and annotation verjo18.iq.usp.br/schisto/ Verjovski-Almeida et al., 2003
TIGR gene index compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=s_mansoni Merrick et al., 2003
ESTs and KEGG pathways rgmg.cpqrr.fiocruz.br/ Oliveira et al., Unpublished
EST of S. mansoni, S. japonicum
and S. haematobium 		www.sanger.ac.uk/Projects/S_mansoni/ Wellcome Trust Sanger Institute, Unpublished
S. mansoni SNPs bioinfo.cpqrr.fiocruz.br/snp/ Simões at al., 2007
Schistosome Related Reagent
Repository - SR3		 www.afbr-bri.com/SR3/ Unpublished
Genome sequence of S. mansoni www.genedb.org/genedb/smansoni/ Unpublished
Genome database for S. mansoni www.shistodb.net Unpublished, under construction
Genome sequence of S. japonicum lifecenter.sgst.cn/sj.do# Unpublished
CDD database 130.14.29.110/Structure/cdd/cdd.shtml Marchler-Bauer et al., 2007
Pfam database www.sanger.ac.uk/Software/Pfam/ Finn et al., 2006
PIR and UNIPROT pir.georgetown.edu/ Wu et al., 2003; Wu et al., 2006a
Swiss-Prot www.expasy.org/sprot/ Wu et al., 2006a
InterPro www.ebi.ac.uk/interpro/ Mulder et al., 2002
KEGG www.genome.jp/kegg/ Kanehisa et al., 2004
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There are two main approaches for the large scale production of cDNA sequences. The conventional method is based on reverse-transcribing mRNA, cloning and sequencing the extremities of the inserted cDNA (Fulton et al., 1995). The alternative approach named ORESTES is to sequence short randomly amplified and cloned cDNAs (Dias et al., 2000) (Figure 1). Both methods can potentially result in large numbers of redundant sequences of cDNAs. The sequences need to be assembled so that transcripts from one gene are grouped together. The assembly process yields, in many cases, full length virtual cDNA sequences. The methods are complimentary. The ORESTES tend to accumulate at the central region of the original mRNA and the conventional method at the 5' and 3' ends. Therefore, by combining both methods, the chance of assembling a full virtual cDNA sequence increases. The ORESTES method allows sampling of a larger number of small cDNA libraries, which is useful especially in cases of difficult to obtain mRNA. The method also normalizes the mini cDNA libraries, making the relative number of ESTs similar irrespective of the different gene transcription levels in the sampled tissue. Normalization permits the identification of rare transcripts. The conventional method, on the other hand, permits an in depth sampling of much larger cDNA libraries. In addition, the conventional method permits the estimation of the relative numbers of each transcript by not normalizing the libraries. Also importantly, the conventional cDNA sequencing method produces frozen stocks of clones, many of which are complete or near complete cDNAs. These cDNA clones are an important resource to the research community.

Figure 1.

Figure 1

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Methods for producing expressed sequence tags (ESTs). The complete cDNA clone is represented by the central line. A – ORESTES. B – Conventional cDNA sequencing. Conventional cDNAs are usually 100 to 500 bp sequences produced from cDNA cloned and sequenced directionally in respect to the 5' or 3' end, and tend to accumulate at the end of the representing mRNA. On the other hand, ORESTES are shorter sequences that tend to accumulate on the central region of the representing mRNA.

The study of the transcriptome can provide many different types of information about an organism. The main type of information generated is the discovery of new genes which can be accomplished efficiently and quickly with a transcriptomic approach, especially for organisms with large genomes, such as schistosomes. In the pre-genome era of schistosome research, this approach was used and yielded a large number of new genes (Franco et al., 1995;Oliveira, 2001) cDNA clones were also widely distributed to the research community. More recently, larger scale projects on S. mansoni have immensely expanded the knowledge and made mining the information much more effective (Oliveira and Johnston, 2001;Hu et al., 2003;Verjovski-Almeida et al., 2003;Verjovski-Almeida et al., 2004; Oliveira, unpublished effort of the RGMG). For S. japonicum one large project was published (Hu et al., 2003). The Wellcome Trust Sanger Institute also maintains at its ftp site EST sequences of S. mansoni and S. haematobium (Table 1).

The annotation, or attribution of possible function, of the sequences can be carried out with each individual read and with assembled sequences. Generally the methods will involve the search for similarities between the EST or contig and a database of DNA or protein sequence with functional annotation. This type of analysis will initially yield information about which genes are known and which are unknown. In order to annotate gene function using similarity search, curated databases of proteins are preferably used. Some of the interesting databases are: CDD (Marchler-Bauer et al., 2007) or Pfam (Finn et al., 2006) for conserved protein domains and families and PIR (Wu et al., 2003), SwissProt or UniProt (Wu et al., 2006a) for annotated proteins, among many others. InterPro contains several databases with information on protein families, domains and functional sites (Mulder et al., 2002), which can be queried with InterProScan (Quevillon et al., 2005). One of the problems with annotating a gene or a protein is the use of a structured vocabulary shared among different organisms. For this reason, it is also useful to annotate a sequence with a Gene Ontology term (Ashburner et al., 2000). One example of an annotated transcriptome database is the Gene Index (Merrick et al., 2003), but others are also available (Table 1).

The results obtained with schistosome EST sequences indicate that under the Molecular Function category, most of the ESTs belong to the binding (nucleic acid binding) and enzyme (mainly hydrolases and transferases) categories (Hu et al., 2003;Verjovski-Almeida et al., 2003). Under Biological Process the most prevalent categories were metabolism or transport. The finding of these categories as the most prevalent was expected, as metabolism is usually the major physiological activity of an organism. Some interesting features were observed, such as the presence of glucose importers, proteins involved in the uptake of amino acids and lipids and storage of lipids. The identification of the metabolic pathways in which parasite gene products participate will contribute to the understanding of their metabolic processes. This investigation may also contribute to the identification of possible drug targets (Fairlamb, 2002). One of the possibilities is to use KEGG that provides biochemical pathways, among other resources (Kanehisa et al., 2004). A visualization of the KEGG pathways obtained with the use of the transcriptome of S. mansoni is available (Oliveira, unpublished; Table 1).

After annotation, lists of genes possibly involved with a certain physiological function in the organism are usually produced. One example is genes involved in sex differentiation. This group of genes is of interest because interfering with sex differentiation or maturation may be one way of preventing the disease by interrupting egg production by the female parasite, as the eggs cause most of the pathology (LoVerde, 2002). In addition, sex differentiation in schistosomes should be an interesting model for study since most platyhelminths are hermaphrodites (Mone and Boissier, 2004). Several homologues of genes involved in sex differentiation, such as fox-1, mog-1, tra-2 and fem-1 were observed (Verjovski-Almeida et al., 2003). Hu and colleagues (2003), focused on genes differentially expressed between sexes and obtained results comparable to those of microarray experiments (Hoffmann et al., 2002). Among the differentially expressed genes are those coding for egg shell proteins, maleless, epididymal secretory protein E1 and transformer-2β.

One interesting approach for analyzing the EST content is the comparison of the frequency of ESTs of one organism with those of model organisms. This will provide information on conserved genes (Hu et al., 2003), but also can be informative in relation to the expected number of ESTs, indicating genes that follow unusual patterns of expression in comparison with other organisms (Mudado and Ortega, 2006).

Interestingly, from the first descriptions with fewer sequences (Franco et al., 1995), to the larger projects (Hu et al., 2003;Verjovski-Almeida et al., 2003), at least 50% of all transcripts identified yield no similar transcripts in GenBank. The number may decrease as the clusters grow in size and with the availability of better gene models with the genomic data. Nevertheless, clearly, schistosomes contain a large set of unique genes. One of the main challenges is the characterization of their function.

Functional genomics will contribute to describe gene function. In this issue recent developments in knocking down gene expression by RNA interference and over expression by the introduction of exogenous DNA are discussed. However, there is a need for observable phenotypes in order to see an effect resulting from knock-out, knock-down or transgene approaches. The function of a gene product may sometimes also be inferred by a guilt-by-association approach (although experimental evidence is necessary to corroborate the indications). Microarray experiments, for example, can reveal genes that are co-expressed in different situations and may, therefore, work in conjunction, or interact to generate a certain phenotype (Quackenbush, 2003;Voy et al., 2006). Proteomics results may also be analyzed in this fashion (Shin et al., 2005). Novel computational approaches may prove to be powerful tools in assigning function to an unknown gene product (Wolfe et al., 2005;Wu et al., 2006b). Microarray (Hoffmann et al., 2001;Hoffmann et al., 2002;Fitzpatrick et al., 2004;Fitzpatrick et al., 2005;Chai et al., 2006;Dillon et al., 2006;Gobert et al., 2006;Moertel et al., 2006;Vermeire et al., 2006) and proteomic (Curwen et al., 2004;Cheng et al., 2005;Knudsen et al., 2005;van Balkom et al., 2005;Braschi et al., 2006;Liu et al., 2006;Van Hellemond et al., 2006) data have been produced for schistosomes, but not yet fully explored with powerful computational methods.

The identification of genes in a genome is still an arduous task for large and complex genomes. ESTs have also been very useful, providing experimental evidence for the elaboration of gene models. EST evidence has been incorporated into various algorithms for gene identification (Stanke et al., 2006). New ESTs may also reveal previously unknown genes. It has been shown for the human genome, for example, that new EST information is useful for identifying transcribed regions in the genome even with the availability of an already large cDNA dataset (de Souza et al., 2000). In the case of schistosomes, for which a finished complete genome sequence is not available, ESTs will also provide evidence for genes that may not have been sequenced.

As previously stated, some effort has been dedicated to producing full length cDNA clones from existing libraries for S. mansoni and S. japonicum. The interests in obtaining full length cDNA clones are several. Full length clones can provide extremely useful information for the production of accurate gene models and provide better annotation (Castelli et al., 2004). Distinct intron/exon boundaries, transcription start sites, antisense RNA are some examples of the richness of the genome that can be further explored (Miura et al., 2006). The existing ESTs and those made available in the future will greatly contribute towards the full understanding of many features of the genome. Full length clones have also been used in novel global approaches towards the identification of candidate antigens for the development of malaria vaccines using a DNA vaccine approach (Shibui et al., 2005). Towards the goal of providing the research community with a set of full length cDNA clones, Faria-Campos and colleagues (2006) identified within the S. mansoni cDNAs clones from the RGMG work, a set of clones that potentially contained the initiating methionine. A similar approach was undertaken for S. japonicum (Hu et al., 2003). The nature of the selection process however yielded mostly short sequences. An effort toward the production of a large number of long full length cDNAs would benefit from the construction of capped cDNA libraries (Suzuki et al., 1997). As well as providing full length cDNAs, a finishing approach to the transcriptome should target ESTs pairs from longer transcripts or not fully covered gene models (Sogayar et al., 2004). Full length cDNAs are the gold standard for defining a transcript and will enhance genome characterization and gene annotation.

ESTs clones have been an invaluable resource for the research community. Obtaining cDNA clones for a gene of interest will not always be trivial. Several efforts towards providing the research community with already cloned cDNAs has made the cloning effort much easier and cheaper. Some of the efforts for human cDNA clones are for example I.M.A.G.E. clones from ATCC or the German Resource Center for Genome Research (Lennon et al., 1996), the Japanese NITE and Riken Resource Centers that distribute full length human cDNA clones, TAIR for Arabidopsis full length cDNA clones (Rhee et al., 2003) and several commercial suppliers. For parasite material there is the MR4 that provides a variety of types of clones (Wu et al., 2001). With the idea of making resources readily available and inexpensive for the research community, recently the SR3 was created and will need community support for its success (Table 1).

The study of the transcriptome using microarrays has been carried out in Schistosoma and is discussed in depth in this issue. In addition, SAGE analysis have also been conducted to some extent in Schistosoma (Verjovski-Almeida et al., 2003), Williams, see this issue of EP). Recently it was shown by long-SAGE that in vitro exposure to nitric oxide modulates the expression of several genes, among them the upregulation of superoxide dismutase (Masserli et al. 2006). EST data was been used for the design of microarrays, both of the cDNA and oligonucleotide types (Hoffmann et al., 2001; Hoffmann et al., 2002; Fitzpatrick et al., 2004; Fitzpatrick et al., 2005; Chai et al., 2006; Dillon et al., 2006; Gobert et al., 2006; Moertel et al., 2006; Vermeire et al., 2006). The identification of SAGE tags will enhance the knowledge of transcription patterns as well as corroborate experimental evidence for transcribed genomic regions.

In addition to finding and annotating genes, ESTs can be mined for other types of information. Simões at al. (2007) have used EST and base call quality information made available by one of the published projects (Verjovski-Almeida et al., 2003) to identify SNPs. The authors identified 15,615 putative SNPs, of which 1,832 resulted in non-synonymous amino acid substitutions. Many of the known antigens and vaccine candidates contained amino acid polymorphisms. This kind of approach will provide valuable information for researchers interested in vaccine development and the identification of drug targets.

In addition to SNPs, differences in splicing can be observed by investigating EST data. Differentially spliced genes, linked to development or sex, for example, are increasingly being observed in schistosomes (Shoemaker et al., 1992;Hamdan and Ribeiro, 1998;De Mendonca et al., 2002;Ram et al., 2004;DeMarco et al., 2006;Bahia et al., 2006). Alternative transcription initiation and polyadenylation sites could also be explored with the use of EST data (Kan et al., 2001;Zavolan et al., 2003;Le, V et al., 2006;Seoighe et al., 2006;Tian et al., 2007).

In conclusion, ESTs have been produced in large scale for S. mansoni and S. japonicum and contributed significantly to the discovery of new genes, annotation of the genome and production of microarrays. There are, however, several other uses that have been less explored, such as the identification of polymorphisms, alternative splice sites and transcription initiation sites, among others.

Acknowledgements

GO receives financial support from NIH grants 5D43TW007012-03 and 5R03TW007358-02; CNPq/FIOCRUZ 400315/2006-8; FAPEMIG REDE-2829/05 and EDT 17001/01.

abbreviations

cDNA

complementary DNA

mRNA

messenger RNA

STS

sequence-tagged sites

ORESTES

Open Reading Frame Expressed Sequence Tags

RGMG

Minas Gerais Genome Network

CDD

Conserved Domain Database

KEGG

Kyoto Encyclopedia of Genes and Genomes

RNA

ribonucleic acid

DNA

deoxyribonucleic acid

SNP

single nucleotide polymorphism

MR4

Malaria Research and Reference Reagent Resource

SR3

Schistosome Related Reagent Repository

SAGE

Serial Analysis of Gene Expression

Footnotes

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