BACKGROUND
malaria, the most devastating parasitic human disease, is due to infections by intracellular protozoan parasites belonging to the plasmodium genus transmitted by anopheles mosquitoes  <cit> .
four plasmodium species are pathogenic to humans, with p. falciparum responsible for 90% of all reported cases of malaria, which causes  <dig>  to  <dig>  million deaths per annum  <cit> .
no efficient vaccine is currently available, despite ongoing efforts over the last decades  <cit> , and alternative drugs and targets are being investigated to fight the drug-resistant parasites that have emerged since the 1960s and are continuously spreading  <cit> .
deciphering of the p. falciparum genome in  <dig>  <cit>  revealed  <dig> – <dig>  genes, 60% of which were initially annotated as hypothetical, since no function could be ascribed to them based on sequence similarity.
the plasmodb database  gathers genomic and post-genomic data regarding p. falciparum and related species, and the last inventory  indicated  <dig>  coding genes,  <dig>   of which were still annotated as hypothetical or hypothetical conserved .
determining gene structures is particularly difficult in the case of p. falciparum, not only because most genes are devoid of characterized orthologs on which gene models could be based, but also because of the very high a-t content of the genome, i.e.
<dig> % on average  <cit> .
gene-coding predictions, based on several algorithms  have however allowed models to be proposed for p. falciparum genes  <cit> , but these gene models require experimental data to be validated.
we previously reported the construction of an est library using highly synchronized p. falciparum parasites of the fcb <dig> strain  to isolate genes selectively expressed during merozoite morphogenesis  <cit> .
the merozoite is the tiny  free form of the parasite that is able to recognize, bind and then invade erythrocytes  <cit> .
this very specialized cell displays a number of remarkable features, including a surface coat composed of highly polymorphic merozoite surface proteins , some of which were shown to be essential for parasite invasion and survival  <cit> .
the merozoite is also equipped with specialized organelles, such as micronemes, rhoptries and dense granules, devoted to invasion.
for example, erythrocyte binding antigens, stored in micronemes, are released prior to invasion and allow host cell recognition, while rhoptry proteins stored in rhoptries are release later and are believed to participate in parasitophorous vacuole formation  <cit> .
shortly after invasion, internalized merozoites differentiate into rings, thus losing their shape, and specific invasive organelles and rings eventually differentiate into haemoglobin-degrading trophozoites, about  <dig> h after invasion  <cit> .
nuclear division takes place at about  <dig> h post-invasion, yielding schizont stages progressively containing up to  <dig> nuclei  <cit> .
merozoites are individualized around each nucleus in the very last hours of the erythrocytic cycle, just prior to their release from infected erythrocytes  <cit> .
protein synthesis, trafficking and organelle assembly to form mature merozoites take place mainly during the final 10– <dig> h of asexual development  <cit> .
for these reasons, the fcb1-schizont-est library was constructed by subtracting transcripts from highly synchronized late stages  by transcripts from mixed remaining stages  by suppression subtractive hybridization  <cit> .
a pilot study of this library, limited to  <dig> clones, led to the identification of  <dig> genes over-expressed in schizont stages, including the well-known late-schizont/merozoite specific genes coding for eba- <dig>  clag/rhoph <dig>  coronin, msp <dig>  msp <dig>  msp <dig>  myosin a, sera and serp proteins, which was in good agreement with the proposed stage specificity of this library  <cit> .
seven inserts randomly selected from this fcb1-schizont-est library were used to probe cdnas amplified from rings, early trophozoites, late trophozoites, early schizonts and late schizonts and all seven genes were consistently detected in samples corresponding to early and late schizonts as compared to a control gene expressed in all stages  <cit> .
molecular analysis of the pfdyn <dig> gene identified during this pilot study  also confirmed its expression in late schizonts  <cit> .
in addition to p. falciparum genes over-expressed in late schizont stages, this pilot study led to the identification of two genes  whose est sequences indicated intron/exon boundaries that differed from those previously predicted and four genes  for which clear strain-dependent polymorphism was identified between fcb <dig> and 3d <dig>  <cit> .
in this context, it was thus of interest to conduct a large-scale analysis of this fcb1-est library.
the expectations were: 1) to obtain an exhaustive collection of genes selectively over-expressed in late schizont stages; 2) to validate or invalidate p. falciparum gene models by aligning all ests with the genome sequence, and 3) to identify genes indicating protein polymorphism between the fcb <dig> and 3d <dig> strains.
while writing this article, version  <dig>  of plasmodb was released, taking into account three new sets of p. falciparum ests, namely those analyzed by watanabe et al.
<cit>  and lu et al.
<cit>  as well as the ests sequenced in the present work.
this article presents and discusses the fcb1-schizont-est data and its contribution to our knowledge of p. falciparum gene structure and polymorphism.
RESULTS
fcb1-schizont-ests clustering on the p. falciparum genome
a total of  <dig>  clones randomly picked from the library were sequenced, yielding  <dig>   usable ests that were matched to the p. falciparum genome using the blast/est2genome method  <cit> .
three successive est2genome clustering analyses were performed.
the first clustering  was carried out using plasmodb version  <dig>   as a source of genomic data for p. falciparum.
plasmodb version  <dig>  provides, in addition to genomic sequences and gene annotations, various gene models built using algorithms such as phat, genefinder, glimmerm and hexamer  <cit> .
the second clustering  was achieved using plasmodb version  <dig> , released on june  <dig>  it yielded very similar results except that alternative gene models were not accessible in this new plasmodb version, which prevented an in-depth analysis of some of the data .
these two clustering analyses allowed the matching of  <dig>  ests  into  <dig> distinct clusters, with a cluster being a group of overlapping ests  matching a specific genomic locus .
the number of ests per cluster ranged from  <dig>  to thousands  .
the remaining unmatched  <dig>  fcb <dig> ests that could correspond to gene fragments that diverge markedly between fcb <dig> and 3d <dig> strains, to genes present in fcb <dig> but absent from 3d <dig>  or to genes encoded in the mitochondrial or apicoplast genomes, were clustered again on the p. falciparum genome , thus lowering the blast score to  <dig>  while  <dig> ests were rejected for being too small  or displaying very low complexity,  <dig> additional fcb1-ests could be significantly aligned with loci on the p. falciparum genome , revealing  <dig> additional clusters .
the remaining unmatched ests were then aligned with protein sequences of the uniprot database  <cit>  using the blast algorithm, revealing additional ests matching msp <dig> variants of k1-type , to ebl- <dig> , the mitochondrial genome  and the tufa gene  on the apicoplast genome  .
the results of the three est2genome clustering analyses on the 3d <dig> genome can be viewed at  and  respectively , and are discussed hereafter.
then results regarding fcb1-schizont-ests that did not match 3d <dig> genomic sequences but did match other p. falciparum sequences in the uniprot database are presented and discussed.
fcb1-schizont-est clusters matching the p. falciparum 3d <dig> genome
a systematic analysis of the  <dig> clusters  matching the 3d <dig> genome was performed to identify and study the corresponding loci.
these were distributed along the  <dig> p. falciparum chromosomes  and corresponded to  <dig> coding genes  and  <dig> non-protein-coding loci, namely:  <dig> distinct rrna loci  and  <dig> subtelomeric regions  .
this table lists the number of fcb1-schizont-ests  mapped on each of the  <dig> p. falciparum chromosomes , detailing those matching protein coding genes , ribosomal loci  or telomeric loci .
the number of protein coding genes covered by the fcb1-schizont-ests is indicated in ,  detailing whether these genes are functionnaly annotated genes , putative genes  or hypothetical genes .
columns  indicate the number of gene models that were confirmed , modified  or identified as displaying some strain dependent polymorphism between fcb <dig> and 3d <dig>  after analysis of 3d7-genomic versus fcb1-est alignments.
"total" corresponds to the total number of cases for the  <dig> chromosomes, "unique" accounts for the fact that many ribosomal-rna-matching ests matched both chromosomes  <dig> and  <dig> and that the  <dig> ests matching the telomeric end of chromosome  <dig> also matched the telomeric end of chromosome  <dig>
protein coding genes
among the  <dig> protein coding genes identified in this study,  <dig>  currently correspond to functionally annotated genes,  <dig>  to putative genes and  <dig>  to hypothetical genes .
functionally annotated genes in plasmodb are genes whose annotations are supported by experimental data  while putative genes are annotated based on significant similarities with functionally annotated genes in other species but lack experimental data in p. falciparum to support these annotations.
finally, hypothetical genes lack similarities with functionally annotated genes in the database and some of them rely solely on mathematical algorithms for identification.
as expected, this new collection of  <dig> protein coding genes includes all  <dig> protein coding genes initially identified from the fcb1-schizont-est library after the pilot study  <cit>  .
available transcriptomic profiles  <cit>  were recovered for each of these  <dig> protein coding genes and plotted on graphs to check whether the stage specificity of the fcb1-schizont-library was in agreement with the results of these other transcriptomic studies .
good concordance was observed, though the fcb1-schizont collection appeared to be slightly younger than expected, corresponding mostly to genes peaking at 37– <dig> h in the bozdech transcriptomic study and in early schizonts in the le roch transcriptomic study.
this shift may be due to differences in the time required to accomplish a full erythrocytic cycle in vitro , which is known to be strain dependent  <cit> .
it may also be due to slight variations in morphological appreciation of the different developmental stages.
nonetheless, most genes were confirmed by both transcriptomic studies, with few exceptions .
the expression of about 80% of genes of the fcb1-schizont collection reported to peak in gametocytes according to the le roch transcriptomic studies, appeared to peak at 37– <dig> h in the bozdech transcriptomic studies .
finally, conflicting expression profiles may correspond to genes differentially regulated in fcb <dig> as compared to 3d <dig>  as previously observed by llinas et al.
for a few genes of hb <dig> and dd <dig> strains  <cit> .
therefore, the fcb1-schizont-est collection appears to be of interest as a complementary dataset for studying merozoite morphogenesis.
the fcb1-schizont-est collection contained well known merozoite specific proteins such as merozoite capping protein- <dig> , merozoite surface proteins msp <dig> , msp <dig> , msp <dig> , msp <dig> , msp <dig> , msp <dig> , msp7-like  and msp9/abra , glurp , pfemp <dig> , eba  and eba- <dig> .
interestingly, we also identified four histone genes: pff0860c , pfc0920w , pff0865w , pff0510w  reported to be involved in nucleosome structure  and assembly  as well as chromatin structure  and assembly .
in addition to these four histone genes,  <dig> genes are also annotated in the gene ontology database by the term chromosome : pfe0450w , pf14_ <dig>  and pfl1930w .
as previously observed  <cit> , we also identified several cytoskeleton associated proteins: myosin a , myosin d , coronin , dynamin- <dig> , dynamin- <dig>  and formin- <dig> .
several rhoptry proteins were also present in this fcb1-schizont dataset:  <dig> of the  <dig> known clag/rhoph <dig> , rhoph <dig> , rhoph <dig> , rap <dig> , rap <dig> , rama  and also pf14_ <dig>  which is an ortholog of the tgron <dig> protein secreted from the rhoptry neck portion in toxoplasma gondii  <cit> .
we also characterized the expression of mal8p <dig> , reported to be an ortholog of ts <dig>  a t. gondii protein detected in rhoptry extracts  <cit> , which is also known to bind ama- <dig>  like tgron <dig> and tgron <dig>  <cit> .
to further characterize the cellular components, molecular functions and biological processes in which the genes of the fcb1-schizont dataset may be involved, go-terms were downloaded from genedb .
go annotations were, however, available only for  <dig> of the  <dig> genes .
we then used gostat software  <cit>  to identify go terms over-represented in the annotations of these  <dig> genes as compared to the complete p. falciparum genome, using a p-value threshold of  <dig>  .
over-represented cellular components were actin cytoskeleton , chromosome , myosin complex , nucleosome  and rhoptry .
similarly, over-represented molecular functions included actin-binding , calcium ion binding  and phospholipid binding .
over-represented biological processes corresponded to cell division , cytokinesis , dna packaging , nucleosome assembly  and entry into host cells .
these go terms are in good agreement with our current knowledge of biological and molecular mechanisms that occur during merozoite morphogenesis.
conversely, a single go term appeared to be under-represented in our study: the term defense response  .
in p. falciparum, this term has been attributed to var genes , which encode pfemp <dig> surface proteins responsible for antigenic variation  <cit> .
since these var genes are known to be transcribed early during parasite development  <cit> , the corresponding ests were not expected to be present in the fcb1-schizont library.
a statistical analysis was performed to identify go terms over- or under-represented in the annotated genes of this collection as compared to their distribution in the annotated genes of the whole genome.
gostat software  <cit>  was used for this analysis, using a p-value threshold of  <dig> .
only  <dig> genes have go annotations among the  <dig> genes of the collection .
this table reports all non-redundant over- or under-represented terms  in the three ontologies .
all terms were over-represented except for the term "defense response"  that was under-represented.
such a statistical analysis of go-terms is still, however, limited by the number of p. falciparum proteins not yet annotated in the gene ontology database.
for example, only two of the  <dig> rhoptry proteins that were identified in the fcb1-schizont list  were annotated by the corresponding go term : rap <dig>  and rap <dig> .
examination of fcb1-3d <dig> alignments
although most fcb1-schizont ests do not span the full length of the corresponding genes, a detailed systematic comparative analysis between fcb <dig> est sequences and corresponding 3d <dig> genomic sequences was performed to check the accuracy of the currently proposed gene models in plasmodb and also to identify protein polymorphism between these two strains.
validation and modification of intron/exon boundaries
in most cases , fcb1-schizont-ests matched protein coding genes in the middle of described exons, without providing any relevant information for gene model validation.
on several occasions , fcb1-schizont-ests matched 5' or 3' borders of genes or spanned exon-exon boundaries.
a gene by gene analysis of these cases allowed confirmation of one or several introns in  <dig> gene models  and to propose modifications in  <dig> gene models .
clustering of fcb1-schizont-ests in plasmodb , which displays alternative gene models, was particularly informative during this analysis and a few remarkable examples of gene model validations and gene model modifications are illustrated in additional files  <dig> and  <dig>  respectively.
the most spectacular gene model correction was found for pfe0240w, whose fcb1-schizont-est data provided evidence of four additional exons and an extended c-terminal end .
this table, derived from additional file  <dig>  lists the  <dig> p. falciparum genes whose gene models were confirmed by fcb1-schizont-ests.
the first  <dig> columns were downloaded from plasmodb version  <dig>  and correspond respectively to: gene accession numbers in plasmodb , their current description in plasmodb , the maximum expression time during the erythrocytic cycle according to the transcriptomic data of  <cit>   and the maximum expression stage according to the transcriptomic data of  <cit>  .
column  indicates the number of ests corresponding to each gene and isolated in this study.
column  details the genetic elements that were confirmed.
asterisks  in this last column refer to examples illustrated in additional file  <dig>  pf-irbc, p. falciparum-infected red blood cells.
this table, derived from additional file  <dig>  lists the  <dig> p. falciparum genes whose gene models were corrected based on fcb1-schizont-ests.
columns  to  are as described in the table  <dig> legend.
the last column  details each modification.
note that three genes in this list were both modified/confirmed : mal8p <dig> , pfe1490c and pfl0290w.
asterisks  in this last column refer to examples illustrated in additional file  <dig>  the model revision proposed for pfi0905w  will need to be confirmed by other experimental data since these  <dig> ests also matched p. falciparum telomerase rna .
pf-irbc, p. falciparum-infected red blood cells.
fcb <dig> versus 3d <dig> protein polymorphism for  <dig> protein coding genes
although the fcb1-schizont-est collection provides useful nucleotide sequence data to indicate single nucleotide polymorphism  specific to the fcb <dig> strain, these data  are not discussed here.
however, during the systematic comparative analysis of fcb1-schizont-ests versus 3d <dig> sequences, we identified  <dig> genes for which some protein polymorphism was observed between fcb <dig> and 3d <dig> encoded proteins .
as illustrated in additional file  <dig> where these  <dig> protein alignments are displayed, in most cases, this protein polymorphism corresponded to a variable number of repeated elements or to amino acid variations in these repeat elements.
malaria protein polymorphism has been suggested to be one of the main strategies of the parasite to evade the host immune mechanism, and antigens that are under natural immune pressure tend to have a higher level of polymorphism  <cit> .
these  <dig> genes therefore seem to be good candidates for encoding such antigens.
however, according to plasmodb, only  <dig> of these  <dig> genes encode proteins harbouring putative signal peptides consistent with their exposure on cell surface: pf10_ <dig> , pf10_ <dig> , pf10_ <dig>  and pfl1385c .
for the other proteins, these results raise questions about the reasons underlying such polymorphism or how these proteins may be exported either to the surface of the parasite or the surface of infected-red blood cells  <cit> .
this table, derived from additional file  <dig>  lists the  <dig> p. falciparum genes for which some protein polymorphism was identified between fcb <dig> and 3d <dig> strains.
columns  to  are as described in the table  <dig> legend.
the last column  details the various cases.
all protein sequence alignments are illustrated in additional file  <dig>  pf-irbc, p. falciparum-infected red blood cells.
3d <dig> non-protein coding loci covered by fcb1-schizont-ests
due to the methodology used to build the fcb1-schizont-est library, i.e.
isolation of total rna followed by selective conversion of polya+ rna into double strand cdna by smart-pcr  <cit> , a very large number of sequences  corresponded to rrna fragments, as previously observed  <cit> .
these non-protein coding, rrna-matching ests represent more that half of the sequenced ests, which is much higher than the 30%, estimated after the pilot study  <cit> .
this may be due to the fact that all picked clones were randomly sequenced in this current high throughput study, while in the pilot study est clones had been size selected prior to sequencing.
the rrna gene organisation in the p. falciparum genome is very unusual as compared to other eukaryotes.
instead of having hundreds of repetitions of rrna units in its genome, p. falciparum has seven complete and incomplete rrna  units on six of its  <dig> chromosomes and  <dig> copies of 5s rrna genes on chromosome  <dig>  <cit>  .
some of these rrna loci are known to be expressed in a developmentally regulated fashion, e.g.
the two a-type rrna units  that are mainly expressed in human infection stages and the two s-type rrnas  that are expressed in insect infection stages  <cit> .
the role played by other rrna loci present in the p. falciparum genome is not entirely clear  <cit> .
approximate position indicated in kb;  also contains atypical 18s and 28s;  18s and  <dig> s are of s-type and 28s is divergent ;  18s is missing from these units;   <dig> tandem repeats of 5s.
data compiled from  <cit>  and plasmodb.
most of these ests clustered with homologous loci of both chromosomes  <dig> and  <dig>
most non-protein coding fcb1-schizont-ests correspond to the two a-type rrna units on chromosomes  <dig> and 7
in this study, a very large majority of the rrna-matching ests clustered on both a-type rrna units, located on chromosomes  <dig> and  <dig>  and most matched both homologous a-type regions on chromosomes  <dig> and  <dig> .
however, a limited number of fcb1-schizont ests clustered on two atypical rrna units, respectively located on chromosome  <dig>  and chromosome  <dig>  .
none of the fcb1-schizont est clusters matched any of the two s-type rrna loci, which was an interesting negative control of our data regarding the developmental stage of the studied parasite population.
fcb1-schizont-est analysis provides evidence of the actual expression of two atypical rrna loci located on chromosomes  <dig> and 8
a total of  <dig> ests matched chromosome  <dig> at the level of mal1_28s, which is annotated as a-type rrna in plasmodb but described as being atypical  by gardner et al.
<cit> .
the four longest ests matching this locus did not match elsewhere in the p. falciparum genome .
these results indicated that the mal1_28s gene was indeed expressed in our experimental population of highly synchronized fcb <dig> parasites.
owing to the small number of truly specific ests corresponding to mal1_28s, the expression level of this gene in the parasite population is obviously much lower than the level rrna gene expression from typical chr <dig> and chr <dig> a-type loci .
while the physiological conditions allowing mal1_28s rrna expression remain to be determined, our experimental data clearly argue in favour of the actual expression of mal1_28s rna which, to the best of our knowledge, has not yet been documented.
the two ests matching chromosome  <dig> were mapped on a non-annotated area located between mal8a_ <dig> s and pf08_temp <dig> , with the latter also being annotated as rrna encoding in plasmodb.
both are non-protein coding ests and do not match elsewhere in the genome.
this strongly suggests that they are indeed encoded by this new locus of unknown function in which, interestingly, several snps are indicated .
the fcb1-schizont-est data therefore supports the transcription of a non-protein coding rna at this new locus of chromosome  <dig>
non-protein coding fcb1-schizont-ests matching sub-telomeric regions
four clusters  and one atypical est  matched sub-telomeric regions in the p. falciparum genome, very close to the chromosome ends .
notably, the  <dig> ests corresponding to cluster_ <dig>  also corresponded to cluster_ <dig> .
an in-depth analysis of these ests, using the blast algorithm optimized for highly similar sequences , indicated that they matched virtually all chromosome ends , in telomere-associated repeat element  regions  <cit> .
to refine this analysis, these ests were aligned to the nucleotide sequence corresponding to a particularly well annotated telomeric end on chromosome  <dig> .
these fcb1-schizont-ests were then found to match the same region, likely corresponding to the tare <dig> or tare <dig> region, between telomere and r-cg <dig> segments  <cit>  .
non-protein coding fcb1-schizont ests matching telomerase rna
an independent analysis of the fcb1-schizont-est library aimed at unravelling the genetic structure of the p. falciparum telomerase rna  revealed  <dig> fcb <dig> ests that mapped to the 5' end of the recently described telomerase rna gene in a template binding region  <cit>  .
it was then noted that four of these fcb1-schizont-ests also corresponded to the cluster 500_ <dig> cluster, matching the hypothetical gene pfi0905w .
the fact that these were non-protein coding ests indicates that they corresponded to the telomerase rna gene rather than to cdna fragments of the pfi0905w gene.
in-depth analysis of fcb1-schizont-ests not matching the 3d <dig> genome reveals similarities to other variants of the msp <dig> gene and to a paralog of eba- <dig>  and eba- <dig>
the msp <dig> gene codes for one of the most polymorphic proteins in p. falciparum.
the analysis of fcb1-schizont-ests using est2genome allowed clustering of  <dig> ests with the n-terminal end of the msp <dig> gene in plasmodb .
however, additional fcb1-schizont-ests matching other msp <dig> variants were discovered by comparing unmatched ests to the uniprot database using the blast algorithm .
<dig> of these fcb1-schizont-ests matched five partly overlapping regions of the k1-type msp <dig>  and  <dig> additional ests matched the c-terminal end of msp <dig> in an iranian isolate  reported to be of k1-type  <cit> .
therefore, a total of  <dig> fcb1-schizont-ests spanned about 50% of the fcb1-msp <dig> gene .
interestingly, in the pilot study of this library, we found that the msp <dig> variant in fcb <dig> was also of k1-type  <cit> .
comparison of fcb1-schizont-ests with the uniprot database also revealed  <dig> ests matching .
these ests partly spanned the c-terminal end of the protein  with  <dig> to 100% identity.
is annotated as ebl- <dig> from the strain 3d <dig> but, in fact, two p. falciparum proteins appear as paralogs of : eba- <dig>  and eba- <dig> .
we thus believe that these ests correspond to a fcb <dig> gene belonging to the ebl family, known to be involved in invasion  <cit> .
a few fcb1-schizont ests mapped on organellar genomes
fcb1-schizont-ests matching the mitochondrial genome  were mapped at the level of the three described genes: putative cytochrome oxidase iii , putative cytochrome oxidase i  and putative cytochrome b .
the single est matching the apicoplast genome , corresponded to the tufa gene .
this latter result, based on a single est in the whole library corresponding to transcription of the apicoplast genome, which is known to occur just prior to the transcription of merozoite specific genes  <cit> , provides an interesting control of the proposed stage specificity of this fcb1-est library.
CONCLUSIONS
extensive analysis of the fcb1-schizont-est library, which was constructed to be enriched in transcripts over-expressed in the last hours of the parasite development in erythrocytes, provided valuable information regarding genes expressed in the late stages of the parasite development.
concerning the stage specificity, both the pilot study including experimental controls  <cit>  and the current work provide support that the genes spanned by these ests were actually expressed late during the parasite development, even though in many cases this conclusion was based on comparative analysis with other transcriptomic studies  <cit> .
the fact that a single est  matched the apicoplast genome, further indicates that all genes corresponding to this fcb1-schizont are expressed later, at least in the fcb <dig> strain.
the transcriptomic profiles determined by  <cit>  and  <cit>  for the  <dig> genes of this fcb1-schizont collection indicate, however, that these genes may be expressed slightly earlier than initially expected: 37– <dig> h  rather than 42– <dig> h .
this observation is in line with the fact that we identified  <dig> genes coding for rhoptry proteins and only two coding for micronemal proteins  and eba- <dig> ).
indeed, it has been shown that rhoptry proteins are expressed prior to micronemal proteins during merozoite morphogenesis  <cit> .
for all of these reasons, we believe that this fcb1-schizont collection is a truly appropriate dataset to focus on early steps of merozoite morphogenesis after apicoplast transcription.
the  <dig> proteins currently annotated as hypothetical or hypothetical conserved are therefore favourable candidates involved in this morphogenesis.
one shortcoming is, however, the fact that in several cases no additional experimental data is currently available to confirm the expression of these candidates.
additional data and molecular analyses are thus required to confirm the involvement of these candidates in merozoite morphogenesis as, for example, was done in the case of pfdyn <dig>  <cit> .
but, interestingly, the total number of genes identified in the fcb1-schizont dataset  is consistent with the number of genes proposed by  <cit>  as corresponding to merozoite specific genes.
an interesting outcome of this study is that intron/exon boundaries were validated in  <dig> genes, revised gene models were proposed for  <dig> genes and fcb <dig> versus 3d <dig> protein polymorphisms were identified for  <dig> genes.
although this information was mostly collected at the gene fragment rather than full-length gene level, it represents a valuable contribution, along with similar data by others  <cit> , to gain greater insight into p. falciparum gene structure and polymorphism.
the most unexpected results concerned ests corresponding to non-protein coding rnas present in this fcb1-schizont collection.
although these ests are limited in number, especially as compared to the very high number of ests matching the well known a-type rrna loci, they provided indications on the actual expression of a 28s rrna gene located on chromosome  <dig> and on the expression of a non-protein coding transcript between mal8a_ <dig> s and pf08_tmp <dig> genes.
ests providing this information do not cluster elsewhere in the p. falciparum genome, so they are strictly specific to these two mainly uncharacterized loci.
the actual physiological conditions under which these loci are expressed are, however, totally unknown.
a second small series of  <dig> non-protein coding ests provided information regarding the actual transcription of tare regions, likely corresponding to tare- <dig> or tare- <dig>  to the best of our knowledge, this has not been shown before.
finally,  <dig> ests matched the 5' end of p. falciparum telomerase rna.
rather than indicating that this rna is expressed, which has recently been reported  <cit> , this result raises the question as to the structure and expression of pfi0905w, located at the very same genomic locus.
the methodology used to built the fcb1-schizont-est library, i.e.
suppressive subtractive hybridization  <cit> , which was selected to ensure stage specificity, has some technical disadvantages.
the first is the very high redundancy in the ests produced, which is directly due to the pcr-amplification step.
the second is the fact that these ests are necessarily digested by an endonuclease to be subtracted  and are therefore of small or very small size.
consequently, most genes identified by this strategy were only partly spanned by fcb1-schizont-ests.
these shortcomings did not affect the analyses in the pilot study  <cit>  and were therefore not expected to limit the present work.
nevertheless, the high throughput analysis of the fcb1-schizont-est library affected the full-length coverage of gene loci by est clusters and was biased by est multicopies.
for example, in the case of msp <dig> and based on the results of the pilot study, we were expecting to yield enough ests to span the entire gene, but, instead, we obtained a large number of highly redundant ests at fragmented locations.
despite this low full-length gene coverage, the in-depth analysis of this fcb1-schizont-est collection should help, together with data by others  <cit> , to gain further insight into p. falciparum gene structure and polymorphism.
in the present work, which involved a high-throughput analysis of this fcb1-schizont-est library, a few unexpected gene expression features were also discovered, such as the actual transcription of atypical rrna loci and subtelomeric regions.

 malaria, the most devastating parasitic human disease, is due to infections by intracellular protozoan parasites belonging to the plasmodium genus transmitted by anopheles mosquitoes. four plasmodium species are pathogenic to humans, with p. falciparum responsible for 90% of all reported cases of malaria, which causes to million deaths per annum. no efficient vaccine is currently available, despite ongoing efforts over the last decades, and alternative drugs and targets are being investigated to fight the drug-resistant parasites that have emerged since the 1960s and are continuously spreading. deciphering of the p. falciparum genome in revealed – genes, 60% of which were initially annotated as hypothetical, since no function could be ascribed to them based on sequence similarity. the plasmodb database gathers genomic and post-genomic data regarding p. falciparum and related species, and the last inventory indicated coding genes, of which were still annotated as hypothetical or hypothetical conserved. determining gene structures is particularly difficult in the case of p. falciparum, not only because most genes are devoid of characterized orthologs on which gene models could be based, but also because of the very high a-t content of the genome, i.e. % on average. gene-coding predictions, based on several algorithms have however allowed models to be proposed for p. falciparum genes, but these gene models require experimental data to be validated. we previously reported the construction of an est library using highly synchronized p. falciparum parasites of the fcb strain to isolate genes selectively expressed during merozoite morphogenesis. the merozoite is the tiny free form of the parasite that is able to recognize, bind and then invade erythrocytes. this very specialized cell displays a number of remarkable features, including a surface coat composed of highly polymorphic merozoite surface proteins, some of which were shown to be essential for parasite invasion and survival. the merozoite is also equipped with specialized organelles, such as micronemes, rhoptries and dense granules, devoted to invasion. for example, erythrocyte binding antigens, stored in micronemes, are released prior to invasion and allow host cell recognition, while rhoptry proteins stored in rhoptries are release later and are believed to participate in parasitophorous vacuole formation. shortly after invasion, internalized merozoites differentiate into rings, thus losing their shape, and specific invasive organelles and rings eventually differentiate into haemoglobin-degrading trophozoites, about h after invasion. nuclear division takes place at about h post-invasion, yielding schizont stages progressively containing up to nuclei. merozoites are individualized around each nucleus in the very last hours of the erythrocytic cycle, just prior to their release from infected erythrocytes. protein synthesis, trafficking and organelle assembly to form mature merozoites take place mainly during the final 10– h of asexual development. for these reasons, the fcb1-schizont-est library was constructed by subtracting transcripts from highly synchronized late stages by transcripts from mixed remaining stages by suppression subtractive hybridization. a pilot study of this library, limited to clones, led to the identification of genes over-expressed in schizont stages, including the well-known late-schizont/merozoite specific genes coding for eba- clag/rhoph coronin, msp msp msp myosin a, sera and serp proteins, which was in good agreement with the proposed stage specificity of this library. seven inserts randomly selected from this fcb1-schizont-est library were used to probe cdnas amplified from rings, early trophozoites, late trophozoites, early schizonts and late schizonts and all seven genes were consistently detected in samples corresponding to early and late schizonts as compared to a control gene expressed in all stages. molecular analysis of the pfdyn gene identified during this pilot study also confirmed its expression in late schizonts. in addition to p. falciparum genes over-expressed in late schizont stages, this pilot study led to the identification of two genes whose est sequences indicated intron/exon boundaries that differed from those previously predicted and four genes for which clear strain-dependent polymorphism was identified between fcb and 3d. in this context, it was thus of interest to conduct a large-scale analysis of this fcb1-est library. the expectations were: 1) to obtain an exhaustive collection of genes selectively over-expressed in late schizont stages; 2) to validate or invalidate p. falciparum gene models by aligning all ests with the genome sequence, and 3) to identify genes indicating protein polymorphism between the fcb and 3d strains. while writing this article, version of plasmodb was released, taking into account three new sets of p. falciparum ests, namely those analyzed by watanabe et al. and lu et al. as well as the ests sequenced in the present work. this article presents and discusses the fcb1-schizont-est data and its contribution to our knowledge of p. falciparum gene structure and polymorphism.  fcb1-schizont-ests clustering on the p. falciparum genome a total of clones randomly picked from the library were sequenced, yielding usable ests that were matched to the p. falciparum genome using the blast/est2genome method. three successive est2genome clustering analyses were performed. the first clustering was carried out using plasmodb version as a source of genomic data for p. falciparum. plasmodb version provides, in addition to genomic sequences and gene annotations, various gene models built using algorithms such as phat, genefinder, glimmerm and hexamer. the second clustering was achieved using plasmodb version, released on june it yielded very similar results except that alternative gene models were not accessible in this new plasmodb version, which prevented an in-depth analysis of some of the data. these two clustering analyses allowed the matching of ests into distinct clusters, with a cluster being a group of overlapping ests matching a specific genomic locus. the number of ests per cluster ranged from to thousands. the remaining unmatched fcb ests that could correspond to gene fragments that diverge markedly between fcb and 3d strains, to genes present in fcb but absent from 3d or to genes encoded in the mitochondrial or apicoplast genomes, were clustered again on the p. falciparum genome, thus lowering the blast score to while ests were rejected for being too small or displaying very low complexity, additional fcb1-ests could be significantly aligned with loci on the p. falciparum genome, revealing additional clusters. the remaining unmatched ests were then aligned with protein sequences of the uniprot database using the blast algorithm, revealing additional ests matching msp variants of k1-type, to ebl-, the mitochondrial genome and the tufa gene on the apicoplast genome. the results of the three est2genome clustering analyses on the 3d genome can be viewed at and respectively, and are discussed hereafter. then results regarding fcb1-schizont-ests that did not match 3d genomic sequences but did match other p. falciparum sequences in the uniprot database are presented and discussed. fcb1-schizont-est clusters matching the p. falciparum 3d genome a systematic analysis of the clusters matching the 3d genome was performed to identify and study the corresponding loci. these were distributed along the p. falciparum chromosomes and corresponded to coding genes and non-protein-coding loci, namely: distinct rrna loci and subtelomeric regions. this table lists the number of fcb1-schizont-ests mapped on each of the p. falciparum chromosomes, detailing those matching protein coding genes, ribosomal loci or telomeric loci. the number of protein coding genes covered by the fcb1-schizont-ests is indicated in, detailing whether these genes are functionnaly annotated genes, putative genes or hypothetical genes. columns indicate the number of gene models that were confirmed, modified or identified as displaying some strain dependent polymorphism between fcb and 3d after analysis of 3d7-genomic versus fcb1-est alignments. "total" corresponds to the total number of cases for the chromosomes, "unique" accounts for the fact that many ribosomal-rna-matching ests matched both chromosomes and and that the ests matching the telomeric end of chromosome also matched the telomeric end of chromosome protein coding genes among the protein coding genes identified in this study, currently correspond to functionally annotated genes, to putative genes and to hypothetical genes. functionally annotated genes in plasmodb are genes whose annotations are supported by experimental data while putative genes are annotated based on significant similarities with functionally annotated genes in other species but lack experimental data in p. falciparum to support these annotations. finally, hypothetical genes lack similarities with functionally annotated genes in the database and some of them rely solely on mathematical algorithms for identification. as expected, this new collection of protein coding genes includes all protein coding genes initially identified from the fcb1-schizont-est library after the pilot study. available transcriptomic profiles were recovered for each of these protein coding genes and plotted on graphs to check whether the stage specificity of the fcb1-schizont-library was in agreement with the results of these other transcriptomic studies. good concordance was observed, though the fcb1-schizont collection appeared to be slightly younger than expected, corresponding mostly to genes peaking at 37– h in the bozdech transcriptomic study and in early schizonts in the le roch transcriptomic study. this shift may be due to differences in the time required to accomplish a full erythrocytic cycle in vitro, which is known to be strain dependent. it may also be due to slight variations in morphological appreciation of the different developmental stages. nonetheless, most genes were confirmed by both transcriptomic studies, with few exceptions. the expression of about 80% of genes of the fcb1-schizont collection reported to peak in gametocytes according to the le roch transcriptomic studies, appeared to peak at 37– h in the bozdech transcriptomic studies. finally, conflicting expression profiles may correspond to genes differentially regulated in fcb as compared to 3d as previously observed by llinas et al. for a few genes of hb and dd strains. therefore, the fcb1-schizont-est collection appears to be of interest as a complementary dataset for studying merozoite morphogenesis. the fcb1-schizont-est collection contained well known merozoite specific proteins such as merozoite capping protein-, merozoite surface proteins msp, msp, msp, msp, msp, msp, msp7-like and msp9/abra, glurp, pfemp, eba and eba-. interestingly, we also identified four histone genes: pff0860c, pfc0920w, pff0865w, pff0510w reported to be involved in nucleosome structure and assembly as well as chromatin structure and assembly. in addition to these four histone genes, genes are also annotated in the gene ontology database by the term chromosome: pfe0450w, pf14_ and pfl1930w. as previously observed, we also identified several cytoskeleton associated proteins: myosin a, myosin d, coronin, dynamin-, dynamin- and formin-. several rhoptry proteins were also present in this fcb1-schizont dataset: of the known clag/rhoph, rhoph, rhoph, rap, rap, rama and also pf14_ which is an ortholog of the tgron protein secreted from the rhoptry neck portion in toxoplasma gondii. we also characterized the expression of mal8p, reported to be an ortholog of ts a t. gondii protein detected in rhoptry extracts, which is also known to bind ama- like tgron and tgron. to further characterize the cellular components, molecular functions and biological processes in which the genes of the fcb1-schizont dataset may be involved, go-terms were downloaded from genedb. go annotations were, however, available only for of the genes. we then used gostat software to identify go terms over-represented in the annotations of these genes as compared to the complete p. falciparum genome, using a p-value threshold of. over-represented cellular components were actin cytoskeleton, chromosome, myosin complex, nucleosome and rhoptry. similarly, over-represented molecular functions included actin-binding, calcium ion binding and phospholipid binding. over-represented biological processes corresponded to cell division, cytokinesis, dna packaging, nucleosome assembly and entry into host cells. these go terms are in good agreement with our current knowledge of biological and molecular mechanisms that occur during merozoite morphogenesis. conversely, a single go term appeared to be under-represented in our study: the term defense response. in p. falciparum, this term has been attributed to var genes, which encode pfemp surface proteins responsible for antigenic variation. since these var genes are known to be transcribed early during parasite development, the corresponding ests were not expected to be present in the fcb1-schizont library. a statistical analysis was performed to identify go terms over- or under-represented in the annotated genes of this collection as compared to their distribution in the annotated genes of the whole genome. gostat software was used for this analysis, using a p-value threshold of. only genes have go annotations among the genes of the collection. this table reports all non-redundant over- or under-represented terms in the three ontologies. all terms were over-represented except for the term "defense response" that was under-represented. such a statistical analysis of go-terms is still, however, limited by the number of p. falciparum proteins not yet annotated in the gene ontology database. for example, only two of the rhoptry proteins that were identified in the fcb1-schizont list were annotated by the corresponding go term: rap and rap. examination of fcb1-3d alignments although most fcb1-schizont ests do not span the full length of the corresponding genes, a detailed systematic comparative analysis between fcb est sequences and corresponding 3d genomic sequences was performed to check the accuracy of the currently proposed gene models in plasmodb and also to identify protein polymorphism between these two strains. validation and modification of intron/exon boundaries in most cases, fcb1-schizont-ests matched protein coding genes in the middle of described exons, without providing any relevant information for gene model validation. on several occasions, fcb1-schizont-ests matched 5' or 3' borders of genes or spanned exon-exon boundaries. a gene by gene analysis of these cases allowed confirmation of one or several introns in gene models and to propose modifications in gene models. clustering of fcb1-schizont-ests in plasmodb, which displays alternative gene models, was particularly informative during this analysis and a few remarkable examples of gene model validations and gene model modifications are illustrated in additional files and respectively. the most spectacular gene model correction was found for pfe0240w, whose fcb1-schizont-est data provided evidence of four additional exons and an extended c-terminal end. this table, derived from additional file lists the p. falciparum genes whose gene models were confirmed by fcb1-schizont-ests. the first columns were downloaded from plasmodb version and correspond respectively to: gene accession numbers in plasmodb, their current description in plasmodb, the maximum expression time during the erythrocytic cycle according to the transcriptomic data of and the maximum expression stage according to the transcriptomic data of. column indicates the number of ests corresponding to each gene and isolated in this study. column details the genetic elements that were confirmed. asterisks in this last column refer to examples illustrated in additional file pf-irbc, p. falciparum-infected red blood cells. this table, derived from additional file lists the p. falciparum genes whose gene models were corrected based on fcb1-schizont-ests. columns to are as described in the table legend. the last column details each modification. note that three genes in this list were both modified/confirmed: mal8p, pfe1490c and pfl0290w. asterisks in this last column refer to examples illustrated in additional file the model revision proposed for pfi0905w will need to be confirmed by other experimental data since these ests also matched p. falciparum telomerase rna. pf-irbc, p. falciparum-infected red blood cells. fcb versus 3d protein polymorphism for protein coding genes although the fcb1-schizont-est collection provides useful nucleotide sequence data to indicate single nucleotide polymorphism specific to the fcb strain, these data are not discussed here. however, during the systematic comparative analysis of fcb1-schizont-ests versus 3d sequences, we identified genes for which some protein polymorphism was observed between fcb and 3d encoded proteins. as illustrated in additional file where these protein alignments are displayed, in most cases, this protein polymorphism corresponded to a variable number of repeated elements or to amino acid variations in these repeat elements. malaria protein polymorphism has been suggested to be one of the main strategies of the parasite to evade the host immune mechanism, and antigens that are under natural immune pressure tend to have a higher level of polymorphism. these genes therefore seem to be good candidates for encoding such antigens. however, according to plasmodb, only of these genes encode proteins harbouring putative signal peptides consistent with their exposure on cell surface: pf10_, pf10_, pf10_ and pfl1385c. for the other proteins, these results raise questions about the reasons underlying such polymorphism or how these proteins may be exported either to the surface of the parasite or the surface of infected-red blood cells. this table, derived from additional file lists the p. falciparum genes for which some protein polymorphism was identified between fcb and 3d strains. columns to are as described in the table legend. the last column details the various cases. all protein sequence alignments are illustrated in additional file pf-irbc, p. falciparum-infected red blood cells. 3d non-protein coding loci covered by fcb1-schizont-ests due to the methodology used to build the fcb1-schizont-est library, i.e. isolation of total rna followed by selective conversion of polya+ rna into double strand cdna by smart-pcr, a very large number of sequences corresponded to rrna fragments, as previously observed. these non-protein coding, rrna-matching ests represent more that half of the sequenced ests, which is much higher than the 30%, estimated after the pilot study. this may be due to the fact that all picked clones were randomly sequenced in this current high throughput study, while in the pilot study est clones had been size selected prior to sequencing. the rrna gene organisation in the p. falciparum genome is very unusual as compared to other eukaryotes. instead of having hundreds of repetitions of rrna units in its genome, p. falciparum has seven complete and incomplete rrna units on six of its chromosomes and copies of 5s rrna genes on chromosome. some of these rrna loci are known to be expressed in a developmentally regulated fashion, e.g. the two a-type rrna units that are mainly expressed in human infection stages and the two s-type rrnas that are expressed in insect infection stages. the role played by other rrna loci present in the p. falciparum genome is not entirely clear. approximate position indicated in kb; also contains atypical 18s and 28s; 18s and s are of s-type and 28s is divergent; 18s is missing from these units; tandem repeats of 5s. data compiled from and plasmodb. most of these ests clustered with homologous loci of both chromosomes and most non-protein coding fcb1-schizont-ests correspond to the two a-type rrna units on chromosomes and 7 in this study, a very large majority of the rrna-matching ests clustered on both a-type rrna units, located on chromosomes and and most matched both homologous a-type regions on chromosomes and. however, a limited number of fcb1-schizont ests clustered on two atypical rrna units, respectively located on chromosome and chromosome. none of the fcb1-schizont est clusters matched any of the two s-type rrna loci, which was an interesting negative control of our data regarding the developmental stage of the studied parasite population. fcb1-schizont-est analysis provides evidence of the actual expression of two atypical rrna loci located on chromosomes and 8 a total of ests matched chromosome at the level of mal1_28s, which is annotated as a-type rrna in plasmodb but described as being atypical by gardner et al.. the four longest ests matching this locus did not match elsewhere in the p. falciparum genome. these results indicated that the mal1_28s gene was indeed expressed in our experimental population of highly synchronized fcb parasites. owing to the small number of truly specific ests corresponding to mal1_28s, the expression level of this gene in the parasite population is obviously much lower than the level rrna gene expression from typical chr and chr a-type loci. while the physiological conditions allowing mal1_28s rrna expression remain to be determined, our experimental data clearly argue in favour of the actual expression of mal1_28s rna which, to the best of our knowledge, has not yet been documented. the two ests matching chromosome were mapped on a non-annotated area located between mal8a_ s and pf08_temp, with the latter also being annotated as rrna encoding in plasmodb. both are non-protein coding ests and do not match elsewhere in the genome. this strongly suggests that they are indeed encoded by this new locus of unknown function in which, interestingly, several snps are indicated. the fcb1-schizont-est data therefore supports the transcription of a non-protein coding rna at this new locus of chromosome non-protein coding fcb1-schizont-ests matching sub-telomeric regions four clusters and one atypical est matched sub-telomeric regions in the p. falciparum genome, very close to the chromosome ends. notably, the ests corresponding to cluster_ also corresponded to cluster_. an in-depth analysis of these ests, using the blast algorithm optimized for highly similar sequences, indicated that they matched virtually all chromosome ends, in telomere-associated repeat element regions. to refine this analysis, these ests were aligned to the nucleotide sequence corresponding to a particularly well annotated telomeric end on chromosome. these fcb1-schizont-ests were then found to match the same region, likely corresponding to the tare or tare region, between telomere and r-cg segments. non-protein coding fcb1-schizont ests matching telomerase rna an independent analysis of the fcb1-schizont-est library aimed at unravelling the genetic structure of the p. falciparum telomerase rna revealed fcb ests that mapped to the 5' end of the recently described telomerase rna gene in a template binding region. it was then noted that four of these fcb1-schizont-ests also corresponded to the cluster 500_ cluster, matching the hypothetical gene pfi0905w. the fact that these were non-protein coding ests indicates that they corresponded to the telomerase rna gene rather than to cdna fragments of the pfi0905w gene. in-depth analysis of fcb1-schizont-ests not matching the 3d genome reveals similarities to other variants of the msp gene and to a paralog of eba- and eba- the msp gene codes for one of the most polymorphic proteins in p. falciparum. the analysis of fcb1-schizont-ests using est2genome allowed clustering of ests with the n-terminal end of the msp gene in plasmodb. however, additional fcb1-schizont-ests matching other msp variants were discovered by comparing unmatched ests to the uniprot database using the blast algorithm. of these fcb1-schizont-ests matched five partly overlapping regions of the k1-type msp and additional ests matched the c-terminal end of msp in an iranian isolate reported to be of k1-type. therefore, a total of fcb1-schizont-ests spanned about 50% of the fcb1-msp gene. interestingly, in the pilot study of this library, we found that the msp variant in fcb was also of k1-type. comparison of fcb1-schizont-ests with the uniprot database also revealed ests matching. these ests partly spanned the c-terminal end of the protein with to 100% identity. is annotated as ebl- from the strain 3d but, in fact, two p. falciparum proteins appear as paralogs of: eba- and eba-. we thus believe that these ests correspond to a fcb gene belonging to the ebl family, known to be involved in invasion. a few fcb1-schizont ests mapped on organellar genomes fcb1-schizont-ests matching the mitochondrial genome were mapped at the level of the three described genes: putative cytochrome oxidase iii, putative cytochrome oxidase i and putative cytochrome b. the single est matching the apicoplast genome, corresponded to the tufa gene. this latter result, based on a single est in the whole library corresponding to transcription of the apicoplast genome, which is known to occur just prior to the transcription of merozoite specific genes, provides an interesting control of the proposed stage specificity of this fcb1-est library.  extensive analysis of the fcb1-schizont-est library, which was constructed to be enriched in transcripts over-expressed in the last hours of the parasite development in erythrocytes, provided valuable information regarding genes expressed in the late stages of the parasite development. concerning the stage specificity, both the pilot study including experimental controls and the current work provide support that the genes spanned by these ests were actually expressed late during the parasite development, even though in many cases this conclusion was based on comparative analysis with other transcriptomic studies. the fact that a single est matched the apicoplast genome, further indicates that all genes corresponding to this fcb1-schizont are expressed later, at least in the fcb strain. the transcriptomic profiles determined by and for the genes of this fcb1-schizont collection indicate, however, that these genes may be expressed slightly earlier than initially expected: 37– h rather than 42– h. this observation is in line with the fact that we identified genes coding for rhoptry proteins and only two coding for micronemal proteins and eba- ). indeed, it has been shown that rhoptry proteins are expressed prior to micronemal proteins during merozoite morphogenesis. for all of these reasons, we believe that this fcb1-schizont collection is a truly appropriate dataset to focus on early steps of merozoite morphogenesis after apicoplast transcription. the proteins currently annotated as hypothetical or hypothetical conserved are therefore favourable candidates involved in this morphogenesis. one shortcoming is, however, the fact that in several cases no additional experimental data is currently available to confirm the expression of these candidates. additional data and molecular analyses are thus required to confirm the involvement of these candidates in merozoite morphogenesis as, for example, was done in the case of pfdyn. but, interestingly, the total number of genes identified in the fcb1-schizont dataset is consistent with the number of genes proposed by as corresponding to merozoite specific genes. an interesting outcome of this study is that intron/exon boundaries were validated in genes, revised gene models were proposed for genes and fcb versus 3d protein polymorphisms were identified for genes. although this information was mostly collected at the gene fragment rather than full-length gene level, it represents a valuable contribution, along with similar data by others, to gain greater insight into p. falciparum gene structure and polymorphism. the most unexpected results concerned ests corresponding to non-protein coding rnas present in this fcb1-schizont collection. although these ests are limited in number, especially as compared to the very high number of ests matching the well known a-type rrna loci, they provided indications on the actual expression of a 28s rrna gene located on chromosome and on the expression of a non-protein coding transcript between mal8a_ s and pf08_tmp genes. ests providing this information do not cluster elsewhere in the p. falciparum genome, so they are strictly specific to these two mainly uncharacterized loci. the actual physiological conditions under which these loci are expressed are, however, totally unknown. a second small series of non-protein coding ests provided information regarding the actual transcription of tare regions, likely corresponding to tare- or tare- to the best of our knowledge, this has not been shown before. finally, ests matched the 5' end of p. falciparum telomerase rna. rather than indicating that this rna is expressed, which has recently been reported, this result raises the question as to the structure and expression of pfi0905w, located at the very same genomic locus. the methodology used to built the fcb1-schizont-est library, i.e. suppressive subtractive hybridization, which was selected to ensure stage specificity, has some technical disadvantages. the first is the very high redundancy in the ests produced, which is directly due to the pcr-amplification step. the second is the fact that these ests are necessarily digested by an endonuclease to be subtracted and are therefore of small or very small size. consequently, most genes identified by this strategy were only partly spanned by fcb1-schizont-ests. these shortcomings did not affect the analyses in the pilot study and were therefore not expected to limit the present work. nevertheless, the high throughput analysis of the fcb1-schizont-est library affected the full-length coverage of gene loci by est clusters and was biased by est multicopies. for example, in the case of msp and based on the results of the pilot study, we were expecting to yield enough ests to span the entire gene, but, instead, we obtained a large number of highly redundant ests at fragmented locations. despite this low full-length gene coverage, the in-depth analysis of this fcb1-schizont-est collection should help, together with data by others, to gain further insight into p. falciparum gene structure and polymorphism. in the present work, which involved a high-throughput analysis of this fcb1-schizont-est library, a few unexpected gene expression features were also discovered, such as the actual transcription of atypical rrna loci and subtelomeric regions. 
together with other collections of p. falciparum ests, usually generated from mixed parasite stages, this collection of fcb1-schizont-ests provides valuable data to gain further insight into the p. falciparum gene structure, polymorphism and expression. in addition, a large number of non-protein coding ests were identified, mainly matching with the two a-type rrna units and to a lower extent, two atypical rrna loci, tare subtelomeric regions and the recently described telomerase rna gene. statistical analysis of go terms, when available, indicated significant enrichment in genes involved in "entry into host-cells" and "actin cytoskeleton". here, we undertook a large scale analysis of a p. falciparum fcb1-schizont-est library previously constructed by suppression subtractive hybridization to study genes expressed during merozoite morphogenesis, with the aim of: 1) obtaining an exhaustive collection of schizont specific ests, 2) experimentally validating or correcting p. falciparum gene models and 3) pinpointing genes displaying protein polymorphism between the fcb and 3d strains. in addition, this analysis demonstrated the actual transcription of several remarkable non-protein coding loci: atypical rrna, tare region and telomerase rna gene. this fcb1-schizont-est analysis confirmed the actual expression of protein coding genes, allowing the correction of structural annotations for a quarter of these sequences. experimental data is therefore required for structural and functional assessments of p. falciparum genes and expression, and polymorphic data are further necessary to exploit genomic information to further qualify therapeutic target candidates. although most ests do not span full-length gene reading frames, detailed sequence comparison of fcb1-ests versus 3d genomic sequences allowed the confirmation of exon/intron boundaries in genes, the detection of new boundaries in genes and identification of protein polymorphism for genes. a total of clones randomly picked from the ssh library were sequenced, yielding usable ests that were then clustered on the p. falciparum genome.
 the plasmodium falciparum genome published in revealed ~ genes, mostly based on in silico predictions. experimental data is therefore required for structural and functional assessments of p. falciparum genes and expression, and polymorphic data are further necessary to exploit genomic information to further qualify therapeutic target candidates. here, we undertook a large scale analysis of a p. falciparum fcb1-schizont-est library previously constructed by suppression subtractive hybridization to study genes expressed during merozoite morphogenesis, with the aim of: 1) obtaining an exhaustive collection of schizont specific ests, 2) experimentally validating or correcting p. falciparum gene models and 3) pinpointing genes displaying protein polymorphism between the fcb and 3d strains.  a total of clones randomly picked from the ssh library were sequenced, yielding usable ests that were then clustered on the p. falciparum genome. this allowed identification of protein coding genes, including previously annotated as hypothetical. statistical analysis of go terms, when available, indicated significant enrichment in genes involved in "entry into host-cells" and "actin cytoskeleton". although most ests do not span full-length gene reading frames, detailed sequence comparison of fcb1-ests versus 3d genomic sequences allowed the confirmation of exon/intron boundaries in genes, the detection of new boundaries in genes and identification of protein polymorphism for genes. in addition, a large number of non-protein coding ests were identified, mainly matching with the two a-type rrna units and to a lower extent, two atypical rrna loci, tare subtelomeric regions and the recently described telomerase rna gene.  this fcb1-schizont-est analysis confirmed the actual expression of protein coding genes, allowing the correction of structural annotations for a quarter of these sequences. in addition, this analysis demonstrated the actual transcription of several remarkable non-protein coding loci: atypical rrna, tare region and telomerase rna gene. together with other collections of p. falciparum ests, usually generated from mixed parasite stages, this collection of fcb1-schizont-ests provides valuable data to gain further insight into the p. falciparum gene structure, polymorphism and expression. 