Update of the Gene Discovery Program in Schistosoma mansoni with the Expressed Sequence Tag Approach

Continuing the Schistosoma mansoni Genome Project 363 new templates were sequenced generating 205 more ESTs corresponding to 91 genes. Seventy four of these genes (81%) had not previously been described in S. mansoni. Among the newly discovered genes there are several of significant biological interest such as synaptophysin, NIFs-like and rho-GDP dissociation inhibitor

Analysis of expressed sequence tags from Trypanosoma cruzi amastigotes

A total of 880 expressed sequence tags (EST) originated from clones randomly selected from a Trypanosoma cruzi amastigote cDNA library have been analyzed. Of these, 40% (355 ESTs) have been identified by similarity to sequences in public databases and classified according to functional categorization of their putative products. About 11% of the mRNAs expressed in amastigotes are related to the translational machinery, and a large number of them (9% of the total number of clones in the library) encode ribosomal proteins. A comparative analysis with a previous study, where clones from the same library were selected using sera from patients with Chagas disease, revealed that ribosomal proteins also represent the largest class of antigen coding genes expressed in amastigotes (54% of all immunoselected clones). However, although more than thirty classes of ribosomal proteins were identified by EST analysis, the results of the immunoscreening indicated that only a particular subset of them contains major antigenic determinants recognized by antibodies from Chagas disease patients.

Production of full-length cDNA sequences by sequencing and analysis of expressed sequence tags from Schistosoma mansoni

The number of sequences generated by genome projects has increased exponentially, but gene characterization has not followed at the same rate. Sequencing and analysis of full-length cDNAs is an important step in gene characterization that has been used nowadays by several research groups. In this work, we have selected Schistosoma mansoni clones for full-length sequencing, using an algorithm that investigates the presence of the initial methionine in the parasite sequence based on the positions of alignment start between two sequences. BLAST searches to produce such alignments have been performed using parasite expressed sequence tags produced by Minas Gerais Genome Network against sequences from the database Eukaryotic Cluster of Orthologous Groups (KOG). This procedure has allowed the selection of clones representing 398 proteins which have not been deposited as S. mansoni complete CDS in any public database. Dedicated sequencing of 96 of such clones with reads from both 5' and 3' ends has been performed. These reads have been assembled using PHRAP, resulting in the production of 33 full-length sequences that represent novel S. mansoni proteins. These results shall contribute to construct a more complete view of the biology of this important parasite.

Analysis and chromosomal mapping of Leishmania (Leishmania) amazonensis amastigote expressed sequence tags

The characterization of expressed sequence tags (ESTs) generated from a cDNA library of Leishmania (Leishmania) amazonensis amastigotes is described. The sequencing of 93 clones generated new L. (L.) amazonensis ESTs from which 32% are not related to any other sequences in database and 68% presented significant similarities to known genes. The chromosome localization of some L. (L.) amazonensis ESTs was also determined in L. (L.) amazonensis and L. (L.) major. The characterization of these ESTs is suitable for the genome physical mapping, as well as for the identification of genes encoding cysteine proteinases implicated with protective immune responses in leishmaniasis.

Microsatellite markers in analysis of resistance to coffee leaf miner in Arabica coffee

The objective of this work was to analyze coffee (Coffea arabica) genotypes resistant to the coffee leaf miner (Leucoptera coffeella) using microsatellite markers. Sixty-six loci were evaluated, of which 63 were obtained from the Brazilian Coffee Expressed Sequence Tag (EST) database. These loci were amplified in bulks of individuals from F5 progenies of 'Siriema' (C. arabica x C. racemosa) resistant and susceptible to the insect, in eight samples of C. racemosa, and in a F6 population of 'Siriema' with 91 individuals segregating for resistance to the leaf miner. Polymorphisms were verified for two simple sequence repeat (SSR) loci in bulks of the susceptible progenies. The two polymorphic alleles were present in around 70% of the susceptible genotypes in F5 and in approximately 90% of the susceptible individuals in F6. However, the polymorphic EST-SSR markers among populations contrasting for resistance to leaf miner were not correlated to the evaluated characteristics. SSR markers show inter- and intraspecific polymorphism in C. arabica and C. racemosa.

MOLECULAR CHARACTERIZATION AND SEQUENCE PHYLOGENETIC ANALYSIS OF SURFACE ANTIGEN 3 (SAG3) GENE OF LOCAL INDIAN ISOLATES (CHENNAI AND IZATNAGAR) OF Toxoplasma gondii

Context and objective:The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess the homologous variations between the different loci of various strains of parasites.Design and setting:The present communication deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3) of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI.Method:The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences.Results:The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain (both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them.Conclusion:Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of view of molecular phylogeny.

Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST). We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative.

Clonal study of avian Escherichia coli strains by fliC conserved-DNA-sequence regions analysis

The clonal relationship among avian Escherichia coli strains and their genetic proximity with human pathogenic E. coli, Salmonela enterica, Yersinia enterocolitica and Proteus mirabilis, was determined by the DNA sequencing of the conserved 5' and 3'regions fliC gene (flagellin encoded gene). Among 30 commensal avian E. coli strains and 49 pathogenic avian E. coli strains (APEC), 24 commensal and 39 APEC strains harbored fliC gene with fragments size varying from 670bp to 1,900bp. The comparative analysis of these regions allowed the construction of a dendrogram of similarity possessing two main clusters: one compounded mainly by APEC strains and by H-antigens from human E. coli, and another one compounded by commensal avian E. coli strains, S. enterica, and by other H-antigens from human E. coli. Overall, this work demonstrated that fliC conserved regions may be associated with pathogenic clones of APEC strains, and also shows a great similarity among APEC and H-antigens of E. coli strains isolated from humans. These data, can add evidence that APEC strains can exhibit a zoonotic risk.

Characterization of a clone from an adult worm cDNA library selected with anti-Schistosoma mansoni human antibodies dissociated from immune complexes: a preliminary report

Considering the scarcity of defined antigens, actually useful and reliable for use in the field studies, we propose an alternative method for selection of cDNA clones with potential use in the diagnosis of schistosomiasis. Human antibodies specific to a protein fraction of 31/32 kDa (Sm31/32), dissociated from immune complexes, are used for screening of clones from an adult worm cDNA library. Partial sequencing of five clones, selected through this strategy, showed to be related to Schistosoma mansoni: two were identified as homologous to heat shock protein 70, one to glutathione S-transferase, one to homeodomain protein, and one to a previously described EST (expressed sequence tag) of S. mansoni. This last clone was the most consistently reactive during the screening process with the anti-Sm31/32 antibodies dissociated from the immune complexes. The complete sequence of this clone was obtained and the translation data yielded only one ORF (open reading frame) that code for a protein with 57 amino acids. Based on this amino acid sequence two peptides were chemically synthesized and evaluated separately against a pool of serum samples from schistosomiasis patients and non-schistosomiasis individuals. Both peptides showed strong reactivity only against the positive pool, suggesting that these peptides may be useful as antigens for the diagnosis of schistosomiasis mansoni.

Nucleotide-sugar transporters: structure, function and roles in vivo

The glycosylation of glycoconjugates and the biosynthesis of polysaccharides depend on nucleotide-sugars which are the substrates for glycosyltransferases. A large proportion of these enzymes are located within the lumen of the Golgi apparatus as well as the endoplasmic reticulum, while many of the nucleotide-sugars are synthesized in the cytosol. Thus, nucleotide-sugars are translocated from the cytosol to the lumen of the Golgi apparatus and endoplasmic reticulum by multiple spanning domain proteins known as nucleotide-sugar transporters (NSTs). These proteins were first identified biochemically and some of them were cloned by complementation of mutants. Genome and expressed sequence tag sequencing allowed the identification of a number of sequences that may encode for NSTs in different organisms. The functional characterization of some of these genes has shown that some of them can be highly specific in their substrate specificity while others can utilize up to three different nucleotide-sugars containing the same nucleotide. Mutations in genes encoding for NSTs can lead to changes in development in Drosophila melanogaster or Caenorhabditis elegans, as well as alterations in the infectivity of Leishmania donovani. In humans, the mutation of a GDP-fucose transporter is responsible for an impaired immune response as well as retarded growth. These results suggest that, even though there appear to be a fair number of genes encoding for NSTs, they are not functionally redundant and seem to play specific roles in glycosylation.

Study of simple sequence repeat markers from coffee expressed sequences associated to leaf miner resistance

The objective of this work was to identify expressed simple sequence repeats (SSR) markers associated to leaf miner resistance in coffee progenies. Identification of SSR markers was accomplished by directed searches on the Brazilian Coffee Expressed Sequence Tags (EST) database. Sequence analysis of 32 selected SSR loci showed that 65% repeats are of tetra-, 21% of tri- and 14% of dinucleotides. Also, expressed SSR are localized frequently in the 5'-UTR of gene transcript. Moreover, most of the genes containing SSR are associated with defense mechanisms. Polymorphisms were analyzed in progenies segregating for resistance to the leaf miner and corresponding to advanced generations of a Coffea arabica x Coffea racemosa hybrid. Frequency of SSR alleles was 2.1 per locus. However, no polymorphism associated with leaf miner resistance was identified. These results suggest that marker-assisted selection in coffee breeding should be performed on the initial cross, in which genetic variability is still significant.

Clustering of Schistosoma mansoni mRNA sequences and analysis of the most transcribed genes: implications in metabolism and biology of different developmental stages

The study of the Schistosoma mansoni genome, one of the etiologic agents of human schistosomiasis, is essential for a better understanding of the biology and development of this parasite. In order to get an overview of all S. mansoni catalogued gene sequences, we performed a clustering analysis of the parasite mRNA sequences available in public databases. This was made using softwares PHRAP and CAP3. The consensus sequences, generated after the alignment of cluster constituent sequences, allowed the identification by database homology searches of the most expressed genes in the worm. We analyzed these genes and looked for a correlation between their high expression and parasite metabolism and biology. We observed that the majority of these genes is related to the maintenance of basic cell functions, encoding genes whose products are related to the cytoskeleton, intracellular transport and energy metabolism. Evidences are presented here that genes for aerobic energy metabolism are expressed in all the developmental stages analyzed. Some of the most expressed genes could not be identified by homology searches and may have some specific functions in the parasite.

A directed approach for the identification of transcripts harbouring the spliced leader sequence and the effect of trans-splicing knockdown in Schistosoma mansoni

Schistosomiasis is a major neglected tropical disease caused by trematodes from the genus Schistosoma. Because schistosomes exhibit a complex life cycle and numerous mechanisms for regulating gene expression, it is believed that spliced leader (SL) trans-splicing could play an important role in the biology of these parasites. The purpose of this study was to investigate the function of trans-splicing in Schistosoma mansoni through analysis of genes that may be regulated by this mechanism and via silencing SL-containing transcripts through RNA interference. Here, we report our analysis of SL transcript-enriched cDNA libraries from different S. mansoni life stages. Our results show that the trans-splicing mechanism is apparently not associated with specific genes, subcellular localisations or life stages. In cross-species comparisons, even though the sets of genes that are subject to SL trans-splicing regulation appear to differ between organisms, several commonly shared orthologues were observed. Knockdown of trans-spliced transcripts in sporocysts resulted in a systemic reduction of the expression levels of all tested trans-spliced transcripts; however, the only phenotypic effect observed was diminished larval size. Further studies involving the findings from this work will provide new insights into the role of trans-splicing in the biology of S. mansoni and other organisms. All Expressed Sequence Tags generated in this study were submitted to dbEST as five different libraries. The accessions for each library and for the individual sequences are as follows: (i) adult worms of mixed sexes (LIBEST_027999: JZ139310 - JZ139779), (ii) female adult worms (LIBEST_028000: JZ139780 - JZ140379), (iii) male adult worms (LIBEST_028001: JZ140380 - JZ141002), (iv) eggs (LIBEST_028002: JZ141003 - JZ141497) and (v) schistosomula (LIBEST_028003: JZ141498 - JZ141974).