Reading Frame Expressed Sequence Tags Research Articles

Trypanosoma rangeli displays a clonal population structure, revealing a subdivision of KP1(−) strains and the ancestry of the Amazonian group

Assessment of the genetic variability and population structure of Trypanosoma rangeli, a non-pathogenic American trypanosome, was carried out through microsatellite and single-nucleotide polymorphism analyses. Two approaches were used for microsatellite typing: data mining in expressed sequence tag /open reading frame expressed sequence tags libraries and PCR-based Isolation of Microsatellite Arrays from genomic libraries. All microsatellites found were evaluated for their abundance, frequency and usefulness as markers. Genotyping of T. rangeli strains and clones was performed for 18 loci amplified by PCR from expressed sequence tag/open reading frame expressed sequence tags libraries. The presence of single-nucleotide polymorphisms in the nuclear, multi-copy, spliced leader gene was assessed in 18 T. rangeli strains, and the results show that T. rangeli has a predominantly clonal population structure, allowing a robust phylogenetic analysis. Microsatellite typing revealed a subdivision of the KP1(−) genetic group, which may be influenced by geographical location and/or by the co-evolution of parasite and vectors occurring within the same geographical areas. The hypothesis of parasite–vector co-evolution was corroborated by single-nucleotide polymorphism analysis of the spliced leader gene. Taken together, the results suggest three T. rangeli groups: (i) the T. rangeli Amazonian group; (ii) the T. rangeli KP1(−) group; and (iii) the T. rangeli KP1(+) group. The latter two groups possibly evolved from the Amazonian group to produce KP1(+) and KP1(−) strains.

Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury.

BackgroundThe sequencing of the D.melanogaster genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of Drosophila protein-coding genes contain one or more alternative exons. A recent transcription map of the Drosophila embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of Drosophila transcriptome.ResultsBioinformatic analysis of 1,303 Drosophila ORESTES clusters identified 68 sequences derived from unannotated regions in the current Drosophila genome version (4.3). Of these, a set of 38 was analysed by polyA+ northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The SP212 gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this locus is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury.ConclusionUsing the ORESTES methodology we identified 17 novel exons from low abundance Drosophila transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data.

Biomphalaria glabrata transcriptome: Identification of cell-signalling, transcriptional control and immune-related genes from open reading frame expressed sequence tags (ORESTES)

Biomphalaria glabrata is the major intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. Much remains to be discovered concerning specific molecules mediating the defence events in these intermediate hosts, triggered by invading schistosomes. An expressed sequence tag (EST) gene discovery strategy known as ORESTES has been employed to identify transcripts that might be involved in snail–schistosome interactions in order to examine gene expression patterns in infected B. glabrata. Over 3930 ESTs were sequenced from cDNA libraries made from both schistosome-exposed and unexposed snails using different tissue types, producing a database of 1843 non-redundant clones. The non-redundant set has been assessed for gene ontology and KEGG pathway assignments. This approach has revealed a number of signalling, antioxidant and immune-related gene homologues that, based on current understanding of molluscan and other comparative systems, might play an important role in the molluscan defence response towards infection.

Hippocampal gene expression analysis using the ORESTES methodology shows that homer 1a mRNA is upregulated in the acute period of the pilocarpine epilepsy model

In the study of temporal lobe epilepsy (TLE) the characterization of genes expressed in the hippocampus is of central importance for understanding their roles in epileptogenic mechanisms. Although several large-scale studies on TLE gene expression have been reported, precise assignment of individual genes associated with this syndrome is still debatable. Here we investigated differentially expressed genes by comparison of mRNAs from normal and epileptic rat hippocampus in the pilocarpine model of epilepsy. For this we used a powerful EST sequencing methodology, ORESTES (Open Reading frame Expressed Sequence Tags), which generates sequence datasets enriched for mRNAs open reading frames (ORFs) rather than simple 5' and 3' ends of mRNAs. Analysis of our sequences shows that ORESTES readily enables the identification of epilepsy associated ORFs. PFAM analysis of protein motifs present in our ORESTES epilepsy database revealed diverse important protein family domains, such as cytoskeletal, cell signaling and protein kinase domains, which could be involved in processes underlying epileptogenesis. More importantly, we show that the expression of homer 1a, known to be coupled to mGluR and NMDA synaptic transmission, is associated with pilocarpine induced status epilepticus (SE). The combined use of the pilocarpine model of epilepsy with the ORESTES technique can significantly contribute to the identification of specific genes and proteins related to TLE. This is the first study applying a large-scale method for rapid shotgun sequencing directed to ORFs in epilepsy research.

Gene Expression Profile Associated with Response to Doxorubicin-Based Therapy in Breast Cancer

This study was designed to identify genes that could predict response to doxorubicin-based primary chemotherapy in breast cancer patients. Biopsy samples were obtained before primary treatment with doxorubicin and cyclophosphamide. RNA was extracted and amplified and gene expression was analyzed using cDNA microarrays. Response to chemotherapy was evaluated in 51 patients, and based on Response Evaluation Criteria in Solid Tumors guidelines, 42 patients, who presented at least a partial response (> or =30% reduction in tumor dimension), were classified as responsive. Gene profile of samples, divided into training set (n = 38) and independent validation set (n = 13), were at first analyzed against a cDNA microarray platform containing 692 genes. Unsupervised clustering could not separate responders from nonresponders. A classifier was identified comprising EMILIN1, FAM14B, and PBEF, which however could not correctly classify samples included in the validation set. Our next step was to analyze gene profile in a more comprehensive cDNA microarray platform, containing 4,608 open reading frame expressed sequence tags. Seven samples of the initial training set (all responder patients) could not be analyzed. Unsupervised clustering could correctly group all the resistant samples as well as at least 85% of the sensitive samples. Additionally, a classifier, including PRSS11, MTSS1, and CLPTM1, could correctly distinguish 95.4% of the 44 samples analyzed, with only two misclassifications, one sensitive sample and one resistant tumor. The robustness of this classifier is 2.5 greater than the first one. A trio of genes might potentially distinguish doxorubicin-responsive from nonresponsive tumors, but further validation by a larger number of samples is still needed.

Gene Expression Profiles Reveal that DCN, DIO1, and DIO2 Are Underexpressed in Benign and Malignant Thyroid Tumors

To investigate the molecular events involved in the pathogenesis and/or progression of thyroid tumors, we compared the gene expression profiles of three thyroid carcinoma cell lines, which represent major tumor subtypes of thyroid cancer and normal thyroid tissue. Using cDNA array methodology, we investigated the expression of 1807 open reading frame expressed sequence tags (ORESTES), selected from head and neck tumor libraries generated through the Brazilian Human Cancer Project-LICR/FAPESP. We found that 505 transcripts were differentially expressed in the thyroid carcinoma cell lines. Using a more stringent criterion, transcripts underexpressed or overexpressed more than fivefold in 1 of 3 or 3 of 3 carcinoma cell lines, a list of 55 ESTs were detected. Five candidate genes were further validated by quantitative polymerase chain reaction (qPCR) in an independent set of 52 thyroid tumors and 22 matched normal thyroid tissues. DCN was found underexpressed in a high percentage of the follicular thyroid adenomas, follicular thyroid carcinomas, and follicular variant of papillary thyroid carcinomas. DIO1 and DIO2 were underexpressed in nearly all papillary thyroid carcinomas. These genes not only could help to better define a tumor signature for thyroid tumors, but may, in part, also become useful as potential targets for thyroid tumor treatment.

Genes differentially expressed in breast cancer (BC) samples as compared to normal breast tissue from women without cancer

9593 Background: We have determined the gene profile correlated to doxorubicin resistance in 38 BC patients (pt) treated with primary chemotherapy and 37 genes were differentially expressed in tumors according to response. Among the genes more expressed in sensitive tumors as compared to resistant tumors were ephrin receptor and c-src; JunD; and CDKN2B. Genes more expressed in resistant tumors were fibroblast growth factor receptor 2 and lymphocyte protein TK; Max (Myc partner); and MMP12 (metalloproteinase). We have now analyzed gene expression of these 38 tumors as compared to normal breast tissue (perilesional) obtained from 5 women without cancer submitted to resection of benign lesions. Methods: A tissue specific cDNA microarray glass slide was assembled based on genes expressed in mammary tissue and BC. Genes hyperexpressed in BC as determined in SAGE libraries were also selected. Sequences representing 496 genes and 195 ESTs were chosen in an ORESTES (open reading frame expressed sequence tags) bank (Instituto Ludwig de Pesquisa sobre o Câncer/FAPESP) or synthesized in PCR reactions and spotted 3 or 6 times on glass slides. Tissue was hand dissected, RNA was extracted, amplified, and co-hybridized with a HB4A (normal mammary epithelial cell line) probe against cDNA microarray slides. Results: Median age of BC pt was 51 y; pt were clinically staged as IIA (3), IIB (2), IIIA (17), IIIB (15) and IV (1 pt). Sixty three molecules were differentially expressed between tumors and normal breast tissue. Genes more expressed in tumors included KRT8 and KRT19 (cytoskeleton proteins); MMP11 and MMP12; transmembrane 4 superfamily member tetraspan NET-7; NME2 (tumor suppressor gene); REL (oncogene); MSH2 (DNA repair); XIAP (inhibitor of apoptosis) associated factor 1. Genes more expressed in normal tissue were CCND3 (cyclin D3); KRT14 and KRT15; LAMA3 and LAMB3 (laminin); maspin (proteinase inhibitor); GSTP1 (detoxifying enzyme); ITGB4 (adhesion molecule); JUN, FOS, MYC (transcription factors) and MAX; and ABCG2 (ATP binding cassette). Conclusions: This gene profile seems to characterize breast malignancy. Supported by FAPESP 01/00146–8 No significant financial relationships to disclose.

From ESTs to ORESTES

A public consortium based in Sao Paolo, Brazil, has generated about 700 000 sequences specific to the open reading frames of the human transcriptome. The work represents ‘a significant contribution by people outside the mainstream US/UK axis,’ said Ian Hubbard, head of the human genome analysis group at the Sanger Centre, UK. Camargo et al. call the sequences ‘open reading frame expressed sequence tags’ ORESTES because they provide data on central coding regions of transcripts. Expressed sequence tags (ESTs) are sequences derived from the ends of cDNA libraries normally generated using primers corresponding to the poly A sequences at the ends of cDNAs. So, ESTs are biased towards the 3′ ends of genes, making prediction of alternative splicing products difficult. ORESTES, however, are generated by a PCR-based strategy using degenerate primers and thus cover all regions of the transcript. The greatest advantage of ORESTES is that they are distributed almost uniformly along human transcripts. Although these sequences are only about 18% of those in the human EST database, they are an excellent complement and will help facilitate the determination of the human transcriptome. (Camargo, A.A. et al.[2001] Proc. Natl. Acad. Sci. U. S. A.98, 12103–12108) AP