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ABSTRACT: Equine infectious anemia (EIA) is an important viral infection affecting horses worldwide. The course of infection is accompanied generally by three characteristic stages: acute, chronic and inapparent. There is no effective EIA vaccine or treatment, and the control of the disease is based currently on identification of EIAV inapparent carriers by laboratory tests. Recombinant envelope protein (rgp90) was expressed in Escherichia coli and evaluated via enzyme-linked immunosorbent assay (ELISA). There was an excellent agreement (95.42%) between the ELISA results using rgp90 and agar gel immunodiffusion test results. AGID is considered the "gold-standard" serologic test for equine infectious anemia (EIA). After 1160 serum samples were tested, the relative sensitivity and specificity of the ELISA were 96.1% and 96.4%, respectively. Moreover, analysis diagnostic accuracy of the ELISA was performed. The ELISA proved robust. Furthermore, good reproducibility was observed for the negative controls and, positive controls for all plates tested.
Journal of virological methods 03/2012; 180(1-2):62-7. · 2.13 Impact Factor
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ABSTRACT: A wide variety of high-throughput microarray platforms have been used to identify molecular targets associated with biological and clinical tumor phenotypes by comparing samples representing distinct pathological states.
The gene expression profiles of human cutaneous melanomas were determined by cDNA microarray analysis. Next, a robust analysis to determine functional classifications and make predictions based on data-oriented hypotheses was performed. Relevant networks that may be implicated in melanoma progression were also considered.
In this study we aimed to analyze coordinated gene expression changes to find molecular pathways involved in melanoma progression. To achieve this goal, ontologically-linked modules with coordinated expression changes in melanoma samples were identified. With this approach, we detected several gene networks related to different modules that were induced or repressed during melanoma progression. Among them we observed high coordinated expression levels of genes involved in a) cell communication (KRT4, VWF and COMP); b) epidermal development (KLK7, LAMA3 and EVPL); and c) functionally related to kallikreins (EVPL, KLK6, KLK7, KLK8, SERPINB13, SERPING1 and SLPI). Our data also indicated that hKLK7 protein expression was significantly associated with good prognosis and survival.
Our findings, derived from a different type of analysis of microarray data, highlight the importance of analyzing coordinated gene expression to find molecular pathways involved in melanoma progression.
BMC Medical Genomics 01/2011; 4:76. · 3.69 Impact Factor
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ABSTRACT: Cancer shows a great diversity in its clinical behavior which cannot be easily predicted using the currently available clinical or pathological markers. The identification of pathways associated with lymph node metastasis (N+) and recurrent head and neck squamous cell carcinoma (HNSCC) may increase our understanding of the complex biology of this disease.
Tumor samples were obtained from untreated HNSCC patients undergoing surgery. Patients were classified according to pathologic lymph node status (positive or negative) or tumor recurrence (recurrent or non-recurrent tumor) after treatment (surgery with neck dissection followed by radiotherapy). Using microarray gene expression, we screened tumor samples according to modules comprised by genes in the same pathway or functional category.
The most frequent alterations were the repression of modules in negative lymph node (N0) and in non-recurrent tumors rather than induction of modules in N+ or in recurrent tumors. N0 tumors showed repression of modules that contain cell survival genes and in non-recurrent tumors cell-cell signaling and extracellular region modules were repressed.
The repression of modules that contain cell survival genes in N0 tumors reinforces the important role that apoptosis plays in the regulation of metastasis. In addition, because tumor samples used here were not microdissected, tumor gene expression data are represented together with the stroma, which may reveal signaling between the microenvironment and tumor cells. For instance, in non-recurrent tumors, extracellular region module was repressed, indicating that the stroma and tumor cells may have fewer interactions, which disable metastasis development. Finally, the genes highlighted in our analysis can be implicated in more than one pathway or characteristic, suggesting that therapeutic approaches to prevent tumor progression should target more than one gene or pathway, specially apoptosis and interactions between tumor cells and the stroma.
BMC Medical Genomics 01/2011; 4:33. · 3.69 Impact Factor
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ABSTRACT: Nodules of the thyroid gland are observed frequently in patients who undergo ultrasound studies. The majority of these nodules are benign, corresponding to goiters or adenomas, and only a small fraction corresponds to carcinomas. Among thyroid tumors, the diagnosis of follicular adenocarcinomas by preoperative fine-needle aspiration biopsy is a major challenge, because it requires inspection of the entire capsule to differentiate it from adenoma. Consequently, large numbers of patients undergo unnecessary thyroidectomy.
Using data from gene expression analysis, the authors applied Fisher linear discriminant analysis and searched for expression signatures of individual samples of adenomas and follicular carcinomas that could be used as molecular classifiers for the precise classification of malignant and nonmalignant lesions.
Fourteen trios of genes were described that fulfilled the criteria for the correct classification of 100% of samples. The robustness of these trios was verified by using leave-1-out cross-validation and bootstrap analyses. The results demonstrated that, by combining trios, better classifiers could be generated that correctly classified >92% of samples.
The strategy of classifiers based on individual signatures was a useful strategy for distinguishing between samples with very similar expression profiles.
Cancer 05/2006; 106(9):1891-900. · 4.77 Impact Factor
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Beatriz S. Stolf Ph.D,
Mariana M. S. Santos M.D,
Daniel F. Simao M.D,
Juan P. Diaz M.D,
Elier B. Cristo M.D,
Roberto Hirata Jr. M.D,
Maria P. Curado M.D,
Eduardo J. Neves M.D,
Luiz P. Kowalski M.D,
Alex F. Carvalho M.D,
Beatriz S. Stolf,
Mariana M. S. Santos,
Daniel F. Simao,
Juan P. Diaz,
Elier B. Cristo,
Roberto Hirata Jr,
Maria P. Curado,
Eduardo J. Neves,
Luiz P. Kowalski, Alex F. Carvalho
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ABSTRACT: BACKGROUND
Nodules of the thyroid gland are observed frequently in patients who undergo ultrasound studies. The majority of these nodules are benign, corresponding to goiters or adenomas, and only a small fraction corresponds to carcinomas. Among thyroid tumors, the diagnosis of follicular adenocarcinomas by preoperative fine-needle aspiration biopsy is a major challenge, because it requires inspection of the entire capsule to differentiate it from adenoma. Consequently, large numbers of patients undergo unnecessary thyroidectomy.METHODS
Using data from gene expression analysis, the authors applied Fisher linear discriminant analysis and searched for expression signatures of individual samples of adenomas and follicular carcinomas that could be used as molecular classifiers for the precise classification of malignant and nonmalignant lesions.RESULTSFourteen trios of genes were described that fulfilled the criteria for the correct classification of 100% of samples. The robustness of these trios was verified by using leave-1-out cross-validation and bootstrap analyses. The results demonstrated that, by combining trios, better classifiers could be generated that correctly classified >92% of samples.CONCLUSIONS
The strategy of classifiers based on individual signatures was a useful strategy for distinguishing between samples with very similar expression profiles. Cancer 2006. © 2006 American Cancer Society.
Cancer 04/2006; 106(9):1891 - 1900. · 4.77 Impact Factor
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Beatriz S Stolf,
Cintia M Abreu,
Maria B Mahler-Araújo,
Márcia Dellamano,
Waleska K Martins,
Marcos Brasilino de Carvalho,
Maria P Curado,
Juan P Díaz,
Artur Fabri,
Helena Brentani, Alex F Carvalho,
Fernando A Soares,
Luiz P Kowalski,
Roberto Hirata,
Luiz F L Reis
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ABSTRACT: Using cDNA microarrays with 3800 cDNA fragments, we determined the expression profile of normal thyroid tissue, goiter, adenoma and papillary carcinoma (10 samples from each class). After background correction and statistical analysis, we identified a set of 160 genes as being differentially expressed in all pair-wise comparisons. Here we demonstrate that, at least on the basis of these differentially expressed genes, a positive correlation between goiter and papillary carcinomas could be observed. We identified a common set of genes whose expression is diminished in both goiter and papillary carcinomas as compared to normal thyroid tissue. Moreover, no genes with inverse correlation in samples from goiter and papillary carcinomas could be detected. Using Real-Time PCR and/or tissue microarrays, we confirmed the altered expression of some of the identified genes. Of notice, we demonstrate that the reduced mRNA levels of p27(kip1) observed in papillary carcinomas as compared to either goiter or normal thyroid tissues (P<0.001) is accompanied by an altered protein distribution within the cell. In papillary carcinomas, P27(KIP1) is preferentially cytoplasmic as opposed to goiter or normal thyroid tissue, where P27(KIP1) is preferentially located in the nucleus. The exploitation of the data presented here could contribute to the understanding of the molecular events related to thyroid diseases and gives support to the notion that common molecular events might be related to the frequent observation of areas of papillary carcinomas in the gland of patients with goiter.
Cancer Letters 10/2005; 227(1):59-73. · 4.24 Impact Factor
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Luciana I Gomes,
Gustavo H Esteves, Alex F Carvalho,
Elier B Cristo,
Roberto Hirata,
Waleska K Martins,
Sarah M Marques,
Luiz P Camargo,
Helena Brentani,
Adriane Pelosof,
Cláudia Zitron,
Rubens A Sallum,
André Montagnini,
Fernando A Soares,
E Jordão Neves,
Luiz F L Reis
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ABSTRACT: Adenocarcinomas of stomach and esophagus are frequently associated with preceding inflammatory alterations of the normal mucosa. Whereas intestinal metaplasia of the gastric mucosa is associated with higher risk of malignization, Barrett's disease is a risk factor for adenocarcinoma of the esophagus. Barrett's disease is characterized by the substitution of the squamous mucosa of the esophagus by a columnar tissue classified histopathologically as intestinal metaplasia. Using cDNA microarrays, we determined the expression profile of normal gastric and esophageal mucosa as well as intestinal metaplasia and adenocarcinomas from both organs. Data were explored to define functional alterations related to the transformation from squamous to columnar epithelium and the malignant transformation from intestinal metaplasia to adenocarcinomas. Based on their expression profile, adenocarcinomas of the esophagus showed stronger correlation with intestinal metaplasia of the stomach than with Barrett's mucosa. Second, we identified two functional modules, lipid metabolism and cytokine, as being altered with higher statistical significance. Whereas the lipid metabolism module is active in samples representing intestinal metaplasia and inactive in adenocarcinomas, the cytokine module is inactive in samples representing normal esophagus and esophagitis. Using the concept of relevance networks, we determined the changes in linear correlation of genes pertaining to these two functional modules. Exploitation of the data presented herein will help in the precise molecular characterization of adenocarcinoma from the distal esophagus, avoiding the topographical and descriptive classification that is currently adopted, and help with the proper management of patients with Barrett's disease.
Cancer Research 09/2005; 65(16):7127-36. · 7.86 Impact Factor
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ABSTRACT: During the last 5 years, the number of papers describing data obtained by microarray technology increased exponentially with about 3000 papers in 2003. Undoubtedly, cancer is by far the disease that received most of the attention as far as the amount of data generated. As array technology is rather new and highly dependent on bioinformatics, mathematics and statistics, a clear understanding of the knowledge and information derived from array-based experiments is not widely appreciated. We shall review herein some of the issues related to the construction of DNA arrays, quantities and heterogeneity of probes and targets, the consequences of the physical characteristics of the probes, data extraction and data analysis as well as the applications of array technology. Our goal is to bring to the general audience, some of the basics of array technology and its possible application in oncology. By discussing some of the basic aspects of the methodology, we hope to stimulate criticism concerning the conclusions proposed by authors, especially in the light of the very low degree of reproducibility already proven when commercially available platforms were compared . Regardless of its pitfalls, it is unquestionable that array technology will have a great impact in the management of cancer and its applications will range from the discovery of new drug targets, new molecular tools for diagnosis and prognosis as well as for a tailored treatment that will take into account the molecular determinants of a given tumor. Hence, we shall also highlight some of the already available and promising applications of array technology on the day-to-day practice of oncology.
Critical Reviews in Oncology/Hematology 06/2005; 54(2):95-105. · 4.41 Impact Factor
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Francis M F Nunes,
Valeria Valente,
Josane F Sousa,
Marco A V Cunha,
Daniel G Pinheiro,
Rafaela M Maia,
Daniela D Araujo,
Maria C R Costa,
Waleska K Martins, Alex F Carvalho, [......],
Andrew J G Simpson,
Marco A Zago,
Ademilson E E Soares,
Marcia M G Bitondi,
Enilza M Espreafico,
Foued S Espindola,
Maria L Paco-Larson,
Zila L P Simoes,
Klaus Hartfelder,
Wilson A Silva
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ABSTRACT: The ongoing efforts to sequence the honey bee genome require additional initiatives to define its transcriptome. Towards this end, we employed the Open Reading frame ESTs (ORESTES) strategy to generate profiles for the life cycle of Apis mellifera workers.
Of the 5,021 ORESTES, 35.2% matched with previously deposited Apis ESTs. The analysis of the remaining sequences defined a set of putative orthologs whose majority had their best-match hits with Anopheles and Drosophila genes. CAP3 assembly of the Apis ORESTES with the already existing 15,500 Apis ESTs generated 3,408 contigs. BLASTX comparison of these contigs with protein sets of organisms representing distinct phylogenetic clades revealed a total of 1,629 contigs that Apis mellifera shares with different taxa. Most (41%) represent genes that are in common to all taxa, another 21% are shared between metazoans (Bilateria), and 16% are shared only within the Insecta clade. A set of 23 putative genes presented a best match with human genes, many of which encode factors related to cell signaling/signal transduction. 1,779 contigs (52%) did not match any known sequence. Applying a correction factor deduced from a parallel analysis performed with Drosophila melanogaster ORESTES, we estimate that approximately half of these no-match ESTs contigs (22%) should represent Apis-specific genes.
The versatile and cost-efficient ORESTES approach produced minilibraries for honey bee life cycle stages. Such information on central gene regions contributes to genome annotation and also lends itself to cross-transcriptome comparisons to reveal evolutionary trends in insect genomes.
BMC Genomics 12/2004; 5:84. · 4.07 Impact Factor
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Sibele I Meireles,
Elier B Cristo, Alex F Carvalho,
Roberto Hirata,
Adriane Pelosof,
Luciana I Gomes,
Waleska K Martins,
Maria D Begnami,
Cláudia Zitron,
André L Montagnini,
Fernando A Soares,
E Jordão Neves,
Luiz F L Reis
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ABSTRACT: High incidence of gastric cancer-related death is mainly due to diagnosis at an advanced stage in addition to the lack of adequate neoadjuvant therapy. Hence, new tools aimed at early diagnosis would have a positive impact in the outcome of the disease. Using cDNA arrays having 376 genes either identified previously as altered in gastric tumors or known to be altered in human cancer, we determined expression signature of 99 tissue fragments representing normal gastric mucosa, gastritis, intestinal metaplasia, and adenocarcinomas. We first validated the array by identifying molecular markers that are associated with intestinal metaplasia, considered as a transition stage of gastric adenocarcinomas of the intestinal type as well as markers that are associated with diffuse type of gastric adenocarcinomas. Next, we applied Fisher's linear discriminant analysis in an exhaustive search of trios of genes that could be used to build classifiers for class distinction. Many classifiers could distinguish between normal and tumor samples, whereas, for the distinction of gastritis from tumor and for metaplasia from tumor, fewer classifiers were identified. Statistical validations showed that trios that discriminate between normal and tumor samples are powerful classifiers to distinguish between tumor and nontumor samples. More relevant, it was possible to identify samples of intestinal metaplasia that have expression signature resembling that of an adenocarcinoma and can now be used for follow-up of patients to determine their potential as a prognostic test for malignant transformation.
Cancer Research 03/2004; 64(4):1255-65. · 7.86 Impact Factor
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BioTechniques 11/2003; 35(4):688-90, 692. · 2.67 Impact Factor
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ABSTRACT: Limiting amounts of RNA is a major issue in cDNA microarray, especially when one is dealing with fresh tissue samples. Here we describe a protocol based on template switch and T7 amplification that led to efficient and linear amplification of 1300x. Using a glass-array containing 368 genes printed in three or six replicas covering a wide range of expression levels and ratios, we determined quality and reproducibility of the data obtained from one nonamplified and two independently amplified RNAs (aRNA) derived from normal and tumor samples using replicas with dye exchange (dye-swap measurements). Overall, signal-to-noise ratio improved when we used aRNA (1.45-fold for channel 1 and 2.02-fold for channel 2), increasing by 6% the number of spots with meaningful data. Measurements arising from independent aRNA samples showed strong correlation among themselves (r(2)=0.962) and with those from the nonamplified sample (r(2)=0.975), indicating the reproducibility and fidelity of the amplification procedure. Measurement differences, i.e, spots with poor correlation between amplified and nonamplified measurements, did not show association with gene sequence, expression intensity, or expression ratio and can, therefore, be compensated with replication. In conclusion, aRNA can be used routinely in cDNA microarray analysis, leading to improved quality of data with high fidelity and reproducibility.
Analytical Biochemistry 11/2003; 321(2):244-51. · 3.00 Impact Factor
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Helena Brentani,
Otávia L Caballero,
Anamaria A Camargo,
Aline M da Silva,
Wilson Araújo da Silva,
Emmanuel Dias Neto,
Marco Grivet,
Arthur Gruber,
Pedro Edson Moreira Guimaraes,
Winston Hide, [......],
Teresa Cristina de Lima E Silva,
Fernando Augusto Soares,
Maria de Fátima Sonati,
Josane de Freitas Sousa,
Diana Queiroz,
Valéria Valente,
André Luiz Vettore,
Fabiola Elizabeth Villanova,
Marco Antonio Zago,
Heloisa Zalcberg
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ABSTRACT: Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define approximately 23,500 genes, of which only approximately 1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that <1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers.
Proceedings of the National Academy of Sciences 11/2003; 100(23):13418-23. · 9.68 Impact Factor
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Beatriz S Stolf, Alex F Carvalho,
Waleska K Martins,
Franco B Runza,
Marcel Brun,
Roberto Hirata,
Eduardo Jordão Neves,
Fernando A Soares,
Juan Postigo-Dias,
Luis P Kowalski,
Luiz F L Reis
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ABSTRACT: Here, we describe the identification of three human genes with altered expression in thyroid diseases. One of them corresponds to insulin-like growth factor binding protein 5 (IGFBP5), which has already been described as over expressed in other cancers and, for the first time, is identified as overexpressed in thyroid tumors. The other genes, named 44 and 199, are ESTs with yet unknown function and were mapped on human chromosomes seven and four, respectively. We determined by RT-PCR the expression level of these genes in ten samples of disease-free thyroid, ten of goiter, nine of papillary carcinoma, ten of adenoma and seven of follicular carcinoma and the significance of observed differences was statistically determined. IGFBP-5 and gene 44 were significantly overexpressed in papillary carcinoma when compared to normal and goiter. Genes 44 and 199 were differentially expressed in follicular carcinoma and adenoma when compared to normal thyroid tissue.
Cancer Letters 04/2003; 191(2):193-202. · 4.24 Impact Factor
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Sibele I Meireles, Alex F Carvalho,
Roberto Hirata,
André L Montagnini,
Waleska K Martins,
Franco B Runza,
Beatriz S Stolf,
Lara Termini,
Chamberlein E M Neto,
Ricardo L A Silva,
Fernando A Soares,
E Jordão Neves,
Luiz F L Reis
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ABSTRACT: Using cDNA fragments from the FAPESP/lICR Cancer Genome Project, we constructed a cDNA array having 4512 elements and determined gene expression in six normal and six tumor gastric tissues. Using t-statistics, we identified 80 cDNAs whose expression in normal and tumor samples differed more than 3.5 sample standard deviations. Using Self-Organizing Map, the expression profile of these cDNAs allowed perfect separation of malignant and non-malignant samples. Using the supervised learning procedure Support Vector Machine, we identified trios of cDNAs that could be used to classify samples as normal or tumor, based on single-array analysis. Finally, we identified genes with altered linear correlation when their expression in normal and tumor samples were compared. Further investigation concerning the function of these genes could contribute to the understanding of gastric carcinogenesis and may prove useful in molecular diagnostics.
Cancer Letters 03/2003; 190(2):199-211. · 4.24 Impact Factor
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Sandro J de Souza,
Anamaria A Camargo,
Marcelo R S Briones,
Fernando F Costa,
Maria Aparecida Nagai,
Sergio Verjovski-Almeida,
Marco A Zago,
Luis Eduardo C. Andrade,
Helaine Carrer,
Hamza F. A. El-Dorry, [......],
Daniel F. Simão,
Josane F Sousa,
Daniella Stecconi,
Fernando Tsukumo,
Valéria Valente,
Heloisa Zalcberg,
Ricardo R Brentani,
Luis F. L. Reis,
Emmanuel Dias-Neto,
Andrew J G Simpson
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ABSTRACT: Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1,181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html).
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Anamaria A Camargo,
Helena P. B. Samaia,
Emmanuel Dias-Neto,
Daniel F. Simão,
Italo A. Migotto,
Marcelo R S Briones,
Fernando F Costa,
Maria Aparecida Nagai,
Sergio Verjovski-Almeida,
Marco A Zago, [......],
Fernando Soares,
Eloisa S. Moreira,
Diana N Nunes,
Ricardo G Correa,
Heloisa Zalcberg, Alex F Carvalho,
Luis F. L. Reis,
Ricardo R Brentani,
Andrew J G Simpson,
Sandro J de Souza
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ABSTRACT: Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription–PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.
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Emmanuel Dias-Neto,
Ricardo Garcia Correa,
Sergio Verjovski-Almeida,
Marcelo R S Briones,
Maria Aparecida Nagai,
Wilson da Silva,
Marco Antonio Zago,
Silvana Bordin,
Fernando Ferreira Costa,
Gustavo Henrique Goldman, [......],
Adriana Brunstein,
Paulo S L de Oliveira,
Philipp Bucher,
C Victor Jongeneel,
Michael J O'Hare,
Fernando Soares,
Ricardo R Brentani,
Luis F. L. Reis,
Sandro J de Souza,
Andrew J G Simpson
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ABSTRACT: Theoretical considerations predict that amplification of expressed gene transcripts by reverse transcription–PCR using arbitrarily chosen primers will result in the preferential amplification of the central portion of the transcript. Systematic, high-throughput sequencing of such products would result in an expressed sequence tag (EST) database consisting of central, generally coding regions of expressed genes. Such a database would add significant value to existing public EST databases, which consist mostly of sequences derived from the extremities of cDNAs, and facilitate the construction of contigs of transcript sequences. We tested our predictions, creating a database of 10,000 sequences from human breast tumors. The data confirmed the central distribution of the sequences, the significant normalization of the sequence population, the frequent extension of contigs composed of existing human ESTs, and the identification of a series of potentially important homologues of known genes. This approach should make a significant contribution to the early identification of important human genes, the deciphering of the draft human genome sequence currently being compiled, and the shotgun sequencing of the human transcriptome.
