Arthur Gruber

Molecular karyotype of clone CL Brener chosen for the Trypanosoma cruzi Genome Project

a Escola Paul&a de Medicina, Rua Botucatu, 862, CEP 04023-062, Stio Pa&o, Brazil b Institute de Quimica da USP, Sao Paula, Brazil ’ Institute de Inuestigaciones en Engenharia Genetica y Biologia Molecular, Buenos Aires. Argentina ’ Instituto de Parasitologia y Biomedicina, Granada, Spain e FIOCRUZ, Rio de Janeiro, Brazil f Instituto de Biofisica, UFRJ, Rio de Janeiro, Brazil g Centro de Biologia Celular, XV, Caracas, Venezuela h Centro de Biologia Molecular, IJAM_ Madrid, Spain

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome

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.

The generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags

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 ≈23,500 genes, of which only ≈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.

A Selective Review of Advances in Coccidiosis Research

Coccidiosis is a widespread and economically significant disease of livestock caused by protozoan parasites of the genus Eimeria. This disease is worldwide in occurrence and costs the animal agricultural industry many millions of dollars to control. In recent years, the modern tools of molecular biology, biochemistry, cell biology and immunology have been used to expand greatly our knowledge of these parasites and the disease they cause. Such studies are essential if we are to develop new means for the control of coccidiosis. In this chapter, selective aspects of the biology of these organisms, with emphasis on recent research in poultry, are reviewed. Topics considered include taxonomy, systematics, genetics, genomics, transcriptomics, proteomics, transfection, oocyst biogenesis, host cell invasion, immunobiology, diagnostics and control.

Genomic analysis of the causative agents of coccidiosis in domestic chickens

Global production of chickens has trebled in the past two decades and they are now the most important source of dietary animal protein worldwide. Chickens are subject to many infectious diseases that reduce their performance and productivity. Coccidiosis, caused by apicomplexan protozoa of the genus Eimeria, is one of the most important poultry diseases. Understanding the biology of Eimeria parasites underpins development of new drugs and vaccines needed to improve global food security. We have produced annotated genome sequences of all seven species of Eimeria that infect domestic chickens, which reveal the full extent of previously described repeat-rich and repeat-poor regions and show that these parasites possess the most repeat-rich proteomes ever described. Furthermore, while no other apicomplexan has been found to possess retrotransposons, Eimeria is home to a family of chromoviruses. Analysis of Eimeria genes involved in basic biology and host-parasite interaction highlights adaptations to a relatively simple developmental life cycle and a complex array of co-expressed surface proteins involved in host cell binding.

EGene: a configurable pipeline generation system for automated sequence analysis

UNLABELLED EGene is a generic, flexible and modular pipeline generation system that makes pipeline construction a modular job. EGene allows for third-party programs to be used and integrated according to the needs of distinct projects and without any previous programming or formal language experience being required. EGene comes with CoEd, a visual tool to facilitate pipeline construction and documentation. A series of components to build pipelines for sequence processing is provided. AVAILABILITY http://www.lbm.fmvz.usp.br/egene/ CONTACT alan@ime.usp.br; argruber@usp.br SUPPLEMENTARY INFORMATION http://www.lbm.fmvz.usp.br/egene/

Biological shape characterization for automatic image recognition and diagnosis of protozoan parasites of the genus Eimeria

We describe an approach of automatic feature extraction for shape characterization of seven distinct species of Eimeria, a protozoan parasite of domestic fowl. We used digital images of oocysts, a round-shaped stage presenting inter-specific variability. Three groups of features were used: curvature characterization, size and symmetry, and internal structure quantification. Species discrimination was performed with a Bayesian classifier using Gaussian distribution. A database comprising 3891 micrographs was constructed and samples of each species were employed for the training process. The classifier presented an overall correct classification of 85.75%. Finally, we implemented a real-time diagnostic tool through a web interface, providing a remote diagnosis front-end.

A survey of the inter- and intraspecific RAPD markers of Eimeria spp. of the domestic fowl and the development of reliable diagnostic tools

Coccidiosis of domestic fowl is a protozoan disease, caused by seven distinct species of the genus Eimeria, which is responsible for important economic losses in poultry production. In order to select RAPD primers for the discrimination of these seven Eimeria species, we carried out an initial screening using samples of E. acervulina, E. tenella and E. maxima. Out of 150 primers tested, 110 generated band profiles specific for each one of these species. A subset of 14 oligonucleotides were also tested for the simultaneous differentiation of the seven species, resulting in 11 discriminative primers. The intraspecific discrimination was assessed for five different species, using samples from different geographic regions including three continents. Numerous primers exhibited highly discriminative band profiles containing strain-specific markers, with a higher variability being observed among strains of E. acervulina than among E. tenella and E. maxima strains. However, no major differences were observed in the band patterns from strains collected in locations near to one another compared to strains originating from distantly located regions. Because RAPD is a technique performed under low stringency conditions, it suffers from poor reproducibility. Aiming at obtaining more reliable markers that might be universally used, we started an effort to convert species-specific RAPD fragments into SCAR markers. An initial conversion of 25 RAPD markers into SCARs, followed by validation of their specificity, resulted in 14 totally new Eimeria species-specific markers that can be used for the molecular diagnosis of the seven species that infect domestic fowl. This work represents a first step in the development of a set of species-specific SCARs that will be useful as tools for molecular diagnosis, genome mapping, and genetic diversity studies.

TRAP: automated classification, quantification and annotation of tandemly repeated sequences

TRAP, the Tandem Repeats Analysis Program, is a Perl program that provides a unified set of analyses for the selection, classification, quantification and automated annotation of tandemly repeated sequences. TRAP uses the results of the Tandem Repeats Finder program to perform a global analysis of the satellite content of DNA sequences, permitting researchers to easily assess the tandem repeat content for both individual sequences and whole genomes. The results can be generated in convenient formats such as HTML and comma-separated values. TRAP can also be used to automatically generate annotation data in the format of feature table and GFF files.

Trypanosoma cruzi defined antigens in the serological evaluation of an outbreak of acute Chagas disease in Brazil (Catolé do Rocha, Paraíba).

Immunoglobulin G and M humoral response to recombinant protein B13 and glycoconjugate LPPG Trypanosoma cruzi defined antigens was evaluated by ELISA in 18 patients in the acute phase of Chagas disease, who were contaminated on the same occasion. LPPG showed 100% positivity detecting both IgM and IgG antibodies, while positivity of 55-65% was observed for B13. An epimastigote alkaline extract (EPI) also showed high sensitivity for acute IgM (100%) and IgG (90%) antibodies. However LPPG had better discriminatory reactivity since with EPI two patients showed negative IgG, and several other sera presented OD values for IgG and IgM antibodies very close to the cutoff. Thus, it is suggested that detection of IgM antibodies by LPPG may be used for diagnosis of the acute phase of Chagas disease. An intense decline of IgG and IgM antibodies to the three antigens was observed in response to anti-T. cruzi chemotherapy in all acute phase patients. After treatment, six (30%) individuals maintained IgG positivity to EPI, LPPG, and B13 with lower reactivity than that measured at the acute phase. For comparison, serology of a group of 22 patients in the chronic phase of Chagas disease and also submitted to chemotherapy was determined. Positive IgM antibodies to EPI, LPPG and B13 were detected in only 5-9% cases. In all chronic-phase patients IgG antibodies highly reactive to the three antigens were present and no significant decrease resulted after benznidazole administration. These observations reinforce previous reports that treatment in the acute phase may reduce or eliminate the parasite.