A molecular study on the evolution of a subtype B variant frequently found in Brazil.

In spite of the remarkable diversity of HIV-1 env genes, several amino acids are extremely conserved, probably due to functional constraints. One example is the proline found at the second position of the GPGR motif. Several viruses, however, bear substitutions at this site, for instance, GWGR subtype B variant. GWGR viruses are described in Brazil since the beginning of the epidemics, but the extent of their dispersion or the geographical origin of the variant remains unknown. In the present study, phylogenetic trees were constructed in order to study the origin and spread of this variant. All GWGR sequences as well as a subset of subtype B sequences available were included in the analyses. Analyses of differential selection were also performed on GWGR and non-GWGR sequences in order to unveil evolutionary novelties due to the action of positive selection. Although the GWGR variant was found at least in 23 countries, its expansion probably has a single origin, and Brazil is the epicenter.

In silico reconstruction of the amino acid metabolic pathways of Trypanosoma cruzi.

Trypanosoma cruzi is the epidemiological agent of Chagas' disease, affecting most of Central and South America, constituting a significant health and socio-economic problem. The parasite has a metabolism largely based on the consumption of amino acids, which participate in a diversity of metabolic pathways, leading to many crucial compounds for the survival of this parasite. Study of its enzymes has the potential to disclose new therapeutic targets and foster the development of new drugs. In this study, we employed computational approaches to reconstruct in silico the amino acid metabolic pathways of T. cruzi, aiming to link genomic information with functional information. For that, protein sequences from 570 EC classes belonging to 25 different pathways in general amino acid metabolism were downloaded from KEGG. A subset of 471 EC classes had at least one sequence deposited. Clustering of the proteins belonging to each EC class was performed using a similarity-based approach implemented in the tool AnEnPi. Reconstruction of the metabolic pathways comprising the amino acid metabolism of T. cruzi was performed by analyzing the output of BLASTP, using as query the dataset of predicted proteins of T. cruzi against all sequences of each individual cluster. This approach allowed us to identify 764 T. cruzi proteins probably involved in the metabolism of amino acids as well as the identification of several putative cases of analogy. Furthermore, we were able to identify several enzymatic activities of T. cruzi that were not previously included in KEGG.

Use of PCR-mediated amplification of Mycobacterium leprae DNA in different types of clinical samples for the diagnosis of leprosy.

DNA of Mycobacterium leprae, obtained by a highly efficient nucleic acid extraction procedure, was used for standardisation of the amplification of an M. leprae-specific repetitive sequence by use of the polymerase chain reaction (PCR). With pure DNA, M. leprae-specific amplification was obtained with as low as 100 ag (1 ag = 10(-18) g) of target DNA, a quantity equal to about one-tenth of the bacterial genome. Optimal processing of different types of clinical samples such as biopsy material, blood and lymph fluid, from multibacillary leprosy patients, was studied. Simple freezing-boiling cycles in the presence of Triton X100, with some additional sample-specific modifications such as pre-treatment with NaOH to eliminate PCR inhibitors, was found to be sufficient to yield amplification of bacterial DNA in samples from paucibacillary patients. Clinical samples from 27 untreated leprosy patients, covering the various clinical forms of the disease, and with a bacterial index ranging from 5+ to 0, were collected and processed for PCR analysis. After hybridisation of the amplified material with a specific sequence, 25 of 27 patients analysed gave positive results for M. leprae in at least one of the samples. The potential of PCR for the diagnosis of leprosy is discussed.

Use of PCR-restriction fragment length polymorphism analysis of the hsp65 gene for rapid identification of mycobacteria in Brazil.

Polymerase chain reaction amplification of part of the gene coding for the heat shock protein hsp65 followed by restriction enzyme analysis (PRA) is a recently described tool for rapid identification of mycobacteria. In this study, the speed and simplicity of PRA for identification of isolates of mycobacteria from patients with clinical symptoms of tuberculosis was evaluated and compared with identification results obtained by commercially available methods. Established PRA patterns were observed for nineteen isolates of Mycobacterium tuberculosis, eleven belonging to the complex M. avium-intracellulare, four of M. kansasii, one of M. fortuitum, one of M. abscessus, three of M. gordonae and one of the recently described species M. lentiflavum, as identified by commercially available methods. Two isolates of M. fortuitum and one of M. gordonae had unique and so far undescribed PRA patterns, suggesting geographically-related intra-species variation within the hsp65 sequence. We propose the inclusion of these new patterns in the PRA identification algorithm and have defined more accurately the molecular weight values of the restriction fragments. This is the first report on the isolation of M. lentiflavum in Brazil suggesting that identification by means of PRA could be useful for detection of mycobacterial species that are usually unnoticed. Where the use of several commercial techniques in combination was necessary for correct identification, PRA demonstrated to be a simple technique with good cost-benefit for characterization of all mycobacterial isolates in this study.

Gene expression, amino acid conservation, and hydrophobicity are the main factors shaping codon preferences in Mycobacterium tuberculosis and Mycobacterium leprae.

Mycobacterium tuberculosis and Mycobacterium leprae are the ethiological agents of tuberculosis and leprosy, respectively. After performing extensive comparisons between genes from these two GC-rich bacterial species, we were able to construct a set of 275 homologous genes. Since these two bacterial species also have a very low growth rate, translational selection could not be so determinant in their codon preferences as it is in other fast-growing bacteria. Indeed, principal-components analysis of codon usage from this set of homologous genes revealed that the codon choices in M. tuberculosis and M. leprae are correlated not only with compositional constraints and translational selection, but also with the degree of amino acid conservation and the hydrophobicity of the encoded proteins. Finally, significant correlations were found between GC3 and synonymous distances as well as between synonymous and nonsynonymous distances.

TcruziDB, an integrated database, and the WWW information server for the Trypanosoma cruzi genome project.

Data analysis, presentation and distribution is of utmost importance to a genome project. A public domain software, ACeDB, has been chosen as the common basis for parasite genome databases, and a first release of TcruziDB, the Trypanosoma cruzi genome database, is available by ftp from ftp://iris.dbbm.fiocruz.br/pub/genomedb/Tcr uziDB as well as versions of the software for different operating systems (ftp://iris.dbbm.fiocruz.br/pub/unixsoft/). Moreover, data originated from the project are available from the WWW server at http://www.dbbm.fiocruz.br. It contains biological and parasitological data on CL Brener, its karyotype, all available T. cruzi sequences from Genbank, data on the EST-sequencing project and on available libraries, a T. cruzi codon table and a listing of activities and participating groups in the genome project, as well as meeting reports. T. cruzi discussion lists (tcruzil@iris.dbbm.fiocruz.br and tcgenics@iris.dbbm.fiocruz.br) are being maintained for communication and to promote collaboration in the genome project.

Identification of transcribed sequences (ESTs) in the Trypanosoma cruzi genome project.

Random single pass sequencing of cDNA fragments, also known as generation of Expressed Sequence Tags (ESTs), has been highly successful in the study of the gene content of higher organisms, and forms an integral part of most genome projects, with the objective to identify new genes and targets for disease control and prevention and to generate mapping probes. In the Trypanosoma cruzi genome project, EST sequencing has also been a starting point, and here we report data on the first 797 sequences obtained, partly from a CL Brener epimastigote non-normalized library, partly on a normalized library. Only around 30% of the sequences obtained showed similarity with Genbank and dbEST databases, half of which with sequences already reported for T. cruzi.

An oligonucleotide probe derived from kDNA minirepeats is specific for Leishmania (Viannia).

Sequence analysis of Leishmania (Viannia) kDNA minicircles and analysis of multiple sequence alignments of the conserved region (minirepeats) of five distinct minicircles from L. (V.) braziliensis species with corresponding sequences derived from other dermotropic leishmanias indicated the presence of a sub-genus specific sequence. An oligonucleotide bearing this sequence was designed and used as a molecular probe, being able to recognize solely the sub-genus Viannia species in hybridization experiments. A dendrogram reflecting the homologies among the minirepeat sequences was constructed. Sequence clustering was obtained corresponding to the traditional classification based on similarity of biochemical, biological and parasitological characteristics of these Leishmania species, distinguishing the Old World dermotropic leishmanias, the New World dermotropic leishmanias of the sub-genus Leishmania and of the sub-genus Viannia.

Use of PCR for the determination of the frequency of the delta F508 mutation in Brazilian cystic fibrosis patients.

The delta F508 mutation in the cystic fibrosis (CF) gene was studied in a population of 18 Brazilian CF patients and their 17 families by use of PCR and differential hybridization with oligonucleotides. In a total of 34 chromosomes considered, 12 (35%) carried the F508 deletion, a frequency much lower than that reported in most other populations. As a consequence, CF in Brazil would be predominantly caused by mutations different from the F508 deletion.

GenoMycDB: a database for comparative analysis of mycobacterial genes and genomes

Several databases and computational tools have been created with the aim of organizing, integrating and analyzing the wealth of information generated by large-scale sequencing projects of mycobacterial genomes and those of other organisms. However, with very few exceptions, these databases and tools do not allow for massive and/or dynamic comparison of these data. GenoMycDB (http://www.dbbm.fiocruz.br/GenoMycDB) is a relational database built for large-scale comparative analyses of completely sequenced mycobacterial genomes, based on their predicted protein content. Its central structure is composed of the results obtained after pair-wise sequence alignments among all the predicted proteins coded by the genomes of six mycobacteria: Mycobacterium tuberculosis (strains H37Rv and CDC1551), M. bovis AF2122/97, M. avium subsp. paratuberculosis K10, M. leprae TN, and M. smegmatis MC2 155. The database stores the computed similarity parameters of every aligned pair, providing for each protein sequence the predicted subcellular localization, the assigned cluster of orthologous groups, the features of the corresponding gene, and links to several important databases. Tables containing pairs or groups of potential homologs between selected species/strains can be produced dynamically by user-defined criteria, based on one or multiple sequence similarity parameters. In addition, searches can be restricted according to the predicted subcellular localization of the protein, the DNA strand of the corresponding gene and/or the description of the protein. Massive data search and/or retrieval are available, and different ways of exporting the result are offered. GenoMycDB provides an on-line resource for the functional classification of mycobacterial proteins as well as for the analysis of genome structure, organization, and evolution.