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.

Personalized diagnosis by cached solutions with hypertension as a study model

Statistical modeling of links between genetic profiles with environmental and clinical data to aid in medical diagnosis is a challenge. Here, we present a computational approach for rapidly selecting important clinical data to assist in medical decisions based on personalized genetic profiles. What could take hours or days of computing is available on-the-fly, making this strategy feasible to implement as a routine without demanding great computing power. The key to rapidly obtaining an optimal/nearly optimal mathematical function that can evaluate the [quot ]disease stage[quot ] by combining information of genetic profiles with personal clinical data is done by querying a precomputed solution database. The database is previously generated by a new hybrid feature selection method that makes use of support vector machines, recursive feature elimination and random sub-space search. Here, to evaluate the method, data from polymorphisms in the renin-angiotensin-aldosterone system genes together with clinical data were obtained from patients with hypertension and control subjects. The disease [quot ]risk[quot ] was determined by classifying the patients' data with a support vector machine model based on the optimized feature; then measuring the Euclidean distance to the hyperplane decision function. Our results showed the association of renin-angiotensin-aldosterone system gene haplotypes with hypertension. The association of polymorphism patterns with different ethnic groups was also tracked by the feature selection process. A demonstration of this method is also available online on the project's web site.

Cystic fibrosis: low frequency of DF508 mutation in 2 population samples from Rio de Janeiro, Brazil.

Blood samples from 44 unrelated cystic fibrosis (CF) patients from Rio de Janeiro, Brazil, were analyzed for the 8 European CF mutations. Six homozygous and 15 heterozygous carriers of the DF508 mutation were found, corresponding to 47.7% of CF patients (allele frequency 0.3068). The G542X and G551D mutations were also observed with allele frequencies of 0.0227 and 0.0114, respectively. An analysis of the DF508 mutation in 291 randomly chosen, healthy individuals was performed, and only 3 heterozygous carriers were identified. These results show that the frequency of the DF508 allele in Rio de Janeiro is much lower than the world average; this may be due to the extremely heterogeneous ethnic admixture of the study population. By combining the results of these 2 different samples (CF patients and random population) and admixture data from Rio de Janeiro, we can estimate the CF incidence in this population to be 1:3542 individuals. However, taking into account the Rio de Janeiro ethnic admixture, we can find an estimate of 1:6902 individuals.

Use of PCR in diagnosis of human american tegumentary leishmaniasis in Rio de Janeiro, Brazil.

In Brazil, the most common etiological agent of American tegumentary leishmaniasis is Leishmania (Viannia) braziliensis. In general, diagnostic techniques envisage the visualization of the parasite, but that technique has a low sensitivity. The main purpose of the present work was to evaluate the PCR as a routine tool for the diagnosis of leishmaniasis. Biopsy specimens from cutaneous or mucosal lesions were taken from 230 individuals from areas where Leishmania is endemic: 216 patients who had a clinical picture suggestive of leishmaniasis and 14 individuals with cutaneous lesions due to other causes. Each specimen was processed for histopathologic examination, culture, touch preparation, and DNA isolation. Oligonucleotides that amplify the conserved region of the minicircle molecules of Leishmania were used in a hot-start PCR. While at least one conventional technique was positive for Leishmania for 62% (134 of 216) of the patients, PCR coupled to hybridization was positive for 94% (203 of 216) of the patients. The 14 patients whose clinical picture was not suggestive of leishmaniasis had negative results by all techniques. The impact of the PCR was striking in mucosal disease. While the disease in only 17% (4 of 24) of the patients could be diagnosed by conventional techniques, PCR was positive for 71% (17 of 24) of the patients. Hybridization showed that all cases of disease were caused by parasites belonging to the Viannia subgenus. Altogether, the results indicate that PCR is a valuable tool for the diagnosis of leishmaniasis on a routine basis and is likely to provide valuable epidemiological information about the disease in countries where it is endemic.

DNA fingerprinting of mycobacterium tuberculis from patients with and without AIDS in Rio de Janeiro

Isolates of Mycobacterium tuberculosis derived from patients with AIDS from a single hospital in Rio de Janeiro were typed using a standardized RFLP technique detecting IS6110 polymorphism. Nineteen isolates were obtained from 15 different patients. Eleven distinct IS6110 patterns were found, with 4 banding patterns shared by 2 patients. The clustering value of 53 percent was much higher in comparison with clustering of M. tuberculosis strains from TB patients without clinical signs for HIV infection from randomly selected health centers. We present these results as preliminary data on M. tuberculosis strain polymorphism in Brazil and on the higher risk for recent transmission amongst patients with AIDS.

Towards the physical map of the Trypanosoma cruzi nuclear genome: construction of YAC and BAC libraries of the reference clone T. cruzi CL-Brener

Strategies to construct the physical map of the Trypanosoma cruzi nuclear genome have to capitalize on three main advantage of the parasite genome, namely (a) its small size, (b) the fact that all chromosomes can be defined, and many of them can be isolated by pulse field gel electrophoresis, and (c) the fact that simple Southern blots of electrophoretic karyotypes can be used to map sequence tagged sites and expressed sequence tags to chromosomal bands. A major drawback to cope with is the complexity of T. cruzi genetics, that hinders the construction of a comprehensive genetic map. As a first step towards physical mapping, we report the construction and partial characterization of a T. cruzi CL-Brener genomic library in yeast artificial chromosomes (YACs) that consists of 2.770 individual YACs with a mean insert size of 365 kb encompassing around 10 genomic equivalents. Two libraries in bacterial artificial chromosomes (BACs) have been constructed, BACI and BACII. Both libraries represent about three genome equivalents. A third BAC library (BAC III) is being constructed. YACs and BACs are invaluable tools for physical mapping. More generally, they have to be considered as a common resource for research in Chagas disease.

Comparison of the polymerase chain reaction and serology for the detection of canine visceral leishmaniasis.

The polymerase chain reaction (PCR) and serology was evaluated for the diagnosis of canine visceral leishmaniasis in Bahia, Brazil in a study of 125 dogs. The PCR was 100% sensitive in 25 dogs that had Leishmania demonstrated by either culture or hamster inoculation. It was 100% specific for 35 dogs from the northeastern United States, all were PCR negative. However, 22 of 54 Brazilian dogs that were culture-hamster inoculation-negative were positive by PCR. The nature of the PCR product was identified by hybridization with specific Leishmania probes. Whereas the sensitivity of serology in relationship to infection, as determined by hamster or culture assay was more than 80%, sensitivity of serology was only 63% when compared with PCR. These results raise questions about the use of serology to detect Leishmania infection in dogs, and suggest that the PCR might serve as a better gold standard to define Leishmania infection than culture or hamster inoculation.

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 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.

Cloning and structure of a mouse interleukin-2 chromosomal gene.

Using non-stringent hybridization with a human interleukin-2 cDNA probe, we have isolated recombinant phages from a mouse genomic DNA library cloned in the EMBL3 phage. The sequence and organization of the mouse interleukin-2 (IL-2) gene was determined. By comparison with the human IL-2 sequence, three introns can be identified with lengths of 99, +/- 2 400, and +/- 1 900 base pairs, respectively. The mouse IL-2 gene codes for a polypeptide of 169 amino acids and contains a putative signal peptide of 20 amino acids. The homology to the human interleukin-2 is 72% at the nucleotide level in the coding part and 65% at the amino acid level. An extraordinary sequence, consisting of 12 consecutive CAG codons coding for glutamine, is found in the first exon.

Molecular cloning of human interleukin 2 cDNA and its expression in E. coli.

A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes. Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA. A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal sequence of 20 amino acids. A mature polypeptide starting with either Met-Ala-Pro or Met-Pro was expressed in E. coli under control of the E. coli trp promoter or using a combination of the phage lambda PL promoter and a ribosome binding site derived from phage Mu. The bacterial IL2 polypeptide had a molecular weight of 15,000 daltons and accounted for more than 10% of the total E. coli proteins in fully induced cells; it was biologically active in the T-cell specific DNA synthesis assay, even after recovery from a SDS-containing polyacrylamide gel.