RESUMO
The complete sequence and genomic characterization of the tyrosine aminotransferase (TAT) gene from Trypanosoma rangeli is reported. The gene was found to be organized in a tandem multicopy gene array. A homologous mRNA species (2.5 kb) was identified in the epimastigote form of the parasite. From the deduced amino acid sequence, the gene encodes a protein of 420 amino acids with a predicted molecular mass of 46.4 kDa and a theoretical pI of 6.23. A high sequence identity was found with the Trypanosoma cruzi, human and rat enzymes. All the essential residues for TAT enzymatic activity are conserved, as well as a pyridoxal-phosphate attachment site typical of class-I aminotransferases. The recombinant enzyme was recognized by a monoclonal antibody against the T. cruzi enzyme. Additionally, the recombinant protein showed enzymatic activity when incubated with L-tyrosine and 2-oxoglutaric acid as substrates.
Assuntos
Trypanosoma/enzimologia , Trypanosoma/genética , Tirosina Transaminase/genética , Sequência de Aminoácidos , Animais , Genes de Protozoários , Genoma , Humanos , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Ratos , Alinhamento de Sequência , Análise de SequênciaRESUMO
Most molecular trees of trypanosomatids are based on point mutations within DNA sequences. In contrast, there are very few evolutionary studies considering DNA (re) arrangement as genetic characters. Waiting for the completion of the various parasite genome projects, first information may already be obtained from chromosome size-polymorphism, using the appropriate algorithms for data processing. Three illustrative models are presented here. First, the case of Leishmania (Viannia) braziliensis/L. (V.) peruviana is described. Thanks to a fast evolution rate (due essentially to amplification/deletion of tandemly repeated genes), molecular karyotyping seems particularly appropriate for studying recent evolutionary divergence, including eco-geographical diversification. Secondly, karyotype evolution is considered at the level of whole genus Leishmania. Despite the fast chromosome evolution rate, there is qualitative congruence with MLEE- and RAPD-based evolutionary hypotheses. Significant differences may be observed between major lineages, likely corresponding to major and less frequent rearrangements (fusion/fission, translocation). Thirdly, comparison is made with Trypanosoma cruzi. Again congruence is observed with other hypotheses and major lineages are delineated by significant chromosome rearrangements. The level of karyotype polymorphism within that "species" is similar to the one observed in "genus" Leishmania. The relativity of the species concept among these two groups of parasites is discussed.
Assuntos
Evolução Molecular , Rearranjo Gênico , Genoma de Protozoário , Trypanosomatina/genética , Animais , Cariotipagem , Leishmania braziliensis/citologia , Leishmania braziliensis/genética , Polimorfismo Genético , Trypanosoma cruzi/citologia , Trypanosoma cruzi/genéticaRESUMO
Forty-eight cDNA clones obtained from different developmental stages of Trypanosoma cruzi and all encoding the C-terminal domain of the major cysteine proteinase (cruzipain) have been sequenced. A number of polymorphisms were detected, seven of them resulting in amino acid replacements. The predicted pI values of the corresponding gene products varied between 7.05 and 8.12. These changes in amino acid sequence, together with previously reported variations in carbohydrate composition at the only N-glycosylation site in the C-terminal domain, may account for most of the heterogeneities found in the mature enzyme.
Assuntos
Cisteína Endopeptidases/genética , Polimorfismo Genético , Trypanosoma cruzi/genética , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Clonagem Molecular , Cisteína Endopeptidases/química , DNA Complementar , DNA de Protozoário/análise , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas de Protozoários , Homologia de Sequência de Aminoácidos , Trypanosoma cruzi/enzimologiaRESUMO
Genes encoding a 29-kDa flagellar calcium-binding protein (F29) in Trypanosoma cruzi, strongly homologous to EF-hand calcium-binding protein-encoding genes previously reported in this parasite, were isolated by immunoscreening. F29 is encoded by a number of very similar genes, highly conserved among different T. cruzi isolates. The genes are located on a pair of homologous chromosomes, arranged in one or two clusters of tandem repeats. PCR amplification of Trypanosoma rangeli genomic DNA, using primers derived from the T. cruzi F29 sequence made it possible to isolate the homologous gene in T. rangeli, encoding a 23-kDa protein called TrCaBP. Gene sequence comparisons showed homology to EF-hand calcium-binding proteins from T. cruzi (82.8%), Trypanosoma brucei brucei (60.2%), and Entamoeba histolytica (28.4%). Northern blot analysis revealed that the TrCaBP gene is expressed in T. rangeli as a polyadenylated transcript. The TrCaBP-encoding genes are present in at least 20 copies per cell, organized in tandem arrays, on large T. rangeli chromosomes in some isolates and on two smaller ones in others. This gene, however, seems to be absent from Leishmania.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Sequência Conservada , Trypanosoma cruzi/genética , Trypanosoma/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ligação ao Cálcio/química , Mapeamento Cromossômico , Clonagem Molecular , DNA de Protozoário/química , Flagelos/química , Dados de Sequência Molecular , Família Multigênica , Reação em Cadeia da Polimerase , Proteínas de Protozoários/análise , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , RNA de Protozoário/análise , Sequências Repetitivas de Ácido Nucleico , Alinhamento de Sequência , Trypanosoma/metabolismo , Trypanosoma cruzi/metabolismoRESUMO
PCR amplification of genomic DNA from the American trypanosome, Trypanosoma rangeli, using as primers oligonucleotides derived from the gene of cruzipain, the major cysteine proteinase (CP) from Trypanosoma cruzi, allowed the production of a probe which was used to obtain three clones encoding a CP with 70% overall identity with cruzipain. The genes are organized in tandem, with a monomere size of approximately 2 kbp, located on two chromosomes which, in some parasite isolates, have a high molecular mass (higher than 5.7 Mbp), and in others are much smaller (about 500 kbp). The low expression of this CP at the protein level correlates well with the low level of specific mRNA found in Northern blots.
Assuntos
Cisteína Endopeptidases/genética , Trypanosoma/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Genoma de Protozoário , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Forty-three Trypanosoma cruzi isolates from Chile and Colombia and three cloned stocks from Bolivia and Brazil were studied at the karyotype level by hybridization with four different parasite gene probes to chromosomes separated by pulsed-field gel electrophoresis. The results showed that classification of parasite isolates based on isoenzyme analysis at 12 or more genetic loci correlated with the classification obtained by molecular karyotype analysis. However, less correlation was found between molecular karyotypes and the zymodemes Z1, Z2Bra, Z2Bol, and Z3 based on analysis at only two genetic loci. All the four probes used in this study allowed differentiation between different T. cruzi stocks but the SAPA and the antigen 13 probes were most informative. Isolates which were unclassified at the isoenzyme level were also studied and in most cases similar hybridization patterns were observed as obtained with one or more isoenzyme-classified isolates. The results demonstrate the potential of using molecular karyotyping as a tool for classification. A few pulsed-field gel electrophoresis hybridization experiments provide the same information as obtained by isoenzyme analysis using a dozen or more enzymes. The clonal theory of T. cruzi propagation is supported by our results since the strong correlation between isoenzyme classification and molecular karyotype is difficult to explain with a sexual mode of replication. No minichromosomes were detected in any of the T. cruzi samples studied. Neither was any strong correlation found between the clinical manifestations of Chagas' disease and the molecular karyotypes of the T. cruzi isolates.
Assuntos
Isoenzimas/análise , Trypanosoma cruzi/classificação , Animais , Chile , Colômbia , Sondas de DNA , DNA de Protozoário/análise , Eletroforese em Gel de Campo Pulsado , Humanos , Isoenzimas/genética , Cariotipagem , Hibridização de Ácido Nucleico , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genéticaRESUMO
The complete sequence of a gene encoding a 46-kDa protein of Trypanosoma cruzi is presented. The first ATG complies with the consensus sequence for initiation of translation. A single band of 2 kb was highlighted by hybridizing a probe from the 46-kDa protein gene to a Northern filter containing total T. cruzi RNA. The gene is present in 50-80 copies per cell and most of them are contained in 2 tandem arrays on large T. cruzi chromosomes (> 2000 kb). A strong homology with rat and human tyrosine aminotransferase was detected. Homology with a Trypanosoma brucei retrotransposon was found in the nonsense strand of the intergenic region.
Assuntos
DNA de Protozoário/genética , Genes de Protozoários , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Tirosina Transaminase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA de Protozoário/isolamento & purificação , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Peso Molecular , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Ratos , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Trypanosoma cruzi/enzimologiaAssuntos
Glicoproteínas/genética , Neuraminidase/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Sequência de Bases , DNA de Protozoário/genética , Genes de Protozoários , Dados de Sequência Molecular , Família Multigênica , Sinais Direcionadores de Proteínas/genética , Homologia de Sequência de Aminoácidos , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/imunologiaRESUMO
In American man can be infected with two trypanosomes: Trypanosoma cruzi, the etiological agent of Chagas' disease and Trypanosoma rangeli, a suspected nonpathogenic parasite. In this communication are presented 4 methods in order to improve the current knowledge about the specific identification of these parasites. Using the SDS-PAGE technique it was possible to differentiate between T. rangeli. and T. cruzi based in at less 4 protein bands with a relative molecular weights of 93, 77-73, 63 and 54-52 KDa. These polypeptides were found only in T. rangeli electrophoretic profiles. An ELISA test showed that the antigenic composition found in the enzyme cisteine proteinase (cruzipain) is specific for T. cruzi epimastigotes. Antigenic analysis by Western blot assay, proved that T. rangeli and not T. cruzi present antigenic bands with a Mr of 142, 63, 54, 51, 49, 43, 39 and 24 KDa. Finally, using the Southern blot procedure, it was confirmed that SAPA, a DNA sequence originally identified in the T. cruzi, genome, is absent in T. rangeli nuclear DNA. These initial observations revealed that it is possible to identify both parasites using the described methods, however further works are required to clarify the biochemical, immunological and molecular relationship between T. rangeli and T. cruzi.
Assuntos
Trypanosoma/isolamento & purificação , Animais , Antígenos de Protozoários/análise , Southern Blotting , Western Blotting , DNA de Protozoário/análise , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Estudos de Avaliação como Assunto , Trypanosoma/classificação , Trypanosoma/genética , Trypanosoma/imunologiaRESUMO
In American man can be infected with two trypanosomes: Trypanosoma cruzi, the etiological agent of Chagas' disease and Trypanosoma rangeli, a suspected nonpathogenic parasite. In this communication are presented 4 methods in order to improve the current knowledge about the specific identification of these parasites. Using the SDS-PAGE technique it was possible to differentiate between T. rangeli. and T. cruzi based in at less 4 protein bands with a relative molecular weights of 93, 77-73, 63 and 54-52 KDa. These polypeptides were found only in T. rangeli electrophoretic profiles. An ELISA test showed that the antigenic composition found in the enzyme cisteine proteinase (cruzipain) is specific for T. cruzi epimastigotes. Antigenic analysis by Western blot assay, proved that T. rangeli and not T. cruzi present antigenic bands with a Mr of 142, 63, 54, 51, 49, 43, 39 and 24 KDa. Finally, using the Southern blot procedure, it was confirmed that SAPA, a DNA sequence originally identified in the T. cruzi, genome, is absent in T. rangeli nuclear DNA. These initial observations revealed that it is possible to identify both parasites using the described methods, however further works are required to clarify the biochemical, immunological and molecular relationship between T. rangeli and T. cruzi
Assuntos
Animais , Trypanosoma/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Antígenos de Protozoários/análise , DNA de Protozoário/análise , Eletroforese em Gel Bidimensional , Estudo de Avaliação , Southern Blotting , Trypanosoma/classificação , Trypanosoma/genética , Trypanosoma/imunologia , Western BlottingRESUMO
We demonstrate that cruzipain, the major cysteine proteinase of Trypanosoma cruzi epimastigotes, is encoded by a large number of tandemly arranged genes. Restriction enzyme analysis of 20 clones containing complete repeat units of the gene, as well as sequencing of 2 of these clones, and comparison with previously published partial sequences, indicated that the sequence is conserved among the repeat units, although polymorphisms clearly exist. The repeat units contain an intergenic region of 528 bp and coding regions for pre- and pro-enzyme, a central domain and a C-terminal extension. The predicted amino acid sequences of these regions indicated a sequence identity of 30, 60, 70 and 36%, respectively, when the T. cruzi sequence was compared with the sequence of a similar cysteine proteinase from Trypanosoma brucei. Studies by pulsed field gel electrophoresis, complemented with restriction analysis, indicated that the clusters are located on 2-4 different chromosomes in several parasite isolates.