RESUMO
Trypanosoma cruzi, the causative agent of Chagas disease, has evolved particular mechanisms of gene regulation. Gene expression is regulated firstly at post-transcriptional level. This feature makes proteomic methods a promising tool for studying adaptative changes in these parasites. In this work we generated a reproducible method for protein analysis by two-dimensional electrophoresis coupled to mass spectrometry, and a protein map for T. cruzi. Western-blot analysis supported the identity of some of the proteins. This work points to proteomic approach as a powerful tool to study differential expression, stress response or drug resistance in T. cruzi.
Assuntos
Doença de Chagas/genética , Proteoma/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Animais , Western Blotting/métodos , Ciclo Celular/genética , Doença de Chagas/metabolismo , Eletroforese em Gel Bidimensional/métodos , Expressão Gênica/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Most of the yeast strains used in fermented beverages and foods are classified as Saccharomyces cerevisiae. However, different strains are suitable for different fermentation processes. The purpose of this work is the proposal of a standardized methodology for the molecular genotyping of S. cerevisiae strains based on polymorphisms at microsatellite loci and/or single nucleotide polymorphisms (SNPs). Single nucleotide variants in the coding region of FLO8, a key regulator of flocculation and pseudohyphae formation, were analyzed in a subset of Uruguayan wine strains. Polymorphism analysis at nine microsatellite loci (selected from 33 loci tested) was performed in a collection of 120 strains, mostly wine strains, from different origins. From a total of 184 different alleles scored, 50 were exclusive alleles that could identify 29 strains. Four selected microsatellite loci are located within or near genes of putative enological interest. The Uruguayan strains are highly diverse and evenly distributed in the phylogenetic reconstructions, suggesting an evolutionary history previous to human use. The Saccharomyces cerevisiae Microsatellites and SNPs Genotyping Database is presented (www.pasteur.edu.uy/yeast). Comparison of standardized results from strains coming from different settings (industrial, clinical, environmental) will provide a reliable and growing source of information on the molecular biodiversity of S. cerevisiae strains.