The untranslated regions of genes from Trypanosoma cruzi: perspectives for functional characterization of strains and isolates

The sequencing of Trypanosoma cruzi genome has been completed and a great deal of information is now available. However, the organization of protozoa genomes is somewhat elusive and much effort must be applied to reveal all the information coded in the nucleotide sequences. Among the DNA segments that needs further investigation are the untranslated regions of genes. Many of the T. cruzi genes that were revealed by the genome sequencing lack information about the untranslated regions. In this paper, some features of these untranslated segments as well as their applications in T. cruzi populations are discussed.

Calcium-dependent metabolic regulations in prokaryotes indicate conserved nature of calmodulin gene.

Role of free calcium and calcium binding protein calmodulin as signal molecule in cellular regulation is well established in eukaryotes. However, reports on Ca(2+)-dependent processes and their inhibition by calcium and/or calmodulin antagonists indicate towards the presence of calmodulin in prokaryotes as well. The common evolutionary origin of pro- and eukaryotes and many examples of evolutionary conservation of structure and functions support the contention of such conservation of the role of Ca2+ and calmodulin. Eukaryotic calmodulin (CaM) contains four structurally and functionally similar Ca2+ domains named I, II, III and IV. Each Ca2+ binding loop consists of 12 amino acid residues with ligands arranged spatially to satisfy the octahedral symmetry of Ca2+ binding. Plant calmodulin differ from vertebrate ones in 13 to 14 amino acid positions of which nine occur at -COOH- terminal half. Differences between protozoan and mammalian CaM also occur mostly in the same half. Isolation and characterization, although to a little extent, of CaM-like proteins from bacteria and cyanobacteria and their comparison with CaMs from diverse origin suggest high degree of conservation. Non-bulky amino acids like glycine, alanine and serine with low specific rotation are present in greater number in the primitive form of calmodulin and have been significantly reduced in highly evolved form of calmodulin, suggesting that their requirement was insignificant and were eliminated from EF hand structure during evolution. However, amino acids like glutamate/glutamine and aspartate/asparagine were highly conserved and did not show any major change in their frequency since their positions are too significant in calcium binding domain. While the number of positively charged amino acids like arginine and leucine was increased, histidine containing weakly ionized group and having a significant buffering capacity was reduced to a major extent, further suggesting that the acidic nature of calmodulin protein has been maintained during evolution. Thus it is now clear that the entire superfamily of Ca2+ binding proteins have arisen from a common genetic ancestry. Two successive tandem duplications of gene encoding a single domain containing protein of 30-40 residues gave rise to a four domain molecule from which this family was then derived.

Caracterización parcial de la calmodulina de Plasmodium falciparum / Partial characterization of Plasmodium falciparum calmoduline

Se describe una alternativa para la purificación parcial de Calmodulina (CaM) a partir de Plasmodium falciparum, que de acuerdo con anticuerpos monoclanes altamente específicos contra CaM, permite separar por lo menos dos formas de la proteína, que aunque difieren en sus pesos moleculares (12.600 y entre 36.000 y 50.000 dalton), son capaces de estimular a la ATPasa de calcio del eritrocito por separado. La forma de menor peso molecular, que es la mejor representada, a diferencia de la CaM purificada de otras fuentes muestra un comportamiento electroforético independiente de la presencia de calcio: lo que conjuntamente con evidencias experimentales que sugieren una disminución en la carga negativa total a pH neutral y en la hidrofobicidad en su estado asociado al calcio, permite plantear la posibilidad de una modificación estructural de la CaM de Plasmodium falciparum, que de todas maneras no interfiere con su función como activador de la ATPasa de calcio. Los estudios cinéticos en presencia de dos inhibidores específicos de CaM (Calmidazolium (CdZ) y W-7) confirman la identidad de la proteína purificada del parásito como CaM y por otro lado revelan que aparentemente la CaM de Plasmodium falciparum es inhibible en menor grado que la CaM de eritrocito en su función estimuladora de la ATPasa de calcio del eritrocito, lo que hace pensar en una mayor afinidad de la proteína del parásito por ésta enzima o en modificación de la zona regulatoria a la que se unen los inhibidores. Se plantea la posibilidad de que la CaM de Plasmodium falciparum no constituya un blanco ideal para el desarrollo de agentes quimioterapeúticos sintéticos contra la malaria, así como de que los inhibidores de CaM, Calmidazolium (CdZ) y W-7, no sean útiles como drogas para tratar la enfermedad