ABSTRACT
Trypanosoma cruzi is the etiological agent of Chagas. Although the nuclear chromatin of this parasite is organized in the form of nucleosome filaments, its chromatin is physically and enzymatically fragile, and no condensation into chromosomes occurs during mitosis. All previous investigations have been carried out with epimastigote form in its proliferate stage. It is not known whether these differences in chromatin structure are also found in the non-proliferate stationary epimastigote forms and in tissue derived trypomastigotes. Our results confirm that chromatin of logarithmic epimastigotes presents limited compaction when increasing salt concentrations from 1 to 100 mM NaCl, and no 30-nm fibers were formed. Contrary to these results, non-proliferative forms of the parasites showed a pattern of compactation similar to that observed in rat liver chromatin, where solenoids of 30-nm fibers are formed at 100-mM NaCl. In accordance with these results, digestion of the nuclear chromatin with DNase I revealed that the chromatin of logarithmic phase epimastigotes was more accessible to the enzyme. We conclude from these results that structural differences in the chromatin exist not only between T. cruzi and higher eukaryotes but also among various forms of the parasite. The functional significance of these differences are currently under investigation.
Subject(s)
Cell Nucleus/ultrastructure , Chromatin/ultrastructure , Life Cycle Stages/physiology , Nucleosomes/ultrastructure , Trypanosoma cruzi/cytology , Animals , Deoxyribonuclease I/metabolism , Sodium Chloride/pharmacology , Trypanosoma cruzi/geneticsABSTRACT
A cDNA clone derived from the Trypanosoma cruzi alpha-tubulin gene was isolated and sequenced (Tc alpha tub; L37345). Tc alpha tub revealed an 87.79 percent and an 85.36 percent identity with the DNA sequence of T. brucei and Leishmania, respectively. This clone was used to study, by Northern blots, alpha-tubulin gene expression in epimastigotes, cell-cultured derived trypomastigotes and extracellular amastigotes. alpha-tubulin MRNA levels were the same in epimastigotes and trypomastigotes, however, there was a drastic decrease in amastigotes. This clone could be useful to elucidate the regulatory mechanisms of alpha-tubulin gene expression during the differentiation of T. cruzi
Subject(s)
Animals , Cloning, Molecular , DNA, Complementary/isolation & purification , Gene Expression/genetics , Nucleotides/genetics , RNA, Messenger/analysis , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Trypanosoma cruzi/genetics , Tubulin/geneticsABSTRACT
Eleven antigenic clones were isolated from a Trypanosoma cruzi cDNA expression library using a pool of Chagasic sera from Venezuelan patients. These clones were tested with 26 Chagasic sera and 11 sera from patients with other diseases. One clone (Clone 2) reacted with the majority of the Chagasic sera and did not react with the non-Chagasic sera. Restriction enzyme digestion of Clone 2 DNA showed that it contained an insert of 5.2 kb. In a Southern blot of a pulse field gel electrophoresis, Clone 2 hybridized to three T. cruzi chromosomal fragments. By Northern blot analyses, it was observed that the clone hybridizes to a major T. cruzi RNA band of 0.97 kb and to two minor bands of 4.8 and 6.3 kb. The expression of these three RNAs was higher in trypomastigotes than in epimastigotes or spheromastigotes. Specific antibodies isolated against the beta-galactosidase-Clone 2 fusion protein expressed in E. coli reacted with a 28 kilodalton protein in T. cruzi
