Organization of satellite DNA in the genome of Trypanosoma cruzi.

Trypanosoma cruzi has about 10(5) copies of a 195 bp repeat, called satellite DNA, which correspond to about 10% of the total DNA. Since very little is known about satellite distribution and function in this and in many other organisms, we studied the genomic organization of satellite DNA in T. cruzi Y, CL Brener, and Silvio X10 cl1 strains. We found that satellite repeats are present in some, but not all, chromosomes in these strains. We also found that the copy number of the satellite repeats is distinct in each one of the strains. We have estimated that the satellite DNA is four to six times more abundant in Y and CL Brener strains as compared to Silvio X10 cl1. Despite these differences, the repeated units are organized in a similar way. Based on restriction nuclease digestion analysis, we found that satellite repeats are grouped in clusters comprising about 30+/-10 kb. The same cluster size was also found in the different chromosomes, as seen by two-dimensional pulsed field-gel electrophoresis. As evidenced by a fluorescence hybridization assay of spread DNA fibers, we additionally found that these clusters are constrained in specific regions of the chromosomes. Sequence analysis of several repeat units of satellite DNA of the three strains revealed a remarkable similarity with conserved nucleotide blocks separated by more variable regions. Such conserved distribution and common sequence may suggest that these repeated units might have a structural role in the T. cruzi chromosomes.

Evidence for multiple hybrid groups in Trypanosoma cruzi.

A role for parasite genetic variability in the spectrum of Chagas disease is emerging but not yet evident, in part due to an incomplete understanding of the population structure of Trypanosoma cruzi. To investigate further the observed genotypic variation at the sequence and chromosomal levels in strains of standard and field-isolated T. cruzi we have undertaken a comparative analysis of 10 regions of the genome from two isolates representing T. cruzi I (Dm28c and Silvio X10) and two from T. cruzi II (CL Brener and Esmeraldo). Amplified regions contained intergenic (non-coding) sequences from tandemly repeated genes. Multiple nucleotide polymorphisms correlated with the T. cruzi I/T. cruzi II classification. Two intergenic regions had useful polymorphisms for the design of classification probes to test on genomic DNA from other known isolates. Two adjacent nucleotide polymorphisms in HSP 60 correlated with the T. cruzi I and T. cruzi II distinction. 1F8 nucleotide polymorphisms revealed multiple subdivisions of T. cruzi II: subgroups IIa and IIc displayed the T. cruzi I pattern; subgroups IId and IIe possessed both the I and II patterns. Furthermore, isolates from subgroups IId and IIe contained the 1F8 polymorphic markers on different chromosome bands supporting a genetic exchange event that resulted in chromosomes V and IX of T. cruzi strain CL Brener. Based on these analyses, T. cruzi I and subgroup IIb appear to be pure lines, while subgroups IIa/IIc and IId/IIe are hybrid lines. These data demonstrate for the first time that IIa/IIc are hybrid, consistent with the hypothesis that genetic recombination has occurred more than once within the T. cruzi lines.