Table_4_Trypanosoma cruzi Genomic Variability: Array Comparative Genomic Hybridization Analysis of Clone and Parental Strain.xlsx

Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits extensive inter- and intrastrain genetic diversity. As we have previously described, there are some genetic differences between the parental G strain and its clone D11, which was isolated by the limiting dilution method and infection of cultured mammalian cells. Electrophoretic karyotyping and Southern blot hybridization of chromosomal bands with specific markers revealed chromosome length polymorphisms of small size with additional chromosomal bands in clone D11 and the maintenance of large syntenic groups. Both G strain and clone D11 belong to the T. cruzi lineage TcI. Here, we designed intraspecific array-based comparative genomic hybridization (aCGH) to identify chromosomal regions harboring copy-number variations between clone D11 and the G strain. DNA losses were more extensive than DNA gains in clone D11. Most alterations were flanked by repeated sequences from multigene families that could be involved in the duplication and deletion events. Several rearrangements were detected by chromoblot hybridization and confirmed by aCGH. We have integrated the information of genomic sequence data obtained by aCGH to the electrophoretic karyotype, allowing the reconstruction of possible recombination events that could have generated the karyotype of clone D11. These rearrangements may be explained by unequal crossing over between sister or homologous chromatids mediated by flanking repeated sequences and unequal homologous recombination via break-induced replication. The genomic changes detected by aCGH suggest the presence of a dynamic genome that responds to environmental stress by varying the number of gene copies and generating segmental aneuploidy.

锥虫属的克氏锥虫（Trypanosoma cruzi），为查加斯病的病原体，表现出广泛的种间和种内基因多样性。正如我们之前所述，亲本G菌株与其克隆D11之间存在一些遗传差异，克隆D11是通过限制稀释法和培养哺乳动物细胞的感染而分离得到的。电泳核型分析和染色体带与特异性标记的南方杂交揭示了克隆D11中存在的小型染色体长度多态性，以及额外的染色体带，同时保持了大型同源群。G菌株和克隆D11均属于克氏锥虫的TcI谱系。在此，我们设计了基于阵列的种内比较基因组杂交（aCGH），以鉴定克隆D11与G菌株之间携带拷贝数变异的染色体区域。克隆D11中的DNA丢失比DNA获得更为广泛。大多数变异被多基因家族的重复序列所包围，这些序列可能参与了复制和删除事件。通过染色体杂交和aCGH检测到几种重排，并通过aCGH得到证实。我们将aCGH获得的基因组序列数据整合到电泳核型中，从而重建了可能产生克隆D11核型的重组事件。这些重排可能由旁侧重复序列介导的姐妹或同源染色单体之间的非等位交换以及通过断裂诱导复制的不等同源重组所引起。aCGH检测到的基因组变化表明，存在一个动态的基因组，该基因组能够通过改变基因拷贝数和产生片段非整倍性来响应环境压力。