Figure 1.
Karyotype polymorphism between the G strain and clone D11.
Panel A) Chromosomal bands were separated by Pulsed-Field Gel Electrophoresis (PFGE) and stained with SYBR Green I. The bands from the G strain were numbered using Arabic numerals (1–19) as in a previous study (Souza et al., 2011) while capital letters (A – U) were used for clone D11, starting from the smallest band. Panel B) Diagrammatic representation of the molecular karyotypes of the G strain and clone D11. The rectangles represent a unique distinguishable band visualized after SYBR Green I staining. The thickness of the rectangles represents the proportional staining of each chromosomal band. The number and letter of chromosomal bands as well as their molecular weight are indicated to the left and right of each strip, respectively.
Figure 2.
Identification of homologous chromosomal bands of similar molecular sizes in the G strain and clone D11.
Hybridization profile of specific chromosomal markers hybridized to one or more bands of similar molecular size in both isolates after chromosome separation by PFGE and Southern-blot hybridization. The markers used are TEUF0099, rDNA18S, TEUF0242 and ADC. Gene identification and GenBank accession number of each marker are shown in Table 1.
Figure 3.
Conservation of large syntenic groups between the G strain and clone D11.
Selected markers belonging to in silico chromosomes TcChr37 (Panel A) and TcChr39 (Panel B) previously defined in clone CL Brener were mapped on chromosomal bands of the G strain and clone D11 separated by PFGE. The diagrammatic representation above each panel indicates the position of the markers on the in silico chromosome. Markers from TcChr37 are THTc, TEUF0001, TEUF0180 and delta-6. Markers from TcChr39 are XM_811753, H49, JL8 and ankyrin. Gene identification and GenBank accession number of each marker are shown in Table 1.
Figure 4.
Identification of possible chromosomal rearrangements in clone D11.
Mapping of markers belonging to in silico chromosomes TcChr7 (Panel A) and TcChr22 (Panel B). Identification of chromosomal rearrangements involving one band in the G strain and two bands in clone D11 (Panel A) or vice versa (Panel B). The positions of the markers used as radiolabeled probes are indicated in the diagrammatic representation of the in silico chromosomes. Markers from TcChr7 are XM_799116, XM_803657, TryP and NLI. Markers from TcChr22 are XM_801648, XM_801647, Bpp-1 and XM_801649. Gene identification and GenBank accession number of each marker are shown in Table 1.
Table 1.
Location of molecular markers on G strain and clone D11 chromosomal bands.
Figure 5.
Identification of a rearrangement involving a large fragment containing the α- tubulin gene in clone D11.
Panel A) Mapping of the α-tubulin gene on chromosomal bands of the G strain and clone D11 showing a translocation event involving large chromosomes. β-tubulin, hypothetical protein XM_804243 and endomembrane protein (XM_ 812238) were also mapped and showed the same hybridization profile. The positions of markers used as probes are indicated in the diagrammatic representation of in silico chromosomes TcChr14. Panel B) Restriction fragment analysis of α-tubulin gene loci was carried out by digesting genomic DNA with PstI (P) or double-digesting it with BglII and PstI (B/P). Phage lambda DNA digested with HaeIII, used as a molecular weight marker, is shown on the left. Panel C) Restriction analysis of whole chromosomes in agarose blocks was performed using the rare-cutting enzymes PacI and SfiI. The molecular weights of fragments recognized by the probe are shown on the left.
Figure 6.
Telomere length polymorphism of the G strain and clone D11.
Panel A) Southern-blot hybridization of restriction fragments generated by HaeIII and MspI probed with the telomeric repeat (TTAGGG). HaeIII-digested phage lambda DNA (used as a molecular weight marker) is shown on the left. Panel B) Analysis of the subtelomeric length of the G strain and clone D11 chromosomes was performed by Southern-blot hybridization of SfiI restriction fragments with the telomeric repeat. The size of the larger subtelomeric fragment of clone D11 is shown on the left.
Table 2.
Allele sizes (bp) for each microsatellite locus amplified for the G strain and clone D11.
Figure 7.
Possible mechanisms of genetic recombination that could give rise to chromosomal polymorphism in T. cruzi.
Panel A) Translocation mechanism: a DNA fragment (210 kb) from a heterologous chromosome (red) is translocated to another chromosome (blue) by homologous recombination, generating “homologous” chromosomes of different sizes. Panel B) Fusion and breakage mechanism: two homologous chromosomes of different sizes are fused, forming a dicentric chromosome which is then broken, generating two chromosomes of similar sizes but with different gene content.