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Interclonal variations in the molecular karyotype of Trypanosoma cruzi: chromosome rearrangements in a single cell-derived clone of the G strain.

Lima FM, Souza RT, Santori FR, Santos MF, Cortez DR, Barros RM, Cano MI, Valadares HM, Macedo AM, Mortara RA, da Silveira JF - PLoS ONE (2013)

Bottom Line: Our results also suggest that telomeric regions are involved in this process.The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes.If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil.

ABSTRACT
Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure.

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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.
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pone-0063738-g006: 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.

Mentions: To examine telomere length polymorphism in the G strain and clone D11, Southern blot hybridization was performed using frequently cutting restriction enzymes (HaeIII and MspI) whose sites are found within the telomeric junction sequence, a T. cruzi telomere signature. Telomeric hexameric repeats (TTAGGG) were used as a probe. The hybridization profile of the G strain was quite different from that of clone D11. The telomeric restriction fragments of the parental strain show a broad spectrum of lengths ranging from 0.5 kb to over 23 kb. With HaeIII, bands that varied from 0.5 to approximately 3 kb were observed, representing a significant fraction of the G strain telomeres (Figure 6A). For clone D11, two distinct classes of telomeric restriction fragment were identified: one composed of fragments larger than 6.5 kb and another composed of fragments smaller than 4.3 kb. The hexameric probe hybridized only to three HaeIII and two MspI restriction fragments of over 6.5 kb, showing that either clone D11 has few large telomeres or these fragments represent internal telomeric sequences. However, only three bands ranging from 2.3 to 4.3 kb were observed for the second class of telomeric restriction fragment. It is noteworthy that in clone D11, fragments<0.5 kb hybridize strongly with the telomeric probe (Figure 6A) suggesting that most of clone D11 telomeres are less than approximately 500 bp long. The telomere shortening in clone D11 may represent a trace of earlier recombinogenic activity.


Interclonal variations in the molecular karyotype of Trypanosoma cruzi: chromosome rearrangements in a single cell-derived clone of the G strain.

Lima FM, Souza RT, Santori FR, Santos MF, Cortez DR, Barros RM, Cano MI, Valadares HM, Macedo AM, Mortara RA, da Silveira JF - PLoS ONE (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646811&req=5

pone-0063738-g006: 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.
Mentions: To examine telomere length polymorphism in the G strain and clone D11, Southern blot hybridization was performed using frequently cutting restriction enzymes (HaeIII and MspI) whose sites are found within the telomeric junction sequence, a T. cruzi telomere signature. Telomeric hexameric repeats (TTAGGG) were used as a probe. The hybridization profile of the G strain was quite different from that of clone D11. The telomeric restriction fragments of the parental strain show a broad spectrum of lengths ranging from 0.5 kb to over 23 kb. With HaeIII, bands that varied from 0.5 to approximately 3 kb were observed, representing a significant fraction of the G strain telomeres (Figure 6A). For clone D11, two distinct classes of telomeric restriction fragment were identified: one composed of fragments larger than 6.5 kb and another composed of fragments smaller than 4.3 kb. The hexameric probe hybridized only to three HaeIII and two MspI restriction fragments of over 6.5 kb, showing that either clone D11 has few large telomeres or these fragments represent internal telomeric sequences. However, only three bands ranging from 2.3 to 4.3 kb were observed for the second class of telomeric restriction fragment. It is noteworthy that in clone D11, fragments<0.5 kb hybridize strongly with the telomeric probe (Figure 6A) suggesting that most of clone D11 telomeres are less than approximately 500 bp long. The telomere shortening in clone D11 may represent a trace of earlier recombinogenic activity.

Bottom Line: Our results also suggest that telomeric regions are involved in this process.The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes.If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil.

ABSTRACT
Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure.

Show MeSH
Related in: MedlinePlus