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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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Related in: MedlinePlus

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.
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pone-0063738-g005: 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.

Mentions: The α- and β-tubulin genes were mapped on two chromosomal bands of 1.35 and 2.00 Mb in the parental G strain whereas in clone D11 they were translocated to two bands of 2.35 and 2.58 Mb (Figure 5A). Since the T. cruzi α- and β-tubulin genes are in physically linked as alternating tubulin repeat units ([24]; GenBank AF091836 and M97956; Bartholomeu DC, personal communication), our results suggest that the complete tubulin repeat unit was translocated to large chromosomes in clone D11. To understand this phenomenon and to investigate to what extent homologous chromosomes can be different in size, the following approaches were used: 1) hybridization of the chromoblots with probes located on the same chromosomal bands in which tubulin genes were mapped; 2) restriction analysis tubulin loci and estimation of copy number of tubulin genes in the G strain and clone D11.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063738-g005: 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.
Mentions: The α- and β-tubulin genes were mapped on two chromosomal bands of 1.35 and 2.00 Mb in the parental G strain whereas in clone D11 they were translocated to two bands of 2.35 and 2.58 Mb (Figure 5A). Since the T. cruzi α- and β-tubulin genes are in physically linked as alternating tubulin repeat units ([24]; GenBank AF091836 and M97956; Bartholomeu DC, personal communication), our results suggest that the complete tubulin repeat unit was translocated to large chromosomes in clone D11. To understand this phenomenon and to investigate to what extent homologous chromosomes can be different in size, the following approaches were used: 1) hybridization of the chromoblots with probes located on the same chromosomal bands in which tubulin genes were mapped; 2) restriction analysis tubulin loci and estimation of copy number of tubulin genes in the G strain and clone D11.

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