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Possible integration of Trypanosoma cruzi kDNA minicircles into the host cell genome by infection
A R Teixeira1, E R Argañaraz, L H Freitas
1Department of Pathology, Faculty of Health Sciences, University of Brasilia, Brazil.
Abstract:
Infection with Trypanosoma cruzi is known to induce the division of peritoneal macrophages in BALB/c mice. We have demonstrated, by cytogenetic analysis, that accessory DNA elements are associated with the metaphase macrophage chromosomes of such infected macrophages. The identification of these accessory DNA elements with T. cruzi DNA is strongly supported by the association of 3H-label with some chromatids in macrophages previously infected with T. cruzi which had been labelled with 3H-methyl-thymidine. The karyotyping consistently showed preferential associations of T. cruzi DNA with chromosomes 3, 6 and 11. A conclusive demonstration of the parasite origin of the integrated DNA came from fluorescein in situ hybridization studies using specific parasite DNAs as probes. In order to determine the identity of the inserted DNA and to investigate the nature of the integration mechanism, Southern blot analyses were performed on DNA extracted from both uninfected and infected (but parasite-free) macrophages. Hybridizations of BamHI, EcoRI and TaqI digests of DNA from T. cruzi-infected host cells all revealed the presence of a 1.7-kb DNA fragment when probed with kDNA. The covalent association of kDNA with that of the host was confirmed by the fact that AluI and Hinf-I digests of DNA from infected host cells produced a number of bands, in a size range of 0.8-3.6 kb, which hybridized with kDNA minicircles. None of these bands was found in DNA purified from cell-free preparations of the parasite and thus it must be concluded that they represent insertion fragments between parasite and host cell DNA. These results strongly suggest that kDNA minicircles from T. cruzi have been integrated into the genome of the host cell following infection.
Insights
Trypanosoma cruzi infection integrates parasite DNA into mouse macrophage chromosomes. This study confirms the integration of kinetoplast DNA minicircles into host cell genomes following infection.
Area of Science:
- Molecular Biology
- Parasitology
- Genetics
Background:
- Trypanosoma cruzi infection is known to stimulate peritoneal macrophage division in BALB/c mice.
- Previous observations suggested accessory DNA elements associated with macrophage chromosomes during infection.
Purpose of the Study:
- To cytogenetically identify and characterize accessory DNA elements in T. cruzi-infected macrophages.
- To determine the origin and integration mechanism of parasite DNA within the host genome.
Main Methods:
- Cytogenetic analysis and karyotyping of infected macrophages.
- Radioactive labeling (3H-methyl-thymidine) to trace DNA.
- Fluorescence in situ hybridization (FISH) with parasite DNA probes.
- Southern blot analysis of host cell DNA digests.
Main Results:
- Cytogenetic analysis revealed T. cruzi DNA associated with macrophage chromosomes, particularly chromosomes 3, 6, and 11.
- FISH confirmed the parasite origin of the integrated DNA.
- Southern blot analysis identified a 1.7-kb fragment hybridizing with kinetoplast DNA (kDNA) in infected cells.
- Digestion with specific enzymes showed unique bands in infected cells, indicating covalent association between parasite and host DNA.
Conclusions:
- T. cruzi infection leads to the integration of parasite DNA into the host macrophage genome.
- Kinetoplast DNA minicircles from T. cruzi are implicated in this integration process.
- The findings provide strong evidence for the integration of parasite DNA into the host cell genome following infection.