Generation of heterozygous knockout cells expressing reduced levels of TcRPA-2.

<p>A. Schematic representation of the TcRPA-2 locus (central panel) or the locus generated after replacement of the RPA-2 gene by gene targeting (bottom panel) with the recombination cassette carrying the hygromycin resistance gene (top panel). Arrows indicate the regions where the primers used in the experiments presented in B anneal. B. DNA extracted from RPA-2 heterozygous knockout cells was amplified using the primers presented in A. 1- Hyg_f (2) + Hyg_r (3) (1.026 bp); 2- Hyg_f (2) + EXT5’_r (4) (2.113 bp); 3- EXT5’_f (1) + Hyg_r (3) (1.994 bp); 4- EXT5’_f (1) + EXT3’_r (4). 5–1 kb plus DNA ladder marker (Invitrogen). The primers EXT5’_f and EXT3’_r anneal in regions outside of the recombination site. Thus, the two bands observed in lane 4 correspond to the amplified regions from the wild-type (2.070 bp) and targeted RPA-2 alleles (3.081 bp), which are present in the RPA-2+/- parasites. C. Cell extracts from control and RPA-2+/- cells were subjected to SDS-PAGE, transferred onto nitrocellulose membranes and incubated with anti-rTcRPA-2 or anti-GAPDH, which was used as a loading control. D. The intensity of the bands obtained with anti-rTcRPA-2 presented on C were normalized using the intensity of the GAPDH bands. Graph shows average and standard deviation of three independents experiments.</p

TcRPA-1 is able to bind ssDNA by OBF1.

<p>A and B. Circular dichroism (CD) spectra of (A) rTcRPA-1 and (B) rTcRPA-2 in the absence (black line) or presence of single stranded DNA of 24 bp (ssDNA24; blue line). A digoxin-labeled single stranded DNA (ssDNA) was maintained alone or in the presence (+) of recombinant TcRPA-1 (rRPA-1) (C) or of increasing concentrations of recombinant TcRPA-2 (rRPA-2) (D). The samples were analyzed by EMSA. E. Schematic representation of entire TcRPA-1 and three truncated mutants corresponding to each OBF domain. F. A digoxigenin-labeled ssDNA was maintained alone (-) or in the presence of recombinant OBF1, OBF2, and OBF3.</p

RPA-2 heterozygous knockout cells present growth and DNA replication impairment and S-phase progression delay.

<p>A. Growth curve of RPA-2+/- and wild type (wt) cells. * indicates the day during the growth curve that samples were taken to perform assays presented in B and C. B. Control and RPA-2+/- cells were maintained in the presence of EdU for 5 minutes and the percentage of labeled cells was determined. Graph shows average and standard deviation of three independents experiments. C. FACS analysis of wild type (wt) and RPA-2+/- cells labeled with propidium iodide. D. Growth culture of wild type (wt) and RPA-2+/- cells after treatment with 75μM of cisplatin or with no treatment (no drug). Statistical analyses were performed using a Student t-test, ** means p < 0.001; n.s. means p > 0.8.</p

Interclonal Variations in the Molecular Karyotype of <i>Trypanosoma cruzi</i>: Chromosome Rearrangements in a Single Cell-Derived Clone of the G Strain

<div><p><i>Trypanosoma cruzi</i> 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 <i>T. cruzi</i> 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 <i>Leishmania infantum,</i> 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 <i>T. cruzi</i> arsenal for responding to environmental pressure.</p></div

The pattern of TcRPA distribution changes after DNA damage.

<p>Cells were treated with HU (A) or irradiated with UV (B). Cells were fixed, permeabilized and incubated with anti-rTcRPA-1 (anti-RPA-1), anti-rTcRPA-2 (anti-RPA-2) and anti-CPD and stained with DAPI. In A, the percentage of cells found with each pattern of RPA distribution is indicated.</p

TcRPA-1 and TcRPA-2 form a complex <i>in vivo</i> and localize at replication foci.

<p>A. Cell extracts were immunoprecipitated with an anti-rTcRPA-2 antibody (anti-RPA2) or with pre-immune serum as a control. Samples eluted from the resin were submitted to SDS-PAGE, transferred onto nitrocellulose membranes and incubated with anti-rTcRPA-1 (anti-RPA-1) or anti-rTcRPA-2 (anti-RPA-2) antibodies. B. Cells were incubated with EdU, fixed, incubated with anti-rTcRPA-1 (anti-RPA-1) or anti-rTcRPA-2 (anti-RPA-2) and stained with DAPI. N-nucleus, k-kinetoplast.</p

RPA-2+/- cells present a higher capacity of differentiation and infection compared with wild type cells.

<p>(A) The same amount of wild type (wt) and heterozygous knockout RPA-2+/- cells were submitted to metacyclogenesis and obtained metacyclic trypomastigotes were quantified. B. The same amount of wild type (wt) and heterozygous knockout RPA-2+/- metacyclic trypomastigotes were used to infect cells. After 24 h, the percentage of infected cells was determined. Statistical analyses were performed using a Student t-test, * means p < 0.01.</p

Identification of homologous chromosomal bands of similar molecular sizes in the G strain and clone D11.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063738#pone-0063738-t001" target="_blank">Table 1</a>.</p

Conservation of large syntenic groups between the G strain and clone D11.

<p>Selected markers belonging to <i>in silico</i> chromosomes TcChr37 (<b>Panel A</b>) and TcChr39 (<b>Panel B</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 <i>in silico</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063738#pone-0063738-t001" target="_blank">Table 1</a>.</p

Identification of possible chromosomal rearrangements in clone D11.

<p>Mapping of markers belonging to <i>in silico</i> chromosomes TcChr7 (<b>Panel A</b>) and TcChr22 (<b>Panel B</b>). Identification of chromosomal rearrangements involving one band in the G strain and two bands in clone D11 (<b>Panel A</b>) or vice versa (<b>Panel B</b>). The positions of the markers used as radiolabeled probes are indicated in the diagrammatic representation of the <i>in silico</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063738#pone-0063738-t001" target="_blank">Table 1</a>.</p