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Repetitive DNA is associated with centromeric domains in Trypanosoma brucei but not Trypanosoma cruzi

Samson O Obado1, Christopher Bot1, Daniel Nilsson2, Bjorn Andersson2 and John M Kelly1*

Author Affiliations

1 Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK

2 Center for Genomics and Bioinformatics, Karolinska Institutet, Berzelius vag, S-171 77 Stockholm, Sweden

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Genome Biology 2007, 8:R37  doi:10.1186/gb-2007-8-3-r37

Published: 12 March 2007

Additional files

Additional data file 1:

The schematic of the 0.51 Mb chromosome shows the position of the 11 kb GC-rich strand-switch domain (yellow oval). The expanded section (70 kb) contains the four ORFs used for chromosome fragmentation (Tc1-Tc4; Additional data file 5). The implied direction of polycistronic transcription is indicated. A 0.9 kb DNA fragment from ORF Tc1 was cloned into the pTEX-CF vector [22]. This was linearized so that the targeting fragment was at one end and telomeric sequences at the other. Plasmid DNA within the vector is marked in red. Site-specific integration (crossed lines) results in the deletion of approximately 100 kb of DNA between the target sequence and the telomere (Additional data file 2). Telomeric sequences supplied by the vector are shown as horizontal arrowheads. Expression of neor is under control of the rDNA promoter (flagged)

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Additional data file 2:

The 11 kb GC-rich strand-switch domains are shown (yellow oval). Arrows (Tc1-Tc4) indicate positions of the target sequences used for fragmentation (Additional data file 5). Parasites were transfected with linearized vector DNA and transformants selected with G418 [22] (see Materials and methods). Chromosomal DNA from wild-type (WT) and transfected (T) parasites was separated by CHEFE with S. cerevisiae markers (M) and analyzed by Southern blotting using plasmid, Tc5 and Tc6 radiolabelled probes. Autoradiographs are shown, together with an ethidium bromide (EtBr) stained gel, overexposed to show the truncated chromosome. In the example shown, integration at the Tc1 locus of the 0.51 Mb chromosome, in the direction of polycistronic transcription, results in deletion of approximately 100 kb of DNA between the target sequence and the telomere. Hybridization identifies the larger (1.2 Mb) and smaller homologues (0.51 Mb) and the truncated product (0.40 Mb). Plasmid DNA is incorporated into truncated chromosomes as part of the integration process (Additional data file 1)

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Additional data file 3:

(a) Schematic showing the 0.51 Mb and 1.2 Mb homologues and their respective truncated products C1 (0.2 Mb) and C2 (0.9 Mb), with the locations of probes Tc2-Tc5 (Additional data file 5). (b) Autoradiograph illustrating the deletion of the right arms of each chromosome homologue following integration of the fragmentation vector at ORF Tc4. Two clones were isolated after transfection and chromosomal DNA from wild-type (WT) and both clones (C1 and C2) were separated by CHEFE and analyzed by Southern blotting using radiolabeled probe Tc4. Hybridization identifies the larger homologue (1.2 Mb), the smaller homologue (0.51 Mb) and their respective truncated products (0.9 Mb and 0.2 Mb). We had previously shown that probe Tc5 is located a similar distance (approximately 50 kb) from the end of both homologues [22]

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Additional data file 4:

T. brucei procyclic cultures were treated with 500 μM etoposide and chromosomal DNA isolated and fractionated using a Bio-Rad CHEF Mapper system. Membranes were hybridized with probes Tb1-Tb17, the GeneDB systemic names of which are shown in Additional data file 5. The positions of probes (arrows) and locations of the putative centromeric regions (yellow ovals) are indicated. Red arrows indicate INGI/DIRE retroelements and green arrows identify putative ORFs and the implied direction of transcription. The locations of the AT-rich repeat arrays are highlighted (striped box) and the %GC contents across the regions are shown. GeneDB systemic names of ORFs adjacent to the arrays are given and allow the repeat arrays to be mapped onto the chromosomal contigs. Lane N, non-treated parasites; lane E, etoposide-treated. Chromosome sizes were calculated using S. cerevisiae (0.2-2.2 Mb) and Hansenula wingei (1.0-3.1 Mb) chromosome markers (Bio-Rad). In most cases, sequence contigs do not extend to the ends of chromosomes. Gaps in the sequence of AT-rich arrays in chromosomes 3, 6 and 8 are indicated by asterixes and double slashes. We have also found, using long-range restriction mapping (Figures 4b and 5bB), that the 'centromeric-domains' of chromosomes 1 and 4 are larger than predicted by the genome sequence

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Additional data file 5:

T. cruzi (Tc1-12) and T. brucei (Tb1-18) probes used in this study, together with their GeneDB systemic names

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