A Telomere-mediated Chromosome Fragmentation Approach to Assess Mitotic Stability and Ploidy Alterations of LeishmaniaChromosomes

Abstract

We have used a telomere-associated chromosome fragmentation strategy to induce internal chromosome-specific breakage of Leishmania chromosomes. The integration of telomeric repeats from the kinetoplastid Trypanosoma brucei into defined positions of the Leishmania genome by homologous recombination can induce chromosome breakage accompanied by the deletion of the chromosomal part that is distal to the site of the break. The cloned telomeric DNA at the end of the truncated chromosomes is functional and it can seed the formation of new telomeric repeats. We found that genome ploidy is often altered upon telomere-mediated chromosome fragmentation events resulting in large chromosomal deletions. In most cases diploidy is either preserved, or partial trisomic cells are observed, but interestingly we report here the generation of partial haploid mutants in this diploid organism. Partial haploid Leishmania mutants should facilitate studies on the function of chromosome-assigned genes. We also present several lines of evidence for the presence of sequences involved in chromosome mitotic stability and segregation during cell cycle in this parasitic protozoan.

Functional mapping of a trypanosome centromere by chromosome fragmentation identifies a 16-kb GC-rich transcriptional “strand-switch” domain as a major feature

“…This approach has been used previously to investigate chromosome structure in other organisms (Farr et al 1992;Itzhaki et al 1992;Tamar and Papadopoulou 2001). Initially, we generated partially monosomic cell lines by targeting the Tcmpx and Tcsod loci, and deleted 200 kb and 50 kb of DNA, respectively.…”

The heat shock protein HSP70 and heat shock cognate protein HSC70 contribute to antimony tolerance in the protozoan parasite Leishmania

“…Polymerase chain reaction fragments, using the cosmid X6 as template for the L tarentolae HSC70 (5Ј-GACGACCACTG CCGCAGAGATG-3Ј and 5Ј-GTCCTTGCTCAGCCGGC CCTTG-3Ј), and the L tarentolae HSP70 (5Ј-CATCGAT-GAGCGTCACAGAG-3Ј and 5Ј-ATGCACCGAGTTGCAC AGTC-3Ј) were cloned in the expression vector pSP72␣-NEO-␣ and were transfected in L tarentolae or L infantum. Pulse field gel electrophoresis of Leishmania chromosomes was done as described previously (Tamar and Papadopoulou 2001) using a Bio-Rad CHEF DR III apparatus. …”

Repetitive Elements in Genomes of Parasitic Protozoa

“…Similarly, in Leishmania, the 272-bp repeat has been implicated in the mitotic stability of artifical chromosomes (59). However, the presence of subtelomeric repeats does not seem to be sufficient for chromosomal stability in this species (181).…”