Mitotic stability of a coding DNA sequence-free version of Leishmania major chromosome 1 generated by targeted chromosome fragmentation.
The deletion of a 260-kb segment containing all the coding DNA sequences (CDS) of chromosome 1 of Leishmania major Friedlin strain was performed through homologous recombination during a transfection experiment. This allowed the selection of a mutant clone containing a linear extra chromosome sizing 155 kb (XC155). The structure of XC155 was determined by restriction analysis and DNA cloning and sequencing of the gel-purified chromosome: it is made of a 'mirror' inverted duplication of the 'right' end of chromosome 1a (approximately 25 kb at each end), and in its central part of a complex tandem amplification of the linearized transfection vector containing the hygromycin resistance gene (over approximately 105 kb). No sequence of the coding region of chromosome 1 (including the 1.6-kb 'switch' region) was found. By contrast, XC155 contains two large (approximately 13 kb) clusters of tandemly repeated subtelomeric sequences (272-bp 'satellite' DNA) as well as telomeric hexamer repeats. This extra chromosome was found to be mitotically stable after >150 generations without selective pressure in vitro. Two sequence elements are considered which may have an effect on mitotic stability and participate to centromeric function in this extra chromosome: the amplification of the input vector and the 272-bp 'satellite' DNA bound by telomeric repeats.
Dubessay
Pascal
P
CNRS UMR5093 Génome et Biologie Moléculaire des Protozoaires Parasites, Laboratoire de Parasitologie-Mycologie, Faculté de Médecine, 163 Rue A. Broussonet, F-34090 Montpellier, France.
Ravel
Christophe
C
Bastien
Patrick
P
Stuart
Ken
K
Dedet
Jean-Pierre
JP
Blaineau
Christine
C
Pagès
Michel
M
D000818:Animals; D002872:Chromosome Deletion; D002875:Chromosomes / Q000235:genetics*; D016054:DNA, Protozoan / Q000235:genetics*; D017125:Genes, Protozoan / Q000235:genetics; D018320:Leishmania major / Q000235:genetics*; D008938:Mitosis / Q000235:genetics*; D011995:Recombination, Genetic; D016615:Telomere / Q000235:genetics
D016054:DNA, Protozoan