Mitotic stability of a coding DNA sequence-free version of Leishmania major chromosome 1 generated by targeted chromosome fragmentation

被引:16
作者
Dubessay, P
Ravel, C
Bastien, P
Stuart, K
Dedet, JP
Blaineau, C
Pagès, M
机构
[1] Fac Med, Lab Parasitol Mycol, CNRS UMR5093 Genome & Biol Mol Protozoaires Paras, F-34090 Montpellier, France
[2] Seattle Biomed Res Inst, Seattle, WA 98109 USA
关键词
mini-chromosome; chromosome stability; recombination; segregation; centromere; satellite DNA;
D O I
10.1016/S0378-1119(02)00506-1
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
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 la (similar to25 kb at each end), and in its central pail of a complex tandem amplification of the linearized transfection vector containing the hygromycin resistance gene (over similar to105 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 (similar to13 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. Published by Elsevier Science B.V.
引用
收藏
页码:151 / 159
页数:9
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