Mitotic stability of a coding DNA sequence-free version of Leishmania major chromosome 1 generated by targeted chromosome fragmentation Pascal Dubessay a , Christophe Ravel a , Patrick Bastien a , Ken Stuart b , Jean-Pierre Dedet a , Christine Blaineau a , Michel Page `s a, * a CNRS UMR5093 ‘Ge ´nome et Biologie Mole ´culaire des Protozoaires Parasites’, Laboratoire de Parasitologie-Mycologie, Faculte ´ de Me ´decine, 163 Rue A. Broussonet, F-34090 Montpellier, France b Seattle Biomedical Research Institute, Seattle, WA, USA Received 3 September 2001; received in revised form 4 January 2002; accepted 19 February 2002 Received by B. Dujon Abstract 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 (,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 ,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 (,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. Published by Elsevier Science B.V. Keywords: Mini-chromosome; Chromosome stability; Recombination; Segregation; Centromere; Satellite DNA 1. Introduction Leishmania is a protozoan parasite responsible for a group of diseases whose symptoms range from mild cuta- neous lesions to fatal visceral involvement. The population at risk is ,350 millions, and 1–2 million cases are esti- mated to occur annually. During the last few years, there has been an increase in the knowledge of the structure and organization of the genome of this organism. Leishmania is thought to be ‘essentially’ diploid (Ravel et al., 1998). The haploid genome is about 35 Mbp in size, and it comprises 34, 35 or 36 heterologous chromosomes according to the species (Britto et al., 1998). Structural studies also demon- strated a high degree of genomic plasticity, observed by the instability of subtelomeric regions (Ravel et al., 1996), frequent aneuploidy (Cruz et al., 1993; Ravel et al., 1998; Tamar and Papadopoulou, 2001) and the amplifica- tion of circular or linear extrachromosomal DNAs, occur- ring either spontaneously (Segovia and Ortiz, 1997) or in response to drug selection (Ouellette and Papadopoulou, 1993). The systematic sequencing of the genome of the model strain Leishmania major Friedlin (LmF) has been undertaken by an international consortium (see www.ebi.a- c.uk/parasites/leish.html) and lead to the publication of the complete sequence of the smallest chromosome, chromo- some 1 (Myler et al., 1999). This chromosome is 285–315 kb in size according to the homologue considered (Sunkin et al., 2000). The most remarkable finding of this sequence was the gene organization in two large separate clusters of 29 and 50 coding DNA sequences (CDSs), each on one DNA strand and in an opposite orientation towards the telomeres (Myler et al., 1999); these clusters occupy the major part of the chromosome (about 250 kb) and are regularly distributed on each side of a short 1.6-kb ‘switch’ Gene 289 (2002) 151–159 0378-1119/02/$ - see front matter. Published by Elsevier Science B.V. PII: S0378-1119(02)00506-1 www.elsevier.com/locate/gene Abbreviations: HYG, hygromycin resistance gene; DHFRTS, dihydrofo- late reductase-thymidilate synthase; PCR, polymerase chain reaction; CDS, coding DNA sequence; THR, telomeric hexamer repeats; LST-R, Leishma- nia subtelomeric repeats; LmF, Leishmania major Friedlin * Corresponding author. Tel.: 1 33-4-6763-5513; fax: 133-4-6763-0049. E-mail address: genpara@sc.univ-montp1.fr (M. Page `s).