GENOMICS 35, 509–516 (1996) ARTICLE NO. 0391 Medium-Range Restriction Maps of Five Chromosomes of Leishmania infantum and Localization of Size-Variable Regions C HRISTOPHE R AVEL,P ATRICK WINCKER ,C HRISTINE B LAINEAU,C ONSTANC ¸A B RITTO, P ATRICK B ASTIEN, AND M ICHEL P AGE ` S 1 Laboratoire Ge ´nome des Parasites, Parasitologie, Faculte ´ de Me ´decine, 163 rue Auguste Broussonet, 34000 Montpellier, France Received January 16, 1996; accepted May 6, 1996 light on the organization of different protozoan ge- The chromosomes of the human protozoan parasite nomes (Triglia et al., 1992; Watanabe and Inselburg, Leishmania exhibit striking size polymorphisms among 1994; Koota et al., 1987; Beals and Boothroyd, 1992; different strains. To define the structural basis for these Morzaria and Young, 1992; Janse et al., 1992; variations, we have constructed full-length restriction Blaineau et al., 1991; Weiden et al., 1991). In Trypa- maps of five chromosomes of 370 to 490 kb in Leish- nosoma brucei and Plasmodium falciparum, karyo- mania infantum clone LEM1317. Rare-cutting sites for typic polymorphisms associated with mechanisms the enzymes AseI, DraI, XbaI, SspI, SpeI, and Sfi I were leading to antigenic diversity have been described mapped by partial and complete digestion of either gel- among strains of the same species (Pays and Stein- purified chromosomes or total DNA. Sixty-eight anony- ert, 1988; Lanzer et al., 1994; Hinterberg et al., mous DNA probes were localized on these maps, as well 1994). With no classical genetic analysis possible for as the mini-exon and dihydrofolate reductase – thymidi- Leishmania, the construction of a complete physical late synthase gene probes. These maps were compared map of its genome would be invaluable for under- with those from other strains of L. infantum and Leish- standing its biology. Such a project has been under- mania donovani. This showed that the distribution of taken as an international effort under the initiative the restriction sites was conserved in these two close of the TDR Program of the World Health Organiza- species. Four regions involved in the size variations of tion. Recently, all 36 physical linkage groups consti- three chromosomes were localized; subtelomeric se- tuting the genome of Leishmania infantum have quences were responsible for size variability in three been identified with the use of 244 chromosome-spe- of four cases. The whole of this study takes a particular cific markers; they range between 0.35 and 3 Mb, and significance in the frame of the project of complete the haploid genome size was estimated as 36{0.5 physical mapping and sequencing of the Leishmania Mb (Wincker et al., 1996). These linkage groups are genome.  1996 Academic Press, Inc. conserved among all Old World Leishmania species, and the study confirmed that homologous chromo- somes display significant size variations within as INTRODUCTION well as between species (Ravel et al., 1995a, Wincker et al., 1996). The construction of preliminary restric- The protozoan parasite Leishmania is responsible tion maps for different homologues of chromosome for a broad spectrum of human diseases, some of I revealed that repetitive sequences, located in one which can be fatal if left untreated. It constitutes a subtelomeric region, were responsible for these size severe public health problem in many tropical coun- variations (Blaineau et al., 1991). The importance tries, with an estimated 15 million people infected of these regions in chromosome instability has been (Ashford et al., 1992). Like in other Trypanoso- documented in different protozoan parasites, sug- matids, the life cycle of the different species compos- gesting that they could play a role in the genetic ing the genus Leishmania has been elucidated; how- diversity of these organisms (Janse et al., 1992; ever, important questions about the biology of this Lanzer et al., 1994; Hinterberg et al., 1994). parasite, like the existence of genetic exchange, a The present study aimed to construct the first com- sexual cycle (Kelly et al., 1991; Bastien et al., 1992), plete medium-range restriction maps of five full-length or the ploidy (Blaineau et al., 1992; Cruz et al., 1993) Leishmania chromosomes. It also sought to define the are still debated. regions involved in chromosome instability. The chro- The development of mapping techniques has shed mosomes under study were chromosomes I, II, III, V, and VI (as defined in Ravel et al., 1995a) and are among the smallest of this organism, ranging between 370 and 1 To whom correspondence should be addressed. Telephone: 33 67 63 55 13. Fax: 33 67 63 00 49. 490 kb. 509 0888-7543/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.