Molecular and Biochemical Parasitology, 55 (1992) 65-74 65 © 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00

MOLBIO 01805

Dynamics and size polymorphisms of minichromosomes in Leishmania major LV-561 cloned lines

Miguel Navar ro a, Rhaiza Maingon b, R a y m o n d Hamers c and Manue l Segovia a aDepartamento de Gdnetica y Microbiologia, Facultad de Medicina, Universidad de Murcia, Murcia, Spain; b Wolfson Unit of

Molecular Genetics, Liverpool School of Tropical Medicine, Liverpool, UK; and Clnstituut voor Moleculaire Biolog&, Vrije Universiteit Brussel, Brussels, Belgium

(Received 9 January 1992; accepted 9 June 1992)

Various lines and cloned lines of Leishmania major of varying degrees of virulence in BALB/c mice possessed size polymorphic multicopy minichromosomes related to previously described LD 1/CD 1 and 715-class DNAs of Leishmania. The minichromosomes were not necessary for virulence. Two of these DNAs (MI80 and M210), coexisting in a single cloned line, showed remarkable dynamics in terms of loss or gain when followed through multiple transfers during in vitro culture and in vivo passage in BALB/c mice. Although there was significant sequence heterogeneity among minichromosomes, M180 sequences were present within large (megabase) and in intermediate (550-760 kb) chromosomes in the L. major lines analysed. MI80 related small DNAs were also detected in Leishmania mexicana and Leishmania donovani isolates, suggesting that the generation of these molecules involves a common, probably functional basic mechanism widespread in Leishmania.

Key words: Leishmania major; Pulsed field gel electrophoresis; Molecular karyotype; Minichromosome

Introduction

Pulsed field electrophoresis (PFE) and hybridisation to coding and chromosome specific sequences have provided evidence for locus amplification, diploidy and genetic recombination for at least some chromosomes in Leishmania [1-3]. Recently, additional circular and small linear DNA molecules have been observed in molecular karyotypes [4]. Various Leishmania possess multicopy circular DNAs containing genes coding for drug resistance [5-8]. Other circular and small

Correspondence address: Manuel Segovia, Departamento de Genetica y Microbiologia, Facultad de Medicina, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain.

Abbreviations: BSA, bovine serum albumin; SDS, sodium dodecyl sulphate; PBS, phosphate-buffered saline; CHEF, contour-clamped homogeneous electric field electrophoresis; MI80, M210, M240, M minichromosomes from L. major LV- 561 numbered by kilobase.

linear DNAs (CD1, LD1, 715 class) unrelated to drug resistance have also been detected [9- 11]. LD1/CD1 related sequences occur within large chromosomes (>1 Mb) in all stocks of Leishmania examined to date [12]. Their exact structural relationship and their functional relationship to leishmanial survival or viru- lence remains to be determined.

We have established cloned lines of L. major that present various degrees of virulence and which show size polymorphic multicopy mini- chromosomes (M180, M210, M240, named according to their sizes in kb) related to LD1/ CD1 and 715-class DNAs. These minichromo- somes showed striking plasticity with regard to size/number, when followed through multiple transfers during in vitro culture and in vivo passage in BALB/c mice. Minichromosome sequences were located in megabase-sized chromosomes, and 760-kb and 550-kb chro- mosomes. Five random M180 derived cloned probes recognised all minichromosomes of

66

Leishmania major. All probes also hybridised to megabase chromosome of L. major, Leish- mania donovani and Leishmania amazonensis; however, only one hybridised to small chro- mosomes present in L. donovani and L. amazonensis. These findings have implications for the mechanism of generation and possible role of these elements. The different clones were characterized as to their virulence and the role of the CD 1/LD 1/715 class of elements will be discussed in this context.

Materials and Methods

L. major cloned lines. Two lines from the same stock (L. major MHOM/IL/67/Jericho- II: LV-561:LRC-137) were maintained in different conditions: LV-561/M was alterna- tively passaged into BALB/c mice to produce lesions, from which parasites were isolated and subsequently cultured in HO-MEN media [13] for up to 3 months before re-inoculation into BALB/c mice. This cycle has been continu- ously repeated in order to maintain a virulent parasite population. The second line, LV-561/ O, was maintained exclusively in HO-MEN media. Virulent parasites were operationally defined as those producing lesion(s) in mice within 8 weeks, after subcutaneous inoculation of 106 parasites. The same inoculum of non- virulent parasites fails to produce lesions in 4 months.

Non-virulent clones (C01,C02) derived from the parental population LV-561/O line and virulent clones (C2, C l l +) from the LV-561/ M line were obtained by plating their respec- tive parental populations onto soft-agar [14]. The cloned line C2 was re-cloned two years later obtaining C2.2, C2.3 (Fig. 1). Cloned line C2.2 was cryopreserved, inoculated into BALB/c mice and cultured in HO-MEN media as indicated (Fig. 1). One of the cryopreserved aliquots was subcultured start- ing from a dilution of 106 in HO-MEN media. The diluted subculture (C2.2D + ) was used to inoculate mice (C2.2DR~_3+) and to obtain further subcultures (C2.2Db + and C2.2Df+). L. donovani MHOM/ET/67/HU-3:LV-9 and L.

amazonensis MPRO/BR/73/M 1845:LV-78 were also studied.

Contour-clamped homogeneous electric field electrophoresis conditions. Agarose plugs containing 1 × l0 s parasites ml=l, harvested at late log phase, were prepared as described [15]. CHEF electrophoresis was performed in a commercial Bio-Rad apparatus (CHEF DM T M lI). All gels were 1.5% (w/v) agarose in 0.5 × TBE (45 mM Tris/45 mM boric acid/1 mM EDTA, pH 8.0). Running conditions were 160 V, at 12°C for 22 h using 60-90 s pulse time followed by 22 h using 120-160 s, except when indicated. Annealed multimers of bacter- iophage 2ci857 Sam 7 (Boehringer, Man- nheim) and Saccharomyces cerevisiae strain YNN295 (Bio-Rad) were used as molecular weight markers.

Densitometry. The relative abundance of ethidiurn bromide-stained bands was calcula- ted using densitometric determination (Joyce- Loebl laser scanner) of CHEF photographic negatives showing a linear response for S. cerevisiae chromosomes and the areas obtained were normalised relative to DNA size. Relative abundance was standardised to chromosome 1 (310 kb) as in [1]. Densitometric determination of signal hybridisation in X-ray films was normalised in turn to the relative abundance of minichromosomes.

DNA probes. Chromosomes of L. major were separated using CHEF, and the 180-kb minichromosome was isolated using electro- elution and labelled by nick translation with [~-32p]dCTP to a specific activity of 2 × 108 dpm /~g-~. The isolated 180-kb minichromo- some (M180) was digested to completion with PstI, ligated to Bluescript plasmid and used to transform competent Escherichia coli (XL1- Blue, Stratagene). Five (out of 35) M-180 cloned fragments were chosen according to their different Pstl fragment sizes: pBSM101 (1.1 kb); pBSM104 (2.1 kb); pBSM107 (0.8 kb); pBSM124 (1.2 kb); pBSM125 (1.0 kb). A synthetic oligodeoxynucleotide 3'-(GGG- ATT)16GGGA-5' derived from a DNA se-

I--

67

L. major

Salb/o mloo ,

L V - 5 6 1 / M Balb/o mloe

1 In v i t r o

I ~ LV561/MR,

B,Jlb~o mloo

In v i t r o

I - - LV561/MR,

Balb/o mloe

[ In v i t r o

I - - LV561/MR,

Balb/o mloo

In v i t r o

I ~ L V 5 6 1 / M R , +

Balb/o mloe

In v i t r o

I cloning 1 9 8 8

I 1 C2 Cl l +...

1 B,,Ib/o mloo

1 In v i t r o (40)

I sub-cloning 1987

I I C 2 . 2 C 2 . 3 C 2 . 4

I, v i t r o LV561/MR,+

C 2 . 2 D R ,

C2.2DP=

C2.2DP=

C 2 . 2 D b *

C 2 . 2 D f +

C 2 . 2 H C2.2R+ C 2 . 2 +

In v i t r o Balb /o mloe cryopreserved (400) (1~o)

L V - 5 6 1 ( 1 9 8 3 ) In v i t r o

oul ture medium

L V - 5 6 1 / 0

cloning 1985

C 0 1 . . .

Ba lb /c mloe

C2.2D + --

10 ed l lu t lon /

10 ed l lu t lon

C 2 . 2 D "

Fig. 1. History of two lines: LV-561/M and LV-561/O, and cloned lines derived from strain L. major LV-561. Lines and clones containing additional minichromosomes are indicated by (+) . The number of subcultures in vitro is indicated in parentheses. Successive serial re-isolations are referred to as R followed by the isolation number. D denotes subcultures at

6 1:10 ; f and b indicate duplicate diluted subcultures. M denotes passage through BALB/c mice, O indicates in vitro culturing in HO-MEN medium.

quence located in L. donovani telomeres and the repeat 5'-(CCCTAA)3-3' were used as telomeric probes [16,17]. M-180 derived spe- cific probes, the telomeric oligomer and cDNA probe mapping to CD1 from Leishmania infantum (ITMAP263) were labelled to 1 x 109 dpm 1 /~g- by priming random hexamers [18].

Blotting and hybridisation. Restriction DNA fragments were transferred to Hybond N nylon

membranes using alkaline conditions as re- commended by the suppliers (Amersham). CHEF gels were exposed to short UV light, followed by incubation in 1 M HC1 (30 min each treatment) prior to alkali transfer. Hybridisation of chromosomal blots to dou- ble-stranded probes was performed in 5 x SSPE (20 × SSPE = 3.0 M NaCl/230 mM NaH2PO4/22 mM EDTA)/5 x Denhardt's solution/0.5% SDS/0.1 mg m1-1 herring sperm DNA at 65°C for 16 h. Filters after

68

hybridization were washed twice in 2 x SSPE/ 0.1% SDS at room temperature for 15 min, followed by two washes in 0.1 x SSPE/0.1% SDS at 65°C for 30 min except as otherwise indicated. Hybridization to the telomeric probe was performed in 5 x SSPE/5 x Denhardt 's solution/0.1% SDS/0.1 mg m l - ~ tRNA for 2 h at 42°C. Filters hybridized to the oligomer were washed three times in 5 x SSPE/0.1% SDS at room temperature.

A 1 2 3 4 5 6

Results

Additional chromosome-sized DNAs coexist in a cloned line of L. major. CHEF analysis of parental (LV-561) and cloned lines of L. major was performed. It was observed that one (Cl l + ) of 6 clones analysed possessed one additional DNA band of approximately 210 kb (M210), when compared to the karyotype of the parental line (Fig. 2A, lane 4 vs. 6). One cryopreserved stock of clone C2.2 (referred to as C2.2+) presented a D N A molecule of 180 kb (M180) in addition to M210 (Fig. 2A, lane 3). Clones C01, C2.2H and the line LV-561/O did not show additional DNAs (Fig. 2A, lanes 1, 2 and 5). Quantitative differences are in particular evident for chromosome 2 where size (clone C2 .2+) or ethidium bromide staining intensity (C2.2 vs. C01) can differ and where chromosome band doubling occurs (Fig. 2). Size variation of chromosome 2 in another line of L. major (LT-252) was shown to be due to amplification of the miniexon locus [1,19] and this could also be the case here in some of these clones.

No differences allowing a distinction be- tween karyotypes of virulent and less virulent clones and lines derived from L. major LV-561 were observed.

Variation in the relative abundance of M210 and M180. The relative abundance of M210 and M180 in clone C2.2+ was measured using densitometry of the ethidium bromide stained gel shown in Fig. 2A. This indicated that the relative abundance of M180, M210 and chromosome 1 was 3:6:2 respectively. How-

B 1 2 3 4 L 5 6 7 8

C 1 2 3 4 Y 5 6 8 7

2200=,,-

1020=,,-

245=.-

Fig. 2. Molecular karyotypes of lines and clones of L. major. Chromosomes from the LV-561 lines and their clones as indicated below (subscripts denote number of subcultures in clones or re-isolations in lines) were separated by CHEF at 160 V and visualised by ethidium bromide staining: (A) CHEF run was pulsed at 60-90 s; 22 h followed by 120 160 s for 22 h. (1) C01; (2) C2.2H; (3) C2.2+; (4) CI1 + ; (5) LV-561/O and (6) LV-561/MR2. (B) CHEF run was at 40 s; 44 h. (1) L. amazonensis (LV-78); (2) C2.2R+; (3) C I I + ; (4) C2.2Db+; (5) C2.2D40+; (6) C2.2D2o+; (7) C-2-2Df+; (8) C-2-2DRj + ; (L) Bacterio- phage lambda ladder. (C) CHEF run was at 80 V; 480 s;

120 h. Lanes were as for (B).

ever a different ratio 16:2:2 (M180: M210:chromosome 1) was observed in a subculture of clone C2.2+ which was ob- tained by a 1:106 dilution, named as C2.2D20 + (Fig. 2B, lane 6). To investigate these multi- copy DNAs in more detail, various subcultures of clone C2.2D+ were chosen at random for CHEF analysis. Karyotypes of duplicate subcultures are shown in Fig. 2B: clone C2.2Db+ possessed an increased number of copies of M210 compared to M180 (2:10:2 for M180:M210:chromosome 1) whereas clone C2.2Df+ presented more copies of M180 relative to M210 (12:2:2, M180:M210:chro- mosome 1; lane 4 vs. 7) (Table I). This plasticity was also apparent for C2.2D+ samples differing only in number of passages in liquid culture (Fig. 2B, lanes 5 and 6 and Fig. 3A, lane 6). Furthermore, a new DNA molecule of 240 kb (M240) was demonstrated in clone C2.2R+, which was analyzed after 150 passages in culture (Fig. 2B, lane 2). C2.2R+ was originally isolated from BALB/ c mice inoculated with C2.2 (whose karyotype was not determined prior to inoculation). When C2.2D+ containing M210 and M180 was used to inoculate mice and parasites were re-isolated, generating clones C2.2DR~_3+, the karyotypes consistently presented only the smaller M180 molecule (Fig. 2B, lane 8, and Figs. 3, 4 and 5).

Three re-isolates (LV-561/MR1 3) obtained during a year of periodic cycles of mice/culture passages of the line LV-561/M, did not have

T A B L E I

Size and relative abundance o f minichromosomes

Cloned line Min ichromosomes

Size (kb) Relative abundance

MI80 M210 M240 Chr l

C2.2+ 180, 210 3 6 - 2 C2.2D20 + 180, 210 16 2 2 C2.2D40 + 180, 210 12 1 - 2 C2.2D60 + 180, 210 1 12 - 2 C2.2Db + 180, 210 2 10 - 2 C2 .2Df+ 180, 210 12 2 - 2 C2.2R + 240 - - 12 2 C2.2DR] + 180 24 - - 2 C l l + 210 - 10 - 2

69

minichromosomes when analysed by CHEF electrophoresis and hybridisation to M180 probes. A fourth isolate (LV-561/MR4+) showed a DNA element of 180 kb which hy- bridised strongly to both telomeric probes (Fig. 3, lane 2 and data not shown). This element did not migrate differently with pulse or temperature variations (Figs. 4A and 5A, lane 2; data not shown). The same DNA element hybridized to the M180 probe, ob- tained from C2.2+ (Fig. 4, lane 2), and the M180 derived probes (Fig. 5A and B, lane 2).

A 1 2 3 4 5 6 7 8

I . L , .

B 1 2 3 4 5 6 7 8

291

194,,-

Fig. 3. Southern blot analysis o f molecular karyotypes with a telomeric probe. (A) Ethidium bromide staining pat tern o f a C H E F gel run at 160 V; 40 s; 44 h, for the following lines and clones: (1) C2 .2DR2+; (2) LV-561 /MR4+; (3) C 2 . 2 D b + ; (4) C 2 . 2 D f + ; (5) C2.2H; (6) C2.2D60+; (7) C 2 . 2 D R 3 + and (8) C 2 . 2 D R ~ + . (B) S o u t h e r n blot hybridization pat tern to the telomeric probe o f the gel in

(A).

70

A kb

comp-_.. 1125=,,-

770 , .

5 8 0 , -

245 , .

B comp

C

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 ~ q i anlm t B I ~ ,~ ,* o

. i n l m ~ b a l l b 0 4 1 D , m m - - - .

7 7 0 , .

580=,-

245, . - I ~ O n

10 1 1 1 2 1 3

770=,.-

s 8 o ~ ,

2451,,--

A

c o n q

1

B comp

C

comp_,,.

D

770=,,-

2 4 5 ~ -

7701,,--

2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

Fig. 4. Hybridisation of chromosomal profiles to the minichromosome M180. (A) Ethidium bromide stained CHEF gel performed at 16°C for the indicated lines and clones: (1) LV-561/MR3; (2) LV-561/MR4+; (3) LV-561/ MRs,left+, isolated from left inguinal node; (4) LV-561/ MR5,right+ , isolated from right inguinal node; (5) CII + ; (6) C2.2Df+; (7) C2.2DR3 +; (8) L. donovani (LV-9) and (9) L. amazonensis (LV-78) (Y) S. cerevisiae chromosomes as molecular weight markers. (B) The gel in (A) was blotted onto nylon and hybridised to radiolabelled total M180 which was isolated from clone C2.2D+. (C) Another CHEF gel (100 V; 40-60 s; 50 h) hybridised to the same

probe as in (B): (10) C2.2R+; (11) C2 ; (12)C2.2H.

To investigate the fate of M180 in BALB/c mice, LV-561/MR4 + was inoculated at the tail base and parasites were isolated (4 weeks later)

2 4 5 , .

Fig. 5. Southern blot analysis using MI80 and CDI derived probes. CHEF conditions (except temperature) were as in Materials and Methods. (A) Another gel using the same samples and CHEF conditions as in Fig. 4A except that the temperature was 6°C, was blotted and hybridized to pBSM101. (B) The gel shown in Fig. 4A was blotted onto nylon and hybridised to pBSM104. (C) The gel shown in Fig. 4A was blotted onto nylon and hybridised to a CD1 1.3 kb cDNA probe derived from L. infantum (cloned line ITMAP263). (1) LV-561/MR3; (2) LV-561/MR4+; (3) LV- 561/MRs,heft+, isolated from left inguinal node; (4) LV- 561/MRs,right+, isolated from right inguinal node; (5) CI 1 + ; (6) C2.2Df+ ; (7) C2.2DR3 + ; (8) L. donovani (LV-

9) and (9) L. amazonensis (LV-78).

from both inguinal lymphatic nodes. It is interesting to observe that the isolate obtained from the left node appeared to contain more copies of minichromosome by ethidium bro- mide staining when compared to the isolate from the right node and to the original inoculated parasite population (Fig. 4A, lanes 3 and 4). Hybridisation with M 180 probes gave unexpected results. The probes hybridised more strongly with the original inoculate than with the left node isolate and did not hybridise at all with the right node isolate, although minichromosomal material was clearly present (Fig. 5A and B lanes 2-4). This difference in the predominant minichromosome in parasites isolated from each node was intriguing and it is currently being studied.

Minichromosomes in cloned lines are a family of DNA elements. M210 and M180 behaved as linear molecules since their mobility did not change with pulse frequency (Fig. 2B and C) nor with temperature variation in CHEF runs (Figs. 4A and 5A; data not shown) [20]. Moreover these molecules hybridised to telo- meric probes (Fig. 3 and data not shown), therefore it can be concluded that M210 and M180 represent true small chromosomes.

To study the relationship between these minichromosomes, ct-32p-labelled M180 was used as a probe (Fig. 4). The strong hybridisa- tion signal obtained at high stringency, indicated that M210 and M240 share homo- logous sequences with M180. In addition, the M 180 probe hybridised to chromosomes of 550 kb, 760 kb and to the 'compression zone' (Fig. 4B). Although clones C2 and C2.2H did not show minichromosomes according to ethidium bromide staining and hybridisation to the M180 probe, their hybridisation pattern above the 550-kb chromosome range was similar to those clones containing them (Fig. 4C). Five M 180-derived probes from C2.2D + (pBSM101, pBSM104, pBSM107, pBSM124, pBSM125) clearly hybridised to M180, M210 and M240 of all L. major cloned lines. All M 180 specific probes clearly hybridised to the 'compression zone' (Fig. 5A and B and data not shown), and pBSM101 and pBSM104 also

71

slightly recognised 550 kb and 760 kb and other chromosomes after prolonged film exposure (data not shown).

The relationship of M180 to other small circular and linear molecules. The presence of mini- chromosomes homologous to M180 was investigated in other Leishmania species in our laboratory. The M180 DNA fragment contained in clone pBSM104 hybridised to multiple chromosome-sized DNAs larger than 760 kb, including the 'compression zone' (Fig. 5B, lane 8), and after long film exposure also to small DNA not detected by ethidium bromide staining in L. donovani (LV-9) (data not shown). On the other hand, even after prolonged film exposure, no hybridisation of probes pBSM101, pBSM107, pBSM124 and pBSM125 to this DNA was observed other than in the 'compression zone' (Fig. 5A and data not shown). The pBSM104 probe also hybridised in L. amazonensis stock (LV-78) to chromosomal DNAs larger than 760 kb (Fig. 5A, lane 9) and weakly to 2 small DNA elements not detected by ethidium bromide staining (data not shown). In contrast and similarly to what was observed in L. donovani, pBSM101 only hybridised to the 'compression zone'. This hybridisation pattern indicated that the minichromosomes (M180, M210 and M240) described here are related to CD1/ LD1 DNAs and the 715-class, previously found in various Leishmania species including the L. donovani line analysed here [21]. This point was investigated using a 1.3-kb cDNA probe [22] mapping to CD1 from L. infantum (ITMAP263) (Fig. 5C) and a CD1 probe of L. mexicana M379 (not shown) [21]. It is con- cluded that the minichromosomes of L. major LV-561 observed here are related to previously described CD1/LD1 and 715-class sequences.

Discussion

This report describes small, linear multicopy DNAs which possess telomeric sequences and therefore are true minichromosomes. These minichromosomes behave in a very dynamic

72

manner and diversify within cloned popula- tions. Clone C2 for instance, when it was karyotyped did not show the presence of these elements. Of 3 stocks derived from the subclone C2.2, one maintained in vitro (C2.2H) showed no minichromosome, one passaged in mice (C2.2R+) displayed a M240, and one cryopreserved (C2.2+) showed M210 and M180 minichromosomes. Several descendant lines of C2.2D + possessed only the 180-kb minichromosome, others showed variable proportions of M I80 and M210 minichromosomes. Ours results suggest that M180 in LV-561/MR4+ arose de novo during in vivo passage. When LV-561/MR4+ was inoculated into mice, parasites were isolated from the inguinal nodes. The isolate from the left node had an increased relative abundance of M180 compared to both the isolate from the right node and the original inoculated LV-561/MR4+. However, the ka- ryotypes did show a differential hybridisation signal to M180 derived probes and telomeric probes, suggesting that the stained band corresponding to 'M180' is heterogeneous. Since LV-561/M is a stock, part of the heterogeneity observed could be due to selec- tion of a minor population containing M 180.

Hybridisation with the CD1 of L. mexicana (M 379) and with a probe derived from CD1 of L. in[antum (ITMAP263) belonging to the common region of all the CD1 elements classifies the linear chromosomes of L. major described in this paper as belonging to the CD1/LD1 family of genetic elements related to the 715-class. The hybridisation observed with the M180 cloned probes and the pattern reported by others [11] suggest that minichro- mosomes have sequences from megabase chromosomes but only part of them belong to a common region between species which could be involved in the generation mechan- ism.

The hybridisation pattern obtained when the minichromosome M180 was used as a probe suggests that there are homologous sequences present in megabase chromosomes and se- quences present in chromosome sizes of 760 and 550 kb. The fact that MI80 and the probes

derived from M180 do not hybridise with the same relative intensity to the compression zone and to the 550- and 760-kb chromosomes could be due either to the presence ot additional sequences in M180, or to differ- ences in relative sequence copy number in different chromosomes. However, similar re- sults may be generated by the presence of inverted repeats in M180 which are prone to 'snapback' and yield weak hybridisation signals.

The presence of M 180 sequences in the large chromosomes suggests that the minichromo- somes originate through a genetic mechanism involving larger chromosomes (as source) as was proposed for the 715 class [11]. In L. mexicana it has been postulated that conver- sion of linear into circular form occurs via a CHl-like sequence [22]. The postulated recom- bination mechanism via CHI-like sequences would not only generate circular elements but would also lead to different sizes of minichro- mosomes by recombination events. The size difference (180, 210, 240 kb) suggests that the basic repeat is about 30 kb as observed for the other CD1/LD1 elements.

It will be interesting to investigate the relationships existing between the small linear DNA elements described here for L. major LV- 561, the small polymorphic chromosomes of L. infantum which also vary by size increments of 30 kb [2], and the 715 class in another line of L. major [ 111.

The increased occurrence of these additional DNAs in all species of Leishmania (our data, and ref. 12) indicates that the generation process might be a common basic feature capable of attributing to the parasite pheno- type advantages for survival and/or rapid adaptability. All clones of LV-561 containing these elements analysed to date were highly virulent, as defined by the limited criteria of appearance of lesions within 8 weeks from inoculation of BALB/c mice. However, it is clear that these elements do not code any function essential for parasite virulence or indeed survival since various clones which did not possess them were almost as virulent as those which did have them. Although M 180 is

highly transcribed (M. Navarro, unpublished), the coding capacity of these DNAs in Leishmania as well as their structure in the 'source' chromosomes, need to be studied. Apart from using expression vectors derived from these elements, newly developed methods [23-25] for DNA transformation of selected Leishmania clones should provide a powerful experimental handle to test this hypothesis.

Acknowledgements

W e t h a n k A . N a v a r r o a n d I. N a v a r r o fo r p a r a s i t e cu l t u r i ng , M s T. K n a p p fo r the syn thes i s a n d D r J. C r a m p t o n fo r p r o v i d i n g the s u b t e l o m e r i c / t e l o m e r i c o l i g o n u c l e o t i d e . W e a re g r a t e f u l to D r M. C h a n c e fo r s t r a in s o f L. donovani (LV-9) and L. amazonensis (LV-78). We also thank J. Liu, D. Muthui and N. Gajendran for hybridization experiments with the CD1 probe. This work was supported by the Spanish FISS programme grant (90-0459) and by the European Economic Communities 'Science and Technology for Development' programme (TS2-0034-UK/AM) and the Bel- gian BIO-10 program. R.M. is a Wolfson Lecturer in Molecular Genetics.

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