Inverted repeat structure and homologous sequences in the LD1 amplicons of Leishmania spp.

MO~LAR AND

ELSEVIER

Molecular and Biochemical Parasitology 68 (1994) 69-80

BIOCHEMICAL PARASITOLOGY

Inverted repeat structure and homologous sequences in the LD 1 amplicons of Leishmania spp. Miguel Navarro a,b, Jianhua Liu a, David Muthui a, Gines Ortiz b, Manuel Segovia b, Raymond Hamers a,, a Instituut voorMoleculaire Biologie, Vrije UniversiteitBrussel, Paardenstraat60, B1640 St.-Genesius-Rode, Brussels, Belgium b Departumento de Gendtica y Microbiologia, Facultad de Medicina, Universidad de Murcia, 30100 Murcia, Spain

Received 7 September 1993; accepted 22 July 1994

Abstract

In the parasitic trypanosomatids of the genus Leishmania, novel circular (CD) and linear (LD) multicopy genetic elements arise de novo either spontaneously or as a result of drug selection. We report that the LD 1 minichromosomes of L. donovani, L. major and L. mexicana (ranging in size from 180 to 230 kb) have an inverted repeat structure and contain homologous sequences located at similar distances from the telomere; one half of the chromosome being the mirror image of the other. They must therefore have originated from a unique conserved source chromosome; the size polymorphism being generated by the point at which inversion occurs. The circular CD 1 elements appear to be circularised segments of the LD 1 elements. These observations lead to a unified concept of how minichromosomes LD 1 and circular CD 1 genetic elements emerge within the Leishmania and contribute to evolution of karyotype. Keywords: Leishmania; Amplified DNA; Minichromosome; Chromosomal rearrangement; Inverted repeat

1. Introduction

The development o f pulsed field gel (PFG) electrophoresis has allowed the study o f different Leishmania karyotypes and the observation o f D N A amplification via the generation o f extra chromosomal D N A s (for review, see [1]). Most circular multicopy D N A s occur when there is selection for drug resis-

Abbreviations: ED1, Linear DNAs 1; CDt, Circular DNAs 1; PFG, pulsed field gel * Corresponding author, Tel.: 32-2-358 34 17; Fax: 32-2-359 03 90; E-mail: [email protected].

Elsevier Science B.V. SSDI 0166-6851(94)00147-2

tance [2-5]. Circular and linear amplicons in ct-difluoromethylornithine-resistant Leishmania donovani [6] as well as in arsenite-resistant Leishmania tarentolae [7] have also been described. Related circular and linear multicopy D N A s appear not only when there is selection for drug resistance, but also spontaneously, as in the case o f Circular D N A 1 (CD 1) in one clone of Leishmania mexicana M379 [8], and in Leishmania infantum I T M A P 2 6 7 as a circle o f 56 kb [9,10]. The CD 1 elements are related in sequence to small linear multicopy chromosomes of variable size, which appear in the absence of known selective pressure in another clone of the same L. mexicana M379 isolate

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M. Navarro et al./Molecular and BiochemicalParasitology68 (1994) 69-80

(LD 1 of 210 kb) [8], in L. donovani L133 (LD 1 of 180 kb) [9], in L. donovani HU-3 (HU-3 minichromosome) [11], in Leishmania major LV-561 [12] (LDlS of 180, 210 and 230 kb), in L. major LT252 (715-class of small linear DNAs) [13] and in Leishmania braziliensis M2903 [9,14] (LD 1 of 250 kb). Related sequences of all these are also present in a large chromosome (1.6 Mb) in all species. It has been postulated that these elements are extrachromosomal amplifications which are generated in a conservative manner from the source megabase chromosome. The observation that the circular CD 1 and linear LD 1 elements of L. mexicana, L. donovani, L. infantum, L. major and L. braziliensis contain shared sequences led us to investigate in detail the structure of linear LD~ elements in several species. In the present work we show that all the LD~s examined have an inverted repeat structure and conserved homologous sequences located at similar distances from the telomere. The length of the central inverted repeat region of these LDlS is responsible for the size polymorphism. The circular C D 1 elements appear to be circularised segments of the LD 1 elements. This is the first report which clearly describes the relationship between circular and linear amplicons of the LD1/CD 1 family.

2. Materials and methods

2.1. Parasites Evans clone No. 8 of L. mexicana M N Y C / B Z / 6 2 / M 3 7 9 was used to isolate LD 1 of 210 kb, while Garnham clone No. 1 from the same isolate was the CD 1 source [8]. L. donovani ITMAP1899/LCRL133 was the source of an 180-kb LD 1 [9]. L. infantum M H O M / M A ( B ) / 6 7 / I T M A P 2 6 7 was used to isolate a CD 1 of 56 kb. These promastigotes were cultured in GLSH medium [15] supplemented with 10% foetal calf serum. Three clones (C2.2DR, C l l , CO.1) from the same stock (L. major M H O M / I L / 6 7 / Jericho-II; LV-561) were used to isolate the 180-, 210- and 230-kb size polymorphism LDls [12]. L. major promastigotes were cultured in HO-MEM media [16] supplemented with 10% foetal calf serum.

2.2. Pulsed field gel electrophoresis and LD 1 physical maps Parasite blocks were prepared as described previously [17] but at a preparative concentration of 10 9 cells m1-1. Pure LDlS were isolated from 1% lowmelting-point agarose gels performed in a Contourclamped homogeneous electric field apparatus (CHEF, Bio-Rad) at 20 s pulse time, 6 V cm -1, 120 ° angle, at 14°C for 20 h. The first band, corresponding to LDls, was cut out of the gels with a razor blade, transferred to an Eppendorf tube and covered with I × T E buffer (10 mM Tris-HC1/1 mM EDTA). Digestion of the isolated LDlS were carried out on agarose pieces incubated in 1 × restriction buffer as described previously [18], using 50100 U of restriction enzymes to produce large fragments. The products were separated in 1% agarose CHEF gels using 2-8-s pulses, 6 V c m -1, for 16 h to resolve fragments between 150 kb and 4 kb. Another programme (2-s pulses, 6 V cm -1, 16 h) was used to separate fragments ranging from 100 kb to 2 kb. Fragment size was estimated against A concatemers and A HindlII fragments embedded in agarose blocks and separated in the same gel. 2.3. Densitometric analysis Gel photographic negatives were analysed using a Hitachi KP-C503GE/K camera, over a Lambert illumination box. The densitometric profiles were obtained with a Kontron Imco 10 image analyser. 2.4. Probes and hybridisation The following probes were used for hybridisation: (1) A telomeric probe of 150 bp of 5 ' - ( C C C T A A ) n - 3 ' repeats; (2) cDNA clones selected from L. major clone C2.2DR 1 + cDNA library [12] in h ZAPII (Stratagene) and hybridising with a pure LD 1 180-kb probe; (3) Cloned fragments of CD 1 L. infantum designated as B8 (3.4 kb BamHI) and El3 (1.3 kb EcoRI) [10]; (4) A cloned cDNA fragment of 1.2 kb of L. infantum CD 1 mapping in the region common to CDls of L. mexicana and L. infantum [12]; (5) A cloned gDNA fragment C37 encompassing the Chilike sequence of CD 1 of Z. mexicana M379 [19]; (6) An L. mexicana LD 1 probe E28 to determine the

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M. Navarro et al. / Molecular and Biochemical Parasitology 68 (1994) 69-80

H y b r i d i s a t i o n of c h r o m o s o m a l blots to doublestranded probes was performed as in [12]. Filters were w a s h e d twice in 2 X SSPE, (20 X SSPE = 3.0

orientation of C D 1 L. m e x i c a n a in L D 1 L. m e x i c a n a [8]. A l l probes were labelled u s i n g r a n d o m primers

[201.

L. major

LDls Asn kb

180210

l

Asn

Sspl

230

180210

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180210

230

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230

180210

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194 145.5

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230

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Fig. 1. Restriction fragments of the three size-polymorphism LDlS of 180, 210, and 230 kb of L. major. After digestion of PFG purified LDxs by different enzymes indicated above, the fragments were separated by a second PFG electrophoresis using 2-8-s pulses for Asn I and Ssp! (top) and 2 s pulses for XbaI (bottom). After transfer to nylon membranes, they were hybridised with the probes indicated below each gel.

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M. Navarro et al. / Molecular and Biochemical Parasitology 68 (1994) 69-80

M NaC1/230 mM NaH2PO4/22 mM EDTA)/0.1% SDS at room temperature for 15 min, followed by two washes in 0.5 × SSPE/0.1% SDS at 65°C for 30 min. The characterisation of inverted repeat sequences was performed by a modification of the method described by Ford et al. (1985). Total DNA of clone C2.2DR1 was denatured with 100 mM NaOH for 60 min at room temperature and then neutralised with 1 M Tris-HC1, pH 7.8 and 100 mM HC1 on ice. The DNA was precipitated and digested with various restriction enzymes.

the size estimated from PFG, indicating the presence of repeated structures. For instance, the XbaI digest LD 1 180 yielded restriction fragments of 9, 12, 18, 25 and 35 kb, totalling 97 kb instead of the expected 180 kb (Fig. 1; Table 1). The fragments, characterised by size, fell into three sets, namely: (1) one fragment, later shown to hybridise to a telomeric probe, was similar in size in LD 1 80 and LD 1 210 but slightly larger in LD 1 230; (2) a series of fragments common to all three chromosomes; (3) one or more fragments characterising each chromosome. In the AsnI digest this latter class of chromosomespecific fragment is represented by a single fragment, of 100, 130 and 140 kb for the CD t elements of 180, 210 and 230 kb, respectively. In the SspI digest there are single fragments of 34 and 60 kb for the LD 1 elements of 180 and 210 kb, respectively, and three fragments (4 kb, 5 kb and 30 kb) for the 230-kb LD 1 elements. Similarly, in the XbaI digest single fragments of 18 kb and 49 kb, respectively, are present in LD 1 180 and LD1 210, and appear to be replaced by 22-kb fragments in LD 1 230. Hence, the size variation of LD 1 correlates with differences within a single region, although this is not self-evident. Other unusual features appear from the analysis of these digests. First, as already mentioned, the sum of

3. Results

3.1. LD l elements of L. major are inverted repeats An initial comparison was made to determine the origin of the size differences between the three LD 1 chromosomes found in L. major LV-561. LD 1 minichromosomes of L. major (180, 210 and 230 kb), isolated from PFG, were digested with the restriction enzymes AsnI, SspI (Fig. 1, top) and XbaI (Fig. 1, bottom) which yield a small number of fragments. Unexpectedly, the sum of the sizes of the restriction fragments obtained always fell short of Table 1 LD1 restriction fragment sizes a Asn I

L. major LV-551 Ssp I

Xba I

Estimated size b Single fragment c Double internal fragments

180 100 28

210 130 28

230 140 28

180 34 18 11 10 6 5.5

210 60 18 11 10 6 5.5

Double telomeric fragments Total size simple structure a Total size symmetric structure e

11 139 178

11 159 208

13 181 222

21 105.5 177

21 131.5 208

230 5 18 11 10 6 5.5 30 23 112.5 220

L. donovani LCR-133 Spe I

L. mexicana M379 Spe I

180 18 25 12 9

210 49 25 12 9

230 22 25 12 9 22

180 19 30 16 14

210 27 58 6.5 6

33 97 176

33 128 206

35 125 226

21 100 180

20 117.5 208

a Sizes are in kb b The estimated size is obtained by comparing the migration of the intact LD1 with the 48.5-kb A ladder. c The single fragment is the unique fragment which gives the lower ethidium bromide staining. d The total size 'simple structure' is the sum of the sizes of the restriction fragments. The total size 'symmetry hypothesis' is obtained by multiplying the sum of the double fragments by a factor 2 and adding to this sum, the size of the single fragment.

M. Navarro et al. / Molecular and Biochemical Parasitology 68 (1994) 69-80

the sizes of the restriction fragments is up to 50% smaller than the physical size estimated from PFG and depends upon the restriction enzyme utilised. The unique chromosome-specific fragments, or, in the case of LD 1 230, one of the fragments which correlate with the differences in size, always appear to fluoresce less intensely after ethidium bromide staining, a feature which can be also detected by densitometry (Fig. 2). Finally, when the digests were probed with a telomeric probe, a single hybridising fragment was obtained with each of the restriction enzymes utilised. One possible explanation for these unusual features is that the LD1 chromosomes have an inverted repeat structure and that the variable size fragments represent the central inversion region. In such a model, only the central fragment should be unique and represented once, whereas all other fragments are represented twice. A calculation based on this assumption, in which the less intensely stained

Ssp I

73

fragment was considered as unique while the other fragments were considered as doubles, yielded an estimate corresponding to the physical size obtained from PFG, irrespective of the restriction enzyme utilised (Table 1). A long range map of the LD t chromosomes could be established using double digestion with restriction enzymes and by analysing the digests with cDNA and genomic DNA probes derived from LD 1 and CD 1 elements of L. major, L. mexicana and L. infantum. In Southern blots, all probes hybridised either to a single fragment or to two fragments shown to be adjacent. For instance, the 3.9 cDNA probe and the telomeric probe hybridised to the same XbaI 33-kb fragment. In the AsnI digest however, the 3.9 cDNA probe hybridised to the ll-kb fragment and to the 28-kb fragment, whereas the telomeric probe only hybridised with the l l-kb fragment. This establishes that the ll-kb and 28-kb fragments

Spe I

I LDI 180

, I LD1 180

L. major

L. donovani

Xba I Spel

~ LD1 180

L. major

, LD1 210

~ L. mexicana

Fig. 2. Densitometric scans of LDls restriction digests. Restriction fragments were separated in 1% agarose PFG. The ethidium bromide-stained gels were photographed using a UV transilluminator and the photographic negatives were scanned. The band showing lower fluorescence intensity is indicated by an arrow in each densitometric tracing.

74

M. Navarro et aL /Molecular and Biochemical Parasitology 68 (1994) 69-80

mation concerning the differences between LD 1 180, LD 1 210 and LD~ 230 of L. major. The B8 probe hybridised with the LD 1 210 and LD 1 230 elements whereas the El3 probe only hybridised with the LD 1 230. Mapping was compatible with the location of 1.2 cDNA as the distal marker in LD~ 180 whereas B8 and E13 mapped respectively as the distal markers in LD 1 210 and LD 1 230. This result indicates that the larger size was not due to internal duplication but to inclusion of additional genomic sequences present in the CD 1 of L. infantum. Moreover, the order of the markers 1.2 cDNA, B8 and El3 is

are adjacent and that in the model proposed the 100-, 130- and 140-kb fragments are derived from the central segment of the chromosome. This of course explains why the size estimates obtained by adding the AsnI restriction fragments were always larger than those obtained using SspI and XbaI restriction fragments where the putative central fragment is much smaller. The deduced map was confirmed by digesting the larger fragments with other enzymes (data not shown). The probes from the well-characterised C D 1 of L. infantum 1.2 cDNA, B8 and El3 gave unique infor-

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0::1. I Fig. 3. Characterisation of the symmetry of the central region of M180. (A) Detailed restriction map of the central Xba fragment of LD 1 180 (one half shown). The location of the sequence hybridising with 1.2 cDNA probe is indicated. The enzyme used were Xba I(X), EcoRI(E), Eco 0109I (EO), Barn H 1 (]3) and Kpn I (K). (B) Southern blot of genomic DNA digested with EcoRI and EcoOl091 and probed with the 1.2 cDNA probe before or after alkaline denaturation and reannealing.

75

M. Navarro et al. /Molecular and Biochemical Parasitology 68 (1994) 69-80

size estimated from P F G and is compatible with the hypothesis of a symmetrical inverted repeat. This hypothesis implies that the central fragment

identical in L . m a j o r and L. infantum. T h e size of the genetic map estimated by both restriction analysis and sequence localisation corresponds to 50% of the

LD1 180 kb

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Fig. 4. Restriction fragments of LDls from L. donovani and L. mexicana. After digestion of LDls with restriction enzymes indicated above each lane (X, XbaI; Sp, SpeI: Sf, SfiI; N, NotI) the products were separated by electrophoresis. The gel was transferred and hybridised with the probes indicated at the bottom.

M. Navarro et al. /Molecular and Biochemical Parasitology 68 (1994) 69-80

76

will, upon further restriction mapping, display the same features as the entire chromosome, namely that the sizes of the restriction fragments will be between 50% and 100% of the original fragment and that both DNA strands of the central region will upon denaturation and renaturation snap back to form hairpin molecules with the behaviour of a doublestranded DNA of half the original size. Both these predictions were verified for the L. major LD 180 chromosome. Map measurements showed that the

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cDNA 1.2 probe hybridised to sequences which must lie quite close to the inversion point. Genomic DNA of L. major clone CS.2DR~ which contains the LD]180 was denatured with alkali and reannealed and then digested with EcoRI and EcoOl09I. The 1.2 eDNA, which hybridised to a 4.2-kb fragment in the native DNA, now hybridises to a 2.1-kb fragment, indicating that the Eco RI and EcoOl09I fragments are perfect or near perfect repeats (Fig. 3).

~

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I 50

I 60

LD1 210 L, major

ss ' '

i iLD1230L'major

(Sf)(X) i u i

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L, mexicana

~i

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L, donovani

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L, mexicana

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[] iNE N N N i . . . . . . .

I

L, major

I 70

, N

Iw

I 90

I0 10

I 80

2,9 eDNA

NA

LD1 I., m~cana Probe

CD1

EEi i

I 110

CD1 L,

infantum

kb

El3

L. infantum Probes

Fig. 5. Restriction maps of LD1/CD ] elements in different species. Only half of the maps arc sbown, with the telomeric sequence on the left and the symmetry axis represented by a dotted line on the right. The LD] restriction maps were constructed from the fragments sizes obtained by infrequent cutters. (A, AsnI; S, Sspl; X,XbaI; Sp, SpeI: St', SfiI; N, NotI; C, ClaI; E, EcoRI). The CD 1 half maps are taken from refs. [8] (L. mexicana) and [10] (L. infantum). The shadow box indicates the approximate position of the sequences hybridising to the DNA probes. For clarity, ClaI sites in L. mexicana LD 1 and the Spel sites in L. mexicana CD 1 have not been indicated. EcoRI sites in L. infantum LD 1 and SpeI sites in L. infantum CD I have not been determined. Restriction sites between brackets in L. mexicana are only present on one arm of the LDlS.

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M. Naoarroet al. / Molecular and BiochemicalParasitology 68 (1994) 69-80

are compatible with an identical localisation in each species.

3.2. The LD I minichromosomes have homologous and conserved structures in L. major, L. mexicana and L. donovani

3.3. The relationship of CD I sequence to LD t sequences

When the LD1 chromosomes of L. mexicana and L. donovani were digested with a set of restriction enzymes, similar features emerged from the restriction analysis (Fig. 4). The sum of the fragments gave a size estimate smaller than the physical size of the whole chromosome (Table 1); a single telomeric fragment was detected after hybridisation and one of the bands in ethidium bromide staining consistently showed a weaker fluorescence (Fig. 2). These results suggest a homologous inverted repeat structure common to the three species. All the probes hybridising with the 180-kb L. major LD x gave positive hybridisation on both LD 1 of L. donovani and L. mexicana. It was thereby possible to construct a map which describes the position range of each sequence by localising hybridising sequences between two restriction sites (Fig. 5). The order of the hybridising sequences was found to be identical in each species and the map distances

CD 1 and LD 1 sequences have been described in two different clones of an L. mexicana isolate [8]. The CD 1 sequence is completely present in LD 1 and the restriction analysis data [8] were utilised to confirm by hybridisation its presence and orientation within the other minichromosomes. These results show that the CD1 sequence is located close to the central inversion point of the 180-kb chromosome of L. major and L. donovani. The CD 1 of L. mexicana and L. infantum share also a region of sequence homology (Fig. 5). From this alignment it can be seen that L. infantum CD 1 extends towards the central region of LD 1 chromosomes as already shown by hybridisation of the B8 and E l 3 respectively with the LD 1 210 and 230 or with LD~ 230 alone; the E l 3 sequence being localised by alignment at 110 kb from the telomere.

L. major

LDls Sap

Aan I kb

180210

230

Asn I

I

180210

230

194 14,5.6 97

180210

230

Sap 180210

I

Aan 230

180210

I 230

Ssp 180210

I 230

e

*e m

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~

23.1 9.4

dP

6.6

,IP

. o

i

4.3

1.2 c D N A

Probe

B8 Probe

E13 P r o b e

Fig. 6. Hybridisationof the gel shown in Fig. 1 with the probes, from L. infantura CD1, indicated at the bottom. Probeswhich by alignment (Fig. 5) fall outside the shorter LD1 sequencesfail to hybridise.

78

M. Navarro et al. /Molecular and BiochemicalParasitology 68 (1994) 69-80

4. Discussion In this paper we provide a unifying insight into the structure of the amplicons of the L D 1 / C D 1 family in the Leishmanias. In the three species examined, L. donovani, L. major and L. mexicana, evidence is provided that the LD 1 elements have a homologous inverted structure composed of two identical unique sequences centered on an inversion point. The sum of the restriction fragments gives a variable size estimate dependent on the restriction enzyme used and always smaller than the physical size estimated from PFG. In limit restriction digests, a fragment can be found which consistently appears to be less abundant by ethidium bromide fluorescence, and a single fragment is found which hybridises to a telomeric probe. All these discrepancies disappear if the hypothesis is made of an inverted repeat structure composed of two unique and identical sequences. In the deduced maps, the telomeric probe hybridises to a single terminal fragment indicating that it is indeed recognising a telomere and not some internal telomeric sequences. This telomeric end has the same size in L. major LD 1 180 and L. major LD 1 210 and is slightly larger in L. major LD 1 230. Therefore, if our conclusions are correct, both ends of the LD1 elements have telomeres identical in size; this in turn suggests that telomere size is somehow regulated by the identical symmetrical structure. The sequences identified by specific DNA probes could be ordered using restriction digestion, and showed that in all three species the LD 1 possess a homologous map conserving the order and location of the hybridisation of sequences. The existence of a central symmetric fragment was demonstrated in the case of LD 1 180 by heating and annealing experiments. The larger LD 1 chromosomes are characterised by incorporation of additional unique genomic material within the central region and not by subtelomeric or other duplications as it is the case for other small chromosomes showing size variations [18]. Hence some sequences from the largest LD~s fail to recognise the smaller ones (Figs. 5 and 6). This could explain the results described by Beverley and co-workers [13] using different probes to analyse the relationship of the 715class, HU-3 and LD~ minichromosomes. One of

these 715 class probes, P19, recognised all minichromosomes but not L. infantum CD 1 while a cloned fragment from L. infantum CD1, the B8 probe, hybridised to LD1, HU3 and some, but not all, 715-class minichromosomes. This probably occurs because P19 is presumably derived from a region closer to the telomere than B8. With the exception of the 715 class and the HU-3 mini-chromosomes the LD 1 elements do not appear to be related to other small chromosomes and their sequences crosshybridise mainly with large megabase chromosomes which have been postulated as the source from which the LD 1 can be repeatedly generated. These findings support the view that an evolutionary highly conserved unique chromosome is acting as a reservoir from which the CD 1 and LD 1 are amplified within the different species. The putative source chromosome is however quite large and it is possible that larger LD 1 elements have been generated which integrate a larger fragment of the source chromosome and which by virtue of size will migrate among the larger chromosome. This could be the case in the L. donovani/L, infantum complex where a single chromosome of intermediate but variable size hybridising with the cDNA 1.2 is detected in several stabilates [9]. The C D 1 elements of L. infantum and L. mexicana appear to be derived from overlapping sections of the LD 1 elements, in which the CD1 of L. mexicana appears to be a perfect or near perfect direct repeat with a map size of approx 26.6 kb and a physical size of 53 kb (Nguyen, Bouton and Hamers, unpublished results) whereas the CD~ of L. infantum, appears to be an inverted repeat and has possibly retained one of the inversion junctions of an LD 1 chromosome. Circularisation could then have occurred through a recombinational event localised between the LD~ inversion point and the telomere. Hence, we suggest that CD 1 sequences originate through recombination events between or within the multicopy LD~s a n d / o r their source chromosome. It is possible that other already characterised circular genetic elements originated from source chromosome regions further away from the telomere and do not contain sequences present in the small LD~ examined. The different-sized LD 1 chromosomes might be generated by homologous recombination involving

M. Navarro et al. / Molecular and Biochemical Parasitology 68 (1994) 69-80

repeat or inverted repeat sequences located in different positions in the source c h r o m o s o m e , as has b e e n described for the H circle f o u n d in L. t a r e n t o l a e [7,22]. At present, the only d o c u m e n t e d repeat seq u e n c e in the L D 1 is the C h i - l i k e s e q u e n c e f l a n k i n g the C D 1 sequence of L. m e x i c a n a [21]. Alternatively, the D N A p o l y m e r a s e could double b a c k to m a k e a hairpin m o l e c u l e w h i c h w o u l d then yield an L D t s y m m e t r i c e l e m e n t in the next duplication. The analysis o f the inversion regions w o u l d give better insight in the possible m e c h a n i s m s .

Acknowledgements W e thank I. Navarro and C. B o u t o n for technical assistance. This w o r k was supported b y a B e l g i a n G o v e r n m e n t grant B I O - 1 0 , F G W O grant 3.0121.93 and b y a Spanish G o v e r n m e n t FIS grant 90-0459. M. Navarro was recipient of a short term E M B O fellowship A S T F 7157. D. M u t h u i was the recipient of a B e l g i a n G o v e r n m e n t A B O S grant. W e also thank Dr. M.T. Castells (Image A n a l y s i s Unit. U n i v e r s i t y of Murcia) for the computerised i m a g e analysis ( C I C Y T IN89-0573).

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