Polymorphisms within minicircle sequence classes in the kinetoplast DNA of Trypanosoma cruzi clones

Molecular and Biochemical Parasitology, 16 (1985) 61-74 Elsevier MBP 00557

POLYMORPHISMS KINETOPLAST

WITHIN DNA OF

MINICIRCLE

SEQUENCE

CLASSES

IN THE

TR Y P A N O S O M A C R U Z I CLONES

ROBERTO A. M A C I N A ~, D A N I E L O. S A N C H E Z ~, JOSE L. A F F R A N C H I N O ~, J U A N C. E N G E L z and A L B E R T O C.C. FRASCH 1'3'*

~Centro de lnvestigaciones Bioenergeticas, Facultad de Medicina, Paraguay 2155, 5°P, 1121 Buenos Aires, 21nstituto National 'M. Fatala Chaben', Paseo Colon 568, 1063 Buenos Aires, and 31nstituto de lnvestigaciones Bioquimicas 'Fundaci6n Campomar', Antonio Machado 151, 1405 Buenos Aires, Argentina (Received 7 November 1984; accepted 1 March 1985)

Four minicircle classes were analyzed using cloned minicircles as probes and single-cell cloned Trypanosoma cruzi parasites. The hybridization conditions used allowed identification of minicircle classes within kinetoplast D N A that were non-homologous to each other. Two of these minicircle classes, detected with probes pTckAWP-2 and -3, were present together in several of the CA 1 and Miranda clones, in spite of the fact that either pTckAWP-2 or both minicircle classes were undetectable in other isolates and clones of the parasite. The other two minicircle classes (pTckM-84 and -88) were located in some Miranda cloned parasites which were characterized by the simple restriction endonuclease pattern of their minicircles. Both pTckM-84 and -88 minicircle classes represented 52-71% of the kinetoplast D N A in the latter group of trypanosomes. Restriction endonuclease mapping allowed the identification of polymorphic minicircles in two of the four minicircle classes analyzed (pTckAWP-2 and pTckM-88). The polymorphisms were observed in part of the molecules of one minicircle class within a single trypanosome clone, as well as when different clones or even some of those obtained from the same isolate were compared. In addition, a different proportion of pTckM-88 type of minicircle sequence class was observed in the kinetoplast DNA from two of the Miranda clones analyzed. These observations demonstrated that similar molecules may evolve independently in sequence in each parasite. The polymorphic minicircles detected may arise from sequence variations before expansion of a future homogeneous minicircle sequence class. Key words: Trypanosoma cruzi; Minicircles; Kinetoplast DNA

INTRODUCTION The

mitochondrial

complexity. animal

DNA

from

several organisms

varies considerably

In some cases, several copies of a single small genome

c e l l s [1]. I n o t h e r c a s e s , a s in h i g h e r p l a n t s , t h e g e n o m e

in s i z e a n d

are present, as in

size and complexity

* To whom all correspondence should be addressed. Abbreviations: kDNA, kinetoplast DNA; kbp, kilobase pairs; SDS, sodium dodecyl sulfate; SSC, sodium chloride/sodium citrate solution. 0166-6851/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

62 far exceed that required for their expected coding capacity [2]. One of the most surprising mitochondrial DNA is the so-called kinetoplast DNA (kDNA), present in members of the order Kinetoplastida [3,4]. The kDNA is made up by two groups of molecules. Those of larger size, the maxicircles, are present in a small copy number per cell, and are the real mitochondrial DNA of these parasites [4-7]. The other component is the minicircle. Smaller in size, they make up the bulk of the kinetoplast structure, comprising from 3 000 to 30000 copies per cell. They constitute a very intriguing DNA as their possible function remains controversial [4,8]. Besides, minicircles make up roughly 10-20% of total cellular DNA. One of the characteristics of these molecules is their heterogeneity. Few trypanosomes in the African group [9] have been described containing homogeneous molecules and few major minicircle classes have been defined in Leishmania tarentolae [ 10,11]. Trypanosoma brucei brucei, however, has as many as 200 different kinds of molecules [9,12], some of which share sequences of perfect homology [ 13]. Among American trypanosomes, the T. cruzi and T. rangeli minicircles contain four repeats per molecule [ 14,15], although outside these conserved regions sequence divergency seems to be the rule [16]. Previous analysis with cloned molecules has demonstrated that four out of five minicircle fragments detected heterogeneous minicircle sequence classes [16]. However, one of these molecules, when used as a probe, only detected its homologous minicircle class, making up 5% of the total kDNA [16]. Another characteristic of the latter minicircle class was its presence in certain T. cruzi isolates but not in others [17]. Isolate-specific minicircles appear to be present in several other T. cruzi stocks [18]. In this paper, we have analyzed four of these homogeneous and isolate-specific minicircle classes in several single-cell cloned T. cruzi parasites and demonstrate polymorphisms in a single minicircle class within a clone, and among clones of T. cruzi. MATERIALS AND METHODS Parasites. The T. cruzi isolates were grown in liquid medium [14]. AWP, RA and UP T. cruzi isolates were obtained from acute cases of Chagas' disease [19]. Eighteen clones from the original T. cruzi stocks CA 1 (-59, -64, -65, -67, -69, -70, -71, -72, -73)

and Miranda (-75, -76, -77, -78, -80, -81, -83, -84, -88) were kindly supplied by Drs. E.L. Segura and J. Dvorak [20]. They were obtained by single-cell cloning from T. cruzi Miranda (isolated from a chronic Chagasic patient) and CA 1 (from a dog infected with CA 1, originally isolated from a patient with chronic Chagas' disease) [20]. kDNA was purified by a quick isolation procedure [15]. Two cloned minicircles (pTckAWP-2 and -3) were obtained from AWP kDNA digested with HindIII and ligated with HindIII-digested pBR322. The other two minicircle probes were cloned in the ClaI site of pBR322 after HpaII digestion of kDNA from Miranda clones M-84 (pTckM-84) and M-88 (pTckM-88). Transformation, colony hybridization and purification of plasmid DNA were performed as before [16]. kDNA isolation and minicircle cloning,

63 Minicircle inserts were isolated from the recombinant clones by means of low melting agarose.

Agarose gel electrophoresis and filter hybridization.

2% agarose gels were run in Tris/borate buffer at 100 V for 3-4 h [15]. Conditions for Southern blotting [21] and hybridization with 'nick-translated' D N A probes [22] were as described [17]. After hybridization, filters were washed 3 times, 45 min each, at 65°C in 0.1 × SSC (1 X SSC = 150 mM NaC1/15 m M sodium citrate)/0.1% sodium dodecyl sulfate (SDS; high stringency) to avoid detection of constant minicircle regions [16]. To estimate minicircle copy number in total k D N A digests [16], the concentration of D N A solutions was measured spectrophotometricaily at 260 nm or by comparison with D N A standards in photographs of gels stained with ethidium bromide. The 260:280 ratio was higher than 1.7. Different amounts of k D N A and from each isolated minicircle insert obtained from the corresponding recombinant clones, were spotted (1-40 ng in 5 pl) onto nitrocellulose filters, hybridized with the desired probe, and the spots excised and counted in a liquid scintillation counter. The assay was linear between the above mentioned amounts of DNA, allowing the percentage of each minicircle class within whole k D N A to be estimated if stringent conditions were used for the hybridization (see Results section). RESULTS

Nine cloned trypanosomes from each of two T. cruzi stocks; CA 1 (-59, -64, -65, -67, -69, -70, -71, -72 and -73) and Miranda (-75, -76, -77, -78, -80, -81, -83, -84 and -88) were studied, k D N A restriction endonuclease patterns allowed identification of three groups of clones among them. In the first group, CA 1 clones-59,-64,-67,-70,-71,-72 and -73 were included due to their similar (or identical) H a e l I I and H p a l I restriction endonuclease patterns (see examples in lanes of CA 1-64, -72 and -73 in the left panel of Fig. 1). Likewise, in the second group of parasites, two CA 1 clones (-65 and -69) and three clones of Miranda (-76, -78 and -83) were included because of their similar H a e l I I and H p a l I patterns, which were different from those of the first group (compare the patterns of the first group of clones with that of CA 1-65 in the left panel of Fig. 1). Both the first and the second groups depicted typical T. cruzi restriction endonuclease patterns with those enzymes that cut within the constant minicircle region [16]. Thus, the main bands observed with H p a l I and HaelII were those corresponding to 1/1, 3/4, 1/2 and 1/4 of the total minicircle length. The third group of clones (Miranda -75, -77, -80, -81, -84 and-88), however, depicted a much simpler pattern than the previous ones with H p a l I and H a e l I I enzymes (panels A and B, Fig. 2). In order to analyze possible variations in restriction endonuclease sites of one minicircle class, it was necessary to localize probes for homogeneous minicircle sequences, that is, for groups of similar molecules within each cloned parasite. Two minicircles cloned in pBR322 (pTckAWP-2 and pTckAWP-3) originally isolated from

64

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~E

pTckAWP-2

' 0,40 kbp

1

Fig. 1. kDNA restriction endonuclease patterns of the first and second group of cloned parasites (see text) and detection of pTckAWP-2 minicircle class, kDNA from the indicated clones was digested with HaeIII and Hpall enzymes and electrophoresed in 2% agarose gels (left panel). After blotting, the filters were hybridized with probes derived from either pTckAWP-2 insert (center panel) or a 0.40 kbp fragment obtained after HpaII digestion of pTckAWP-2 insert (left panel) (see map in Fig. 4). The upper arrow indicates the new band detected in HaeIII and HpaII kDNA digests from clone CA 1-73. The lower arrow indicates the 0.66 kbp band present in Hpall kDNA digests from CA 1-65 parasite clone.

k D N A of A W P T. cruzi stock, were f o u n d to be present within the first a n d the s e c o n d g r o u p o f the cloned parasites a n a l y z e d as classified before (see e x a m p l e s in Fig. 3). The highly stringent h y b r i d i z a t i o n c o n d i t i o n s used, as defined u n d e r M a t e r i a l s a n d M e t h ods, allowed identification o f discrete b a n d s in k D N A restriction e n d o n u c l e a s e digests with b o t h p r o b e s ( p T c k A W P - 2 a n d -3) (Fig. 3). T h e m a i n b a n d s detected were similar in size to those g e n e r a t e d from the c o r r e s p o n d i n g minicircle p r o b e (Fig. 4). F o r e x a m p l e , p T c k A W P - 2 p r o b e d e t e c t e d m a i n l y 0.90, 0.35 a n d 0.20 kilobase pairs (kbp) f r a g m e n t s in H a e I I I digests o f k D N A (the last f r a g m e n t h a r d l y visible in the a u t o r a d i o g r a m o f Fig. 1, center panel), which were similar to the f r a g m e n t size o b s e r v e d after H a e I I I digestion o f the minicircle insert c o n t a i n e d in p T c k A W P - 2 (see m a p in Fig. 4). S i m i l a r r e s u l t s o b t a i n e d with H p a I I a n d H i n d I I I for p T c k A W P - 2 a n d H a e I I I a n d H p a I I for p T c k A W P - 3 , allow us to c o n c l u d e that the b a n d s detected with each p r o b e in total k D N A digests c o r r e s p o n d e d to minicircles similar (or identical) to each of the minicircle p r o b e s used ( c o m p a r e the m a p s in Fig. 4 w i t h the m a i n b a n d s detected in the a u t o r a d i o g r a m s o f Figs. 1 a n d 3 for p T c k A W P - 2 a n d Figs. 3 and 5 for p T c k A W P - 3 ) . It s h o u l d be m e n t i o n e d that u n d e r the h y b r i d i z a t i o n c o n d i t i o n s used, p T c k A W P - 2 a n d -3 d i d not c r o s s - h y b r i d i z e (not shown).

65

Fig. 2. Analysis

of kDNA digests and pTckM-84

group (see text). kDNA from the indicated EcoRI/HaeIII

and HaeIII

from the indicated cloned minicircle

parasite probes

and -88 minicircle classes in cloned parasites

from the third

clones of T. cruzi was digested with HpaII (panel A) or EcoRI,

(panel B), and run on 2% agarose

gel electrophoresis.

In panel C, the kDNAs

clones were digested with HaeIII, run on 2% agarose gel and hybridized pTckM-84

or pTckM-88

(left and right panels, respectively).

M, Miranda

with the clones.

pTckAWP-2 and -3 minicircle classes proved undetectable in the third group of cloned trypanosomes (Miranda -75, -77, -80, -81, -84, -88) (not shown). Jn order to isolate probes for this latter group of parasites, we cloned the HpaII band of linearized minicircles from kDNA of Miranda -84 and -88, in the ClaI site of pBR322. Restriction endonuclease analysis and cross-hybridization experiments of 29 recombinants allow-

66

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,

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Fig. 3. Detection of pTckAWP-2 and -3 minicircle classes in k D N A from cloned parasites and analysis of polymorphic restriction endonuclease sites, k D N A from the indicated T. cruzi clones was digested with HpalI and H p a l I / H i n d l l I (for pTckAWP-2 minicircle class detection) or with HpalI (for pTckAWP-3" minicircle class detection), and run on 2% agarose gels. The blotted gels were hybridized with the indicated probes. The upper arrow shows the 0.66 kbp Hpall fragment detected in clones CA 1-69, M-76, M-78 and M-83, but absent in clones CA 1-59,-67 and -70 (see text). The middle arrow indicates the 0.40 kbp HpalI band which is cut by HindllI to a fragment of 0.35 kbp (lower arrow) in clones CA 1-59, -67 and -70, but not present in the other cloned parasites (see text). M, Miranda clones.

ed identification of two classes of minicircles; one representative of each group was named pTckM-84 and pTckM-88, respectively. Similar analyses to those performed with the previously mentioned probes were carried out. Thus, H a e I I I cut pTckM-84 minicircle insert once (fragment size 1.44 kbp) and pTckM-88 minicircle insert twice (fragment sizes 1.08 and 0.36 kbp). Bands of the expected length were the only ones detected with the corresponding probe in HaeIII-digested k D N A from each cloned parasite of this third group (see lane M-80 in panel C, Fig. 2 and Fig. 4). This, and the results obtained with HpaII, EcoRI and TaqI (not shown) allow us to conclude that pTckM-84 and -88 detected in this third group of parasites a minicircle class similar or identical to themselves. Both probes did not cross-hybridize under the stringent conditions used (not shown). Once probes for homogeneous (or nearly homogeneous, see below) minicircle classes were obtained for the three groups of cloned trypanosomes, analysis of possible

67

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I AwP CA1-59,- 64. -67.

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¢~p

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1

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I

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[

I

0.4

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0.6

-7o.-r~. -72

, CA1-65, -69

J

M-76,-78 -83

I CAI-Z3

I

I

1.2 kbp

IHa

Jjlp ll"

I

1.6 p ~

jEr

jl"

IHla

lyl

I

IE ir

I~, M.75,.80,.81 '

, IHo

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Fig. 4. Restrictionendonucleasemapping of pTckAWP-2,-3, pTckM-84and -88 minicircleclasses,and their homologues in the cloned parasites (see text). Triangles and dotted lines indicate sites absent in either most or part of the moleculesof the same minicircle class, respectively.Hi, HindlII; Hp, Hpall; Ha, HaelII; T, TaqI; E, EcoRI. Sizes (in kbp) of the HaelII and HpalI fragments in pTckAWP-2 are (from left to right starting from the first restriction endonuclease site): 0.20, 0.35, 0.90 and 0.27, 0.78, 0.40, respectively.M, Miranda clones. polymorphisms in these minicircle classes was undertaken, pTckAWP-2 detected in H a e l I I and H p a l I k D N A digests of most clones from the first group oftrypanosomes (CA 1-59, -64, -67, -70, -71, -72) the bands expected to arise from pTckAWP-2 minicircle class, i.e,, 0.90, 0.35 and 0.20 kbp for H a e l I I and 0.78, 0.40 and 0.27 kbp for H p a l I digests (see lanes of CA 1-64 and -72 in Fig. 1). However, in one clone belonging to this same first group (CA 1-73), new bands of 1.25 kbp for H a e l I I and 1.15 kbp for H p a l I k D N A digests were observed (Fig. 1, upper arrow). The H a e l I I and H p a l I new bands were the size expected if one H a e l I I restriction endonuclease site (between fragments of 0.90 and 0.35 kbp, right side of the map in Fig. 4), and if a H p a l I site (between fragments of 0.78 and 0.40 kbp, right side of the map in Fig. 4)were missing, respectively. Confirmation that the new H a e l I I and H p a l I fragments were indeed the consequence of the corresponding restriction endonuclease sites lacking in some of the pTckAWP-2 class of molecules in clone CA 1-73 was obtained after hybridization of the same blotted gel shown in Fig. 1 with a 0.40 kbp subprobe from pTckAWP-2 cut with H p a l I ( H p a l I fragment on the right side of the map in Fig. 4). This 0.40 kbp subprobe detected in k D N A digests of CA 1-73 its homologous fragment together with the new H p a l I and H a e l I I bands (upper arrow, left panel in Fig. 1). A final proof

68

II, l l !

a

PRO BE

pTckAWP.~.

--

,,--

pTc u WP-3

Fig. 5~ Detection of isolate-specific minicircle classes, kDNA from the indicated T. eruzi isolates was digested with HaeIII, electrophoresed and stained with ethidium bromide (left panel) or blotted and hybridized with probes derived from either pTckAWP-2or pTckAWP-3(center and right panels, respectively).

confirming the previous results was obtained after isolation of a 1.15 kbp fragment from CA 1-73 k D N A digested with HpaII, having sequence homologies with pTckAWP-2 under stringent conditions, but lacking the above mentioned HaeIII and H p a I I restriction endonuclease sites (not shown). When a similar analysis was performed in the second group of cloned trypanosomes (CA 1-65, -69 and Miranda -76, -78, -83), two new restriction endonuclease sites proved to be missing in the pTckAWP-2 minicircle class. The HpaII site on the left side of the map in Fig. 4 was absent in some of the minicircles from the pTckAWP-2 class, giving rise in this second group of clones to a new band of 0.66 kbp (lower arrow in lane CA 1-65 in Fig. 1 and upper arrow in lanes CA 1-69 and Miranda -76, -78 and -83 in Fig. 3). This new band was also confirmed to be due to the absence o f a HpaII site (the one between fragments of 0.40 and 0.27 kbp, Fig. 4) using the 0.40 kbp subprobe from pTckAWP-2, which detected its homologous band together with the 0.66 kbp fragment (lane CA 1-65 in Fig. 1, lower arrow), and after cloning in a plasmid vector, a 0.66 kbp HpaII fragment homologous to pTckAWP-2 from CA 1-65 kDNA, lacking the HpaII site (not shown). The other restriction endonuclease site missing in this second

69 group of cloned parasites was the single HindlII site. This enzyme cut the 0.40 kbp HpalI fragment (middle arrow in Fig. 3) to a fragment of 0.35 kbp (lower arrow in Fig. 3) and a smaller one not visible in the autoradiogram. This was not the case for all the clones of this second group of parasites as shown in Fig. 3 (middle arrow). While the previously described polymorphic restriction endonuclease sites were absent in some of the pTckAWP-2 class of minicircles, the HindlII site was observed to be absent in most (or all) molecules of this second group of parasites. Appropriate control experiments, which include hybridization of the blotted gels used to detect variable sites in pTckAWP-2 class of minicircles, with pTckAWP-3 probe, rule out partial kDNA digestion as a possible explanation (not shown). The other minicircle class analyzed within the kDNA of the first and second group of cloned trypanosomes was pTckAWP-3. In addition to the bands expected from the restriction endonuclease map of pTckAWP-3 minicircle insert (Fig. 4), new bands were also observed in kDNA digests with this probe (see examples in the right panel of Fig. 3). For example, in HpalI digests, two bands should be seen (1.10 and 0.34 kbp, Fig. 4). However, new weak bands of 0.90 and 1.44 kbp were also visible (the larger one in Fig. 3 must correspond to circular molecules). This result suggested the presence of variable restriction endonuclease sites. This possible explanation was, however, difficult to confirm for the pTckAWP-3 class of minicircles because most of the new HpalI and HaelII fragments (see also Fig. 5) fall in the size range of 1/1, 3/4, 1/2 and 1/4 of the minicircle length, making it difficult to differentiate between 'new bands' and background. Similar experiments to the ones described above were performed to analyze the possible polymorphisms in pTckM-84 and -88 minicircle classes among the third group of cloned parasites (Miranda -75, -77, -80, -81, -84, -88) (data not shown). Most of the restriction endonuclease sites were conserved, thus confirming the identity of the cloned molecules with the minicircle classes detected in whole kDNA digests with both probes. However, either one or both of the two EcoRI sites found in pTckM-88 molecules were absent in part of the minicircles from Miranda -77 parasites, but present in all the other clones of this group (Fig. 4). Following previous results suggesting that molecules that constituted homogeneous minicircle classes were present in certain T. cruzi isolates but absent in several others [16], we have confirmed these observations for pTckAWP-2, -3, pTckM-84 and -88. Thus, pTckAWP-2 and -3 were present in the first and second groups of cloned parasites and in the AWP stock of T. cruzi (Figs. 3 and 5). However, in other non-cloned parasites, either pTckAWP-2 (Tul 0) or both minicircles (Peru, Y and Sonya) proved undetectable (Fig. 5). Similarly, pTckM-84 and -88, detected in the third group of cloned trypanosomes, were undetectable in parasites of the first and second groups of clones as well as in other non-cloned parasites available in our laboratory (Fig. 2, panel C). We finally addressed the question of whether a minicircle class could also vary in number among cloned trypanosomes (see Materials and Methods). No differences

70 could be detected between pTckAWP-2 and -3 within the k D N A of each cloned parasite, and among cloned trypanosomes, values being 10% (from 5 to 15%) of the total k D N A for each class of molecules (see also Discussion). On the other hand, variations were observed in pTckM-84 and -88 minicircle classes. First, pTckM-84 class of minicircles was present in a higher proportion than pTckM-88, values being 47, 41 and 42% for the pTckM-84 class and 24, 11 and 17% for the pTckM-88 minicircle class in total k D N A of the Miranda clones -75, -84 and -88, respectively. Second, duplicate experiments confirmed that the differences found in the proportion of pTckM-88 minicircle class among Miranda clones -75 (24%) and -84 (11%) were reproducible. Thus, both molecule classes add up to 52-71% of the total k D N A in the different clones, which explained the much simpler restriction endonuclease pattern observed in the third group of the cloned trypanosomes analyzed. The values obtained for the pTckM-84 and -88 minicircle classes suggest that other molecules must be present in the k D N A of this third group of parasite clones. These other minicircles not related to pTckM-84 and -88 minicircle classes may be contained in faint bands present in H p a I I digests (not visible in the photograph of Fig. 2, panel A). DISCUSSION With few exceptions, trypanosomatids have different degrees of minicircle heterogeneity [9], which makes it difficult to follow the possible variations of the molecules a m o n g parasites in order to understand how they are maintained and how they evolve. In T. cruzi, one of the minicircle classes studied previously was homogeneous and specific for some non-cloned parasites [16,17]. Later, it was concluded that most of the T. cruziisolates so far studied may have isolate-specific minicircle subpopulations [18]. In the present work, we have analyzed four cloned molecules which make up different homogeneous minicircle sequence classes within single-cell cloned parasites. Two of the molecules studied, pTckAWP-2 and -3 made up from 5 to 15% of the total k D N A (not shown). In spite of having carried out four separate evaluations, no significant variations were found between both minicircle classes and in each minicircle class among the T. cruzi clones. In fact, differences among clones were no greater than fluctuations among experiments. Two other minicircles, pTckM-84 and -88, were obtained from another group of six cloned trypanosomes which were characterized by their simple k D N A restriction endonuclease pat~tern. In these latter parasites, both types of minicircle classes add up to 52-71% of the total kDNA. The reason for these different degrees of heterogeneity between clones of T. cruzi is still obscure. In African trypanosomes, examples of parasites having only homogeneous molecules have been described [23]. In some cases, a maxicircle alteration was present in this latter group of trypanosomes [9]. Although this may not be true for T. cruzi, as these trypanosomes are dependent on mitochondrial function [24], it is suggestive that those T. cruziclones depicting a simpler minicircle pattern (Miranda -75, -77, -80, -81, -84, -88) grow more slowly (up to half rates) than other clones analyzed in this study [20]. Analysis of the

71 maxicircle structure of these parasites is now under way to determine whether their minicircle patterns correlate with maxicircle alterations. The existence of specific minicircle classes seems characteristic o f parasites o f the order Kinetoplastida as several authors have recently reached similar conclusions for the Leishmania species [25-27]. However, attempts to establish whether isolates of the same Leishmania species carry different molecules have not yet been successful [28]. If the continuous generation of new minicircle families is a general p h e n o m e n o n a m o n g k D N A molecules, the use of cloned minicircles and highly stringent hybridization conditions m a y allow location o f isolate-specific minicircles in all trypanosomatids. Isolation of these types of probes may be useful for taxonomic purposes to avoid the complications involved in the use of k D N A digests for typing isolates of the same species [15,28,29]. Isolate-specific minicircles seem due to their fast evolution, leading to sequences with a higher replication rate (Fig. 6) [16,17]. Mechanisms such as gene conversion [30] together with an unequal segregation o f molecules [31,32] could also contribute to their generation. Here we have demonstrated variability in the p r o p o r t i o n and p o l y m o r p h i s m s in the structure of some minicircle classes in cloned T. cruzi parasites. The polymorphic sites observed were present in most molecules of cloned parasites (the H i n d l I I site of p T c k A W P - 2 minicircle class in parasite clones C A 1-65, -69 and M-76, -78, -83), or, more frequently, in part of the molecules from a single minicircle class. Due to the fact that several molecules o f the same minicircle class had identical p o l y m o r p h i c restriction endonuclease sites, it is feasible that the p o l y m o r p h i c molecules were generated in the cells before expansion in n u m b e r (Fig. 6). This is also

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Fig. 6. A possible model for minicircle evolution. In each T. cruzi parasite, isolate-specific minicircles appear, starting from a hypothetical trypanosome with a single type of molecule, and after an unknown number of generations. I n some cases, closelyrelated parasites may carry similar moleculesdiffering in some regions but preserving the overall structure (denoted as E and e). After several generations, some of these isolate-specific minicircles may grow in number over the other molecules,giving rise to the above mentioned populations.

72 suggested by the fact that some clones had characteristic p o l y m o r p h i c molecules n o t present in o t h er clones (see Fig. 4). W h e t h e r m u t a t i o n s , i n s e r t i o n s / d e l e t i o n s or o t h e r alterations are responsible for the p o l y m o r p h i c molecules c a n n o t be stated at present. It is n o t e w o r t h y , however, that in at least two cases (Fig. 4), two p o l y m o r p h i c restriction e n d o n u c l e a s e sites were present within a 50-100 bp region, thus suggesting a larger a l t e r a t i o n rather than a single point m u t a t i o n . F u r t h e r analysis of these polym o r p h i s m s m a y give some clue as to h o w this r e m a r k a b l e variability is generated. ACKNOWLEDGEMENTS We t h a n k Dr. A . O . M . S t o p p a n i for his advice and e n c o u r a g e m e n t ; D r . E.M. G o n z a l e z - C a p p a for p r o v i d i n g us with A W P , R A and U P T. c r u z i isolates; Drs. E.L. Segura an d J. D v o r a k for the Peru, Y and S o n y a stocks, as well as for the C A 1 an d M i r a n d a clones o f T. cruzi; Dr. J . J . C a z z u l o for his critical r ead i n g of the m a n u s c r i p t ; L. Vifias and R. M a d r i d for their excellent technical assistance a n d M a r i a D o l o r e s T u r r o f o r typing the manuscript. This work was s u p p o r t e d by grants f r o m U N D P / W o r l d B a n k / W H O Special Prog r a m for Research and T r a i n i n g in T r o p i c a l Diseases and Secretaria de Ciencia y T6cnica, A r g e n t i n a . R . A . M . , D.O.S. and J . L . A . are Research Fellows an d J . C . E . and A . C . C . F . are M e m b e r s o f the C a r r e r a del I n v e s t i g a d o r Cientifico f r o m the N a t i o n a l Research C o u n cil ( C O N I C E T ) , A r g e n t i n a . REFERENCES 1

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