Barnabé, Christian

Bosseno, Marie-France

Brenière, Simone Frédérique

Tibayrenc, Michel

Urdaneta-Morales, S.

Name not available

Copy number differences in the 195 BP repeated satellite DNA from Trypanosoma cruzi and Trypanosoma rangeli : potential use for epidemiologic surveys

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-,.- $'&?$Inst. Oswaldo Cruz, Rio de Janeiro, 
Vol. 88 (1): 163-165, janhar. 1993 
I 
Research Note 
Z S S I V :  O Q ~ A - D 2 ? 4  
COPY NUMBER DIFFERENCES IN THE 195 BP REPEATED SATELLITE DNA 
FROM TRYPANOSOMA CRUZ! AND TRYPANOSOMA RANGELI: POTENTIAL USE 
FOR EPIDEMIOLOGIC SURVEYS 
s. F. BRENIERE; M. F. BOSSENO; C. BARNABE; S. URDANETA-MORALES* & M. TIBAYRENC 
UMR CNRS ORSTOM 9926 "GCnétique Moléculaire des Parasites et des Vecteurs", ORSTOM, 91 1, 
Av. Agropolis B. P. 5045, 34032 Montpellier cédex 1, France *Universidad Central de Venemela, 
Centro de Biologia Celular, Apdo. 47058, Caracas 1041A, Venezuela 
Ttypanosoma cruzì, the agent of Chagas' 
disease, and the apparently non-pathogenic 
species Trypanosoma rangeli can be both en- 
countered in human and in the same mam- 
miferous hosts. On fresh isolates, the discrimi- 
nation between both species can be sometimes 
difficult. Hence the development of reliable 
molecular markers able to distinguish between 
the two species is sorely needed (F. Guhl et 
al., 1987 Parasitol., 94: 475-484). Toward 
reaching $is goal, we report here further char- 
acterization of the 195 bp DNA repeat previ- 
ously described in T. cruzì (P. Sloof et al., 
1983, J. Mol. Biol., 167: 1-21). A closely re- 
lated satellite DNA sequence was evidenced 
in T. rangeli, but apparently with a much lower 
number of repeats, hence providing a potential 
epidemiological tool for discrimination between 
the two taxa. 
Fifteen different T. crud stocks as well 7 
T. rangeli stocks were studied. These stocks 
represent for each taxon a high genetic vari- 
ability, as verified by isoenzyme analysis at 11 
polymorphic loci, according to the methods 
described by M. Tibayrenc & F. J. Ayala, 
(1988, Evolution, 42: 277-292). Oligonuclotide 
primers TCZl and TCZ2 previo.usly described 
by D. R. Moser et al. (1989, J.  Clin. Microbiol., 
27: 1477-1482), were used for DNA amplifi- 
cation of the 195 bp DNA units from purified 
total DNA (F. Veas et al., 1991, Cell. Mol. 
Biol., 37: 73-84), according to the PCR ampli- 
fication procedure described by S. F. Brenière 
et al. (1992, Am. J. Trop. Med. Hyg., 46: 335- 
341). 
This work was supported by a grant from TDR Chagas' 
disease no 880190. 
Received 13 August 1992. 
Accepted 22 October 1992. 
As expected, all T. cruzì stocks gave a major 
amplified band of 195 bp on ethidium bromide- 
stained 2% agarose gels. For the 7 T. rangeli 
stocks studied, PCR products exhibiting a com- 
parable molecularweight were obtained (Fig. 1). 
These 195 bp length PCR fragments were not 
observed with other Kinetoplastida species used 
as control (Leishmania, T. brucei), a result al- 
ready notedby D. R. Moseret al. (1989, loc. cif.). 
The minimal amount of template DNA required 
to obtain a positive PCR amplification was 10 fg 
for one T. cruzì stockand 1 O pg for dne T. rangeli 
stock. 
The sequence homology of T. cmzi and T. 
rangeli was evaluated by cross-hybridization 
of T. cruzì and T. rangeli probes with sequen- 
tial diluted PCR products, electrophoresed and 
transfered onto nylon membranes ( S .  F. 
Brenière et al., 1992, loc. cit). Both probes 
were prepared as follows: the PCR fragments 
of one T. cruzì stock and one T. rangeli stock 
were purified by electrophoresis on 0.8% pre- 
parative low melting ultra pure agarose gels 
(Bethesda Research Laboratories, Uxbridge 
UK). The fragments were eluted from the 
agarose using Glass beads (Gene Clean kit) 
according to instructions of the manufacturer , 
(Bio 101, La Jolla, CA USA). Non radioactive 
labeling of purified products was performed 
by using ECL gene detection system based on 
were incubated at 42 OC in hybridization buffer 
(0.1 ml/cm2) for 15 mn and during the same 
time, each purified probe was labeled for 10 
at 42 OC overnight in a rotative oven. A simi- 
lar sensitivity was obtained with either homolo- 
b, c). This result favours the hypothesis of 
strong sequence homology between 195 bp 
PCR products from T. cruzì- and T. rangeli. 
am QJ 
Q, 
80 'S 
enhanced chemiluminescence (Amersham, 2 r m q  *melm 
Buckinghamshire, TJK). Briefly membranes Q S  
~ ~ 
mn at 37 OC. The hybridization was performed 
gous or heterologous hybridizations (Fig. 2a, 
4- 
a3 
o') 
P 
CG w 
va 
m 
164 
c 
. .  
. d Th -_ 
-. 
S. F. Breniere et al. 
- -  -
Fig. 1 : electrophoresed and ethidium bromide stained 2% agarose gel exhibiting PCR products from: lanes 1 2 , 4  
and 5,  1 pg of total DNA of reference Trypanosoma rangeli stocks (R2008, Basel, Riera and R2010 respectively); 
lanes 3 and 7, 1 pg of total DNA of T. cmzi stock SC43 cl 1; lane 6, 100 pg of total DNA of T. cwzi stock OPS21; 
lane 8, molecular weight marker PBR 322- Alu I. The arrow indicate the major amplified band of 195 bp. 
Fig. 2: a - Electrophoresed and ethidium bromide stained 0.8% agarose gel exhibiting - lane 1 to 8: serial dilutions 
( IA ,  1/2 to 1/128) of the PCR products from Trypanosoma rangeli Basel stock total DNA (1 mg); lanes 11 to 17 
and 10: serial dilutions (IA9 1/2 to 1/128) of the PCR products from T. cruzì NR stock total DNA (100 fg) 
respectively; lane 9: molecular weight marker PBR 322- Alu I. b and c -hybridizations of the blotted gel with NR 
T. crud and Basel T. rangeli non radioactive probes (5 ng/ml and 20 ng/ml respectively), high washing stringency 
was monitored twice in 6 M urea, 0.5 x SSC buffer for 20 mn at 42 "C and then twice in 2 x SSC buffer fo,r 10 mn. 
The expositions were performed 10 mn on autoradiography films (Hyperfilm TM-MP Amersham, Buckinghamshire, 
UK). . I. . 
.+ . ._". 
.,>-.w-. ..... ?Y, . . . , ... L_ I . .  
LI *'\ Pa d ~ m . h t .  Oswaldo Cmz, Rio de Janeiro, Vol. 88 (I), jan./mar. 1993 165 
We have dot-blotted serially-diluted total 
DNA extracts from the total range of 15 T. 
cmzi stocks and 7 T. rangeli stocks as well as 
one T. cmzi marinkellei stock, 2 Leishmania 
stocks and 3 T. bmcei stocks, according to F. 
Veas et al. (1990, ZOC. cit.). All T. cmzi stocks 
gave a positive hybridization at a minimal 
concentration ranging from 0.01 to 10 ng, ei- 
ther with T. cruLi or T. rangeli probes (see 
above). Some T. rangeli stocks gave a positive 
hybridization but with a minimal concentra- 
tion of 500 ng total DNA, while others re- 
mained negative, even with a concentration as 
high as 1 pg total DNA. With 500 ng total 
DNA, non hybridization was observed for the 
others species used as controls. 
These results confirm the presence in T. 
cmzi of a satellite DNA composed of 195 bp 
repeat, and show that this DNA is constantly 
observed over a range of stocks of this parasite 
that are genetically highly diversified. More- 
over, the presence of repeats that are either 
identical or similar was shown also on a set of 
T. rangeli stocks whose genetic heterogeneity 
is also very high. Nevertheless, the number os 
repeats appeared as far lower in the case of T. 
rangeli than for T. cruzì; we propose the hy- 
pothesis that the T. rangeli sequence is an 
ancestral one that has been duplicated and 
amplified in the species T. cruzi. Whatever be 
the explanation of it, this result (far higher 
number of repeats in T. cruzi than in T. rangeli) 
seems to be valid for any T. cmzi or T. rangeli 
stock, since it has been verified for two set of 
stocks belonging to the two taxa, that are ge- 
netically extremely diversified this potentially 
provides a convenient tool to discriminate 
between the two species in the course of epi- 
demiological surveys, by simple dot-blot hy- 
bridizations. Dïscrimzatiön of both taxa by 
restriction endonuclease patterns of kDNA (A. 
C. C. Frasch, 1981, Mol. Biochem. Parasitol., 
4: 163-170; A. M. Gonçalves et al., 1991, Mem. 
Inst. Osvaldo Cruz, 86: 477-478) and PCR 
amplification of mini-exon genes (V. K. 
Murthy, 1992, Mol. Cell. Probes, 6: 237-243) 
have been proposed. These authors do not 
contemplate the intraspecific genetic variabil- 
ity of both taxa which is particulary high (R. 
Kreutzer & O. E. Sousa, 1981, Am. J. Trop. 
Med. Hyg., 30: 308-317; M. Tibayrenc & F. J. 
Ayala, 1988, loc. cit.), nevertheless, the mark- 
ers used in these experiments should be valid 
for discrimination of the taxa. Studies, in the 
light of population genetics, of the different 
proposed markers with extensive samples of 
stocks pertaining to both taxa, are highly rec- 
ommended to investigate their possible corre- 
lation. 
In our dot-blot experiments, the only T. 
cnizi marinkellei stock, which is considered a 
subspecies of T. cruzi, and hence, closely re- 
lated to T. cruzi sensu stricto showed no hy- 
bridization signal. The close genetic relation- 
ship between T. cmzi and T. cruzì mareikeIIei 
is questioned by this unexpected result. This 
opens the more general and difficult problem 
of a reliable identification of the so-called T. 
cruzi-like organisms, whose epidemiological 
relevance and actual taxonomic relationship 
with T. cruzì remain unclear. Work is in hand 
to explore this problem by complete popula- 
tion genetics analysis. . 
Acknowledgement: to Prof. Dominique Le 
Ray (Tropical Medicine Institute in Antwerp, 
Belgium), who kindly provided three of the T. 
rangeli stocks). 


Copy number differences in the 195 BP repeated satellite DNA from Trypanosoma cruzi and Trypanosoma rangeli : potential use for epidemiologic surveys

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