Molecular & Biochemical Parasitology 120 (2002) 305
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Short communication

Comparative study of Trypanosoma rangeli and Trypanosoma cruzi

telomeres
Miguel A. Chiurillo a,b, Andreina Peralta a, Jose L. Ramrez a,b,*
b

a
Instituto de Biologia Experimental, Universidad Central de Venezuela, Apartado 47525, 1041-A Caracas, Venezuela
Instituto de Estudios Avanzados, Ministerio de Ciencia y Tecnologia, Carretera Nacional Hoyo de la Puerta, al lado de la Universidad Simon Bolvar,
Municipio Baruta, Caracas, Venezuela

Received 8 October 2001; accepted in revised form 2 January 2002

Keywords: Trypanosoma rangeli telomeric structure; Comparison with Trypanosoma cruzi

Trypanosoma cruzi causes Chagas disease, a debilitating and often lethal illness endemic in some Latin
American countries. T. cruzi shares reservoirs and
vectors with Trypanosoma rangeli, a related protozoan
pathogenic to Reduvid bugs but harmless to humans [1].
Under light microscope examination these parasites are
difficult to distinguish and, due to common antigenic
determinants, serological diagnosis can be inconclusive
[2,3].
Both parasites can infect the mammalian host, though
using different ways T. rangeli is inoculated by the bite
of the Reduvid bug, whereas T. cruzi enters by
contamination of the Reduvids bite-site with feces
containing this parasite. Once in the blood stream, T.
rangeli does not multiply and is rapidly engulfed and
eliminated by host macrophages [4]. On the contrary, T.
cruzi survives the macrophage attack and multiplies
within these, and other cells, causing an acute phase that
may eventually lead to the lethal chronic stage of the
disease [5].
A molecular explanation of this differential behavior
in the mammalian host is far from having been reached.
Authors have pointed out T. cruzi cell surface glycoproteins [6
/9], not yet found in T. rangeli, as important

Abbreviations: SIRE, short interspersed repetitive element; CHEF,

clamped homogeneous gel electrophoresis; UTR, untranslated gene
region; SZ23, SIRE associated sequence; VSG, variant surface
glycoprotein.

Note: Nucleotide sequence data reported in this paper have been
submitted to the GenBankTM data base with the accession numbers
AF126016
/AF126022.
* Corresponding author. Tel.: 58-212-962-1644; fax: 58-212962-1120.
E-mail address: jramirez@reacciun.ve (J.L. Ramrez).

determinants of virulence. Among these proteins, members of the sialidase/transialidase superfamily have been
extensively studied [10]. In a previous work, we showed
that gene sequences from this superfamily are a structural part of T. cruzi telomeres, and we have speculated
that these telomeric copies could be associated with the
generation of sequence variation [11]. Considering the
close biological association of these two parasites, we
wanted to know whether their telomeres share structural
features. To achieve this goal, we cloned and characterized T. rangeli telomeres and compared them with their
T. cruzi counterparts.
T. rangeli telomeric cloning was done as described
elsewhere [11]. We selected the Western Venezuela T.
rangeli isolate DOG-82 [12] and to ensure maximum
integrity of its DNA this was recovered from agarose
blocks after digestion with b Agarase I (Gibco-BRL).
We used six different telomeric adaptors covering all
possible permutations of the hexameric repeat 5?TTAGGG-OH 3?, and plasmid bluescript KS  double
digested with Hin dIII and Pst I endonucleases. Twelve
positive recombinants were further characterized, nine
of which were partially sequenced.
Restriction analysis revealed insert sizes ranging from
714 to 3363 bp with the Pst I cloning site at one end. The
lack of internal PstI sites in larger recombinants rules
out the possibility that they are molecular chimera.
Sequence analysis from theT3 side in pBSK  revealed
that all recombinants had the same adaptor sequence
ending in 5?-CCCTAACCC-OH 3?, therefore the last
nine nucleotides of T. cruzi overhang are 5?GGGTTAGGG-OH 3?. The presence of this terminal
sequence confirms our previous observations of a
generic fixation of the DNA sequence at the terminus,
T. rangeli being identical to T. cruzi and Trypanosoma

0166-6851/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 6 - 6 8 5 1 ( 0 2 ) 0 0 0 0 5 - 1

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Fig. 1. T. rangeli telomere characterization. (A) Clustal W alignment of SubTR sequences from recombinants TrTel 1, 3, 4, 5, 6, 7 and 10. Gaps are
indicated by dash ( */). Lowercase bold letters indicate nucleotide divergence. Arrows mark the primers use to generate the SubTR probe. Black dots
indicate continuation of the sequence. (B) Schematic representation of Trypanosoma telomere organization. Arrows indicate sense of genes.
Discontinuous blocks mean that the structure extends towards the centromere. T. brucei telomere organization is based on sequence AF335471.

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Fig. 2. Chromosomal localization of SubTR sequences. Chromosomal

bands were separated by pulsed field electrophoresis. (A) Ethidium
bromide stained CHEF gel. (B) Southern blot of the gel A after
hybridization with radioactively labeled T. rangeli SubTR probe.
Lanes are: 1, MW marker Saccharomyces cerevisiae (New England
BioLabs Inc.); 2, T. rangeli DOG-82 (MCAN/VE/82/DOG-82); 3, T.
rangeli triatomino 1 (IRHO/VE/98/Triat 1); 4, T. rangeli San Agustin
(AJ012417), 5, T. rangeli mono (MMON/VE/98/MONO); 6, T. rangeli
JROH (MHOM/VE/92/JROH); 7, T. cruzi CL Brener; 8, T. cruzi JMP
(MHOM/VE/91/JMP); 9, Leishmania amazonensis LTB 0016
(MHOM/BR/77/LTB 0016); 10, Leishmania donovani LSB-51.1
(MHOM/SD/00/Khartoum).

brucei, whereas in Leishmania donovani [13] and L.

major (submitted), the telomeres end in 5?GGTTAGGGT-OH 3?. We have speculated that this
sequence preservation could be a reflection of the
mechanisms of telomeric synthesis (submitted).
Next to the hexameric repeats (more chromosomal
internal), all recombinants had a 516
/639 bp homologous region (subTR) with no hits in GenBank and
with a 50
/57% G/C content. In most recombinants
this region harbored the Pst I cloning site. We found no
common features between T. rangeli and T. brucei
subtelomeric regions [14,15]; the latter are A/T-rich
(near 70%) and contain many repeated sequences. On
the other hand, T. cruzi subtelomeric regions have
similar G/C content (50
/56%), and stretches of
similarity with T. rangeli subTR (up to 50%) (Fig.
1A), suggesting that insertion of gp85 sequences within
T. cruzi subtelomeric regions occurred as a later event
after the separation of the two species.
According to sequence variations in subTR we
included recombinants 1, 6 and 7 in one group,
recombinants 5 and 10 containing an insert with
TTGG or TTGC repeats were included in a second
group, and a third group was made by recombinants 3
and 4. Sequence identity was 99% within groups and
64% between groups.

307

Distal to the hexameric repeats, some recombinants

contained satellite like repetitions such as TATATA,
CACA and GAGA. Out of the larger recombinants,
only in TrTel 4 did we find any sequence similarity in
GenBank and this was with T. cruzi gp85 and gp82
genes (accession numbers: AF128843, L14824, M64836
and U02615). Based on our results, and previous results
of T. cruzi and T. brucei , a general scheme of
Trypanosomatidae telomeres is presented in Fig. 1B.
To determine which T. rangeli chromosomal bands
shared the subTR region, we labeled a probe consisting
of an amplification product of this region within
recombinant TrTel 5 (Fig. 1B) labeled with dCTP
a32P. As shown in Fig. 2, all chromosomal bands from
different T. rangeli isolates gave a positive hybridization
signal, whereas no signal was detected for T. cruzi or
Leishmania . Thus the SubTR sequence seems to be an
exclusive feature of T. rangeli chromosomes, and
consequently the PCR assay based on this sequence
can be used in the detection of T. rangeli infections.

Acknowledgements
This project received support from CONICIT Grant
No. G 9900036 and CONICIT Post-Doctoral fellowship
No. 9900034. Thanks to Mr. Ian McClure for revising
the English spelling and to Dr. Nestor Anez for parasite
isolates.

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