Characterization of a candidate Trypanosoma rangeli small nucleolarRNA gene and its application in a PCR-based parasite detectionq

Liliana Morales,a,1 Ibeth Romero,a,1 Hugo Diez,a Patricia Del Portillo,b Marleny Montilla,c

Santiago Nicholls,c and Concepci�oon Puertaa,*

a Laboratorio de Parasitolog�ııa Molecular, Departamento de Microbiolog�ııa, Facultad Ciencias, Universidad Javeriana, Carrera 7 No 43-82,

Lab. 113, Bogot�aa, Colombiab Corporaci�oon Corpogen, Carrera 5 No 66-88, Bogot�aa, Colombia

c Laboratorio de Parasitolog�ııa, Instituto Nacional de Salud, Av. El Dorado, Carrera 50 Can, Zona 6, Bogot�aa, Colombia

Received 2 October 2001; received in revised form 13 June 2002; accepted 14 February 2003

Abstract

In this study, we report the isolation and characterization of a candidate Trypanosoma rangeli small nucleolar RNA (snoRNA)

gene, and the development of a PCR assay for detection of the parasite based on its nucleotide sequence. This gene, isolated from a

T. rangeli genomic sub-library, was named snoRNA-cl1 and is encoded by a multi-copy gene of 801 bp in length. Computer se-

quence analysis of snoRNA-cl1 showed the presence of two sequence motifs, box C and box D, as well as of two long stretches

that perfectly complement the universal core region of the mature rRNA 28S, suggesting that cl1 encodes for a Box C/D snoRNA

from the parasite. Hybridization analysis using cl1 as probe, showed a weak hybridization signal with Trypanosoma cruzi DNA,

demonstrating the existence of differences in this locus between these two species. Two oligonucleotide primers from this gene, which

specifically amplified a 620-bp fragment in KP1 (+) and KP1 ()) strains of T. rangeli, were used in a PCR assay. The amplificationallowed the detection of 1 pg of DNA in the presence of heterologous DNA and no amplification was observed with different

T. cruzi strains (groups I and II). In addition, the PCR assay reported here is able to detect T. rangeli in the presence of T. cruzi

DNA, and is useful for detection of the parasite in samples from infected vectors.

� 2003 Elsevier Science (USA). All rights reserved.

Index Descriptors and Abbreviations: Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma vivax, Leptomona collosoma,

Leishmania tarentolae, Leishmania amazonensis, Leishmania panamensis, Crithidia fasciculata, Rhodnius prolixus, snoRNA, small nucleolar RNA,

kinetoplast, KP1 (+), KP1 ()), PCR, polymerase chain reaction.

1. Introduction

Trypanosomatids are early divergent protozoan para-

sites which include several species of medical interest. Inspite of its lack of pathogenecity to man, Trypanosoma

rangeli is a serious concern for the epidemiology and

diagnosis of Chagas disease because: (i) Trypanosoma

cruzi and T. rangeli have similar geographical distribu-

tions (D�Alessandro, 1976; D�Alessandro and Saravia,

1992), (ii) They share the same host range and often

have identical insect vectors (D�Alessandro, 1976;D�Alessandro and Saravia, 1992), (iii) Mixed infectionsmay occur in both vertebrate and invertebrate hosts(D�Alessandro, 1976), (iv) The two trypanosomes havemorphologically comparable developmental stages

(Vallejo et al., 1988), and (v) There is a high immuno-

logic cross reactivity between them (Afchain et al., 1979;

Basso et al., 1991). Because of these reasons, research on

T. rangeli has been focused on the development of a

rapid and sensitive test to differentiate between these

organisms.Several techniques have been used in distinguishing

these trypanosomes, but none is completely satisfactory

when used alone. Indirect diagnosis methods are sensi-

Experimental Parasitology 102 (2002) 72–80

www.elsevier.com/locate/yexpr

qThe sequence data reported herein has been submitted to GenBank

and assigned the Accession No. AY028385.* Corresponding author. Fax: +57-1-3208320, ext. 4021.

E-mail address: cpuerta@javeriana.edu.co (C. Puerta).1 Same participation in this work.

0014-4894/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.doi:10.1016/S0014-4894(03)00027-4

tive but they are not specific (Cotrim et al., 1990; Guhlet al., 1987; Vasquez et al., 1997). In addition, these

techniques do not indicate if the infection is active and

they are not reliable for immunodepressed patients.

Other approaches such as resistance to complement-

mediated lysis and reaction with monoclonal antibodies

(Acosta et al., 1991), lectin agglutination lysis (de Mi-

randa Santos and Pereira, 1984), nuclear or kinetoplast

DNA probes (Greig et al., 1990; Macedo et al., 1993;Vallejo et al., 1994), and the restriction pattern of ki-

netoplast DNA (Vallejo et al., 1993) require previous

propagation of the parasite.

To overcome these problems, enzymatic amplification

of DNA by PCR has been successfully developed. Sev-

eral PCR assays can identify both T. rangeli and T. cruzi

using repetitive DNA sequences as targets: miniexon

genes (Fernandes et al., 2001; Murthy et al., 1992), genescoding for a flagellar protein (Silber et al., 1997), mini-

circles from kinetoplast DNA (Dorn et al., 1999; Sturm

et al., 1989; Vallejo et al., 1999), 24S a (large subunits)ribosomal RNA (Souto et al., 1999) and cysteine pro-

teinase (Tanaka, 1997). Some of these tests have the

problem that they amplify bands of similar size in both

trypanosome species. In addition, miniexon-based PCRs

amplify polymorphic regions which could be a disad-vantage in epidemiological field studies. Besides, mini-

circle-based PCRs have the problem that they are biased

to T. cruzi identification and require the presence of at

least 75% of T. rangeli in order to detect this parasite

(Dorn et al., 1999; Vargas et al., 2000). At present, only

two PCR assays have been designed to exclusively detect

T. rangeliminiexon genes (Grisard et al., 1999) and P542

element, a repetitive genomic DNA sequence (Vargaset al., 2000).

In this paper, we report a T. rangeli clone that seems

to encode the first C/D box small nucleolar RNA

(snoRNA) gene from T. rangeli, a type of snoRNA im-

plicated in the 20-O-ribose methylation of the rRNA.Based on the nucleotide sequence of the 50 and 30 non-coding regions flanking this gene, we developed a PCR

that specifically identifies T. rangeli DNA and is ame-nable for use on biological samples from triatomines.

2. Materials and methods

2.1. Parasites

Epimastigotes of KP1 (+) T. rangeli strains: H14from Honduras (MHOM/Hond/H14, Acosta et al.,

1991), and Choachi from Colombia (IRHO/CO/82/

Choachi), and KP1()) T. rangeli strains: C23 (MAOT/CO/82/C23, Zu~nniga et al., 1997), 5048 (MHOM/CO/99/5048), and T. rangeli (Tre), Colombian strains were

used in this study. They were grown at 28 �C in mod-ified REI medium supplemented with 2% (v/v) heat-

inactivated fetal bovine serum. T. cruzi epimastigotes,strains Y, Munanta, Ikiakarora, Shubacbarina, B.M.

L�oopez, M. Rangel, D. marsupialis R-56, Tulahuen and

D. marsupialis No. 3 (Rodriguez et al., 1998), and Saul

Parra (MHOM/CO/87/SPR) were grown at 28 �C in

liver infusion triptone (LIT) medium supplemented

with 10% (v/v) heat-inactivated fetal bovine serum.

DNA preparations from other trypanosomatids were

obtained from the National Health Institute (Bogot�aa,Colombia).

2.2. Triatomine samples

Three groups of 10 Rhodnius prolixus nymphs were

used. The first group was allowed to feed upon BALB/c

male mice infected with the Saul Parra strain of T. cruzi,

the second upon non-infected BALB/c male mice andthe third group was injected with the Choach�ıı strainof T. rangeli. Fifteen days post-infection the insects

were sacrificed and dissected; haemolymph and faecal

samples were obtained and diluted 1:1 with guanidine–

EDTA buffer. The DNA was isolated by phenol–chlo-

roform–isoamilic alcohol extraction and ethanol

precipitation (Vallejo et al., 1999).

2.3. Sub-library construction

Total DNA of T. rangeli C23 strain was isolated

using standard methods (Requena et al., 1988). The

isolated DNA was digested with NotI and the resulting

fragments were resolved by electrophoresis in 0.8%

agarose gels.

After ethidium bromide staining, fragments ofapproximately 1 kb were cut, purified using Glassmax

(Gibco-BRL) and cloned into the NotI site of pBlue-

script plasmid (Stratagene, La Jolla, CA).

2.4. Southern blot analysis

Total DNA (1–4 lg) were digested separately withrestriction endonucleases HindIII, NotI, SphI, StyI, andTaqI, resolved in 0.8% agarose gels and transferred to a

nylon membrane (BioRad) by standard procedures

(Sambroock et al., 1989). The hybridization conditions

were carried out using the methodology previously

described by Puerta et al. (1994). The inserts of the

recombinant clones were labeled by the random primer

method using [a-32P]dCTP (Feinberg and Vogelstein,1983). The final wash of the filters was performed in0.1� SSC/0.1% SDS at 65 �C for 1 h and the filters wereexposed to Curix RP2 medical X-ray film (Kodak).

2.5. Nucleotide sequence

Both strands of the insert of cl1 were sequenced using

fluorescent dye terminator chemistry (ABI, Foster City,

L. Morales et al. / Experimental Parasitology 102 (2002) 72–80 73

CA) in a 377 Automatic DNA sequencer (ABI Prism377). Homology searches were performed in the Gen-

Bank and EMBL databases using the FASTA program

(Pearson, 1990) and sequences were aligned using

MULTALIN (Corpet, 1988) and LALIGN programs

(Pearson, 1990).

2.6. Northern blot analysis

Cytoplasmic RNA was isolated using standard pro-

cedures (Mara~nnon et al., 2000). RNA (5 lg) were size-fractionated on 1% agarose/formaldehyde gels and

transferred to a nylon membrane using a 40mM NaOH

solution. Hybridization was carried out overnight at

42 �C in 50% formamide/5� SSC/0.1% SDS/5� Den-

hart�s/0.05M Na2HPO4=NaH2PO4 buffer/0.25mgml�1

salmon sperm DNA. The insert of clone cl1 was used asprobe.

2.7. Chromosomal blot analysis

For pulsed field gel electrophoresis (PFGE) analysis,

agarose blocks containing about 5� 107 parasites wereprepared as described by Clark et al. (1990) and stored

at 4 �C in 0.5M EDTA, pH 9.5. 1/5 of each block waselectrophoresed in a LKB 2015 Pulsaphor System

apparatus (Pharmacia LKB, Sweden), using 1% agarose

gels and 0.5� TBE buffer (40mM Tris, 45mM boric

acid, and 1mM EDTA, pH 8.3) at 13 �C. The runningconditions were 200, 350, 550, and 700 s pulse times over

a period of 80 h at 84V. The resolved chromosomes

were transferred to a nylon filter and hybridized with the

insert of clone cl1 as probe.

2.8. PCR primers and amplification

The PCR primers were designed based on the cl1insert DNA sequence, using the OligoTM versi�oon 4.0program for Macintosh: TrF (50-CGCCCCGTCTTGCCCTGT-30) and TrR2 (50-CGCAGCAAGGACAGGAGGGA-30). All reactions were performed in a finalvolume of 25 ll containing 100 ng of purified DNA fromthe different parasite strains or 10 ll of DNA extractedfrom R. prolixus haemolymph or feces, 1� reaction

buffer (10mM Tris–HCl, pH 9.0, 50mM KCl, and 0.1%de Triton X-100), 1.25U of Taq DNA polymerase

(Promega), 1.5mM of MgCl2, 0.2mM of each deoxy-

nucleoside triphosphate, and 20 pmol of each primer.

The reaction was carried out on a MJ Research PTC-

100 DNA thermal cycler, using the following profile:

94 �C/5min, 35 cycles of 95 �C/30 s, 63 �C/1min and72 �C/30 s, and a final incubation of 72 �C for 5min. Thesamples were kept at 4 �C in the thermal cycler untilremoved for analysis. Ten microliter of the reaction

products were electrophoresed in 1% agarose gels

stained with ethidium bromide.

3. Results

3.1. Isolation and characterization of the cl1 clone

A size-selected T. rangeli genomic sub-library was

constructed and used to identify sequences specific to

this parasite. This was done by randomly isolating

several clones from the library and using them as probes

in Southern blots using T. rangeli and T. cruzi DNA.Among those, a clone named cl1 was selected on the

basis of it giving a higher hybridization signal with

T. rangeli DNA, in spite of the fact that there was a

higher amount of T. cruzi DNA (Fig. 1A).

Further Southern blot analysis of T. rangeli DNA

digested separately with NotI, SphI, and StyI endonuc-

leases, shows that cl1 hybridizes with a fragment of

0.8 kb in length which is organized in tandem array inthe genome (Fig. 1B). In order to determine the chro-

mosomal location of the insert of cl1, the T. rangeli

chromosomes were separated by pulsed-field electro-

phoresis, blotted and hybridized with the labeled cl1

insert. The results indicated that the cluster is located on

a single chromosome of 1Mb in T. rangeli. When this

same probe was used with T. cruzi chromosomes, a

single band of 1.8Mb was observed, but again, thehybridization signal was weaker (Fig. 1C).

Fig. 1. Genomic organization of the snoRNA-cl1 gene. Southern blot

of genomic and chromosomal DNA from T. cruzi and T. rangeli DNA

hybridized with the radiolabeled cl1 clone insert. (A) Four microgram

from the T. cruzi D. marsupialis No. 3 strain (Tc) and 2lg of DNAfrom epimastigotes of the T. rangeli C23 strain (Tr) were digested with

different restriction endonucleases. k Phage DNA digested with Hin-

dIII was used as marker and the sizes of the fragments are indicated.

(B) One microgram of DNA from epimastigotes of the T. rangeli C23

strain was digested with different restriction endonucleases. k PhageDNA digested with HindIII was used as marker and the sizes of the

fragments are indicated. (C) Pulsed-field gel electrophoresis of chro-

mosomes from T. rangeli C23 strain (Tr) and the T. cruzi Brazilian

Y strain (Tc). The numbers at the left indicate in Mb the Saccharo-

myces cerevisiae chromosomes.

74 L. Morales et al. / Experimental Parasitology 102 (2002) 72–80

3.2. Characterization of Box C/D snoRNA-cl1

The nucleotide sequence of the insert of the cl1 clone

is shown in Fig. 2A. A search for homology with the

FASTA program from NCBI revealed a 79.5% identity

of 87 nucleotides (nt), with a nucleotide sequence which

has been identified as a box C/D snoRNA in Leptomona

collosoma. Further bioinformatics analysis of the trans-

cribed region (capital letters in Fig. 2A) shows a 48%identity with box C/D snoRNAs from Trypanosoma

brucei, T. cruzi, and Leishmania tarentolae (Fig. 2B).

In addition, computer analysis confirmed that this pu-

tative gene, named here snoRNA-cl1, contains the

consensus C, C0, D0, and D boxes.Furthermore, snoRNA-cl1 possess two regions of

11–12 nt that are complementary to a universal core

region of the mature 28S rRNA, a characteristic of theC/D box snoRNAs (Fig. 2C). SnoRNA-cl1 have also

the potential to interact with 28S rRNA from T. cruzi

and T. brucei, and with 18S rRNA from T. rangeli,

T. cruzi, and T. brucei (data not shown).

In order to confirm the expression of the putative

snoRNA gene, a Northern blot analysis was carried out,

using the cl1 insert as probe. After stringent hybridiza-

tion conditions, a band of approximately 90 nt wasdetected in the total RNA of epimastigotes from

T. rangeli, but not from T. cruzi RNA (Fig. 3).

3.3. A putative snoRNA-cl1 homologue in T. cruzi

In order to investigate if T. cruzi has a similar

snoRNA, FASTA analysis with the transcribed region

of the putative snoRNA-cl1 gene was performed. Theresults of this search showed a 79.5% identity with a

genomic fragment of unknown function from T. cruzi,

reported by Aguero et al. (2000) (Accession No.

AQ445586). Further searches for the presence of charac-

teristic motifs of C/D box snoRNAs, indicated the

existence of C, C0, D, and D0 boxes, as well as of theinternal guide sequence that is able to base pair with a

specific segment of the mature 28S rRNA (Fig. 4). Theseresults suggest that this sequence is transcribed in a box

C/D snoRNA in T. cruzi. Interestingly, the upstream

and downstream regions flanking the transcribed region

of these snoRNAs in T. cruzi and T. rangeli did not

show homology.

3.4. PCR amplification of snoRNA-cl1 gene

Based on the above results, two oligonucleotides

(TrF and TrR2, underlined in Fig. 2A) were designed

to specifically amplify a 620-bp fragment from the

Fig. 2. (A) Nucleotide sequence of the snoRNA-cl1 gene. The putative

transcribed region is in capital letters. The numbers at the left hand in-

dicate the nucleotide position. The oligonucleotides used for PCR am-

plification are underlined. (B) Alignment of the snoRNA coding

sequences from T. rangeli snoRNA-cl1 (Tr: Accession No. AY028385),

L. collosoma snoRNA-G2 (Lc: Accession No. AF331656), T. cruzi

snoRNA-94nt (Tc: Accession No. AF016400), T. brucei snoRNA-94nt

(Tb: Accession No. Z50171), and L. tarentolae snoRNA-92nt (Lt: Ac-

cession No. AF016399). Gaps introduced into sequences are denoted

by dashes. The conserved boxes C/C0 and D/D0 of the snoRNAs areoverlined-capital letters. (C) Schematic representation of potential base

pairing interactions between snoRNA-cl1 and its target sites on 28S

rRNA. The conserved motifs D/D0 of the snoRNA are underlined-capital letters. Regions that are complementary to rRNA are depicted

by bars. The potential guided methylation sites are marked by filled

circles. The positions on the rRNA are indicated.

Fig. 3. Northern blot analysis of total RNA from epimastigotes of the

T. rangeli C23 strain (Tr) and the T. cruzi strain (Tc) in the logarithmic

phase of growth. The filter was hybridized with the radiolabeled

snoRNA-cl1gene.

L. Morales et al. / Experimental Parasitology 102 (2002) 72–80 75

T. rangeli genome. As shown in Fig. 5, we obtained aband of the predicted size in the four T. rangeli strains

analyzed, but no amplification was observed for T.

cruzi.

To examine the specificity of these oligonucleotides,

further PCR assays were done using DNA from T. cruzi

(groups I and II, Satellite Meeting, 1999), L. panamensis,

L. amazonensis, Trypanosoma vivax, and Crithidia sp

strains as well as with non-related human DNA, and noamplification products were observed (Fig. 6). The same

results were obtained with eight more T. cruzi strains

(data not shown). In order to confirm the integrity of T.

cruzi DNA, we also performed a PCR using H2A his-

tone genes as target (Puerta et al., 1994) and the ex-

pected fragment was amplified in all T. cruzi strains

(data not shown).

The sensitivity of the TrF and TrR2 primers in a PCRassay was determined using serial dilutions of purified T.

rangeli DNA in the presence of 50 ng of heterologous

human DNA. The limit for the visualization of the

amplified product on ethidium bromide-stained gels was

1 pg of target DNA (Fig. 7), equivalent to 5 parasites

(Kooy et al., 1989).

To investigate if the PCR assay reported here was

able to identify T. rangeli in mixed infections, we con-

ducted a PCR using both T. rangeli and T. cruzi DNAs

as targets in the same reaction tube. The results shown

in Fig. 8 indicated that the presence of T. cruzi DNA in

the samples does not interfere with the amplification of

T. rangeli DNA. Again, the quality of the T. cruzi DNA

was verified by a PCR test using the H2A histone genes

as target (data not shown).

3.5. Amplification of snoRNA-cl1 gene in biological

samples

To determine whether the snoRNA-cl1 gene from T.

rangeli could be amplified in biological samples, we

analyzed haemolymph and faecal samples of non-

infected R. prolixus and R. prolixus infected with

T. rangeli or T. cruzi. As seen in Fig. 9, TrF/TrR2

Fig. 5. Ethidium bromide-stained 1% agarose gel containing 10 ll ofthe PCR products obtained from DNAs of T. rangeli 5048 (1), H14 (2),

C23 (3), Tre (4), strains and T. cruzi Shubacbarina (5), and Y strains

(6). The negative control (NC) used distilled water as the template. kPhage DNA digested with HindIII was used as marker and the sizes of

the fragments are indicated.

Fig. 6. Specificity of the PCR using TrF/TrR2 primers. Ethidium

bromide-stained 1% agarose gel containing 10ll of the PCR productsobtained from DNAs of T. rangeli Choachi (1), T. rangeli 5048 strains

(2), T. cruzi Y (3), T. cruzi No. 3 strains (4), L. panamensis (5), L.

amazonensis (6), T. vivax (7), Crithidia sp (8), human (9), and negative

control (10).

Fig. 7. Sensitivity of the PCR using TrF/TrR2 primers. Amplification

of serial dilutions of T. rangeli Tre DNA in the presence of 50 ng of

human DNA. Ten microliter of each reaction mixture was run on a 1%

agarose gel. Lanes: 1, 10 ng; 2, 1 ng; 3, 100 pg; 4, 10 pg; 5, 1 pg; 6,

100 fg; 7, 10 fg; and 8, negative control (NC).

Fig. 8. TrF/TrR2 PCR amplification using DNA from T. rangeli Tre

strain (Tr) and T. cruzi B.M. Lopez strain (Bml) as targets in the same

reaction tube. Lanes: 1, 100 ng of Tr and Bml; 2, 200 ng of Tr and

100 ng of Bml; 3, 100 ng of Tr and 200 ng of Bml; 4, 100 ng of Tr; 5,

100 ng of Bml; and 6, negative control (NC).

Fig. 4. Alignment of the snoRNA transcribed sequences from T.

rangeli snoRNA-cl1 (Tr: Accession No. AY028385), L. collosoma

snoRNA-G2 (Lc: Accession No. AF331656), and the putative T. cruzi

snoRNA-cl1 (Tc: Accession No. AQ445586). Gaps introduced into

sequences are denoted by dashes. The conserved boxes C/C0 and D/D0

of the snoRNAs are overlined-capital letters. The regions of comple-

mentarity with rRNA are underlined.

76 L. Morales et al. / Experimental Parasitology 102 (2002) 72–80

primers only amplified the haemolymph sample from T.

rangeli infected vectors. No amplification products were

observed in feces from T. cruzi-infected and non-in-

fected vectors.

4. Discussion

In the search for a specific DNA fragment of T.

rangeli, able to distinguish T. cruzi from T. rangeli, we

identified a genomic clone which encodes for a putative

small nucleolar RNA (snoRNA) from T. rangeli. Try-

panosomes possess unique RNA processing mechanisms

including trans-splicing of pre-mRNA (Agabian, 1990),

RNA editing of mithochondrial transcripts (Simpson

and Shaw, 1989) and further cleavage of 28S rRNA into

28Sa, 28Sb, sr1, sr2, sr4, and sr6 RNAs (Hasan et al.,1984; White et al., 1986). Pre-rRNA processing and ri-

bosome assembly occur primarily in the cell nucleolus,

which contains a complex populations of small RNAs,

known as small nucleolar RNAs (snoRNAs). The

snoRNAs can be divided into two classes on the basis of

common sequence boxes. One group of snoRNAs con-

tains two sequence motifs, box C (50-PuUGAUGA-30,where Pu is purine) and box D (50-CUGA-30), that bindan abundant nucleolar protein, fibrillarin (Maxwell and

Fournier, 1995), and the other group has the H (An-

AnnA) and ACA motifs. A few snoRNAs in each family

are involved in pre-RNA processing but the majority of

them are implicated in nucleotide modification. Box C/

D snoRNAs guide 20-O-ribose methylation (Samarskyet al., 1998) and the H/ACA snoRNAs the pseudouri-

dine formation (Balakin et al., 1996).Methylations of RNA, at the 20-O-ribose position,

involve numerous types of nucleoside modifications

which can affect the three-dimensional structure of

rRNAs and their interaction with proteins. However,

the precise function of these modifications remains un-

known (Bachellerie and Cavaille, 1997; Bachellerie et al.,1995).

Although rRNA methylation is generally found in

invariant sequences among yeast and vertebrates, the

discovery of archeal snoRNAs (Omer et al., 2000; Potter

et al., 1995) and a fibrillarin homologue protein in a

related archaeon (Amiri, 1994) indicate that methylation

of RNA and snoRNAs may be more widespread, and

may include archeas and early eukaryotes. Indeed thefollowing observations have been reported in the ancient

eukaryotic trypanosomatids: (i) more than 100 sites of

20-O-ribose methylation were mapped in Crithidia fas-

ciculata (Gray, 1979), (ii) three small nucleolar

RNAs were found in the spliced-leader associated RNA

locus in T. brucei, L. tarentolae, and T. cruzi (Roberts

et al., 1998), (iii) a fibrillarin and seventeen fibrillarin-

associated snoRNAs have been identified in T. brucei

(Dunbar et al., 2000), and (iv) four additional snoRNAs

has been identified in L. collosoma. Remarkably, some

of them appear to be homologous of antisense snoR-

NAs of yeast and vertebrates. Most of these snoRNAs

from trypanosomatids have the potential to guide

methylation.

In this study we present evidence that cl1 encodes for

the first T. rangeli snoRNA. The transcribed regioncontains the canonical C, C0, D, and D0 boxes, thatcharacterize the C/D box of snoRNAs. Like other try-

panosomatid snoRNAs studied, the genomic organiza-

tion of snoRNA-cl1 is characterized by multicopy

tandemly arranged (Dunbar et al., 2000; Roberts et al.,

1998; Xu et al., 2001). Interestingly, each copy of the

snoRNA-2 tandem from L. collosoma and snoRNA-

cl1 tandem from T. rangeli has a single snoRNA trans-cribed region. This contrasts with other trypanosomatid

snoRNAs, in which each tandem repeat encodes for

more than one snoRNA.

Conserved among all C/D box snoRNAs, the C and

D elements are required for snoRNA nucleolar locali-

zation, accumulation, maturation and fibrillarin asso-

ciation (Maxwell and Fournier, 1995; Samarsky et al.,

1998).The snoRNA-cl1 that we have identified possesses C

and C0 boxes with a nucleotide usage resembling the T.brucei snoRNAs TBR13 and TBR1, (Dunbar et al.,

2000), whereas the D and D0 boxes are identical to thisin yeast and higher eukaryotes (Maxwell and Fournier,

1995; Samarsky et al., 1998). The C/D box snoRNAs

involved in ribose methylation also contain an internal

guide sequence that is able to base pair with a specificsegment of rRNA (Bachellerie and Cavaille, 1997;

Bachellerie et al., 1995). Our computer sequence analysis

showed that snoRNA-cl1 has two potential regions of

complementarity with 28S rRNA from trypanosoma-

tids, across a duplex of 11–12 bp, thus being able to

guide methylation of two different sites in 28S rRNA

molecule. This may be expected since the relatively small

Fig. 9. Detection of T. rangeli in biological samples from triatomines by

PCR assay using TrF/TrR2. Ten microliter of PCR products obtained

from DNA isolates from haemolymph of R. prolixus infected with

T. rangeli Choachi strain (Ch), from feces of R. prolixus infected with

T. cruzi Saul Parra strain (Sp) or from feces of non-infected vectors

(UN) was run on a 1% agarose gel. The positive controls were DNA

isolated from cultures T. rangeli C23 and 5048 strains. The negative

control (NC) used distilled water as the template. k Phage DNAdigested withHindIII was used as marker and the sizes of the fragments

are indicated.

L. Morales et al. / Experimental Parasitology 102 (2002) 72–80 77

genome size of the parasite may have forced manysnoRNAs to guide methylation at two sites, similar to

what happens in Archaea and L. collosoma, where nu-

merous snoRNAs appear to be double guides (Omer

et al., 2000). Interestingly, computer analyses also

showed that snoRNA-cl1 can potentially interact with

18S rRNA from trypanosomatids; therefore, we cannot

exclude the possibility that, as happens with snoRNA-2

from L. collosoma (Levitan et al., 1998), snR51 fromyeast (Lowe and Eddy, 1999) and TBR14 from T. brucei

(Dunbar et al., 2000), snoRNA-cl1 has also the potential

to guide methylation in two different rRNA molecules.

FASTA analysis indicated that snoRNA-cl1 gene has

a high identity with snoRNA-G2 from L. collosoma,

showing identical upstream D and D0 regions. ThesnoRNA-G2 guides ribose methylation of A3697 and

A3709 on 28S rRNA (Xu et al., 2001). Moreover, thesnoRNA snR71 from yeast and U29 from human, in

charge of A3709 methylation, have guiding sequences

similar to that found in snoRNA-cl1 and G2. Based on

these findings, we believe that the function of the T.

rangeli snoRNA-cl1 is methylation of A3697 and A3709 on

the parasite 28S rRNA. However, further studies are

required in order to precisely identify the methylation

sites in both 28S and 18S rRNA.Computer analysis of the reported sequence also

suggests that T. cruzi contains a unit that encodes for a

homologous snoRNA-cl1. The homology between these

genes only takes place through the 87 nts of the trans-

cribed region, thus explaining the weak hybridization

signal obtained in Southern blot analysis. In order to

confirm the presence of a snoRNA-cl1 like gene in

T. cruzi, further analyses using the transcribed regionof snoRNA-cl1 as probe will be necessary.

Based on the non-homologous regions of both T.

rangeli and T. cruzi snoRNA-cl1 genes, we developed a

PCR assay which specifically ampliflies a 620 bp frag-

ment from T. rangeli DNA, even in the presence of T.

cruzi DNA. Different molecular studies have shown that

T. rangeli can be divided into two groups: One group

includes parasites from Central America and thenorthern part of South America, which contain the

minicircle KP1 and the other includes parasites from

southern Brazil, which are KP1 ()) (Grisard et al., 1999;Macedo et al., 1993; Vallejo et al., 1994). Recent studies

have shown that domestic strains of T. rangeli strains

harbor the minicircle KP1 whereas the sylvatic strains

do not (Vallejo et al., 2002). Although some extent of

sequence variability of snoRNA-cl1 genes among strainscan not be excluded, this PCR has the advantage that it

is able to detect T. rangeli DNA from KP1 (+) and KP1

()) strains. This result, together with the fact that TrF/TrR2 were designed based on the sequence of an im-

portant gene, that is implicated in the rRNA methyla-

tion, render this PCR a good target for studying T.

rangeli strains from different geographic origins.

Molecular diagnoses, such as PCR assays, must takeinto account the fact that the presence of host DNA in

the biological samples reduces the sensitivity of the test

(Grisard et al., 1999). TrF/TrR2 primers are able to

amplify the equivalent of 5 parasites in the presence of

heterologous DNA.

By varying the ratio of T. cruzi and T. rangeli DNA

in the same tube reaction, we also showed that T. rangeli

can be detected even in the presence of twice the amountof T. cruzi DNA. In addition, the PCR reported here is

able to detect the presence of the parasite in its natural

invertebrate host.

Therefore, the PCR assay reported here has the

following advantages: (i) as the TrF/TrR2 primers do

not have annealing sites on T. cruziDNA, the reaction is

based on the presence or absence of a T. rangeli am-

plified fragment which renders this assay a clear test fordiagnosis, (ii) DNA from KP1 (+) and KP1 ()) strainsamplify a fragment of the same size, suggesting that the

annealing site of TrF/TrR2 primers are not polymor-

phic, and (iii) in artificially mixed T. cruzi and T. rangeli

DNAs, this PCR test does not bias the diagnosis to-

wards T. cruziDNA. For all these reasons, this PCR test

could be useful for human diagnosis as well as for epi-

demiological field studies.

Acknowledgment

This work was supported by Facultad de Ciencias de

la Universidad Javeriana (Bogot�aa, Colombia).

References

Acosta, I., Romanha, A.J., Cosenza, H., Krettli, A.U., 1991. Trypa-

nosomatids isolates from Honduras: differentiation between Try-

panosoma cruzi and T. rangeli. American Journal of Tropical

Medicine and Hygiene 44, 676–683.

Afchain, D., Le Ray, D., Capron, A., 1979. Antigenic make-up of

Trypanosoma cruzi culture forms: identification of a specific

component. Journal of Parasitology 65, 507–514.

Agabian, N., 1990. Trans-splicing of nuclear pre-mRNAs. Cell 61,

1157–1160.

Aguero, F., Verdon, R., Frasch, A.C.C., Sanchez, D.O., 2000. A

random sequencing approach for the analysis of the Tryapanosoma

cruzi genome: general structure, large gene and repetitive DNA

families and gene discovery. Genome Research 10, 1996–2005.

Amiri, K.A., 1994. Fibrallarin-like proteins occur in the domain

Archaea. Journal of Bacteriology 176, 2124–2127.

Bachellerie, J.P., Cavaille, J., 1997. Guiding ribose methylation of

rRNA. Trends in Biochemical Science 22, 257–261.

Bachellerie, J.P., Michot, B., Nicoloso, M., Balakin, A., Ni, J.,

Fournier, M.J., 1995. Antisense snoRNAs: a family of nucleolar

RNAs with long complementarities to rRNA. Trends in Bio-

chemical Science 20, 261–264.

Balakin, A., Smith, L., Fournier, M., 1996. The RNA world of the

nucleolus; two major families of small nucleolar RNAs defined by

different box elements with related functions. Cell 86, 823–834.

78 L. Morales et al. / Experimental Parasitology 102 (2002) 72–80

Basso, B., Moretti, E.R.A., Votiero-Cima, E., 1991. Immune response

and Trypanosoma cruzi infection in Trypanosoma rangeli-immu-

nized mice. American Journal of Tropical Medicine and Hygiene

44, 413–419.

Corpet, F., 1988. Multiple sequence alignment which hierarchical

clustering. Nucleic Acids Research 16, 10881–10890.

Clark, G.C., Lai, E.Y., Fulton, C., Cross, G.A.M., 1990. Electropho-

retic karyotype and linkage groups of the amoeboflagellate

Naegleria gruberi. Journal of Protozoology 37, 400–408.

Cotrim, P.C., Paranhos, G.S., Mortara, R.A., Wanderley, J., Rassi,

A., Camargo, M.E., da Silveira, J.F., 1990. Expression in E. coli of

a dominant Immunogen of Trypanosoma cruzi recognized by

human chagasic sera. Journal of Clinical Microbiology 28, 519–

524.

D�Alessandro, A., 1976. Biology of Trypanosoma Herpetosoma rangeli

Tejera, 1920. In: Lumsden, W.H.R., Evans, D.A. (Eds.), Biology of

Kinetoplastida, vol. I. Academic Press, London, New York, and

San Francisco, pp. 328–403.

D�Alessandro, A., Saravia, N., 1992. Trypanosoma rangeli. In: Lums-

den, W.H.R., Evans, D.A. (Eds.), Parasitic Protozoa, vol. II.

Academic Press, London, New York, and San Francisco, pp. 1–54.

de Miranda Santos, I.K.F., Pereira, M.E.A., 1984. Lectins discrimi-

nate between pathogenic and nonpathogenic South American

Trypanosomes. American Journal of Tropical Medicine and

Hygiene 33, 839–844.

Dorn, P.L., Engelke, D., Rodas, A., Rosales, R., Melgar, S., Brahney,

B., Flores, J., Monroy, C., 1999. Utility of the polymerase chain

reaction in detection of Trypanosoma cruzi in Guatemalan Chagas�disease vectors. American Journal of Tropical Medicine and

Hygiene 60, 740–745.

Dunbar, D., Wormsley, S., Lowe, T., Baserga, S., 2000. Fibrallarin-

associated box C/D small nucleolar RNAs in Trypanosoma brucei.

The Journal of Biological Chemistry 275, 14767–14776.

Feinberg, A.P., Vogelstein, B., 1983. A technique for radiolabeling

DNA restriction endonuclease fragments to high specific activity.

Analytical Biochemistry 132, 6–13.

Fernandes, O., Santos, S.S., Cupolillo, E., Mendoca, B., Derre, R.,

Junqueira, A.C.V., Santos, L.C., Sturm, N.R., Naiff, R.D., Barret,

T.V., Campell, D.A., Coura, J.R., 2001. A mini-exon multiplex

polymerase chain reaction to distinguish the major groups of

Trypanosoma cruzi and T. rangeli in the Brazilian Amazon.

Transactions of the Royal Society of Tropical Medicine and

Hygiene 95, 97–99.

Gray, M.W., 1979. The ribosomal RNA of the trypanosomatid

protozoan Crithidia fasciculata: physical characteristics and

methylated sequences. Canadian Journal of Biochemistry 57,

914–926.

Greig, S., Ashall, F., Hudson, L., 1990. Use of total parasite DNA

probes for the direct detection of Trypanosoma cruzi and Trypan-

osoma rangeli in domiciliary Rhodnius prolixus. Transactions of the

Royal Society of Tropical Medicine and Hygiene 84, 59–60.

Grisard, E.C., Campbell, D.A., Romanha, A.J., 1999. Mini-exon gene

sequence polymorphism among Trypanosoma rangeli strains iso-

lated from distinct geographical regions. Parasitology 118, 375–

382.

Guhl, F., Hudson, L., Marinkelle, C., Jaramillo, C.A., Bridge, D.,

1987. Clinical Trypanosoma rangeli infection as a complication of

Chagas disease. Parasitology 94, 475–484.

Hasan, G., Turner, M.J., Cordingley, J.S., 1984. Ribosomal RNA

genes of Trypanosoma brucei: mapping the regions specifying the

six small ribosomal RNAs. Gene 27, 75–86.

Kooy, R.F., Ashall, F., van der Ploeg, M., Overdulve, J.P., 1989. On

the DNA content of Trypanosoma cruzi. Molecular and Bio-

chemical Parasitology 36, 73–76.

Levitan, A., Xu, Y., Ben-Dov, C., Ben-Shlomo, H., Zhang, Y.,

Michaeli, S., 1998. Characterization of a novel trypanosomatid

small nucleoler RNA. Nucleic Acids Research 26, 1775–1783.

Lowe, T.M., Eddy, S.R., 1999. A computational screen for methy-

lation guide snoRNAs in yeast. Science 283, 1168–1171.

Macedo, A.M., Vallejo, G.A., Chiairi, E., Pena, S.D.J., 1993. DNA

fingerprinting reveals relationships between strains of Trypanosoma

rangeli and Trypanosoma cruzi. In: Pena, S.D.J., Chakraborty, R.,

Epplen, J.T., Jeffreys, A.J. (Eds.), DNA Fingerprinting: State of

Science. Birkhauser, Basel, pp. 321–329.

Mara~nnon, C., Thomas, M.C., Puerta, C., Alonso, C., Lopez, M.C.,

2000. The stability and maturation of the H2A histone mRNAs

from Trypanosoma cruzi are implicated in their post-transcriptional

regulation. Biochimica et Byiophysica Acta 1490, 1–10.

Maxwell, E.S., Fournier, M.J., 1995. The small nucleolar RNAs.

Annual Review of Biochemistry 35, 897–934.

Murthy, V.K., Dibbern, K.M., Cambpell, D.A., 1992. PCR amplifi-

cation of mini-exon genes differentiates Trypanosoma cruzi from

Trypanosoma rangeli. Molecular and Cellular Probes 6, 237–243.

Omer, A.D., Lowe, T.M., Russell, A.G., Ebhardt, H., Eddy, S.R.,

Dennis, P.P., 2000. Homologs of small nucleolar RNAs in

Archaea. Science 288, 517–522.

Pearson, W.R., 1990. Rapid and sensitive sequence comparison with

FASTP and FASTA. Methods in Enzymology 183, 63–98.

Potter, S., Durovic, P., Dennis, P.P., 1995. Ribosomal RNA precursor

processing by a eukaryotic U3 small nucleolar RNA-like molecule

in an archaeon. Science 268, 1056–1060.

Puerta, C., Martin, J., Alonso, C., Lopez, M.C., 1994. Isolation and

characterization of the gene enconding histone H2A from Trypan-

osoma cruzi. Molecular and Biochemical Parasitology 64, 1–10.

Requena, J.M., Lopez, M.C., Jimenez-Ruiz, A., de la Torre, J.,

Alonso, C., 1988. A head-to-tail tandem organization of hsp70

genes in Trypanosoma cruzi. Nucleic Acid Research 16, 1393–1406.

Roberts, T.G., Sturm, N.R., Yee, B.K., Michael, C., Hartshorne, T.,

Agabian, N., Campbell, D.A., 1998. Three small nucleolar RNAs

identified from the spliced leader-associated RNA locus in kineto-

plastid protozoans. Molecular and Cellular Biology 18, 4409–4417.

Rodriguez, P., Montilla, M., Nicholls, S., Zarante, I., Puerta, C., 1998.

Isoenzimatic characterization of Colombian strains of Trypano-

soma cruzi. Memorias do Instituto Oswaldo Cruz 93, 739–740.

Samarsky, D.A., Fournier, M.J., Singer, R.H., Bertrand, E., 1998. The

snoRNA box C/D motif directs nucleolar targeting and also

couples snoRNA synthesis and localization. Embo Journal 17,

3747–3757.

Sambroock, J., Maniatis, T., Fritsh, E.F., 1989. Gel electrophoresis of

DNA. In: Molecular Cloning. A Laboratory Manual, second ed.

Cold Spring Harbor Laboratory, New York.

Satellite Meeting, 1999. Standardization of the nomenclature of

Trypanosoma cruzi strains. Memorias do Instituto Oswaldo Cruz

94, 429–432.

Silber, A.M., Bua, J., Porcel, B.M., Segura, E.L., Ruiz, A.M., 1997. T.

cruzi: specific detection of parasites by PCR infected humans and

vectors using a set of primers (BP1/BP2) targeted to a nuclear DNA

sequence. Experimental Parasitology 85, 225–232.

Simpson, L., Shaw, J., 1989. RNA editing and the mitochondrial

cryptogenes of kinetoplastid protozoa. Cell 57, 355–366.

Souto, R.P., Vargas, N., Zingales, B., 1999. Trypanosoma rangeli:

discrimination from Trypanosoma cruzi based on a variable domain

from the large subunit ribosomal RNA gene. Experimental

Parasitology 91, 306–314.

Sturm, N.R., Degrave, W., Morel, C., Simpson, L., 1989. Sensitive

detection and schizodeme classification of Trypanosoma cruzi cells

by amplification of kinetoplast minicircle DNA sequences: used in

diagnosis of Chagas� diseases. Molecular and Biochemical Parasi-tology 33, 205–214.

Tanaka, T., 1997. Differential diagnosis of Trypanosoma cruzi and T.

rangeli infection by PCR of cysteine proteinase genes. Kan-

senshogaku Zasshi 71, 903–909.

Vallejo, G.A., Macedo, A.M., Chiari, E., Pena, S.D.J., 1994. The

Kinetoplast DNA from Trypanosoma rangeli contains two distinct

L. Morales et al. / Experimental Parasitology 102 (2002) 72–80 79

classes of minicircle with different size and molecular organization.

Molecular and Biochemical Parasitology 67, 245–253.

Vallejo, G.A., Guhl, F., Carranza, J.C., Lozano, L.E., S�aanchez, J.L.,

Jaramillo, J.C., Gualtero, D., Casta~nneda, N., Silva, J.C., Steindel,

M., 2002. kDNA markers define two major Trypanosoma rangeli

lineages in Latin-America. Acta Tropica 81, 77–82.

Vallejo, G., Guhl, F., Chiari, E., Macedo, A., 1999. Species specific

detection of Trypanosoma cruzi and Trypanosoma rangeli in

vector and mammalian hosts by polymerase chain reaction

amplification of kinetoplast minicircle DNA. Acta Tropica 72,

203–212.

Vallejo, G.A., Chiari, E., Macedo, A.M., Pena, S.D.J., 1993. A simple

laboratory method for distinguishing between Trypanosoma cruzi

and Trypanosoma rangeli. Transactions of the Royal Society of

Tropical Medicine and Hygiene 87, 165–166.

Vallejo, G.A., Marinkelle, C.J., Guhl, F., de Sanchez, N., 1988.

Behavior of the infection and morphologic differentiation of

Trypanosoma cruzi and T. rangeli in the intestine of the vector

Rhodnius prolixus. Revista Brasileira de Biologia (Rio de Janeiro,

Brasil) 48, 577–587.

Vargas, N., Souto, R., Carranza, J., Vallejo, G.A., Zingales, B., 2000.

Amplification of a specific repetitive DNA sequence for Trypa-

nosoma rangeli identification and its potential application in

epidemiological investigations. Experimental Parasitology 96,

147–159.

Vasquez, J.E., Krusnell, J., Orn, A., Sousa, O.E., Harris, R.A., 1997.

Serological diagnosis of Trypanosoma rangeli infected patients. A

comparison of different methods and its implications for the

diagnosis of Chagas� disease. Scandinavian Journal of Immunology45, 322–330.

White, T.C., Rudenko, G., Borst, P., 1986. Three small RNAs within

the 10 kb trypanosome rRNA transcription unit are analogous to

domain VII of other eukaryotic 28S rRNAs. Nucleic Acids

Research 14, 9471–9489.

Xu, Y., Liu, L., Lopez-Estra~nno, C., Michaeleli, S., 2001. Expression

studies on clustered trypanosomatid box C/D small nucleolar

RNAs. The Journal of Biological Chemistry 276, 14289–14298.

Zu~nniga, C., Palau, T., Penin, P., Gamallo, C., de Diego, J.A., 1997.

Characterization of a Trypanosoma rangeli strain of Colombian

origin. Memorias do Instituto Oswaldo Cruz 92, 523–530.

80 L. Morales et al. / Experimental Parasitology 102 (2002) 72–80