MOLECULAR iZ%HEMICAL PARASITOLOGY

Molecular

and Biochemical

Parasitology

71 (1995) 273-278

Short communication

Molecular karyotype of clone CL Brener chosen for the Trypanosoma cruzi Genome Project
Maria I. Cano a, Arthur Gruber b, Martin Vazquez , Arantxa Corks d, Mariano J. Levin , Antonio Gonzhlez d, Wim Degrave e, Edson Rondinelli f, Bianca Zingales b, Jo& Luis Ramirez g, Carlos Alonso h, Jo& M. Requena h, Jo& Franc0 da Silveira a3 *
a Escola Paul&a de Medicina, Rua Botucatu, 862, CEP 04023-062, Stio Pa&o, Brazil b Institute de Quimica da USP, Sao Paula, Brazil Institute de Inuestigaciones en Engenharia Genetica y Biologia Molecular, Buenos Aires. Argentina Instituto de Parasitologia y Biomedicina, Granada, Spain e FIOCRUZ, Rio de Janeiro, Brazil f Instituto de Biofisica, UFRJ, Rio de Janeiro, Brazil g Centro de Biologia Celular, XV, Caracas, Venezuela h Centro de Biologia Molecular, IJAM_ Madrid, Spain Received 10 February 1995; accepted 28 March 1995

Keywords: Trypanosoma cruzi; Clone CL Brener; Genome project; Molecular

karyotype;

Chromosome

mapping

The establishment of detailed physical and genetic maps of the Trypanosoma cruzi genome has been hampered by the existence of a large range of parasite strains with distinct biological and immunological characteristics, the lack of sexual stages and a seemingly variable or poorly defined number of chromosomes [l-8]. In fact, size fractionation of chromosomal bands by pulsed-field gel electrophoresis (PFGE) and hybridization to different DNA probes suggested either limited [3] or marked [4-81 chromosomal size heterogeneity among strains and clones of T. cruzi.

* Corresponding author. Tel.: (5.5-11) 576-4.532; 571-1095; e-mail: epmpara@brfapesp.bitnet

Fax: (55-11)

In the Trypanosomatid Genome Network Planning Meeting (April 1994, FIOCRUZ, Rio de Janeiro, Brazil) a T. cruzi clone derived from the CL strain was selected. This clone was named CL Brener, after Professor Zigman Brener who isolated the original CL strain in Brazil in 1963 [9]. Clone CL Brener meets all the desired characteristics of a model T. cruzi isolate: it is derived from Triatoma infestans, a strictly domiciliary vector, differentiates in LIT medium, infects mammalian cell monolayers, has preferential parasitism of heart and muscle cells, presents defined parasitemia curves and promotes mortality in mice, and is highly susceptible to wellknown drugs (Zingales, B., Pereira, M.E.S., Brener, Z., unpublished). Here we report the analysis of the molecular karyotype of clone CL Brener, and the

0166-6851/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-6851(95)00066-6

Telomere

1.9 1 II Ii

P4

1.8

2.2

3.0

Hsp60 Ubiquitin H49 and CRA tx-tubulin rDNA 24% 812 and El13 Hsp70 Actin

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chromosomal location of a number of homologous cloned sequences, including housekeeping genes and polymorphic sequences. We have used improved PFGE conditions that allowed to achieve good separation of chromosomal bands ranging from 0.45 to 4.0 Mb, and a high resolution of the megabase long bands (Fig. 1). PFGE was done in a Pharmacia Gene Navigator apparatus using a hexagonal electrode array. Agarose blocks containing epimastigotes were lysed and prepared as described [4]. Approx. lo7 cells per well were used, and the separations were carried out in 1.2% agarose gels (Rapid Agarose, Gibco/BRL) in 0.5 X TBE (45 mM Tris/45 mM boric acid/ 1 mM EDTA, pH 8.3) in running buffer (0.5 X TBE) maintained at constant temperature (13C). The best separation was achieved by the use of 5 phases of homogeneous pulses (N/S, E/W) with interpolation for 132 h at 80 V: phase 1, pulse time 90 s (run time 30 h); phase 2, 200 s (30 h); phase 3, 350 s (24 h); phase 4, 500 s (24 h) and phase 5, 800 s (24 h). After electrophoresis, gels were stained with ethidium bromide (0.5 mg ml- ), photographed and transferred onto nylon filters (Hybond N, Amersham) [6]. Chromosomes from Saccharomyces cereuisiae (Gibco/BRL) and Hunsenulu wingei (Gibco/BRL) were used as molecular mass standards. The CL Brener molecular karyotype obtained under the above mentioned conditions indicates the existence of 12 chromosomal bands: 12 megabase bands ranging from 3.5 to 1.0 Mb and 8 intermediate chromosomal bands between 1.0 to 0.45 Mb. The same chromosomal pattern was obtained in the original CL strain (not shown). Contrasting with previous studies [3-81, we succeeded in resolving the compression zone (ranging from 1.6 to 4.0 Mb), and were not able to detect bands smaller than 0.45 Mb or minichromosomes [4], even under conditions where we resolved bands in the molecular mass range of 50-900 kb. Arbitrarily, we numbered the

chromosomal bands using roman numerals (I-XX), starting from the smallest 0.45-Mb band. The stability of the molecular karyotype of clone CL Brener was confirmed by the fact that no changes were detected after more than 55 electrophoretical runs in 8 independent chromosomal preparations obtained from parasites subcultured for 6 months in axenic medium. In hybridization experiments using a T. brucei telomere sequence [lo] as probe, all chromosomal bands were labelled by the probe, confirming their integrity and identity as chromosomes (Fig. 1A). All bands were, moreover, sensitive to Bal-31 nuclease digestion, indicating that the chromosomal bands represent linear DNA molecules (data not shown). The intensity of the ethidium bromide fluorescence was not evenly distributed along all chromosomal bands showing a good correlation with the hybridization pattern obtained by the telomeric probe (Fig. 1A). The increased fluorescence and hybridization signal of some bands may indicate band comigration of two or more chromosomes or aneuploidy. To get a rough estimate of the number of chromosomes per band, autoradiograms obtained by hybridization with the telomeric probe were scanned and the relative area of band I was chosen arbitrarily as a standard for minimal ploidy. Since this probe only hybridizes to the chromosomal ends, it is expected that regardless of their size, all chromosomal bands should yield the same signal intensity. Therefore, an estimate of the number of chromosomes per band, defined here as chromosome equivalents, was obtained dividing the densitometric value of the area of each band by the area of band I (Fig. 1B). The cumulative sum of these values gave 64 chromosome equivalents per epimastigote cell. This result is in agreement with previous PFGE studies with other strains and clones that suggested that the total number of chromosomes in T. cruzi exceeds 40 [6]. The value here presented, however, should not be taken

Fig. 1. Molecular karyotype of clone CL Brener. (Panel A) Ethidium bromide-stained pattern (Et Br) of chromosomal bands after separation by PFGE and Southern blot hybridization with T. brucei telomere sequences. The sizes (Mb) and the numbers of the chromosomal bands are indicated. (Panel B) Scanning of the autoradiogram shown in panel A with Shimadzu CS-9000 Dual Wavelength flying-spot scanner. The roman and arabic numerals indicate, respectively, the chromosomal bands and the corresponding chromosome equivalents of each band (see text). (Panel C) Southern blot hybridization with i? crzui cloned sequences. Membranes were hybridized in 50% formamide at 42 for 18 h and washed twice (30 min) in 0.1 X SSC (15 mM NaCl/l.S mM Na, citrate) at 56C.

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as definitive, since (i) chromosomal band I was arbitrarily chosen as the unit of chromosomal equivalents and (ii) the representativeness of telomeric sequences in the different chromosomes is not known. Thus, we suggest that most of the chromosomal bands contain two or more chromosomes representing either homologous chromosomes and/or heterologous chromosomes with similar sizes. To estimate the complexity (in Mb) of each chromosomal band of clone CL Brener, the number of chromosome equivalents of each band was multiplied by its corresponding molecular size. Adding all these values, the total content of nuclear DNA of clone CL Brener was estimated to be approx. 87 Mb. According to direct microfluorometry analysis and taking in account that kinetoplast DNA corresponds to about 30% of the total parasite DNA, the nuclear genome size of T. cruzi strains and clones has been estimated to be 94.5-151 Mb [ll]. Therefore, the nuclear DNA content for the CL Brener clone, obtained from our data, is similar to the values estimated for other T. cruzi strains. The karyotype of clone CL Brener was also ana-

lysed using as probes a panel of homologous genes and repetitive DNA sequences (Table 1) in Southern blot hybridizations of PFGE-separated chromosomes (Fig. lC>. Two middle repeated DNA sequences named SIRE and C6 (C6 shown in Fig. lC> hybridized to all chromosomal bands, and could be used as generic chromosomal markers. A minisatellite sequence [12] and a retrotransposon-like sequence (locus Bll) were mapped on eleven and ten chromosomal bands, respectively, sharing six bands in common. We believe that repeated sequences may provide useful polymorphic markers for chromosoma1 mapping. Table 1 compiles the chromosomal location of 23 T. cruzi homologous probes with relevant information. Besides the repeated sequences mentioned above, we analysed housekeeping genes, genes encoding structural RNAs, multicopy genes encoding stage-specific surface antigens and single-copy genes bearing repetitive motifs. It was observed that eight genes have unique chromosomal locations which can be used, therefore, as specific mapping tools for the identification of single chromosomes, Several mark-

Table 1 Localization
LOCUS

of Z. cruzi cloned sequences GenBank accession II16295 x75033 KO0393 L14824 JO4667 L11287 X52323 U15615 X02838 LO9564 JO4016 X83599 K02632 X67473 X07083 LO7759 U15616 _ JO3945 U20234 X69655 M25364 M28885 No.

on chromosomal Function

bands of clone CL Brener Chromosomal bands

C6 SIRE Minisatellite Gp 82 Gp 85 Gp 90 TcP2 p Bll lF8 H49 CRA sz5 SL-RNA Hsp 60 Hsp 70 B12 B13 (Y Tubulin Ubiquitin Actin MIP KAP 24Sa rDNA

Interspersed repetitive element Shortinterspersed repetitive element Satellite repetitive element 82-kDa surface antigen 85-kDa surface antigen 90-kDa surface antigen Ribosomal protein P2p Retrotransposon-like element Calcium-binding protein Flagellar repetitive antigen Cytoplasmic repetitive antigen Non-repetitive SIRE-associated sequence Spliced leader sequence 60-kDa heat-shock protein 70-kDa heat-shock protein 230-kDa repetitive antigen 140/116kDa repetitive antigen a-Tubulin Ubiquitin Actin Peptidyl-prolyl c&tram isomerase Kinetoplast-associated protein 24So rRNA

All All v, VIII, x, XII, XIV-XX III, v, VI, VIII, IX, x, XII, I, II, IV, v, VI, VIII, IX, x, III, v, VI, VIII, IX, x, XII, IX, x, XII, XIV, xv, XVI IX, x, XII, XIV, XV, XVI, II, v XVI, XVII XVI, XVII III, VII v, XVI XX IX, x XIII XIII xv XVIII VI IX VI, VIII XIV

xv, XVI, XVIII XII, XV-XVIII xv, XVI, XVIII XIX

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ers, such as CRA and H49 (both at bands XVI and XVII>, Hsp70 (bands IX and X) and KAP (bands VI and VIII) were mapped at neighboring chromosomal bands, which may represent size-polymorphic homologous chromosomes as had been previously suggested [6]. Complex hybridization patterns were obtained using as probes members from different T. cruzi multigene families such as: gp85/sialidase family (gp90, gp85, gp82) [14,15] and ribosomal P protein family (TcP2/3) [16] (Fig. lC, Table 1). A further observation shown in Table 1 concerns the possibility of establishing linkage maps between several loci coding for proteins or structural RNAs mapped on the same chromosomal bands (Table 1). For example, B12 and B13 genes, coding for two different antigens [13], were mapped on chromosoma1 band XIII, and in fact the physical linkage of these genes was confirmed by restriction analysis of cloned T. cruzi genomic DNA (Gruber, A., Pereira, C. and Zingales, B., unpublished). The hybridization patterns obtained with the gp90 and gp82 probes were very similar, suggesting that some of the members of these families could be linked. This suggestion was also confirmed by isolation of genomic clones carrying linked copies of the gp90 and gp82 genes (data not shown). Demonstration of the linkage relationships between the other loci shown in Table 1 will require a molecular analysis of the genes involved because, as discussed above, we cannot rule out the existence in a single band of heterologous chromosomes of similar sizes. Mapping of many other genes and probes will be necessary to construct relevant physical linkage groups for the T. cruzi genome, and to identify genetic markers of interest which may be linked to particular phenotypic traits in this organism. At present we have constructed T. cruzi clone CL Brener nuclear DNA libraries in yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACS) vectors [ 171 which will allow a deep analysis of linkage groups and the elucidation of the physical map of single chromosomes by assembling of contigs.

ments of Prof. Daniel Cohen and members of CEPH/Fondation Jean Dausset (Paris, France). We are indebted to Prof. Walter Colli and Ian MClure for helpful discussions and critical reading of the manuscript, and to Paulo E. Ribolla for his assistance in the scanning. We are grateful to Drs. Angela Cruz and Thelma Slezynger, and Prof. George Cross for providing us the following probes: T. brucei telomerit probe, ubiquitin and gp85, respectively. This work was supported by CYTED-Biotechnology Subprogram (Spain), FAPESP (Brazil), CNPq/PADCT (Brazil), Fundacion Antorchas, UNIDO/ICGEBARG91-01, T. cruzi Genome Project of University of Buenos Aires and UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases.

References [I] Brener, Z. (1973) Biology

[2] of Trypanosoma cruzi. Annu. Rev. Microbial. 27, 347-383. Tibayrenc, M., Ward, P., Moya, A. and Ayala, F.J. (1986) Natural populations of Trypanosoma cruzi, the agent of Chagas disease, have a multiclonal structure. Proc. Natl. Acad. Sci. USA 83, 115-119. Gibson, W.C. and Miles, M.A. (1986) The karyotype and ploidy of Trypanosoma cruzi. EMBO J. 5, 1299-1305. Engman, D.M., Reddy, L.V., Donelson, J.E. and Kirchhoff, L.V. (1987) Trypanosoma cruzi exhibits inter-and intra-strain heterogeneity in karyotype and chromosomal gene location. Mol. Biochem. Parasitol. 22, 115-123. Aymerich, S. and Goldenberg, S. (1989) The karyotype of Trypanosoma cruzi Dm28c: comparison with other T.cruzi strains and trypanosomatids. Exp. Parasitol. 69, 107-l 15. Henriksson, J., Aslund, L., Macina, R.A., Franke de Cazzulo, B.M., Cazzulo, J.J., Frasch, A.C.C. and Pettersson, U. (1990) Chromosomal localization of seven cloned antigen genes provides evidence of diploidy and further demonstration of karyotype variability in Trypanosoma cruzi. Mol. Biochem. Parasitol. 42, 213-224. Henriksson, J., Pettersson, U. and Solari, A. (1993) Trypanosoma cruzi: Correlation between karyotype variability and isoenzyme classification. Exp. Parasitol. 77, 334-348. Wagner, W. and So, M. (1990) Genomic variation of Try panosoma cruzi: Involvement of multicopy genes. Infect. Immun. 58,3217-3224. Brener, Z. and Chiari, E. (1963) Variaees morfolbgicas observadas em diferentes amostras de Trypanosoma cruzi. Rev. Inst. Med. Trop. S%o Paulo 5, 220-224. Van der Ploeg, L.H.T., Liu, A.Y.C. and Borst, P. (1984) Structure of the growing telomeres of trypanosomes. Cell 36, 459-468.

[3] [4]

[5]

[6]

[7]

[8]

[9]

Acknowledgements
[lo]

We thank to Dr. Rafael Range1 Aldao for help and encouragement. We acknowledge help and com-

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[ll] Kooy, R.F., Ashall, F., Van der Ploeg, M. and Overdulve, J.P. (1989) On the DNA content of Typanosoma cruzi. Mol. B&hem. Parasitol. 36, 73-76. [12] Gonzalez, A., Prediger, E., Huecas, M.E., Nogueira, N. and Lizardi, P. (1984) Minichromosomal repetitive DNA in Trypanosoma cruzi: its use in a high-sensitivity parasite detection assay. Proc. Natl. Acad. Sci. USA 81, 3356-3360. [13] Gruber, A. and Zingales, B. (1993) Trypanosoma cruzi: characterization of two recombinant antigens with potential application in the diagnosis of Chagas disease. Exp. Parasitol. 76, 1-12. [14] Araya, J.E., Cano, MI., Yoshida, N. and Franc0 da Silveira, J. (1994) Cloning and characterization of a gene for the stage-specific 82-kDa surface antigen of metacyclic trypomastigotes of Trypanosoma cruzi. Mol. Biochem. Parasitol. 65, 161-169.

[1.5] Takle, G.B. and Cross, G.A.M. (1991) An 8%kilodalton surface antigen gene family of Tvpanosoma cruzi encodes polypeptides homologous to bacterial neuraminidases. Mol. Biochem. Parasitol. 48, 185-198. [16] Vazquez, M.P., Schijman, A.G. and Lcvin, M.J. (1994) A short interspersed repetitive element provides a new 3 acceptor site for trans-splicing in certain ribosomal P2b protein genes of Trypanosoma cruzi. Mol. Biochem. Parasitol. 64, 327-336. [17] Levin, M.J., Ferrari, I., Vazquez, M., Franc0 da Silveira, J., Cano, MI., Degrave, W., Requena, J.M., Alonso, C., Zingales, B., Gonzalez, Hernandez, R., Ramirez, J.L., Aldao, R.R., Saumier, M., Billaut, A., LePaslier, D. and Cohen, D. (1994) Toward the physical map of the Ttypanosoma cruzi nuclear genome. Mem. Inst. Oswald0 Cruz Rio de Janeiro 89, 17-18 (Suppl.1).