The transcription promoter of the spliced leader gene from Trypanosoma cruzi

Gene 188 (1997) 157–168

The transcription promoter of the spliced leader gene from Trypanosoma cruzi Luiz R. Nunes, Maria Ruth C. Carvalho, Alison M. Shakarian1, Gregory A. Buck * Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Box 980678, MCV Station, Richmond, VA 23298-0678, USA Received 30 May 1996; revised 29 August 1996; accepted 2 September 1996; Received by C.M. Kane

Abstract A putative promoter element responsible for transcription of the spliced leader (SL) gene of Trypanosoma cruzi was identified by overlapping deletion and linker scanning analyses of the upstream flanking sequences using the bacterial chloramphenicol acetyltransferase (CAT ) gene as a reporter in transient transfections of cultured epimastigotes. Deletion or substitution of a proximal sequence element (PSE ) between positions −53 and −40 relative to the transcription start point eliminated CAT gene expression. Comparison of SL genes from several strains of T. cruzi revealed two alternative sequence patterns for the putative SL PSE, both composed of a short run of purines followed by a run of pyrimidines. Moreover, an examination of these sequences supports the subdivision of T. cruzi isolates into two divergent groups. Double-stranded oligonucleotides containing the sequence of the PSE exhibited specific gel mobility shifts after incubation with T. cruzi nuclear extracts, suggesting that a transcription factor binds this site. Finally, experiments designed to increase the level of CAT expression from the SL promoter suggest that it is not a strong promoter in cultured T. cruzi epimastigotes.[ 1997 Elsevier Science B.V. All rights reserved. Keywords: Proximal sequence element; PSE; Trypanosomatid; Gene expression

1. Introduction Recent estimates suggest that more than 16 million people throughout Latin America are infected with T. cruzi, causative agent of Chagas’ disease, and up to 60 000 new cases occur annually ( WHO, 1991; Warren, 1988). The spectrum of symptoms observed in chronic Chagas’ disease ranges from relatively benign and asymptomatic to the often fatal chronic Chagasic cardiomyopathy. Although it is becoming increasingly evident that T. cruzi is diverse and can be subdivided into several clearly distinct groups; i.e., zymodemes, schizodemes, etc. (Solari et al., 1992; Bogliolo et al., 1986; Carneiro et al., 1990; Morel et al., 1980; Ready and * Corresponding author. Tel +1 804 8282318; Fax +1 804 8289946; e-mail: [email protected] 1 Present address: Laboratory of Parasitic Diseases, NIH-NIAID, 9000 Rockville Pike, Bethesda, MD, 20892, USA. Abbreviations: bp, base pair(s); CAT, chloramphenicol acetyltransferase; pRNA, processor RNA; PSE, proximal sequence element; R, purine; SL, spliced leader; SL RNA, spliced leader RNA; U-RNA, small nuclear RNA; UTR, untranslated region; Y, pyrimidine.

Miles, 1980; Tibayrenc and Ayala, 1988), there is no proven correlation between the disease and such groups (Sanchez et al., 1990; Breniere et al., 1989; Apt et al., 1987; Carneiro et al., 1991). Since there is no effective treatment for chronic Chagas’ disease, it remains an important cause of morbidity and mortality in endemic regions of Latin America ( Tanowitz et al., 1992; Kirchhoff, 1993), and identification of processes in which the parasite and its mammalian hosts differ is essential for the development of new chemotherapeutic strategies. In the last decade, it has been well documented that the trypanosomatids and their mammalian hosts differ at the level of gene structure and transcription. For example, parasite protein-coding genes are frequently or always transcribed as polycistronic RNAs that are processed to maturity by trans-splicing (Nilsen, 1992; Agabian et al., 1987), are not always transcribed by RNA Polymerase II (Pol II ) (Zomerdijk et al., 1991), and seem to lack TATA boxes and other known eukaryotic promoter elements (Ben Amar et al., 1991; Nakaar et al., 1994; Brown et al., 1992; Ajioka and Swindle, 1993).Transcription promoters have been difficult to identify in trypanosomes because of the polycistronic

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nature of the transcripts of protein-coding genes. However, in all trypanosomatids, the spliced leader (SL) genes, which encode the small abundant SL RNAs that donate their capped 5∞ 39 nt SL to mature mRNAs in the trans-splicing reaction, are transcribed as short monocistronic RNAs from several hundred tandem direct repeats, and each repeat is presumed to bear a transcription promoter (Agami and Shapira, 1992; Saito et al., 1994; Bellofatto et al., 1988; Roditi, 1992; Agabian et al., 1987; McCarthy Burke et al., 1989). Thus, we have used the SL gene promoter to drive expression of a reporter gene after transfection into cultured T. cruzi epimastigotes (Lu and Buck, 1991). Similar systems have recently been used to define SL gene promoter elements in Leptomonas (Hartree and Bellofatto, 1995) and Leishmania (Saito et al., 1994; Agami et al., 1994). These promoters will be essential for the genetic dissection of these parasites, and because of the evident differences from known transcription systems, represent potential future therapeutic targets.In this communication, we use a transient expression system to dissect the T. cruzi SL gene promoter. We identify a proximal sequence element (PSE) that is located between positions −53 and −40 and seems to be required for optimal SL gene promoter activity. The sequence of this PSE is partially but not completely conserved among different T. cruzi strains, and two different subgroups of PSEs can be observed. The putative PSE is specifically recognized and bound by a putative transcription factor present in T. cruzi nuclear extracts. Finally, experiments designed to enhance levels of reporter gene activity from the SL promoter suggest that this promoter is not strongly expressed in T. cruzi epimastigotes.

2.2. Plasmid constructions and electroporation The construction of pTcSL-CAT, bearing the CAT reporter gene driven by the SL gene promoter region, has been previously described (Lu and Buck, 1991). Plasmid pG2SL-CAT was created by ligating the BamHI released SL-CAT fragment of pTcSL-CAT into BamHI linearized pGEM 2 (Promega). Plasmid pG2SL-CAT3∞ was constructed by ligating a 137-bp BamHI fragment containing the 3∞ end of the 35.2 gene (GenBank U57983) into the BamHI site downstream from the CAT gene in pG2SL-CAT. To create plasmid pG2SLCAT3∞5∞, a 360-bp EcoRI/PvuII fragment spanning from the 3∞ end of 35.2 down to the first 130 bp of its immediately downstream gene was isolated, blunt-ended with Klenow fragment polymerase as described (Maniatis et al., 1982) and ligated into the similarly blunt-ended BamHI site at the 3∞ end of CAT in pG2SLCAT. Plasmid pG2SL-CATA was constructed with the aid of mutagenic primer pA ( Table 2), which introduces a G to A transition at position G40 of the SL RNA, the essential first nucleotide of the splice donor site. This primer also spans the HindIII site between positions +47 and +52, at the 3∞ end of the SL repeat cloned in pG2SL-CAT. Using this primer with primer pGem, a full length copy of the SL repeat bearing the G40 to A40 transition at the splice donor site was amplified from pG2SL-CAT by PCR. This mutated version of the SL repeat was digested with HindIII and ligated into HindIII cleaved pG2SL-CAT. All constructs were verified by sequence analysis. Electroporations were performed with 50 mg of plasmid DNA as previously described (Lu and Buck, 1991). 2.3. Serial deletions and linker scanning mutants of the SL gene promoter

2. Materials and methods 2.1. Organisms and culture conditions Epimastigotes of T. cruzi strains ( Table 1) CL ( Filardi and Brener, 1975), CL Brener (Cano et al., 1995), Basileu ( Filardi and Brener, 1975), Y, DM28c ( Filardi and Brener, 1975), Colombiana (Filardi and Brener, 1975), X10 clone 4 ( Tibayrenc and Miles, 1983), Can III, Esmeraldo (Miles et al., 1981), G2, G3, Peru (Nussenzweig and Goble, 1995), 150 zd (Carneiro et al., 1990), 222 zc (Carneiro et al., 1990), 229 za (Carneiro et al., 1990) SO3 cl5 and NRcl39 (Solari et al., 1992) were grown essentially as previously described (Zwierzynski et al., 1989) in LIT medium supplemented to 10% with fetal calf serum at 28°C under gentle agitation. The cells were passaged to fresh media every 7 days.

Constructs D1, D2, and D3, bearing deletions in the upstream region in pG2SL-CATA were generated by PCR amplification using primer pA in conjunction with primers pSL5∞1, pSL5∞2 and pSL5∞3 ( Table 2) to amplify sequentially reduced portions of the SL repeat from pG2SL-CATA. These primers were designed with internal HindIII sites to permit cleavage and ligation into HindIII cleaved pG2SL-CAT. The D4 construct was cloned as an artifact of amplification in a PCR reaction involving primers pA and pGem. To generate the linker scanning mutations, block substitutions of 8 nt were introduced into the sequence spanning from positions −71 to +52 of construct D3. Mutants LS1 to LS16 were constructed by the oligonucleotide directed mutagenesis method of Morrison and Desrosiers (1993). Generation of each mutation required a pair of complementary mutagenic primers LSN and LSN∞ in conjunction with an upstream primer pGem, and downstream primer pCAT ( Table 2). The amplified

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L.R. Nunes et al. / Gene 188 (1997) 157–168 Table 1 T. cruzi strains and isolates Isolate

Location

Host

Zymodeme groupinga

SL gene group

CL brener CL NR cl39 S03 cl5 150zd Basileu Y 222zc 229za CanIII Esmeraldo X10 cl4 G2 G3 Dm28c Colombiana Peru Tulahue´n

Brazil (Rio Grande do Sul ) Brazil (Rio Grande do Sul ) Chile (Atacama) Bolivia Brazil (Minas Gerais) Brazil (Minas Gerais) Sa˜o Paulo Brazil (Minas Gerais) Brazil (Minas Gerais) Brazil (Bele´m) Brazil (Bahia) Brazil (Bele´m) Brazil (Santa Catarina) Brazil (Santa Catarina) Venezuela Colombia Peru Chile

triatome triatome man triatome man (chronic) man (acute) man (acute) man (chronic) man (chronic) man (acute) man (acute) man marsupial marsupial marsupial man (chronic) man triatome

Z1/ZB Z1/ZB Z2/cl39b Z2/cl39b ZD NDc Z2/ZA ZC Z2/ZA Z3 Z2 Z1 Z1 Z1 ZB Z1/ZB Z2 Z1

I I I I I I I I I I I IIa IIa IIa IIa/b IIb IIb IIb

a Zymodeme classification is shown as described by Miles and co-workers ( Z1, Z2 or Z3) (Ready and Miles, 1980; Miles et al., 1978) and/or according to Carneiro et al. (ZA, ZB, ZC or ZD) (Carneiro et al., 1990). b Classification according to Solari et al. (1992). c ND, not determined.

mutated fragments were digested with HindIII and ligated into HindIII linearized pG2SL-CAT. For mutants LS15 and LS16, which bear mutations in the HindIII site of the SL RNA sequence, we used primer pBsp in place of pCAT as the downstream primer, permitting use of a unique BspEI site in pG2SL-CAT to clone this fragment. All constructs were verified by sequence analysis. 2.4. Gel mobility shift assays The target double-stranded nucleotide (ds55) encompassing sequences from −60 to −31 of the SL gene was prepared by annealing oligos p55 and p55∞ ( Table 2). Equimolar quantities of each oligo were mixed in the presence of 10 mM Tris, pH 7.9, 10 mM MgCl and 2 1 mM DTT. The reaction was incubated at 65°C for 15 min, 37°C for 15 min, and 24°C for 15 min. The doublestranded oligonucleotides were electrophoresed in a 12% polyacrylamide gel, isolated by electroelution as described (Maniatis et al., 1982), and labeled with T4 polynucleotide kinase and [c-32P]ATP (Maniatis et al., 1982).32 mg of total protein from nuclear extracts prepared as previously described ( Zwierzynski et al., 1989) were incubated in a final volume of 20 ml with 2 ng of labeled ds55 for 30 min at room temperature in the presence of 2 mM Tris pH 7.9, 50 mM KCl, 10% glycerol, 1 mM EDTA, 1 mM DTT, 5 mg poly dI:dC and 2 mg oligo p5∞dIII, a random sequence oligonucleotide that helped compete out single-stranded DNA binding proteins present in the nuclear extract. Unlabeled oligonucleotide competitor, when present, was added at

the beginning of the incubation period. We used as competitors, oligonucleotides ds55, dsRandom (annealed pRandom and pRandom∞) ds3 (annealed p3 and p3∞) and ds4 (annealed p4 and p4∞). After incubation, the reactions were electrophoresed in an 8% polyacrylamide gel (29:1, acrylamide/bis-acrylamide) for 30 min at 8 V/cm at room temperature in 1×TBE. The voltage was then increased to 120 V/cm and the electrophoresis was allowed to proceed for two additional h. The gels were wrapped with plastic wrap and exposed to X-ray film for 16–18 h at −70°C with intensifying screens. 2.5. DNA extraction and PCR amplification of the SL repeats Total DNA from the several T. cruzi strains was extracted as described (McCarthy Burke et al., 1989), and an entire copy of the SL repeat from each strain was amplified by PCR using primers complementary to the highly conserved region encompassing the SL. Primer SL1 is complementary to the +1 to +20 sequence of the SL, and primer SL2 has the sequence of the +25 to +44 position of the SL RNA ( Table 2). PCR was performed with either AmpliTaq DNA Polymerase (Perkin-Elmer-La Roche) or SequiTherm ( Epicentre), by standard protocols using 100 ng of total DNA from T. cruzi. The cycling profile included 30 cycles of amplification (1 min/94°C, 1 min/55°C and 1 min/72°C ). The amplified fragments were electrophoresed in a 2% NuSieve agarose gel as described (Maniatis et al., 1982), electroeluted, concentrated with a

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L.R. Nunes et al. / Gene 188 (1997) 157–168 Table 2 Ologonucleotides used in this worka

aOligonucleotides listed 5∞ to 3∞.

Centricon 30 microconcentrator (Amicon), and submitted to the MCV-VCU DNA Core Laboratory for automated sequencing. The sequence for the Tulahue´n strain has been previously described (De Lange et al., 1984).

3. Results and discussion 3.1. Relative activity of the SL gene promoter We have previously shown that a construct bearing a fragment of the T. cruzi SL gene (pTcSL-CAT, Fig. 1)

will drive expression of a CAT reporter when electroporated into cultured epimastigotes, whereas neither the vector alone nor the fragment inserted in the reverse orientation do, suggesting that the fragment bears an orientation-dependent transcription promoter (Lu and Buck, 1991). We have also performed a preliminary characterization of the T. cruzi rRNA gene promoter, and have shown that in these transient transfection systems, there is no detectable CAT expression in the absence of bona fide T. cruzi DNA sequences, even in the presence of a functional 3∞ splice acceptor ( TylerCross et al., 1995; Lu and Buck, 1991). Herein, we

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Fig. 1. Activity of the T. cruzi SL gene promoter. Reporter vectors were constructed and tested for CAT activity after transfection into cultured T. cruzi epimastigotes. Constructions are as follows: pTcSL-CAT expresses CAT from promoter sequences present in a HindIII fragment bearing an entire repeat of the SL gene from the CL strain (Lu and Buck, 1991); pG2SL-CAT contains the SL repeat and the CAT gene, but not the SV40 sequences, from pTcSL-CAT, religated into pGEM2Z; pG2SL-CAT3∞ contains the 3∞-UTR and poly(A) addition site of 35.2, a T. cruzi gene of unknown function; pG2SL-CAT3∞5∞ is similar to pG2SL-CAT3∞, but contains an extra 220 bp corresponding to intergenic sequences (IGS) and 5∞UTR and splice site (5∞ss) of the 35.2 gene; pG2SL-CATA contains a G-A transition mutation at the splice donor site of the SL RNA. The transcription start point (arrow) and the 39 nt SL are indicated. The shaded boxes next to the transcription start point represent the SL 39-mer, solid boxes represent the SL RNA coding region and the remaining boxes are as indicated in the figure. The diagrams are not to scale. The average values and standard deviations presented were derived from 4 independent experiments, each performed in duplicate. Values were background subtracted and normalized to the numbers obtained with the original construct pTcSL-CAT.

exploited this transient transfection system to identify putative promoter elements of this SL gene fragment that are responsible for the transcription of the T. cruzi SL gene. Electroporation of pTcSL-CAT into T. cruzi epimastigotes yields easily detectable CAT activity, but expression is low compared to that from pTcpa-CAT, the analogous construct containing the rRNA promoter of T. cruzi (Tyler-Cross et al., 1995). Expression levels from pTcSL-CAT may be low because: (i) SV40 polyadenylation sequences immediately downstream from CAT in pTcSL-CAT interfere with CAT expression; (ii) absence of a specific T. cruzi polyadenylation signal sequence leads to improper 3∞ processing; (iii) presence of a functional 5∞ splice donor site in the CAT mRNA directs the transcript into a ‘dead-end’ product in which the 39-mer SL from the transcript is removed from the primary transcript by splicing mechanisms; or (iv) the SL promoter is a weak promoter in T. cruzi epimastigotes. To examine these possibilities, we constructed a series of derivatives from pTcSL-CAT and tested them by electroporation into T. cruzi epimastigotes (Fig. 1).First, we eliminated the SV40 sequences of pTcSL-CAT by transferring the SL-CAT fragment into the BamHI site of pGEM2, creating a new derivative, pG2SL-CAT. Next, to determine if specific T. cruzi polyadenylation signals would generate higher CAT activity, we constructed two derivatives of pG2SL-CAT containing known 3∞ processing signals from T. cruzi genes. The first, pG2SL-CAT3∞, contained the 3∞-untranslated region (UTR) of 35.2, a tandemly

repeated gene from T. cruzi of unknown function that has been previously described (McCarthy Burke et al., 1989). Since it has been suggested that 3∞ processing of mRNAs in trypanosomatids is linked to trans-splicing of the proximal downstream gene (LeBowitz et al., 1993), we also designed the construct pG2SL-CAT3∞5∞, which contains an additional 220 bp corresponding to the intergenic region and the 5∞-UTR of the downstream 35.2 gene copy. Finally, to prevent the SL-CAT mRNA from participating as a potential SL donor in the transsplicing pathway, we introduced a G to A transition in the crucial ‘GT’ dinucleotide at the 5∞ splice site in pG2SL-CAT, generating the construct pG2SLCATA.All derivatives promoted CAT activity when electroporated into T. cruzi epimastigotes, although consistent variations were observed ( Fig. 1). Thus, deletion of the SV40 polyadenylation site in pG2SL-CAT led to a drop in CAT activity. Although addition of a specific 3∞ domain in pG2SL-CAT3∞ did not restore CAT activity to the same levels observed with pTcSL-CAT, addition of both a 3∞ poly(A) site and a 5∞ splice site downstream from the CAT reporter in pG2SL-CAT3∞5∞ did. This observation is consistent with the proposal (LeBowitz et al., 1993) that polyadenylation and transsplicing are linked and interdependent in trypanosomes. The G to A transition in the splice donor site in pG2SLCATA also yielded an incremental enhancement in CAT activity, consistent with the idea that some of the SL-CAT mRNA from pTcSL-CAT might be diverted into a ‘dead-end’ splicing product.As described below, the CAT activity obtained from these constructs cannot

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be quantified with high confidence since we were unable to establish an internal control for these transient transfections. However, multiple repetitions of each transfection yielded equivalent results, and none of the constructs yielded activity greater than that of the original pTcSL-CAT (Fig. 1). Together, these results argue that the SL promoter is not a strong promoter in T. cruzi epimastigotes, although other possibilities cannot be completely ruled out. It would be surprising to find that a transcript as abundant and critical as the SL RNA depends upon a weak promoter for its expression. However, the SL gene repeats are present in multiple copies in the genomes of trypanosomatids, and low transcriptional activity may not be a limiting factor. It is also possible that regulatory mechanisms play a role in controlling the levels of activity of the SL gene promoter. 3.2. Identification of the minimal promoter region of the SL genes Since the above results suggested that the SV40 sequences present in pTcSL-CAT might influence the level of CAT expression, we characterized the SL gene promoter in the pG2SL-CATA construct, which lacks all SV40 related sequences but shows levels of CAT activity similar to those observed with pTcSL-CAT. Serial overlapping deletions of the SL promoter region were generated in this construct using PCR, and the deleted plasmids were electroporated into T. cruzi epimastigotes and assayed for CAT activity ( Fig. 2). Constructs D1, D2 and D3 exhibited full promoter activity, but construct D4, which was deleted upstream of −47 relative to the known SL gene transcription start point, was incapable of driving CAT expression. Since D3 lacked sequences upstream of −71, these observations suggested the presence of at least one important promoter element between positions −71 and −47. To confirm this possibility and further map the

position of promoter elements present within this region we performed a linker scanning or block substitution analysis of the SL sequences present in vector D3. 3.3. Block substitution of the minimal promoter region of the SL promoter A panel of 16 block substitutions spanning from −71 to +52 of the SL repeat was generated by a PCR based site-directed mutagenesis approach essentially as described in Section 2. Each block substitution replaced an 8-bp wild-type sequence with an 8-bp NotI site. T. cruzi epimastigotes were transfected with each of the mutant constructs by electroporation and CAT activity was examined ( Fig. 3). It was not possible to measure CAT RNA directly in these transient transfections (not shown) probably due to the low levels of expression from the SL gene promoter. Moreover, we were unable to establish an internal control with alternative reporter genes (see below). Therefore, each transfection was repeated at least four times in duplicate to ensure reproducibility. Although consistent partial effects were observed with several mutants, we focus herein on those mutants that reproducibly abolished CAT expression in these transient assays.LS3 and LS4 were the only block substitutions upstream from the transcription start point that essentially abolished CAT activity. The altered bases in these two constructs span positions −53 to −40 of the SL repeat, suggesting the presence of a critical T. cruzi SL gene basal promoter element at this site. Since randomly initiated transcription of plasmidborne reporter genes occurs in other trypanosomatid systems, it was possible that the abolition of CAT activity we observed was due to trans-splicing of nonspecifically initiated transcripts, and that the LS3 and LS4 mutations abolish expression by compromising fortuitous trans-splicing at proximal AG dinucleotides. Although substitution mutant LS3 eliminates an AG dinucleotide at position −48, we believe that these

Fig. 2. Activity of the 5∞ deletions of the SL gene promoter. Mutations D1, D2, and D3 are deleted for sequences upstream from positions −234, −152 and −71, respectively. Mutation D4 is deleted for the sequences between positions −313 and −47. The relative average levels of CAT activity were obtained from 4 different experiments performed in duplicate, background subtracted and normalized to the activity obtained with pG2SL-CAT standard. Symbols are as for Fig. 1.

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Fig. 3. Linker scanning of the minimal promoter region. Only the altered bases in each construct are indicated. CAT activity values obtained from 4 independent experiments are shown. Each experiment was performed in duplicate, the numbers were background subtracted and normalized to the values obtained with D3 of pG2SL-CATA. (±) indicates low CAT activity that was only detectable by extended exposure of TLC plates. The transcription start point (+1) is indicated.

mutations are affecting transcription initiation for two main reasons. First, we have previously shown that similar plasmid constructs containing the CAT gene, but not a specific T. cruzi promoter, fail to permit CAT expression in our transient assays, even when a bona fide T. cruzi 3∞ splice acceptor is present upstream from a reporter gene ( Tyler-Cross et al., 1995). We have also observed this lack of expression in the presence of a 3∞ splice acceptor in a variety of constructs, using selectable (neo) and non-selectable (cat) reporter genes in both stable and transient transfections (unpublished data). Thus, expression via random non-specific initiation is apparently not occurring at detectable levels in our transient transfection system. Second, mutant LS4, which completely abolishes CAT activity, retains all of the potential 3∞ AG dinucleotide splice acceptors including one at −48 and another at −58 in the SL gene promoter fragment. The LS4 substitutions are downstream (−47 to −40) from and unlikely to affect activity of either of these potential 3∞ splice acceptors. Together, these observations argue that the LS3 and LS4 mutations abolish transcription initiation, and we conclude that the sequence between −53 and −40 of the T. cruzi SL gene bears a putative basal promoter element. Identification of putative promoter elements for the T. cruzi SL gene was based herein on the presence or absence of CAT activity in repeated experiments. However, it deserves comment that other mutations, notably LS1, LS5, LS7 and LS9, showed reproducible incremental decrease or enhancement of CAT activity, possibly indicating the presence of other transcription response elements in corresponding regions. However, due to the high variability in the levels of CAT activity obtained with some of the LS constructs, verification of these response elements will only be possible with the development of an alternative reporter gene and an internal standard against which CAT expression can be

normalized. As mentioned above, attempts to establish such internal control using the T. cruzi rRNA gene promoter and the bacterial luciferase (LUC ) reporter gene have provided unreliable results (L.R.N. and G.A.B., unpublished), possibly due to promoter interference, and the establishment of such a control awaits the availability of other T. cruzi promoters.Levels of CAT activity were also severely reduced in mutants LS10–15 which span from +1 to +48 of the SL gene (Fig. 3), and the presence of additional promoter elements within this sequence cannot be ruled out. However, thin layer chromatography showed low but detectable CAT activity in cells transfected with these constructs (data not shown), suggesting that the SL promoter remains active and that the reduction of CAT activity in these mutants may be due to other factors or functions of the SL RNA. Although the function(s) of these portions of the SL RNA have yet to be demonstrated, they may include RNA processing, transport, localization, maturation and/or stability as recently suggested for the SL sequences in Leishmania (Perry and Agabian, 1991; Saito et al., 1994), and thus could have an effect on expression of the reporter gene, cat.The putative SL gene promoter element between −53 and −40 of the T. cruzi SL gene is similar in location and size to elements recently described for the SL genes of the distantly related trypanosomatids, Leishmania and Leptomonas (Saito et al., 1994; Agami et al., 1994; Hartree and Bellofatto, 1995). The SL gene promoter elements in these three trypanosomatids are all striking in their resemblance to the proximal sequence elements (PSEs) responsible for promoting transcription of the processor RNA (pRNA) genes of higher eukaryotes. pRNAs include U RNAs and other small RNAs associated with RNA processing and transport in higher eukaryotes (Hernandez, 1992). Known pRNA gene promoters are structurally similar, bearing 11–30-bp

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PSEs approximately 40–80 bp upstream from their transcription start points, and these PSEs are part of the basal promoter required for transcription of these genes. The PSEs are the primary promoter elements required for basal expression of pRNA genes transcribed by RNA Pol II, while some pRNAs transcribed by Pol III also bear an essential TATA-box between the PSE and the transcription start point (for review, see Hernandez, 1992). Interestingly, the SL RNA, as a small RNA involved in maturation of trypanosomatid mRNA, clearly fits the definition of a pRNA. Thus, it may not be a coincidence that the SL gene promoters structurally resemble known promoters of the pRNA genes.There is controversy over which RNA polymerase is responsible for transcription of the SL genes in trypanosomatids. Grondal et al. (1989) have proposed that the SL genes of T. brucei are transcribed by Pol III based on three lines of evidence: (i) relative sensitivity to 1,10-phenanthroline and Mn+2 concentration; (ii) presence of a poly( T ) putative Pol III terminator; and (iii) presence within the SL RNA coding region of sequences resembling the A and B boxes of eukaryotic Pol III promoters. In contrast, studies with specific inhibitors have suggested that the SL genes of T. brucei (Laird et al., 1985), Leishmania tarentolae (Saito et al., 1994) and T. cruzi (G.A.B, unpublished data) are transcribed by Pol II. The presence of a single PSE located in the upstream region of the SL genes and absence of a corresponding TATA-box is also consistent with the Pol II dependent pRNA promoters in higher eukaryotes (Hernandez, 1992). 3.4. A putative transcription factor recognizes the T. cruzi SL gene basal promoter element Gel shift analyses were performed with T. cruzi nuclear extracts to identify factors responsible for activation of the SL gene promoter. A 30-bp double-stranded oligonucleotide (ds55) containing the sequence between positions −60 and −31 of the SL gene was 5∞ 32P-labeled, incubated with nuclear extract, and electrophoresed in a nondenaturing polyacrylamide gel (Fig. 4). One major shifted band appeared when using the oligonucleotide in the absence of a specific competitor (Fig. 4, lane NC ). This band was strongly depleted in the presence of excess unlabeled specific competitor (ds55), but largely unaffected by the presence of an excess of unlabeled oligonucleotide with random sequence (dsRandom). Thus, a factor present in T. cruzi nuclear extracts is apparently capable of specifically binding to the putative PSE identified by linker scanning analysis, and this factor is a candidate for an SL gene transcription factor.To verify the specificity of the putative transcription factor for the PSE identified by transient transfection, oligonucleotides ds3 and ds4 bearing the sequences of linker scanning mutants LS3 and LS4,

which abolished CAT expression in transient transfections, were also tested as competitors in gel shift experiments (Fig. 4). Neither of these oligonucleotides completely eliminated the shifted band, indicating that the mutated versions of the PSE that are not transcriptionally active are also inefficiently recognized by this putative transcription factor. However, at high concentrations, ds3 partially competed the gel shift, suggesting that an important component of the recognition sequence for this factor remains in the sequences adjacent to the altered sequence in linker scanning mutant LS3. Together, these results are consistent with the in vivo results from transient transfection of the overlapping deletion and linker scanning mutants, and strongly support the conclusion that the sequence between −53 and −40 is a promoter element that is specifically recognized and bound by a T. cruzi transcription factor.Our attempts to generate a DNAse I ‘footprint’ with this factor showed strong ‘protection’ of the −53 to −40 putative basal element (not shown). However, this effect was also observed in the absence of nuclear extract, and thus may be due to structure of the sequence of the PSE, a characteristic previously associated with some promoter sequences (Parvin et al., 1995; Kim et al., 1995; Kneidl et al., 1995) (see below). Our inability to verify a specific footprint on this putative promoter element is consistent with other recent reports describing difficulties in obtaining footprints on putative promoter elements in other trypanosomatids (Janz et al., 1994; Brown and Van der Ploeg, 1994).The structure and organization of transcription factors in trypanosomatids remain unexplored, although Brown et al. (1992) recently provided evidence suggesting that a singlestranded DNA binding protein might act as a transcription factor for the PARP promoter of T. brucei. Our attempts to clone the TATA box binding protein of T. cruzi by PCR amplification with degenerate oligonucleotides, as previously described for other organisms (Meade and Stringer, 1991; Peterson and Tjian, 1993), were not successful (L.R.N., unpublished), suggesting that even this highly conserved transcription factor is quite different or even absent in trypanosomes. Thus, we are currently attempting to clone and characterize the gene encoding the SL PSE binding factor to further understand the mechanism of transcription initiation and regulation in T. cruzi. 3.5. The spliced leader gene promoter region identifies at least two groups of T. cruzi A fragment containing approximately 80 bp of the region upstream from the transcription start point of the SL gene repeats was PCR amplified and sequenced from several strains of T. cruzi isolated from various hosts and localities and representing various zymodeme groups (Table 1). Surprisingly, these sequences

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Fig. 4. Gel mobility shift assay. T. cruzi nuclear extracts were incubated with 2 ng of 32P-end-labeled specific oligonucleotide ds55 and the specified excess of unlabeled competitor (ds55, dsRandom, ds3 or ds4), electrophoresed in an 8% nondenaturing polyacrylamide gel and exposed to X-ray film overnight at −70°C. Lane P (probe alone), labeled specific ds55 oligo, without nuclear extract or competitors; lane NC (no competitor), labeled specific ds55 oligo incubated in the presence of nuclear extract without unlabeled competitor; lanes ds55 contain the indicated excesses of unlabeled ds55 oligo as a competitor; lanes dsRandom contain the indicated excesses of unlabeled dsRandom oligonucleotide as a competitor; lanes ds3 contain the indicated excesses of unlabeled ds3 oligo as a competitor, and lanes ds4 contain the indicated excesses of unlabeled ds4 oligo as a competitor The arrow indicates the shift related to a binding activity specific to the sequence of the SL PSE.

demonstrated two clearly distinct sequence patterns (Fig. 5). All sequences in Group I are identical across this ~80-bp region, but the sequences of Group II exhibit limited variability that may partition into two

subgroups, IIa and IIb. Interestingly, strain DM28c exhibits a sequence that appears to be a hybrid between subgroups IIa and IIb, consistent with there being genetic exchange in these organisms. Groups I and II

Fig. 5. SL gene promoter sequences from T. cruzi strains. (A) SL gene sequences upstream from several T. cruzi strains were PCR amplified using primer pairs SL1 and SL2 and the PCR fragments were directly sequenced. Sequences between ~−80 and +10 relative to the transcription start point are shown. The boxed area indicates the putative PSE. Assignment to Group I, IIa or IIb based on the sequence of the promoter region and the PSEs is indicated. (B) A polypurine-polypyrimidine sequence present in the upstream region of two U2 RNA genes from T. cruzi ( Y strain) is shown in comparison with the putative SL PSE of the Y and X10 clone 4 strains. (–) indicates identity with the CL sequence; (:) indicates gaps. The sequences for the CL and Tulahue´n strains of T. cruzi were previously described (De Lange et al., 1984; McCarthy Burke et al., 1989). The GenBank accession numbers for these sequences are as follows: CL, U57984; CL Brener, U39748; NR c1one 39, U57985; SO3 clone 5, U57986; 150zd, U39749; Basileu, U39750; Y, U39760; 222zc, U39732; 229za, U39752; CanIII, U39753; Esmeraldo, U39754; X10 clone 4, U39755; G2, U39756; G3, U39757; Dm28c, U57987; Colombiana, U39758; Peru, U39759; Tulahue´n, K02632.

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exhibit ~30% dissimilarity over the 80 bases upstream from the transcription start point of the gene ( Fig. 5), in contrast to the high level of conservation of the transcribed regions (McCarthy Burke et al., 1989; A.M.S., M.R.C.C. and G.A.B., data not shown). Although a preponderance of the Group II strains were isolated from marsupials, there is little obvious correlation between these two groups and previously reported zymodeme or schizodeme groups, geographical area of isolation, or clinical manifestation of the disease ( Table 1). However, these two groups may correspond with two groups proposed by Souto and Zingales (1993), based on PCR amplification of a T. cruzi specific rRNA sequence.The sequences of the putative PSE from the two T. cruzi subgroups are only ~70% conserved. This divergence is strikingly high for PSEs from different isolates of the same species, since previously characterized PSEs, even from different pRNA genes in the same species, show higher conservation rates (Hernandez, 1992). Moreover, preliminary results show that our construct D3 drives expression best in those strains with similar PSEs (L.R.N., M.R.C.C., and G.A.B., data not shown), suggesting that the basal promoter sequences of the T. cruzi SL gene are strain or group-specific. The existence of two functionally distinct PSE subtypes among T. cruzi strains would indicate previously unsuspected high levels of genetic divergence among T. cruzi strains.In spite of the high degeneracy displayed by the sequence of these elements, the putative SL gene PSEs of both groups of T. cruzi display a R Y sequence ( Fig. 5A). Interestingly, a 6–8 5–6 similar sequence is also found upstream from the transcription start point of the T. cruzi U2 snRNA genes (Fig. 5B). Similar R Y regions are known to form n n unusual DNA structures such as turns and bends (Crothers et al., 1990), and have been associated with promoter recognition in other systems (Parvin et al., 1995; Kim et al., 1995; Kneidl et al., 1995; Ohyama and Hirota, 1993). Thus, it is tempting to speculate that the presence of the R Y sequences may indicate the presn n ence of important promoter elements for each of these T. cruzi genes. 3.6. The SL promoter of T. cruzi in relation to other trypanosomatids The T. cruzi SL gene promoter shows similarities and differences in comparison to the SL gene promoters of Leptomonas and Leishmania. Agami et al. (1994) recently reported that the SL gene promoter in L. amazonensis is composed of two distinct elements: a PSE-like element between positions −70 and −30; and an initiator-like element located between −10 and −1. Saito et al. (1994) demonstrated that substitution of the sequence between −67 and −58 of the SL gene of L. tarentolae abolished transcription of this gene and pro-

posed a core element at this position, in view of its high conservation across Leishmania species. In spite of the high conservation of this putative Leishmania SL gene promoter element, it does not display significant homology to the putative T. cruzi PSE, although there is also a pyrimidine rich stretch at the 3∞ end of the L. amazonensis PSE. This observation is not surprising, since sequences of PSEs of homologous genes show little conservation across genera (Hernandez, 1992). The putative L. seymouri SL gene promoter is also composed of a PSE-like element between −20 and −70 and an initiator-like element between −10 and −1. The proposed L. seymouri PSE is bipartite, composed of two blocks between positions −20 and −40, and −50 to −70, both reported to be bound by a single transcription factor (Hartree and Bellofatto, 1995). Again, no significant sequence homology was observed between this element and the proposed T. cruzi PSE, although the upstream block of the L. seymouri PSE is composed mostly of purine residues, while the downstream block is pyrimidine rich. Thus, it is clear that the SL gene promoter of T. cruzi, L. seymouri and Leishmania each bear a PSE located −20 to −70 bp upstream from the transcription start point, and although sequence homology among them is low, their sequences display structural similarity. However, in contrast to Leptomonas and Leishmania, we found no evidence for an initiatorlike element near the transcription start point in the SL gene promoter of T. cruzi ( Fig. 3).

4. Conclusions (1) Expression of the SL genes in T. cruzi is dependent upon a putative PSE located between positions −53 and −40 in relation to the transcription start site. (2) The T. cruzi SL PSE is recognized by a putative transcription factor present in nuclear extracts from this parasite. This observation is the first evidence for a transcription factor in this organism. (3) Comparison of the ~80 bp upstream from the transcription start point of the SL gene, including the putative PSE identified herein, categorizes T. cruzi strains into two clear groups and suggests an unexpected dichotomy in this trypanosomatid species. (4) In contrast to the relative abundance of the SL RNA, the activity of the SL gene promoter in our constructs is unexpectedly low. The presence of multiple copies of the SL gene in the parasite genome, or as of yet unconfirmed regulatory mechanisms suggested by our observations, may account for this apparent discrepancy.

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Acknowledgement This work was supported by funds from the National Institutes of Health and the American Heart Association. L.R.N. was supported by a fellowship from the Brazilian agency CNPq. All oligonucleotide synthesis and sequencing was performed in the MCV-VCU DNA Core Laboratory.

References Agabian, N., Perry, K.L. and Murphy, W.M. (1987) RNA joining and trypanosome gene expression. In: Inouye, M. and Dudock, B.S. ( Eds.), Molecular Biology of RNA: New Perspectives. Academic Press, New York, NY, pp. 151–171. Agami, R. and Shapira, M. (1992) Nucleotide sequence of the spliced leader RNA gene from Leishmania mexicana amazonensis. Nucleic Acids Res. 20, 1804. Agami, R., Aly, R., Halman, S. and Shapira, M. (1994) Functional analysis of cis-acting DNA elements required for expression of the SL RNA gene in the parasitic protozoan Leishmania amazonensis. Nucleic Acids Res. 22, 1959–1965. Ajioka, J. and Swindle, J. (1993) The calmodulin-ubiquitin associated genes of Trypanosoma cruzi: their identification and transcription. Mol. Biochem. Parasitol. 57, 127–136. Apt, W., Aguilera, X., Arribada, A., Gomez, L., Miles, M.A. and Widmer, G. (1987) Epidemiology of Chagas’ disease in northern Chile: isozyme profiles of Trypanosoma cruzi from domestic and sylvatic transmission cycles and their association with cardiopathy. Am. J. Trop. Med. Hyg. 37, 302–307. Bellofatto, V., Cooper, R. and Cross, G.A. (1988) Discontinuous transcription in Leptomonas seymouri: presence of intact and interrupted mini-exon gene families. Nucleic Acids Res. 16, 7437–7456. Ben Amar, M.F., Jefferies, D., Pays, A., Bakalara, N., Kendall, G. and Pays, E. (1991) The actin gene promoter of Trypanosoma brucei. Nucleic Acids Res. 19, 5857–5862. Bogliolo, A.R., Chiari, E., Silva-Pereira, R.O. and Silva-Pereira, A.A. (1986) A comparative study of Trypanosoma cruzi enzyme polymorphisms in South America. Braz. J. Med. Biol. Res. 19, 673–683. Breniere, S.F., Carrasco, R., Revollo, S., Aparicio, G., Desjeux, P. and Tibayrenc, M. (1989) Chagas’ disease in Bolivia: clinical and epidemiological features and zymodeme variability of Trypanosoma cruzi strains isolated from patients. Am. J. Trop. Med. Hyg. 41, 521–529. Brown, S.D., Huang, J. and Van der Ploeg, L.H.T. (1992) The promoter for the procyclic acidic repetitive protein (PARP) genes of Trypanosoma brucei shares features with RNA polymerase I promoters. Mol. Cell. Biol. 12, 2644–2652. Brown, S.D. and Van der Ploeg, L.H.T. (1994) Single-stranded DNAprotein binding in the procyclic acidic repetitive protein (PARP) promoter of Trypanosoma brucei. Mol. Biochem. Parasitol. 65, 109–122. Cano, M., Gruber, A., Vazquez, M., Corte´s, A., Levin, M.J., Gonza´lez, A., Degrave, W., Rondinelli, E., Zingales, B., Ramirez, J.L., Alonso, C., Requena, J.M. and Da Silveira, J.F. (1995) Molecular karyotype of clone CL Brener chosen for the Trypanosoma cruzi Genome Project. Mol. Biochem. Parasitol. 71, 273–278. Carneiro, M., Chiari, E., Goncalves, A.M., Silva Pereira, A.A., Morel, C.M. and Romanha, A.J. (1990) Changes in the isoenzyme and kinetoplast DNA patterns of Trypanosoma cruzi strains induced by maintenance in mice. Acta Tropica 47, 35–45. Carneiro, M., Romanha, A.J. and Chiari, E. (1991) Biological charac-

167

terization of Trypanosoma cruzi strains from different zymodemes and schizodemes. Mem. Inst. Oswaldo Cruz 86, 387–393. WHO (1991) Control of Chagas’ Disease. Technical Report Series, World Health Organization. Crothers, D.M., Haran, T.E. and Nadeau, J.G. (1990) Intrinsically bent DNA. J. Biol. Chem. 265, 7093–7096. De Lange, T., Berkvens, T.M., Veerman, H.J., Frasch, A.C., Barry, J.D. and Borst, P. (1984) Comparison of the genes coding for the common 5∞ terminal sequence of messenger RNAs in three trypanosome species. Nucleic Acids Res. 12, 4431–4443. Filardi, L.S. and Brener, Z. (1975) Cryopreservation of Trypanosoma cruzi bloodstream forms. J. Protozool. 22, 398–401. Grondal, E.J.M., Evers, R., Kosubek, K. and Cornelissen, A.W.C.A. (1989) Characterization of the RNA polymerases of Trypanosoma brucei: trypanosomal mRNAs are composed of transcripts derived from both RNA polymerase II and III. EMBO J. 8, 3383–3389. Hartree, D. and Bellofatto, V. (1995) Essential components of the mini-exon gene promoter in the trypanosomatid Leptomonas seymouri. Mol. Biochem. Parasitol. 71, 27–39. Hernandez, N. (1992) Transcription of vertebrate snRNA genes and related genes. In: McNight, S.L. and Yamamoto, K.R. ( Eds.), Transcriptional Regulation. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 281–313. Janz, L., Hug, M. and Clayton, C. (1994) Factors that bind to RNA polymerase I promoter sequences of Trypanosoma brucei. Mol. Biochem. Parasitol. 65, 99–108. Kim, J., Klooster, S. and Shapiro, D.J. (1995) Intrinsically bent DNA in a eukaryotic transcription factor recognition sequence potentiates transcription activation. J. Biol. Chem. 270, 1282–1288. Kirchhoff, L.V. (1993) Chagas disease. American trypanosomiasis. Infect. Dis. Clin. N. Am. 7, 487–502. Kneidl, C., Dinkl, E. and Grummt, F. (1995) An intrinsically bent region upstream of the transcription start site of the rRNA genes of Arabidopsis thaliana interacts with an HMG-related protein. Plant Mol. Biol. 27, 705–713. Laird, P.W., Kooter, J.M., Loosbroek, N. and Borst, P. (1985) Mature mRNAs of Trypanosoma brucei possess a 5∞ cap acquired by discontinuous RNA synthesis. Nucleic Acids Res. 13, 4253–4266. LeBowitz, J.H., Smith, H.Q., Rusche, L. and Beverley, S.M. (1993) Coupling of poly(A) site selection and trans-splicing in Leishmania. Genes Dev. 7, 996–1007. Lu, H.-Y. and Buck, G.A. (1991) Expression of an exogenous gene in Trypanosoma cruzi epimastigotes. Mol. Biochem. Parasitol. 44, 109–114. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. McCarthy Burke, C., Taylor, Z.A. and Buck, G.A. (1989) Characterization of the spliced leader genes and transcripts in Trypanosoma cruzi. Gene 82, 177–189. Meade, J.C. and Stringer, J.R. (1991) PCR amplification of DNA sequences from the transcription factor IID and cation transporting ATPase genes in Pneumocystis carinii. J. Protozool. 38, 66S–68S. Miles, M., Souza, A., Povoa, M., Shaw, J.J., Lainson, R. and Toye, P. (1978) Isozymic heterogeneity of Trypanosoma cruzi in the first autochthonous patients with Chagas’ disease in Amazonian Brazil. Nature 272, 819–821. Miles, M.A., Povoa, M.M., de Souza, A.A., Lainson, R., Shaw, J.J. and Ketteridge, D.S. (1981) Chagas’ disease in the Amazon basin: II. The distribution of Trypanosoma cruzi zymodemes 1 and 3 in Para state, north Brazil. Trans. R. Trop. Med. Hyg. 77, 76–83. Morel, C., Chiari, E., Camargo, E.P., Mattei, D.M., Romanha, A.J. and Simpson, L. (1980) Strains and clones of Trypanosoma cruzi can be characterized by pattern of restriction endonuclease products of kinetoplast DNA minicircles. Proc. Natl. Acad. Sci. USA 77, 6810–6814. Morrison, H.G. and Desrosiers, R.C. (1993) A PCR-based strategy

168

L.R. Nunes et al. / Gene 188 (1997) 157–168

for extensive mutagenesis of a target DNA sequence. BioTechniques 14, 454–457. Nakaar, V., Dare, A.O., Hong, D., Ullu, E. and Tschudi, C. (1994) Upstream tRNA genes are essential for expression of small nuclear and cytoplasmic RNA genes in trypanosomes. Mol. Cell. Biol. 14, 6736–6742. Nilsen, T.W. (1992) Trans-splicing in protozoa and helminths. Infect. Agents Dis. 1, 212–218. Nussenzweig, V. and Goble, F.C. (1995) Further studies on the antigenic constitution of strains of Trypanosoma (Schizotrypanum) cruzi. Exp. Parasitol. 18, 224–230. Ohyama, T. and Hirota, Y. (1993) A possible function of DNA curvature in transcription. Nucleic Acids Symp. Series, Volume 29, pp. 153–154. Parvin, J.D., McCormick, R.J., Sharp, P.A. and Fisher, D.E. (1995) Pre-bending of a promoter sequence enhances affinity for the TATAbinding factor. Nature 373, 724–727. Perry, K. and Agabian, N. (1991) mRNA processing in the Trypanosomatidae. Experientia 47, 118–128. Peterson, G. and Tjian, R. (1993) Cross-species polymerase chain reaction: cloning of TATA box-binding proteins. Meth. Enzymol. 218, 493–507. Ready, P.D. and Miles, M.A. (1980) Delimination of Trypanosoma cruzi zymodemes by numerical taxonomy. Trans. R. Soc. Trop. Med. Hyg. 74, 238–242. Roditi, I. (1992) Trypanosoma vivax: linkage of the mini-exon (spliced leader) and 5S ribosomal RNA genes. Nucleic Acids Res. 20, 1995. Saito, R.M., Elgort, M.G. and Campbell, D.A. (1994) A conserved upstream element is essential for transcription of the Leishmania tarentolae mini-exon gene. EMBO J. 13, 5460–5469. Sanchez, G., Wallace, A., Olivares, M., Diaz, N., Aguilera, X., Apt, W. and Solari, A. (1990) Biological characterization of Trypanosoma cruzi zymodemes: in vitro differentiation of epimastigotes and infec-

tivity of culture metacyclic trypomastigotes to mice. Exp. Parasitol. 71, 125–133. Solari, A., Mun˜oz, S., Venegas, J., Wallace, A., Aguilera, X., Apt, W., Brenie`re, S.F. and Tibayrenc, M. (1992) Characterization of Chilean, Bolivian, and Argentinian Trypanosoma cruzi populations by restriction endonuclease and isoenzyme analysis. Exp. Parasitol. 75, 187–195. Souto, R.P. and Zingales, B. (1993) Sensitive detection and strain classification of Trypanosoma cruzi by amplification of a ribosomal RNA sequence. Mol. Biochem. Parasitol. 62, 45–52. Tanowitz, H.B., Kirchhoff, L.V., Simon, D., Morris, S.A., Weiss, L.M. and Wittner, M. (1992) Chagas’ disease. Clin. Microbiol. Rev. 5, 400–419. Tibayrenc, M. and Ayala, F. (1988) Isoenzyme variability in Trypanosoma cruzi, the agent of Chagas’ disease: genetical, taxonomic and epidemiological significance. Evolution 42, 277–292. Tibayrenc, M. and Miles, M.A. (1983) A genetic comparison between Brazilian and Bolivian zymodemes of Trypanosoma cruzi. Trans. R. Soc. Trop. Med. Hyg. 77, 76–83. Tyler-Cross, R.E., Short, S.L., Floeter-Winter, L.M. and Buck, G.A. (1995) Transient expression mediated by the Trypanosoma cruzi rRNA promoter. Mol. Biochem. Parasitol. 72, 23–31. Warren, K.S. (1988) The global impact of parasitic diseases. In: Englund, P.T. and Sher, A. ( Eds.), The Biology of Parasitism. Alan R. Liss, New York, NY, pp. 3–12. Zomerdijk, J.C.B.M., Kieft, R., Shiels, P.G. and Borst, P. (1991) Alpha-amanitin-resistant transcription units in trypanosomes: a comparison of promoter sequences for a VSG gene expression site and for the ribosomal RNA genes. Nucleic Acids Res. 19, 5153–5158. Zwierzynski, T.A., Widmer, G. and Buck, G.A. (1989) In vitro 3∞ end processing and poly(A) tailing of RNA in Trypanosoma cruzi. Nucleic Acids Res. 17, 4647–4660.