Modulation of oat arginine decarboxylase gene expression and genome organization in transgenic Trypanosoma cruzi epimastigotes Marı ´ a P. Serra, Carolina Carrillo, Ne ´ lida S. Gonza ´ lez and Israel D. Algranati Fundacio ´ n Instituto Leloir, Buenos Aires, Argentina Trypanosoma cruzi, the etiological agent of Chagas’ disease, is a parasitic protozoan with a digenetic life cycle involving an insect vector and a mammalian host. The parasite undergoes major morphological and biochemical changes during the different stages of its life cycle. The epimastigote form is noninfective and proliferates extracellularly in the insect gut where it differentiates into metacyclic trypomastigotes, which can then infect the mammalian host cells and replicate intracellularly after transforming into amastigotes [1–4]. Epimastigotes from different wild-type strains of T. cruzi are able to grow continuously in vitro in a rich culture medium [5], but proliferation stops after a few passages in a semidefined medium, which contains only traces of polyamines [6,7]. T. cruzi remain viable for several weeks in the defined medium and are able to resume normal growth only upon the addition of exo- genous polyamines to the culture [7]. These results confirm previous reports from our and other laborat- ories indicating that T. cruzi epimastigotes are unable Keywords free episome; genome organization; heterologous ADC gene expression; plasmid integration; Trypanosoma cruzi transformation Correspondence I. D. Algranati, Fundacio ´ n Instituto Leloir, Avenue Patricias Argentinas 435 (1405), Buenos Aires, Argentina Fax: +5411 5238 7501 Tel: +5411 5238 7500 E-mail: ialgranati@leloir.org.ar (Received 24 November 2005, accepted 12 December 2005) doi:10.1111/j.1742-4658.2005.05098.x We have previously demonstrated that wild-type Trypanosoma cruzi epi- mastigotes lack arginine decarboxylase (ADC) enzymatic activity as well as its encoding gene. A foreign ADC has recently been expressed in T. cruzi after transformation with a recombinant plasmid containing the complete coding region of the oat ADC gene. In the present study, upon modulation of exogenous ADC expression, we found that ADC activity was detected early after transfection; subsequently it decreased to negligible levels between 2 and 3 weeks after electroporation and was again detected  4 weeks after electroporation. After this period, the ADC activity increased markedly and became expressed permanently. These changes of enzymatic activity showed a close correlation with the corresponding levels of ADC transcripts. To investigate whether the genome organization of the transgenic T. cruzi underwent any modification related to the expression of the heterologous gene, we performed PCR amplification assays, restriction mapping and pulse-field gel electrophoresis with DNA samples or chromo- somes obtained from parasites collected at different time-points after trans- fection. The results indicated that the transforming plasmid remained as free episomes during the transient expression of the foreign gene. After- wards, the free plasmid disappeared almost completely for several weeks and, finally, when the expression of the ADC gene became stable, two or more copies of the transforming plasmid arranged in tandem were integra- ted into a parasite chromosome (1.4 Mbp) bearing a ribosomal RNA locus. The sensitivity of transcription to a-amanitin strongly suggests involvement of the protozoan RNA polymerase I in the transcription of the exogenous ADC gene. Abbreviations ADC, arginine decarboxylase; G418, geneticin; ODC, ornithine decarboxylase; PFGE, pulse-field gel electrophoresis. 628 FEBS Journal 273 (2006) 628–637 ª 2006 The Authors Journal compilation ª 2006 FEBS to synthesize putrescine, as a result of the absence of ornithine and arginine decarboxylases (ODC and ADC) [7–10], which catalyse the first steps of both possible pathways of putrescine biosynthesis [11]. In accordance with this conclusion we have observed that the addition of ornithine or arginine to the culture medium cannot support the continuous growth of T. cruzi in the defined medium, as proliferation in the presence of these amino acids is arrested at the same time as in unsupplemented cultures [9]. In all these cases, growth can be resumed by the addition of putrescine, cadaverine or spermidine. On the other hand, all our attempts to detect ODC or ADC enzy- matic activities in various strains of wild-type T. cruzi epimastigotes, by adding radioactive ornithine or arginine to intact parasites or cell extracts, gave negli- gible values [7,9,10]. These results could be attributed neither to a deficient uptake of the amino acids by the parasites [12] nor to the presence of ODC or ADC inhibitors in the protozoan internal medium [7,10]. Studies carried out in order to correlate the parasite growth under different conditions with the correspond- ing intracellular levels of polyamines have shown that the proliferation of wild-type T. cruzi epimastigotes depends exclusively on the endogenous concentrations of spermidine or aminopropylcadaverine [13,14]. In fact, when polyamine-depleted cultures of T. cruzi epi- mastigotes in synthetic media were supplemented with putrescine, together with cyclohexylamine (a known inhibitor of putrescine conversion into spermidine) [15,16], the parasites were unable to resume growth, even though the putrescine levels increased markedly inside the protozoa, because the spermidine concentra- tions remained low [13]. We have recently investigated whether the failure to detect ODC and ADC activities in wild-type T. cruzi epimastigotes could be caused by the absence of the corresponding genes in the parasite genome. Bioinfor- matic analyses based on available data from the T. cruzi genome project, and hybridization assays with specific probes homologous to conserved regions of ODC or ADC genes from many organisms, have indi- cated the absence of ODC- and ADC-like nucleotide sequences in the wild-type T. cruzi genome [10,17]. As the described results show that wild-type T. cruzi behaves as a natural deletion mutant for ODC and ADC genes, we used these polyamine auxotrophic par- asites as recipients of foreign ODC or ADC genes to study their expression and the eventual suppression of polyamine auxotrophy [7,10]. We have previously transformed wild-type T. cruzi epimastigotes with a recombinant plasmid containing the oat ADC cDNA coding region [10]. In the present work we used ADC-transgenic protozoa to follow the time-course modulation of foreign gene expression and to investigate whether this modulation can be explained by the concomitant changes occurring in the parasite genome organization. Results and Discussion Expression of the oat ADC gene in T. cruzi In order to study the expression of the foreign ADC gene in T. cruzi epimastigotes, we transformed wild- type parasites with the recombinant plasmid, pADC-8 [10], bearing the complete coding region of the oat ADC gene cloned in the sense orientation, in the vector pRIBOTEX. This vector contains a ribosomal promo- ter region, derived from a T. cruzi rRNA locus, ligated upstream of the multiple cloning sequence [18]. It has been previously shown that this promoter region of pRIBOTEX and related vectors induces the transcrip- tion of genes cloned downstream of the promoter and their chromosomal integration [19]. After 48 h of transfection, geneticin (G418) was added to the cul- tures of transformed parasites to select plasmid- containing protozoa. The time course of ADC gene expression was followed by northern hybridization analysis and measurements of the new enzymatic activ- ity. Total RNA was extracted from transformed para- sites at different time-points after electroporation and analysed with a labelled probe specific for the oat ADC gene (Fig. 1A), using a ribosomal RNA probe as a loading control (Fig. 1B). The relative intensities of the hybridization bands corresponding to ADC tran- scripts and ribosomal RNA showed that the concen- tration of ADC mRNA ( 2.2 kb long) reached an early maximum at 1 week after transfection, decreased markedly between 2 and 4 weeks after electroporation, and then increased again, attaining high levels after ‡ 6 weeks (Fig. 1C). Transgenic T. cruzi showed a significant ADC activity as early as 48 h after transfection, as previ- ously reported [10]. This activity has been character- ized by its products, the reaction stoichiometry and the specific inhibition by a-difluoromethylarginine [10]. In the present work we observed that, initially, the enzymatic activity of cell extracts increased up to a maximum value at  1 week after electroporation and then decreased to almost undetectable levels after 2–3 weeks (Fig. 1D). These results, and the sim- ilar time-course variation of ADC RNA levels, con- firm previously published data indicating an initially transient expression of the foreign gene [10]. ADC mRNA and the corresponding enzymatic activity M. P. Serra et al. Foreign ADC gene expression in T. cruzi FEBS Journal 273 (2006) 628–637 ª 2006 The Authors Journal compilation ª 2006 FEBS 629 increased again when transformed T. cruzi were cul- tured for longer time-periods in the presence of G418 (Fig. 1A–D). At this point, the ADC gene became permanently expressed at rather high levels in the transgenic parasites. It is worthy of note that although these parasites contain high enzymatic activities of ADC, they are unable to overcome T. cruzi auxotrophy for polyam- ines, as previously reported [10]. These results indicate that agmatine cannot fulfill the physiological roles of polyamines, and at the same time strongly suggest that T. cruzi does not contain agmatinase activity that would convert agmatine into putrescine. We have also observed that transformed parasites cultured in the absence of G418 showed only a tran- sient period of ADC activity (Fig. 1D). Previous results from our laboratory have shown that the complete elimination of untransformed parasites by G418 under our experimental conditions occurs in  1 month; therefore, in the 1 month time-period between transfection and elimination, the parasite cul- tures are variable mixtures of transformed and untrans- formed cells. However, the correlation of RNA transcripts with the enzymatic activity levels for each time-point is relevant and allowed us to detect the tran- sient and stable periods of exogenous gene expression. Genome organization of transformed parasites In order to explain the described changes of ADC mRNA levels and the corresponding enzymatic activi- ties after T. cruzi transformation, we investigated the genome organization of transgenic parasites at differ- ent time-points after transfection. To ascertain whe- ther the recombinant plasmid used for transformation was integrated into the parasite genome or remained free as extrachromosomal elements, we performed PCR amplification assays using two different sets of primers, as described in the Experimental procedures. If the recombinant plasmid remained as a free A B D C Fig. 1. Time-course of arginine decarboxylase (ADC) RNA levels and enzymatic activities in transfected Trypanosoma cruzi. Northern blot analysis of total RNA samples (20 lg) prepared from ADC-transformed T. cruzi epimastigotes harvested at different time-points after electro- poration. Hybridization bands with ADC- and rRNA-specific probes are shown in (A) and (B), respectively. (C) Relative intensities of ADC mRNA bands normalized to the rRNA loading controls. Lane 1, RNA from wild-type T. cruzi; lanes 2, 3, 4, 5 and 6 correspond to RNA pre- pared from transgenic parasites 2 days and 1, 2, 4 and 24 weeks after transfection, respectively. (D) ADC-specific activities in transgenic T. cruzi epimastigotes at different time-points postelectroporation. All samples correspond to parasites harvested at the early logarithmic phase of growth, and ADC activity values are the average of assays carried out in duplicate. Transformed parasites were cultured in the absence of geneticin (G418) (s) or with the antibiotic added 48 h after electroporation (d). Foreign ADC gene expression in T. cruzi M. P. Serra et al. 630 FEBS Journal 273 (2006) 628–637 ª 2006 The Authors Journal compilation ª 2006 FEBS episome (either a circular single copy or a multimeric form), only one of both PCR reactions should pro- duce a DNA segment of 2030 bp when using primers T7 (specific for the promoter region of pRIBOTEX vector) and ADC2 (specific for an internal segment of the ADC coding region) (Fig. 2A). On the other hand, if total integration of one plasmid copy has occurred by homologous recombination, presumably at a ribosomal RNA locus of the parasite genome, the amplification assay with primers T7 and RIB (spe- cific for the ribosomal locus of the wild-type T. cruzi genome) should give a DNA segment of 890 bp (Fig. 2B), and no other product from the PCR reac- tion with the set of primers T7 and ADC2. Further- more, we could expect that the integration in the parasite genome of two or more units of tandemly amplified plasmid molecules should give rise to both segments (2030 and 890 bp) by the described PCR amplifications (Fig. 2C). In order to study these possi- bilities, total DNA was obtained from samples of transfected parasites collected at different time-points after electroporation, and all these preparations were used in PCR amplification reactions with the two sets of primers described above. Gel electrophoresis analy- ses of the PCR products indicated that during the first 15 days after transfection, the transforming plas- mid remained as free episomes, as only a 2030 bp DNA segment was detected after both PCR assays (Fig. 3A, lanes 5 and 6). Two weeks after electropora- tion, the free plasmid had almost disappeared (Fig. 3A, lanes 7 and 8). The faint PCR band of  3500 bp detected in Fig. 3A (lane 8), in addition to the expected 2030 bp PCR product, might be gener- ated by a partial DNA rearrangement inside the pADC-8 plasmid molecule. During the subsequent period, the integration of ‡ 2 units of the transfected plasmid into the parasite genome seemed to occur, as shown by the production of both DNA segments after ‡ 4 weeks of transformation (Fig. 3A, lanes 9– 12). We obtained the same results (890 and 2030 bp segments) with DNA samples from transformed T. cruzi 6 months after electroporation (Fig. 3A, Fig. 2. Schematic diagrams to predict the fate of the recombinant plasmid after parasite transformation. (A) Free episome elements; (B) integration of one plasmid copy into the parasite genome; (C) integration of two plasmid copies arranged in tandem. The hatched horizontal segments represent the expected PCR amplification products for each type of genome organization when the corresponding DNA samples were assayed as described in the Experimental procedures. The primer annealing sites are indicated by arrows. The cutting sites of NheI and SalI enzymes used for the restriction mapping are also shown. M. P. Serra et al. Foreign ADC gene expression in T. cruzi FEBS Journal 273 (2006) 628–637 ª 2006 The Authors Journal compilation ª 2006 FEBS 631 lanes 13 and 14), indicating that a stable genome structure has probably been reached. However, we cannot exclude that a small portion of the recombin- ant plasmid could remain free, even after stable trans- formation. The described patterns of genome organization after transfection are in good correlation with the time- course of ADC gene expression (transcription and translation) found in the transformed parasites and depicted in Fig. 1. The integration of the transforming plasmid ‡ 4 weeks after electroporation has also been demon- strated by digestion of total DNA from transfected T. cruzi with restriction enzymes that cut twice inside the pADC-8 sequence, as shown in Fig. 2A–C. The subsequent hybridization analyses using the described labelled probe, specific for the ADC gene, gave the results predicted. The radioactive bands obtained in the corresponding Southern blot assay (Fig. 3B) could only be explained by the integration, into the parasite genome, of ‡ 2 units of the plasmid pADC-8 arranged in a head-to-tail tandem, as previously suggested for the pRIBOTEX vector [18]. In order to confirm the tandem arrangement of the integrated copies of pADC-8 plasmid, we performed two different digestion reactions of DNA from para- sites, harvested after 6 months of transfection, with the restriction enzymes SstII or BstBI, each with a single cutting site near the 5¢ or the 3¢ end of the pADC-8 sequence, respectively, but outside the ADC ORF. After hybridization of the digestion products with the ADC-specific probe, both experiments showed a com- mon hybridizing fragment similar in size to that of the pADC-8 plasmid ( 7.9 kbp), as depicted in Fig. 4A. This result strongly supports the conclusion that the stable transformed protozoa contain two or more cop- ies of plasmid pADC-8 in a head-to-tail tandem integ- rated without rearrangements nor gaps. The additional 23 kbp-labelled band, seen in lane 2, was probably caused by the fact that the restriction enzyme, BstBI, only produced partial digestion of the DNA samples. On the other hand, the absence of a second hybridiza- tion band in lane 1 might be the result of insufficient sensitivity of our assay. Rehybridization of the mem- brane shown in Fig. 4A with a labelled probe specific for the neomycin-resistance gene gave the same radio- A B Fig. 3. Genome organization of transformed Trypanosoma cruzi assayed by PCR amplification (A) or by Southern hybridization after digestion with restriction enzymes (B). (A) Total DNA was prepared from transgenic parasites harvested after different times of transfection. Each DNA sample was used as template in PCR amplification reactions with two pairs of primers – (a) T7 and ADC2, and (b) T7 and RIB – under the conditions described in the Experimental procedures. PCR products were analysed by electrophoresis on agarose gels containing ethi- dium bromide and observed under UV light. Lanes 1 and 2 correspond to the assay carried out with DNA from untransformed wild-type T. cruzi as template; lanes 3 and 4, correspond to the assay carried out with purified recombinant plasmid pADC-8; and lanes 5–14 corres- pond to the assay carried out with DNA samples obtained from transformed parasites after 2 days and 2, 4, 6 and 24 weeks after transfec- tion. Lanes 1, 3, 5, 7, 9, 11 and 13 show the PCR products obtained using the primer pair T7 and RIB. Lanes 2, 4, 6, 8, 10, 12 and 14 show the PCR products obtained using the primer pair T7 and ADC2. Lane 15, 1 kb DNA ladder standard. (B) DNA obtained from arginine decarb- oxylase (ADC)-transformed parasites harvested 6 months after transfection was completely digested with NheIorSalI restriction enzymes. After gel electrophoresis, the digestion products were blotted onto a nylon membrane and hybridization analysis was carried out with the radioactive ADC-specific probe. Southern hybridization bands correspond to digestion with NheI (lane 1) or SalI (lane 2). Foreign ADC gene expression in T. cruzi M. P. Serra et al. 632 FEBS Journal 273 (2006) 628–637 ª 2006 The Authors Journal compilation ª 2006 FEBS active bands (Fig. 4B), providing further support for the integration of entire molecules of the transforming plasmid, pADC-8. To investigate further the fate of the transforming plasmid after electroporation and the integration site within the parasite genome, we performed pulse-field gel electrophoresis (PFGE) of chromosomes obtained at different time-points after parasite transformation, followed by hybridization assays with the ADC-specific probe. The results were in complete agreement with those obtained by PCR and restriction digestion experiments shown in Fig. 3A,B. Early after electropo- ration, the ADC gene appeared almost exclusively in a hybridization band at the origin of the gel, indicating that the transforming plasmid remained as extrachro- mosomal elements during this period (Fig. 5A, lane 2). It is relevant to mention that when a small amount of purified pADC-8 plasmid was added to intact wild- type parasites before the preparation of chromosome- containing agar blocks for the PFGE experiments, we obtained a very similar pattern of radioactive bands after hybridization (Fig. 5A, lane 5). One week after transformation we were able to detect only a faint band corresponding to the ADC gene at the origin of the gel (Fig. 5A, lane 3) suggesting an almost complete destruction of free pADC-8 plasmid. After 3 months, a small amount of episome was still detectable and the ADC gene was mainly at a radioactive band with the same mobility as the 1.4 Mbp parasite chromosome, which bears a ribosomal RNA locus, as shown in Fig. 5A (lane 4) and Fig. 5B [19]. Therefore, insertion of the exogenous gene was probably not specific at an ADC-like sequence, but rather targeted to the parasite ribosomal RNA locus by the ribosomal promoter region included in the vector pRIBOTEX [18,19]. Effect of a-amanitin on exogenous ADC transcription in transformed T. cruzi We also studied the a-amanitin sensitivity of the ADC gene transcription. For this purpose we carried out dot-blot hybridization analyses with membranes con- taining DNA spots (5 lg each) corresponding to inter- nal fragments of ADC or cruzipain genes. The latter (used as a control) is a housekeeping gene that encodes the main cysteine proteinase of T. cruzi [20]. Prelimin- ary experiments have shown that transcription of the A B Fig. 4. Arrangement of plasmid pADC-8 copies integrated into the transformed parasite genome. DNA from stably transformed para- sites collected 6 months after transfection was digested with the restriction enzymes SstII (lane 1) or BstBI (lane 2). After gel electro- phoresis of the digestion products and blotting onto a nylon mem- brane, hybridization analysis was performed with the specific probes for arginine decarboxylase (ADC ) (A) or neomycin-resistance (neo) (B) genes. All other details are as described in the Experimen- tal procedures. A B Fig. 5. Southern blot analysis of chromosomes prepared from untransformed and transformed parasites collected at different time-points after transfection. Trypanosoma cruzi chromosomes were separated by pulse-field gel electrophoresis (PFGE) and analysed by Southern blot hybridization with labelled arginine decarboxylase (ADC) (A) or rRNA (B) specific probes. Lanes 1, 2, 3 and 4 in panel A correspond to parasites before transformation, or 48 h, 1 week or 3 months after transfection, respectively. Lane 5 is a control of chromosomes from wild-type parasites with the addition of 10 ng of purified pADC-8 plasmid. Panel B shows a duplicate of lane 4 in panel A hybridized with the radioactive rRNA 24Sa probe. M. P. Serra et al. Foreign ADC gene expression in T. cruzi FEBS Journal 273 (2006) 628–637 ª 2006 The Authors Journal compilation ª 2006 FEBS 633 cruzipain gene is inhibited by a-amanitin (I. D. Algra- nati, unpublished results). The hybridization assays were performed with radioactive RNA synthesized by transformed parasites that were permeabilized and then incubated in the presence of different concentra- tions of a-amanitin. Figure 6 shows that transcription of the ADC gene did not decrease, even at very high levels of a-amanitin (500 lgÆmL )1 ), while the synthesis of cruzipain mRNA was markedly reduced. It has been reported that trypanosomes contain three differ- ent RNA polymerases: RNA Pol I (which synthesizes ribosomal RNA); RNA Pol II (responsible for the transcription of most protein-coding genes); and RNA Pol III (which transcribes tRNA and 5SRNA). RNA Pol II is the only one sensitive to a-amanitin [21]. According to these data, our results strongly suggest the involvement of RNA Pol I in ADC gene transcrip- tion and RNA pol II in cruzipain transcription. Our conclusion, that the ADC gene of transformed para- sites was transcribed by RNA Pol I, is also in agree- ment with the fact that the transforming plasmid bearing the foreign gene contains a strong rRNA pro- moter region. Conclusions Our studies, on the modulation of oat ADC gene expression in T. cruzi epimastigotes, have shown an early period of transient expression during which the transforming recombinant plasmid remained as a free element undergoing transcription and translation (Figs 1 and 3). This episome was probably almost completely degraded between 2 and 4 weeks after transfection. However, the continuous selection pres- sure of the antibiotic, G418, allowed stable expression of the ADC gene, presumably after recombination and integration into the parasite genome of two or more pADC-8 copies arranged in tandem. ADC transcripts and the corresponding enzymatic activities followed a similar pattern of modulation (Fig. 1). When the selec- tion drug, G418, was omitted after parasite transfor- mation, we could only detect transient expression of the ADC gene. Previous results obtained in our laboratory with the ODC gene, and data reported by other authors with several genes, have indicated a fast plasmid integration after transfection when using the same pRIBOTEX or a related expression vector, pTREX [18,19]. On the contrary, in the present work we found a late plasmid integration into the T. cruzi genome and a concomit- ant stable gene expression, despite the fact that we also used the pRIBOTEX vector. We speculate that an as-yet not well understood mechanism requiring plasmid duplication during integration, the particular structure of plasmid pADC-8 and ⁄ or the involvement of putative intermediate forms of the transforming plasmid might explain the different pattern of expres- sion observed in our experiments. Experimental procedures Materials and reagents Brain–heart infusion, liver infusion broth, tryptose and yeast extracts were obtained from Difco Laboratories (Detroit, MI, USA). Minimal essential medium (SMEM), amino acids and vitamins were obtained from Gibco BRL (Gaithersburg, MD, USA). Bases, haemin, pyridoxal 5¢-phosphate, poly- amines, Hepes buffer and antibiotics were purchased from Sigma (St Louis, MO, USA). Fetal calf serum was from Natocor (Carlos Paz, Cordoba, Argentina), and L-[U- 14 C] arginine (305 CiÆmol )1 ), [ 32 P]dCTP[aP] (3000 CiÆmmol )1 ) and [ 32 P]UTP[aP] (3000 Ci mmol )1 ) were from Amersham Life Sciences (Bucks., UK). Fig. 6. Sensitivity to a-amanitin of the transcription of arginine decarboxylase (ADC) and cruzipain genes in transformed Trypano- soma cruzi. Permeable parasites were preincubated at 0 °C for 5 min in the absence or presence of different concentrations of a-amanitin. Transcription was performed for 30 min at 30 °C in the presence of [ 32 P]UTP[aP], and purified radioactive RNA was ana- lysed by dot-blot hybridization, as described in the Experimental procedures. The amount of specific RNA hybridized to each dot was measured by scintillation counting and expressed as a percent- age of the corresponding value obtained in the absence of a-aman- itin ADC-specific (s) or cruzipain-specific (d) transcripts. All values are the average of experiments carried out in duplicate. Foreign ADC gene expression in T. cruzi M. P. Serra et al. 634 FEBS Journal 273 (2006) 628–637 ª 2006 The Authors Journal compilation ª 2006 FEBS Parasite cultures T. cruzi epimastigotes, strains Tulahuen 2 [22] and RA [23], were cultured at 28 °C in rich (BHT) or semidefined (SDM- 79) media [6] supplemented with haemin (20 mgÆL )1 ), 10% (v ⁄ v) heat inactivated fetal bovine serum and antibiotics (100 lgÆmL )1 streptomycin and 100 UÆmL )1 penicillin). Parasite growth was followed by cell counting. The doub- ling time for wild-type T. cruzi proliferation was 18–24 h. All cultures were diluted weekly to 8–12 · 10 6 cellsÆmL )1 using fresh medium with the indicated additions. Parasite extracts and ADC assay T. cruzi were harvested by centrifugation for 5 min at 3500 g, and after washing with NaCl ⁄ P i they were resus- pended at 1 · 10 9 cellsÆmL )1 in the reaction solution con- taining 50 mm Hepes buffer, pH 7.5, 0.5 mm EDTA, 1 mm dithiothreitol and 0.1 mm pyridoxal 5¢-phosphate. Cells were disrupted by three cycles of freeze–thawing, followed by a brief sonication to break the DNA. After centrifuga- tion at 12 000 g for 15 min at 4 °C, the supernatant fluid was used to measure the enzyme activity in a total volume of 50 lL with the addition of radioactive arginine (0.25 lCi, 1 mm final concentration). All measurements of ADC activity were carried out using cell extracts from transformed T. cruzi collected at the early or mid-logarith- mic phase of growth (cell concentration 20–30 · 10 6 para- sites per mL), as the enzymatic specific activity decreased markedly at late exponential or stationary phase (I. D. Algranati, results not shown). Therefore, ADC activities were obtained using parasite cultures diluted with fresh medium, to 10–15 · 10 6 cells per mL, 24 h before the collection of each sample. The enzymatic assays were carried out under linear conditions for protein concentra- tion and reaction time. ADC activities were calculated by measuring the radioactive CO 2 released during the reac- tions [24]. Protein concentrations of enzyme preparations were determined by Bradford’s method [25], with BSA as the standard. Construction of the recombinant plasmid pADC-8 and parasite transfection A cDNA fragment containing the complete coding region of the oat ADC gene was cloned in the pRIBOTEX expres- sion vector [18], as previously described [10]. The recombin- ant plasmid pADC-8 (with the ADC coding region inserted in the sense orientation) was selected after analysis by restriction mapping and nucleotide sequencing. Wild-type T. cruzi epimastigotes collected at the early exponential phase of growth were transfected by electroporation using 3 · 10 8 parasites resuspended in 350 lL of liver infusion tryptose medium [26] without fetal bovine serum. After the addition of 20–100 lg of pADC-8 recombinant plasmid, electroporation was performed essentially as described by Hariharan et al . [27] using 2 mm gap cuvettes. Parasites were then diluted with rich medium containing 10% fetal calf serum and incubated at 28 °C for 48 h before the addi- tion of G418 (500 lgÆmL )1 ) in order to select transformed T. cruzi during the subsequent period of culture. Control electroporation assays were carried out with buffer solution or pRIBOTEX vector instead of the recombinant plasmid. Samples of parasite culture were collected at different time- points after electroporation to measure ADC enzymatic activities and to prepare DNA and RNA for hybridization analyses. Southern and northern blot hybridization Total DNA from wild-type and transformed parasites was prepared according to Medina-Acosta & Cross [28]. With this method it is possible to recover genomic DNA as well as free episomes. After digestion with different restriction enzymes, the DNA fragments were separated by electro- phoresis on 1% agarose gels and transferred to nylon mem- branes (Hybond N + ; Amersham). Total RNA from parasites, before and after transforma- tion, was obtained using TRIzol LS reagent (Invitrogen, Carlsbad, CA, USA) [29]. Samples containing 20 l gof total RNA were fractionated by electrophoresis on a 1% agarose gel, containing 2.2 m formaldehyde, and blotted onto nylon membranes. Southern and northern hybridization assays were per- formed with 32 P-labelled probes specific for oat ADC [10], ribosomal RNA or neomycin resistance (neo) genes. The radioactive specific probe for the latter gene was prepared by PCR amplification with the plasmid pADC-8 as tem- plate and the forward and reverse primers pNeo 1 (5¢- CCGGAATTCTGAATGAACTGCAGGACGAGGCAG-3¢) and pNeo 2 (5¢-CCGGAATTCCGGCCATTTTCCACCAT GATATTC-3¢), respectively. The labelled probe specific for rRNA 24Sa was obtained by PCR amplification of a DNA segment of this gene with the forward and reverse primers 75 (5¢-GCAGATCTTGGTTGGCGTAG-3¢) and 76 (5¢-GGTTCTCTGTTGCCCCTTTT-3¢), respectively, kindly provided by A. Schijman (INGEBI, Buenos Aires, Argentina). PCR analyses of DNA from transformed T. cruzi Samples containing 50–100 ng of total DNA prepared from parasites collected at different time-points after electropora- tion were amplified by PCR using two sets of primers: (a) the forward primer T7 promoter primer (Promega, Madison, WI, USA) and the reverse primer ADC2 (5¢-CCGGAATT CCAGCTTGGAAGAGAGATCGCGGAT-3¢) with a nuc- leotide sequence complementary to an internal segment of M. P. Serra et al. Foreign ADC gene expression in T. cruzi FEBS Journal 273 (2006) 628–637 ª 2006 The Authors Journal compilation ª 2006 FEBS 635 the ADC coding region [30], and (b) T7 primer and the reverse primer, RIB, complementary to an internal sequence of a parasite ribosomal RNA locus [19]. PCR amplifications were performed for 30 cycles at the following cycle parame- ters: 94 °C, 30 s; 50 °C, 1 min; and 72 °C, 2 min. PCR prod- ucts were separated by electrophoresis on agarose gels and detected by ethidium bromide staining. PFGE Agarose blocks containing 2 · 10 7 untransformed or transformed parasites harvested at different time-points after transfection were prepared as described by Cano et al. [31], and chromosomes were separated by PFGE in a Bio-Rad Lab (Hercules, CA, USA) apparatus using 1% agarose gels, 0.5 · TBE electrophoresis buffer (89 mm Tris-borate, pH 8.2, 2 mm EDTA) and the running condi- tions indicated by Lorenzi et al. [19]. After blotting onto nylon membranes (Hybond N + ; Amersham), hybridization analyses were carried out with radioactive probes specific for oat ADC or rRNA 24Sa genes. Transcription in transformed T. cruzi Parasites collected at the early logarithmic phase of growth 6 months after transfection with the pADC-8 recombinant plasmid were permeabilized with palmitoyl-l-a-lysophos- phatidylcholine (Sigma), as previously described [32,33]. After transcription in the presence of [ 32 P]UTP[aP] and dif- ferent concentrations of a-amanitin, radioactive RNA was isolated [29] and then hybridized to dot-blots prepared with 5 lg of DNA segments corresponding to ADC [10] or cruzi- pain [20] genes. The latter DNA was obtained by PCR amplification using a recombinant plasmid containing the cruzipain gene as template and primers A (5¢-ATGT CTGGCTGGGCGCG-3¢; forward) and B (5¢-GAGGCG ACGATGACGGC-3¢; reverse). Radioactive spots on the membranes were cut and counted in a scintillation counter. Acknowledgements We thank Dr Sara H. Goldemberg for helpful discus- sions and Edith Trejo and Carlos Zadikian for techni- cal assistance. We are indebted to Drs J. J. Cazzulo and C. Labriola for their generous gifts of a cruzipain- containing plasmid and primers A and B. This work was partially supported by grants from The National Research Council (CONICET, Argentina) and the University of Buenos Aires. 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