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

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-

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-

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

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

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

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

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-

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-

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-

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

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-

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FEBS Journal 273 (2006) 628–637 ª 2006 The Authors Journal compilation ª 2006 FEBS 637