Parasitol Res (2011) 108:731–739

DOI 10.1007/s00436-010-2190-6

ORIGINAL PAPER

Evidence of RNA editing in Leishmania braziliensis

promastigotes
César Ramírez & Concepción Puerta & Jose M. Requena

Received: 14 August 2010 / Accepted: 6 October 2010 / Published online: 4 December 2010
# Springer-Verlag 2010

Abstract RNA editing in trypanosomatids is an elaborate detect full-size transcripts by Northern blotting in promas-
form of post-transcriptional processing that inserts and tigotes of L. braziliensis, led us to the suggestion that the
deletes uridines in many mitochondrial pre-mRNAs, pro- strain used in this study (M2904) lacks of critical RNA
viding the genetic information needed to create functional guides for a complete edition of ND8 transcripts.
transcripts. The process has been extensively analyzed in
Trypanosoma brucei, Crithidia fasciculata, and Leishmania
tarentolae. However, few data exist on this mechanism in Introduction
pathogenic Leishmania species. Here, we show evidence
that this process also operates in Leishmania braziliensis, Protozoan parasites of the genera Leishmania are causative
being the first time that RNA editing has been described in agents of a group of diseases, known as leishmaniases.
a species of the Viannia subgenus. A partially edited These range from the spontaneously healing cutaneous
transcript corresponding to the NADH dehydrogenase lesions arising from Leishmania major infection to muco-
subunit 8 (ND8) gene was identified in L. braziliensis cutaneous leishmaniasis usually associated with Leishmania
promastigotes. Sequence analysis allowed the identification braziliensis and the often fatal visceralising disease, caused
of the maxicircle-encoded cryptogene, which shows a high by Leishmania donovani, in the Indian sub-continent, and
degree of sequence conservation with the corresponding Leishmania infantum (Leishmania chagasi), in Latin
cryptogenes in other Leishmania species. Although an America and the Mediterranean basin (Bañuls et al.
edition pattern could be postulated for the ND8 transcripts 2007). World Health Organization (WHO) epidemiologi-
in L. braziliensis, attempts to isolate completely edited cal data indicate that there are over two million new cases
transcripts by RT-PCR were not fruitful; instead, many of leishmaniasis each year in 88 countries, with 367
transcripts with partial and unexpected editing patterns million people at risk (http://www.who.int/health-topics/
were isolated. This data, together with our inability to leishmaniasis.htm). Leishmania parasites undergo a dige-
netic life cycle, differentiating from the promastigote form
Electronic supplementary material The online version of this article in the insect vector, the phlebotomine sand fly, to the
(doi:10.1007/s00436-010-2190-6) contains supplementary material, amastigote form in the lysosomal compartment of the
which is available to authorized users. macrophages of mammals.
C. Ramírez : C. Puerta Leishmania, and related trypanosomatids of the genera
Laboratorio de Parasitología Molecular, Trypanosoma, derive from one of the earlier evolutionary
Departamento de Microbiología, Pontificia Universidad Javeriana,
lines diverging from the common trunk of eukaryotes
Bogotá, Colombia
(Moreira et al. 2004). As a reflection of their evolutionary
C. Ramírez : J. M. Requena (*) distance to most of eukaryotes, they possess several
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), distinctive features and surprising molecular mechanisms
Universidad Autónoma de Madrid,
(Donelson et al. 1999). Perhaps one of the most fascinating
c/ Nicolás Cabrera 1,
28049 Madrid, Spain molecular mechanisms operating in these organisms is RNA
e-mail: [email protected] editing of their mitochondrial transcripts (Estevez and
732 Parasitol Res (2011) 108:731–739

Simpson 1999). RNA editing is a unique post-transcriptional Apart from T. brucei, L. tarentolae, and C. fasciculata,
RNA maturation process that in trypanosomatids involves in which the mitochondrial RNA editing has been studied
the addition or removal of uridine (U) residues at precise extensively, there are few reports of editing in other species
sites of mitochondrial transcripts. This process creates of the class Kinetoplastea (Simpson et al. 2000). Until
initiation and termination codon, corrects frameshifts, and recently, mammalian-infecting Leishmania species were
builds entire open-reading frames from nonsense DNA not analyzed for the presence of RNA editing. Ibrahim and
sequences. The mitochondrial genome of trypanosomatids co-workers (Ibrahim et al. 2008) described that L.
is also peculiar; it consists of a network of two classes of donovani coxII gene is edited in an identical manner as
topologically interlocked circular DNA molecules: maxi- the homologous gene in L. tarentolae. Recently, a more
circles and minicircles (Schneider 2001; Lukes et al. 2005). complete picture of the RNA editing process in this
There exist about 50 copies of the maxicircle DNA (20– species has been reported (Nebohacova et al. 2009). In L.
40 kb in size, depending on the species), they are similar in major, after analysis of the maxicircle sequence and its
composition and contain the complement of genes typically comparison with the L. tarentolae maxicircle sequence, it
found in the mitochondrial genomes. Minicircles are smaller has been suggested that the RNA editing process should
molecules (0.65–2.5 kb), heterogeneous in sequence, that are be equivalent to that described in L. tarentolae (Yatawara
found in 5,000–10,000 copies per organelle. Some of the et al. 2008). More recently, a study on the editing process
maxicircle genes (termed cryptogenes) encode transcripts in Leishmania mexicana amazonensis has been published
that need to be remodeled by RNA editing in order to (Maslov 2010). However, no data have yet been reported
convert them into translatable mRNAs. The genetic infor- on the existence of RNA editing in any Leishmania
mation dictating RNA editing of particular transcripts is species of the subgenus Viannia. Thus, this study provides
specified by short transcripts called guide RNAs (gRNAs), the first experimental evidence of active RNA editing in L.
most of them encoded in the minicircle DNAs (Arts and braziliensis, a species of the subgenus Viannia, which
Benne 1996). cause cutaneous and mucocutaneous leishmaniasis in
The first description of RNA editing was provided by Latin America (Martinez et al. 2010).
Benne et al. (1986) and was based on the finding of a major
transcript of the cytochrome oxidase subunit II (coxII)
containing four nucleotides non-encoded in the DNA and Materials and methods
that corrected the frameshift in the Trypanosoma brucei
coxII gene. Soon thereafter, additional examples of RNA Parasites
editing were reported in T. brucei and in two other
trypanosomatids, the saurian parasite Leishmania tarentolae Promastigotes of L. braziliensis (MHOM/BR/75/M2904)
and the insect parasite Crithidia fasciculata, which became were grown at 26°C in Schneider's medium supplemented
model organisms for studying RNA editing (Arts and with 20% heat-inactivated fetal calf serum and 100 ng/ml
Benne 1996; Simpson and Shaw 1989). Twelve of the 17 of 6-biopterin (Sigma–Aldrich). This strain was provided
pre-mRNAs encoded in the mitochondrial genomes of by the Centro Internacional de Entrenamiento e Inves-
these trypanosomatids were shown to be edited (Stuart tigaciones Médicas (Cali, Colombia).
et al. 1997). Some pre-mRNAs are extensively edited
(pan-edited) and this remodeling may affect more than RNA extraction and RT-PCR
50% of the final sequence. However, the extent of editing
within a given gene may be different between species Total cell RNA was isolated from L. braziliensis promas-
(Seiwert 1995; Simpson et al. 2000). Another remarkable tigotes using Total Quick RNA extraction kit (Talent, Italy).
feature is the co-existence in the mitochondrial steady- First strand of cDNA synthesis was carried out on L.
state RNA population of completely unedited, partially braziliensis total RNA using oligo-dT primer and a cDNA
edited, and completely edited forms for each gene synthesis kit (Pharmacia LKB). Amplification of edited
transcript (Simpson and Shaw 1989). RNA editing is transcripts was performed by RT-PCR on poly(T)-primed
believed to proceed in the 3′ to 5′ direction; however, cDNA using specific primers: LbND8-D, 5′-TTTTA
partially edited RNAs with aberrant patterns of editing are GTGTT TTTAA TAATT ATATG-3′; and LbND8-R, 5′-
frequently observed at the transition of unedited to edited TACTC GTATA ATAAT TAAAC AAAAC-3′. RT-PCR
sequences, in which editing sites contain wrong number of products were separated on agarose gels, visualized under
Us, non-edited sites are edited, and 5′ sites are edited UV transilluminator and purified using the Favorgen gel
before complete editing of 3′ sites. The frequency of this purification kit (Biotech Corp., Taiwan). Finally, the
class of aberrant transcripts is both transcript and species amplification product was cloned into the pCR2.1 T-vector
dependent (Benne 1994). (Invitrogen).
Parasitol Res (2011) 108:731–739 733

Sequence analysis Since the existence of RNA editing in L. braziliensis had

not been documented until now, we considered of interest to
Sequences were determined using the Big Dye Terminators analyze further the clone. Thus far, within the Leishmania
v3.1 kit (Applied Biosystem) by automatic sequencing at genus, experimental demonstration of active RNA editing of
the Servicio de Genómica (Parque Tecnológico de Madrid, mitochondrial transcripts has been obtained for L. tarentolae
Universidad Autónoma de Madrid). Sequence of clone 600 (Simpson and Shaw 1989) L. donovani (Ibrahim et al. 2008)
D has been deposited in the European Nucleotide Archive and L. m. amazonensis (Maslov 2010).
(EMBL-EBI) with the accession number FR686353. In order to check the hypothesis that the 600D cDNA
Sequence analyses were performed using the BioEdit might represent an edited transcript, we searched in the L.
program (Hall 1999). Maxicircle DNA sequences were braziliensis database for the putative cryptogene. A 2083-
obtained from the L. braziliensis database at GeneDB nt-long sequence, centered on the 155 conserved nucleo-
(www.genedb.org). The assembled sequence used in this tides of the 600D clone, was assembled from shotgun reads
study (see Supplementary material) was derived from the found in the L. braziliensis database (see Supplementary
following shotgun reads: brazil20h04.q1k, brazil27a07.q1k, material). Afterwards, a manual alignment between the
brazil81e11.p1k, and brazil802h07.q1k. 600D sequence and the assembled region was carried out
considering the fact that the GAC-sequences of both
Northern blot analysis cryptogene and cDNA must be identical, since RNA editing
affects only the U residues. As shown in Fig. 1, a perfect
L. braziliensis total RNA (4 μg) was fractionated by co-linearity was found between both sequences, suggesting
electrophoresis in formaldehyde (6%)-agarose (1.5%) gel that indeed the clone 600D was derived from an edited
as previously described (Folgueira and Requena 2007). transcript. Moreover, the edition was found to be extensive
After electrophoresis, gels were blotted onto positively in the edited region of transcript 600D (from nucleotide 156
charged Nylon membranes (Roche Diagnostics, Germany). to 301; Fig. 1). The editing entails addition of 81 uridines
The filters were hybridized with the 600D probe (see and removal of 20 uridines at 40 total sites, resulting in an
below), which was labeled with digoxigenin-dUTP using edited region that is 52% larger than the pre-edited region.
the DIG High Prime DNA Labeling Kit (Roche). These data suggested that the 600D transcript was a result
of massive editing, comparable to extreme cases of editing
such as those affecting to COIII (Feagin et al. 1988) and
Results and discussion MURF3 transcripts (Koslowsky et al. 1990) of T. brucei.
The 600D cDNA ended with a non-encoded poly(A) tail, a
In the process of cloning the 3′-UTRs of L. braziliensis typical characteristic of mitochondrial mRNAs in trypano-
HSP70 genes, we isolated a cDNA clone (named 600D) somatids (Van der Spek et al. 1990). Another typical feature
containing a sequence that did not correspond to the of maxicircle genes encoding extensively edited mRNAs is
searched regions. An analysis for sequence homology in the existence of a guanidine (G) versus cystine (C) strand
the L. braziliensis database (GeneDB) yielded no results bias, and the coding sequence located invariably in the G-
when the 600D sequence was compared with either rich strand (Stuart 1991). This bias exists also in the 600D
predicted genes or chromosome contigs, pointing to a cDNA sequence, which has a G%−C% value of 11.8.
cloning artifact. However, when a BLAST search against L. In order to identify whether a protein is encoded in the
braziliensis unassembled shotgun reads was performed, a 600D cDNA, we analyzed possible ORFs. The results were
disconcerting result was obtained. Of the 349 nucleotides rather disappointing, since stop codons were observed in all
composing the 600D cDNA sequence, only the first 155 three frames. Thus, we assumed that the 600D cDNA might
nucleotides were found to be present (100% identical) in a correspond to a partial edited transcript, and searches
large number of unassembled entries. However, none of the against the UniProtKB database (http://www.uniprot.org/)
database entries showed significant homology with the rest were carried out using the amino acid sequences deduced in
of the sequence. An important clue about the nature of this the three frames. Remarkably, short stretches from frames 1
cDNA clone was obtained after searching in the GenBank and 3 yielded significant sequence homology with the
database: the sequence gave a high score of homology with Q7M3U1 entry, which corresponds to the NADH dehydro-
the FJ416603 entry, which contains a partial sequence of genase subunit 8 (ND8,G1) of L. tarentolae. In this species,
the L. donovani maxicircle (Nebohacova et al. 2009). the protein is encoded by the G1 cryptogene that needs to
Again, the homology was restricted to the 5′-end of the be extensively edited to yield a translatable mRNA (Gao
600D sequence. This finding prompted us to analyze et al. 2001; Thiemann et al. 1994). Similarly, in T. brucei,
whether the clone 600D might be a cDNA copy of an the homologous cryptogene, named CR1, is edited by the
edited transcript from the L. braziliensis mitochondria. addition of 259 uridines and the removal of 46 uridines
734 Parasitol Res (2011) 108:731–739

Fig. 1 Editing pattern of the

600D cDNA deduced from
the alignment between the
600D cDNA and L. braziliensis
maxicircle DNA sequence
(LbMax). Edited positions (due
to uridine insertions) in the
600D sequence are shaded in
green. Positions in the genomic
sequences, removed during
editing (uridine deletions), are
shaded in red

(Souza et al. 1992). In both species, only short stretches Assuming that the 600D cDNA was probably an editing
at both ends of the ND8 transcripts are not edited. intermediate of the ND8 transcript, we analyzed sequence
However, despite these tremendous editions, the final conservation between L. tarentolae G1cryptogene and the
amino acid sequences are highly conserved (Thiemann putative homologous region in L. braziliensis. In addition,
et al. 1994). we included in the comparison analysis the sequences for

Fig. 2 Alignment of the ND8

cryptogene of L. tarentolae
(LtND8c; (Thiemann et al.
1994)), DNA sequence for L.
braziliensis maxicircle (LbMax;
this work), L. donovani ND8
cryptogene (LdND8c; positions
1501–1920 from the GenBank
entry with accession number
FJ416603), and L. amazonensis
ND8 cryptogene (LaND8c;
Maslov 2010). Nucleotide
positions with sequence
conservation greater than or
equal to 75% are shaded.
Dashes denote gaps introduced
to maximize alignment. The
nucleotides that form part of the
initiation and termination
codons, which are created by
editing, are underlined
Parasitol Res (2011) 108:731–739 735

this cryptogen in L. donovani (Nebohacova et al. 2009) and similarity) with the ND8 protein from other Leishmania
L. amazonensis (Maslov 2010), which have been recently species (Fig. 4). This analysis showed the existence of a
determined. Figure 2 shows the alignment of ND8 probable frameshift (denoted by “+” in Fig. 3) in the edited
cryptogenes from these four Leishmania species, evidencing region of the 600D cDNA, suggesting that a misedition took
remarkable sequence conservation, which suggests both a place in the original transcript. RNA editing intermediates
selective pressure to maintain the DNA sequence and, with unexpected patterns of editing in which uridines are
probably, a functional importance for these cryptogenes. inserted at sites not normally edited are frequently observed
This high sequence conservation prompted us to deduce a (Sturm and Simpson 1990). Another noticeable finding of
theoretical edited transcript for L. braziliensis ND8 based on the deduced ND8 sequence, also occurring in the L.
the edited ND8 transcript sequence in L. tarentolae (Gao tarentolae sequence, is that the tryptophan residue is coded
et al. 2001). As shown in Fig. 3, it was possible to deduce a by the UGA stop codon. Reassignation of the UGA codon to
hypothetical edition pattern that fits perfectly to the tryptophan occurs in many mitochondria including the ones
cryptogene sequence and, more importantly, codes for an from trypanosomatids (Schneider 2001). In summary, these
amino acid sequence sharing 93% of identity (97% of data suggest that ND8 transcript in L. braziliensis needs to be

Fig. 3 Editing of L. braziliensis

ND8 mRNA (LbND8h) and
comparison with the homolo-
gous transcript of L. tarentolae
(LtND8). The sequence for
the L. braziliensis ND8 crypto-
gene (LbMax) is also shown.
Genomically encoded nucleoti-
des are shown in upper case,
inserted uridines with lower
case (u), and deleted residues
are indicated with asterisks.
Experimentally deduced editing
events, according to the 600D
cDNA, are shaded in green,
whereas those inferred by
sequence comparison with
LtND8 transcript (Gao et al.
2001; Thiemann et al. 1994) are
shaded in yellow. Gaps (shown
with dashes) were introduced
to maximize alignment. The
initiation (AuG) and the stop
(uAA) of the L. braziliensis
ND8 transcript, created by
editing, are indicated in bold.
The predicted amino acid
sequence for L. braziliensis
ND8 (LbND8p) is also shown.
A putative mis-edited position in
the 600D cDNA sequence is
denoted by plus sign, “+”. The
position of the oligonucleotides,
designed for RT-PCR amplifica-
tion of edited ND8 mRNAs, is
shown by underlining in the
LbND8h sequence
736 Parasitol Res (2011) 108:731–739

Fig. 4 Alignment of the predicted

amino acid sequences of ND8
proteins from T. brucei (TbND8;
GenBank accesión number
AAA91499), L. tarentolae
(LtND8; (Thiemann et al. 1994)),
L. braziliensis (LbND8), and L.
amazonensis (Maslov 2010).
Amino acid positions with
sequence conservation greater
than or equal to 75%
are shaded

pan-edited to become a functional mRNA and the 600D oligonucleotide (5′ TACTC GTATA ATAAT TAAAC
cDNA represents only a partial, misedited intermediate. AAAAC 3′), located in the edited region of the 600D cDNA.
Based on the edition pattern deduced for the L. braziliensis These oligonucleotides, and oligo(dT)-primed cDNA from L.
ND8 transcript (Fig. 3), we designed a forward oligonucleotide braziliensis, were used to specifically PCR-amplify edited
(5′ TTTTA GTGTT TTTAA TAATT ATATG 3′), located in the ND8 sequences. After cloning of the RT-PCR product, ten
5′-end, non-edited sequence of the cryptogene, and a reverse cDNA clones were analyzed by restriction analysis. The inserts

Fig. 5 Alignment of cDNA

sequences derived from partial-
ly edited L. braziliensis ND8
transcripts. In the sequence of
the clone 600D, nucleotides
expected to be deleted during
editing are shaded in blue, those
added during editing are shaded
in green, and a misedited nucle-
otide present in the cDNA 600D
is shaded in red. In the sequence
of clone Cl-F, an adenine seems
to have been deleted during
editing (position shaded in
yellow). The position of the
oligonucleotides, designed for
RT-PCR amplification of edited
ND8 mRNAs, is shown by
underlining in the sequence
of clone Cl-B
Parasitol Res (2011) 108:731–739 737

of the clones had a size lower than the expected for the cytochrome b and subunit III of cytochrome oxidase in L.
completely edited RNA, which would be around 550 nt tarentolae (Sturm and Simpson 1990). In summary, our
(Fig. 3). Four of them (clones B, C, F, and H) were sequenced observations suggest that the editing process, at least for the
(Fig. 5). As expected from their insert size, all clones central domains of the ND8 transcript, is quite indiscriminate,
correspond to partial edited intermediates. The extent of adding and deleting variable numbers of uridines at many
edition found in these clones was lower than that observed in sites, and that the editing in these central domains does not
the clone 600 D. Nevertheless, the 3′-ends of the sequences progress in a strictly consecutive 3′ to 5′ direction.
showed an edition pattern identical to that found in clone 600 Since it was not obtained evidence of complete edition
D; however, in upstream regions, the sequences were either of the ND8 transcripts in L. braziliensis by RT-PCR
unedited or misedited. The existence of “unexpected” editing amplification, we carried out a Northern blot analysis using
patterns, which are inconsistent with strictly progressive 3′ to the insert of clone 600D as probe (Fig. 6). RNA samples
5′ editing has been previously documented (Sturm et al. 1992; were obtained from promastigotes growing either at the
Sturm and Simpson 1990). In two of the clones (F and H; logarithmic or stationary phase; also RNA samples were
Fig. 5), the uridine predicted to be misedited in the 600D obtained from promastigotes submitted to heat shock
transcript (Fig. 3; see above), was missing. This finding treatment. In all lanes, a hybridization band of about 0.3–
reinforced the conclusion that this uridine residue was 0.4 kb was observed, but transcripts with higher size,
misadded by the editing process in the 600D transcript. accounting for a complete edited transcript, were not found.
Since all ND8-derived cDNA clones had a different In summary, our data suggest that ND8 transcripts in L.
sequence and, more importantly, they contained many braziliensis (MHOM/BR/75/M2904) promastigotes are
miseditions, it can be postulated that incomplete editing mostly edited in a partial manner. This strain, chosen for
intermediates must be abundantly generated during the L. braziliensis genome sequencing (Laurentino et al. 2004;
editing process of the ND8 transcript in L. braziliensis; it is Peacock et al. 2007), has been propagated axenically for long
likely that the editing of this transcript is very prone to time (in our hands during the last two years) and probably it
error. Abundant, imprecisely edited transcripts have been has experienced a loss of critical gRNAs needed to achieve a
found in the editing process of many trypanosomatid complete editing of ND8 transcript. Apparently, in cultured
mitochondrial RNAs (Decker and Sollner-Webb 1990). In trypanosomes, the NADH dehydrogenase complex is not
fact, partially edited mRNAs are substantially more
abundant than mature mRNAs for extensively edited 1 2 3 4 5
transcripts (Koslowsky et al. 1991). Remarkably, for all
the ND8-derived cDNAs (Fig. 5), the 3′ portion was
identical and correctly edited, the central region was
partially edited (and misedited in some positions) and the
5′ portion was unedited. These features suggest the
existence of several editing domains in the L. braziliensis ND8
ND8 transcript; additionally, these findings are consistent
with a 3′ to 5′ global direction of the editing. However,
within a given domain (i.e., the central region of ND8
cDNAs), editing appeared to be both indiscriminate and
disorganized. Thus, in this region, there are incompletely
edited sites that contain either too many or too few uridines
relative to the edited sequences. Furthermore, some sites
were found to be incompletely edited in more than one way rRNA
in different clones, suggesting that there is no single way to
edit a site during the editing process. For example, clone
Cl-B (Fig. 5) contained an insertion of 11 uridines in a site
where only one uridine must be inserted. Also, this clone
contained insertions at sites where edition is not expected to Fig. 6 Northern blot analysis of ND8 transcripts in L. braziliensis
occur. Perhaps more striking was the observation that an promastigotes. Four micrograms of total RNA from promastigotes
encoded adenine, present in the cryptogene, seems to have growth at 26°C (lane 1) or incubated for 1 h at 32°C (lane 2), 35°C
been deleted during editing process, as deduced from the (lane 3), or 37°C (lane 4), and from promastigotes at the stationary
phase of growth (lane 5) were electrophoresed as described in
sequence of the Cl-F cDNA (Fig. 5). However, clones “Materials and methods”. The insert of 600D clone was used as
displaying unexpected patterns in which purine residues are probe. As load control, the ethidium bromide-stained agarose gel is
deleted have been found in partially edited mRNAs for shown (bottom panel)
738 Parasitol Res (2011) 108:731–739

functional and not used for energy production (Benne 1994). this species may be a labile genetic trait. However, to verify
Disruption of RNA editing by prolonged cell culture has whether gRNA loss did occur in L. braziliensis strain
been documented in L. tarentolae (Thiemann et al. 1994) M2904, a complete characterization of both maxicircle and
and L. donovani (Nebohacova et al. 2009). In L. tarentolae, minicircle sequences needs to be done, and a comparison with
species in which this phenomenon has been studied more those of recent isolates of this species should be carried out.
extensively, transcripts of the G1–G5 cryptogenes are pan- Furthermore, determination of possible differences in RNA
edited in a recently isolated strain (LEM125), but not in the editing between promastigotes and amastigotes, using serial
UC strain which has been axenically cultured for many analysis of gene expression (Ouakad et al. 2007; Li et al.
years. At least 32 minicircle-encoded gRNAs, present in 2008), would help to deciphering the actual importance of
LEM125, were found to be absent in UC strain (Thiemann et this process in Leishmania. This is likewise the emphasis in
al. 1994). The L. tarentolae G1 transcript is the homologous the next stage of our research.
to the L. braziliensis ND8 transcript, reported in this work.
As shown in Fig. 2, both cryptogenes are highly conserved, Acknowledgements Genome sequence data from the Sanger Institute
sequencing projects (GeneDB) were invaluable for this work and their
suggesting that similar edition pattern must occur to obtain
provision in the public domain is gratefully acknowledged. This work
the mature ND8 mRNA. In fact, the RNA edition of G1/ was funded by grants from the Ministerio de Ciencia e Innovación
ND8 transcripts, experimentally demonstrated, is highly (BFU2009-08986), the Spanish Ministry of Science and Innovation and
conserved in both species (Fig. 3) and yields nearly identical the Instituto de Salud Carlos III within the Network of Tropical Diseases
Research (RICET RD06/0021/0008 - FEDER) to JMR, and the
amino acid sequences for the deduced proteins (Fig. 4). The
Colciencias Research project No. 1203-405-20233 to CP. CR is the
complete edition of G1 transcript in L. tarentolae requires the recipient of a predoctoral fellowship from Colciencias (Colombia). Also,
sequential use of 13 gRNAs (Gao et al. 2001; Thiemann an institutional grant from Fundación Ramón Areces is acknowledged.
et al. 1994). The edition of the partially edited ND8 in the
600D cDNA would need of the use of gRNAs equivalent to
L. tarentolae gND8-I, -II, -III, and -IV RNA guides. References
Therefore, it is likely that the gND8-V homologous in L.
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