Experimental Parasitology 106 (2004) 110118

www.elsevier.com/locate/yexpr

Arbitrary-primed PCR for genomic ngerprinting

and identication of dierentially regulated genes in Indian
isolates of Leishmania donovani
G. Sreenivas,a Ruchi Singh,a A. Selvapandiyan,b N.S. Negi,c
Hira L. Nakhasi,b and Poonam Salotraa,*
a
Institute of Pathology (ICMR), Safdarjung Hospital, New Delhi 110029, India
b
Division of Emerging and Transfusion Transmitted Diseases, OBRR/CBER/FDA, Rockville, MD 20892, USA
c
Department of Medicine, Safdarjung Hospital, New Delhi 110029, India

Received 23 June 2003; received in revised form 30 January 2004; accepted 30 March 2004
Available online 30 April 2004

Abstract

The arbitrary-primed PCR (AP-PCR) technique was employed with the twin goals of identifying genetic polymorphisms within
the Indian isolates and to identify dierentially expressed gene sequences. The parasite isolates from Indian Kala-azar patients could
be dierentiated from Leishmania donovani isolates from distinct geographic regions. Moreover, dierences within the Indian iso-
lates could also be identied. A majority (17/19) of the Indian isolates gave identical AP-PCR pattern, while two isolates gave
consistently divergent pattern. The distinctive AP-PCR fragments obtained with Indian isolates were used as probes in Northern
blot analysis. Three such fragments were found to represent transcribed sequences that were dierentially expressed in the two stages
of the parasite. These sequences led to cloning and characterization of Leishmania Centrin gene and a novel gene termed A-1 that is
over-expressed in amastigote stage of the parasite. The study demonstrates the utility of random genome sampling methods in
genomic ngerprinting and in identifying dierentially transcribed sequences that could potentially contribute to parasite virulence.
2004 Elsevier Inc. All rights reserved.

Index Descriptors and Abbreviations: Leishmania donovani; AP-PCR, arbitrary-primed polymerase chain reaction; Genomic ngerprinting; Poly-
morphic fragment; Dierentially expressed genes

1. Introduction 25 countries (Desjeux, 1998; Pintado and Lopez-Velez,

Leishmania currently infects about 12 million people
in 88 countries, with 600,000 new clinical cases reported
annually with 350 million at risk (WHO, 1990). The two
major clinical forms of leishmaniasis, cutaneous and
visceral, are the result of infection by dierent species of
the parasite. Visceral leishmaniasis (VL), fatal if left
untreated, is caused by the members of Leishmania
donovani complex that include L. d. donovani, L. d. in-
fantum, and L. d. chagasi. The problem of VL has be-
come more serious because of coinfection with HIV that
is becoming increasingly frequent with cases reported in
*
Corresponding author. Fax: +91-11-2619-8401.
E-mail address: salotra@vsnl.com (P. Salotra).
2001). More than 90% of the VL cases in the world are
reported from Bangladesh, Brazil, India, and Sudan. The
causative organism in India is L. d. donovani, where it is
endemic in the Northeastern regions of the country,
primarily in the state of Bihar (Sundar et al., 2001). The
disease often turns epidemic claiming lives of thousands
and causing severe morbidity to hundreds of thousands.
The ocial estimate of 430,000 VL cases in Bihar state of
India over the past 11 years may represent only a fraction
of the real numbers. The actual number is believed to be
at least ve times as great (Monograph, 1996). Control of
leishmaniasis is complicated by the multiplicity of
Leishmania species and their diverse clinical manifesta-
tions (Amaral et al., 2002). Understanding Leishmania
genetic diversity is important for epidemiological and

0014-4894/$ - see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.exppara.2004.03.011
 G. Sreenivas et al. / Experimental Parasitology 106 (2004) 110118
taxonomic studies as well as for precise diagnosis. Fur-
ther, accurate identication of pathogenic agent at the
level of subspecies and strain provides a rm basis for all
studies of medical relevance. The characterization of
Leishmania species and strains can be accomplished by
clinical, epidemiological, and biochemical criteria. A
number of biochemical methods have been developed
including isoenzyme proling and use of monoclonal
antibodies (Kreutzer and Christensen, 1980; McMahon-
Pratt and David, 1981; Noyes et al., 1996). In recent
years, genetic diversity in L. donovani has been assessed
using an array of molecular markers (ElTai et al., 2000;
Mauricio et al., 1999, 2001). Methods such as PCR-
SSCP allow scanning of genes for single base dierences
that could generate useful genetic markers (ElTai et al.,
2001). Similarly parasite typing with species or complex
specic PCR primers could distinguish parasite isolates
from Sudan, Mexico and Brazil (Amalia and Gustavo,
2002; Andersen et al., 1996; ElTai et al., 2000, 2001; Is-
hikawa et al., 2002). However, existing studies have not
yet looked at clinical isolates of L. donovani from Indian
population. Mapping of genetic diversity within
L. donovani species is likely to have an impact on a range
of problems such as drug resistance, response to
vaccines, and transmissibility through vector.
In the Leishmania life cycle, the dierentiation from
promastigote to amastigote stage is associated with
dramatic morphological and biochemical changes that
have been correlated with changes in gene expression
(Handman, 1999; Molyneux and Killick-Kendrick,
1987). A number of studies have been conducted
searching for changes in gene expression amongst
Leishmania stages, using methods such as dierential or
subtractive hybridization, AP-PCR, dierential display,
and cDNA microarrays derived from spliced leader li-
braries (Almeida et al., 2002; Bellatin et al., 2002; Cha-
rest and Matlashewski, 1994; Coulson and Smith, 1990;
Heard et al., 1996; Liu et al., 2000; Pogue et al., 1995a,b;
Wu et al., 2000). We have exploited AP-PCR technique
to map genetic dierences within the Indian isolates of
L. donovani and to identify DNA polymorphisms that
distinguish geographic isolates. Further, we utilized the
polymorphic DNA fragments to identify, isolate, and
characterize dierentially expressed gene sequences.
2. Materials and methods
2.1. Parasites
The reference stocks of Leishmania used in this study
were; L. donovani 1S from Sudan (WHO designation,
MHOM/SD/00/1S-C12D), L. donovani WR657 from
India (MHOM/IN/80/DD8), L. donovani WR684
from Ethiopia (MHOM/ET/67/82), L. donovani AG83
from India (MHOM/IN/83/AG83), and L. d. infantum
111
from Spain (MCAN/SP/00/XXX). In addition, clinical
isolates of L. donovani prepared from 19 patients of
kala-azar were included in the study.
2.2. Promastigotes and axenic amastigotes from clinical
samples
Bone marrow samples were collected from KA pa-
tients originating from Bihar, reporting to Safdarjung
Hospital, New Delhi. Prior consent from patients was
obtained for collecting the bone marrow samples fol-
lowing the guidelines by the Institutional Ethical com-
mittee. The patients presented with clinical symptoms of
KA such as fever, hepatosplenomegaly, anaemia, and
leucopenia. The patients were evaluated clinically and
conrmed by demonstration of amastigotes in bone
marrow aspirates. The bone marrow samples were col-
lected in medium M199, pH 7.4, and 25 mM Hepes
supplemented with 10% heat inactivated fetal calf serum
with 100 lg/ml streptomycin and 100 U/ml penicillin and
incubated at 26 C. The promastigotes thus obtained
were propagated and used to isolate DNA. Cytodier-
entiation into axenic amastigotes was accomplished by
inducing promastigotes to gradually adapt to altered
growth conditions (Joshi et al., 1993). Initially, pH of
the medium (RPMI-1640) was set to 5.5 and the tem-
perature was raised gradually to 37 C in a 5% CO2
environment. Once adapted to grow in these conditions,
the parasites were cycled between the two stages every
5th day.
2.3. Isolation of nucleic acids
Genomic DNA and total RNA were prepared from
minimally passaged parasites from Kala-azar isolates.
Mid log phase parasites (1
109 cells) were washed twice
with cold PBS and used for DNA isolation by Wizard
Genomic DNA kit (Promega). Total RNA from
promastigotes and axenic amastigotes was prepared
from parasites at identical time points using Trizol re-
agent (Life technologies).
2.4. AP-PCRs
AP-PCRs were carried out in a 50 ll reaction mixture
containing 200 lM each of dNTP, 2 mM MgCl2 , 100 ng
oligonucleotide, 100 ng template DNA (unless stated
otherwise), and 2.5 U Taq DNA polymerase overlaid
with mineral oil. The sequence of AP primers used and
the PCR conditions were same as those reported earlier
(AP8-TGCCGAGCTG; AP9-GTTGCGATCC; AP-10-
AGGTGACCGT; and AP16-CAGCACCCAC: Pogue
et al., 1995a,b). Amplication products from AP-PCRs
were analyzed by electrophoresis in 1% agarose gels and
by staining with ethidium bromide. The pattern of AP-
PCR amplication fragments is sensitive to the amount
112 G. Sreenivas et al. / Experimental Parasitology 106 (2004) 110118
of genomic DNA template and the amount of AP oligo
(Welsh and McClelland, 1990). The amount of template
and AP oligo were optimized for obtaining reproducible
amplication from all Leishmania species tested. In case
of oligo AP-9, amplication reactions were also carried
out with 50 and 150 ng DNA concentration to obtain
additional unique DNA fragments. Polymorphic DNA
bands were sliced from the agarose gels and puried
with QiaQuick gel extraction kit (Qiagen) following the
protocol supplied.
2.5. Southern and Northern blot analysis
Genomic DNA (5 lg per lane) was digested with the
restriction enzyme and blotted on to charged nylon
membrane (Hybond , Amersham). Total RNA (15 lg
per lane) isolated from mid log-phase axenic promasti-
gote and amastigote cultures of Indian isolates, dena-
tured by formaldehyde/formamide treatment was
separated in 1% agaroseformaldehyde gels in 1
Mops
buer according to method described previously (Joshi
et al., 1993). Southern and Northern blots were hy-
bridized with 32 P-labeled probes prepared in a random
octamer labeling reaction using NEBlot kit (New En-
gland Biolabs). Blots were visualized by autoradiogra-
phy using Biomax lms (Kodak). The intensity of the
hybridized bands was quantied from autoradiograms
using AlphaEase software (Alphaimager) and average of
three experiments was taken. Normalization of the
RNA added in each lane was done by reprobing the
blots with 32 P-labeled a-tubulin.
2.6. RT-PCR
Total RNA from axenic amastigote or promastigote
parasites was used in a reverse transcription reaction to
synthesize rst strand cDNA using oligo(dT) (Invitro-
gen). This was followed by the PCR with specic
primers from the coding region of the gene of interest
involving cycles performed at 94 C for 30 s, 50 C for
30 s, and 72 C for 1 min.
2.7. Nucleotide sequence analysis
The polymorphic AP-PCR DNA fragments and the
various PCR amplied products were cloned into pCR
II-TOPO vector system (Invitrogen) and sequenced on
automated sequencer (PerkinElmer).
3. Results
3.1. Parasite isolation and characterization
Leishmania parasites were isolated from bone marrow
aspirates of Indian patients of Kala-azar (n 19) and
propagated as promastigotes for 35 passages in vitro
before DNA isolation. All parasite isolates were char-
acterized as L. donovani by isoenzyme analysis using a
panel of enzymes (6PGD, G6PDH, NH, ME, and
MDH) according to the standard method (Kreutzer and
Christensen, 1980) and by immunouorescence assay
(Noyes et al., 1996) using a panel of species-specic
monoclonal antibodies against L. donovani, Leishmania
major, and Leishmania tropica, obtained as a kind gift
from WHO.
3.2. Finger printing of L. donovani isolates by AP-PCR
A subset of AP-PCR primers that were shown to
yield consistent amplication products (Pogue et al.,
1995a,b) was employed to obtain amplication patterns
from genomic DNA of L. donovani isolates prepared
from bone marrow samples of Indian Kala-azar pa-
tients. We report here the comparison of genomic n-
gerprints of 19 isolates of L. donovani, all from patients
originating from Bihar, an area endemic for Kala-azar
in India. A large majority of isolates (17/19) displayed
identical amplication prole (isolates K1K17) while
two isolates (K18 and K19) showed a divergent pattern
with oligo AP-9 (Fig. 1A). The two isolates K18 and
K19 that deviated from the typical pattern showed a
divergent prole when amplied with another oligo, AP-
10 (Fig. 1B).
The amplication proles from the 19 Indian isolates
of the two categories identied above were compared to
L. donovani clonal isolates from Sudan (Ld1S), India
(AG83), Ethiopia (WR684), and L. infantum (Spain)
Fig. 1. Fingerprinting of Indian isolates of L. donovani by AP-PCR.
Genomic DNA (100 ng) from parasites isolated from bone marrow
samples of Kala-azar patients (K1K19) was subjected to AP-PCR
analysis. Lanes 119 show pattern with isolates LdK1LdK19, using
oligo AP-9 (A) and oligo AP-10 (B); M, 1 kb ladder.
 G. Sreenivas et al. / Experimental Parasitology 106 (2004) 110118 113

Fig. 2. Genomic ngerprinting of L. donovani isolates from distinct geographic locations. (A) AP-PCR with oligo AP-8. L. d. donovani DD8 from
India; L. d. donovani Ethiopia; L. d. infantum from Spain; L. donovani Sudan; L. donovani AG83 India; and clinical isolates LdK1, LdK2, LdK18,
and LdK19. (B) AP-PCR using oligo AP-16. L. d. donovani from Ethiopia, L. d. infantum from Spain, L. donovani from Sudan, L. donovani AG83
from India, and clinical isolates LdK1, LdK2, LdK18, and LdK19.

using oligos AP-8 and AP-16. Representative data for
two isolates of each category has been shown (Figs. 2A
and B). The prole with Indian isolates was distinct
from L. donovani isolates from Ethiopia, Sudan or
L. infantum from Spain (Figs. 2A and B). The AP-PCR
prole of the predominant isotype of KA isolates was
similar to that of the reference strains of Indian origin.
The two isolates K18 and K19 consistently showed a
divergent pattern while the remaining 17 isolates (K1
K17) exhibited identical genomic ngerprint with all
oligos examined. Isolate LdK1 belonging to the pre-
dominant isotype was used in all subsequent studies.
The amplication patterns in AP-PCR are sensitive to
the primer and template DNA concentration (Welsh
and McClelland, 1990). When the template DNA con-
centration was altered, remarkably dierent patterns
were obtained with AP-9. Comparison of LdK1 with
geographic isolates with AP-9 under dierent reaction
conditions yielded distinct amplication proles (Figs.
35). Dierences in the AP-PCR proles were also evi-
dent between clinical isolates with minimum in vitro
passaging as compared to LdAG83 strain that has been
frequently passaged in vitro over several years (lanes
LdK1, LdAG83, Fig. 3).
3.3. Identication of polymorphic fragments in Indian
isolates
It has been shown that AP-PCR is an attractive tool
in assigning specic ngerprint to Ld geographic isolates
that are morphologically and biochemically indistin-
guishable (Pogue et al., 1995a,b). Using a typical isolate
LdK1, attempt was made to detect unique DNA se-
quences in AP-PCR to identify transcribed sequences
corresponding to dierentially expressed genes. Com-
parison of the AP-PCR ngerprints of Indian isolates
with geographic isolates revealed unique polymorphic
DNA fragments of size 1.6 kb and a minor band of
1.2 kb (termed K-1.6 and K-1.2, respectively) in Indian
isolates following amplication reaction with oligo AP-9
using 100 ng DNA (lane LdK1, Fig. 3A). On varying the
template DNA concentration to 150 ng, a distinct am-
plication prole was obtained and an additional poly-
morphic fragment of 1.4 kb (K-1.4) was identied (lane
LdK1, Fig. 4A). In AP-PCR with 50 ng DNA, another
polymorphic fragment of size 0.45 kb (K-0.45) was
identied (lane LdK1, Fig. 5A).
3.4. Northern hybridization with cloned unique DNA
fragments
We wished to investigate if the polymorphic fragments
that were obtained by AP-PCR encompass transcribed
sequences, considering the low frequency of introns and
high gene density in trypanasomatid parasites. The four
distinctive DNA fragments obtained from Indian iso-
lates of L. donovani were cloned into pCRII-TOPO and
used as probes in Northern blot analysis of total RNA
derived from promastigotes and axenically grown am-
astigotes of isolate LdK1. The probe containing the K-
1.2 fragment hybridized to a 1.7 kb RNA (Fig. 3B) that
gave 2-fold higher expression in promastigote stage in
comparison with amastigote stage. The K-1.6 DNA
fragment did not yield any positive signal on Northern
114 G. Sreenivas et al. / Experimental Parasitology 106 (2004) 110118

Fig. 3. Identication of transcribed sequences from the polymorphic fragments in AP-PCR with AP-9. (A) AP-PCR prole using 100 ng genomic
DNA as template with oligo AP-9 in Ld Sudan, Ld Ethiopia, L. infantum from Spain, Ld AG83 from India, and clinical isolate LdK1. M, 1 kb
ladder. The arrows point to polymorphic fragments identied in K1. (B) Northern hybridization with total RNA isolated from pro and amastigotes
using K-1.2 as probe. Total RNA (15 lg) from promastigotes (lane P) or amastigotes (lane A) was resolved in 1% formaldehyde/formamide gels.

analysis, indicating that it did not represent a transcribed
sequence (data not shown). The K-1.4 probe hybridized
with a 1.7 kb RNA that displayed 2.4-fold higher abun-
dance in amastigotes compared to promastigotes
(Fig. 4B). The K-0.45 probe hybridized with 1.8 kb RNA
that showed 1.5-fold higher expression in promastigote
stage (Fig. 5B). The dierential expression on Northern
was repeated at least three times to conrm its authen-
ticity. Further, it was validated by taking RNA from two
separate parasite preparations of L. donovani to preclude
biological variation due to clonal nature of gene ex-
pression.
3.5. Cloning of dierentially expressed genes
Two of the three fragments representing transcribed
sequences that exhibited dierential expression in
Northern analysis were selected for cloning of full length
genes. The K-1.2 fragment that hybridized to 1.7 kb
RNA in Northern (Fig. 3B) led to identication of
Centrin gene in Leishmania. Cloning, sequencing, and
functional analysis of this gene was carried out and re-
ported elsewhere (Selvapandiyan et al., 2001).
The nucleotide sequence of the K-1.4 clone that hy-
bridized with a 1.7 kb RNA (Fig. 4B) revealed that it
contained 30 portion of a novel ORF (termed A1). To
identify the 50 end of this partial ORF, a reverse tran-
scription PCR with conserved spliced leader sequence as
forward primer and A-1 gene specic internal primer
was performed. The reaction yielded a 0.73 kb product
with pro- as well as amastigote derived total RNA. The
RT-PCR product was cloned into pCRII-TOPO and its
nucleic acid sequence determined. The full length A-1
ORF was PCR amplied from the LdK1 genomic DNA,
 G. Sreenivas et al. / Experimental Parasitology 106 (2004) 110118 115

Fig. 4. Identication of polymorphic amplied DNA fragments followed by Northern analysis. (A) AP-PCR prole with oligo AP-9 using 150 ng
genomic DNA as template from LdK1, Ld Sudan, L. d. infantum from Spain, Ld Ethiopia, L. tropica, and L. major. The arrow points to polymorphic
fragment identied in K1. (B) Northern hybridization with total RNA isolated from pro and amastigotes using K-1.4 as probe. Other details same as
in Fig. 3.

cloned into pCRII-TOPO and the nucleotide sequence
conrmed (GenBank Accession No. AY326448). Se-
quence analysis indicated that the A-1 gene contained a
528 bp ORF coding for a 20 kDa protein. A search of
GenBank database with BLAST revealed that the A-1
sequence was highly homologous to unannotated se-
quence of chromosome-29 clone LB00711 of L. major
strain Friedlin (GenBank Accession No. AC133777.2).
The database matching conrmed that the clone was
genuine Leishmania genomic fragment, but did not
specify if it represented expressed or intergenic sequence.
Searches for homologues of A-1 in L. major nucleotide
sequence database (http://www.ebi.ac.uk:parasites:le-
ish.html) did not reveal members with signicant ho-
mology. Comparison of the deduced aminoacid
sequence of the A-1 by BLAST analysis in GenBank or
in Leishmania database did not show any matches with
signicant homology. Expression and characterization
of this gene was undertaken (manuscript under
preparation).
3.6. Genomic background of Centrin and A-1
We sought to determine the genomic background of
Centrin and A-1 in Leishmania parasites to explore the
possibility of using these genes as tools to dierentiate
dierent strains/species of Leishmania. To that end we
carried out a Southern blot analysis, using genomic
DNA from dierent strains of Leishmania digested with
NcoI or StuI restriction enzymes and probed with cen-
trin gene. The results showed that the DNA from
Leishmania mexicana had polymorphism at an NcoI site
(Fig. 6A). A comparative Southern blot of DNA sam-
ples probed with the coding region of A1 ORF displayed
polymorphism with SacI (Fig. 6B) where as L. tropica
showed polymorphism with BglII (Fig. 6B).
4. Discussion
We describe in this report, the genomic ngerprinting
of Indian isolates of L. donovani obtained from Kala-
azar patients originating from the endemic area, Bihar,
by amplifying the genomic DNA with single arbitrary
primers. The arbitrary PCR employed low-temperature
stringency to allow sampling of diverse portions of
Leishmania genome without any apparent bias.
The PCR with random oligos is capable of producing
unique genetic ngerprints from closely related organ-
isms and this attribute has been previously utilized to
dierentiate several groups of parasites in trypanoso-
matid (Guizani et al., 2002; Pogue et al., 1995a,b; Scho-
nian et al., 1996; Tibayrenc et al., 1993; Waitumbi and
Murphy, 1993), coccidial (Procunier et al., 1993; Shirley
and Bumstead, 1994) or schistosome parasites (Neto
et al., 1993). We obtained distinct and reproducible
pattern of amplied DNA fragments with various
arbitrary primers examined. The Indian isolates pro-
duced categorically divergent PCR proles compared to
L. donovani from distinct geographic regions. The pattern
116 G. Sreenivas et al. / Experimental Parasitology 106 (2004) 110118

Fig. 5. Identication of polymorphic amplied DNA fragments followed by Northern analysis. (A) AP-PCR prole using with oligo AP-9 and 50 ng
genomic DNA from Ld K1, Ld Ethiopia, Ld Sudan, and L. d. infantum. The arrow points to polymorphic fragment identied in K1. (B) Northern
hybridization with total RNA isolated from pro and axenic amastigotes using K-0.45 as probe. Other details same as in Fig. 3.

Fig. 6. Southern blot analysis of the genomic DNA. Five micrograms of genomic DNA from Ld India, Ld Ethiopia, Ld Sudan, L. infantum, L.
mexicana, L. major or L. tropica was digested with the restriction enzymes indicated, resolved on 1% agarose gels and probed with the coding region
of the Centrin (A) or A-1 (B).

of amplied fragments obtained was identical in a large between the genetic variability within the L. donovani
majority of Indian isolates. Two of the 19 isolates exhibited isolates and the endemicity status of the region from which
a pattern dissimilar from the rest with each of the primers the isolates were obtained, since all 19 isolates originated
tested. However, no correlation could be ascertained from an area in Bihar that is hyper endemic for KA.
 G. Sreenivas et al. / Experimental Parasitology 106 (2004) 110118
Our results demonstrate that unique and reproducible
ngerprints can be assigned to distinct geographic iso-
lates within L. donovani species by AP-PCR. The re-
markable similarity obtained within the Indian isolates
testies to the relevance of AP-PCR technique in ge-
nomic ngerprinting. Interestingly, the predominant
class of isolates showed AP-PCR proles quite similar to
standard reference strains of Indian origin, AG83 and
DD8. However, the standard reference isolates could be
distinguished from the predominant isotype of KA iso-
lates recently placed in culture, by AP-PCR under cer-
tain conditions. The deviant pattern with WHO
reference strains in comparison with the clinical isolates
that were minimally passaged in vitro indicates the ad-
vantage of using newly prepared isolates for the purpose
of identifying dierentially transcribed sequences based
on the unique amplied DNA fragments since the
Leishmania parasites are known to accumulate sequence
polymorphism on longtime in vitro culture.
Studies on intraspecic polymorphism in 23 Sudanese
isolates of L. donovani revealed at least three dierent
proles in the internal transcribed spacer1 (ITS-1) re-
gion, although PCR ngerprinting using single primers
gave highly similar fragment patterns among the 23
isolates (ElTai et al., 2000). In the context of genetic
polymorphism that was shown to exist among Indian
isolates, it remains to be seen how this diversity trans-
lates into deducing relevant medical properties/pheno-
typic eects such as infectivity, virulence, relationship
between pathology, and refractoriness to chemotherapy.
At present genetic markers for none of the above attri-
butes exist. The occurrence of polymorphism within the
same geographic area is a signicant step towards
identifying such markers.
To understand Leishmania pathogenesis and to de-
velop means of disease prevention and treatment,
functional analysis of new genes is required. Compari-
son of the changes in gene expression among dierent
growth stages and isolation of the developmentally
regulated genes would help to elucidate the mechanisms
of gene regulation so as to provide the information
needed to disrupt the progression of the disease. Earlier,
arbitrarily primed polymerase chain reaction approach
has been exploited in surveying the Leishmania genome
for gene sequences of interest and several classes of
dierentially expressed genes from dierent isolates of
L. donovani strain have been identied and some of them
revealed unique expression (Pogue et al., 1995a,b; Sel-
vapandiyan et al., 2001).
The relative rarity of introns and short intergenic
regions of Leishmania genome allowed us to ask if the
polymorphic fragments could be used to isolate partial
coding regions that are dierentially transcribed be-
tween the parasite life stages. In present study, four such
fragments were identied and used as probes in North-
ern analysis and three were observed to represent tran-
117
scribed sequences. Moreover, the fragments hybridized
to RNA species that exhibited dierential expression in
the two stages of the parasite, pro and amastigote. One
such polymorphic fragment lead to identication of
human homologue of Centrin in Leishmania. Centrin
was found to be a growth regulated gene in L. donovani
and mutant parasites over-expressing the centrin that
lacked N-terminal Ca2 binding domain showed a
negative dominant eect on the parasite growth in
Leishmania (Selvapandiyan et al., 2001).
Another unique fragment hybridized to RNA species
that showed signicantly higher expression in amasti-
gote stage of the parasite. The full coding region from
this fragment was assembled from the sequence of the
RT-PCR product using conserved spliced leader se-
quence as forward primer. The nucleotide sequence
comparison by BLAST analysis in GenBank indicated
that it is a novel ORF. Functional characterization of A-
1 gene is currently in progress. The dierential expres-
sion of A-1 gene at the phenotype and species level
suggest that its intensive study should lead to important
insights into the dierentiation process and hence viru-
lence. It is of interest to see if these expression patterns
are exclusive to viscerotropic species. Finally, successful
genetic manipulation of these genes could lead to further
dening their function and contribution to the Leish-
mania life cycle.
Acknowledgments
This work was supported by a grant under Indo-US
Vaccine Action Program. R.S. is grateful to CSIR for
nancial support.
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