Vol 27 No 2 December - The Indian Society for Parasitology

106 AMA1 and DBP polymorphism in P.vivax
JPD : Vol. 27 (2), 2003
Figure-1: PCR product and RFLP patterns of AMA-1 (HVR)
gene digested with Pvu-II restriction enzyme of few
representative isolates of P. vivax collected from Northern region
of India. (Lane-1 Negative control, Lane-2 DNA molecular
weight Marker (100 bp ladder plus). Lane 3-6 RFLP patterns of
representative isolates, Lane-7 PCR Product)
Figure-2: PCR product and RFLP patterns of DBP (Central
Polymorphic region) gene digested with Eco R-1 restriction
enzyme of few representative isolates of P. vivax collected from
Northern region of India. (Lane-1 DNA molecular weight
Marker {100 bp ladder}; Lane-2 PCR product of isolates 3; Lane
3-8 RFLP pattern of shown isolates.
DISCUSSION
The results of present study confirm the earlier
observation (Oliveira et al 1996) that AMA-1 gene is
largely conserved because of functional constraints of
this gene in the biology of parasite. The absence of size
variation in this segment of AMA-1 gene has also been
observed in Philippines and Indonesian isolates of
P.vivax by sequencing techniques (Cheng and Saul
1994). The sequencing of full length of AMA-1 gene
also showed limited polymorphism (Oliveira et al
1996, Cheng and Saul 1994). Figtree et al (2000) in a
study on isolates collected from different geographical
regions including Africa, China, India, Indonesia,
Philippines, Papua New Guinea, Solomon Island and
Thailand also did not observe any size variation in 219
isolates in the same region of the gene. These
investigators observed dimorphism in 4 nucleotides
(680 - 684) in all isolates except those from Africa by
sequencing analysis. However, Cheng and Saul (1994)
has also shown the presence of polymorphism in
variable region particularly at the 5' end of AMA-1
gene.
The MP-1 family of erythrocyte binding proteins of
Plasmodium species has a highly conserved structure
(Adams et al 1992). In the present study, no size
variation was observed in DBP gene among these
clinical isolates. This is in accordance with findings of
Tsuboi et al (1994) who did not observed size variation
among 50 clinical isolates collected from hyper
endemic area of Papua New Guinea. Recently another
study from Korea also reported no size variation
among 30 clinical isolates (Kho et al 2001)
PCR amplified product was subjected to enzymatic
digestion with Eco-R1 to check the restriction fragment
length polymorphism. Digestion resulted into two
fragments of 1.6 Kb and 700 bp in all the isolates
showing no difference in RFLP pattern of different
isolates. However, in contrast to our study, Tsuboi et al
(1994) studied the same region of DBP (RIV) and
reported variation in RFLP patterns with Eco-R 1.
Further they observed the presence of three different
types of insertions within R-IV on the basis of
nucleotide sequence analysis i.e., 30 bp, 6 bp and both
30 bp and 6 bp. On the basis of these inserted
nucleotide sequences, Tsuboi et al (1994) has
categorized strains into three allelic types (group 1, 2
& 3). Several studies conducted in diverse
geographical region have reported polymorphism in
principal Duffy binding domain (R-II) of DBP gene.