- Email: [email protected]
Genetic polymorphism of the Duffy receptor binding domain of Plasmodium vivax in Colombian wild isolates
MOLECULAR
iii%mMIcAL PARtisIToLoGy
ELSEVTER
Molecular and Biochemical Parasitology 78 (1996) 269-272
Short communication
Genetic polymorphism
of the Duffy receptor binding domain of Plasmodium vivax in Colombian wild isolates’
Elizabeth Imtitt~ro
Ampudia,
de Inmunolo~ic~,
Hospital
Manuel
Alfonso Patarroyo, Luis Angel Murillo*
SCM Juun de Dim.
Unicersidud
Nacionol
Manuel de Colomhiu
Elkin Patarroyo, , Santaf~
de Bogotci
Color~~hiu
Received 7 December 1995: revised 1I March 1996: accepted 12 March 1996
Keword.s: DBP:
PCR; P. uitu~; Malaria;
Duffy receptor;
Polymorphism
-
The Plusmodium vivax Duffy receptor binding protein (DBP) is a member of the protein family localized in the Plasmodium parasite micronemes that binds to erythrocytes carrying this receptor. Given its importance in the parasite life cycle, it is being considered as a potential candidate in vaccine development against P. viva infection [1,2]. Its molecular weight is 140 kDa and the primary structure has been previously reported for the Sal-I strain [3,4]. This protein is divided into five important regions: a leader peptide sequence, two cysteine rich regions separated by a non-homologous hydrophilic region, and a transmembrane domain (Fig. la). The amino terminal cysteine-rich region has been implicated in the binding process to red blood cells (RBCs) carrying the Duffy receptor in a way analagous to what has been demonstrated with the EBA-175 protein Ahbrrt~icrtiorw: PCR, polymerase chain reaction; DBP, DuKy receptor binding protein; RBC, red blood cells. * Corresponding author: ’ Note: Accession numbers: U50575-U50591.
of P. falciparum [557]. A previous report showed natural variation within this critical ligand domain in the P. vivnx Sal-I strain [8]. Polymorphism studies within this region are highly significant for the rational design vaccines against asexual blood stages of P. vivus. Twenty samples were obtained from two different regions of Colombia where malaria is markedly seasonal: one in the South-Western Pacific side (Tumaco) and the other on the Eastern side (Villavicencio) of the Andes (with an annual prevalence rate of P. vivax of 36 and 64X, respectively). Purified Belem strain DNA was used as a positive control. DNA was extracted from each sample and used as a template for PCR. A 1150-bp fragment, corresponding to nucleotide positions 738-1888 (aa 1722555) of the amino terminal cysteine-rich region of the Dutfy receptor, was amplified. All samples gave a positive hybridization signal when tested with the PCR amplified fragment from Belem strain as a probe (Fig. lb). An extra amplified band of 800 bp was observed in 4 of the 20 samples which did
0166-6851/96/$15.00 f-3 1996 Elsevier Science B.V. All rights reserved PII Sl66-6851(96)0261 l-4
270
E. Ampudia et al. 1 Molecular and Biochemical Parasitology 78 (1996) 269-272
not hybridize with the control probe. All fragments were cloned in pMOS blue (Amersham, UK) and sequenced using Sequanase v.2.0 (United States Biochemical). In order to detect any possible nucleotide mis-incorporation due to Taq polymerase activity, 3 clones of each one of the amplification products were sequenced. Based on these results, ten variant families (named I to X) were identified on the basis of grouping mutations in the nucleotides and their corresponding amino acids (indicated in Fig. 2). Five families were identified in each of the two geographical areas studied. The positions of cysteine residues were conserved between the Colombian samples and the reference (Belem) strain. Most of the predicted amino acid changes correspond to replacements with glutamic acid, lysine or aspartic acid. From a total of 383 amino acids, 19 variations were found, corresponding to a polymorphism of 5%. Of these, seven variations in positions 37 1, 384, 385, 386, 417, 424, and 437 of the Villavicencio strains were also found in a previous study in Papua New Guinea [8]. Two of these variations, at 384 and 424, were also found in the samples obtained from Tumaco. Only three amino acid variations were shared, at positions 220, 384 and 424, in all Villavicencio and Tumaco samples. It is important to note that all of the samples from Villavicencio had two common variations at position 191 (Ala to Glu) and 417 (Arg to Lys). One of these changes (417 Arg to Lys) is also present in the Papua New Guinea sample [S]. Tumaco samples all showed an exclusive and unique change from Arg to Asp at position 192 (Fig. 2b). One Tumaco sample, assigned to family II, also showed novel variations at amino acids 369, 428 and 515. It is clear from these results that this region has overall low polymorphism rate (5%)) but that certain amino acids are repeatedly found to be modified. These variations in the P. z!iua_u Duffy binding domain, where the putative binding sequence is localized, are not considered to be the result in alternate erythrocyte specificities. This is demonstrated by the fact that different polymorphic variants of the Duffy binding domain bind in an identical fashion to RBCs [3,9]. It seems more likely that they are a result of immune selection, a phenomenon commonly seen
with other immunogenic malarial proteins [8,10]. From studies of this kind - showing such large genetic variation at key points in few samples one can hypothesize that natural populations of malaria parasites consist of a large and diverse population of genetically distinct clones. No evidence for the over-representation of a single genotype was seen in this study which would indicate
Fig. la.
Kpnl
Fig. lb.
Fig. 1. (A) Schematic representation of the Sal-I strain Duffy receptor. Region II amplified by PCR is shown by arrows ($). (B) Genomic DNA (100 ng) of 21 samples (including Belem positive control lane 5) obtained from P. uirax-infected patients from Tumaco and Villavicencio (Colombia) lanes 6-15, was amplified by PCR (94”C/min, 42”C/min, 72”C/1.5 min, 25 cycles) using oligomers flanking the N-terminal cystein-rich region (Liz-l: 5’-TGGGGAGGAAAAAGATG3’ and Liz-2: S’AACGGAACAAACGCATAAC). After amplification, 15 ,uI were analyzed on a 0.8% agarose gel, transferred Zeta-Probe membranes (BIO RAD) and hybridized using the Belem amplification product as a probe. As controls, human DNA (lane 3) and genomic P. falciparum DNA (FCB-2 strain) (lane 4) were amplified with the same oligomers, electrophoresed and hybridized as above. After hybridizations, all membranes were washed with 2X SSC for 10 min at room temperature. followed by one wash with 6X SSC pH 7.4 + SDS 0.1% for 10 min at room temperature, a second wash with the same solution at 55°C for 10 min and exposed for 6 h at - 70°C to XAR-5 film with intensifying screens, lane 1: molecular weight marker, lane 2: negative control.
E. Ampudia
et ai.
/ Molecular
Parasitology
and Biochemical
78 (1996)
1-71
269-272
VILLAVICENCIO v 189 19, 192 211 220 A&+ GCA AAC CTI GGG
Flg.Za
I --II --111--N --v ._.
_A_ ___ __. _A_ .__ _A_ _c_ .._ ._. _A_ ._. _.. .._ _A_ __. __. ___ ._. _A_ _.. _A_
189 191 AAA GCA ._. “I .._ “II S,,,, G__ IX G__ .._ X
369 371 376 TAT AAA PAT
__. _.. ___ _._
_._ __. _A_ _._ _A_
._. G-e
___ .
._.
___
_G_
A__
__.
__.
_..
.._
__T ___
__A
.._ A.. ___ A__
371 AA4 .._ .._ ___ .__
376 384 365 AAT GAT w\A .._ ___ __A __A ___
___ __. _G_ _G_ ...
___ ___ _._ ... ._.
424 426 437 503 605 511 515 TTA GTA TGG ATA GTA GTT ffiT _._
.__
___
___
___
.._
._A
A__
._.
__A
.._
___
__A
.__
C__
.._
___
_A_
v
424 TTA
___
A__
_._ .._
_.. ___ A__
___ ._. . . .
_..
.._
386 417 AAG AAT ___ ___ ___ _._
.._
__A
v
v
192 211 220 369 AAC CTT GGG TAT
.__ G-e ___ G-e .__ G__ G.. ___ G-e
v
364 585 366 417 GAT GAA PAG AAT
C__
._.
C__
___
426 437 503 505 511 515 GTA TGG ATA GTA GTT GGT
A__ A__ ___
Sal-I I =1,2.3 II III IV V
Sal-i
.._ .__ .._ .__
.._ ___ C_. C__ C__
___ __. ___ _A_ ___ _A_ .__ _A_
VI VII VIII IX x
503
606 V
511 V
%?.-I
.._
4,BELEM
=1,4.5 8,BELEM = 4, 6, BELEM = 9.10, BELEM =4 1.7 EELEM
= 1524,299Z 3067,3061,2224 q 2893,938l. 3751 = 1514,3067,9381, 2893 = 2224.3061 =5037
VILIAVICENCIO 169 .K
Fig.zbI
191 A
192 N
E
211 L
7 220 G
569 Y
371 K
_
E
_
_
376 N
v
384 D
365 E
366 K
417 N
_
_
K
v
424 L
426 437 “WI
_
515 G
_
I =1,2,3,4,BELEM II Ill IV V
169 191 192 211 270 KANLGYKNDEKNLVWIVVG VI
VI, VIII
.
E
Ix
E.
x
-
IJ
.
,,
_
v 364
369
371
376
.
.
.
_
_
N
-
K
G
K
G
.
.
365
366
417
v 424
426
437
503
_
.
.
,
_
505
511
= 1,4. 5. 8. BELEM = 4 6. BELEM = 9: 10, BELEM =4,1.7 BELEM
516 Sal-l
-
D
E
D.
_
N.
-
D
E
-
_-..I _
.
_
.
_
. _.I.._
.
_ 0
-
-
.
L
.
D
.
.
.
L
_
_
L
_D
VI VII VIII IX x
= 1524,2992.3C67.3061,2224 = 2893. 9361, 3751 = 1514,3067,9381. 2893 = 2224,3061 15037
Fig. 2. Nucleotide sequence (A) and amino acid variations (B) from the DNA samples of Tumaco and Villavicencio. The numeration above indicates the amino acid position of each one of the variations. The changes in common between Tumaco and Villavicencio samples is showed by an arrow (v ).
the presence of a predominant strain. It is likely, therefore, that during mixed infections, random recombination could occur allowing the production of novel genotypes [I I]. In summary, the presence of certain polymorphic residues within the Duffy binding domain, which do not appear to affect its erythrocyte binding capacity, suggests that these residues are subject to immune pressure. However, conserved residues from within this region, which are less likely to be subject to
immune pressure and possibly involved in erythrocyte binding, could be important for the rational design of a P. civax vaccine.
Acknowledgements This research has been supported by the Presidency and The Ministery of Public Health of Colombia, Colciencias and the German Leprosy
272
E. Ampudia et al. / Molecular and Biochemical Parasitology 78 (1996) 269-272
Relief Association. Peter David MD, PhD from the Pasteur Institute provided the P. uiuax Belem strain DNA, and Alfonso Narvaez, Stella Buitrago and Gladys Garcia helped in the supply of P. vivax samples.
References
VI
Adams. J.H., Hudson, D.E., Torii. M.. Ward, G.E.. Wellems, T.E., Aikawa, M. and Miller, L.H. (1990) The Duffy receptor family of Plasmodium knowlesi is located within the micronemas of invasive malaria merozoites. Cell 63, 141-153. 121Prickett, M.D., Smarz, T.R. and Adams. J.H. (1994) Dimorphism and intergenic recombination within the microneme protein (MP-1) gene family of Plasmodium knowlesi. Mol. Biochem. Parasitol. 63. 37-48. P. (1989) [31 Barnwell. J.W., Nichols, M.E. and Rubinstein, In vitro evaluation of role of the Duffy blood in erythrocyte invasion by Plasmodium civas. J. Exp. Med. 169. 17955 1802. [41 Fang. X.D., Kaslow, D.C., Adams, J.H. and Miller, L.H. (1991) Cloning of the Plasmodium uiaa.u Duffy receptor. Mol. Bichem. Parasitol. 44. 1255132.
VI Adams,
J.H., Sim, B.K., Dolan, S.A., Fang, X., Kaslow. D.C. and Miller, L.H. (1992) A family of erythrocyte binding proteins of malaria. Proc. Natl. Acad. Sci. USA 89, 708557089. WIChitnis. C. and Miller, L.H. (1994) Identification of the erythrocyte binding domains of Plasmodium Gt!as and Plasmodium kno,rlesi proteins involved in erythrocyte invasion. J. Exp. Med. 180, 497-506. K., Hadley, T.J. [71Sim. B.K.L., Chitnis. C.E., Wasnioswka, and Miller, L.H. (1994) Receptor and ligands domains for invasion of erythrocytes by Plasmodium ,falciparum. Science 264. 1941-1944. Fl Tsubol, K., Kappe. S.H.I., Al-Yaman. F., Prieckett. M.D., Alpers, M. and Adams. J.H. (1994) Natural variation within the principal adhesion domain of the Plasmodium ciuar Duffy binding protein. Infect. Immun. 5581-5586. of the [91Chitnis, C.E. and Miller, L.H. (1994) Identification erythrocyte binding domains of Plasmodium z1ioa.yand Plasmodium knobvlesi proteins involved in erythrocyte invasion. J. Exp. Med. 180. 497-506. [lOI Marshall, V.M., Coppel. R.L., Martin, R.K.. Oduola. A.M.J., Anders, R.F. and Kemp. D.J. (1991) A Plasmodirrm ,/bkiparum MSA-2 gene apparently generated by intragenic recombination between the two allelic families. Mol. Biochem. Parasitol. 45, 3499352. D. (1991) Malaria parasites: randomly inter[I 11Walliker. breeding or ‘clonal’ populations? Parasitol. Today 7(9), 232 -235.
iii%mMIcAL PARtisIToLoGy
ELSEVTER
Molecular and Biochemical Parasitology 78 (1996) 269-272
Short communication
Genetic polymorphism
of the Duffy receptor binding domain of Plasmodium vivax in Colombian wild isolates’
Elizabeth Imtitt~ro
Ampudia,
de Inmunolo~ic~,
Hospital
Manuel
Alfonso Patarroyo, Luis Angel Murillo*
SCM Juun de Dim.
Unicersidud
Nacionol
Manuel de Colomhiu
Elkin Patarroyo, , Santaf~
de Bogotci
Color~~hiu
Received 7 December 1995: revised 1I March 1996: accepted 12 March 1996
Keword.s: DBP:
PCR; P. uitu~; Malaria;
Duffy receptor;
Polymorphism
-
The Plusmodium vivax Duffy receptor binding protein (DBP) is a member of the protein family localized in the Plasmodium parasite micronemes that binds to erythrocytes carrying this receptor. Given its importance in the parasite life cycle, it is being considered as a potential candidate in vaccine development against P. viva infection [1,2]. Its molecular weight is 140 kDa and the primary structure has been previously reported for the Sal-I strain [3,4]. This protein is divided into five important regions: a leader peptide sequence, two cysteine rich regions separated by a non-homologous hydrophilic region, and a transmembrane domain (Fig. la). The amino terminal cysteine-rich region has been implicated in the binding process to red blood cells (RBCs) carrying the Duffy receptor in a way analagous to what has been demonstrated with the EBA-175 protein Ahbrrt~icrtiorw: PCR, polymerase chain reaction; DBP, DuKy receptor binding protein; RBC, red blood cells. * Corresponding author: ’ Note: Accession numbers: U50575-U50591.
of P. falciparum [557]. A previous report showed natural variation within this critical ligand domain in the P. vivnx Sal-I strain [8]. Polymorphism studies within this region are highly significant for the rational design vaccines against asexual blood stages of P. vivus. Twenty samples were obtained from two different regions of Colombia where malaria is markedly seasonal: one in the South-Western Pacific side (Tumaco) and the other on the Eastern side (Villavicencio) of the Andes (with an annual prevalence rate of P. vivax of 36 and 64X, respectively). Purified Belem strain DNA was used as a positive control. DNA was extracted from each sample and used as a template for PCR. A 1150-bp fragment, corresponding to nucleotide positions 738-1888 (aa 1722555) of the amino terminal cysteine-rich region of the Dutfy receptor, was amplified. All samples gave a positive hybridization signal when tested with the PCR amplified fragment from Belem strain as a probe (Fig. lb). An extra amplified band of 800 bp was observed in 4 of the 20 samples which did
0166-6851/96/$15.00 f-3 1996 Elsevier Science B.V. All rights reserved PII Sl66-6851(96)0261 l-4
270
E. Ampudia et al. 1 Molecular and Biochemical Parasitology 78 (1996) 269-272
not hybridize with the control probe. All fragments were cloned in pMOS blue (Amersham, UK) and sequenced using Sequanase v.2.0 (United States Biochemical). In order to detect any possible nucleotide mis-incorporation due to Taq polymerase activity, 3 clones of each one of the amplification products were sequenced. Based on these results, ten variant families (named I to X) were identified on the basis of grouping mutations in the nucleotides and their corresponding amino acids (indicated in Fig. 2). Five families were identified in each of the two geographical areas studied. The positions of cysteine residues were conserved between the Colombian samples and the reference (Belem) strain. Most of the predicted amino acid changes correspond to replacements with glutamic acid, lysine or aspartic acid. From a total of 383 amino acids, 19 variations were found, corresponding to a polymorphism of 5%. Of these, seven variations in positions 37 1, 384, 385, 386, 417, 424, and 437 of the Villavicencio strains were also found in a previous study in Papua New Guinea [8]. Two of these variations, at 384 and 424, were also found in the samples obtained from Tumaco. Only three amino acid variations were shared, at positions 220, 384 and 424, in all Villavicencio and Tumaco samples. It is important to note that all of the samples from Villavicencio had two common variations at position 191 (Ala to Glu) and 417 (Arg to Lys). One of these changes (417 Arg to Lys) is also present in the Papua New Guinea sample [S]. Tumaco samples all showed an exclusive and unique change from Arg to Asp at position 192 (Fig. 2b). One Tumaco sample, assigned to family II, also showed novel variations at amino acids 369, 428 and 515. It is clear from these results that this region has overall low polymorphism rate (5%)) but that certain amino acids are repeatedly found to be modified. These variations in the P. z!iua_u Duffy binding domain, where the putative binding sequence is localized, are not considered to be the result in alternate erythrocyte specificities. This is demonstrated by the fact that different polymorphic variants of the Duffy binding domain bind in an identical fashion to RBCs [3,9]. It seems more likely that they are a result of immune selection, a phenomenon commonly seen
with other immunogenic malarial proteins [8,10]. From studies of this kind - showing such large genetic variation at key points in few samples one can hypothesize that natural populations of malaria parasites consist of a large and diverse population of genetically distinct clones. No evidence for the over-representation of a single genotype was seen in this study which would indicate
Fig. la.
Kpnl
Fig. lb.
Fig. 1. (A) Schematic representation of the Sal-I strain Duffy receptor. Region II amplified by PCR is shown by arrows ($). (B) Genomic DNA (100 ng) of 21 samples (including Belem positive control lane 5) obtained from P. uirax-infected patients from Tumaco and Villavicencio (Colombia) lanes 6-15, was amplified by PCR (94”C/min, 42”C/min, 72”C/1.5 min, 25 cycles) using oligomers flanking the N-terminal cystein-rich region (Liz-l: 5’-TGGGGAGGAAAAAGATG3’ and Liz-2: S’AACGGAACAAACGCATAAC). After amplification, 15 ,uI were analyzed on a 0.8% agarose gel, transferred Zeta-Probe membranes (BIO RAD) and hybridized using the Belem amplification product as a probe. As controls, human DNA (lane 3) and genomic P. falciparum DNA (FCB-2 strain) (lane 4) were amplified with the same oligomers, electrophoresed and hybridized as above. After hybridizations, all membranes were washed with 2X SSC for 10 min at room temperature. followed by one wash with 6X SSC pH 7.4 + SDS 0.1% for 10 min at room temperature, a second wash with the same solution at 55°C for 10 min and exposed for 6 h at - 70°C to XAR-5 film with intensifying screens, lane 1: molecular weight marker, lane 2: negative control.
E. Ampudia
et ai.
/ Molecular
Parasitology
and Biochemical
78 (1996)
1-71
269-272
VILLAVICENCIO v 189 19, 192 211 220 A&+ GCA AAC CTI GGG
Flg.Za
I --II --111--N --v ._.
_A_ ___ __. _A_ .__ _A_ _c_ .._ ._. _A_ ._. _.. .._ _A_ __. __. ___ ._. _A_ _.. _A_
189 191 AAA GCA ._. “I .._ “II S,,,, G__ IX G__ .._ X
369 371 376 TAT AAA PAT
__. _.. ___ _._
_._ __. _A_ _._ _A_
._. G-e
___ .
._.
___
_G_
A__
__.
__.
_..
.._
__T ___
__A
.._ A.. ___ A__
371 AA4 .._ .._ ___ .__
376 384 365 AAT GAT w\A .._ ___ __A __A ___
___ __. _G_ _G_ ...
___ ___ _._ ... ._.
424 426 437 503 605 511 515 TTA GTA TGG ATA GTA GTT ffiT _._
.__
___
___
___
.._
._A
A__
._.
__A
.._
___
__A
.__
C__
.._
___
_A_
v
424 TTA
___
A__
_._ .._
_.. ___ A__
___ ._. . . .
_..
.._
386 417 AAG AAT ___ ___ ___ _._
.._
__A
v
v
192 211 220 369 AAC CTT GGG TAT
.__ G-e ___ G-e .__ G__ G.. ___ G-e
v
364 585 366 417 GAT GAA PAG AAT
C__
._.
C__
___
426 437 503 505 511 515 GTA TGG ATA GTA GTT GGT
A__ A__ ___
Sal-I I =1,2.3 II III IV V
Sal-i
.._ .__ .._ .__
.._ ___ C_. C__ C__
___ __. ___ _A_ ___ _A_ .__ _A_
VI VII VIII IX x
503
606 V
511 V
%?.-I
.._
4,BELEM
=1,4.5 8,BELEM = 4, 6, BELEM = 9.10, BELEM =4 1.7 EELEM
= 1524,299Z 3067,3061,2224 q 2893,938l. 3751 = 1514,3067,9381, 2893 = 2224.3061 =5037
VILIAVICENCIO 169 .K
Fig.zbI
191 A
192 N
E
211 L
7 220 G
569 Y
371 K
_
E
_
_
376 N
v
384 D
365 E
366 K
417 N
_
_
K
v
424 L
426 437 “WI
_
515 G
_
I =1,2,3,4,BELEM II Ill IV V
169 191 192 211 270 KANLGYKNDEKNLVWIVVG VI
VI, VIII
.
E
Ix
E.
x
-
IJ
.
,,
_
v 364
369
371
376
.
.
.
_
_
N
-
K
G
K
G
.
.
365
366
417
v 424
426
437
503
_
.
.
,
_
505
511
= 1,4. 5. 8. BELEM = 4 6. BELEM = 9: 10, BELEM =4,1.7 BELEM
516 Sal-l
-
D
E
D.
_
N.
-
D
E
-
_-..I _
.
_
.
_
. _.I.._
.
_ 0
-
-
.
L
.
D
.
.
.
L
_
_
L
_D
VI VII VIII IX x
= 1524,2992.3C67.3061,2224 = 2893. 9361, 3751 = 1514,3067,9381. 2893 = 2224,3061 15037
Fig. 2. Nucleotide sequence (A) and amino acid variations (B) from the DNA samples of Tumaco and Villavicencio. The numeration above indicates the amino acid position of each one of the variations. The changes in common between Tumaco and Villavicencio samples is showed by an arrow (v ).
the presence of a predominant strain. It is likely, therefore, that during mixed infections, random recombination could occur allowing the production of novel genotypes [I I]. In summary, the presence of certain polymorphic residues within the Duffy binding domain, which do not appear to affect its erythrocyte binding capacity, suggests that these residues are subject to immune pressure. However, conserved residues from within this region, which are less likely to be subject to
immune pressure and possibly involved in erythrocyte binding, could be important for the rational design of a P. civax vaccine.
Acknowledgements This research has been supported by the Presidency and The Ministery of Public Health of Colombia, Colciencias and the German Leprosy
272
E. Ampudia et al. / Molecular and Biochemical Parasitology 78 (1996) 269-272
Relief Association. Peter David MD, PhD from the Pasteur Institute provided the P. uiuax Belem strain DNA, and Alfonso Narvaez, Stella Buitrago and Gladys Garcia helped in the supply of P. vivax samples.
References
VI
Adams. J.H., Hudson, D.E., Torii. M.. Ward, G.E.. Wellems, T.E., Aikawa, M. and Miller, L.H. (1990) The Duffy receptor family of Plasmodium knowlesi is located within the micronemas of invasive malaria merozoites. Cell 63, 141-153. 121Prickett, M.D., Smarz, T.R. and Adams. J.H. (1994) Dimorphism and intergenic recombination within the microneme protein (MP-1) gene family of Plasmodium knowlesi. Mol. Biochem. Parasitol. 63. 37-48. P. (1989) [31 Barnwell. J.W., Nichols, M.E. and Rubinstein, In vitro evaluation of role of the Duffy blood in erythrocyte invasion by Plasmodium civas. J. Exp. Med. 169. 17955 1802. [41 Fang. X.D., Kaslow, D.C., Adams, J.H. and Miller, L.H. (1991) Cloning of the Plasmodium uiaa.u Duffy receptor. Mol. Bichem. Parasitol. 44. 1255132.
VI Adams,
J.H., Sim, B.K., Dolan, S.A., Fang, X., Kaslow. D.C. and Miller, L.H. (1992) A family of erythrocyte binding proteins of malaria. Proc. Natl. Acad. Sci. USA 89, 708557089. WIChitnis. C. and Miller, L.H. (1994) Identification of the erythrocyte binding domains of Plasmodium Gt!as and Plasmodium kno,rlesi proteins involved in erythrocyte invasion. J. Exp. Med. 180, 497-506. K., Hadley, T.J. [71Sim. B.K.L., Chitnis. C.E., Wasnioswka, and Miller, L.H. (1994) Receptor and ligands domains for invasion of erythrocytes by Plasmodium ,falciparum. Science 264. 1941-1944. Fl Tsubol, K., Kappe. S.H.I., Al-Yaman. F., Prieckett. M.D., Alpers, M. and Adams. J.H. (1994) Natural variation within the principal adhesion domain of the Plasmodium ciuar Duffy binding protein. Infect. Immun. 5581-5586. of the [91Chitnis, C.E. and Miller, L.H. (1994) Identification erythrocyte binding domains of Plasmodium z1ioa.yand Plasmodium knobvlesi proteins involved in erythrocyte invasion. J. Exp. Med. 180. 497-506. [lOI Marshall, V.M., Coppel. R.L., Martin, R.K.. Oduola. A.M.J., Anders, R.F. and Kemp. D.J. (1991) A Plasmodirrm ,/bkiparum MSA-2 gene apparently generated by intragenic recombination between the two allelic families. Mol. Biochem. Parasitol. 45, 3499352. D. (1991) Malaria parasites: randomly inter[I 11Walliker. breeding or ‘clonal’ populations? Parasitol. Today 7(9), 232 -235.