Complete nucleotide sequence of the 6 kb element and conserved cytochrome b gene sequences among...

Research note

Complete nucleotide sequence of the 6 kb element andconserved cytochrome b gene sequences among

Indian isolates of Plasmodium falciparumq

Indu Sharmaa, D.S. Rawatb, S.T. Pashab, S. Biswasc, Y.D. Sharmaa,*

aDepartment of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi ± 110029, IndiabDivision of Biochemistry & Biotechnology, National Institute of Communicable Diseases, 22 Sham Nath Marg, Delhi, India

cMalaria Research Centre, 22 Sham Nath Marg, Delhi -110054, India

Received 19 January 2001; received in revised form 2 April 2001; accepted 2 April 2001

Abstract

The malaria parasite contains a nuclear genome with 14 chromosomes and two extrachromosomal DNA molecules of 6 kb and 35 kb in

size. The smallest genome, known as the 6 kb element or mitochondrial DNA, has been sequenced from several Plasmodium falciparum

isolates because this is a potential drug target. Here we describe the complete nucleotide sequence of this element from an Indian isolate of P.

falciparum. It is 5967 bp in size and shows 99.6% homology with the 6 kb element of other isolates. The element contains three open reading

frames for mitochondrial proteins-cytochrome oxidase subunit I (CoI), subunit III (CoIII) and cytochrome b (Cyb) which were found to be

expressed during blood stages of the parasite. We have also sequenced the entire cyb gene from several Indian isolates of P. falciparum. The

rate of mutation in this gene was very low since 12 of 14 isolates showed the identical sequence. Only one isolate showed a maximum change

in ®ve amino acids whereas the other isolate showed only one amino acid change. However, none of the Indian isolates showed any change in

those amino acids of cyb which are associated with resistance to various drugs as these drugs are not yet commonly used in India. q 2001

Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Malaria; 6 kb element; Mitochondria; Cytochrome b; Drug resistance; Gene expression

Malaria remains uncontrolled to-date, so malaria control

programs need effective antimalarial drugs and vaccines. To

identify newer potential targets for drugs and vaccines, the

sequencing of the malarial genome has been initiated. The

malaria parasite contains a total of 14 chromosomes in its

nucleus and also has two extra chromosomal DNA mole-

cules: i.e. the 35 kb circle and the 6 kb element. While the 35

kb circle is known as plastid-like genome because of the

presence of characteristic DNA sequences and genes, the 6

kb element is known as the mitochondrial genome since it

contains mitochondrial protein coding genes (Feagin, 2000;

Williamson et al., 1996; van Lin et al., 2000). This 6 kb

mitochondrial DNA of the malaria parasite, unlike its coun-

terparts in higher eukaryotes, contains large number of frag-

mented ribosomal RNA genes (Feagin et al., 1997; Rehkopf

et al., 2000). Its mode of replication is also not by the usual

D-loop structures seen in higher eukaryotes, but by the

recombination dependent replication process as well as by

rolling circle method (Williamson et al., 1996; Presier et al.,

1996). The 6 kb element is present in a relatively high copy

number (20±150 copies per cell) and exists as linear

tandemly repeated molecule. Because the mitochondria in

malaria is a potential drug target, its genome, i.e. 6 kb

element has been sequenced from several isolates of Plas-

modium falciparum [ Feagin, 1992 (Accession No M76611,

C-10 from Gambia) and Conway et al., 2000 (Accession No

AJ276844, NF54 from The Netherlands; AJ276845, K1

from Thailand; AJ276846, T9/96 from Thailand and

AJ276847, 7G8 from Brazil]. In the present study, we

International Journal for Parasitology 31 (2001) 1107±1113

0020-7519/01/$20.00 q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

PII: S0020-7519(01)00218-1

www.parasitology-online.com

q The nucleotide sequence reported in this paper has been submitted to the

EMBL database. The complete nucleotide sequence of the 6 kb element of

P. falciparum from the Indian isolate (PF PH10) is available under the

accession number AJ298788. The nucleotide sequence of the cyb gene

from 13 other Indian isolates of P. falciparum have been deposited under

accession numbers: AJ298775 (clinical isolate 317); AJ298776 (clinical

isolate 353); AJ298777 (clinical isolate 392); AJ298778 (clinical isolate

393); AJ298779 (clinical isolate 406); AJ298780 (clinical isolate 407);

AJ298781 (clinical isolate 410); AJ298782 (clinical isolate 411);

AJ298783 (chloroquine resistant parasite line M20); AJ298784 (chloro-

quine resistant parasite line M22); AJ298785 (chloroquine resistant parasite

line FSH); AJ298786 (chloroquine sensitive parasite line D9) and

AJ298787 (chloroquine sensitive parasite line NE).

* Corresponding author. Tel.: 191-11-6967045; fax: 191-11-6852286.

E-mail address: [email protected] (Y.D. Sharma).

describe the complete nucleotide sequence of the 6 kb

element from an Indian isolate of P. falciparum. All three

open reading frames (ORF) for mitochondrial proteins

present in this element were found to be expressed during

blood stages of the parasite. We also describe here the

sequence of the P. falciparum cyb gene from several other

Indian isolates in order to study the sequence variation

linked with the resistance towards various drugs.

The entire 6 kb element of P. falciparum (clinical isolate

PF PH10) was ampli®ed in sections from the parasite DNA.

The primers for PCR were designed from the published

sequence of the 6 kb element of P. falciparum from the

Gambian isolate C 10 (Feagin, 1992). The sequence of the

primers is shown in Table 1. The PCR conditions were as

described (Kant and Sharma, 1996). The annealing tempera-

tures for various sets of primers were as follow: 628C for

R106 1 R113; 608C for P110 1 R101, P112 1 R113,

P108 1 R103, P103 1 R105; 588C for P110 1 P102,

P111 1 R109, P101 1 P102, P108 1 R109, P109 1 R103,

R106 1 R107, R106 1 R110, cybF 1 R113; 568C for

P111 1 R101, P214 1 R103, P108 1 P106, P103 1 P106,

R106 1 R114, cybF 1 P106, cybF 1 R105; 558C for

P103 1 R113, P105 1 R106; 548C for mtcyb c-

ter 1 R114; 528C for P210 1 P106, P103 1 PVCOI-R. The

various sized PCR products generated here were sequenced

directly by cycle sequencing using various internal and other

primers described in Table 1. These PCR products were also

cloned into pGEMT easy vector (Promega) and sequenced

using universal as well as internal primers. Separately, we

have also ampli®ed the entire cyb gene of P. falciparum from

eight clinical isolates and ®ve parasite lines (three chloro-

quine resistant and two chloroquine sensitive) using primers

CybF and R113. These PCR products were sequenced by

cycle sequencing. Sequencing was performed using the auto-

mated nucleotide sequencers (Applied Biosystem model

373A and 310 Genetic Analyzer) and manual sequencer.

The complete sequence was analysed using PCGENE,

DNASIS, Clone manager, Blast, Clustal W, and Lasergene

(DNASTAR Inc). For Reverse transcription PCR (RT-PCR),

total RNA was extracted from cultured P. falciparum by the

method of Chomczymski and Sacchi (1987). One microgram

of total RNA was reverse transcribed to cDNA by using

Thermo Script reverse transcriptase, oligo (dT) primer,

RNaseOUT, DTT, dNTPs and 1 £ reverse transcriptase

buffer from GIBCO BRL following their instructions (Life

Technologies, Gibco BRL). After cDNA synthesis, DNA-

PCR was carried out by using primer set P101 and P102 for

the coIII gene, R103 and P109 for the coI gene, and cybF and

P106 for the cyb gene. PCR conditions were as described

(Kant and Sharma, 1996). For mRNA in-situ hybridisation,

thin smears of P. falciparum cultures were prepared on Poly-

L-Lysine coated slides (Sigma Co) and subjected to hybridi-

sation according to Thompson and Sinden (1994). mRNA

hybridisation was carried out using the sense and antisense

riboprobes for the coI, coIII and cyb genes. The riboprobes

were generated from the clones where part of the coI, coIII

and cyb genes, obtained by PCR, were cloned into pGEMT

easy vector. The in-vitro transcription from these clones,

using T7 and SP6 promoters, was carried out in the presence

of ¯uorescein 11-UTP to generate labelled probes. The

primers used to amplify part of the coI and coIII genes

were the same as in the RT-PCR whereas the primer set

cybF and R105 was used for generating the 258 bp fragment

from the cyb gene. In-situ hybridisation was performed by

using the RNA color kit from Amersham and following their

instructions (Amersham International plc). Slides were

mounted with DPX and viewed under 100 £ oil immersion

in a Nikon microscope.

The complete nucleotide sequence of the 6 kb element of

P. falciparum from the Indian isolate PF PH10 revealed a

total of 5967 nucleotides (Accession No. AJ298788). The

sequence contains 68.29% (4075 bp) A 1 T and 31.71%

(1892 bp) G 1 C contents. The sequence was 99.6% homo-

logous to that of the Gambian isolate PF C10 (Feagin,

1992). The sequence showed 89.5% homology to the 6 kb

element of the Plasmodium vivax and 89% to the rodent

malaria parasite Plasmodium yoelii (Sharma et al., 1998;

Vaidya et al., 1989). In fact, the P. yoelii sequence showed

more homology to P. vivax (91.7%) than to P. falciparum.

The sequence differences were con®rmed by performing

independent PCR reactions and sequencing these products.

This element is packed with a large number of genes located

on both strands of the element. There were three mitochon-

drial protein genes (coI, coIII and cyb) and about 20 frag-

I. Sharma et al. / International Journal for Parasitology 31 (2001) 1107±11131108

Table 1

Primer sequences in the 6 kb element of P. falciparuma

S No Primer No. Sequence (5 0 ! 3 0)

1 R110 GGC ATG GTT ACG AGA ATT TAA GG

2 P110 CCT CAC GAG TCG ATC AGG AA

3 P111 GTA GAA CAA TAT TGA GTT GAC GG

4 P112 CTC ATT CCA ATG GAA CCT TGT TC5 P101 ACT AGA GAT TTC AAA ACT CAT TC

6 R101 GAA TGA GTT TTG AAA TCT CTA GT

7 P102 GTA TCT TAT CCT TCA TTA ACA TCA8 P214 GAT GTT AAT GCA GGA TAT GAA AC

9 P108 ACT GGG TAT AGA ACT CCA GGC

10 R109 CCT CCG AAT AAT CCT GGC ATT

11 P109 AAT GCC AGG ATT ATT CGG AGG12 R103 ATC TCC TGC AAA TGT TGG GTC

13 P103 GAC CCA ACA TTT GCA GGA GAT

14 P210 TTA GGT AAT GCT GCC ATT GAT

15 PVCOI-R TTA AAA ATT AGT AAA CCA AAT16 P105 GCA AGT CGA TAT ACA CCA GAT

17 R105 GAG AAG CAC CTG TTG CGT GC

18 R106 GGA GGA TAT ACT GTG AGT GAT C19 P106 CCT CCT ATA TGA CAC TCA CTA G

20 mtcyb c-ter CTG GTT TAG TAA TTG GTA TTA TTA T

21 R107 GTT CCG CTC AAT TAC TCA GAA ATG

22 P115 CAT AGA ATG CAC TCA TAA ACC23 R113 TAA TAT AGT ATA TAT CGA AGC ATC CAT C

24 R114 GTG GTA TAG TCA TAT CTC CAT

25 cybF GTT TGG ATC CAT GAA CTT TTA CTC TAT TAAT

a Source: Acc. No. M76611 (Gambian isolate PF C10).

mented rRNA genes. The coI gene is 1597 bp long contain-

ing 70.99% A 1 T and encodes 508 amino acids of protein.

It differs at ®ve different nucleotide positions from that of

the other P. falciparum isolates. These changes are at

nucleotide positions 231 (T! C), 824 (T! A), 1386

(A! G), 1489 (T! A) and 1500 (T! C). While nucleo-

tide changes at positions 231, 824 and 1500 do not change

the encoded amino acid, the base change at positions 1386

and 1489 leads to change in amino acids from Phe! Tyr

and Ser! Thr, respectively. Changes in these two amino

acids found in the Indian isolate seem to be unique since

they are conserved in other P. falciparum isolates as well as

in P. vivax and P. yoelii. As compared with coI, the coIII

gene is smaller in size, i.e. 840 bp with 75.12% A 1 T and

encodes 279 amino acids. It has variation at only three

nucleotide positions as compared with the other isolates.

But all these changes lead to an amino acid change, i.e.

changes at nucleotide positions 235 (T! C), 452 (C! G)

and 802 (A! G) leads to the change in amino acids Ser!Pro, Thr! Ser, and Ile! Val, respectively. The cyb gene is

of 1131 bp in size containing 72.24% A 1 T. It encodes a

376 amino acid long protein. This gene however shows

more variation than the coI and coIII genes. It differs in

seven nucleotides from the cyb gene of other isolates.

There is a net change in ®ve amino acids while two point

mutations (T! C at nucleotide position 273 and A! T at

nucleotide position 861) caused silent mutations. The

changes at nucleotide position 275 (A! G) lead to His!Arg, at 377 (A! T) to Tyr! Phe, at 494 (C! G) to Thr!Arg, at 862 (T! A) to Leu! Ile and at 929 (T! A) to

Phe! Tyr. Expression of these mitochondrial protein genes

by the parasite was con®rmed by detecting their RNA tran-

scripts using the RT-PCR and mRNA in-situ hybridisation

techniques. The data suggest that all the three mitochondrial

genes (coI, coIII and cyb) are being transcribed during the

erythrocytic stages of the parasite (Fig. 1). While RT-PCR

detected the presence of mRNA of all these genes, the in-

situ hybridisation experiments located these transcripts in

infected erythrocytes (Fig. 1). Fig. 1B shows that these

genes were expressed by ring, trophozoite and schizont

stages of the parasite. Expressions of these mitochondrial

genes during asexual stages have also been reported by

Feagin and Drew (1995).

Separately, we also sequenced the entire cyb gene from

eight other clinical isolates and ®ve parasite lines (chloro-

quine resistant lines, i.e. PF M20, PF M22 and PF FSH as well

as chloroquine sensitive lines, i.e. PF NE and PF D9). The

sequence comparison shows that except for one isolate (PF

392), all other isolates and parasite lines had identical nucleo-

tide and deduced amino acid sequences (Table 2). There was

only one nucleotide change in the cyb gene of PF392 at

nucleotide position 977 (T! C) which resulted in changing

the amino acid Phe to Thr. In comparison with the PF C10

Gambian isolate, the nucleotide sequence of all 13 samples

was identical except at nucleotide position 6 (C! T), 8

(T! A) and 12 (C! T). While the nucleotide change at 6

and 12 does not alter the encoded amino acid, the base change

at position 8 changes the encoded amino acid from Phe to

Tyr. The cyb gene from these isolates therefore did not show

much variation as seen in the above mentioned isolate PF

PH10 in comparison with the PF C10 Gambian isolate. The

sequence pair distance analysis shows that except for isolate

PF PH10 all other isolates from India were identical (Fig. 2).

In comparison with each other, the PF PH10 showed highest

variation with 98.4% homology followed by PF 392 which

showed 99.7% homology. The PF PH10 isolate however

showed more homology (98.7%) with the PF C10 isolate

than the Indian isolates. The other Indian isolates showed

99.7% homology with PF C10 except PF 392, which showed

99.5% homology. Partial sequences of the cyb gene available

in the databases, from different geographical locations,

showed different homologies with the PF PH10. Netherlands

(3D7 Acc. No AF069605), Kenya (Acc. No AF069606),

Santa Lucia (Acc. No AF069607) isolates showed 99.2%

identity, Malaysian isolate (Acc. No AF069608) showed

99.1% and isolate from China (Acc. No. AF069609) showed

99.0% identity with cyb gene of PF PH10 in an overlap of

1035 bp.

The malaria parasite genome sequencing projects have

been initiated to discover newer strategies to control the

disease. Sequencing of P. falciparum chromosome 2 and 3

is already completed while sequencing of the remaining 12

chromosomes is in rapid progress (Gardner et al., 1998;

Bowman et al., 1999). The extrachromosomal DNA mole-

cules have also been sequenced from this parasite (Feagin,

1992; Wilson et al., 1996). Interest in sequencing the extra-

chromosomal DNA molecules has been due to the fact that

both of them are potential drug targets. The smallest

genome of the 6 kb element contains the mitochondrial

genes as potential drug targets, and has therefore been

sequenced from several isolates. Here, for the ®rst time,

we report the complete nucleotide sequence of the 6 kb

element from an Indian isolate. This sequence showed

99.6% homology with P. falciparum isolate PF C10 and

89.5% to P. vivax (Feagin, 1992; Sharma et al., 1998).

The nucleotide sequence differences are not concentrated

to any particular gene or part of the DNA segment but

distributed throughout the 6 kb element.

We also observed the expression of mitochondrial protein

genes of this 6 kb element which is suggestive of the

presence of active mitochondria in the malaria parasite.

Mitochondria of the malaria parasite generate a membrane

potential through an electron transport system and as such is

a probable target for antimalarial drugs like primaquine and

atovaquone (Ginsburg et al., 1986; Vaidya et al., 1993). The

new antimalarial atovaquone disrupts the mitochondrial

membrane potential (Srivastava et al., 1997). It has recently

been shown that the antimalarial activity of atovaquone

involves interaction with cytochrome b encoded by the 6

kb element of the mitochondrial DNA. Amino acid residues

causing this drug resistance have been de®ned as they are

located at putative drug-binding sites (Syafruddin et al.,

I. Sharma et al. / International Journal for Parasitology 31 (2001) 1107±1113 1109


Fig. 1. Expression of P. falciparum mitochondrial protein genes. (A) The RT-PCR on the P. falciparum RNA from erythrocytic stages was carried out for coI,

coIII and cyb genes using speci®c primers and the template obtained with (lane 1) and without (lane 2) reverse transcription. Marker lane for coIII and cyb is

shown on right hand side and for coI on left hand side. (B) mRNA in-situ hybridisation was carried out on thin smears of the P. falciparum parasites by using

antisense and sense riboprobes for coI, coIII and cyb genes. R, ring; T, trophozoite; S, schizont. Scale bar, 10 mm. Arrowheads indicate the parasite.


Tab

le2

Seq

uen

cean

alysi

so

fcy

bg

ene

of

P.

falc

ipa

rum

from

clin

ical

isola

tes

and

par

asit

eli

nes

a

S.

No

Iso

late

Chan

ge

inn

ucl

eoti

de

and

amin

oac

idse

quen

ce

Nu

cleo

tid

ep

osi

tio

n6

812

273

275

377

494

861

862

929

977

1P

FC

10

Ind

ian

Iso

late

sC

TC

TG

AC

AT

TT

2P

FP

H1

0C

TC

CA

TG

TA

AT

(none)

(His!

Arg

)(P

he!

Tyr)

(Thr!

Arg

)(L

eu!

Ile)

(Phe!

Tyr)

(none)

3P

F3

17

TA

TT

GA

CA

TT

T

(No

ne)

(Ph

e!

Tyr)

(none)

4P

F3

53

TA

TT

GA

CA

TT

T

(No

ne)

(Ph

e!

Tyr)

(none)

5P

F3

92

TA

TT

GA

CA

TT

C

(No

ne)

(Ph

e!

Tyr)

(none)

(Phe!

Ser

)

6P

F3

93

TA

TT

GA

CA

TT

T

(No

ne)

(Ph

e!

Tyr)

(none)

7P

F4

06

TA

TT

GA

CA

TT

T

(No

ne)

(Ph

e!

Tyr)

(none)

8P

F4

07

TA

TT

GA

CA

TT

T

(No

ne)

(Ph

e!

Tyr)

(none)

9P

F4

10

TA

TT

GA

CA

TT

T

(No

ne)

(Ph

e!

Tyr)

(none)

10

PF

41

1T

AT

TG

AC

AT

TT

(No

ne)

(Ph

e!

Tyr)

(none)

11

PF

M2

0T

AT

TG

AC

AT

TT

(No

ne)

(Ph

e!

Tyr)

(none)

12

PF

M2

2T

AT

TG

AC

AT

TT

(No

ne)

(Ph

e!

Tyr)

(none)

13

PF

FS

HT

AT

TG

AC

AT

TT

(No

ne)

(Ph

e!

Tyr)

(none)

14

PF

D9

TA

TT

GA

CA

TT

T

(No

ne)

(Ph

e!

Tyr)

(none)

15

PF

NE

TA

TT

GA

CA

TT

T

(No

ne)

(Ph

e!

Tyr)

(none)

aO

nly

the

po

siti

on

of

var

ian

tn

ucl

eoti

des

/(am

ino

acid

s)is

show

n.

1999; Korsinczky et al., 2000; Srivastava et al., 1999). The

sequencing of the entire cyb gene from eight clinical isolates

and ®ve parasite lines, however, did not show any mutation

in these residues. This is expected since the drug has not yet

been used in the ®eld in India. Indeed, 12 of 14 Indian

isolates have identical nucleotide and amino acid sequence

of the cyb gene. Furthermore, there was no sequence diver-

gence in those amino acids, which are involved in providing

resistance against various other drugs. The sequences of Qi

and Qo centres were particularly conserved. The point

mutations in these centres have been found to be associated

with drug resistance (Vaidya et al., 1993). The cyb gene

sequence of all the chloroquine resistant and sensitive

lines was also the same as the wild type thereby indicating

that chloroquine resistance does not involve this gene. The

highly conserved sequence of the cyb gene among the

majority of isolates is surprising since we have seen more

variation in nuclear encoded proteins (viz. CSP, MSA-1,

MSA-2, TRAP) among Indian isolates (Bhutani et al.,

1998; Ranjit and Sharma, 1999). Furthermore, the mito-

chondrial encoded cyb gene is evolving rapidly in mammals

with approximately a 10-fold greater mutation rate than

nuclear genes (Contamine and Picard, 2000). However,

the recent ®ndings suggest that the rate of mutations in

mitochondrial DNA can be suppressed by the import of

cytoplasmic tRNA (Kolesnikova et al., 2000).

Acknowledgements

This work was supported by grants (to YDS) from the

Department of Biotechnology (Government of India) and

The Council of Scienti®c and Industrial research. One

of us (IS) received Senior Research Fellowship from

CSIR.

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Complete nucleotide sequence of the 6 kb element and conserved cytochrome b gene sequences among...

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