Molecular and Biochemical Parasitology 97 (1998) 259–263

Short communication

Complete nucleotide sequence of the Plasmodium 6i6ax 6 kbelement�

Indu Sharma a, S. Tazeen Pasha b, Yagya D. Sharma a,*a Department of Biotechnology, All India Institute of Medical Sciences, Delhi 110029, India

b Di6ision of Biochemistry and Biotechnology, National Institute of Communicable Diseases, 22 Sham Nath Marg,New Delhi 10054, India

Received 1 August 1998; received in revised form 14 September 1998; accepted 21 September 1998

Keywords: Mitochondria; PCR; Human; Malaria parasite; Plasmodium 6i6ax

The malarial parasite contains three genomes;the nuclear genome with fourteen chromosomesand two extra chromosomal DNA molecules: the35 kb circular DNA and the 6 kb element [1]. The6 kb element represents the mitochondrialgenome. Mitochondria in the malaria parasitehave been visualized by labelling with Rhodamine123 [2]. The mitochondrial DNA is present inabout 20–150 copies cell−1 and its mode of repli-cation is not by the usual D-loop structure seen inhigher eukaryotes but by the recombination-de-pendent replication process as well as by rollingcircle method which are similar to that of thebacteriophages [3,4]. Mitochondria of malaria

parasites are proposed to be potential drug targets[5]. The mitochondrial genome has been com-pletely sequenced from the human malaria para-site Plasmodium falciparum and the rodentmalaria parasite P. yoelii [6,7]. Here we describethe complete nucleotide sequence of the 6 kbelement from the human malaria parasite P.6i6ax.The 6 kb element of P. 6i6ax encodes three mito-chondrial proteins and 13 fragmented ribosomalRNAs.

The 6 kb element was amplified by PCR from apatient’s blood infected with P. 6i6ax. The possi-bility of a mixed infection with P. falciparum wasruled out by nested PCR for species specific detec-tion of 18S rRNA gene [8]. Further confirmationwas made by checking PCR amplification for P.falciparum specific knob-associated histidine-richprotein (KHARP) and thrombospondin-relatedanonymous protein (TRAP) genes, which gave noPCR product in this sample [9]. Oligonucleotide

* Corresponding author. Tel.: +91-11-6593617; fax: +91-11-6852286.� Note: Nucleotide sequence data reported in this paper are

available in the EMBL, GenBank™ and DDJB data basesunder the Accession number Y17721.

0166-6851/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved.

PII: S 0166 -6851 (98 )00140 -6

I. Sharma et al. / Molecular and Biochemical Parasitology 97 (1998) 259–263260

Fig. 1. Organization of P.6i6ax 6 kb element. Genes encoding mitochondrial proteins (CoI, CoIII, and Cyb) are shown as strippedarrows. The direction of arrows indicate the direction of transcription. The fragmented large subunit (LSU) and small subunit (SSU)rRNA genes are indicated. The direction of transcription of rRNA genes is same as that of mitochondrial genes for each strand, andis indicated by simple arrows. The location of primers, used to amplify the 6 kb element in two halves, is shown by arrow heads.

primers based on the sequence of the P. falci-parum element were designed to amplify the 6 kbDNA in two parts (forward primer P101-5% ACTAGA GAT TTC AAA ACT CAT TC-3% andreverse primer P106-5% CCT CCT ATA TGACAC TCA CTA G-3% to amplify one half andforward primer R101- 5% GAA TGA GTT TTGAAA TCT CTA GT-3% and reverse primer R106-5% GGA GTA TAT ACT GTG AGT GAT C-3%to amplify the remaining half of the 6 kb ele-ment). The PCR products from two to threeindependent amplifications were sequenced on anautomated nucleotide sequencer (Applied Biosys-tem, USA, Model A 373) using dideoxy chainterminators and specific primers. In order to ob-tain the sequences in the proximity of the abovementioned four primers, the PCR products fromtwo independent amplifications were cloned intopGEMT vector and their ends were sequenced byusing M13 universal primers.

The size of P. 6i6ax mitochondrial DNA wasfound to be 5989 bp. The sequence containedthree mitochondrial protein genes; the cy-tochrome c oxidase subunit I and III (CoI andCoIII) and cytochrome b (Cyb). There were atotal of 13 different ribosomal RNA genes; sevenfor large and six for small subunit rRNA. Theorganization of these genes is shown in Fig. 1.

The nucleotide sequence of P. 6i6ax 6 kb ele-ment showed more homology to P. yoelii (91.7%)than P. falciparum (90.1%) [6,7]. The sequencedifferences between P.6i6ax and P.falciparum ele-ments were found in both coding (89.94% identity

in a 4674 nucleotide overlap) and non-coding(92.3% identity in a 1315 nucleotide overlap) re-gions. The rate of sequence variation was maxi-mum in protein-coding region (87.35% identity ina 3472 nucleotide overlap) and minimum in non-protein coding region containing rRNA genes(97.09% identity in a 1309 nucleotide overlap).The rate of sequence variation was similar forsmall (97.01% identity in a 435 nucleotide over-lap) and large (97.13% identity in a 874 nucleotideoverlap) subunits of rRNA genes. In fact, most ofthe rRNA genes showed minor differences exceptlarge subunit B (86.8% identity in a 91 nucleotideoverlap) and small subunit A (93.8% identity in a97 nucleotide overlap) genes. The rRNA genesalso showed a minimum number of nucleotidedeletions (one each in large subunit B and Dgenes) and additions (one in small subunit F andone in large subunit G). The P. 6i6ax elementshowed a total of 19 deletions and 41 additions ofnucleotides as compared to P. falciparum. Themaximum number of 14 nucleotide deletion oc-curred in non-coding region. This region alsoshowed a large number of 15 additional nucle-otides. The maximum number of 18 nucleotideadditions were however observed in CoI genewhich had not shown any deletions. The CoIIIgene showed six additions and three deletionswhile the number of nucleotides in Cyb generemained same.

Although there was a lower level of nucleotidesequence homology in the protein coding regionsof P. 6i6ax and P. falciparum genes, the encoded

I. Sharma et al. / Molecular and Biochemical Parasitology 97 (1998) 259–263 261

Fig. 2. Mitochodrial protein sequence homology across the species of malarial parasites. Homology for CoI and Cyb is shown inFig. 2a and b respectively. The identical amino acids are shown by stars. Gaps are introduced for the maximization of homologyand are indicated by dashes. PV, P.6i6ax ; PY, P.yoelii ; PF, P. falciparum.

protein’s homology was still higher due to differ-ent codon usage. The amino acid sequence homol-ogy of P. 6i6ax Cyb and CoI to P. falciparum andP. yoelii is shown in Fig. 2. The homology datafor P. 6i6ax CoIII have been described earlier [10].

At the amino acid (aa) level, the CoI of P. 6i6axshowed 92.7% identity in a 508 aa overlap to P.falciparum. Like P. falciparum, the CoI of P. 6i6axalso started with methionine residue. The majordifferences in CoI were observed at N-terminus

I. Sharma et al. / Molecular and Biochemical Parasitology 97 (1998) 259–263262

Fig. 2. (Continued)

region where the P. 6i6ax CoI contains six extraaa as compared to P. falciparum and 37 extra aacompared to P. yoelii. All transmembrane do-mains and seven histidine residues, involved inbinding the redox centers were also present in theP. 6i6ax molecule [11].

The P. 6i6ax Cyb was 376 aa long which issimilar to P. falciparum and P. yoelii (Fig. 2b).However, the aa sequence homology of P. 6i6axCyb was closer to P. yoelii (93.6% identity) thanP. falciparum (91% identity). The four histidineresidues (His 78, His 92, His 173, and His 187)involved in interaction with heme were conservedacross the species (Fig. 2b). Most of the druginteracting sites were also conserved in P. 6i6axexcept a change from isoleucine to leucine atresidue 14 [12].

While this manuscript was under preparation,the nucleotide sequence of 6 kb element from P.6i6ax of Salvador I strain has been deposited inthe data base (accession number AF055587).There is about 99.2% homology between the 6 kbelements of the Indian and Salvador strains. TheCyb gene of the Indian strain is six aa shorter atC-terminus end because of a non-sense mutationat nucleotide position 5384 (A–T). In case of CoI,there were three point mutations; two of themcausing an amino acid change and third one wasa silent mutation.

Acknowledgements

The financial assistance (to YDS) came from the

I. Sharma et al. / Molecular and Biochemical Parasitology 97 (1998) 259–263 263

the Department of Biotechnology (DBT) andCouncil of Scientific, and Industrial Research(CSIR). One of us (IS) received Junior ResearchFellowship from CSIR. We thank Dr Arvind Rai,Dr M.R. Ranjit, Dr K. Kita, D.S. Rawat, DrRanjana Anand, and Manoj Kumar for their help.

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