Publication: Molecular cloning of a Plasmodium falciparum gene interrupted by 15 introns encoding a functional primase 53 kDa subunit as demonstrated by expression in a baculovirus system
Issued Date
1996-11-28
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ISSN
03051048
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2-s2.0-0029829543
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Mahidol University
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SCOPUS
Bibliographic Citation
Nucleic Acids Research. Vol.24, No.20 (1996), 3934-3941
Suggested Citation
Suttiphan Prasartkaew, Natasha M. Zijlstra, Prapon Wilairat, J. Prosper Overdulve, Erik De Vries Molecular cloning of a Plasmodium falciparum gene interrupted by 15 introns encoding a functional primase 53 kDa subunit as demonstrated by expression in a baculovirus system. Nucleic Acids Research. Vol.24, No.20 (1996), 3934-3941. doi:10.1093/nar/24.20.3934 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/17531
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Title
Molecular cloning of a Plasmodium falciparum gene interrupted by 15 introns encoding a functional primase 53 kDa subunit as demonstrated by expression in a baculovirus system
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Abstract
The gene encoding the primase small subunit was isolated from genomic DNA of strain K1 of the human malarial parasite Plasmodium falciparum. Isolation of a complete cDNA clone revealed the presence of 15 introns in the genomic sequence. This is unprecedented for Plasmodium genes, which usually contain no or only 1 or 2 introns. The gene is present as a single copy and the cDNA contains an open reading frame of 1356 nt encoding a protein of 452 amino acids. A single mRNA of 2.1 kb was identified by Northern blotting. Comparison of the amino acid sequence with five eukaryotic small primase subunits revealed the presence of eight conserved regions. Sequence alignments allowed the identification of putative motifs A, B and C that are essential features of the catalytic centre of DNA polymerases, RNA polymerases and reverse transcriptases. Also, similarity of a C-terminal region of ~ 100 amino acids to a conserved region in herpes virus primases, α-like DNA polymerases and RNA polymerase II was noted. The complete gene was expressed as a fusion product containing an N-terminal polyhistidine tag using a baculovirus expression vector. The protein was overproduced in insect cells and purified. Activity assays demonstrated the ability of the p53 subunit to initiate de novo primer formation.