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.
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 approximately 100 amino acids to a conserved region in herpes virus primases, alpha-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.
Prasartkaew
S
S
Institute of Infectious Diseases and Immunology, Department of Parasitology and Tropical Veterinary Medicine, Utrecht University, The Netherlands.
Zijlstra
N M
NM
Wilairat
P
P
Overdulve
J P
JP
de Vries
E
E
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D019915:DNA Primase; D018076:DNA, Complementary / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000235:genetics; D017125:Genes, Protozoan / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology*; D012316:RNA Nucleotidyltransferases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000235:genetics; D016415:Sequence Alignment; D017421:Sequence Analysis
D018076:DNA, Complementary; D011994:Recombinant Proteins; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase