MOLECULAR-CLONING OF CTP - PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE FROM PLASMODIUM-FALCIPARUM

CTP:phosphocholine cytidylyltransferase (CCT) is the rate-limiting and regulatory enzyme in the synthesis of phosphatidylcholine, the major membrane phospholipid, in Plasmodium. The structural gene encoding CCT was isolated from the human malaria parasite Plasmodium falciparum. T his was achieved using the PCR to amplify genomic DNA with degenerate primers constructed on the basis of conserved regions identified withi n yeast and rat liver CCT molecules, and using the PCR product to scre en a genomic library. The P. falciparum CCT gene encodes a protein of 370 amino acids (42. 6 kDa) and displays 41-43% similarity (28-29% ide ntity) to CCT molecules of the other The central domain of CCT, propos ed as the catalytic domain of the CTP-transfer reaction, shows 68-72% similarity and 48-55% identity among P. falciparum, human, rat and yea st enzymes. This gene is present in a single copy, as determined by So uthern-blotting of genomic DNA, and located on chromosome 13 of P. fal ciparum. Large transcripts were detected by Northern analysis and indi cate that this gene is expressed in the asexual intraerythrocytic stag es. The coding region of the P. falciparum CCT gene was inserted into an Escherichia coli expression vector to confirm the function of the C CT product. The recombinant CCT expressed in E. coli is catalytically active, as evidenced by the conversion of phosphocholine to CDP-cholin e.