THE CLONING AND SEQUENCING OF CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE cDNA FROM MURINE HEMOPOIETIC CELLS

CTP:phosphocholine cytidylyltransferase (CT) catalyzes a rate limiting reaction in the synthesis of phosphatidylcholine in higher eucaryotes. CT is subject to regulation by interconversion between an inactive cytosolic form and an active membrane bound form. This translocation is controlled by lipid activators and phosphorylation. At present, only the sequence of the rat liver CT cDNA and the yeast CT gene (Saccharomyces cerivisiae) have been reported. Comparison of the primary amino acid sequence shows a significant homology only in the central domain of the two proteins (64%). Since the study of CT has mainly focused on the mammalian enzyme, we have cloned and sequenced the CT cDNAs from a mouse hemopoeitic cell line. The strategy for isolating the mouse CT cDNA clone employed the polymerase chain reaction (PCR) in three sets of reactions. The initial set of reactions utilized two degenerate primers whose sequence was derived from the central conserved region of rat CT. A 224 bp fragment of the mouse cDNA was amplified, cloned and sequenced. In the next two sets of reactions, oligonucleotide primers specific for this 224 bp region of the mouse CT were used in conjunction with (1) the forward or (2) the reverse sequencing primer of the vector. These reactions, (1) and (2), amplified DNA fragments corresponding to the 5' and the 3' ends of the mouse CT cDNA, respectively. These two overlapping clones encoded a 1101 bp open reading frame. To check for PCRincorporated errors in the nucleotide sequence, a third set of reactions was performed using two mouse specific primers corresponding to the 5' and 3' untranslated regions. The amplified DNA (1281 bps) was cloned and sequenced. The sequences of 3 complete mouse clones were compared to determine the final mouse CT sequence. The human CT cDNA was cloned and sequenced using an identical approach. Comparison of the mouse, human and rat CT protein sequences demonstrated a high degree of conservation among these mammalian enzymes. When compared to rat CT, 4