POLYMERASE CHAIN REACTION-BASED CLONING OF ALKYL-DIHYDROXYACETONEPHOSPHATE SYNTHASE COMPLEMENTARY-DNA FROM GUINEA-PIG LIVER

Peroxisomes are indispensable organelles for ether lipid biosynthesis in mammalian tissues, and the deficiency of these organelles in a numb er of peroxisomal disorders leads to deficiencies in ether phospholipi ds. We have previously purified the committed enzyme for ether lipid b iosynthesis, i.e. alkyl-dihydroxyac etonephosphate synthase, to homoge neity. We have now determined the N-terminal amino acid sequence, as w ell as additional internal sequences obtained after cyanogen bromide c leavage of the enzyme. With primers directed against the N-terminal se quence and against a cyanogen bromide fragment sequence, a 1100-bp cDN A fragment was obtained by conventional polymerase chain reaction usin g first-strand cDNA from guinea pig liver as a template, The 5' and 3' ends of the cDNA were obtained by rapid amplification of cDNA ends, T he open reading frame encodes a protein of 658 amino acids, containing the N terminal amino acid sequence as well as the cyanogen bromide cl eavage fragment sequences, The derived amino acid sequence includes a mature protein 600 amino acids long and a presequence 58 amino acids l ong. The latter contains a stretch of amino acids known as peroxisomal targeting signal 2, The size of the mRNA was estimated to be around 4 200 nucleotides. Recombinant His-tagged alkyl-dihydroxyacetonephosphat e synthase expressed in Escherichia coil was enzymatically active.