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Polymerase chain reaction-based cloning of alkyl-dihydroxyacetonephosphate synthase complementary DNA from guinea pig liver.

Abstract
Peroxisomes are indispensable organelles for ether lipid biosynthesis in mammalian tissues, and the deficiency of these organelles in a number of peroxisomal disorders leads to deficiencies in ether phospholipids. We have previously purified the committed enzyme for ether lipid biosynthesis, i.e. alkyl-dihydroxyacetone-phosphate synthase, to homogeneity. We have now determined the N-terminal amino acid sequence, as well as additional internal sequences obtained after cyanogen bromide cleavage of the enzyme. With primers directed against the N-terminal sequence and against a cyanogen bromide fragment sequence, a 1100-bp cDNA fragment was obtained by conventional polymerase chain reaction using 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. The open reading frame encodes a protein of 658 amino acids, containing the N-terminal amino acid sequence as well as the cyanogen bromide cleavage fragment sequences. The derived amino acid sequence includes a mature protein 600 amino acids long and a presequence 58 amino acids long. The latter contains a stretch of amino acids known as peroxisomal targeting signal 2. The size of the mRNA was estimated to be around 4200 nucleotides. Recombinant His-tagged alkyl-dihydroxyacetonephosphate synthase expressed in Escherichia coli was enzymatically active.
AuthorsE C de Vet, A W Zomer, G J Lahaut, H van den Bosch
JournalThe Journal of biological chemistry (J Biol Chem) Vol. 272 Issue 2 Pg. 798-803 (Jan 10 1997) ISSN: 0021-9258 [Print] United States
PMID8995366 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • DNA, Complementary
  • Peptide Fragments
  • Transferases
  • Alkyl and Aryl Transferases
  • alkylglycerone-phosphate synthase
  • Cyanogen Bromide
Topics
  • Alkyl and Aryl Transferases
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cyanogen Bromide (metabolism)
  • DNA, Complementary (chemistry)
  • Guinea Pigs
  • Liver (enzymology)
  • Microbodies (enzymology)
  • Molecular Sequence Data
  • Peptide Fragments (chemistry)
  • Polymerase Chain Reaction (methods)
  • Transferases (genetics)

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