Complete cDNA and genomic sequences encoding the Onchocerca volvulus S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine biosynthesis, have been isolated and characterized. The deduced amino acid sequence encodes a 42 kDa proenzyme with a moderate level of sequence homology to eukaryotic SAMDCs. Enzymically active O. volvulusSAMDC was expressed at a high level in an Escherichia colimutant strain lacking endogenous SAMDC. The recombinant enzyme was purified to homogeneity using DEAE-cellulose, methylglyoxal bis(guanylhydrazone)–Sepharose and Superdex S-200 chromatography. It was determined that the recombinant proenzyme is cleaved to produce 32 and 10 kDa subunits. The sequence of the N-terminal portion of the large subunit was determined and comparison with the sequence of the proenzyme revealed that the precise cleavage site lies between Glu86 and Ser87. Gel-filtration experiments demonstrated that these two subunits combine to form an active heterotetramer. Comparison of the cDNA and genomic sequences revealed that the SAMDC mRNA undergoes both cis- and trans-splicing in its 5´-untranslated region (UTR). Anchored PCR on O. volvulusmRNA confirmed the cDNA sequence and identified two distinct trans-spliced products, a 22-nucleotide spliced-leader sequence and a 138 bp sequence containing the 22 nucleotide spliced-leader sequence. Genomic Southern-blot analysis suggests that the O. volvulusSAMDC is encoded by a single-copy gene. This gene spans 5.3 kb and is comprised of nine exons and eight introns. The first intron is located in the 5´-UTR and processing of this intron has a potential regulatory function. The 5´-flanking region of the gene contains potential transcriptional regulatory elements such as a TATA box, two CAAT boxes and AP-1-, C/EBP-, ELP-, H-APF-1-, HNF-5- and PEA3-binding sites.

This content is only available as a PDF.

Author notes

The nucleotide sequences reported in this paper have been submitted to the GenBankTM/EMBL Data Bank with the accession numbers X95713 and X95714 for the cDNA and gene sequences respectively.