A NOVEL TRANS-SPLICED MESSENGER-RNA FROM ONCHOCERCA-VOLVULUS ENCODES A FUNCTIONAL S-ADENOSYLMETHIONINE DECARBOXYLASE

Complete cDNA and genomic sequences encoding the Onchocerca volvulus S -adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine b iosynthesis, have been isolated and characterized. The deduced amino a cid sequence encodes a 42 kDa proenzyme with a moderate level of seque nce homology to eukaryotic SAMDCs. Enzymically active O. volvulus SAMD C was expressed at a high level in an Escherichia coli mutant strain l acking endogenous SAMDC. The recombinant enzyme was purified to homoge neity using DEAF-cellulose, methylglyoxal bis(guanylhydrazone)-Sepharo se and Superdex S-200 chromatography. It was determined that the recom binant proenzyme is cleaved to produce 32 and 10 kDa subunits. The seq uence of the N-terminal portion of the large subunit was determined an d comparison with the sequence of the proenzyme revealed that the prec ise cleavage site lies between Glu(86) and Ser(87). Gel-filtration exp eriments demonstrated that these two and genomic sequences encoding th e subunits combine to form an active heterotetramer. Comparison of the cDNA and genomic sequences revealed that the SAMDC mRNA undergoes bot h cis- and trans-splicing in its 5'-untranslated region (UTR). Anchore d PCR on O. volvulus mRNA confirmed the cDNA sequence and identified t wo distinct trans-spliced products, a 22-nucleotide spliced-leader seq uence and a 138 bp sequence containing the 22 nucleotide spliced-leade r sequence. Genomic Southern-blot analysis suggests that the O. volvul us SAMDC 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 loca ted in the 5'-UTR and processing of this intron has a potential regula tory function. The 5'-flanking region of the gene contains potential t ranscriptional 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.