A novel trans-spliced mRNA 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 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. volvulus SAMDC was expressed at a high level in an Escherichia coli mutant 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. volvulus mRNA 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. volvulus 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 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.
Da'Dara
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Bernhard Nocht Institute for Tropical medicine, Department of Biochemistry, Hamburg, Germany.
Henkle-Dührsen
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K
Walter
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D012437:Adenosylmethionine Decarboxylase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000737:chemistry; D005091:Exons; D017238:Genes, Helminth; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D017181:Onchocerca volvulus / Q000201:enzymology* / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000201:enzymology
D018076:DNA, Complementary; D004792:Enzyme Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012437:Adenosylmethionine Decarboxylase