Onchocerca volvulus superoxide dismutase genes: identification of functional promoters for pre-mRNA transcripts which undergo trans-splicing.
The genes encoding three forms of superoxide dismutase, the cytosolic and extracellular CuZn superoxide dismutases and the mitochondrial Mn superoxide dismutase, were isolated from an Onchocerca volvulus lambda fix II genomic library. Genomic Southern blot analyses indicate single-copy genes in the O. volvulus genome. The O. volvulus cytosolic and extracellular CuZnSOD genes (Ov-sod-1 and Ov-sod-2) are separated by 0.8 kb of sequence and are convergently transcribed. Since the transcripts from all three sod genes are trans-spliced, the transcription start point of each gene was determined in a heterologous system that lacks trans-splicing machinery by in vitro transcription using Drosophila embryo nuclear extracts, followed by primer extension experiments. The ability of the 5' flanking region of the genes encoding the three Ov-SODs to promote transcription was further examined in transient transfections of Chinese hamster ovary cells. In firefly luciferase reporter assays, the Ov-sod-1 and -2 and the MnSOD (Ov-sod-3) gene promoters showed minimal, strong, and moderate levels of activity in these cells, respectively. Both Ov-sod-2 and -3 gene promoter regions showed an initial increase in activity in response to 5' deletions. The results from the in vitro transcription experiments and the luciferase reporter assays were consistent and suggest the presence of Inr-like elements in the promoter regions of the Ov-sod genes.
Tawe
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Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht Str. 74, Hamburg, Germany.
Walter
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RD
Henkle-Dührsen
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K
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D016466:CHO Cells; D006224:Cricetinae; D018509:DNA, Helminth / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D017930:Genes, Reporter; D016678:Genome; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D017181:Onchocerca volvulus / Q000201:enzymology / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D018510:RNA, Helminth / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D015183:Restriction Mapping; D013482:Superoxide Dismutase / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D018509:DNA, Helminth; D012322:RNA Precursors; D018510:RNA, Helminth; D012333:RNA, Messenger; D008156:Luciferases; D013482:Superoxide Dismutase