Heterologous expression of the cytotoxin restriction in Aspergillus nidulans and Aspergillus niger.
The cDNA clone of restrictocin was placed under the control of the glucoamylase promoter from Aspergillus awamori and was transformed into Aspergillus nidulans and Aspergillus niger. Site-specific changes were introduced into cDNA constructs and these were transformed into A. nidulans. The secretion signal sequence was deleted from one form of the gene and three mutations introduced single amino acid substitutions into the protein. Culture conditions were optimized for maximum expression levels of restrictocin. The activities of the expressed proteins were characterized with an in vitro rabbit reticulocyte assay. Protein synthesis in this assay was inhibited 50% by 2.5 ng/ml wild-type restrictocin, 3.5 ng/ml E95G, 30 ng/ml E95C, and 600 ng/ml H136L. Toxic effects of restrictocin were observed in the A. nidulans expression system with reduced levels of cellular protein and messenger RNA upon induction of restrictocin expression as well as the formation of the alpha-fragment product of ribosomal RNA cleavage. Toxic effects were most highly pronounced in strains expressing restrictocin without the signal sequence, less so in strains expressing native restrictocin, and absent in strains expressing H136L restrictocin.
Brandhorst
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T
Department of Biochemistry, University of Wisconsin at Madison 53706.
Yang
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R
Kenealy
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WR
D000485:Allergens; D052179:Antigens, Plant; D001230:Aspergillus / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D001233:Aspergillus nidulans / Q000187:drug effects / Q000235:genetics; D001234:Aspergillus niger / Q000187:drug effects / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003603:Cytotoxins / Q000096:biosynthesis* / Q000235:genetics / Q000633:toxicity; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000633:toxicity; D015966:Gene Expression Regulation, Fungal; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000378:metabolism* / Q000633:toxicity; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012260:Ribonucleases / Q000096:biosynthesis* / Q000235:genetics / Q000633:toxicity; D014170:Transformation, Genetic; D014404:Tubulin / Q000032:analysis
C081947:ASPF1 protein, Aspergillus fumigatus; D000485:Allergens; D052179:Antigens, Plant; D003603:Cytotoxins; D005656:Fungal Proteins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014404:Tubulin; C036196:MITF protein, Aspergillus restrictus; D012260:Ribonucleases; D005087:Glucan 1,4-alpha-Glucosidase