title: A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii.
abstract: Although Chlamydomonas reinhardtii serves as the most popular algal model system, no efficient enzymatic selection marker for the nuclear transformation of wild-type cells is available. We sequenced an aminoglycoside 3'-phosphotransferase gene (aph) from Streptomyces rimosus. Though the derived protein sequence is homologous to members of APH type V, it constitutes a new type, named APHVIII. Since the aphVIII gene has a codon bias similar to that of the nuclear genome of green algae, the aphVIII coding sequence was fused to the 5'- and 3'-untranslated regions of the C. reinhardtii rbcS2 gene. C. reinhardtii transformants were capable of inactivating the antibiotics paromomycin, kanamycin, and neomycin, to which wild-type cells are sensitive. After addition of the 5'-region of hsp70A as a second promoter and insertion of the rbcS2 intron I, the transformation rate increased to two transformants per 1 x 10(5) cells, which is close to the efficiency of transforming auxotrophic strains with the homologous marker arg7. Transformation with the promoter-less aphVIII led to random gene fusion at high frequency. In an aphVIII-based reporter gene assay we have found a so far unknown promoter activity of the 3'-untranslated region of rbcS2, that may promote antisense RNA synthesis from the rbcS2 gene in vivo. We conclude that the aphVIII gene is a useful marker for nuclear transformation and promoter tagging of C. reinhardtii wild-type and probably other green algae.
authors.affiliation: Biological Institute, St. Petersburg State University, Oranienbaumskoye sch., 2, St. Petersburg, 198904 Russia. irina.sizova@is6522.spb.edu
authors.forename: I
authors.initials: I
authors.lastname: Sizova
authors.forename: M
authors.initials: M
authors.lastname: Fuhrmann
authors.forename: P
authors.initials: P
authors.lastname: Hegemann
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D007612:Kanamycin / Q000494:pharmacology; D019868:Kanamycin Kinase / Q000235:genetics*; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D010303:Paromomycin / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D020121:5' Untranslated Regions; D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D018840:HSP70 Heat-Shock Proteins; D007612:Kanamycin; D010303:Paromomycin; D019868:Kanamycin Kinase; D012273:Ribulose-Bisphosphate Carboxylase; D009355:Neomycin