A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii

Gene. 2001 Oct 17;277(1-2):221-9. doi: 10.1016/s0378-1119(01)00616-3.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5' Untranslated Regions / genetics
  • Amino Acid Sequence
  • Animals
  • Anti-Bacterial Agents / pharmacology
  • Chlamydomonas reinhardtii / drug effects
  • Chlamydomonas reinhardtii / genetics*
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Drug Resistance / genetics
  • Gene Expression Regulation, Enzymologic
  • HSP70 Heat-Shock Proteins / genetics
  • Kanamycin / pharmacology
  • Kanamycin Kinase / genetics*
  • Molecular Sequence Data
  • Neomycin / pharmacology
  • Paromomycin / pharmacology
  • Plasmids / genetics
  • Promoter Regions, Genetic / genetics
  • Ribulose-Bisphosphate Carboxylase / genetics
  • Sequence Alignment
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid
  • Streptomyces / enzymology
  • Streptomyces / genetics*
  • Transformation, Genetic


  • 5' Untranslated Regions
  • Anti-Bacterial Agents
  • DNA, Bacterial
  • HSP70 Heat-Shock Proteins
  • Kanamycin
  • Paromomycin
  • Kanamycin Kinase
  • Ribulose-Bisphosphate Carboxylase
  • Neomycin

Associated data

  • GENBANK/AF182845