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

Although Chlamydomonas reinhardtii serves as the most popular algal model s ystem, no efficient enzymatic selection marker for the nuclear transformati on of wild-type cells is available. We sequenced an aminoglycoside 3'-phosp hotransferase gene (aph) from Streptomyces rimosus. Though the derived prot ein sequence is homologous to members of APH type V, it constitutes a new t ype, 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 paro momycin, kanamycin, and neomycin, to which wild-type cells are sensitive. A fter addition of the 5'-region of hsp70A as a second promoter and insertion of the rbcS2 intron I, the transformation rate increased to two transforma nts per I X 10(5) cells, which is close to the efficiency of transforming a uxotrophic strains with the homologous marker arg7. Transformation with the promoter-less aphVIII led to random gene fusion at high frequency. In an a phVIII-based reporter gene assay we have found a so far unknown promoter ac tivity of the 3'-untranslated region of rbcS2, that may promote antisense R NA 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. r einhardtii wild-type and probably other green algae. (C) 2001 Published by Elsevier Science B.V.