The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas

A chimeric gene composed of the coding sequence of the ble gene from Streptoalloteichus hindustanus fused to the 5' and 3' untranslated regions of the Chlamydomonas reinhardtii nuclear gene RBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome of C. reinhardtii by co-transformation with the ARG7 marker yields Arg+ transformants of which approximately 80% possess the ble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (PmR) phenotype. Western blot analysis using antibodies against the ble gene product confirms the presence of the protein in the PmR transformants and genetic analysis demonstrates the co-segregation of the ble gene with the phenotype in progeny arising from the mating of a PmR transformant to wild-type strains. Direct selection of PmR transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome of C. reinhardtii and provides a useful dominant marker for nuclear transformation.