Stable nuclear transformation of Chlamydomonas reinhardtii with a Streptomyces rimosus gene as the selective marker

Abstract

The aminoglycoside 3′-phosphotransferase typeVIII ( APHVIII) encoding gene ( aphVIII) from Streptomyces rimosus was introduced by glass-bead high-efficiency transformation into the nuclear genome of green unicellular alga Chlamydomonas reinhardtii for induction of transformants resistant to aminoglycoside antibiotics. The aphVIII structural sequence was flanked by S. rimosus regulatory sequences which failed to direct expression in C. reinhardtii. The pSU937 plasmid containing these sequences was able to transform C. reinhardtii strain cw15 arg7–8 mt + for paromomycin resistance (Pm R) at a frequency (1.3–1.9) × 10 -7, probably as a result of in vivo gene fusion and expression of the aphVIII gene from regulatory elements of nuclear DNA. Evidence for the real C. reinhardtii transformation includes blot-hybridization with a probe specific for aphVIII and demonstration of APHVIII enzyme activity in crude cell extracts of transformants. Integrated Streptomyces DNA sequences, APHVIII enzyme activity and the aminoglycoside-resistance phenotype were stable through mitosis in the presence and absence of selection. The Pm R phenotype was inherited by the meiotic progeny of transformants from crosses with wild-type strains.