STABLE NUCLEAR TRANSFORMATION OF CHLAMYDOMONAS-REINHARDTII WITH A STREPTOMYCES-RIMOSUS GENE AS THE SELECTIVE MARKER

The aminoglycoside 3'-phosphotransferase typeVIII (APHVIII) encoding g ene (aphVIII) from Streptomyces rimosus was introduced by glass-bead h igh-efficiency transformation into the nuclear genome of green unicell ular alga Chlamydomonas reinhardtii for induction of transformants res istant 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 sequ ences was able to transform C. reinhardtii strain cw15 arg7-8 mt(+) fo r paromomycin resistance (Pm-R) at a frequency (1.3-1.9) x 10(-7), pro bably 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 c rude cell extracts of transformants. Integrated Streptomyces DNA seque nces, APHVIII enzyme activity and the aminoglycoside-resistance phenot ype were stable through mitosis in the presence and absence of selecti on. The Pm-R phenotype was inherited by the meiotic progeny of transfo rmants from crosses with wild-type strains.