MAINTENANCE AND EXPRESSION OF HETEROLOGOUS GENES IN CHLOROPLAST OF CHLAMYDOMONAS-REINHARDTII

The chloroplast genome of Chlamydomonas reinhardtii has been transform ed with a chimeric gene consisting of the chloroplast atpA promoter an d the bacterial gene for aminoglycoside adenine transferase (aadA). Th e atpA-aadA cassette has been placed within the chloroplast DNA EcoRI restriction enzyme fragment 14, or within the chloroplast BamH1 fragme nt 10. The chimeric constructs were introduced into the chloroplast by particle bombardment. Integration of the cassette into chloroplast DN A then occurred via homologous recombination of sequences flanking the cassette with their corresponding chloroplast sequences. We demonstra te that the chloroplast atpA promoter in atpA-aadA routinely recombine s with its endogenous counterpart, resulting in heteroplasmic chloropl ast DNA populations that may persist for many generations. The heterol ogous gene does not require a 3' inverted repeat sequence for its expr ession. The atpA-aadA gene copy number, which is dictated here by its position in the chloroplast genome, is proportional to the steady stat e level of atpA-aadA mRNA. However, neither genomic position, gene cop y number, or MRNA level have a significant effect on cellular resistan ce to spectinomycin, nor activity of the aadA gene product in vitro. T hese results suggest that, in the case of aadA, the limiting step for expression of this gene is at the translational or post-translational level. The atpA-aadA cassette should prove a useful model for future s tudies on the maintenance and expression of heterologous genes in C. r einhardtii chloroplasts.