Maintenance and expression of heterologous genes in chloroplast ofChlamydomonas reinhardtii

The chloroplast genome ofChlamydomonas reinhardtii has been transformed with a chimeric gene consisting of the chloroplastatpA promoter and the bacterial gene for aminoglycoside adenine transferase (aadA). TheatpA-aadA cassette has been placed within the chloroplast DNAEcoRI restriction enzyme fragment 14, or within the chloroplastBamH1 fragment 10. The chimeric constructs were introduced into the chloroplast by particle bombardment. Integration of the cassette into chloroplast DNA then occurred via homologous recombination of sequences flanking the cassette with their corresponding chloroplast sequences. We demonstrate that the chloroplastatpA promoter inatpA-aadA routinely recombines with its endogenous counterpart, resulting in heteroplasmic chloroplast DNA populations that may persist for many generations. The heterologous gene does not require a 3 inverted repeat sequence for its expression. TheatpA-aadA gene copy number, which is dictated here by its position in the chloroplast genome, is proportional to the steady state level ofatpA-aadA mRNA. However, neither genomic position, gene copy number, or mRNA level have a significant effect on cellular resistance to spectinomycin, nor activity of theaadA gene productin vitro. These results suggest that, in the case ofaadA, the limiting step for expression of this gene is at the translational or post-translational level. TheatpA-aadA cassette should prove a useful model for future studies on the maintenance and expression of heterologous genes inC. reinhardtii chloroplasts.