Expression of a foreign gene in Chlamydomonas reinhardtii chloroplast

The active digestion of uniparental chloroplast DNA in a single zygote of Chlamydomonas reinhardtii is revealed by using the optical tweezer

“…The bacterial aadA gene encodes aminoglycoside adenyl transferase and is expressed in chloroplasts conferring resistance to spectinomycin and streptomycin. Chloroplast transformation was carried out with a particle gun as described in a previous report (17). The aadA cassette was inserted into the noncoding region downstream from the rbcL gene in the wild-type chloroplast genome by homologous recombination.…”

“…Transformed strain L03c was used for this purpose. The cpDNA of L03c strain was transformed with the bacterial aadA gene, by using a particle gun (17). The behaviors of mt ϩ and mt Ϫ cpDNA were monitored separately by PCR amplification of the aadA sequences by using zygotes resulting from the cross between L03c gametes and wild-type 137c gametes of the opposite mating type.…”

“…Using this technique, we isolated a single zygote under direct observation, according to the presence or absence of fluorescent mt Ϫ cp nucleoids. The mt ϩ and mt Ϫ cpDNA molecules were labeled separately by using bacterial aadA (aminoglycoside adenyl transferase) sequences (16,17). Single zygotes obtained in this manner were subjected to highly sensitive nested-PCR analysis.…”

An mt+ gamete-specific nuclease that targets mt− chloroplasts during sexual reproduction in C. reinhardtii

“…Whether the active digestion of mt − cp-DNA occurs before or after chloroplast fusion is an important consideration in understanding the molecular mechanism that controls the preferential digestion and protection of cpDNA in young zygotes. To address this question, a chloroplast transformant (AG8 mt + ) was used (Ishikura et al 1999). The cpDNA of this strain was transformed by particle gun bombardment with the uidA gene, resulting in the accumulation of GUS (␤-glucuronidase) proteins in mt + chloroplasts.…”

“…Next, we used a tagged chloroplast transformant, AG8 mt + (Ishikura et al 1999), to investigate whether the preferential digestion of mt − cpDNA occurred before or after the fusion of mt + and mt − chloroplast membranes. Our results revealed that preferential digestion was completed before chloroplast fusion.…”