Promotion of autonomous replication of plasmids within plastids themselves would provide a function-based assay in a homologous system for locating plastid origins of replication. Free monomeric plasmids are known to persist for a short period after transformation in C. reinhardtii (Boynton et al. 1988). This persistence might reflect replication and/or excision of complete plasmids integrated by single recombination events into the plastid genome. Homologous recombination between 16 bp direct repeats gave rise to an 868-bp DNA minicircle in N. ta-bacum plastids that was unstable but persisted as a multimeric series for several months during growth and development of transplastomic plants (Staub and Ma-liga 1994). The 868 bp excised sequence extends from the trnI(GAU) intron to the trnA(UGC) intron in the large inverted repeat and is located about 300 bases from the 82 bp region containing Ori A (Kunnimalaiyaan and Nielsen 1997a).
In C. reinhardtii, rescue of an atpB photosynthetic mutant with a partial function atpB allele led to amplification of the transforming plasmid in the form of large tandem arrays that appeared to be episomal (Suzuki et al. 1997). The maintenance of these episomal tandem arrays in plastids required a region of homology with the resident plastid genome. Revealingly, plastid transformation was not observed if there was no homology between resident plastid genome and transforming plasmid. Moreover, the inclusion of C. reinhardtii Ori A promoted integration rather than persistent autonomous replication (Suzuki et al. 1997). These results demonstrate that small plasmids containing mapped plastid replication origins do not contain sufficient sequence information for replication and partition to exist as stable autonomous replicons in plastids. Recombination appears to play a role in plasmid maintenance in plastids and this may also be true of WT plastid genomes (see Section 7 below).
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