GENE AMPLIFICATION CAN CORRECT A PHOTOSYNTHETIC GROWTH DEFECT CAUSED BY MESSENGER-RNA INSTABILITY IN CHLAMYDOMONAS CHLOROPLASTS

Chlamydomonas reinhardtii chloroplast transformants that lack an inver ted repeat normally found at the 3' end of the chloroplast atpB gene h ave a slow phototrophic growth phenotype due to reduced accumulation o f atpB mRNA and the chloroplast ATPase beta subunit. We have recovered transformants exhibiting more robust phototrophic growth at a moderat e frequency (similar to 1% relative to slow-growing transformants). Qu antitative DNA blot analysis indicated that in one class of these robu st photosynthetic transformants, the introduced plasmid DNA is maintai ned at high copy number-similar to 25 copies per chloroplast genome or 2000 copies per cell. Partial restriction digests resulted in a ladde r with at least 15 visible fragments, indicating that most of the tran sforming DNA is organized as a long head-to-tail tandem repeat. Total atpB transcription and accumulation of atpB mRNA and the ATPase beta s ubunit were increased approximately fivefold relative to transformants that carry a single copy of the truncated atpB gene. The amplified DN A was stably maintained at high copy number under mixotrophic growth c onditions. It was inherited uniparentally from the mt(+) parent, and i ts synthesis was sensitive to 5-fluoro-2'-deoxyuridine, an inhibitor o f chloroplast DNA synthesis. Therefore, we conclude that the tandem re peat is maintained in the chloroplast. Restriction enzymes that fail t o digest the transforming plasmid but have recognition sites in chloro plast DNA did not alter the electrophoretic mobility of the tandem rep eat, suggesting that it is not integrated in the chloroplast genome. W e conclude that the tandem repeat is probably episomal and hypothesize that its replication is independent of the chloroplast genome.