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

Chlamydomonas reinhardtii chloroplast transformants that lack an inverted repeat normally found at the 3' end of the chloroplast atpB gene have a slow phototrophic growth phenotype due to reduced accumulation of atpB mRNA and the chloroplast ATPase beta subunit. We have recovered transformants exhibiting more robust phototrophic growth at a moderate frequency (similar to 1% relative to slow-growing transformants). Quantitative DNA blot analysis indicated that in one class of these robust photosynthetic transformants, the introduced plasmid DNA is maintained at high copy number-similar to 25 copies per chloroplast genome or 2000 copies per cell. Partial restriction digests resulted in a ladder with at least 15 visible fragments, indicating that most of the transforming DNA is organized as a long head-to-tail tandem repeat. Total atpB transcription and accumulation of atpB mRNA and the ATPase beta subunit were increased approximately fivefold relative to transformants that carry a single copy of the truncated atpB gene. The amplified DNA was stably maintained at high copy number under mixotrophic growth conditions. It was inherited uniparentally from the mt(+) parent, and its synthesis was sensitive to 5-fluoro-2'-deoxyuridine, an inhibitor of chloroplast DNA synthesis. Therefore, we conclude that the tandem repeat is maintained in the chloroplast. Restriction enzymes that fail to digest the transforming plasmid but have recognition sites in chloroplast DNA did not alter the electrophoretic mobility of the tandem repeat, suggesting that it is not integrated in the chloroplast genome. We conclude that the tandem repeat is probably episomal and hypothesize that its replication is independent of the chloroplast genome.