THE PLANT CELL, Vol 6, Issue 2 187-200, Copyright © 1994 by American Society of Plant Biologists
Gene Amplification Can Correct a Photosynthetic Growth Defect Caused by mRNA Instability in Chlamydomonas Chloroplasts
K. L. Kindle, H. Suzuki and D. B. Stern
Plant Science Center, Biotechnology Building, Cornell University, Ithaca, New York 14853
Chlamydomonas reinhardtii chloroplast transformants that lack an inverted
repeat normally found at the 3[prime] 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 (~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-~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[prime]-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.
