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THE PLANT CELL, Vol 2, Issue 11 1059-1070, Copyright © 1990 by American Society of Plant Biologists
Transcriptional Analysis of Endogenous and Foreign Genes in Chloroplast Transformants of Chlamydomonas
A. D. Blowers, G. S. Ellmore, U. Klein and L. Bogorad
The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138
Transcription from modified chloroplast genes has been studied in vitro,
but only with the recently developed ability to stably introduce foreign
DNA into Chlamydomonas reinhardtii chloroplast chromosomes in situ has it
become possible to do so in vivo. Cloned chloroplast DNA sequences, into
which had been inserted chimeric genes composed of the GUS coding sequence
reporter under transcriptional control of chloroplast promoters for the C.
reinhardtii atpA, atpB, and rbcL genes, were introduced into the cells on
microprojectiles. These constructs become integrated into chloroplast
chromosomes by homologous recombination. RNA gel blot analyses demonstrated
that a single major [beta]-glucuronidase (GUS)-hybridizing transcript
accumulates in each chloroplast transformant. We have found that: (1)
Transcription of the chimeric gene begins at the same site as in the
corresponding endogenous chloroplast gene; (2) the rates of transcription
in vivo of the atpA:GUS and atpB:GUS genes relative to one another and to
other genes are the same as those for the endogenous atpA and atpB genes,
respectively, indicating that these promoters are fully functional despite
being fused to a foreign gene and being at an alien location on the
chloroplast chromosome; (3) in contrast to the atpA and atpB promoters, the
rbcL promoter directs transcription of the rbcL:GUS gene at only 1% of the
expected rate, suggesting that other features are required for optimal
activity of this promoter; and (4) 22 base pairs upstream of the 5[prime]
end of the atpB:GUS transcript in the atpB promoter element is sufficient
to confer wild-type levels of promoter activity.
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