THE PLANT CELL, Vol 1, Issue 1 123-132, Copyright © 1989 by American Society of Plant Biologists
Studies on Chlamydomonas Chloroplast Transformation: Foreign DNA Can Be Stably Maintained in the Chromosome
A. D. Blowers, L. Bogorad, K. B. Shark and J. C. Sanford
The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138
As shown originally by Boynton and co-workers (Boynton, J.E., Gillham,
N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R.,
Randolph-Anderson, B.L., Robertson, D., Klein, T.M., Shark, K.B., and
Sanford, J.C. [1988]. Science 240, 1534-1538), a nonphotosynthetic,
acetate-requiring mutant strain of Chlamydomonas reinhardtii with a
2.5-kilobase pair deletion in the chloroplast Bam 10 restriction fragment
region that removes the 3[prime] half of the atpB gene and a portion of one
inverted repeat can be transformed to photosynthetic competency following
bombardment with microprojectiles coated with wild-type Bam10 DNA. We have
found that assorted other circular plasmids, single-strand DNA circles, or
linear, duplex DNA molecules containing the wild-type atpB gene can also
complement the same mutant. DNA gel blot hybridization analysis of all such
transformants indicates that the complementing DNA has integrated into the
chromosome at the atpB locus and suggests that a copy-correction mechanism
operating between the inverted repeats maintains sequence identity in this
region. Sequences from the intact inverted repeat may be recruited to
restore the incomplete copy when exogenous DNA with only a portion of the
deleted sequence is introduced. Furthermore, a foreign, unselected-for,
chimeric gene flanked by chloroplast DNA sequences can be integrated and
maintained stably in the chloroplast chromosome. The bacterial neomycin
phosphotransferase structural gene fused to the maize chloroplast promoter
for the large subunit gene of ribulose-1,5-biphosphate carboxylase (rbcL)
has been integrated into the inverted repeat region of the Bam10
restriction fragment. RNA transcripts that hybridize to the introduced
foreign gene have been identified.
