Molecular and Cellular Biology, May 2001, p. 3472-3481, Vol. 21, No. 10
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.10.3472-3481.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


Section of Molecular Cell and Developmental Biology and Institute for Cellular and Molecular Biology, School of Biological Sciences, University of Texas at Austin, Austin, Texas 78712
Received 25 August 2000/Returned for modification 10 November 2000/Accepted 27 February 2001
Introns 2 and 4 of the psbA gene of Chlamydomonas
reinhardtii chloroplasts (Cr.psbA2 and
Cr.psbA4, respectively) contain large free-standing open
reading frames (ORFs). We used transformation of an
intronless-psbA strain (IL) to test whether these introns undergo homing. Each intron, plus short exon sequences, was cloned into
a chloroplast expression vector in both orientations and then
cotransformed into IL along with a spectinomycin resistance marker (16S
rrn). For Cr.psbA2, the sense construct gave
nearly 100% cointegration of the intron whereas the antisense
construct gave 0%, consistent with homing. For Cr.psbA4,
however, both orientations produced highly efficient cointegration of
the intron. Efficient cointegration of Cr.psbA4 also
occurred when the intron was introduced as a restriction fragment
lacking any known promoter. Deletion of most of the ORF, however,
abolished cointegration of the intron, consistent with homing. The
Cr.psbA4 constructs also contained a
3-(3,4-dichlorophenyl)-1,1-dimethylurea resistance marker in exon 5, which was always present when the intron integrated, thus demonstrating
exon coconversion. Remarkably, primary selection for this marker gave
>100-fold more transformants (>10,000/µg of DNA) than did the
spectinomycin resistance marker. A trans homing assay was
developed for Cr.psbA4; the ORF-minus intron integrated
when the ORF was cotransformed on a separate plasmid. This assay was
used to identify an intronic region between bp
88 and
194 (relative
to the ORF) that stimulated homing and contained a possible bacterial
(
10,
35)-type promoter. Primer extension analysis detected a
transcript that could originate from this promoter. Thus, this mobile,
self-splicing intron also contains its own promoter for ORF expression.
The implications of these results for horizontal intron transfer and
organelle transformation are discussed.
Present address: Department of Biochemistry, University of Texas
Health Science Center at San Antonio, San Antonio, TX 78284-7760.
Present address: San Diego Supercomputer Center, University of
California at San Diego, La Jolla, CA 92093-0537.
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