Mobile self-splicing group I introns from the psbA gene of Chlamydomonas reinhardtii: highly efficient homing of an exogenous intron containing its own promoter.
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/microgram 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.
Odom
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OW
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, USA.
Holloway
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SP
Deshpande
N N
NN
Lee
J
J
Herrin
D L
DL
D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D017343:Genes, Plant; D017125:Genes, Protozoan; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics*; D045332:Photosystem II Protein Complex; D011401:Promoter Regions, Genetic
D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex