Mobile Self-Splicing Group I Introns from the psbA Gene of Chlamydomonas reinhardtii : Highly Efficient Homing of an Exogenous Intron Containing Its Own Promoter

Abstract

ABSTRACT 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.