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 read ing frames (ORFs). We used transformation of an intron-less-psbA strain (IL ) to test whether these introns undergo homing, Each intron, pins short exo n sequences was cloned into a chloroplast expression vector in both orienta tions and then cotransformed into IL along with a spectinomycin resistance marker (16S rrn). For Cr.psbA2, the sense construct gave nearly 100% cointe gration of the intron whereas the antisense construct gave 0%, consistent w ith homing. For Cr.psbA4, however, both orientations produced highly effici ent cointegration of the intron. Efficient cointegration of Cr.psbA4 also o ccurred when the intron was introduced as a restriction fragment lacking an y known promoter, Deletion of most of the ORF, however, abolished cointegra tion of the intron, consistent with homing. The Cr.psbA4 constructs also co ntained 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 > 10 0-fold more transformants (>10,000/mug of DNA) than did the spectinomycin r esistance marker. A trans homing assay; was developed for Cr.psbA4; the ORF -minus intron integrated when the ORF was cotransformed on a separate plasm id. 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 b acterial (-10, -35)-type promoter. Primer extension analysis detected a tra nscript that could originate from this promoter. Thus, this mobile, self-sp licing intron also contains its own promoter for ORF. expression. The impli cations of these results for horizontal intron transfer and organelle trans formation are discussed.