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

Introns 2 and 4 of the psbA gene of Chlamydomonas reinhardtii chloroplasts (Cr.psbA2 andCr.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 (16Srrn). 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. TheCr.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.

ACKNOWLEDGMENTS

We thank Udo Johanningmeier for the IL strain, Michel Goldschmidt-Clermont for the atpX vector, and Lib Harris and the Chlamydomonas Genetics Center for plasmid DNAs.

This research was supported by grants from the USDA (NRICGP 96-35301-3420 and 99-35301-7847) and the Robert A. Welch Foundation (F-1164).