Mobile self-splicing group I introns from the psbA gene of Chlamydomonas reinhardtii: highly efficient homing of an exogenous intron containing its own promoter

Molecular and Cellular Biology
O W OdomD L Herrin

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/microgram of ...Continue Reading

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Citations

Sep 7, 2005·Photosynthesis Research·David L Herrin, Jöerg Nickelsen
Nov 26, 2002·The Plant Journal : for Cell and Molecular Biology·Fei LiDavid L Herrin
Nov 27, 2007·The Plant Journal : for Cell and Molecular Biology·Obed W OdomDavid L Herrin
Aug 13, 2005·Protein Expression and Purification·Hyong-Ha KimDavid L Herrin
Oct 7, 2017·Nucleic Acids Research·Marina CavaiuoloOlivier Vallon

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