Fig. 1.

Plasmid assay for group II intron retrohoming in X. laevis oocyte nuclei and D. melanogaster embryos. The assay uses the recipient plasmid pBRR3-ltrB, which contains the Ll.LtrB intron target site (ligated ltrB exon 1 and 2 sequences; E1 and E2) cloned upstream of a promoterless tet^R gene in a pBR322-based vector carrying an amp^R marker (20). The plasmid is microinjected into X. laevis oocyte nuclei or D. melanogaster embryos, followed by Ll.LtrB RNPs containing lariat or linear intron RNA with a phage T7 promoter inserted in intron RNA domain IV. Site-specific integration of the intron into the target site introduces the T7 promoter upstream of the promoterless tet^R gene. After extraction, nucleic acids are electroporated into E. coli HMS174(DE3), which expresses T7 RNA polymerase, and colonies containing retrohoming products are selected by plating on LB medium containing tetracycline and ampicillin. Integration efficiencies are determined as the ratio of (Tet^R + Amp^R)/Amp^R colonies. T1 and T2 are E. coli rrnB transcription terminators, and Tφ is a phage T7 transcription terminator.