starter cultures of LB medium overnight at 37uC, inoculated into 0.5-l LB medium in ultra-yield flasks, and autoinduced by growing at 37uC for 3 h, followed by 18uC for 24 h [66]. Cells were harvested by centrifugation (Beckman JLA-8.1000; 4,0006 g, 15 min, 4uC), resuspended in 1 M NaCl, 20 mM Tris-HCl, pH 7.5, 20% glycerol, and 0.1 mg/ml lysozyme (Sigma-Aldrich, St. Louis, MO), kept on ice for 15 min, and lysed by 3 freeze-thaw cycles on dry ice followed by sonication (Branson 450 Sonifier, Branson Ultrasonics, Danbury, CT; three or four 10 sec bursts on ice at an amplitude of 60%, with 10 sec between bursts). After pelleting cell debris (Beckman JA-14 rotor, 10,000 rpm, 30 min, 4uC), nucleic acids were precipitated from the supernatant with 0.4% polyethylenimine (PEI) and constant stirring for 20 min at 4uC, followed by centrifugation (Beckman JA-14 rotor, 14,000 rpm, 30 min, 4uC). Proteins were precipitated from the supernatant by adding ammonium sulfate to 50% saturation with constant stirring for 1 h at 4uC. The precipitated proteins were pelleted (Beckman JA-14 rotor, 14,000 rpm, 30 min, 4uC), dissolved in 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, and run through a 10-ml amylose column (FPLC; Amylose High-Flow resin; New England BioLabs, Ipswich, MA), which was washed with 3 column volumes of 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol and eluted with 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol containing 10 mM maltose. Fractions containing the MalE-LtrA fusion were incubated with TEV protease (80 mg/ml, 18 h, at 4uC), and imidazole was added to a final concentration of 40 mM. LtrA freed of the MalE tag was then purified by FPLC through a Ni- NTA equilibrated with 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, 40 mM imidazole. The Ni-NTA column, which takes advantage of endogenous histidine residues in LtrA’s C- terminal domain, was washed with 3 column volumes of 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, 40 mM imidaz- ole, and eluted in 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, 300 mM imidazole. Finally, the peak LtrA fractions from the Ni-NTA column were further purified through two tandem 1- ml heparin Sepharose columns (New England BioLabs). The columns were equilibrated with 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, loaded directly with LtrA protein from the Ni-NTA column, washed with 5-column volumes of 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, and eluted with a 20-column volume gradient of 0.5 to 1 M NaCl, 20 mM Tris- HCl, pH 7.5, 10% glycerol. The protein elutes approximately midway through the gradient at ,750 mM NaCl. The purified protein was concentrated to 30 mM, exchanged into 100 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol by dialysis, flash- frozen in liquid nitrogen, and stored at 280uC. Retrohoming assays and analysis of intron-integration junctions Drosophila embryos were microinjected with ,300 pl recipient plasmid pBRR3-ltrB at 1.4 mg/ml in solution with 500 mM MgCl2 and 17 mM dNTPs, followed within 5 min by ,300 pl of Ll.LtrB lariat or linear RNPs at 2.6 mg/ml in 10 mM KCl, 5 mM MgCl2, 40 mM HEPES, pH 8.0. The RNPs consist of LtrA protein bound to Ll.LtrB lariat or linear intron RNA with a phage T7 promoter sequence inserted in intron domain IV, and the recipient plasmid contains the Ll.LtrB intron target site (ligated exon 1 and 2 sequences of the ltrB gene; E1 and E2) cloned upstream of a promoterless tetR gene in a pBR322-based vector carrying an AmpR marker. Site-specific integration of the intron into the target site introduces the T7 promoter upstream of the promoterless tetR gene, thereby activating that gene. Eighty embryos were injected and incubated at 30uC for 1 h for each Linear Group II Intron Retrohoming in Drosophila assay. The pooled embryos were incubated in lysis buffer (20 mM Tris-HCl, pH 8.0, 5 mM EDTA, 400 mM NaCl, 1% SDS (w/v)) with 400 mg/ml proteinase K (Molecular Biology Grade; Sigma- Aldrich) for 1 h at 55uC, and then extracted with phenol-CIA. Nucleic acids were ethanol precipitated and dissolved in 12 ml of distilled water. For assays of retrohoming efficiency, a 4-ml portion of the nucleic acid preparation was electroporated into electrocompetent E. coli HMS174(DE3) F2, hsdR, recA, rifr (Novagen, EMD Chemicals, Gibbstown, NJ), which expresses T7 RNA polymerase. Cells were plated at different dilutions on 2% agar containing LB medium with ampicillin (50 mg/ml) plus tetracycline (25 mg/ml) or the same concentration of ampicillin alone. Colonies were counted after overnight incubation at 37uC, and the integration efficiency was calculated as the ratio of (AmpR+TetR)/AmpR colonies. For analysis of intron-integration junctions, a 1-ml portion of the nucleic acid preparation was used as template for PCR using Phusion High Fidelity PCR Master Mix with HF buffer (New England BioLabs). The 59-junction PCRs were done with primers P1 (59-CTGATCGATAGCTGAAACGC) and LtrB933a (59- AGGGAGGTACCGCCTTGTTCACATTAC), and the 39 junc- tion PCRs were done with primers P3 (59-CAGTGAATTTT- TACGAACGAACAATAAC) and P4 (59-AATGGACGA- TATCCCGCA). The PCR was done for 25 cycles for all strains, except for parallel assays of wild-type Or-R and the polQ mutant, which required 35 PCR cycles to obtain sufficient PCR product from the mutant. The PCR products were purified using a MinElute PCR purification Kit (Qiagen), cloned into a TOPO TA cloning vector (pCRII-TOPO; Invitrogen, Carlsbad, CA), and transformed into chemically competent E. coli (One Shot TOP10; Invitrogen). The cloned PCR products were then amplified from randomly picked colonies by colony PCR using Phusion High Fidelity PCR Master Mix with HF buffer and primers M13 F(-20) (59- GTAAAACGACGGCCAGT) and M13 R(-26) (59-CAG- GAAACAGCTATGAC) for 25 cycles, and sequenced using primers M13 R(-24) (59-GGAAACAGCTATGACCATG) or M13 F(-20) [67]. Biochemical assays of group II intron RT template switching Biochemical assays were done by incubating purified LtrA protein (40 nM) with synthetic oligonucleotide substrates that correspond to the 59 end of the Ll.LtrB intron (60-nt Ll.LtrB RNA; 40 nM) with an annealed 59-32P-labeled DNA primer correspond- ing to nascent cDNA (45-nt Pri c; 44 nM) in the presence of exon 1 RNA or DNA (40-nt E1; 40 nM) in 20 ml of reaction medium containing 450 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol (DTT) and 200 mM dNTPs. The reaction components were assembled on ice with substrate added last and then incubated at 30uC for 30 min. Reactions were terminated by phenol-CIA extraction. Portions of the reaction product (3 ml) were added to an equal volume of gel loading buffer II (95% formamide, 18 mM EDTA and 0.025% each of SDS, xylene cyanol, and bromophenol blue; Ambion, Austin, TX), denatured at 98uC for 7 min, and analyzed by electrophoresis in a denaturing 10 or 15% polyacrylamide gel, which was visualized by scanning with a PhosphorImager (Typhoon Trio, GE Healthcare, Piscataway, NJ). 32P-labeled DNA products were excised from the gel, amplified by PCR, as described (Sabine Mohr, Scott Kuersten, and A.M.L., manuscript in preparation), and cloned into the TOPO-TA pCR2.1 vector (Invitrogen), according to the manufacturer’s protocol. Random colonies were picked and the cloned PCR products were amplified by colony PCR using Phusion High Fidelity PCR Master Mix/HF buffer with primers PLoS Genetics | www.plosgenetics.org 13 February 2012 | Volume 8 | Issue 2 | e1002534