SHM vectors used to monitor SHM in AID-expressing cell lines (A) pmutEGFP vector: EGFP (enhanced green fluorescent protein), a variant of the GFP gene, is placed under the control of the CMV promoter (human cytomegalovirus immediate-early promoter/enhancer); pUC–prokaryotic origin of replication; AMPR –ampicillin resistance gene; SV40 ori–eukaryotic origin of replication; KanR/NeoR–kanamycin/neomycin resistance gene (B) pEM7-EGFP vector: EGFP is placed under the control of both an eukaryotic (CMV) and prokaryoric (EM7) promoter; pUC–prokaryotic origin of replication; SV40 ori–eukaryotic origin of replication; KanR/NeoR–kanamycin/neomycin resistance gene (C) Sequence contexts of premature stop codons in the EGFP gene.
figure
posted on 2013-02-21, 11:14 authored by Tihana Jovanic, Benjamin Roche, Géraldine Attal-Bonnefoy, Olivier Leclercq, François RougeonThe upper row corresponds to the wild-type sequences and the lower row to the mutated sequences. Three variants of the EGFP gene inactivated with a premature termination codon introduced by site-directed mutagenesis were used 1) The G of the TAG termination codon at the position 182 is embedded within the RGYW hotspot motif and is thus a potential target of AID 2) The TAG stop codon at position 52 lies within two RGYW motifs 3) TAA codon at position 52 is used to monitor A∶T mutations only. RGYW motifs are shown in bold letters. The mutated nucleotides are underlined.
