Figure 2

(A) Diagram of N-terminal deletions of OXR1. In these assays, the full-length OXR1 construct is represented by both pMV1263, which starts with methionine 225 in exon 7 (the expected start site of OXR1 in pMV520) and pMV1260, which encodes both this species and a slightly larger OXR1 species (by virtue of the presence of an initiating methionine start site inserted prior to codon 200 in exon 7). (B) Papillation assay for full-length OXR1 species (pMV1260 and pMV1263) and three N-terminal deletions starting within exon 8 (pMV1266), exon 9 (pMV1269) and exon 12 (pMV1275). Plasmids were expressed in strain MV4709 (mutM mutY lacZ cc104) and plated on minimal lactose plates containing 100 μg/ml carbenicillin with and without 1 mM IPTG. Aliquots of 10-fold serial dilutions are plated beginning with undiluted cells on the left. No OXR1 controls containing pBR322 showed patterns that were identical to pMV1269 and p1275 (not shown). (C) Papillation assay for full-length OXR1 species (pMV1260 and pMV1263) and three N-terminal deletions starting within exon 8 (pMV1266), exon 9 (pMV1269) and exon 12 (pMV1275). Plasmids were expressed in strain MV4709 (mutM mutY lacZ cc104) containing pMS421 (LacI-producer) and plated on minimal lactose plates containing 100 μg/ml carbenicillin, 40 μg/ml streptomycin, 20 μg/ml spectinomycin and 1 mM IPTG. (D) SDS-PAGE of extracts containing plasmids expressing OXR1 N-terminal deletions. Plasmid pMV1263 makes the full-length OXR1 species encoded by pMV520, while pMV1260 makes both this species and a slightly larger one that starts at position Lys-200 in exon 7. In addition, the arrows denote positions of observed protein bands for pMV1269 and pMV1272. (E) SDS-PAGE comparing full length OXR1 (pMV520) and OXR1 fragment starting at exon 9 (pMV1269). Arrows denote positions of induced protein bands; full length OXR1 is present as a doublet (see text for details).