UV-induced mutagenesis in silent and constitutively expressed genes. (A) Schematic representation of the strategy used to appraise UV-induced mutagenesis. Genomic fragments from the indicated genes were chosen to contain the possible mutation sites GATT (T <> C) dimer in the transcribed strand) or AATC (dimer in the nontranscribed stand), both converted to AATT if a mutation occurs during replication, allowing digestion with Tsp509I. Nucleotide numbering is according to NCBI. Fragment sizes (after digestion, ligation of an adapter, and amplification by nested PCR) are shown next to each potential mutation site. For positive controls, longer sequences (black bars) containing a genuine AATT site were amplified with a different primer. For TP53, the positive control was created by site-directed mutagenesis of the downstream GATT site. (B) Mutagenesis in the silent Myosin gene, in cells irradiated or not irradiated with 5 J/m2 UV. Top left, A 98-bp mutated fragment was detected in all 3 cell types, and induced by UV in B lymphocytes (n = 12 donors) and XP-E lymphoblasts. Bottom left, Calibration curve used to appraise mutation frequency. Amplification of a 112-bp control band was detected after diluting the positive control genomic fragment with the test fragment down to a ratio of 5 × 10−6. Right, After quantification of ethidium bromide fluorescence, the mutation frequency in each cell type was determined on the basis of the calibration curve. Data are representative of 3 distinct experiments and shown as mean ± SEM. (C) Mutagenesis in the nontranscribed strand (NTS) of the DHFR gene. Top left, 2 mutated fragments of 186 bp and 113 bp were detected in UV-irradiated XP-E B lymphoblasts but not in WT lymphoblasts or B lymphocytes (n = 10 donors). Bottom left, Calibration curve for the DHFR control fragment. Right, Quantification of mutation frequency based on the calibration curve. Data are representative of 3 distinct experiments, and shown as mean ± SEM. (D) Mutagenesis in the transcribed strand (TS) of the TP53 gene. Top left, Amplification of a 265-bp and an 89-bp fragment was expected if a mutation occurred, but only background levels were detected, irrespective of UV irradiation. Bottom left, Calibration curve for the TP53 control fragment. This control, generated by site-directed mutagenesis, has the same size (265 bp) as the expected fragment and is detectable down to a dilution of 5 × 10−6. Right, Quantification of mutation frequency in the TP53 fragment.
