Appendix I
Details of individual mutations in the lacI gene of untreated and treated (3.5 Gy) Big Blue/wild-type and Big Blue/Ogg1-/- mice. Mutations are summarized in Table 1. Mutants are listed by position in the lacI gene, and single base substitutions are listed before other mutations. + in the CpG column indicate that the site of the mutation is part of a 5’-CpG-3’ sequence.
Big Blue wild-type, untreated.
pfu mutants clonal exp. mut. freq. (x10-5) gc to ta (x10-5) gc to at (x10-5)
230.000 11 2 4.8 0 (0.0) 7 (3.0)
295.000 13 2 4.4 2 (0.7) 8 (2.7)
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Base position Sequence (5’-3’) CpG wt aa mut aa codon
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42 ACG ® ATG + Thr Met 5
56 GCA ® ACA + Ala Thr 10
92 CGC ® TGC + Arg Cys 22
131 CGG ® TGG + Arg Cys 35
227 GCC ® CCC Ala Pro 67
297 CTG ® CCG Leu Pro 90
382 CGC ® CAC + Arg His 119
692 GCC ® ACC Ala Thr 227
791 CGC ® TGC + Arg Cys 255
940 TTG ® TTC Leu Phe 304
620-623 4 nt deletion reading frame distortion
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92 CGC ® TGC + Arg Cys 22
129 ACG ® AAG + Thr Lys 34
131 CGG ® TGG + Arg Trp 35
186 GCA ® GGA Ala Gly 53
198 GCG ® GTG + Ala Val 57
269 GCG ® ACG + Ala Thr 81
329 CGA ® TGA + Arg STP 101
380 CGC ® AGC + Arg Ser 118
594 TTA ® TAA Leu STP 189
659 CAG ® TAG Gln STP 211
783 GGC ® GAC Gly Asp 252
944 CAA ® TAA Gln STP 306
1011 Insert A Reading frame distortion
Big Blue Ogg1-/-, untreated.
pfu mutants clonal exp. mut. freq. (x10-5) gc to ta (x10-5) gc to at (x10-5)
220.000 8 2 3.6 3 (1.4) 3 (1.4)
300.000 15 4 5.0 4 (1.3) 9 (3.0)
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Base position Sequence (5’-3’) CpG wt aa mut aa codon
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42 ACG ® AAG + Thr Lys 5
188 CAA ® AAA Gln Lys 55
242 GCC ® CCC + Ala Pro 73
269 GCG ® ACG + Ala Thr 81
273 GCG ® GAG + Ala Glu 82
284 CGC ® TGC + Arg Cys 86
382 CGC ® CAC + Arg His 118
635 1 nt deletion Reading frame distortion
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113 CAC ® TAC His Tyr 29
180 CGC ® CAC + Arg His 51
197 GCG ® CCG Ala Pro 57
198 GCG ® GTG + Ala Val 57
269 GCG ® ACG + Ala Thr 81
273 GCG ® GAG + Ala Glu 82
308 GTG ® ATG + Val Met 94
318 TCG ® TAG + Ser STP 97
329 CGA ® TGA + Arg STP 101
380 CGC ® AGC + Arg Ser 118
381 CGC ® CAC + Arg His 118
569 CAA ® TAA Gln STP 181
671 GAA ® TAA Glu STP 217
792 CGC ® CAC + Arg His 255
1010 ACC ® CCC Thr Pro 328
Big Blue wild-type, 3.5 Gy.
pfu mutants clonal exp. Mut. freq. (x10-5) gc to ta (x10-5) gc to at (x10-5)
250.000 12 2 4.8 3 (1.2) 7 (2.8)
270.000 14 17 5.2 2 (0.7) 8 (3.0)
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Base position Sequence (5’-3’) CpG wt aa mut aa codon
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44 TTA ® GTA Leu Val 6
92 CGC ® AGC + Arg Ser 22
93 CGC ® CTC + Arg Leu 22
120 TCT ® TTT Ser Phe 31
180 CGC ® CAC + Arg His 51
269 GCG ® ACG + Ala Thr 81
284 CGC ® TGC + Arg Cys 86
357 GCG ® GTG + Ala Val 110
377 CAA ® TAA Gln STP 117
681 GGC ® GTC Gly Val 217
770 CAG ® TAG Gln STP 248
223 1 nt deletion Reading frame distortion
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31 G ® A initiation
56 GCA ® ACA + Ala Thr 10
95 GTG ® ATG + Val Met 23
197 GCG ® CCG Ala Pro 57
198 GCG ® GTG + Ala Val 57
530 CGA ® TGA + Arg STP 168
566 CAG ® TAG Gln STP 180
594 TTA ® TAA Leu STP 189
670 GAA ® TAA Glu STP 217
744 CCC ® CGC Pro Arg 239
777 GCG ® GTG + Ala Val 250
843 GGA ® GAA Gly Glu 272
1005 AGA ® ATA Arg Ile 326
-22 1 nt deletion promoter
Big Blue Ogg1-/-, 3.5 Gy.
pfu mutants clonal exp. mut. freq. (x10-5) gc to ta (x10-5) gc to at (x10-5)
215.000 22 2 10.2 9 (4.2) 7 (3.3)
230.000 23 0 10.0 9 (3.9) 8 (3.5)
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Base position Sequence (5’-3’) CpG wt aa mut aa codon
______
42 ACG ® AAG + Thr Lys 5
93 CGC ® CTC + Arg Leu 22
96 GTG ® GCG Val Ala 23
103 AAC ® AAA Asn Lys 25
129 ACG ® AAG + Thr Lys 34
173 CCC ® TCC Pro Ser 49
180 CGC ® CAC + Arg His 51
186 GCA ® GTA Ala Thr 53
198 GCG ® GAG + Ala Glu 57
206 CAG ® TAG Gln STP 60
231 ACC ® AAC Thr Asn 69
269 GCG ® ACG + Ala Thr 81
329 CGA ® TGA + Arg stop 101
382 CGC ® CAC + Arg Ala 118
783 GGC ® GTC Gly Val 252
1004 AGA ® TGA Arg Thr 326
1005 AGA ® ATA Arg Ile 326
1011 ACC ® AAC Thr Asn 328
153 1 nt deletion Reading frame distortion
337 1 nt deletion Reading frame distortion
293-294 2 nt deletion Reading frame distortion
529-530 GC ® AT + 2 nt substitution 167-168
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-12 A ® G Initiation
83 ACC ® GCC Thr Ala 19
86 GTT ® ATT + Val Ile 20
116 GTT ® AAA + Val Lys 30
117 GTT ® GCT Val Ala 30
169 TAC ® TAA Tyr STP 47
188 CAA ® AAA Gln Lys 54
260 CAA ® TAA Gln STP 78
269 GCG ® ACG + Ala Thr 81
273 GCG ® GAG + Ala Glu 82
318 TCG ® TAG + Ser STP 97
357 GCG ® GAG + Ala Glu 110
375 GCG ® GTG + Ala Val 116
382 CGC ® CAC + Arg His 118
382 CGC ® CAC + Arg His 118
561 GGT ® TGT Gly Cys 178
631 TGG ® TGA Trp STP 201
731 GAG ® TAG Glu STP 235
791 CGC ® AGC + Arg Ser 255
1037 GCC ® TCC + Ala Ser 337
59 9 nt insertion 3 aa’s addition
623 5 nt insertion Reading frame distortion
809 1 nt insertion Reading frame distortion
Appendix II
Mutation frequencies have been compared assuming a binomial distribution of mutations between treated and untreated mice. The ratio of the mutation frequencies are given with 95% confidence intervals. The test is described in more detail in Materials and methods. The comparison is only done for treated vs. nontreated animals of the same genotype.
Mutation frequencies (Figure 4A)
Wild-type (treated vs. nontreated)
ratio=1.1 (95% C.I. 0.6 - 2.0, P=0.861)
Ogg1-/- (treated vs. nontreated)
ratio=2.3 (95% C.I. 1.4 - 4.0, P=0.001)
Frequencies of GC to AT transitions (Figure 4 B, upper panel)
Wild-type (treated vs. nontreated)
ratio=1.0 (95% C.I. 0.5 - 2.2, P=1.000)
Ogg1-/- (treated vs. nontreated)
ratio=1.4 (95% C.I. 0.6 - 3.2 P=0.551)
Frequencies of GC to TA transversions (Figure 4 B, lower panel)
Wild-type (treated vs. nontreated)
ratio=2.5 (95% C.I 0.4 - 26.5 P=0.445)
Ogg1-/- (treated vs. nontreated)
ratio=3.0 (95% C.I. 1.2 - 8.5, P=0.016)