Evidence for mutation showers

Wang et al. 10.1073/pnas.0610902104.

Supporting Information

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SI Table 3
SI Table 4
SI Text
SI Figure 3
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SI Figure 3

Fig. 3. Mutation shower distribution outside of lacI. The distribution of mutations in mutants with ectomutations is shown as a histogram with 1.8-kb bins extending downstream from lacI. The region between the flanking and remote regions that was not sequenced is denoted. The nucleotide numbering shown corresponds to that of the downstream regions. Where corresponding upstream regions were sequenced, those mutation counts were added to the counts for the downstream regions. Where corresponding upstream regions were not sequenced, the counts in the downstream regions were doubled to estimate the combined upstream/downstream counts.

SI Figure 4

Fig. 4. The lLIZ shuttle vector (45,530 bp) of the Big Blue mouse is derived from wild-type l-phage (48,502 bp) (NCBI GenBank accession no. NC_001416) by a deletion of three nonconsecutive segments (5,109 bp, 2,323 bp, and 17 bp) and insertion of the LIZ plasmid (4,475 bp) containing the lacI gene promoter and coding sequence, operator sequence, and a-portion of the lacZ gene in the reversed orientation (1).

1. Kohler SW, Provost GS, Fieck A, Kretz PL, Bullock WO, Putman DL, Sorge JA, Short JM (1991) Environ Mol Mutagen 18:316-321.

SI Text

Ectomutations Are Less Frequent in the Remote Regions. Only two ectomutations are found in the 8.5 kb of the remote region sequenced per mutant. This is likely to reflect a lower frequency of ectomutations 15-20 kb away from the initially observed domuplets in the lacI region relative to flanking regions.

As in the flanking regions, mutations in the remote regions are unlikely to disrupt the lambda lytic cycle. Analyses of l-phage protein alignments and types of mutations identified lead to the conclusion that l-phage and the lacZa proteins are generally quite tolerant to missense changes. Amino acid alignments of 10 to 20 homologous proteins reveal that the l-amino acid sequences are not highly conserved, showing only ≈3-7% highly conserved amino acids. Second, the two mutations identified in the remote regions are missense changes. In other experiments, four additional mutations were identified in the remote regions (one mutation in UR, three mutations in DR), three of which are missense changes and one is a silent change (manuscript in preparation). Therefore, among these six mutations, five are missense and one is silent (5:1 ratio). In the extreme, if none of the amino acids mattered we would expect a ratio of 3:1, based on the target size of missense and silent mutations (data not shown). The ratio of missense to nonsense mutations seen in the regions with essential genes also provides evidence supporting the tolerance to mutations (data not shown). Third, ≈60% of UF is composed of the J gene (≈2.2 kb), which is essential for phage lytic growth and the lacZa gene (0.6 kb), which is essential for identification of a blue plaque. Yet in the 3.6-kb UF region, six ectomutations are found, whereas only eight ectomutations are found in the 7.1-kb DF region. Although there happened to be more in the UF region, this is not statistically significant (P = 0.5). Thus, the 3.6-kb segment is not behaving differently within the sample size than if its target was truly on the order of 3.6 kb. Also, the lacZa segment is also in the UF region (0.6 kb). Thus 2.8 of the 3.8-kb UF includes genes for which deleterious mutations would prevent blue plaques from being identified. Five missense and two silent ectomutations were identified within these essential genes for a total of 10 missense to three silent mutations; however, there is no difference in the frequency of changes seen.

Two Germline Mutations in the Big Blue Shuttle Vector Were Identified. A total of 195 mutants were selected for analysis of additional sequence outside the lacI region of the Big Blue shuttle vector. A deletion of 507 nt was observed at bp 19683. This is located in the a-portion of lacZ. A 43-bp direct repeat is located at the deletion junctions of the 507-bp deletion, indicative of a strand slippage mechanism for the deletion. The mutation was observed in 13 of the 195 mutants and in eight animals and six tissues, consistent with a germ-line mutation in one of the forty copies of the Big Blue lLIZ shuttle vector integrated on chromosome 4. If this were a germ-line mutation, and if the ≈40 copies of the lacI genes in Big Blue (37) were packaged at similar frequencies, ≈2.5% of all plaques would contain the mutation. A second germ-line mutation C1064T was also found in 11 of the 195 mutants (4 singlets, 4 domuplets and 3 TBMs). This mutation results in a silent change (CAC1064 to CAT, His to His) in the A gene of the UR region.