Estimates of Mutation Clusters per Genome of Lac+ Stress-Induced Mutantsa.

aIn all of the studies cited, the frequency of one or more classes of chromosomal unselected secondary mutations were ascertained among Lac+ stress-induced mutants, the number of base-pairs that could be mutated to produce the mutant phenotype assayed was estimated (Text S1), and the number of mutations expected per all of the basepairs in the genome was then extrapolated. These estimates are based on the assumption that all Lac+ stress-induced mutants had an equal probability of accumulating secondary mutations, i.e., that a single mutable population produces stress-induced mutants. Other models and their consequences are discussed in the Discussion.bDirect transfer (DT) and purify-and-patch (PP) methods for identifying secondary mutants among Lac+ mutants are described in the text.cPhenotype assayed for when screening for secondary mutants. Mal?, unable to ferment maltose; Xyl?, unable to ferment xylose; Aux, auxotrophic mutants.dThe approximate numbers of basepairs that when mutated can lead to the phenotypes screened are estimated in Text S1, except for Salmonella auxotrophs, which we estimate by comparison with E. coli to involve 84 genes of a total size of about 99,000bp, one third of which, or 33,000bp, would be predicted to give a phenotype when mutated (see Text S1).eThe mutations observed per basepair targeted are extrapolated to the 4,639,221 bp E. coli genome. For S. enterica we took a genome size of 4,857,432 [82]. These figures represent the number of predicted mutation clusters (of one or more mutations) in addition to the Lac+ mutation in these cells.fThese are the combined data from two strains. Each strain served as a negative control, in which there was no cleavage of DNA with the endonuclease I-SceI, for experiments in which the frequency of secondary mutations was assayed in cells that express I-SceI and carry an I-SceI cutsite, and which we show experience DNA cleavage. The two negative-control strains, SMR6276 and SMR6277, either express the enzyme but have no cutsite (��Enzyme only�� strain) or have neither the cutsite nor the I-SceI gene under the control of the chromosomally engineered PBAD promoter (��PBAD only�� strain), and the data from each strain separately are shown in Table 3.