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.a. 在所引用的全部研究中，研究人员均在Lac+应激诱导突变体中确定了一类或多类染色体未选择次级突变的发生频率；同时对可发生突变以产生所检测突变表型的碱基对（base-pair）数目进行了估算（详见补充材料S1），并进一步外推得到了全基因组每个碱基对预期的突变数目。上述估算基于以下假设：所有Lac+应激诱导突变体积累次级突变的概率均等，即单一可突变种群可产生应激诱导突变体。其他模型及其相关推论详见讨论部分。 b. 本文详述了用于在Lac+突变体中筛选次级突变体的直接转移法（Direct Transfer, DT）与纯化修复法（Purify-and-Patch, PP）。 c. 次级突变体筛选时所检测的表型包括：Mal?（无法发酵麦芽糖）、Xyl?（无法发酵木糖）以及Aux（营养缺陷型突变体）。 d. 除沙门氏菌营养缺陷型外，其余可发生突变以产生待筛选表型的碱基对近似数目均在补充材料S1中进行了估算。针对沙门氏菌营养缺陷型，我们通过与大肠杆菌（E. coli）比对进行估算：其基因组中共有84个基因，总长度约99000 bp，其中约三分之一（即33000 bp）的区域发生突变时可产生相应表型（详见补充材料S1）。 e. 将所检测到的每目标碱基对突变数外推至长度为4639221 bp的大肠杆菌（E. coli）基因组。对于肠炎沙门氏菌（S. enterica），我们采用的基因组大小为4857432 bp[82]。上述数值代表这些细胞中除Lac+突变外，预期存在的突变簇（包含1个或多个突变）数目。 f. 本部分数据为两株菌株的合并结果。在表达核酸内切酶I-SceI且携带I-SceI酶切位点、经证实可发生DNA切割的细胞中检测次级突变频率的实验中，每株菌株均作为阴性对照，此时不会发生I-SceI介导的DNA切割。两株阴性对照菌株分别为SMR6276与SMR6277：其一仅表达该酶但无对应酶切位点（记为“仅酶株”），其二既无酶切位点，也未携带染色体工程化PBAD启动子调控的I-SceI基因（记为“仅PBAD株”）；单株菌株的单独数据详见表3。