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We compared radiosensitivity in a "single-hit" cell line with TP53 mutation converted to "double-hit" using RNA interference targeting FHIT.
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Mutations converted to the wild type allele in a given strain are indicated by a symbol.
Surprisingly, the originally stable ben1-1 mutation was converted to a partial loss-of-function mutation in the triple mutant background.
The PMA1 URA3 variant was created from the pma1Δ::KanMX4/PMA1 strain; the ura3Δ0 mutation was converted to URA3 via PCR amplification of URA3 from pRS306 and transformation.
All point mutations were converted to binary data (0 = no mutation, 1 = somatic mutation) for each sample, and a matrix with sample names in rows and loci in columns was generated.
From a directly measured 4.3 viable mutations per genome per generation, the viable deleterious mutation rate was converted to 2.6 by assuming a deleterious fraction of 0.6 (based on direct estimates from other viruses).
The mutation at 23973th nucleotide resulted in mutating Histidine at 53th residue to be Asparagine (H53N); 24171th nucleotide mutation changed Isoleucine at 119th to Valine (I119V); and 245789th nucleotide mutation converted Glu-313 to Lys-313 (E313K).
MCR can rapidly reveal recessive somatic phenotypes as heterozygous mutations are efficiently converted to the homozygous state.
When CB2000 in which the recA, dnaB, and yfjK mutations had been converted to wild type was mixed with Founder Δe14 (Ara−), survival of the two strains was similar (the CB2000 reversion strain had less than a 0.5 log advantage [ Figure 3D,E]).
When CB2000 (Ara−) was mixed in a 1 1 ratio with CB2000 in which the recA, dnaB, and yfjK mutations had been converted to wild type, and irradiated at 2000 and 3000 Gy, CB2000 exhibited at least a 2 log advantage in survival at the higher IR dose.
For 4CL1, one non-sense mutation was found in 6-9-G10 6-9-G10 6-9-G10tation converted a tryptophan to a stop codon.
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