Viral DNA encoding nerve agent model compound-resistant hBChE variants was sequenced to identify sites where mutagenesis was present.
The sequences of the primers used for site-specific mutagenesis are presented in Table S1.
Details of oligonucleotides used in RT PCR, cloning and site-directed mutagenesis are presented in Supplementary Table S1.
Detailed information on mutagenesis is presented in Additional file 3. Mutant G301A contains an Ala instead of Gly at position 301, mutant I330T a Thr instead of Ile at position 330, mutant C331Y a Tyr instead of Cys at position 331, and mutant G301A, I330T, C331Y has all three mutations.
Others of the five DNA polymerases might dampen mutagenesis simply by being present and competing with the mutagenic DNA polymerases, and influencing the dynamics of DNA polymerase choice thereby.
Two theories have been postulated; either secondary resistance mutations develop de novo during therapy by mutagenesis or they are present in minor subclones prior to therapy.
A possible mechanism, which involves these water molecules and is consistent with reported site-directed mutagenesis experiments, is presented in Figure 6.
The relative mutation frequency for the panel of viruses tested in the non-HSV DNA mutagenesis assay are presented in Table 2. Consistent with recent data from Hwang et al. [ 13], PAAr5 showed a minor increase in mutation frequency, compared with wild type HSV-1 and HSV-2 (Table 2).
Site-directed mutagenesis, random mutagenesis, and site-saturation mutagenesis were utilized to increase the OP-degrading activity of Dr-OPH.
However, at present it is difficult to compare these results with our study as different methodologies were used and most likely different mechanisms of mutagenesis were involved.
