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The three modifier mutations recovered in msl1 cluster around the PEHE domain.
Using eT1, the forward mutation frequency for MMC was observed to be 2% with an equal number of lethal mutations recovered in the first and second broods (A. M. Rose, unpublished results).
This indicates that the mutations recovered in the oncogenes are likely the direct consequence of the alkylation treatment and that the local sequence effects seen in E. coli also appear to occur in mammalian cells.
We validated the resistance profile for the vast majority of candidate mutations recovered in our primary screen, and found that less than 10% of the mutations were false positives (8).
Mutations recovered in genes encoding elongation factors have revealed their involvement in a diverse range of biological pathways including the pathogenesis of numerous cancers, embryonic patterning, haematopoiesis, and also neuronal development, migration and degeneration.
Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.
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Our design also reduced the probability that mutations recovered would arise in shared elements of neurotransmitter release machinery or determinants of cholinergic and GABAergic signaling, as these lines would be predicted to exhibit locomotor dysfunction on solid substrate.
A second class of mutations, recovered only during evolution in higher sublethal concentrations of the antibiotic, deleted the C-terminal end of the ATP synthase shaft.
In contrast, silent, missense, and splice junction mutations were recovered in an equivalent proportion as predicted (Table 3).
In contrast missense, stop and splice junction mutations were recovered in a slightly lower proportion than predicted (Table 2).
Osakabe et al. (2010) reported ZFNs with relatively low somatic mutagenesis frequencies (0.26 3%), and yet heritable mutations were recovered in two of nine lines analyzed.
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