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As 81% of our TS alleles were mis-sense mutations, searching for mis-sense mutations in mutant genome exon sequences should lead to finding the causative mutations in most TS mutants.
Black line indicates location of GGG to ATA mutations in mutant transposon ITR.
This approach has been previously described as beneficial for weeding out deleterious mutations in mutant strains [ 51, 52].
The mutations in mutant fus and pro44 are transitions, which are typical for EMS-induced changes (Bautz and Freese 1960).
These criteria were validated by the Sanger sequencing of 20 "true-positive" mutations and 22 "false-positive" mutations in mutant clone F-43.
It is an adaptation of the TILLING (Targeting Induced Local Lesion In Genomes) technique that is used to detect point mutations in mutant populations [ 1].
Similar(52)
Recent advances in sequencing technology have permitted rapid and affordable resequencing of fungal genomes, and this has enabled identification of causative mutations in mutants generated in genetic screens (McCluskey et al. 2011; Pomraning et al. 2011; Nowrousian et al. 2012; Bielska et al. 2014; Tan et al. 2014; Yao et al. 2014; Zhang et al. 2014).
The study of mutants to elucidate gene functions has a long and successful history; however, to discover causative mutations in mutants that were generated by random mutagenesis often takes years of laboratory work and requires previously generated genetic and/or physical markers, or resources like DNA libraries for complementation.
These results demonstrate that the lack of a functional pro41 is the causative mutation in mutant pro23.
In the case of the fus mutant, exactly one mutation was found that satisfied these conditions, and this turned out to be the causative mutation (Table 3; see section Analysis of the causative mutation in mutant fus).
This deleted region contained part of the open reading frame (ORF) of the developmental gene pro41, and the variant was established as the causative mutation of mutant pro23 (as mentioned the section Analysis of the causative mutation in mutant pro23).
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