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Lack of correlation between growth rate and ribosomal biogenesis gene expression was also observed in a compendium of 196 deletion mutants, for which both growth rates and genome-wide expression profiles were reported [19].
In addition to the genes identified by the insertional mutagenesis screen, mixture model analysis allowed us to identify a small number of genes, in mutants, for which expression patterns could not be accounted for using the three parasite states – genes that may play a mechanistic role in switching from the tachyzoite to bradyzoite stage.
We also contacted investigators for updated information on specific mutants, added a few loci from recent work in our laboratory, removed several genes where the estimated genetic location conflicted with the known physical location, and excluded mutants for which seed stocks were no longer available.
The mutants for which this was observed were P246F, L413F, L410W, L410F + S300E, and L410F + S365L.
Dark gray shading indicates mutants for which there were also timing defects.
gDNA digestions were performed with MunI, with the exception of N8 mutants for which the gDNA was digested with SpeI.
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We tested this model by designing a mutant for which the orientation of the two protein subdomains forming the nucleotide-binding pocket is directly controlled by the crosslinker, and confirmed successful reversible photoswitching in vitro.
We think that ATA is still a potent inhibitor for the oseltamivir-resistant strain when compared with oseltamivir in inhibiting the H274Y mutant, for which an over 700- fold increase of IC50 is needed [33].
This result was corroborated by assays using recombinant human KIT intracellular domain with the D816V mutation (Table 1) and its murine equivalent D814V mutant, for which masitinib had an IC50 of 3.0±0.1 µM (Figure 3C and Table 1).
In contrast to the arr21 knockout mutant, for which no phenotype was detected [30], over-expression of the constitutively active ARR21C protein results in abnormal development with tissues resembling in vitro callus [31].
In initial experiments, we tried labeling the Trp346Phe mutant for which we have a three-dimensional crystal structure; however, this labeled protein proved to be too unstable, and we could not accumulate enough pure protein for a spectrum.
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