Transfection of individual mutants revealed only a single interface at TM I, most prominently marked by N55C and W56C (Fig 2A).
In fact a superimposition of subunits from the six KaiC mutant structures reveals only minor adjustments in the geometry of the P-site loop region (i.e. in the spacing of the S431 and T426 residues, Fig. 4B).
Probing with Rpb1 Ab reveals both endogenous and recombinant Rpb1 whereas HA reveals only recombinant Y1F mutant.
Sequencing of purified 5-FOA-resistant mutants revealed that only 13 out of 55 sequenced mutants had a mutation in the pyrF gene (orotate decarboxylase), only 42 out of 69 had a mutation in the pyrE2 gene, and none out of 61 had mutation in the pyrE1 gene (orotate phosphoribosyl transferases).
However, a subsequent genetic study of spontaneously acquired TMC-207 resistant mutants revealed that only 15 out of 53 mutants harbored such mutations in atpE (Huitric et al, 2010) suggesting that the drug has either additional targets or resistance mechanisms, such as drug efflux.
Importantly, miRNAs which are unrelated in sequence may function in concert as demonstrated by the phenotypes of individual miRNA mutants, which are revealed only in genetic backgrounds where miRNA levels are globally reduced [ 26].
Four house-keeping or reference genes that did not confer any mis-expression in nub 1 mutants were also analyzed, revealing only one Oct site in the distal promoter region of one of these genes.
Sequence analysis revealed only the mutant sequence.
Discarding the common mutations revealed only 19 mutations unique to the HI mutant, a number small enough for downstream experimental validation (Table 1).
The quantification results confirmed the visual impression and revealed only minor differences in hyperphosphorylation of BicD between most mutants.
