Analyses of the ICL knockdown mutant in single-strain infections confirmed this and demonstrated that silencing of ICL during chronic infections impaired persistence of Mtb to the extent that the pathogen was cleared from the lungs of most mice.
However, even though complementation of icl-TetON in vitro and in vivo was incomplete, the analyses of this mutant in single-strain and multi-strain mouse infections demonstrated that ICL activity is not only essential for Mtb to establish an infection in mice but also to persist during chronic infections (Figure 10).
All mutants showing an RNAi phenotype were revealed for identity and then crossed with ced-3 lf) ced-3 lfin single or in comutantson and tested for enhancer phenotypes.
Our analysis demonstrates the power of barseq screening to robustly provide growth-rate phenotypes for dozens of mutants in single mice, and opens up the possibility for large-scale reverse genetic screens for multiple areas of Plasmodium biology.
In natural populations, each new resistance gene appears as a mutant in a single individual and then spreads by natural selection throughout the population in subsequent generations.
Unfortunately, the high demands of animal experiments complicate the analysis of many conditional knockdown mutants in single-strain infections.
Sequencing a mixture of 100 template molecules, one of which is mutant, in a single tube would yield an electropherogram in which the dominant signal at the mutated position would be the wild-type base.
Heme binds to the Y75A HasAp mutant in a single orientation, which is evident from the Fobs – Fcalc electron density map in Figure 6A.
Although gene redundancy may affect the outcome of a screen, it should be noted that mutants affected in single catalase or other antioxidant activities do show an oxidative stress phenotype.
For example, the two genes YDR342C/HXT7 and YDR343C/HXT6, which encode glucose transporters and are involved in glucose signal transduction pathways [ 13, 14], were up-regulated to 3-4 fold in double mutants yet none in single mutants.
