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As daf-18 mutations suppress the lifespan phenotype of daf-2 and age-1 mutants, it has been proposed that the role of DAF-18 in lifespan regulation relies on inhibition of the insulin/IGF-1 signaling pathway (Mihaylova et al., 1999).
Moreover, as an optimal strategy to prevent the emergence of escape mutants, it has been recently proposed to combine a polymerase III driven shRNA targeting the shared exon of tat and rev in combination with a TAR decoy and a chimeric VA1-ribozyme targeting human CCR5 [50].
Importantly, to prevent degradation of sarcoglycan mutants, it has been shown that it is also possible to intervene upstream of the proteasome.
Since neither primary nor secondary siRNAs are detected in those mutants, it has been suggested that Ago-1 is required for production/stability of siRNAs [ 19].
Based on the analysis of cutin biosynthesis mutants, it has been demonstrated that plant cuticle is required for the regulation of ABA biosynthesis and osmotic stress tolerance.
Since homologous pairing between recipient and donor sequences is still observed in vivo in rad54 mutants, it has been suggested that the primary function of Rad54 is postsynaptic rather than during the search for homology (Sugawara et al. 2003).
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By RNA interference approaches and the use of a dominant negative Mad2 mutant it has been shown that the meiotic SAC depends on Mad2 as well [10] [12].
By using a temperature-sensitive degron-tagged cdc48-td mutant, it has been shown that Cdc48 is required for the execution of Start commitment point in Saccharomyces cerevisiae by degrading the G1-cyclin-dependent kinase inhibitor Far1 [45].
While p.G469E was categorized as an intermediate-activity mutant, it has the lowest kinase activity within this category (1.8 fold increase) [ 13].
Indeed, in the specific case of the Chlamydomonas vfl2 mutant, it has previously been reported that even though the vfl2 mutation causes centrioles to detach from the spindle poles, the acentriolar spindles are nonetheless still bipolar [ 19].
Although there is no guarantee of any measureable phenotype in the resulting mutant, it has been calculated from pilot studies that 5000 DNA samples are sufficient to identify at least two alleles with 90% confidence (29), and ENU archives much larger than this are now available for screening (28, 30).
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com