The Notch repressor complex in Drosophila: in vivo analysis of Hairless mutants using overexpression experiments

Novel Genome-Engineered H Alleles Differentially Affect Lateral Inhibition and Cell Dichotomy Processes during Bristle Organ Development

“…We note that H cwt displays a weak H bristle phenotype in trans over H null alleles [30]. However, because the respective H* alleles were based on H cDNA, H cwt was still used as control [32]. For loss of function alleles, H attP /TM6B (BL94608) [30], H LD /TM6B (BL93865) [30], H 22 /TM6B (BL93661) [33] and Su(H) attP /CyO (BL93861) [28] were used.…”

“…The alleles H FA , H LLAA and H WA were generated through genome engineering as previously described [30,35]. Mutant H cDNAs [32] were cloned into the pGE-attB GMR vector [35], to be integrated using the H attP allele as a platform by site-specific recombination as outlined earlier [30,31]. Genotypes were confirmed by PCR and sequencing where applicable.…”

“…Unexpectedly, the three mutant variants (H FA , H LLAA and H WA ) behaved very similarly in overexpression analyses, indicating that all three had lost most of their H repressive activity [25,32]. Their residual binding to Su(H), however, was uncovered by a combined overexpression of the H mutant variants together with Su(H), causing phenotypes similar to the wild-type H isoform [32]. Apparently, a subtle weakening of the cohesion within the H-Su(H) repressor complex was sufficient to disrupt its activity.…”

“…We expected a linear decrease in the activity of the H protein variants, reflecting their residual Su(H) binding capacity as determined by prior isothermal titration calorimetry [25]. Unexpectedly, the three mutant variants (H FA , H LLAA and H WA ) behaved very similarly in overexpression analyses, indicating that all three had lost most of their H repressive activity [25,32]. Their residual binding to Su(H), however, was uncovered by a combined overexpression of the H mutant variants together with Su(H), causing phenotypes similar to the wild-type H isoform [32].…”

“…We aimed at introducing conservative changes to those amino acids predicted important for the binding by structural analyses. To this end, we replaced amino acids F237, L245 and L247, as well as W258, with Alanine in the CSL-ID of H and assayed the binding properties and remaining biological activity of the respective mutants in overexpression assays [32] (Figure 1b). We expected a linear decrease in the activity of the H protein variants, reflecting their residual Su(H) binding capacity as determined by prior isothermal titration calorimetry [25].…”