Generation of New Hairless Alleles by Genomic Engineering at the Hairless Locus in Drosophila melanogaster
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Authors: | Heiko Praxenthaler Thomas K Smylla Anja C Nagel Anette Preiss Dieter Maier |
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Institution: | Universität Hohenheim, Institut für Genetik (240), Garbenstr. 30, 70599, Stuttgart, Germany, ; National Institutes of Health (NIH), UNITED STATES, |
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Abstract: | Hairless (H) is the major antagonist within the Notch signalling pathway of Drosophila melanogaster. By binding to Suppressor of Hairless Su(H)] and two co-repressors, H induces silencing of Notch target genes in the absence of Notch signals. We have applied genomic engineering to create several new H alleles. To this end the endogenous H locus was replaced with an attP site by homologous recombination, serving as a landing platform for subsequent site directed integration of different H constructs. This way we generated a complete H knock out allele H
attP, reintroduced a wild type H genomic and a cDNA-construct (H
gwt, H
cwt) as well as two constructs encoding H proteins defective of Su(H) binding (H
LD, H
iD). Phenotypes regarding viability, bristle and wing development were recorded, and the expression of Notch target genes wingless and cut was analysed in mutant wing discs or in mutant cell clones. Moreover, genetic interactions with Notch (N
5419) and Delta (Dl
B2) mutants were addressed. Overall, phenotypes were largely as expected: both H
LD and H
iD were similar to the H
attP null allele, indicating that most of H activity requires the binding of Su(H). Both rescue constructs H
gwt and H
cwt were homozygous viable without phenotype. Unexpectedly, the hemizygous condition uncovered that they were not identical to the wild type allele: notably H
cwt showed a markedly reduced activity, suggesting the presence of as yet unidentified regulatory or stabilizing elements in untranslated regions of the H gene. Interestingly, H
gwt homozygous cells expressed higher levels of H protein, perhaps unravelling gene-by-environment interactions. |
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