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Generation of New Hairless Alleles by Genomic Engineering at the Hairless Locus in Drosophila melanogaster
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 ne...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598140/ https://www.ncbi.nlm.nih.gov/pubmed/26448463 http://dx.doi.org/10.1371/journal.pone.0140007 |
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author | Praxenthaler, Heiko Smylla, Thomas K. Nagel, Anja C. Preiss, Anette Maier, Dieter |
author_facet | Praxenthaler, Heiko Smylla, Thomas K. Nagel, Anja C. Preiss, Anette Maier, Dieter |
author_sort | Praxenthaler, Heiko |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4598140 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-45981402015-10-20 Generation of New Hairless Alleles by Genomic Engineering at the Hairless Locus in Drosophila melanogaster Praxenthaler, Heiko Smylla, Thomas K. Nagel, Anja C. Preiss, Anette Maier, Dieter PLoS One Research Article 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. Public Library of Science 2015-10-08 /pmc/articles/PMC4598140/ /pubmed/26448463 http://dx.doi.org/10.1371/journal.pone.0140007 Text en © 2015 Praxenthaler et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Praxenthaler, Heiko Smylla, Thomas K. Nagel, Anja C. Preiss, Anette Maier, Dieter Generation of New Hairless Alleles by Genomic Engineering at the Hairless Locus in Drosophila melanogaster |
title | Generation of New Hairless Alleles by Genomic Engineering at the Hairless Locus in Drosophila melanogaster
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title_full | Generation of New Hairless Alleles by Genomic Engineering at the Hairless Locus in Drosophila melanogaster
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title_fullStr | Generation of New Hairless Alleles by Genomic Engineering at the Hairless Locus in Drosophila melanogaster
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title_full_unstemmed | Generation of New Hairless Alleles by Genomic Engineering at the Hairless Locus in Drosophila melanogaster
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title_short | Generation of New Hairless Alleles by Genomic Engineering at the Hairless Locus in Drosophila melanogaster
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title_sort | generation of new hairless alleles by genomic engineering at the hairless locus in drosophila melanogaster |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598140/ https://www.ncbi.nlm.nih.gov/pubmed/26448463 http://dx.doi.org/10.1371/journal.pone.0140007 |
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