Structural and Functional Dissection of the 5' Region of the Notch Gene in Drosophila melanogaster

• Published in: Genes 2019-12, Vol.10 (12), p.1037
• Main Authors: Volkova, Elena I, Andreyenkova, Natalya G, Andreyenkov, Oleg V, Sidorenko, Darya S, Zhimulev, Igor F, Demakov, Sergey A
• Format: Article
• Language: English
• Subjects: 5' Untranslated Regions - genetics; Animals; Cell differentiation; Chromosome Structures - genetics; Chromosomes; CRISPR; CRISPR-Cas Systems; Drosophila melanogaster - genetics; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Gene Expression Regulation - genetics; Genomes; Homologous recombination; Homologous Recombination - genetics; Insects; Morphogenesis; Mutation; Nervous system; Notch protein; Phages; Plasmids; Polytene; Polytene Chromosomes - genetics; Proteins; Receptors, Notch - genetics; Receptors, Notch - metabolism; Regulation; RNA polymerase; Structure-Activity Relationship; Structure-function relationships; X chromosomes
• ISSN: 2073-4425; 2073-4425
• DOI: 10.3390/genes10121037

Notch is a key factor of a signaling cascade which regulates cell differentiation in all multicellular organisms. Numerous investigations have been directed mainly at studying the mechanism of Notch protein action; however, very little is known about the regulation of activity of the gene itself. Here, we provide the results of targeted 5'-end editing of the gene in its native environment and genetic and cytological effects of these changes. Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) system in combination with homologous recombination, we obtained a founder fly stock in which a 4-kb fragment, including the 5' nontranscribed region, the first exon, and a part of the first intron of , was replaced by an attachment Phage (attP) site. Then, fly lines carrying a set of six deletions within the 5'untranscribed region of the gene were obtained by 31-mediated integration of transgenic constructs. Part of these deletions does not affect gene activity, but their combinations with transgenic construct in the first intron of the gene cause defects in the Notch target tissues. At the polytene chromosome level we defined a DNA segment (~250 bp) in the 5'-nontranscribed region which when deleted leads to disappearance of the 3C6/C7 interband and elimination of CTC-Factor (CTCF) and Chromator (CHRIZ) insulator proteins in this region.