TY  - JOUR
AB  - Drosophila melanogaster has served as an excellent genetic model to decipher the molecular basis of the circadian clock. Two key proteins, PERIOD (PER) and TIMELESS (TIM), are particularly well explored and a number of various arrhythmic, slow, and fast clock mutants have been identified in classical genetic screens. Interestingly, the free running period (tau, τ) is influenced by temperature in some of these mutants, whereas τ is temperature-independent in other mutant lines as in wild-type flies. This, so-called “temperature compensation” ability is compromised in the mutant timeless allele “ritsu” (tim-rit), and, as we show here, also in the tim-blind allele, mapping to the same region of TIM. To test if this region of TIM is indeed important for temperature compensation, we generated a collection of new mutants and mapped functional protein domains involved in the regulation of τ and in general clock function. We developed a protocol for targeted mutagenesis of specific gene regions utilizing the CRISPR/Cas9 technology, followed by behavioral screening. In this pilot study, we identified 20 new timeless mutant alleles with various impairments of temperature compensation. Molecular characterization revealed that the mutations included short in-frame insertions, deletions, or substitutions of a few amino acids resulting from the non-homologous end joining repair process. Our protocol is a fast and cost-efficient systematic approach for functional analysis of protein-coding genes and promoter analysis in vivo. Interestingly, several mutations with a strong temperature compensation defect map to one specific region of TIM. Although the exact mechanism of how these mutations affect TIM function is as yet unknown, our in silico analysis suggests they affect a putative nuclear export signal (NES) and phosphorylation sites of TIM. Immunostaining for PER was performed on two TIM mutants that display longer τ at 25°C and complete arrhythmicity at 28°C. Consistently with the behavioral phenotype, PER immunoreactivity was reduced in circadian clock neurons of flies exposed to elevated temperatures.
AU  - Singh, Samarjeet
AU  - Giesecke, Astrid
AU  - Damulewicz, Milena
AU  - Fexova, Silvie
AU  - Mazzotta, Gabriella M.
AU  - Stanewsky, Ralf
AU  - Dolezel, David
DA  - 2019-12-03
DO  - 10.3389/fphys.2019.01442
KW  - circadian clock
KW  - reverse genetics
KW  - screening
KW  - candidate genes
KW  - temperature compensation
KW  - CRISPR-CAS9
KW  - Drosophila melanogaster
LA  - eng
N1  - Frontiers in Physiology 10 (2019) 1442, 1-23
N1  - Finanziert durch den Open-Access-Publikationsfonds der Westfälischen Wilhelms-Universität Münster (WWU Münster)
N1  - This work was supported by the National Science Foundation of the Czech Republic (GACR project 17-01003S).
N1  - The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys.2019.01442/full#supplementary-material
PY  - 2019-12-03
TI  - New "Drosophila" Circadian Clock Mutants Affecting Temperature Compensation Induced by Targeted Mutagenesis of "Timeless"
UR  - https://nbn-resolving.org/urn:nbn:de:hbz:6-48079548066
Y2  - 2024-11-22T01:17:58
ER  -