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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Physiol. | doi: 10.3389/fphys.2019.01442

New Drosophila circadian clock mutants affecting temperature compensation induced by targeted mutagenesis of timeless

Samarjeet Singh1, 2,  Milena Damulewicz1, 3, Silvie Fexova1,  Gabriella M. Mazzotta1, 4 and  David Dolezel1, 5*
  • 1Centre for Biology, Academy of Sciences of the Czech Republic, Czechia
  • 2University of South Bohemia in České Budějovice, Czechia
  • 3Jagiellonian University, Poland
  • 4University of Padova, Italy
  • 5Faculty of Science, University of South Bohemia, Czechia

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’ (timrit), and, as we show here, also in the timblind 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 twenty 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 suggest they affect a putative nuclear export signal 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.

Keywords: Circadian clock, Reverse Genetics, screening, candidate genes, Temperature compensation, CRISPR-Cas9, NHEJ, Drosophila melanogaster, HDR

Received: 30 Apr 2019; Accepted: 07 Nov 2019.

Copyright: © 2019 Singh, Damulewicz, Fexova, Mazzotta and Dolezel. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Mx. David Dolezel, Centre for Biology, Academy of Sciences of the Czech Republic, České Budějovice, 370 05, South Bohemia, Czechia, dolezel@entu.cas.cz