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      Analysing the evolutional and functional differentiation of four types of Daphnia magna cryptochrome in Drosophila circadian clock

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          Abstract

          Cryptochrome (CRY) plays an important role in the input of circadian clocks in various species, but gene copies in each species are evolutionarily divergent. Type I CRYs function as a photoreceptor molecule in the central clock, whereas type II CRYs directly regulate the transcriptional activity of clock proteins. Functions of other types of animal CRYs in the molecular clock remain unknown. The water flea Daphnia magna contains four Cry genes. However, it is still difficult to analyse these four genes. In this study, we took advantage of powerful genetic resources available from Drosophila to investigate evolutionary and functional differentiation of CRY proteins between the two species. We report differences in subcellular localisation of each D. magna CRY protein when expressed in the Drosophila clock neuron. Circadian rhythm behavioural experiments revealed that D. magna CRYs are not functionally conserved in the Drosophila molecular clock. These findings provide a new perspective on the evolutionary conservation of CRY, as functions of the four D. magna CRY proteins have diverse subcellular localisation levels. Furthermore, molecular clocks of D. magna have been evolutionarily differentiated from those of Drosophila. This study highlights the extensive functional diversity existing among species in their complement of Cry genes.

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          An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases.

          Germ-line transformation via transposable elements is a powerful tool to study gene function in Drosophila melanogaster. However, some inherent characteristics of transposon-mediated transgenesis limit its use for transgene analysis. Here, we circumvent these limitations by optimizing a phiC31-based integration system. We generated a collection of lines with precisely mapped attP sites that allow the insertion of transgenes into many different predetermined intergenic locations throughout the fly genome. By using regulatory elements of the nanos and vasa genes, we established endogenous sources of the phiC31 integrase, eliminating the difficulties of coinjecting integrase mRNA and raising the transformation efficiency. Moreover, to discriminate between specific and rare nonspecific integration events, a white gene-based reconstitution system was generated that enables visual selection for precise attP targeting. Finally, we demonstrate that our chromosomal attP sites can be modified in situ, extending their scope while retaining their properties as landing sites. The efficiency, ease-of-use, and versatility obtained here with the phiC31-based integration system represents an important advance in transgenesis and opens up the possibility of systematic, high-throughput screening of large cDNA sets and regulatory elements.
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            Molecular architecture of the mammalian circadian clock.

            Circadian clocks coordinate physiology and behavior with the 24h solar day to provide temporal homeostasis with the external environment. The molecular clocks that drive these intrinsic rhythmic changes are based on interlocked transcription/translation feedback loops that integrate with diverse environmental and metabolic stimuli to generate internal 24h timing. In this review we highlight recent advances in our understanding of the core molecular clock and how it utilizes diverse transcriptional and post-transcriptional mechanisms to impart temporal control onto mammalian physiology. Understanding the way in which biological rhythms are generated throughout the body may provide avenues for temporally directed therapeutics to improve health and prevent disease. Copyright © 2013 Elsevier Ltd. All rights reserved.
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              Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31.

              The phiC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites. Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos. Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47%. In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells. Two lines of Drosophila were created by integrating an attP site into the genome with a P element. phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring. A total of 100% of these progeny carried a precise integration event at the genomic attP site. These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the phiC31 integrase system and will likely benefit research in Drosophila and other insects.
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                Author and article information

                Contributors
                sugimto@bio.sc.niigata-u.ac.jp
                atsushi.sugie@bri.niigata-u.ac.jp
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                20 June 2019
                20 June 2019
                2019
                : 9
                : 8857
                Affiliations
                [1 ]ISNI 0000 0001 0671 5144, GRID grid.260975.f, Center for Transdisciplinary Research, , Niigata University, ; Niigata, Japan
                [2 ]ISNI 0000 0001 0671 5144, GRID grid.260975.f, Brain Research Institute, , Niigata University, ; Niigata, Japan
                [3 ]ISNI 0000 0001 0671 5144, GRID grid.260975.f, Department of Cell Science, , Faculty of Graduate School of Science and Technology, Niigata University, ; Niigata, Japan
                [4 ]ISNI 0000 0001 0671 5144, GRID grid.260975.f, School of Medicine, , Niigata University, ; Niigata, Japan
                Article
                45410
                10.1038/s41598-019-45410-w
                6586792
                31222139
                a0d22805-57cc-411e-a289-8be163924918
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 11 October 2018
                : 6 June 2019
                Funding
                Funded by: FundRef https://doi.org/10.13039/501100001691, MEXT | Japan Society for the Promotion of Science (JSPS);
                Award ID: 18K14835
                Award ID: 18J00367
                Award ID: 18K05552
                Award ID: 17H04983
                Award Recipient :
                Funded by: Grant-in-Aid for Young Scientists (A)
                Categories
                Article
                Custom metadata
                © The Author(s) 2019

                Uncategorized
                evolutionary genetics,molecular evolution
                Uncategorized
                evolutionary genetics, molecular evolution

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