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Molecular cloning and circadian expression profile of insect neuropeptide PDF in black blowfly, Phormia regina

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Abstract

Pigment-dispersing factor PDF is an 18-amino acid insect neuropeptide that mediates a circadian rhythmicity in locomotor activity. PDF is coded in a precursor protein together with another neuropeptide named PDF-associated peptide, PAP. PDF is highly conserved among insects, whereas the homology of PAPs is very low with considerably varied amino acid sequences. Since such dissimilarity has suggested that the function of PAP peptide is not associated with that of PDF, we have attempted to analyze the sequences of PDF precursor proteins among a series of species of insects and hypothesized that PDF precursors are classified into at least three different classes: Drosophila-Musca, Meimuna-Romalea, and Gryllus. In order to exemplify this hypothesis, we here describe the molecular cloning of the pdf-gene of the black blowfly Phormia regina and an in silico screening for the pdf-gene in the genome databank of the mosquito Anopheles gambie, both species belonging to the Diptera. It was found that deduced amino acid sequences of PDF peptides are almost completely conserved among all Dipterans and also the amino acid sequences of PAPs are considerably highly preserved (55–82 similarity) among the species of Diptera. The results confirmed the validity of grouping the PDF precursor proteins. In situ hybridization was carried out in fly brains to identify the precise locations of pdf-expressing cells and to examine any daily cycling of pdf mRNA. Intense signals for pdf mRNA were identified in the medulla, but not in the pars lateralis where PDF neurons were strongly immunostained by the antibody raised against PDF peptide. Hybridization was also performed for the brain samples at two hour intervals throughout the day. Although very intense hybridization signals were observed at ZT8 even in some neurites, no prominent rhythmicity of pdf mRNA expression was observed.

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Authors and Affiliations

  1. Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Kyushu University, Fukuoka, 812-8581, Japan

    Ayami Matsushima, Satoru Yokotani, Xiaohui Lui, Kazunori Sumida, Takeshi Honda, Seiji Sato, Atsushi Kaneki, Yukimasa Takeda, Yoshiro Chuman, Daisuke Asai, Takeru Nose & Yasuyuki Shimohigashi

  2. Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo, 060-0810, Japan

    Yoshiro Chuman

  3. Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto, 606-8585, Japan

    Mamiko Ozaki

  4. Faculty of Health and Welfare, Seinan Jo Gakuin University, Kitakyushu, 803-0835, Japan

    Hitoshi Onoue

  5. Department of Physiological Sciences, Faculty of Medical Sciences, Kyushu University, Fukuoka, 812-8581, Japan

    Yushi Ito

  6. Division of Biology, Faculty of Science, Fukuoka University, Fukuoka, 814-0180, Japan

    Yoshiya Tominaga & Miki Shimohigashi

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  1. Ayami Matsushima
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  2. Satoru Yokotani
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  3. Xiaohui Lui
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  8. Yukimasa Takeda
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  9. Yoshiro Chuman
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  11. Daisuke Asai
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  16. Yasuyuki Shimohigashi
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Matsushima, A., Yokotani, S., Lui, X. et al. Molecular cloning and circadian expression profile of insect neuropeptide PDF in black blowfly, Phormia regina . Int J Pept Res Ther 10, 419–430 (2003). https://doi.org/10.1007/s10989-004-2396-5

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  • DOI: https://doi.org/10.1007/s10989-004-2396-5

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