cDNA cloning of the housefly pigment‐dispersing factor (PDF) precursor protein and its peptide comparison among the insect circadian neuropeptides

Abstract

Pigment-dispersing factor (PDF), an 18-amino acid neuropeptide, is a principal circadian neurotransmitter for the circadian rhythms of the locomotor activity in flies. Recently, two completely different types of PDF precursor were clarified; that of the cricket Gryllus bimaculatus and that of the last-summer cicada Meimuna opalifera. The G. bimaculatus PDF precursor is extraordinarily short and comprises a nuclear localization signal (NLS), while the M. opalifera PDF precursor is of ordinary length, comparable to that seen for the precursors of crustacean beta-PDH homologues. Although their PDF peptide regions were exactly the same, the regions containing a signal peptide combined with a PDF-associated peptide (PAP) were remarkably different from each other. Such a grouping suggested a fundamental role for the PAP peptide in the circadian clock, perhaps associated with PDF function. In the present study, the cDNA cloning of PDF from the adult brains of the housefly Musca domestica was carried out and it was found that an isolated clone (527 bp) encodes a PDF precursor protein of ordinary length. The PDF peptide shows a high sequence identity (78%-94%) and similarity (89%-100%) to insect PDFs and also to the crustacean beta-PDH peptides. In particular, there is only a single amino acid difference between the PDFs of Musca and Drosophila; at position 14 Ser for Musca PDF and Asn for Drosophila PDF. A characteristic Ser10 in Drosophila was retained in Musca, indicating the presence of a structural profile unique to these PDFs. The results of sequence analyses suggest that Musca and Drosophila PDFs are to be considered members of a single group that has evolved structurally. When the primary structure of the PAP regions was compared, the Musca PDF precursor also belonged to the same group as that to which the Drosophila PDF precursor belongs.

Comparative Analysis of Pdf-Mediated Circadian Behaviors BetweenDrosophila melanogasterandD. virilis

“…IHC using anti-Pdf showed both large and small LN vequivalent neuronal groups in the adult brain of M. domestica (Pyza and Meinertzhagen 1997;Pyza et al 2003). In contrast to this result, in situ hybridization revealed MdPdf mRNA expression only in the l-LN v -like neurons (Matsushima et al 2004). We confirmed these results independently (data not shown, n ¼ 6).…”

“…Comparison of cDNA sequence (Matsushima et al 2004) with PCR-amplified genomic DNA one revealed a 63-bp phase-0 intron (i.e., between two codons) in the M. domestica Pdf (MdPdf ) gene (GenBank accession no. FJ043031).…”

Structural isoforms of the circadian neuropeptide PDF expressed in the optic lobes of the cricket Gryllus bimaculatus: Immunocytochemical evidence from specific monoclonal antibodies

“…Additional antibodies have also been raised against insect PDFs, however. For instance, Park et al (2000) prepared a rat antibody against Drosophila PDF, and we have prepared rabbit polyclonal antibodies against PDF in the housefly Musca domestica Matsushima et al, 2003Matsushima et al, , 2004. For almost all these antibodies, precise epitope structures have yet to be clarified.…”

Metamorphosis of a clock: Remodeling of the circadian timing system in the brain of Rhodnius prolixus (Hemiptera) during larval‐adult development

“…It recognizes the insect homologs of PDH (known as pigment dispersing factors, PDFs) and has been extensively used in tracing axonal projections of putative clock cells in various insects (e.g., Helfrich-Förster and Homberg, 1993;Kaneko et al, 1997;Kula et al, 2006), including Rhodnius (Vafopoulou et al, 2010). PDFs have been cloned from seven insect species (references and sequence alignments in Matsushima et al, 2004); insect PDFs possess 78-89% (average 83%) sequence identity with Uca PDH. A BLAST search for other arthropod proteins containing partial sequence identity to the uPDH sequence retrieved all the PDH/PDF peptides, but only a small number of other proteins whose sequence identities to PDH were below 60%.…”