Molecular structure, expression patterns, and localization of the circadian transcription modulator CYCLE in the cricket, Dianemobius nigrofasciatus

Abstract

CYCLE (CYC), also known as BMAL1 in vertebrate nomenclature, is a transcription modulator of the circadian genes period and timeless of Drosophila melanogaster. We cloned a cDNA encoding a CYC homologue from the head of the ground cricket, Dianemobius nigrofasciatus (Dncyc), the first CYC from Hemimetabola. The deduced sequence corresponded to a 601 amino-acid polypeptide, with well-defined bHLH, PAS-A, PAS-B, PAC, and BTCR domains. The amino-acid sequence showed 70.7% identity with the CYC protein of Athalia rosae, 63.8% with D. melanogaster, and 52% identity with the human homologue. A cyc transcript of around 3.6kb occurs in the brain, midgut, testis, fatbody, and muscle. An additional band of around 1.1kb gave a hybridization signal in the head. No temporal oscillation in cyc mRNA abundance was observed in the head of the adult cricket when investigated by Northern blot analysis. CYC-like immunohistochemical reactivity (ir) and its dimerization partner CLOCK (CLK)-ir appeared in the pars intercerebralis (PI), tritocerebrum, dorsolateral protocerebrum, and subesophageal ganglion (SOG), but no CYC-ir was observed in the optic lobe (OL) that showed CLK-ir. The deutocerebrum showed a unique CLK-ir but no CYC-ir pattern. Double-labelling experiments showed that both antigens were co-localized in the mandibular and maxillary neuromeres of the SOG. CYC-ir showed no daily oscillation in intensity and the staining pattern was always cytoplasmic. CLK-ir occurred in the nucleus at ZT 16, but was cytoplasmic at other ZT times. A neuronal network equivalent to adult system occurred in the second nymphal stadium.