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

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.

Co-localization and unique distributions of two clock proteins CYCLE and CLOCK in the cephalic ganglia of the ground cricket, Allonemobius allardi.

CYCLE (CYC) and CLOCK (CLK) are transcriptional activators of the circadian clock genes, period (per) and timeless (tim), binding at E-boxes of their upstream regulatory region in Drosophila. CYC-like and CLK-like immunohistochemical reactivities (CYC-ir and CLK-ir) were investigated in the ground cricket, Allonemobius allardi, in which immunohistochemical reactivities for three circadian clock proteins (PERIOD, Doubletime, and Cryptochrome), two neuropeptides (crustacean cardioactive peptide and diapause hormone), and arylalkylamine-N-acetyltransferase had previously been mapped in the brain-subesophageal ganglion (SOG) complex. CYC-ir and CLK-ir occurred predominantly in the cytoplasm of the neurons distributed mainly in the central brain, SOG, and corpora cardiaca. Double-labeling experiments showed that CYC-ir and CLK-ir were co-localized only in the mandibular and maxillary neuromeres of the SOG. The neuronal processes in the dorsolateral region of the protocerebrum partially shared the immunoreactivities, whereas most of the other immunoreactivities were unique. The optic lobe showed reactivity to anti-CYC at small proximal frontodorsal cells and to anti-CLK at small proximal frontoventral cells. The frontal ganglion exhibited CYC-ir in the cell bodies that lacked CLK-ir. No difference in their number, distribution, or staining intensity was found between sampling under light:dark regimes of 16:8 and 12:12. The levels of both CYC-ir and CLK-ir showed no oscillation throughout a 24-h period. The co-localization pattern suggests that the midline cells of the SOG share most of the circadian-related immunoreactivities, thus constituting the heart of the circadian clock in A. allardi.