TIMELESS: A link between fly's circadian and photoperiodic clocks?

Potential involvement of circadian clock genes in so far unknown mechanism of photoperiodic time measurement is an important question of insect life-cycle regulation science. Here we report about the cloning of full-length cDNA of the structural homologue of the Drosophila's timeless gene in Chymomyza costata. Its expression was compared in two strains: a wild-type strain, responding to short days by entering larval diapause and a npd-mutant strain, showing no photoperiodic response. The timeless mRNA transcripts were not detectable by Northern blot analysis in the fly heads of npd-mutants, while they were detectable and showed typical daily oscillations in the wild-type strain. After disrupting the normal process of timeless transcription in the wild-type strain by injection of timeless double-strandRNA into early embryos of wild-type (RNAi method: Kennerdell & Carthew 1998, 2000), a certain proportion of the individuals adopted a npd-mutant phenotype, showing no-diapause in response to short-daylength. Cloning of genomic DNA fragments revealed that npd-mutants carry a different allele, timelessnpd, with a 13-bp insertion in an intron positioned within the 5'-leader sequence. Genetic linkage analysis showed that the 13-bp insertion (a marker for timelessnpd) and the absence of response to short days (a marker for npd-phenotype) are strictly co-inherited in the F2 progeny of the reciprocal crosses between wild-type and npd-mutant flies. Such results indicated that the locus npd could code for the timeless gene in C. costata and its product might thus represent a molecular link between circadian and photoperiodic clock systems in this fly.