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

Jaroslav Pavelka,Vladimir Kostal,Kimio Shimada

doi:10.14411/eje.2003.041

Abstract

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.

Highlights

Circadian clock systems allow organisms to anticipate daily changes in environmental factors and appropriately tune their physiological state
The photoperiodic time measurement system perceives seasonal change of day-length or night-length, which signals for seasonally cycling deterioration of conditions for life/development and allows organisms to prepare in advance, by entering diapause state in insects, for example (Denlinger, 2000, 2002; Saunders, 2002)
Two strains of Chymomyza costata were used for experiments: a wild-type (Sapporo) strain originally collected in Sapporo (43°N), Japan in 1983; a non-photoperiodic-diapause-mutant strain (NPD) which was isolated by Riihimaa & Kimura (1988) from wild-type flies collected in Tomakomai (42.3°N), Japan

Summary

Introduction

Circadian clock systems allow organisms to anticipate daily changes in environmental factors and appropriately tune their physiological state. Part of the scientific community accepts that certain ele ments with circadian oscillatory nature may be function ally involved in photoperiodic clocks. The evidence so far gath ered is not conclusive enough (Tauber & Kyriacou, 2001; Veerman, 2001)

Methods

Results

Conclusion