We have isolated three alleles of a novel Drosophila clock gene, double-time (dbt). Short- (dbtS) and long-period (dbtL) mutants alter both behavioral rhythmicity and molecular oscillations from previously identified clock genes, period and timeless. A third allele, dbtP, causes pupal lethality and eliminates circadian cycling of per and tim gene products in larvae. In dbtP mutants, PER proteins constitutively accumulate, remain hypophosphorylated, and no longer depend on TIM proteins for their accumulation. We propose that the normal function of DOUBLETIME protein is to reduce the stability and thus the level of accumulation of monomeric PER proteins. This would promote a delay between per/tim transcription and PER/TIM complex function, which is essential for molecular rhythmicity.

The cloning of double-time (dbt) is reported. DOUBLETIME protein (DBT) is most closely related to human casein kinase Iepsilon. dbtS and dbtL mutations, which alter period length of Drosophila circadian rhythms, produce single amino acid changes in conserved regions of the predicted kinase. dbtP mutants, which eliminate rhythms of per and tim expression and constitutively overproduce hypophosphorylated PER proteins, abolish most dbt expression. dbt mRNA appears to be expressed in the same cell types as are per and tim and shows no evident oscillation in wild-type heads. DBT is capable of binding to PER in vitro and in Drosophila cells, suggesting that a physical association of PER and DBT regulates PER phosphorylation and accumulation in vivo.

Eclosion, or emergence of adult flies from the pupa, and locomotor activity of adults occur rhythmically in Drosophila melanogaster, with a circadian period of about 24 hours. Here, a clock mutation, timeless (tim), is described that produces arrhythmia for both behaviors. The effects of tim on behavioral rhythms are likely to involve products of the X chromosome-linked clock gene period (per), because tim alters circadian oscillations of per RNA. Genetic mapping places tim on the left arm of the second chromosome between dumpy (dp) and decapentaplegic (dpp).