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Abstract

The 1st circadian “clock” gene identified was the X-linked period (per) gene in Drosophila melanogaster. In the pioneering initial report, Konopka and Benzer (1971) characterized 3 alleles of per that shortened (per ^S; ~19 h), lengthened (per ^L; ~29 h), or abolished (per ⁰) circadian behavioral rhythms. They also showed that transheterozygotes carrying the per ^S and per ^L mutations exhibit robust behavioral rhythms with nearly normal periods of ~23 h, highlighting the semidominant nature of many clock mutants. In this study, per ⁰ flies bearing a doubly mutated per transgene that carries both the per ^S and per ^L alleles (per ⁰; per ^S/L) were analyzed for behavioral and molecular rhythms. Unlike singly mutated versions, the per ⁰;per ^S/L transgenic flies are arrhythmic in constant dark conditions and exhibit little, if any, entrainment to daily light—dark cycles. In a wildtype per ⁺ background, expression of per ^S/L abolishes behavioral rhythms, indicating that it functions in a transdominant negative fashion. Biochemical analysis of head extracts revealed that only hyperphosphorylated isoforms of the PERS/L protein are detected throughout a daily cycle, and the levels remain constant. Intriguingly, little if any PERS/L is observed in key pacemaker neurons that control daily activity rhythms, consistent with the notion that hyperphosphorylated isoforms of PER are unstable. Nonetheless, PERS/L is detected in ectopic cells in the brain, in which it exhibits an unusual localization, mainly staining the periphery of the nucleus. These results suggest that posttranslational mechanisms play a key role in limiting the accumulation of PER to specific cells. On a broader scope, our results indicate that the semidominant effects of period-altering alleles observed in trans are not necessarily preserved in the cis-configuration and that novel phenotypes can emerge.

Keywords

Drosophila circadian rhythms period protein phosphorylation perS perL

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Cis -Combination of the Classic per S and per L Mutations Results in Arrhythmic Drosophila with Ectopic Accumulation of Hyperphosphorylated PERIOD Protein

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