Mutagenesis of the clock gene period in Drosophila melanogaster

The period gene is an essential component of the circadian oscillator in D. melanogaster. A coding polymorphism in the Thr-Gly region of the gene, may contribute to the temperature compensation of the clock. The protein sequences flanking the repeat are believed to interact with the repeat and are structurally similar to it. Several in vitro deletion and insertion mutations of the Thr-Gly region were designed to test which regions flanking the repeat are required for temperature compensation. The locomotor activity analysis of flies transformed with these mutagenised constructs revealed that the removal of sequence 3' to the repeat unexpectedly causes shortening of the period. Insertion of sequence which disrupts the predicted beta turns in the middle of the repeat, or deletions of the repeat produced flies with long periods and unusual patterns of temperature compensation. These may be a result of disrupted interactions between the repeat and its flanking regions. The period changes in the deletion genotypes are likely to be a result of altered PER stability. The long period Thr-Gly mutants were not additive to the effects of the classic perLI mutant (which has delayed nuclear entry), signifying that the mutations affected a different part of the PER molecular cycle. Additionally, the LD activity patterns of the short and long period Thr-Gly mutants are different from those observed for perS and perLI, as the mutants show increasing shifts of activity into the night at high temperatures. The DD activity patterns concur with the observed LD patterns and reveal that at high temperatures the subjective night is shortened for most of the Thr-Gly genotypes. This suggests that the Thr-Gly deletions may disrupt an interaction with another factor that plays a part in establishing locomotor activity patterns.