Short-period mutations of per affect a double-time-dependent step in the Drosophila circadian clock

Circadian (24 hour) PERIOD (PER) protein oscillation is dependent on the double-time (dbt) gene, a casein kinase Ivarepsilon homolog [1-3]. Without dbt activity, hypophosphorylated PER proteins over-accumulate, indicating that dbt is required for PER phosphorylation and turnover [3,4]. There is evidence of a similar role for casein kinase Ivarepsilon in the mammalian circadian clock [5,6]. We have isolated a new dbt allele, dbt(ar), which causes arrhythmic locomotor activity in homozygous viable adults, as well as molecular arrhythmicity, with constitutively high levels of PER proteins, and low levels of TIMELESS (TIM) proteins. Short-period mutations of per, but not of tim, restore rhythmicity to dbt(ar) flies. This suppression is accompanied by a restoration of PER protein oscillations. Our results suggest that short-period per mutations, and mutations of dbt, affect the same molecular step that controls nuclear PER turnover. We conclude that, in wild-type flies, the previously defined PER'short domain' [7,8] may regulate the activity of DBT on PER.     

Embryonic expression of the period clock gene in the central nervous system of Drosophila melanogaster.

We have examined the temporal and spatial expression of the 4.5-kb mRNA that is transcribed from the period locus of Drosophila melanogaster and is the best candidate for the per gene product. Both Northern blot analyses and hybridizations in situ to tissue sections reveal significant expression of the 4.5-kb mRNA in embryos. This expression is limited to the central nervous system of the developing embryo and is localized within the brain and ventral ganglia. The 4.5-kb mRNA is enriched in adult heads (by Northern blotting) although we were not able to detect specific localization (in situ). In addition to the physiological role the 4.5-kb mRNA might have in maintaining biological rhythms, we now suggest that it has a developmental role for establishing mechanisms that are necessary for eventual expression of clock functions.     

A latitudinal cline in a Drosophila clock gene.

The clock gene period determines biological rhythmicity in Drosophila melanogaster and encodes a protein characterized by an alternating series of threonine-glycine pairs. The minisatellite region encoding the threonine-glycine repeat is polymorphic in length in natural Drosophila melanogaster populations. In this paper we report the geographical analysis of this polymorphism within Europe and North Africa. A robust clinal pattern is observed along a north-south axis. We suggest the possibility that the length polymorphism could be maintained by thermal selection because the threonine-glycine region has been shown to provide thermostability to the circadian phenotype.     

Circadian rhythms in Drosophila melanogaster: analysis of period as a function of gene dosage at the per (period) locus

Mutations at the per (period) locus of Drosophila melanogaster affect the period of its circadian rhythms. An analysis of published data on strains having duplications and deletions of the per locus indicates that the period is a logarithmic function of the level of the per gene product. The analysis also indicates that period is relatively insensitive to the level of that gene product; a presumed 300% increase in the gene product level produces only a 4.6% decrease in the period. The period of a strain transformed with per+ DNA conforms to the same logarithmic relationship if the level of mRNA in the transformant, which is one-tenth that in the wild type, is considered equivalent to a gene dosage one-tenth the wild type dose of 2, or 0.2. The periods of strains having various doses of mutant per alleles which shorten (pers) or lengthen (per1) the period can be fitted to the same logarithmic function. The analysis may provide an explanation for the partial dominance of pers over per+ and the dominance of per+ over per1, since it suggests that the per1 gene product is nearly inactive while the pers gene product is more than 34 times as active as the wild type product. Analysis of periods of strains heterozygous at the per locus suggests that the per gene product may be a multimeric protein. Three possible roles for the per gene product in circadian rhythmicity are discussed, including a role in synchronizing rhythm-producing cells.     

Drosophila clock mutations affect the morphology of a brain neurosecretory cell group

Mutations that eliminate circadian rhythmicity in two species of Drosophila affect the location of cells in a neurosecretory cell group. This is the first example of mutations which affect the morphology of an identifiable cell group in the Drosophila brain.     

The clock gene period in the medfly Ceratitis capitata

We have isolated the clock gene period (per) from the medfly Ceratitis capitata, one of the most economically important insect pest species. The overall pattern of conserved, non-conserved and functional domains that are observed within dipteran and lepidopteran per orthologues is preserved within the coding sequence. Expression analysis from fly heads revealed a daily oscillation in per mRNA in both light : dark cycles and in constant darkness. However PER protein levels from head extracts did not show any significant evidence for cycling in either of these two conditions. When the Ceratitis per transgene under the control of the Drosophila per promoter and 3'UTR was introduced into Drosophila per -null mutant hosts, the transformants revealed a low level of rescue of behavioural rhythmicity. Nevertheless, the behaviour of the rhythmic transformants showed some similarities to that of ceratitis, suggesting that Ceratitis per carries species-specific information that can evidently affect the Drosophila host's downstream rhythmic behaviour.     

The Drosophila dCREB2 gene affects the circadian clock

We report the role of dCREB2, the Drosophila homolog of CREB/CREM, in circadian rhythms. dCREB2 activity cycles with a 24 hr rhythm in flies, both in a light:dark cycle and in constant darkness. A mutation in dCREB2 shortens circadian locomotor rhythm in flies and dampens the oscillation of period, a known clock gene. Cycling dCREB2 activity is abolished in a period mutant, indicating that dCREB2 and Period affect each other and suggesting that the two genes participate in the same regulatory feedback loop. We propose that dCREB2 supports cycling of the Period/Timeless oscillator. These findings support CREB's role in mediating adaptive behavioral responses to a variey of environmental stimuli (stress, growth factors, drug addiction, circadian rhythms, and memory formation) in mammals and long-term memory formation and circadian rhythms in Drosophila.     

Phosphorylation of period is influenced by cycling physical associations of double-time, period, and timeless in the Drosophila clock.

The clock gene double-time (dbt) encodes an ortholog of casein kinase Iepsilon that promotes phosphorylation and turnover of the PERIOD protein. Whereas the period (per), timeless (tim), and dClock (dClk) genes of Drosophila each contribute cycling mRNA and protein to a circadian clock, dbt RNA and DBT protein are constitutively expressed. Robust circadian changes in DBT subcellular localization are nevertheless observed in clock-containing cells of the fly head. These localization rhythms accompany formation of protein complexes that include PER, TIM, and DBT, and reflect periodic redistribution between the nucleus and the cytoplasm. Nuclear phosphorylation of PER is strongly enhanced when TIM is removed from PER/TIM/DBT complexes. The varying associations of PER, DBT and TIM appear to determine the onset and duration of nuclear PER function within the Drosophila clock.     

Reproductive isolation and the period gene of Drosophila

The identification of genes of large effect on ecologically important traits is an important aim of molecular ecology. The period gene of Drosophila is a candidate for a gene with a large influence on premating isolation between Drosophila species, as it determines species specific aspects of courtship behaviour. Strains of D. melanogaster are available which have been genetically transformed with the period gene of either D. melanogaster or D. simulans. Here we show that D. melanogaster females do not discriminate between two such strains. This suggests that period may only make a small contribution to total premating isolation between these species. We discuss the use of genetically transformed strains in assessing the influence of single genes on complex traits.     

The period gene of Drosophila carries species-specific behavioral instructions.

We have analyzed and compared the circadian locomotor activity rhythms of Drosophila melanogaster and D.pseudoobscura. The rhythms of D.pseudoobscura are stronger and the periods shorter than those of D.melanogaster. We have also transformed D.melanogaster flies with a hybrid gene containing the coding region of the D.pseudoobscura period (per) gene. Behavioral assays of flies containing this hybrid gene show that the per protein encoded by the D.pseudoobscura per gene is able to rescue the rhythmic deficiencies of arrhythmic, pero1 D.melanogaster. More important, the rhythms of some of these strains are stronger and the periods shorter than those of D.melanogaster (and those of transformants which carry the equivalent D.melanogaster per gene construct) and hence resemble those of D.pseudoobscura. The results suggest that the primary amino acid sequence of the per gene encodes species-specific behavioral instructions that are detectable when only the per gene is transferred to a different species.     

Intra‐ and interspecific divergence in the nuclear sequences of the clock gene period in species of the Drosophila buzzatii cluster

We have measured nucleotide variation in the CLOCK/CYCLE heterodimer inhibition domain (CCID) of the clock X‐linked gene period in seven species belonging to the Drosophila buzzatii cluster, namely D. buzzatii, Drosophila koepferae, Drosophila antonietae, Drosophila serido, Drosophila gouveai, Drosophila seriema and Drosophila borborema. We detected that the purifying selection is the main force driving the sequence evolution in period, in agreement with the important role of CCID in clock machinery. Our survey revealed that period provides valuable phylogenetic information that allowed to resolve phylogenetic relationships among D. gouveai, D. borborema and D. seriema, which composed a polytomic clade in preliminary studies. The analysis of patterns of intraspecific variation revealed two different lineages of period in D. koepferae, probably reflecting introgressive hybridization from D. buzzatii, in concordance with previous molecular data.     

Transfer of dye among salivary gland cells is not affected by genetic variations of the period clock gene in Drosophila melanogaster

Larval salivary gland cells of Drosophila melanogaster were injected with a fluorescent dye to assess strengths of intercellular communication among such cells, as influenced by mutations at the period locus and by a per transgene. This clock gene had been reported to increase the extent of dye transfer when mutated such that it shortens the period of biological rhythms; the previous study also showed that a per-null mutant decreased the strength of transfer among salivary gland cells. Our re-examination of this feature of larval physiology—in observer-blind analyses, using the per ^s and per ^o mutants as well as two per-normal strains—revealed no appreciable differences in extents of dye transfer among these four genotypes. These results are discussed in the context of emerging findings which suggest that the period gene's product controls pacemaker functioning as an intracellularly acting entity.     

Different period gene repeats take 'turns' at fine-tuning the circadian clock

The repetitive region of the circadian clock gene period in Drosophila pseudoobscura consists predominantly of a pentapeptide sequence whose consensus is NSGAD. In D. melanogaster, this region is replaced by a dipeptide Thr-Gly repeat, which plays a role in the thermal stability of the circadian phenotype. The Thr-Gly repeat has been shown to form a type II or III beta-turn, whose conformational monomer is (Thr-Gly)3. Here we report, using conformational analyses, that both an NSGAD pentapeptide, and a polymer of the same sequence, form type II beta-turns. Thus two peptide sequences, whose amino-acid composition is very different, nevertheless form the same secondary structure. The implications of these structures for clock function are discussed.     

Molecular evolution of a repetitive region within the per gene of Drosophila

The clock gene period (per) controls a number of biological rhythms in Drosophila. In D. melanogaster, per has a repetitive region that encodes a number of alternating threonine-glycine residues. We sequenced and compared this region from several different Drosophila species belonging to various groups within the Drosophila and Sophophora subgenera. This part of per shows a great variability in both DNA sequence and length. Furthermore, analysis of the data suggests that changes in the length of this variable region might be associated with amino acid replacements in the more conserved flanking sequences.      

Molecular evolution of the period gene in Drosophila athabasca

We measured nucleotide variability within and between the three semispecies of the Drosophila athabasca complex, at the period (per) gene by using a polymerase chain reaction-based four-cutter restriction- enzyme analysis. The levels of polymorphism varied considerably between the three semispecies. Our results for per, combined with previous data for X-linked allozymes, suggest that the X chromosome in the western- northern semispecies is less variable than expected under an equilibrium-neutral model. Both the pattern of divergence between the semispecies and a cladistic clustering of per haplotypes support the previously hypothesized grouping of eastern A and eastern B as the two most recently diverged semispecies. A 21-bp in-frame segment in the region of per which shares sequence similarity with the neuronal development gene single minded is deleted in all eastern A and eastern B flies examined but is present in all of the western-northern flies and all other published per sequences. Despite these hints that there may be significant differences at the per gene between the semispecies, especially the western-northern group versus the two eastern groups, there is no compelling evidence that per is involved in the mating song differences between the semispecies.