Temporal and spatial expression patterns of transgenes containing increasing amounts of the Drosophila clock gene period and a lacZ reporter: mapping elements of the PER protein involved in circadian cycling.

Rhythmic oscillations of the PER protein, the product of the Drosophila period (per) gene, in brain neurons of the adult fly are strongly involved in the control of circadian rhythms. We analyzed temporal and spatial expression patterns of three per-reporter fusion genes, which share the same 4 kb regulatory upstream region but contain increasing amounts of per's coding region fused in frame to the bacterial lacZ gene. The fusion proteins contained either the N-terminal half (SG), the N-terminal-two-thirds (BG), or nearly all (XLG) of the PER protein. All constructs led to reporter signals only in the known per-expressing cell types within the anterior CNS and PNS. Whereas the staining intensity of SG files was constantly high at different Zeitgeber times, the in situ signals in BG and XLG files cycled with approximately 24 hr periodicity in the PER-expressing brain cells in wild-type and per01 loss of function files. Despite the rhythmic fusion-gene expression within the relevant neurons of per01 BG files, their locomotor activity in light/dark cycling conditions and in constant darkness was identical to that of per01 controls, uncoupling protein cycling from rhythmic behavior. The XLG construct restored weak behavioral rhythmicity to (otherwise) per01 files, indicating that the C-terminal third of PER (missing in BG) is necessary to fulfill the biological function of this clock protein.

Dosage compensation of the period gene in Drosophila melanogaster.

The period (per) gene is located on the X chromosome of Drosophila melanogaster. Its expression influences biological clocks in this fruit fly, including the one that subserves circadian rhythms of locomotor activity. Like most X-linked genes in Drosophila, per is under the regulatory control of gene dosage compensation. In this study, we assessed the activity of altered or augmented per+ DNA fragments in transformants. Relative expression levels in male and female adults were inferred from periodicities associated with locomotor behavioral rhythms, and by histochemically assessing beta-galactosidase levels in transgenics carrying different kinds of per-lacZ fusion genes. The results suggest that per contains multipartite regulatory information for dosage compensation within the large first intron and also within the 3' half of this genetic locus.

A promoterless period gene mediates behavioral rhythmicity and cyclical per expression in a restricted subset of the Drosophila nervous system.

Transgenic flies carrying a 7.2 kb piece of DNA from the period (per) gene were analyzed for the presence of circadian locomotor activity rhythms and fluctuations of per-encoded mRNA and protein. The 5' end of this genomic fragment is within the first intron, which precedes the coding region. This promotorless fragment could rescue circadian behavioral rhythms and mediate spatial expression of PER in a subset of wild-type per cells within the CNS and PNS. In one behaviorally rhythmic line, PER protein was found in only "per lateral neurons." In the rhythmic transgenics, per mRNA and protein levels undergo circadian cycling, as previously described for wild type. Cycling of PER in brain cells of flies carrying the same 7.2 kb piece of per DNA under the control of a heat shock promoter corroborated the hypothesis that per's molecular cyclings and behavioral rhythmicity are causally related.

An ultrashort clock mutation at the period locus of Drosophila melanogaster that reveals some new features of the fly's circadian system.

A rhythm mutant of Drosophila melanogaster was induced by chemical mutagenesis and isolated by testing for locomotor activity rhythms, which in the new variant had periods of approximately 16 hr. The sex-linked mutation responsible for this ultrashort period causes 20-hr rhythms when heterozygous with a normal X. This semidominance notwithstanding, the new mutation was revealed to be an allele of the period (per) gene by noncomplementation with per-null variants, in the sense that females heterozygous for perT (as the ultrafast-clock allele is called) and per- exhibited periods that were much shorter than in the case of perT/+. These tests also revealed in a clearer manner than in previous cases that two "doses" of a fast-clock per mutation lead to appreciably shorter periods than those exhibited by one-dose females whose other per allele is a loss-of-function variant. In light-dark cycles (LD 12:12), flies carrying perT in a genotypic condition leading to free-running periods that are 8 hr faster than normal nevertheless entrained, by phase-shifting that large number of hours each day; the evening peak of locomotor activity was, however, many hours earlier than normal. The use of a newly developed device for monitoring Drosophila eclosion automatically showed that perT exhibits a very marginal emergence rhythm at 25 degrees C, but periodicity of ca. 17-18 hr at 19 degrees. Staining of the per-encoded protein (PER) in sections of perT versus normal pharate adults revealed for the first time that the immunohistochemically detected signal cycles in its intensity in wild-type, in a manner that is similar to the PER rhythm previously demonstrated in adults. The staining cycle in pharate adults expressing perT differed from that of wild-type. Temperature compensation of the adult activity rhythm of perT was found to be faulty, in that periods became appreciably shorter as the flies were heated. However, the mutant exhibited a normal degree of period lengthening when its locomotor activity was monitored in the presence of heavy water. The perT mutation interacted with the long-period Andante allele of the dusky locus in a manner that was anomalous (in comparison to dyAnd interactions with per+ or another short-period per mutation). This and other unique features of perT are discussed from the standpoint of the new mutation's heuristic value, including that which may stimulate a deeper understanding of the period gene's action at the molecular level.

Behavior in light-dark cycles of Drosophila mutants that are arrhythmic, blind, or both.

Certain of the rhythm mutations in Drosophila melanogaster lead to arrhythmic locomotor activity (and aperiodic eclosion) in constant conditions. In light-dark (LD) cycles, however, such mutants exhibit clear fluctuations between high levels of activity when the lights are on and much lower ones when they are off. Our data, in contrast to some previous conclusions, strongly suggest that period0 (per0) adults are, in LD conditions, merely being "forced" into exhibiting periodic behavior. These experiments involved application of 8-, 12-, 16-, and 24-hr LD cycles, in which the arrhythmic mutant could have any of these periodicities imposed upon it, whereas wild-type flies tended to exhibit periods of about 24 hr in cycling conditions whose T values were > 8 hr different from 24. In phase-shift experiments, it was found that Drosophila expressing genotypes associated with rhythmicity achieved a 5-hr advance over a 2-day period following an advanced lights-on; per0 adults altered the phase of their locomotor peaks more rapidly. Against a background of the fact that eyeless or blind flies exhibit normal entrainment, it was hypothesized that double-mutant flies--carrying such visual mutations and per0 as well--should not synchronize to LD cycles, if the forced rhythms seen in the latter single-mutant type are mediated solely by light input through the external photoreceptors. Since an appreciable proportion of the double mutants did synchronize (to LD 12:12), it is thus suggested that the visual cues involved in forcing rhythmicity could be input through the same extraocular photoreceptors that, in general, subserve the fly's rhythm system.

Expression of the period clock gene within different cell types in the brain of Drosophila adults and mosaic analysis of these cells' influence on circadian behavioral rhythms.

The product of the period (per) gene of Drosophila melanogaster is continuously required for the functioning of the circadian pacemaker of locomotor activity. We have used internally marked mosaics to determine the anatomical locations at which per expression is required for adult rhythmicity, and thus where the fly's circadian pacemaker is likely located in this holometabolous insect. We first provide a detailed description of the distribution and nature of per-expressing cells in the fly's CNS. Using an antibody to the per gene product, or to that of a reporter of per expression, in conjunction with an antibody to the embryonic lethal-abnormal visual system (elav) gene product--which is used as a marker of neuronal identity--we have experimentally confirmed previously proposed assignments of per-expressing cells to the neuronal and glial classes. Thus, we found that per expression and elav immunoreactivity colocalized in large cells located in the lateral cortex of the central brain, as well as in more dorsally located cells in the posterior central brain. In contrast, we found that cells located at the margins of the cortex and the neuropil, and within the neuropil, as well as smaller cortical cells found throughout the brain's cortex, were elav negative, supporting the notion that they are glial in nature. Using internally marked mosaics, we find that the pacemaker is located in brain but is not exclusive to the eyes, the ocelli, or the optic lobes, which is consistent with previous reports obtained in this and other insects of this class. Although the pacemaker may be a paired structure, we show that the functioning of one of them is sufficient for rhythmicity. Finally, we report that glial expression is sufficient for some behavioral rhythmicity to be manifest. However, the rhythmicities of animals for which per expression was confined to glia were weak, suggesting that neuronal per expression as well may be required for normal pacemaker function.