Three mutants have been isolated in which the normal 24-hour rhythm is drastically changed.One mutant is arrhythmic; another has a period of 19 hr; a third has a period of 28 hr. Both the eclosion rhythm of a population and the locomotor activity of individual flies are affected. All these mutations appear to involve the same functional gene on the X chromosome.Rhythmic variations in behavior are displayed by many organisms, ranging from single cells to man (1). When the rhythm persists under constant conditions, and has a period of around one day, depending little on temperature, the rhythm is called circadian (2). Many experiments have attempted to probe the mechanism (3), but the nature of the underlying oscillation remains unknown (4). Perturbations by inhibitors of RNA or protein synthesis suggest that such molecules are involved (5-8). Biochemical systems that oscillate with much shorter periods have been demonstrated both in vivo and in vitro (9, 10), but their relation to circadian rhythms is not clear.An approach that has been successful in unravelling mechanisms in some systems is the use of genetic alterations. Since the expression of a rhythm requires an integrated system, mutation of the genes responsible for development and function of the system could lead to abnormal rhythms. Various aspects of circadian rhythms have indeed been shown to be sensitive to genetic makeup (11-18). For genetic dissection of circadian rhythms in an organism having a nervous system, Drosophila offers certain advantages. Much is already known about the rhythm of eclosion (emergence of the adult fly from the pupa), and genetic methodology is readily available. This paper describes the first result of such an analysis. MATERIALS AND METHODSIsolation of mutants D. melanogaster of the C-S (Canton-Special) wild strain was maintained on cornmeal medium. Mutagenesis by ethyl methane sulfonate was according to Lewis and Bacher (19), the treated males being mated to virgin attached-X females, so that each F1 progeny male carri&Va treated X chromosome received from his father. Each male was mated individually to attached-X females, producing a stock of males bearing identical X chromosomes, plus normal-rhythm attached-X females. The stocks were reared at constant temperature under LD 12:12 (12 hr of 50 foot-candles or more of white fluorescent light, 12 hr of darkness each day).To detect X-linked rhythm mutants, the stocks were examined for ones in which males emerged abnormally. The normal females in each bottle served as an internal control, at least twice as many emerging during the light as during the dark period. In a few bottles, males emerged in approximately equal numbers during day and night. Each mutant candidate was examined in more detail by raising pupae in LD 12:12, then monitoring the adult eclosion rhythm in constant darkness. From a total of about 2000 F1 males, three rhythm mutants were obtained. Determination of eclosion and locomotor activity rhythmsEclosion rhythms, free-running in constant darkness, w...

The circadian oscillators of genetically short-period and long-period Drosophila exhibit reciprocal behaviour in four distinct ways: (1) with respect to the dependence of period on temperature, (2) in the change of period during constant darkness after ten days of constant light, (3) in the change of period during the second ten days of darkness as compared with the period during the first ten days, and (4) in the period change resulting from exposure to low-intensity constant light. The homeostatic control of the dependence of period length on temperature is impaired in the mutants as compared with wild-type flies. The normal Drosophila pacemaker may comprise two mutually coupled oscillators, whereas the mutants may represent a reduction in activity of one or the other constituent oscillator.

Mutations at the period (per) locus of Drosophila melanogaster disrupt several biological rhythms. Molecular cloning of DNA sequences encompassing the per+ locus has allowed germ-line transformation experiments to be carried out. Certain subsegments of the per region, transduced into the genome of arrhythmic pero flies, restore rhythmicity in circadian locomotor behavior and the male's courtship song.

P-element-mediated transformations involving DNA fragments from the period (per) clock gene of Drosophila melanogaster have shown that several subsegments of the locus restore rhythmicity to per0 or per- mutants. Such fragments overlap in a genomic region complementary to one transcript, a 4.5-kb RNA which is probably the per message, in that it is necessary and (in terms of expression from this X-chromosomal locus) sufficient for the fly's circadian rhythms. It is also at least necessary for the high-frequency oscillations normally produced by courting males as they vibrate their wings. The entirety of the 4.5-kb transcript is not necessary for rather strong rhythmicity; nor does it seem to be sufficient, in transformants, for wild-type behavioral phenotypes. A 0.9-kb RNA, homologous to genomic region immediately adjacent to the source of the 4.5-kb species, oscillates in its abundance over the course of a day; but coverage of this transcript source in several transformants carrying a per0 mutation--which eliminates the 0.9-kb RNA's oscillation--does not restore rhythmicity. All of the independently isolated arrhythmic mutations tested were covered by the same array of overlapping per+-derived DNA fragments, implying that the only portion of the locus which has mutated to arrhythmicity is complementary to the 4.5-kb transcript.