Loss of circadian behavioral rhythms and per RNA oscillations in the Drosophila mutant timeless.

Eclosion, or emergence of adult flies from the pupa, and locomotor activity of adults occur rhythmically in Drosophila melanogaster, with a circadian period of about 24 hours. Here, a clock mutation, timeless (tim), is described that produces arrhythmia for both behaviors. The effects of tim on behavioral rhythms are likely to involve products of the X chromosome-linked clock gene period (per), because tim alters circadian oscillations of per RNA. Genetic mapping places tim on the left arm of the second chromosome between dumpy (dp) and decapentaplegic (dpp).

Light-induced degradation of TIMELESS and entrainment of the Drosophila circadian clock.

Two genes, period (per) and timeless (tim), are required for production of circadian rhythms in Drosophila. The proteins encoded by these genes (PER and TIM) physically interact, and the timing of their association and nuclear localization is believed to promote cycles of per and tim transcription through an autoregulatory feedback loop. Here it is shown that TIM protein may also couple this molecular pacemaker to the environment, because TIM is rapidly degraded after exposure to light. TIM accumulated rhythmically in nuclei of eyes and in pacemaker cells of the brain. The phase of these rhythms was differentially advanced or delayed by light pulses delivered at different times of day, corresponding with phase shifts induced in the behavioral rhythms.

Positional cloning and sequence analysis of the Drosophila clock gene, timeless.

The Drosophila genes timeless (tim) and period (per) interact, and both are required for production of circadian rhythms. Here the positional cloning and sequencing of tim are reported. The tim gene encodes a previously uncharacterized protein of 1389 amino acids, and possibly another protein of 1122 amino acids. The arrhythmic mutation tim01 is a 64-base pair deletion that truncates TIM to 749 amino acids. Absence of sequence similarity to the PER dimerization motif (PAS) indicates that direct interaction between PER and TIM would require a heterotypic protein association.

Block in nuclear localization of period protein by a second clock mutation, timeless.

In wild-type Drosophila, the period protein (PER) is found in nuclei of the eyes and brain, and PER immunoreactivity oscillates with a circadian rhythm. The studies described here indicate that the nuclear localization of PER is blocked by timeless (tim), a second chromosome mutation that, like per null mutations, abolishes circadian rhythms. PER fusion proteins without a conserved domain (PAS) and some flanking sequences are nuclear in tim mutants. This suggests that a segment of PER inhibits nuclear localization in tim mutants. The tim gene may have a role in establishing rhythms of PER abundance and nuclear localization in wild-type flies.

Isolation of timeless by PER protein interaction: defective interaction between timeless protein and long-period mutant PERL.

The period (per) gene likely encodes a component of the Drosophila circadian clock. Circadian oscillations in the abundance of per messenger RNA and per protein (PER) are thought to arise from negative feedback control of per gene transcription by PER. A recently identified second clock locus, timeless (tim), apparently regulates entry of PER into the nucleus. Reported here are the cloning of complementary DNAs derived from the tim gene in a two-hybrid screen for PER-interacting proteins and the demonstration of a physical interaction between the tim protein (TIM) and PER in vitro. A restricted segment of TIM binds directly to a part of the PER dimerization domain PAS. PERL, a mutation that causes a temperature-sensitive lengthening of circadian period and a temperature-sensitive delay in PER nuclear entry, exhibits a temperature-sensitive defect in binding to TIM. These results suggest that the interaction between TIM and PER determines the timing of PER nuclear entry and therefore the duration of part of the circadian cycle.

Rhythmic expression of timeless: a basis for promoting circadian cycles in period gene autoregulation.

The clock gene timeless (tim) is required for circadian rhythmicity in Drosophila. The accumulation of tim RNA followed a circadian rhythm, and the phase and period of the tim RNA rhythm were indistinguishable from those that have been reported for per. The tim RNA oscillations were found to be dependent on the presence of PER and TIM proteins, which demonstrates feedback control of tim by a mechanism previously shown to regulate per expression. The cyclic expression of tim appears to dictate the timing of PER protein accumulation and nuclear localization, suggesting that tim promotes circadian rhythms of per and tim transcription by restricting per RNA and PER protein accumulation to separate times of day.

Life's 24-hour clock: molecular control of circadian rhythms in animal cells.

Our sleep-wake cycles and many other approximately 24-hour rhythms of behavior and physiology persist in the absence of environmental cues. Genetic and biochemical studies have shown that such rhythms are controlled by internal molecular clocks. These are assembled from the cycling RNA and protein products of a small group of genes that are conserved throughout the animal kingdom.

Regulation of nuclear entry of the Drosophila clock proteins period and timeless.

Two genes, period (per) and timeless (tim), are essential for circadian rhythmicity in Drosophila. The encoded proteins (PER and TIM) physically interact. Here, it is shown that TIM and PER accumulate in the cytoplasm when independently expressed in cultured (S2) Drosophila cells. However, the proteins move to the nuclei of these cells if coexpressed. Domains of PER and TIM have been identified that block nuclear localization of the monomeric proteins. In vitro protein interaction studies indicate that the sequence inhibiting the nuclear accumulation of PER forms a binding site for TIM. The results indicate a mechanism for controlled nuclear localization in which suppression of cytoplasmic localization is accomplished by direct interaction of PER and TIM. No other clock functions are required for nuclear localization. The findings suggest that a checkpoint in the circadian cycle is established by requiring cytoplasmic assembly of a PER/TIM complex as a condition for nuclear transport of either protein.

Circadian rhythms in Drosophila can be driven by period expression in a restricted group of central brain cells.

Neural tissues controlling circadian rhythmicity have been identified in a variety of organisms and are often closely associated with the visual system. In Drosophila, the clock gene period (per), which is required for circadian rhythms, is expressed in many neurons and glia throughout the eye and brain. We asked whether biological rhythms could be generated if per expression were restricted to a subset of these cells that is involved in photoreception. Here we demonstrate that expression of per under the control of the glass promoter confers both behavioral and molecular rhythmicity. glass is required for development of Drosophila photoreceptors, and this promoter is active in eyes, ocelli, and certain cells of the central brain. When we genetically removed all external photoreceptor cells, rhythms persisted in these transgenic animals. This suggests that a few central brain cells producing glass and per are capable of generating biological rhythms.

A TIMELESS-independent function for PERIOD proteins in the Drosophila clock.

The mutation timeless(UL) generates 33 hr rhythms, prolonged nuclear localization of PERIOD/TIMELESS(UL) protein complexes, and protracted derepression of period (per) and timeless (tim) transcription. Light-induced elimination of TIM(UL) from nuclear PER/TIM(UL) complexes gives strong downregulation of per and tim expression. Thus, in the absence of TIM, nuclear PER can function as a potent negative transcriptional regulator. Two additional studies support this role for PER: (1) Drosophila expressing PER that constitutively localizes to nuclei produce dominant behavioral arrhythmicity, and (2) constitutively nuclear PER represses dCLOCK/CYCLE-mediated transcription of per in cultured cells without TIM. Conversion of PER/TIM heterodimers to nuclear PER proteins appears to be required to complete transcriptional repression and terminate each circadian molecular cycle.

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.

New short period mutations of the Drosophila clock gene per.

Earlier work has indicated that the period length of Drosophila circadian behavioral rhythms is dependent on the abundance of the period (per) gene product. Increased expression of this gene has been associated with period shortening for both the circadian eclosion (pupal hatching) rhythm and circadian locomotor activity rhythms of adult Drosophila. In this study it is shown that a wide variety of missense mutations, affecting a region of the per protein consisting of approximately 20 aa, predominantly generate short period phenotypes. The prevalence of such mutations suggests that short period phenotypes may result from loss or depression of function in this domain of the per protein. Possibly mutations in the region eliminate a regulatory function provided by this segment, or substantially increase stability of the mutant protein.

Circadian regulation of gene expression systems in the Drosophila head.

Mechanisms composing Drosophila's clock are conserved within the animal kingdom. To learn how such clocks influence behavioral and physiological rhythms, we determined the complement of circadian transcripts in adult Drosophila heads. High-density oligonucleotide arrays were used to collect data in the form of three 12-point time course experiments spanning a total of 6 days. Analyses of 24 hr Fourier components of the expression patterns revealed significant oscillations for approximately 400 transcripts. Based on secondary filters and experimental verifications, a subset of 158 genes showed particularly robust cycling and many oscillatory phases. Circadian expression was associated with genes involved in diverse biological processes, including learning and memory/synapse function, vision, olfaction, locomotion, detoxification, and areas of metabolism. Data collected from three different clock mutants (per(0), tim(01), and Clk(Jrk)), are consistent with both known and novel regulatory mechanisms controlling circadian transcription.

Single amino acid substitutions in EGF-like elements of Notch and Delta modify Drosophila development and affect cell adhesion in vitro.

Notch locus EGF-like element mutations spl, altering eye development, and AxE2, affecting wing and sensilla development, are modified by mutations at Delta. It is shown that two allele-specific suppressors of spl involve single amino acid substitutions in the 4th (Dlsup5) and 9th (Dlsup4) EGF-like elements of the Delta protein. Cultured cells producing spl or AxE2 aggregate with cells producing wild-type Delta or Dlsup5 protein, and Dlsup5-producing cells adhere to cells producing wild-type Notch protein. However, spl,AxE2, and Dlsup5 are each defective in promoting these cell affinities, as none of the mutant proteins can compete with the corresponding wild-type proteins for formation of cell aggregates. Thus, widely separated EGF-like elements of Notch and Delta appear to participate in functional molecular interactions between the proteins. Dlsup5 does not improve adhesiveness of spl in vitro, so suppression in vivo may involve altered developmental signaling by spl-Dlsup5 complexes, rather than modified cell adhesion.

Mammalian circadian autoregulatory loop: a timeless ortholog and mPer1 interact and negatively regulate CLOCK-BMAL1-induced transcription.

We report the cloning and mapping of mouse (mTim) and human (hTIM) orthologs of the Drosophila timeless (dtim) gene. The mammalian Tim genes are widely expressed in a variety of tissues; however, unlike Drosophila, mTim mRNA levels do not oscillate in the suprachiasmatic nucleus (SCN) or retina. Importantly, hTIM interacts with the Drosophila PERIOD (dPER) protein as well as the mouse PER1 and PER2 proteins in vitro. In Drosophila (S2) cells, hTIM and dPER interact and translocate into the nucleus. Finally, hTIM and mPER1 specifically inhibit CLOCK-BMAL1-induced transactivation of the mPer1 promoter. Taken together, these results demonstrate that mTim and hTIM are mammalian orthologs of timeless and provide a framework for a basic circadian autoregulatory loop in mammals.

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.

In situ localization of the per clock protein during development of Drosophila melanogaster.

The per locus influences biological rhythms in Drosophila melanogaster. In this study, per transcripts and proteins were localized in situ in pupae and adults. Earlier genetic studies have demonstrated that per expression is required in the brain for circadian locomotor activity rhythms and in the thorax for ultradian rhythmicity of the Drosophila courtship song. per RNA and proteins were detected in a restricted group of cells in the eyes and optic lobes of the adult brain and in many cell bodies in the adult and pupal thoracic ganglia. per products were also found in the pupal ring gland complex, a tissue involved in rhythmic aspects of Drosophila development. Abundant expression was seen in gonadal tissue. No biological clock phenotypes have been reported for this tissue in any of the per mutants, per protein mapped to different subcellular locations in different tissues. The protein accumulated in or around nuclei in some cells and appeared to be cytoplasmic in others.

Sequence of the notch locus of Drosophila melanogaster: relationship of the encoded protein to mammalian clotting and growth factors.

The Notch locus is essential for proper differentiation of the ectoderm in Drosophila melanogaster. Notch corresponds to a 37-kilobase transcription unit that codes for a major 10.4-kilobase polyadenylated RNA. The DNA sequence of this transcription unit is presented, except for portions of the two largest intervening sequences. DNA sequences also were obtained from three Notch cDNA clones, allowing the 5' and 3' ends of the gene to be mapped, and the structures and locations of nine RNA coding regions to be determined. The major Notch transcript encodes a protein of 2,703 amino acids. The protein is probably associated with cell surfaces and carries an extracellular domain composed of 36 cysteine-rich repeating units, each of about 38 amino acids. The gene appears to have evolved by repeated tandem duplications of the DNA coding for the 38-amino-acid-long protein segments, followed by insertion of intervening sequences. These repeating protein segments are quite homologous to portions of mammalian clotting factors IX and X and to the product of the Caenorhabditis elegans developmental gene lin-12. They are also similar to mammalian growth hormones, typified by epidermal growth factor.

Restriction of P-element insertions at the Notch locus of Drosophila melanogaster.

P elements move about the Drosophila melanogaster genome in a nonrandom fashion, preferring some chromosomal targets for insertion over others (J. C. J. Eeken, F. H. Sobels, V. Hyland, and A. P. Schalet, Mutat. Res. 150:261-275, 1985; W. R. Engels, Annu. Rev. Genet. 17:315-344, 1983; M. D. Golubovsky, Y. N. Ivanov, and M. M. Green, Proc. Natl. Acad. Sci. USA 74:2973-2975, 1977; M. J. Simmons and J. K. Lim, Proc. Natl. Acad. Sci. USA 77:6042-6046, 1980). Some of this specificity may be due to recognition of a particular DNA sequence in the target DNA; derivatives of an 8-base-pair consensus sequence are occupied by these transposable elements at many different chromosomal locations (K. O'Hare and G. M. Rubin, Cell 34:25-36, 1983). An additional level of specificity of P-element insertions is described in this paper. Of 14 mutations induced in the complex locus Notch by hybrid dysgenesis, 13 involved P-element insertions at or near the transcription start site of the gene. This clustering was not seen in other transposable element-induced mutations of Notch. DNA sequences homologous to the previously described consensus target for P-element insertion are not preferentially located in this region of the locus. The choice of a chromosomal site for integration appears to be based on more subtle variations in chromosome structure that are probably associated with activation or expression of the target gene.

Extrachromosomal DNA forms of copia-like transposable elements, F elements and middle repetitive DNA sequences in Drosophila melanogaster. Variation in cultured cells and embryos.

Drosophila melanogaster embryos and cells in culture were screened for the presence of unintegrated covalently closed circular DNA forms that hybridize to copia-like transposable elements, the F element and uncharacterized dispersed middle repetitive DNA elements. Our results indicate that the majority of copia-like elements (including copia, 297, 412, mdg1, mdg3 and gypsy), the F elements, and 9 of 12 middle repetitive DNA elements are present as free DNA forms in cultured cells and embryos. An 18 base-pair inverted repeat has been reported to flank the long direct repeat of mdg3, implying that mdg3 is not an orthodox copia-like element; however, we have sequenced two independently isolated mdg3 clones and shown that the inverted repeat is not part of the element. The relative abundance with which free DNA forms are found varies between the cultured cells used, and between cultured cells and embryos. This variation, which can be up to 20-fold for some elements, does not correlate well with either the amount of element-specific poly(A)+ RNA present per cell or the number of element-specific sequences integrated in the genome.