Interlocked feedback loops within the Drosophila circadian oscillator.

Drosophila Clock (dClk) is rhythmically expressed, with peaks in mRNA and protein (dCLK) abundance early in the morning. dClk mRNA cycling is shown here to be regulated by PERIOD-TIMELESS (PER-TIM)-mediated release of dCLK- and CYCLE (CYC)-dependent repression. Lack of both PER-TIM derepression and dCLK-CYC repression results in high levels of dClk mRNA, which implies that a separate dClk activator is present. These results demonstrate that the Drosophila circadian feedback loop is composed of two interlocked negative feedback loops: a per-tim loop, which is activated by dCLK-CYC and repressed by PER-TIM, and a dClk loop, which is repressed by dCLK-CYC and derepressed by PER-TIM.

How a circadian clock adapts to seasonal decreases in temperature and day length.

We show that a thermosensitive splicing event in the 3' untranslated region of the mRNA from the period (per) gene plays an important role in how a circadian clock in Drosophila adapts to seasonally cold days (low temperatures and short day lengths). The enhanced splicing of this intron at low temperatures advances the steady state phases of the per mRNA and protein cycles, events that significantly contribute to the preferential daytime activity of flies on cold days. Because the accumulation of PER is also dependent on the photosensitive TIMELESS (TIM) protein, long photoperiods partially counteract the cold-induced advances in the oscillatory mechanism by delaying the daily increases in the levels of TIM. Our findings also indicate that there is a temperature-dependent switch in the molecular logic governing cycles in per mRNA levels.

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.

Analysis of period mRNA cycling in Drosophila head and body tissues indicates that body oscillators behave differently from head oscillators.

The period (per) gene is thought to be part of the Drosophila circadian pacemaker. The circadian fluctuations in per RNA and protein that constitute the per feedback loop appear to be required for pacemaker function, and have been measured in head neuronal tissues that are necessary for locomotor activity and eclosion rhythms. The per gene is also expressed in a number of neuronal and nonneuronal body tissues for which no known circadian phenomena have been described. To determine whether per might affect some circadian function in these body tissues, per RNA cycling was examined. These studies show that per RNA cycles in the same phase and amplitude in head and body tissues during light-dark cycles. One exception to this is the lack of per RNA cycling in the ovary, which also appears to be the only tissue in which PER protein is primarily cytoplasmic. In constant darkness, however, the amplitude of per RNA cycling dampens much more quickly in bodies than in heads. Taken together, these results indicate that circadian oscillators are present in head and body tissues in which PER protein is nuclear and that these oscillators behave differently.

per mRNA cycling is locked to lights-off under photoperiodic conditions that support circadian feedback loop function.

Circadian fluctuations in per mRNA and protein are central to the operation of a negative feedback loop that is necessary for setting the free-running period and for entraining the circadian oscillator to light-dark cycles. In this study, per mRNA cycling and locomotor activity rhythms were measured under different light and dark cycling regimes to determine how photoperiods affect the molecular feedback loop and circadian behavior, respectively. These experiments reveal that per mRNA peaks in abundance 4 h after lights-off in photoperiods of < or = 16 h, that, phase shifts in per mRNA cycling and behavioral rhythmicity occur rapidly after flies are transferred from one photoperiod to another, and that photoperiods longer than 20 h abolish locomotor activity rhythms and leave per mRNA at a median constitutive level. These results indicate that the per feedback loop uses lights-off as a phase reference point and suggest (along with previous findings for per01 and tim01) that per mRNA cycling is not regulated via simple negative feedback from the per protein.

Two alternatively spliced transcripts from the Drosophila period gene rescue rhythms having different molecular and behavioral characteristics.

The period (per) and timeless (tim) genes encode key components of the circadian oscillator in Drosophila melanogaster. The per gene is thought to encode three transcripts via differential splicing (types A, B, and C) that give rise to three proteins. Since the three per mRNA types were based on the analysis of cDNA clones, we tested whether these mRNA types were present in vivo by RNase protection assays and reverse transcriptase-mediated PCR. The results show that per generates two transcript types that differ only by the presence (type A) or absence (type B') of an alternative intron in the 3' untranslated region. Transgenic flies containing transgenes that produce only type B' transcripts (perB'), type A transcripts (perA), or both transcripts (perG) rescue locomotor activity rhythms with average periods of 24.7, 25.4, and 24.4 h, respectively. Although no appreciable differences in type A and type B' mRNA cycling were observed, a slower accumulation of PER in flies making only type A transcripts suggests that the intron affects the translation of per mRNA.

A circadian enhancer mediates PER-dependent mRNA cycling in Drosophila melanogaster.

Genes expressed under circadian-clock control are found in organisms ranging from prokaryotes to humans. In Drosophila melanogaster, the period (per) gene, which is required for clock function, is transcribed in a circadian manner. We have identified a circadian transcriptional enhancer within a 69-bp DNA fragment upstream of the per gene. This enhancer drives high-amplitude mRNA cycling under light-dark-cycling or constant-dark conditions, and this activity is per protein (PER) dependent. An E-box sequence within this 69-bp fragment is necessary for high-level expression, but not for rhythmic expression, indicating that PER mediates circadian transcription through other sequences in this fragment.

Activating inhibitors and inhibiting activators: a day in the life of a fly.

The circadian clock keeps time through an intracellular oscillator that requires rhythmic gene expression. In Drosophila melanogaster, the core of this oscillator is composed of a circadian feedback loop in which the transcription of the period and timeless genes is repressed by their own protein products. In the past year, our understanding of clock organization and function in Drosophila has been advanced by breakthroughs that define when, where and how this feedback loop operates. These studies, along with those in other organisms, suggest that circadian feedback loops are widespread and that genes within these feedback loops are conserved between Drosophila and mammals.

dCLOCK is present in limiting amounts and likely mediates daily interactions between the dCLOCK-CYC transcription factor and the PER-TIM complex.

In Drosophila melanogaster four circadian clock proteins termed PERIOD (PER), TIMELESS (TIM), dCLOCK (dCLK), and CYCLE (CYC/dBMAL1) function in a transcriptional feedback loop that is a core element of the oscillator mechanism. dCLK and CYC are members of the basic helix-loop-helix (bHLH)/PAS (PER-ARNT-SIM) superfamily of transcription factors and are required for high-level expression of per and tim and repression of dClk, whereas PER and TIM inhibit dCLK-CYC-mediated transcription and lead to the activation of dClk. To understand further the dynamic regulation within the circadian oscillator mechanism, we biochemically characterized in vivo-produced CYC, determined the interactions of the four clock proteins, and calculated their absolute levels as a function of time. Our results indicate that throughout a daily cycle the majority of the dCLK present in adult heads stably interacts with CYC, indicating that CYC is the primary in vivo partner of dCLK. dCLK-CYC dimers are bound by PER and TIM during the late evening and early morning, suggesting the formation of a tetrameric complex with impaired transcriptional activity. Although dCLK is present in limiting amounts and CYC is by far the most abundant of the four clock proteins that have been examined, PER and TIM appear to interact preferentially with dCLK. Our results suggest that dCLK is the main component regulating the daily abundance of transcriptionally active dCLK-CYC complexes.

Spec2 genes of Strongylocentrotus purpuratus. Structure and differential expression in embryonic aboral ectoderm cells.

Members of the Spec gene family are expressed during embryonic development of the sea urchin, Strongylocentrotus purpuratus. The family encodes proteins related to the calmodulin/troponin C/myosin light chain group of calcium binding proteins and one gene, Spec1, has been studied extensively in our laboratory. In this paper, we analyze other members of the family, collectively termed Spec2 genes. We make use of several hybridization probes derived from Spec1 and Spec2 cDNA clones, which recognize different members of the family. Genomic DNA gel blot and slot blot analyses show that there are approximately eight Spec genes in the S. purpuratus genome. The structures of three Spec2 genes, Spec2a, Spec2c and Spec2d, are described. A 60 kb (kb = 10(3) bases or base-pairs) region of the genome contains the linked Spec1-Spec2c genes and two separate 20 kb regions contain the Spec2a and Spec2d genes. Six members of a repetitive sequence family are dispersed at various locations among the genes. The transcriptional initiation sites of the three Spec2 genes are mapped, and 400 to 500 base-pairs of 5'-flanking DNA sequenced. All three Spec2 genes initiate transcription approximately 120 base-pairs upstream from the 3' end of the first exon. In contrast, the 5' end of the Spec1 transcript begins about 107 base-pairs farther upstream, so it contains 5' untranslated sequences that correspond to non-transcribed 5'-flanking sequences of the Spec2 genes. There is little similarity among the sequences upstream from the CAP site of the Spec2 genes except the TATA consensus sequence and a repeating trinucleotide, AAC. Measurements of Spec mRNA levels during embryogenesis show that Spec1 mRNA begins to accumulate at the early blastula stage and is the most abundant; Spec2a/Spec2c mRNAs begin accumulating several hours later at the late blastula-early gastrula stage and reach about 40 to 60% the levels of Spec1; and Spec2d mRNAs accumulate mostly during the gastrula and pluteus stages with levels reaching only 2% those of Spec1. In situ hybridization with probes that recognize either all Spec2 mRNAs or only Spec2d mRNAs show that, like Spec1, these mRNAs are restricted to aboral ectoderm cells and their precursors. The Spec gene family represents a group of related genes whose mRNAs all accumulate in the same cell type but at different times and to different levels during embryogenesis.

Structure of the Spec1 gene encoding a major calcium-binding protein in the embryonic ectoderm of the sea urchin, Strongylocentrotus purpuratus.

We have identified and characterized the structure of the Spec1 gene in the sea urchin Strongylocentrotus purpuratus. In earlier studies we demonstrated that a small family of messenger RNAs, termed Spec mRNAs for S. purpuratus ectodermal mRNAs, begins to accumulate 20 hours after fertilization in ectoderm cells of the sea urchin embryo. The Spec mRNAs code for a group of low molecular weight proteins belonging to the troponin C superfamily. Spec1 transcripts, the predominant mRNAs of the family, are heterogeneous in their 3' untranslated sequences but code for a single protein, recently shown to be a calcium-binding protein. Spec complementary DNA clones were used to isolate genomic clones from two lambda libraries. These genomic clones comprise a 41 kb (kb = 10(3) bases or base-pairs) region of the S. purpuratus genome and contain a Spec1 gene closely linked to another Spec gene, Spec2c. The Spec1 gene is 10.3 kb in length and contains six exons. The genomic clones containing the Spec1 gene can be placed into two groups based on restriction fragment length differences and differences in hybridization strengths using probes derived from Spec1 3' untranslated regions. Evidence that these groups probably correspond to two alleles of the Spec1 gene was obtained by probing genomic DNA blots of sperm DNA from different individuals with 3' untranslated sequences of Spec1 complementary DNA clones. These blots show that two of the Spec1 mRNAs we have characterized, and probably a third, are alleles of the Spec1 gene. Thus, there appears to be a single polymorphic Spec1 gene in the sea urchin genome. We used S1 protection and primer extension procedures to map the 5' end of the Spec1 gene. Results from these experiments indicate that the initiation of transcription of the Spec1 mRNA begins at an A residue 220 bases from the 3' end of the first exon. Adding support to this claim, cannonical T-A-T-A and C-A-A-T sequences, indicative of many eukaryotic promoters, are found 23 bases and 60 bases upstream from this site, respectively. Analysis of sequences within a few kb of the Spec1 gene show that there are five members of a repetitive sequence family near the gene, three upstream and two downstream. The 5' leader sequence of another Spec mRNA, Spec2a, also contains a member of this repeat family.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental state and the circadian clock interact to influence the timing of eclosion in Drosophila melanogaster.

In Drosophila melanogaster, the emergence of adults from their pupal cases (eclosion) is gated by the circadian clock such that it occurs during a window of approximately 8-10 h starting 1-2 h before lights-on in 12-h light:12-h dark cycles (LD). This gate is shifted several hours earlier by the clock mutant per(s), indicating that the clock controls the phase of eclosion under these conditions. Both the day and the time of eclosion are determined by the interplay between developmental state and the circadian clock. At a certain phase of the circadian cycle, the circadian clock, either directly or through some circadian clock-controlled mechanism, measures development state, and those pharate adults that have reached a certain developmental state by this phase eclose during the first available gate, while those that have not wait until a subsequent gate. Using wing pigmentation as a late developmental state marker, an early boundary for when the circadian clock assesses developmental state occurs roughly at the time when lights go out during LD cycles. This event is shifted several hours earlier in per(s), showing that it is under circadian control. A fly's developmental state at the time of developmental assessment also influences when eclosion will occur (during the gate) in that flies whose wings have become pigmented early (12-24 h before assessment) will eclose earlier in the gate than those whose wings become pigmented late (0-12 h before assessment). These data suggest that the circadian clock (or some clock-controlled mechanism) measures developmental state (wing pigmentation) in wild-type flies between lights-off and expression of the first clock-regulated marker approximately 4-5 h before eclosion and that the developmental state of the fly determines both which gate is chosen for eclosion and when eclosion occurs during that gate.

The period E-box is sufficient to drive circadian oscillation of transcription in vivo.

The minimum element from the Drosophila period promoter capable of driving in vivo cycling mRNA is the 69 bp circadian regulatory sequence (CRS). In cell culture, an 18 bp E-box element from the period promoter is regulated by five genes that are involved in the regulation of circadian expression in flies. This E-box is a target for transcriptional activation by bHLH-PAS proteins dCLOCK (dCLK) and CYCLE (CYC), this activation is inhibited by PERIOD (PER) and TIMELESS (TIM) together, and inhibition of dCLK/CYC by PER and TIM is blocked by CRYPTOCHROME (CRY) in the presence of light. Here, the same 18 bp E-box region generated rhythmic expression of luciferase in flies under both light-dark cycling and constant conditions. Flies heterozygous for the Clke(jrk) mutation maintained rhythmic expression from the E-box although at a lower level than wild type. Homozygous mutant Clk(jrk) animals had drastically lowered and arrhythmic expression. In a per01 background, expression from the E-box was high and not rhythmic. Transcription mediated by the per E-box was restricted to the same spatial pattern as the CRS. The per E-box DNA element and cognate binding proteins can confer per-like temporal and spatial expression. This demonstrates in vivo that the known circadian genes that form the core of the circadian oscillator in Drosophila integrate their activities at a single DNA element.

Specific sequences outside the E-box are required for proper per expression and behavioral rescue.

A 69 bp circadian regulatory sequence (CRS) upstream of the per gene is sufficient to drive circadian transcription, mediate proper spatial expression, and rescue behavioral rhythmicity in per01 flies. Within the CRS, an E-box is required for transcriptional activation by two basic-helix-loop-helix (bHLH) PERARNT-SIM (PAS) transcription factors, dCLOCK (dCLK) and CYCLE (CYC). To define sequences within the CRS that are required for spatial expression, circadian expression, and behavioral rhythmicity, a series of mutants that alter blocks of 3 to 12 nucleotides across the entire CRS were used to drive lacZ or per expression in vivo. As expected, the E-box within the CRS is necessary for high-level expression and behavioral rhythmicity, but sequences outside the E-box are also required for transcriptional activation, proper spatial expression, and behavioral rhythmicity. These results indicate that the dCLK-CYC target site extends beyond the E-box and that factors other than dCLK and CYC modulate per transcription.

Transcriptional feedback loop regulation, function, and ontogeny in Drosophila.

The Drosophila circadian oscillator is composed of interlocked period/timeless (per/tim) and Clock (Clk) transcriptional feedback loops. These feedback loops drive rhythmic transcription having peaks at dawn and dusk during the daily cycle and function in the brain and a variety of peripheral tissues. To understand how the circadian oscillator keeps time and controls metabolic, physiological, and behavioral rhythms, we must determine how these feedback loops regulate rhythmic transcription, determine the relative importance of the per/tim and Clk feedback loops with regard to circadian oscillator function, and determine how these feedback loops come to be expressed in only certain tissues. Substantial insight into each of these issues has been gained from experiments performed in our lab and others and is summarized here.

From biological clock to biological rhythms.

The genetic and molecular analysis of circadian timekeeping mechanisms has accelerated as a result of the increasing volume of genomic markers and nucleotide sequence information. Completion of whole genome sequences and the use of differential gene expression technology will hasten the discovery of the clock output pathways that control diverse rhythmic phenomena.

Temporal and spatial expression of an adult cuticle protein gene from Drosophila suggests that its protein product may impart some specialized cuticle function.

An adult cuticle protein gene (Dacp-1) from Drosophila melanogaster has been isolated and characterized. This gene was classified as an adult cuticle protein gene because it maintains the conserved structure of other cuticle protein genes, the sequence of its conceptual translation product contains a repeated motif that is found almost exclusively in a subset of adult cuticle proteins from Locust migratoria, and the gene is expressed in the epidermis underlying the head and thoracic cuticle. The bulk of Dacp-1 expression starts approximately 72 hr after pupariation, peaks approximately 12 hr after eclosion, and decreases thereafter to undetectable levels by 3 days after eclosion. The stage specificity and spatial restriction of Dacp-1 expression as well as the physical properties of its conceptual translation product suggest that it may be involved in some specialized function such as thickening of the adult cuticle.

Unusual sequence conservation in the 5' and 3' untranslated regions of the sea urchin spec mRNAs.

The Spec1 and Spec2 mRNAs (Strongylocentrotus purpuratus ectoderm mRNAs) represent a small gene family that encodes 10-12 members of the troponin C superfamily of calcium-binding proteins. These mRNAs and proteins accumulate in the aboral (dorsal) ectoderm of sea urchin embryos and larvae. Using genomic and cDNA clones, we have compared the sequences of four Spec mRNAs: Spec1, Spec2a, Spec2c, and Spec2d. The mRNAs all have at least 120 bases of 5' untranslated leader, approximately 450 bases of open reading frame, and 900 bases (Spec1) or 1250 bases (Spec2a, 2c, 2d) of 3' untranslated trailer. Unexpectedly, when long stretches of 5' untranslated regions or 3' untranslated regions are compared to one another, they are found to be less divergent than the protein-coding regions. Comparing Spec2d, the most divergent member of the family, with the other Spec mRNAs shows that while the protein-coding regions are 60-62% matched, the untranslated regions are greater than 80% matched. Comparisons among Spec1, Spec2a, and Spec2c demonstrate similar but less dramatic conservation of untranslated regions. Our data imply that the Spec gene family has evolved differently from most gene families, with mutations accumulating most rapidly in intron regions, less rapidly in protein-coding regions, and least rapidly in 5' and 3' untranslated regions.

Drosophila photoreceptors contain an autonomous circadian oscillator that can function without period mRNA cycling.

Circadian oscillations in period (per) mRNA and per protein (PER) constitute, in part, a feedback loop that is required for circadian pacemaker function in Drosophila melanogaster. Oscillations in PER are required for oscillations in per mRNA, but the converse has not been rigorously tested because of a lack of measurable quantities of per mRNA and protein in the same cells. This circadian feedback loop operates synchronously in many neuronal and non-neuronal tissues, including a set of lateral brain neurons (LNs) that mediate rhythms in locomotor activity, but whether a hierarchy among these tissues maintains this synchrony is not known. To determine whether per mRNA cycling is necessary for PER cycling and whether cyclic per gene expression is tissue autonomous, we have generated per01 flies carrying a transgene that constitutively expresses per mRNA specifically in photoreceptors, a cell type that supports feedback loop function. These transformants were tested for different aspects of feedback loop function including per mRNA cycling, PER cycling, and PER nuclear localization. Under both light/dark (LD) cycling and constant dark (DD) conditions, PER abundance cycles in the absence of circadian cycling of per mRNA. These results show that per mRNA cycling is not required for PER cycling and indicate that Drosophila photoreceptors R1-R6 contain a tissue autonomous circadian oscillator.