A PER/TIM/DBT interval timer for Drosophila's circadian clock.

Circadian rhythms in Drosophila are supported by a negative feedback loop, in which PERIOD (PER) and Timeless (TIM) shut down their own transcription as they translocate once a day from the cytoplasm of clock-containing cells to the nucleus. Period length is partially determined by an interval of cytoplasmic retention of the TIM and PER proteins. To study this process, we examined PER/TIM/Doubletime (DBT) physical interactions and nuclear translocation by imaging individual cultured Drosophila cells. Using live cell video microscopy and green fluorescent protein (GFP) tags, we observed dynamic patterns of stability and localization for DBT, PER, and TIM that resembled those previously found in vivo. These studies suggest that a cytoplasmic interval timer regulates nuclear translocation of these proteins. The cultured cell assay provides a potent system to study interactions among new and known genes involved in the generation of circadian behavior.

Responses of plants and invertebrate trophic groups to contrasting herbicide regimes in the Farm Scale Evaluations of genetically modified herbicide-tolerant crops.

Effects of genetically modified herbicide-tolerant (GMHT) and conventional crop management on invertebrate trophic groups (herbivores, detritivores, pollinators, predators and parasitoids) were compared in beet, maize and spring oilseed rape sites throughout the UK. These trophic groups were influenced by season, crop species and GMHT management. Many groups increased twofold to fivefold in abundance between early and late summer, and differed up to 10-fold between crop species. GMHT management superimposed relatively small (less than twofold), but consistent, shifts in plant and insect abundance, the extent and direction of these effects being dependent on the relative efficacies of comparable conventional herbicide regimes. In general, the biomass of weeds was reduced under GMHT management in beet and spring oilseed rape and increased in maize compared with conventional treatments. This change in resource availability had knock-on effects on higher trophic levels except in spring oilseed rape where herbivore resource was greatest. Herbivores, pollinators and natural enemies changed in abundance in the same directions as their resources, and detritivores increased in abundance under GMHT management across all crops. The result of the later herbicide application in GMHT treatments was a shift in resource from the herbivore food web to the detritivore food web. The Farm Scale Evaluations have demonstrated over 3 years and throughout the UK that herbivores, detritivores and many of their predators and parasitoids in arable systems are sensitive to the changes in weed communities that result from the introduction of new herbicide regimes.

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.

Time zones: a comparative genetics of circadian clocks.

The circadian clock is a widespread cellular mechanism that underlies diverse rhythmic functions in organisms from bacteria and fungi, to plants and animals. Intense genetic analysis during recent years has uncovered many of the components and molecular mechanisms comprising these clocks. Although autoregulatory genetic networks are a consistent feature in the design of all clocks, the weight of evidence favours their independent evolutionary origins in different kingdoms.

A Drosophila PIAS homologue negatively regulates stat92E.

Transcriptional activation by, and therefore the physiologic impact of, activated tyrosine-phosphorylated STATs (signal transducers and activators of transcription) may be negatively regulated by proteins termed PIAS (protein inhibitors of activated stats), as shown by previous experiments with mammalian cells in culture. Here, by using the genetic modifications in Drosophila, we demonstrate the in vivo functional interaction of the Drosophila homologues stat92E and a Drosophila PIAS gene (dpias). To this end we use a LOF allele and conditionally overexpressed dpias in JAK-STAT pathway mutant backgrounds. We conclude that the correct dpias/stat92E ratio is crucial for blood cell and eye development.

A role for the segment polarity gene shaggy/GSK-3 in the Drosophila circadian clock.

Tissue-specific overexpression of the glycogen synthase kinase-3 (GSK-3) ortholog shaggy (sgg) shortens the period of the Drosophila circadian locomotor activity cycle. The short period phenotype was attributed to premature nuclear translocation of the PERIOD/TIMELESS heterodimer. Reducing SGG/GSK-3 activity lengthens period, demonstrating an intrinsic role for the kinase in circadian rhythmicity. Lowered sgg activity decreased TIMELESS phosphorylation, and it was found that GSK-3 beta specifically phosphorylates TIMELESS in vitro. Overexpression of sgg in vivo converts hypophosphorylated TIMELESS to a hyperphosphorylated protein whose electrophoretic mobility, and light and phosphatase sensitivity, are indistinguishable from the rhythmically produced hyperphosphorylated TIMELESS of wild-type flies. Our results indicate a role for SGG/GSK-3 in TIMELESS phosphorylation and in the regulated nuclear translocation of the PERIOD/TIMELESS heterodimer.

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.

Effect of improved housing on illness in children under 5 years old in northern Malawi: cross sectional study.

OBJECTIVE: To evaluate the effects of a Habitat for Humanity housing improvement programme in northern Malawi on the prevalence of childhood illnesses. DESIGN: Household based cross sectional study. SETTING: Rural communities centred near the small northern Malawi town of Ekwendeni. SUBJECTS: 318 children under 5 years old. MAIN OUTCOME MEASURES: Prevalence of respiratory, gastrointestinal, and malarial infections according to maternal recall, laboratory, or clinical data. RESULTS: Children living in improved homes were less likely to have respiratory, gastrointestinal, or malarial illnesses (odds ratio 0.56, 95% confidence interval 0.35 to 0.91) after confounding factors were controlled for. The reductions in individual diseases were not significant. CONCLUSION: Improved housing significantly reduced the burden of disease among children under 5 years old.

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.

Specific genetic interference with behavioral rhythms in Drosophila by expression of inverted repeats.

We describe a new experimental technique that allows for a tissue-specific reduction of gene activity in the Drosophila nervous system. On the basis of the observation that certain gene functions can be ubiquitously blocked by injecting double-stranded RNA into Drosophila embryos, we employed a method to interfere with an individual gene function permanently in a predetermined cell type. This was achieved by the formation of an inverted-repeat RNA sequence in the tissue of interest under control of the GAL4/UAS binary expression system. As an example, we show that inverted-repeat-mediated interference with the period gene produces a hypomorphic period phenotype. A selective decrease of period RNA appears to be a component of the cellular response.

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.

Isolation and analysis of six timeless alleles that cause short- or long-period circadian rhythms in Drosophila.

In genetic screens for Drosophila mutations affecting circadian locomotion rhythms, we have isolated six new alleles of the timeless (tim) gene. Two of these mutations cause short-period rhythms of 21-22 hr in constant darkness, and four result in long-period cycles of 26-28 hr. All alleles are semidominant. Studies of the genetic interactions of some of the tim alleles with period-altering period (per) mutations indicate that these interactions are close to multiplicative; a given allele changes the period length of the genetic background by a fixed percentage, rather than by a fixed number of hours. The tim(L1) allele was studied in molecular detail. The long behavioral period of tim(L1) is reflected in a lengthened molecular oscillation of per and tim RNA and protein levels. The lengthened period is partly caused by delayed nuclear translocation of TIM(L1) protein, shown directly by immunocytochemistry and indirectly by an analysis of the phase response curve of tim(L1) flies.

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.

The effectiveness of weekly iron supplementation in pregnant women of rural northern Malawi.

A weekly iron/folate supplement was compared with a standard daily iron/folate supplement in pregnant women living in rural Malawi. Women were enrolled as they attended the local antenatal clinic, stratified by grade of anaemia and then randomized to receive either 60 mg iron/0.25 mg folate per day (n = 211) or 120 mg iron/0.50 mg folate once a week (n = 202). Supplementation was continued for a minimum of 8 weeks (10 weeks on average) and was self administered by the women at home. Initial haemoglobin values for the daily (mu = 105.7 g/l) and weekly (mu = 104.4 g/l) groups as well as final haemoglobin values (107.5 g/l and 105.6 g/l, respectively) did not differ significantly between the two groups. Haemoglobin values increased by similar levels in both groups with the subset of anaemic women increasing by an average of 6.3 g/l in the daily group (n = 70) and 5.9 g/l in the weekly group (n = 66) for all women. For compliant, anaemic women, the increases were 7.4 g/l and 6.6 g/l for the daily and weekly groups, respectively. Compliance, as indicated by self reporting and by regular counts of remaining tablets, was significantly higher in the weekly group (76% compared with 60%, P < 0.05), however compliance was identical in both groups when assessed by a stool test for elemental iron. Reported side effects were significantly reduced in the weekly group (6% compared with 17%, P < 0.05). We conclude that a weekly iron supplement given to pregnant women in rural Malawi has similar haematologic effects, and an improved side effect profile, in comparison with a standard daily supplement when administered through an existing primary healthcare programme, although both regimens are relatively unsuccessful in the reduction of anaemia prevalence during pregnancy.

Identification and characterization of a p53 homologue in Drosophila melanogaster.

The tumor suppressor gene p53 in mammalian cells plays a critical role in safeguarding the integrity of genome. It functions as a sequence-specific transcription factor. Upon activation by a variety of cellular stresses, p53 transactivates downstream target genes, through which it regulates cell cycle and apoptosis. However, little is known about p53 in invertebrates. Here we report the identification and characterization of a Drosophila p53 homologue gene, dp53. dp53 encodes a 385-amino acid protein with significant homology to human p53 (hp53) in the region of the DNA-binding domain, and to a lesser extent the tetramerization domain. Purified dp53 DNA-binding domain protein was shown to bind to the consensus hp53-binding site by gel mobility analysis. In transient transfection assays, expression of dp53 in Schneider cells transcriptionally activated promoters that contained consensus hp53-responsive elements. Moreover, a mutant dp53 (Arg-155 to His-155), like its hp53 counterpart mutant, exerted a dominant-negative effect on transactivation. Ectopic expression of dp53 in Drosophila eye disk caused cell death and led to a rough eye phenotype. dp53 is expressed throughout the development of Drosophila with highest expression levels in early embryogenesis, which has a maternal component. Consistent with this, dp53 RNA levels were high in the nurse cells of the ovary. It appears that p53 is structurally and functionally conserved from flies to mammals. Drosophila will provide a useful genetic system to the further study of the p53 network.

Cycling vrille expression is required for a functional Drosophila clock.

We identified a novel regulatory loop within Drosophila's circadian clock. A screen for clock-controlled genes recovered vrille (vri), a transcription factor essential for embryonic development. vri is expressed in circadian pacemaker cells in larval and adult brains. vri RNA levels oscillate with a circadian rhythm. Cycling is directly regulated by the transcription factors dCLOCK and CYCLE, which are also required for oscillations of period and timeless RNA. Eliminating the normal vri cycle suppresses period and timeless expression and causes long-period behavioral rhythms and arrhythmicity, indicating that cycling vri is required for a functional Drosophila clock. We also show that dCLOCK and VRI independently regulate levels of a neuropeptide, pigment dispersing factor, which appears to regulate overt behavior.

Molecular control of circadian behavioral rhythms.

Several genes have been recognized in Drosophila that regulate circadian rhythms. Homologues of these genes have now been found in mice and humans, suggesting a mechanism that is conserved throughout the animal kingdom. For some of these genes and their products, molecular oscillations are produced in certain cells of the Drosophila and mammalian brain. Two genes, period and timeless, are transcribed with a circadian rhythm that is regulated by activities derived from their encoded proteins, PER and TIM. Nuclear localization of these proteins downregulates per and tim transcription by suppressing the activities of two transcription factors, dCLOCK and dBMAL1. Cycles in this feedback regulation are promoted by events that regulate the accumulation, physical interaction, and nuclear translocation of PER and TIM proteins. PER and TIM must physically associate to enter the nucleus and their cytoplasmic interaction is delayed by a kinase encoded by the clock gene, double-time. This kinase directs PER phosphorylation, which leads to PER degradation. Effects of the kinase are blocked once PER is complexed to TIM. These interactions prolong the interval of per and tim transcription by ensuring that PER/TIM complexes from only after TIM has accumulated for several hours.