Ezio Rosato

Natural Selection Favors a Newly Derived timeless Allele in Drosophila melanogaster

Circadian and other natural clock-like endogenous rhythms may have evolved to anticipate regular temporal changes in the environment. We report that a mutation in the circadian clock gene timeless in Drosophila melanogaster has arisen and spread by natural selection relatively recently in Europe. We found that, when introduced into different genetic backgrounds, natural and artificial alleles of the timeless gene affect the incidence of diapause in response to changes in light and temperature. The natural mutant allele alters an important life history trait that may enhance the flys adaptation to seasonal conditions.

Light-dependent interaction between Drosophila CRY and the clock protein PER mediated by the carboxy terminus of CRY

BACKGROUND The biological clock synchronizes the organism with the environment, responding to changes in light and temperature. Drosophila CRYPTOCHROME (CRY), a putative circadian photoreceptor, has previously been reported to interact with the clock protein TIMELESS (TIM) in a light-dependent manner. Although TIM dimerizes with PERIOD (PER), no association between CRY and PER has previously been revealed, and aspects of the light dependence of the TIM/CRY interaction are still unclear. RESULTS Behavioral analysis of double mutants of per and cry suggested a genetic interaction between the two loci. To investigate whether this was reflected in a physical interaction, we employed a yeast-two-hybrid system that revealed a dimerization between PER and CRY. This was further supported by a coimmunoprecipitation assay in tissue culture cells. We also show that the light-dependent nuclear interactions of PER and TIM with CRY require the C terminus of CRY and may involve a trans-acting repressor. CONCLUSIONS This study shows that, as in mammals, Drosophila CRY interacts with PER, and, as in plants, the C terminus of CRY is involved in mediating light responses. A model for the light dependence of CRY is discussed.

A Molecular Basis for Natural Selection at the timeless Locus in Drosophila melanogaster

Diapause is a protective response to unfavorable environments that results in a suspension of insect development and is most often associated with the onset of winter. The ls-tim mutation in the Drosophila melanogaster clock gene timeless has spread in Europe over the past 10,000 years, possibly because it enhances diapause. We show that the mutant allele attenuates the photosensitivity of the circadian clock and causes decreased dimerization of the mutant TIMELESS protein isoform to CRYPTOCHROME, the circadian photoreceptor. This interaction results in a more stable TIMELESS product. These findings reveal a molecular link between diapause and circadian photoreception.

Seasonal behavior in Drosophila melanogaster requires the photoreceptors, the circadian clock, and phospholipase C

Drosophila melanogaster locomotor activity responds to different seasonal conditions by thermosensitive regulation of splicing of a 3′ intron in the period mRNA transcript. Here we demonstrate that the control of locomotor patterns by this mechanism is primarily light-dependent at low temperatures. At warmer temperatures, when it is vitally important for the fly to avoid midday desiccation, more stringent regulation of splicing is observed, requiring the light input received through the visual system during the day and the circadian clock at night. During the course of this study, we observed that a mutation in the no-receptor-potential-AP41 (norpAP41) gene, which encodes phospholipase-C, generated an extremely high level of 3′ splicing. This cannot be explained simply by the mutations effect on the visual pathway and suggests that norpAP41 is directly involved in thermosensitivity.

Analysis of locomotor activity rhythms in Drosophila

The genetic, molecular and anatomical dissection of the circadian clock in Drosophila and other higher organisms relies on the quantification of rhythmic phenotypes. Here, we introduce the methods currently in use in our laboratories for the analysis of fly locomotor activity rhythms. This phenotype provides a relatively simple, automated, efficient, reliable and robust output for the circadian clock. Thus it is not surprising that it is the preferred readout for measuring rhythmicity under a variety of conditions for most fly clock laboratories. The procedure requires at least 10 days of data collection and several days for analysis. In this protocol we advise on fly maintenance and on experimental design when studying the genetics of behavioral traits. We describe the setup for studying locomotor activity rhythms in the fruit fly and we introduce the statistical methods in use in our laboratories for the analysis of periodic data.

Comparative analysis of circadian clock genes in insects

After a slow start, the comparative analysis of clock genes in insects has developed into a mature area of study in recent years. Brain transplant or surgical interventions in larger insects defined much of the early work in this area, before the cloning of clock genes became possible. We discuss the evolution of clock genes, their key sequence differences, and their likely modes of regulation in several different insect orders. We also present their expression patterns in the brain, focusing particularly on Diptera, Lepidoptera, and Orthoptera, the most common non‐genetic model insects studied. We also highlight the adaptive involvement of clock molecules in other complex phenotypes which require biological timing, such as social behaviour, diapause and migration.

A constitutively active cryptochrome in Drosophila melanogaster

Light-activated cryptochrome (CRY) regulates circadian photoresponses in Drosophila melanogaster. Removing the carboxy (C) terminus to create CRYΔ produces, in yeast, a light-independent, constitutively active form. Here we show that flies overexpressing CRYΔ have a longer free-running period of locomotor activity, as well as altered cycling kinetics of the clock proteins timeless (TIM) and period (PER). Moreover, at the cellular level, they show a reduction in the level of TIM and in the nuclear localization of TIM and PER in two significant clusters of behavioral pacemaker cells: the large and the small ventral lateral neurons (LNvs). These effects are similar to those seen in wild-type flies under continuous light and suggest a regulatory role for the C terminus of CRY on the photosensitive, photolyase-like part of the protein.

A second timeless gene in Drosophila shares greater sequence similarity with mammalian tim

R.C. and C.P.K. were supported by grants from the European Community and CRUI-MURST-British Council, R.C. by grants from MURST-progetti nazionali and Ministero per le Politiche Agricole, and E.R. by a David Phillips Fellowship from BBSRC.

The circadian clock of the fly: a neurogenetics journey through time.

Forty years ago, a mutagenesis screening in the fruit fly, Drosophila melanogaster, led to the discovery of period, the first gene to be involved in the endogenous 24-h rhythmicity of an organism. Since then circadian clocks have been identified in fungi, cyanobacteria, plants, and other animals. Although the molecular components are not conserved across the main divisions of life, it appears that in every organism, a common design, based upon a transcription-translation feedback loop (TTL), is in place to regulate endogenous 24 h cycles. The TTL model has informed chronobiology research for the majority of the past 30 years with spectacular results. However, new evidence and the rediscovery of old observations suggest that this model is coming to age. Here, we provide a comprehensive review of the current TTL model in Drosophila highlighting its accomplishments and its limitations. We conclude by offering our personal view on the organization and the evolution of circadian clocks.

The period Gene Thr-Gly Polymorphism in Australian and African Drosophila melanogaster Populations: Implications for Selection

The period gene is a key regulator of biological rhythmicity in Drosophila melanogaster. The central part of the gene encodes a dipeptide Thr-Gly repeat that has been implicated in the evolution of both circadian and ultradian rhythms. We have previously observed that length variation in the repeat follows a latitudinal cline in Europe and North Africa, so we have sought to extend this observation to the southern hemisphere. We observe a parallel cline in Australia for one of the two major length variants and find higher levels of some Thr-Gly length variants, particularly at the tropical latitudes, that are extremely rare in Europe. In addition we examined >40 haplotypes from sub-Saharan Africa and find a very different and far more variable profile of Thr-Gly sequences. Statistical analysis of the periodicity and codon content of the repeat from all three continents reveals a possible mechanism that may explain how the repeat initially arose in the ancestors of the D. melanogaster subgroup of species. Our results further reinforce the view that thermal selection may have contributed to shaping the continental patterns of Thr-Gly variability.