Many higher animals have evolved the ability to use the Earth’s magnetic field, particularly for orientation. However, the biophysical mechanism by which magnetoreception is achieved remains elusive. One theoretical model (the radical pair mechanism - RPM) proposes that the geomagnetic field is perceived by chemical reactions involving the blue-light photoreceptor Cryptochrome (CRY). Recent evidence supports the RPM in Drosophila melanogaster and reveals a mechanistic link with the circadian clock. Here I have confirmed, albeit with rather different results, that a low frequency electromagnetic field (AC-EMF) along with a Static Field (SF) exposure does affect circadian and activity behaviour in the fruit fly. Furthermore, I have developed two new assays to investigate the effects of EMF in Drosophila melanogaster, negative geotaxis and an additional light wavelength preference assay, revealing a net CRY-dependent response. My data support the idea of CRY mediated magnetoreception, thereby indirectly supporting the RPM. Furthermore, I provide some striking new results that challenge our view that only the canonical clock neurons contribute to behavioural rhythms in Drosophila melanogaster.

Centrosomes are the major microtubule (MT) organising centres in most animal cells, and they play important roles in many cellular processes. Centrosomes comprise a pair of centrioles surrounded by a matrix of proteins known as the pericentriolar material (PCM). Centrosomes assemble around the mother centriole when the PCM is recruited to nucleate and organise the MTs. For decades, the PCM has been viewed as an amorphous cloud of proteins. Although there are hundreds of PCM proteins, a small number of these are thought to assemble an underlying PCM scaffold that extends around the mitotic centriole and subsequently recruits other mitotic PCM proteins. In flies, Centrosomin (Cnn) and Spindle-defective protein 2 (Spd-2) are such essential proteins, and Asterless (Asl) helps recruit these proteins to the mother centriole. However, it is unclear how these proteins assemble into a scaffold at the centrosome, and none of these core PCM assembly proteins have been characterised at a structural level. In this thesis, I have identified and recombinantly expressed functional domains of Cnn, Spd-2 and Asl in order to characterise them in vitro. In collaboration with colleagues in the laboratory, the biological significance of these domains has been investigated in vivo. I define a Phospho-Regulated Multimerisation (PReM) domain within Cnn that is phosphorylated by Polo kinase in vitro, that dimerises via a leucine zipper, and that can form higher order oligomers when phospho-mimicking mutations are incorporated into the protein in vitro. In vivo, forms of Cnn that cannot be phosphorylated in the PReM domain cannot efficiently assemble a Cnn scaffold, while phospho-mimicking forms can spontaneously assemble into scaffold structures. I show that the Centrosomin-Motif-2 (CM2) domain of Cnn is also required to promote Cnn scaffold assembly. The crystal structure of CM2 reveals an unusual asymmetric dimer that further assembles into a tetramer in crystallo. Strikingly, Cnn-CM2 demonstrates a zinc-binding activity that stabilises the dimer, and preliminary in vivo studies indicate that the tetramer interface is essential for Cnn scaffold assembly in vivo. Finally, I show that Cnn-CM2 exhibits a weak interaction with the first of three ASPM, Spd-2, Hydin (ASH) domains in Spd-2, and that this domain facilitates Spd-2 expansion into the PCM region. The structure of two of the Spd-2 ASH domains has been investigated using X-ray crystallography and Nuclear Magnetic Resonance spectroscopy, revealing an Immunoglobulin (Ig)-like protein fold. These studies provide the first atomic insights into the molecular mechanism underlying mitotic PCM assembly, and reveal how the assembly of a Cnn scaffold may be controlled to ensure robust centrosome assembly in vivo.

Drosophila melanogaster lovesong is a genetically programmed behaviour that is important because of its role in mate recognition. Previous work on D. melanogaster lovesong identified 10 genes and several general areas within the neural tissue that influenced song production. The aims of this project were to identify more genes and more specific neurons involved in lovesong production. This study describes the use of the GAL4 enhancer trap technology to identify genes and neurons involved in the production of D. melanogaster lovesong. The initial screen made use of the binary nature of the system to test the songs of the enhancer trap lines themselves and of flies expressing transformer or tetanus toxin in subsets of cells defined by the enhancers. Defects were found in both the number of cycles per pulse and the inter pulse interval. Lines picked up in the screen were used to drive the expression of GFP to allow identification of the cells in which the enhancers were active. For the most interesting lines, the surrounding DNA was rescued and candidate genes identified that may have had an influence on song. Mutants of these genes were obtained and tested for their abilities to sing. Ultimately, several brain structures were identified that appear to influence song production and ten genes were identified that had mutants with song defects. Some of these produced aberrant song when the enhancer trap line was placed over a deletion, some when the mutations were placed over a deletion, some when the mutations were placed over the enhancer trap lines and some when tested directly as homozygotes or hemizygotes. Two of these genes, argos and elav, have a known interaction and several of the candidate genes were known to interact with Ras85D.

The circadian central oscillator of Drosophila melanogaster consists of at least two interlocked negative transcriptional feedback loops. This has been taken to be a general model for higher eukaryotes with the core components conserved but their regulation altered. The work presented here indicates that in Musca domestica, a dipteran closely related to Drosophila, one of these regulatory loops, involving PERIOD (PER) and TIMELESS (TIM), functions in a completely different manner. This study shows that in contrast to Drosophila, Musca PER remains constant in western studies in any lighting condition, whereas like Drosophila TIM cycles in both LD and DD and is constantly degraded in LL. In addition within the central brain immunostaining revealed that even in the small set of cells thought to contain the central pacemaker PER staining was restricted exclusively to the cytoplasm. However following the Drosophila model PER was observed to cycle in the cytoplasm of these cells. Although TIM co-localises with PER in these cells, unlike PER, TIM does become nuclear. This indicates that the negative feedback model illustrated by analysis of the Drosophila is inadequate to explain clock function in Musca. A putative Musca PER nuclear export sequence which functions in other species was tested in GFP constructs but not shown to be involved in altered localisation. In contrast in peripheral tissue such as photoreceptor cells both PER and TIM cycle and both proteins become nuclear late at night as in Drosophila. Stability of Musca PER in LL and an altered relationship between transgenic Musca PER and Drosophila DOUBLETIME indicates an altered relationship between PER and the DBT kinase that may be responsible for PER stability. Thus although it can be seen that a different model is required for other insect species how these proteins act remains to be elucidated.

Development can be described as a network of genetic interactions. These interactions can be highly conserved over large evolutionary distances or they can vary between closely related species resulting in morphological differences. What are the forces promoting change in such interactions and how do they evolve The interaction between the transcription factor Bicoid (Bed) and the hunchback (hb) promoter was compared between M. domestica and D. melanogaster (Bonneton et al., 1997 Shaw etal., 2002). This interaction is conserved in function despite differences in both the Bed homeodomain and the hb promoter sequences. Functional tests of the components of this interaction suggest that they are co-evolving in each species to maintain function in spite of sequence divergence. In this thesis, two further Bed regulated genes, tailless (til) and caudal (cad), were studied to provide a comparison to the Bcd-hb promoter interaction and to investigate the consequences of regulatory sequence change within a network of interactions. The til promoter sequences are unalignable between M. domestica and D. melanogaster yet are similar in function. As with the Bed-hb promoter interaction functional tests indicate that the interaction is diverging between the species at the molecular level. The interaction between Bed and the cad mRNA was also investigated. cad sequence and expression data indicate that the function and regulation of cad is conserved between M. domestica and D. melanogaster. However, the M. domestica cad 3' regulatory sequence is unalignable with that of D. melanogaster. In conclusion, the Bed-dependent regulatory sequences are evolving relatively quickly but all indications are that function is conserved. This raises questions about the structure of regulatory sequences, the flexibility of non-coding regulatory sequences to change and the evolution of interactions and of non-coding regions in general.

No description available.

No description available.

The nonA gene of Drosophila melanogaster influences vision, courtship song and viability, and encodes a protein inferred to bind single-stranded nucleic acids. This study describes the molecular and functional characterisation of the nonA gene of Drosophila virilis. The main goal of the project was to establish whether the D. virilis nonA gene rescued the viability and behavioural defects of nonA(-) mutants, and whether it carried species-specific information concerning the lovesong. The D. virilis nonA gene was cloned and sequenced. The overall structure of the D. virilis nonA gene was similar to that of D. melanogaster. Nucleotide and amino acid sequence comparisons revealed a highly diverged and repetitive N-terminus, followed by a conserved C-terminal region. Study of the promoter region highlighted islands of homology containing putative transcription factor binding sites. In addition, the D. virilis gene was found to display an unusual codon usage bias in comparison to other D. virilis or D. melanogaster genes. Finally, protein secondary structure predictions revealed differences between the D. virilis and D. melanogaster polypeptides. P element-mediated transformation was used to assess the ability of the D. virilis nonA clone to rescue viability, visual and song defects of D. melanogaster flies lacking nonA (nonA(-)) and an adjacent and partially overlapping lethal gene l(l)il9e. Complete rescue of the viability indicated that the transgene contained an entire functional D. virilis l(l)il9e gene. Visual studies assessing the transformant flies' optomotor response suggested that the D. virilis transgene completely rescued this phenotype. Lovesong analysis initially presented difficulties in distinguishing between incomplete rescue or transfer of species-species song characteristics. However, extensive statistical analyses suggested that some D. virilis song traits may be conveyed to transformants by the D. virilis nonA gene, but the large differences between the D. virilis and D. melanogaster lovesongs are probably due to the influence of many other genes.

The period gene (per) of Drosophila melanogaster, which lies at the core of the fly's circadian clock, controls a number of biological rhythms, including the circadian periodicity of locomotor activity and adult emergence from the pupal case. Levels of per gene products cycle with a 24 hour period, as required for a clock molecule. Furthermore, a temporal delay between the peak expression of per mRNA and protein suggests the existence of a negative feedback loop through which Per protein regulates the synthesis of its own mRNA. In an attempt to determine the extent to which clock molecules and mechanisms are conserved among dipterans, I cloned the per homologue from the housefly, Musca domestica. The Musca per gene encodes a protein of 1072 amino acids in which areas of high similarity with Drosophila per are interspersed by non-conserved stretches. Areas of high conservation include the amino terminus, the PAS domain and the region surrounding the per5 mutation site of D. melanogaster. Successively I assessed Musca per functionality in the D. melanogaster circadian machinery. Behavioural analysis of transgenic per0 fruit flies expressing the Musca per homologue, demonstrates that the housefly per is able to replace endogenous per functions in the host's clock system. Expression of per products was investigated in both housefly and transgenic fruitfly. Housefly per transcript levels display daily changes in abundance, similar to those observed in wild-type D. melanogaster. Circadian oscillations were also found in per levels in D. melanogaster transformants, albeit with a lower amplitude than those described for wild type. Expression of Per protein in both housefly and D. melanogaster transformants does not show the daily fluctuation characteristic of wild type D. melanogaster Per. This comparative study suggests that the current model based on the negative feedback loop may be inadequate to explain the molecular mechanism underlying the circadian clock.

The period (per)gene of Drosophila encodes a fundamental part of the circadian clock. The PER protein interacts with other proteins in a negative feedback loop which includes cycling of PER and other clock proteins. These proteins subsequently act as transcriptional regulators of other proteins in the loop. It is assumed therefore that they also act at the transcriptional level to affect the production of downstream genes which then control rhythmic phenotypes such as circadian activity patterns, pupal eclosion and lovesong cycle. The work presented here concentrates on a polymorphic '(Thr-Gly)' region of per, part of which encodes for a series of threonine-glycine (Thr-Gly) repeats. Clock genes from orders as diverse as mammals, fungi and cyanobacteria have identifiable (Thr-Gly) regions albeit encoding for smaller (Thr-Gly) repeats, therefore the possibility exists that the region has some fundamental importance to clock function. The role of the repeat and its interaction with sequences immediately 5' to it, is investigated with D.psuedoobscural/D.melanogaster interspecific chimaeric per transgenes. These molecular manipulations reveal a 'coevolved' functional unit which consists of the repeat and the approximately 60 amino acid upstream flanking sequence. Using these interspecific chimaeric per genes also allowed the subsequent mapping of species-specific behaviour differences between D.pseudoobscura and D.melanogaster to discrete regions of the per gene. The role of the polymorphic (Thr-Gly) repeat in temperature compensation (the process whereby clock function is maintained over a range of temperatures) is investigated with a series of (Thr-Gly) invitro mutated transgenes, and the results suggest a selective explanation for the latitudinal cline in (Thr-Gly) length variations in European populations of D.melanogaster. These (Thr-Gly) variants are also shown to affect the behavioural patterns displayed in light/dark cycles.