As a strategy to survive to the upcoming winter, many insects enter diapause (a typical overwintering response that results on their developmental arrest). Drosophila melanogaster undergoes an adult or reproductive diapause that can be easily spotted by looking at the stage of development of the females’ ovaries. The possibility of the circadian clock influencing this phenotype was proposed to explain photoperiodic differences in induction levels. Nevertheless, to the date the debate is still on. In this thesis, I looked at several canonical clock mutants and assessed their impact on diapause, finding that 1) depending on the temperature in which they were reared the effects on the adult flies varied enormously 2) most of the clock mutants gave a strong effect in one or other growing conditions. In particular, Pdf0 and ClkJrk mutants behave in completely opposite ways. A second part of the project consisted on looking at the effects of period temperature-sensitive splicing in diapause. Using splicing locked transgenic flies provided by Isaac Edery, I found that expression of the summer isoform impaired the ability of the flies to undergo diapause. Hence, I cloned the different splicing variants into a pUAST vector and generated UAS lines to perform a neuroanatomical dissection of the phenotype. Also, related with the previous project, I decided to look if any miRNA could be regulating diapause by affecting any of the splicing variants. I found several possible miRNAs that could target the summer (intron-containing) non-splicing isoform. I found that one particular, miRNA-276b, was having a huge effect on diapause. Using a sponge particularly against this miRNA (which would result in its downregulation) diapause levels halved compared to all the controls that were performed in parallel.

The study of breeding system evolution has been prolific given the broad subjects that it embraces, from the social environment and sexual selection to courtship behaviour and parental care. The general objective of this dissertation was to test empirically ideas shown in recent models of breeding system evolution. Using plovers Charadrius spp. as model organisms, which show remarkable variation in breeding systems, I investigate how adult sex ratio (ASR) and operational sex ratio (OSR) are linked together and relate to two major components of breeding systems: courtship behaviour and parental care. Experimental studies have led to equate ASR and OSR, but they are not necessarily the same and the way they correlate in nature is unknown. Here I show that in a wild polygamous plover population these ratios seem to be uncorrelated (Chapter 2). Differences in the social environment and the degree of mating competition between monogamous and polygamous populations could also be reflected in their courtship behaviour; higher courtship rates in a polygamous plover than in a monogamous closely-related plover support this prediction (Chapter 3). OSR may be more variable throughout time than ASR, and therefore be less reliable as a cue to the social environment. In support of this prediction ASR was better than OSR at predicting the duration of female brood care in a polygamous plover where females have variable care (Chapter 4). Sex-specific timing of breeding could differ according to the social environment and the level of mating competition in a population, I compared six populations of five closely-related plover species with varying breeding systems (Chapter 5); only one polygamous population showed sex-specific differences in arrival date and the length of time spent in the breeding grounds. Finally, I discuss the contribution of these results to the understanding of breeding system evolution and suggest potential future lines of research.

No description available.

Flowering plants account for the 30 crops that provide 95 % of the food for humans. The reproduction of this group depends on the production of two twin sperms. The establishment of the male germline lineage requires the transcription factor DUO POLLEN 1 (DUO1). DUO1 is required for both the cell cycle progression and sperm cell differentiation. This thesis focused on the origin of DUO1 and its target regulation. Much work was dedicated in searching the evolutionary origin of DUO1 in the R2R3 MYB clade. Based on the analysis of sequences homologous to DUO1 and its sister clade GAMYB, the earliest DUO1 homolog was identified in the green algae. The DUO1 clade did not proliferate after multiple polyploidy events, possibly restricted by its male germline-specific role supported by transcriptome data. The ancestral DUO1 experienced a major MYB domain sequence change in the bryophytes and a second change in the C-terminus in the angiosperms. The MYB domain changes caused a change in the target DNA sequence, which has then been conserved among Embryophyta DUO1 homologs. Another change also happened in the region where a miR159 binding site is present in most angiosperm DUO1 homologs. Sequence and functional analysis showed that this change evolved long before the emergence of miR159. The changes in the C-terminus of DUO1 led to a higher target promoter activation capability in the angiosperm homologs, which was confirmed by functional tests of the angiosperm and bryophyte DUO1. This C-terminal region contains the transactivation domain (TAD) of DUO1 and certain functionally important motifs were highlighted in the study. While these motifs indicated that DUO1 was a member of a TAD family, it was also demonstrated that unknown sequences carry critical features for activation. Together these results mapped the evolution history of DUO1 in the Streptophyta lineage.

Insects are emerging as a key lineage for the study of epigenetic phenomena. This is due to the variety of polyphenisms found in insects. In this thesis, the caste polymorphism of the buff-tailed bumblebee Bombus terrestris and the phase polymorphism of the desert locust Schistocerca gregaria are studied to elucidate the underlying epigenetic mechanisms. I establish the presence of allele-specific expression and methylation in B. terrestris. I used next-generation RNA-sequencing to establish the DNA methylation, alternative splicing, and gene expression patterns of B. terrestris worker reproduction. The presence of allele-specific methylation and allele-specific expression were then determined in the same context. Correlations with the aforementioned epigenetic mechanisms were drawn. One major finding was that a higher degree of methylation was witnessed in more highly expressed genes. Higher methylation levels were also associated with more differentially expressed genes and isoforms between workers of a different reproductive state. However, the association between allele-specific expression and allele-specific methylation was weak. The relationship between alternative splicing and the circadian clock in S. gregaria was investigated. The first evidence of genes with differential circadian isoform expression patterns is reported. Finally, I analysed whether genome-wide alternative splicing levels are an important component in ascertaining the varying levels of eusociality found in the Hymenoptera. Fewer splicing events per gene with multiple isoforms was found in more highly eusocial species compared with solitary and more primitively eusocial species. Thus this is the first evidence of an association between level of sociality and alternative splicing.

On the assumption that the replication of plasmids is under negative control, an attempt was made to isolate temperature-sensitive mutants of an Flac+ factor having uncontrolled replication. It was anticipated that such mutants would be lethal to the host at 420°C but give a high frequency of Lac revertants due to loss of the F particle. Although several temperature-sensitive mutants were found, none were of this type. In some of the isolated mutants, the reversion frequency was enhanced by the presence of the F particle. On detailed study, one of these mutants proved to have a chromosomal lesion similar to those classified as defective in the initiation of chromosome replication. Known initiation-defective mutants (CRT83 and CRT46) also show an enhanced reversion frequency when infected with F or some F prime factors. In many of the revertants isolated from F+ and Flac+ derivatives of CRT83, the F factor is integrated into the chromosome. Furthermore, such revertants still carry the temperature-sensitive mutation, showing that the reversion is by suppression. It is supposed that in CRT83 and CRT46, the F replication is not temperature-sensitive and when the F particle is integrated into the chromosome the whole genome can replicate at non-permissive temperature under the control of the F replication system. However, the integration of the F particle into the chromosome alone is apparently not sufficient for the suppression of temperature-sensitivity. Studies of DNA synthesis of F+, Flac+ and Hfr derivatives of CRT46 and CRT83 showed that the temperature-sensitive defect of these mutants can be complemented partially by the F factor. Several mating experiments involving CRT83 and CRT46 showed that these mutants carry an additional mutation, mapping near ilv which renders the cells partially defective in recombinant production.

Mutants of A. nidulans which grow normally at 37°C but not at 20°C have been isolated. They are designated cs (cold-sensitive). Growth tests of 75 cs mutants demonstrate that they have a range of defects. Genetic analysis of some of these strains shows that mutation to cold-sensitivity can arise in many loci and affect many functions. Two of the cs mutants (cs 13 and cs 48) have a specific nutritional requirement for growth at 20°C (isoleucine and choline respectively). One strain, cs 67, exhibits extranuclear inheritance of cold-sensitivity. After growth at 37°C followed by incubation at 20°C, four of the cs mutants produce altered ribosome sedimentation profiles. These strains are designated arp and all four produce profiles containing a reduced ratio of the amounts of large to small ribosomal subunits. In at least three of the arp strains the altered profile results from decreased production of large ribosomal subunits at 20°C. In each of the arp strains there is a single mutation which determines both cold-sensitivity and production of an altered ribosome profile. These four mutations define three loci, one of which (arp A) is in linkage group VII. The other two loci (arp B and arp C) are in linkage group VIII but are not closely linked. Both mutations at the arp A locus also confer resistance to a high concentration of actidione. Other mutants were selected for actidione-resistance and their analysis has identified a further six loci which determine resistance to this drug. The properties of the arp strains are compared with those of mutants affecting ribosome synthesis in bacteria and in other eukaryotes.

The release of a haemolytic toxin from some urinopathogenic strains of E. coli is unique in that it is the only reported polypeptide to be truly secreted from this Gram-negative organism. This secretion system comprises four genes on a contiguous, approximately 7.5Kb DNA fragment, encoding the toxin itself (HlyA, 107KD), two membrane localised export proteins (HlyB,D) and the cytoplasmic HlyC protein, whose only apparent function is to post-translationally activate the toxin. Recently, another unlinked gene, tolC, encoding a minor outer membrane protein has also been reported to be required for the export process together with HlyB,D. A novel feature of this system is the presence of a C-terminal targeting signal to direct HlyA from the cell. In this study I show that the HlyA C-terminal targeting signal can be harnessed to secrete the majority of both the mammalian prochymosin and the E. coli cytoplasmic, LacZ, proteins in an HlyB,D dependent manner. I have also shown that the efficiency of secretion dramatically decreases when either the C-terminal domain is reduced in size, from 23KD to 4KD, or as the "passenger" domain increases in size. These results suggest that the HlyA signal domain is composed of sequences required for both efficiency of secretion and targeting and that the secretion process is inhibited by heterologous passenger domains, the effect increasing with size presumably due to the adoption of more stable folded conformations. This study has also been concerned with the investigation into the nature of the novel hlyA targeting signal by deploying a series of in vitro mutagenesis methods (hydroxylamine, site directed and "saturation"), to introduce point mutations. This work has generated a bank of mutants, the analysis of which, has highlighted several residues essential for efficient secretion and also indicated a minimal region containing the signal motif. However, this information together with comparisons with other molecules carrying similar targeting signals have not yet identified a common signal motif.

The aim of the project was to investigate expression of the plasmodial-specific gene, P46 (Sweeney et al., 1987), during the apogamic amoebal-plasmodial transition of Physarum polycephalum. The intention was to establish the onset of transcription of the P46 gene in relation to the extended cell cycle that occurs during development (Bailey et al., 1987), and also in relation to commitment to plasmodial development. Initially, it was intended to use in situ hybridization to analyse expression of the P46 gene in individual developing cells. This method proved to be insufficiently sensitive for this purpose, and P46 expression was consequently investigated using isolated RNA. Following hybridization experiments to establish an appropriate method for the quantitative analysis of gene expression during development, expression of the P46 gene was investigated by northern analysis of RNA isolated from developing cells. It was possible to conclude from the results that expression of P46 begins in uninucleate cells that have become committed to plasmodial development. The hybridization assay was insufficiently sensitive to establish the onset of transcription more precisely, and the polymerase chain reaction (PCR) was used in subsequent experiments, to amplify P46 transcripts prior to their detection with radioactively-labelled probes. Using the PCR it was possible to conclude that P46 expression begins within a few hours of commitment. Amplification and sequence analysis of genomic DNA revealed an intron, of 182 base pairs in length, within the P46 gene. Amplification of amoebal RNA generated a fragment of a size corresponding to the genomic P46 fragment, suggesting unprocessed P46 transcripts are transcribed in amoebae.

The MYO1 gene (5.55 kb) from Saccharomyces cerevisiae has been completely sequenced. The predicted polypeptide has a molecular mass of 213 kDa and is shown to be a type II myosin. The N-terminal domain displays high sequence similarity to those of other myosins, whereas the C-terminal region contains the seven and twenty eight residue repeating motifs characteristic of type II myosins. Six proline residues are located within the tail region, at approximately two thirds from the start of the tail. Gene disruption has been used to investigate the biological function of a 4kDa C-terminal fragment of the yeast myosin. Disruption of MYO1 by the URA3 gene gave rise to cells defective in both cytokinesis and nuclear migration, similar to the null phenotype. When the MYO1 gene was disrupted at the same location with the TRP1 gene, the resulting cells displayed no defect in either cytokinesis or nuclear migration. It is predicted that 16 residues in the TRP1 sequence form a fusion protein with the myosin which restores its activity. Over-expression of the MYO1 gene product from a galactose inducible promoter is shown to be lethal. The purified yeast myosin, visualised by electron microscopy, consists of two globular head regions attached to an extended tail, similar to mammalian type II myosins.