Innate Immunity in Insects, Function and Regulation of Hemolin from Hyalophora cecropia

Insects are useful models for the study of innate immune reactions and development. The distinction between recognition mechanisms preceding the breakdown of apoptotic cells during metamorphosis, and the breakdown of cells in response to infections, is unclear. Hemolin, a Lepidopteran member of the immunoglobulin superfamily, is a candidate molecule in self/nonself recognition. This thesis investigates hemolin function and hemolin gene regulation at a molecular level. We investigated the binding and cell adhesion properties of hemolin from H. cecropia and demonstrated that the proteins could homodimerize in presence of calcium. Moreover, a higher molecular weight membrane form of hemolin was present on hemocytes. These results, taken together with an earlier finding that soluble hemolin inhibits hemocyte adhesion, indicated that the secreted hemolin could modulate hemocyte aggregation in a competitive manner in the blood. In addition, hemolin was expressed in different tissues and at different developmental stages. Since hemolin is expressed both during development and during the immune response, its different regulatory factors must act in concert. We found that the third intron contains an enhancer, through which Dif, C/EBP and HMGI synergistically activate a reporter construct in vitro. We concluded that the enhancer is used during infection, since the κB-site is crucial for an immune response. Interestingly, we also found that the active form of the steroid hormone, ecdysone, induces the hemolin gene transcription in vivo, and in addition, acts synergistically during bacterial infection. Preliminary in vivo results indicate a secondary effect of ecdysone and the importance of hormone receptor elements in the upstream promoter region of hemolin. To explore the use of Drosophila as a genetic tool for understanding hemolin function and regulation, we sought to isolate the functional homologue in this species. A fly cDNA library in yeast was screened using H. cecropia hemolin as bait. The screen was not successful. However, it did lead to the discovery of a Drosophila protein with true binding specificity for hemolin. Subsequent characterization revealed a new, highly conserved gene, which we named yippee. Yippee is distantly related to zinc finger proteins and represents a novel family of proteins present in numerous eukaryotes, including fungi, plants and humans. Notably, when the Drosophila genome sequence was revealed, no hemolin orthologue could be detected. Finally, an extensive Drosophila genome chip analysis was initiated. The goal was to investigate the Drosophila immune response, and, in contrast to earlier studies of artificially injected flies, to examine a set of natural microbes, orally and externally applied. In parallel experiments viruses, bacteria, fungi and parasites were compared to unchallenged controls. We obtained a unique set of genes that were up-regulated in the response to the parasite Octosporea muscadomesticae and to the fungus Beauveria bassiana. We expect both down-regulated and up-regulated genes to serve as a source for the discovery of new effector molecules, in particular those that are active against parasites and fungi.

Adaptation and Constraint in the Plant Reproductive Phase

Conservatism is a central theme of organismic evolution. Related species share characteristics due to their common ancestry. Some concern have been raised among evolutionary biologists, whether such conservatism is an expression of natural selection or of a constrained ability to adapt. This thesis explores adaptations and constraints within the plant reproductive phase, particularly in relation to the evolution of fleshy fruit types (berries, drupes, etc.) and the seasonal timing of flowering and fruiting. The different studies were arranged along a hierarchy of scale, with general data sets sampled among seed plants at the global scale, through more specific analyses of character evolution within the genus Rhamnus s.l. L. (Rhamnaceae), to descriptive and experimental field studies in a local population of Frangula alnus (Rhamnaceae). Apart from the field study, this thesis is mainly based on comparative methods explicitly incorporating phylogenetic relationships. The comparative study of Rhamnus s.l. species included the reconstruction of phylogenetic hypotheses based on DNA sequences. Among geographically overlapping sister clades, biotic pollination was not correlated with higher species richness when compared to wind pollinated plants. Among woody plants, clades characterized by fleshy fruit types were more species rich than their dry-fruited sister clades, suggesting that the fleshy fruit is a key innovation in woody habitats. Moreover, evolution of fleshy fruits was correlated with a change to more closed (darker) habitats. An independent contrast study within Rhamnus s.l. documented allometric relations between plant and fruit size. As a phylogenetic constraint, allometric effects must be considered weak or non-existent, though, as they did not prevail among different subclades within Rhamnus s.l. Fruit size was correlated with seed size and seed number in F. alnus. This thesis suggests that frugivore selection on fleshy fruit may be important by constraining the upper limits of fruit size, when a plant lineage is colonizing (darker) habitats where larger seed size is adaptive. Phenological correlations with fruit set, dispersal, and seed size in F. alnus, suggested that the evolution of reproductive phenology is constrained by trade-offs and partial interdependences between flowering, fruiting, dispersal, and recruitment phases. Phylogenetic constraints on the evolution of phenology were indicated by a lack of correlation between flowering time and seasonal length within Rhamnus cathartica and F. alnus, respectively. On the other hand, flowering time was correlated with seasonal length among Rhamnus s.l. species. Phenological differences between biotically and wind pollinated angiosperms also suggested adaptive change in reproductive phenology.

Group I Introns and Homing Endonucleases in T-even-like Bacteriophages

Homing endonucleases are rare-cutting enzymes that cleave DNA at a site near their own location, preferentially in alleles lacking the homing endonuclease gene (HEG). By cleaving HEG-less alleles the homing endonuclease can mediate the transfer of its own gene to the cleaved site via a process called homing, involving double strand break repair. Via homing, HEGs are efficiently transferred into new genomes when horizontal exchange of DNA occurs between organisms. Group I introns are intervening sequences that can catalyse their own excision from the unprocessed transcript without the need of any proteins. They are widespread, occurring both in eukaryotes and prokaryotes and in their viruses. Many group I introns encode a HEG within them that confers mobility also to the intron and mediates the combined transfer of the intron/HEG to intronless alleles via homing. Bacteriophage T4 contains three such group I introns and at least 12 freestanding HEGs in its genome. The majority of phages besides T4 do not contain any introns, and freestanding HEGs are also scarcely represented among other phages. In the first paper we looked into why group I introns are so rare in phages related to T4 in spite of the fact that they can spread between phages via homing. We have identified the first phage besides T4 that contains all three T-even introns and also shown that homing of at least one of the introns has occurred recently between some of the phages in Nature. We also show that intron homing can be highly efficient between related phages if two phages infect the same bacterium but that there also exists counteracting mechanisms that can restrict the spread of introns between phages. In the second paper we have looked at how the presence of introns can affect gene expression in the phage. We find that the efficiency of splicing can be affected by variation of translation of the upstream exon for all three introns in T4. Furthermore, we find that splicing is also compromised upon infection of stationary-phase bacteria. This is the first time that the efficiency of self-splicing of group I introns has been coupled to environmental conditions and the potential effect of this on phage viability is discussed. In the third paper we have characterised two novel freestanding homing endonucleases that in some T-even-like phages replace two of the putative HEGs in T4. We also present a new theory on why it is a selective advantage for freestanding, phage homing endonucleases to cleave both HEG-containing and HEG-less genomes.