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Innate Immunity in Insects, Function and Regulation of Hemolin from <i>Hyalophora cecropia</i>

<p>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 <i>Lepidopteran</i> member of the immunoglobulin superfamily, is a candidate molecule in self/nonself recognition. This thesis investigates hemolin function and <i>hemolin </i>gene regulation at a molecular level.</p><p>We investigated the binding and cell adhesion properties of hemolin from <i>H. cecropia </i>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.</p><p>Since <i>hemolin</i> 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 <i>in vitro</i>. 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 <i>hemolin</i> gene transcription <i>in vivo</i>, and in addition, acts synergistically during bacterial infection. Preliminary <i>in vivo</i> results indicate a secondary effect of ecdysone and the importance of hormone receptor elements in the upstream promoter region of <i>hemolin</i>.</p><p>To explore the use of <i>Drosophila</i> 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 <i>H. cecropia</i> hemolin as bait. The screen was not successful. However, it did lead to the discovery of a <i>Drosophila</i> protein with true binding specificity for hemolin. Subsequent characterization revealed a new, highly conserved gene, which we named <i>yippee</i>. 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 <i>Drosophila</i> genome sequence was revealed, no hemolin orthologue could be detected.</p><p>Finally, an extensive <i>Drosophila</i> genome chip analysis was initiated. The goal was to investigate the<i> Drosophila</i> 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 <i>Octosporea muscadomesticae</i> and to the fungus <i>Beauveria bassiana</i>. 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.</p>

Identiferoai:union.ndltd.org:UPSALLA/oai:DiVA.org:su-3
Date January 2001
CreatorsRoxström-Lindquist, Katarina
PublisherStockholm University, Stockholm University
Source SetsDiVA Archive at Upsalla University
LanguageEnglish
Detected LanguageEnglish
TypeDoctoral thesis, monograph, text

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