Present in almost all living organisms, cytochrome P450s form one of the biggest enzyme superfamilies. They are versatile biocatalysts, capable of performing a range of biochemical reactions and are involved in a wide spectrum of biological functions. The vinegar fly, Drosophila melanogaster, has 85 P450s in its sequenced genome. Six of these have been found to catalyse the synthesis of the important insect molting hormone, 20-hydroxyecdysone and a handful have been implicated in insecticide resistance. The other P450s remained largely uncharacterised. / In the first half of this thesis, the expression patterns of P450s in the D. melanogaster genome were characterised by in situ hybridisation at the third instar larval stage. Most P450s have defined expression patterns at this stage of development. A majority of P450s are expressed in the midgut, Malpighian tubules and fat body, tissues that are involved in the metabolism of xenobiotics. Other P450s are expressed in specific tissues, such as the prothoracic glands, the salivary glands and the gonads, where they might have roles in development or reproduction. In particular, Cyp6g2 is expressed in the corpus allatum (CA), where it could play a role in juvenile hormone synthesis. An RNAi lethality screen using lines that were available from the Vienna Drosophila RNAi Centre identified a number of P450s which are essential for development and viability. / In the second half of the thesis, the transcriptional regulation of a P450 involved in insecticide resistance, Cyp6g1, was investigated. Cyp6g1 was regulated by two discrete cis-regulatory modules/enhancers, one controlling expression in the Malpighian tubules and one controlling expression in the midgut and fat body. Phenobarbital induction of Cyp6g1 is tissue-specific and is mediated by a fragment in the 5’ regulatory region that interacts with both enhancers. Characterisation of the long terminal repeat (LTR) of the Accord transposable element in the 5’ region of Cyp6g1, present in insecticide resistant populations, shows that the Accord LTR contains cis-regulatory elements which increase expression of Cyp6g1 in the fat body, midgut and Malpighian tubules, and contribute to insecticide resistance in these populations. / This study shows that the diverse tissue distribution of different P450s in D. melanogaster is related to the diverse biological functions of the enzymes encoded. This is exemplified by the detailed examination of the regulation of the insecticide resistance-conferring P450, Cyp6g1. Its expression pattern reflects its detoxification function in the fly. The role of transposable element insertions in changing gene expression patterns and contributing to selectable variation in genomes is also demonstrated through the Cyp6g1 study.

Sucrose synthase (Susy) is a key enzyme of sucrose metabolism, catalysing the reversible conversion of sucrose and UDP to UDP-glucose and fructose. Therefore, its activity, localization and function have been studied in various plant species. It has been shown that Susy can play a role in supplying energy in companion cells for phloem loading (Fu and Park, 1995), provides substrates for starch synthesis (Zrenner et al., 1995), and supplies UDP-glucose for cell wall synthesis (Haigler et al., 2001). Analysis of the Arabidopsis genome identifies six Susy isoforms. The expression of these isoforms was investigated using promoter-reporter gene constructs (GUS) and real time RT-PCR. Although these isoforms are closely related at the protein level they have radically different spatial and temporal patterns of expression in the plant with no two isoforms showing the same distribution. More than one isoform is expressed in all organs examined. Some of them have high but specific expression in particular organs or developmental stages whilst others are constantly expressed throughout the whole plant and across various stages of development. The in planta function of the six Susy isoforms were explored through analysis of T-DNA insertion mutants and RNAi lines. Plants without the expression of individual isoforms show no differences in growth and development, and are not significantly different from wild type plants in soluble sugars, starch and cellulose contents under all growth conditions investigated. Analysis of T-DNA insertion mutant lacking Sus3 isoform that was exclusively expressed in stomata cells only had a minor influence on guard cell osmoregulation and/or bioenergetics. Although none of the sucrose synthases appear to be essential for normal growth under our standard growth conditions, they may be necessary for growth under stress conditions. Different isoforms of sucrose synthase respond differently to various abiotic stresses. It has been shown that oxygen deprivation up regulates Sus1 and Sus4 and increases total Susy activity. However, the analysis of the plants with reduced expression of both Sus1 and Sus4 revealed no obvious effects on plant performance under oxygen deprivation. Low temperature up regulates Sus1 expression but the loss of this isoform has no effect on the freezing tolerance of non acclimated and cold acclimated plants. These data provide a comprehensive overview of the expression of this gene family which supports some of the previously reported roles for Susy and indicates the involvement of specific isoforms in metabolism and/or signalling. / Saccharose spielt eine zentrale Rolle in höheren Pflanzen. Es zählt zu den wichtigsten Kohlenhydraten und wird als Nährstoff, Speicherstoff (z.B. in Zuckerrüben, Zuckerrohr, Mohrrüben) oder auch als potentielles Signalmolekül verwendet. Saccharose ist eines der primären Endprodukte der Photosynthese in den grünen Blättern der Pflanzen, kann aber auch in nicht-photosynthetisch aktiven Geweben (z.B. in keimenden Samen) synthetisiert und verstoffwechselt werden. Die Saccharosesynthase (Susy) stellt ein Schlüsselenzym im Saccharosestoffwechsel dar. Es katalysiert die reversible Umwandlung von Saccharose zu UDP-Glukose und Fruktose. Die Aktivität, die Lokalisierung und die Funktionen der Susy wurden bereits in verschiedenen Pflanzenarten untersucht. Dabei hatte sich herausgestellt, daß die Susy eine wichtige Rolle in der Bereitstellung von Energie für Transportprozesse spielt. Außerdem stellt Susy die Substrate für die Stärkesynthese in Speichergeweben, sowie fast alle Substrate für die Zellwandsynthese bereit. Eine Untersuchung des Genoms von Arabidopsis thaliana ergab, daß die Ackerschmalwand sechs Isoformen der Susy besitzt. Die Expression dieser Isoformen wurde mittels Echtzeit RT-PCR analysiert. Obwohl die verschiedenen Isoformen auf Proteinebene in ihrer Sequenz sehr ähnlich sind, zeigen sie Unterschiede in ihrem zeitlichen und räumlichen Auftreten innerhalb der Pflanze. Einige der Isoformen sind hoch exprimiert in speziellen Organen oder Entwicklungsstufen der Pflanze. Andere hingegen sind gleichmäßig in der ganzen Pflanze und über verschiedene Entwicklungsstufen hinaus exprimiert. In allen untersuchten Organen der Pflanze ist mehr als eine Isoform exprimiert. Um die spezifische Funktion der einzelnen Isoformen aufzuklären, wurden für alle sechs Saccharosesynthasen Mutanten-Linien isoliert und analysiert. Alle Pflanzen, bei denen die Expression einer bestimmten Isoform fehlte, zeigten im Vergleich zu Wildtyppflanzen keine signifikanten Unterschiede in Wachstum und Entwicklung. Des Weiteren waren die Gehalte an Stärke, Saccharose und Zellulose in Blättern und Wurzeln im Vergleich zu Wildtyppflanzen unverändert. Mutanten, denen die ausschließlich in Schließzellen lokalisierte Isoform Sus3 fehlte, zeigten nur geringe Veränderungen in der Osmoregulation und/oder der Bioenergetik der Schließzellen. Daraus kann gefolgert werden, dass in dem Ackerunkraut Arabidopsis keine der Saccharosesynthasen essentiell für normales Wachstum unter Standardbedingungen ist. Es ist jedoch möglich, dass Saccharosesynthasen unter Stressbedingungen benötigt werden. Es war bereits bekannt, dass einzelne Isoformen der Susy auf Stress reagieren und in ihrer Expression verändert sind. Es konnte gezeigt werden, daß Sauerstoffmangel zu einer Erhöhung der Expression der Isoformen Sus1 und Sus4 und zu einer Zunahme der Susy Gesamtaktivität führt. Die Analyse von Pflanzen mit reduzierter Expression von Sus1 und Sus4 zeigte jedoch, dass Sauerstoffmangel keinen offensichtlichen Einfluss auf das Wachstum dieser Pflanzen hat. Niedrige Temperaturen führen zu einer Erhöhung der Sus1 Expression, aber auch ein Verlust dieser Isoform hat keinen Einfluss auf die Gefriertoleranz von normalen oder an Kälte akklimatisierten Pflanzen. Diese Ergebnisse bieten einen umfassenden Einblick in die Expression der Genfamilie der Saccharosesynthase; sie untermauern die genannten Funktionen der Saccharosesynthase und weisen auf eine mögliche Beteiligung mehrerer Isoformen am Saccharosestoffwechsel und/oder der Signaltransduktion hin.

We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.

We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.

Antifreeze proteins (AFPs) protect marine teleosts from freezing in icy seawater by binding to nascent ice crystals and preventing their growth. It has been suggested that the gene dosage for AFPs in fish reflects the degree of exposure to harsh winter climates. The starry flounder, _Platichthys stellatus_, has been chosen to examine this relationship because it inhabits a range of the Pacific coast from California to the Arctic. This flatfish is presumed to produce type I AFP, which is an alanine-rich, amphipathic alpha-helix. Genomic DNA from four starry flounder was Southern blotted and probed with a cDNA of a winter flounder liver AFP. The hybridization signal was consistent with a gene family of approximately 40 copies. Blots of DNA from other starry flounder indicate that California fish have far fewer gene copies whereas Alaska fish have far more. This analysis is complicated by the fact that there are three different type I AFP isoforms. The first is expressed in the liver and secreted into circulation, the second is a larger hyperactive dimer also thought to be expressed in the liver, and the third is expressed in peripheral tissues. To evaluate the contribution of these latter two isoforms to the overall gene signal on Southern blots, hybridization probes for the three isoforms were isolated from starry flounder DNA by genomic cloning. Two clones revealed linkage of genes for different isoforms, and this was confirmed by genomic Southern blotting, where hybridization patterns indicated that the majority of genes were present in tandem repeats. The sequence and diversity of all three isoforms was sampled in the starry flounder genome by PCR. All coding sequences derived for the skin and liver isoforms were consistent with the proposed structure-function relationships for this AFP, where the flat hydrophobic side of the helix is conserved for ice binding. There was greater sequence diversity in the skin and hyperactive isoforms than in the liver isoform, suggesting that the latter evolved recently from one of the other two. The genomic PCR primers are currently being used to sample isoform diversity in related right-eyed flounders to test this hypothesis. / Thesis (Master, Biochemistry) -- Queen's University, 2009-01-30 13:38:08.346

The B cell repertoire in the neonatal immune system (IS) is characterised by reactivity towards self-components, including other immunoglobulin (Ig) V-regions. These properties have been suggested to be a requirement for the development of a normal immune system. DNA sequencing of two interacting Ig idiotypes, derived from neonatal, preimmune mice, demonstrated that such idiotypic connectivity is germ- line encoded and devoid of VDJ junctional diversity. The serum levels of the same Ig idiotypes were studied in normal mice and demonstrated that the expression in serum fluctuated over time in a pattern compatible with a complex dynamic system. In contrast, similar analyses in autoimmune mice or humans demonstrated fluctuations in Ig titers that differed significantly from the healthy individuals. These findings suggested that pathological autoimmunity may be associated with fundamental alterations in the dynamics of natural antibody (ab) expression. This was further investigated in the nonobese diabetic (NOD) mouse, an animal model for human Type I diabetes. Suppression of the early B cell development in the NOD mouse prevented the development of diabetes, suggesting a role for B cells/Igs in the development of diabetes in these mice. Furthermore, neonatal injections of polyclonal Ig preparations or single, monoclonal natural abs inhibited disease induction. The prevention of diabetes development by one such natural ab was demonstrated to be dependent on both the dose injected and the timing of administration. Studies of the B cell repertoire development in NOD mice, compared to normal mice, by DNA-sequence analyses of IgVH rearrangements utilising genes from the most D-proximal Vh family, Vh7183, supported the idea of an aberrant B cell repertoire in this mouse model. Thus, the adult NOD mouse retained a neonatal pattern of Vh7183 rearrangements. This pattern could, however, be "normalised" by neonatal injection of a natural antibody, previously demonstrated to prevent the development of T cell dependent autoimmunity in the NOD mouse. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1993, härtill 6 uppsatser</p> / digitalisering@umu

We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.

A família gênica Forkhead P {FOXP) tem sido alvo de muitos estudos envolvendo evolução do cérebro e comportamento animal. Destacam-se particularmente as investigações com o gene FOXP2, que indicam que mudanças neste gene estariam associadas com a evolução da vocalização em algumas espécies de mamíferos, incluindo o Homo sapiens. Recentemente, estudos de desordem intrínseca de proteínas (IDPs) tem ganhado ênfase no contexto evolut ivo, visto que uma correlação posit iva entre regiões de desordem e altas taxas evolutivas tem sido observada. Através de um conjunto de abordagens que inclui predizer o conteúdo de desordem e os motivos lineares de interação, bem como as taxas evolutivas, buscamos desvendar a historia evolutiva dos genes da subfamília FOXP. Concentramos nossas análises sobre regiões desordenadas das proteínas FOXPl, FOXP2, FOXP3 e FOXP4 encontradas em 77 espécies de tetrápodes. Tais regiões proteicas são normalmente negligenciadas em estudos dessa natureza, pois se localizam fora de seus tra dicionais domínios conservados, normalmente associados à função principal da proteína. Sít ios apontados estando sob seleção positiva e relaxamento da restrição seletiva mostraram-se hotspots importantes para mudanças que podem impactar na capacidade de interação das proteínas. Encontramos que os maiores valores de w são mais prevalentes em regiões desordenadas que em ordenadas. Ainda, alto e similar valor de desordem (70%) foi encontrado nas 77 proteínas ortólogas de FOXPl , FOXP2, e FOXP4, indicando a manutenção de um "padrão geral" sobre um longo tempo evolutivo. Portanto, a variabilidade tanto de aminoácidos quanto de motivos lineares dentro das regiões de desordem foi marcante. A proteína FOXP3 apresentou menor nível de desordem (30%), mas signif icante sinal de seleção positiva em alguns sítios. Composição idênt ica de resíduo de aminoácido e/ou motivos lineares em espécies filogeneticamente distantes, indica clara convergência molecular, provavelmente associada a pressões seletivas similares. Sucessivamente, nossos achados mostraram uma clara diferença na composição de motivos lineares entre mamíferos e não mamíferos, dando suporte para a importância dos estudos de evolução da interatividade proteica para as compreensões de características taxa-específicas. / Forkhead Family P (FOXP) has been target of many studies about brain and behavior evo lution among species. FOXP2 receives special attention in academic society, due associations with vocalízation evolution in mammals, including Homo sapiens. Recently, intrinsically disorder proteins studies have gained emphasis in the evolutionary context, as positive correlation between disorder regions and higher evolutionary rate has been observed. Through a set of approaches, including disorder and linear motif predictions, as well as estimate evolutionary rates, we aimed to unveil the evolutionary history of FOXP subfamily genes. We focused our ana lysis over disordered regions of FOXPl, FOXP2, FOXP3 and FOXP4 proteins retrieved in 77 tetrapods. Such protein regions are usually neglected in studies of this nature, for being localized out of the traditional conserved domains, usua lly associated with the main function of the protein. Sites indicated as under relaxation of selective constrains or positive selection have shown to be important hotspots for changes that can impact in protein interaction capability. Higher w va lues are prevalent in disordered regions than in ordered ones. Still, high and similar disorder proportion (~70%) was found among 77 orthologues proteins of FOXPl, FOXP2 and FOXP4, indicating general pattern of disorder maintenance, along tetrapod's evolutionary tree. However, amino acid and linear motifs variability within disordered regions was observed. FOXP3 protein presented lower disorder leveis (~30%), when compared with other paralogues, but signal of positive selection was observed in some sites. ldentical composition of amino acid residues and/or linear motifs is, probably, associated with similar selective pressure. Successively, ou r results showed clear differences in linear motif composition between mammals and non-mammals, supporting the importance of evolutionary studies on protein interaction for the understanding of taxa-specifics characteristics.

A família gênica Forkhead P {FOXP) tem sido alvo de muitos estudos envolvendo evolução do cérebro e comportamento animal. Destacam-se particularmente as investigações com o gene FOXP2, que indicam que mudanças neste gene estariam associadas com a evolução da vocalização em algumas espécies de mamíferos, incluindo o Homo sapiens. Recentemente, estudos de desordem intrínseca de proteínas (IDPs) tem ganhado ênfase no contexto evolut ivo, visto que uma correlação posit iva entre regiões de desordem e altas taxas evolutivas tem sido observada. Através de um conjunto de abordagens que inclui predizer o conteúdo de desordem e os motivos lineares de interação, bem como as taxas evolutivas, buscamos desvendar a historia evolutiva dos genes da subfamília FOXP. Concentramos nossas análises sobre regiões desordenadas das proteínas FOXPl, FOXP2, FOXP3 e FOXP4 encontradas em 77 espécies de tetrápodes. Tais regiões proteicas são normalmente negligenciadas em estudos dessa natureza, pois se localizam fora de seus tra dicionais domínios conservados, normalmente associados à função principal da proteína. Sít ios apontados estando sob seleção positiva e relaxamento da restrição seletiva mostraram-se hotspots importantes para mudanças que podem impactar na capacidade de interação das proteínas. Encontramos que os maiores valores de w são mais prevalentes em regiões desordenadas que em ordenadas. Ainda, alto e similar valor de desordem (70%) foi encontrado nas 77 proteínas ortólogas de FOXPl , FOXP2, e FOXP4, indicando a manutenção de um "padrão geral" sobre um longo tempo evolutivo. Portanto, a variabilidade tanto de aminoácidos quanto de motivos lineares dentro das regiões de desordem foi marcante. A proteína FOXP3 apresentou menor nível de desordem (30%), mas signif icante sinal de seleção positiva em alguns sítios. Composição idênt ica de resíduo de aminoácido e/ou motivos lineares em espécies filogeneticamente distantes, indica clara convergência molecular, provavelmente associada a pressões seletivas similares. Sucessivamente, nossos achados mostraram uma clara diferença na composição de motivos lineares entre mamíferos e não mamíferos, dando suporte para a importância dos estudos de evolução da interatividade proteica para as compreensões de características taxa-específicas. / Forkhead Family P (FOXP) has been target of many studies about brain and behavior evo lution among species. FOXP2 receives special attention in academic society, due associations with vocalízation evolution in mammals, including Homo sapiens. Recently, intrinsically disorder proteins studies have gained emphasis in the evolutionary context, as positive correlation between disorder regions and higher evolutionary rate has been observed. Through a set of approaches, including disorder and linear motif predictions, as well as estimate evolutionary rates, we aimed to unveil the evolutionary history of FOXP subfamily genes. We focused our ana lysis over disordered regions of FOXPl, FOXP2, FOXP3 and FOXP4 proteins retrieved in 77 tetrapods. Such protein regions are usually neglected in studies of this nature, for being localized out of the traditional conserved domains, usua lly associated with the main function of the protein. Sites indicated as under relaxation of selective constrains or positive selection have shown to be important hotspots for changes that can impact in protein interaction capability. Higher w va lues are prevalent in disordered regions than in ordered ones. Still, high and similar disorder proportion (~70%) was found among 77 orthologues proteins of FOXPl, FOXP2 and FOXP4, indicating general pattern of disorder maintenance, along tetrapod's evolutionary tree. However, amino acid and linear motifs variability within disordered regions was observed. FOXP3 protein presented lower disorder leveis (~30%), when compared with other paralogues, but signal of positive selection was observed in some sites. ldentical composition of amino acid residues and/or linear motifs is, probably, associated with similar selective pressure. Successively, ou r results showed clear differences in linear motif composition between mammals and non-mammals, supporting the importance of evolutionary studies on protein interaction for the understanding of taxa-specifics characteristics.

We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.