A diapausa é um fenômeno amplamente presente nos artrópodes e é considerada como primordial para o sucesso evolutivo da Classe Insecta, pois possibilita a sobrevivência em condições adversas, como estações frias e secas. Sabe-se que durante a diapausa ocorre o silenciamento de muitos genes e que outros são unicamente expressos nesta fase. Embora existam evidências de que o processo da diapausa tenha se mantido conservado durante a evolução das espécies, ainda há lacunas no conhecimento sobre o nível de conservação dos padrões metabólicos. Um bom modelo para se estudar a diapausa é Tetrapedia diversipes, uma espécie bivoltina de abelha solitária. Os indivíduos que nascem na primeira geração seguem o desenvolvimento desde ovo até adulto em tempo bem menor do que aqueles que nascem na segunda geração; estes retardam o desenvolvimento na fase larval. Além disso, essa espécie é de fácil obtenção no seu ambiente natural, pois apresenta alta taxa de nidificação em ninhos-armadilha. O objetivo deste trabalho foi comparar o perfil de expressão de genes entre as larvas da 1ª geração (que não entram em diapausa), larvas da 2ª geração (que entrariam em diapausa) e das larvas em diapausa. Foram identificados 196 genes diferencialmente expressos, destes 87 foram anotados. Muitos destes genes já foram descritos na literatura como relacionados à diapausa em outras espécies, no entanto, o padrão de expressão não é conservado. Os genes aqui identificados foram divididos em cinco grupos: relacionados à desintoxicação celular, cutícula e citoesqueleto, metabolismo de lipídeos e esteróis, ciclo celular e outros genes relacionados à diapausa / The diapause is broadly distributed among the arthropods and has had an important role for the evolutionary success of the Class Insecta, mainly because this process permits insects to explore adverse conditions, such as cold and dry seasons. It is known that there are many genes being silenced and others being uniquely expressed during diapause. And although there are evidences that the diapause process has remained conserved during the evolution of species, it is still not clear how conserved are the metabolic patterns involved in this behavior. Tetrapedia diversipes is a solitary bee and a good model to study diapause. Individuals from the first generation do not enter in diapause and develop faster than individuals from the second generation, which enter in diapause during the winter. Moreover, this species is easy to capture in natural conditions due to the high rate of nesting in trap nests. The aim of this work was to compare the gene expression profile among non-diapause larvae from first and second generation (about to enter diapause) and larvae already in diapause, trough transcriptome data. One hundred ninety-four genes were identified as differentially expressed and 87 of them were annotated. Many of these genes have already been described as related to diapause in others species, but the expression pattern was not conserved. These genes were divided in five groups: related to cellular detoxification, cuticle and cytoskeleton, lipids and steroids metabolism, cell cycle and other genes related to diapause

Contrasting physiological requirements for T. brucei survival between procyclic (vector) and bloodstream (mammal) forms necessitate different molecular processes and therefore changes in protein expression. Transcriptional regulation is unusual in T. brucei because the arrangement of genes is polycistronic; however, genes which are transcribed together are subsequently cleaved into separate mRNAs by trans-splicing and are individually regulated. During the process of trans-splicing, a 39-nucleotide splice-leader RNA is added to the 5´ end of mRNA. In this study, gene regulation in trypanosomes will be examined in the context of the 7-methylguanosine cap attached to the 5´ end of the splice-leader. Interestingly, in addition to the capping enzymes identified in other eukaryotes, trypanosomatids have an additional guanylyltransferase and methyltransferase in the form of a bifunctional enzyme (TbCGM1). TbCGM1 was found to be essential in bloodstream form T. brucei, although the purpose of this bifunctional capping enzyme remains unclear. Null mutants of a related enzyme, monomeric methyltransferase TbCMT1, did not show an effect on cell viability in culture, however, the enzyme proved to be important for virulence in vivo. Complementary to the study of T. brucei capping enzymes, we worked to develop a method to allow structural analysis of the 5´mRNA cap by mass spectrometry. Following pre-mRNA processing, regulation of the mature mRNAs is a tightly controlled cellular process. While multiple stage-specific transcripts have been identified, previous studies using RNA-seq found that the changes in overall transcript level do not necessarily reflect the abundance of the corresponding proteins. We hypothesized that in addition to mRNA stability, mRNA recruitment to ribosomes may play a significant role in the regulation of gene expression in T. brucei. To approach this question, we performed RNA-seq of total, subpolysomal, and polysomal mRNA. This transcriptomic data was then correlated with published proteomic studies to obtain a global picture of the relative translation efficiencies and their relationship to steady-state protein levels between bloodstream and procyclic form T. brucei.

Gene expression in eukaryotes is dependent on the N-7 methylguanosine cap, located at the 5’ end of RNA pol II transcripts, which marks pre-mRNA for processing, stabilisation and translation initiation. The enzymes that catalyse the formation of the N-7 methylguanosine cap are recruited to RNA pol II at the initial stages of transcription. The final step in this process, N-7 methylation of the guanosine cap, is catalysed by the RNA guanine-7 methyltransferase, RNMT. RNA guanine-7 methylation is an essential process for cell viability and its up-regulation has been associated with cell transformation. However, the mechanistic details of RNMT function in mammalian cells remain elusive. In order to gain better understanding of the molecular mechanisms associated with RNA guanine-7 methylation, cellular RNMT complexes were purified from human cells and constituent proteins were identified using mass spectrometry. A novel component of the RNA guanine-7 methyltransferase complex was identified and designated as RAM (RNMT activating mini-protein). The vast majority of RNMT is found in a complex with RAM and vice versa.RAM is an RNA-binding protein, promoting recruitment of RNA to RNMT. RAM increases recombinant and cellular RNMT cap methyltransferase activity and is required for cap methylation in vivo. We therefore, describe RAM as an “obligate activator” of the human cap methyltransferase. As expected of a protein essential for cap methylation, RAM is required for gene expression, and RAM depletion results in loss of cell viability. Current studies are being focused on determining RAM/RNMT crystal structure as well as determining how the RNA guanine-7 methyltransferase complex is regulated within cells.

The phenotypic variability in human populations is partly the result of gene polymorphisms and differential gene expression. Studying the variability of gene expression across human populations is essential to understanding the molecular basis for diversity. However, key issues remain unanswered with respect to human expression variability. For example, the role of gene methylation in expression variability is uncertain, nor is it clear what role tissue-specific factors may have. Moreover, the contribution that expression variability has in aging and development is unknown. Here we classified human genes based on their expression variability in normal human breast and brain samples and identified functional aspects associated with high and low expression variability. Interestingly, both high variability and low variability gene sets are enriched for developmentally essential genes. There is limited overlap between the variably expressed genes of different tissues, indicating that tissue-specific rather than individual-specific factors are at work. We also find that methylation likely has a key role in controlling expression variability insofar as genes with low expression variability are likely to be non-methylated. Importantly, we find that genes with high population expression variability are likely to have age-, but not sex-dependent expression. Taken together, our work indicates that gene expression variability is tissue-specific, methylation-dependent, and is an important component of the natural aging process.

Drosophila melanogaster is unique amongst model organisms in that males utilize achiasmatic meiosis, where formation of the synaptonemal complex (SC) and recombination are absent. Most organisms require the SC and chiasmata for the successful completion of meiosis and production of viable gametes, making D. melanogaster an ideal system for the study of meiotic variation. The goal of my research was to examine in detail the origin and evolution of male achiasmatic meiosis in Diptera. This was done in three parts: 1) assessing the presence and absence of meiosis genes across dipteran species, 2) analyzing the rate of evolution of Drosophila achiasmatic meiosis genes, and 3) evaluating differences in expression and splicing of meiosis genes between D. melanogaster males and females. I queried genome and transcriptome data from eleven dipteran species for both canonical and achiasmatic meiosis genes. Surprisingly, I found that a set of meiosis-specific genes was lost prior to the gain of Drosophila male achiasmy genes, suggesting that the latter were a later addition to an already non-canonical meiotic process. To assess the evolution of fourteen Drosophila achiasmatic meiosis genes, I performed phylogenetic, rate, selection and co-evolution analyses. My results show that, although these genes appear to be evolving under purifying selection, they are all evolving rapidly compared to their paralogs and paralogous genes throughout the Drosophila genome. Some groups of these genes are also co-evolving, supporting their potential for encoding members of protein complexes. These results suggest that male achiasmy is globally influencing the rapid evolution of these genes, even though their functions within meiosis vary greatly. Lastly, I investigated the expression and splicing of meiosis genes between male and female D. melanogaster. As expected, many meiosis genes with sex-limited roles showed biased expression for the sex that utilized them. However, some genes were expressed equally in both sexes or higher in the opposite sex. I also found evidence that sex-biased splicing may have a role in regulating protein production for some meiosis genes. These results indicate that the regulation of meiotic gene expression is more complex than originally thought and that multiple mechanisms, including alternative splicing, are utilized to control protein production. The combination of results from all parts of this work highlight some of the major events that occurred prior to and during the evolution of Drosophila male achiasmy and lay groundwork for future studies examining the details of this unusual evolutionary path.

Das humane MLC1 (auch als KIAA0027 oder WKL1 benannt) ist ein 377 AS umfassendes Protein, welches vornehmlich in neuralen Geweben exprimiert wird. Aufgrund von Strukturanalysen und Homologievergleichen wurde eine Funktion als Ionenkanal mit acht Transmembrandomänen postuliert. Loss-of-function-Mutationen des MLC1-Gens lassen sich mit dem Auftreten der Megalenzephale Leukenzephalopathie mit subkortikalen Zysten korrelieren. Ferner konnte anhand einer Stammbaumanalyse gezeigt werden, dass die C1121A-Mutation in einer größeren Familie mit dem Auftreten der Periodischen Katatonie nach Leonhardt (PK) kosegregierte, wobei Folgeuntersuchungen zur Assoziation von MLC1-Mutationen und dem Auftreten der PK widersprüchliche Ergebnisse erbrachten. Zur weiteren Aufklärung der biologischen Funktion von MLC1 war es das Ziel der vorliegenden Arbeit, in zwei experimentellen Ansätzen nähere Kenntnisse zum transkriptionellen Expressionsmuster von MLC1 in vivo zu gewinnen, und anschließend durch Herstellung eines polyklonalen Antikörpers gegen das humane MLC1 den Grundstein für weitergehende Untersuchungen zur funktionellen Bedeutung von MLC1 zu legen. Mittels In Situ-Hybridisierung humaner und muriner Gewebeschnitte aus Hippocampus und Cerebellum konnte gezeigt werden, dass die MLC1/Mlc1-Transkription in diesen Geweben vornehmlich in den Bergmann-Gliazellen der Purkinjezellschicht des Cerebellums sowie – in schwächerem Umfang – in verstreut liegenden und in der subgranulären Zone des Gyrus dentatus gehäuften Astrozyten des murinen Hippocampus nachweisbar war. Im zweiten Schritt der Analyse wurden humane post-mortem cDNA-Proben aus verschiedenen Gehirnregionen und zusätzlich einigen nicht-neuralen Geweben von zwei Menschen gewonnen, mittels quantitativer Real-time-PCR die Genexpression von MLC1 bestimmt und mithilfe des Expressionsniveaus von ausgewählten Housekeeping-Genen (GAPDH, L13a, β-Aktin, ARP und Cyclophilin) normalisiert. Es zeigte sich, dass in allen getesteten Hirnregionen eine deutliche MLC1-Expression festzustellen war, deren Maxima im Cerebellum und Frontalhirn und deren Minima im Putamen bzw. im nicht-neuralen Plexus chorioideus lagen. Zudem konnte eine nicht-neurale Expression auf sehr geringem Niveau für Lunge und Milz nachgewiesen werden. Zur Gewinnung eines polyklonalen Antikörpers gegen humanes MLC1 wurden mittels computergestützter Verfahren ein 117 AS langes Vakzinierungsprotein entworfen, welches immunogene Abschnitte des N-Terminus (61 AS) und C-Terminus (54 AS) enthielt. Die kodierende Sequenz wurde unter Verwendung des Impact-CN®-Expressionssystems in einen pTYB-Vektor kloniert, in ER2566-Zellen exprimiert, das Protein affinitätschromatographisch über Chitin-Säulen isoliert und aufgereinigt und mittels Bradford-Assay und SDS-Gelelektrophorese nachgewiesen. Leider konnte trotz vielfältiger Variation der Versuchsparameter kein eindeutiger Nachweis einer ausreichenden Expression des MLC1-Proteins in den ER2566-Zellen erbracht werden, die für die anschließende Vakzinierung von Kaninchen zur Gewinnung des polyklonalen Antiserums erforderlich gewesen wäre. Die Gründe hierfür sind unklar, denkbar sind beispielsweise eine suboptimale Codon-Frequenz, eine schlechte Proteinlöslichkeit, intrazelluläre mRNA-Degradation, proteolytische Abbauvorgänge oder eine Hemmung der Proteinbiosynthese durch die biologische Funktion des Proteins. Zusammenfassend konnten die im Rahmen dieser Arbeit erzielten Ergebnisse einen Beitrag zur Erweiterung des Wissens zur MLC1-Expression leisten. Dabei entsprachen die Befunde zur humanen MLC1-Expression weitgehend den diesbezüglichen Beobachtungen zur regionalen und zellulären Expressionsstärkenverteilung aus dem Mausmodell, welche eine funktionelle Bedeutung von MLC1 im Rahmen von neuralen Schrankenstrukturen nahelegten (vgl. Schmitt et al. 2003). Mittels der zwischenzeitlich von anderen Arbeitsgruppen (über andere experimentelle Verfahren) erzeugten Antikörper gegen MLC1 konnte gezeigt werden, dass funktionelles MLC1 vermutlich als zellmembranständiges Dimer vorliegt und seine biologische Funktion u.a. durch Interaktion mit dem DGC (=Dystrophin-assoziierten Glykoprotein-Komplex) in den Caveolae ausübt. Es bleibt eine Aufgabe für die Zukunft, die genauen molekularen Mechanismen dieser Prozesse und ihre mögliche therapeutische Beeinflussbarkeit zur Behandlung der MLC zu erforschen. Auch die Frage der potenziellen extraneuralen MLC1-Expression, für die in dieser Arbeit Hinweise gefunden wurden, mag ein interessanter Ansatzpunkt für zukünftige Forschungsarbeiten sein. / Studies on the expression of megalencephalic leukoencephalopathy with subcortical cysts 1 (MLC1/Mlc1) in human and murine tissues

No description available.

No description available.

Recently, it has been shown that genetic variation that perturbs the regulation of gene expression is widespread in eukaryotic genomes. Regulatory variation (RV) is expected to be an important driver of phenotypic differences, evolutionary change, and susceptibility to complex genetic diseases. Because trans-acting regulators of gene expression control mRNA levels of multiple genes simultaneously, we hypothesise that RV that affects these components will have a shared-influence upon the expression levels of multiple genes. Since genes are regulated in trans by combinations of basal and tissue specific factors, we further hypothesise that RV in these components may have different effects in each tissue. We used microarrays to identify 755 genes that were affected by RV in at least one of the brain, kidney and liver of two inbred mouse strains, C57BL/6J and DBA/2J. Just 2% were affected in all three tissues, suggesting that the influence of RV is predominantly tissue specific. To study shared-RV, we measured the expression levels of these 755 genes in the same 3 tissues from a panel of recombinant inbred mice, and identified groups of correlated genes that are putatively under the influence of shared trans-acting RV. Using methods that we developed for studying the effects of RV in multiple tissues, we identified 212 genes that are correlated in all three tissues, which include 10 groups of at least 3 genes. We developed a novel method called coherency analysis to show that RV consistently affected the expression levels of these groups of genes in different genetic backgrounds. Strikingly, the relative up- or down-regulation of genes in each group was markedly different in the three tissues of the same mouse, suggesting that the influence of RV itself is not tissue specific as previously expected, but that RV can influence genes with differing outcomes in each tissue. These observations are compatible with RV affecting combinations of basal and tissue specific regulatory factors. This is the first cross-tissue investigation into the influence of shared-RV in multiple tissues, which has important implications in humans, where access to the phenotypically relevant tissue may be necessarily limited.

Recently, it has been shown that genetic variation that perturbs the regulation of gene expression is widespread in eukaryotic genomes. Regulatory variation (RV) is expected to be an important driver of phenotypic differences, evolutionary change, and susceptibility to complex genetic diseases. Because trans-acting regulators of gene expression control mRNA levels of multiple genes simultaneously, we hypothesise that RV that affects these components will have a shared-influence upon the expression levels of multiple genes. Since genes are regulated in trans by combinations of basal and tissue specific factors, we further hypothesise that RV in these components may have different effects in each tissue. We used microarrays to identify 755 genes that were affected by RV in at least one of the brain, kidney and liver of two inbred mouse strains, C57BL/6J and DBA/2J. Just 2% were affected in all three tissues, suggesting that the influence of RV is predominantly tissue specific. To study shared-RV, we measured the expression levels of these 755 genes in the same 3 tissues from a panel of recombinant inbred mice, and identified groups of correlated genes that are putatively under the influence of shared trans-acting RV. Using methods that we developed for studying the effects of RV in multiple tissues, we identified 212 genes that are correlated in all three tissues, which include 10 groups of at least 3 genes. We developed a novel method called coherency analysis to show that RV consistently affected the expression levels of these groups of genes in different genetic backgrounds. Strikingly, the relative up- or down-regulation of genes in each group was markedly different in the three tissues of the same mouse, suggesting that the influence of RV itself is not tissue specific as previously expected, but that RV can influence genes with differing outcomes in each tissue. These observations are compatible with RV affecting combinations of basal and tissue specific regulatory factors. This is the first cross-tissue investigation into the influence of shared-RV in multiple tissues, which has important implications in humans, where access to the phenotypically relevant tissue may be necessarily limited.