Transposable elements (TEs) are interspersed repetitive sequences that are present in most genomes. Miniature inverted repeat transposable elements (MITEs) are the most numerous Class II elements in higher eukaryotes. Little is known about their origin, transposition and function. In this study, three novel MITE families (Kiddo, MDM1 and MDM2) were identified in the rice genome. They bear terminal inverted repeats (TIRs) and show target site duplications (TSDs) at the insertion sites. Each family is present in hundreds of copies with length that range from 200 bp to 400 bp. An evolutionary relationship between Mutator elements and MDM1 and MDM2 family was established. The absence of an observed transposition event, together with the mutated ancestral elements identified by in silico analysis, led to a conclusion that Kiddo and its autonomous elements are not presently active. To overcome laborious and time consuming manual analysis of MITEs on a genomic scale, MAK, a computational tool kit, was developed to automatically retrieve MITE sequences, their neighboring genes and ancestral elements from genome sequences. MAK has been functionally tested and is now available to the research community. Studies on the effect of MITE (Kiddo and MDM1) insertions into a rice ubiquitin (rubq2) promoter revealed a two-edged role of MITEs on gene regulation. While Kiddo and MDM1 contribute ~40% to rubq2 promoter activity, they also induce progressive silencing of this promoter. The evolutionary implications of the two-edged role of MITEs in gene regulation are discussed.

A regulação da expressão gênica é vital para todos os organismos se adaptarem rapidamente às mudanças ambientais. Estes mecanismos adaptativos são altamente complexos e a maioria deles não está completamente esclarecida. O fosfato inorgânico (Pi), um nutriente essencial para todos os organismos, é requerido em importantes processos celulares como a biosíntese de ácidos nucléicos e a sinalização metabólica. O sistema de aquisição de Pi no fungo filamentoso Neurospora crassa inclui pelo menos quatro genes regulatórios: nuc-2, preg, pgov e nuc-1. Em condições limitantes de Pi, NUC- 2, uma proteína com domínio de repetição de anquirina, inibe o funcionamento do complexo PREG-PGOV, ativando assim o fator de transcrição NUC-1 e a expressão de genes envolvidos na captação de fosfato, como fosfatases, fosfato permeases e nucleases. Visando entender a funcionalidade do gene nuc-2 na regulação da expressão gênica em resposta aos níveis de Pi exógeno, foram construídas duas bibliotecas de subtração de cDNA entre as linhagens selvagem St.L.74A e nuc-2A de N. crassa, cultivadas em Pilimitante. Obtivemos 52 transcritos induzidos e 16 reprimidos pela proteína NUC-2. A categorização funcional destas sequências revelou genes envolvidos em diversos processos celulares, como transporte, regulação transcricional, transdução de sinal, metabolismo, síntese protéica e desenvolvimento. Entre os genes modulados negativamente pela proteína NUC-2, foi identificado um gene que codifica a proteína MAK-2 (mitogen-activated protein kinase-2), envolvida em vias de sinalização intracelular. O papel funcional deste gene no monitoramento do Pi extracelular foi avaliado por microarranjos de oligonucleotídeos, comparando as linhagens selvagem e mutante mak-2, cultivadas em baixa concentração de Pi. Foram identificados 4.214 genes regulados pela proteína MAK- 2, dentre eles a ciclina codificada pelo gene preg. Além disto, genes regulados em função da concentração de Pi foram identificados, mostrando o envolvimento de 3.174 transcritos. Os resultados obtidos neste trabalho revelam novos aspectos moleculares envolvidos na adaptação à disponibilidade de Pi extracelular, sugerindo que o gene mak-2 constitui um novo componente da via de sinalização e monitoramento de fosfato em N. crassa. / Gene expression regulation is crucial for all organisms to rapidly adapt to environmental changes. These adaptive mechanisms are highly complex and most of them have not been completely elucidated. The inorganic phosphate (Pi), an essential nutrient for all organisms, is required for important cellular processes, such as nucleic acids biosynthesis and metabolic signaling. The Pi acquisition system in the filamentous fungus Neurospora crassa includes at least four regulatory genes: nuc-2, preg, pgov and nuc-1. Under limiting Pi conditions, NUC-2, an ankyrin-like repeat protein, inhibits the functioning of the PREG-PGOV complex, allowing the activation of the transcription factor NUC-1 and the expression of genes involved in phosphate acquisition, such as phosphatases, phosphate permeases and nucleases. Aiming at a better comprehension of the nuc-2 functionality in gene expression regulation in response to exogenous Pi levels, two cDNA subtraction libraries were constructed comparing N. crassa wild type St.L.74A and nuc-2A strains, grown under Pi starvation. We obtained 52 NUC-2 up- and 16 downregulated genes. Functional categorization of these sequences revealed genes involved in several cellular processes, such as cellular transport, transcriptional regulation, metabolism, protein synthesis and development. Among the NUC-2 negatively modulated genes, we identified the MAK-2 (mitogen-activated protein kinase-2) protein coding gene, involved in the intracellular signaling pathway. The functional role of this gene in the extracellular Pi sensing was evaluated by oligonucleotide microarrays, comparing wild type and mak-2 strains responses under Pi starvation. We identified 4.214 MAK-2 regulated genes, among them the cyclin coding gene, preg. Furthermore, 3.174 genes regulated in response to Pi levels were identified. In a nutshell, the results obtained in this work reveal novel molecular aspects of the adaptation to extracellular Pi availability, suggesting that the mak-2 gene constitutes a novel component of the N. crassa phosphate sensing and signaling pathway.

A regulação da expressão gênica é vital para todos os organismos se adaptarem rapidamente às mudanças ambientais. Estes mecanismos adaptativos são altamente complexos e a maioria deles não está completamente esclarecida. O fosfato inorgânico (Pi), um nutriente essencial para todos os organismos, é requerido em importantes processos celulares como a biosíntese de ácidos nucléicos e a sinalização metabólica. O sistema de aquisição de Pi no fungo filamentoso Neurospora crassa inclui pelo menos quatro genes regulatórios: nuc-2, preg, pgov e nuc-1. Em condições limitantes de Pi, NUC- 2, uma proteína com domínio de repetição de anquirina, inibe o funcionamento do complexo PREG-PGOV, ativando assim o fator de transcrição NUC-1 e a expressão de genes envolvidos na captação de fosfato, como fosfatases, fosfato permeases e nucleases. Visando entender a funcionalidade do gene nuc-2 na regulação da expressão gênica em resposta aos níveis de Pi exógeno, foram construídas duas bibliotecas de subtração de cDNA entre as linhagens selvagem St.L.74A e nuc-2A de N. crassa, cultivadas em Pilimitante. Obtivemos 52 transcritos induzidos e 16 reprimidos pela proteína NUC-2. A categorização funcional destas sequências revelou genes envolvidos em diversos processos celulares, como transporte, regulação transcricional, transdução de sinal, metabolismo, síntese protéica e desenvolvimento. Entre os genes modulados negativamente pela proteína NUC-2, foi identificado um gene que codifica a proteína MAK-2 (mitogen-activated protein kinase-2), envolvida em vias de sinalização intracelular. O papel funcional deste gene no monitoramento do Pi extracelular foi avaliado por microarranjos de oligonucleotídeos, comparando as linhagens selvagem e mutante mak-2, cultivadas em baixa concentração de Pi. Foram identificados 4.214 genes regulados pela proteína MAK- 2, dentre eles a ciclina codificada pelo gene preg. Além disto, genes regulados em função da concentração de Pi foram identificados, mostrando o envolvimento de 3.174 transcritos. Os resultados obtidos neste trabalho revelam novos aspectos moleculares envolvidos na adaptação à disponibilidade de Pi extracelular, sugerindo que o gene mak-2 constitui um novo componente da via de sinalização e monitoramento de fosfato em N. crassa. / Gene expression regulation is crucial for all organisms to rapidly adapt to environmental changes. These adaptive mechanisms are highly complex and most of them have not been completely elucidated. The inorganic phosphate (Pi), an essential nutrient for all organisms, is required for important cellular processes, such as nucleic acids biosynthesis and metabolic signaling. The Pi acquisition system in the filamentous fungus Neurospora crassa includes at least four regulatory genes: nuc-2, preg, pgov and nuc-1. Under limiting Pi conditions, NUC-2, an ankyrin-like repeat protein, inhibits the functioning of the PREG-PGOV complex, allowing the activation of the transcription factor NUC-1 and the expression of genes involved in phosphate acquisition, such as phosphatases, phosphate permeases and nucleases. Aiming at a better comprehension of the nuc-2 functionality in gene expression regulation in response to exogenous Pi levels, two cDNA subtraction libraries were constructed comparing N. crassa wild type St.L.74A and nuc-2A strains, grown under Pi starvation. We obtained 52 NUC-2 up- and 16 downregulated genes. Functional categorization of these sequences revealed genes involved in several cellular processes, such as cellular transport, transcriptional regulation, metabolism, protein synthesis and development. Among the NUC-2 negatively modulated genes, we identified the MAK-2 (mitogen-activated protein kinase-2) protein coding gene, involved in the intracellular signaling pathway. The functional role of this gene in the extracellular Pi sensing was evaluated by oligonucleotide microarrays, comparing wild type and mak-2 strains responses under Pi starvation. We identified 4.214 MAK-2 regulated genes, among them the cyclin coding gene, preg. Furthermore, 3.174 genes regulated in response to Pi levels were identified. In a nutshell, the results obtained in this work reveal novel molecular aspects of the adaptation to extracellular Pi availability, suggesting that the mak-2 gene constitutes a novel component of the N. crassa phosphate sensing and signaling pathway.

MEd / Department of Curriculum Studies

Monoklonale Antikörper (mAk) werden bereits erfolgreich zur therapeutischen Depletion verschiedener Zellpopulationen in vivo verwendet, die Mechanismen der Depletion sind jedoch unklar geblieben. In dieser Arbeit wurden im Mausmodell die molekularen Grundlagen der CD4+ T-Zelldepletion (CD4 TZD) nach einmaliger Gabe (i.p.) von 100 µg des anti-CD4-mAk YTS191.1 untersucht. Dabei konnte eine starke Korrelation zwischen Depletion und der Modulation des CD4-Moleküls von der Oberfläche beobachtet werden. Gleichzeitig zeigten sich organabhängige Unterschiede, sowohl im zeitlichen Verlauf, als auch in der Effizienz der Depletion. Im Thymus konnten weder Depletion noch Modulation detektiert werden, in Milz und Lymphknoten (Lk) war die CD4 TZD nach starker CD4-Modulation bereits nach 48 h mit 80-90 % maximal, in den Peyer-Plaques jedoch niedriger und verzögert (50-60 % nach 72 h). Anhand C3-defizienter Mäuse konnte ferner kein wesentlicher Beitrag von Komplement an der CD4 TZD beobachtet werden. Im Gegensatz dazu konnte durch die Verwendung verschiedener FcGamma-Rezeptor (FcGammaR)-defizienter Mäuse (FcGammaRI, FcGammaRII, FcGammaRIII, FcGammaRI/III und FcRGamma) wie auch durch die Blockade des FcGammaRIV eine starke, zudem organabhängige Beteiligung von FcGammaR an der CD4 TZD gezeigt werden. Während in der Milz die CD4 TZD von FcGammaRIV vermittelt wurde, waren in den Lk und Peyer-Plaques FcGammaRI/III involviert. Diese Befunde korrelierten mit der starken Expression von FcGammaRIV in Milz, Lunge, Darm, Niere und Leber, während in den Lk nur eine schwache und in Thymus und Peyer-Plaques keine Expression detektiert werden konnte. Innerhalb der Milz konnten erstmalig F4/80hoch Makrophagen als FcGammaRIV+ identifiziert und somit als potenzielle Effektorzellen der CD4 TZD bestimmt werden. Der direkte Vergleich der Depletion von CD4+ T-Zellen mit der Depletion von ICOS+ T-Zellen verdeutlichte darüber hinaus, dass die Effizienz der Zelldepletion nicht nur von den Eigenschaften des verwendeten mAk, sondern auch von denen des Zielmoleküls abhängig ist. / Monoclonal antibodies (mAb) are efficiently used for the therapeutic depletion of various cells in vivo yet the mechanisms of depletion are still unclear. In this work, the molecular principles of CD4+ T cell depletion (CD4 Tcd) by a single application of 100 µg of the anti-CD4 mAb YTS191.1.1 were investigated in the mouse. A strong correlation between the depletion and the surface modulation of the CD4 molecule could be observed. At the same time, organ-dependent differences in the kinetics as well as in the efficiency of depletion could be detected. In the thymus, neither modulation nor depletion were detectable. In the spleen and the lymph nodes (Ln), the modulation was strong and the depletion was maximal (80-90%) 48 h after mAb treatment. Interestingly, both modulation and depletion were decreased and delayed (50-60% after 72 h) in the Peyer`s patches. By using C3-deficient mice, no major contribution of complement to the CD4 Tcd was seen. On the contrary, with the help of different FcGamma-receptor (FcGammaR)-deficient mice (FcGammaRI, FcGammaRII, FcGammaRIII, FcGammaRI/III, and FcRGamma) and through the blockade of FcGammaRIV, a strong organ dependent involvement of FcGammaR could be shown. While the depletion in the spleen was clearly dependent on FcGammaRIV, in the Ln and the Peyer`s patches, FcGammaRI/III were involved. These findings correlated with the strong expression of FcGammaRIV in the spleen, the lung, the colon, the kidney, and the liver, while in the Ln the expression was weak and undetectable in the thymus and the Peyer`s patches. For the first time, F4/80high macrophages in the spleen could be identified as also being FcGammaRIV+, and are therfore considered as the potential effector cells of the CD4 Tcd. The direct comparison of the depletion of T cells via CD4 or ICOS pointed out that the target cell depletion is not only dependent on the properties of the mAb used, but also on those of the target molecule.