Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-06T19:20:01Z No. of bitstreams: 2 Dissertação - Igor Gomes de Oliveira - 2011.pdf: 1076733 bytes, checksum: 1faa803ec01cef7512bec500010c985b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-06T19:26:27Z (GMT) No. of bitstreams: 2 Dissertação - Igor Gomes de Oliveira - 2011.pdf: 1076733 bytes, checksum: 1faa803ec01cef7512bec500010c985b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-06T19:26:27Z (GMT). No. of bitstreams: 2 Dissertação - Igor Gomes de Oliveira - 2011.pdf: 1076733 bytes, checksum: 1faa803ec01cef7512bec500010c985b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2011-03-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Clinical studies have shown that the combinations of chemotherapeutic drugs gemcitabine (GEM) plus cisplatin (CIS) and gemcitabine (GEM) plus doxorubicin (DXR) exert important cytotoxic activity against several types of cancer in advanced stages as well as metastatic cancer. CIS, DXR and GEM have different mechanisms of action. GEM is a pro-drug that must be phosphorylated by deoxycytidine kinase to evolve into its active form. Both gemcitabine diphosphate and gemcitabine triphosphate inhibit processes required for DNA synthesis. CIS induces a variety of DNA structural changes, mainly intra- and interstrand cross-links between adjacent purine bases. DXR acts as a topoisomerase II inhibitor. This study compares the genetic toxicity effects induced by GEM+CIS and GEM+DXR co-treatments with the single drug treatments. We used the Somatic Mutation And Recombination Test (SMART), which simultaneously detects and quantifies mutagenic and recombinogenic toxicological endpoints through loss of heterozygosity of two genetic markers involved in the metabolic pathways of the Drosophila melanogaster wing hairs formation. Using the standard cross, the third-stage larvae were chronically treated with different concentrations of GEM (0.008, 0.010, 0.012 and 0.014 mM) combined with CIS (0.05 mM) or DXR (0.2 mM). Comparing with GEM single treatment, GEM+CIS and GEM+DXR co-treatments induced a synergistic effect manifested as an increment in the mutant clones frequencies. Homologous recombination was the main genotoxic effect observed. / As combinações dos quimioterápicos gemcitabina (GEM) com cisplatina (CIS) e gemcitabina (GEM) com doxorrubicina (DXR) têm demonstrado uma importante atividade citotóxica contra vários tipos de câncer em estágio avançado e/ou metastático em diversos estudos clínicos. CIS, DXR e GEM possuem diferentes mecanismos de ação, sendo este um fator importante para o sucesso da associação entre quimioterápicos. A GEM é uma pró-droga análoga de desoxicitidina que deve ser fosforilada pela desoxicitidina quinase para se tornar ativa. Ambos, gemcitabina difosfato e gemcitabina trifosfato, atuam inibindo os processos necessários para a síntese de DNA. Já a CIS induz uma variedade de mudanças estruturais, principalmente por meio de ligações cruzadas intra e intercadeias entre bases purínicas adjacentes do DNA. A DXR age como um inibidor da topoisomerase II, formando um complexo entre esta proteína e o DNA, inibindo os processos necessários para a síntese do DNA. Este estudo teve como objetivo comparar os efeitos de toxicidade genética induzidos pelos tratamentos utilizando os combinados de GEM+CIS e GEM+DXR com os tratamentos usando as drogas isoladas. Utilizamos o Teste de Mutação e Recombinação Somática (SMART), que detecta e quantifica, simultaneamente, parâmetros mutagênicos e recombinogênicos através da perda de heterozigose de dois marcadores genéticos envolvidos nas vias metabólicas da formação dos pêlos da asa de Drosophila melanogaster. Usando o cruzamento padrão, as larvas de terceiro estágio foram tratadas cronicamente com diferentes concentrações de GEM (0,008, 0,010, 0,012 e 0,014 mM), combinado com CIS (0,05 mM) ou DXR (0,2 mM). Comparando os combinados GEM+CIS e GEM+DXR com o tratamento isolado com GEM, notou-se que nos combinados houve um efeito sinérgico demonstrado pelo aumento nas frequências de clones mutantes destes em comparação com o tratamento isolado com GEM. A recombinação homóloga foi o principal tipo de efeito genotóxico observado nas combinações.

O metabolismo das células S2 (Schneider 2) de Drosophila melanogaster ainda não é totalmente conhecido. Existem poucos estudos específicos sobre o metabolismo de células S2, sejam elas selvagens ou recombinantes (rS2), como por exemplo aquelas transfectadas para a expressão da glicoproteína do vírus da raiva (GPV). Como o genoma da Drosophila melanogaster já foi mapeado, as principais enzimas que atuam nos processos metabólicos em geral já foram identificadas e estão à disposição no KEGG (Kyoto Encyclopedia of Genes and Genomes). Assim, o KEGG apresenta todas as possíveis vias metabólicas com as enzimas que podem ser codificadas. Diante deste quadro, foi proposto um modelo metabólico baseado em um conjunto de vias de assimilação de glicose e glutamina e foram encontrados os modos elementares característicos do sistema através do programa Metatool. Em seguida, foi definido o modelo matemático mediante o equacionamento desses modos elementares. Esse processo se repetiu até se encontrar um conjunto de vias metabólicas que, através da modelagem matemática, respondesse coerentemente a um conjunto de dez ensaios em diferentes condições de concentrações iniciais de glicose, glutamina e oxigênio dissolvido. Chegou-se então, a um metabolismo básico para a rS2 contendo 33 vias metabólicas englobando a glicólise, a via das pentoses, o ciclo de Krebs e a fosforilação oxidativa. Dados anteriores indicavam elevada flexibilidade metabólica dessa célula, o que foi prevista através de algumas reações propostas como reversíveis nas vias de degradação e síntese de glutamina. Essa proposta de metabolismo resultou em 37 modos elementares. Outra característica interessante da modelagem foi a utilização da produção de purinas e pirimidinas para a estimativa do crescimento celular. Depois de realizada a modelagem, as mesmas condições iniciais dos ensaios foram simuladas através de um programa de simulação do comportamento cinético das células rS2 desenvolvido em MATLAB. Esse simulador foi utilizado também para simulação com diferentes meios e condições iniciais de cultivo. Chegando-se a um ajuste geral entre valores experimentais e simulados com coeficiente de correlação de 0,88. / The metabolism of the S2 cells (Schneider 2) Drosophila melanogaster is not yet fully known. There have been few specific studies on the metabolism of S2 cells, whether recombinant or wild (rS2), such as those transfected for expressing the rabies virus glycoprotein (RVGP). As the genome of Drosophila melanogaster have been mapped, the key enzymes that act on the metabolic processes in general have been identified and are available in the KEGG (Kyoto Encyclopedia of Genes and Genomes). Thus, KEGG presents all possible pathways with the enzymes that can be encoded. Given this context, it was proposed a metabolic model based on a set metabolic glucose and glutamine assimilation pathways and were found characteristic elementary modes of the system through the Metatool program. Then the mathematical model was defined by addressing these elementary modes. This process was repeated until a set of metabolic pathways, by mathematical modelling, consistently responded to a set of ten experiments (in various conditions). We came to a basic metabolism for rS2 containing 33 pathways comprising glycolysis, pentose, Krebs cycle and oxidative phosphorylation. Previous data indicate that rS2 is a cell with high metabolic flexibility, which was confirmed by some reactions in the process proposed as reverse breakdown and synthesis of glutamine. The proposed metabolism resulted in 37 elementary modes. Another interesting model characteristic was the use of the production of purines and pyrimidines for the estimation of cell growth. After the modelling performed, the same initial runs conditions were simulated using a software of Simulation of the Kinetic behaviour of rS2 cells, developed in MATLAB. This simulator was also used for simulation of other experiments with different initial conditions and methods of cultivation. Coming to a general adjustment of experimental and simulated values with correlation coefficient of 0.88.

Une surprolifération cellulaire associée à de l’aneuploïdie est un marqueur couramment retrouvé dans les cancers et une faible instabilité génétique peut-être un élément aggravant (sinon déclencheur) de la tumorigénèse. Récemment, il a été montré sur un modèle de cellules cancéreuses en culture qu’une forte aneuploïdie compromet la prolifération cellulaire en entraînant la mort de ces dernières. Au cours de ma thèse, nous avons souhaité tester si cette hypothèse se vérifiait in vivo en utilisant comme modèle, les tumeurs du système nerveux central de la larve de D. melanogaster. Nous avons fait le choix d’utiliser des mutants pour des gènes impliqués dans la formation du fuseau mitotique et la ségrégation des chromosomes (Sas-4 ou AurA) afin d’induire ces tumeurs. Pour générer l’aneuploïdie, nous avons choisi d’associer les mutations sas-4 ou aurA avec des mutations pour des gènes essentiels du SAC, Mad2 ou BubR1ken. Nous avons ensuite analysé par immunofluorescence et microscopie l’effet de la perte du SAC sur la prolifération des Nb. Pour sas-4, la perte du SAC cause l’apparition d’une forte aneuploïdie et une baisse du nombre de Nb associée à une forte réduction de taille des cerveaux. Cela compromet totalement la capacité des cerveaux mutants à induire des tumeurs lorsqu’on les injecte dans l’abdomen de mouches adultes saines. Dans le cas d’aurA, ni hausse de l’aneuploïdie dans le tissu ni baisse de la prolifération des Nbs n’ont été observés. Par ailleurs, la même forte proportion de mouches injectées avec des cerveaux aurA ou aurA mad2 développant une tumeur a été constaté. Afin de mieux comprendre pourquoi le mutant aurA ne réagit pas comme le mutant sas-4 à la déplétion du SAC, nous avons entrepris une analyse détaillée des mutants aurA et aurA mad2. Nous avons d’abord observé que, malgré la perte du SAC, 1) il existe toujours un délai en mitose dans aurA mad2 et 2) il existe un délai entre la satisfaction du SAC et l’entrée en anaphase dans aurA. Comme l’entrée en anaphase est dépendante de la dégradation de la CycB et de la Sécurine via l’APC/C, nous avons analysé le comportement de la CycB (couplé à une étiquette GFP) par vidéo-microscopie en temps réel et observé un défaut de la régulation de la dégradation de cette dernière dans le mutant aurA ainsi que dans le double mutant aurA mad2. Ces observations nous ont permis de proposer un nouveau rôle pour la kinase AurA dans la régulation de la dégradation de la CycB en fin de mitose. / Cellular overproliferation associated with aneuploidy is a common hallmark of cancers. Low genetic instability may be a contributing factor of tumorigenesis. Recently, it was shown on a cellular cancer model in culture that strong aneuploidy compromises cell proliferation by causing cell death. During my thesis, we have test if this hypothesis was verified in vivo by using as a model, the tumours of the larval central nervous system of D. melanogaster. We decided to use mutants involved in mitotic spindle formation and chromosome segregation (Sas-4 or AurA) to induce these tumours. To generate aneuploidy, we chose to associate these mutations with mutations in genes essential for the SAC, Mad2 or BubR1ken. We then analysed the effect of the SAC depletion on the Nb proliferation. For sas-4, loss of the SAC leads to high aneuploidy and a decrease in Nb number associated with brain size reduction. It completely undermines the ability of mutant brain to induce tumors when injected into the abdomen of healthy adult flies. In the case of aurA, nor increase of aneuploidy in tissue or decrease in nb proliferation have been observed. Moreover, the same proportion of flies injected with aurA or aurA mad2 brains developed tumours. To better understand why the aurA mutant not react as the sas-4 mutant to the SAC depletion, we undertook a detailed analysis of aurA and aurA mad2 mutants. We first observed that despite the SAC depletion, 1) there is always a delay in mitosis in aurA mad2 and 2) there is a delay between SAC satisfaction and anaphase onset in aurA. Since anaphase onset is dependent of the CycB and Securine degradation via the APC / C, we analysed the behaviour of the CycB (coupled with a GFP tag) by real-time videomicroscopy and observed a defect in the regulation of CycB degradation in aurA and in the double aurA mad2 mutant. These observations lead us to propose a new role for AurA kinase in regulating the degradation of CycB at the end of mitosis.

Les rétrovirus endogènes sont des parasites génétiques qui s’insèrent dans l’ADN génomique. Bien que leurs insertions délétères soient éliminées par la sélection naturelle, ils prolifèrent et sont une source de plasticité génomique. L’étude de l’impact de leur mobilité sur le génome hôte est rendue difficile par le faible taux de transposition de ces éléments, réprimés par des petits ARN appelés piARNs. Nous avons développé une approche génétique permettant d’inactiver ce contrôle et de déterminer l’impact sur le génome de la drosophile, d’une transposition réplicative. Nous avons remarqué la mise en place d’autres mécanismes de répression des rétrovirus endogènes lors de la perte des piARNs pouvant ainsi limiter leur propagation. Nous avons aussi identifié de nouveaux sites d’intégrations des rétrovirus endogènes après un cycle de transposition réplicative. Cependant, le taux de transposition reste faible. Ce projet combinant différentes approches (génétique, séquençage à haut débit et bioinformatique) a permis de démontrer que la voie des piARNs n’est pas cruciale pour le maintien de l’intégrité du génome, et que d’autres mécanismes semblent intervenir afin de maintenir sa stabilité. / Endogenous retrovirsuses are genetic parasites which are inserted in the genomic DNA. Although their deleterious insertions are eliminated by natural selection, they proliferate and are a source of genomic plasticity. The study of the impact of their mobility on the host genome is made difficult by the transposition’s low rate of these elements, suppressed by a class of small RNA, called piRNA. We have developed a genetic approach to inactivate this control and determine the impact on Drosophila’s genome, after one replicative transposition. We noticed the establishment of other endogenous retroviruses repression mechanisms that are awakened after the loss of the piRNA and they are able to limit their spread. We identified new integrations sites of endogenous retrovirus after one replicative transposition. But we noticed that the transposition rate still low. This project combines different approaches (genetics, high-throughput sequencing and bioinformatics) and show the piRNA pathway is not essential to maintain genome integrity but other mechanisms involving small RNA can be implicated in the genome stability.

Des perturbations de l’horloge circadienne imposées par nos modes de vie désynchronisés «24/7» peuvent être néfastes à long terme. Ces horloges contrôlent des rythmes biologiques avec une périodicité d’environ 24h et se synchronisent aux cycles journaliers de lumière-obscurité. Les perturbations de l’horloge peuvent influencer le vieillissement normal et celui de patients atteints de la maladie de Parkinson (MP). Ces patients présentent souvent des symptômes non moteurs incluant des troubles du sommeil et/ou des rythmes circadiens, qui se manifestent avant les déficits moteurs ou cognitifs. L’objectif de ce travail de thèse a été d’explorer l’impact des perturbations de l’horloge circadienne sur le déclin fonctionnel lié à l’âge au cours du vieillissement normal et dans des modèles de MP chez la drosophile. Les résultats obtenus confirment l’impact négatif de l’arythmie circadienne sur la longévité, et révèlent que l’inactivation du gène d’horloge Clock (Clk) augmente le niveau de stress oxydatif dans le cerveau et accélère le déclin des capacités locomotrices au cours du vieillissement. Ce dernier effet a pu être relié à une fonction de Clk dans les neurones d’horloge qui contrôlent les rythmes d’activité-repos en obscurité constante, et leurs connections à un groupe spécifique de neurones dopaminergiques. Nous avons aussi observé que des décalages horaires chroniques mènent également à une accélération du déclin locomoteur lié à l’âge, qui peut être sauvée en adaptant les rythmes lumière-obscurité à la période endogène des drosophiles. Enfin, notre travail souligne l’impact négatif de l’arythmie circadienne sur le déclin locomoteur lié à l’âge dans un modèle de MP. / Long-term disruption of circadian clocks, as occurs often in our "24/7 societies", can be detrimental. These clocks control self-sustained biological rhythms with ~24h periodicity and synchronize to the daily light-dark cycle. Clock disruption may impact normal brain aging as well as Parkinson disease (PD) pathogenesis: PD patients frequently exhibit non-motor symptoms including sleep and/or circadian rhythm disruptions that occur before motor or cognitive deficits, and decrease quality of life. The objective of this PhD work was thus to explore the impacts of circadian disruptions on age-related functional decline, both during normal aging and in a fly model of PD-like conditions using well established Drosophila models. Our results confirmed the negative impact of circadian arrhythmia on longevity and revealed that inactivation of a specific circadian gene, Clock (Clk), increases brain oxidative stress levels and accelerates locomotor decline during aging. The latter effect could be associated with Clk function in the clock neurons that drive circadian rest-activity rhythms in constant darkness, and their connections with a specific cluster of dopaminergic neurons. We also observed that chronic jet lag led to an accelerated age-related locomotor decline that could be rescued by adapting the light-dark to the flies’ endogenous period. Finally, our work demonstrated the negative impact of environmentally imposed circadian arrhythmia on age-related locomotor decline in a fly model of PD.

Les acides gras (AGs) sont impliqués dans de nombreuses fonctions biologiques, allant de la composition des membranes cellulaires au stockage de l’énergie, en passant par la biosynthèse des hormones. En terme de santé publique, les conséquences d'une surconsommation en AGs sont très préoccupantes, l’OMS estimant que 2.8 millions de décès par an sont dus à l'obésité et à ses effets secondaires. Si le métabolisme lipidique est relativement bien connu, les mécanismes sous-jacents à la détection et à la préférence pour les AGs restent peu étudiés. Quelques études ont montré que la préférence des mammifères pour les AGs est modifiée par une exposition précoce à ces composés, mais peu de choses sont connues concernant les effets à long terme (intergénérationnel) d'une exposition aux AGs sur leur perception et le comportement alimentaire.La majorité des observations a été réalisée sur les mammifères ; les invertébrés, comme la Drosophile, ont été délaissés malgré leurs avantages (durée du cycle de développement, facilité d'élevage, outils génétiques) et malgré la bonne conservation des acteurs du métabolisme lipidique au cours de l’évolution. Il a été récemment montré dans notre laboratoire, que les larves et les adultes de Drosophila melanogaster sont capables de détecter et de discriminer les AGs en fonction de leur degré d’insaturation, et que leurs préférences vis-à-vis des AGs varient: les larves sont attirées par les AGs insaturés et repoussées par les AGs saturés alors que les adultes sont repoussés par les AGs insaturés et sont indifférents aux AGs saturés. Il a été suggéré que cette évolution des préférences pourrait refléter des besoins métaboliques différents chez la larve et chez l'adulte.Durant ma thèse, j'ai étudié les conséquences, intra- et intergénérationnelles, d’une exposition à un milieu enrichi en AG saturé (acide stéarique = C18:0) ou en AG insaturé (acide oléique = C18:1), sur les préférences larvaires et adultes (choix du site d'oviposition) ainsi que sur différents traits de vie. L'évolution des préférences larvaires et adultes pour ces deux AGs à l’aide de deux procédures de sélection a également été obordée.Les résultats obtenus montrent que, si les processus de sélections ne modifient pas durablement les préférences des individus envers les deux AGs utilisés, le comportement d'individus exposés, soit ponctuellement durant leur développement, soit de manière permanente sur une à dix générations, est affecté par cette exposition. Le choix du site de ponte par les femelles est modifié spécifiquement suite à une exposition au C18:0 ou au C18:1 durant leur développement larvaire. Si l'influence d'une expérience sensorielle précoce sur les préférences alimentaires de l'adulte avait déjà été mise en évidence chez certains mammifères et quelques insectes holométaboles (dont le système nerveux est presque complètement remanié durant la métamorphose), c'est la première fois qu'un tel phénomène est clairement démontré chez la Drosophile. Nous avons pu montrer qu’une exposition permanente à chacun des AGs modifie durablement à la fois les préférences d'oviposition et certains traits de vie majeurs (durée de développement, sex-ratio, fécondité et survie adulte). Ces résultats suggèrent que la Drosophile est capable de s'adapter à des nourritures variées et que cette plasticité, vraisemblablement déterminée génétiquement, pourrait expliquer le succès de cette espèce généraliste. Outre leur intérêt "écologique", ces observations démontrent l'intérêt du modèle Drosophile pour l'étude de la plasticité intra- et inter-générationelle des préférences envers les AGs. / Fatty acids (FAs) are involved in many biological functions, from the cell membrane composition to energy storage, through hormone biosynthesis. The consequences of FAs overconsumption are of great concern in terms of public health since the WHO estimates that 2.8 million annual deaths due to obesity and its side effects. If lipid metabolism is relatively well known, the mechanisms underlying the detection and preference for FAs remain little studied. While some studies have shown that the preference of mammals for FAs is modified by early exposure to these compounds, little is known about FAs long-term effects on both their perception and food preference.The majority of studies have been conducted in mammals, invertebrates being neglected despite the benefits (life cycle, size, ease of breeding, genetic tools) of some models, such as Drosophila, and despite the good conservation of lipid metabolism actors during evolution. It has recently been shown that both Drosophila melanogaster larvae and adults are able to detect and discriminate FAs according to their unsaturation. Moreover, larval and adult preferences are different: the larvae are attracted by unsaturated FAs (UFAs) and repelled by saturated FAs (SFAs) while adults are repelled by the UFAs and indifferent to SFAs. It has been suggested that these preferences change may reflect different metabolic requirements between larvae and adults.During my PhD, I studied the effects of intra -and inter- generational exposure to a medium enriched either with a SFA (stearic acid = C18: 0) either with a UFA (oleic acid = C18: 1) on larval and adult preferences (oviposition site selection) toward these FAs, as well as on different life traits. On the other hand, I tested the evolution of both larval and adult preferences for these two FAs after two selection procedures, using these preferences as a selection criterion.My results show that if the selection processes do not permanently modify the individual preferences for both FAs considered, the behavior of individuals exposed either occasionally during development, either permanently from one to ten generations, is affected by this exposure. In particular, the egg-laying site choice by females is specifically modified by exposure to C18:0 and C18:1 during larval development. If the influence of early sensory experience on food preferences of adults had already been demonstrated in mammals and some holometabolous insects (whose nervous system is almost completely remodeled during metamorphosis), this is the first time that such a phenomenon is clearly demonstrated in Drosophila. On the other hand, continuous exposure to each of these FAs permanently alters both oviposition preferences and major life traits (development time, sex ratio, fecundity and adult survival). These results suggest that Drosophila is able to adapt to different foods, and this plasticity, probably genetically determined, may explain the success of this generalist species. In addition to their ecological interest, these results also demonstrate the usefulness of this model for the study of intra -and inter- generational preferences plasticity towards FAs.

Drosophila are used extensively for genetic, developmental and now molecular biology research. At present, germline transformation of these organisms can only be achieved by microinjection of P-element vectors into the pole cells of young embryos. The technique of microinjection however, requires a delicate touch and is quite laborious. Therefore, the development of a rapid and simple technique was investigated. Electroporation, like microinjection, is a physical means of introducing DNA into a cell and is therefore potentially applicable to all cell types. Electroporation involves the use of an electrical current to create pores in the membrane of a cell. Macromolecules, such as DNA may enter a cell via these pores. Electroporation is a quick, reproducible, and efficient method for transforming cells. Through studies of the survival and permeability of Drosophila melanogaster embryos exposed to electrical currents, it was discovered that although the survival of the embryos decreased steadily as field strength increased, the embryos did not become permeable to a water soluble dye unless a pulse of 10 kV/cm was applied. Few embryos survived this extreme voltage required for dye uptake. Attempts to introduce DNA into dechorionated Drosophila embryos utilizing this technique however, produced no transformants. These results suggested that the remaining protective coatings of the dechorionated embryo were obstructing efficient pore formation, thus preventing DNA penetration. In view of these results, methods to eliminate the wax layer, present between the chorion and vitelline membrane of laid eggs, were examined. Wax removal by detergent solubilization, solvent extraction and melting by heating were investigated, yet did not produce a satisfactory procedure. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The nucleotide sequence of the single Drosophila meianogaster tRNA gene contained in the recombinant plasmid, pDtl20R was determined by the Maxam and Gilbert method. This plasmid hybridizes to the 90 BC site on the Val Drosophila polytene chromosomes, a minor site of tRNA4 hybridization. The Val nucleotide sequence of the tRNA4 gene present in pDtl20R differs at four Val positions from the sequence expected from that of tRNA4 . The four differences occur at nucleotides 16, 29, 41 and 57 in the coding region. Comparison of the DNA sequence of pDtl20R to that of the plasmid pDt92R, which also hybridizes to the 90 BC site, indicates that the Drosophila fragments contained in these two plasmids are either alleles or repeats. The implications of these findings are discussed. An in vitro transcription system was developed from a Drosophila Schneider II cell line. This homologous cell-free extract support specific and accurate transcription of various Drosophila tRNA Val genes. The major product of transcription is a tRNA precursor which is processed to a tRNA sized species. Transfer RNA valine genes originating from different sites on the Drosophila chromosomes are transcribed at different rates. Comparison of the sequences in the internal promoter regions of the various genes indicates that the few differences within the coding regions may not be responsible for the observed difference in the rates of transcription. This conclusion is substantiated by studies with hybrid genes constructed during the course of this work. Preliminary evidence indicates that the Val tRNA gene which is transcribed at the highest rate may be preceded in its 5'-flanking region by a positively modulating sequence. Val The precursor RNAs directed by various tRNA genes are also processed at different rates. Transcription and processing experiments with hybrid genes suggest that nucleotide changes within the coding region, which do not affect the rate of transcription, influence the rate of processing. Time course and competition experiments demonstrate that at least two kinetic steps are required for the formation of a stable transcription complex. Studies with an in vitro constructed mutant missing in nucleotides 51-61 in the tRNA coding region suggests that this deleted region (which is highly conserved in eukaryotic tRNAs) may be involved in the primary interaction required for tRNA gene transcription. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

No description available.

The physiological response to environmental cues involves complex interorgan communication via endocrine factors and hormones, but the underlying mechanisms are poorly understood. In particular, little is known about how animals coordinate systemic growth and developmental timing in response to environmental changes. The morphogen Hedgehog (Hh), which is well studied in tissue patterning and homeostasis, has only recently been implicated in the regulation of lipid and sugar metabolism. Interestingly, Hh is present in systemic circulation in both, ies and mammals. Here, we demonstrate that systemic Hh is produced in the midgut and secreted in association with the lipoprotein particle lipophorin (Lpp) into the hemolymph to mediate the interorgan communication between the midgut and two tissues, the fat body and the prothoracic gland (PG). We show that midgut hh expression is regulated by dietary sugar and amino acid levels, and RNAi-mediated knock-down of circulating Hh leads to starvation sensitivity. We demonstrate that circulating Hh is required to inhibit systemic growth and developmental progression. In insects, developmental transitions are regulated by steroid hormones, which are produced by the PG. Nutritional regulation of growth is, in part, mediated by the Drosophila fat body. Strikingly, canonical Hh pathway components are present in both tissues, the fat body and the PG. To understand the Hh-mediated function during nutritional stress, we ectopically activated or inhibited the Hh signaling pathway specifically in the fat body and the PG. Our results show that systemic Hh exerts its function through these two target tissues. Hh signaling in the fat body is required for survival during periods of nutrient deprivation, and ectopic activation of fat body Hh signaling causes an inhibition of systemic growth. Hh signaling in the PG slows down developmental progression by inhibiting steroid hormone biosynthesis. In conclusion, we propose that the midgut senses the uptake of dietary sugar and amino acids and secrets Hh in association with Lpp particles into circulation to relay information about the feeding status to the developing animal. Therefore, circulating Hh functions as a hormone and signals in an endocrine manner to the fat body and the prothoracic gland to coordinate systemic growth and developmental timing in response to changes in nutrient availability.