Call number: LD2668 .T4 1978 G37 / Master of Science

There are a vast number of factors that influence nucleosome formation, and consequently gene regulation. These factors include histone modifications, nucleotide composition, transcriptional region elements, and specific nucleotide motifs, among others. Although the amount we know now is limited, we are creating new techniques and discoveries to assist us in continued understanding of chromatin. To make a significant contribution to the field of chromatin, I conducted two hypothesis driven sets of experiments that address the topic of chromatin structure. First, I created a technique for tissue specific nucleosome isolation with the goal of observing the effect of single nucleotide polymorphisms (SNPs) on nucleosome formation. Second, I created and tested a method to recover lost in vitro nucleosome reconstitution data, which can improve this type of data, commonly used for observing nucleosome positioning. The first experiment needs a more specific antibody to complete the last step and function as designed. The second experiment shows that our nucleosome recovery method, when applied conservatively, can recover 90% of the lost nucleosome data.

Reactive oxygen species (ROS) such as H2O2 can damage cellular components and are formed as a byproduct of mitochondrial oxidative metabolism. Studies using the nematode C. elegans have found that increasing ROS during development or early adulthood can extend lifespan, while increasing ROS during later adulthood normally decreases lifespan. NADPH provides the reducing power for several cellular antioxidants and is synthesized in a two-step reaction from NAD+ with the first step being catalyzed by NAD kinase (NADK). In this study, it questioned the effects of knocking down C. elegans cytoplasmic NADK, nadk-1 globally or in a neuron or intestine-specific manner starting from early development on oxygen consumption and ROS levels. It was hypothesized that knocking down the cytoplasmic NADK in C. elegans alters NADP+/NADPH ratios. This led to the prediction that ROS and O2 consumption levels would be decreased, and thioredoxin reductase activity levels will be increased in global and tissue-specific knockdowns. Three different strains of nematodes were used for this study: N2 wild type, intestinal RNAi sensitive strain VP303, and neuronal RNAi sensitive strain TU3401. The nadk-1 RNAi was introduced to the nematodes through their food source. To measure ROS levels, nematodes were treated with a tert-butylhydroperoxide insult to induce ROS generation, along with H2DCFDA dye to fluoresce when oxidized by ROS, pipetted into a 96-well plate, and ran through a microplate reader. For the oxygen consumption measurements, the nematodes were run through a Clark O2 electrode. Finally, Thioredoxin Reductase activity levels were measured using an assay kit that would inhibit testing groups thioredoxin reductase to only measure glutathione reductase levels and subtract it from uninhibited testing groups. The nematodes were pipetted into a 96-well plate and ran through a microplate reader. All experiments were normalized to average protein levels of C. elegans and analyzed using a t-test. Whole body knockdown of nadk-1 to decrease cytoplasmic NADPH levels decreased oxygen consumption and tert-butyl hydroperoxide-stimulated ROS levels, which was phenocopied by intestine-specific or neuron-specific knockdown. Thioredoxin reductase measurements following nadk-1 knockdown showed a trend toward increased activity. These results establish that nadk-1 does have antioxidant properties but does not extend lifespan as is the case of other NADPH generating enzyme knockdowns. .

No description available.

Development of the central nervous system requires the precise coordination of intrinsic genetic programs to instruct cell fate, synaptic connectivity and function. The MEF2 family of transcription factors (TFs) plays many essential roles in neural development; however, the mechanisms of gene regulation by MEF2 in neurons remain unclear. This dissertation focuses on the molecular mechanisms by which MEF2 binds to the genome, activates enhancers, and regulates gene expression within the developing nervous system. We find that one MEF2 family member in particular, MEF2D, is an essential regulator of the development and function of retinal photoreceptors, the primary sensory neurons responsible for vision. Despite being expressed broadly across many tissues, in the retina MEF2D binds to retina-specific enhancers and regulates photoreceptor-specific transcripts, including critical retinal disease genes. Functional genome-wide analyses demonstrate that MEF2D achieves tissue-specific binding and action through cooperation with a retina-specific TF, CRX. CRX recruits MEF2D away from canonical MEF2 binding sites by promoting MEF2D binding to retina-specific enhancers that lack a strong consensus MEF2 binding sequence. MEF2D and CRX then synergistically co-activate these enhancers to regulate a cohort of genes critical for normal photoreceptor development. These findings demonstrate that MEF2D, a broadly expressed TF, contributes to retina-specific gene expression in photoreceptor development by binding to and activating tissue-specific enhancers cooperatively with CRX, a tissue-specific co-factor. A major unresolved feature of MEF2D function in the retina is that the number of MEF2D binding sites significantly exceeds the number of genes that are dependent on MEF2D for expression. We investigated causes of this discrepancy in an unbiased manner by characterizing the activity of MEF2D-bound enhancers genome-wide. We find that many MEF2D-bound enhancers are inactive. Furthermore, less than half of active MEF2D-bound enhancers require MEF2D for activity, suggesting that significant redundancies exist for TF function within enhancers. These findings demonstrate that observed TF binding significantly overestimates direct TF regulation of gene expression. Taken together, our results suggest that the broadly expressed TF MEF2D achieves tissue specificity through competitive recruitment to enhancers by tissue-specific TFs and activates a small subset of enhancers to regulate genes.

Hereditary myopathy with lactic acidosis (HML, OMIM#255125) is an autosomal recessive disorder which originates from Västerbotten and Ångermanland in the Northern part of Sweden. HML is characterized by severe exercise intolerance which manifests with tachycardia, dyspnea, muscle pain, cramps, elevated lactate and pyruvate levels, weakness and myoglobinuria. The symptoms arise from malfunction of the energy metabolism in skeletal muscles with defects in several important enzymes involved in the TCA cycle and the electron transport chain. All affected proteins contain iron-sulfur (Fe-S) clusters, which led to the suggestion that the disease was caused by malfunctions in either the transportation, assembly or processing of Fe-S clusters. The aim of my thesis was to identify the disease causing gene of HML and to investigate the underlying disease-mechanisms. In paper I we identified a disease-critical region on chromosome 12; a region containing 16 genes. One of the genes coded for the Fe-S cluster assembly protein ISCU and an intronic base pair substitution (g.7044G>C) was identified in the last intron of this gene. The mutation gave rise to the insertion of intron sequence into the mRNA, leading to a protein containing 15 abberant amino acids and a premature stop. In paper II we investigated why a mutation in an evolutionary well conserved protein with a very important cellular role, which in addition is expressed in almost all tissues, gives rise to a muscle-restricted phenotype. Semi-quantitative RT-PCR analysis showed that the mutant transcript constituted almost 80% of total ISCU mRNA in muscle, while in both heart and liver the normal splice form was dominant. We could also show that, in mice, complete absence of Iscu protein was coupled with early embryonic death, further emphasizing the importance of the protein in all tissues. These data strongly suggested that tissue-specific splicing was the main mechanism responsible for the muscle-specific phenotype of HML. In paper III the splicing mechanisms that give rise to the mutant ISCU transcript was further investigated. We identified three proteins; PTBP1, IGF2BP1 and RBM39, that could bind to the region containing the mutation and could affect the splicing pattern of ISCU in an in vitro system. PTBP1 repressed the inclusion of the intronic sequence, while IGF2BP1 and RBM39 repressed the total ISCU mRNA level though the effect was more pronounced for the normal transcript. Moreover, IGF2BP1 and RBM39 were also able to reverse the effect of PTBP1. IGF2BP1, though not a splicing factor, had higher affinity for the mutant sequence. This suggested that the mutation enables IGF2BP1 binding, thereby preventing the PTBP1 induced repression seen in the normal case. In conclusion, we have determined the genetic cause of HML, identifying a base pair substitution in the last intron of the ISCU gene that gives rise to abnormally spliced transcript. The muscle-specific phenotype was also analyzed and tissue-specific splicing was identified as the main disease-mechanism. Furthermore, nuclear factors with ability to affect the splicing pattern of the mutant ISCU gene were identified. This work has thoroughly investigated the fundamental disease mechanisms, thus providing deeper understanding for this hereditary myopathy.

Made available in DSpace on 2015-03-26T13:36:46Z (GMT). No. of bitstreams: 1 texto completo.pdf: 3795682 bytes, checksum: 12bec11cea96f50faf5ddfa5a06024dc (MD5) Previous issue date: 2007-03-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The soybean SBP2 (sucrose binding protein) promoter is capable to drive vascular tissue-specific expression of reporter genes in tobacco transgenic lines. This vascular-specific activity of the SBP2 promoter is confined to a distal region (-2000 to -700 sequences) designated CRD-A (cis-regulatory domain-A). Here, we first confirmed the tissue-specific activity of CRD-A through gain-of-function experiments, in which the CRD-A sequences were directly fused to 5´end of GUS cDNA, -136pSBP2-GUS and -92pSBP2-GUS constructs. In tobacco, CRD-A was able to reduce GUS activity in all organs analyzed, recapitulating in some cases the tissue-specific pattern of the full promoter. In addition, CRD-A promoted GUS transcription in the absence of the proximal TATA-containing region, which suggests that CRD-A may contain cis-regulatory elements to sustain basal transcription. In fact, this region (-2000 a -700) harbors several TATA box-like sequences, positions -790, -783 and -761, that potentially may function as alternative TATA boxes. To delimit the cis-regulatory elements responsible for the tissue-specific activity of SBP2 promoter, the -2000 to -700 sequence was divided into five fragments, which were fused to -92pSBP2-GUS construct and used to obtain transgenic lines. Histochemical analysis revealed that all the CRDA sub-fragments reduced the SBP2 promoter activity, as their fusion to the 5 end of -92pSBP2 altered its constitutive expression pattern. These results identified the presence of several potentially cis-regulatory domains. The region encompassing the sequences -1765 to -945 may contain strong shoot apex expression-repressing elements, capable to totally abolish expression, whereas the region -944 to -705 may harbor weaker repressing elements that restricted GUS expression to the vascular tissue. We also found several root expression silencers, operating in the root meristem (-1765 to -705) and in the root elongation zone (-1765 to -1485 and -1211 to -945). Furthermore, the region delimited by positions -1765 to -1485 also exhibited a strong root expressionrepressing element whose effect may be attenuated by cis-regulatory elements present in the -1485 to -705 region. Finally, a cis-element that confines GUS expression to the inner phloem of stem was identified in the region delimited by positions -1485 to -1212. The function of the identified cis-elements was evaluated through electrophoretic mobility shift assay (EMSA) that revealed sequence-specific interactions between putative transfactors from soybean and tobacco nuclear extracts and the -1765/-1485 (fragII) fragment from GmSBP2. To determine whether the SBP2 protein accumulation correlated with the tissuespecific promoter activity, a SBP2-GFP fusion was expressed in tobacco transgenic lines under the control of SBP2 promoter. Fluorescence analysis revealed that the SBP2 protein was, indeed, located in the vascular tissue, which was consistent with SBP2 promoter activity and the involvement of SBP2 in physiological process dependent of sucrose translocation. / O promotor do gene SBP2 (sucrose binding protein) de soja é capaz de dirigir a expressão tecido vascular-específica de genes repórteres em plantas transgênicas de tabaco. Esta regulação se deve à presença de domínios cisregulatórios distais (CRD-A, posição -2000 a -700) presentes no promotor. Neste trabalho, a atividade tecido-específica de CRD-A foi confirmada por meio de experimentos de ganho-de-função, nos quais o fragmento CRD-A foi diretamente fusionado ao gene repórter GUS e às construções -136pSBP2-GUS e -92pSBP2-GUS e sua atividade avaliada no sistema heterólogo de tabaco. CRDA foi capaz de reduzir a atividade de GUS em todos os órgãos analisados, restaurando, em alguns casos, o padrão tecido-específico do promotor completo. Além disso, observou-se que CRD-A é capaz de promover a transcrição de GUS, independente de promotor mínimo, indicando a presença de cis-elementos capazes de promoverem a transcrição basal. De fato, nessa região (-2000 a -700) foram identificados vários elementos TATA box, localizados nas posições -790, -783 e -761, que podem potencialmente funcionar como TATA boxes alternativos. No intuito de delimitar os cis-elementos responsáveis pelo padrão tecido-específico do promotor SBP2, a seqüência -2000 a -700 foi dividida em cinco fragmentos, os quais foram inseridos na construção -92pSBP2-GUS, e utilizados para obtenção de plantas transgênicas. Análises histoquímicas revelaram que todos os fragmentos foram capazes de reduzir a atividade do promotor SBP2, uma vez que sua inserção na extremidade 5 de -92pSBP2 alterou o padrão de expressão constitutiva do mesmo. Com base nestes resultados, diversas regiões potencialmente regulatórias foram identificadas. A região compreendida entre -1765 e -945 deve conter fortes elementos repressores para o ápice caulinar, capazes de abolir totalmente a atividade do promotor, enquanto que a região entre -944 e -705 demonstrou conter elementos repressores mais fracos, que restringiram a expressão ao tecido vascular. Foram encontrados vários elementos silenciadores para a raiz, tanto para o meristema radicular (região entre -1765 e -705), quanto para a zona de alongamento (de -1765 a -1485 e de -1211 a -945). Além disso, a região de -1765 a -1485 também apresenta um forte repressor para raiz, cujo efeito deve ser atenuado por ciselementos presentes entre -1485 e -705. Por fim, foi identificado um elemento responsável por restringir a expressão apenas ao floema interno no caule, na região entre -1485 e -1212. A funcionalidade dos cis-elementos identificados foi avaliada através do ensaio de mudança na mobilidade eletroforética (EMSA), tendo sido observada a interação seqüência-específica entre possíveis transfatores presentes em extratos nucleares de soja e de tabaco e o fragmento -1765/-1485 (fragII) de GmSBP2. A fim de verificar se o acúmulo da proteína SBP2 correlaciona-se com a atividade do promotor em tecidos específicos, foi obtida a proteína quimérica SBP2-GFP, sob o controle do promotor SBP2, em tabacos transgênicos. A análise de fluorescência revelou que a proteína SBP2 está, de fato, localizada na região de tecido vascular, consistente com o padrão de atividade do gene repórter e com seu envolvimento nos processos fisiológicos dependentes de translocação de sacarose.

Master of Science / Department of Entomology / Kristopher S. Silver / Kun Yan Zhu / The yellow fever mosquito, Aedes aegypti, is a significant vector of several viral diseases, including Zika, dengue fever, yellow fever, and chikungunya. Since vaccines are not currently available for these viruses, control of the disease vectors by using insecticides is the most common practice for preventing disease. As a result, Ae. aegypti has developed resistance against many of the most commonly used insecticides, including organophosphates and pyrethroids. The rise in resistance in vector mosquitoes requires the search for new control strategies, such as RNA interference (RNAi), to manage mosquito populations. Vacuolar H[sup plus]+-ATPase (V-ATPase), a multi-subunit enzyme involved in many cellular processes, including membrane energization, acidification of organelles, and entry of dengue virus into the cytoplasm, is a potential target for RNAi, though little is known about its genetic structure or expression patterns in Ae. aegypti. In this study, I performed genome-wide surveys to identify the genes encoding different subunits of the V-ATPase protein complex, partially characterized the molecular properties and expression patterns of selected V-ATPase subunit genes, and tested the feasibility of using oral-based delivery of nanoparticles formed from double-stranded RNA (dsRNA) and chitosan to suppress the expression of selected V-ATPase subunit genes in Ae. aegypti. My genome-wide surveys revealed that Ae. aegypti V-ATPase consists of 13 different subunits (A, B, C, D, E, F, G, H, a, c, c”, d, e) encoded by 14 genes. Analysis of exon-intron arrangements for each gene demonstrated that each V-ATPase subunit gene has between one (subunit c) and 12 (subunit C) exons, with most genes (11) having 3 to 6 exons. Subsequent phylogenetic analysis of the deduced amino acid sequences of each subunit showed that V-ATPase subunits A, B, C, F, G, H, and a exhibited high levels of conservation among all the examined species, but subunits D, E, c, c”, d, and e showed high conservation only among dipteran species. Analysis of the expression profiles in different tissues and developmental stages of three specific V-ATPase subunits (A, D, and H) showed that whereas the expression of these genes varied between tissues and developmental stages, the patterns of expression of subunits A, D, and H were very similar. The highest mRNA expression level was observed in Malpighian tubules in fourth-instar larvae. Interestingly, expression of subunits A, D, or H in different tissues of adults was highest in male hindgut versus Malpighian tubules in females. Feeding mosquito larvae with chitosan nanoparticles made with dsRNA complementary to subunits A, D, or H resulted in significant suppression of mRNA transcript levels of each of these subunits. Peak suppression of V-ATPase A, D, or H transcripts occurred on the fifth day, where the gene transcript level was suppressed by 66.0, 27.3, or 70.4%, respectively, as compared with those of the control. Additionally, feeding of dsRNA/chitosan nanoparticles targeting subunit D caused mortality starting on day 3, with cumulative larval mortality reaching 14.8% on the sixth day. These results suggest that oral delivery of dsRNA/chitosan nanoparticles can substantially suppress target gene expression in Ae. aegypti larvae. However, increasing RNAi efficiency in targeting V-ATPase subunit genes in mosquito larvae appears to be necessary in order to obtain higher larval mortality using oral delivery of dsRNA/chitosan nanoparticles.

Les anthocyanes constituent une composante importante de la qualité des fruits rouges, particulièrement pour le raisin noir, et pour la couleur des vins qui en dérivent. La biosynthèse des anthocyanes est déterminée par des facteurs génétiques et affectée par des facteurs environnementaux, notamment la lumière. Pour la plupart des cépages, la pellicule des baies est le tissu principal ou exclusif accumulant les anthocyanes. Notre travail analyse les effets de la lumière et du génotype sur la biosynthèse des anthocyanes. Le matériel végétal que nous avons utilisé était constitué de cépages teinturiers, qui accumulent des anthocyanes dans la pellicule et dans la pulpe, et de populations hybrides. Dix-neuf anthocyanes mono-glycosylées ont été identifiés dans sept tissus colorés du cépage teinturier Yan-73 (V. vinifera). La composition et la concentration en anthocyanes varient selon les organes et le stade de développement. Les anthocyanes de la pellicule incluent principalement les dérivés de la malvidine, alors que les dérivés de la péonidine sont les plus abondants dans la pulpe. Les dérivés de malvidine et de péonidine prédominent dans le rachis, les pédicelles des baies, les limbes foliaires, les nervures et les pétioles, et dans l‘écorce à la base du cep. Les concentrations des anthocyanes dans les pellicules, la pulpe, le rachis et les pédicelles augmentent rapidement à partir de la véraison, ou une semaine après la véraison. Elles sont élevées dans les limbes foliaires jeunes et sénescents, et faibles dans les feuilles en expansion et les feuilles adultes. Elles ne varient pas beaucoup au cours de la saison dans les nervures et les pétioles, ou dans l‘écorce. Les cépages ont pu être distingués selon leur réponse à des traitements d‘exclusion de la lumière imposés de la nouaison à la maturité en entourant les grappes par des boîtes opaques. Le cépage non teinturier ―Gamay‖ à peau rouge accumule très peu d‘anthocyanes dans la pellicule en absence de lumière. Au contraire, les cultivars teinturiers, ‗Yan-73‘ et ‗Gamay Fréaux‘ (mutant teinturier de ‘Gamay’) accumulent des anthocyanes aussi bien dans la pellicule que dans la pulpe et présentent une coloration sombre même en absence de lumière... / Anthocyanins are an important component of red fruit quality, especially for grape berries, and for the color of the wines made from these berries. Anthocyanin biosynthesis is determined by genetic factors and affected by environmental factors, especially sunlight. For most grape cultivars, the berry skin is the main or only tissue accumulating anthocyanins. The present work investigates the effects of light and grape genotype on anthocyanin biosynthesis. We used teinturier grape cultivars (also called dyers, which synthesize anthocyanins in both skin and pulp) and its hybrid population as plant materials. Nineteen monoglucoside anthocyanins were identified in seven colored tissues of the teinturier cultivar Yan-73 (V. vinifera). Anthocyanin composition and concentration varied among grape organs and with developmental stage. Skin anthocyanins were mainly composed of malvidin derivatives, while peonidin derivatives were the most abundant anthocyanins in the pulp. Both malvidin and peonidin derivatives were predominant in rachis, berry pedicels, leaf lamina, vein and petioles, and living bark at the base of the shoot. The concentration of anthocyanins in berry skin, pulp, rachis and pedicels rapidly increased starting from veraison on, or one week after veraison. Anthocyanin concentrations were high in young and senescing leaf lamina and low in expanding and mature lamina. They did not vary much throughout the growing season in the leaf veins and petiole tissues, or in the bark. Grape cultivars could be distinguished by their response to sunlight exclusion treatments imposed from fruit set to maturity by surrounding the clusters with opaque boxes. The red-skinned non-teinturier cultivar Gamay could barely accumulate anthocyanins in berry skin under sunlight exclusion. In contrast, teinturier cultivars, Yan-73 and Gamay Fréaux (teinturier mutant of Gamay) accumulated anthocyanins in both skin and pulp and showed dark color even under sunlight exclusion...

Many of the cutting-edge innovations on the CRISPR/Cas9 system involve the use of complex sgRNA libraries. For example, such libraries have recently been used in functional genomics applications to screen for specific genes in both cell lines and living organisms, as well as in subcellular imaging to fluorescently label chromatin in living cells. However, chemically synthesizing customized libraries can be cost prohibitive, requires precise genomic information, and can takes several weeks. We developed a rapid and efficient enzymatic method to generate sgRNA libraries from arbitrary DNA substrates, significantly reducing the cost of library generation. A type IIS restriction enzyme binding site was incorporated directly into the sgRNA scaffold sequence without affecting the catalytic properties of the Cas9 complex, resulting in a simple method that generates high fidelity sgRNA libraries. Additionally, the library is constructed on the surface of streptavidin coated magnetic beads and a strand displacing polymerase is used to elute DNA from the beads, reducing the loss of material and simplifying purification between steps. We used this method to generate two sgRNA libraries. The first targets the entire E. coli genome, and the second targets genes expressed in the developing zebrafish heart. This second library will be used in a forward genetic screen in zebrafish to identify genes underlying heart defects. These complex sgRNA libraries show the utility of the method in generating libraries from DNA from any species, ranging from prokaryotes like bacteria, to eukaryotes including plants and animals.