No description available.

GcvB est un ARN bactérien conservé de 200 nucléotides, qui régule négativement l’expression de plusieurs gènes impliqués dans l’import et la biosynthèse des acides aminés. Bien que le rôle physiologique de GcvB ne soit pas complètement élucidé, il contribuerait vraisemblablement à équilibrer les ressources nutritionnelles en conditions de croissance rapide. GcvB inhibe la traduction des ARNm cibles en s’appariant avec des séquences à l’intérieur ou en amont du site de liaison du ribosome. Dans cette étude, la caractérisation d’un nouveau locus régulé par GcvB a permis de dévoiler des aspects singuliers du mode de fonctionnement de cet ARN régulateur. Nous avons découvert que GcvB réprime yifK - un gène très conservé, codant pour un transporteur d’acides aminés putatif - en ciblant un élément activateur de la traduction sur l’ARNm. Deux motifs ACA dans la séquence cible sont les déterminants principaux de la fonction activatrice. Le remplacement de l’un ou l’autre avec des triplets aléatoires, provoque une diminution de 10 fois du niveau d’expression de yifK, quelque soit l’allèle de GcvB (délétion ou changement de séquence permettant la reconnaissance de la cible mutante). Il apparait ainsi que l’efficacité de GcvB à réguler négativement sa cible serait liée a sa capacité d’antagoniser l’élément activateur. Lorsque l’activateur est éliminé, l’action de GcvB n’est plus un facteur limitant pour l’expression de yifK. Dans son ensemble, cette étude apporte une meilleure compréhension de la fonction de GcvB et révèle un nouvel aspect du processus d’initiation de la traduction. En plus du contrôle par GcvB, le locus yifK est régulé au niveau transcriptionnel par Lrp (leucine-responsive regulatory protein) et par HdfR (YifA) un régulateur transcriptionnel peu connu qui requerrait le produit du gène adjacent orienté de façon divergente, yifE, pour son expression ou activité. Enfin, la transcription initiée au niveau du promoteur yifK s’étend dans l’opéron d’ARNt argX-hisR-leuT-proM adjacent, donnant lieu à un transcrit primaire qui est à la fois un lARNm et un précurseur des ARNt. Cet ARN chimère est rapidement maturé par l’ARNase E. / GcvB is a conserved 200 nucleotide RNA that downregulates several genes involved in amino acid uptake or biosynthesis in bacteria. The physiological role of GcvB action is not entirely clear, but it is likely aimed at balancing of nutritional resources under fast growth conditions. GcvB inhibits translation of target messenger RNAs by pairing with sequences inside or upstream of ribosome binding sites. In the present study, characterization of a novel GcvB-regulated locus revealed some unique features in the mode of functioning of this regulatory RNA. We found that GcvB represses yifK - a highly conserved locus encoding a putative amino acid transporter - by targeting a translational enhancer element. Two ACA motifs within the target sequence are the main determinants of the enhancer activity. Replacing either of these motifs with random triplets caused up to a 10-fold decrease in yifK expression regardless of the GcvB allele (deleted or suitably modified to recognize the mutated target). It thus appears that GcvB effectiveness as a regulator results from countering the enhancer activity. When the enhancer is removed, GcvB action no longer constitutes a rate-limiting factor for yifK expression. Overall, this study is relevant not only to a better understanding of GcvB function but it also provides insight into an elusive aspect of the translation initiation process. Besides the GcvB control, the yifK locus is regulated at the transcriptional level by the leucine responsive regulator Lrp, and by HdfR (YifA) a poorly known transcriptional regulator, that appears to require the product of the adjacent, divergently oriented gene, yifE, for expression or activity. Transcription initiating at the yifK promoter extends into the adjacent argX-hisR-leuT-proM tRNA operon yielding an unusual primary transcript which both a messenger RNA and a tRNA precursor. This chimeric RNA si rapidly processed by RNAse E.

A influência dos tensoativos lauril sulfato de sódio, cloreto de cetil trimetil amônio e álcool láurico etoxilado com 12 moles de óxido de etileno sobre o stratum corneum da pele de muda das cobras Bothrops jararaca e Spilotis pullatus foi avaliada através das técnicas biofísicas de PAS-FTIR, FT-Raman e DSC. Foram utilizadas soluções dos tensoativos em concentrações acima e abaixo da cmc e tratamentos por 4 e 8 horas (stratum corneum íntegro) e por 12 horas (stratum corneum após a remoção mecânica das camadas superficiais de células com fita adesiva). A presença do lauril sulfato de sódio e do cloreto de cetil trimetilamônio no stratum corneum foi detectada e a principal alteração observada para ambos foi o aumento do conteúdo hídrico do tecido; o álcool láurico etoxilado com 12 moles de óxido de etileno não promoveu aumento do conteúdo hídrico, apresentando ação de extração de lipídeos. A remoção mecânica das camadas superficiais de células com fita adesiva mostrou ser um procedimento útil para tratamento do stratum corneum da pele de muda de cobra, tornando a membrana mais adequada para estudos da ação de promotores de permeação. / The influence of the surfactants sodium lauryl sulfate, cetyl trimethyl ammonium chloride and lauryl alcohol ethoxylate (12 moles ethylene oxide) was evaluated on the stratum corneum of shed snake skin (Bothrops jararaca and Spilotis pullatus) through the biophysical techniques PAS-FTIR, FT-Raman and DSC. The surfactants were used in solutions above and below the cmc and the stratum corneum samples were pre-treated for periods of 4 and 8 hours (whole stratum corneum) and for 12 hours (stratum corneum after tape stripping procedure). The presence of the surfactants sodium lauryl sulfate and cetyl trimetyl ammonium chloride could be detected in the stratum corneum and the main change promoted by both was the increase of the water content of the membrane; the lauryl alcohol ethoxylate (12 moles ethylene oxide) did not promote increase in the hydration but showed some action as far as lipid extraction. The tape stripping procedure showed to be useful for the treatment of the stratum corneum of shed snake skin, making the membrane more adequate than the whole stratum corneum for the studies of the action of permeation enhancers.

<p>Pre-mRNA splicing and alternative pre-mRNA splicing are key regulatory steps controlling geneexpression in higher eukaryotes. The work in this thesis was focused on a characterization of thesignificance of exonic and intronic splicing enhancer elements for pre-mRNA splicing.</p><p>Previous studies have shown that removal of introns with weak and regulated splice sitesrequire a splicing enhancer for activity. Here we extended these studies by demonstrating thattwo "strong" constitutively active introns, the adenovirus 52,55K and the Drosophila Ftzintrons, are absolutely dependent on a downstream splicing enhancer for activity <i>in vitro</i>.</p><p>Two types splicing enhancers were shown to perform redundant functions as activators ofSplicing. Thus, SR protein binding to an exonic splicing enhancer element or U1 snRNP bindingto a downstream 5'splice site independently stimulated upstream intron removal. The datafurther showed that a 5'splice site was more effective and more versatile in activating splicing.Collectively the data suggest that a U1 enhancer is the prototypical enhancer element activatingsplicing of constitutively active introns.</p><p>Adenovirus IIIa pre-mRNA splicing is enhanced more than 200-fold in infected extracts. Themajor enhancer element responsible for this activation was shown to consist of the IIIa branchsite/polypyrimidne tract region. It functions as a Janus element and blocks splicing in extractsfrom uninfected cells while functioning as a splicing enhancer in the context of infected extracts.</p><p>Phosphorylated SR proteins are essential for pre-mRNA splicing. Large amount recombinantSR proteins are needed in splicing studies. A novel expression system was developed to expressphosphorylated, soluble and functionally active ASF/SF2 in <i>E. Coli</i>.</p>

Chromatin insulators are short DNA sequences that, together with enhancers and silencers, orchestrate gene transcription through DNA-protein interactions in eukaryotic genomes. It has been proposed that insulators operate at the chromatin level by generating functionally independent higher-order chromatin domains. Insulators may maintain the integrity of such domains using two properties: blocking enhancer-promoter interactions and blocking heterochromatin spreading. The gypsy insulator of Drosophila was identified as a region of the gypsy retrovirus responsible for the production of tissue-specific mutations in many genes. The Suppressor of Hairy wing [Su(Hw)] protein contains 12 zinc fingers that specifically bind the gypsy insulator. Upon DNA binding, Su(Hw) recruits a second protein, Modifier of Mdg4 67.2 [Mod(mdg4) 67.2], and the interaction of both proteins is required for insulator function in vivo. We have found that three different arrays of gypsy retrovirus insertions in a yellow transgene result in unique yellow phenotypes, showing that the enhancer-blocking activity of the Drosophila gypsy insulators depends on the relative orientation of the gypsy retroviruses on the chromosome. We also observed from transgenic lines with gypsy retrovirus or insulator insertions that interaction of insulators may be regulated by active enhancers according to the relative positions of the insulators flanking the enhancers. Moreover, we show that gypsy insulators can positively modulate yellow activation and result in wild-type levels of expression when placed upstream of enhancers in yellow transgenes in which enhancers are placed out of context by &#;-DNA spacers and fail to reproduce the expression levels of yellow in wings and body cuticle. Our results provide evidence indicating that this phenomenon is independent of the boundary activity. Genetic analysis using mod(mdg4)67.2 mutant lines containing gypsy retrovirus insertions revealed that the gypsy insulator may be placed close to the yellow promoter region and be intimately involved in transcriptional activation and repression. Therefore, we suggest that insulators may also function by mediating long range interactions between enhancers and promoters.

Pre-mRNA splicing and alternative pre-mRNA splicing are key regulatory steps controlling geneexpression in higher eukaryotes. The work in this thesis was focused on a characterization of thesignificance of exonic and intronic splicing enhancer elements for pre-mRNA splicing. Previous studies have shown that removal of introns with weak and regulated splice sitesrequire a splicing enhancer for activity. Here we extended these studies by demonstrating thattwo "strong" constitutively active introns, the adenovirus 52,55K and the Drosophila Ftzintrons, are absolutely dependent on a downstream splicing enhancer for activity in vitro. Two types splicing enhancers were shown to perform redundant functions as activators ofSplicing. Thus, SR protein binding to an exonic splicing enhancer element or U1 snRNP bindingto a downstream 5'splice site independently stimulated upstream intron removal. The datafurther showed that a 5'splice site was more effective and more versatile in activating splicing.Collectively the data suggest that a U1 enhancer is the prototypical enhancer element activatingsplicing of constitutively active introns. Adenovirus IIIa pre-mRNA splicing is enhanced more than 200-fold in infected extracts. Themajor enhancer element responsible for this activation was shown to consist of the IIIa branchsite/polypyrimidne tract region. It functions as a Janus element and blocks splicing in extractsfrom uninfected cells while functioning as a splicing enhancer in the context of infected extracts. Phosphorylated SR proteins are essential for pre-mRNA splicing. Large amount recombinantSR proteins are needed in splicing studies. A novel expression system was developed to expressphosphorylated, soluble and functionally active ASF/SF2 in E. Coli.

The physiological process of cold acclimation protects plants native to the temperate regions of the world from the deleterious effects of low and freezing temperatures. This is achieved by a series of transcriptional, regulatory, and metabolic changes that enable continued growth and survival. Within minutes of exposure to temperatures below ca. 10°C, a complex cascade of transcriptional events is initiated to accomplish these changes. The initial alarm phase favors the rapid induction of a library of stress proteins with protective functions (e.g. COR proteins). This is followed by a cold hardened phase, characterized by maximal freezing tolerance, which continues until either the stress is removed, or the plant's metabolic and/or developmental state can no longer support maximal resistance. We have studied some of the important transcription factors and transcriptional changes associated with the initial alarm and later hardened phases of cold acclimation in the herbaceous annual Arabidopsis thaliana and the woody perennial Populus spp. We confirmed the functionality of the CBF-mediated signaling cascade in Poplar overexpressing AtCBF1, but noted that regulon composition and endogenous poplar CBF ortholog induction appeared to be tissue-specific. The lack of statistically significant DRE enrichment in the Poplar AtCBF1 regulons led us to investige cis-element abundance in the cold-associated transcription factor regulons of publicly available microarray data from Arabidopsis, leading to the development of a gene voting method of microarray analysis that we used to test for regulatory associations between transcription factors and their downstream cis-elements and gene targets. This analysis resulted in a new transcriptional model of the ICE1-mediated signaling cascade and implicated a role for phytochrome A. Application of this same method to microarray data from arabidopsis leaves developed at low temperature allowed us to identify a new cis-element, called DDT, which possessed enhancer-blocking function during the alarm stage of cold stress, but was enriched in the promoters of genes upregulated during the later cold hardened stages. As leaf growth and development at low temperature correlated with the enhancement freeze tolerance in Arabidopsis, we compared the transcriptomes of rapidly growing and fully grown poplar leaves at night (when both low temperatures and PhyA status might play important roles in nature), in the hopes of comparing this data with that of cold-treated leaves in the future. We identified the nocturnal mode of leaf growth in Populus deltoides as predominantly proliferative as opposed to expansive, and potentially linked to cellular carbohydrate status.

No description available.

GcvB is a conserved 200 nucleotide RNA that downregulates several genes involved in amino acid uptake or biosynthesis in bacteria. The physiological role of GcvB action is not entirely clear, but it is likely aimed at balancing of nutritional resources under fast growth conditions. GcvB inhibits translation of target messenger RNAs by pairing with sequences inside or upstream of ribosome binding sites. In the present study, characterization of a novel GcvB-regulated locus revealed some unique features in the mode of functioning of this regulatory RNA. We found that GcvB represses yifK - a highly conserved locus encoding a putative amino acid transporter - by targeting a translational enhancer element. Two ACA motifs within the target sequence are the main determinants of the enhancer activity. Replacing either of these motifs with random triplets caused up to a 10-fold decrease in yifK expression regardless of the GcvB allele (deleted or suitably modified to recognize the mutated target). It thus appears that GcvB effectiveness as a regulator results from countering the enhancer activity. When the enhancer is removed, GcvB action no longer constitutes a rate-limiting factor for yifK expression. Overall, this study is relevant not only to a better understanding of GcvB function but it also provides insight into an elusive aspect of the translation initiation process. Besides the GcvB control, the yifK locus is regulated at the transcriptional level by the leucine responsive regulator Lrp, and by HdfR (YifA) a poorly known transcriptional regulator, that appears to require the product of the adjacent, divergently oriented gene, yifE, for expression or activity. Transcription initiating at the yifK promoter extends into the adjacent argX-hisR-leuT-proM tRNA operon yielding an unusual primary transcript which both a messenger RNA and a tRNA precursor. This chimeric RNA si rapidly processed by RNAse E.

The study examined interactions between morogo plants, arbuscular mycorrhizal fungi (AMF) and Fusarium species. Morogo refers to traditional leafy vegetables that, together with maize porridge, are dominant staple foods in rural areas of the Limpopo Province such as the Dikgale Demographic Surveillance Site (DDSS). Morogo plants grow either as weeds (often among maize), occur naturally in the field or are cultivated as subsistence crops by rural communities. Botanical species of morogo plants consumed in the DDSS were determined. Colonisation of morogo plant roots by AMF and Fusarium species composition in the immediate soil environment were investigated in four of eight DDSS subsistence communities, Isolated AMF were shown to belong to the genera Acaulospora and Glomus. Twelve Fusarium species were isolated from soil among which Fusariurn verticilliodes and Fusarium proliferaturn occurred predominantly. Greenhouse pot trials were conducted to examine the effect of AMF on morogo plant growth (cowpea; Mgna unguiculata) and Fusarium proliferatum levels in soil, Interaction between plants and AMF, as well as tripartite interactions of cowpea plants, AMF and Fusarium proliferatum were investigated. Non-inoculated cowpea plants served as controls for the following inoculations of cowpea in pots: (i) Fusarium proliferatum; (ii) commercial AMF from Mycoroot (PTY) Ltd. (a mixture of selected indigenous Glomus spp referred to commercial AMF for the purpose of this study); (iii) indigenous AMF obtained from DDSS soil (referred to iocal AMF for the purpose of this study); (iv) commercial AMF plus Fusarium proliferatum; (v) local AMF plus Fusariurn proliferatum. Results showed reduced root colonization by local as well as commercial AMF when Fusarium proliferatum were present. Local AMF significantly enhanced cowpea growth while commercial AMF apparently reduced the level of Fusarium proliferatum in the rhizosphere and surrounding soil. Results suggest that AMF may have potential as biological growth enhancers and bioprotective agents against Fusarium proliferatum. / Thesis (M. Environmental Science (Water Science))--North-West University, Potchefstroom Campus, 2007.