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<i>In silico</i> Studies of Early Eukaryotic EvolutionCanbäck, Björn January 2002 (has links)
<p>A question of great interest in evolutionary biology is how and why the eukaryotic cell evolved. Several hypotheses have been proposed, ranging from an early emergence of a primitive eukaryotic cell, to various fusion models like the hydrogen hypothesis. Within this context, relevant bacterial gene families and genomes are examined in this thesis.</p><p>The mitochondrion, the energy producing organelle in the eukaryotic cell, is generally believed to be of α-proteobacterial descent. To learn more about mitochondrial evolution, and therefore eukaryotic evolution, the genomes of the α-proteobacteria <i>Bartonella henselae</i> and <i>Bartonella quintana</i> were sequenced. Software was developed and used in the annotation of these genomes.</p><p>Several gene products of nuclear-encoded genes are exported to the mitochondrion. Many of these genes are thought to originate from the emerging organelle. An analysis of the more than 400 genes encoding proteins targeted to the yeast mitochondrion indicates that one set of genes originated from the bacterial symbiont, while the eukaryotic host contributed another. Thus, the mitochondrial proteome has a dual origin.</p><p>The hydrogen hypothesis postulates that the glycolytic genes belong to the group of genes that were transferred from symbiont to host. These genes are thoroughly analysed from a phylogenetic perspective. Contrary to the predictions of the hydrogen hypothesis, the results provide no support for a close relationship between nuclear genes encoding glycolytic enzymes and their α-proteobacterial homologs. </p><p>In general, it is thought that intensive gene transfers may limit our ability to reconstruct gene and species evolution, especially among microbes. A phylogenetic analysis of a large cohort of genes from the AT-rich genome of the γ-proteobacterium <i>Buchnera aphidicola</i> (Sg) resulted in a high fraction of atypical tree topologies, previously interpreted as horizontal gene transfers. By applying methods that accommodate for asymmetric nucleotide substitutions, it is shown that many well-supported gene topologies are drastically altered, so that they now agree with the rRNA topology. The conclusion is that atypical topologies may not necessarily be evidence for horizontal gene transfers. </p>
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Structural and functional studies of glycoside hydrolase family 12 enzymes from Trichoderma reesei and other cellulolytic microorganismsSandgren, Mats January 2003 (has links)
<p>Cellulose is the most abundant organic compound on earth. A wide range of highly specialized microorganisms, have evolved that utilize cellulose as carbon and energy source. Enzymes called cellulases, produced by these cellulolytic organisms, perform the major part of cellulose degradation.</p><p>In this study the three-dimensional structure of four homologous glycoside hydrolase family 12 cellulases will be presented, three fungal enzymes; <i>Humicola grisea</i> Cel12A, <i>Hypocrea schweinitzii </i>Cel12A, <i>Trichoderma reesei</i> Cel12A, and one bacterial; <i>Streptomyces sp. 11AG8</i> Cel12A. The structural and biochemical information gathered from these and 15 other GH family 12 homologues has been used for the design of variants of these enzymes. These variants have biochemically been characterized, and thereby the positions and the types of mutations have been identified responsible for the biochemical differences between the homologous enzymes, e.g., thermal stability and activity. The three-dimensional structures of two <i>T. reesei</i> Cel12A variants, where the mutations have significant impact on the stability or the activity of the enzyme have been determined. Four ligand complex structures of the WT <i>H. grisea</i> Cel12A enzyme, that have made it possible to characterize the interactions between substrate and enzyme, have also been determined. </p><p>The structural and biochemical studies of these closely related GH family 12 enzymes, and their variants, have provided insight on how specific residues contribute to protein thermal stability and enzyme activity. This knowledge can in the future serve as a structural toolbox, i.e., to design Cel12A enzymes with specific properties and features by introducing subtle changes in structural components of the enzymes. These can then be utilized to develop new industrial products or fine-tune enzymes in already existing applications.</p>
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Cyanobacterial Hydrogen Metabolism : Transcriptional Regulation of the Hydrogenases in Filamentous StrainsAxelsson, Rikard January 2003 (has links)
<p>Cyanobacteria are a heterogeneous group of phototrophic microorganisms. Many cyanobacteria have the capacity to fix atmospheric nitrogen. During the process of nitrogen fixation, molecular hydrogen is produced. Three enzymes are directly involved in hydrogen metabolism in cyanobacteria. A nitrogenase, evolving hydrogen during nitrogen-fixation, an uptake hydrogenase, recycling the hydrogen produced by nitrogenase, and a bidirectional hydrogenase that has the capacity to both take up and produce hydrogen. The main objective in this thesis was to examine the transcriptional regulation of both the uptake and the bidirectional hydrogenase in filamentous cyanobacteria.</p><p>The transcriptional regulation of the uptake hydrogenase was demonstrated to be influenced by external conditions in <i>Nostoc muscorum</i> and <i>Nostoc punctiforme</i>. Nickel, molecular hydrogen, and anaerobic conditions all induced the relative amount of uptake hydrogenase transcript. In addition, a transcript could be detected in nitrogen-fixing, but not in non-nitrogen fixing conditions.</p><p>The transcriptional regulation of the bidirectional hydrogenase in <i>N. muscorum</i> and <i>Anabaena</i> PCC 7120 was also examined. The relative amount of transcript from the bidirectional hydrogenase in both strains was demonstrated to increase during anaerobic conditions. Moreover, experiments using <i>N. muscorum</i> demonstrated that addition of nickel also increase the amount of transcript. However, no change in the relative amount of transcript from the bidirectional hydrogenase could be observed by additional hydrogen or during a shift from non-nitrogen fixing to nitrogen fixing conditions.</p><p>The genes responsible for maturation of the hydrogenase were identified, cloned and sequenced in <i>N. punctiforme</i>. The transcription of the genes was examined and all genes were located on a single transcript. Like the uptake hydrogenase, a transcript could be detected under nitrogen-fixing but not under non-nitrogen fixing conditions. </p><p>Initial studies, using microarrays, were used to analyse and compare the transcription of a large set of <i>Anabaena</i> PCC 7120 genes under non-nitrogen and nitrogen-fixing conditions. Both up- and down-regulated genes could be identified.</p><p>This thesis advances the knowledge about the transcriptional regulation of the hydrogenases in filamentous cyanobacteria and can be used as a platform for further experiments aiming at a modified hydrogen metabolism.</p>
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EXT Proteins in Heparan Sulfate BiosynthesisBusse, Marta January 2006 (has links)
<p>Heparan sulfate (HS) is a long unbranched polysaccharide composed of alternating glucosamine and hexuronic (glucuronic or iduronic) acid residues. Modification by sulfate groups in various positions generates a highly heterogeneous molecule. HS is synthesized as a proteoglycan by virtually all cells, and play pivotal functions in signaling and developmental patterning, but also in pathogenic events such as tumor metastasis and microbial adhesion.</p><p>This thesis deals with the properties of enzymes involved in HS chain elongation. Polymerization of the HS chain is believed to be catalyzed by the EXT family of proteins. In humans, the EXT family consists of five members: EXT1, EXT2, EXTL1, EXTL2 and EXTL3; their respective functions in HS biosynthesis are not fully understood. </p><p>In this study, for the first time, successful in vitro HS polymerization on oligosaccharide acceptor substrates was demonstrated, using recombinant EXT1 and EXT1/EXT2 complex. EXT1 formed longer chains than EXT1/EXT2 and their mechanisms of sugar incorporation were different. </p><p>Suppression of EXT1 or EXT2 expression by siRNA in a human cell line resulted in reduction of HS chain length. In contrast, cells transfected with EXTL3 siRNA produced longer HS chains. Overexpression of soluble EXT1, alone or co-expressed with EXT2, resulted in increased chain length, whereas overexpression of soluble EXT2 or EXTL3 has no detectable effect on HS chain elongation. </p><p>Structural analysis of HS from fibroblasts isolated from mice with a hypomorphic mutation in Ext1 showed that they produced significantly shorter HS chains then the wild-type fibroblasts (20 and 70 kDa, respectively). The disaccharide composition of the HS produced by the mutant cells was virtually indistinguishable from that of the wild-type HS, however, the mutant HS chains contained higher proportions of unmodified regions. Mutant cells responded less efficiently than wild-type cells to low concentrations of FGF2, as analyzed by ERK phosphorylation assay.</p>
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In silico Studies of Early Eukaryotic EvolutionCanbäck, Björn January 2002 (has links)
A question of great interest in evolutionary biology is how and why the eukaryotic cell evolved. Several hypotheses have been proposed, ranging from an early emergence of a primitive eukaryotic cell, to various fusion models like the hydrogen hypothesis. Within this context, relevant bacterial gene families and genomes are examined in this thesis. The mitochondrion, the energy producing organelle in the eukaryotic cell, is generally believed to be of α-proteobacterial descent. To learn more about mitochondrial evolution, and therefore eukaryotic evolution, the genomes of the α-proteobacteria Bartonella henselae and Bartonella quintana were sequenced. Software was developed and used in the annotation of these genomes. Several gene products of nuclear-encoded genes are exported to the mitochondrion. Many of these genes are thought to originate from the emerging organelle. An analysis of the more than 400 genes encoding proteins targeted to the yeast mitochondrion indicates that one set of genes originated from the bacterial symbiont, while the eukaryotic host contributed another. Thus, the mitochondrial proteome has a dual origin. The hydrogen hypothesis postulates that the glycolytic genes belong to the group of genes that were transferred from symbiont to host. These genes are thoroughly analysed from a phylogenetic perspective. Contrary to the predictions of the hydrogen hypothesis, the results provide no support for a close relationship between nuclear genes encoding glycolytic enzymes and their α-proteobacterial homologs. In general, it is thought that intensive gene transfers may limit our ability to reconstruct gene and species evolution, especially among microbes. A phylogenetic analysis of a large cohort of genes from the AT-rich genome of the γ-proteobacterium Buchnera aphidicola (Sg) resulted in a high fraction of atypical tree topologies, previously interpreted as horizontal gene transfers. By applying methods that accommodate for asymmetric nucleotide substitutions, it is shown that many well-supported gene topologies are drastically altered, so that they now agree with the rRNA topology. The conclusion is that atypical topologies may not necessarily be evidence for horizontal gene transfers.
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176 |
Structural and functional studies of glycoside hydrolase family 12 enzymes from Trichoderma reesei and other cellulolytic microorganismsSandgren, Mats January 2003 (has links)
Cellulose is the most abundant organic compound on earth. A wide range of highly specialized microorganisms, have evolved that utilize cellulose as carbon and energy source. Enzymes called cellulases, produced by these cellulolytic organisms, perform the major part of cellulose degradation. In this study the three-dimensional structure of four homologous glycoside hydrolase family 12 cellulases will be presented, three fungal enzymes; Humicola grisea Cel12A, Hypocrea schweinitzii Cel12A, Trichoderma reesei Cel12A, and one bacterial; Streptomyces sp. 11AG8 Cel12A. The structural and biochemical information gathered from these and 15 other GH family 12 homologues has been used for the design of variants of these enzymes. These variants have biochemically been characterized, and thereby the positions and the types of mutations have been identified responsible for the biochemical differences between the homologous enzymes, e.g., thermal stability and activity. The three-dimensional structures of two T. reesei Cel12A variants, where the mutations have significant impact on the stability or the activity of the enzyme have been determined. Four ligand complex structures of the WT H. grisea Cel12A enzyme, that have made it possible to characterize the interactions between substrate and enzyme, have also been determined. The structural and biochemical studies of these closely related GH family 12 enzymes, and their variants, have provided insight on how specific residues contribute to protein thermal stability and enzyme activity. This knowledge can in the future serve as a structural toolbox, i.e., to design Cel12A enzymes with specific properties and features by introducing subtle changes in structural components of the enzymes. These can then be utilized to develop new industrial products or fine-tune enzymes in already existing applications.
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177 |
Cyanobacterial Hydrogen Metabolism : Transcriptional Regulation of the Hydrogenases in Filamentous StrainsAxelsson, Rikard January 2003 (has links)
Cyanobacteria are a heterogeneous group of phototrophic microorganisms. Many cyanobacteria have the capacity to fix atmospheric nitrogen. During the process of nitrogen fixation, molecular hydrogen is produced. Three enzymes are directly involved in hydrogen metabolism in cyanobacteria. A nitrogenase, evolving hydrogen during nitrogen-fixation, an uptake hydrogenase, recycling the hydrogen produced by nitrogenase, and a bidirectional hydrogenase that has the capacity to both take up and produce hydrogen. The main objective in this thesis was to examine the transcriptional regulation of both the uptake and the bidirectional hydrogenase in filamentous cyanobacteria. The transcriptional regulation of the uptake hydrogenase was demonstrated to be influenced by external conditions in Nostoc muscorum and Nostoc punctiforme. Nickel, molecular hydrogen, and anaerobic conditions all induced the relative amount of uptake hydrogenase transcript. In addition, a transcript could be detected in nitrogen-fixing, but not in non-nitrogen fixing conditions. The transcriptional regulation of the bidirectional hydrogenase in N. muscorum and Anabaena PCC 7120 was also examined. The relative amount of transcript from the bidirectional hydrogenase in both strains was demonstrated to increase during anaerobic conditions. Moreover, experiments using N. muscorum demonstrated that addition of nickel also increase the amount of transcript. However, no change in the relative amount of transcript from the bidirectional hydrogenase could be observed by additional hydrogen or during a shift from non-nitrogen fixing to nitrogen fixing conditions. The genes responsible for maturation of the hydrogenase were identified, cloned and sequenced in N. punctiforme. The transcription of the genes was examined and all genes were located on a single transcript. Like the uptake hydrogenase, a transcript could be detected under nitrogen-fixing but not under non-nitrogen fixing conditions. Initial studies, using microarrays, were used to analyse and compare the transcription of a large set of Anabaena PCC 7120 genes under non-nitrogen and nitrogen-fixing conditions. Both up- and down-regulated genes could be identified. This thesis advances the knowledge about the transcriptional regulation of the hydrogenases in filamentous cyanobacteria and can be used as a platform for further experiments aiming at a modified hydrogen metabolism.
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178 |
EXT Proteins in Heparan Sulfate BiosynthesisBusse, Marta January 2006 (has links)
Heparan sulfate (HS) is a long unbranched polysaccharide composed of alternating glucosamine and hexuronic (glucuronic or iduronic) acid residues. Modification by sulfate groups in various positions generates a highly heterogeneous molecule. HS is synthesized as a proteoglycan by virtually all cells, and play pivotal functions in signaling and developmental patterning, but also in pathogenic events such as tumor metastasis and microbial adhesion. This thesis deals with the properties of enzymes involved in HS chain elongation. Polymerization of the HS chain is believed to be catalyzed by the EXT family of proteins. In humans, the EXT family consists of five members: EXT1, EXT2, EXTL1, EXTL2 and EXTL3; their respective functions in HS biosynthesis are not fully understood. In this study, for the first time, successful in vitro HS polymerization on oligosaccharide acceptor substrates was demonstrated, using recombinant EXT1 and EXT1/EXT2 complex. EXT1 formed longer chains than EXT1/EXT2 and their mechanisms of sugar incorporation were different. Suppression of EXT1 or EXT2 expression by siRNA in a human cell line resulted in reduction of HS chain length. In contrast, cells transfected with EXTL3 siRNA produced longer HS chains. Overexpression of soluble EXT1, alone or co-expressed with EXT2, resulted in increased chain length, whereas overexpression of soluble EXT2 or EXTL3 has no detectable effect on HS chain elongation. Structural analysis of HS from fibroblasts isolated from mice with a hypomorphic mutation in Ext1 showed that they produced significantly shorter HS chains then the wild-type fibroblasts (20 and 70 kDa, respectively). The disaccharide composition of the HS produced by the mutant cells was virtually indistinguishable from that of the wild-type HS, however, the mutant HS chains contained higher proportions of unmodified regions. Mutant cells responded less efficiently than wild-type cells to low concentrations of FGF2, as analyzed by ERK phosphorylation assay.
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The multi-faceted RNA molecule : Characterization and Function in the regulation of Gene ExpressionEnsterö, Mats January 2008 (has links)
In this thesis I have studied the RNA molecule and its function and characteristics in the regulation of gene expression. I have focused on two events that are important for the regulation of the transcriptome: Translational regulation through micro RNAs; and RNA editing through adenosine deaminations. Micro RNAs (miRNAs) are ~22 nucleotides long RNA molecules that by semi complementarity bind to untranslated regions of a target messenger RNA (mRNA). The interaction manifests through an RNA/protein complex and act mainly by repressing translation of the target mRNA. I have shown that a pre-cursor miRNA molecule have significantly different information content of sequential composition of the two arms of the pre-cursor hairpin. I have also shown that sequential composition differs between species. Selective adenosine to inosine (A-to-I) RNA editing is a post-transcriptional process whereby highly specific adenosines in a (pre-)messenger transcript are deaminated to inosines. The deamination is carried out by the ADAR family of proteins and require a specific sequential and structural landscape for target recognition. Only a handful of messenger substrates have been found to be site selectively edited in mammals. Still, most of these editing events have an impact on neurotransmission in the brain. In order to find novel substrates for A-to-I editing, an experimental setup was made to extract RNA targets of the ADAR2 enzyme. In concert with this experimental approach, I have constructed a computational screen to predict specific positions prone to A-to-I editing. Further, I have analyzed editing in the mouse brain at four different developmental stages by 454 amplicon sequencing. With high resolution, I present data supporting a general developmental regulation of A-to-I editing. I also present data of coupled editing events on single RNA transcripts suggesting an A-to-I editing mechanism that involve ADAR dimers to act in concert. A different editing pattern is seen for the serotonin receptor 5-ht2c.
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Normal human T cells as a model system for the study of molecular events at the G₀/G₁ interfaceKim, Suil. January 1996 (has links)
Thesis (Ph. D.)--University of Michigan.
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