| 1. |
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| 2. |
- Ceron, Julian, et al.
(författare)
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Caenorhabditis elegans comes of age
- 2008
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Ingår i: Genome Biology. - : Springer Science and Business Media LLC. - 1465-6906 .- 1474-760X. ; 9:6, s. 312-
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 3. |
- Djupedal, Ingela, et al.
(författare)
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Molecular biology - The paradox of silent heterochromatin
- 2008
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 320:5876, s. 624-625
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 4. |
- Ekwall, Karl
(författare)
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'Arc' escorts siRNAs in heterochromatin assembly
- 2007
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Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 14:3, s. 178-179
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- RNA interference (RNAi) is important in directing heterochromatin assembly at centromeres in fission yeast, which is crucial for maintaining a stable genome through mitotic and meiotic divisions. In this issue, Buker et al. describe a new Argonaute siRNA chaperone (ARC) that converts duplex RNA to single-stranded RNA. This is a previously unknown step in the RNAi-directed heterochromatin-formation pathway.
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| 5. |
- Buch, Charlotta
(författare)
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Dynamic protein trafficking of the nuclear membrane and in peroxisomes
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The cell nucleus is enclosed by the nuclear envelope (NE), a double lipid membrane separating the nucleoplasm from the cytoplasm. Transport of macromolecules between the nucleus and the cytoplasm takes places through nuclear pore complexes (NPCs) in a selective and energy dependent manner. The inner nuclear membrane (INM) contains transmembrane proteins that interact with the nuclear lamina and chromatin. In addition to being a barrier between the nucleoplasm and cytoplasm, an emerging view is that the NE has an active role in chromatin organization and gene regulation.In order to study structural and functional organization of the NE in live cells, we have used green fluorescent protein (GFP)-labeled proteins and laser scanning confocal microscopy (LSCM). In order to investigate dynamic properties of specific proteins or protein complexes we have used photobleaching techniques. In order to understand the organization of the NPC it is essential to study components necessary for NPC biogenesis and maintenance. We have investigated the possible alterations in the NPC in cells naturally lacking one of the integral membrane proteins of the NPC, gp210. Despite the lack of gp210, we observed no difference in distribution or density of pores. Neither did cell cycle progression nor generation time differ between cells having or lacking gp210. In addition, targeting or dynamic properties of the NPC proteins POM121, Nup107 or Nup153 were unaltered in the absence of gp210. We conclude that gp210 can not be essential for NPC biogenesis or maintaining stability of the NPC.The steps involved in onset of nuclear apoptosis are unclear. We studied nuclear alterations during apoptosis. We show that the nucleocytoplasmic barrier is disrupted early in apoptosis at the same time as chromatin collapses against the nuclear periphery but prior to nucleosomal DNA fragmentation. In addition, the disruption of nucleocytoplasmic transport correlates with caspase-3 dependent cleavage of POM121 at aspartate-531.The INM is estimated to contain ~70 uncharacterized transmembrane proteins. We characterized a novel putative mammalian NE protein that we termed Samp1. We show that Samp1 is an integral membrane protein specifically localized to the inner nuclear membrane during interphase. Interestingly, during mitosis a sub fraction of Samp1 distributed in the polar region of the mitotic spindle, colocalizing with tubulin and a lipid marker. However, another inner nuclear membrane protein, emerin, was excluded from this area. Thus Samp1 appears to define a specific membrane domain associated with the mitotic machinery.The distribution of peroxisomal fatty acid metabolizing enzymes have been reported to vary in different tissues. We investigated whether photobleaching techniques could be used to study the distribution of peroxisomal matrix proteins. We used GFP-labeled peroxisomal proteins and fluorescence recovery after photobleaching to show that peroxisomal matrix proteins become “trapped” inside peroxisomes after import. Thus we conclude that fluorescence loss in photobleaching can be used to distinguish between a strictly cytoplasmic localization and a dual localization when a protein is present both in the cytoplasm and in peroxisomes. Using this technique we determined that GFP-BAAT (bile acid-CoA:amino acid N-acyltransferase) is exclusively localized to the cytoplasm in HeLa cells.
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| 6. |
- Djupedal, Ingela
(författare)
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Characterization of RNA polymerase II subunit Rpb7 in silencing and transcription
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The DNA in eukaryotes is arranged in fibres of chromatin. The chromatin may be more or less compacted and the degree of condensation of the chromatin affects the accessibility of the DNA. The accessibility of the DNA, in turn, affects transcription and gene regulation. Genes within inaccessible DNA are commonly repressed whereasgenes within accessible DNA are active and expressed. This thesis concerns the interplay between chromatin and transcription with focus on the function of the RNA polymerase II (pol II) subunit Rpb7. We have demonstrated that processing of centromeric transcripts by the ribonuclease III family protein Dcr1 is required for heterochromatin formation at the centromeres of Schizosaccharomyces pombe. A point mutation in the pol II subunit Rpb7 caused a specific defect in centromeric heterochromatin formation. We have shown i) that the centromeric transcripts that accumulate in dcr1delta cells are products of pol II, ii) the rpbG150D mutation is deficient in recognition and/or initiation of transcription from the centromeric promoter. Transcription by pol II within the centromeres was surprising since insertion of marker genes within these loci normally results in repression of pol II transcription. Here, paradoxically, pol II transcription was required for the construction of the inaccessible heterochromatin structure. Our analysis of sRNA in S. pombe revealed that most centromeric siRNA are originating from two clusters, which are repeated several times within the centromeres. This lead us to propose a model in which centromeric transcripts fold into double stranded structures that are processed by Dcr1. The resulting siRNAs may contribute with the starting signal for the RNAi feedback loop required for heterochromatin formation at the centromeres. Finally, we demonstrate that the genome-wide association of Rpb7 is nearly identical to that of the core pol II subunit Rpb2, indicating a general role for Rpb7 in transcription. We further show that the occupancy pattern of Rpb4, a pol II subunit that forms a subcomplex together with Rpb7, differs from those of Rpb2 and Rpb7. Rpb4 may therefore have a less general function in transcription than Rpb7. Hence, transcription by pol II is required not only for gene expression but also for repression via formation of inaccessible heterochromatin.
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| 7. |
- Efimenko, Evgeni
(författare)
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The study of sensory cilia development in caenorhabditis elegans
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cilia and flagella are widespread eukaryotic subcellular components that are conserved from green algae to mammals. In different organisms they function in cell motility, movement of extracellular fluids and sensory reception. While the function and structural description of cilia and flagella are well established, very little is known about the developmental mechanisms by which cilia are generated and shaped and how their components are assembled into functional machineries. To answer these questions, we used sensory cilia development in the nematode Caenorhabditis elegansas a model system.The work described here developed from the initial discovery of the ciliogenic properties of the gene daf-19, which encodes the sole C. elegans member of the RFX-type transcription factors. All members of the RFX transcription factor family are characterized by the presence of a conserved DNA binding domain, which recognizes special motifs (X-boxes) in promoters of its target genes. By using a genome search approach for X-box promoter motif-containing genes (xbx genes) we identified a list of about 750 xbx genes (candidates). This list comprises some already known ciliary genes as well as new genes, many of which we hypothesize to be important for cilia development and functioning.A computational search for X-box motifs in the C. briggsae genome has demonstrated strong conservation of this motif between closely related nematode species. To find out whether RFX-type transcription factors can also regulate ciliogenic pathways in other organisms, we applied a similar search strategy to distant species such as the fruit fly Drosophila. Using X-box consensus sequences with varying degrees of refinement and subsequent gene expression analysis, we were able to identify a set of Drosophila xbx genes. Intriguingly, the majority of fly xbx genes that have homologs in C. elegans were down regulated in dRfx fly mutants, suggesting an evolutionary conserved role for RFX-type transcription factors in the regulation of ciliary genes.Using X-box matches as a prediction tool we were able to identify novel ciliary genes, dyf-2 and dyf-11, in the C. elegans genome. We cloned these genes by transgenic rescue of mutant phenotypes and by sequencing of mutant alleles. Loss of DYF-2 and DYF-11 functions selectively affects the assembly and motility of different intraflagellar transport (IFT) components, resulting in compromised protein transport within cilia, and subsequently in defective cilia structures and sensory functions. Importantly, the mouse orthologs of DYF-2 and DYF-11 also localize to cilia, pointing to evolutionarily conserved roles for these proteins in cilia biogenesis.In conclusion, our studies of the regulation of sensory cilia formation demonstrated how contributions of multiple factors are integrated into a developmental module that leads to the formation of the primary sensory organs, cilia. In addition, data obtained during the course of this study provide a useful resource for researchers interested in further identification and study of new genes implicated in cilia biogenesis and will have a significant impact on the understanding and treatment of cilia-based pathologies in humans.
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| 8. |
- Fagerström-Billai, Fredrik
(författare)
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Genome wide analysis of the Ssn6-Tup11/Tup12 co-repressor complex in the fission yeast Schizosaccharomyces pombe
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this study, we have investigated the fission yeast Ssn6-Tup11 /Tup 12 transcriptional corepressor which is involved in regulation of many genes important for a wide variety of processes. In contrast to the well characterised budding yeast Tup1 protein there are two paralogous proteins present in fission yeast, namely Tup11 and Tup12. We have shown that the two proteins can interact with each other and are expressed at similar levels, which is in line with a reported redundant function. Sequence analysis shows that the intermediate proposed histone interacting domain is highly variable between Tup11 and Tup12 indicating a diversification. Interestingly, we show that tup11 and tup12 mutants have different phenotypes on media containing KC1 and CaC12. Consistent with this functional difference, we identify a number of target genes by genome wide expression profiling that are differentially affected by tup11 - and tup12. Many of these genes are Tup12 dependent and correlate with genes that have previously been shown to respond to a range of different environmental stress conditions. The observed different physiological roles of Tup11 and Tup12 can not be explained by differential recruitment of Ssn6 which can interact independently with both Tup11 and Tup12. Most interestingly we show that the Ssn6 protein is essential in fission yeast and therefore must have a distinct role separated from Tup11 and Tup12. Surprisingly, a conditional ssn6HA-ts mutant displays the same growth phenotype as tup12, indicating a role in Tup12 dependent stress response. Consistent with the diverse phenotypes of the individual co-repressor proteins, we identify a group of genes that requires Ssn6 for their regulation which is overlapping but distinct from the group of genes that depend on Tup11 or Tup12. Genome wide chromatin immunoprecipitation shows that Ssn6 is almost invariably found in the same genomic locations as Tup11 and/or Tup12. All three co-repressor subunits are generally bound to genes that are selectively regulated by Ssn6 or Tup11/12, and thus, likely in the context of a co-repressor complex containing all three subunits. The co-repressor binds to both the intergenic and coding regions of genes, but differential localization of the co-repressor within genes does not appear to account for the selective dependence of target genes on the Ssn6 or Tup11/12 subunits. Ssn6, Tup11, and Tup12 are preferentially found at genomic locations at which histones are deacetylated, primarily by the Clr6 class I HDAC. A subset of co-repressor target genes, including direct target genes affected by Ssn6 overexpression, is in addition associated with the function of class II (Clr3) and III (Hst4 and Sir2) HDACs. Interestingly, many specific Hst4 repressed ORF targets involved in amino acid biosynthesis are also direct targets for the Ssn6-Tup11/12 co-repressor, suggesting an association with the class ill sirtuins which has not been reported previously.
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| 9. |
- Ferreira, Monica E.
(författare)
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Studies of transcription factor domains and their interactions with other transcription factors
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The studies in this thesis deal with different questions concerning interactions of functional domains of factors involved in transcriptional regulation. The first study of this thesis is focused on the target factor binding mechanism of transcriptional activators. Many activators in evolutionary distant species are classified as acidic based on a high content of acidic residues in the activation domain and intrinsically unstructured in solution. Our results indicate that such activation domains interact with target factors through coupled binding and folding of the activation domain after an initial ionic interaction, and demonstrate the generality of this binding mechanism. We propose that target interaction through coupled binding and folding of the recruiting domain is important for the role of activators as regulators of transcription. In the following study we show that deletion of two regions that mediate interaction with activators in vitro prevents promoter recruitment of the SWI/SNF chromatinremodeling complex in vivo, and causes strongly reduced transcriptional activity of the corresponding genes. This study validates direct interaction between the Swi1- and Snf5 activator binding domains of the S. cerevisiae SWI/SNF complex and activators previously demonstrated in vitro, and importantly indicates that the activator binding domains are essential for the ability of SWI/SNF to function as co-activator. In the last study we investigate which domains are involved in distinct in vivo function of the paralogous co-repressors Tup11 and Tup12 of the Ssn6/Tup complex in S. pombe. Tup11 and Tup12 have been shown to differ in importance in context of a common complex for subsets of Ssn6/Tup target genes, and it was proposed that this might depend on divergence in the histone-interaction domain. Here we show that distinct in vivo roles of Tup12 do not depend on differences in the highly diverged histoneinteraction domain, but mainly on differences in the overall highly conserved WD40 repeat domain, which putatively mediates interaction with repressors and target factors such as histone modifying complexes and components of the transcriptional machinery. We propose that clusters of amino acids, putatively located in blade 3 of the WD40 repeat domain, could be important for interaction with distinct target factors of Tup11 and Tup12. Furthermore, we show that the stoichiometry of the Ssn6/Tup complex is likely to change under CaCl2 stress, by a mechanism involving changes in the relative cellular levels of the complex components.
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| 10. |
- Hårdeman, Fredrik
(författare)
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Exploring the metagenome of the Baltic Sea sediment
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Environmental microorganisms are fundamental to ecosystem function, acting as drivers in processes such as primary production, organic matter remineralisation, pollution remediation and global biogeochemical cycling. However, the study of the bacterial communities requires the application of advanced culture-independent methods considering that only a small fraction of the community is otherwise accessed. The goal of this thesis was to investigate the bacterial community structures and functions of Baltic Sea coastal sediments. To assess the distribution and identity of metabolically active bacteria along a vertical redox gradient, a polyphasic method was applied including: reverse transcriptase-PCR (transcription) and bromodeoxyuridine immunocapture (replication) for 16S rRNA gene analyses through both clone library sequence analysis and terminal restriction fragment length polymorphism (T-RFLP). It was demonstrated that the bacterial communities were highly diverse and significantly different at different redox layers. Phylogenetic analysis identified several novel bacterial groups, some with potentially important ecological roles, notably the first genetic evidence of active anammox bacteria, demonstrating that the bacterial community of the Baltic Sea sediment includes several largely unexplored groups. A metagenomic approach was used to access the bacterial diversity. Considering that the Baltic Sea sediment contained a diverse and largely unexplored bacterial community and also represent a permanently cold environment. This community is likely to harbor bacteria with enzymes adapted to low temperatures that would have a potential biotechnological value. The capacity of functional metagenomics for bioprospecting was demonstrated though the construction of a fosmid library of the prokaryotic genomic pool and expression screening, which enabled the identification of several novel lipolytical enzymes. A novel lipase, h1Lip1 (DQ118648) was isolated, overexpressed, purified and characterized for catalytic activity, substrate specificity, apparent temperature optimum and thermo-stability, demonstrating that the enzyme was low temperature active. 3D protein structure modelling of the lipase supported the presence of an alpha/beta-hydrolase fold, a catalytic triad and a lid structure, covering the active site. Comparative structure analyses and site directed-mutagenesis further showed the importance of a region within the N-terminal and lid for substrate affinity and thermal stability. In conclusion, these targeted molecular strategies demonstrate that the Baltic Sea sediments contain a highly diverse and unique bacterial community that also represents a useful source of biotechnologically interesting molecules.
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