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  • 1.
    Alkemar, Gunnar
    et al.
    Södertörn University, Avdelning Naturvetenskap. Stockholm Universtiy.
    Nygård, Odd
    Södertörn University, Avdelning Naturvetenskap.
    A possible tertiary rRNA interaction between expansion segments ES3 and ES6 in eukaryotic 40S ribosomal subunits2003In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 9, no 1, 20-24 p.Article in journal (Refereed)
    Abstract [en]

    Eukaryotic 16S-like ribosomal RNAs contain 12 so-called expansion segments, i.e., sequences not included in the RNA secondary structure core common to eubacteria, archaea, and eukarya. Two of these expansion segments, ES3 and ES6, are juxtaposed in the recent three-dimensional model of the eukaryotic 40S ribosomal subunit. We have analyzed ES3 and ES6 sequences from more than 2900 discrete eukaryotic species, for possible sequence complementarity between the two expansion segments. The data show that ES3 and ES6 could interact by forming a helix consisting of seven to nine contiguous base pairs in almost all analyzed species. We, therefore, suggest that ES3 and ES6 form a direct RNA-RNA contact in the ribosome.

  • 2.
    Alkemar, Gunnar
    et al.
    Södertörn University, School of Life Sciences. Stockholm University.
    Nygård, Odd
    Södertörn University, School of Life Sciences.
    Probing the secondary structure of expansion segment ES6 in 18S ribosomal RNA2006In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 45, no 26, 8067-8078 p.Article in journal (Refereed)
    Abstract [en]

    Expansion segment ES6 in 18S ribosomal RNA is, unlike many other expansion segments, present in all eukaryotes. The available data suggest that ES6 is located on the surface of the small ribosomal subunit. Here we have analyzed the secondary structure of the complete ES6 sequence in intact ribosomes from three eukaryotes, wheat, yeast, and mouse, representing different eukaryotic kingdoms. The availability of the ES6 sequence for modification and cleavage by structure sensitive chemicals and enzymatic reagents was analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The experimental results were used to restrict the number of possible secondary structure models of ES6 generated by the folding software MFOLD. The modification data obtained from the three experimental organisms were very similar despite the sequence variation. Consequently, similar secondary structure models were obtained for the ES6 sequence in wheat, yeast, and mouse ribosomes. A comparison of sequence data from more than 6000 eukaryotes showed that similar structural elements could also be formed in other organisms. The comparative analysis also showed that the extent of compensatory base changes in the suggested helices was low. The in situ structure analysis was complemented by a secondary structure analysis of wheat ES6 transcribed and folded in vitro. The obtained modification data indicate that the secondary structure of the in vitro transcribed sequence differs from that observed in the intact ribosome. These results suggest that chaperones, ribosomal proteins, and/or tertiary rRNA interactions could be involved in the in vivo folding of ES6.

  • 3.
    Alkemar, Gunnar
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Stockholm University.
    Nygård, Odd
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits2004In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 10, no 3, 403-411 p.Article in journal (Refereed)
    Abstract [en]

    The 18S rRNA of the small eukaryotic ribosomal subunit contains several expansion segments. Electron microscopy data indicate that two of the largest expansion segments are juxtaposed in intact 40S subunits, and data from phylogenetic sequence comparisons indicate that these two expansion segments contain complementary sequences that could form a direct tertiary interaction on the ribosome. We have investigated the secondary structure of the two expansion segments in the region around the putative tertiary interaction. Ribosomes from yeast, wheat, and mouse-three organisms representing separate eukaryotic kingdoms-were isolated, and the structure of ES3 and part of the ES6 region were analyzed using the single-strand-specific chemical reagents CMCT and DMS and the double-strand-specific ribonuclease V1. The modification patterns were analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The investigated sequences were relatively exposed to chemical and enzymatic modification. This is in line with their indicated location on the surface at the solvent side of the subunit. The complementary ES3 and ES6 sequences were clearly inaccessible to single-strand modification, but available for cleavage by double-strand-specific RNase V1. The results are compatible with a direct helical interaction between bases in ES3 and ES6. Almost identical results were obtained with ribosomes from the three organisms investigated.

  • 4.
    Askarian Nameghi, Shahnaz
    Södertörn University College, School of Life Sciences.
    Genotyping Escherichia coli isolates by Pulsed-Field Gel Electrophoresis2007Independent thesis Advanced level (degree of Magister), 20 points / 30 hpStudent thesis
    Abstract [en]

    Transmission of bacterial strains between patients is a serious problem in hospitals and with the increasing rate of antibiotic resistance the problem has farther escalated. Enterobacteriaceae produced extended-spectrum beta-lactamses (ESBLs), especially Escherichia coli (E-coli), are increasingly important nosocomial pathogens (7, 8). These bacteria are often multiple resistant and are responsible for many intestinal infections and urinary tract infections (2, 5). With the more frequent use of invasive devices in hospital care, these types of nosocomial infections have increased, particularly in seriously ill patients.

    In order to diminish transmission of bacterial strains between patients and to study the epidemiology of these bacteria, it is of great importance to develop rapid and specific methods to be able to subtype on strain-level, i.e. to create a fingerprint of the isolates. The method may be based on phenotypic or genotypic characteristics of the microorganism. Any typing method must have high reproducibility and discrimination power to differentiate unrelated strains and also to demonstrate relationship of organisms deriving from the same source. In the present project, a Pulsed-Field Gel Electrophoresis (PFGE) assay for genotyping clinical E. coli isolates was used. PFGE can be used as a genotyping tool and is widely used to type bacteria and trace nosocomial infection. However, the method is time-consuming and relatively expensive in compare with other methods like PCR. In this study, a total of 93 strains were collected. The study was aimed to investigate the genotypes of the collected isolates and to identify and potential the outbreak strains.

    The isolates investigated were genotypically diverse shown by a variety of PFGE banding patterns. However, clusters of closely related isolates involved in outbreaks were also identified.

    In conclusion, when analyzing a large number of strains, a combination of a rapid phenotyping or genotyping method and a powerful genotyping method like PFGE would be an appropriate strategy for studying clonal relationship among isolates e.g. for detecting cross-transmission of nosocomial pathogens.

  • 5. Ban, L.
    et al.
    Didon, Andrea
    Södertörn University, School of Life Sciences.
    Jonsson, L. M. V.
    Glinwood, R.
    Delp, Gabriele
    Södertörn University, School of Life Sciences.
    An improved detection method for the Rhopalosiphum padi virus (RhPV) allows monitoring of its presence in aphids and movement within plants2007In: Journal of Virological Methods, ISSN 0166-0934, E-ISSN 1879-0984, Vol. 142, no 1-2, 136-142 p.Article in journal (Refereed)
    Abstract [en]

    Rhopalosiphumpadi virus (RhPV) is an insect RNA virus that infects aphids, reducing their lifespan and fecundity. It can be transmitted vertically between aphids and horizontally via the plant. An improved detection method for the virus in aphids and plants using RT-PCR was developed; this allowed individual aphids to be tested for RhPV. Testing of R. padi aphids collected from different sites in Sweden revealed the presence of RhPV in wild aphid populations for the first time in Europe. Virus could be detected in several life stages of R. padi, including sexual individuals and eggs, establishing an over-wintering route for the virus. Using RT-PCR, systemic transport of the virus in plants was tracked. Virus spread from the aphid feeding site to all parts of the plant, including roots, within 7 days, and could be acquired by virus-free aphids feeding on the same plant.

  • 6. Bao, W J
    et al.
    Thullberg, M
    Zhang, H Q
    Onischenko, A
    Strömblad, Staffan
    Södertörn University, Avdelning Naturvetenskap.
    Cell attachment to the extracellular matrix induces proteasomal degradation of p21(CIP1) via Cdc42/Rac1 signaling2002In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 22, no 13, 4587-4597 p.Article in journal (Refereed)
    Abstract [en]

    The cyclin-dependent kinase 2 (Cdk2) inhibitors p21(CIP1) and p27(KIP1) are negatively regulated by anchorage during cell proliferation, but it is unclear how integrin signaling may affect these Cdk2 inhibitors. Here, we demonstrate that integrin ligation led to rapid reduction of p21(CIp1) and p27(KIP1) protein levels in three distinct cell types upon attachment to various extracellular matrix (ECM) proteins, including fibronectin (FN), or to immobilized agonistic anti-integrin monoclonal antibodies. Cell attachment to FN did not rapidly influence p21(CIp1) mRNA levels, while the protein stability of p21(CIp1) was decreased. Importantly, the down-regulation of p21(CIP1) and p27(KIP1) was completely blocked by three distinct proteasome inhibitors, demonstrating that integrin ligation induced proteasomal degradation of these Cdk2 inhibitors. Interestingly, ECM-induced proteasomal proteolysis of a ubiquitination-deficient p21(CIP1) mutant (p21K6R) also occurred, showing that the proteasomal degradation of p21(CIP1) was ubiquitin independent. Concomitant with our finding that the small GTPases Cdc42 and Rac1 were activated by attachment to FN, constitutively active (ca) Cdc42 and ca Rac1 promoted down-regulation of p21(CIP1). However, dominant negative (dn) Cdc42 and do Rac1 mutants blocked the anchorage-induced degradation of p21(CIP1), suggesting that an integrin-induced Cdc42/Rac1 signaling pathway activates proteasomal degradation of p21(CIP1). Our results indicate that integrin-regulated proteasomal proteolysis might contribute to anchorage-dependent cell cycle control.

  • 7.
    Bartish, Galyna
    et al.
    Södertörn University, School of Life Sciences. Stockholm University.
    Nygård, Odd
    Södertörn University, School of Life Sciences.
    Importance of individual amino acids in the Switch I region in eEF2 studied by functional complementation in S. cerevisiae2008In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 90, no 5, 736-748 p.Article in journal (Refereed)
    Abstract [en]

    Elongation factor 2 (eEF2) is a member of the G-protein super family. G-proteins undergo conformational changes associated with binding of the guanosine nucleotide and hydrolysis of the bound GTP. These structural rearrangements affects the Switch I region (also known as the Effector loop). We have studied the role of individual amino acids in the Switch I region (amino acids 25-73) of S. cerevisiae eEF2 using functional complementation in yeast. 21 point mutations in the Switch I region were created by site-directed mutagenesis. Mutants K49R, E52Q, A53G, F55Y, K60R, Q63A, T68S, 169M and A73G were functional while mutants R54H, F55N, D57A, D57E, D57S, R59K, R59M, Q63E, R65A, R65N, T68A and T68M were inactive. Expression of mutants K49R, A53G, Q63A, 169M and A73G was associated with markedly decreased growth rates and yeast cells expressing mutants A53G and 169M became temperature sensitive. The functional capacity of eEF2 in which the major part Switch I (amino acids T56 to 169) was converted into the homologous sequence found in EF-G from E. coli was also studied. This protein chimera could functionally replace yeast eEF2 in vivo. Yeast cells expressing this mutant grew extremely slowly, showed increased cell death and became temperature sensitive. The ability of the mutant to replace authentic eEF2 in vivo indicates that the structural rearrangement of Switch I necessary for eEF2 function is similar in eukaryotes and bacteria. The effect of two point mutations in the P-loop was also studied. Mutant A25G but not A25V could functionally replace yeast eEF2 even if cells expressing the mutant grew slowly. The A25G mutation converted the consensus sequences AXXXXGK[T/S] in eEF2 to the corresponding motif GXXXXGK[T/S] found in all other G-proteins, suggesting that the alanine found in the P-loop of peptidyltranslocases are not essential for function.

  • 8. Bauer, S H J
    et al.
    Månsson, Martin
    Södertörn University, Avdelning Naturvetenskap.
    Hood, D W
    Richards, J C
    Moxon, E R
    Schweda, Elke K H
    Södertörn University, Avdelning Naturvetenskap.
    A rapid and sensitive procedure for determination of 5-N-acetyl neuraminic acid in lipopolysaccharides of Haemophilus influenzae: a survey of 24 non-typeable H-influenzae strains2001In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 335, no 4, 251-260 p.Article in journal (Refereed)
    Abstract [en]

    In view of the importance of 5-N-acetyl neuraminic acid in bacterial pathogenesis, a sensitive, reproducible and reliable method for the determination of 5-N-acetyl neuraminic acid levels in lipopolysaccharide (LPS) is described and applied to 24 different non-typeable Haemophilus influenzae (NTHi) strains. The method involves analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of terminal 5-N-acetyl neuraminic acid residues released by neuraminidase treatment of O-deacylated LPS. The procedure is relatively fast and the instrumental effort is moderate. The results of the procedure were compared with data obtained by H-1 NMR and electrospray ionisation-mass spectrometry (ESI-MS). The analysis of LPS from 24 NTHi strains showed that 5-N-acetyl neuraminic acid was found to be a common constituent of LPS in NTHi. Only one strain (NTHi 432) did not show any sialylation. Molar ratios (LPS /5-N-acetyl neuraminic acid) ranged between 5/1 and 500/1. Several strains in which no 5-N-acetyl neuraminic acid could be determined by other methods including 1H NMR and ESI-MS were shown to contain 5-N-acetyl neuraminic acid by this HPAEC-PAD procedure. The method was applied to determine levels of terminal 5-N-acetyl neuraminic acid in LPS from NTHi strains grown under different conditions and mutant strains containing inactive LPS biosynthetic genes.

  • 9. Beckman, M
    et al.
    Kihlmark, Madeleine
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Iverfeldt, K
    Hallberg, Einar
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Degradation of GFP-labelled POM121, a non-invasive sensor of nuclear apoptosis, precedes clustering of nuclear pores and externalisation of phosphatidylserine2004In: Apoptosis (London), ISSN 1360-8185, E-ISSN 1573-675X, Vol. 9, no 3, 363-368 p.Article in journal (Refereed)
    Abstract [en]

    The nuclear pore membrane protein POM121 is specifically degraded during apoptosis by a caspase-3-dependent process enabling early detection of apoptosis in living cells expressing POM121-GFP. Here we further investigated temporal aspects of apoptotic degradation of POM121-GFP. We demonstrate that decreased POM121-GFP fluorescence precedes annexin V-labelling of apoptotic cells. This indicates that degradation of the nuclear pore complex starts prior to redistribution of plasma membrane phosphatidylserine, which serves as a signal for phagocytotic elimination of apoptotic cells. Furthermore, a caspase-resistant GFP-labelled mutant of POM121 resisted degradation even in late apoptosis and was detected in clustered nuclear pores. Thus, it can be concluded that loss of POM121-GFP is a specific sensor of the activation of caspase-3-dependent proteolysis at the nuclear pores.

  • 10.
    Beckman, Marie
    et al.
    Södertörn University, School of Life Sciences, Molecular biology.
    Freeman, Craig
    Parish, Christopher R.
    Small, David H.
    Activation of cathepsin D by glycosaminoglycans2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 24, 7343-7352 p.Article in journal (Refereed)
    Abstract [en]

    We have previously shown that heparin can increase the activity of the proenzyme form of Alzheimer's beta-site amyloid precursor protein cleaving enzyme 1 (BACE1). Cathepsin D (CD) is a member of the aspartic protease family and has sequence similarity to BACE1. Therefore, we examined whether heparin and other glycosaminoglycans (GAGs) can influence the activity of CD. Heparin and other GAGs were found to stimulate the activity of recombinant proCD. Desulfation of heparin almost abolished the stimulation, indicating that sulfate groups were important for the stimulatory effect. In addition, the stimulation was dependent on the length of the GAG chain, as larger GAGs were more potent in their ability to stimulate proCD than shorter fragments. In the presence of heparin, limited autocatalytic proteolysis of the proenzyme was increased, suggesting that heparin increases the activity of proCD by accelerating the conversion of proCD, which has little activity, to pseudoCD, an active form lacking residues 1-26 of the prodomain. Furthermore, the activity of spleen-derived mature CD, which lacks the entire 44 amino acid residue prodomain, was also increased by heparin, indicating that the catalytic domain of CD contains at least one region to which GAGs bind and stimulate enzyme activity. Because heparin also stimulated the activity of pseudoCD, proenzyme activation was probably accelerated by the interaction of heparin with the catalytic domain of pseudoCD. However, it is possible that heparin may also activate the proenzyme directly. On the basis of this study, we propose that GAGs may regulate CD activity in vivo.

  • 11. Benach, J
    et al.
    Filling, C
    Oppermann, U C T
    Roversi, P
    Bricogne, G
    Berndt, Kurt D
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Jörnvall, H
    Ladenstein, R
    Structure of bacterial 3 beta/17 beta-hydroxysteroid dehydrogenase at 1.2 angstrom resolution: A model for multiple steroid recognition2002In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 50, 14659-14668 p.Article in journal (Refereed)
    Abstract [en]

    The enzyme 3beta/17beta-hydroxysteroid dehydrogenase (3beta/17beta-HSD) is a steroid-inducible component of the Gram-negative bacterium Conramonas testosteroni. It catalyzes the reversible reduction/ dehydrogenation of the oxo/beta-hydroxy groups at positions 3 and 17 of steroid compounds, including hormones and isobile acids. Crystallographic analysis at 1.2 Angstrom resolution reveals the enzyme to have nearly identical subunits that form a tetramer with 222 symmetry. This is one of the largest oligomeric structures refined at this resolution. The subunit consists of a monomer with a single-domain structure built around a seven-stranded beta-sheet flanked by six alpha-helices. The active site contains a Ser-Tyr-Lys triad, typical for short-chain dehydrogenases/reductases (SDR). Despite their highly diverse substrate specificities, SDR members show a close to identical folding pattern architectures and a common catalytic mechanism. In contrast to other SDR apostructures determined, the substrate binding loop is well-defined. Analysis of structure-activity relationships of catalytic cleft residues, docking analysis of substrates and inhibitors, and accessible surface analysis explains how 3beta/17beta-HSD accommodates steroid substrates of different conformations.

  • 12. Bergh, F T
    et al.
    Flinn, Elisabeth M
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Svaren, J
    Wright, Anthony P
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Horz, W
    Comparison of nucleosome remodeling by the yeast transcription factor Pho4 and the glucocorticoid receptor2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 12, 9035-9042 p.Article in journal (Refereed)
    Abstract [en]

    Chromatin reorganization of the PHO5 and murine mammary tumor virus (MMTV) promoters is triggered by binding of either Pho4 or the glucocorticoid receptor (GR), respectively. In order to compare the ability of Pho4 and GR to remodel chromatin and activate transcription, hybrid promoter constructs were created by insertion of the MMTV B nucleosome sequence into the PHO5 promoter and then transformed into a yeast strain expressing GR, Activation of either Pho4 (by phosphate depletion) or GR (by hormone addition) resulted in only slight induction of hybrid promoter activity. However, simultaneous activation of both Pho4 and GR resulted in synergistic activation to levels exceeding that of the wild type PHO5 promoter. Under these conditions, Pho4 completely disrupted the nucleosome containing its binding site. In contrast, GR had little effect on the stability of the MMTV B nucleosome. A minimal transactivation domain of the GR fused to the Pho4 DNA-binding domain is capable of efficiently disrupting the nucleosome with a Pho4-binding site, whereas the complementary hybrid protein (Pho4 activation domain, GR DNA-binding domain) does not labilize the B nucleosome. Therefore, we conclude that significant activation by Pho4 requires nucleosome disruption, whereas equivalent transcriptional activation by GR is not accompanied by overt perturbation of nucleosome structure. Our results show that the DNA-binding domains of the two factors play critical roles in determining how chromatin structure is modified during promoter activation.

  • 13. Bernard, Pascal
    et al.
    Schmidt, Christine Katrin
    Vaur, Sabine
    Dheur, Sonia
    Drogat, Julie
    Genier, Sylvie
    Ekwall, Karl
    Södertörn University, School of Life Sciences.
    Uhlmann, Frank
    Javerzat, Jean-Paul
    Cell-cycle regulation of cohesin stability along fission yeast chromosomes2008In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 27, no 1, 111-121 p.Article in journal (Refereed)
    Abstract [en]

    Sister chromatid cohesion is mediated by cohesin, but the process of cohesion establishment during S-phase is still enigmatic. In mammalian cells, cohesin binding to chromatin is dynamic in G1, but becomes stabilized during S-phase. Whether the regulation of cohesin stability is integral to the process of cohesion establishment is unknown. Here, we provide evidence that fission yeast cohesin also displays dynamic behavior. Cohesin association with G1 chromosomes requires continued activity of the cohesin loader Mis4/Ssl3, suggesting that repeated loading cycles maintain cohesin binding. Cohesin instability in G1 depends on wpl1, the fission yeast ortholog of mammalian Wapl, suggestive of a conserved mechanism that controls cohesin stability on chromosomes. wpl1 is nonessential, indicating that a change in wpl1-dependent cohesin dynamics is dispensable for cohesion establishment. Instead, we find that cohesin stability increases at the time of S-phase in a reaction that can be uncoupled from DNA replication. Hence, cohesin stabilization might be a pre-requisite for cohesion establishment rather than its consequence.

  • 14. Beskow, Anne
    et al.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences.
    Comparative analysis of regulatory transcription factors in Schizosaccharomyces pombe and budding yeasts2006In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 23, no 13, 929-935 p.Article in journal (Refereed)
    Abstract [en]

    Regulatory transcription factors (rTFs), which bind specific DNA sequences in the regulatory regions of genes and subsequently activate or repress transcription, play a central role in programming genomic expression. The number of rTFs in a species might therefore reflect its functional complexity. For simple organisms like yeast, a relatively small number of rTFs might be expected that is fairly constant between yeast species. We show that the budding yeast, Saccharomyces cerevisiae, contains 201 rTfs, which is one of the largest rTF numbers found in yeast species for which genome sequences are available. This is a much higher number than the 129 rTFs found in the fission yeast, Schizosaccharomyces pombe, which is currently the yeast with the lowest number of rTFs. Comparative analysis of several different budding yeast species shows that most of the 'extra' rTFs found in S. cerevisiae were probably acquired as a result of a whole genome duplication (WGD) event that occurred in an ancestor of a subset of budding yeast species. However, we also show that budding yeast species that have not been affected by the WGD contain a greater number of rTFs than S. pombe (mean = 145). Thus, two or more mechanisms have led to the 60% increase in rTFs in S. cerevisiae compared to S. pombe. This difference may correlate with a more extensive functional divergence in budding yeasts compared to fission yeasts. The relatively small number of rTFs in S. pombe make this organism an attractive model for global studies of mechanisms that programme gene expression.

  • 15.
    Bjerling, Pernilla
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Uppsala University / University of Copenhagen, Denmark .
    Ekwall, Karl
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Egel, R
    Thon, G
    A novel type of silencing factor, Clr2, is necessary for transcriptional silencing at various chromosomal locations in the fission yeast Schizosaccharomyces pombe2004In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 32, no 15, 4421-4428 p.Article in journal (Refereed)
    Abstract [en]

    The mating-type region of the fission yeast Schizosaccharomyces pombe comprises three loci: mat1, mat2-P and mat3-M. mat1 is expressed and determines the mating type of the cell. mat2-P and mat3-M are two storage cassettes located in a 17 kb heterochromatic region with features identical to those of mammalian heterochromatin. Mutations in the swi6(+), clr1(+), clr2(+), clr3(+), clr4(+) and clr6(+) genes were obtained in screens for factors necessary for silencing the mat2-P-mat3-M region. swi6(+) encodes a chromodomain protein, clr3(+) and clr6(+) histone deacetylases, and clr4(+) a histone methyltransferase. Here, we describe the cloning and characterization of clr2(+). The clr2(+) gene encodes a 62 kDa protein with no obvious sequence homologs. Deletion of clr2(+) not only affects transcriptional repression in the mating-type region, but also centromeric silencing and silencing of a PolII-transcribed gene inserted in the rDNA repeats. Using chromatin immunoprecipitation, we show that Clr2 is necessary for histone hypoacetylation in the mating-type region, suggesting that Clr2 acts upstream of histone deacetylases to promote transcriptional silencing.

  • 16.
    Björk, Petra
    et al.
    Stockholm University.
    Baurén, Göran
    Stockholm University.
    Jin, ShaoBo
    Stockholm University.
    Tong, Yong-Guang
    Karolinska Institutet.
    Bürglin, Thomas R.
    Karolinska Institutet.
    Hellman, Ulf
    Ludwig Institute for Cancer Research.
    Wieslander, Lars
    Stockholm University.
    A novel conserved RNA-binding domain protein, RBD-1, is essential for ribosome biogenesis2002In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 13, no 10, 3683-3695 p.Article in journal (Refereed)
    Abstract [en]

    Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20-30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

  • 17. Blacque, O E
    et al.
    Perens, E A
    Boroevich, K A
    Inglis, P N
    Li, C M
    Warner, A
    Khattra, J
    Holt, R A
    Ou, G S
    Mah, A K
    McKay, S J
    Huang, P
    Swoboda, Peter
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Jones, S J M
    Marra, M A
    Baillie, D L
    Moerman, D G
    Shaham, S
    Leroux, M R
    Functional genomics of the cilium, a sensory organelle2005In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 15, no 10, 935-941 p.Article in journal (Refereed)
    Abstract [en]

    Cilia and flagella play important roles in many physiological processes, including cell and fluid movement, sensory perception, and development [1]. The biogenesis and maintenance of cilia depend on intraflagellar transport (IFT), a motility process that operates bidirectionally along the ciliary axoneme [1, 2]. Disruption in IFT and cilia function causes several human disorders, including polycystic kidneys, retinal dystrophy, neurosensory impairment, and Bardet-Bledl syndrome (BBS) [3-5]. To uncover new ciliary components, including IFT proteins, we compared C. elegans ciliated neuronal and nonciliated cells through serial analysis of gene expression (SAGE) and screened for genes potentially regulated by the cillogenic transcription factor, DAF-19 [6]. Using these complementary approaches, we identified numerous candidate ciliary genes and confirmed the ciliated-cell-specific expression of 14 novel genes. One of these, C27H5.7a, encodes a ciliary protein that undergoes IFT. As with other IFT proteins, its ciliary localization and transport is disrupted by mutations in IFT and bbs genes. Furthermore, we demonstrate that the ciliary structural defect of C. elegans dyf-13(mn396) mutants is caused by a mutation in C27H5.7a. Together, our findings help define a ciliary transcriptome and suggest that DYF-13, an evolutionarily conserved protein, is a novel core IFT component required for cilia function.

  • 18. Bratic, Ivana
    et al.
    Hench, Jürgen
    Södertörn University, School of Life Sciences.
    Henriksson, Johan
    Södertörn University, School of Life Sciences, Molecular biology.
    Antebi, Adam
    Bürglin, Thomas R.
    Trifunovic, Aleksandra
    Mitochondrial DNA level, but not active replicase, is essential for Caenorhabditis elegans development2009In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 6, 1817-1828 p.Article in journal (Refereed)
    Abstract [en]

    A number of studies showed that the development and the lifespan of Caenorhabditis elegans is dependent on mitochondrial function. In this study, we addressed the role of mitochondrial DNA levels and mtDNA maintenance in development of C. elegans by analyzing deletion mutants for mitochondrial polymerase gamma (polg-1(ok1548)). Surprisingly, even though previous studies in other model organisms showed necessity of polymerase gamma for embryonic development, homozygous polg-1(ok1548) mutants had normal development and reached adulthood without any morphological defects. However, polg-1 deficient animals have a seriously compromised gonadal function as a result of severe mitochondrial depletion, leading to sterility and shortened lifespan. Our results indicate that the gonad is the primary site of mtDNA replication, whilst the mtDNA of adult somatic tissues mainly stems from the developing embryo. Furthermore, we show that the mtDNA copy number shows great plasticity as it can be almost tripled as a response to the environmental stimuli. Finally, we show that the mtDNA copy number is an essential limiting factor for the worm development and therefore, a number of mechanisms set to maintain mtDNA levels exist, ensuring a normal development of C. elegans even in the absence of the mitochondrial replicase.

  • 19.
    Bredenberg, Johan
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Nilsson, L
    Karolinska Institutet.
    Conformational states of the glucocorticoid receptor DNA-binding domain from molecular dynamics simulations2002In: Proteins: Structure, Function, and Genetics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 49, no 1, 24-36 p.Article in journal (Refereed)
    Abstract [en]

    Molecular dynamics simulations (MD) have been performed on variant crystal and NMR-derived structures of the glucocorticoid receptor DNA-binding domain (GR DBD). A loop region five residues long, the so-called D-box, exhibits significant flexibility, and transient perturbations of the tetrahedral geometry of two structurally important Cys4 zinc finger are seen, coupled to conformational changes in the D-box. In some cases, one of the Cys ligands to zinc exchanges with water, although no global distortion of the protein structure is observed. Thus, from MD simulation, dynamics of the D-box could partly be explained by solvent effects in conjunction with structural reformation of the zinc finger.

  • 20. Brenden, N.
    et al.
    Rietz, C.
    Böhme, Jan
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    E expression is needed on both bone marrow derived cells and thymic epithelium to increase IL-4 production and achieve protection in NOD bone marrow chimeras1999In: Cytokine, ISSN 1043-4666, E-ISSN 1096-0023, Vol. 11, no 10, 766-772 p.Article in journal (Refereed)
    Abstract [en]

    The NOD mouse is an animal model for insulin-dependent diabetes with many similarities to the human disease. NOD mice which are transgenic for the Ea gene, allowing expression of the E molecule, are protected from diabetes and rarely develop insulitis. We have constructed bone marrow chimeras between transgenic and non-transgenic NOD mice to study the correlation of E expression on bone marrow derived cells and thymic epithelium vs the production of IL-4 and IFN-γ. We show that NOD-E→NOD-E and NOD-E→NOD chimeras have elevated levels of IL-4 compared to NOD→NOD and NOD→NOD-E chimeras in the thymus. However, in the periphery the protected NOD-E→NOD-E show much higher IL-4 levels than any of the other chimeras. This drop in peripheral IL-4 production seen in NOD-E→NOD, NOD→NOD-E and NOD→NOD chimeras correlates with the increased insulitis seen in these mice compared to NOD-E→NOD-E. In contrast, there were no differences in IFN-γ production between the chimeras. We suggest that the precommitted, regulatory T cells, selected in an E-expressing thymic environment, need continuous interaction with E-expressing primary antigen presenting cells in the periphery for optimal IL-4 production. Decrease in IL-4 production correlates with increased insulitis.

  • 21.
    Buch, Charlotta
    et al.
    Södertörn University, School of Life Sciences.
    Hunt, Mary C.
    Alexson, Stefan E. H.
    Hallberg, Einar
    Södertörn University, School of Life Sciences.
    Localization of peroxisomal matrix proteins by photobleaching2009In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 388, no 2, 355-359 p.Article in journal (Refereed)
    Abstract [en]

    The distribution of some enzymes between peroxisomes and cytosol, or a dual localization in both these compartments, can be difficult to reconcile. We have used photobleaching in live cells expressing green fluorescent protein (GFP)-fusion proteins to show that imported bona fide peroxisomal matrix proteins are retained in the peroxisome. The high mobility of the GFP-fusion proteins in the cytosol and absence of peroxisomal escape makes it possible to eliminate the cytosolic fluorescence by photobleaching, to distinguish between exclusively cytosolic proteins and proteins that are also present at low levels in peroxisomes. Using this technique we found that GFP tagged bile acid-CoA:amino acid N-acyltransferase (BAAT) was exclusively localized in the cytosol in HeLa cells. We conclude that the cytosolic localization was due to its carboxyterminal non-consensus peroxisomal targeting signal (-SQL) since mutation of the -SQL to -SKL resulted in BAAT being efficiently imported into peroxisomes.

  • 22.
    Carlson, Tomas
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Skorupinska-Tudek, K
    Hertel, J
    Chojnacki, T
    Olsson, J M
    Swiezewska, E
    Single polyprenol and dolichol isolation by semipreparative high-performance liquid chromatography technique2000In: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 41, no 7, 1177-1180 p.Article in journal (Refereed)
    Abstract [en]

    A new method of separation of single polyprenols (or dolichols) from a mixture of isoprenoid alcohols is described. Application of a high-performance liquid chromatography (HPLC) apparatus equipped with a semipreparative ODS column resulted in preparation of long-chain (dihydro)polyprenols of high purity (>95%). This approach substantially decreases the time scale of the conventional chromatographical preparative procedure. The method can be widely used in chemical and biochemical projects, where single polyprenols or dolichols are required.

  • 23. Chamakuri, Srinivas
    et al.
    Guduru, Shiva Krishna Reddy
    Pamu, Sreedhar
    Chandrasekar, Gayathri
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies.
    Kitambi, Satish Srinivas
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies. Karolinska institutet.
    Arya, Prabhat
    A Modular Approach to Build Macrocyclic Diversity in Aminoindoline Scaffolds Identifies Antiangiogenesis Agents from a Zebrafish Assay2013In: European Journal of Organic Chemistry, ISSN 1434-193X, E-ISSN 1099-0690, no 19, 3959-3964 p.Article in journal (Refereed)
    Abstract [en]

    A modular approach to explore the macrocyclic chemical space around an aminoindoline scaffold is developed. This is achieved by incorporating an amino acid moiety and subsequent stitching technology. Through screening of a zebrafish assay, several antiangiogenesis agents are identified.

  • 24.
    Cierlik, Izabela Anna
    Södertörn University College, School of Life Sciences.
    Regulation of callose synthases and beta-1,3-glucanases during aphid infestation on barley cv. Clipper2008Independent thesis Basic level (professional degree), 20 points / 30 hpStudent thesis
    Abstract [en]

    Plant resistance hypothesis says that under a period of time when a plant is exposed to powerful herbivore attack it will prioritise defence as a major metabolic function. In theory, induced plant defence (resistance) will provide opportunities for this organism to “invest” in other functions, in example growth when attackers are absent.

    One of the compounds taking part in plant defence is callose. This β-1,3-glucan is synthesised by callose synthase and broken down by β-1,3-glucanase. Deposition of callose occurs as a reaction to aphid attack an varies, depending on cultivars, and aphid species. In this experiment barley (Hordeum vulgare) cultivar Clipper is being infested with two types of aphids: Russian wheat aphid (RWA, Diuraphis noxia) and bird cherry-oat aphid (BCA, Rhopalosiphium padi) over a time period. Infestation by those two insects results in different callose formation and deposition level.

    Six sequences encoding for putative callose synthase genes and nine sequences encoding for β-1,3-glucanase were examined by RT-PCR and Real – Time PCR methods for different expression patterns.

    The results did not show any significant regulation of gene expression during RWA and BCA attack for any of these genes. Thus the pathway regulating aphid – induced callose deposition in barley reminds unresolved.

  • 25. Cunnea, P M
    et al.
    Miranda-Vizuete, A
    Bertoli, G
    Simmen, T
    Damdimopoulos, A E
    Hermann, Stefan
    Södertörn University, Avdelning Naturvetenskap.
    Leinonen, S
    Huikko, M P
    Gustafsson, J A
    Sitia, R
    Spyrou, G
    ERdJ5, an endoplasmic reticulum (ER)-resident protein containing DnaJ and thioredoxin domains, is expressed in secretory cells or following ER stress2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 2, 1059-1066 p.Article in journal (Refereed)
  • 26. Czene, S
    et al.
    Testa, E
    Nygren, Jonas
    Södertörn University, Avdelning Naturvetenskap.
    Belyaev, I
    Harms-Ringdahl, M
    DNA fragmentation and morphological changes in apoptotic human lymphocytes2002In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 294, no 4, 872-878 p.Article in journal (Refereed)
    Abstract [en]

    Cell suspensions enriched in cells at various stages of apoptosis were obtained by separation of irradiated human peripheral blood lymphocytes on density gradients at different post-irradiation times. The state of DNA fragmentation in the cells was determined by comet assay and pulsed field gel electrophoresis. The morphologically distinguishable features of apoptosis such as chromatin condensation and cell shrinkage correlated with discrete stages of DNA fragmentation. It was found that greater than or equal to50kbp fragmentation of DNA occurs already in cells of normal density whereas the subsequent DNA fragmentation onto fragments <50 kbp occurs in parallel with cell shrinkage and simultaneous increase in cell density. The observed stages of DNA fragmentation seem to be separated in time that could allow in case of abortive apoptosis formation of chromosomal aberrations.

  • 27.
    Dahlman, Lena
    et al.
    Umeå universitet.
    Mattsson, Jan-Eric
    Westberg, Martin
    Naturhistoriska riksmuseet.
    Palmkvist, Kristin
    Umeå universitet.
    Choice of photobiont is more important than geographical origin or morphology in determining resource levels and metabolic capacity of lichens.2000Conference paper (Other academic)
  • 28. Deadman, Mary E.
    et al.
    Lundström, Susanna L.
    Schweda, Elke K. H.
    Södertörn University, School of Life Sciences.
    Moxon, E. Richard
    Hood, Derek W.
    Specific amino acids of the glycosyltransferase LpsA direct the addition of glucose or galactose to the terminal inner core heptose of Haemophilus influenzae lipopolysaccharide via alternative linkages2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 40, 29455-29467 p.Article in journal (Refereed)
    Abstract [en]

    Lipopolysaccharide is the major glycolipid of the cell wall of the bacterium Haemophilus influenzae, a Gram-negative commensal and pathogen of humans. Lipopolysaccharide is both a virulence determinant and a target for host immune responses. Glycosyltransferases have high donor and acceptor substrate specificities that are generally limited to catalysis of one unique glycosidic linkage. The H. influenzae glycosyltransferase LpsA is responsible for the addition of a hexose to the distal heptose of the inner core of the lipopolysaccharide molecule and belongs to the glycosyltransferase family 25. The hexose added can be either glucose or galactose and linkage to the heptose can be either beta 1-2 or beta 1-3. Each H. influenzae strain uniquely produces only one of the four possible combinations of linked sugar in its lipopolysaccharide. We show that, in any given strain, a specific allelic variant of LpsA directs the anomeric linkage and the added hexose, glucose, or galactose. Site-directed mutagenesis of a single key amino acid at position 151 changed the hexose added in vivo from glucose to galactose or vice versa. By constructing chimeric lpsA gene sequences, it was shown that the 3' end of the gene directs the anomeric linkage (beta 1-2 or beta 1-3) of the added hexose. The lpsA gene is the first known example where interstrain variation in lipopolysaccharide core structure is directed by the specific sequence of a genetic locus encoding enzymes directing one of four alternative possible sugar additions from the inner core.

  • 29.
    Delp, Gabriele
    et al.
    Södertörn University, School of Life Sciences, Biology. Södertörn University, School of Life Sciences, Molecular biology.
    Gradin, Therese
    Södertörn University, School of Life Sciences, Biology. Södertörn University, School of Life Sciences, Molecular biology.
    Ahman, Inger
    Jonsson, Lisbeth M. V.
    Södertörn University, School of Life Sciences, Biology. Södertörn University, School of Life Sciences, Molecular biology.
    Microarray analysis of the interaction between the aphid Rhopalosiphum padi and host plants reveals both differences and similarities between susceptible and partially resistant barley lines2009In: Molecular Genetics and Genomics, ISSN 1617-4615, E-ISSN 1617-4623, Vol. 281, no 3, 233-248 p.Article in journal (Refereed)
    Abstract [en]

    The bird cherry-oat aphid (Rhopalosiphum padi L.) is an important pest on cereals causing plant growth reduction without specific leaf symptoms. Breeding of barley (Hordeum vulgare L.) for R. padi resistance shows that there are several resistance genes, reducing aphid growth. To identify candidate sequences for resistance-related genes, we performed microarray analysis of gene expression after aphid infestation in two susceptible and two partially resistant barley genotypes. One of the four lines is a descendant of two of the other genotypes. There were large differences in gene induction between the four lines, indicating substantial variation in response even between closely related genotypes. Genes induced in aphid-infested tissue were mainly related to defence, primary metabolism and signalling. Only 24 genes were induced in all lines, none of them related to oxidative stress or secondary metabolism. Few genes were down-regulated, with none being common to all four lines. There were differences in aphid-induced gene regulation between resistant and susceptible lines. Results from control plants without aphids also revealed differences in constitutive gene expression between the two types of lines. Candidate sequences for induced and constitutive resistance factors have been identified, among them a proteinase inhibitor, a serine/threonine kinase and several thionins.

  • 30.
    Dionisi, Hebe
    et al.
    Patagonian Natl Res Ctr CENPAT CONICET, Buenos Aires, DF, Argentina.
    Matos, Marina
    Patagonian Natl Res Ctr CENPAT CONICET, Buenos Aires, DF, Argentina.
    Anselmino, Luciano
    Patagonian Natl Res Ctr CENPAT CONICET, Buenos Aires, DF, Argentina.
    Lozada, Mariana
    Patagonian Natl Res Ctr CENPAT CONICET, Buenos Aires, DF, Argentina.
    Mac Cormack, Walter
    Argentinean Antarctic Inst, Buenos Aires, DF, Argentina / Natl Univ Buenos Aires, Buenos Aires, DF, Argentina.
    Carroll, Jolynn
    Univ Tromsö, N-9001 Tromsö, Norway / Akvaplan Niva AS, FRAM High North Res Ctr Climate & Environm, Tromsö, Norway.
    Lundgren, Leif
    Stockholm University.
    Sjöling, Sara
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Biology.
    Chavarria, Krystle
    Lawrence Berkeley Natl Labs, Berkeley, CA USA.
    Henrissat, Bernard
    Ctr Natl Rech Sci, Marseille, France.
    Jansson, Janet
    Lawrence Berkeley Natl Labs, Berkeley, CA USA.
    Mining alginate lyases in sediment metagenomes from four geographically distant cold coastal environments2014In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 31, S69-S70 p.Article in journal (Refereed)
  • 31.
    Djupedal, Ingela
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Kos-Braun, Isabelle C.
    University of Edinburgh, Edinburgh, UK / Universität Heidelberg, Heidelberg, Germany.
    Mosher, Rebecca A.
    University of Cambridge, Cambridge, UK.
    Söderholm, Niklas
    Karolinska Institutet.
    Simmer, Femke
    University of Edinburgh, Edinburgh, UK.
    Hardcastle, Thomas J.
    University of Cambridge, Cambridge, UK.
    Fender, Aurelie
    Uppsala universitet.
    Heidrich, Nadja
    Uppsala universitet.
    Kagansky, Alexander
    University of Edinburgh, Edinburgh, UK.
    Bayne, Elizabeth
    University of Edinburgh, Edinburgh, UK.
    Wagner, E. Gerhart H.
    Uppala universitet.
    Baulcombe, David C.
    University of Cambridge, Cambridge, UK.
    Allshire, Robin C.
    University of Edinburgh, Edinburgh, UK.
    Ekwall, Karl
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA2009In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 28, no 24, 3832-3844 p.Article in journal (Refereed)
    Abstract [en]

    The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 50-monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably well-conserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1D cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity. The EMBO Journal (2009) 28, 3832-3844. doi: 10.1038/emboj.2009.351; Published online 26 November 2009

  • 32. Dunleavy, Elaine M.
    et al.
    Pidoux, Alison L.
    Monet, Marie
    Bonilla, Carolina
    Richardson, William
    Hamilton, Georgina L.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institute.
    McLaughlin, Paul J.
    Allshire, Robin C.
    A NASP (N1/N2)-related protein, Sim3, binds CENP-A and is required for its deposition at fission yeast Centromeres2007In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 28, no 6, 1029-1044 p.Article in journal (Refereed)
    Abstract [en]

    A defining feature of centromeres is the presence of the histone H3 variant CENP-A(Cnp1). It is not known how CENP-A(Cnp1) is specifically delivered to, and assembled into, centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast, we identified Sim3. Sim3 is homologous to known histone binding proteins NASP(Human) and N1/N2(Xenopus) and aligns with Hif1(S. cerevisiae), defining the SHNi-TPR family. Sim3 is distributed throughout the nucleoplasm, yet it associates with CENP-A(Cnp1) and also binds H3. Cells defective in Sim3 function have reduced levels of CENP-A(CnP1) at centromeres (and increased H3) and display chromosome segregation defects. Sim3 is required to allow newly synthesized CENP-A(Cnp1) to accumulate at centromeres in S and G2 phase-arrested cells in a replication-independent mechanism. We propose that one function of Sim3 is to act as an escort that hands off CENP-A(Cnp1) to chromatin assembly factors, allowing its incorporation into centromeric chromatin.

  • 33.
    Durand-Dubief, Mickael
    et al.
    Södertörn University, School of Life Sciences.
    Ekwall, Karl
    Södertörn University, School of Life Sciences.
    Heterochromatin tells CENP-A where to go2008In: Bioessays, ISSN 0265-9247, E-ISSN 1521-1878, Vol. 30, no 6, 526-529 p.Article in journal (Refereed)
    Abstract [en]

    The centromere is the region of the chromosome where the kinetochore forms. Kinetochores are the attachment sites for spindle microtubules that separate duplicated chromosomes in mitosis and meiosis. Kinetochore formation depends on a special chromatin structure containing the histone H3 variant CENP-A. The epigenetic mechanisms that maintain CENP-A chromatin throughout the cell cycle have been studied extensively but little is known about the mechanism that targets CENP-A to naked centromeric DNA templates. In a recent report published in Science,((1)) such de novo centromere assembly of CENP-A is shown to be dependent on heterochromatin and the RNA interference pathway.

  • 34.
    Durand-Dubief, Mickael
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Sinha, Indranil
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Fagerström-Billai, Fredrik
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Bonilla, Carolina
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Wright, Anthony
    Södertörn University, School of Life Sciences. Karolinska Instiutet.
    Grunstein, Michael
    University of California, Los Angeles, CA, USA.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation and retrotransposon silencing2007In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 26, no 10, 2477-2488 p.Article in journal (Refereed)
    Abstract [en]

    Expression profiling, ChiP-CHIP and phenotypic analysis were used to investigate the functional relationships of class III NAD(+)-dependent HDACs (Sirtuins) in fission yeast. We detected significant histone acetylation increases in Sirtuin mutants at their specific genomic binding targets and were thus able to identify an in vivo substrate preference for each Sirtuin. At heterochromatic loci, we demonstrate that although Hst2 is mainly cytoplasmic, a nuclear pool of Hst2 colocalizes with the other Sirtuins at silent regions (cen, mat, tel, rDNA), and that like the other Sirtuins, Hst2 is required for rDNA and centromeric silencing. Interestingly we found specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation. Hst2 directly represses genes involved in transport and membrane function, whereas Hst4 represses amino-acid biosynthesis genes and Tf2 retrotransposons. A specific role for Hst4 in Tf2 50 mRNA processing was revealed. Thus, Sirtuins share functions at many genomic targets, but Hst2 and Hst4 have also evolved unique functions in gene regulation.

  • 35. Dzieciatkowska, Monika
    et al.
    Schweda, Elke K. H.
    Södertörn University, School of Life Sciences.
    Moxon, E. Richard
    Richards, James C.
    Li, Jianjun
    Characterization of intact lipopolysaccharides from the Haemophilus influenzae strain RM 118 using electrophoresis-assisted open-tubular liquid chromatography-mass spectrometry2008In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 29, no 10, 2171-2181 p.Article in journal (Refereed)
    Abstract [en]

    We have applied an electrophoresis-assisted open-tubular LC-MS method for analyzing intact lipopolysaccharides (LPSs) from Haemophilus influenzae strain RM 118 (Rd). We were able to obtain structural information on both core oligosaccharides (OSs) and the lipid A moiety including the sialylation, glycylation, and the distribution of fatty acid residues on the disaccharide backbone of lipid A. The fragmentation patterns of sodiated and protonated LPS molecules were investigated for determining the location of sialic acid. It was found that the tandem mass spectra of sodiated ions provided unambiguous evidence of both sialylated lactose and sialylated lacto-N-neotetraose. In contrast, the fragment ions of protonated ions only offered the evidence for the existence of sialylated lacto-N-neotetraose. The lipid A of Gram-negative bacteria, as the principal endotoxic component of LP S, plays a major role in the pathogenesis of bacterial infections. We have previously characterized lipid. A species after mild acid hydrolysis of LPS during which lipid A precipitates. In this study, intact LPS was directly introduced to a tandem mass spectrometer. In-source dissociation strategy was employed, followed by multiple-stage MS/MS on the ions originating from the lipid part to obtain structural information. This is the first time that the structure of lipid A of H. influenzae was characterized by MS/MS on intact LPS molecules without any prior chemical modifications. In the same way information on the OS can be obtained by MS/MS by focusing on ions originating from core OS.

  • 36. Edvardsson, Anna
    et al.
    Shapiguzov, Alexey
    Petersson, Ulrika A.
    Södertörn University, School of Life Sciences. Stockholm University.
    Schröder, Wolfgang P.
    Vener, Alexander V.
    Immunophilin AtFKBP13 sustains all peptidyl-prolyl isomerase activity in the thylakoid lumen from Arabidopsis thaliana deficient in AtCYP20-22007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 33, 9432-9442 p.Article in journal (Refereed)
    Abstract [en]

    The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates.

  • 37.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institute.
    'Arc' escorts siRNAs in heterochromatin assembly2007In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 14, no 3, 178-179 p.Article in journal (Other academic)
    Abstract [en]

    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.

  • 38.
    Ekwall, Karl
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    The RITS complex - A direct link between small RNA and heterochromatin2004In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 13, no 3, 304-305 p.Article in journal (Refereed)
  • 39.
    Ekwall, Karl
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    The roles of histone modifications and small RNA in centromere function2004In: Chromosome Research, ISSN 0967-3849, E-ISSN 1573-6849, Vol. 12, no 6, 535-542 p.Article in journal (Refereed)
    Abstract [en]

    Here, epigenetic regulation of centromeric chromatin in fission yeast (Schizosaccharomyces pombe) is reviewed, focussing on the role of histone modifications and the link to RNA interference (RNAi). Fission yeast centromeres are organized into two structurally and functionally distinct domains, both of which are required for centromere function. The central core domain anchors the kinetochore structure while the flanking heterochromatin domain is important for sister centromere cohesion. The chromatin structure of both domains is regulated epigenetically. In the central core domain, the histone H3 variant Cnp1(CENP-A) plays a key role. In the flanking heterochromatin domain, histones are kept underacetylated by the histone deacetylases (HDACs) Clr3, Clr6 and Sir2, and methylated by Clr4 methyltransferase (HMTase) to create a specific binding site for the Swi6 protein. Swi6 then directly mediates cohesin binding to the centromeric heterochromatin. Recently, a surprising link was made between heterochromatin formation and RNAi. Centromeric flanking repeats are transcribed and the transcripts processed by the RNAse III-like enzyme, Dicer (Dcr1), to produce small interfering RNAs ( siRNA), which direct formation of heterochromatin via the RNA-induced Initiation of Transcriptional Silencing (RITS) protein complex. Consequently Dicer, Argonaute (Ago1), an RNA-dependent RNA polymerase (Rdp1) and several hitherto uncharacterized Csp ( centromere suppressor of position effect) gene products implicated in the RNAi pathway at centromeres are required for sister chromatid cohesion.

  • 40. El-Beqqali, Aziza
    et al.
    Kussak, Anders
    Södertörn University, School of Life Sciences.
    Abdel-Rehim, Mohamed
    Fast and sensitive environmental analysis utilizing microextraction in packed syringe online with gas chromatography-mass spectrometry - Determination of polycyclic aromatic hydrocarbons in water2006In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1114, no 2, 234-238 p.Article in journal (Refereed)
    Abstract [en]

    A new sensitive, selective, fast and accurate technique for online sample preparation was developed. Microextraction in a packed syringe (MEPS) is a new miniaturised, solid-phase extraction (SPE) technique that can be connected online to GC or LC without any modifications. In MEPS approximately 1 mg of the solid packing material is inserted into a syringe (100-250 ml) as a plug. Sample preparation takes place on the packed bed. The bed can be coated to provide selective and suitable sampling conditions. The new method is very promising. It is very easy to use, fully automated, of low cost and rapid in comparison with previously used methods. The determination of polycyclic hydrocarbons (PAHs) in water was performed using MEPS as sample preparation method online with gas chromatography and mass spectrometry (MEPS-GC-MS). The results from MEPS as sample preparation were compared with other techniques such as stir bar sorptive extraction (SBSE) and solid-phase microextraction (SPME). The method was validated and the standard curves were evaluated by the means of quadratic regression and weighted by inverse of the concentration: 1/x for the calibration range 5-1000 ng/L. The MEPS applied polymer (silica-C8) could be used more than 400 times before the syringe was discarded. The extraction recovery was about 70%. The results showed close correlation coefficients (R > 0.998) for all analytes in the calibration range studied. The accuracy of MEPS-GC-MS was between 90 and 113% and the inter-day precision (n = 3 days), expressed as the relative standard deviation (RSD%), was 8-16%. MEPS reduced the handling time by 30 and 100 times compared to SPME and SBSE, respectively.

  • 41. El-Beqqali, Aziza
    et al.
    Kussak, Anders
    Södertörn University, School of Life Sciences.
    Blomberg, Lars
    Abdel-Rehim, Mohamed
    Microextraction in packed syringe/liquid chromatography/electrospray tandem mass spectrometry for quantification of acebutolol and metoprolol in human plasma and urine samples2007In: Journal of Liquid Chromatography & Related Technologies, ISSN 1082-6076, E-ISSN 1520-572X, Vol. 30, no 4, 575-586 p.Article in journal (Refereed)
    Abstract [en]

    The aim of the present investigation was to develop a simple, fast, and sensitive method for the determination of acebutolol and metoprolol in human plasma and urine samples. The determination of acebutolol and metoprolol in plasma and urine was performed using micro extraction in packed syringe (MEPS) as a sample preparation method, online with high performance liquid chromatography and tandem mass spectrometry (LC-MS/MS). In MEPS the sampling sorbent was 1 mg polystyrene polymer, which was inserted in a 250 mu L syringe. The lower limits of quantification (LLOQ) for acebutolol and metoprolol were set to 1.0 ng/mL. The accuracy of quality control samples (QC) varied by +/- 10%, and precision (R.S.D.) had a deviation of 1.4-12% for plasma and urine samples. The calibration curve was obtained within the concentration range 1.0-100 ng/mL in both plasma and urine. The regression correlation coefficients (R-2) for plasma and urine samples were >= 0.999 for all runs. The present method is miniaturized, fully automated, robust, and can be easily used for pharmacokinetic and pharmacodynamic studies of acebutolol and metoprolol.

  • 42.
    Elgan, Tobias H.
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Berndt, Kurt D.
    Södertörn University, School of Life Sciences. Karolinska Insitute.
    Quantifying Escherichia coli Glutaredoxin-3 Substrate Specificity Using Ligand-induced Stability2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 47, 32839-32847 p.Article in journal (Refereed)
    Abstract [en]

    Traditionally, quantification of protein-ligand affinity is performed using kinetic or equilibrium measurements. However, if the binding reaction proceeds via a stable covalent complex, these approaches are often limited. By exploiting the fact that the conformational stabilization of a protein is altered upon ligand binding due to specific interactions, and using an array of selectively chosen ligand analogs, one can quantify the contribution individual interactions have on specificity. We have used ligand-induced stability as a basis to dissect the interaction between glutaredoxin-3 (Grx3) and one of its native substrates, the tripeptide glutathione. Taking advantage of the fact that Grx3 can be trapped in a covalent mixed disulfide to glutathione or to selected synthetic glutathione analogs as part of the natural catalytic cycle, individual contributions to binding of specific molecular groups can be quantified by changes in ligand-induced stability. These changes in conformational stability are interpreted in terms of interaction energies (i.e. specificity) of the particular groups present on the ligand analog. Our results illustrate that although Grx3 recognizes glutathione predominantly through independent and additive ionic interactions at the N- and C-terminal of glutathione, van der Waals interactions from the unique gamma-glutamate moiety of glutathione also play an important role. This study places us closer to understanding the complex task of accommodating multiple substrate specificities in proteins of the thioredoxin superfamily and underscores the general applicability of ligand-induced stability to probe substrate specificity.

  • 43.
    Elgán, Tobias H.
    et al.
    Södertörn University, School of Life Sciences. Karlolinska instituet.
    Planson, A. -G
    Beckwith, J.
    Güntert, P.
    Berndt, Kurt D.
    Södertörn University, School of Life Sciences, Chemistry. Karolinska institutet.
    Determinants of activity in glutaredoxins: An in vitro evolved Grx1-like variant of Escherichia coli Grx32010In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 430, no 3, 487-495 p.Article in journal (Refereed)
    Abstract [en]

    The Escherichia coli glutaredoxins 1 and 3 (Grx1 and Grx3) are structurally similar (37% sequence identity), yet have different activities in vivo. Unlike Grx3, Grx1 efficiently reduces protein disulfides in proteins such as RR (ribonucleotide reductase), whereas it is poor at reducing S-glutathionylated proteins. An E. coli strain lacking genes encoding thioredoxins 1 and 2 and Grx1 is not viable on either rich or minimal medium; however, a M43V mutation in Grx3 restores growth under these conditions and results in a Grx1-like protein [Ortenberg, Gon, Porat and Beckwith (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 7439-7944]. To uncover the structural basis of this change in activity, we have compared wild-type and mutant Grx3 using CD and NMR spectroscopy. Ligand-induced stability measurements demonstrate that the Grx3(M43V/C65Y) mutant has acquired affinity for RR. Far-UV CD spectra reveal no significant differences, but differences are observed in the near-UV region indicative of tertiary structural changes. NMR 1H- 15N HSQC (heteronuclear single quantum coherence) spectra show that approximately half of the 82 residues experience significant (Δδ &gt; 0.03 p.p.m.) chemical shift deviations in the mutant, including nine residues experiencing extensive (Δδ ≥ 0.15 p.p.m.) deviations. To test whether the M43V mutation alters dynamic properties of Grx3, H/D (hydrogen/deuterium) exchange experiments were performed demonstrating that the rate at which backbone amides exchange protons with the solvent is dramatically enhanced in the mutant, particularly in the core of the protein. These data suggest that the Grx1-like activity of the Grx3(M43V/C65Y) mutant may be explained by enhanced intrinsic motion allowing for increased specificity towards larger substrates such as RR.

  • 44. Engskog, Mikael K. R.
    et al.
    Yildirim, Håkan H.
    Södertörn University, School of Life Sciences.
    Li, Jianjun
    Richards, James C.
    Deadman, Mary
    Hood, Derek W.
    Schweda, Elke K. H.
    Södertörn University, School of Life Sciences, Chemistry.
    A dual role for the lex2 locus: identification of galactosyltransferase activity in non-typeable Haemophilus influenzae strains 1124 and 20192009In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 344, no 5, 632-641 p.Article in journal (Refereed)
    Abstract [en]

    Lipopolysaccharide (LPS) of Haemophilus influenzae comprises a conserved tri-L-glycero-D-manno-heptosyl inner-core moiety (L-alpha-D-Hepp-(1 -> 2)-[PEtn -> 6]-L-alpha-D-Hepp-(1 -> 3)-[beta-D-Glclp-(1 -> 4)]-L-alpha-D-Hepp-(1 -> 5)-alpha-Kdop) to which addition of beta-D-Glcp to O-4 of Glcl in serotype b strains is controlled by the gene lex2B. In non-typeable H. influenzae strains 1124 and 2019, however, a beta-D-Galp is linked to O-4 of Glcl. In order to test the hypothesis that the 1ex2 locus is involved in the expression Of beta-D-Galp-(1 -> 4-beta-D-Glcp-(1 -> - from Hepl, 1ex2B was inactivated in strains 1124 and 2019, and LPS glycoform populations from the resulting mutant strains were investigated. Detailed structural analyses using NMR techniques and electrospray-ionisation mass spectrometry (ESIMS) on O-cleacylated LPS and core oligosaccharide material (OS), as well as ESIMS" on permethylated dephosphorylated OS, indicated both lex2B mutant strains to express only beta-D-Glcp extensions from Hepl. This provides strong evidence that Lex2B functions as a galactosyltransferase adding a beta-D-Galp to O-4 of Glcl in these strains, indicating that allelic polymorphisms in the lex2B sequence direct alternative functions of the gene product.

  • 45. Eriksson, AnnaCarin
    et al.
    Glaser, Elzbieta
    Sjöling, Sara
    A general processing proteinase of spinach leaf mitochondria is a membrane bound enzyme1993In: Plant Mitochondria: with emphasis on RNA editing and cytoplasmic male sterility / [ed] Brennicke, Axel and Kuch, U, VCH Verlag , 1993, 233-241 p.Chapter in book (Other academic)
    Abstract [en]

    The book summarizes our current knowledge in the molecular biology of plant mitochondria. It covers such topics as: - RNA editing - mitochondrial gene organization and expression - protein synthesis and transport - cytoplasmic male sterility Specific emphasis is placed on RNA editing. Different systems known to date in mitochondria and plastids are compared. In addition, their connection with the molecular biology involved with the functional analysis is delineated. Another major topic is the molecular biology of mitochondrial genomes of cytoplasmic male sterile (cms) plants. The similarities observed in different cms systems not only promote our understanding of those processes which lead to male sterile plants but are also helpful for breeding strategies. Concise and timely, this book is a unique collaboration of researchers from different fields.

  • 46.
    Eriksson, Charlotta
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    Rustum, Cecilia
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Stockholm University.
    Hallberg, Einar
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Dynamic properties of nuclear pore complex proteins in gp210 deficient cells2004In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 572, no 1-3, 261-265 p.Article in journal (Refereed)
    Abstract [en]

    Gp210, an integral membrane protein of the nuclear pore complex (NPC), is believed to be involved in NPC biogenesis. To test this hypothesis, we have investigated dynamic properties of the NPC and distribution of NPC proteins in NIH/ 3T3 cells lacking gp210. POM121 (the other integral NPC protein) and NUP107 (of the NUP107/160 complex) were correctly distributed at the nuclear pores in the absence of gp210. Furthermore, fluorescence recovery after photobleaching experiments showed that POM121 and NUP107 remained stably associated at the NPCs. We conclude that gp210 cannot be required for incorporation of POM121 or NUP107 or be required for maintaining NPC stability.

  • 47. Eriksson, S
    et al.
    Nygren, Jonas
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Ahnström, G
    Matrix association of early- and late-replicating chromatin studied by single-cell electrophoresis2002In: Biochimica et Biophysica Acta. Molecular Cell Research, ISSN 0167-4889, E-ISSN 1879-2596, Vol. 1590, no 1-3, 103-108 p.Article in journal (Refereed)
    Abstract [en]

    CHO-K1 cells were synchronized at the G(1)/S border by mitotic shake-off and aphidicolin incubation. Pulse-labeling with tritium was done at 30 min, 2 or 5 h into the S-phase, with chase incubations for different times in non-radioactive medium. The cells were subjected to neutral microelectrophoresis to extend the DNA into "comets," after which the label was visualized through autoradiography. At zero chase time, all label was positioned in the head. The displacement of label into the tails increased with time, reaching a maximum at about 5 h after the pulse. A lag phase of 2 - 3 It was observed for the early-labeled cells before the displacement started. Also, more label was released after overnight serum starvation, but this was reversed through a 3-h incubation at normal growth conditions. It was found that late-replicating chromatin is organized in larger domains than early-replicating chromatin, and DNA polymerase seems to be an important organizer. Early-replicating chromatin has other important attachments to the nuclear matrix, dependent on metabolic activity.

  • 48.
    Fagerström-Billai, Fredrik
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Wright, Anthony P H
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Functional comparison of the Tup11 and Tup12 transcriptional corepressors in fission yeast2005In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 25, no 2, 716-727 p.Article in journal (Refereed)
    Abstract [en]

    Gene duplication is considered an important evolutionary mechanism. Unlike many characterized species, the fission yeast Schizosaccharomyces pombe contains two paralogous genes, tup11(+) and tup12(+), that encode transcriptional corepressors similar to the well-characterized budding yeast Tup1 protein. Previous reports have suggested that Tup11 and Tup12 proteins play redundant roles. Consistently, we show that the two Tup proteins can interact together when expressed at normal levels and that each can independently interact with the Ssn6 protein, as seen for Tup1 in budding yeast. However, tup11(-) and tup12(-) mutants have different phenotypes on media containing KCl and CaCl2. Consistent with the functional difference between tup11(-) and tup12- mutants, we identified a number of genes in genome-wide gene expression experiments that are differentially affected by mutations in the tup11(+) and tup12(+) genes. Many of these genes are differentially derepressed in tup11(-) mutants and are over-represented in genes that have previously been shown to respond to a range of different stress conditions. Genes specifically derepressed in tup12(-) mutants require the Ssn6 protein for their repression. As for Tupl.2, Ssn6 is also required for efficient adaptation to KCI- and CaCl2-mediated stress. We conclude that Tup11 and Tup12 are at least partly functionally diverged and suggest that the Tup12 and Ssn6 proteins have adopted a specific role in regulation of the stress response.

  • 49.
    Ferreira, Monica E.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska institutet.
    Studies of transcription factor domains and their interactions with other transcription factors2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    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.

  • 50.
    Ferreira, Monica E.
    et al.
    Södertörn University, School of Life Sciences, Molecular biology.
    Berndt, Kurt D.
    Södertörn University, School of Life Sciences, Chemistry.
    Nilsson, Johan
    Södertörn University, School of Life Sciences, Molecular biology.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences, Molecular biology.
    WD40 Domain Divergence Is Important for Functional Differences between he Fission Yeast Tup11 and Tup12 Co-Repressor Proteins2010In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, no 6, e11009- p.Article in journal (Refereed)
    Abstract [en]

    We have previously demonstrated that subsets of Ssn6/Tup target genes ave distinct requirements for the Schizosaccharomyces pombe homologs of he Tup1/Groucho/TLE co-repressor proteins, Tup11 and Tup12. The very igh level of divergence in the histone interacting repression domains f the two proteins suggested that determinants distinguishing Tup11 and up12 might be located in this domain. Here we have combined hylogenetic and structural analysis as well as phenotypic haracterization, under stress conditions that specifically require up12, to identify and characterize the domains involved in up12-specific action. The results indicate that divergence in the epression domain is not generally relevant for Tup12-specific function. nstead, we show that the more highly conserved C-terminal WD40 repeat omain of Tup12 is important for Tup12-specific function. Surface amino cid residues specific for the WD40 repeat domain of Tup12 proteins in ifferent fission yeasts are clustered in blade 3 of the propeller-like tructure that is characteristic of WD40 repeat domains. The Tup11 and up12 proteins in fission yeasts thus provide an excellent model system or studying the functional divergence of WD40 repeat domains.

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