| 1. |
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| 2. |
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| 3. |
- Andreasson, Erik, et al.
(författare)
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The MAP kinase substrate MKS1 is a regulator of plant defence responces
- 2005
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Ingår i: EMBO Journal. - : Wiley. - 1460-2075 .- 0261-4189. ; 24:14, s. 2579-2589
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Tidskriftsartikel (refereegranskat)abstract
- Arabidopsis MAP kinase 4 (MPK4) functions as a regulator of pathogen defense responses, because it is required for both repression of salicylic acid (SA)-dependent resistance and for activation of jasmonate (JA)-dependent defense gene expression. To understand MPK4 signaling mechanisms, we used yeast two-hybrid screening to identify the MPK4 substrate MKS1. Analyses of transgenic plants and genome-wide transcript profiling indicated that MKS1 is required for full SA-dependent resistance in mpk4 mutants, and that overexpression of MKS1 in wild-type plants is sufficient to activate SA-dependent resistance, but does not interfere with induction of a defense gene by JA. Further yeast two-hybrid screening revealed that MKS1 interacts with the WRKY transcription factors WRKY25 and WRKY33. WRKY25 and WRKY33 were shown to be in vitro substrates of MPK4, and a wrky33 knockout mutant was found to exhibit increased expression of the SA-related defense gene PR1. MKS1 may therefore contribute to MPK4-regulated defense activation by coupling the kinase to specific WRKY transcription factors.
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| 4. |
- Ansell, R, et al.
(författare)
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The two isoenzymes for yeast NAD+-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation.
- 1997
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Ingår i: The EMBO journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 16:9, s. 2179-87
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Tidskriftsartikel (refereegranskat)abstract
- The two homologous genes GPD1 and GPD2 encode the isoenzymes of NAD-dependent glycerol 3-phosphate dehydrogenase in the yeast Saccharomyces cerevisiae. Previous studies showed that GPD1 plays a role in osmoadaptation since its expression is induced by osmotic stress and gpd1 delta mutants are osmosensitive. Here we report that GPD2 has an entirely different physiological role. Expression of GPD2 is not affected by changes in external osmolarity, but is stimulated by anoxic conditions. Mutants lacking GPD2 show poor growth under anaerobic conditions. Mutants deleted for both GPD1 and GPD2 do not produce detectable glycerol, are highly osmosensitive and fail to grow under anoxic conditions. This growth inhibition, which is accompanied by a strong intracellular accumulation of NADH, is relieved by external addition of acetaldehyde, an effective oxidizer of NADH. Thus, glycerol formation is strictly required as a redox sink for excess cytosolic NADH during anaerobic metabolism. The anaerobic induction of GPD2 is independent of the HOG pathway which controls the osmotic induction of GPD1. Expression of GPD2 is also unaffected by ROX1 and ROX3, encoding putative regulators of hypoxic and stress-controlled gene expression. In addition, GPD2 is induced under aerobic conditions by the addition of bisulfite which causes NADH accumulation by inhibiting the final, reductive step in ethanol fermentation and this induction is reversed by addition of acetaldehyde. We conclude that expression of GPD2 is controlled by a novel, oxygen-independent, signalling pathway which is required to regulate metabolism under anoxic conditions.
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| 5. |
- Antoun, Ayman, et al.
(författare)
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How initiation factors tune the rate of initiation of protein synthesis in bacteria.
- 2006
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 25:11, s. 2539-50
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Tidskriftsartikel (refereegranskat)abstract
- The kinetics of initiator transfer RNA ( tRNA) interaction with the messenger RNA ( mRNA)-programmed 30S subunit and the rate of 50S subunit docking to the 30S preinitiation complex were measured for different combinations of initiation factors in a cell-free Escherichia coli system for protein synthesis with components of high purity. The major results are summarized by a Michaelis-Menten scheme for initiation. All three initiation factors are required for maximal efficiency ( k(cat)/K-M) of initiation and for maximal in vivo rate of initiation at normal concentration of initiator tRNA. Spontaneous release of IF3 from the 30S preinitiation complex is required for subunit docking. The presence of initiator tRNA on the 30S subunit greatly increases the rate of 70S ribosome formation by increasing the rate of IF3 dissociation from the 30S subunit and the rate of 50S subunit docking to the IF3-free 30S preinitiation complex. The reasons why IF1 and IF3 are essential in E. coli are discussed in the light of the present observations.
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| 6. |
- Antoun, Ayman, et al.
(författare)
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The roles of initiation factor 2 and guanosine triphosphate in initiation of protein synthesis
- 2003
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 22:20, s. 5593-5601
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Tidskriftsartikel (refereegranskat)abstract
- The role of IF2 from Escherichia coli was studied in vitro using a system for protein synthesis with purified components. Stopped flow experiments with light scattering show that IF2 in complex with guanosine triphosphate (GTP) or a non-cleavable GTP analogue (GDPNP), but not with guanosine diphosphate (GDP), promotes fast association of ribosomal subunits during initiation. Biochemical experiments show that IF2 promotes fast formation of the first peptide bond in the presence of GTP, but not GDPNP or GDP, and that IF2–GDPNP binds strongly to post-initiation ribosomes. We conclude that the GTP form of IF2 accelerates formation of the 70S ribosome from subunits and that GTP hydrolysis accelerates release of IF2 from the 70S ribosome. The results of a recent report, suggesting that GTP and GDP promote initiation equally fast, have been addressed. Our data, indicating that eIF5B and IF2 have similar functions, are used to rationalize the phenotypes of GTPase-deficient mutants of eIF5B and IF2.
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| 7. |
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| 8. |
- Arkov, AL, et al.
(författare)
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Mutations in RNAs of both ribosomal subunits cause defects in translation termination
- 1998
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Ingår i: EMBO JOURNAL. - : OXFORD UNIV PRESS. - 0261-4189. ; 17:5, s. 1507-1514
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Mutations in RNAs of both subunits of the Escherichia coil ribosome caused defects in catalysis of peptidyl-tRNA hydrolysis in a realistic in vitro termination system. Assaying the two codon-dependent cytoplasmic proteins that drive termination, RF1 and R
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| 9. |
- Bacal, Julien, et al.
(författare)
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Mrc1 and Rad9 cooperate to regulate initiation and elongation of DNA replication in response to DNA damage
- 2018
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Ingår i: EMBO Journal. - : Wiley-VCH Verlagsgesellschaft. - 0261-4189 .- 1460-2075. ; 37:21
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Tidskriftsartikel (refereegranskat)abstract
- The S-phase checkpoint maintains the integrity of the genome in response to DNA replication stress. In budding yeast, this pathway is initiated by Mec1 and is amplified through the activation of Rad53 by two checkpoint mediators: Mrc1 promotes Rad53 activation at stalled forks, and Rad9 is a general mediator of the DNA damage response. Here, we have investigated the interplay between Mrc1 and Rad9 in response to DNA damage and found that they control DNA replication through two distinct but complementary mechanisms. Mrc1 rapidly activates Rad53 at stalled forks and represses late-firing origins but is unable to maintain this repression over time. Rad9 takes over Mrc1 to maintain a continuous checkpoint signaling. Importantly, the Rad9-mediated activation of Rad53 slows down fork progression, supporting the view that the S-phase checkpoint controls both the initiation and the elongation of DNA replication in response to DNA damage. Together, these data indicate that Mrc1 and Rad9 play distinct functions that are important to ensure an optimal completion of S phase under replication stress conditions.
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| 10. |
- Baumeister, Ulf, et al.
(författare)
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Association of Csk to VE-cadherin and inhibition of cell proliferation
- 2005
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Ingår i: EMBO Journal. - : Wiley-Blackwell Publishing Inc.. - 0261-4189 .- 1460-2075. ; 24:9, s. 1686-1695
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Tidskriftsartikel (refereegranskat)abstract
- Vascular endothelial cadherin (VE-cadherin) mediates contact inhibition of cell growth in quiescent endothelial cell layers. Searching for proteins that could be involved in VE-cadherin signaling, we found the cytosolic C-terminal Src kinase (Csk), a negative regulator of Src family kinases. We show that Csk binds via its SH2 domain to the phosphorylated tyrosine 685 of VE-cadherin. VE-cadherin recruits Csk to cell contacts and both proteins can be co-precipitated from cell lysates of transfected cells and endothelial cells. Association of VE-cadherin and Csk in endothelial cells increased with increasing cell density. CHO cells expressing the tyrosine replacement mutant VE-cadherin-Y685F grow to higher cell densities than cells expressing wild-type VE-cadherin. Overexpression of Csk in these cells under an inducible promoter inhibits cell proliferation in the presence and absence of VE-cadherin, but not in the presence of VE-cadherin-Y685F. Reduction of Csk expression by RNA interference enhances endothelial cell proliferation. Our results suggest that the phosphorylated tyrosine residue 685 of VE-cadherin and probably the binding of Csk to this site are involved in inhibition of cell growth triggered by cell density.
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| 11. |
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| 12. |
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| 13. |
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| 14. |
- Bernard, Pascal, et al.
(författare)
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Cell-cycle regulation of cohesin stability along fission yeast chromosomes
- 2008
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 27:1, s. 111-121
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 15. |
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| 16. |
- Bianco, Paolo, et al.
(författare)
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Regulation of stem cell therapies under attack in Europe: for whom the bell tolls
- 2013
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Ingår i: EMBO Journal. - : Wiley. - 1460-2075 .- 0261-4189. ; 32:11, s. 1489-1495
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- At the time of writing, the Italian Parliament is debating a new law that would make it legal to practice an unproven stem cell treatment in public hospitals. The treatment, offered by a private non-medical organization, may not be safe, lacks a rationale, and violates current national laws and European regulations. This case raises multiple concerns, most prominently the urgent need to protect patients who are severely ill, exposed to significant risks, and vulnerable to exploitation. The scientific community must consider the context-social, financial, medical, legal-in which stem cell science is currently situated and the need for stringent regulation. Additional concerns are emerging. These emanate from the novel climate, created within science itself, and stem cell science in particular, by the currently prevailing model of 'translational medicine'. Only rigorous science and rigorous regulation can ensure translation of science into effective therapies rather than into ineffective market products, and mark, at the same time, the sharp distinction between the striving for new therapies and the deceit of patients.
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| 17. |
- Bilkei-Gorzo, Orsolya, et al.
(författare)
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The E3 ubiquitin ligase RNF115 regulates phagosome maturation and host response to bacterial infection
- 2022
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Ingår i: EMBO Journal. - : EMBO. - 0261-4189 .- 1460-2075.
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Tidskriftsartikel (refereegranskat)abstract
- Phagocytosis is a key process in innate immunity and homeostasis. After particle uptake, newly formed phagosomes mature by acquisition of endolysosomal enzymes. Macrophage activation by interferon gamma (IFN-gamma) increases microbicidal activity, but delays phagosomal maturation by an unknown mechanism. Using quantitative proteomics, we show that phagosomal proteins harbour high levels of typical and atypical ubiquitin chain types. Moreover, phagosomal ubiquitylation of vesicle trafficking proteins is substantially enhanced upon IFN-gamma activation of macrophages, suggesting a role in regulating phagosomal functions. We identified the E3 ubiquitin ligase RNF115, which is enriched on phagosomes of IFN-gamma activated macrophages, as an important regulator of phagosomal maturation. Loss of RNF115 protein or ligase activity enhanced phagosomal maturation and increased cytokine responses to bacterial infection, suggesting that both innate immune signalling from the phagosome and phagolysosomal trafficking are controlled through ubiquitylation. RNF115 knock-out mice show less tissue damage in response to S. aureus infection, indicating a role of RNF115 in inflammatory responses in vivo. In conclusion, RNF115 and phagosomal ubiquitylation are important regulators of innate immune functions during bacterial infections.
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| 18. |
- Billker, Oliver, et al.
(författare)
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Distinct mechanisms of internalization of Neisseria gonorrhoeae by members of the CEACAM receptor family involving Rac1- and Cdc42-dependent and -independent pathways
- 2002
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Ingår i: EMBO Journal. - : European Molecular Biology Organization. - 0261-4189 .- 1460-2075. ; 21:4, s. 560-571
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Tidskriftsartikel (refereegranskat)abstract
- Opa adhesins of pathogenic Neisseria species target four members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. CEACAM receptors mediate opsonization-independent phagocytosis of Neisseria gonorrhoeae by human granulocytes and each receptor individually can mediate gonococcal invasion of epithelial cells. We show here that gonococcal internalization occurs by distinct mechanisms depending on the CEACAM receptor expressed. For the invasion of epithelial cell lines via CEACAM1 and CEACAM6, a pathogen-directed reorganization of the actin cytoskeleton is not required. In marked contrast, ligation of CEACAM3 triggers a dramatic but localized reorganization of the host cell surface leading to highly efficient engulfment of bacteria in a process regulated by the small GTPases Rac1 and Cdc42, but not Rho. Two tyrosine residues of a cytoplasmic immune receptor tyrosine-based activating motif of CEACAM3 are essential for the induction of phagocytic actin structures and subsequent gonococcal internalization. The granulocyte-specific CEACAM3 receptor has properties of a single chain phagocytic receptor and may thus contribute to innate immunity by the elimination of Neisseria and other CEACAM-binding pathogens that colonize human mucosal surfaces.
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| 19. |
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| 20. |
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| 21. |
- Björk, Glenn R, et al.
(författare)
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A primordial tRNA modification required for the evolution of life?
- 2001
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 20:1-2, s. 231-239
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Tidskriftsartikel (refereegranskat)abstract
- The evolution of reading frame maintenance must have been an early event, and presumably preceded the emergence of the three domains Archaea, Bacteria and Eukarya. Features evolved early in reading frame maintenance may still exist in present-day organisms. We show that one such feature may be the modified nucleoside 1-methylguanosine (m(1)G37), which prevents frameshifting and is present adjacent to and 3' of the anticodon (position 37) in the same subset of tRNAs from all organisms, including that with the smallest sequenced genome (Mycoplasma genitalium), and organelles. We have identified the genes encoding the enzyme tRNA(m(1)G37)methyltransferase from all three domains. We also show that they are orthologues, and suggest that they originated from a primordial gene. Lack of m(1)G37 severely impairs the growth of a bacterium and a eukaryote to a similar degree. Yeast tRNA(m(1)G37)methyltransferase also synthesizes 1-methylinosine and participates in the formation of the Y-base (yW). Our results suggest that m(1)G37 existed in tRNA before the divergence of the three domains, and that a tRNA(m(1)G37)methyltrans ferase is part of the minimal set of gene products required for life.
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| 22. |
- Björklund, Stefan, et al.
(författare)
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An S-phase specific release from a transcriptional block regulates the expression of mouse ribonucleotide reductase R2 subunit
- 1992
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Ingår i: EMBO Journal. - 0261-4189 .- 1460-2075. ; 11:13, s. 4953-4959
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RR) activity in mammalian cells is closely linked to DNA synthesis. The RR enzyme is composed of two non-identical subunits, proteins R1 and R2. Both proteins are required for holoenzyme activity, which is regulated by S-phase specific de novo synthesis and breakdown of the R2 subunit. In quiescent cells stimulated to proliferate and in elutriated cell populations enriched in the various cell cycle phases the R2 protein levels are correlated to R2 mRNA levels that are low in G0/G1-phase cells but increase dramatically at the G1/S border. Using an R2 promoter-luciferase reporter gene construct we demonstrate an unexpected early activation of the R2 promoter as cells pass from quiescence to proliferation. However, due to a transcriptional block, this promoter activation only results in very short R2 transcripts until cells enter the S-phase, when full-length R2 transcripts start to appear. The position for the transcriptional block was localized to a nucleotide sequence approximately 87 bp downstream from the first exon/intron boundary by S1 nuclease mapping of R2 transcripts from modified in vitro nuclear run-on experiments. These results identify blocking of transcription as a mechanism to control cell cycle regulated gene expression.
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| 23. |
- Blomqvist, Sandra Rodrigo, 1974, et al.
(författare)
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Epididymal expression of the forkhead transcription factor Foxi1 is required for male fertility.
- 2006
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Ingår i: The EMBO journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 25:17, s. 4131-41
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Tidskriftsartikel (refereegranskat)abstract
- An essential aspect of male reproductive capacity is the immediate availability of fertilization-ready spermatozoa. To ensure this, most mammals rely on post-testicular sperm maturation. In epididymis, germ cells are matured and stored in a quiescent state that readily can be altered to produce active spermatozoa. This depends on active proton secretion into the epididymal lumen. We have identified Foxi1 as an important regulator of gene expression in narrow and clear cells-the major proton secretory cells of epididymal epithelia. Foxi1 appears to be required for the expression of the B1-subunit of the vacuolar H+ -ATPase proton pump and for carbonic anhydrase II as well as the chloride/bicarbonate transporter pendrin. Using transfection experiments, we have identified a Foxi1 binding cis-element in the ATP6V1B1 (encoding the B1-subunit) promoter that is critical for reporter gene activation. When this site is mutated to eliminate Foxi1 binding, activation is also abolished. As a consequence of defect Foxi1-dependent epididymal sperm maturation, we demonstrate that spermatozoa from Foxi1 null males fail to reach the female genital tract in sufficient number to allow fertilization.
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| 24. |
- Blume-Jensen, Peter, et al.
(författare)
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Activation of the human c-kit product by ligand-induced dimerization mediates circular actin reorganization and chemotaxis
- 1991
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Ingår i: EMBO Journal. - 0261-4189 .- 1460-2075. ; 10:13, s. 4121-4128
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Tidskriftsartikel (refereegranskat)abstract
- The proto-oncogene c-kit is allelic with the murine white spotting (W) locus and encodes a transmembrane protein tyrosine kinase that is structurally related to the receptors for platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1). Recently the ligand for the c-kit product, stem cell factor (SCF), was identified in both transmembrane and soluble forms. In order to examine the mechanism for receptor activation by SCF and biological properties of the activated c-kit product, we transfected the wild-type human c-kit cDNA into porcine aortic endothelial cells. We found that the receptor was down-regulated and transmitted a mitogenic signal in response to stimulation with soluble SCF. We also demonstrate that SCF induces dimerization of the c-kit product in intact cells, and that dimerization of the receptor is correlated with activation of its kinase. Activation of the c-kit product by SCF was found to induce circular actin reorganization indistinguishable from that mediated by the PDGF beta-receptor in response to PDGF-BB. Furthermore, soluble SCF was a potent chemotactic agent for cells expressing the c-kit product, a property which might be of importance during embryonic development.
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| 25. |
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| 26. |
- Bohmert, K., et al.
(författare)
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AGO1 defines a novel locus of Arabidopsis controlling leaf development
- 1998
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 17:1, s. 170-180
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Tidskriftsartikel (refereegranskat)abstract
- An allelic series of the novel argonaute mutant (ago1-1 to ago1-6) of the herbaceous plant Arabidopsis thaliana has been isolated, The ago1 mutation pleotropically affects general plant architecture, The apical shoot meristem generates rosette leaves and a single stem, but axillary meristems rarely develop, Rosette leaves lack a leaf blade but still show adaxial/abaxial differentiation, Instead of cauline leaves, filamentous structures without adaxial/abaxial differentiation develop along the stem and an abnormal inflorescence bearing infertile flowers with filamentous organs is produced, Two independent T-DNA insertions into the AGO1 locus led to the isolation of two corresponding genomic sequences as well as a complete cDNA. The AGO1 locus was mapped close to the marker mi291a on chromosome 1. Antisense expression of the cDNA resulted in a partial mutant phenotype, Sense expression caused some transgenic lines to develop goblet-like leaves and petals, The cDNA encodes a putative 115 kDa protein with sequence similarity tea translation products of a novel gene family present in nematodes as,yell as humans, No specific function has been assigned to these genes, Similar proteins are not encoded by the genomes of yeast or bacteria, suggesting that AGOI belongs to a novel class of genes with a function specific to multicellular organisms.
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| 27. |
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| 28. |
- Borenäs, Marcus, et al.
(författare)
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ALK ligand ALKAL2 potentiates MYCN-driven neuroblastoma in the absence of ALK mutation
- 2021
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Ingår i: EMBO Journal. - : John Wiley & Sons. - 0261-4189 .- 1460-2075. ; 40:3
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Tidskriftsartikel (refereegranskat)abstract
- High‐risk neuroblastoma (NB) is responsible for a disproportionate number of childhood deaths due to cancer. One indicator of high‐risk NB is amplification of the neural MYC (MYCN) oncogene, which is currently therapeutically intractable. Identification of anaplastic lymphoma kinase (ALK) as an NB oncogene raised the possibility of using ALK tyrosine kinase inhibitors (TKIs) in treatment of patients with activating ALK mutations. 8–10% of primary NB patients are ALK‐positive, a figure that increases in the relapsed population. ALK is activated by the ALKAL2 ligand located on chromosome 2p, along with ALK and MYCN, in the “2p‐gain” region associated with NB. Dysregulation of ALK ligand in NB has not been addressed, although one of the first oncogenes described was v‐sis that shares > 90% homology with PDGF. Therefore, we tested whether ALKAL2 ligand could potentiate NB progression in the absence of ALK mutation. We show that ALKAL2 overexpression in mice drives ALK TKI‐sensitive NB in the absence of ALK mutation, suggesting that additional NB patients, such as those exhibiting 2p‐gain, may benefit from ALK TKI‐based therapeutic intervention.
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| 29. |
- Botelho, Salomé Calado, et al.
(författare)
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TIM23-mediated insertion of transmembrane alpha-helices into the mitochondrial inner membrane
- 2011
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 30:6, s. 1003-1011
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Tidskriftsartikel (refereegranskat)abstract
- While overall hydrophobicity is generally recognized as the main characteristic of transmembrane (TM) alpha-helices, the only membrane system for which there are detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells. Here, we provide comparable data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the TM segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated difference between the two insertion systems.
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| 30. |
- Bountra, K., et al.
(författare)
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Structural basis for antibacterial peptide self-immunity by the bacterial ABC transporter McjD
- 2017
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Ingår i: Embo Journal. - : EMBO. - 0261-4189 .- 1460-2075. ; 36:20, s. 3062-3079
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Tidskriftsartikel (refereegranskat)abstract
- Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self-immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward-occluded and a new nucleotide-bound state, high-energy outward-occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross-linking in E.coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi-drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic-level build-up.
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| 31. |
- Boutté, Yohann, et al.
(författare)
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Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis
- 2010
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 29:3, s. 546-58
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Tidskriftsartikel (refereegranskat)abstract
- Cytokinesis represents the final stage of eukaryotic cell division during which the cytoplasm becomes partitioned between daughter cells. The process differs to some extent between animal and plant cells, but proteins of the syntaxin family mediate membrane fusion in the plane of cell division in diverse organisms. How syntaxin localization is kept in check remains elusive. Here, we report that localization of the Arabidopsis KNOLLE syntaxin in the plane of cell division is maintained by sterol-dependent endocytosis involving a clathrin- and DYNAMIN-RELATED PROTEIN1A-dependent mechanism. On genetic or pharmacological interference with endocytosis, KNOLLE mis-localizes to lateral plasma membranes after cell-plate fusion. Fluorescence-loss-in-photo-bleaching and fluorescence-recovery-after-photo-bleaching experiments reveal lateral diffusion of GFP-KNOLLE from the plane of division to lateral membranes. In an endocytosis-defective sterol biosynthesis mutant displaying lateral KNOLLE diffusion, KNOLLE secretory trafficking remains unaffected. Thus, restriction of lateral diffusion by endocytosis may serve to maintain specificity of syntaxin localization during late cytokinesis.
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| 32. |
- Breyer, F., et al.
(författare)
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TPL-2 kinase induces phagosome acidification to promote macrophage killing of bacteria
- 2021
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Ingår i: Embo Journal. - : EMBO. - 0261-4189 .- 1460-2075. ; 40:10
-
Tidskriftsartikel (refereegranskat)abstract
- Tumour progression locus 2 (TPL-2) kinase mediates Toll-like receptor (TLR) activation of ERK1/2 and p38 alpha MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase-independent regulatory function for TPL-2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL-2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL-2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V-ATPase proton pump subunits. Furthermore, TPL-2 stimulated the phosphorylation of DMXL1, a regulator of V-ATPases, to induce V-ATPase assembly and phagosome acidification. Consistent with these results, TPL-2 catalytic activity was required for phagosome acidification and the efficient killing of Staphylococcus aureus and Citrobacter rodentium following phagocytic uptake by macrophages. TPL-2 therefore controls innate immune responses of macrophages to bacteria via V-ATPase induction of phagosome maturation.
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| 33. |
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| 34. |
- Bryant, Helen E, et al.
(författare)
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PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination.
- 2009
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Ingår i: The EMBO journal. - : Wiley. - 1460-2075 .- 0261-4189. ; 28:17, s. 2601-15
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Tidskriftsartikel (refereegranskat)abstract
- If replication forks are perturbed, a multifaceted response including several DNA repair and cell cycle checkpoint pathways is activated to ensure faithful DNA replication. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1) binds to and is activated by stalled replication forks that contain small gaps. PARP1 collaborates with Mre11 to promote replication fork restart after release from replication blocks, most likely by recruiting Mre11 to the replication fork to promote resection of DNA. Both PARP1 and PARP2 are required for hydroxyurea-induced homologous recombination to promote cell survival after replication blocks. Together, our data suggest that PARP1 and PARP2 detect disrupted replication forks and attract Mre11 for end processing that is required for subsequent recombination repair and restart of replication forks.
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| 35. |
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| 36. |
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| 37. |
- Byström, Anders S, et al.
(författare)
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The nucleotide sequence of an Escherichia coli operon containing genes for the tRNA(m1G)methyltransferase, the ribosomal proteins S16 and L19 and a 21-K polypeptide
- 1983
-
Ingår i: EMBO Journal. - : Oxford University Press. - 0261-4189 .- 1460-2075. ; 2:6, s. 899-905
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Tidskriftsartikel (refereegranskat)abstract
- The nucleotide sequence of a 4.6-kb SalI-EcoRI DNA fragment including the trmD operon, located at min 56 on the Escherichia coli K-12 chromosome, has been determined. The trmD operon encodes four polypeptides: ribosomal protein S16 (rpsP), 21-K polypeptide (unknown function), tRNA-(m1G)methyltransferase (trmD) and ribosomal protein L19 (rplS), in that order. In addition, the 4.6-kb DNA fragment encodes a 48-K and a 16-K polypeptide of unknown functions which are not part of the trmD operon. The mol. wt. of tRNA(m1G)methyltransferase determined from the DNA sequence is 28 424. The probable locations of promoter and terminator of the trmD operon are suggested. The translational start of the trmD gene was deduced from the known NH2-terminal amino acid sequence of the purified enzyme. The intercistronic regions in the operon vary from 9 to 40 nucleotides, supporting the earlier conclusion that the four genes are co-transcribed, starting at the major promoter in front of the rpsP gene. Since it is known that ribosomal proteins are present at 8000 molecules/genome and the tRNA-(m1G)methyltransferase at only approximately 80 molecules/genome in a glucose minimal culture, some powerful regulatory device must exist in this operon to maintain this non-coordinate expression. The codon usage of the two ribosomal protein genes is similar to that of other ribosomal protein genes, i.e., high preference for the most abundant tRNA isoaccepting species. The trmD gene has a codon usage typical for a protein made in low amount in accordance with the low number of tRNA-(m1G)methyltransferase molecules found in the cell.
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| 38. |
- Cabeen, Matthew T., et al.
(författare)
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Bacterial cell curvature through mechanical control of cell growth
- 2009
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 28:9, s. 1208-1219
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Tidskriftsartikel (refereegranskat)abstract
- The cytoskeleton is a key regulator of cell morphogenesis. Crescentin, a bacterial intermediate filament-like protein, is required for the curved shape of Caulobacter crescentus and localizes to the inner cell curvature. Here, we show that crescentin forms a single filamentous structure that collapses into a helix when detached from the cell membrane, suggesting that it is normally maintained in a stretched configuration. Crescentin causes an elongation rate gradient around the circumference of the sidewall, creating a longitudinal cell length differential and hence curvature. Such curvature can be produced by physical force alone when cells are grown in circular microchambers. Production of crescentin in Escherichia coli is sufficient to generate cell curvature. Our data argue for a model in which physical strain borne by the crescentin structure anisotropically alters the kinetics of cell wall insertion to produce curved growth. Our study suggests that bacteria may use the cytoskeleton for mechanical control of growth to alter morphology. The EMBO Journal (2009) 28, 1208-1219. doi: 10.1038/emboj.2009.61; Published online 12 March 2009
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| 39. |
- Cameron, AD, et al.
(författare)
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Crystal structure of human glyoxalase .1. Evidence for gene duplication and 3D domain swapping
- 1997
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Ingår i: EMBO JOURNAL. - : OXFORD UNIV PRESS. - 0261-4189. ; 16:12, s. 3386-3395
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The zinc metalloenzyme glyoxalase I catalyses the glutathione-dependent inactivation of toxic methylglyoxal. The structure of the dimeric human enzyme in complex with S-benzyl-glutathione has been determined by multiple isomorphous replacement (MIR) and r
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| 40. |
- CAMPBELL, D, et al.
(författare)
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ELECTRON-TRANSPORT REGULATES EXCHANGE OF 2 FORMS OF PHOTOSYSTEM-II D1 PROTEIN IN THE CYANOBACTERIUM SYNECHOCOCCUS
- 1995
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Ingår i: EMBO Journal. - 0261-4189 .- 1460-2075. ; 14:22, s. 5457-5466
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Tidskriftsartikel (refereegranskat)abstract
- Synechococcus sp, PCC 7942 modulates photosynthetic function by transiently replacing the constitutive D1 photosystem II protein, D1:1, with an alternate form, D1:2, to help counteract photoinhibition under excess light, We show that a temperature drop from 37 to 25 degrees C also drives D1:1/D1:2 exchange under constant, moderate light, Chilling or light-induced D1 exchange results from rapid loss of psbAI message coding for D1:1 and accumulation of psbAII and psbAIII messages coding for D1:2, During chilling, a large pool of a novel form, D1:2*, transiently accumulates, distinguishable from normal D1 by an increase in apparent molecular mass, D1:* is not phosphorylated and is probably a functionally inactive, incompletely processed precursor, After acclimation to 25 degrees C, D1:2* disappears and D1:1 again predominates, although substantial D1:2 remains, Partial inhibition of electron transport under constant, moderate light also triggers the D1 exchange process, These treatments all increase excitation pressure on photosystem II relative to electron transport, Therefore, information from photosynthetic electron transport regulates D1 exchange without any requirement for a change in light intensity or quality, possibly via a redox sensing mechanism proximal to photosystem II.
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| 41. |
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| 42. |
- Cava, Felipe, et al.
(författare)
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Distinct pathways for modification of the bacterial cell wall by non-canonical D-amino acids
- 2011
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 30:16, s. 3442-3453
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Tidskriftsartikel (refereegranskat)abstract
- Production of non-canonical D-amino acids (NCDAAs) in stationary phase promotes remodelling of peptidoglycan (PG), the polymer that comprises the bacterial cell wall. Impairment of NCDAAs production leads to excessive accumulation of PG and hypersensitivity to osmotic shock; however, the mechanistic bases for these phenotypes were not previously determined. Here, we show that incorporation of NCDAAs into PG is a critical means by which NCDAAs control PG abundance and strength. We identified and reconstituted in vitro two (of at least three) distinct processes that mediate NCDAA incorporation. Diverse bacterial phyla incorporate NCDAAs into their cell walls, either through periplasmic editing of the mature PG or via incorporation into PG precursor subunits in the cytosol. Production of NCDAAs in Vibrio cholerae requires the stress response sigma factor RpoS, suggesting that NCDAAs may aid bacteria in responding to varied environmental challenges. The widespread capacity of diverse bacteria, including non-producers, to incorporate NCDAAs suggests that these amino acids may serve as both autocrine- and paracrine-like regulators of chemical and physical properties of the cell wall in microbial communities.
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| 43. |
- Cevher, Murat A., et al.
(författare)
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Nuclear deadenylation/polyadenylation factors regulate 3 ' processing in response to DNA damage
- 2010
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 29:10, s. 1674-1687
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Tidskriftsartikel (refereegranskat)abstract
- We previously showed that mRNA 3' end cleavage reaction in cell extracts is strongly but transiently inhibited under DNA-damaging conditions. The cleavage stimulation factor-50 (CstF-50) has a role in this response, providing a link between transcription-coupled RNA processing and DNA repair. In this study, we show that CstF-50 interacts with nuclear poly(A)-specific ribonuclease (PARN) using in vitro and in extracts of UV-exposed cells. The CstF-50/PARN complex formation has a role in the inhibition of 3' cleavage and activation of deadenylation upon DNA damage. Extending these results, we found that the tumour suppressor BARD1, which is involved in the UV-induced inhibition of 3' cleavage, strongly activates deadenylation by PARN in the presence of CstF-50, and that CstF-50/BARD1 can revert the cap-binding protein-80 (CBP80)mediated inhibition of PARN activity. We also provide evidence that PARN along with the CstF/BARD1 complex participates in the regulation of endogenous transcripts under DNA-damaging conditions. We speculate that the interplay between polyadenylation, deadenylation and tumour-suppressor factors might prevent the expression of prematurely terminated messengers, contributing to control of gene expression under different cellular conditions.
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| 44. |
- Chen, Peng, et al.
(författare)
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A "gain of function" mutation in a protein mediates production of novel modified nucleosides.
- 2005
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 24:10, s. 1842-1851
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Tidskriftsartikel (refereegranskat)abstract
- The mutation sufY204 mediates suppression of a +1 frameshift mutation in the histidine operon of Salmonella enterica serovar Typhimurium and synthesis of two novel modified nucleosides in tRNA. The sufY204 mutation, which results in an amino-acid substitution in a protein, is, surprisingly, dominant over its wild-type allele and thus it is a "gain of function" mutation. One of the new nucleosides is 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) modified by addition of a C(10)H(17) side chain of unknown structure. Increased amounts of both nucleosides in tRNA are correlated to gene dosage of the sufY204 allele, to an increased efficiency of frameshift suppression, and to a decreased amount of the wobble nucleoside mnm(5)s(2)U34 in tRNA. Purified tRNA(Gln)(cmnm(5)s(2)UUG) in the mutant strain contains a modified nucleoside similar to the novel nucleosides and the level of aminoacylation of tRNA(Gln)(cmnm(5)s(2)UUG) was reduced to 26% compared to that found in the wild type (86%). The results are discussed in relation to the mechanism of reading frame maintenance and the evolution of modified nucleosides in tRNA.
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| 45. |
- Cisneros, David A., et al.
(författare)
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Minor pseudopilin self-assembly primes type II secretion pseudopilus elongation
- 2012
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 31:4, s. 1041-1053
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Tidskriftsartikel (refereegranskat)abstract
- In Gram-negative bacteria, type II secretion systems (T2SS) assemble inner membrane proteins of the major pseudopilin PulG (GspG) family into periplasmic filaments, which could drive protein secretion in a piston-like manner. Three minor pseudopilins PulI, PulJ and PulK are essential for protein secretion in the Klebsiella oxytoca T2SS, but their molecular function is unknown. Here, we demonstrate that together these proteins prime pseudopilus assembly, without actively controlling its length or secretin channel opening. Using molecular dynamics, bacterial two-hybrid assays, cysteine crosslinking and functional analysis, we show that PulI and PulJ nucleate filament assembly by forming a staggered complex in the plasma membrane. Binding of PulK to this complex results in its partial extraction from the membrane and in a 1-nm shift between their transmembrane segments, equivalent to the major pseudopilin register in the assembled PulG filament. This promotes fully efficient pseudopilus assembly and protein secretion. Therefore, we propose that PulI, PulJ and PulK self-assembly is thermodynamically coupled to the initiation of pseudopilus assembly, possibly setting the assembly machinery in motion.
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| 46. |
- Dai, Qi, et al.
(författare)
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Drosophila Ebi mediates Snail-dependent transcriptional repression through HDAC3-induced histone deacetylation
- 2008
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 27:6, s. 898-909
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Tidskriftsartikel (refereegranskat)abstract
- The Drosophila Snail protein is a transcriptional repressor that is necessary for mesoderm formation. Here, we identify the Ebi protein as an essential Snail co-repressor. In ebi mutant embryos, Snail target genes are derepressed in the presumptive mesoderm. Ebi and Snail interact both genetically and physically. We identify a Snail domain that is sufficient for Ebi binding, and which functions independently of another Snail co-repressor, Drosophila CtBP. This Ebi interaction domain is conserved among all insect Snail-related proteins, is a potent repression domain and is required for Snail function in transgenic embryos. In mammalian cells, the Ebi homologue TBL1 is part of the NCoR/SMRT–HDAC3 (histone deacetylase 3) co-repressor complex. We found that Ebi interacts with Drosophila HDAC3, and that HDAC3 knockdown or addition of a HDAC inhibitor impairs Snail-mediated repression in cells. In the early embryo, Ebi is recruited to a Snail target gene in a Snail-dependent manner, which coincides with histone hypoacetylation. Our results demonstrate that Snail requires the combined activities of Ebi and CtBP, and indicate that histone deacetylation is a repression mechanism in early Drosophila development.
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| 47. |
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| 48. |
- Davidson, Iain F., et al.
(författare)
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Rapid movement and transcriptional re-localization of human cohesin on DNA
- 2016
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Ingår i: EMBO Journal. - : EMBO. - 0261-4189 .- 1460-2075. ; 35:24, s. 2671-2685
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Tidskriftsartikel (refereegranskat)abstract
- The spatial organization, correct expression, repair, and segregation of eukaryotic genomes depend on cohesin, ring-shaped protein complexes that are thought to function by entrapping DNA. It has been proposed that cohesin is recruited to specific genomic locations from distal loading sites by an unknown mechanism, which depends on transcription, and it has been speculated that cohesin movements along DNA could create three-dimensional genomic organization by loop extrusion. However, whether cohesin can translocate along DNA is unknown. Here, we used single-molecule imaging to show that cohesin can diffuse rapidly on DNA in a manner consistent with topological entrapment and can pass over some DNA-bound proteins and nucleosomes but is constrained in its movement by transcription and DNA-bound CCCTC-binding factor (CTCF). These results indicate that cohesin can be positioned in the genome by moving along DNA, that transcription can provide directionality to these movements, that CTCF functions as a boundary element for moving cohesin, and they are consistent with the hypothesis that cohesin spatially organizes the genome via loop extrusion.
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| 49. |
- Davidson, Marta B., et al.
(författare)
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Endogenous DNA replication stress results in expansion of dNTP pools and a mutator phenotype
- 2012
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Ingår i: EMBO Journal. - : European Molecular Biology Organization. - 0261-4189 .- 1460-2075. ; 31:4, s. 895-907
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Tidskriftsartikel (refereegranskat)abstract
- The integrity of the genome depends on diverse pathways that regulate DNA metabolism. Defects in these pathways result in genome instability, a hallmark of cancer. Deletion of ELG1 in budding yeast, when combined with hypomorphic alleles of PCNA results in spontaneous DNA damage during S phase that elicits upregulation of ribonucleotide reductase (RNR) activity. Increased RNR activity leads to a dramatic expansion of deoxyribonucleotide (dNTP) pools in G1 that allows cells to synthesize significant fractions of the genome in the presence of hydroxyurea in the subsequent S phase. Consistent with the recognized correlation between dNTP levels and spontaneous mutation, compromising ELG1 and PCNA results in a significant increase in mutation rates. Deletion of distinct genome stability genes RAD54, RAD55, and TSA1 also results in increased dNTP levels and mutagenesis, suggesting that this is a general phenomenon. Together, our data point to a vicious circle in which mutations in gatekeeper genes give rise to genomic instability during S phase, inducing expansion of the dNTP pool, which in turn results in high levels of spontaneous mutagenesis. The EMBO Journal (2012) 31, 895-907. doi: 10.1038/emboj.2011.485; Published online 10 January 2012
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| 50. |
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