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  • 51. Bergknut, Magnus
    et al.
    Kucera, Adam
    Frech, Kristina
    Andersson, Erika
    Engwall, Magnus
    Rannug, Ulf
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Koci, Vladimir
    Andersson, Patrik L
    Haglund, Peter
    Tysklind, Mats
    Identification of potentially toxic compounds in complex extracts of environmental samples using gas chromatography-mass spectrometry and multivariate data analysis.2007In: Environ Toxicol Chem, ISSN 0730-7268, Vol. 26, no 2, p. 208-17Article in journal (Other academic)
  • 52. Bertrand, Yann
    et al.
    Topel, Mats
    Elvang, Annelie
    Melik, Wessam
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Magnus
    First Dating of a Recombination Event in Mammalian Tick-Borne Flaviviruses2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 2, p. e31981-Article in journal (Refereed)
    Abstract [en]

    The mammalian tick-borne flavivirus group (MTBFG) contains viruses associated with important human and animal diseases such as encephalitis and hemorrhagic fever. In contrast to mosquito-borne flaviviruses where recombination events are frequent, the evolutionary dynamic within the MTBFG was believed to be essentially clonal. This assumption was challenged with the recent report of several homologous recombinations within the Tick-borne encephalitis virus (TBEV). We performed a thorough analysis of publicly available genomes in this group and found no compelling evidence for the previously identified recombinations. However, our results show for the first time that demonstrable recombination (i.e., with large statistical support and strong phylogenetic evidences) has occurred in the MTBFG, more specifically within the Louping ill virus lineage. Putative parents, recombinant strains and breakpoints were further tested for statistical significance using phylogenetic methods. We investigated the time of divergence between the recombinant and parental strains in a Bayesian framework. The recombination was estimated to have occurred during a window of 282 to 76 years before the present. By unravelling the temporal setting of the event, we adduce hypotheses about the ecological conditions that could account for the observed recombination.

  • 53.
    Beskow, Anne
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Grimberg, Kristian Björk
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bott, Laura C.
    Karolinska Institutet, Institutionen för cell- och molekulärbiologi.
    Salomons, Florian A.
    Karolinska Institutet, Institutionen för cell- och molekulärbiologi.
    Dantuma, Nico P.
    Karolinska Institutet, Institutionen för cell- och molekulärbiologi.
    Young, Patrick
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    A conserved unfoldase activity for the p97 AAA-ATPase in proteasomal degradation2009In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 394, no 4, p. 732-46Article in journal (Refereed)
    Abstract [en]

    The multifunctional AAA-ATPase p97 is one of the most abundant and conserved proteins in eukaryotic cells. The p97/Npl4/Ufd1 complex dislocates proteins that fail the protein quality control in the endoplasmic reticulum to the cytosol where they are subject to degradation by the ubiquitin/proteasome system. Substrate dislocation depends on the unfoldase activity of p97. Interestingly, p97 is also involved in the degradation of specific soluble proteasome substrates but the exact mode of action of p97 in this process is unclear. Here, we show that both the central pore and ATPase activity of p97 are necessary for the degradation of cytosolic ubiquitin-fusion substrates. Addition of a flexible extended C-terminal peptide to the substrate relieves the requirement for p97. Deletion mapping reveals a conserved length dependency of 20 residues for the peptide, which allows p97-independent degradation to occur. Our results suggest that initiation of unfolding may be more complex than previously anticipated and that the 19S regulatory complex of the proteasome can require preprocessing of highly folded, ubiquitylated substrates by the p97(Ufd1/Npl4) complex. Our data provide an explanation for the observation that p97 is only essential for a subpopulation of soluble substrates and predict that a common characteristic of soluble p97-dependent substrates is the lack of an initiation site to facilitate unfolding by the 26S proteasome.

  • 54. Bettencourt, R
    et al.
    Assefaw-Redda, Y
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Faye, I
    The insect immune protein hemolin is expressed during oogenesis and embryogenesis2000In: Mechanisms of development, ISSN 0925-4773, Vol. 95, no 1-2, p. 301-304Article in journal (Refereed)
  • 55.
    Bettencourt, Raul
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hemolin, a versatile immune protein from the Cecropia moth1999Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Insects have become useful models for the study of innate immune mechanisms, due to their lack of antibodies and receptors involved in adaptive immune response. However, the molecules and mechanisms involved in primordial immune recognition are still poorly understood. Hemolin, originally cloned from Hyalophora cecropia, is a soluble and membrane associated Ig-related molecule which meets the criteria for a pattern recognition molecule. It is constitutively expressed in the hemolymph and up-regulated after bacterial injection. It was shown to bind specifically to the Lipid A moiety of LPS from bacteria and to associate with aggregates formed by hemocytes and bacteria. The aim of the present studies was to characterize the binding of hemolin to insect cells and further investigate the mechanisms behind. Earlier results shown in Manduca sexta were confirmed and it was shown that soluble hemolin has a deaggregating effect on naive hemocytes of H. cecropia. Moreover, hemolin increases phagocytic activity of hemocytes, especially when combined with LPS. This enhanced phagocytosis was correlated to the activity of protein kinase C and tyrosine phosphorylation. We revealed a number of cell adhesion characteristics of hemolin 1) a membrane associated form, (2) Ca2+-dependent homophilic binding, (3) glycosylation (4) importance of Ca2+ and carbohydrates on the binding to hemocytes. Based on our present results and the hemolin crystal structure, we have proposed a model for the interactions between soluble hemolin and its membrane form. The close relatedness to NCAMs prompted us to investigate its expression in other tissues than those originally described. It was found that hemolin is present in the retinal eye discs of pupae, in developing follicles during oogenesis and in embryos of H. cecropia. It was inferred that hemolin has a role in developmental processes in addition to its putative immune functions in insects. From a phylogenetic point of view, hemolin function is consistent with the assumption that non-self recognition molecules of the IgSF arose from cell-adhesion molecules with multiple functions.

  • 56.
    Bettencourt, Raul
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Terenius, Olle
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Faye, Ingrid
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hemolin gene silencing by ds-RNA injected into Cecropia pupae is lethal to next generation embryos2002In: Insect Molecular Biology, ISSN 0962-1075, Vol. 11, no 3, p. 267-271Article in journal (Refereed)
    Abstract [en]

    There is increasing evidence of an intimate connection between participants in the innate immune system and in development. Molecules involved in the determination of dorso-ventral polarity in Drosophila have related counterparts in the signalling pathways for immune gene activation in both insects and mammals. Hemolin from the Giant silkmoth, Hyalophora cecropia, identified as a bacteria-inducible molecule and a member of the immunoglobulin superfamily, is present as protein and transcripts in oocytes and embryos. We used RNA interference (RNAi) to investigate H. cecropia gene function in vivo and demonstrated that Hemolin is crucial for the normal development of embryos. When RNAi-females were mated, no larvae emerged from their eggs and when dissected, the eggs revealed malformed embryos. Western blot analysis confirmed the lack of Hemolin gene products. We conclude that Hemolin is necessary for development, since the silencing of Hemolin gene expression leads to embryonic lethality

  • 57. Bianchi, Vera
    et al.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Pontis, Elisabeth
    Reichard, Peter
    Interruption of the ferredoxin (flavodoxin) NADP+ oxidoreductase gene of Escherichia coli does not affect anaerobic growth but increases sensitivity to paraquat.1995In: J Bacteriol, ISSN 0021-9193, Vol. 177, no 15, p. 4528-31Article in journal (Other academic)
  • 58.
    Biverstal, Anna
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Cyclobutane pyrimidine dimers do not fully explain the mutagenicity induced by UVA in Chinese hamster cells2008In: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 648, no 02-jan, p. 32-39Article in journal (Refereed)
    Abstract [en]

    UVA generates low levels of cyclobutane pyrimidine dimers (CPDs). Here we asked the question whether CPDs could fully explain the level of mutations induced by UVA. Relative mutagenicities of UVA and UVC were calculated at equal levels of CPDs in cell lines, deficient in different aspects of repair. Survival and gene mutations in the hprt locus were analyzed in a set of Chinese hamster ovary (CHO) cell lines, i.e., wild-type, Cockayne syndrome B protein-deficient (CSB), XRCC3-deficient and XRCC1-deficient adjusted to the same level of CPDs which was analyzed as strand breaks as a result of DNA cleavage by T4 endonuclease V at CPD sites. Induced mutagenicity of UVA was approximately 2 times higher than the mutagenicity of UVC in both wild-type and XRCC1-deficient cells when calculated at equal level of CPDs. Since this discrepancy could be explained by the fact that the TT-dimers, induced by UVA, might be more mutagenic than C-containing CPDs induced by UVC, we applied acetophenone, a photosensitizer previously shown to generate enhanced levels of TT-CPDs upon UVB exposure. The results suggested that the TT-CPDs were actually less mutagenic than the C-containing CPDs. We also found that the mutagenic effect of UVA was not significantly enhanced in a cell line deficient in the repair of CPDs. Altogether this suggests that neither base excision- nor nucleotide excision-repair was involved. We further challenge the possibility that the lesion responsible for the mutations induced by UVA was of a more complex nature and which possibly is repaired by homologous recombination (HR). The results indicated that UVA was more recombinogenic than UVC at equal levels of CPDs. We therefore suggest that UVA induces a complex type of lesion, which might be an obstruction during replication fork progression that requires HR repair to be further processed. 

  • 59. Blundred, Rachel
    et al.
    Myers, Katie
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Goldman, Alastair S H
    Bryant, Helen E
    Human RECQL5 overcomes thymidine-induced replication stress.2010In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 9, no 9, p. 964-75Article in journal (Refereed)
    Abstract [en]

    Accurate DNA replication is essential to genome integrity and is controlled by five human RecQ helicases, of which at least three prevent cancer and ageing. Here, we have studied the role of RECQL5, which is the least characterised of the five human RecQ helicases. We demonstrate that overexpressed RECQL5 promotes survival during thymidine-induced slowing of replication forks in human cells. The RECQL5 protein relocates specifically to stalled replication forks and suppresses thymidine-induced RPA foci, CHK1 signalling, homologous recombination and gammaH2AX activation. It is unlikely that RECQL5 promotes survival through translesion synthesis as PCNA ubiquitylation is also reduced. Interestingly, we also found that overexpressing RECQL5 relieves cells of the cell cycle arrest normally imposed by thymidine, but without causing mutations. In conclusion, we propose that RECQL5 stabilises the replication fork allowing replication to overcome the effects of thymidine and complete the cell cycle.

  • 60.
    Brehwens, Karl
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The changing dose rate: A new factor of importance in radiobiology?2011Licentiate thesis, monograph (Other academic)
  • 61.
    Brehwens, Karl
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bajinskis, Ainars
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Staaf, Elina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haghdoost, Siamak
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Cederwall, Bo
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    A NEW DEVICE TO EXPOSE CELLS TO CHANGING DOSE RATES OF IONISING RADIATION2012In: Radiation Protection Dosimetry, ISSN 0144-8420, E-ISSN 1742-3406, Vol. 148, no 3, p. 366-371Article in journal (Refereed)
    Abstract [en]

    In many exposure scenarios to ionising radiation, the dose rate is not constant. Despite this, most in vitro studies aimed at investigating the effects of ionising radiation are carried out exposing samples at constant dose rates. Consequently, very little data exist on the biological effects of exposures to changing dose rates. This may be due to technical limitations of standard irradiation facilities, but also to the fact that the importance of research in this area has not been appreciated. We have recently shown that cells exposed to a decreasing dose rate suffer higher levels of cytogenetic damage than do cells exposed to an increasing or a constant dose rate. To further study the effects of changing dose rates, a new device was constructed that permits the exposure of cell samples in tubes, flasks or Petri dishes to changing dose rates of X-rays. This report presents the technical data, performance and dosimetry of this novel device.

  • 62.
    Brehwens, Karl
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bajinskis, Ainars
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Staaf, Elina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haghdoost, Siamak
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Changing dose rates influences the cellular effect of ionising radiation2011In: Strahlentherapie und Onkologie (Print), ISSN 0179-7158, E-ISSN 1439-099X, Vol. 187, p. 107-108Article in journal (Refereed)
  • 63.
    Brehwens, Karl
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Staaf, Elina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haghdoost, Siamak
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    González, Abel J
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Cytogenetic damage in cells exposed to ionizing radiation under conditions of a changing dose rate2010In: Radiation Research, ISSN 0033-7587, E-ISSN 1938-5404, Vol. 173, no 3, p. 283-9Article in journal (Refereed)
    Abstract [en]

    The current international paradigm on the biological effects of radiation is based mainly on the effects of dose with some consideration for the dose rate. No allowance has been made for the potential influence of a changing dose rate (second derivative of dose), and the biological effects of exposing cells to changing dose rates have never been analyzed. This paper provides evidence that radiation effects in cells may depend on temporal changes in the dose rate. In these experiments, cells were moved toward or away from an X-ray source. The speed of movement, the time of irradiation, and the temperature during exposure were controlled. Here we report the results of the first experiments with TK6 cells that were exposed at a constant dose rate, at an increasing dose rate, or at a decreasing dose rate. The average dose rate and the total dose were same for all samples. Micronuclei were scored as the end point. The results show that the level of cytogenetic damage was higher in cells exposed to a decreasing dose rate compared to both an increasing and a constant dose rate. This finding may suggest that the second derivative of dose may influence radiation risk estimates, and the results should trigger further studies on this issue.

  • 64. Bryant, Helen E
    et al.
    Petermann, Eva
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jemth, Ann-Sofie
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Loseva, Olga
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Issaeva, Natalia
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Fernandez, Serena
    McGlynn, Peter
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination.2009In: The EMBO journal, ISSN 1460-2075, Vol. 28, no 17, p. 2601-15Article in journal (Refereed)
    Abstract [en]

    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.

  • 65. Brzozowska, Kinga
    et al.
    Johannes, Christian
    Obe, Gunter
    Hentschel, Reinhard
    Morand, Josselin
    Moss, Ray
    Wittig, Andrea
    Sauerwein, Wolfgang
    Liniecki, Julian
    Szumiel, Irena
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Effect of temperature during irradiation on the level of micronuclei in human peripheral blood lymphocytes exposed to X-rays and neutrons.2009In: International Journal of Radiation Biology, ISSN 0955-3002, E-ISSN 1362-3095, p. 1-9Article in journal (Refereed)
    Abstract [en]

    Objectives: It has been reported that the level of cytogenetic damage in human peripheral blood lymphocytes (PBL) is higher following irradiation at 37 degrees C than at 0-4 degrees C. The mechanisms of this cytogenetic temperature effect are not fully known. The aim of our study was to check whether the effect was related to the indirect or direct action of radiation. Materials and methods: PBL were kept at 37 degrees C and 0 degrees C for 20 min and exposed to 2 Gy of X-rays. In some experiments PBL were isolated and 0.5 M dimethyl sulfoxide (DMSO) was added for 5 min before exposure. PBL were also irradiated at 37 degrees C and 0 degrees C with 1 Gy of 6 MeV neutrons. Micronuclei were scored as the endpoint. Following exposure to X-rays the level of initial DNA damage was also measured by the alkaline and neutral comet assay. Results: The frequency of micronuclei in cells exposed at 37 degrees C to X-rays or neutrons was higher than that after exposure at 0 degrees C. No effect of temperature was seen when PBL were exposed to X-rays in the presence of DMSO. No effect of temperature was observed on the level of DNA damage measured with the alkaline or neutral comet assay. Conclusions: The results of experiments with DMSO indicate that the temperature effect is due to the indirect action of radiation, i.e., via reactive oxygen species. However, this is not supported by the results with neutrons and the comet assay. Possible reasons for the discrepancies are discussed.

  • 66. Brzozowska, Kinga
    et al.
    Pinkawa, Michael
    Eble, Michael J.
    Muller, Wolfgang-Ullrich
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Kriehuber, Ralf
    Schmitz, Sabine
    In vivo versus in vitro individual radiosensitivity analysed in healthy donors and in prostate cancer patients with and without severe side effects after radiotherapy2012In: International Journal of Radiation Biology, ISSN 0955-3002, E-ISSN 1362-3095, Vol. 88, no 5, p. 405-413Article in journal (Refereed)
    Abstract [en]

    Background : A high cellular radiosensitivity may be connected with a risk for development of severe side effects after radiotherapy and indicate cancer susceptibility. Hence, a fast and robust in vitro test is desirable to identify radiosensitive individuals. Materials and methods : The study included 25 prostate cancer patients with severe side effects (S) and 25 patients without severe side effects (0) after radiotherapy as well as 23 male healthy age-matched donors. Blood samples were exposed to 0.5 Gy or 1 Gy of gamma-rays. The initial level of double-strand breaks (dsb) and repair kinetics measured by phosphorylation of histone H2A (gamma-H2AX-assay), apoptosis (Annexin V-assay) and the induction of chromatid aberrations after irradiation in the G2-phase of the cell cycle (G2-assay) were analysed. Results : A significant higher chromatid aberration yield was found in lymphocytes from prostate cancer patients when compared to healthy donors. We found no significant differences between patients S and patients 0. Conclusions : There is no obvious correlation between clinical and cellular radiosensitivity in lymphocytes of prostate cancer patients when all chosen in vitro assays are considered. Although 25% of the patients showed both severe side effects and increased radiation-induced chromosomal sensitivity, predictive value of G2-assay is doubtful.

  • 67.
    Buch, Charlotta
    et al.
    Södertorns högskola.
    Hunt, Mary C
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Alexson, Stefan E H
    Karolinska Institutet.
    Hallberg, Einar
    Södertorns Högskola.
    Localization of peroxisomal matrix proteins by photobleaching.2009In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 388, p. 355-9Article 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.

  • 68. Bugaeva, Elizaveta Y.
    et al.
    Shpanchenko, Olga V.
    Felden, Brice
    Isaksson, Leif A.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Dontsova, Olga A.
    One SmpB molecule accompanies tmRNA during its passage through the ribosomes2008In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 582, no 10, p. 1532-1536Article in journal (Refereed)
    Abstract [en]

    tmRNA and SmpB are the main participants of trans-translation, a process which rescues the ribosome blocked during translation of non-stop mRNA. While a one-to-one stoichiometry of tmRNA to the ribosome is generally accepted, the number of SmpB molecules in the complex is still under question. We have isolated tmRNA-ribosome complexes blocked at different steps of the tmRNA path through the ribosome and analyzed the stoichiometry of the complexes. Ribosome, tmRNA and SmpB were found in equimolar amount in the tmRNA-ribosome complexes stopped at the position of the 2nd, 4th, 5th or the 11th codons of the coding part of the tmRNA. 

  • 69. Bugaeva, Elizaveta Y
    et al.
    Surkov, Serhiy
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Golovin, Andrey V
    Ofverstedt, Lars-Göran
    Skoglund, Ulf
    Isaksson, Leif A
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bogdanov, Alexey A
    Shpanchenko, Olga V
    Dontsova, Olga A
    Structural features of the tmRNA-ribosome interaction2009In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 15, no 12, p. 2312-2320Article in journal (Refereed)
    Abstract [en]

    Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA-ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.

  • 70.
    Cardoso Palacios, Carlos
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Site-specific recombination in P2-like coliphages2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The scope of these studies has been to investigate the site-specific recombination systems of P2-like coliphages, both in an evolutionary perspective by a comparative analysis of related phages as well as in a functional perspective.

    Surveys of P2-like phages in Escherichia coli isolated from nature reveal the existence of seven discrete immunity classes and three integration sites, one of them previously unknown. Phylogenetic analysis of the repressor proteins and other analyses show that homologous recombination plays a role in the appearance of new immunities. Other studies of P2-like prophages from sequenced genomes from public databases show that the P2-like phages grow in different γ-proteobacteria. Based on the type of immunity and site-specific recombination system they can be roughly subdivided in two distinct subgroups and some new host integration sites could be identified. Some of the host attachment sites have a high identity to the sequences in the human genome, making them interesting as potential tools for targeted gene insertions into unmodified human cells.

    The functional studies have been focused on the identification of the determinants for site specificity, which is important for the use of the enzyme for targeted gene insertions into unmodified genomes. Two approaches have been used. In one, we have performed a structure-function analysis of P2 Int that has identified several presumptive residues involved in specific binding to the core sequence, all of them located in the same alpha-helix. This knowledge could be a base for an in vitro evolution of the integrase to enable it to accept new DNA targets with a high affinity. With respect to the excisionases from P2-like coliphages integrating in different sites, we found that they share some common features when they bind and bend to their DNA targets, but there are also significant differences, especially those related to the number of binding sites and the distribution of these and the IHF binding sites in the attP regions. In the other approach we have started to characterize the site-specific recombination system of another P2-like phage, ΦD145, that has a host target with a high identity to a site in the human genome. This looks promising since the human sequence can be used in vivo in E. coli with a rather high efficiency.

  • 71.
    Cardoso Palacios, Carlos
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sylwan, L.
    Haggård-Ljungquist, E.
    A structure-function analysis of P2 integrasManuscript (Other academic)
  • 72.
    Cardoso-Palacios, Carlos
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sylwan, Lina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Mandali, Sridhar
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Frumerie, Clara
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    A structure-function analysis of P2 integraseManuscript (preprint) (Other academic)
    Abstract [en]

    Bacteriophage P2 integrase catalyzes site-specific recombination between the phage DNA and the host chromosome thereby promoting integration or excision of the phage genome. P2 integrase belongs to the large tyrosine family of integrases that shows little sequence identity besides some conserved boxes and patches in the catalytic domain. However, the overall structure of the tyrosine family of integrases seems to be similar. Phage integrases have the potential as tools for site-specific gene insertions into eukaryotic genomes provided that target sequences are available. To elucidate the possibility of evolving the P2 integrase to accept new targets, we have in this work initiated a structure-function analysis of the P2 integrase using two approaches based on a comparison of the predicted secondary structure of P2 integrase with that determined for the lambda integrase. First, we have made hybrids between P2 integrase and the related WΦ integrase that has a different host DNA target, to locate the region promoting specificity between the integrases. This, however, has not been possible, the N-terminal domains can be exchanged without losing biological activity and this will not affect the specificity. All other hybrids made were biological inactive. Next we have made an alanine scanning of the alpha helices believed to be involved in specific interactions with the target, and four amino acids have been identified as candidates for sequence-specific interactions with the core.

  • 73. Chadt, J
    et al.
    Sykora, D
    Nilsson, Robert
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Monitoring of dimethyl sulphate-induced N3-methyladenine, N7-methylguanine and O6-methylguanine DNA adducts using reversed-phase high performance liquid chromatography and mass spectrometry 2008In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 867, p. 43-48Article in journal (Refereed)
    Abstract [en]

    This work describes the determination of N3-methyladenine, N7-methylguanine and O6-methylguanine adducts in dimethyl sulphate-treated salmon-testes DNA employing reversed-phase high performance liquid chromatography (RP-HPLC) with UV–vis detection, followed by mass-spectrometric verification using electrospray ionisation in positive mode ESI(+). Within validation parameters, accuracy, precision, calibration parameters, limit of detection (LOD) and quantitation (LOQ) as well as stability of standard stock solutions were tested and presented for UV/vis detection. The limit of detection (LOD) was found to be 0.1 ng/mL for N3-methyladenine and 0.2 ng/mL for both N7-methylguanine and O6-methylguanine (S/N = 3). The limit of quantitation (LOQ) was found to be 0.5 ng/mL for all measured compounds, (S/N = 10). Quantitative results were obtained for each substance based on eight-point calibration. Intra- and inter-day precisions were within 1.73–6.96 and 2.26–7.58%, respectively, and correlation coefficients of calibration curves (R2) ranged from 0.9992 to 0.9997. Relative proportion of N7-methylguanine was accounted for 61.53 ± 2.97% (R.S.D. = 4.8), N3-methyladenine for 38.19 ± 2.99% (R.S.D. = 9.6) and O6-methylguanine for 0.29 ± 0.02% (R.S.D. = 5.1), respectively. The application of the above-mentioned techniques provides a valuable contribution for simultaneous determination of methylated DNA adducts, and may represent a suitable approach for similar monitoring/screening studies.

  • 74. Chan, Norman
    et al.
    Pires, Isabel M
    Bencokova, Zuzana
    Coackley, Carla
    Luoto, Kaisa R
    Bhogal, Nirmal
    Lakshman, Minalini
    Gottipati, Ponnari
    Oliver, F Javier
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hammond, Ester M
    Bristow, Robert G
    Contextual synthetic lethality of cancer cell kill based on the tumor microenvironment.2010In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 70, no 20, p. 8045-54Article in journal (Refereed)
    Abstract [en]

    Acute and chronic hypoxia exists within the three-dimensional microenvironment of solid tumors and drives therapy resistance, genetic instability, and metastasis. Replicating cells exposed to either severe acute hypoxia (16 hours with 0.02% O(2)) followed by reoxygenation or moderate chronic hypoxia (72 hours with 0.2% O(2)) treatments have decreased homologous recombination (HR) protein expression and function. As HR defects are synthetically lethal with poly(ADP-ribose) polymerase 1 (PARP1) inhibition, we evaluated the sensitivity of repair-defective hypoxic cells to PARP inhibition. Although PARP inhibition itself did not affect HR expression or function, we observed increased clonogenic killing in HR-deficient hypoxic cells following chemical inhibition of PARP1. This effect was partially reversible by RAD51 overexpression. PARP1(-/-) murine embryonic fibroblasts (MEF) showed a proliferative disadvantage under hypoxic gassing when compared with PARP1(+/+) MEFs. PARP-inhibited hypoxic cells accumulated γH2AX and 53BP1 foci as a consequence of altered DNA replication firing during S phase-specific cell killing. In support of this proposed mode of action, PARP inhibitor-treated xenografts displayed increased γH2AX and cleaved caspase-3 expression in RAD51-deficient hypoxic subregions in vivo, which was associated with decreased ex vivo clonogenic survival following experimental radiotherapy. This is the first report of selective cell killing of HR-defective hypoxic cells in vivo as a consequence of microenvironment-mediated "contextual synthetic lethality." As all solid tumors contain aggressive hypoxic cells, this may broaden the clinical utility of PARP and DNA repair inhibition, either alone or in combination with radiotherapy and chemotherapy, even in tumor cells lacking synthetically lethal, genetic mutations.

  • 75.
    Chatzakos, Vicky
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Regulation and functions of bioactive N-acyl amino acids.2010Licentiate thesis, monograph (Other academic)
  • 76.
    Chatzakos, Vicky
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Studies of bioactive lipids in cancer2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Lipids are a broad class of molecules that, besides being a major form of energy storage and components of cell membranes, act as bioactive signalling molecules. N-acyl taurines are structurally related to endocannabinoids that are known to exert antiproliferative actions in a variety of cancer cells. We have evaluated the cytotoxicity of N-oleoyl taurine and N-arachidonoyl taurine and found N-acyl taurines to reduce proliferation of prostate cancer cells.

    The sphingolipids ceramide and sphingosine act as tumour suppressors. We found selenite treatment to cause reduced viability, induction of neutral sphingomyelinase activity and accumulation of ceramide in the liver cancer cells. Inhibition of sphingosine kinase 1 (SK1) sensitized the cells to selenite treatment with regards to ceramide accumulation, arrest of cells in the G1/S phases of the cell cycle and formation of reactive oxygen species. Whereas combined selenite treatment and SK1 inhibition synergistically reduced the number of viable cells, the non-tumorigenic hepatocyte cell line, remained unaffected.

    Furthermore, we studied the involvement of sphingolipid metabolism in bladder cancer cells treated with Bacillus Calmette-Guérin (BCG), and found BCG to induce formation of nitric oxide and upregulation of nitric oxide synthase 2 as well as SK1 protein levels. Additionally, pharmacological inhibition of SK1 enhanced the viability reduction, ceramide accumulation and induction of apoptosis observed following BCG treatment.

    In conclusion, our findings have shown that N-acyl taurines exert antiproliferative effects on prostate cancer cells. Furthermore, sphingolipids were shown to be involved in cytotoxic treatment with selenite and BCG in liver cancer and bladder cancer cells respectively, and inhibition of SK1 increased the cytotoxicity. Our findings raise the possibility that modulation of ceramide-metabolizing enzymes could be used to enhance the effects of selenite and BCG in these cancer cell types.

  • 77.
    Chatzakos, Vicky
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Rothlind, Emmie
    Karolinska Institutet.
    Izza Hamzo, Maria
    Karolinska Institutet.
    Wiklund, Peter
    Karolinska Institutet.
    Flygare, Jenny
    Karolinska Institutet.
    De Verdier, Petra
    Karolinska Institutet.
    Bacillus Calmette-Guérin treatment specifically induces SK1 protein expression inurothelial bladder-cancer cellsManuscript (preprint) (Other academic)
    Abstract [en]

    Bladder instillation with Bacillus Calmétte-Guerin (BCG) is an established treatmentmodality for superficial high-risk urinary bladder-cancer and carcinoma in situ (CIS), but theanti-tumor mechanisms following BCG-instillations remain largely unknown. In this study weexamined the effects of BCG-treatment on SK1 protein expression in the murine bladdercancer cell line MBT2. To simulate in vivo BCG-instillations, where the immune systemplays a vital role for successful BCG-treatment, we also stimulated MBT2 cells withsupernatant from BCG-treated (SupBCG) or un-treated Raw 264.7 macrophages. BCGtreatment as well as SupBCG treatment induced SK1 protein expression. Treatment with thesphingosine-kinase 1 (SK1) inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SK1-II) induced reduced viability in a dose dependent manner. However, combined treatment withBCG diminished this viability reduction suggesting a protective effect of BCG, but not ofSup-BCG, from SK1-II induced toxicity. Apoptosis was detected, as PARP-cleavage, in cellstreated with SupBCG whereas BCG-only or supernatant from untreated macrophages did notinduce apoptosis. A substantial increase in ceramides C18, C18:1 and C20:1 was observedafter BCG-treatment. These ceramide subspecies, as well as ceramides C14, C16, C20, C22and C22:1, were also induced by 20 or 30M of SK1-II, but the induction was abrogated byco-treatement with BCG. Following treatment with Sup-BCG, the levels of all the abovementioned ceramide subspecies were increased. Levels of ceramides C14, C16, C18, C18:1,C20, C20:1, C22 and C22:1 were more than two times higher in response to combinedtreatment with Sup-BCG and 10M of SK1-II as compared to treatment with Sup-BCG orSK1-II alone. Taken together these data suggest the sphingolipid metabolism as an importantpathway in the response to BCG-therapy.

  • 78.
    Chatzakos, Vicky
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Rundlöf, Anna-Klara
    Karolinska Institutet.
    Ahmed, Dilruba
    Karolinska Institutet.
    De Verdier, Petra J.
    Karolinska Institutet.
    Flygare, Jenny
    Karolinska Institutet.
    Inhibition of sphingosine kinase 1 enhances cytotoxicity, ceramide levels and ROS formation in liver cancer cells treated with selenite2012In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 84, no 5, p. 712-721Article in journal (Refereed)
    Abstract [en]

    High doses of selenite have been shown to induce cell death in acute myeloid leukemia and lung cancercells. In this study, we combined selenite treatment with modulators of sphingolipid metabolism in thehepatocellular carcinoma cell line Huh7. Treatment with 20 mM of selenite reduced the viability of Huh7cells by half and increased the levels of long chain C14-, C16-, C18- and C18:1- ceramides by two-fold.Inhibition of neutral sphingomyelinase with 3-O-methylsphingosine significantly reduced the cytotoxiceffect of selenite. In line with this result, selenite caused a 2.5-fold increase in the activity of neutralsphingomyelinase. The sphingosine kinase 1 (SK1) inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophe-nyl)thiazole (SK1-II) sensitized the cells to the cytotoxic effects of selenite. Preincubation with 10 mM ofSK1-II prior to treatment with 10 mM of selenite caused induction of apoptosis and gave rise to a 2.5-foldincrease in C14-, C16-, C18- and C18:1- ceramides. Instead, 50 mM of SK1-II combined with 10 mM ofselenite caused accumulation of cells in G1/S phases, but less apoptosis and accumulation of ceramides.The formation of reactive oxygen species (ROS) after treatment with 10 mM of selenite was maximallyenhanced by 1 mM of SK1-II. Moreover, combined treatment with SK1-II and 10 mM of selenitesynergistically reduced the number of viable Huh7 cells, while the non-tumorigenic hepatocyte cell lineMIHA remained unaffected by the same treatment. These results raise the possibility that a combinationof selenite and SK1 inhibitors could be used to treat liver cancer cells, that are regarded as drug resistant,at doses that are non-toxic to normal liver cells.

  • 79.
    Chatzakos, Vicky
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Slätis, Katharina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Djureinovic, Tatjana
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hunt, Mary C.
    N-Acyl Taurines are Anti-Proliferative in Prostate Cancer Cells2012In: Lipids, ISSN 0024-4201, E-ISSN 1558-9307, Vol. 47, no 4, p. 355-361Article in journal (Refereed)
    Abstract [en]

    Endocannabinoids have been implicated in cancer development and cause heterogenous effects in tumor cells, by inducing apoptosis, reducing migration, causing anti-angiogenic activity and alterations in the cell cycle resulting in growth arrest. Recently, several novel amides of fatty acids that are structurally related to endocannabinoids have been isolated from mammalian sources, although the functions of these fatty amides are not well studied. One group of these novel fatty acid amides are the N-acyl taurines (fatty acids conjugated to the amino acid taurine). This study examined if N-acyl taurines, specifically N-arachidonoyl taurine and N-oleoyl taurine could function in a similar way to endocannabinoids and result in cell cycle alterations or growth arrest in the human prostate adenocarcinoma cell line PC-3. PC-3 cells were treated with various concentrations of N-arachidonoyl taurine and N-oleoyl taurine and cell proliferation and viability was measured using resazurin and colony formation assays. Effects of N-acyl taurines on the cell cycle was measured using FACS analysis. Treatment with N-arachidonoyl taurine and N-oleoyl taurine resulted in a significant reduction in proliferation of PC-3 cells, even at concentrations as low as 1 mu M. Treatment with N-oleoyl taurine resulted in an increased number of cells in the subG1 population, suggesting apoptosis, and a lower number of cells in S-phase of the cell cycle. In summary, our results show that novel biologically active lipids, the N-acyl taurines, result in reduced proliferation in PC-3 cells.

  • 80.
    Chen, Yao
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sjölinder, Mikael
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Wang, Xiao
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Altenbacher, Georg
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hagner, Matthias
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Berglund, Pernilla
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Gao, Yumin
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lu, Ting
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jonsson, Ann-Beth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sjölinder, Hong
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Thyroid hormone enhances nitric oxide mediated bacterial clearance and promotes survival after meningococcal infection2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 7, article id e41445Article in journal (Refereed)
    Abstract [en]

    Euthyroid sick syndrome characterized by reduced levels of thyroid hormones (THs) is observed in patients with meningococcal shock. It has been found that the level of THs reflects disease severity and is predictive for mortality. The present study was conducted to investigate the impact of THs on host defense during meningococcal infection. We found that supplementation of thyroxine to mice infected with Neisseria meningitidis enhanced bacterial clearance, attenuated the inflammatory responses and promoted survival. In vitro studies with macrophages revealed that THs enhanced bacteria-cell interaction and intracellular killing of meningococci by stimulating inducible nitric oxide synthase (iNos)-mediated NO production. TH treatment did not activate expression of TH receptors in macrophages. Instead, the observed TH-directed actions were mediated through nongenomic pathways involving the protein kinases PI3K and ERK1/2 and initiated at the membrane receptor integrin alpha v beta 3. Inhibition of nongenomic TH signaling prevented iNos induction, NO production and subsequent intracellular bacterial killing by macrophages. These data demonstrate a beneficial role of THs in macrophage-mediated N. meningitidis clearance. TH replacement might be a novel option to control meningococcal septicemia.

  • 81.
    Chen, Yunying
    et al.
    Karolinska Institutet.
    Wermeling, Fredrik
    Karolinska Institutet.
    Sundqvist, Johanna
    Karolinska Institutet.
    Jonsson, Ann-Beth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Tryggvason, Karl
    Karolinska Institutet.
    Pikkarainen, Timo
    Karolinska Institutet.
    Karlsson, Mikael C I
    Karolinska Institutet.
    A regulatory role for macrophage class A scavenger receptors in TLR4-mediated LPS responses.2010In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 40, no 5, p. 1451-60Article in journal (Refereed)
    Abstract [en]

    Recognition of microbial components by TLR, key sensors of infection, leads to induction of inflammatory responses. We found that, in vivo, TLR4 engagement by LPS induces up-regulation of the class A scavenger receptors (SR) macrophage receptor with a collagenous structure (MARCO) and SR-A, which occurs, at least in the case of MARCO, via both MyD88-dependent and -independent pathways. When challenging mice with a low dose of LPS followed by a high dose, class A SR-deficient mice showed a higher survival rate than WT mice. This was paired with increased production of IL-10 and anti-LPS Ab, as well as increased activation status of marginal zone B cells. However, the receptors were not crucial for survival when challenging mice i.p. with Neisseria meningitidis or Listeria monocytogenes, but they were found to contribute to microbial capture and clearance. This indicates physiological significance for the up-regulation of class A SR during early stages of bacterial infection. Thus, we believe that we have revealed a mechanism where SR regulate the activation status of the immune system and are involved in balancing a proper immune response to infection. This regulation could also be important in maintaining tolerance since these receptors have been shown to be involved in regulation of self-reactivity.

  • 82. Cheng, Wen-Hsing
    et al.
    Muftic, Diana
    Muftuoglu, Meltem
    Dawut, Lale
    Morris, Christa
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Shiloh, Yosef
    Bohr, Vilhelm A
    WRN is required for ATM activation and the S-phase checkpoint in response to interstrand cross-link-induced DNA double-strand breaks.2008In: Mol Biol Cell, ISSN 1939-4586, Vol. 19, no 9, p. 3923-33Article in journal (Refereed)
  • 83. Chouaia, Bessem
    et al.
    Rossi, Paolo
    Montagna, Matteo
    Ricci, Irene
    Crotti, Elena
    Damiani, Claudia
    Epis, Sara
    Faye, Ingrid
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sagnon, N'Fale
    Alma, Alberto
    Favia, Guido
    Daffonchio, Daniele
    Bandi, Claudio
    Molecular Evidence for Multiple Infections as Revealed by Typing of Asaia Bacterial Symbionts of Four Mosquito Species2010In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 76, no 22, p. 7444-7450Article in journal (Refereed)
    Abstract [en]

    The recent increased detection of acetic acid bacteria (AAB) of the genus Asaia as symbionts of mosquitoes, such as Anopheles spp. and Aedes spp., prompted us to investigate the diversity of these symbionts and their relationships in different mosquito species and populations. Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with four mosquito species, Anopheles stephensi, Anopheles gambiae, Aedes aegypti, and Aedes albopictus, which are important vectors of human and/or animal pathogens. Denaturing gradient gel electrophoresis (DGGE) analysis based on the 16S rRNA gene revealed the presence of several bacterial taxa, among which Asaia sequences were among the dominant in most of the samples. A collection of 281 Asaia isolates in cell-free media was established from individuals belonging to the four species. The isolates were typed by internal transcribed spacer (ITS)-PCR, tRNA-PCR, BOX-PCR, and randomly amplified polymorphic DNA (RAPD)-PCR, revealing that different Asaia strains are present in different mosquito populations, and even in single individuals.

  • 84. Coteur, Geoffroy
    et al.
    Mellroth, Peter
    De Lefortery, Coline
    Gillan, David
    Dubois, Philippe
    Communi, David
    Steiner, Håkan
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Peptidoglycan recognition proteins with amidase activity in early deuterostomes (Echinodermata).2007In: Dev Comp Immunol, ISSN 0145-305X, Vol. 31, no 8, p. 790-804Article in journal (Refereed)
  • 85.
    Croitoru, Victor
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Study on the Function of Translation Initiation Factor IF12006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Initiation is the first step in protein biosynthesis representing a fundamental event in cell life which determines fidelity, efficiency and regulation of gene expression. In addition to the ribosome and mRNA, three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. Several minor functions have been attributed to the smallest of these factors, IF1. However, the main function of IF1 remains to be elucidated.

    In order to investigate the role of this protein in the initiation process we have mutated the corresponding gene infA. Using a high-copy plasmid and site-directed mutagenesis, the six arginine residues of IF1 were separately altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. This strategy was followed by deletion of the chromosomal infA gene. All variants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Several of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type allele. Some of these mutants displayed reduced growth rates and a partial cold-sensitive phenotype.

    The influence of the leucine group of mutants in IF1 on the expression of two reporter genes with different initiation and/or +2 codons has been investigated. Our results do not indicate any involvement of IF1 in recognition of the +2 codon immediately following the start codon, thus representing the A-site. In addition, this group of mutants has no changed efficiency of decoding at the near-cognate initiation codons UUG and GUG. However, one cold-sensitive IF1 mutant shows a general overexpression of both reporter genes, in particular at low temperatures. Overall, the results do not support the hypothesis that IF1 could possess codon discriminatory functions while blocking the A-site of the ribosome.

    In this study we also identify that IF1 has RNA chaperone activity both in vitro and in vivo. The chaperone assays are based on splicing of the group I intron in the thymidylate synthase gene (td) from phage T4. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.

  • 86.
    Croitoru, Victor
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bucheli-Witschel, Margarete
    Hägg, Peter
    Abdulkarim, Farhad
    Isaksson, Leif A.
    Generation and characterization of functional mutants in the translation initiation factor IF1 of Escherichia coli2004In: European Journal of Biochemistry, ISSN 1742-4658, Vol. 271, no 3, p. 534-544Article in journal (Refereed)
  • 87.
    Croitoru, Victor
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bucheli-Witschel, Margarete
    Isaksson, Leif A.
    In vivo involvement of mutated initiation factor IF1 in gene expression control at the translational level2005In: FEBS Letters, ISSN 0014-5793, Vol. 579, no 5, p. 995-1000Article in journal (Refereed)
  • 88.
    Croitoru, Victor
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Semrad, Katharina
    Prenninger, Silvia
    Rajkowitsch, Lukas
    Vejen, Max
    Laursen, Brian Sogaard
    Sperling-Petersen, Hans Uffe
    Isaksson, Leif A.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    RNA chaperone activity of translation initiation factor IF12006In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 88, no 12, p. 1875-1882Article in journal (Refereed)
    Abstract [en]

    Translation initiation factor IF1 is an indispensable protein for translation in prokaryotes. No clear function has been assigned to this factor so far. In this study we demonstrate an RNA chaperone activity of this protein both in vivo and in vitro. The chaperone assays are based on in vivo or in vitro splicing of the group I intron in the thymidylate synthase gene (td) from phage T4 and an in vitro RNA annealing assay. IF1 wild-type and mutant variants with single amino acid substitutions have been analyzed for RNA chaperone activity. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Furthermore, both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.

  • 89.
    Dahlgren, Ann
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Function and regulation of release factor one in Escherichia coli2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    During translation termination the stop codons UAA and UAG are recognised by release factor one (RF1). RF1 binds to the ribosome and mediates the hydrolysis of the nascent peptide from the peptidyl-tRNA. In this process, RF1 interacts directly or indirectly with several ribosomal proteins (r-proteins). To study this interaction in vivo we have used a mutant allele of RF1, prfA1. The mutation causes a temperature sensitive (Ts) phenotype and increased readthrough of the stop codons. A mutant form of r-protein S4 that suppresses this Ts phenotype was isolated. The S4 mutant also increases the readtrough of UAG caused by prfA1. In the mutated S4 allele, rpsD101, Tyr51 is changed to Asp. That change introduces a negatively charged amino acid in a part of S4 that belongs to the positively charged RNA binding surface. This might affect the binding of S4 to the 16S rRNA, to another r-protein, or to RF1, and in that way influence on the function of RF1.

    One of the few things known about the regulation of RF1 is that expression is decreased with increasing generation time. prfA is the second gene in the hemA-operon located at 27 minutes on the E. coli chromosomal map. We have shown that the growth rate regulation of RF1 is exerted at one of the promoters preceding the hemA gene, PhemA1. The promoter is also growth phase regulated and it is turned off in stationary phase. We have characterised two mutations, asuA1 and asuA2, that increase expression of RF1. The asuA2 mutation is a G to A change one nucleotide downstream of the -10 region of PhemA1. Besides increasing expression of RF1 this mutation also abolishes the growth rate and growth phase regulations we have found. The growth phase regulation is partly dependent on ppGpp. We present two models concerning the effect of ppGpp on PhemA1, and what the asuA2 mutation does to it. The prfA gene has low translation initiation efficiency due to a long spacing between the Shine-Dalgarno (SD) sequence and the start codon. The asuA1 mutation creates a new start codon with a shorter distance to the SD sequence. Most likely this increases the efficiency of translation initiation. That PhemA1 is turned off in stationary phase suggests additional regulation of RF1. Our results indicate a putative promoter for the prfA gene within hemA. This promoter does not seem to be growth rate or growth phase regulated.

  • 90.
    Dahlgren,, Ann
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Kaczanowska and, Magdalena
    Rydén Aulin, Monica
    Studies of the expression of release factor one in Escherichia coliManuscript (Other academic)
  • 91.
    Dahlgren, Ann
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Kaczanowska, M
    Rydén-Aulin, Monica
    Studies of the expression of release factor one in Escherichia coli: PhemA1 and identification of a putative promoterManuscript (Other academic)
  • 92.
    Dahlgren, Ann
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Rydén-Aulin, Monica
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    A novel mutation in ribosomal protein S4 thataffects the function of a mutated RF12000In: Biochimie, ISSN 0300-9084, Vol. 82, no 8, p. 683-691Article in journal (Refereed)
    Abstract [en]

    Release factors (RF) 1 and 2 trigger the hydrolysis of the peptide from the peptidyl-tRNA during translation termination. RF1 binds to the ribosome in response to the stop codons UAG and UAA, whereas RF2 recognizes UAA and UGA. RF1 and RF2 have been shown to bind to several ribosomal proteins. To study this interaction in vivo, prfA1, a mutant form of RF1 has been used. A strain with the prfA1 mutation is temperature sensitive (Ts) for growth at 42 °C and shows an increased misreading of UAG and UAA. In this work we show that a point mutation in ribosomal protein S4 can, on the one hand, make the RF1 mutant strain Ts+; on the other hand, this mutation increases the misreading of UAG, but not UAA, caused by prfA1. The S4 mutant allele, rpsD101, is a missense mutation (Tyr51 to Asp), which makes the cell cold sensitive. The behaviour of rpsD101 was compared to the well-studied S4 alleles rpsD12, rpsD14, and rpsD16. These three mutations all confer both a Ts (44 °C) phenotype and show a ribosomal ambiguity phenotype, which rpsD101 does not. The three alleles were sequenced and shown to be truncations of the S4 protein. None of the three mutations could compensate for the Ts phenotype caused by the prfA1 mutation. Hence, rpsD101 differs in all studied characteristics from the three above mentioned S4 mutants. Because rpsD101 can compensate for the Ts phenotype caused by prfA1 but enhances the misreading of UAG and not UAA, we suggest that S4 influences the interaction of RF1 with the decoding center of the ribosome and that the Ts phenotype is not a consequence of increased readthrough.

  • 93.
    Dahlgren, Ann
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Rydén-Aulin, Monica
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Effects of two cis-acting mutations on the regulation and expression of release factor one in Eschericchia coli2004In: Biochimie, ISSN 0300-9084, Vol. 86, no 7, p. 431-438Article in journal (Refereed)
    Abstract [en]

    Together with release factor (RF) 2, RF1 recognises the stop codons and triggers the hydrolysis of the nascent peptide from peptidyl-tRNA during translation termination. prfA, the gene that codes for RF1, is located at 27 min on the Escherichia coli map as the second gene in the hemA-operon. The concentration of RF1 has been shown to increase with increased growth rate, but it is not known where and how this control is exerted. In this study we show that the growth rate regulation of RF1, at least in part, is controlled at PhemA1, one of two promoters preceding the hemA gene. We have also characterised two mutations, asuA1 and asuA2, that are antisuppressors to the tRNA suppressor Su2. Our data indicate that the antisuppressor phenotype is caused by an increased amount of RF1. The asuA2 mutation is a G to an A change just downstream of the –10 region of PhemA1, it leads to a higher concentration of RF1 in the cell and abolishes the growth rate regulation. This indicates that the sequence between the –10 region and the transcription start site is important for growth rate control. The increase in concentration of RF1 caused by asuA1 is most likely at the translational level. The efficiency of translation initiation of prfA is low due to a long distance between the start codon and the Shine-Dalgarno (SD) sequence. The asuA1 mutation creates a new start codon with a more optimal distance to the SD sequence. This leads to an increased expression of RF1, probably due to increased initiation efficiency

  • 94. Damiani, Claudia
    et al.
    Ricci, Irene
    Crotti, Elena
    Rossi, Paolo
    Rizzi, Aurora
    Scuppa, Patrizia
    Capone, Aida
    Ulissi, Ulisse
    Epis, Sara
    Genchi, Marco
    Sagnon, N'Fale
    Faye, Ingrid
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Kang, Angray
    Chouaia, Bessem
    Whitehorn, Cheryl
    Moussa, Guelbeogo W.
    Mandrioli, Mauro
    Esposito, Fulvio
    Sacchi, Luciano
    Bandi, Claudio
    Daffonchio, Daniele
    Favia, Guido
    Mosquito-Bacteria Symbiosis: The Case of Anopheles gambiae and Asaia2010In: Microbial Ecology, ISSN 0095-3628, E-ISSN 1432-184X, Vol. 60, no 3, p. 644-654Article in journal (Refereed)
    Abstract [en]

    The symbiotic relationship between Asaia, an alpha-proteobacterium belonging to the family Acetobacteriaceae, and mosquitoes has been studied mainly in the Asian malaria vector Anopheles stephensi. Thus, we have investigated the nature of the association between Asaia and the major Afro-tropical malaria vector Anopheles gambiae. We have isolated Asaia from different wild and laboratory reared colonies of A. gambiae, and it was detected by PCR in all the developmental stages of the mosquito and in all the specimens analyzed. Additionally, we have shown that it localizes in the midgut, salivary glands and reproductive organs. Using recombinant strains of Asaia expressing fluorescent proteins, we have demonstrated the ability of the bacterium to colonize A. gambiae mosquitoes with a pattern similar to that described for A. stephensi. Finally, fluorescent in situ hybridization on the reproductive tract of females of A. gambiae showed a concentration of Asaia at the very periphery of the eggs, suggesting that transmission of Asaia from mother to offspring is likely mediated by a mechanism of egg-smearing. We suggest that Asaia has potential for use in the paratransgenic control of malaria transmitted by A. gambiae.

  • 95.
    Dang, Li
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lisowska, Halina
    Shakeri Manesh, Sara
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sollazzo, Alice
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Deperas-Kaminska, Marta
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. Jan Kochanowski University, Poland.
    Staaf, Elina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haghdoost, Siamak
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Brehwens, Karl
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. Jan Kochanowski University, Poland.
    Radioprotective effect of hypothermia on cells - a multiparametric approach to delineate the mechanisms2012In: International Journal of Radiation Biology, ISSN 0955-3002, E-ISSN 1362-3095, Vol. 88, no 7, p. 507-514Article in journal (Refereed)
    Abstract [en]

    Purpose: Low temperature (hypothermia) during irradiation of cells has been reported to have a radioprotective effect. The mechanisms are not fully understood. This study further investigates the possible mechanisms behind hypothermia-mediated radioprotection. Materials and methods: Human lymphoblastoid TK6 cells were incubated for 20 min at 0.8 or 37 degrees C and subsequently exposed to 1 Gy of gamma- or X-rays. The influence of ataxia telangiectasia mutated (ATM)-mediated double-strand break signalling and histone deacetylase-dependent chromatin condensation was investigated using the micronucleus assay. Furthermore, the effect of hypothermia was investigated at the level of phosphorylated histone 2AX (gamma H2AX) foci, clonogenic cell survival and micronuclei in sequentially-harvested cells. Results: The radioprotective effect of hypothermia (called the temperature effect [TE]) was evident only at the level of micronuclei at a single fixation time, was not influenced by the inhibition of ATM kinase activity and completely abolished by the histone deacetylase inhibition. No TE was seen at the level of gamma H2AX foci and cell survival. Conclusions: We suggest that low temperature during irradiation can induce a temporary cell cycle shift, which could lead to a reduced micronucleus frequency. Future experiments focused on cell cycle progression are needed to confirm this hypothesis.

  • 96. Deperas-Kaminska, Marta
    et al.
    Zaytseva, Ekaterina M.
    Deperas-Standylo, Joanna
    Mitsyn, Gennady V.
    Molokanov, Alexander G.
    Timoshenko, Gennady N.
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Inter-chromosomal variation in aberration frequencies in human lymphocytes exposed to charged particles of LET between 0.5 and 55 keV/mu m2010In: International Journal of Radiation Biology, ISSN 0955-3002, E-ISSN 1362-3095, Vol. 86, no 11, p. 975-985Article in journal (Refereed)
    Abstract [en]

    Purpose: To investigate the distribution of chromosomal aberrations in chromosomes 2, 8 and 14 induced by charged particles, using the fluorescence in situ hybridisation (FISH) technique. Methods: Irradiation of peripheral blood from six healthy volunteers (four male and two female) was performed at the accelerators of the Joint Institute for Nuclear Research (JINR) in Dubna (Russia). Whole blood samples were irradiated with 2 and 3 Gy of protons (170 MeV/nucleon (n), linear energy transfer (LET) approximate to 0.5 keV/mu m), 3.5 Gy of C-12 ions (480 MeV/n, LET = 10.6 keV/mu m), 3 Gy of C-12 ions 500 MeV/n, LET = 12 keV/mu m), 4 Gy of Li-7 ions (30 MeV/n, LET approximate to 20 keV/mu m) and 3 Gy of B-11 ions (32 MeV/n, LET approximate to 55 keV/mu m). Chromosomal aberrations were analysed in metaphase and prematurely condensed chromosomes (PCC) induced in G(2)-cells using calyculin A. Chromosomes 2, 8 and 14 were painted in different colours and aberrations scored with the help of an image-analysis system. Results: Chromosome 2 was generally less sensitive than expected on the basis of its DNA content. A higher than expected frequency of exchanges was found in chromosomes 8 and 14. On average, the dicentric frequency in chromosome 2 was higher than the translocation frequency, whereas variable dicentric to translocation ratios were observed in chromosomes 8 and 14. When aberrations in all painted chromosomes were summed up the ratio was close to 1. The frequency of complex aberrations correlated with LET. Conclusion: In lymphocytes of donors studied in this work chromosome 2 appears to be consistently less sensitive to protons and heavy ions than chromosomes 8 and 14. Complex aberrations appear to be a potential marker of radiation quality.

  • 97. Dietz, Rune
    et al.
    Teilmann, Jonas
    Andersen, Signe M.
    Riget, Frank
    Olsen, Morten T.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Movements and site fidelity of harbour seals (Phoca vitulina) in Kattegat, Denmark, with implications for the epidemiology of the phocine distemper virus2013In: ICES Journal of Marine Science, ISSN 1054-3139, E-ISSN 1095-9289, Vol. 70, no 1, p. 186-195Article in journal (Refereed)
    Abstract [en]

    Twenty-seven harbour seals were caught and tagged at the island of Anholt in central Kattegat, Denmark, the epicentre of the phocine distemper virus (PDV) outbreaks in 1988 and 2002 that killed 50-60% of the populations. The satellite tagging shows that harbour seals from Anholt moved widely across Kattegat with a maximum distance of 249 km from the tagging haul-out site. Overall, females travelled over a wider area compared with males [90% kernel home range (KHR) females, 5189 km(2); males, 3293 km(2)). KHR calculated for yearlings (6414 km(2)) is larger than for subadults (2534 km(2)), which again is larger than for adult seals (1713 km(2)), showing a strong site fidelity, indicating limited gene flow between haul-out sites. Distances moved and home range sizes increased across autumn, peaked in February-March, and decreased through spring. During the breeding season in spring, all seals were very stationary around Anholt. The onset of the PDV epizootics in 1988 and 2002 took place when the Anholt harbour seals congregate on the Island during April. Anholt seal were also documented to have contact with infected seal locations at Hesselo, L so, and the Swedish west coast, although this contact takes place during winter prior to the documented summer outbreaks.

  • 98. Duro, Eris
    et al.
    Lundin, Cecilia
    Ask, Katrine
    Sanchez-Pulido, Luis
    MacArtney, Thomas J.
    Toth, Rachel
    Ponting, Chris P.
    Groth, Anja
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Rouse, John
    Identification of the MMS22L-TONSL Complex that Promotes Homologous Recombination2010In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 40, no 4, p. 632-644Article in journal (Refereed)
    Abstract [en]

    Budding yeast Mms22 is required for homologous recombination (HR)-mediated repair of stalled or broken DNA replication forks. Here we identify a human Mms22-like protein (MMS22L) and an MMS22L-interacting protein, NF kappa BIL2/TONSL. Depletion of MMS22L or TONSL from human cells causes a high level of double-strand breaks (DSBs) during DNA replication. Both proteins accumulate at stressed replication forks, and depletion of MMS22L or TONSL from cells causes hypersensitivity to agents that cause S phase-associated DSBs, such as topoisomerase (TOP) inhibitors. In this light, MMS22L and TONSL are required for the HR-mediated repair of replication fork-associated DSBs. In cells depleted of either protein, DSBs induced by the TOP1 inhibitor camptothecin are resected normally, but the loading of the RAD51 recombinase is defective. Therefore, MMS22L and TONSL are required for the maintenance of genome stability when unscheduled DSBs occur in the vicinity of DNA replication forks.

  • 99.
    Ederth, Josefine
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The Role of the Escherichia coli RNA Polymerase β´Jaw in Transcription2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Transcription, the central step in gene expression and regulation, is carried out by the DNA-dependent RNA polymerase (RNAP). Bacterial RNAP is a complex molecular machine in which the network of interacting parts and their movements, including contacts to nascent RNA and the DNA template, are at best partially understood.

    In this work, the aim has been to elucidate the role of the RNAP jaw-domain, which is part of the RNAPs largest subunit ( β´), in transcription. The jaw-domain is interesting because of its close proximity to downstream DNA in the complex. Downstream DNA is known as a component involved in all three steps of transcription. However, the key role it plays in regulating the enzyme´s activity is far from understood.

    Initially, two novel mutations in the β´ jaw-domain were isolated and characterized in vivo. It was shown that the jaw-domain has specific effects on regulation of the ColE1 plasmid copy number, likely via different effects on initiation at the RNA Iand RNA II- promoters.

    Further, in vitro characterisations indicate that contacts of the jaw-domain to downstream DNA, at the leading edge of the transcription complex, contribute to regulation during all three phases of transcription. The results provide insight into the role of the jaw-domain-downstream DNA contact in transcription initiation and pausing, and suggest possible explanations for the previously reported effects on ColEI plasmid copy number.

    The RNAP jaw-domain is situated at ~30 Å distance from the active site. Suppressor mutations were selected to gain further knowledge of how the jaw-domain can exert such profound effects on the enzyme´s activity. Allele specific suppressor mutations were found in the rifampicin-pocket, only a few Ångströms away from the active site. It is shown that the suppressor substitutions compensate for the jawdeletion RNAPs defects in transcriptional pausing and inability to grow at elevated temperatures. These same substitutions exacerbate the jaw-deletion´s defect at transcription initiation, which suggests that the elongation defects are responsible for the temperature sensitive growth phenotype of the jaw-deletion strain. In light of the RNAP crystal structures available, a possible mechanism for counteracting effects on the active site mediated by the suppressor mutations and the jaw-deletion is suggested.

  • 100. Eldridge, Bill
    et al.
    Cooley, R. Neil
    Odegrip, Richard
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    McGregor, Duncan P.
    FitzGerald, Kevin J.
    Ullman, Christopher G.
    An in vitro selection strategy for conferring protease resistance to ligand binding peptides2009In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 22, no 11, p. 691-698Article in journal (Refereed)
    Abstract [en]

    One drawback to the use of peptides as therapeutics has been their susceptibility to proteolysis. Here, we have used an in vitro display technology, CIS display, to enhance the proteolytic resistance of ligand-binding peptides by selection of protecting motifs from a large peptide library. The premise to this selection was that certain linear peptides within a library could form structures capable of preventing the access of proteases to defined cleavage sites without affecting ligand binding. A diverse 12-mer peptide library was inserted between a FLAG epitope motif and a thrombin cleavage site and this construct was fused to the bacterial initiator protein RepA for CIS display selection. After five rounds of selection, protection motifs were isolated that were capable of preventing proteolytic cleavage of the adjacent thrombin site. Some of the selected peptides were also resistant to more promiscuous proteases, such as chymotrypsin and trypsin, which were not used in the selection. The observed resistance to thrombin, trypsin and chymotrypsin translated into increased resistance to plasma proteases in vitro and to an increase in circulating half-lives in rats. This method can be applied to enhancing the in vivo stability of therapeutic peptides.

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