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  • 751.
    Wirebrand, Lisa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Madhushani, Anjana W. K.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Irie, Yasuhiko
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF6002018In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 20, no 1, p. 186-199Article in journal (Refereed)
    Abstract [en]

    The dmp-system encoded on the IncP-2 pVI150 plasmid of Pseudomonas putida CF600 confers the ability to assimilate (methyl)phenols. Regulation of the dmp-genes is subject to sophisticated control, which includes global regulatory input to subvert expression of the pathway in the presence of preferred carbon sources. Previously we have shown that in P. putida, translational inhibition exerted by the carbon repression control protein Crc operates hand-in-hand with the RNA chaperon protein Hfq to reduce translation of the DmpR regulator of the Dmp-pathway. Here we show that Crc and Hfq co-target four additional sites to form riboprotein complexes within the proximity of the translational initiation sites of genes encoding the first two steps of the Dmp-pathway to mediate two-layered control in the face of selection of preferred substrates. Furthermore, we present evidence that Crc plays a hitherto unsuspected role in maintaining the pVI150 plasmid within a bacterial population, which has implications for (methyl)phenol degradation and a wide variety of other physiological processes encoded by the IncP-2 group of Pseudomonas-specific mega-plasmids.

  • 752.
    Wirebrand, Lisa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Österberg, Sofia
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    López-Sánchez, Aroa
    Govantes, Fernando
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    PP4397/FlgZ provides the link between PP2258 c-di-GMP signalling and altered motility in Pseudomonas putida2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 12205Article in journal (Refereed)
    Abstract [en]

    Bacteria swim and swarm using rotating fagella that are driven by a membrane-spanning motor complex. Performance of the fagella motility apparatus is modulated by the chemosensory signal transduction system to allow navigation through physico-chemical gradients – a process that can be fne-tuned by the bacterial second messenger c-di-GMP. We have previously analysed the Pseudomonas putida signalling protein PP2258 that has the capacity to both synthesize and degrade c-di-GMP. A PP2258 null mutant displays reduced motility, implicating the c-di-GMP signal originating from this protein in control of P. putida motility. In Escherichia coli and Salmonella, the PilZ-domain protein YcgR mediates c-di-GMP responsive control of motility through interaction with the fagellar motors. Here we provide genetic evidence that the P. putida protein PP4397 (also known as FlgZ), despite low sequence homology and a diferent genomic context to YcgR, functions as a c-di-GMP responsive link between the signal arising from PP2258 and alterations in swimming and swarming motility in P. putida.

  • 753. Wolfstetter, Georg
    et al.
    Shirinian, Margret
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Stute, Christiana
    Grabbe, Caroline
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hummel, Thomas
    Baumgartner, Stefan
    Palmer, Ruth H
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Holz, Anne
    Fusion of circular and longitudinal muscles in Drosophila is independent of the endoderm but further visceral muscle differentiation requires a close contact between mesoderm and endoderm.2009In: Mechanisms of Development, ISSN 0925-4773, E-ISSN 1872-6356, Vol. 126, no 8-9, p. 721-736Article in journal (Refereed)
    Abstract [en]

    In this study we describe the morphological and genetic analysis of the Drosophila mutant gürtelchen (gurt). gurt was identified by screening an EMS collection for novel mutations affecting visceral mesoderm development and was named after the distinct belt shaped visceral phenotype. Interestingly, determination of visceral cell identities and subsequent visceral myoblast fusion is not affected in mutant embryos indicating a later defect in visceral development. gurt is in fact a new huckebein (hkb) allele and as such exhibits nearly complete loss of endodermal derived structures. Targeted ablation of the endodermal primordia produces a phenotype that resembles the visceral defects observed in huckebein(gürtelchen) (hkb(gurt)) mutant embryos. It was shown previously that visceral mesoderm development requires complex interactions between visceral myoblasts and adjacent tissues. Signals from the neighbouring somatic myoblasts play an important role in cell type determination and are a prerequisite for visceral muscle fusion. Furthermore, the visceral mesoderm is known to influence endodermal migration and midgut epithelium formation. Our analyses of the visceral phenotype of hkb(gurt) mutant embryos reveal that the adjacent endoderm plays a critical role in the later stages of visceral muscle development, and is required for visceral muscle elongation and outgrowth after proper myoblast fusion.

  • 754. Wu, Z.
    et al.
    Milton, Debra L..
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nybom, P.
    Sjö, A.
    Magnusson, K. E.
    Vibrio cholerae hemagglutinin/protease (HA/protease) causes morphological changes in cultured epithelial cells and perturbs their paracellular barrier function1996In: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 21, no 2, p. 111-123Article in journal (Refereed)
    Abstract [en]

    In this report, we describe the cytotoxic activity of the cholera hemagglutinin/protease (HA/protease). A concentrated protein sample from the 37 degrees C overnight culture supernatant of CVD110, a delta ctxA, delta zot, delta Ace and hlyA::(ctxB mer) mutant of El Tor biotype Ogawa serotype strain E7946 caused morphological changes in cultured MDCK-I epithelial cells and altered their arrangement of filamentous actin (F-actin) and Zonula occludens-associated protein ZO-1. The drastic morphological changes can be inhibited by Zincov, a specific bacterial metalloprotease inhibitor. The cytotoxic fractions of the sample after FPLC gelfiltration fractionation showed two visible protein bands with molecular weights of approximately 34- and 32 kDa. Microsequencing of these two proteins revealed that they were the cholera HA/protease.

  • 755.
    Xu, Fu
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Johansson, Marcus J. O.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    SSD1 suppresses phenotypes induced by the lack of Elongator-dependent tRNA modifications2019In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 15, no 8, article id e1008117Article in journal (Refereed)
    Abstract [en]

    The Elongator complex promotes formation of 5-methoxycarbonylmethyl (mcm5 ) and 5-carbamoylmethyl (ncm5 ) side-chains on uridines at the wobble position of cytosolic eukaryotic tRNAs. In all eukaryotic organisms tested to date, the inactivation of Elongator not only leads to the lack of mcm5 /ncm5 groups in tRNAs, but also a wide variety of additional phenotypes. Although the phenotypes are most likely caused by a translational defect induced by reduced functionality of the hypomodified tRNAs, the mechanism(s) underlying individual phenotypes are poorly understood. In this study, we show that the genetic background modulates the phenotypes induced by the lack of mcm5 /ncm5 groups in Saccharomyces cerevisiae. We show that the stress-induced growth defects of Elongator mutants are stronger in the W303 than in the closely related S288C genetic background and that the phenotypic differences are caused by the known polymorphism at the locus for the mRNA binding protein Ssd1. Moreover, the mutant ssd1 allele found in W303 cells is required for the reported histone H3 acetylation and telomeric gene silencing defects of Elongator mutants. The difference at the SSD1 locus also partially explains why the simultaneous lack of mcm5 and 2- thio groups at wobble uridines is lethal in the W303 but not in the S288C background. Collectively, our results demonstrate that the SSD1 locus modulates phenotypes induced by the lack of Elongator-dependent tRNA modifications.

  • 756.
    Xu, Fu
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Zhou, Yang
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus J. O.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Identification of factors that promote biogenesis of tRNACGASer.2018In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 15, no 10, p. 1286-1294Article in journal (Refereed)
    Abstract [en]

    A wide variety of factors are required for the conversion of pre-tRNA molecules into the mature tRNAs that function in translation. To identify factors influencing tRNA biogenesis, we previously performed a screen for strains carrying mutations that induce lethality when combined with a sup61-T47:2C allele, encoding a mutant form of tRNACGASer. Analyzes of two complementation groups led to the identification of Tan1 as a protein involved in formation of the modified nucleoside N4-acetylcytidine (ac4C) in tRNA and Bud13 as a factor controlling the levels of ac4C by promoting TAN1 pre-mRNA splicing. Here, we describe the remaining complementation groups and show that they include strains with mutations in genes for known tRNA biogenesis factors that modify (DUS2, MOD5 and TRM1), transport (LOS1), or aminoacylate (SES1) tRNACGASer. Other strains carried mutations in genes for factors involved in rRNA/mRNA synthesis (RPA49, RRN3 and MOT1) or magnesium uptake (ALR1). We show that mutations in not only DUS2, LOS1 and SES1 but also in RPA49, RRN3 and MOT1 cause a reduction in the levels of the altered tRNACGASer. These results indicate that Rpa49, Rrn3 and Mot1 directly or indirectly influence tRNACGASer biogenesis.

  • 757.
    Xu, Hao
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Functional aspects of modified nucleosides in tRNA2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Transfer ribonucleic acids (tRNAs) are extensively modified, especially their anticodon loops. Modifications at position 34 (wobble base) and 37 in these loops affect the tRNAs’ decoding ability, while modifications outside the anticodon loops, e.g. m1A58 of tRNAMeti, may be crucial for tRNA structure or stability. A number of gene products are required for the formation of modified nucleosides, e.g. at least 26 proteins (including Elongator complex) are needed for U34 modifications in yeast, and methyl transferase activity of the Trm6/61p complex is needed to form m1A58. The aim of the studies which this thesis is based upon was to investigate the functional aspects of tRNA modifications and regulation of the modifying enzymes’ activity.

    First, the hypothesis that ncm5U34, mcm5U34, or mcm5s2U34 modifications may be essential for reading frame maintenance was investigated. The results show that mcm5 and s2 group of mcm5s2U play a vital role in reading frame maintenance. Subsequent experiments showed that the +1 frameshifting event at Lys AAA codon occurs via peptidyl-tRNA slippage due to a slow entry of the hypomodified tRNA-Lys.

    Moreover, the hypothesis that Elp1p N-terminal truncation may regulate Elongator activity was investigated. Cleavage of Elp1p was found to occur between residue 203 (Lys) and 204 (Ala) and to depend on the vacuolar protease Prb1p. However, including trichloroacetic acid (TCA) during protein extraction abolished the appearance of truncated Elp1p, showing that its truncation is a preparation artifact.

    Finally, in glioma cell line C6, PKCα was found to interact with TRM61. RNA silencing of TRM6/61 causes a growth defect that can be partially suppressed by tRNAMeti overexpression. PKCα overexpression reduces the nuclear level of TRM61, likely resulting in reduced level of TRM6/61 complex in the nucleus. Furthermore, lower expression of PKCα in the highly aggressive GBM (relative to its expression in less aggressive Grade II/III glioblastomas) is accompanied by increased expression of TRM6/61 mRNAs and tRNAMeti, highlighting the clinical relevance of the studies.

  • 758.
    Xu, Hao
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bygdell, Joakim
    Wingsle, Gunnar
    Byström, Anders S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Yeast Elongator protein Elp1p does not undergo proteolytic processing in exponentially growing cells2015In: MicrobiologyOpen, ISSN 2045-8827, E-ISSN 2045-8827, Vol. 4, no 6, p. 867-878Article in journal (Refereed)
    Abstract [en]

    In eukaryotic organisms, Elongator is a six-subunit protein complex required for the formation of 5-carbamoylmethyl (ncm5) and 5-methylcarboxymethyl (mcm5) side chains on uridines present at the wobble position (U34) of tRNA. The open reading frame encoding the largest Elongator subunit Elp1p has two in-frame 5′ AUG methionine codons separated by 48 nucleotides. Here, we show that the second AUG acts as the start codon of translation. Furthermore, Elp1p was previously shown to exist in two major forms of which one was generated by proteolysis of full-length Elp1p and this proteolytic cleavage was suggested to regulate Elongator complex activity. In this study, we found that the vacuolar protease Prb1p was responsible for the cleavage of Elp1p. The cleavage occurs between residues 203 (Lys) and 204 (Ala) as shown by amine reactive Tandem Mass Tag followed by LC-MS/MS (liquid chromatography mass spectrometry) analysis. However, using a modified protein extraction procedure, including trichloroacetic acid, only full-length Elp1p was observed, showing that truncation of Elp1p is an artifact occurring during protein extraction. Consequently, our results indicate that N-terminal truncation of Elp1p is not likely to regulate Elongator complex activity.

  • 759.
    Yasmin, Lubna
    et al.
    Umeå University, Faculty of Medicine, Medical Biosciences, Pathology.
    Jansson, Anna L
    Umeå University, Faculty of Medicine, Medical Biosciences, Pathology.
    Panahandeh, Tooba
    Umeå University, Faculty of Medicine, Medical Biosciences, Pathology.
    Palmer, Ruth H
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Francis, Matthew S
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Medical Biosciences, Pathology.
    Delineation of exoenzyme S residues that mediate the interaction with 14-3-3 and its biological activity.2006In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, no 3, p. 638-646Article in journal (Refereed)
  • 760.
    Yasmin, Lubna
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Veesenmeyer, Jeffrey L
    Diaz, Maureen H
    Francis, Matthew S
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Ottmann, Christian
    Palmer, Ruth H
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hauser, Alan R
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Electrostatic interactions play a minor role in the binding of ExoS to 14-3-3 proteins2010In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 427, no 2, p. 217-224Article in journal (Refereed)
    Abstract [en]

    14-3-3 proteins belong to a family of conserved molecules expressed in all eukaryotic cells that play an important role in a multitude of signalling pathways. 14-3-3 proteins bind either to phosphoserine/phosphothreonine residues or to sequence-specific non-phosphorylated motifs in more than 200 interaction partners [Pozuelo Rubio, Geraghty, Wong, Wood, Campbell, Morrice and Mackintosh (2004) Biochem. J. 379, 395-408]. These interactions result in cell-cycle regulation, apoptosis, stress responses, cell metabolism and malignant transformation. One example of a phosphorylation-independent interaction is the binding of 14-3-3 to ExoS (exoenzyme S), a bacterial ADP-ribosyltransferase toxin of Pseudomonas aeruginosa. In the present study, we have utilized additional biochemical and infection analyses to define further the structural basis of the interaction between ExoS and 14-3-3. An ExoS leucine-substitution mutant dramatically reduced the interaction potential with 14-3-3 suggesting that Leu422, Leu423, Leu426 and Leu428 of ExoS are important for its interaction with 14-3-3, its enzymatic activity and cytotoxicity. However, ExoS substitution mutants of residues that interact with 14-3-3 through an electrostatic interaction, such as Ser416, His418, Asp424 and Asp427, showed no reduction in their interaction potential with 14-3-3. These ExoS substitution mutants were also as aggressive as wild-type ExoS at inducing cell death and to modify endogenous ExoS target within the cell. In conclusion, electrostatic interaction between ExoS and 14-3-3 via polar residues (Ser416, His418, Asp424 and Asp427) appears to be of secondary importance. Thus the interaction between the 'roof' of the groove of 14-3-3 and ExoS relies more on hydrophobic interaction forces, which probably contributes to induce cell death after ExoS infection and activation.

  • 761. Yim, Lucía
    et al.
    Moukadiri, Ismaïl
    Björk, Glenn
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Armengod, M-Eugenia
    Further insights into the tRNA modification process controlled by proteins MnmE and GidA of Escherichia coli.2006In: Nucleic Acids Res, ISSN 1362-4962, Vol. 34, no 20, p. 5892-905Article in journal (Refereed)
    Abstract [en]

    In Escherichia coli, proteins GidA and MnmE are involved in the addition of the carboxymethylaminomethyl (cmnm) group onto uridine 34 (U34) of tRNAs decoding two-family box triplets. However, their precise role in the modification reaction remains undetermined. Here, we show that GidA is an FAD-binding protein and that mutagenesis of the N-terminal dinucleotide-binding motif of GidA, impairs capability of this protein to bind FAD and modify tRNA, resulting in defective cell growth. Thus, GidA may catalyse an FAD-dependent reaction that is required for production of cmnmU34. We also show that GidA and MnmE have identical cell location and that both proteins physically interact. Gel filtration and native PAGE experiments indicate that GidA, like MnmE, dimerizes and that GidA and MnmE directly assemble in an alpha2beta2 heterotetrameric complex. Interestingly, high-performance liquid chromatography (HPLC) analysis shows that identical levels of the same undermodified form of U34 are present in tRNA hydrolysates from loss-of-function gidA and mnmE mutants. Moreover, these mutants exhibit similar phenotypic traits. Altogether, these results do not support previous proposals that activity of MnmE precedes that of GidA; rather, our data suggest that MnmE and GidA form a functional complex in which both proteins are interdependent.

  • 762. Zammit, Carla M.
    et al.
    Mangold, Stefanie
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Jonna, Venkateswara Rao
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Mutch, Lesley A.
    Watling, Helen R.
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Watkin, Elizabeth L. J.
    Bioleaching in brackish waters-effect of chloride ions on the acidophile population and proteomes of model species2012In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 93, no 1, p. 319-329Article in journal (Refereed)
    Abstract [en]

    High concentrations of chloride ions inhibit the growth of acidophilic microorganisms used in biomining, a problem particularly relevant to Western Australian and Chilean biomining operations. Despite this, little is known about the mechanisms acidophiles adopt in order to tolerate high chloride ion concentrations. This study aimed to investigate the impact of increasing concentrations of chloride ions on the population dynamics of a mixed culture during pyrite bioleaching and apply proteomics to elucidate how two species from this mixed culture alter their proteomes under chloride stress. A mixture consisting of well-known biomining microorganisms and an enrichment culture obtained from an acidic saline drain were tested for their ability to bioleach pyrite in the presence of 0, 3.5, 7, and 20 g L(-1) NaCl. Microorganisms from the enrichment culture were found to out-compete the known biomining microorganisms, independent of the chloride ion concentration. The proteomes of the Gram-positive acidophile Acidimicrobium ferrooxidans and the Gram-negative acidophile Acidithiobacillus caldus grown in the presence or absence of chloride ions were investigated. Analysis of differential expression showed that acidophilic microorganisms adopted several changes in their proteomes in the presence of chloride ions, suggesting the following strategies to combat the NaCl stress: adaptation of the cell membrane, the accumulation of amino acids possibly as a form of osmoprotectant, and the expression of a YceI family protein involved in acid and osmotic-related stress.

  • 763.
    Zare, Aman
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Regulation of gene expression in fruit flies: how does it start, and will it be remembered?2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    One of the most distinctive features of eukaryotic chromosomes is the bundling of DNA together with functionally associated RNA and proteins in chromatin. This allows huge amounts of DNA to be packed inside the very tiny space of the nucleus, and alterations in the structure of chromatin enable access to the DNA for transcription (“reading” genes by production of RNA copies). Much of the current knowledge of chromatin structure and regulation comes from studies of Drosophila melanogaster. When the chromatin structure is open the transcription of a gene can start after recruitment of the necessary factors. The main enzyme for gene transcription is Polymerase II (Pol II). For successful gene transcription, Pol II must not only be recruited to the gene’s promoter, but also escape from a pausing state which occurs soon after transcription initiation. CBP/P300 is one of the co-activators involved in transcriptional activation. In the studies this thesis is based upon, my colleagues and I (hereafter we) discovered a new function for CBP in transcription activation. Using high throughput sequencing techniques, we found that CBP directly stimulates recruitment of Pol II to promoters, and facilitates its release from the paused state, enabling progression to the elongation stage of transcription.

    For cells to remember their identity following division during development, the transcriptional state of genes must be transmitted. Intensively studied players involved in this memory are the Polycomb group (PcG) proteins, responsible for maintaining the repressed state of important developmental genes. The core members are Polycomb repressive complex 1 and 2 (PRC1 and PRC2), which are recruited in flies through poorly known mechanisms to target genes by so-called Polycomb response elements (PREs). Using Drosophila mutant cell lines, we showed that (in contrast to previous models) some PREs can recruit PRC1 even when PRC2 is absent. We also observed that at many PREs, PRC1 is needed for recruitment of PRC2 and concluded that targeting PRC complexes to PREs is a much more flexible and variable process than previously thought.

    Some phenotypic effects of environmental changes can be transferred to subsequent generations. Previous efforts to identify the mechanisms involved have focused on material (mainly, but not only, DNA) transferred through germ cells. However, organisms’ microbiomes are also transferred to the next generation. Thus, to investigate possible contributions of microbiomes to such transfer, we used fruit flies as the microbiomes they inherit can be easily controlled. We altered some parents’ environmental conditions by lowering the temperature, then grew offspring that received microbiomes from cold-treated and control parents in control conditions and compared their transcriptional patterns. Our results suggest that most of the crosstalk between the microbiome and the fly happens in the gut, and that further investigation of this previously unsuspected mode of inheritance is warranted.

  • 764.
    Zare, Aman
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, Anna-Mia
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Karlsson, Edvin
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Division of CBRN Security and Defence, FOI-Swedish, Defence Research Agency, Umeå, Sweden.
    Delhomme, Nicolas
    Stenberg, Per
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Division of CBRN Security and Defence, FOI-Swedish, Defence Research Agency, Umeå, Sweden.
    The gut microbiome participates in transgenerational inheritance of low temperature responses in Drosophila melanogaster2018In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 592, no 24, p. 4078-4086Article in journal (Refereed)
    Abstract [en]

    Environmental perturbations induce transcriptional changes, some of which may be inherited even in the absence of the initial stimulus. Previous studies have focused on transfers through the germ-line although microbiota is also passed on to the offspring. Thus, we inspected the involvement of the gut microbiome in transgenerational inheritance of environmental exposures in Drosophila melanogaster. We grew flies in the cold versus control temperatures and compared their transcriptional patterns in both conditions as well as in their offspring. F2 flies grew in control temperature while we controlled their microbiota acquisition from either F1 sets. Transcriptional status of some genes was conserved transgenerationally, and a subset of these genes, mainly expressed in the gut, was transcriptionally dependent on the acquired microbiome. This article is protected by copyright. All rights reserved.

  • 765.
    Zeng, Qing-Yin
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Development of molecular techniques for fungal diagnostic research2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Fungi are present everywhere in indoor and outdoor environments. Many fungi are toxigenic or pathogenic that may cause various public health concerns. Rapid detection, quantification and characterization of fungi in living and working environments are essential for exposure risk assessment to safe guard public health.

    Rapid and accurate detection and identification of fungi using molecular method require specific markers. In this thesis, partial mt SSU and LSU rDNA were amplified and sequenced from 31 fungal species of 16 genera. Sequence alignments showed that fungal mt SSU and LSU rDNA contained sufficient amount of variation for the development of markers that can discriminate even among closely related species. Forty-eight probes were designed and were verified as highly specific to 25 fungal species commonly detected in living and working environments. These specific probes would have potential applications in clinical diagnosis and public health-related environmental monitoring.

    Nested PCR is a highly sensitive and specific method. Based on the nuclear 18S rDNA sequence variation pattern, three nested PCR systems were developed to detect the conifer tree pathogen Gremmeniella abietina, an ascomycete fungus that causes stem canker and shoot dieback in many conifer species. The three nested PCR systems showed high specificity and sensitivity. These methods could have broad applications in forest protection and disease management programs.

    Quantitative real-time PCR offers the ability of simultaneous detection and quantification of DNA of a specific microbe in one reaction. Based on the 18S rDNA sequence, two real-time PCR assays were developed to detect and quantify Wallemia sebi, a deuteromycete fungus commonly found in agricultural environments and is suspected to be a causative agent of farmer’s lung disease. Both PCR systems proved to be highly specific and sensitive for W. sebi detection even in a high background of other fungal DNAs. Application of the real-time PCR methods in the quantification of W. sebi in the aerosols of a farm revealed a high concentration of W. sebi spores (107/m3). The study indicates that W. sebi is a dominant fungus in agriculture environments.

    Cladosporium spores are important aeroallergens, and prolonged exposure to elevated spore concentrations can provoke chronic allergy and asthma. A TaqMan probe and a SYBR Green I based real-time PCR assay were developed to detect and quantify Cladosporium in aerosols. The two real-time PCR systems proved to be highly specific and sensitive for Cladosporium. These methods were employed to quantify Cladosporium in aerosols of five different indoor environments. High spore concentration of Cladosporium (107/m3) was observed in a cow barn. Cladosporium spore concentration in paper and pulp factory and countryside house also exceeded threshold value for clinical significance. Prolonged exposure in these environments could impose certain health risk. Thus, monitoring Cladosporium spore concentration in indoor environments is important for indoor air quality control.

  • 766.
    Zeng, Qing-Yin
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Hansson, Per
    Wang, Xiao-Ru
    Specific and sensitive detection of the conifer pathogen Gremmeniella abietina by nested PCR2005In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 5, p. 65-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Gremmeniella abietina (Lagerb.) Morelet is an ascomycete fungus that causes stem canker and shoot dieback in many conifer species. The fungus is widespread and causes severe damage to forest plantations in Europe, North America and Asia. To facilitate early diagnosis and improve measures to control the spread of the disease, rapid, specific and sensitive detection methods for G. abietina in conifer hosts are needed. RESULTS: We designed two pairs of specific primers for G. abietina based on the 18S rDNA sequence variation pattern. These primers were validated against a wide range of fungi and 14 potential conifer hosts. Based on these specific primers, two nested PCR systems were developed. The first system employed universal fungal primers to enrich the fungal DNA targets in the first round, followed by a second round selective amplification of the pathogen. The other system employed G. abietina-specific primers in both PCR steps. Both approaches can detect the presence of G. abietina in composite samples with high sensitivity, as little as 7.5 fg G. abietina DNA in the host genomic background. CONCLUSION: The methods described here are rapid and can be applied directly to a wide range of conifer species, without the need for fungal isolation and cultivation. Therefore, it represents a promising alternative to disease inspection in forest nurseries, plantations and quarantine control facilities.

  • 767.
    Zeng, Qing-Yin
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Rasmuson-Lestander, Åsa
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Wang, Xiao-Ru
    Extensive set of mitochondrial LSU rDNA-based oligonucleotide probes for the detection of common airborne fungi.2004In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 237, no 1, p. 79-87Article in journal (Refereed)
    Abstract [en]

    Fungi exist in every indoor and outdoor environment. Many fungi are toxigenic or pathogens that may cause various public health concerns. Rapid and accurate detection and identification of fungi require specific markers. In this study, partial mitochondrial large subunit rDNA was amplified and sequenced from 32 fungal strains representing 31 species from 14 genera. Based on the sequence variation pattern, 26 oligonucleotide probes were designed for their discrimination. The specificity of the probes was evaluated through homology search against GenBank database and hybridization examination on 38 fungal strains. The 26 probes were verified as highly specific to 20 fungal species. A two-step detection procedure through PCR followed by probe hybridization gave ten-fold increase in detection sensitivity than single-step PCR assay and would be a practical approach for environmental sample screening. The probes developed in this study can be applied in clinical diagnosis and environmental monitoring of fungal agents.

  • 768.
    Zeng, Qing-Yin
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Wang, Xiao-Ru
    Blomquist, Göran
    Development of mitochondrial SSU rDNA-based oligonucleotide probes for specific detection of common airborne fungi2003In: Molecular and Cellular Probes, ISSN 1044-7431, E-ISSN 1095-9327, Vol. 17, no 6, p. 281-288Article in journal (Refereed)
    Abstract [en]

    In this study we sequenced partial mitochondrial small subunit rDNA from 32 fungal strains representing 31 species from 16 genera. Most of these species are common airborne fungi and pathogens that may cause various public health concerns. Sequence alignment showed several conserved and highly variable regions. The variable regions were deployed to design oligonucleotide probes for each fungal species. The specificity of the designed probes was first examined through homology search against GenBank database then further verified through hybridization experiments to 38 fungal strains. A total of 23 probes were verified as specific to 15 fungal species commonly detected in living and working environments. These new probes will have potential applications in clinical diagnosis and public health-related environmental monitoring.

  • 769.
    Zeng, Qing-Yin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Westermark, Sven-Olof
    Rasmuson-Lestander, Åsa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wang, Xiao-Ru
    Detection and quantification of Cladosporium in aerosols by real-time PCR2006In: Journal of Environmental Monitoring, ISSN 1464-0325, E-ISSN 1464-0333, Vol. 8, no 1, p. 153-160Article in journal (Refereed)
    Abstract [en]

    Cladosporium is one of the most common airborne molds found in indoor and outdoor environments. Cladosporium spores are important aeroallergens, and prolonged exposure to elevated spore concentrations can provoke chronic allergy and asthma. To accurately quantify the levels of Cladosporium in indoor and outdoor environments, two real-time PCR systems were developed in this study. The two real-time PCR systems are highly specific and sensitive for Cladosporium detection even in a high background of other fungal DNAs. These methods were employed to quantify Cladosporium in aerosols of five different indoor environments. The investigation revealed a high spore concentration of Cladosporium (10(7) m(-3)) in a cow barn that accounted for 28-44% of the airborne fungal propagules. In a countryside house that uses firewood for heating and in a paper and pulp factory, Cladosporium was detected at 10(4) spores m(-3), which accounted for 2-6% of the fungal propagules in the aerosols. The concentrations of Cladosporium in these three indoor environments far exceeded the medical borderline level (3000 spores m(-3)). In a power station and a fruit and vegetable storage, Cladosporium was found to be a minor component in the aerosols, accounted for 0.01-0.1% of the total fungal propagules. These results showed that monitoring Cladosporium in indoor environments is more important than in outdoor environments from the public health point of view. Cladosporium may not be the dominant fungi in some indoor environments, but its concentration could still be exceeding the threshold value for clinical significance. The methods developed in this study could facilitate accurate detection and quantification of Cladosporium for public health related risk assessment.

  • 770.
    Zeng, Qing-Yin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). National Institute for Working Life.
    Westermark, Sven-Olof
    Rasmuson-Lestander, Åsa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wang, Xiao-Ru
    Detection and quantification of Wallemia sebi in aerosols by real-time PCR, conventional PCR, and cultivation2004In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 70, no 12, p. 7295-7302Article in journal (Refereed)
    Abstract [en]

    Wallemia sebi is a deuteromycete fungus commonly found in agricultural environments in many parts of the world and is suspected to be a causative agent of farmer's lung disease. The fungus grows slowly on commonly used culture media and is often obscured by the fast-growing fungi. Thus, its occurrence in different environments has often been underestimated. In this study, we developed two sets of PCR primers specific to W. sebi that can be applied in either conventional PCR or real-time PCR for rapid detection and quantification of the fungus in environmental samples. Both PCR systems proved to be highly specific and sensitive for W. sebi detection even in a high background of other fungal DNAs. These methods were employed to investigate the presence of W. sebi in the aerosols of a farm. The results revealed a high concentration of W. sebi spores, 10(7) m(-3) by real-time PCR and 10(6) m(-3) by cultivation, which indicates the prevalence of W. sebi in farms handling hay and grain and in cow barns. The methods developed in this study could serve as rapid, specific, and sensitive means of detecting W. sebi in aerosol and surface samples and could thus facilitate investigations of its distribution, ecology, clinical diagnosis, and exposure risk assessment.

  • 771.
    Zhang, Jingpu
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Rasmuson-Lestander, Åsa
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Expression preference of the S-adenosylmethionine synthetase (SamS) gene in Drosophila melanogaster1997In: Dev Rep Biol, Vol. 6, p. 7-17Article in journal (Other (popular science, discussion, etc.))
  • 772. Zhang, Shu-sheng
    et al.
    Park, Chae Gyu
    Zhang, Pei
    Bartra, Sara Schesser
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Plano, Gregory V
    Klena, John D
    Skurnik, Mikael
    Hinnebusch, B Joseph
    Chen, Tie
    Plasminogen activator Pla of Yersinia pestis utilizes murine DEC-205 (CD205) as a receptor to promote dissemination2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 46, p. 31511-21Article in journal (Refereed)
    Abstract [en]

    Yersinia pestis, a Gram-negative bacterium that causes bubonic and pneumonic plague, is able to rapidly disseminate to other parts of its mammalian hosts. Y. pestis expresses plasminogen activator (PLA) on its surface, which has been suggested to play a role in bacterial dissemination. It has been speculated that Y. pestis hijacks antigen-presenting cells, such as macrophages (MPhis) and dendritic cells, to be delivered to lymph nodes to initiate dissemination and infection. Both alveolar MPhis and pulmonary dendritic cells express a C-type lectin receptor, DEC-205 (CD205), which mediates antigen uptake and presentation. However, no ligand has been identified for DEC-205. In this study, we show that the invasion of alveolar MPhisby Y. pestis depends both in vitro and in vivo on the expression of PLA. DEC-205-expressing MPhis and transfectants, but not their negative counterparts, phagocytosed PLA-expressing Y. pestis and Escherichia coli K12 more efficiently than PLA-negative controls. The interactions between PLA-expressing bacteria and DEC-205-expressing transfectants or alveolar MPhis could be inhibited by an anti-DEC-205 antibody. Importantly, the blockage of the PLA-DEC-205 interaction reduced the dissemination of Y. pestis in mice. In conclusion, murine DEC-205 is a receptor for PLA of Y. pestis, and this host-pathogen interaction appears to play a key role in promoting bacterial dissemination.

  • 773. Zhou, Guang-Qian
    et al.
    Zhang, Youyi
    Ferguson, David J P
    Chen, Sa
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Rasmuson-Lestander, Åsa
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Campbell, Frederick C
    Watt, Suzanne M
    The Drosophila ortholog of the endolysosomal membrane protein, endolyn, regulates cell proliferation.2006In: J Cell Biochem, ISSN 0730-2312, Vol. 99, no 5, p. 1380-96Article in journal (Refereed)
    Abstract [en]

    Endolyn (CD164) is a sialomucin that regulates the proliferation, adhesion, and migration of human haematopoietic stem and progenitor cells. This molecule is predominately localized in endocytotic compartments, where it may contribute to endolysosomal biogenesis and trafficking. In order to more closely define the function of endolyn from an evolutionary view-point, we first analyzed endolyn orthologs in species ranging from insects, fish, and birds to mammals. The predicted molecular structures of the endolyn orthologs from these species are well conserved, particularly with respect to significant O-linked glycosylation of the extracellular domain, and the high degree of amino acid similarities within their transmembrane and cytoplasmic domains, with the latter possessing the lysosomal target signal, YXXphi. Focusing on Drosophila, our studies showed that the subcellular distribution of endolyn in non-polarized Drosophila S2 cells resembles that of its human counterpart in hematopoietic cells, with its predominant localization being within intracellular vesicles, while a small fraction occurs on the cell surface. Both Y --> A and L --> A mutations in the YHTL motif perturbed the normal subcellular distribution of Drosophila endolyn. Interestingly, embryonic and early larval development was often arrested in endolyn-deficient Drosophila mutants. This may partly be due to the role of endolyn in regulating cell proliferation, since knock-down of endolyn expression in S2 cells resulted in up to 50% inhibition of cell growth, with a proportion of cells undergoing apoptosis. Taken together, these results demonstrate that endolyn is an evolutionarily conserved sialomucin fundamentally involved in cell proliferation in both the human and Drosophila melanogaster. 2006 Wiley-Liss, Inc.

  • 774.
    Zhou, Yang
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Regulation of pre-mRNA splicing and mRNA degradation in Saccharomyces cerevisiae2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Messenger RNAs are transcribed and co-transcriptionally processed in the nucleus, and transported to the cytoplasm. In the cytoplasm, mRNAs serve as the template for protein synthesis and are eventually degraded. The removal of intron sequences from a precursor mRNA is termed splicing and is carried out by the dynamic spliceosome. In this thesis, I describe the regulated splicing of two transcripts in Saccharomyces cerevisiae. I also describe a study where the mechanisms that control the expression of magnesium transporters are elucidated.

    The pre-mRNA retention and splicing (RES) complex is a spliceosome-associated protein complex that promotes the splicing and nuclear retention of a subset of pre-mRNAs. The RES complex consists of three subunits, Bud13p, Snu17p and Pml1p. We show that the lack of RES factors causes a decrease in the formation of N4-acetylcytidine (ac4C) in tRNAs. This phenotype is caused by inefficient splicing of the pre-mRNA of the TAN1 gene, which is required for the formation of ac4C in tRNAs. The RES mutants also show growth defects that are exacerbated at elevated temperatures. We show that the temperature sensitive phenotype of the bud13Δ and snu17Δ cells is caused by the inefficient splicing of the MED20 pre-mRNA. The MED20 gene encodes a subunit of the Mediator complex. Unspliced pre-mRNAs that enter the cytoplasm are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway, which targets transcripts that contain premature translation termination codons. Consistent with the nuclear retention function of the RES complex, we find that NMD inactivation in the RES mutants leads to the accumulation of both TAN1 and MED20 pre-mRNAs. We also show that the cis-acting elements that promote RES-dependent splicing are different between the TAN1 and MED20 pre-mRNAs.

    The NMD pathway also targets transcripts with upstream ORFs (uORFs) for degradation. The ALR1 gene encodes the major magnesium importer in yeast, and its expression is controlled by the NMD pathway via a uORF in the 5’ untranslated region. We show that the ribosome reaches the downstream main ORF by a translation reinitiation mechanism. The NMD pathway was shown to control cellular Mg2+ levels by regulating the expression of the ALR1 gene. We further show that the NMD pathway targets the transcripts of the vacuolar Mg2+ exporter Mnr2p and the mitochondrial Mg2+ exporter Mme1p for degradation.

    In summary, we conclude that the RES complex has a role in the splicing regulation of a subset of transcripts. We also suggest a regulatory role for the NMD pathway in maintaining the cellular Mg2+ concentration by controlling the expression of Mg2+ transporters.

  • 775.
    Zhou, Yang
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Chen, Changchun
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus J. O.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The pre-mRNA retention and splicing complex controls tRNA maturation by promoting TAN1 expression2013In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 11, p. 5669-5678Article in journal (Refereed)
    Abstract [en]

    The conserved pre-mRNA retention and splicing (RES) complex, which in yeast consists of Bud13p, Snu17p and Pml1p, is thought to promote nuclear retention of unspliced pre-mRNAs and enhance splicing of a subset of transcripts. Here, we find that the absence of Bud13p or Snu17p causes greatly reduced levels of the modified nucleoside N-4-acetylcytidine (ac(4)C) in tRNA and that a lack of Pml1p reduces ac(4)C levels at elevated temperatures. The ac(4)C nucleoside is normally found at position 12 in the tRNA species specific for serine and leucine. We show that the tRNA modification defect in RES-deficient cells is attributable to inefficient splicing of TAN1 pre-mRNA and the effects of reduced Tan1p levels on formation of ac(4)C. Analyses of cis-acting elements in TAN1 pre-mRNA showed that the intron sequence between the 5' splice site and branchpoint is necessary and sufficient to mediate RES dependency. We also show that in RES-deficient cells, the TAN1 pre-mRNA is targeted for degradation by the cytoplasmic nonsense-mediated mRNA decay pathway, indicating that poor nuclear retention may contribute to the tRNA modification defect. Our results demonstrate that TAN1 pre-mRNA processing has an unprecedented requirement for RES factors and that the complex controls the formation of ac(4)C in tRNA.

  • 776.
    Zhou, Yang
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The nonsense-mediated mRNA decay pathway controls the expression of magnesium transporters in Saccharomyces cerevisiaeManuscript (preprint) (Other academic)
  • 777.
    Zhou, Yang
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus J O
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). BRF Krutet, Norra Majorsgatan, Umea, Sweden; University of Tartu, Institute ofTechnology, Nooruse, Tartu, Estonia.
    The pre-mRNA retention and splicing complex controls expression of the Mediator subunit Med202017In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 14, no 10, p. 1411-1417Article in journal (Refereed)
    Abstract [en]

    The heterotrimeric pre-mRNA retention and splicing (RES) complex, consisting of Bud13p, Snu17p and Pml1p, promotes splicing and nuclear retention of a subset of intron-containing pre-mRNAs. Yeast cells deleted for individual RES genes show growth defects that are exacerbated at elevated temperatures. Although the growth phenotypes correlate to the splicing defects in the individual mutants, the underlying mechanism is unknown. Here, we show that the temperature sensitive (Ts) growth phenotype of bud13Δ and snu17Δ cells is a consequence of inefficient splicing of MED20 pre-mRNA, which codes for a subunit of the Mediator complex; a co-regulator of RNA polymerase II transcription. The MED20 pre-mRNA splicing defect is less pronounced in pml1Δ cells, explaining why they grow better than the other 2 RES mutants at elevated temperatures. Inactivation of the cytoplasmic nonsense-mediated mRNA decay (NMD) pathway in the RES mutants leads to accumulation of MED20 pre-mRNA, indicating that inefficient nuclear retention contributes to the growth defect. Further, the Ts phenotype of bud13Δ and snu17Δ cells is partially suppressed by the inactivation of NMD, showing that the growth defects are augmented by the presence of a functional NMD pathway. Collectively, our results demonstrate an important role of the RES complex in maintaining the Med20p levels.

  • 778. Zindy, Frederique
    et al.
    Nilsson, Lisa
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Nguyen, Luc
    Meunier, Cecile
    Smeyne, Richard
    Rehg, Jerold
    Eberhart, Charles
    Sherr, Charles
    Roussel, Martine
    Hemangiosarcomas, medulloblastomas, and other tumors in Ink4c/p53-null mice2003In: Cancer Research, ISSN 0008-5472, Vol. 63, no 17, p. 5420-5427Article in journal (Refereed)
  • 779.
    Zwack, Erin
    et al.
    University of Pennsylvania, USA.
    Snyder, Annelise
    University of Pennsylvania, USA.
    Wynosky-Dolfi, Meghan
    University of Pennsylvania, USA.
    Ruthel, Gordon
    University of Pennsylvania, USA.
    Philip, Naomi
    University of Pennsylvania, USA.
    Marketon, Melanie
    Indiana University, USA.
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bliska, James
    SUNY Stony Brook, USA.
    Brodsky, Igor
    University of Pennsylvania, USA.
    Inflammasome activation in response to the Yersinia type III secretion system requires hyperinjection of translocon proteins YopB and YopD2015In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 6, no 1, article id e02095-14Article in journal (Refereed)
    Abstract [en]

    Type III secretion systems (T3SS) translocate effector proteins into target cells in order to disrupt or modulate host cell signaling pathways and establish replicative niches. However, recognition of T3SS activity by cytosolic pattern recognition receptors (PRRs) of the nucleotide-binding domain leucine rich repeat (NLR) family, either through detection of translocated products or membrane disruption, induces assembly of multiprotein complexes known as inflammasomes. Macrophages infected with Yersinia pseudotuberculosis strains lacking all known effectors or lacking the translocation regulator YopK induce rapid activation of both the canonical NLRP3 and noncanonical caspase-11 inflammasomes. While this inflammasome activation requires a functional T3SS, the precise signal that triggers inflammasome activation in response to Yersinia T3SS activity remains unclear. Effectorless strains of Yersinia as well as ΔyopK strains translocate elevated levels of T3SS substrates into infected cells. To dissect the contribution of pore formation and translocation to inflammasome activation, we took advantage of variants of YopD and LcrH that separate these functions of the T3SS. Notably, YopD variants that abrogated translocation but not pore-forming activity failed to induce inflammasome activation. Furthermore, analysis of individual infected cells revealed that inflammasome activation at the single-cell level correlated with translocated levels of YopB and YopD themselves. Intriguingly, LcrH mutants that are fully competent for effector translocation but produce and translocate lower levels of YopB and YopD also fail to trigger inflammasome activation. Our findings therefore suggest that hypertranslocation of YopD and YopB is linked to inflammasome activation in response to the Yersinia T3SS.

  • 780.
    Zweifel, Ulla-Li
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Science and Technology, Umeå Marine Sciences Centre (UMF).
    Wikner, Johan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Science and Technology, Umeå Marine Sciences Centre (UMF).
    Hagström, Åke
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Science and Technology, Umeå Marine Sciences Centre (UMF).
    Lundberg, Erik
    Umeå University, Faculty of Science and Technology, Umeå Marine Sciences Centre (UMF).
    Norrman, Bo
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Science and Technology, Umeå Marine Sciences Centre (UMF).
    DYNAMICS OF DISSOLVED ORGANIC-CARBON IN A COASTAL ECOSYSTEM1995In: Limnology and Oceanography, ISSN 0024-3590, E-ISSN 1939-5590, Vol. 40, no 2, p. 299-305Article in journal (Refereed)
    Abstract [en]

    In the Bothnian Sea, there was a marked seasonal variation of dissolved organic C (DOC) in 1990-1992, with a large increase in DOC concentrations in summer at two stations. The accumulation of DOC at the coastal station persisted for 5 months, reaching peak values 24-31% above the mean winter value (288 mu M). At the offshore station DOC concentrations were elevated throughout the water column in July, reaching 14% above the mean winter value (291 mu M). The DOC concentration at the Coastal station was significantly correlated to water flow in an adjacent river, suggesting that the source of the summer DOC increase was largely explained by riverine input. Bioassays indicated that a large portion (22-99%) of the introduced DOC was degradable by bacteria after inorganic nutrients were added. A negative correlation between DOC and phosphate concentration was also found, suggesting that the system was P deficient in summer. The accumulation of DOC in summer was thus possibly caused by slow bacterial degradation due td phosphate deficiency and transient accumulation of refractory DOG. An annual C balance at the coastal station indicated an insufficient supply of C from phytoplankton production to support the C demand of the system; at the offshore station the budget was close to balanced. The results suggest that riverine DOC had a major impact on coastal DOC dynamics and that it was partly used in the microbial food web in the bay.

  • 781.
    Åberg, Anna
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Balsalobre, Carlos
    (p)ppGpp regulates type 1 fimbriation of Escherichia coli by modulating the expression of the site-specific recombinase FimB.2006In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 60, no 6, p. 1520-1533Article in journal (Refereed)
    Abstract [en]

    In this report we have examined the role of the regulatory alarmone (p)ppGpp on expression of virulence determinants of uropathogenic Escherichia coli strains. The ability to form biofilms is shown to be markedly diminished in (p)ppGpp-deficient strains. We present evidence (i) that (p)ppGpp tightly regulates expression of the type 1 fimbriae in both commensal and pathogenic E. coli isolates by increasing the subpopulation of cells that express the type 1 fimbriae; and (ii) that the effect of (p)ppGpp on the number of fimbrial expressing cells can ultimately be traced to its role in transcription of the fimB recombinase gene, whose product mediates inversion of the fim promoter to the productive (ON) orientation. Primer extension analysis suggests that the effect of (p)ppGpp on transcription of fimB occurs by altering the activity of only one of the two fimB promoters. Furthermore, spontaneous mutants with properties characteristic of ppGpp(0) suppressors restore fimB transcription and consequent downstream effects in the absence of (p)ppGpp. Consistently, the rpoB3770 allele also fully restores transcription of fimB in a ppGpp(0) strain and artificially elevated levels of FimB bypass the need for (p)ppGpp for type 1 fimbriation. Our findings suggest that the (p)ppGpp-stimulated expression of type 1 fimbriae may be relevant during the interaction of pathogenic E. coli with the host.

  • 782.
    Åberg, Anna
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Balsalobre, Carlos
    Regulation of the fimB promoter: a case of differential regulation by ppGpp and DksA in vivo2008In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 67, no 6, p. 1223-1241Article in journal (Refereed)
  • 783.
    Åström, S U
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nordlund, M E
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Erickson, F L
    Hannig, E M
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Genetic interactions between a null allele of the RIT1 gene encoding an initiator tRNA-specific modification enzyme and genes encoding translation factors in Saccharomyces cerevisiae1999In: Molecular General Genetics, ISSN 0026-8925, E-ISSN 1432-1874, Vol. 261, no 6, p. 967-976Article in journal (Refereed)
    Abstract [en]

    The Saccharomyces cerevisiae gene RIT1 encodes a phospho-ribosyl transferase that exclusively modifies the initiator tRNA (tRNAMet(i)) by the addition of a 2'-O-ribosyl phosphate group to Adenosine 64. As a result, tRNAMet(i) is prevented from participating in the elongation steps of protein synthesis. We previously showed that the modification is not essential for the function of tRNAMet(i) in the initiation of translation, since rit1 null strains are viable and show no obvious growth defects. Here, we demonstrate that yeast strains in which a rit1 null allele is combined with mutations in any of the genes for the three subunits of eukaryotic initiation factor-2 (eIF-2), or with disruption alleles of two of the four initiator methionine tRNA (IMT) genes, show synergistic growth defects. A multicopy plasmid carrying an IMT gene can alleviate these defects. On the other hand, introduction of a high-copy-number plasmid carrying the TEF2 gene, which encodes the eukaryotic elongation factor 1alpha (eEF-1alpha), into rit1 null strains with two intact IMT genes had the opposite effect, indicating that increased levels of eEF-1alpha are deleterious to these strains, presumably due to sequestration of the unmodified met-tRNAMet(i) for elongation. Thus, under conditions in which the components of the ternary met-tRNAMet(i):GTP:eIF-2 complex become limiting or are functionally impaired, the presence of the 2'-O-ribosyl phosphate modification in tRNAMet(i) is important for the provision of adequate amounts of tRNAMet(i) for formation of this ternary complex.

  • 784.
    Åström, Stefan U
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Rit1, a tRNA backbone-modifying enzyme that mediates initiator and elongator tRNA discrimination1994In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 79, no 3, p. 535-546Article in journal (Refereed)
    Abstract [en]

    Using a genetic screen in yeast aimed at identifying cellular factors involved in initiator and elongator methionine tRNA discrimination in the translational process, we have identified a mutation that abolish the requirement for elongator methionine tRNA. The gene affected, which we call the ribosylation of the initiator tRNA gene or RIT1, encodes a 2'-O-ribosyl phosphate transferase. This enzyme modifies exclusively the initiator tRNA in position 64 using 5'-phosphoribosyl-1'-pyrophosphate as the modification donor. As the initiator tRNA participates both in the initiation and elongation of translation in a rit1 strain, we conclude that the 2'-O-ribosyl phosphate modification discriminates the initiator tRNAs from the elongator tRNAs during protein synthesis. The modification enzyme was shown to recognize the stem-loop IV region that is unique in eukaryotic cytoplasmic initiator tRNAs.

  • 785.
    Åström, Stefan U.
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    von Pawel-Rammingen, Ulrich
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The yeast initiator tRNAMet can act as an elongator tRNA(Met) in vivo1993In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 233, no 1, p. 43-58Article in journal (Refereed)
    Abstract [en]

    Saccharomyces cerevisiae uses two different methionine accepting tRNAs during protein synthesis. One, tRNA(iMet), is used exclusively during the initiation of translation whereas the other, tRNA(mMet), is used during the elongation of translation. To study the unique features of each methionine tRNA species, we constructed yeast strains with null alleles of the five elongator methionine tRNA (EMT) genes and strains with null alleles of the four initiator methionine tRNA (IMT) genes, respectively. Consequently, growth of these strains was dependent either on a tRNA(mMet) or a tRNA(iMet), respectively, encoded from a plasmid-derived gene. For both null mutants, the plasmid carrying the wild-type gene can be selected against and exchanged for another plasmid derived EMT or IMT gene (wild-type or mutant). A high gene dosage of the wild-type IMT gene could restore growth to the elongator-depleted strain. However, wild-type EMT genes in a high gene dosage never restored growth of the initiator depleted strain. Thus, the elongator tRNA(Met) is much more restricted to participate in the initiation of translation than the initiator tRNA(Met) is restricted to participate in the elongation process. Using the two null mutants, we have identified tRNA(mMet) mutants, which show reduced elongator activity, and tRNA(iMet) mutants, with improved elongator activity in the elongator depleted strain. Also, tRNA(mMet) mutants that function as an initiator tRNA in the initiator depleted strain were identified. From this mutant analysis, we showed that the conserved U/rT at position 54 of the elongator tRNA(Met) is an important determinant for an elongator tRNA. The most important determinant for an initiator was shown to be the acceptor stem and especially the conserved A1.U72 base-pair. Mutant tRNAs, with reduced activity in either process, were investigated for enhanced activity during overproduction of the alpha and beta-subunits of the eukaryotic initiation factor 2 (eIF-2) or the eukaryotic elongation factor 1 alpha (eEF-1 alpha). The data suggest that the U/rT of the elongator at position 54 is important for eEF-1 alpha recognition and that the acceptor stem of the initiator is important for eIF-2 recognition.

  • 786.
    Öhman, Samuel
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    A metagenomic approach to detect low abundance pathogens in complex samples2017Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
  • 787.
    Östberg, Yngve
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Pinne, Marija
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Benz, Roland
    Rosa, Patricia
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Elimination of channel-forming activity by insertional inactivation of the p13 gene in Borrelia burgdorferi2002In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 184, no 24, p. 6811-6819Article in journal (Refereed)
    Abstract [en]

    P13 is a chromosomally encoded 13-kDa integral outer membrane protein of the Lyme disease agent, Borrelia burgdorferi. The aim of this study was to investigate the function of the P13 protein. Here, we inactivated the p13 gene by targeted mutagenesis and investigated the porin activities of outer membrane proteins by using lipid bilayer experiments. Channel-forming activity was lost in the p13 mutant compared to wild-type B. burgdorferi, indicating that P13 may function as a porin. We purified native P13 to homogeneity by fast performance liquid chromatography and demonstrated that pure P13 has channel-forming activity with a single-channel conductance in 1 M KCl of 3.5 nS, the same as the porin activity that was lost in the p13 mutant. Further characterization of the channel formed by P13 suggested that it is cation selective and voltage independent. In addition, no major physiological effects of the inactivated p13 gene could be detected under normal growth conditions. The inactivation of p13 is the first reported inactivation of a gene encoding an integral outer membrane protein in B. burgdorferi. Here, we describe both genetic and biophysical experiments indicating that P13 in B. burgdorferi is an outer membrane protein with porin activity.

  • 788.
    Österberg, Sofia
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Metabolism-dependent taxis and control of motility in Pseudomonas putida2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Bacteria living in soil and aquatic habitats rapidly adapt to changes in physico-chemical parameters that influence their energy status and thus their ability to proliferate and survive. One immediate survival strategy is to relocate to more metabolically optimal environments. To aid their movement through gradients (a process called taxis), many bacteria use whip like flagella organelles. Soil-dwelling Pseudomonas putida possesses a polar bundle of flagella that propel the bacterium forward in directed swimming motility. P. putida strains are generally fast growing, have a broad metabolic capacity, and are resistant to many harmful substances – qualities that make them interesting for an array of industrial and biotechnological application. This thesis identifies some of the factors that are involved in controlling the flagella driven motility of P. putida.

    In the first part of the thesis, I present evidence that P. putida displays energy-taxis towards metabolisable substrates and that the surface located Aer2 receptor (named after its similarities to the Escherichia coli Aer receptor) is responsible for detecting the changes in energy-status and oxygen-gradients that underlie this response. Aer2 is expressed simultaneously with the flagella needed for taxis responses and its expression is ensured during nutrient scares conditions through the global transcriptional regulators ppGpp and DksA.

    In addition to Aer2, P. putida possesses two more Aer-like receptors (Aer1 and Aer3) that are differentially expressed. Like Aer2, Aer1 and Aer3 co-localize to one cell pole. Although the signals to which Aer1 and Aer3 respond are unknown, analysis of Aer1 uncovered a role in motility control for a protein encoded within the same operon. This protein, called PP2258, instigated the work described in the second part of my thesis on the involvement of the second messenger c-di-GMP in regulation of P. putida motility. Genetic dissection of the catalytic activities of PP2258 revealed that it has the unusual capacity to both synthesize and degrade c-di-GMP. Coupling of the c-di-GMP signal originating from PP2258 to motility control was traced to the c-di-GMP binding properties of the protein PP4397. In the last part of the thesis, I present possible mechanisms for how these different components might interact to create a signal transduction cascade – from the surface located Aer1 receptor to PP2258 and the c-di-GMP responsive PP4397, and from there to the flagella motors – to ultimately determine flagella performance and the motility status of P. putida.

  • 789.
    Österberg, Sofia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    del Peso-Santos, Teresa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Regulation of alternative sigma factor use2011In: Annual Review of Microbiology, ISSN 0066-4227, E-ISSN 1545-3251, Vol. 65, p. 37-55Article in journal (Refereed)
    Abstract [en]

    Alternative bacterial sigma factors bind the catalytic core RNA polymerase to confer promoter selectivity on the holoenzyme. The different holoenzymes are thus programmed to recognize the distinct promoter classes in the genome to allow coordinated activation of discrete sets of genes needed for adaptive responses. To form the holoenzymes, the different sigma factors must be available to compete for their common substrate (core RNA polymerase). This review highlights (a) the roles of antisigma factors in controlling the availability of alternative sigma factors and (b) the involvement of diverse regulatory molecules that promote the use of alternative sigma factors through subversion of the domineering housekeeping σ(70). The latter include the nucleotide alarmone ppGpp and small proteins (DksA, Rsd, and Crl), which directly target the transcriptional machinery to mediate their effects.

  • 790.
    Österberg, Sofia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Skoog, Lisa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    PP4397 provides the link between PP2258 c-di-GMP signalling and altered motility in Pseudomonas putidaManuscript (preprint) (Other academic)
  • 791.
    Österberg, Sofia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Skärfstad, Eleonore
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The σ-factor FliA, ppGpp and DksA coordinate transcriptional control of the aer2 gene of Pseudomonas putida2010In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 12, no 6, p. 1439-1451Article in journal (Refereed)
    Abstract [en]

    Here the σ-factor requirement for transcription of three similar, but differentially regulated, aer genes of Pseudomonas putida KT2440 is investigated. Previous work has shown that the three Aer proteins, like chemoreceptors, colocalize to a single pole in a CheA-dependent manner. Lack of Aer2 - the most abundant of these three proteins - mediates defects in metabolism-dependent taxis and aerotaxis, while lack of Aer1 or Aer3 has no apparent phenotype. We show, using wild-type and mutant P. putida derivatives combined with P. putida reconstituted FliA- (σ28) and σ70-dependent in vitro transcription assays, that transcription of aer2 is coupled to motility through the flagella σ-factor FliA, while σ70 is responsible for transcription of aer1 and aer3. By comparing activities of the wild-type and mutant forms of the aer2 promoter, we present evidence (i) that transcription from FliA-dependent Paer2 is enhanced by changes towards the Escherichia coli consensus for FliA promoters rather than towards that of P. putida, (ii) that the nature of the AT-rich upstream region is important for both output and σ70 discrimination of this promoter, and (iii) that Paer2 output is directly stimulated by the bacterial alarmone ppGpp and its cofactor DksA.

  • 792.
    Österberg, Sofia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Åberg, Anna
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Herrera Seitz, M. Karina
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Genetic dissection of a motility-associated c-di-GMP signalling protein of Pseudomonas putida2013In: Environmental Microbiology Reports, ISSN 1758-2229, E-ISSN 1758-2229, Vol. 5, no 4, p. 556-565Article in journal (Refereed)
    Abstract [en]

    Lack of the Pseudomonas putidaPP2258 protein or its overexpression results in defective motility on solid media. The PP2258 protein is tripartite, possessing a PAS domain linked to two domains associated with turnover of c-di-GMP - a cyclic nucleotide that controls the switch between motile and sessile lifestyles. The second messenger c-di-GMP is produced by diguanylate cyclases and degraded by phosphodiesterases containing GGDEF and EAL or HD-GYP domains respectively. It is common for enzymes involved in c-di-GMP signalling to contain two domains with potentially opposing c-di-GMP turnover activities; however, usually one is degenerate and has been adopted to serve regulatory functions. Only a few proteins have previously been found to have dual enzymatic activities - being capable of both synthesizing and hydrolysing c-di-GMP. Here, using truncated and mutant derivatives of PP2258, we show that despite a lack of complete consensus in either the GGDEF or EAL motifs, the two c-di-GMP turnover domains can function independently of each other, and that the diguanylate cyclase activity is regulated by an inhibitory I-site within its GGDEF domain. Thus, motility-associated PP2258 can be added to the short list of bifunctional c-di-GMP signalling proteins.

  • 793.
    Öztokatli, Hande
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hörnberg, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Berghard, Anna
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bohm, Staffan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Retinoic acid receptor and CNGA2 channel signaling are part of a regulatory feedback loop controlling axonal convergence and survival of olfactory sensory neurons2012In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26, no 2, p. 617-627Article in journal (Refereed)
    Abstract [en]

    Little is known about the identities and functions of extracellular signaling molecules that work in concert with neuronal activity to regulate refinement and maintenance of the mouse olfactory sensory map. We show that expression of a dominant negative retinoic acid receptor (RAR) in olfactory sensory neurons (OSNs) increased the number of glomeruli that incorrectly contained OSN axons expressing different odorant receptors. This phenotype became apparent postnatally, coincided with increased cell death, and was preceded by increased Neuropilin-1 and reduced Kirrel-2 expressions. Kirrel-2-mediated cell adhesion influences odorant receptor-specific axonal convergence and is regulated by odorant receptor signaling via the olfactory cyclic nucleotide-gated (CNG) ion channel. Accordingly, we found that inhibited RAR function correlated with reduced CNG channel expression. Naris occlusion experiments and analysis of CNG channel-deficient mice further indicated that RAR-regulated CNG channel levels influenced the intrinsic neuronal activity required for cell survival in the absence of odor stimulation. Finally, we showed that CNG channel activity regulated expression of the retinoic acid-degrading enzyme Cyp26B1. Combined, these results identify a novel homeostatic feedback mechanism involving retinoic acid metabolism and CNG channel activity, which influences glomerular homogeneity and maintenance of precisely connected OSNs.

13141516 751 - 793 of 793
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