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  • 301.
    Jacobsson, Linn
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Skottheim Honn, John
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Ekström, Karin
    Rasmuson-Lestander, Åsa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Empty spiracles represses twin-of-eyeless in the Drosophila embryonic headManuscript (preprint) (Other academic)
    Abstract [en]

    The specification of the eye-antennal disc primordium in the Drosophila embryo requires the expression of two paralogous Pax6 genes: twin of eyeless (toy) and eyeless (ey). toy is considered to be the first eye specification gene expressed in the regulatory network that governs eye formation and the gene that, in turn, activates eyeless. What regulates toy expression is, however, still unclear. We show, by misexpression and mutant analysis, that the head-specific gene empty spiracles alters the expression pattern of Toy in the head region around the visual primordia.

  • 302. Jafarzadeh, Meisam
    et al.
    Soltani, Bahram Mohammad
    Ekhteraei-Tousi, Samaneh
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Behmanesh, Mehrdad
    Hsa-miR-497 as a new regulator in TGF beta signaling pathway and cardiac differentiation process2018In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 675, p. 150-156Article in journal (Refereed)
    Abstract [en]

    Cardiosphere-derived cells (CDCs) contain cardiac stem cell subpopulations and are introduced as useful source for cardiac differentiation and therapy. Furthermore, research has highlighted miRNAs important role in various biological processes and cardiogenesis. Here, we intended to investigate the effect of hsa-miR-497 (miR-497) on TGF beta signaling pathway genes expression during the process of CDCs differentiation to cardiomyocytes. CDCs were successfully differentiated to the cardiac-like cells. In this study, we found that after cardiac differentiation induction, miR-497 expression was significantly decreased. Computational miRNA target prediction analyses revealed that TGF beta signaling pathway is a possible target of miR-497. Therefore, miR-497 was overexpressed in CDCs before the induction of differentiation. TGF beta 1, TGF beta R1, TGF beta R2, and SMAD3 genes expression level was decreased after miR-497 overexpression. Also, immunocytochemistry and cell morphology analysis indicated that miR-497 overexpression affecting cardiac differentiation process. Finally, direct interaction of miR-497 with 3'-UTR sequence of TGF beta R1 was supported through dual luciferase assay, consistent with miR-497 reported negative effect on SMAD3 expression. Accordingly, here a model of miR-497 involvement in regulation of TGF beta signaling pathway is introduced in which, side branches of TGF beta signaling pathway downregulate miR-497 to ensure upregulation of TGF beta R1 and TGF beta R2 and finally stronger TGF beta signaling.

  • 303.
    Jahns, Anika C.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Lundskog, Bertil
    Umeå University, Faculty of Medicine.
    Berg, Johanna
    Umeå University, Faculty of Medicine.
    Jonsson, Rebecca
    Umeå University, Faculty of Medicine.
    McDowell, Andrew
    Patrick, Sheila
    Golovleva, Irina
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics.
    Palmer, Ruth H.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Alexeyev, Oleg A.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Microbiology of folliculitis: a histological study of 39 cases2014In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 122, no 1, p. 25-32Article in journal (Refereed)
    Abstract [en]

    Folliculitis is a common inflammatory skin syndrome. Several microbial organisms have been put forward as causative agents, but few studies visualized microbes directly in inflamed hair follicles. This retrospective study investigated bacterial and fungal colonization of inflamed hair follicles in patients with clinically diagnosed non-infectious folliculitis. Skin biopsies from 39 folliculitis patients and 27 controls were screened by fluorescence in situ hybridization (FISH) using broad-range bacterial and fungal probes and by immunofluorescence microscopy using a monoclonal antibody towards Gram-positive bacteria. Specific monoclonal and polyclonal antibodies towards Staphylococcus spp. and Propionibacterium acnes were applied for further species identification. Inflamed follicles were associated with bacterial colonization in 10 samples (26%) and fungal colonization in three samples (8%). Staphylococcus spp. were observed in inflamed follicles in seven samples (18%). Two samples were positive for P. acnes, which were identified as either type II or type IB/type III. Both Staphylococcus spp. and P. acnes were seen in macrocolonies/biofilm structures. In conclusion, one-third of patients with clinically diagnosed, non-infectious folliculitis exhibited microbial colonization with predominance of Staphylococcus spp.

  • 304.
    Jahns, Anika C
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Oprica, Cristina
    Karolinska Institute, Södersjukhuset, Stockholm.
    Vassilaki, Ismini
    Karolinska University Hospital, Stockholm.
    Golovleva, Irina
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics.
    Palmer, Ruth H
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Alexeyev, Oleg A.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Simultaneous visualization of Propionibacterium acnes and Propionibacterium granulosum with immunofluorescence and fluorescence in situ hybridization2013In: Anaerobe, ISSN 1075-9964, E-ISSN 1095-8274, Vol. 23, p. 48-54Article in journal (Refereed)
    Abstract [en]

    Propionibacterium acnes (P. acnes) and Propionibacterium granulosum (P. granulosum) are common skin colonizers that are implicated as possible contributing factors in acne vulgaris development. We have established direct visualization tools for the simultaneous detection of these closely related species with immunofluorescence assay and fluorescence in situ hybridization (FISH). As proof of principle, we were able to distinguish P. acnes and P. granulosum bacteria in multi-species populations in vitro as well as in a mock skin infection model upon labelling with 16S rRNA probes in combinatorial FISH as well as with antibodies. Furthermore, we report the co-localization of P. acnes and P. granulosum in the stratum corneum and hair follicles from patients with acne vulgaris as well as in healthy individuals. Further studies on the spatial distribution of these bacteria in skin structures in various skin disorders are needed.

  • 305.
    Jenny, Lundkvist
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    GC-TOFMS Based Metabolomics for Health Biomarker Profiling in Dried Blood Spots2015Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
  • 306. Jishage, Miki
    et al.
    Kvint, Kristian
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nyström, Thomas
    Regulation of sigma factor competition by the alarmone ppGpp2002In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 16, no 10, p. 1260-1270Article in journal (Refereed)
    Abstract [en]

    Many regulons controlled by alternative ç factors, including çs and ç32, are poorly induced in cells lacking the alarmone ppGpp. We show that ppGpp is not absolutely required for the activity of çs-dependent promoters because underproduction of ç70, specific mutations in rpoD (rpoD40 and rpoD35), or overproduction of Rsd (anti-ç70) restored expression from çs-dependent promoters in vivo in the absence of ppGpp accumulation. An in vitro transcription/competition assay with reconstituted RNA polymerase showed that addition of ppGpp reduces the ability of wild-type ç70 to compete with ç32 for core binding and the mutant ç70 proteins, encoded by rpoD40 and rpoD35, compete less efficiently than wild-type ç70. Similarly, an in vivo competition assay showed that the ability of both sigma(32) and sigma(S) to compete with sigma(70) is diminished in cells lacking ppGpp. Consistently, the fraction of çs and ç32 bound to core was drastically reduced in ppGpp-deficient cells. Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of sigma factors in accordance with cellular demands during physiological stress.

  • 307.
    Johansson, Anna-Mia
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Chromosome-wide gene regulatory mechanisms in Drosophila melanogaster2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In Drosophila there are two different chromosome-wide targeting systems, the dosage compensation system that equalizes the transcriptional output from X-linked genes between males and females, and the regulation of the 4th chromosome mediated by the POF protein.

     

    The best studied of these two mechanisms is the dosage compensation system. To attain dosage compensation in Drosophila at least five different proteins, encoded by the male-specific lethal genes msl1, msl2, msl3, mle and mof, are required. These proteins together with two non-coding RNAs (roX1 and roX2) form a dosage compensation complex (MSL complex), which binds exclusively to the X chromosome in Drosophila males and up-regulates the transcription approximately two times.

     

    In this thesis I show that roX1 and roX2 are most likely the only non-coding RNAs within the MSL complex. As expected, the roX transcripts were enriched within the MSL complex. Interestingly, one additional transcript was identified within the MSL complex. This transcript did not associate with the X chromosome and is therefore not believed to be involved in up-regulation of the X-linked genes. This transcript encodes for the rate limiting component in the MSL complex, the MSL2 protein. A model is proposed in which free, partial or complete, MSL complex feed-back regulates the amount of msl2 transcript, and thereby limits the MSL complex production.

     

    The second chromosome-wide regulatory system in flies acts on an autosome, the heterochromatic 4th chromosome. This regulation is a balancing mechanism between at least two different proteins, the chromosome 4 specific protein painting of fourth (POF) and heterochromatin protein 1 (HP1). POF binds to nascent RNAs transcribed from the 4th chromosome and HP1 target the same set of genes at the chromatin level. POF stimulates the transcribed genes, while HP1 represses them; together they create the most optimal condition for these genes. This type of balancing mechanism may be a more general way to fine-tune transcription at a chromosome-wide level and raises the question about autosomal gene regulation as a general mechanism.

  • 308.
    Johansson, Anna-Mia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Allgardsson, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Stenberg, Per
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    msl2 mRNA is bound by free nuclear MSL complex in Drosophila melanogaster2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 15, p. 6428-6439Article in journal (Refereed)
    Abstract [en]

    In Drosophila, the global increase in transcription from the male X chromosome to compensate for its monosomy is mediated by the male-specific lethal (MSL) complex consisting of five proteins and two non-coding RNAs, roX1 and roX2. After an initial sequence-dependent recognition by the MSL complex of 150-300 high affinity sites, the spread to the majority of the X-linked genes depends on local MSL-complex concentration and active transcription. We have explored whether any additional RNA species are associated with the MSL complex. No additional roX RNA species were found, but a strong association was found between a spliced and poly-adenylated msl2 RNA and the MSL complex. Based on our results, we propose a model in which a non-chromatin-associated partial or complete MSL-complex titrates newly transcribed msl2 mRNA and thus regulates the amount of available MSL complex by feedback. This represents a novel mechanism in chromatin structure regulation.

  • 309.
    Johansson, Anna-Mia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Genome-wide mapping of Painting of fourth on Drosophila melanogaster salivary gland polytene chromosomes2014In: Genomics Data, ISSN 1025-6059, E-ISSN 2213-5960, Vol. 2, p. 63-65Article in journal (Refereed)
    Abstract [en]

    The protein Painting of fourth (POF) in Drosophila melanogaster specifically targets and stimulates expression output from the heterochromatic 4th chromosome, thereby representing an autosome specific protein [1,2]. Despite the high specificity for chromosome 4 genes, POF is occasionally observed binding to the cytological region 2L:31 in males and females [3] and two loci on the X-chromosome, PoX1 and PoX2 only in females [4]. Here we provide a detailed description of the experimental design and analysis of the tiling array data presented by Lundberg and colleagues in G3: Genes, Genomes, Genetics 2013 [4], where the female specific POF binding to PoX1 and PoX2 loci on the X chromosome was reported. We show the genome-wide high resolution binding profile of the POF protein where these different POF binding sites are detected. The complete data set is available at http://www.ncbi.nlm.nih.gov/geo/ (accession: GSE45402).

  • 310.
    Johansson, Anna-Mia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Stenberg, Per
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Allgardsson, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    POF Regulates the Expression of Genes on the Fourth Chromosome in Drosophila melanogaster by Binding to Nascent RNA2012In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 32, no 11, p. 2121-2134Article in journal (Other academic)
    Abstract [en]

    In Drosophila, two chromosome-wide compensatory systems have been characterized: the dosage compensation system that acts on the male X chromosome and the chromosome-specific regulation of genes located on the heterochromatic fourth chromosome. Dosage compensation in Drosophila is accomplished by hypertranscription of the single male X chromosome mediated by the male-specific lethal (MSL) complex. The mechanism of this compensation is suggested to involve enhanced transcriptional elongation mediated by the MSL complex, while the mechanism of compensation mediated by the painting of fourth (POF) protein on the fourth chromosome has remained elusive. Here, we show that POF binds to nascent RNA, and this binding is associated with increased transcription output from chromosome 4. We also show that genes located in heterochromatic regions spend less time in transition from the site of transcription to the nuclear envelope. These results provide useful insights into the means by which genes in heterochromatic regions can overcome the repressive influence of their hostile environment.

  • 311. Johansson, Björn P
    et al.
    Levander, Fredrik
    von Pawel-Rammingen, Ulrich
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Berggård, Tord
    Björck, Lars
    James, Peter
    The protein expression of Streptococcus pyogenes is significantly influenced by human plasma2005In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 4, no 6, p. 2302-2311Article in journal (Refereed)
    Abstract [en]

    During the courser of infection, the common human pathogen Streptococcus pyogenes encounters plasma. We show that plasma causes S. pyogenes to rapidly remodel its cellular metabolism and virulence pathways. We also identified a variant of the major virulence factor, M1 protein, lacking 13 amino acids at the NH2-terminus in bacteria grown with plasma. The pronounced effect of plasma on protein expression, suggests this is an important adaptive mechanism with implications for S. pyogenes pathogenicity.

  • 312.
    Johansson, Linda U M
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Solera, Dafne
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bernardo, Lisandro M D
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Moscoso, Joana A
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    sigma54-RNA polymerase controls sigma70-dependent transcription from a non-overlapping divergent promoter.2008In: Molecular microbiology, ISSN 1365-2958, Vol. 70, no 3, p. 709-23Article in journal (Refereed)
    Abstract [en]

    Divergent transcription of a regulatory gene and a cognate promoter under its control is a common theme in bacterial regulatory circuits. This genetic organization is found for the dmpR gene that encodes the substrate-responsive specific regulator of the sigma(54)-dependent Po promoter, which controls (methyl)phenol catabolism. Here we identify the Pr promoter of dmpR as a sigma(70)-dependent promoter that is regulated by a novel mechanism in which sigma(54)-RNA polymerase occupancy of the non-overlapping sigma(54)-Po promoter stimulates sigma(70)-Pr output. In addition, we show that DmpR stimulates its own production through Po activity both in vivo and in vitro. Hence, the demonstrated regulatory circuit reveals a novel role for sigma(54)-RNA polymerase, namely regulation of a sigma(70)-dependent promoter, and a new mechanism that places a single promoter under dual control of two alternative forms of RNA polymerase. We present a model in which guanosine tetra-phosphate plays a major role in the interplay between sigma(54)- and sigma(70)-dependent transcription to ensure metabolic integration to couple sigma(70)-Pr output to both low-energy conditions and the presence of substrate.

  • 313.
    Johansson, Marcus J O
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Determining if an mRNA is a Substrate of Nonsense-Mediated mRNA Decay in Saccharomyces cerevisiae2017In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 1507, p. 169-177Article in journal (Refereed)
    Abstract [en]

    Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic quality control mechanism which triggers decay of mRNAs harboring premature translation termination codons. In this chapter, I describe methods for monitoring the influence of NMD on mRNA abundance and decay rates in Saccharomyces cerevisiae. The descriptions include detailed methods for growing yeast cells, total RNA isolation, and Northern blotting. Although the chapter focuses on NMD, the methods can be easily adapted to assess the effect of other mRNA decay pathways.

  • 314. Johansson, Marcus J O
    et al.
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The Saccharomyces cerevisiae TAN1 gene is required for N4-acetylcytidine formation in tRNA.2004In: RNA, ISSN 1355-8382, Vol. 10, no 4, p. 712-9Article in journal (Refereed)
    Abstract [en]

    The biogenesis of transfer RNA is a process that requires many different factors. In this study, we describe a genetic screen aimed to identify gene products participating in this process. By screening for mutations lethal in combination with a sup61-T47:2C allele, coding for a mutant form of, the nonessential TAN1 gene was identified. We show that the TAN1 gene product is required for formation of the modified nucleoside N(4)-acetylcytidine (ac(4)C) in tRNA. In Saccharomyces cerevisiae, ac(4)C is present at position 12 in tRNAs specific for leucine and serine as well as in 18S ribosomal RNA. Analysis of RNA isolated from a tan1-null mutant revealed that ac(4)C was absent in tRNA, but not rRNA. Although no tRNA acetyltransferase activity by a GST-Tan1 fusion protein was detected, a gel-shift assay revealed that Tan1p binds tRNA, suggesting a direct role in synthesis of ac(4)C(12). The absence of the TAN1 gene in the sup61-T47:2C mutant caused a decreased level of mature, indicating that ac(4)C(12) and/or Tan1p is important for tRNA stability.

  • 315.
    Johansson, Marcus J O
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Dual function of the tRNA(m(5)U54)methyltransferase in tRNA maturation2002In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 8, no 3, p. 324-335Article in journal (Refereed)
    Abstract [en]

    A 5-methyluridine (m(5)U) residue at position 54 is a conserved feature of bacterial and eukaryotic tRNAs. The methylation of U54 is catalyzed by the tRNA(m5U54)methyltransferase, which in Saccharomyces cerevisiae is encoded by the nonessential TRM2 gene. In this study, we identified four different strains with mutant forms of tRNA(Ser)CGA. The absence of the TRM2 gene in these strains decreased the stability of tRNA(Ser)CGA and induced lethality. Two alleles of TRM2 encoding catalytically inactive tRNA(m5U54)methyltransferases were able to stabilize tRNA(Ser)CGA in one of the mutants, revealing a role for the Trm2 protein per se in tRNA maturation. Other tRNA modification enzymes interacting with tRNA(Ser)CGA in the maturation process, such as Pus4p, Trm1 p, and Trm3p were essential or important for growth of the tRNA(Ser)CGA mutants. Moreover, Lhp1p, a protein binding RNA polymerase III transcripts, was required to stabilize the mutant tRNAs. Based on our results, we suggest that tRNA modification enzymes might have a role in tRNA maturation not necessarily linked to their known catalytic activity.

  • 316.
    Johansson, Marcus J O
    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).
    Transfer RNA modifications and modifying enzymes in Saccharomyces cerevisiae.2005In: Fine-Tuning of RNA Functions by Modification and Editing. / [ed] Henri Grosjean, Springer Berlin/Heidelberg, 2005, p. 87-120Chapter in book (Refereed)
    Abstract [en]

    Transfer RNAs are adaptor molecules, which decode mRNA into protein and, thereby, play a central role in gene expression. During the maturation of a primary tRNA transcript, specific subsets of the four normal nucleosides adenosine, cytidine, guanosine, and uridine are modified. The formation of a modified nucleoside can require more than one gene product and may involve several enzymatic steps. In the last few years, the identification of gene products required for formation of modified nucleosides in tRNA has dramatically increased. In this review, proteins involved in modification of cytoplasmic tRNAs in Saccharomyces cerevisiae are described, emphasizing phenotypic characteristics of modification deficient strains and genetic approaches used to determine the in vivo role of modified nucleosides/modifying enzymes.

  • 317.
    Johansson, Marcus J O
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Esberg, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    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).
    Eukaryotic wobble uridine modifications promote a functionally redundant decoding system.2008In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 28, no 10, p. 3301-3312Article in journal (Refereed)
    Abstract [en]

    The translational decoding properties of tRNAs are modulated by naturally occurring modifications of their nucleosides. Uridines located at the wobble position (nucleoside 34 [U34]) in eukaryotic cytoplasmic tRNAs often harbor a 5-methoxycarbonylmethyl (mcm(5)) or a 5-carbamoylmethyl (ncm(5)) side chain and sometimes an additional 2-thio (s2) or 2'-O-methyl group. Although a variety of models explaining the role of these modifications have been put forth, their in vivo functions have not been defined. In this study, we utilized recently characterized modification-deficient Saccharomyces cerevisiae cells to test the wobble rules in vivo. We show that mcm5 and ncm5 side chains promote decoding of G-ending codons and that concurrent mcm5 and s2 groups improve reading of both A- and G-ending codons. Moreover, the observation that the mcm5U34- and some ncm5U34-containing tRNAs efficiently read G-ending codons challenges the notion that eukaryotes do not use U-G wobbling.

  • 318.
    Johansson, Marcus J O
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Xu, Fu
    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).
    Elongator-a tRNA modifying complex that promotes efficient translational decoding2018In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1861, no 4, p. 401-408Article in journal (Refereed)
    Abstract [en]

    Naturally occurring modifications of the nucleosides in the anticodon region of tRNAs influence their translational decoding properties. Uridines present at the wobble position in eukaryotic cytoplasmic tRNAs often contain a 5-carbamoylmethyl (ncm5) or 5-methoxycarbonylmethyl (mcm5) side-chain and sometimes also a 2-thio or 2'-O-methyl group. The first step in the formation of the ncm5 and mcm5 side-chains requires the conserved six-subunit Elongator complex. Although Elongator has been implicated in several different cellular processes, accumulating evidence suggests that its primary, and possibly only, cellular function is to promote modification of tRNAs. In this review, we discuss the biosynthesis and function of modified wobble uridines in eukaryotic cytoplasmic tRNAs, focusing on the in vivo role of Elongator-dependent modifications in Saccharomyces cerevisiae. 

  • 319.
    Johansson, Marcus JO
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Jacobson, Allan
    University Massachusetts, School of Medicine, Dept Mol Genet & Microbiol, Worcester, MA .
    Nonsense-mediated mRNA decay maintains translational fidelity by limiting magnesium uptake2010In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 24, no 14, p. 1491-1495Article in journal (Refereed)
    Abstract [en]

    Inactivation of the yeast nonsense-mediated mRNA decay (NMD) pathway stabilizes nonsense mRNAs and promotes readthrough of premature translation termination codons. Although the latter phenotype is thought to reflect a direct role of NMD factors in translation termination, its mechanism is unknown. Here we show that the reduced termination efficiency of NMD-deficient cells is attributable to increased expression of the magnesium transporter Alr1p and the resulting effects of elevated Mg2+ levels on termination fidelity. Alr1p levels increase because an upstream ORF in ALR1 mRNA targets the transcript for NMD. Our results demonstrate that NMD, at least in yeast, controls Mg2+ homeostasis and, consequently, translational fidelity.

  • 320. Joint, Ian
    et al.
    Tait, Karen
    Callow, Maureen E.
    Callow, James A.
    Milton, Debra L.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Williams, Paul
    Cámara, Miguel
    Cell-to-cell communication across the prokaryote-eukaryote boundary2002In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 298, no 5596, p. 1207-1207Article in journal (Refereed)
  • 321.
    Jonasdotter, Sandra
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Construction of a Mass Cytometry Panel and Characterization of a Control Cell Line for AML samples with FLT3-ITD2016Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
  • 322.
    Jäger, Gunilla
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Chen, Peng
    Björk, Glenn R.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Transfer RNA Bound to MnmH Protein Is Enriched with Geranylated tRNA - A Possible Intermediate in Its Selenation?2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 4, article id e0153488Article in journal (Refereed)
    Abstract [en]

    The wobble nucleoside 5-methylaminomethyl-2-thio-uridine (mnm(5)s(2)U) is present in bacterial tRNAs specific for Lys and Glu and 5-carboxymethylaminomethyl-2-thio-uridine (cmnm(5)s(2)U) in tRNA specific for Gln. The sulfur of (c) mnm(5)s(2)U may be exchanged by selenium (Se)-a reaction catalyzed by the selenophosphate-dependent tRNA 2-selenouridine synthase encoded by the mnmH (ybbB, selU, sufY) gene. The MnmH protein has a rhodanese domain containing one catalytic Cys (C97) and a P-loop domain containing a Walker A motif, which is a potential nucleotide binding site. We have earlier isolated a mutant of Salmonella enterica, serovar Typhimurium with an alteration in the rhodanese domain of the MnmH protein (G67E) mediating the formation of modified nucleosides having a geranyl (ge)-group (C10H17-fragment) attached to the s(2) group of mnm(5)s(2)U and of cmnm(5)s(2)U in tRNA. To further characterize the structural requirements to increase the geranylation activity, we here report the analysis of 39 independently isolated mutants catalyzing the formation of mnm(5)ges(2)U. All these mutants have amino acid substitutions in the rhodanese domain demonstrating that this domain is pivotal to increase the geranylation activity. The wild type form of MnmH(+) also possesses geranyltransferase activity in vitro although only a small amount of the geranyl derivatives of (c) mnm(5)s(2)U is detected in vivo. The selenation activity in vivo has an absolute requirement for the catalytic Cys97 in the rhodanese domain whereas the geranylation activity does not. Clearly, MnmH has two distinct enzymatic activities for which the rhodanese domain is pivotal. An intact Walker motif in the P-loop domain is required for the geranylation activity implying that it is the binding site for geranylpyrophosphate (GePP), which is the donor molecule in vitro in the geranyltransfer reaction. Purified MnmH from wild type and from the MnmH(G67E) mutant have bound tRNA, which is enriched with geranylated tRNA. This in conjunction with earlier published data, suggests that this bound geranylated tRNA may be an intermediate in the selenation of the tRNA.

  • 323.
    Jäger, Gunilla
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Leipuviene, Ramune
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Pollard, Michael
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Qian, Qiang
    Björk, Glenn
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The conserved Cys-X1-X2-Cys motif present in the TtcA protein is required for the thiolation of cytidine in position 32 of tRNA from Salmonella enterica serovar Typhimurium.2004In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 3, p. 750-757Article in journal (Refereed)
    Abstract [en]

    The modified nucleoside 2-thiocytidine (s(2)C) has so far been found in tRNA from organisms belonging to the phylogenetic domains Archaea and BACTERIA: In the bacteria Escherichia coli and Salmonella enterica serovar Typhimurium, s(2)C is present in position 32 of only four tRNA species-, and. An in-frame deletion of an S. enterica gene (designated ttcA, for "two-thio-cytidine") was constructed, and such a mutant has no detectable s(2)C in its tRNA. The TtcA protein family is characterized by the existence of both a PP-loop and a Cys-X(1)-X(2)-Cys motif in the central region of the protein but can be divided into two distinct groups based on the presence and location of additional Cys-X(1)-X(2)-Cys motifs in terminal regions of the sequence. Mutant analysis showed that both cysteines in this central conserved Cys-X(1)-X(2)-Cys motif are required for the formation of s(2)C. The DeltattcA1 mutant grows at the same rate as the congenic wild-type strain, and no growth disadvantage caused by the lack of s(2)C was observed in a mixed-population experiment. Lack of s(2)C32 did not reduce the selection rate at the ribosomal aminoacyl-tRNA site (A-site) for at any of its cognate CGN codons, whereas A-site selection at AGG by was dependent on the presence of s(2)C32. The presence of s(2)C32 in peptidyl- or in peptidyl- interfered with decoding in the A-site. The presence of s(2)C32 in decreased the rate of translation of the CGA codon but not that of the CGU codon.

  • 324.
    Jäger, Gunilla
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nilsson, Kristina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Björk, Glenn R.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The Phenotype of Many Independently Isolated+1 Frameshift Suppressor Mutants Supports a Pivotal Role of the P-Site in Reading Frame Maintenance2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 4, p. e60246-Article in journal (Refereed)
    Abstract [en]

    The main features of translation are similar in all organisms on this planet and one important feature of it is the way the ribosome maintain the reading frame. We have earlier characterized several bacterial mutants defective in tRNA maturation and found that some of them correct a +1 frameshift mutation; i.e. such mutants possess an error in reading frame maintenance. Based on the analysis of the frameshifting phenotype of such mutants we proposed a pivotal role of the ribosomal grip of the peptidyl-tRNA to maintain the correct reading frame. To test the model in an unbiased way we first isolated many (467) independent mutants able to correct a +1 frameshift mutation and thereafter tested whether or not their frameshifting phenotypes were consistent with the model. These 467+1 frameshift suppressor mutants had alterations in 16 different loci of which 15 induced a defective tRNA by hypo- or hypermodifications or altering its primary sequence. All these alterations of tRNAs induce a frameshift error in the P-site to correct a +1 frameshift mutation consistent with the proposed model. Modifications next to and 39 of the anticodon (position 37), like 1-methylguanosine, are important for proper reading frame maintenance due to their interactions with components of the ribosomal P-site. Interestingly, two mutants had a defect in a locus (rpsI), which encodes ribosomal protein S9. The C-terminal of this protein contacts position 32-34 of the peptidyl-tRNA and is thus part of the P-site environment. The two rpsI mutants had a C-terminal truncated ribosomal protein S9 that destroys its interaction with the peptidyl-tRNA resulting in +1 shift in the reading frame. The isolation and characterization of the S9 mutants gave strong support of our model that the ribosomal grip of the peptidylt-RNA is pivotal for the reading frame maintenance.

  • 325.
    Kahn, Tatyana G.
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Dorafshan, Eshagh
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Schultheis, Dorothea
    Zare, Aman
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Stenberg, Per
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Division of CBRN Defense and Security, Swedish Defense Research Agency, FOI, Umea, 906 21, Sweden.
    Reim, Ingolf
    Pirrotta, Vincenzo
    Schwartz, Yuri B.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Interdependence of PRC1 and PRC2 for recruitment to Polycomb Response Elements2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 21, p. 10132-10149Article in journal (Refereed)
    Abstract [en]

    Polycomb Group (PcG) proteins are epigenetic repressors essential for control of development and cell differentiation. They form multiple complexes of which PRC1 and PRC2 are evolutionary conserved and obligatory for repression. The targeting of PRC1 and PRC2 is poorly understood and was proposed to be hierarchical and involve tri-methylation of histone H3 (H3K27me3) and/or monoubiquitylation of histone H2A (H2AK118ub). Here, we present a strict test of this hypothesis using the Drosophila model. We discover that neither H3K27me3 nor H2AK118ub is required for targeting PRC complexes to Polycomb Response Elements (PREs). We find that PRC1 can bind PREs in the absence of PRC2 but at many PREs PRC2 requires PRC1 to be targeted. We show that one role of H3K27me3 is to allow PcG complexes anchored at PREs to interact with surrounding chromatin. In contrast, the bulk of H2AK118ub is unrelated to PcG repression. These findings radically change our view of how PcG repression is targeted and suggest that PRC1 and PRC2 can communicate independently of histone modifications.

  • 326. Kaksonen, Anna H.
    et al.
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Karnachuk, Olia
    Tuovinen, Olli H.
    Puhakka, Jaakko A.
    Biological iron oxidation and sulfate reduction in the treatment of acid mine drainage at low temperatures2008In: Psychrophiles: from biodiversity to biotechnology / [ed] Rosa Margesin, Franz Schinner, Jean-Claude Marx, Charles Gerday, Berlin, Heidelberg: Springer-Verlag , 2008, p. 429-454Chapter in book (Other academic)
  • 327.
    Karlsborn, Tony
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Physiological consequences of Elongator complex inactivation in Eukaryotes2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Mutations found in genes encoding human Elongator complex subunits have been linked to neurodevelopmental disorders such as familial dysautonomia (FD), rolandic epilepsy and amyotrophic lateral sclerosis. In addition, loss-of-function mutations in genes encoding Elongator complex subunits cause defects in neurodevelopment and reduced neuronal function in both mice and nematodes. The Elongator complex is a conserved protein complex comprising six subunits (Elp1p-Elp6p) found in eukaryotes. The primary function of this complex in yeast is formation of the 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) side chains found on wobble uridines (U34) in tRNAs. The aim of this thesis is to investigate the physiological consequences of Elongator complex inactivation in humans and in the yeast Saccharomyces cerevisiae.

    Inactivation of the Elongator complex causes widespread defects in a multitude of different cellular processes in S. cerevisiae. Thus, we investigated metabolic alterations resulting from Elongator complex inactivation. We show that deletion of the S. cerevisiae ELP3 gene leads to widespread metabolic alterations. Moreover, all global metabolic alterations observed in the elp3Δ strain are not restored in the presence of elevated levels of hypomodified tRNAs that normally have the modified nucleoside mcm5s2U. Collectively, we show that modified wobble nucleosides in tRNAs are required for metabolic homeostasis.

    Elongator mutants display sensitivity to DNA damage agents, but the underlying mechanism explaining this sensitivity remains elusive. We demonstrate that deletion of the S. cerevisiae ELP3 gene results in post-transcriptional reduction of Ixr1p levels. Further, we show that the reduced Ixr1p levels prevent adequate Rnr1p levels upon treatment with DNA damage agents. These findings suggest that reduced Ixr1p levels could in part explain why Elongator mutants are sensitive to DNA damage agents.

    Depletion of Elongator complex subunits results in loss of wobble uridine modifications in plants, nematodes, mice and yeast. Therefore, we investigated whether patients with the neurodegenerative disease familial dysautonomia (FD), who have lower levels of the ELP1 protein, display reduced amounts of modified wobble uridine nucleosides. We show that tRNA isolated from brain tissue and fibroblast cell lines derived from FD patients have 64–71% of the mcm5s2U nucleoside levels observed in total tRNA from non-FD brain tissue and non-FD fibroblasts. Overall, these results suggest that the cause for the neurodegenerative nature of FD could be translation impairment caused by reduced levels of modified wobble uridine nucleosides in tRNAs. Thus, our results give new insight on the importance of modified wobble uridine nucleosides for neurodevelopment.

  • 328.
    Karlsborn, Tony
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Mahmud, A K M Firoj
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Tükenmez, Hasan
    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).
    Loss of ncm5 and mcm5 wobble uridine side chains results in an altered metabolic profile2016In: Metabolomics, ISSN 1573-3882, E-ISSN 1573-3890, Vol. 12, no 12, article id 177Article in journal (Refereed)
    Abstract [en]

    Introduction: The Elongator complex, comprising six subunits (Elp1p-Elp6p), is required for formation of 5-carbamoylmethyl (ncm(5)) and 5-methoxycarbonylmethyl (mcm(5)) side chains on wobble uridines in 11 out of 42 tRNA species in Saccharomyces cerevisiae. Loss of these side chains reduces the efficiency of tRNA decoding during translation, resulting in pleiotropic phenotypes. Overexpression of hypomodified tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu), which in wild-type strains are modified with mcm(5)s(2)U, partially suppress phenotypes of an elp3 Delta strain. Objectives: To identify metabolic alterations in an elp3 Delta strain and elucidate whether these metabolic alterations are suppressed by overexpression of hypomodified tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu). Method: Metabolic profiles were obtained using untargeted GC-TOF-MS of a temperature-sensitive elp3 Delta strain carrying either an empty low-copy vector, an empty high-copy vector, a low-copy vector harboring the wild-type ELP3 gene, or a high-copy vector overexpressing tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu). The temperature sensitive elp3 Delta strain derivatives were cultivated at permissive (30 degrees C) or semi-permissive (34 degrees C) growth conditions. Results: Culturing an elp3 Delta strain at 30 or 34 degrees C resulted in altered metabolism of 36 and 46 %, respectively, of all metabolites detected when compared to an elp3D strain carrying the wild-type ELP3 gene. Overexpression of hypomodified tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu) suppressed a subset of the metabolic alterations observed in the elp3 Delta strain. Conclusion: Our results suggest that the presence of ncm(5)- and mcm(5)-side chains on wobble uridines in tRNA are important for metabolic homeostasis.

  • 329.
    Karlsborn, Tony
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Tukenmez, Hasan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Mahmud, A. K. M. Firoj
    Xu, Fu
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Xu, Hao
    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).
    Elongator, a conserved complex required for wobble uridine modifications in Eukaryotes2014In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 11, no 12, p. 1519-1528Article in journal (Refereed)
    Abstract [en]

    Elongator is a 6 subunit protein complex highly conserved in eukaryotes. The role of this complex has been controversial as the pleiotropic phenotypes of Elongator mutants have implicated the complex in several cellular processes. However, in yeast there is convincing evidence that the primary and probably only role of this complex is in formation of the 5-methoxycarbonylmethyl (mcm(5)) and 5-carbamoylmethyl (ncm(5)) side chains on uridines at wobble position in tRNA. In this review we summarize the cellular processes that have been linked to the Elongator complex and discuss its role in tRNA modification and regulation of translation. We also describe additional gene products essential for formation of ncm(5) and mcm(5) side chains at U-34 and their influence on Elongator activity.

  • 330. Karlsson, Roger
    et al.
    Aspenström, Pontus
    Byström, Anders S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    A chicken beta-actin gene can complement a disruption of the Saccharomyces cerevisiae ACT1 gene1991In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 11, no 1, p. 213-217Article in journal (Refereed)
    Abstract [en]

    Recently it was demonstrated that beta-actin can be produced in Saccharomyces cerevisiae by using the expression plasmid pY beta actin (R. Karlsson, Gene 68:249-258, 1988), and several site-specific mutants are now being produced in a protein engineering study. To establish a system with which recombinant actin mutants can be tested in vivo and thus enable a correlation to be made with functional effects observed in vitro, a yeast strain lacking endogenous yeast actin and expressing exclusively beta-actin was constructed. This strain is viable but has an altered morphology and a slow-growth phenotype and is temperature sensitive to the point of lethality at 37 degrees C.

  • 331. Karnachuk, Olia V
    et al.
    Pimenov, Nikolay V
    Yusupov, Sandjar K
    Frank, Yulia A
    Kaksonen, Anna H
    Puhakka, Jaakko A
    Ivanov, Mikhail V
    Lindström, E Börje
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Tuovinen, Olli H
    Sulfate reduction potential in sediments in the Norilsk mining area, northern Siberia2005In: Geomicrobiology Journal, ISSN 0149-0451, E-ISSN 1521-0529, Vol. 22, no 1-2, p. 11-25Article in journal (Refereed)
    Abstract [en]

    The purpose of this study was to characterize the distribution and activity of sulfate-reducing bacteria in tailings and sediments impacted by effluents from mining and smelting operations in the Norilsk area in northern Siberia. The Norilsk mining complex involves three smelter operations, a hydrometallurgical plant, and extensive tailings areas located in the permafrost zone. Sulfate reduction rates measured with a (35)SO(4)(2-) tracer technique under various in-situ conditions ranged from 0.05 to 30 nmol S cm(-3) day(-1). Acetate and glucose addition greatly stimulated sulfate reduction, whereas lactate had less effect. The most pronounced stimulation of sulfate reduction (6.5-fold) was observed with phosphate amendment. Most-probable-number (MPN) counts of sulfate-reducing bacteria in media with glucose, ethanol, lactate, and acetate as electron donors were generally highest at around 10(7) cells ml(-1). The actual MPN counts varied with the sample, electron donor, and incubation conditions (pH 7.2 vs. pH 3.5; 28 degrees C vs. 4 degrees C). Enrichment cultures of sulfate-reducing bacteria were established from a sample that showed the highest rate of sulfate reduction. After multiple serial transfers, the dominant sulfate-reducers were identified by fluorescence in situ hybridization using genus and group-specific 16S rRNA-targeted oligonucleotide probes. Desulfobulbus spp. prevailed in ethanol and lactate enrichments and the Desulfosarcina-Desulfococcus group dominated in acetate and benzoate enrichments. Psychrophilic Desulfotalea-Desulfofustis and moderately psychrophilic Desulforhopalus spp. were identified in enrichments incubated at 4 degrees C, but they were also found in mesophilic enrichments.

  • 332.
    Kasari, Villu
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Margus, Tonu
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Atkinson, Gemma C.
    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).
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). 3 University of Tartu, Institute of Technology, Tartu, Estonia.
    Ribosome profiling analysis of eEF3-depleted Saccharomyces cerevisiae2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 3037Article in journal (Refereed)
    Abstract [en]

    In addition to the standard set of translation factors common in eukaryotic organisms, protein synthesis in the yeast Saccharomyces cerevisiae requires an ABCF ATPase factor eEF3, eukaryotic Elongation Factor 3. eEF3 is an E-site binder that was originally identified as an essential factor involved in the elongation stage of protein synthesis. Recent biochemical experiments suggest an additional function of eEF3 in ribosome recycling. We have characterised the global effects of eEF3 depletion on translation using ribosome profiling. Depletion of eEF3 results in decreased ribosome density at the stop codon, indicating that ribosome recycling does not become rate limiting when eEF3 levels are low. Consistent with a defect in translation elongation, eEF3 depletion causes a moderate redistribution of ribosomes towards the 5' part of the open reading frames. We observed no E-site codon-or amino acid-specific ribosome stalling upon eEF3 depletion, supporting its role as a general elongation factor. Surprisingly, depletion of eEF3 leads to a relative decrease in P-site proline stalling, which we hypothesise is a secondary effect of generally decreased translation and/or decreased competition for the E-site with eIF5A.

  • 333. Kauppi, Anna
    et al.
    Nordfelth, Roland
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Hägglund, U
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Elofsson, Mikael
    Chemistry.
    Salicylanilides are potent inhibitors of type III secretion in Yersinia2003In: Advances in Experimental Medicine and Biology, Vol. 529, p. 97-100Article in journal (Refereed)
  • 334.
    Kauppi, Anna
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nordfelth, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Uvell, Hanna
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Targeting bacterial Virulence:  Inhibitors of type III secretion in Yersinia2003In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 10, no 3, p. 241-249Article in journal (Refereed)
    Abstract [en]

    Agents that target bacterial virulence without detrimental effect on bacterial growth are useful chemical probes for studies of virulence and potential candidates for drug development. Several gram-negative pathogens employ type III secretion to evade the innate immune response of the host. Screening of a chemical library with a luciferase reporter gene assay in viable Yersinia pseudotuberculosis furnished several compounds that inhibit the reporter gene signal expressed from the yopE promoter and effector protein secretion at concentrations with no or modest effect on bacterial growth. The selectivity patterns observed for inhibition of various reporter gene strains indicate that the compounds target the type III secretion machinery at different levels. Identification of this set of inhibitors illustrates the approach of utilizing cell-based assays to identify compounds that affect complex bacterial virulence systems.

  • 335. Keeney, Jill B
    et al.
    Chapman, Karen B
    Lauermann, Vit
    Voytas, Daniel F
    Åström, Stefan U
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    von Pawel-Rammingen, Ulrich
    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).
    Boeke, Jeff D
    Multiple molecular determinants for retrotransposition in a primer tRNA1995In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 15, no 1, p. 217-226Article in journal (Refereed)
    Abstract [en]

    Retroviruses and long terminal repeat-containing retroelements use host-encoded tRNAs as primers for the synthesis of minus strong-stop DNA, the first intermediate in reverse transcription of the retroelement RNA. Usually, one or more specific tRNAs, including the primer, are selected and packaged within the virion. The reverse transcriptase (RT) interacts with the primer tRNA and initiates DNA synthesis. The structural and sequence features of primer tRNAs important for these specific interactions are poorly understood. We have developed a genetic assay in which mutants of tRNA(iMet), the primer for the Ty1 retrotransposon of Saccharomyces cerevisiae, can be tested for the ability to serve as primers in the reverse transcription process. This system allows any tRNA mutant to be tested, regardless of its ability to function in the initiation of protein synthesis. We find that mutations in the T psi C loop and the acceptor stem regions of the tRNA(iMet) affect transposition most severely. Conversely, mutations in the anticodon region have only minimal effects on transposition. Further study of the acceptor stem and other mutants demonstrates that complementarity to the element primer binding site is a necessary but not sufficient requirement for effective tRNA priming. Finally, we have used interspecies hybrid initiator tRNA molecules to implicate nucleotides in the D arm as additional recognition determinants. Ty3 and Ty1, two very distantly related retrotransposons, require similar molecular determinants in this primer tRNA for transposition.

  • 336. Keller, Ulrich B
    et al.
    Old, Jennifer B
    Dorsey, Frank C
    Nilsson, Jonas A
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Nilsson, Lisa
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    MacLean, Kirsteen H
    Chung, Linda
    Yang, Chunying
    Spruck, Charles
    Boyd, Kelli
    Reed, Steven I
    Cleveland, John L
    Myc targets Cks1 to provoke the suppression of p27Kip1, proliferation and lymphomagenesis.2007In: EMBO J, ISSN 0261-4189, Vol. 26, no 10, p. 2562-74Article in journal (Refereed)
    Abstract [en]

    Reduced levels of the cyclin-dependent kinase inhibitor p27(Kip1) connote poor prognosis in cancer. In human Burkitt lymphoma and in precancerous B cells and lymphomas arising in Emu-Myc transgenic mice, p27(Kip1) expression is markedly reduced. We show that the transcription of the Cks1 component of the SCF(Skp2) complex that is necessary for p27(Kip1) ubiquitylation and degradation is induced by Myc. Further, Cks1 expression is elevated in precancerous Emu-Myc B cells, and high levels of Cks1 are also a hallmark of Emu-Myc lymphoma and of human Burkitt lymphoma. Finally, loss of Cks1 in Emu-Myc B cells elevates p27(Kip1) levels, reduces proliferation and markedly delays lymphoma development and dissemination of disease. Therefore, Myc suppresses p27(Kip1) expression, accelerates cell proliferation and promotes tumorigenesis at least in part through its ability to selectively induce Cks1.

  • 337. Keller, Ulrich
    et al.
    Nilsson, Jonas A
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Maclean, Kirsteen H
    Old, Jennifer B
    Cleveland, John L
    Nfkb 1 is dispensable for Myc-induced lymphomagenesis.2005In: Oncogene, ISSN 0950-9232, Vol. 24, no 41, p. 6231-40Article in journal (Refereed)
    Abstract [en]

    Rel/NF-kappaB transcription factors are critical arbiters of immune responses, cell survival, and transformation, and are frequently deregulated in cancer. The p50 NF-kappaB 1 component of Rel/NF-kappaB DNA-binding dimers regulates genes involved in both cell cycle traverse and apoptosis. Nfkb 1 loss accelerates B cell growth and leads to increased B cell turnover in vivo, phenotypes akin to those manifested in B cells of Emu-Myc transgenic mice, a model of human Burkitt lymphoma. Interestingly, Emu-Myc B cells express reduced levels of cytoplasmic and nuclear NF-kappaB 1 and have reduced Rel/NF-kappaB DNA-binding activity, suggesting that Myc-mediated repression of NF-kappaB 1 might mediate its proliferative and apoptotic effects on B cells. Furthermore, Nfkb 1 expression was reduced in the majority of Emu-Myc lymphomas and was also suppressed in human Burkitt lymphoma. Nonetheless, loss of Nfkb 1 did not appreciably affect Myc's proliferative or apoptotic responses in B cells and had no effect on lymphoma development in Emu-Myc mice. Therefore, Nfkb 1 is dispensable for Myc-induced lymphomagenesis.. Oncogene (2005) 24, 6231-6240

  • 338. Keyser, P
    et al.
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Rosell, S
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Virulence blockers as alternatives to antibiotics: type III secretion inhibitors against Gram-negative bacteria2008In: Journal of Internal Medicine, Vol. 264, no 1, p. 17-29Article in journal (Refereed)
    Abstract [en]

    In recent years mounting problems related to antibiotic-resistant bacteria have resulted in the prediction that we are entering the preantibiotic era. A way of preventing such a development would be to introduce novel antibacterial medicines with modes of action distinct from conventional antibiotics. Recent studies of bacterial virulence factors and toxins have resulted in increased understanding of the way in which pathogenic bacteria manipulate host cellular processes. This knowledge may now be used to develop novel antibacterial medicines that disarm pathogenic bacteria. The type III secretion system (T3SS) is known to be a potent virulence mechanism shared by a broad spectrum of pathogenic Gram-negative bacteria that interact with human, animal and plant hosts by injecting effector proteins into the cytosol of host cells. Diseases, such as bubonic plague, shigellosis, salmonellosis, typhoid fever, pulmonary infections, sexually transmitted chlamydia and diarrhoea largely depend on the bacterial proteins injected by the T3SS machinery. Recently a number of T3SS inhibitors have been identified using screening-based approaches. One class of inhibitors, the salicylidene acylhydrazides, has been subjected to chemical optimization and evaluation in several in vitro and ex vivo assays in multiple bacterial species including Yersinia spp., Chlamydia spp., Salmonella spp. and Pseudotuberculosis aeruginosa. Reports published up to date indicate that T3SS inhibitors have the potential to be developed into novel antibacterial therapeutics.

  • 339.
    Khoshnood, Behzad
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Dacklin, Ingrid
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Grabbe, Caroline
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    A proteomics approach to identify targets of the ubiquitin-like molecule Urm1 in Drosophila melanogaster2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 9, article id e0185611Article in journal (Refereed)
    Abstract [en]

    By covalently conjugating to target proteins, ubiquitin-like modifiers (UBLs) act as important regulators of target protein localization and activity, thereby playing a critical role in the orchestration of cellular biology. The most ancient and one of the least studied UBLs is Urm1, a dual-function protein that in parallel to performing similar functions as its prokaryotic ancestors in tRNA modification, also has adopted the capacity to conjugate to cellular proteins analogous to ubiquitin and other UBL modifiers. In order to increase the understanding of Urm1 and its role in multicellular organisms, we have used affinity purification followed by mass spectrometry to identify putative targets of Urm1 conjugation (urmylation) at three developmental stages of the Drosophila melanogaster lifecycle. Altogether we have recovered 79 Urm1-interacting proteins in Drosophila, which include the already established Urm1 binding partners Prx5 and Uba4, together with 77 candidate urmylation targets that are completely novel in the fly. Among these, the majority was exclusively identified during either embryogenesis, larval stages or in adult flies. We further present biochemical evidence that four of these proteins are covalently conjugated by Urm1, whereas the fifth verified Urm1-binding protein appears to interact with Urm1 via non-covalent means. Besides recapitulating the previously established roles of Urm1 in tRNA modification and during oxidative stress, functional clustering of the newly identified Urm1-associated proteins further positions Urm1 in protein networks that control other types of cellular stress, such as immunological threats and DNA damage. In addition, the functional characteristics of several of the candidate targets strongly match the phenotypes displayed by Urm1(n123) null animals, including embryonic lethality, reduced fertility and shortened lifespan. In conclusion, this identification of candidate targets of urmylation significantly increases the knowledge of Urm1 and presents an excellent starting point for unravelling the role of Urm1 in the context of a complex living organism.

  • 340.
    Kim, Maria
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Ekhteraei-Tousi, Samaneh
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lewerentz, Jacob
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The X-linked 1.688 satellite in Drosophila melanogaster promotes specific targeting by Painting of Fourth2018In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 208, no 2, p. 623-632Article in journal (Refereed)
    Abstract [en]

    Repetitive DNA, represented by transposons and satellite DNA, constitutes a large portion of eukaryotic genomes, being the major component of constitutive heterochromatin. There is a growing body of evidence that it regulates several nuclear functions including chromatin state and the proper functioning of centromeres and telomeres. The 1.688 satellite is one of the most abundant repetitive sequences in Drosophila melanogaster, with the longest array being located in the pericentromeric region of the X-chromosome. Short arrays of 1.688 repeats are widespread within the euchromatic part of the X-chromosome, and these arrays were recently suggested to assist in recognition of the X-chromosome by the dosage compensation male-specific lethal complex. We discovered that a short array of 1.688 satellite repeats is essential for recruitment of the protein POF to a previously described site on the X-chromosome (PoX2) and to various transgenic constructs. On an isolated target, i.e., an autosomic transgene consisting of a gene upstream of 1.688 satellite repeats, POF is recruited to the transgene in both males and females. The sequence of the satellite, as well as its length and position within the recruitment element, are the major determinants of targeting. Moreover, the 1.688 array promotes POF targeting to the roX1-proximal PoX1 site in trans Finally, binding of POF to the 1.688-related satellite-enriched sequences is conserved in evolution. We hypothesize that the 1.688 satellite functioned in an ancient dosage compensation system involving POF targeting to the X-chromosome.

  • 341.
    Kim, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Faucillion, Marie-Line
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    RNA-on-X 1 and 2 in Drosophila melanogaster fulfill separate functions in dosage compensation2018In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 14, no 12, article id e1007842Article in journal (Refereed)
    Abstract [en]

    In Drosophila melanogaster, the male-specific lethal (MSL) complex plays a key role in dosage compensation by stimulating expression of male X-chromosome genes. It consists of MSL proteins and two long noncoding RNAs, roX1 and roX2, that are required for spreading of the complex on the chromosome and are redundant in the sense that loss of either does not affect male viability. However, despite rapid evolution, both roX species are present in diverse Drosophilidae species, raising doubts about their full functional redundancy. Thus, we have investigated consequences of deleting roX1 and/or roX2 to probe their specific roles and redundancies in Dmelanogaster. We have created a new mutant allele of roX2 and show that roX1 and roX2 have partly separable functions in dosage compensation. In larvae, roX1 is the most abundant variant and the only variant present in the MSL complex when the complex is transmitted (physically associated with the X-chromosome) in mitosis. Loss of roX1 results in reduced expression of the genes on the X-chromosome, while loss of roX2 leads to MSL-independent upregulation of genes with male-biased testis-specific transcription. In roX1 roX2mutant, gene expression is strongly reduced in a manner that is not related to proximity to high-affinity sites. Our results suggest that high tolerance of mis-expression of the X-chromosome has evolved. We propose that this may be a common property of sex-chromosomes, that dosage compensation is a stochastic process and its precision for each individual gene is regulated by the density of high-affinity sites in the locus.

  • 342.
    Kisand, Veljo
    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).
    Andersson, Nina
    Umeå University, Faculty of Science and Technology, Umeå Marine Sciences Centre (UMF).
    Wikner, Johan
    Umeå University, Faculty of Science and Technology, Umeå Marine Sciences Centre (UMF). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bacterial freshwater species successfully immigrate to the brackish water environment in the northern Baltic2005In: Limnology and Oceanography, ISSN 0024-3590, E-ISSN 1939-5590, Vol. 50, no 3, p. 945-956Article in journal (Refereed)
    Abstract [en]

    We studied the distribution and seasonal dynamics of five species from the genus Flavobacterium and one species from the genus Marinomonas over the course of a year along a northern Baltic Sea river-marine transect. All of the species had been previously demonstrated as important consumers of riverine dissolved organic carbon. Quantitative DNA-DNA hybridization data showed that two of the Flavobacterium spp. and the Marinomonas sp. had highest abundance in the river water (maximum 20,000 cells ml-1), with maximum relative abundance of 0.5-2.5% of the bacterial community. These species declined in abundance from the river to the estuary and the offshore site. Abundance and dynamics in the estuarine environment suggested successful immigration of freshwater bacteria, accompanied by growth in the brackish water environment. Two of the three abundant species showed high cell numbers also during late autumn to early spring in the estuary, indicating a selective advantage when riverine dissolved organic carbon was the main carbon source. The remaining three species showed more episodic abundance close to the detection limit of the method, providing weaker evidence of occurrence in the freshwater environment. Some bacterioplankton consuming riverine organic carbon in the brackish water environment in the northern Baltic are therefore freshwater species, with a selective advantage during winter.

  • 343.
    Kisand, Veljo
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Cuadros, Rocio
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    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).
    Phylogeny of culturable estuarine bacteria catabolizing riverine organic matter in the northern Baltic Sea2002In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 68, no 1, p. 379-388Article in journal (Refereed)
    Abstract [en]

    The objective of our study was to isolate and determine the phylogenetic affiliation of culturable estuarine bacteria capable of catabolizing riverine dissolved organic matter (RDOM) under laboratory conditions. Additions of RDOM consistently promoted the growth of estuarine bacteria in carbon-limited dilution cultures, with seasonal variation in growth rates and yields. At least 42 different taxa were culturable on solid agar media and, according to quantitative DNA-DNA hybridizations, constituted 32 to 89% of the total bacterial number in the enriched treatments. Five species in the Cytophaga-Flexibacter-Bacteroides group and one in the gamma-proteobacteria phylogenetic group (Marinomonas sp.) were numerically dominant during the stationary phase of the RDOM-enriched dilution cultures but not in the control cultures. Four of the isolates in Cytophaga-Flexibacter-Bacteroides group were putatively affiliated with the genus Flavobacterium. All dominating isolates were determined to be new species based on comparison to the current databases. The same group of species dominated independently of the season investigated, suggesting a low diversity of bacteria catabolizing RDOM in the estuary. It also suggested a broad tolerance of the dominating species to seasonal variation in hydrography, chemistry, and competition with other species. Taken together, our results suggest that a limited group of bacteria, mainly in the Flavobacterium genus, played an important role in introducing new energy and carbon to the marine system in the northern Baltic Sea.

  • 344.
    Kisand, Veljo
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    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).
    Combining culture-dependent and -independent methodologies for estimation of richness of estuarine bacterioplankton consuming riverine dissolved organic matter2003In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 69, no 6, p. 3607-3616Article in journal (Refereed)
    Abstract [en]

    Three different methods for analyzing natural microbial community diversity were combined to maximize an estimate of the richness of bacterioplankton catabolizing riverine dissolved organic matter (RDOM). We also evaluated the ability of culture-dependent quantitative DNA-DNA hybridization, a 16S rRNA gene clone library, and denaturing gradient gel electrophoresis (DGGE) to detect bacterial taxa in the same sample. Forty-two different cultivatable strains were isolated from rich and poor solid media. In addition, 50 unique clones were obtained by cloning of the bacterial 16S rDNA gene amplified by PCR from the community DNA into an Escherichia coli vector. Twenty-three unique bands were sequenced from 12 DGGE profiles, excluding a composite fuzzy band of the Cytophaga-Flavobacterium group. The different methods gave similar distributions of taxa at the genus level and higher. However, the match at the species level among the methods was poor, and only one species was identified by all three methods. Consequently, all three methods identified unique subsets of bacterial species, amounting to a total richness of 97 operational taxonomic units in the experimental system. The confidence in the results was, however, dependent on the current precision of the phylogenetic determination and definition of the species. Bacterial consumers of RDOM in the studied estuary were primarily both cultivatable and uncultivable taxa of the Cytophaga-Flavobacterium group, a concordant result among the methods applied. Culture-independent methods also suggested several not-yet-cultivated beta-proteobacteria to be RDOM consumers.

  • 345. Kisand, Veljo
    et al.
    Wikner, Johan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Limited resolution of 16S rDNA DGGE caused by melting properties and closely related DNA sequences2003In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 54, no 2, p. 183-191Article in journal (Refereed)
    Abstract [en]

    The phylogenetic affiliation of 91 operational taxonomic units, randomly sampled from three aquatic microcosm experiments, was investigated by two PCR based and one culture dependent method. The occurrence of multiple melting domains and poor coupling between Tin and DGGE retardation was demonstrated to cause poor resolution at the species level in PCR-DGGE analysis of microbial communities. We also showed that the problem of multiple melting domains was particularly prone for brackish water bacterioplankton in the Flavobacterium genus, providing characteristic band morphology for this genus. Banding patterns from DGGE analysis may therefore be misinterpreted in terms of the species richness in natural bacterial communities, when using commonly applied universal primers. (C) 2003 Elsevier Science B.V. All rights reserved.

  • 346. Kitamura, Aya
    et al.
    Nishimoto, Madoka
    Sengoku, Toru
    Shibata, Rie
    Jäger, Gunilla
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Grosjean, Henri
    Yokoyama, Shigeyuki
    Bessho, Yoshitaka
    Characterization and Structure of the Aquifex aeolicus Protein DUF752 A BACTERIAL tRNA-METHYLTRANSFERASE (MnmC2) FUNCTIONING WITHOUT THE USUALLY FUSED OXIDASE DOMAIN (MnmC1)2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 52, p. 43950-43960Article in journal (Refereed)
    Abstract [en]

    Post-transcriptional modifications of the wobble uridine (U34) of tRNAs play a critical role in reading NNA/G codons belonging to split codon boxes. In a subset of Escherichia coli tRNA, this wobble uridine is modified to 5-methylaminomethyluridine (mnm5U34) through sequential enzymatic reactions. Uridine 34 is first converted to 5-carboxymethylaminomethyluridine (cmnm5U34) by the MnmE-Mnm Genzyme complex. The cmnm5U34 is further modified to mnm5U by the bifunctional MnmC protein. In the first reaction, the FAD-dependent oxidase domain (MnmC1) converts cmnm5U into 5-aminomethyluridine (nm5U34), and this reaction is immediately followed by the methylation of the free amino group into mnm5U34 by the S-adenosylmethionine-dependent domain (MnmC2). Aquifex aeolicus lacks a bifunctional MnmC protein fusion and instead encodes the Rossmann-fold protein DUF752, which is homologous to the methyltransferase MnmC2 domain of Escherichia coli MnmC (26% identity). Here, we determined the crystal structure of the A. aeolicus DUF752 protein at 2.5 Å resolution, which revealed that it catalyzes the S-adenosylmethionine-dependent methylation of nm5U in vitro, to form mnm5U34 in tRNA. We also showed that naturally occurring tRNA from A. aeolicus contains the 5-mnm group attached to the C5 atom of U34. Taken together, these results support the recent proposal of an alternative MnmC1-independent shortcut pathway for producing mnm5U34 in tRNAs.

  • 347.
    Klinth, Jeanna E
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics. 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). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Centre for Biomedical Engineering and Physics (CMTF).
    Pinkner, Jerome S
    Hultgren, Scott J
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Uhlin, Bernt Eric
    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). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Axner, Ove
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Centre for Biomedical Engineering and Physics (CMTF).
    Impairment of the biomechanical compliance of P pili: a novel means of inhibiting uropathogenic bacterial infections?2012In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 41, no 3, p. 285-295Article in journal (Refereed)
    Abstract [en]

    Gram-negative bacteria often initiate their colonization by use of extended attachment organelles, so called pili. When exposed to force, the rod of helix-like pili has been found to be highly extendable, mainly attributed to uncoiling and recoiling of its quaternary structure. This provides the bacteria with the ability to redistribute an external force among a multitude of pili, which enables them to withstand strong rinsing flows, which, in turn, facilitates adherence and colonization processes critical to virulence. Thus, pili fibers are possible targets for novel antibacterial agents. By use of a substance that compromises compliance of the pili, the ability of bacteria to redistribute external forces can be impaired, so they will no longer be able to resist strong urine flow and thus be removed from the host. It is possible such a substance can serve as an alternative to existing antibiotics in the future or be a part of a multi-drug. In this work we investigated whether it is possible to achieve this by targeting the recoiling process. The test substance was purified PapD. The effect of PapD on the compliance of P pili was assessed at the single organelle level by use of force-measuring optical tweezers. We showed that the recoiling process, and thus the biomechanical compliance, in particular the recoiling process, can be impaired by the presence of PapD. This leads to a new concept in the search for novel drug candidates combating uropathogenic bacterial infections-"coilicides", targeting the subunits of which the pilus rod is composed.

  • 348.
    Kolterud, Åsa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The Role of Lhx2 During Organogenesis: - Analysis of the Hepatic, Hematopoietic and Olfactory Systems2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    During embryonic development a variety of tissues and organs such as the lung, eye, and kidney are being formed. The generation of functional organs is regulated by reciprocal cell-cell interactions. Via the secretion of soluble molecules one type of cells affect the fate of their neighboring cells. A central issue in organogenesis is how a cell interprets such extrinsic signals and adopts a specific fate, and how the cell in response to this signal establishes reciprocal signaling. Transcription factors play a critical role in this process and my thesis focuses on the role of the LIM-homeodomain transcription factor, Lhx2, in the development of three different organ systems, the liver, the hematopoietic system and the olfactory system.

    The liver is formed from endoderm of the ventral foregut and mesenchyme of the septum transversum (st) and its development depends upon signaling interactions between these two tissues. As the liver becomes a distinct organ it is colonized by hematopoietic cells and serves as hematopoietic organ until birth. The fetal liver provides a microenvironment that supports the expansion of the entire hematopoietic system (HS) including the hematopoietic stem cells (HSCs). Liver development in Lhx2-/- embryos is disrupted leading to a lethal anemia due to insufficient support of hematopoiesis. To further investigate the role of Lhx2 in liver development I analyzed gene expression from the Lhx2 locus during liver development in wild-type and Lhx2-/- mice. Lhx2 is expressed in the liver associated st mesenchymal cells that become integrated in the liver and contribute to a subpopulation of hepatic stellate cells in adult liver. Lhx2 is not required for the formation of these mesenchymal cells, suggesting that the phenotype in Lhx2-/- livers is due to the presence of defective mesenchymal cells. The putative role of Lhx2 in the expansion of the HS was examined by introducing Lhx2 cDNA into embryonic stem cells differentiated in vitro. This approach allowed for the generation of immortalized multipotent hematopoietic progenitor cell (HPC) lines that share many characteristics with normal HSCs. The Lhx2-dependent generation of HSC-like cell lines suggests that Lhx2 plays a role in the maintenance and/or expansion of the HS. To isolate genes putatively linked to Lhx2 function, genes differentially expressed in the HPC lines were isolated using a cDNA subtraction approach. This allowed for the identification of a few genes putatively linked to Lhx2 function, as well as several stem cell-specific genes. The antagonist of Wnt signalling, Dickkopf-1 (Dkk-1), was identified in the former group of genes as it showed a similar expression pattern in the fetal liver, as that of Lhx2 and expression of Dkk-1 in fetal liver and in HPC lines appeared to be regulated by Lhx2. This suggests that Dkk-1 plays a role in liver development and/or HSC physiology during embryonic development.

    During development of the olfactory epithelium (OE) neuronal progenitors differentiate into mature olfactory sensory neurons (OSNs) that are individually specified into over a thousand different subpopulations, each expressing a unique odorant receptor (OR) gene. The expression of Lhx2 in olfactory neurons suggested a potential role for Lhx2 in the development of OSNs. To address this OE from Lhx2-/- and wild-type mice was compared. In the absence of functional Lhx2 neuronal differentiation was arrested prior to onset of OR expression. Lhx2 is thus required for the development of OSN progenitors into functional, individually specified OSNs.

    Thus, Lhx2 trigger a variety of cellular responses in different organ systems that play important roles in organ development in vivo and stem cell expansion in vitro.

  • 349.
    Kolterud, Åsa
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Alenius, Mattias
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Carlsson, Leif
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Bohm, Staffan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    The Lim homeobox gene Lhx2 is required for olfactory sensory neuron identity2004In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 131, no 21, p. 5319-5326Article in journal (Refereed)
    Abstract [en]

    Progenitor cells in the mouse olfactory epithelium generate over a thousand subpopulations of neurons, each expressing a unique odorant receptor (OR) gene. This event is under the control of spatial cues, since neurons in different epithelial regions are restricted to express region-specific subsets of OR genes. We show that progenitors and neurons express the LIM-homeobox gene Lhx2 and that neurons in Lhx2-null mutant embryos do not diversify into subpopulations expressing different OR genes and other region-restricted genes such as Nqo1 and Ncam2. Lhx2-/- embryos have, however, a normal distribution of Mash1-positive and neurogenin 1-positive neuronal progenitors that leave the cell cycle, acquire pan-neuronal traits and form axon bundles. Increased cell death in combination with increased expression of the early differentiation marker Neurod1, as well as reduced expression of late differentiation markers (Galphaolf and Omp), suggests that neuronal differentiation in the absence of Lhx2 is primarily inhibited at, or immediate prior to, onset of OR expression. Aberrant regional expression of early and late differentiation markers, taken together with unaltered region-restricted expression of the Msx1 homeobox gene in the progenitor cell layer of Lhx2-/- embryos, shows that Lhx2 function is not required for all aspects of regional specification of progenitors and neurons. Thus, these results indicate that a cell-autonomous function of Lhx2 is required for differentiation of progenitors into a heterogeneous population of individually and regionally specified mature olfactory sensory neurons.

  • 350.
    Kolterud, Åsa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wandzioch, Ewa
    Falileeva, Ludmilla
    Carlsson, Leif
    Identification and characterization of genes specifically expressed in hematopoietic progenitor/stem cells immortalized by Lhx2Manuscript (Other academic)
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