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Biomedical subjects

M Bjørås

Publications and source records attributed to M Bjørås.

At least 19 recordsLinked to original sources
PubMed2005

Antimutator role of DNA glycosylase MutY in pathogenic Neisseria species.

Genome alterations due to horizontal gene transfer and stress constantly generate strain on the gene pool of Neisseria meningitidis, the causative agent of meningococcal (MC) disease. The DNA glycosylase MutY of the base excision repair pathway is involved in the protection against oxidative stress. MC MutY expressed in Escherichia coli exhibited base excision activity towards DNA substrates containing A:7,8-dihydro-8-oxo-2'-deoxyguanosine and A:C mismatches. Expression in E. coli fully suppressed the elevated spontaneous mutation rate found in the E. coli mutY mutant. An assessment of MutY activity in lysates of neisserial wild-type and mutY mutant strains showed that both MC and gonococcal (GC) MutY is expressed and active in vivo. Strikingly, MC and GC mutY mutants exhibited 60- to 140-fold and 20-fold increases in mutation rates, respectively, compared to the wild-type strains. Moreover, the differences in transitions and transversions in rpoB conferring rifampin resistance observed with the wild type and mutants demonstrated that the neisserial MutY enzyme works in preventing GC-->AT transversions. These findings are important in the context of models linking mutator phenotypes of disease isolates to microbial fitness.

Base Pair Mismatch↗
PubMed2003

Influence of DNA torsional rigidity on excision of 7,8-dihydro-8-oxo-2'-deoxyguanosine in the presence of opposing abasic sites by human OGG1 protein.

The human protein OGG1 (hOGG1) targets the highly mutagenic base 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) and shows a high specificity for the opposite DNA base. Abasic sites can arise in DNA in close opposition to 8-oxodG either during repair of mismatched bases (i.e. 8-oxodG/A mismatches) or, more frequently, as a consequence of ionizing radiation exposure. Bistranded DNA lesions may remain unrepaired and lead to cell death via double-strand break formation. In order to explore the role of damaged-DNA dynamics in recognition/excision by the hOGG1 repair protein, specific oligonucleotides containing an 8-oxodG opposite an abasic site, at different relative distances on the complementary strand, were synthesized. Rotational dynamics were studied by means of fluorescence polarization anisotropy decay experiments and the torsional elastic constant as well as the hydrodynamic radius of the DNA fragments were evaluated. Efficiency of excision of 8-oxodG was tested using purified human glycosylase. A close relation between the twisting flexibility of the DNA fragment and the excision efficiency of the oxidative damage by hOGG1 protein within a cluster was found.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2001

Highly efficient base excision repair (BER) in human and rat male germ cells.

The quality of germ cell DNA is critical for the fate of the offspring, yet there is limited knowledge of the DNA repair capabilities of such cells. One of the main DNA repair pathways is base excision repair (BER) which is initiated by DNA glycosylases that excise damaged bases, followed by incision of the generated abasic (AP) sites. We have studied human and rat methylpurine-DNA glycosylase (MPG), uracil-DNA glycosylase (UNG), and the major AP endonuclease (HAP1/APEX) in male germ cells. Enzymatic activities and western analyses indicate that these enzymes are present in human and rat male germ cells in amounts that are at least as high as in somatic cells. Minor differences were observed between different cellular stages of rat spermatogenesis and spermiogenesis. Repair of methylated DNA was also studied at the cellular level using the Comet assay. The repair was highly efficient in both human and rat male germ cells, in primary spermatocytes as well as round spermatids, compared to rat mononuclear blood cells or hepatocytes. This efficient BER removes frequently occurring DNA lesions that arise spontaneously or via environmental agents, thereby minimising the number of potential mutations transferred to the next generation.

Animals↗
PubMed2000

Cell-cycle regulation, intracellular sorting and induced overexpression of the human NTH1 DNA glycosylase involved in removal of formamidopyrimidine residues from DNA.

Endonuclease III (Nth) of Escherichia coli is a DNA glycosylase essential for the removal of oxidised pyrimidine base residues from DNA. Several eukaryotic homologues have recently been identified and shown to have biochemical properties similar to those of Nth. However, some of the eukaryotic counterparts also appear to remove imidazole ring-opened purine residues (faPy), a property not shared by the enzymes of bacterial origin. Here, we show that the human enzyme also possesses efficient faPy DNA glycosylase activity as indicated both from studies of the purified protein and induced overexpression of the human NTH1 cDNA in HeLa cells. We constructed green fluorescent protein-tagged hNTH1 fusion proteins to study the cellular localisation of hNTH1 and found strong and exclusive sorting to the nucleus. Studies with synchronised cells showed that the expression of hNTH1 is regulated during the cell cycle with increased transcription during early and mid S-phase.

Amino Acid Sequence↗
PubMed1999

The Saccharomyces cerevisiae homologues of endonuclease III from Escherichia coli, Ntg1 and Ntg2, are both required for efficient repair of spontaneous and induced oxidative DNA damage in yeast.

Endonuclease III from Escherichia coli is the prototype of a ubiquitous DNA repair enzyme essential for the removal of oxidized pyrimidine base damage. The yeast genome project has revealed the presence of two genes in Saccharomyces cerevisiae, NTG1 and NTG2, encoding proteins with similarity to endonuclease III. Both contain the highly conserved helix-hairpin-helix motif, whereas only one (Ntg2) harbors the characteristic iron-sulfur cluster of the endonuclease III family. We have characterized these gene functions by mutant and enzyme analysis as well as by gene expression and intracellular localization studies. Targeted gene disruption of NTG1 and NTG2 produced mutants with greatly increased spontaneous and hydrogen peroxide-induced mutation frequency relative to the wild type, and the mutation response was further increased in the double mutant. Both enzymes were found to remove thymine glycol and 2, 6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (faPy) residues from DNA with high efficiency. However, on UV-irradiated DNA, saturating concentrations of Ntg2 removed only half of the cytosine photoproducts released by Ntg1. Conversely, 5-hydroxycytosine was removed efficiently only by Ntg2. The enzymes appear to have different reaction modes, as judged from much higher affinity of Ntg2 for damaged DNA and more efficient borhydride trapping of Ntg1 to abasic sites in DNA despite limited DNA binding. Northern blot and promoter fusion analysis showed that NTG1 is inducible by cell exposure to DNA-damaging agents, whereas NTG2 is constitutively expressed. Ntg2 appears to be a nuclear enzyme, whereas Ntg1 was sorted both to the nucleus and to the mitochondria. We conclude that functions of both NTG1 and NTG2 are important for removal of oxidative DNA damage in yeast.

Amino Acid Sequence↗
PubMed1998

Inducible expression of the GLT-1 glutamate transporter in a CHO cell line selected for low endogenous glutamate uptake.

Inducible expression of the mammalian glial cell glutamate transporter GLT-1 has been established in a CHO cell line selected for low endogenous Na+-dependent glutamate uptake by [3H]aspartate suicide selection. Culturing the cells in doxycycline-containing medium, to activate GLT-1 expression via the Tet-On system, increased uptake of the GLT-1 substrate D-aspartate 280-fold, and increased cell size. Applying glutamate to whole-cell clamped, doxycycline-treated cells evoked a transporter-mediated current with characteristics appropriate for GLT-1. This cell line provides a useful tool for further examination of the electrical, biochemical and pharmacological properties of GLT-1, the most abundant glutamate transporter in the brain.

ATP-Binding Cassette Transporters↗
PubMed1998

Production, isolation and purification of bacteriocins expressed by two strains of Neisseria meningitidis.

The systemic Neisseria meningitidis strain P241 and the healthy pharyngeal carrier strain BT878 produce bacteriocin-like substances during growth. A method has been devised for obtaining the active substances in solution. The activity was recovered by freeze-thaw extraction of dialyzed Todd-Hewitt agar medium into which the bacteriocins had diffused during growth of the producer strains. The bacteriocins were purified more than 50-fold by ammonium-sulphate precipitation and hydrophobic interaction chromatography. They are quite stable to heat and remain active 100% after 30 min at 100 degrees C. However, the protein nature of the bacteriocins has been confirmed by their sensitivity to alpha-chymotrypsin. Gel filtration indicated an Mr of 100-110 kDa, whereas SDS-polyacrylamide gel electrophoresis produced a common band by Coomassie staining corresponding to an Mr of 47-48 kDa, suggesting a dimer form of the active protein component.

Bacteriocins↗
PubMed1997

DNA damage induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in HL-60 cells and purified DNA in vitro.

Chlorinated tap water often contains 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), which is a potent directly acting bacterial mutagen. We have investigated the induction of DNA damage by MX in a promyelocytic human leukaemia cell line (HL-60 cells). Exposure of HL-60 cells to 100-300 microM MX resulted in increased levels of DNA single-strand breaks and/or alkali-labile sites (SSBs) as detected by alkaline filter elution. When adding inhibitors of DNA break repair (AraC plus hydroxyurea), increased levels of DNA SSBs were observed at very low concentrations (1-3 microM) of MX, as observed by both alkaline filter elution and the single-cell gel electrophoresis assay. Increased DNA SSBs could also be observed if DNA repair inhibitors were added immediately after exposure to 10 microM MX, indicating that low concentrations of MX cause a relatively stable modification of DNA that may be recognized and incised by DNA repair enzyme activities. Further studies with DNA break repair inhibitors indicated that HL-60 cells exposed to 10 microM MX for 1 h repaired 50% of their initial DNA damage during a 2-h period and the repair appeared to be complete at 22 h. Analysis of MX-treated DNA by sequencing methods indicated that MX preferentially reacts with guanines in DNA.

Cytarabine↗
PubMed1996

Base excision of oxidative purine and pyrimidine DNA damage in Saccharomyces cerevisiae by a DNA glycosylase with sequence similarity to endonuclease III from Escherichia coli.

One gene locus on chromosome I in Saccharomyces cerevisiae encodes a protein (YAB5_YEAST; accession no. P31378) with local sequence similarity to the DNA repair glycosylase endonuclease III from Escherichia coli. We have analyzed the function of this gene, now assigned NTG1 (endonuclease three-like glycosylase 1), by cloning, mutant analysis, and gene expression in E. coli. Targeted gene disruption of NTG1 produces a mutant that is sensitive to H2O2 and menadione, indicating that NTG1 is required for repair of oxidative DNA damage in vivo. Northern blot analysis and expression studies of a NTG1-lacZ gene fusion showed that NTG1 is induced by cell exposure to different DNA damaging agents, particularly menadione, and hence belongs to the DNA damage-inducible regulon in S. cerevisiae. When expressed in E. coli, the NTG1 gene product cleaves plasmid DNA damaged by osmium tetroxide, thus, indicating specificity for thymine glycols in DNA similarly as is the case for EndoIII. However, NTG1 also releases formamidopyrimidines from DNA with high efficiency and, hence, represents a glycosylase with a novel range of substrate recognition. Sequences similar to NTG1 from other eukaryotes, including Caenorhabditis elegans, Schizosaccharomyces pombe, and mammals, have recently been entered in the GenBank suggesting the universal presence of NTG1-like genes in higher organisms. S. cerevisiae NTG1 does not have the [4Fe-4S] cluster DNA binding domain characteristic of the other members of this family.

Amino Acid Sequence↗
PubMed1996

Cloning and expression of a neuronal rat brain glutamate transporter.

Glutamate is the major excitatory transmitter in the mammalian central nervous system. Glutamate transporters, which keep the extracellular glutamate concentration low, are required both for normal brain function and for protecting neurons against harmful glutamatergic overstimulation. We have isolated the cDNA for a rat brain glutamate transporter (REAAC1) which has 90% amino acid and 86% nucleotide identity to the rabbit EAAC1. When REAAC1 was expressed in HeLa cells using a recombinant vaccinia-T7 virus expression system, a sodium dependent glutamate uptake was observed. The affinity of the carrier to various substrates was typical of brain "high affinity' glutamate uptake: threo-3-hydroxyaspartate, (R)-aspartate, (S)-glutamate and (S)-trans-pyrrolidine-2,4-dicarboxylic acid were strong inhibitors, but not (R)-glutamate or gamma-aminobutyrate. High resolution, non-radioactive in situ hybridization histochemistry in rat brain revealed the mRNA in several types of glutamatergic as well as non-glutamatergic neurons, but not in glial cells.

ATP-Binding Cassette Transporters↗
PubMed1995

Purification and properties of the alkylation repair DNA glycosylase encoded the MAG gene from Saccharomyces cerevisiae.

The MAG gene of Saccharomyces cerevisiae encodes an alkylation repair DNA glycosylase whose sequence is homologous to the AlkA DNA glycosylase from Escherichia coli. To investigate the biochemical properties of MAG in comparison to AlkA, MAG was expressed in E. coli and purified to electrophoretic homogeneity. N-Terminal sequencing of the purified protein identified the translational start site which corresponded to that predicted previously from the nucleotide sequence. Polyclonal antibodies raised against MAG inhibited the enzymatic activity of MAG, but not that of AlkA, and vice versa, implying that the structures of the active site regions of these enzymes are antigenic, but sufficiently different to have different epitopes. Kinetic analysis of base excision from DNA exposed to [3H]methyl-N-nitrosourea and [3H]dimethyl sulfate showed that MAG was as effective as AlkA in removing 3-methyladenine, 7-methylguanine, and 7-methyladenine. However, the purified MAG enzyme did not catalyze the excision of O2-methylthymine, which is a major substrate for AlkA. Furthermore, 3-methylguanine was excised 20-40 times more slowly by MAG than by AlkA. The kinetics of 3-methylguanine excision by MAG were found to be similar to the low rate of 3-methylguanine excision catalyzed by 3-methyladenine DNA glycosylase I (Tag) of E. coli. Expression of MAG in alkA mutant cells did not effectively restore alkylation resistance of the mutant as did AlkA itself. It thus appears that MAG is a less versatile enzyme than AlkA in spite of the sequence relationship and may have a similar function in yeast as the nonhomologous Tag enzyme in E. coli.

Alkylation↗
PubMed1995

The base excision repair pathway.

The base excision repair pathway has evolved to protect cells from the deleterious effects of endogenous DNA damage induced by hydrolysis, reactive oxygen species and other intracellular metabolites that modify DNA base structure. However, base excision repair is also important to resist lesions produced by ionizing radiation and strong alkylating agents, which are similar to those induced by endogenous factors.

Adenine↗
PubMed1994

Differential expression of two glial glutamate transporters in the rat brain: an in situ hybridization study.

The distributions of the mRNAs encoding the L-glutamate transporters GLT1 and GLAST were examined in the rat brain by in situ hybridization using 35S-labelled oligonucleotide probes. Probes directed to GLT1 produced dense labelling in the hippocampus, neocortex and neostriatum, and weak labelling in the cerebellum. In contrast, GLAST mRNA appeared to be greatly enriched in the cerebellum compared to other brain regions. While the intensity of the labelling for GLAST and GLT1 varied among different regions, their cellular distributions appeared to coincide inasmuch as both mRNAs were mainly expressed by glial cells. Labelling occurred, inter alia, in glial cells throughout the hippocampus, and in Golgi epithelial cells in the Purkinje cell layer of the cerebellum.

Amino Acid Transport System X-AG↗
PubMed1994

Increased removal of 3-alkyladenine reduces the frequencies of hprt mutations induced by methyl- and ethylmethanesulfonate in Chinese hamster fibroblast cells.

We have previously reported the isolation of mammalian cell lines expressing the 3-methyladenine DNA glycosylase I (tag) gene from E. coli. These cells are 2-5 fold more resistant to the toxic effects of methylating agents than normal cells (15). Kinetic measurements of 3-methyladenine removal from the genome in situ show a moderate (3-fold) increase in Tag expressing cells relative to normal as compared to a high (50-fold) increase in exogenous alkylated DNA in vitro by cell extracts. Excision of 7-methylguanine is as expected, unaffected by the tag+ gene expression. The frequency of mutations formed in the hypoxanthine phosphoribosyl transferase (hprt) locus was investigated after methylmethanesulfonate (MMS), ethylmethanesulfonate (EMS), N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) exposure. Tag expression reduced the frequency of MMS and EMS induced mutations to about half the normal rate, whereas the mutation frequency in cells exposed to NMU or NEU is not affected by the tag+ gene expression. These results indicate that after exposure to compounds which produce predominantly N-alkylations in DNA, a substantial proportion of the mutations induced is formed at 3-alkyladenine residues in DNA.

Adenine↗
PubMed1993

Excision of 3-methylguanine from alkylated DNA by 3-methyladenine DNA glycosylase I of Escherichia coli.

Escherichia coli has two DNA glycosylases for repair of DNA damage caused by simple alkylating agents. The inducible AlkA DNA glycosylase (3-methyladenine [m3A] DNA glycosylase II) removes several different alkylated bases including m3A and 3-methylguanine (m3G) from DNA, whereas the constitutively expressed Tag enzyme (m3A DNA glycosylase I) has appeared to be specific for excision of m3A. In this communication we have reexamined the substrate specificity of Tag by using synthetic DNA rich in GC base pairs to facilitate detection of any possible methyl-G removal. In such DNA alkylated with [3H]dimethyl sulphate, we found that m3G was excised from double-stranded DNA by both glycosylases, although more efficiently by AlkA than by Tag. This was further confirmed using both N-[3H]methyl-N-nitrosourea- and [3H]dimethyl sulphate-treated native DNA, from which Tag excised m3G with an efficiency that was about 70 times lower than for AlkA. These results can explain the previous observation that high levels of Tag expression will suppress the alkylation sensitivity of alkA mutant cells, further implying that m3G is formed in quantity sufficient to represent an important cytotoxic lesion if left unrepaired in cells exposed to alkylating agents.

Alkylation↗
PubMed1992

Cloning and expression of a rat brain L-glutamate transporter.

Synaptic transmission of most vertebrate synapses is thought to be terminated by rapid transport of the neurotransmitter into presynaptic nerve terminals or neuroglia. L-Glutamate is the major excitatory transmitter in brain and its transport represents the mechanism by which it is removed from the synaptic cleft and kept below toxic levels. Here we use an antibody against a glial L-glutamate transporter from rat brain to isolate a complementary DNA clone encoding this transporter. Expression of this cDNA in transfected HeLa cells indicates that L-glutamate accumulation requires external sodium and internal potassium and transport shows the expected stereospecificity. The cDNA sequence predicts a protein of 573 amino acids with 8-9 putative transmembrane alpha-helices. Database searches indicate that this protein is not homologous to any identified protein of mammalian origin, including the recently described superfamily of neurotransmitter transporters. This protein therefore seems to be a member of a new family of transport molecules.

Amino Acid Sequence↗