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E Seeberg

Publications and source records attributed to E Seeberg.

At least 37 records · Page 2Linked to original sources
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

Opposite base-dependent reactions of a human base excision repair enzyme on DNA containing 7,8-dihydro-8-oxoguanine and abasic sites.

The guanine modification 7,8-dihydro-8-oxoguanine (8-oxoG) is a potent premutagenic lesion formed spontaneously at high frequencies in the genomes of aerobic organisms. We have characterized a human DNA repair glycosylase for 8-oxoG removal, hOGH1 (human yeast OGG1 homologue), by molecular cloning and functional analysis. Expression of the human cDNA in a repair deficient mutator strain of Escherichia coli (fpg mutY) suppressed the spontaneous mutation frequency to almost normal levels. The hOGH1 enzyme was localized to the nucleus in cells transfected by constructs of hOGH1 fused to green fluorescent protein. Enzyme purification yielded a protein of 38 kDa removing both formamidopyrimidines and 8-oxoG from DNA. The enzymatic activities of hOGH1 was analysed on DNA containing single residues of 8-oxoG or abasic sites opposite each of the four normal bases in DNA. Excision of 8-oxoG opposite C was the most efficient and was followed by strand cleavage via beta-elimination. However, significant removal of 8-oxoG from mispairs (8-oxoG: T >G >A) was also demonstrated, but essentially without an associated strand cleavage reaction. Assays with abasic site DNA showed that strand cleavage was indeed dependent on the presence of C in the opposite strand, irrespective of the prior removal of an 8-oxoG residue. It thus appears that strand incisions are made only if repair completion results in correct base insertion, whereas excision from mispairs preserves strand continuity and hence allows for error-free correction by a postreplicational repair mechanism.

Amino Acid Sequence↗
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

Different efficiencies of the Tag and AlkA DNA glycosylases from Escherichia coli in the removal of 3-methyladenine from single-stranded DNA.

Escherichia coli possesses two different DNA repair glycosylases, Tag and AlkA, which have similar ability to remove the alkylation product 3-methyladenine from double-stranded DNA. In this study we show that these enzymes have quite different activities for the excision of 3-methyladenine from single-stranded DNA, AlkA being 10-20 times more efficient than Tag. We propose that AlkA and perhaps other glycosylases as well may have an important role in the excision of base damage from single-stranded regions transiently formed in DNA during transcription and replication.

Adenine↗
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

Oxidation of thymine to 5-formyluracil in DNA: mechanisms of formation, structural implications, and base excision by human cell free extracts.

Oxidative agents produce several different types of base modifications in DNA, and only a few of these have been properly characterized with respect to mechanisms of formation and biological implications. We have established a procedure using neutral thermal hydrolysis and reverse phase high-performance liquid chromatography to determine the content of the oxidation product 5-formyluracil (5-foU) in DNA. With this method, it is shown that 5-foU residues are formed with high frequency from thymine by quinone-sensitized UV-A photooxidation. Since 5-foU is also induced by ionizing radiation, it appears to be formed under conditions where thymidine radical cations are generated and react with molecular oxygen. It was previously shown that 5-foU is formed directly from [methyl-3H]thymine residues in radioactively labeled DNA by two consecutive transmutations of 3H to 3He. The theoretical basis for the kinetics of such conversion is presented in this paper, and the calculated yields are confirmed experimentally by measuring the content of 5-foU in [methyl-3H]thymine-labeled DNA aged for different time periods. Such DNA contains virtually only 5-(hydroxymethyl)uracil and 5-foU, apart from normal bases, and is therefore very useful for the investigation of repair enzyme activities involved in the repair of 5-foU-containing DNA. Using this substrate, a DNA glycosylase activity was identified in human cell extracts for the removal of 5-foU.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
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↗
PubMed1995

Spectrum of mutations induced by methyl and ethyl methanesulfonate at the hprt locus of normal and tag expressing Chinese hamster fibroblasts.

This work describes the isolation and characterization of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) induced 6-thioguanine-resistant mutants in normal and Escherichia coli tag gene expressing Chinese hamster fibroblast, RJKO, cells. It was previously shown that increased removal of 3-alkylated adenine, effected by 3-methyladenine DNA glycosylase I (Tag), reduces the frequencies of hprt mutations induced by alkylating agents which produce mostly N-alkylation (MMS and EMS) to half the normal rate. In order to identify which type of mutation is suppressed by increased 3-alkyladenine repair we have determined the DNA base sequence changes of the hprt cDNA in 61 independent MMS- and EMS-induced mutant clones. For both cell types and irrespective of the agent used, the majority of mutations were GC to AT transitions originating in the non-transcribed strand. Only 6/55 base substitutions occurred at AT base pairs: five AT to GC transitions and one AT to CG transversion. Six mutations were found to be deletions. These results indicate that 3-alkylated adenines in DNA are not directly premutagenic. The fact that the mutation frequency is reduced by increased 3-alkyladenine removal might be explained by postulating the existence in mammalian cells of an SOS-like response turned on by cytotoxic lesions like 3-alkyladenine, or, alternatively, that increased removal of 3-alkyladenine increases the number of single-strand breaks in DNA, which stalls DNA replication and allows a prolonged time for DNA repair by the alkyltransferase.

Alkylation↗
PubMed1994

DNA glycosylase activities for thymine residues oxidized in the methyl group are functions of the AlkA enzyme in Escherichia coli.

The alkA gene of Escherichia coli encodes a DNA glycosylase involved in base excision repair of DNA alkylation damage. In an attempt to define the reactions of the AlkA enzyme with methylated DNA, we discovered that the enzyme released substantial amounts of radioactivity from [methyl-3H]thymidine-labeled DNA even without any exposure of the DNA to methylating agents. The excised material was identified by chromatography as two different oxidized derivatives of thymine, 5-hydroxymethyluracil and 5-formyluracil. These products are formed in such DNA by one and two consecutive decays, respectively, of the tritiums of the labeled methyl group. Kinetic analysis showed that both the apparent Km and Vmax values for 5-formyluracil removal are within the same range as found for 3-methyladenine removal, suggesting that this catalytic property of AlkA is also significant under in vivo conditions. Removal of 5-hydroxymethyluracil proceeds at a rate that is 1-3 orders of magnitude slower. Since both 5-formyluracil and 5-hydroxymethyluracil are major products formed in DNA by exposure to ionizing radiation, these results implicate the alkA gene function also in the repair of oxidative DNA damage. Neither of the two other enzymes involved in the repair of oxidative DNA damage in E. coli, i.e. endonuclease III and formamidopyrimidine DNA glycosylase, has any affinity for oxidized unsaturated pyrimidines in DNA.

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↗
PubMed1994

Identification of cytidine deaminase as inhibitor of granulocyte-macrophage colony formation.

Mature human blood granulocytes produce regulatory factors that inhibit colony formation by human and murine granulocyte-macrophage colony-forming cells (GM-CFC). The inhibition of GM-CFC by granulocyte extract (GRE) was strongly enhanced by the addition of thymidine (3 to 6 x 10(-5) M for human cells) and by the presence of fetal calf serum (FCS) in the growth medium. Deoxycytidine and deoxyuridine produced effects similar to those of thymidine, but at higher concentrations (2 to 4 x 10(-4) M). It was further observed that GRE prevented the antiproliferative effects of cytosine arabinoside (Ara-C) and azadeoxycytidine, suggesting that GRE contained cytidine deaminase (CDD) activity, since CDD is known to abolish the effects of these nucleoside analogs. Accordingly, the GRE was tested for and shown to contain an enzymatic activity that converted deoxycytidine to deoxyuridine, confirming the presence of CDD activity in GRE. The GM-CFC inhibition factor was found to copurify with CDD activity during three succeeding chromatographic separations, indicating that CDD was indeed the inhibiting factor itself. This conclusion was further substantiated by gel filtration experiments demonstrating a molecular weight (MW) of approximately 50 kd, which corresponds to the MW previously published for CDD activity. Furthermore, addition of tetrahydrouridine (THU), a known specific inhibitor of CDD, abolished the suppressive effect of GRE on GM-CFC, which independently confirmed the identification of CDD as an inhibitor of GM-CFC. The growth-regulating property of CDD could be explained by depletion of deoxycytidine nucleotides necessary for DNA synthesis or by a direct effect of CDD binding to specific receptors on progenitor cells.

Animals↗
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↗