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H Kersten

Publications and source records attributed to H Kersten.

At least 37 records · Page 2Linked to original sources
PubMed1989

The tyrT locus of Escherichia coli exhibits a regulatory function for glycine metabolism.

The tyrT locus in Escherichia coli codes for two gene copies of tRNA(1Tyr). Both genes are organized in one operon, which has a unique structure. The two tRNA genes are separated by a spacer segment highly homologous to a part of a unit which is repeated three times in the distal portion of the locus. This operon also contains coding capacity for a small basic protein. A genomic deletion of this locus was constructed and marked by a kanamycin resistance cassette. Deletion mutants exhibited a characteristic phenotype when cells were shifted from rich medium to minimal medium. The cells entered a transient lag phase, apparently resulting from specific glycine starvation. This phenotype involved stringent response and was therefore not observed in relA derivatives. The genomic deletion was complemented in trans by a plasmid-borne tyrT locus. From deletion mapping, it can be concluded that a product of the tyrT operon is responsible for complementation. However, neither the tRNA(1Tyr) nor the proposed basic protein is the complementation-competent entity.

Amino Acids↗
PubMed1989

Mutations in the Escherichia coli fnr and tgt genes: control of molybdate reductase activity and the cytochrome d complex by fnr.

In eubacteria, the tRNA transglycosylase (Tgt) in specific tRNAs exchanges a guanine in the anticodon for 7-aminomethyl-7-deazaguanine, which is finally converted to queuosine. The tgt gene of Escherichia coli has been mapped at 9 min on the genome, and mutant pairs containing an intact or mutated tgt allele were obtained after transduction of the tgt locus by P1 bacteriophages into a genetically defined E. coli strain (S. Noguchi, Y. Nishimura, Y. Hirota, and S. Nishimura, J. Biol. Chem. 257:6544-6550, 1982). These tgt mutants grew anerobically with fumarate as an electron acceptor, while nitrate or trimethylamine N-oxide could not be reduced. Furthermore, molybdate reductase activity was almost lacking and the characteristic absorption maxima, corresponding to cytochrome a1 and the cytochrome d complex, were not detectable in low-temperature reduced-minus-oxidized difference spectra in anaerobically grown cells. Transduction of the mutated tgt locus into another E. coli recipient resulted in tgt mutants without anaerobic defects. Transformation of the original tgt mutants with an fnr gene-containing plasmid reversed the anaerobic defects. Clearly, the original tgt mutants harbor a second mutation, affecting the anaerobic regulator protein Fnr. The results suggest that fnr is involved in anaerobic control of components of the cytochrome d complex and of the redox system that transfers electrons to molybdate. F' plasmids containing a fused lacI-lacZ gene with the nonsense codon UAG at different positions in the lacI part were transferred to E. coli strains with a mutated or nonmutated tgt locus but intact in fnr. A twofold increase in the frequency of incorrect readthrough of the UAG codon, dependent on the codon context, was observed in the tgt mutant and is suggested to be caused by a tRNA(Tyr) with G in place of queuosine.

Base Sequence↗
PubMed1988

New function of vitamin B12: cobamide-dependent reduction of epoxyqueuosine to queuosine in tRNAs of Escherichia coli and Salmonella typhimurium.

Queuosine (Q), 7-[(4,5-cis-dihydroxy-2-cyclopentene-1-yl)-amino)methyl)-7- deazaguanosine, and Q derivatives usually replace guanosine in the anticodon of tRNAs(GUN) of eubacteria and of cytoplasmic and mitochondrial tRNAs of lower and higher eucaryotes except yeasts. Q appears to be synthesized de novo exclusively in eubacteria, and the free-base queuine serves as a nutrient factor for eucaryotes. Recently, a Q derivative, oQ, containing a 2,3-epoxy-4,5-dihydroxycyclopentane ring, has been identified in Escherichia coli tRNA(Tyr). Here we show that oQ is formed when E. coli or Salmonella typhimurium is grown in glucose-salt medium. The formation of oQ was independent of molecular oxygen, and oQ-tRNAs were converted to Q-tRNAs by adding cobalamin to the growth medium. Under strictly anaerobic conditions, considerable amounts of Q were present in E. coli and S. typhimurium tRNAs when the bacteria were grown in the presence of cobalt ions with glycerol as the carbon source and fumarate as the electron acceptor. Under these conditions, the biosynthesis of cobalamin was induced. The results suggest that oQ is derived from ribose and that oQ is finally reduced to Q by a cobamide-dependent enzyme.

Aerobiosis↗
PubMed1988

The nutrient factor queuine: biosynthesis, occurrence in transfer RNA and function.

Queuine, 7-(( (4,5-cis-dihydroxy-2-cyclopenten-1-yl)-amino]-methyl)-7-deazagu ani ne is synthesized de novo only in eubacteria and is preseent in place of guanine 34 in specific tRNAs containing anticodones GUN where N is one of the four canonical nucleotides. The biosynthetic pathway starting with GTP shares common steps with that of pteridines and riboflavin, and involves iron ions and a 'vitamin B12' coenzyme. Lower and higher eukaryotes are supplied with queuine by nutrition or the intestinal flora. The modification of tRNA with queuine is tissue specific and depends on the metabolic state of cells and tissues. Starvation for queuine and/or Q-deficiency in tRNA causes a few specific changes in the pattern of protein synthesis involving lactate dehydrogenases and cytochromes.

Animals↗
PubMed1986

tRNA (adenine-N1)-methyltransferase from Dictyostelium discoideum. Purification, characterization and developmental changes in activity.

An enzyme activity transferring methyl groups from S-adenosylmethionine to endogenous tRNA was detected in the cytosol of aggregative Dictyostelium discoideum amoebae. This enzyme was purified more than 1000-fold and was characterized as a tRNA (adenine-N1-)-methyltransferase. Kinetic analysis yielded a K0.5 for S-adenosylmethionine of 0.27 microM and competitive inhibition by S-adenosylhomocysteine showed an I0.5 of 0.26 microM. The tRNA methyltransferase activity was stimulated by monovalent cations and the pH optimum was 7.3. tRNAs isolated from D. discoideum as well as from other eucaryotic sources could be methylated only to a minor extent. In contrast, Escherichia coli tRNA accepted up to 0.6 mol methyl group/mol tRNA, suggesting that the target nucleotide is unmethylated in procaryotic tRNA, but is commonly methylated in tRNAs from eucaryotic organisms. The activity of the methyltransferase increased 4-6-fold during cell differentiation from the vegetative to the aggregative stage.

Cations↗
PubMed1986

[Adaptation of cell metabolism to environmental changes: regulation of gene expression of transfer RNA and unusual nucleic acid building-blocks].

In the living cell transfer ribonucleic acids (tRNAs) serve for the transfer of information from genes to proteins. In this article evidence will be presented showing that changes of particular tRNA modifications cause alterations in gene expression, when an organism is exposed to a metabolic stress, e.g. limitation of oxygen or nutrients. tRNA modifications seem to be important to adapt cells to environmental changes. These mechanisms of adaptation are considered to have developed as survival strategies in microorganisms especially when oxygen accumulated in the atmosphere.

Adaptation, Physiological↗
PubMed1985

Differential turnover of tRNAs of the queuosine family in Dictyostelium discoideum and its possible role in regulation.

In its natural environment the protist Dictyostelium discoideum grows on bacteria and queuosine-containing tRNAs of the bacteria serve as source of the nutrient factor queuine. This deazaguanine derivative is inserted into tRNAAsp, tRNAAsn, tRNAHis and tRNATyr of the amoebae. The axenic strain AX-2 of D. discoideum grows equally well in a defined medium with or without exogenous queuine. When queuine is omitted, changes occur in lactate levels, lactate dehydrogenase patterns and cytochromes and the amoebae cannot differentiate after a metabolic stress. In this report we show that growing cells contain two-fold higher levels of tRNAAsp and tRNATyr when sufficiently supplied with queuine, than those lacking queuine. In tRNAAsp a new, as yet unidentified, derivative of queuine has been discovered. When RNA synthesis is totally inhibited by actinomycin, tRNAAsp and tRNATyr remain stable in queuine-containing, but not in queuine-lacking cells. In contrast, tRNAAsn and tRNAHis become partially degraded in both conditions. We suggest that free queuine can be obtained from endogeneous tRNA and that differential salvage of queuine by tRNAs of the Q-family plays a role in the regulation of genes encoding components of redox chains.

Amino Acids↗
PubMed1984

Specific changes in lactate levels, lactate dehydrogenase patterns and cytochrome b559 in Dictyostelium discoideum caused by queuine.

Higher eukaryotes contain tRNA transglycosylases that incorporate the guanine derivative queuine from the nutritional environment into specific tRNAs by exchange with guanine at position 34. Alterations in the queuosine content of specific tRNAs are suggested to be involved in regulatory mechanisms of major routes of metabolism during differentiation. Dictyostelium discoideum has been applied as a model to investigate the function of queuine or queuine-containing tRNAs. Axenic strains are supplied with queuine by peptone, but they grow equally well in a defined queuine-free medium. Queuine-lacking amoebae, starved in suspension culture for 24 h, lose their ability to differentiate into stalk cells and spores, whereas amoebae sufficiently supplied with queuine will overcome this metabolic stress and undergo further development when plated on agar. The results presented here show that D(-)-lactate occurs in the slime mould in millimolar amounts and that its level is remarkably decreased in queuine-lacking cells after 24 h of starvation in suspension culture. On isoelectric-focusing polyacrylamide gels, nine different forms of NAD-dependent D(-)-lactate dehydrogenase can be separated from extracts of vegetative cells, and six forms from extracts of the starved cells. Under queuine limitation, one form is missing in the starved cells. Low amounts of L(+)-lactate are usually found in vegetative amoebae but significantly less in queuine-lacking cells. Five forms of NAD-dependent L(+)-lactate dehydrogenase are detectable in extracts from vegetative, queuine-treated cells, and slight alterations occur in queuine-deficient amoebae. In the starved cells only one form of L(+)-lactate dehydrogenase is found, irrespective of the supply of queuine to the cells. A cytochrome of type b with an absorption maximum at 559 nm accumulates during starvation only in queuine-lacking cells; it might be a component of an NAD-independent lactic acid oxidoreductase as is cytochrome b 557 in yeast and be responsible for the reduced level of lactate in cells lacking queuine in tRNA.

Cytochrome b Group↗
PubMed1984

An antisuppressor mutation of Schizosaccharomyces pombe affects the post-transcriptional modification of the "wobble" base in the anticodon of tRNAs.

The screening of antisuppressor mutants of the yeast Schizosaccharomyces pombe has been successfully accomplished with high resolution liquid chromatographic methods for the analysis of tRNA nucleosides. Antisuppressor mutations reduce or abolish the function of nonsense suppressor-tRNAs or other informational suppressors. Nonradioactive or 35S-labeled unfractionated tRNA from various strains was digested to nucleosides and analyzed by high performance liquid chromatography. The mutant sin3 has lost the nucleoside 5-(methoxycarbonylmethyl)-2-thiouridine from its tRNA in comparison to parental strains. In eukaryotes this nucleoside is found at the first position of the anticodon (wobble position) in several isoacceptor tRNAs that preferentially recognize codons ending with adenosine. The sin3 mutation reduces the efficiency of UGA and UAA suppressor tRNASer and suppressor tRNALeu. The genetic cosegregation of modification loss, antisuppressor phenotype, and a change in cell size is demonstrated. This indicates that a single mutation in the structural gene for a tRNA modification enzyme causes the three different phenotypes.

Anticodon↗
PubMed1984

Queuosine modification in tRNA and expression of the nitrate reductase in Escherichia coli.

In eubacteria the modified nucleoside queuosine is present in tRNAAsn, tRNAAsp, tRNAHis and tRNATyr. A precursor of queuine, pre-queuine, is synthesized from GTP, inserted into the first position of the anticodon of the corresponding tRNAs by a specific tRNA-guanine transglycosylase and further modified to queuosine. Isogenic pairs of Escherichia coli, containing or lacking the tRNA-transglycosylase (JE 7335, tgt+ lacZ+ and JE 7337, tgt- lacZ+; JE 7334, tgt+ lacZ- and JE 7336, tgt- lacZ-), have been employed to study the function of queuosine in tRNA. Compared with the tgt+ strain (JE 7335), the tgt- mutant (JE 7337) grown under anaerobic conditions, is defective with respect to the nitrate respiration system, in which electrons are transported from D(-)-lactate via quinone and cytochrome bNO3-(556) to nitrate. Low temperature cytochrome spectra of the anaerobically grown tgt- mutant show a lowered amount of type b cytochromes involving the spectrum of cytochrome bNO3-(556). In the case of the anaerobically grown tgt- mutant three proteins are missing in the protein pattern of cytoplasmic membranes. Their mol. wts. correspond to those of the subunits of the nitrate reductase complex. In contrast to the tgt+ strains (JE 7334, JE 7335) both tgt- mutants (JE 7336, JE 7337) cannot grow on lactate under anaerobic conditions with nitrate offered as electron acceptor and NO3- is not reduced to NO2-. A possible link between Q-modification of tRNAs, the synthesis of proteins of the nitrate reductase complex and the synthesis of menaquinone or ubiquinone is discussed.

Aerobiosis↗
PubMed1983

Alteration of tRNA modification in eukaryotes: causes and consequences.

To evaluate the role of the modified nucleosides in tRNA function, especially their involvement in regulatory mechanisms of development, differentiation, or neoplastic transformation we use the following organisms: eubacteria, the slime mold D. discoideum, the topminnow Xiphophorus, and mice. Ribosylthymine, a common modified nucleoside at position 54 in tRNAs of prokaryotes and the major class of eukaryotic elongator tRNAs, is involved in the binding to the ribosomal A-site and is important for the proper functioning of tRNA during translation. Alterations in the extent of this modification occur early in the development of D. discoideum. The fully methylated species are found on polysomes, actively synthesizing protein. The partially methylated tRNAs accumulate in the nuclei, and might be involved in regulatory mechanisms at the transcriptional level. The Q base, a modified deazaguanine derivative, is present at position 34, the first position of the anticodon of tRNAAsn, tRNATyr, and tRNAHis. Alterations in the extent of this modification occur in corresponding tRNAs during the first minutes after the onset of development in D. discoideum and before final differentiation into spores, indicating that Q is important for developmental processes. Changes in the modification of G34 to Q34 in specific tRNAs of the melanophoric system of the topminnow Xiphophorus further support the view that Q is necessary in differentiation. In plasmacytomas and in Ehrlich ascites tumor cells of mice, the amount of unmodified G34 in corresponding tRNAs is correlated to the growth rate, density, or age of the tumor cells.

Animals↗
PubMed1982

Regulation of tRNA methyltransferase activities by spermidine and putrescine. Inhibition of polyamine synthesis and tRNA methylation by alpha-methylornithine or 1,3-diaminopropan-2-ol in Dictyostelium.

Inhibitors of polyamine synthesis (alpha-methylornithine and 1,3-diaminopropan-2-ol) were used to study the relationship between polyamine synthesis and specific methylations of tRNA in Dictyostelium discoideum during vegetative growth. Polyamine concentrations were found to be 10 mM for putrescine, 1.6 mM for spermidine and 7 mM for 1,3-diaminopropane throughout the growth stage. On treatment of growing amoebae with alpha-methylornithine or with 1,3-diaminopropan-2-ol (each at 5 mM), the syntheses of putrescine, spermidine and 1,3-diaminopropane were arrested within 4h. After polyamine synthesis had ceased, the incorporation of methyl groups into tRNA was considerably decreased under conditions that had no effect on the incorporation of uridine into tRNA, or on net syntheses of protein and of DNA. The following nucleosides in tRNA were concerned: 1 methyladenosine, 5-methylcytidine, 7-methylguanosine, 2-methylguanosine, N2N2-dimethylguanosine and 5-methyluridine (ribosylthymine). The corresponding tRNA methyltransferases, determined in Mg2+-free enzyme extracts, proved to be inactive unless polyamines were added. Putrescine and/or spermidine at concentrations of 10 mM or 1-2 mM respectively stimulate the transmethylation reaction in vitro to a maximal rate and to an optimal extent at exactly the same concentrations as found in vegetative cells. In contrast, 1,3-diaminopropane, which is formed from spermidine, does not affect the methylation of tRNA in vitro at physiological concentrations. Putrescine and/or spermidine stabilize the tRNA methyltransferases in crude extracts in the presence but not in the absence of the substrate tRNA. The results support the view that S-adenosylmethionine-dependent transmethylation reactions can be regulated by alterations of polyamine concentrations in vivo.

Diamines↗
PubMed1981

Measurements of polyamines and their acetylated derivatives in cell extracts and physiological fluids by use of an amino acid analyzer.

A fast and sensitive method for the determination of free polyamines and their acetylated derivatives is presented. The separation is carried out on a Durrum DC-6A cation-exchange resin with an automated amino acid analyzer. The determination is based on a step wise elution with a sodium chloride-sodium citrate buffer system. Detection is done by fluorescence of the o-phthaldialdehyde-polyamine conjugates. The sensitivity is in the picomole range. No prior purification step is needed. The method has been applied to cell extracts and urine samples.

Amino Acids↗