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PubMed1997

Determination of steady-state levels of oxidative DNA base modifications in mammalian cells by means of repair endonucleases.

The alkaline elution technique in combination with various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) was used to quantify steady-state (background) levels of oxidative base modifications in various types of mammalian cells. In human lymphocytes the number of base modifications sensitive to Fpg protein, which include 8-hydroxyguanine, was 0.25 +/- 0.05 per 10(6) base pairs. Even lower levels (0.07 +/- 0.02 per 10(6) bp) were observed in HeLa cells. The numbers of sites sensitive to the other repair endonucleases were below the detection limit (0.05 per 10(6) bp). In a direct comparison, the background level of Fpg-sensitive modifications determined by alkaline elution was much lower than the background level of 8-hydroxydesoxyguanosine (8-oxodG) determined after enzymatic DNA hydrolysis by HPLC and electrochemical detection. However, the number of additional Fpg-sensitive modifications induced by a photosensitizer plus light was similar to the additional number of 8-oxodG residues determined by HPLC with electrochemical detection. This indicates that the enzyme assay does not systematically underestimate the number of lesions and points to an artefactual generation of 8-oxodG during DNA isolation and hydrolysis.

Chromatography, High Pressure Liquid↗
PubMed1998

Effect of smoking cessation on oxidative DNA modification estimated by 8-oxo-7,8-dihydro-2'-deoxyguanosine excretion.

BACKGROUND: Reactive oxygen species from, e.g. tobacco smoke are suggested to be involved in carcinogenesis by oxidative modification of DNA. The urinary excretion rate of the oxidized nucleoside 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) has been validated as a biomarker of the rate of oxidative DNA modification with mechanistic relation to carcinogenesis. In cross-sectional studies, the urinary excretion rate of 8-oxodG has been shown to be elevated in smokers compared with non-smokers. PURPOSE: In this randomised, controlled smoking cessation study, we investigated whether cigarette smoking per se causes oxidative DNA modification. METHODS: Of the 182 healthy smokers included, 100 were randomized to quit smoking after baseline samples had been taken, and 82 were randomized to continue usual smoking. Before the start of the study and after 4 weeks, the subjects collected 24-h urine samples that were analysed for 8-oxodG content by high-pressure liquid chromatography with electrochemical detection. The subjects randomized to smoking cessation were followed up after 26 weeks. RESULTS: Four weeks of smoking cessation resulted in a 21% decrease in 8-oxodG excretion rate (from mean +/- SD, 30.5 +/- 13.9 to 24.1 +/- 10.5 nmol/24 h, P < 0.001) in 58 quitters included in per-protocol data analysis. Sixty-five continued smokers included in per-protocol analysis showed a 9% decrease in 8-oxodG excretion rate (from 31.6 +/- 13.2 to 28.7 +/- 12.6 nmol/24 h, P = 0.026). After 4 weeks, the 8-oxodG excretion rate was 16% (95% confidence interval 4 to 28%) higher in the continued smokers than in the quitters (P = 0.0085, ANCOVA), demonstrating the effect of smoking per se. A 23% (P < 0.005) decrease in 8-oxodG excretion rate was sustained for 26 weeks in 27 quitters who completed the study. CONCLUSION: Smoking cessation significantly reduces the urinary excretion rate of 8-oxodG, giving direct and controlled evidence that cigarette smoking causes an increased rate of oxidative DNA modification. This could represent a mechanism by which tobacco smoke is carcinogenic.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Influence of glutathione levels and heat-shock on the steady-state levels of oxidative DNA base modifications in mammalian cells.

The effects of thiols, ascorbic acid and thermal stress on the basal (steady-state) levels of oxidative DNA base modifications were studied. In various types of untreated cultured mammalian cells, the levels of total glutathione were found to be inversely correlated with the levels of DNA base modifications sensitive to the repair endonuclease Fpg protein, which include 8-hydroxyguanine (8-oxoG). A depletion of glutathione by treatment with buthionine sulphoximine increased the steady-state level in AS52 Chinese hamster cells by approximately 50%. However, additional thiols in the culture medium did not reduce the level of Fpg-sensitive base modifications: 0-10 mM N-acetylcysteine had no effect, whereas cysteine ethylester even increased the oxidative DNA damage at concentrations >0.1 mM. Similarly, ascorbic acid (0-20 mM) failed to reduce the steady-state levels. When AS52 cells were grown at elevated temperature (41 degrees C), the steady-state level of the oxidative DNA modifications increased by 40%, in spite of a concomitant 1.6-fold increase of the cellular level of total glutathione. Depletion of glutathione at 41 degrees C nearly doubled the already elevated level of oxidative damage. A constitutive expression of the heat-shock protein Hsp27 in L929 mouse fibrosarcoma cells at 37 degrees C increased the glutathione level by 60%, but had little effect on the level of oxidative DNA damage.

Animals↗
PubMed1988

A comparison between different types of covalent DNA modifications (I-compounds, persistent carcinogen adducts and 5-methylcytosine) in regenerating rat liver.

I-Compounds have been recently identified as adduct-like nonpolar covalent DNA modifications that are detectable by 32P-postlabeling assay in tissues of untreated experimental animals and increase with age. Additional I-compounds have now been observed in liver DNA of male Sprague-Dawley rats when the chromatographic conditions were modified to allow for the detection of more polar adducts exhibiting low affinity to polyethyleneimine (PEI)--cellulose anion-exchange thin-layer material. The total I-compound level in 10-month-old animals was as high as one modification in approximately 10(7) nucleotides. This represented a minimum estimate since 100% recovery of all rat liver I-compounds in 32P-labeled form presumably was not achieved by the procedures used. The I-compound pattern was reproducible and variation of I-compound levels among individual animals of the same age was small. We have used regenerating rat liver herein as a model to compare the properties of I-compounds with those of persistent 2-acetylaminofluorene (AAF)-induced DNA adducts and of 5-methylcytosine (m5C), a normal enzymatic DNA modification. Eight- to 10-month-old male Sprague-Dawley rats were given 2-AAF (50 mg/kg in DMSO) or vehicle (DMSO) by i.p. injection. Partial hepatectomy was performed 6 weeks later (i.e. after AAF adduct levels had stabilized) and regenerating liver samples were taken 1 week after the operation for DNA analysis. Consistent with the restoration of cell and tissue loss, the overall levels of I-compounds and 2-AAF adducts were reduced to approximately 47% and approximately 45% respectively of control in regenerating liver by dilution with newly synthesized DNA, while the m5C level was not affected. Thus, in regenerating liver, I-compounds resembled carcinogen--DNA adducts and not m5C. This supports our hypothesis that the formation of these DNA modifications may be due to the binding to DNA of small amounts of reactive electrophilic by-products of normal metabolic activities, leading to the slow accumulation of I-compounds in tissue DNA with ageing.

2-Acetylaminofluorene↗
PubMed1988

Sequence, genomic distribution and DNA modification of a Mu1 element from non-mutator maize stocks.

The increased mutation rate of Mutator stocks of maize has been shown to be the result of transposition of Mu elements. One element, Mu1, is present in 10-60 copies in Mutator stocks and approximately 0-3 copies in non-Mutator stocks. The sequence, structure and genomic distribution of an intact Mu1 element cloned from the non-Mutator inbred line B37 has been determined. The sequence of this element, termed Mu1.4-B37, is identical to Mu1 and it is flanked by 9-bp direct repeats indicative of a target site duplication. Mu1.4-B37 is not in the same genomic location in all stocks, which further suggests that it transposed into its genomic location in B37. We previously reported that in genomic DNA this element is modified such that certain methylation-sensitive restriction enzymes will not cut sites within the element. This is similar to that observed for Mu elements in Mutator stocks that have lost activity. We report herein that the Mu1.4-B37 element loses its modification and becomes accessible to digestion when placed in an active Mutator stock by genetic crosses. This suggests that factors conditioning unmodified elements are dominant in the initial cross between Mutator and non-Mutator stocks. In F2 individuals that have subsequently lost Mutator activity the Mu1.4-B37 element again becomes modified as do most of the Mu elements in the stock. Thus, the modification state of the Mu1.4-B37 element and the other Mu1-like elements correlates with Mutator activity. We hypothesize that factor(s) within an active Mutator stock may inhibit the modification of Mu elements, and that this activity is missing in non-Mutator stocks and may become limiting in certain Mutator stocks resulting in DNA modification.

Base Sequence↗
PubMed2006

Proteomics studies of post-translational modifications in plants.

Post-translational modifications of proteins greatly increase protein complexity and dynamics, co-ordinating the intricate regulation of biological events. The global identification of post-translational modifications is a difficult task that is currently accelerated by advances in proteomics techniques. There has been significant development in sample preparation methods and mass spectrometry instrumentation. To reduce the complexity and to increase the amount of modified proteins available for analysis, proteins are usually subjected to prefractionation such as chromatographic purification and affinity enrichment. In this review, the post-translational modification studies in plants are summarized. The sample preparation strategies applied to each study are also described. These include affinity-based enrichment methods, immobilized metal affinity chromatography and immunoprecipitation used for phosphorylation and ubiquitination studies, respectively, and the phase partitioning approach for glycosylphosphatidylinositol modification studies.

Chromatography, Affinity↗
PubMed1983

Long-range conformational transition in yeast tRNAPhe, induced by the Y-base removal and detected by chloroacetaldehyde modification.

Chemical modification was used to study the conformational changes occurring in yeast tRNAPhe after the Y-base excision. The chemical probe was the adenine- and cytosine-specific reagent chloroacetaldehyde. Comparison of the modification patterns in tRNAPhe and tRNAPhe-Y shows that seven bases, adenines 35, 36 and 38 in the anticodon loop and adenines 73, 76 and cytosines 74, 75 in the 3'-terminus were modified in both tRNAs with a quantitative difference in the modification level of the anticodon loop bases. The most interesting, however, is the qualitative difference consisting in modification of cytosine-60 in the T psi C loop of tRNAPhe-Y. Some aspects of the mechanism of this long-distance conformational transition are briefly discussed.

Acetaldehyde↗
PubMed1983

Chemical modifications of the sigma subunit of the E. coli RNA polymerase.

The function of arginine, cysteine and carboxylic amino acid (glutamic and aspartic) residues of sigma was studied using chemical modification by group specific reagents. Following modification of 3 arginine residues with phenylglyoxal or 3 cysteine residues with N-ethylmaleimide (NEM) sigma activity was lost. Analysis of the kinetic data for inactivation indicated that one arginine or cysteine residue is essential for sigma activity. At low NEM concentration alkylation was limited to a non-critical cysteine which was identified as cysteine-132. Modification of arginine or cysteine residues had no observable effect on the binding of the inactivated sigma to the core polymerase. Modification of aspartic and/or glutamic acid residues with the water-soluble carbodiimides 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) or 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMC) resulted in loss of sigma activity. The inactivation data indicated that one carboxylic amino acid residue is essential for sigma activity. Sigma modified with EDC, CMC or EDC in the presence of glycine was inactive in supporting promoter binding and initiation by core polymerase. Reaction with EDC plus (3H)glycine resulted in the incorporation of glycine into sigma. The (3H)glycine-sigma was unable to form a stable holoenzyme complex.

Amino Acids, Dicarboxylic↗
PubMed1986

Highly selective chemical modification of cruciform loops by diethyl pyrocarbonate.

Diethyl pyrocarbonate reacts with the single-stranded loops of cruciform structures with great selectivity. Adenine bases are carbethoxylated, as a result of which the backbone may be cleaved with piperidine, and the level of chemical modification at each base may be determined. We have studied the ColE1 and (A-T)34 cruciforms of pColIR315 and pXG540. In each case we observe maximal modification at the most central adenosine of the loop, and an overall pattern of modification corresponding to a total loop size of about six bases. The results may be interpreted in terms of a model in which the loop has a defined tertiary structure. No modification was detected at either cruciform four-way junction, suggesting that this region is fully base-paired.

Base Sequence↗
PubMed1993

A novel method for the determination of post-transcriptional modification in RNA by mass spectrometry.

A method is described for the detection, chemical characterization and sequence placement of post-transcriptionally modified nucleotides in RNA. Molecular masses of oligonucleotides produced by RNase T1 hydrolysis can be measured by electrospray mass spectrometry with errors of less than 1 Da, which provides exact base composition, and recognition of modifications resulting from incremental increases in mass. Used in conjunction with combined liquid chromatography-mass spectrometry and gene sequence data, modified residues can be completely characterized at the nucleoside level, and assigned to sequence sites within oligonucleotides defined by selective RNase cleavage. The procedures are demonstrated using E.coli 5S rRNA, in which all RNase T1 fragments predicted from the rDNA sequence are identified solely on the basis of their molecular masses, and using E.coli 16S rRNA for analysis of post-transcriptional modification, including placement of 3-methyluridine at position 1498. The principles described are generally applicable to other covalent structural modifications of RNA which produce a change in mass, such as those resulting from editing, photochemical cross-linking, or xenobiotic modification.

Base Composition↗
PubMed1998

Effect of a mutation in the anticodon of human mitochondrial tRNAPro on its post-transcriptional modification pattern.

Although the gene sequences of all 22 tRNAs encoded in the human mitochondrial genome are known, little information exists about their sequences at the RNA level. This becomes a crucial limitation when searching for a molecular understanding of the growing number of maternally inherited human diseases correlated with point mutations in tRNA genes. Here we describe the sequence of human mt-tRNAPropurified from placenta. It shows absence of editing events in this tRNA and highlights the presence of eight post-transcriptional modifications. These include T54, never found so far in an animal mt-tRNA, and m1G37, a modification known to have fundamental functional properties in a number of canonical tRNAs. Occurrence of m1G37 was further investigated in an analysis of the substrate properties of in vitro transcripts of human mt-tRNAProtowards pure Escherichia coli methylguanosine transferase. This enzyme properly methylates G37 in mt-tRNA and is sensitive to the presence of a second G at position 36, neighboring the target nucleotide for methylation. Since mutation of nt 36 was shown to be correlated with myopathy, the potential consequences of non-modification or under-modification of mt-tRNA nucleotides in expression of the particular myopathy and of mitochondrial diseases in general are discussed.

Anticodon↗
PubMed2003

Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells.

Double-stranded short interfering RNAs (siRNA) induce post-transcriptional silencing in a variety of biological systems. In the present study we have investigated the structural requirements of chemically synthesised siRNAs to mediate efficient gene silencing in mammalian cells. In contrast to studies with Drosophila extracts, we found that synthetic, double-stranded siRNAs without specific nucleotide overhangs are highly efficient in gene silencing. Blocking of the 5'-hydroxyl terminus of the antisense strand leads to a dramatic loss of RNA interference activity, whereas blocking of the 3' terminus or blocking of the termini of the sense strand had no negative effect. We further demonstrate that synthetic siRNA molecules with internal 2'-O-methyl modification, but not molecules with terminal modifications, are protected against serum-derived nucleases. Finally, we analysed different sets of siRNA molecules with various 2'-O-methyl modifications for stability and activity. We demonstrate that 2'-O-methyl modifications at specific positions in the molecule improve stability of siRNAs in serum and are tolerated without significant loss of RNA interference activity. These second generation siRNAs will be better suited for potential therapeutic application of synthetic siRNAs in vivo.

Endoribonucleases↗
PubMed2005

The Small Subunit rRNA Modification Database.

The Small Subunit rRNA Modification Database provides a listing of reported post-transcriptionally modified nucleosides and sequence sites in small subunit rRNAs from bacteria, archaea and eukarya. Data are compiled from reports of full or partial rRNA sequences, including RNase T1 oligonucleotide catalogs reported in earlier literature in studies of phylogenetic relatedness. Options for data presentation include full sequence maps, some of which have been assembled by database curators with the aid of contemporary gene sequence data, and tabular forms organized by source organism or chemical identity of the modification. A total of 32 rRNA sequence alignments are provided, annotated with sites of modification and chemical identities of modifications if known, with provision for scrolling full sequences or user-dictated subsequences for comparative viewing for organisms of interest. The database can be accessed through the World Wide Web at http://medlib.med.utah.edu/SSUmods.

Base Sequence↗
PubMed2006

Post-transcriptional nucleotide modification and alternative folding of RNA.

Alternative foldings are an inherent property of RNA and a ubiquitous problem in scientific investigations. To a living organism, alternative foldings can be a blessing or a problem, and so nature has found both, ways to harness this property and ways to avoid the drawbacks. A simple and effective method employed by nature to avoid unwanted folding is the modulation of conformation space through post-transcriptional base modification. Modified nucleotides occur in almost all classes of natural RNAs in great chemical diversity. There are about 100 different base modifications known, which may perform a plethora of functions. The presumably most ancient and simple nucleotide modifications, such as methylations and uridine isomerization, are able to perform structural tasks on the most basic level, namely by blocking or reinforcing single base-pairs or even single hydrogen bonds in RNA. In this paper, functional, genomic and structural evidence on cases of folding space alteration by post-transcriptional modifications in native RNA are reviewed.

Base Sequence↗
PubMed2006

The tale beyond the tail: histone core domain modifications and the regulation of chromatin structure.

Histone post-translational modifications occur, not only in the N-terminal tail domains, but also in the core domains. While modifications in the N-terminal tail function largely through the regulation of the binding of non-histone proteins to chromatin, based on their location in the nucleosome, core domain modifications may also function through distinct mechanisms involving structural alterations to the nucleosome. This article reviews the recent developments in regards to these novel histone modifications and discusses their important role in the regulation of chromatin structure.

Animals↗
PubMed2003

Promotion of triplex formation by morpholino modification: thermodynamic and kinetic studies.

We examined the effect of morpholino (MOR) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The binding constant of the pyrimidine motif triplex formation at pH 6.8 with MOR-modified TFO was approximately 60 times larger than that observed with unmodified TFO. Kinetic data demonstrated that the observed increase in the binding constant at neutral pH by the MOR backbone modification resulted mainly from the considerable increase in the association rate constant. The present results certainly support the idea that the MOR backbone modification of TFO could be a key modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

Base Sequence↗
PubMed2003

Decoding property of C5 uridine modification at the wobble position of tRNA anticodon.

Post-transcriptional modification at the first (wobble) position of the tRNA anticodon participates in precise decoding of the genetic code. We recently identified a novel taurine-containing modified uridine (tau m5U; 5-taurinomethyluridine) at the wobble position of mammalian mitochondrial tRNAs and found lack of this modification in mutant mitochondrial tRNAs from human pathogenic cells of the mitochondrial encephalomyopathies, investigate molecular pathogenesis of the diseases, decoding activity of wobble uridines with or without C5 modification was measured using E. coli cell-free translation system. It has been revealed that C5 modification has a functional role for stabilizing U:G wobble base pair.

Amino Acid Sequence↗
PubMed2000

Posttranscriptional modification of transfer RNA in the submarine hyperthermophile Pyrolobus fumarii.

In the RNA of hyperthermophiles, which grow optimally between 80 degrees C and 106 degrees C, posttranscriptional modification has been identified as a leading mechanism of structural stabilization. Particularly in the Archaeal evolutionary domain these modifications are expressed as a structurally diverse array of modification motifs, many of which include ribose methylation. Using mass spectrometric techniques we have examined the posttranscriptional modifications in unfractionated tRNA from the remarkable organism Pyrolobus fumarii, which grows optimally at 106 degrees C, but up to 113 degrees C (Blöchl et al. (1997), Extremophiles, 1, 14-21). Twenty-six modified nucleosides were detected, 11 of which are methylated in ribose. A new RNA nucleoside, 1,2'-O-dimethylguanosine (m1Gm) was characterized and the structure confirmed by chemical synthesis.

Chromatography, High Pressure Liquid↗