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At least 163 records · Page 9Linked to original sources
PubMed2003

DNA modification and functional delivery into human cells using Escherichia coli DH10B.

The availability of almost the complete human genome as cloned BAC libraries represents a valuable resource for functional genomic analysis, which, however, has been somewhat limited by the ability to modify and transfer this DNA into mammalian cells intact. Here we report a novel comprehensive Escherichia coli-based vector system for the modification, propagation and delivery of large human genomic BAC clones into mammalian cells. The GET recombination inducible homologous recombination system was used in the BAC host strain E.coli DH10B to precisely insert an EGFPneo cassette into the vector portion of a approximately 200 kb human BAC clone, providing a relatively simple method to directly convert available BAC clones into suitable vectors for mammalian cells. GET recombination was also used for the targeted deletion of the asd gene from the E.coli chromosome, resulting in defective cell wall synthesis and diaminopimelic acid auxotrophy. Transfer of the Yersinia pseudotuberculosis invasin gene into E.coli DH10B asd(-) rendered it competent to invade HeLa cells and deliver DNA, as judged by transient expression of green fluorescent protein and stable neomycin-resistant colonies. The efficiency of DNA transfer and survival of HeLa cells has been optimized for incubation time and multiplicity of infection of invasive E.coli with HeLa cells. This combination of E.coli-based homologous recombination and invasion technologies using BAC host strain E.coli DH10B will greatly improve the utility of the available BAC libraries from the human and other genomes for gene expression and functional genomic studies.

Chromosomes, Artificial, Bacterial↗
PubMed2003

Effects of a piperidine ligand on DNA modification by antitumor cisplatin analogues.

Replacement of the ammine group in antitumor cisplatin by a heterocyclic ligand (piperidine, piperazine, or 4-picoline) results in reduction of cytotoxicity in human ovarian cancer cells. DNA is generally believed to be a major pharmacological target of antitumor platinum complexes. Therefore, we examined conformation of oligodeoxyribonucleotide duplexes containing a cross-link of cis-[PtCl(2)(NH(3))(piperidine)], their recognition by high mobility group proteins, and nucleotide excision repair; that is, some of the processes that may mediate antitumor effects of platinum drugs. The replacement does not affect the DNA binding mode including conformational alterations and excision of the cross-links. The results suggest that in certain cancer cells the lower cytotoxicity of cis-[PtCl(2)(NH(3))(piperidine)] might be partially associated with reduced affinity of the high mobility group proteins to the major intrastrand cross-links of this analogue relative to the same adducts of cisplatin. Besides this and a number of other biochemical factors, the reduced intracellular accumulation with subsequent effects on the level of DNA platination in the cells may also contribute to the reduced cytotoxicity of cis-[PtCl(2)(NH(3))(piperidine)]. The results support the view that the concept based on the design of the complexes structurally derived from cisplatin that do not present an altered DNA binding mode may be less effective in the search for new platinum drugs that would overcome cisplatin resistance.

Antineoplastic Agents↗
PubMed1994

Detection of sulfur mustard-induced DNA modifications.

Sulfur mustard is acutely toxic to the skin, eyes, and respiratory tract, and is considered carcinogenic to humans by the IARC. Since all of these toxicities are thought to be initiated by DNA alkylation, the level of DNA damage should serve as a biomarker for exposure. To develop methods of detecting this damage, DNA was modified by [14C]-labeled sulfur mustard and DNA adducts were released by mild acid hydrolysis. Radioactivity co-eluted on HPLC analysis with marker 7-(2-hydroxyethylthioethyl) guanine and 3-(2-hydroxyethylthio-ethyl) adenine synthesized from 2-chloroethyl 2-hydroxy-ethyl sulfide. Unambiguous identification of the major adduct, 7-(2-hydroxy-ethylthioethyl) guanine, was provided by gas chromatography combined with mass spectrometric detection. The most abundant adduct, 7-(2-hydroxyethyl-thioethyl) guanine, accounted for 61% of the total alkylation and could be detected as a fluorescent HPLC peak with a detection limit of 10 pmol. To demonstrate the applicability of this method to biological samples, DNA was extracted from the white blood cells of human blood exposed to 131 microM sulfur mustard in vitro and shown to contain 470 pmol of 7-(2-hydroxyethylthio-ethyl) guanine per mg of DNA.

Animals↗
PubMed1990

DNA modification in vivo by derivatives of glucose: enhancement by glutathione depletion.

When BHK or HTC cells are cultured for 20 min with [U-14C]glucose in the presence of agents that deplete reduced glutathione, DNA banded from the cells in cesium salt gradients containing guanidium HCl is radioactively labeled. This depletion-dependent labeling required live cells. It was not caused by reactive contaminants in the radioactive glucose preparations, by carbohydrate or protein comigration into the DNA band, or by metabolism of glucose into deoxyribose. Labeling levels are similar whether depletion is achieved by oxidation (with the drug diamide) or by inhibition of synthesis (with methionine sulfoximine). A temporal association between GSH repletion and the appearance of D-lactate, the putative unique product of GSH-dependent glyoxylase action on pyruvaldehyde, suggests possible involvement of 3-carbon dicarbonyls.

Cell Line↗
PubMed1987

O2- and O4-ethylthymine and the ethylphosphotriester dTp(Et)dT are highly persistent DNA modifications in slowly dividing tissues of the ethylnitrosourea-treated rat.

Male Wistar rats received a single i.p. injection of [3H]ethylnitrosourea (140 mg/kg) and were killed after 2 h, 1, 3, 6, 28 or 56 days. DNA of the following organs was isolated and analysed for the presence of 12 different ethylated bases and ethylphosphotriesters: brain, lung, liver, spleen, kidney, intestine, testis and bone marrow. At 2 h after injection the extent of DNA ethylation was found to be heterogeneous: highest in liver and lowest (3-4 times lower) in testis and brain. The rates at which O2- and O4-ethylthymine and the ethylphosphotriester dTp(Et)dT were lost, were very low in all organs except intestine and spleen. Most likely, loss in the latter organs is exclusively due to cell turnover. The rate of O6-ethylguanine repair strongly varied from organ to organ: high in liver, very low in testis and brain and intermediate in the other organs. In none of the DNAs were significant amounts of imidazole ring-opened 7-ethylguanine found. Our results strengthen the notion that a substantial part of carcinogen-induced DNA damage is of a highly persistent nature and might contribute to the carcinogenic process long after the original exposure has occurred.

Animals↗
PubMed1991

Sensitive detection of DNA modifications induced by cisplatin and carboplatin in vitro and in vivo using a monoclonal antibody.

An assay that is based upon a monoclonal antibody (ICR4) is described that enables the quantitation of cisplatin-induced adducts on DNA down to 3 nmol Pt/g DNA (i.e., 1 Pt adduct/10(6) bases), the level necessary to produce toxic effects in cells in vitro and in vivo, using just a few micrograms of DNA. Detection is possible below this level (although probably not necessary for in vivo studies) but the cross-reactivity of unmodified DNA sequences complicates absolute quantitation of adducts. Therefore, it will be possible to investigate the distribution of clinically useful platinum drugs in patients undergoing chemotherapy. Rats of strain F344 appeared to be the best, among several tested, for the production of antibodies to modified DNA, and they were used for the production of hybridomas. Fifteen hybridomas which secreted antibodies that bound to DNA that was highly modified with cisplatin but not to normal DNA were obtained. One (ICR4) was chosen for further characterization because of its relatively strong binding to DNA modified to a moderate level with cisplatin. The characterization included the development of a sensitive competitive enzyme-linked immunoabsorbent assay and the use of DNA that had been reacted with cisplatin both in vitro and in vivo. The levels of platination of both types of DNA samples were determined by atomic absorbance spectroscopy. For DNA that had been exposed to cisplatin in vitro, 50% inhibition of antibody binding was caused by about 15 fmol of total DNA-bound Pt/assay well. At moderate levels of platination, heating of the DNA solution at 100 degrees C for 5 min increased its immunoreactivity such that 50% inhibition was caused by 2.5 fmol Pt adducts/well. Pt adducts on DNA extracted from cells that had been treated with cisplatin were less immunoreactive than DNA treated with cisplatin in vitro, but after heating the immunoreactivity increased such that 50% inhibition in the assay was caused by 2 fmol Pt adduct/well. This sensitivity was invariant over a wide range of levels of platinum adduct frequency. DNA adducts formed by the second generation anticancer drug carboplatin were recognized similarly to the adducts formed by cisplatin, but those formed by the clinically inactive trans-diamminedichloroplatinum(II) or chloro(diethylenetriamine)-platinum(II)-chloride were not significantly immunoreactive. Control DNA cross-reacted in the competitive assay but the immunoreactivity per mol base was 10(7) times lower than the immunoreactivity of cisplatin adducts.

Animals↗
PubMed1995

Induction of mutations by replication of malondialdehyde-modified M13 DNA in Escherichia coli: determination of the extent of DNA modification, genetic requirements for mutagenesis, and types of mutations induced.

The mutagenicity of the lipid peroxidation product, malondialdehyde (MDA), was measured in the lacZ alpha forward mutation assay using a recombinant M13 phage, M13MB102. Single-stranded M13MB102 DNA was reacted with MDA at neutral pH and the modified DNA was transformed into strains of Escherichia coli induced for the SOS response. Increasing concentrations of MDA led to an increase in lacZ alpha-mutations coincident with an increase in the level of the major MDA-deoxyguanosine adduct. Spontaneous and MDA-induced M13MB102 mutants were collected and the lacZ alpha target region was subjected to automated DNA sequence analysis. The most common sequence changes induced by MDA were base-pair substitutions (76%). Of these, 43% (29/68) were transversions, most of which were G-->T (24/29). Transitions account for 57% of the base-pair substitutions (39/68) and were comprised exclusively of C-->T (22/39) and A-->G (17/39). Frameshift mutations were identified in 16% of the induced mutants and were comprised of mainly single base additions occurring in runs of reiterated bases (11/14). The diversity of base-pair substitution and frameshift mutations induced by MDA at low levels of adduction suggests it may be an important contributor to endogenous mutagenesis and carcinogenesis in aerobic organisms.

Bacteriophage M13↗
PubMed1993

DNA modification and repair by 2-nitropropane is extensive in hepatocytes of rats compared to those of humans and mice.

Hepatocytes from rats, mice or humans were exposed to either 2-nitropropane or propane 2-nitronate and the extent of unscheduled DNA synthesis (UDS) was measured. At a concentration of 2-nitropropane (0.1 mM) that produced a marked induction of UDS in rat hepatocytes, a negative or minimal response was seen in hepatocytes from mice and humans. A 10-fold higher concentration of 2-nitropropane was required in mouse hepatocytes for an equivalent UDS response to that seen in rat-liver cells. All three species showed UDS after exposure of hepatocytes to propane 2-nitronate at 0.1 mM although the effect in human cells was variable. In rat hepatocytes the induction of UDS by 2-nitropropane was not inhibited by the antioxidant dimethyl sulphoxide (1%). In these cells, 2-nitropropane produced an electrochemically active modified deoxynucleoside, similar to that reported to be formed in livers of rats which had received 2-nitropropane in vivo. A smaller but significant production of this altered deoxynucleoside was found in mouse hepatocytes but not in human hepatocytes treated identically. The level of 8-oxo-7,8-dihydrode-oxyguanosine was not increased in hepatocytes from any of the three species exposed to 2-NP under the same conditions. It is suggested that the UDS caused by 2-nitropropane may be in response to damage other than that produced by oxygen radicals.

Adult↗
PubMed1994

Regulation of xylose utilization in Bacillus licheniformis: Xyl repressor-xyl-operator interaction studied by DNA modification protection and interference.

Xylose utilization in Bacillus licheniformis is inducible by xylose. We establish here that the Xyl repressor recognizes and binds an xyl operator sequence located 12 nucleotides downstream from the transcription start site of the xyl operon. DNA-retardation experiments employing xyl regulatory DNA and soluble protein extracts indicate complex formation in the presence of Xyl repressor. Two repressor-operator complexes are distinguished by different gel mobilities. They yield the same in situ copper-phenanthroline footprint. This result suggests that a single xyl operator may be bound by different oligomers of Xyl repressor. Methylation and hydroxyl radical cleavage protection of the xyl operator by Xyl repressor binding and ethylation interference of Xyl repressor binding to the xyl operator reveals symmetrical interaction of the repressor with two half sites of the operator, which show palindromic symmetry and are located on the same side of the B-form DNA structure.

Amino Acid Sequence↗
PubMed1996

Sequence-specific DNA modification in Acetobacter xylinum.

Two cryptic plasmids have been discovered in Acetobacter xylinum B42 and in its derivative PEA-1, a cellulose defective mutant. These two plasmids were designated pAX1 and pAX2 (50 and 105 kb in size, respectively). A restriction map was constructed for pAX1. Attempts to cure these plasmids were unsuccessful. Enzyme restriction analysis showed that these plasmids contain protected EcoRI and ApoI sites. Using Southern blot and hybridization techniques, the protection was extended to chromosomal DNA. Enzyme restriction analysis of several plasmids, from different origins and containing different incompatibility groups, isolated from strain PEA-1 also showed EcoRI and ApoI protection. The presence of modifications on specific sequences was not found in A. xylinum 8747. These results strongly suggest the presence of a modification system in A. xylinum B42 that recognizes the tetranucleotide 5'-AATT.

Base Sequence↗
PubMed1989

DNA modification by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant mutagenic heterocyclic amine by weight in cooked foods. This mutagen was found to produce DNA adducts in all ten tested organs of rats using the 32P-postlabeling method. The level of DNA adducts in the pancreas, kidney and liver increased dose-dependently and feeding time-dependently up to four weeks. When diet containing 0.05% PhIP was given to rats for four weeks, levels of PhIP-DNA adducts were relatively high in the lung, pancreas and heart, being around 20 per 10(7) nucleotides, and lowest in the liver, being 2.20 per 10(7) nucleotides. Thus, PhIP showed a unique feature in the formation of DNA adducts compared to other mutagenic and carcinogenic heterocyclic amines, which produce the highest level of DNA adducts in the liver.

Animals↗
PubMed2004

MicroRNAs guide asymmetric DNA modifications guiding asymmetric organs.

In plants and animals, microRNAs have been shown to regulate target genes by inhibiting translation or altering target mRNA stability. In this issue of Developmental Cell, Bao et al. extend the known mechanisms of action of microRNAs to RNA-directed DNA methylation, a mechanism previously associated only with siRNA-mediated gene silencing.

Arabidopsis↗
PubMedDate not supplied

Differential effects of thiols on DNA modifications via alkylation and Michael addition by alpha-acetoxy-N-nitrosopyrrolidine.

The hepatocarcinogen NPYR is metabolically activated by alpha-hydroxylation mediated by cytochrome P-450 enzymes to yield a 4-oxobutylating agent and 2-butenal (crotonaldehyde). Both are reactive intermediates capable of modifying DNA with guanine either by simple alkylation or by Michael type addition, respectively. In order to assess the roles of these pathways in NPYR tumorigenesis, we are interested in identifying agents which can selectively modify one of these two pathways. In this study, we examined the effects of three thiols--(mesna), glutathione (Glu), and N-acetylcysteine (Nac)--on DNA adduct formation by alpha-acetoxyNPYR, a stable precursor of alpha-hydroxyNPYR. Calf thymus DNA isolated from incubation of alpha-acetoxyNPYR with or without thiol was hydrolyzed and analyzed for the adducts formed by alkylation (adducts 1 and 2) and Michael addition (adducts 3-5). The results showed that the addition of mesna completely blocked the formation of the crotonaldehyde-derived adducts 3-5, whereas it exerted little effect on the formation of the alkylated adducts 1 and 2. These results indicate the preferential conjugation of mesna with crotonaldehyde. In contrast, Nac had little selectivity on adduct formation; levels of adducts 1 to 5 were were reduced by 36-75%. These results suggest that Nac conjugated with both alkylating agent and crotonaldehyde. Similar to mesna, Glu blocked the formation of the crotonaldehyde-derived adducts (adducts 3-5) efficiently. However, unlike mesna, Glu inhibited the formation of adduct 1, while it did not inhibit the formation of adduct 2, although both adducts are presumably derived from the 4-oxobutylating agent.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcysteine↗