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PubMed1988

Hydroxyl radical-induced strand break formation in single-stranded polynucleotides and single-stranded DNA in aqueous solution as measured by light scattering and by conductivity.

Combining conductivity measurements and molecular weight determination by means of low-angle laser light scattering, we have found for the polyribonucleotides (polyuridylic acid (poly(U], polyadenylic acid (poly(A], polycytidylic acid (poly(C] and polyguanylic acid (poly(G] and for single-stranded DNA (ssDNA) that, on average, 8.5 counterions per single-strand break (ssb) are liberated under salt-free conditions. This relationship allows us to estimate, from conductivity measurements alone, G-values of single-strand break formation (G(ssb] for the polydeoxyribonucleotides (polydeoxyriboadenylic acid (poly(dA], polydeoxyribocytidylic acid (poly(dC], polydeoxyribothymidylic acid (poly(dT], polydeoxyribouridylic acid (poly(dU] and polydeoxyriboguanylic acid (poly(dG]. The following G(ssb) values (units of mumol J-1) have been obtained for anoxic conditions: poly(dA), 0.23; poly(dC), 0.14; poly(dT), 0.06; poly(dU), 0.046 and poly(dG), 0.009. Time-resolved conductivity measurements in pulse radiolysis enable us to measure the rate of strand break formation. The rate has been found to be similar for poly(dA) and ssDNA over a range of pH values. Poly(dC) and poly(dU) exhibit conductivity increase components with half-lives similar to those of poly(dA) and ssDNA at corresponding pH values. The implications of these results are discussed.

DNA Damage↗

PubMed1971

Antibodies to polynucleotides in human sera: antigenic specificity and relation to disease.

The specificities of anti-polynucleotide antibodies found in human sera were studied using several immunological procedures. Anti-native DNA (NDNA) antibodies and certain anti-double-stranded RNA (DSRNA) antibodies were found to react with single-stranded DNA (SDNA), and anti-NDNA antibodies were observed to react more avidly with SDNA than with NDNA in most sera tested. Antibodies to NDNA showed no preferential reactivity with NDNA or SDNA derived from mammalian tissue, bacterial, or viral sources. Precipitating antibodies reactive with individual bases, with common determinants of bases, and with common determinants of SDNA and NDNA were detected utilizing synthetic polydeoxyribonucleotides. Antibodies to DSRNA were also heterogeneous and reactive with both Poly A . Poly U and Poly I . Poly C in addition to reactivity with Poly A and SDNA. In contrast, antibodies to a ribonucleo-protein determined by hemagglutination and by precipitation showed no reaction with NDNA, SDNA, or DSRNA. Serial studies of serum specimens from patients with systemic lupus erythematosus (SLE) indicated that anti-NDNA antibodies were closely associated with disease activity. Titers of antibodies to SDNA or DSRNA were also frequently increased during these periods but in addition showed peaks during quiescent periods. Anti-NDNA antibodies were detected in most patients' sera at sometime during the course of the disease. Three patients were observed with active SLE, who did not develop anti-NDNA antibodies, even in the presence of severe renal disease. Evidence that other antigen-antibody systems may also play a role in the pathogenesis of the renal disease was particularly apparent in these patients. Anti-ribonucleoprotein antibodies were not well correlated with the peaks of antibody activity of other polynucleotide antibodies, suggesting that an independent immunogen was responsible for induction of these antibodies. The close association of certain populations of anti-polynucleotide antibodies during the course of active SLE, the presence of cross-reacting antigenic determinants of SDNA, NDNA, and DSRNA, the preferential avidity of anti-NDNA antibodies for SDNA, and the frequent increase of anti-SDNA antibodies in SLE and other diseases associated with active tissue destruction suggest that SDNA is a ubiquitous antigen that may stimulate the formation of antibodies reactive with a variety of polynucleotides.

Animals↗

PubMed1994

Co-chromatography of a tamoxifen epoxide-deoxyguanylic acid adduct with a major DNA adduct formed in the livers of tamoxifen-treated rats.

Tamoxifen is a potent liver carcinogen in rats and has been shown to form covalent DNA adducts in the livers of several species of rodent. We have shown previously by 32P-postlabeling (Carcinogenesis, 13, 2197-2203) that > 85% of the total adducts detected and resolved by multi-directional TLC migrate as a single spot. In the present study, this material was further analysed by reverse-phase HPLC and resolved into two approximately equal components. Tamoxifen 1,2-epoxide, a postulated metabolite of tamoxifen, was reacted with DNA and polydeoxyribonucleotides and the products analysed. 32P-Postlabeling revealed three major adduct spots on TLC which comigrated with the three major adduct spots seen with DNA from livers of tamoxifen-treated rats. Moreover, the major epoxide adduct, which contained guanine as the modified base, eluted on HPLC as a single major peak coincident with one of the major peaks derived from the liver DNA of tamoxifen-treated rats. These results demonstrate that approximately 40% of the tamoxifen-DNA adducts formed in vivo are chromatographically indistinguishable with the major product of the reaction of tamoxifen epoxide with guanine residues in DNA and provide important clues to the mechanism of activation of tamoxifen to a genotoxic carcinogen.

Animals↗

PubMed1974

Proton magnetic resonance studies of ultraviolet-irradiated apurinic acid.

In apurinic acid, a single-stranded polydeoxyribonucleotide easily obtained upon depurination of DNA, the proton resonances arising from thymine and cytosine are readily observable in aqueous solution of 25 degrees C. Two methyl thymine resonances, centered at 1.88 ppm and separated by 0.045 ppm, are observed. We attribute the downfield methyl resonance to thymines with no pyrimidine nearest neighbors and the upfield methyl resonance to thymines having pyrimidine neighbors in the 3' and/or 5' positions. Upon ultraviolet irradiation, the upfield methyl and thymine H-6 resonances decrease in amplitude and two methyl resoances appear at 1.63 and 1.52 ppm, corresponding, respectively, to cytosine-thymine and thymine-thymine cyclobutane dimers. Photoreversal eliminates these two minor methyl resonances from the pmr spectrum. We conclude that apurinic acid provides a suitable model system for pmr studies of chemically modified pyrimidine bases in DNA.

Apurinic Acid↗

PubMed1993

Synthetic gene for the hepatitis C virus nucleocapsid protein.

A synthetic gene encoding the hepatitis C virus (HCV) nucleocapsid protein was constructed and expressed in E. coli. To synthesize this gene, we developed a new method that results in the enzymatic synthesis of long polydeoxyribonucleotides from oligodeoxyribonucleotides. The method, designated as the 'Exchangeable Template Reaction' (ETR), uses oligonucleotides as templates for DNA polymerase. A special mechanism was designed to exchange the templates during the polymerase reaction. The mechanism relies on the formation of a single-stranded 3'-protrusion at the 'growing point' of the elongating DNA such that it can be subsequently annealed, in a sequence-specific manner, with the next synthetic oligonucleotide. When annealed to the 3'-protrusion, the added oligonucleotide becomes a template for DNA polymerase, and the protruding 3'-end of the double-stranded DNA is used as the primer. The HCV nucleocapsid gene was assembled with DNA ligase from three fragments synthesized by ETR. The data verify that this method is efficient. The main advantage of ETR is the ability to combine more than two oligonucleotides in one tube together with polymerase and an enzymatic activity that produces a 3'-protrusion (e.g., BstXI) rather than the sequential addition of each component. The data demonstrate that as many as five oligonucleotides can be used simultaneously, resulting in a synthesized DNA fragment of designed sequence. The synthetic gene expressed in E. coli produced a 27 kDa protein that specifically interacted with antibodies from sera obtained from HCV-infected individuals.

Amino Acid Sequence↗

PubMed1976

Purification and properties of a light-inducible nuclease from Euglena gracilis.

The nuclease described by Carell, E.F., Egan, J.M. and Pratt, E.A. [Arch. Biochem. Biophys. (1970) 138, 26-31] has been purified 1000-fold from Euglena gracilis strain Z. The enzyme catalyzes the hydrolysis of both polyribonucleotides and polydeoxyribonucleotides. The relative rates of hydrolysis of synthetic and natural polynucleotides was found to be: poly (U) 100, poly (dT) 33, denatured calf-thymus DNA 33, yeast tRNA 9, E. coli total RNA 6, poly (dA dT) 5, poly (A) less than 1, poly (C) less than .05, and poly (G) less than .05. The enzyme attacks polynucleotides in an endonucleolytic fashion, yielding products terminated with a 3'-phosphate. Poly (U) appears to be hydrolyzed completely to 3'-UMP; both RNA and DNA appear to have some phosphodiester bonds resistant to enzyme catalyzed hydrolysis. Because of its mode of action and its inducibility by light, we propose the name endonuclease L for this enzyme.

Animals↗

PubMed1977

A kinetic study of 1H leads to 3H exchange in C(8) H-groups of purinic residues in DNA.

The kinetic study of 1H leads to 3H exchange in C(8) H-groups of purinic residues of DNAs with different G-C content as well as in corresponding dNMP mixtures have been carried out. The present results show that 1H--3H exchange in DNA is retarded (as compared to the exchange in dNMP mixtures) to a lesser extent (Kret =2.4-2.8) than in RNA (Kret=6-8). The degree of retardation in these polymers is practically independent of their nucleotide composition. Assuming the ylide mechanism of exchange reaction it is suggested that the lower rate of 1H leads to 3H exchange in C(8) H-groups of purinic residues in polynucleotides of A-form (RNA and other polyribonucleotides) as compared to those of B-form (DNA and other polydeoxyribonucleotides) might be accounted for by decreased availability of C(8) H-groups for OH-ions of the solvent due to a different microenviroment of these groups in A- and B-type helixes.

DNA↗

PubMed1981

Circular dichroism spectra of twelve short DNA restriction fragments of known sequence: a comparison of measured and calculated spectra.

The CD spectra of twelve DNA restriction fragments ranging in size from 12 to 360 base pairs are reported. Since the sequences of these fragments are known, it is possible to calculate their CD spectra from a set of nearest neighbor contributions derived from a combination of synthetic polydeoxyribonucleotides. While the calculations lead to good agreement in the negative band at approximately 245 nm, they generally reproduce the positive band at approximately 270 nm only poorly. The experimentally observed positive band consists of two peaks centered around 270 and 285 nm. The comparison of calculated and measured spectra reveals that end effects lead to increased disagreement for fragments smaller than approximately 40 base pairs. The disagreement between calculated and measured spectra can be partially attributed to the fraction of next nearest neighbors in the DNAs, which are also in the spectral components. Thus, the sequence specific CD contributions in the long wavelength region of the spectra extend at least to next nearest neighbor nucleotides and may extend beyond.

Base Composition↗

PubMed2005

Surrogate splicing for functional analysis of sesquiterpene synthase genes.

A method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns is described. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product. The method was optimized by examining the expression of an intron-containing beta-glucuronidase gene agroinfiltrated into petunia (Petunia hybrida) leaves, and its utility was demonstrated by defining the function of two previously uncharacterized terpene synthases. A tobacco (Nicotiana tabacum) terpene synthase-like gene containing six predicted introns was characterized as having 5-epi-aristolochene synthase activity, while an Arabidopsis (Arabidopsis thaliana) gene previously annotated as a terpene synthase was shown to possess a novel sesquiterpene synthase activity for alpha-barbatene, thujopsene, and beta-chamigrene biosynthesis.

Alternative Splicing↗

PubMed2006

Review article: updates in the pathogenesis and therapy of hepatic sinusoidal obstruction syndrome.

Hepatic sinusoidal obstruction syndrome is frequently linked to high-dose chemotherapy/total-body irradiation in recipients of haematopoietic stem cell transplantation, long-term use of azathioprine after organ transplantation and other chemotherapeutic agents. The incidence of hepatic sinusoidal obstruction syndrome varies from 0% to 70%, and is decreasing. Disease risk is higher in patients with malignancies, hepatitis C virus infection, those who present late, when norethisterone is used to prevent menstruation, and when broad-spectrum antibiotics and antifungals are used during and after the conditioning therapy. Hepatic sinusoidal obstruction syndrome presents with tender hepatomegaly, hyperbilirubinaemia and ascites, and diagnosis is mainly clinical (Seattle and Baltimore Criteria). Imaging excludes biliary obstruction and malignancy, but cannot establish accurate diagnosis. Hepatic sinusoidal obstruction syndrome may be prevented by avoiding the highest risk regimens, using non-myelo-ablative regimens, and reducing total-body irradiation dose. Treatment is largely symptomatic and supportive, because 70-80% of patients recover spontaneously. Tissue plasminogen activator plus heparin improves outcome in <30% of cases. Defibrotide, a polydeoxyribonucleotide, is showing encouraging results. Transjugular intrahepatic porto-systemic shunt relieves ascites, but does not improve outcome. Liver transplantation may be an option in the absence of malignancy. Prognosis is variable and depends on disease severity, aetiology and associated conditions. Death is most commonly caused by renal or cardiopulmonary failure.

Animals↗

PubMed1975

DNA polymerases of rat liver. Partial characterization and effect of various inhibitors.

Three distinct DNA-dependent DNA polymerase activities have been partially purified from normal rat liver. Soluble activities are separable into two distinct fractions (P1 and P2) by phosphocellulose chromatography. A low-molecular-weight DNA polymerase was isolated from purified nuclei. The enzymes were characterized according to chromatographic and sedimentation behavior, enzymological properties, and response to various inhibitors. The results indicate that fraction P1 corresponds to the high-molecular-weight enzyme and suggest that polymerase P2 may be derived from partial dissociation of the high-molecular-weight enzyme. The molecular weight of polymerase P1 was estimated to be about 250 000 by Sephadex column chromatography. Both fraction P2 and nuclear DNA polymerase appeared to be low-molecular-weight enzymes. However, the molecular size of these activities was apparently different. The estimated molecular weights of nuclear and P2 enzyme are about 40 000 and 25 000, respectively. As with the nuclear enzyme, polymerase P2 (but not P1) appeared to be free of detectable exonuclease activity. All of these polymerases showed a marked preference for initiated polydeoxyribonucleotide templates. The rat liver polymerases differed in their ability to use poly[d(A-T)-A1 primer-template, as is shown by the ratios of their activity with this synthetic polymer to that with activated DNA: 0.5, 2.75, and 1.34 for P1, P2, and nuclear polymerase, respectively. Denatured DNA was a poor template for both enzymes P1 and P2, but it was inert as template for the nuclear enzyme. Although each of these polymerases required all four deoxynucleoside triphosphates for maximal activity, they catalyzed a high rate of synthesis in the absence of one or more deoxynucleoside triphosphates. Such a 'limited' synthesis was much more extensive for polymerase P2 and nuclear enzyme than for P1 was the most sensitive of the three to sulphydryl reagents, ehtidium bromide, heparin, and single-stranded DNA. The responses of P2 and nuclear enzymes to various inhibitors were very similar. However, these two enzymes respond differently to heat and high ionic strength.

Animals↗

PubMed1977

Purification and properties of a DNA-dependent ATPase induced by bacteriophage T4.

A DNA-dependent ATPase formed after T4 phage infection is purified to apparent homogeneity. The molecular weight of the purified enzyme is 50 000 when determined by glycerol gradient centrifugation and by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The enzyme at an earlier stage in purification (prior to DEAE-cellulose chromatography) exists as a complex with a molecular weight of 100000. However, molecular weight determinations by Sephadex gel chromatography give considerably decreased molecular weights for the complex and for the enzyme after DEAE-cellulose chromatography. The enzyme is stimulated to varying degrees by a variety of single-stranded polydeoxyribonucleotides or by single-stranded DNA, but no chemical change in the polynucleotide has been detected as a result of the enzyme action.

Adenosine Triphosphatases↗

PubMed1983

Association of a polyuridylate-specific endoribonuclease with small nuclear ribonucleo-proteins which had been isolated by affinity chromatography using antibodies from a patient with systemic lupus erythematosus.

Immunoglobulins, containing antibodies against U1-snRNP, have been prepared from a patient with systemic lupus erythematosus. After coupling these antibodies to a Sepharose matrix, U-snRNPs have been isolated and purified from rat liver nuclei by use of immunoaffinity chromatography. The resulting RNPs had the typical protein pattern of U-sn RNPs and a sedimentation coefficient of 12 S. The U-snRNP preparation was associated with an endoribonuclease which required Mg2+ for optimal activity. The enzyme, with an pH optimum of 6.2, degraded only poly(U). Other single-stranded polyribo- and polydeoxyribonucleotides, tRNA, as well as double-stranded RNA and DNA were not digested. The products of a terminal digestion are (U)6-12 with 3'-OH and 5'-P termini. The possible involvement of this endoribonuclease in the splicing of hnRNA is discussed.

Animals↗

PubMed1983

Exodeoxyribonuclease from rat brain specific for single-stranded DNA.

An exodeoxynuclease with single-stranded DNA specificity has been isolated from rat brain and purified to electrophoretic homogeneity. Approximately 1100-fold purification with a yield of 16% has been achieved by chromatography on DNA-agarose, hydroxyapatite and Sephadex G-200. The enzyme has a pH optimum at 8.4, requires Mg2+ or Mn2+ as a cofactor and has a molecular weight of about 60000. It hydrolyzes homologous, heterologous, synthetic and depurinated substrates at the same rate liberating nucleoside 5'-monophosphates but does not attack ultraviolet-irradiated polydeoxyribonucleotides. This DNase is localized predominantly in neuronal cell nuclei and appears to be lacking in rat liver tissue.

Animals↗

PubMed1989

Investigation of RecA--polynucleotide interactions from the measurement of LexA repressor cleavage kinetics. Presence of different types of complex.

The proteolysis of the LexA repressor in the presence of RecA and various polynucleotides was studied by measuring the fluorescence decrease of LexA upon cleavage. The results were compared with the DNA binding of RecA to investigate the presence of multiple DNA-RecA complexes. All single-stranded polydeoxyribonucleotides (DNA) efficiently stimulated the proteolysis and the maximum activation was reached in the presence of three or four nucleotides of polynucleotide per monomer of RecA. The stimulative effect was decreased in the presence of larger amounts of poly(dA), poly(dT) or heat-denatured DNA, whereas the excess of single-stranded DNAs chemically modified with chloroacetaldehyde did not present such an inhibitory effect, despite the fact that a second DNA molecule is likely to interact with RecA as monitored by the intrinsic fluorescence of these DNA species. The complicated cleavage promotion and inhibition pattern is tentatively explained by a three-state model assuming that RecA may interact with three single-stranded DNA molecules. According to this model, occupation of the first site would be necessary and sufficient for cleavage promotion, the second site would be neutral with respect to cleavage and the occupation of the third site would inhibit LexA cleavage at least partially. Double-stranded natural DNA did not stimulate cleavage, even under conditions where RecA binds quantitatively to the DNA. No polyribonucleotides (RNA) examined showed a significant stimulative effect either, nor did they appear to interact with RecA.

Bacterial Proteins↗

PubMed1983

Formation of an adenine-thymine photoadduct in the deoxydinucleoside monophosphate d(TpA) and in DNA.

A photoadduct is formed between the adenine (A) and thymine (T) bases of the deoxydinucleoside monophosphate d(TpA) when it is irradiated at 254 nanometers in aqueous solution. Treatment of the photoadduct with acid converts it specifically into a fluorescent hydrolysis product, C7H7N3O, incorporating the position-8 carbon of adenine and the methyl group of thymine. Isolation of the fluorescent hydrolysis product from acid hydrolyzates of oligo- and polydeoxyribonucleotides has shown that the photoadduct is formed by ultraviolet irradiation of d(pTpA), d(TpApT), d(TpApTpA), poly(dA-dT), and both single- and double-stranded DNA.

Adenine↗

PubMed1970

Uptake of synthetic polynucleotides by competent cells of Bacillus subtilis.

A survey was made of the capacity of competent cells of Bacillus subtilis to take up synthetic polynucleotides. Polydeoxyribonucleotides but not polyribonucleotides are taken up by the cells. Both types of polynucleotide failed to compete with transforming deoxyribonucleic acid in the transformation assay whereas both stimulated amino acid incorporation. The latter phenomenon appears to be nonspecific as there is no correlation between the codon composition of the polynucleotides employed and the amino acids whose incorporation was stimulated.

Amino Acids↗

PubMed1980

Purification and properties of deoxyribonucleic acid binding factor isolated from the surface of Streptococcus sanguis cells.

Deoxyribonucleic acid (DNA) binding factor (BF) was found in surface fluids from competent and noncompetent cells of Streptococcus sanguis strains Challis, Wicky, and Blackburn. Fluids from noncompetent cells exhibited about 10% BF activity compared with extracts from competent cells. BF from competent Wicky cells was purified to homogeneity by electrophoresis and immunodiffusion. Purified BF preparations exhibited slight endonucleolytic activity, directed mainly against single-stranded DNA. Nucleolytic and DNA binding activities present in purified BF could be separated by polyacrylamide gel electrophoresis. Purified BF was sensitive to proteolytic enzymes and to phospholipase D, and its activity was stimulated in the presence of low Triton X-100 concentrations. The protein component of BF is a single, monomeric polypeptide with a molecular weight of 56,000 and an isoelectric point of pH 5.8. Binding of purified BF to DNA was a very rapid process at the optimum temperature, pH, and ionic strength and led to the formation of fast-sedimenting complexes. Purified BF was tested for several properties. It exhibited higher affinity to single- than to double-stranded DNA. It bound poorly to glucosylated phage T4 and single-stranded, synthetic polydeoxyribonucleotides and did not bind to RNA. It protected single-stranded DNA against nuclease S(1) action but did not protect native DNA against deoxyribonuclease I action. No evidence was found for unwinding activity, using double-stranded DNA as a substrate.

Bacterial Proteins↗