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PubMedDate not supplied

Selection of cDNAs for phosphodiesterases that hydrolyze guanosine 3';5'-monophosphate in Escherichia coli.

A genetic selection procedure has been developed which makes the growth of E. coli dependent on expression of a cGMP phosphodiesterase cDNA. E. coli does not contain a cGMP phosphodiesterase, and guanine auxotrophs cannot extract the guanine from cGMP. If a functional cGMP phosphodiesterase is introduced, then guaA auxotrophs will grow on cGMP as a guanine source. The method also selects GMP synthetase cDNAs, which complement the guanine auxotrophy directly. Expression of a Dictyostelium discoideum or human heart cyclic nucleotide phosphodiesterase cDNA permits growth of the E. coli guaA auxotroph in the presence of cGMP. Several commercial cDNA libraries were corrupt and contained phosphodiesterase and/or GMP synthetase sequences that were from a contaminating DNA source.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

UV absorption complicates PCR decontamination.

UV irradiation is widely used to inactivate contaminating DNA in PCR. Highly UV-absorbent deoxyribonucleoside triphosphates in PCR mixtures reduce the efficiency of UV decontamination. Optimal decontamination may be achieved by irradiating the PCR mixture without the deoxyribonucleoside triphosphates.

Artifacts↗
PubMed1992

Magnesium-dependent thermostability of DNase I.

DNase I can be used to remove contaminating DNA from RNA samples. Heat treatment has been recommended as a method to inactivate DNase I enzymatic activity, thereby allowing subsequent reverse transcription and PCR amplification of DNase I treated RNA. We have found that inactivation of DNase I by heat treatment is strongly dependent on Mg2+ concentration. In addition, deoxyribonucleolytic activity of "inactivated" enzyme may be restored by changes in Mg2+ concentration following heat treatment. Caution should be exercised when using heat treatment alone as a method of DNase I inactivation.

Deoxyribonuclease I↗
PubMed1992

Purification process monitoring in monoclonal antibody preparation: contamination with viruses, DNA and peptide growth factors.

Administration in vivo of monoclonal antibodies to humans is challenged by considerations regarding their safety. Contamination with viruses, potentially oncogenic nucleic acids and biologically active components like growth factors and hormones forms a serious point of concern in this respect. We have investigated the potential risk of viral contamination by measuring the reduction of 12 different viruses (after spiking) in the standard downstream purification process of ascitic fluid. Depending on the type of virus added and the purification step employed, the reduction of infectious virus particles varies considerably. The overall reduction ranges from about 10(3), observed for a member of the family of Papovaviridae, to more than 10(12) for members of the families of Herpesviridae and Orthomyxoviridae. Using hybridization analysis with a mouse (genomic) DNA probe, we show that the amount of residual DNA in ascitic fluids may also vary considerably, ranging from 75 ng/ml to 1 microgram/ml. In crude preparations produced in cell culture, much lower DNA concentrations are found (0.3 ng/ml). When standard downstream purification procedures are applied to ascitic fluid, a significant reduction of residual DNA levels is observed in the purified monoclonal antibody preparations and in intermediate fractions. The overall reduction factors vary from about 10(3) to 10(4), which is also confirmed by spiking experiments with either purified DNA or crude chromatin-like DNA. Using in-vitro cellular assays, we further show that peptide growth factors like PDGF and TGF beta are present in considerable amounts in ascitic fluids. The observed biological activities, however, are completely eliminated during the purification steps applied.

Animals↗
PubMed1979

Isolation and characterization of germ line DNA from mouse sperm.

Mouse germ line DNA was isolated from sperm by a physicochemical procedure that preferentially destroys contaminating somatic cell DNA. The use of reducing conditions and chelating agents in combination with phenol permitted extraction of molecular weight DNA from mature sperm nuclei with approximately 80% efficiency. Less than 0.1% somatic cell DNA contamination remained in sperm DNA prepared by this method. Germ line DNA was characterized by determination of its ultraviolet absorbance spectrum, buoyant density in cesium chloride, and melting profile on a hydroxyapatite column. Contamination by mitochondrial DNA was assessed by cesium chloride/ethidium bromide gradient centrifugation. The significance of the mouse germ line DNA isolation procedure is discussed with respect to the possible genetic transmission of mammary tumor virus and leukemia virus, the origin of antibody diversity, and the origin of testicular teratomas.

Animals↗
PubMed1977

Absence of ethidium bromide induced nicking and degradation of mitochondrial DNA in mouse L-cells.

The in vivo effects of ethidium bromide on the integrity of mitochondrial DNA have been studied in a mouse L-cell system in which this DNA may be nearly exclusively radiolabelled. This allows the detection of mitochondrial DNA in the presence of contaminating nuclear DNA and eliminates the need for extensive purification of mitochondria or the use of deoxyribonuclease. The mitochondrial DNA in treated cells rapidly attains a high negative superhelix density and is not substantially nickel or degraded over the course of several days.

DNA, Mitochondrial↗
PubMed1978

In vitro packaging of bacteriophate T7 DNA synthesized in vitro.

An in vitro DNA packaging system was used to encapsulate T7 DNA that had been synthesized by extracts prepared from gently lysed Escherchia coli infected with bacteriophage T7 carrying amber mutations in gene 3 or in both genes 3 and 6. Isopycnic centrifugation of density-labeled wild-type DNA was employed in an effort to separate product from template; suppressor-free indicator bacteria were used to eliminate contributions from endogenous DNA or contaminating phage. Additional controls indicated that fragmented DNA is packaged in vitro only with very low efficiency and that the frequency of recombination during packaging is too low to affect interpretation of these experiments. T7 DNA replicated by extracts prepared using T7 mutants deficient in both genes 3 and 6 could be packaged in vitro with an efficiency comparable to that found when highly purified virion T7 DNA was used. When T7 deficient in the gene 3 endonuclease but with normal levels of the gene 6 exonuclease was used, fast-sedimentingconcatemer-like DNA structures were formed during in vitro DNA synthesis. Electron microscopy revealed many branched and highly complex DNA structures formed during this reaction. This concatemer-like DNA was encapsulated in vitro with an efficiency significantly greater than that found for DNA the length of a single T7 genome.

Coliphages↗
PubMedDate not supplied

In vitro metabolism and DNA adduct formation from the mutagenic environmental contaminant 2-nitrofluoranthene.

The metabolism and DNA adduct formation by the mutagenic environmental contaminant 2-nitrofluoranthene (2-NFA) were studied. Incubation under aerobic conditions with liver microsomes of rats pretreated with 3-methylcholanthrene yielded trans-7,8-dihydroxy-7,8-dihydro-2-nitrofluoranthene, trans-9,10-dihydroxy-9,10-dihydro-2-nitrofluoranthene, and 7-, 8-, and 9-phenolic metabolites. When the epoxide hydrolase inhibitor 3,3,3-trichloropropylene was present in the incubation, only phenolic metabolites were detected. Under hypoxic conditions, 2-aminofluoranthene was obtained, together with a trace of the ring-oxidized metabolites. The activated metabolite, N-hydroxy-2-aminofluoranthene, was prepared in situ and reacted with calf thymus DNA. Upon enzymatic hydrolysis of the DNA and purification by HPLC, a C8-substituted deoxyguanosine adduct, N-(deoxyguanosin-8-yl)-2-aminofluoranthene, was identified by mass and proton NMR spectral analysis. This adduct was also formed at a level of 10 pmol/mg of DNA when 2-NFA was metabolized by xanthine oxidase, 6 pmol/mg of DNA from incubation with liver microsomes of rats pretreated with 3-methylcholanthrene, and 3-pmol/mg of DNA from metabolism by liver microsomes of rats pretreated with phenobarbital.

Animals↗
PubMed1978

Transcription of rat liver deoxyribonucleic acid in vitro at low ionic strength.

1. When RNA polymerase is in excess over DNA, the single-stranded breaks of DNA can be recognized as initiation sites for the ezyme. On the other hand stabel initiation complexes (resistant to inhibition by heparin) are the most abundant under these conditions. The formation of these complexes needs double-stranded DNA. It seems that RNA sequences rich in cytidine are preferentially synthesized; since rat liver DNA is A + T-rich, the transcription thus appears not to be random with respect to the base composition of DNA. 2. When the template is in excess over the polymerase, the single-stranded gaps of DNA are preferentially transcribed by rat liver RNA polymerase B and native DNA regions by Escherichia coli RNA polymerase. 3. With a large excess of DNA over the polymerase, the enzyme activity is markedly inhibited. This inhibition is proportional to the concentration of double-stranded DNA ends, but it also depends on the presence of a contaminant of DNA, removed when DNA is banded in a CsCl gradient. This contaminant could be polyphosphates. Low concentrations of spermine completely reverse this inhibition, by enhancing the rate of RNA chain elongation. 4. Double-stranded RNA is synthesized in great abundance when RNA polymerase is in excess over native DNA. Besides a majority of symmetrical sequences, stable 'hairpins' can be found. Whereas the synthesis of symmetrical sequences is more prevalent in polymerase excess, it seems that the proportion of stable 'hairpins' in RNA is independent of the polymerase/DNA ratio.

Animals↗
PubMed2026

Short-read genome skimming enables molecular barcoding of old myxomycete collections.

This study evaluates the effectiveness of Illumina-based genome skimming for barcoding myxomycete herbarium collections ranging from 29 to 91 years in age. We successfully retrieved partial sequences of the standard marker gene (nucSSU) in all cases, as well as additional markers (mtSSU, EF1a, and COI) for certain collections. Altogether, 28 genes were recognized in the studied material. In a 33-year-old specimen of Lindbladia tubulina, the assembly reached an N50 of 4.19 kb, enabling the recovery of extended functional loci. The input genomic DNA quantity emerges as the primary determinant of sequencing success. Samples with high DNA yields provide representative amounts of contigs coming confirmedly (matching sequences in the NCBI nucleotide database) or potentially (no-hit fraction) from myxomycetes, regardless of specimen age. In addition to target DNA, we revealed distinct signals of both anthropogenic contamination (human DNA and skin microflora) and natural substrate inhabitants, including oribatid mites and bacteria from dead wood, soil, and grass litter. Thus, even in old collections, metagenomic data still carry information regarding the substrate upon which the myxomycete developed. The results demonstrate that short-read genome skimming may help to integrate historical type material of myxomycetes into contemporary phylogenetic research. This method overcomes the length-dependent limitations of traditional Sanger sequencing, thus providing a roadmap for the future of museomics in myxomycetology.

Amoebozoa↗
PubMed1976

A double antibody solid phase assay for DNA autoantibodies for clinical use.

DNA antoantibodies in serum will bind to antigen-coated polystyrene tubes and can be detected by radiolabelled anti-immunoglobulin. The method is quantitative, gives information on the antibody class and is not readily subject to interference by factors such as the size of the DNA, minor contamination of double-stranded with single-stranded DNA and the presence of materials which can combine "non-specifically" with the antigen. Double-stranded DNA gave good discrimination between SLE and rheumatoid arthritis but with single-stranded preparations approximately 40% of the RA patients showed elevated antibody values. Individual results obtained with the two antigens were compared and correlated with the Farr test and ANA titres. Surprisingly, a proportion of the SLE sera gave high background binding to tubes coated with gelatine.

Antibodies, Antinuclear↗
PubMed1975

Serological analysis of human deoxyribonucleic acid polymerases. Preparation and properties of antiserum to deoxyribonucleic acid polymerase I from human lymphoid cells.

The preparation and properties of an antiserum to human DNA polymerase I (6 to 8 S) are described. Care was taken in the purification of the antigen to remove certain other DNA polymerases found in human cells. An incubation of antigen and antiserum lasting about 48 hours is necessary to achieve maximal inhibition. About 1 mug of the antipolymerase immunoglobulin G, prepared in rats, neutralizes 60% of the activity present in 54 ng of the enzyme. Tritrations varying both antiserum and enzyme demonstrate clear regions of antigen and antibody excess. Inhibition of enzyme activity is about the same whether the templateprimer is (dA)n-(dT)12-18, or partially digested DNA. An assay was developed which measures the remaining activity in the supernatant after precipitation of enzyme-antibody complexes with goat anti-rat immunoglobulin G. In this assay, 2.2 mug of the antipolymerase immunoglobulin G quantitatively bind 33 ng of DNA polymerase I. With use of the direct neutralization assay and the immuno-precipitation test, we found little, if any, antigenic relationship between DNA polymerase I and DNA polymerase II (3.4 S). Similarly, little, if any, relationship was found to the DNA polymerases from five RNA tumor viruses. The activities of RNA-directed DNA polymerases from the blood leukocytes of two patients with acute myelogenous leukemia and from the placentas of rhesus monkeys were not inhibited in neutralization assays which were shortened because these enzymes were thermolabile. In identically shortened neutralization assays, the antipolymerase immunoglobulin G neutralized up to 76% of the activity of DNA polymerase I. In addition to its utility in distinguishing cellular DNA polymerases, the rat antiserum should be useful reagent for testing of novel DNA polymerases isolated in small quantities from human tumors for contamination with DNA polymerase I. This enzyme is present in abundance in proliferating tissue and often confuses the biochemical characterization of these novel enzymes.

Animals↗
PubMed1992

JC virus DNA is present in many human brain samples from patients without progressive multifocal leukoencephalopathy.

Sections of normal and diseased brain and kidney tissues were screened for the presence of JC virus (JCV) DNA by using the polymerase chain reaction. As expected, all samples obtained from patients with progressive multifocal leukoencephalopathy (PML) tested positive when multiple JCV-specific primer and probe combinations were used. Unexpectedly, more than 50% of non-PML-affected brains were also found to harbor low levels of JCV DNA. To confirm that the positive signals seen in the tissue sections were not the result of contamination, amplified DNA was cloned and sequenced and in some cases was shown to represent strains of JCV not identified previously. Two predominant regulatory region configurations of JCV have been detected in the human host: archetype JCV, which is excreted in the urine of normal and immunocompromised individuals, and "PML-type" JCV found in diseased brains. This latter group of variants appears to derive from archetype JCV by the deletion and duplication of sequences within the promoter-enhancer region. In the present study, the archetype strain of JCV was identified only in normal kidney samples; JCV DNA found in non-PML-affected brain specimens and in kidney tissue from patients with PML resembled that of strains isolated from PML-affected brain tissue. Our findings indicate that JCV reaches the brain more frequently than previously thought and may persist at this site without causing demyelinating disease. A subsequent episode of prolonged immunodeficiency or a direct interaction with an immunocompromising agent (e.g., human immunodeficiency virus type 1) might activate the latent JCV infection and lead to the development of PML.

Base Sequence↗
PubMed1992

A quick and inexpensive method for removing polysaccharides from plant genomic DNA.

A quick and inexpensive method has been demonstrated to remove polysaccharide contamination from plant DNA. Isolated plant genomic DNA with polysaccharide contaminants was dissolved in TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA) with NaCl ranging from 0.5-3.0 M, then precipitated with two volumes of ethanol. Most of the polysaccharides were removed effectively in a single high-salt precipitation at 1.0-2.5 M NaCl. At 3.0 M NaCl, the salt precipitated out of solution. Purified DNA was easily digested by either HindIII or EcoRI and was satisfactory as a template for PCR. The results show that high-salt precipitation effectively removed polysaccharides and their inhibitory effects on restriction enzyme and Taq polymerase activity.

DNA↗
PubMed1992

Heat-soaked PCR: an efficient method for DNA amplification with applications to forensic analysis.

Heat-soaked PCR (HS-PCR) is a method for enhancing amplification performed by heating the DNA sample at 94 degrees C in 90 microliters 1.1 x buffer for 30 min. A 10-microliters bolus of concentrated (10x) deoxynucleotides, Taq DNA polymerase and primers prepared without buffer is then added just prior to thermal cycling. We have investigated the application of this method in a variety of forensically important DNA samples and compared it with regular PCR (R-PCR). DNA samples extracted from bone, postmortem tissues, bloodstains and hair contained low concentrations of human DNA or were contaminated with either non- human DNA or hemoglobin degradation products. Optimal conditions for HS-PCR were determined for the 3' ApoB VNTR locus and applied to a centromeric repeat element and to a single-copy locus. HS-PCR consistently and reproducibly enhanced product yield and specificity over R-PCR at all three loci in the entire set of DNA samples. HS-PCR was also effective in overcoming the inhibitory effect of hemoglobin at concentrations that fully impeded R-PCR.

DNA↗
PubMed1978

Bovine thymus poly(adenosine diphosphate ribose) polymerase.

About 1,300-fold purification of poly(adenosine diphosphate ribose) polymerase has been achieved from the extract of bovine thymus with a recovery of 10 to 20%. The final preparation has a purity of 99%, and the enzyme is composed of a single peptide with a molecular weight of 130,000. The purified enzyme required NAD+, Mg2+, a thiol compound, DNA, and histones for full activity. Whereas DNA is essential for activation of the enzyme, histones are not. The observed stimulation of the reaction by histones is shown to be due to masking of the inhibitory effect of contaminating denartured DNA in native DNA preparation. The concentration of DNA required for half-maximal enzyme activity (apparent Km for DNA) is proportional to the concentration of enzyme in the reaction mixture. The minimum estimation of the number of nucleotide pairs of DNA required for half-maximal activation of one enzyme molecule is 220 to 240 for bulk of calf thymus DNA, while the value is 10 for a calf thymus DNA fraction, "active DNA," which was separated from the enzyme fraction in a stage of the purification. These results suggest that the enzyme is activated by binding to a specific site on calf thymus DNA. The apparent Km for NAD+ and the maximum velocity of the enzyme are estimated to be 60 micrometer and 0.91 mumolper min per mg, respectively.

Animals↗