scholarly journals Presence of two deoxyribonucleic acid polymerases in bull spermatozoa

1978 ◽  
Vol 175 (2) ◽  
pp. 595-600 ◽  
Author(s):  
M Philippe ◽  
P Chevaillier
Keyword(s):  
Ionic Strength ◽  
Dna Polymerase ◽  
Ethidium Bromide ◽  
Nuclear Dna ◽  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Polymerase Activity ◽  
Exogenous Dna ◽  
Middle Piece ◽  
Low Ionic Strength

A DNA polymerase-endogenous template complex was isolated from nuclear heads of bull spermatozoa. The buoyant density of the complex was 1.15 g/cm 3. The sedimentation coefficient of the nuclear DNA polymerase isolated from the complex was higher at low ionic strength, but approached 3.4S when centrifuged in a medium containing 2M-KCl. Activated exogenous DNA increased polymerase activity. Only very low activities were detected with synthetic templates such as poly(A).(dT)12-18 and poly(dT).poly(A). The nuclear reaction was stimulated by 150mM-KCl and was slightly inhibited by N-ethylmaleimide; it was resistant to actinomycin D, netropsin and ethidium bromide. Another DNA polymerase, highly sensitive to ethidium bromide, was extracted from the mitochondira-rich middle-piece fraction. Its sedimentation coefficient was close to 9S, but fell to approx. 4S in high-ionic-strength medium.

scholarly journals Effect of Phenylhydrazine-induced Hemolytic Anemia on Nuclear RNA Polymerase Activity of the Mouse Spleen

Blood ◽  
1973 ◽  
Vol 42 (2) ◽  
pp. 257-266 ◽  
Author(s):  
Jerry L. Spivak ◽  
Dennis Toretti ◽  
Herbert W. Dickerman
Keyword(s):  
Ionic Strength ◽  
Rna Polymerase ◽  
Hemolytic Anemia ◽  
Nuclear Dna ◽  
Polymerase Activity ◽  
Tenfold Increase ◽  
Rna Polymerase Activity ◽  
Low Ionic Strength ◽  
Dna Template ◽  
Nuclear Rna Polymerase

Abstract The DNA-dependent RNA polymerase of nuclei from lymphoid-rich and erythroid-rich mouse spleens was compared in regard to requirements and conditions of the reactions. The inhibition by either actinomycin D or pancreatic DNase indicated that a DNA template was required for the observed reactions. Following the induction of a hemolytic anemia by the administration of phenylhydrazine, there was a tenfold increase in the nuclear polymerase activity per milligram nuclear DNA of the developing erythropoietic spleen when the assays were done at low ionic strength and more than a threefold increase at high ionic strength. The peak rise in polymerase activity precedes the maximal development of the erythropoietic spleen by 3 days.

Detection and characterization of DNA polymerase activity in Toxoplasma gondii

Parasitology ◽  
1993 ◽  
Vol 107 (2) ◽  
pp. 135-139 ◽  
Author(s):  
A. Makioka ◽  
B. Stavros ◽  
J. T. Ellis ◽  
A. M. Johnson
Keyword(s):  
Toxoplasma Gondii ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Sedimentation Coefficient ◽  
Polymerase Activity ◽  
Magnesium Ions ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Approximate Molecular Weight

SUMMARYA DNA polymerase activity has been detected and characterized in crude extracts from tachzoites of Toxoplasma gondii. The enzyme has a sedimentation coefficient of 6·4 S, corresponding to an approximate molecular weight of 150000 assuming a globular shape. Like mammalian DNA polymerase α, the DNA polymerase of T. gondii was sensitive to N-ethylmaleimide and inhibited by high ionic strength. However, the enzyme activity was not inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases α, δ and ε and also cytosine-β-D-arabinofuranoside-5′-triphosphate which is an inhibitor of α polymerase. The activity was inhibited by 2′,3′-dideoxythymidine-5′-triphosphate which is an inhibitor of mammalian DNA polymerase β and γ. Magnesium ions (Mg2+) were absolutely required for activity and its optimal concentration was 6 mM. The optimum potassium (K+) concentration was 50 mM and a higher concentration of K+ markedly inhibited the activity. Activity was optimal at pH 8. Monoclonal antibodies against human DNA polymerase did not bind to DNA polymerase of T. gondii. Thus the T. gondii enzyme differs from the human enzymes and may be a useful target for the design of toxoplasmacidal drugs.

scholarly journals Evidence against a Simple Tethering Model for Enhancement of Herpes Simplex Virus DNA Polymerase Processivity by Accessory Protein UL42

2002 ◽  
Vol 76 (20) ◽  
pp. 10270-10281 ◽  
Author(s):  
Murari Chaudhuri ◽  
Deborah S. Parris
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Ionic Strength ◽  
Real Time ◽  
Dna Polymerase ◽  
Dissociation Rate ◽  
High Ionic Strength ◽  
Apparent Affinity ◽  
Simplex Virus ◽  
Low Ionic Strength

ABSTRACT The DNA polymerase holoenzyme of herpes simplex virus type 1 (HSV-1) is a stable heterodimer consisting of a catalytic subunit (Pol) and a processivity factor (UL42). HSV-1 UL42 differs from most DNA polymerase processivity factors in possessing an inherent ability to bind to double-stranded DNA. It has been proposed that UL42 increases the processivity of Pol by directly tethering it to the primer and template (P/T). To test this hypothesis, we took advantage of the different sensitivities of Pol and Pol/UL42 activities to ionic strength. Although the activity of Pol is inhibited by salt concentrations in excess of 50 mM KCl, the activity of the holoenzyme is relatively refractory to changes in ionic strength from 50 to 125 mM KCl. We used nitrocellulose filter-binding assays and real-time biosensor technology to measure binding affinities and dissociation rate constants of the individual subunits and holoenzyme for a short model P/T as a function of the ionic strength of the buffer. We found that as observed for activity, the binding affinity and dissociation rate constant of the Pol/UL42 holoenzyme for P/T were not altered substantially in high- versus low-ionic-strength buffer. In 50 mM KCl, the apparent affinity with which UL42 bound the P/T did not differ by more than twofold compared to that observed for Pol or Pol/UL42 in the same low-ionic-strength buffer. However, increasing the ionic strength dramatically decreased the affinity of UL42 for P/T, such that it was reduced more than 3 orders of magnitude from that of Pol/UL42 in 125 mM KCl. Real-time binding kinetics revealed that much of the reduced affinity could be attributable to an extremely rapid dissociation of UL42 from the P/T in high-ionic-strength buffer. The resistance of the activity, binding affinity, and stability of the holoenzyme for the model P/T to increases in ionic strength, despite the low apparent affinity and poor stability with which UL42 binds the model P/T in high concentrations of salt, suggests that UL42 does not simply tether the Pol to DNA. Instead, it is likely that conformational alterations induced by interaction of UL42 with Pol allow for high-affinity and high-stability binding of the holoenzyme to the P/T even under high-ionic-strength conditions.

scholarly journals Contributions of MexAB-OprM and an EmrE Homolog to Intrinsic Resistance of Pseudomonas aeruginosa to Aminoglycosides and Dyes

2003 ◽  
Vol 47 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Xian-Zhi Li ◽  
Keith Poole ◽  
Hiroshi Nikaido
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Ionic Strength ◽  
Ethidium Bromide ◽  
Luria Broth ◽  
Drug Susceptibility ◽  
Intrinsic Resistance ◽  
Aminoglycoside Resistance ◽  
Active Efflux ◽  
Low Ionic Strength ◽  
Strength Medium

ABSTRACT Of the six putative small multidrug resistance (SMR) family proteins of Pseudomonas aeruginosa, a protein encoded by the PA4990 gene (emrE Pae) shows the highest identity to the well-characterized EmrE efflux transporter of Escherichia coli. Reverse transcription-PCR confirmed the expression of emrE Pae in the wild-type strain of P. aeruginosa. Using isogenic emrE Pae, mexAB-oprM, and/or mexB deletion mutants, the contributions of the EmrE protein and the MexAB-OprM efflux system to drug resistance in P. aeruginosa were assessed by a drug susceptibility test carried out in a low-ionic-strength medium, Difco nutrient broth. We found that EmrEPae contributed to intrinsic resistance not only to ethidium bromide and acriflavine but also to aminoglycosides. In this low-ionic-strength medium, MexAB-OprM was also shown to contribute to aminoglycoside resistance, presumably via active efflux. Aminoglycoside resistance caused by these two pumps could not be demonstrated in high-ionic-strength media, such as Luria broth or Mueller-Hinton broth. The EmrE-dependent efflux of ethidium bromide was confirmed by a continuous fluorescence assay.

scholarly journals The binding of echinomycin to deoxyribonucleic acid

1976 ◽  
Vol 157 (3) ◽  
pp. 721-740 ◽  
Author(s):  
L P G Wakelin ◽  
M J Waring
Keyword(s):  
Ionic Strength ◽  
Binding Constant ◽  
Buoyant Density ◽  
Specific Interaction ◽  
Effect Of Temperature ◽  
Heat Denaturation ◽  
Peptide Antibiotic ◽  
Base Pairs ◽  
Calf Thymus ◽  
Low Ionic Strength

Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.

scholarly journals Characteristic alteration in the nuclear DNA polymerase activity during the cell division cycle ofSaccharomyces cerevisiae

1984 ◽  
Vol 12 (7) ◽  
pp. 3143-3154 ◽  
Author(s):  
Eiko Tsuchiya ◽  
Kosei Kimura ◽  
Tokichi Miyakawa ◽  
Sakuzo Fukui
Keyword(s):  
Cell Division ◽  
Dna Polymerase ◽  
Nuclear Dna ◽  
Polymerase Activity ◽  
Cell Division Cycle

scholarly journals Chromatographic Separation, and Characteristics of Nucleic Acids from HeLa Cells

1961 ◽  
Vol 44 (5) ◽  
pp. 899-910 ◽  
Author(s):  
Lennart Philipson
Keyword(s):  
Sodium Chloride ◽  
Ionic Strength ◽  
Bovine Serum Albumin ◽  
Nucleic Acids ◽  
Hela Cells ◽  
Sedimentation Coefficient ◽  
High Ionic Strength ◽  
Salt Gradient ◽  
Low Ionic Strength ◽  
Phenol Extract

The application of the phenol-duponol method to extraction of nucleic acids from HeLa cells is described. Chromatography of the phenol extract on an esterified bovine serum albumin column with a salt gradient of sodium chloride gives separation of soluble RNA, DNA, and two different high molecular RNA fractions. Ultracentrifugation of the DNA eluted from the column gives a sedimentation coefficient (s20o,w) of 38, which agrees with ultracentrifugation data on the phenol extract. The eluted RNA appears polydisperse at low ionic strength, but at high ionic strength and after alcohol precipitation two fractions with the sedimentation coefficients of 16 and 25 to 29, respectively, were obtained.

scholarly journals RNA-instructed DNA polymerase activity in a cytoplasmic particulate fraction in brains from Guamanian patients.

1975 ◽  
Vol 142 (2) ◽  
pp. 483-494 ◽  
Author(s):  
M V Viola ◽  
M Frazier ◽  
L White ◽  
J Brody ◽  
S Spiegelman
Keyword(s):  
Dna Polymerase ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Polymerase Activity ◽  
Particulate Fraction ◽  
Product Analysis ◽  
Visna Virus ◽  
Number Of Patients ◽  
Tumor Virus ◽  
Lateral Sclerosis

Nervous system tissues from a number of patients with idiopathic neurological disorders were examined for biochemical evidence of RNA tumor virus infection. RNase-sensitive DNA polymerase activity was found in a cytoplasmic particulate fraction from two patients with Guamanian amyotrophic lateral sclerosis (ALS) but not in brains from two normal U.S. individuals. The buoyant density of the enzyme-containing fraction was 1.16-1.18 g/ml and could be converted to a denser region of the gradient (1.24 g/ml) by treatment with the nonionic surfactant, Sterox. The cation and detergent requirements for the endogenous RNase-sensitive DNA polymerase reaction were determined. The early (5 min) endogenous reverse transcriptase product was analyzed by cesium sulfate gradient centrifugation. RNase- and heat-sensitive RNA-DNA hybrids were detected in the product analysis of two ALS, one Parkinsonism-dementia (PD) brain, and two brains from asymptomatic Chamorros but not in brains from normal U.S. individuals and a number of patients with neuro-psychiatric disorders. The DNA product was a 4.5S heteropolymer that hybridized more extensively to RNA extracted from the enzyme-containing pellet from PD brain as compared to a similar fraction from normal U.S. brain. The DNA product appeared to be unrelated to Rausvher or visna virus 70S RNA as determined by RNA-[-3H]DNA hybridization.

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