ABSTRACT
In the presence of the Nt.BspD6I nicking endonuclease DNA polymerase Bst stimulates intensive template/primer-independent DNA synthesis. Template/primer-independent DNA synthesis could be the reason for appearing nonspecific DNA products in many DNA amplification reactions particularly in the reactions with using nicking endonucleases. Search of the modes for inhibition template/primer-independent DNA synthesis becomes an urgent task because of broadening the DNA amplification methods with using nicking endonucleases. We report here that the E. coli single-stranded DNA binding protein has no effect on the template/primer-independent DNA synthesis. In the absence of the nicking endonuclease the single-stranded DNA binding protein encoded by bacteriophage T4 gene 32 completely inhibits template/primer-independent DNA synthesis. This protein does not inhibit synthesis of specific DNA product in the presence of nicking endonuclease but remarkably decreases the amount of nonspecific products.
Subject(s)
DNA-Binding Proteins/chemistry , DNA/chemistry , Deoxyribonuclease I/chemistry , Escherichia coli Proteins/chemistry , Viral Proteins/chemistry , DNA/biosynthesis , DNA-Binding Proteins/metabolism , Deoxyribonuclease I/metabolism , Escherichia coli Proteins/metabolism , Viral Proteins/metabolismABSTRACT
The radiation reaction of the mouse brain microvascular system was investigated 15 months after chronic (dose-rate was 3 cGy/day) and acute (dose-rate 3.2 Gy/min) gamma-irradiation. Late pathological changes in the microvascular system after the acute irradiation (3 Gy) were not found. In the same time, after chronic irradiation with equivalent dose (the accumulated dose was 3 Gy) the depletion of endothelial cell population, and multiple foci of minor pathological changes of endotheliocyte ultrastructure at as many as 50% of the population were revealed. It may be more likely to lead to reduction of tolerance of brain microvascular system to an acute radiation (10 Gy) in the late period after chronic exposure. The mechanism of impairment of brain microcirculation after low chronic irradiation will have to understand.
Subject(s)
Brain/radiation effects , Cerebrovascular Circulation/radiation effects , Microcirculation/radiation effects , Animals , Data Interpretation, Statistical , Endothelium, Vascular/cytology , Endothelium, Vascular/radiation effects , Follow-Up Studies , Mice , Mice, Inbred BALB C , Microscopy, Electron , Radiation Dosage , Time FactorsABSTRACT
A method has been developed to prepare random DNA fragments using PCR. First, two cycles are carried out at 16 degrees C with the Klenow's fragment and oligonucleotides (random primers) with random 3'-sequences and the 5'-constant part containing the site for cloning with the site-specific endonuclease. The random primers can link to any DNA site, and random DNA fragments are formed during DNA synthesis. During the second cycle, after denaturation of the DNA and addition of the Klenow's fragment, the random primers can link to newly synthesized DNA strands, and after DNA synthesis single-stranded DNA fragments are produced which have a constant primer sequence at the 5'-end and a complementary to it sequence at the 3'-end. During the third cycle, the constant primer is added and double-stranded fragments with the constant primer sequences at both ends are formed during DNA synthesis. Incubation for 1 h at 37 degrees C degrades the oligonucleotides used at the first stage due to endonuclease activity of the Klenow's fragment. Then routine PCR amplification is carried out using the constant primer. This method is more advantageous than hydrodynamic methods of DNA fragmentation widely used for "shotgun" cloning.
Subject(s)
DNA Fragmentation , Polymerase Chain Reaction/methods , Base Sequence , DNA/chemistry , DNA Primers , Nucleic Acid ConformationABSTRACT
Site-specific endonuclease NspLKI has been isolated and purified to functionally pure state from soil bacterium Nocardia species LK by successive chromatography on columns with phosphocellulose, HTP hydroxyapatite, and heparin-Sepharose. The isolated enzyme recognizes the 5'-GG downward arrowCC-3' sequence on DNA and cleaves it as indicated by the arrow, i.e., it is an isoschizomer of HaeIII. The final enzyme yield is 1.105 units per gram of wet biomass. The enzyme is active in the temperature range of 25-60 degrees C with an optimum at 48-55 degrees C; it does not lose activity on storage for three days at room temperature. An optimal buffer is HRB containing 10 mM Tris-HCl, pH 7.4, 200 microgram/ml albumin, 10 mM MgCl2, and 100 mM NaCl.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/metabolism , Chromatography, Ion Exchange , DNA/metabolism , Electrophoresis, Agar Gel , Enzyme Stability , Hydrogen-Ion Concentration , Substrate Specificity , TemperatureABSTRACT
Strain Bacillus species ZE with a maximal yield of the ClaI isoschizomer was chosen from 12 natural thermophilic strains producing ClaI isomers. The yield is 110 times higher than that of the ClaI prototype endonuclease from Caryophanon latum L. The enzyme is sensitive to DNA dam-methylation and is highly stable under storage.
