ABSTRACT
Ability of site-specific nickase BspD6I (Nt.BspD6I) to oligomerize at concentrations > or = 0.5 microM (> or = 0.035 mg/mL) is studied. Three states of Nt.BspD6I are registered via electrophoretic studies both in the presence and in the absence of DNA. Estimation of their molecular mass allows assigning them as a monomer, a dimer and a trimer. Both dimeric and monomeric Nt.BspD6I are shown to hydrolyze its DNA substrate with the identical specificity. Calculation of the electrostatic potential distribution on the Nt.BspD6I globule surface shows that the protein molecule is a dipole. The Nt. BspD6I oligomeric forms are likely to be the result of ionic protein interactions.
Subject(s)
DNA-Binding Proteins/chemistry , Deoxyribonuclease I/chemistry , Protein Structure, Tertiary , Bacillus/enzymology , DNA/chemistry , Protein MultimerizationABSTRACT
Nicking endonuclease Nt.BspD6I is a heterodimeric restriction endonuclease, one subunit of which exhibits specific nicking activity. It gets bound to double-stranded DNA and makes a break (nick) in one chain at a distance of 4 nucleotides from the binding site. In this work, for visualization of the specific binding and protein landing site an atomic force microscopy was used. In five minutes after incubation of DNA solution with nicking endonuclease, the DNA molecules with associated proteins which located at the expected binding site and "shared" DNA strand into two segments (approximately, 1/3 and 2/3 of length) were observed in the images. In addition, near the binding site DNA molecule had a height corresponding to a single-stranded DNA molecule, which was in good agreement with single-stranded cleavage by nickase in the course of complex formation.
Subject(s)
Bacillus/enzymology , DNA, Single-Stranded/chemistry , DNA/chemistry , Deoxyribonuclease I/chemistry , Binding Sites , DNA/ultrastructure , DNA, Single-Stranded/ultrastructure , Microscopy, Atomic Force , Protein Subunits/chemistry , Substrate SpecificityABSTRACT
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
Whether X-ray refractional introscopy can be used to examine biological objects was studied. The characteristic emission of an X-ray tube provided refractional radiograms of the rat heart and limb, which allow one to make out the details of the structure of soft tissues and bones invisible on absorption radiograms under the same conditions. High-contrast refractional images of calcified formations were seen in the real-time mode when they were registered with a two-coordinate TV detector. The potentialities of the new method and medical diagnostic means are discussed.
Subject(s)
Bone and Bones/diagnostic imaging , Heart/diagnostic imaging , Radiography/methods , Animals , Equipment Design , Forelimb , Radiography/instrumentation , Rats , X-Ray DiffractionABSTRACT
It is shown that X-ray pattern rich in reflections that we have received earlier from concentrated canine duodenal juice is the sum of the patterns arising from both mucin macromolecules and from unknown component of the juice. The functional role of the duodenal juice regularity observed is discussed.
Subject(s)
Duodenum/physiology , Intestinal Secretions/chemistry , Animals , Dogs , Electrophoresis, Polyacrylamide Gel , X-Ray DiffractionABSTRACT
A site-specific endonuclease which recognizes the sequence 5'-CCTNAGG-3' was purified to homogeneity from the thermophilic strain Bacillus sp. R7. The endonuclease (BspR7I) is monomeric protein with an apparent molecular weight of 37 kD. The enzyme is active over a wide range of NaCl concentrations, pH, and temperatures. BspR7I cleaves DNA substrates according to the scheme: 5'-CC decreases TNAGG-3' 3'-GGANT increases CC-5', hence the endonuclease represents an isoschizomer of Bsu361.
Subject(s)
Bacillus/enzymology , Deoxyribonucleases, Type II Site-Specific/isolation & purification , Chromatography, Gel , Enzyme Stability , Molecular Weight , Substrate SpecificityABSTRACT
Two site-specific endonucleases, BspOVI and BspOVII, were isolated from a thermophilic strain Bacillus species OV. The activities of both enzymes are maximal at 48 degrees C and do not depend on ATP and S-adenosyl-L-methionine. BspOVI recognizes the sequence [sequence: see text] and cleaves it as indicated by arrows. Thus, BspOVI is a IIN-subclass endonuclease isoshizomer of Eam1105I. BspOVI is very stable during storage. The enzyme can be used for direct T/A cloning of PCR products. BspOVII recognizes and cleaves the sequence [sequence: see text]; thus, BspOVII is an isoshizomer of CIaI. The cleavage by BspOVII is blocked by dam methylation of adenine inside the recognition site.
Subject(s)
Bacillus/enzymology , Deoxyribonucleases, Type II Site-Specific/metabolism , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Substrate SpecificityABSTRACT
Site-specific endonuclease R. AspMI was isolated and purified to apparent functional homogeneity from Acinetobacter species (strain M). The enzyme recognizes symmetrical DNA sequence 5'-AGG decreases CCT-3' and cleaves it at the site indicated by the arrow forming blind DNA ends. The endonuclease is an isoschizomer of the StuI endonuclease. Cleavage of the DNA site was inhibited by dcm-methylation. AspMI is approximately equal to 30 kD monomer.
Subject(s)
Acinetobacter/enzymology , Deoxyribonucleases, Type II Site-Specific/isolation & purification , Chromatography, Gel , DNA Methylation , Deoxyribonucleases, Type II Site-Specific/genetics , Enzyme Stability , Molecular Weight , Plasmids , Restriction Mapping , Substrate SpecificityABSTRACT
The site-specific DNA-methylase M.BspST5I has been isolated from Bacillus species ST5 and purified to functional purity. M.BspST5I protects DNA from endonuclease R.BspST5I which recognizes the nonpalindromic sequence [formula: see text] on DNA. MpBspST5I belongs to adenine-specific methylases.
Subject(s)
Bacillus/enzymology , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism , Chromatography, Gel , DNA Methylation , Site-Specific DNA-Methyltransferase (Adenine-Specific)/isolation & purification , Substrate SpecificityABSTRACT
The structure of both native mucins and subunits obtained by thiol reduction was studied by small-angle X-ray diffraction. X-ray pattern from the subunits significantly differs from that of the native mucins in the number, spacings and width of the reflections observed. Decrease in the number of the reflections and increase of their width pointed out that isolated subunits are less regular structure than the whole mucin molecule. This may be due to the two reasons: 1) conformation of the subunits in native molecule differs from that of in isolate state; 2) native mucin molecule is not a simple polymer, formed by the subunits.
Subject(s)
Mucins/chemistry , Sulfhydryl Compounds/chemistry , Animals , Chromatography, Gel , Indicators and Reagents , Mucins/isolation & purification , Protein Conformation , Rats , X-Ray DiffractionABSTRACT
A site-specific endonuclease R.BspST5I has been isolated in a functionally pure state from the thermophilic strain of Bacillus species ST5. The enzyme recognizes sequence 5'-GCATC-3' on the DNA and splits it at a distance of five nucleotides from the 3'-end of the recognition site as well as at distances of nine or ten nucleotides at the complementary filament depending on the hydrolyzed sequence of the DNA. The enzyme is a isomer of endonuclease SfaNI from Streptococcus faecalis ND547.
Subject(s)
Bacillus/enzymology , DNA Restriction Enzymes/isolation & purification , Base Sequence , Chromatography, Gel , DNA/metabolism , DNA Restriction Enzymes/metabolism , Electrophoresis, Agar Gel , Molecular Sequence DataABSTRACT
A method for separating into definite sets of a complex mixture of fragments obtained by DNA cleavage with IIS- or IIN-types of restriction endonucleases producing single-stranded termini of different sequences at the fragment ends has been developed. The method is based on the ligation of short double-stranded adapters with single-stranded termini complementary to the termini of a selected set of fragments followed by PCR-amplification with the primer which represents a strand of the adapters. Using endonucleases BcoKI and Bli7361 recognizing sequences CTCTTC and GGTCTC and producing three- and four-nucleotide 5'-termini, respectively, it has been shown that amplification of a set of fragments occurs only when the adapters are attached to DNA fragments with DNA-ligase. Several applications of the SAGF-method are suggested: for obtaining individual bands in DNA fingerprinting; for reducing the kinetic complexity of DNA in the representational difference analysis (RDA method) of complex genomes; for cataloguing DNA fragments, and for constructing physical genomic maps.
Subject(s)
DNA/genetics , Polymerase Chain Reaction/methods , Animals , Base Sequence , Chick Embryo , DNA Fingerprinting , DNA Restriction Enzymes , Molecular Sequence DataABSTRACT
The site-specific endonuclease R . BspIS4I and methylase M . BspIS4I have been isolated and purified to functional purity from the thermophilic strain of Bacillus species IS4. R . BspIS4I recognizes sequence [sequence: see text] on the DNA and cleaves it as indicated by the arrows to form single-stranded 4-nucleotide 5'-protruding termini. The enzyme is an isoschizomer of BbvII. M . BspIS4I is related to adenine-specific methylase.
Subject(s)
Bacillus/enzymology , Site-Specific DNA-Methyltransferase (Adenine-Specific)/isolation & purification , Base Sequence , Chromatography, Ion Exchange , DNA, Bacterial/metabolism , Electrophoresis, Agar Gel , Molecular Sequence Data , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolismABSTRACT
A site-specific endonuclease capable of recognizing the sequence 5'-AAGCTT-3' was detected and purified to homogeneity from the thermophilic strain of Bacillus species KT8. The endonuclease has a molecular mass of 34 kDa and is found in solution in a monomeric form. The activity of BspKT8 does not depend on ATP and is not stimulated by S-adenosyl-L-methionine. The enzyme displays the highest activity with a broad range of temperatures (37 degrees-48 degrees C). Since DNA cleavage occurs in accordance with the scheme: [formula: see text] the enzyme can be assigned to the class-II of restriction endonucleases and represents an isoschizomer of HindIII.
Subject(s)
Bacillus/enzymology , DNA Restriction Enzymes/isolation & purification , Chromatography, Gel , Chromatography, Ion Exchange , DNA Restriction Enzymes/metabolism , Electrophoresis, Agar Gel , Hot Temperature , Substrate SpecificityABSTRACT
The fliC gene of the E. coli B38 flagellin has been cloned and its nucleotide sequence determined using the terminator method. According to the sequencing data, the flagellin contains 565 amino acid residues which exceeds by 65 residues the number of amino acid residues in the earlier decoded E. coli K12 flagellin. Strong homology was observed in the two flagellins among the 160 initial and 89 tail-ended residues, whereas the central, variable parts showed no homology. Similar to the K12 flagellin, the B38 flagellin has no serines, cysteines or tryptophans. The variable part of the fliC E. coli B38 gene contains a Chi-site which initiates the genetic recombination in E. coli and related species.
Subject(s)
Escherichia coli/genetics , Flagellin/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Bacterial , Molecular Sequence Data , Recombination, Genetic , Sequence Homology, Amino AcidABSTRACT
A new site-specific endonuclease BspLU11III was purified to homogeneity from a thermophilic strain Bacillus species LU11. BspLU11III recognizes the 5'-GGGAC-3' sequence on the double-stranded DNA and cleaves the 10/14 and 11/15 nucleotides in different strands away from the recognition site. The enzyme exists in solution as a monomer with a molecular mass of about 93 kDa. When incubated with S-adenosyl-L-methionine, BspLU11III displays a DNA-methyltransferase activity. The adenine residue is methylated inside the recognition site 5'-GGGAC-3' in the only strand. The restriction activity does not change in the presence of ATP but is stimulated by 80 microM S-adenosyl-L-methionine (4-fold). Magnesium cations are needed for the restriction activity. Sodium chloride stimulates the "star" activity of BspLU11III. According to its properties, BspLU11III can be classified as a type IV endonuclease.
Subject(s)
Bacillus/enzymology , DNA Restriction-Modification Enzymes/metabolism , Base Sequence , Chromatography, Gel , DNA/metabolism , DNA Restriction-Modification Enzymes/isolation & purification , Electrophoresis, Agar Gel , Molecular Sequence Data , Molecular Weight , Substrate SpecificityABSTRACT
The site-specific endonuclease R.BspKT6I and the cognate site-specific methylase M.BspKT6I have been isolated from the thermophilic strain of Bacillus species KT6 using gel-filtration on Sephadex G100 followed by chromatography on heparin-Sepharose and hydroxyapatite. Endonuclease BspKT6I is an isomer but not an isoschizomer of Sau3AI and MboI. It recognized on the DNA molecule the GAT decreases C sequence and cleaves it; however, unlike Sau3AI and MboI it produces 3'-protruding dinucleotides. The site cleavage is inhibited by dam-methylation. The sticky ends resulting from the BspKT6I cleavage are identical and complementary to the ends formed after the PvuI cleavage. The isolated from the B. species KT6 methylase protects the DNA from subsequent cleavage by BspKT6I. Adenine is a methylated base.
Subject(s)
Bacillus/enzymology , Deoxyribonucleases, Type II Site-Specific/metabolism , Chromatography, Gel , DNA/metabolism , Deoxyribonucleases, Type II Site-Specific/isolation & purification , Electrophoresis, Agar Gel , Site-Specific DNA-Methyltransferase (Adenine-Specific)/isolation & purification , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism , Substrate SpecificityABSTRACT
Low-angle X-ray diffraction patterns of canine duodenal juice precipitates taken at two stages of digestion, after 20 h fasting and after food stimulation have been obtained. Both diffraction patterns are due to glycoproteins. However, the glycoproteins contained in the precipitate after food stimulation occur in the native form while in fasting animals they are partially degraded.
Subject(s)
Duodenum/physiology , Adaptation, Physiological , Animals , Chemical Precipitation , Digestion , Dogs , Duodenum/chemistry , Duodenum/metabolism , Fasting , Glycoproteins/chemistry , Intestinal Secretions/physiology , X-Ray DiffractionABSTRACT
The new site-specific endonuclease BspKT5I free from interfering impurities has been isolated from thermophilic soil bacteria Bacillus species KT5 by successive chromatography on blue agarose, hydroxyapatite and heparin-Sepharose. BspKT5I on double-stranded DNA recognizes the sequence 5'-CTGAAG16N decreases 3'-GACTTC14N increases and cleaves the DNA at the recognition site as indicated by the arrows to form dinucleotide 3'-protruding termini. The isolated endonuclease is an isoschisomer of Eco57I. However, unlike Eco57I, it is not stimulated by S-adenosylmethionine (SAM) and can therefore be related to subclass IIs but not to IV, as Eco57I. In addition, endonuclease BspKT5I, unlike Eco57I, has no methylase activity.
Subject(s)
Bacillus/enzymology , DNA Restriction Enzymes/metabolism , Base Sequence , Chromatography, Gel , DNA/metabolism , DNA Restriction Enzymes/isolation & purification , Electrophoresis, Agar Gel , Hydrolysis , Molecular Sequence Data , Substrate SpecificityABSTRACT
The strain, producing new site-specific endonuclease BcoKI has been found at the screening of the thermophilic bacteria isolated from tobacco. A phenotype characteristic of the strain is given. It has been identified as a new strain Bacillus coagulans. BcoKI, a class-IIS restriction endonuclease has been obtained by three consecutive chromatographies on blue agarose hydroxyapatite and heparin-Sepharose. BcoKI, an isoschizomer of Ksp6321, recognizes the six base non-palindromic sequence 5'CTCTTC3' and cleaves one nucleotide 3' of the 3' cytosine on this strand and four nucleotide 5' of the 5' guanine on the opposite strand to generate a three base 5' overhang.
