ABSTRACT
The site-specific endonuclease BspLU4I was discovered in the thermophilic Bacillus species LU4 strain and purified to functionally pure state by chromatography on blue agarose, hydroxyapatite HTP, and heparin-Sepharose columns. Analysis of cleavage patterns of different DNAs with known nucleotide sequences demonstrated that the enzyme recognizes the CPyCGPuG site on the DNA. Cleavage points in the sequence were determined with the elongated primer method. It was shown that the endonuclease is an isoschizomer of AvaI. The final yield of the enzyme is 2.25.10(6) units per g wet biomass.
Subject(s)
Bacillus/enzymology , Deoxyribonucleases, Type II Site-Specific/biosynthesis , Base Sequence , Binding Sites/genetics , DNA/genetics , DNA/metabolism , Deoxyribonucleases, Type II Site-Specific/isolation & purification , Deoxyribonucleases, Type II Site-Specific/metabolism , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Molecular Weight , Silicon Dioxide/chemistry , Substrate Specificity , TemperatureABSTRACT
A new method for hybridization analysis of nucleic acids is proposed on the basis of the ability of site-specific nickases to cleave only one DNA strand. The method is based on the use of a labeled oligonucleotide with the recognition site of the nickase hybridized with the target (DNA or RNA) at an optimal temperature of the enzyme (55 degrees C). The two shorter oligonucleotides formed after the cleavage with the nickase do not complex with the target. Thus, a multiple cleavage of the labeled oligonucleotide takes place on one target molecule. The cleavage of the nucleotide is recorded either by polyacrylamide gel electrophoresis (when a radioactive labeled oligonucleotide is used) or by fluorescence measurements (if the oligonucleotide has the structure of a molecular beacon). The new method was tested on nickase BspD6I and a radioactive oligonucleotide complementary to the polylinker region of the viral DNA strand in bacteriophage M13mp19. Unfortunately, nickase BspD6I does not cleave DNA in the RNA-DNA duplexes and therefore cannot be used for detection of RNA targets.
Subject(s)
DNA/chemistry , Deoxyribonuclease I/chemistry , Bacteriophages/chemistry , DNA, Viral/chemistry , Electrophoresis, Polyacrylamide Gel , Hydrolysis , Nucleic Acid Heteroduplexes/chemistry , Nucleic Acid Hybridization/methods , RNA/chemistry , Spectrometry, FluorescenceABSTRACT
Staphylococcus species strain D5 containing two site-specific endonucleases, SspD5 I and SspD5 II, was found during screening of a bacterial strain collection from soil. These endonucleases were purified to functional homogeneity by sequential chromatography. Site-specific endonuclease SspD5 I recognizes sequence 5;-GGTGA(8N/8N) downward arrow-3; on DNA. Unlike Hph I, it cleaves DNA at a distance of 8 nucleotides from the recognized sequence on both chains producing blunt-end DNA fragments, while endonuclease Hph I cleaves DNA forming mononucleotide 3;-OH protruding ends. Thus, endonuclease SspD5 I is a new type II site-specific endonuclease and a neoschizomer of endonuclease Hph I. The advantage of this new endonuclease is that the blunt-end DNA products of this enzyme can be inserted without additional treatment into vector DNAs cleaved with endonucleases yielding DNA blunt-ends. Endonuclease SspD5 II recognizes site 5'-ATGCA T-3' and thus is an isoschizomer of endonuclease Nsi I. The molecular mass of SspD5 I is about 35 kD and that of SspD5 II is 40 kD. The enzymes exhibit maximal activity at 37 degrees C. The optimal buffer for the reaction is HRB (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM NaCl, and 1 mM dithiothreitol).
Subject(s)
Deoxyribonucleases, Type II Site-Specific/chemistry , Deoxyribonucleases, Type II Site-Specific/genetics , Staphylococcus/enzymology , Base Sequence , Chromatography, Agarose , Chromatography, Gel , DNA/metabolism , Deoxyribonucleases, Type II Site-Specific/isolation & purification , Kinetics , Molecular Sequence Data , Substrate SpecificityABSTRACT
Fragments of the 16S rRNA of Thermus thermophilus representing the 3' domain (nucleotides 890-1515) and the 5' domain (nucleotides 1-539) have been prepared by transcription in vitro. Incubation of these fragments with total 30S ribosomal proteins of T. thermophilus resulted in formation of specific RNPs. The particle assembled on the 3' RNA domain contained seven proteins corresponding to Escherichia coli ribosomal proteins S3, S7, S9, S10, S13, S14, and S19. All of them have previously been shown to interact with the 3' domain of the 16S RNA and to be localized in the head of the 30S ribosomal subunit. The particle formed on the 5' RNA domain contained five ribosomal proteins corresponding to E. coli proteins S4, S12, S17, S16, and S20. These proteins are known to be localized in the main part of the body of the 30S subunit. Both types of particle were compact and had sedimentation coefficients of 15.5 S and 13 S, respectively. Together with our recent demonstration of the reconstitution of the RNA particle representing the platform of the T. thermophilus 30S ribosomal subunit [Agalarov, S.C., Zheleznyakova, E.N., Selivanova, O.M., Zheleznaya, L.A., Matvienko, N.I., Vasiliev, V.D. & Spirin, A.S. (1998) Proc. Natl Acad. Sci. USA 95, 999-1003], these experiments establish that all three main structural lobes of the small ribosomal subunit can be reconstituted independently of each other and prepared in the individual state.
Subject(s)
Ribosomal Proteins/chemistry , Thermus thermophilus/chemistry , Centrifugation, Density Gradient , Electrophoresis, Gel, Two-Dimensional , Nucleic Acid Conformation , Plasmids/metabolism , Protein Structure, Tertiary , RNA/chemistry , Sucrose/pharmacology , Thermus thermophilus/metabolism , Transcription, GeneticABSTRACT
A fragment of the 16S RNA of Thermus thermophilus corresponding to the central domain (nucleotides 547-895) has been prepared by transcription in vitro. Incubation of this fragment with the total 30S ribosomal proteins has resulted in the formation of a compact 12S ribonucleoprotein particle. This particle contained five T. thermophilus proteins corresponding to Escherichia coli ribosomal proteins S6, S8, S11, S15, and possibly S18, all of which were previously shown to interact with the central domain of the 16S RNA and to be localized in the platform (side bulge) of the 30S ribosomal subunit. When examined by electron microscopy, isolated particles have an appearance that is similar in size and shape to the corresponding morphological features of the 30S subunit. We conclude that the central domain of the 16S RNA can independently and specifically assemble with a defined subset of ribosomal proteins into a compact ribonucleoprotein particle corresponding to the platform (side bulge) of the 30S subunit.
Subject(s)
RNA, Bacterial/chemistry , RNA, Ribosomal, 16S/chemistry , Ribosomal Proteins/chemistry , Thermus thermophilus/genetics , Base Sequence , Escherichia coli , Microscopy, Electron , Molecular Sequence Data , Nucleic Acid Conformation , Protein Folding , RNA, Bacterial/ultrastructure , RNA, Ribosomal/chemistry , RNA, Ribosomal/ultrastructure , RNA, Ribosomal, 16S/ultrastructure , Ribosomal Proteins/ultrastructure , Thermus thermophilus/ultrastructure , Transcription, GeneticABSTRACT
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.
