Location of the bases modified by M.BcoKIA and M.BcoKIB methylases in the sequence 5 -CTCTTC-3 /5 -GAAGAG-3.

The strain Bacillus coagulans K contains two DNA-methyltransferases, M.BcoKIA and M.BcoKIB, which recognize the sequence 5 -CTCTTC-3 /5 -GAAGAG-3 and possess N4-methylcytosine and N6-methyladenine specificities, respectively. A special construct containing the recognition site of BcoKI and sites of four IIS restriction endonucleases (IIS restriction endonuclease cassette) was designed to locate the nucleotides modified by the methylases. The modified bases were determined as: 5 -m(4)CTCTTC-3 /5 -GAAGAm(6)G-3 .

Two new site-specific endonucleases from Staphylococcus species strain D5.

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).

Site-specific DNA-methylase M.BspST5I methylates only one strand of the recognized site.

We recently isolated a site-specific adenine DNA-methylase, M.BspST5I, which methylates only one strand of the recognized site GCATC. The methylated base is indicated by an asterisk.

Peculiarities of gene expression of the EcoRII modification-restriction system.

The restriction-modification genes of the EcoRII system have been cloned into plasmids under control of phage-specific promoters T7 and SP6. The transcription was induced by cell infection with the recombinant M13 phages with the corresponding genes of phage RNA-polymerases under control of the Plac-promoter in the presence of IPTG. The induction yields significant amounts of EcoRII DNA-methylase for both phage-specific promoters. In both cases no increase in EcoRII endonuclease expression could be achieved. We hypothesize that the expression of the endonuclease gene is regulated on the translational level.

[Novel site-specific endonuclease from Acinetobacter species M strain]. / Novaia sait-spetsificheskaia éndonukleaza iz shtamma Acinetobacter species M.

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.

[A new site-specific adenine DNA-methyltransferase from Bacillus species ST5]. / Novaia sait-spetsificheskaia adeninovaia DNK-metiltransferaza iz shtamma Bacillus species ST5.

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.

[A new site-specific endonuclease from Bacillus species ST5]. / Novaia sait-spetsificheskaia éndonukleaza iz Bacillus species ST5.

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.

[A method of selective PCR-amplification of genomic DNA fragments (SAGF method)]. / Metod izbiratel'noi PCR-amplifikatsii fragmentov genomnoi DNK (metod SAGF).

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.

[Site-specific endonuclease and methylase from thermophilic bacteria of Bacillus species IS4]. / Sait-spetsificheskie éndonukleaza i metilaza iz termofil'noi bakterii Bacillus species IS4.

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.

BspLUII III, a bifunctional restriction and modification enzyme from a thermophilic strain Bacillus species LUII.

BspLUII III, an isomer of FinI (1) and BsmFI (2), was found to cleave DNA at two points 10, 11 and 14, 15 bp in the different strands away from the recognition site, and in the presence of SAM it exhibits the adenine specific methyltransferase activity.Images

[A new site specific endonuclease-methylase from a thermophilic strain of Bacillus species LU11]. / Novaia sait-spetsificheskaia éndonukleaza-metilaza iz termofil'nogo shtamma Bacillus species LU11.

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.

[A new site-specific endonuclease BspKT51]. / Novaia sait-spetsificheskaia éndonukleaza BspKT51.

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.

[Isolation of site-specific endonucleases and methylases from Bacillus cereus 83]. / Vydelenie sait-spetsificheskikh éndonukleazy i metilazy iz Bacillus cereus 83.

The site-specific endonuclease R Bce83I and methylase M Bce83I were isolated from Bacillus cereus 83 by three consecutive chromatographies on blue agarose, hydroxyapatite and heparin-Sepharose. R Bce83I recognizes [formula: see text] sequences on the DNA and cleaves the DNA as indicated by the arrows. The endonuclease is stimulated by S-adenosyl-L-methionine and may consequently be referred to type IV restriction enzymes.

[Two site-specific endonucleases from the thermophilic strain Bacillus species LU11]. / Dve sait-spetsificheskie éndonukleazy iz termofil'nogo shtamma Bacillus species LU11.

Upon screening of natural strains of thermophilic bacteria, a strain has been found which contains two restriction endonucleases. One of those, BspLU11II, is an isoschizomer of XbaI, while the other one, BspLU11I, recognizes the new palindromic sequence 5'-A decreases CATGT-3' and cleaves it as indicated by the arrow. Functionally pure enzymes were obtained by stepwise chromatography with blueagarose, hydroxyapatite and heparin-Sepharose. The restriction endonuclease BspLU11I produces sticky ends identical to those produced by the restriction endonuclease NcoI; hence a combination of BspLU11I and NcoI can be used for enzymatic selection of recombinant DNA. The recognition sequence of BspLU11I contains the ATG codon and can be used to construct expression vectors for chemically synthesized genes.

[New site site-specific endonuclease and methylase from Bacillus licheniformis 736]. / Novye sait-spetsificheskie éndonukleaza i metilaza iz Bacillus licheniformis 736.

The site-specific endonuclease R Bli736I and methylase M Bli736I have been isolated from the Bacillus licheniformis strain 736 by blue-agarose, hydroxyapatite-Ultragel and heparin-Sepharose chromatography. The enzymes are free from interfering impurities. R Bli736I recognizes the 5'-GGTCTCN-3' decreases and decreases 5'-NNNNNGAGACC-3' sequences on the DNA and cleaves the DNA as indicated by arrows to form single-stranded 4-nucleotide 5'-protruding termini. This enzyme is an isoschizomer of Eco3II isolated from E. coli.