Cloning and Characterization of a New Site-Specific Methyl-Directed ElmI Endonuclease Recognizing and Cleaving C5-methylated DNA Sequence 5'-G(5mC)

Putative open reading frames of MD-endonucleases have been identified in Enterobacteria genomes as a result of the search for amino acid sequences homologous to MD-endonuclease BisI. A highly conserved DNA primary structure of these open reading frames in different genera of Enterobacteria (Escherichia, Klebsiella and Cronobacter) has allowed researchers to create primers for PCR screening, which was carried out on Enterobacteria DNA collected from natural sources. The DNA fragment, about 440 bp in length, was amplified by use of the genomic DNA of a wild E.coli LM N17 strain as a template and was inserted into the pMTL22 vector. Endonuclease activity was detected in an E.coli ER 2267 strain transformed with the obtained construction. A new enzyme named ElmI was purified by chromatographic techniques from the recombinant strain biomass. It was discovered that similarly to BisI this enzyme specifically cleaves the methylated DNA sequence 5'-GCNGC- 3' before the central nucleotide "N" if this sequence contains two 5-methylcytosines. However, unlike BisI, ElmI more efficiently cleaves this sequence if more than two cytosine residues are methylated.

[A new DNA methyltransferase M.BstC8I forms 5'-G(M5C)NNGC-3'. Studying of restriction endonuclease sensitivity to M.BstC8I-methylation].

Bacillus stearothermophilus C8 was grown up on the Luria agar at 37 degrees C. A new DNA-methylase was determined in cellular lysate. The methylation of the DNAs of bacteriophages lambda and T7 in the region of 5'-G(m5C)NNGC-3' blocked the activity of BstC8I. Specificity of M.BstC8I was analyzed on methylated lambda DNA. For this purpose, we used computer modeling and the data on the sensitivity of restrictases BstC8I, BsuRI, AjnI, and PvuII to methylation. The sensitivity of some restrictases to new methylation was studied. The results may be used for DNA methylation studying.

[Definition of the specificity of DNA-methyltransferase M.Bsc4I in cell lysate by blocking of restriction endonucleases and computer modeling].

The specificity of DNA-methyltransferase M.Bsc4I was defined in cellular lysate of Bacillus schlegelii 4. For this purpose, we used methylation sensitivity of restriction endonucleases, and also modeling of methylation. The modeling consisted in editing sequences of DNA using replacements of methylated bases and their complementary bases. The substratum DNA processed by M.Bsc4I also were used for studying sensitivity of some restriction endonucleases to methylation. Thus, it was shown that M.Bsc4I methylated 5'-Cm4CNNNNNNNGG-3' and the overlapped dcm-methylation blocked its activity. The offered approach can appear universal enough and simple for definition of specificity of DNA-methyltransferases.

[Microorganisms of Lake Baikal and Lake Nyasa as indicators of anthropogenic influence: prospects of use in biotechnology]. / Mikroorganizmy ozer Baikal i N'iasa kak indikatory antropogennogo vliianiia i perspektiva ikh ispol'zovaniia v biotekhnologii.

Restriction endonucleases (RENs) were detected in 650 microbial strains isolated from water columns and bottom sediments of deep rift lakes, Baikal (Russia) and Nyasa (Southeastern Africa). They enzymes included unique (Fan I, Aca I, and Sse 91) and very rare (Bsi I, and Cci N I) species not typical of aquatic ecosystems. Water columns, deep cores, and bottom sediments of pure areas of the lakes contained no microorganisms with new RENs. Thus, inshore areas of Lake Baikal exposed to anthropogenic influence may contain mutant bacterial strains expressing RENs that have not been described previously.

BtrI, a novel restriction endonuclease, recognises the non-palindromic sequence 5'-CACGTC(-3/-3)-3'.

The recognition sequence and cleavage positions of a new restriction endonuclease BTR:I isolated from Bacillus stearothermophilus SE-U62 have been determined. BTR:I belongs to a rare type IIQ of restriction endonucleases, which recognise non-palindromic nucleotide sequences and cleave DNA symmetrically within them.

[New rare cutting restriction endonuclease SmII from Streptococcus milleri recognises 5'-ATTTAAAT-3']. / Novaia redkorasshchepliaiushchaia éndonukleaza restritsii SmII iz Streptococcus milleri uznaet 5'-ATTTAAAT-3'.

New restriction endonuclease (restrictase) Smil of type II was detected in the bacterial strain Streptococcus milleri. Cellular lysate enzyme cut T7 and adenovirus-2 DNAs at site 5'-ATTT decreases AAAT-3' but not lambda DNA which does not contain this sequence. Intense aeration inhibited the growth of S. milleri. The content of restrictase in the cells was the greatest during the logarithmic growth phase. A total of 20,000 units of Smil were isolated from 4 g of cells by cellular extract fractionation with ammonium sulfate and subsequent chromatography on columns with Bio Gel A 0.5 m, heparin agarose, and phosphocellulose. Purified enzyme cut the synthetic oligonucleotide duplex in the center of the recognized site 5'-ATTT decreases AAAT-3'. Smil restrictase is a true isoschisomer of rare-cutting Swal enzyme. Smil belongs to a small group of enzymes which recognize octanucleotide sites and can be used for large-block fragmentation of DNA. Comparison of specificities of rare-cutting and other restrictases suggests that the enzymes recognizing octanucleotides can evolutionally originate from enzymes recognizing both hexanucleotides and tetranucleotides.

Actinobacillus and Streptococcus: producers of isoschizomers of the restriction endonucleases R.HphI, R.SauI, R.NheI, R.MboI and R.SwaI.

New restriction endonucleases have been found in microorganisms isolated from the microflora of human teeth. The strain-producers are Actinobacillus suis and Streptococcus milleri. The new enzymes are isoschizomers of the prototypes as follows: AsuHPI - HphI; AsuSAI - SauI; AsuNHI - NheI; AsuMBI and SmiMBI - MboI; SmiI - rare-cutter SwaI.

[Restriction endonuclease Sse9I from Sporosarcina sp. strain 9D recognizes the 5'-AATT-3' DNA sequence]. / Endonukleaza restriktsii Sse9I iz shtamma Sporosarcina sp. 9D, uznaiushchaia posledovatel'nost' DNK 5'-AATT-3'.

A new restriction endonuclease Sse9I was isolated from the bacterial strain Sporosarcina sp. 9D. The enzyme belongs to Type II restrictases and recognizes the tetranucleotide sequence 5'-AATT-3'. The enzyme cleaves DNA before the first adenine residue, so it is a true isoschizomer of Tsp509I restrictase. However, unlike the prototype, Sse9I digests DNA at 55 degrees C and loses its activity after 20 min storage at 65 degrees C.

[Restriction endonuclease Sse9I (correction of Sse91) from Sporosarcina strain SP.9D recognizes the 5'-AATT-3' DNA sequence]. / Endonukleaza restriktsii Sse91 iz shtamma Sporosarcina SP.9D uznaet posledovatel'nost' DNK 5'-AATT-3'.

Sse91, a type II restriction endonuclease, has been isolated from Sporosarcina species 9D. The recognition sequence and cleavage point of restriction endonuclease Sse91 have been determined as 5'-decrease AATT-3'. The new enzyme is an isoschizomer of Tsp5091, but its optimal incubation temperature is 55 degrees C and it is inactivated at 65 degrees C for 20 min.

BstAPI, an ApaBI isoschizomer, cleaves DNA at 5'-GCANNNN NTGC-3'.

Cleavage positions of Bst API, a new restriction endonuclease (ENase) that recognizes palindromic interrupted DNA sequence, have been determined. Recognition sequences and cleavage sites comparison shows that Bst API shares similarity with a number of type II restriction enzymes.

[AccBSI - a novel restriction endonuclease from Acinetobacter calcoaceticus BS]. / AccBSI - novaia endonukleaza restriktsii iz Acinetobacter calcoaceticusBS.

The recognition site of a new restriction endonuclease from Acinetobacter calcoaceticus BS was determined. This is a nonpalindromic sequence. AccBSI restrictase cleaves DNA chains in the middle of the recognition sequence; therefore, ligation of its digestion fragments restored AccBSI recognition sites and generated palindromic sequences recognized by SacI and SacII restrictases.

BstF5I, an unusual isoschizomer of FokI.

BstF5I, a new restriction endonuclease (ENase) from Bacillus stearothermophilus F5, has been discovered. This enzyme recognizes 5'-GGATG-3' and cleaves DNA, generating a 2-base 3'extension: 5'-GGATG NN[symbol: see text]-3' 3'-CCTAC[symbol: see text]NN-5' BstF5I is an isoschizomer of FokI and seems to be evolutionarily close to other nonpalindromic-recognizing ENases from thermophilic bacilli.

CciNI, an isoschizomer of NotI from Curtobacterium citreum recognizes 5'-GC decreases GGCCGC-3'.

CciNI, an isoschizomer of NotI, has been isolated from Curtobacterium citreum. The enzyme cleaves within the recognition sequence 5'-GC decreases GGCCGC-3' as indicated by the arrow.

Bsp24I, a new unusual restriction endonuclease.

A new restriction endonuclease, Bsp24I, from Bacillus species 24, recognizing: [formula: see text] has been isolated. Its specificity and cleavage points were determined.

[Determination of restriction endonucleases in Streptomyces and Nocardia colonies]. / Opredelenie éndonukleaz restriktsii v koloniiakh mikroorganizmov Streptomyces i Nocardia.

A simple technique is proposed for the detection of restriction endonucleases in Streptomyces and Nocardia cells. The analysis was performed directly in the cells collected from colonies cultivated on Petri dishes with an inoculation loop. The cells were treated with lysozyme, EDTA and Triton X-100. The lysates were tested for restriction endonucleases. The technique enables the detection of enzymes Nco I, Not I, Nru I, Sfr 3031, and Sfi I in the lysates of the respective strains-producers.

[Detection of aquatic microorganisms from the Black Sea--producers of restriction endonucleases]. / Vyiavlenie vodnykh mikroorganizmov chernogo moria--produtsentov éndonukleaz restriktsii.

300 clones of microorganisms isolated at different stations and from different depths in the Black Sea were screened for restriction endonucleases production. The production of restriction endonucleases was found in 17 clones screened. Three of them were identified to be Alteromonas haloplanktis B1. Restriction endonuclease AhaB1 is an isoshizomer of Sau961. An identified Alteromonas haloplanktis clone B8 produces AhaB8I restriction endonuclease the prototype to which is KpnI. Of the clones isolated three are Moraxella species B4 producing MapB4I restriction endonuclease analogous to BanI, three are Bacillus species producing BspB2I and one is Micrococcus lylae 113 producing Mly1131 analogue of NarI, six Moraxella species B6 produce MspB6I. The isolated producer strains may be used for isolation of above mentioned restriction endonucleases.

II-Q restriction endonucleases--new class of type II enzymes.

Unique restriction endonucleases Bpu 10l and Bsil have been isolated from Bacillus pumilas and Bacillus sphaericus, respectively. The recognition sequences and cleavage points of these enzymes have been determinated as 5'-CC1TNAGC-3'/3'-GGANT1CG-5' for Bpu 10l and 5'-C1TCGTG-3'/3'-GAGCA1C-5' for Bsil. Restriction endonucleases Bpu 10l and Bsil represent a new class of enzymes which recognize non-palindromic nucleotide sequences and hydrolize DNA within the recognition sequence. Bpu 10l and Bsil recognition sequences may be regarded as quasipalindromic and the enzymes may be designated as type II-Q restriction endonucleases.

[BsiI--a new unusual restriction endonuclease]. / BsiI--novaia neobychnaia éndonukleaza restriktsii.

The restriction endonuclease BsiI from Bacillus sphaericus was isolated. The recognition sequence and cleavage point of enzyme BsiI have been determined as (sequence: see text). This restriction endonuclease is not an isoschizomer of any known restriction endonucleases and differs from other enzymes: it hydrolyses DNA into unsymmetrical recognition sequence.

[Type II SsrI restriction endonuclease from Staphylococcus saprophyticus]. / SsrI-endonukleaza restriktsii vtorogo klassa iz Staphylococcus saprophyticus.

The recognition sequence and cleavage site for restriction endonuclease SsrI have been determined, the latter being 5'-GTT decreases AAC-3'. The enzyme was isolated from Staphylococcus saprophyticus strain and may be used in DNA investigation instead of its isoshizomer HpaI.

[Determination of substrate specificity of restriction endonuclease FauI]. / Ustanovlenie substratnoi spetsifichnosti éndonukleazy restriktsii FauI.

The recognition sequence and cleavage point of restriction endonuclease FauI have been determined as 5'-CCCGC(4/6). Not being isoschisomer of any known restriction endonuclease, this enzyme may be used in genetic engineering.