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.

Substrate specificity and biochemical properties of M3.BstF5I DNA methyltransferase from the BstF5I restriction-modification system.

Optimal conditions for DNA methylation by the M3.BstF5I enzyme from Bacillus stearothermophilus and kinetic parameters of lambda phage DNA modification and that of a number of oligonucleotide substrates are established. Comparison of M1.BstF5I and M3.BstF5I kinetic parameters revealed that with similar temperature optima and affinity for DNA, M3.BstF5I has nearly fourfold lower turnover number (0.24 min(-1)) and modifies the hemimethylated recognition site with lower efficiency under optimal conditions than the unmethylated one. In contrast to another three methylases of the BstF5I restriction-modification system, the M3.BstF5I enzyme is able to optionally modify the noncanonical 5'-GGATC-3' DNA sequence with a rate more than one order of magnitude lower than the methylation rate of the canonical 5'-GGATG-3' recognition site.

[Purification of recombinant DNA methyltransferase M2.BstSE from nickase-modification system NM.BstSEI and study of the enzyme properties].

The operon of nickase-modification system from Bacillus stearothermophilus SE-589 (recognition site 5'-GAGTC-3') includes two DNA methyltransferase genes: bstSEIM1 and bstSEIM2. Gene encoding DNA methyltransferase M2.BstSEI was cloned in pJW vector and expressed in E. coli cells. The enzyme M2.BstSEI has been isolated by chromatographic purification. M2.BstSEI displays maximum activity at 55 degrees C and pH 7.5. The enzyme modifies adenine in DNA sequence 5'-GAGTC-3' and has substrate specificity 5'-GASTC-3'. The kinetic parameters of methylation reaction have been determined. The catalytic constant--2.2 min(-1), the Michaelis constant on T7 DNA--9.8 nM and on SAM--5.8 microM.

Isolation and characterization of biochemical properties of DNA methyltransferase FauIA modifying the second cytosine in the nonpalindromic sequence 5'-CCCGC-3'.

A gene encoding DNA methyltransferase (methylase) FauIA of the restriction-modification system FauI from Flavobacterium aquatile (recognizing sequence 5'-CCCGC-3') was cloned in pJW vector. The latter was used for transformation of E. coli RRI cells followed by subsequent thermoinduction and biomass elaboration. Highly purified DNA methyltransferase FauIA preparation was obtained using chromatography on different sorbents. The molecular mass of the isolated enzyme of about 39 kD corresponds to its theoretical value. The enzyme was characterized by temperature and pH optima of 33 degrees C and pH 7.5, respectively. Methylation of a synthetic oligonucleotide by FauIA methylase followed by its cleavage with various restrictases and analysis of the resultant restriction fragments revealed that FauIA methylase modified the second cytosine residue in the sequence 5'-CCCGC-3'. Kinetic analysis revealed Km and catalytic constant values of 0.16 microM and 0.05 min(-1), respectively.

M.BstF5I-2 and M.BstF5I-4 DNA methyltransferases from BstF5I restriction-modification system of Bacillus stearothermophilus F5.

The BstF5I restriction-modification system from Bacillus stearothermophilus F5 includes four site-specific DNA methyltransferases, thus differing from all known restriction-modification systems. Here we demonstrated for the first time that one bacterial cell can possess two pairs of methylases with identical substrate specificities (methylases BstF5I-1 and BstF5I-3 recognize GGATG, whereas methylases BstF5I-2 and BstF5I-4 recognize CATCC) that modify adenine residues on both DNA strands. Different chromatographic methods provide homogenous preparations of methylases BstF5I-2 and BstF5I-4. We estimated the principal kinetic parameters of the reaction of transfer of methyl group from the donor S-adenosyl-L-methionine to the recognition site 5;-CATCC-3; catalyzed by BstF5I-2 and BstF5I-4 DNA [N6-adenine]-methyltransferases from the BstF5I restriction-modification system.

[Cytogenetic analysis of insertions into drosophila interband polytene chromosomes]. / Tsitogeneticheskii analiz insertsii v sostave mezhdiskov politennykh khromosom drosofily.

Using the method of P-element-mediated enhancer detection, 29 Drosophila melanogaster lines were obtained that carried P-1ArB vector insertions in chromosomes 2 and 3. The expression of the reporter gene lacZ at different developmental stages of the transformed lines was determined in color reactions for beta-galactosidase. Regions of vector integration were located using in situ hybridization. Subsequent electron-microscopic mapping of the transformed regions was performed using four lines (nos. 12, 41, 2, and 3). In the lines 12 and 41, lacZ was expressed in most tissues of embryos, larvae, and imagoes (including salivary glands), whereas in lines 2 and 3 its expression was observed only in embryos. Lines 12 and 2 showed the presence of insertions in the 85D9/10 and 86B4/6 interband regions, respectively. The absence of a novel band in line 3 could be associated with transposon integration into the band. In line 41, puffing of the transformed region was observed, which did not allow us to determine the presence of any novel structures in it. The novel structures in lines 12 and 2 looked like single bands with a similar DNA packing ratio of about 30. These bands were obviously polygenic, because the inserted vector contained four functionally different genes.