Restriction-methylation enzymes BstN1 from Bacillus stearothermophilus were isolated and purified. These enzymes are related to a new class of restriction-methylation enzymes of the second type, whose modifying component is N4-cytosine-DNA-methylase. Both enzymes recognize the DNA sequence CC(A/T)GG. Restrictase BstN1 is a protein made up of one subunit with a molecular mass of 25 kDa. The molecular mass of native DNA-methylase BstN1 is about 55 kDa. The temperature optima for restrictase and methylase BstN1 are around 60 degrees C. Possible uses of BstN1 restriction-methylation enzymes for the analysis of cytosine methylation in bacterial and higher plant DNA are discussed.

Unformly 32P-labelled phage-specific tRNAAsn was isolated from bacteriophage T5-infected E. coli cells its oligonucleotide fragments were fractionated by thin-layer chromatography on cellulose and the tRNA's primary structure was determined as follows: (formula; see text). Main features of the structure are: displacement of the constant residue A14 by U; absence of G27 X A43 and U30 X psi40 pairing in the anticodon stem; possibility of additional pairing in D-loop. Comparison of T5 tRNAAsn with other asparagine tRNAs is presented.

A method for simultaneous isolation of four enzymes of modification-restriction of DNA from Haemophilus parainfluenzae is proposed. The properties of HpaI and HpaII DNA-methylases were investigated.

The distribution of 5-methylcytosine in Eco RI-Bam HI fragments of phage lambda DNA in vitro methylated by Eco RII methylase has been studied. The general picture of distribution of methylated sites in phage lambda DNA is slightly different from the statistical distribution. However, the sites have been found, where the distribution of 5-methylcytosine is not accidental. A complete absence of 5-methylcytosine in the J-fragment, a genome lambda area essential for site-specific recombination, has been found. The absence of Eco RII is supposed to be the best protection of this area of phage genome from the increased mutagenesis, characteristic for nucleotide sequences methylated by DNA-methylated Eco RII and Eco RII type.

The restriction endonuclease Eco RII was isolated and purified to homogeneity. The isolation procedure involved the use of the E. coli strain B834/pSK323, containing the recombinant plasmide pSK323 which provides for the oversynthesis of Eco RII enzymes. Data from gel filtration and Na-DS electrophoresis suggest that the restriction endonuclease Eco RII is a protein made up of two subunits, each with molecular weight of 44 000.

DNA of bacteriophage T5 was hydrolyzed with restriction endonucleases HindIII and BamHI, and subjected to the combined hydrolysis with BamHI+EcoRI and BamHI+ +HindIII. Fragments obtained were cloned in the plasmid pBR322. About 17% of T5 genome were recovered in recombinant plasmids. Cloned fragments were localized on the physical map of the phage by restriction analysis and Southern hybridization. With the aim of direct cloning of T5 promoters, PstI/HindIII fragments were inserted into pBR322 followed by selection of recombinants on ApsTCr phenotype. Binding of BsuRI and AluI fragments of hybrid plasmids with E. coli RNA polymerase was studied by nitrocellulose filter assay. The fragments, which were capable to form heparin resistant complexes were identified.

The method of incorporation of an isotopic label into DNA by means of DNA-methyltransferases (DNA-methylases) is proposed. DNA was no degraded and retained its biological activity in the DNA-methylase reaction. The specific activity of labelled DNA preparations can be increased, using the mixtures of different DNA-methylases in the enzymatic reaction. An isotopic label was incorporated into DNA, using DNA methylases M. .EcoRII, M.Eco dam and M.EcoMRE600 dcmI. An average activity of 1 microgram of labelled DNA preparations produced by S-adenosylmethionine (methyl-3H) with specific activity of 15 CU/mmol mas about 1 x 10(5) cpm.