Effect of the M-EcoRII methyltransferase-encoding gene on the phenotype of Nicotiana tabacum transgenic cells.

The EcoRII DNA methyltransferase (M-EcoRII; MTase) modifies a cytosine in the DNA sequence CCWGG which contains a CNG methylation motif characteristic of plant DNA. The gene (ecoRIIM) encoding this MTase has been cloned into the T-DNA of the wild-type Agrobacterium Ti-plasmid pTiC58 downstream from the plant expression nopaline synthase-encoding gene promoter. Nicotiana tabacum cells have been transformed with Agrobacterium tumefaciens harbouring this recombinant Ti-plasmid. The primary transformed tabacco tissue line has given rise to novel stable lines which are morphologically distinctive. Southern hybridization analysis of all transformed tissue lines has shown the presence, in each of them, of ecoRIIM. The tissue studied differed in morphology in callus culture, dependence on phytohormones and the ability to synthesize nopaline.

[A method for detecting new strains producing DNA modification and restriction enzymes--isoschizomers and isomethylomers of known restrictases and methylases]. / Metod poiska novykh produtsentov fermentov modifikatsii i restriktsii DNK--izoshizomerov i izometilomerov izvestnykh restriktaz i metilaz.

The paper describes a technique for the detection of new strains producing enzymes which mediate DNA modification and restriction, and isoschizomers and isomethylomers of the known restriction endonucleases and methylases. Three Bacillus subtilis strains whose DNA carries a BamH1 modification have been found. Two of these strains exert the restrictase activity with an R BamH1 specificity.

[Isolation of modification-restriction enzymes HpaI and HpaII]. / Vydelenie fermentov modifikatsii-restriktsii HpaI i HpaII.

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.

DNA methylation of bacterial viruses T3 and T7 by different DNA methylases in Escherichia coli K12 cells.

We have investigated the susceptibility of the genomes of the related bacteriophages T3 and T7 to the three major DNA methyltransferases (EcoK, dam, dcm) of their host, Escherichia coli K12. In vivo the EcoK host specificity enzyme only methylates the DNA of ocr- phages. This is due to an inhibition of the enzyme by the phage ocr+ gene product, which had previously been shown to be an inhibitor of the restriction endonuclease. EcoK-specific DNA methylation protects the ocr- viruses after one growth cycle on these host cells against the action of corresponding restriction endonuclease EcoK. Owing to the unique S-adenosyl-L-methionine hydrolase (sam+) activity of the T3-coded ocr+ protein, the T3 DNA is absolutely devoid of the methylated bases 6-methylaminopurine and 5-methylcytosine. In contrast to this, T7 derivatives and sam- derivatives of T3 carry a small number of about 2-4 molecules 6-methylaminopurine and 5-methylcytosine per genome. The presence of 6-methylaminopurine is due to dam methylation, though the majority of dam sites remain unmethylated. In vivo as well as in vitro the ocr+ protein has no influence on the activities of the dam and dcm methylase. The experiments gave some evidence for the existence of a second cytosine methylase in E. coli K12. Besides dam and dcm recognition sites being undermethylated, their absolute number in T3 and T7 DNAs is far below the expected value. Moreover, one of the two dcm sites present in T7 (Studier strain) is missing in our T7 strain owing to a 1300-base-pair deletion in gene 0.7.

[The ability of E. coli DNA methylases to modify denatured DNA]. / O sposobnosti DNK-metilaz E. coli modifitsirovat' denaturirovannye DNK.

Adenine and cytosine DNA methylases from different strains of E. coli are able to methylate denaturated and single-stranded DNAs.

[Distribution of 5-methylcytosine in phage lambda genome methylated by DNA methylase Eco RII]. / Raspredelenie 5-metiltsitozina v genome faga lambda, metilirovannom DNK-metilazoi Eco RII.

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.

[Use of DNA-methylases as reagents for the production of isotopically labeled DNAs]. / Ispol'zovanie DNK-metilaz v kachestve reagenta dlia polucheniia mechennykh izotopami DNK.

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.

[Isolation and properties of DNA-cytosine methyltransferase from Escherichia coli C]. / Vydelenie i svoistva DNK-tsitozinmetiltransferazy iz Escherichia coli C.

The method of isolation and partial purification of DNA-cytosine-methyltransferase (DC-methylase) from E. coli C is described. The enzyme underwent approximately 100-fold purification. The obtained preparation of DC-methylase can be additionally considerably purified by sedimentation in sucrose gradient. Native molecular weight of DC-methylase from E. coli C. is 70,000. The activity of enzyme does not depend on the Mg2+ ions. DC-methylase E. coli C provides DNA of lambda phage in vitro with full resistance against restriction endonuclease EcoRII. In DNA methylated by DC-methylase the modified cytosine, mainly in C-MC and C-MC-T sequences, corresponds to the pyrimidine sequences of specific site EcoRII. DNA of lambda.B phage contains approximately 80 sites for modification by DC-methylase E. coli C. The results obtained point to the same specificity in vitro of DNA-cytosine-methylase E. coli C and DNA-methylase EcoRII.

[Isolation and properties of DNA-cytosine-methyltransferase EcoRII and E. coli K12]. / Vydelenie i svoistva DNK-tsitozin-metiltransferaz EcoRII i E. coli K12.

The methods of isolation and partial purification of two DNA-cytosine-methylases (DC-methylases) EcoRII and E. coli K12 are described. After chromatography on phosphocellulose the enzymes were purified 100-fold, the yield being 30%. Further purification of the enzymes was performed by sedimentation in a sucrose concentration gradient. Both enzymes have native molecular weights of 50,000; DC-methylase from E. coli K12 may simultaneously occur in the forms with molecular weights of 70,000, 90,000 and 110,000. Both DC-methylases modify identical nucleotide sequences of DNA, have equal numbers (90) of methylation sites in phage lambda DNA and provide in vitro a complete protection of phage lambda DNA against restriction endonuclease EcoRII. DC-methylases E. Coli K12 and EcoRII differ in their chromatographic behaviour on phosphocellulose and capacity to form compexes with the cell DNA-adenine-methylase.

[Sensitivity of chromosomal and plasmid E. coli DNA to restriction endonuclease Eco RII]. / Chuvstvitel'nost' khromosomnykh i plazmidnykh DNK shtammov E. coli k restriktsionnoi endonykleaze Eco RII.

It was shown that E. coli C, E. coli MRE 600 DNA, and also plasmid DNA of Col E1, RSF 2124 from E. coli K-12, and plasmid DNA from E. coli MRE 600 were completely resistant against restriction endonuclease R. Eco RII. Plasmid DNAs of Col E1, RSF 2124 amplificated for 4 hours in the presence of chloramphenicol are sensitive to R. Eco RII but after 16-hour amplification in the presence of chloramphenicol these DNAs acquire complete resistance against R. Eco RII. These data point to the slower rate of modification of DNA in vivo by DC-methylases of Eco RII type in comparison with DNA methylase Eco RII.