[Properties and specificity of action of DNA methylases from blood lymphocytes of healthy cattle and cattle with chronic lymphoid leukemia]. / Svoistva i spetsifichnost' deistviia DNK-metilaz limfotsitov krovi zdorovykh i bol'nykh khronicheskim limfoleikozom korov.

Using cell extract fractionation with ammonium sulfate and subsequent chromatography on DEAE- and DNA-cellulose and Blue Sepharose, two cytosine DNA-methylases were isolated from blood lymphocytes of cows suffering from lympholeukosis; one cytosine DNA-methylase was isolated from blood lymphocytes of healthy animals. The DNA-methylases from normal lymphocytes was purified 383-fold; the enzyme has a specific activity of 2.3 u./mg, Mr of 114 000 Da and a pH optimum of 7.6. The molecular mass of DNA-methylases from leukemic lymphocytes is about 130,000 Da. The enzymes isolated from leukemic lymphocytes, i.e., DNA-methylase I (568-fold purification, specific activity 14.2 u./mg) and DNA-methylase II (524-fold purification, specific activity 13.1 u./mg) possess different action optima at pH 7.8 and 6.7, respectively. The total DNA-methylase activity of leukemic lymphocytes is about 4 times that of normal lymphocytes. All the DNA-methylases under study methylate in vitro bacterial and animal DNAs of different base composition; bacterial DNAs are the best (GC content is approximately 70%), while homologous DNAs- the worst acceptors of CH3-groups. Heat-denaturated DNAs are methylated more intensively than initial DNAs. The optimal NaCl concentration in the reaction mixture is approximately 50 mM; EDTA (greater than 10 mM) inhibits the reaction. DNA-methylase I of leukemic and normal lymphocytes show the same pH optimum and specificity of action. In vitro methylation of bacterial DNA by these DNA-methylases in the presence of [3H-methyl]S-adenosylmethionine results in the similar type of label distribution among pyrimidine isopliths in the DNA. DNA-methylase II from leukemic lymphocytes methylates about two times more Pu-C-Pu sequences in th same DNA than does DNA-methylase I from leukemic and normal lymphocytes and thus reveals a different specificity. The changes in the type of DNA methylation as well as the appearance of an additional DNA-methylase possessing a different specificity of action in leukemic lymphocytes may be responsible for cell transformation and transcriptional changes in chronic lympholeukosis.

[Non-enzymatic DNA methylation by S-adenosylmethionine results in the formation of minor thymine residues and 5-methylcytosine from cytosine]. / Neénzimaticheskoe metilirovanie DNK pod deistviem S-adenozilmetionina s obrazovaniem iz tsitozina ostatkov minornogo timina i 5-metiltsitozina.

It was found that nonenzymatic DNA methylation proceeds in water solution in the presence of S-adenosylmethionine (AdoMet). The main reaction products are thymine and 5-methylcytosine residues. It was shown that labelled thymine residues are formed also upon DNA incubation in the presence of [methyl-14C]methionine as well as [methyl-14C]cobalamine. Only cytosine reacts with AdoMet resulting in thymine production. AdoMet may be a potential mutagen that induces GC----AT transitions during DNA replication in the cell.

[Determination of DNA methylase activity in animal tissue extracts]. / Opredelenie DNK-matilaznoi aktivnosti v ékstraktakh zhivotnykh tkanei.

An express method for measuring the level of in vitro DNA methylation in homogenates and nuclei from animal tissues as well as during initial steps of DNA methylase isolation and purification when methylase activity is low and hardly testable by other methods has been suggested. The method is based on the measuring the radioactivity incorporated in filter adsorbed DNA (acid-insoluble material) 3H-label from S-adenosile-L-methionine as a result of in vitro DNA methylation. The advantage of the method consists in the replacement of a long-duration repeated deproteinization procedure traditionally used by a relatively simple procedure (15 min incubation of the mixture at 80 degrees C with 10 volumes of the 8M urea, 5 mM EDTA, 5% n-butanol, 2% sodium dodecilsulfate, 1 M sodium chloride solution) and the absence of any loss of DNA. The method is fit for the fast serial assay of DNA methylase activity taking into consideration that about one third of the total acid-insoluble radioactivity is due to the radioactivity in 5-methylcytosine residues in DNA.

[Intragenome distribution of 5-methyl cytosine and reassociation kinetics of cow blood lymphocyte DNA under normal conditions and in chronic lymphoid leukemia]. / Vnutrigenomnoe raspredelenie 5-metiltsitozina i kinetika reassotsiatsii DNK limfotsitov krovi korov v norme i pri khronicheskom limfoleikoze.

The DNA from cow blood lymphocytes is methylated in a varying degree: the maximal content of 5-methyl cytosine (2,3 mol%) is found in the "instantly" renaturating sequences (Cot lett than 10(-4)), a relatively large amount (1,4 mol%)--in moderately repeated sequences (Cot = 10(-4)--400) and the minimal amount (0,9 mol%) in the unique sequences (Cot greater than 400). In their reassociation kinetics, GC-content and other physico-chemical properties the blood lymphocyte DNA of the controls and of animals with chronic lymphoid leukemia appear to be similar. Consequently, the genome organization of leukemic animals does not change significantly; a considerable decrease of 5-methyl cytosine of lymphocyte DNA in lymphoid leukemia parallels the decrease of genome methylation in the leukemic cells. This decrease does not affect the unique sequences, but involves all types of repeated sequences (moderately and frequently repeated ones and palindromes). It is assumed that the specific disturbances in genome methylation under lymphoid leukemia may be a cause of transcription deficiences and cell transformations.

[Tissue specificity of the decrease of cattle lymphocyte DNA methylation during chronic lymphoid leukemia]. / Tkanevaia spetsifichnost' umen'sheniia metilirovaniia DNK limfotsitov krupnogo rogatogo skota pri khronicheskom limfoleikoze.

It has been found that the content of m5C in the DNA preparations tested have been revealed. The DNAs from normal and leukemic lymphocytes of blood, lymphonodi and spleen differ in ther acceptor ability in the reaction of heterologous methylation in vitro, induced by DNA-methylase from Enterobacter cloacea in the presence of [3H-methyl]S-adenosyl methionine: the ratio of radioactivities in methylated cytosine and adenine residues (m5C/m6A) in leukemic lymphocyte DNA is much lower than in healthy animals' lymphocytes. The decrease in the methylation of DNAs from various lymphoid organs of animals with chronic lymphoid leukemia is well correlated with the impairment. No significant changes of the m5C level and the acceptor ability of the in vitro reaction of heterologous methylation of cow lymph lymphocyte DNA have been observed. The data obtained may be interpreted in terms of tissue (cell) specificity or differences in the degree of DNA methylation under conditions of chronic lymphoid leukemia. It is assumed that the changes in DNA methylation may underlie the disturbances in the regulation of activity of the leukemic cell genetic mechanisms.

[Changes in the specificity of DNA methylation in cattle blood lymphocytes under chronic lymphoid leukemia]. / Izmenenie spetsifichnosti metilirovaniia DNK v limfotsitakh krovi krupnogo rogatogo skota pri khronicheskom limfoleikoze.

Under conditions of chronic (spontaneous) lympholeucosis the amount of 5-methylcytosine in cattle blood lymphocyte DNA is decreased approximately by 30%. No other changes in the DNA (e. g. GC-content, Tm, amount of pyrimidine sequences differing in their lengths and composition) were observed. Thus, the decrease in the amount of 5-methylcytosine in lymphocyte DNA is due to a decrease in DNA methylation. This decrease is non-random and involves mainly the Pu-m5C-Pu sequences without affecting the long pyrimidine blocks. In nuclear extracts from lymphocytes of healthy animals the DNA-methylase activity having an optimum at pH 6,0 was found; the DNA-methylase activities found in the nuclear extracts of leukaemic cow lymphocytes had their optima at pH 5,5 and 7,5. In vitro the DNA-methylase activities of leukaemic lymphocytes nuclei methylate the cytosine residues of DNA in other sequences than enzyme(s) of the extracts from normal lymphocyte nuclei. Changes in the pattern of DNA-methylase activities as well as the decrease and distortions in the character of DNA methylation may underlie the disturbances in the regulation of transcription of genome and cause the transformation of cells under conditions of lympholeucosis.

[Conditions of dextranase formation by Penicillium funiculosum 15]. / Usloviia obrazovaniia dekstranazy Penicillium funiculosum 15

The influence of the following factors on the synthesis of extracellular dextranase by Pen. funiculosum 15 has been studied: the quantity and age of the inoculum, pH of the cultivation medium, stimulants of the microbial growth, cultivation temperature and time. The optimal amount of dextranase has been found to form under the following conditions: inoculum--3 day mycelium constituting 4%, cultivation time--4 to 7 days, temperature--28 to 29 degrees C, initial pH of the medium--6.0.