[The physicochemical properties of the DNAse of Mycoplasma fermentans PG-18]. / Fiziko-khimicheskie svoistva DNK-azy Mycoplasma fermentans PG-18.

Physical and chemical properties, as well as polypeptide structure of DNAse from Mycoplasma fermentans PG-18, have been determined. The enzyme in a native form exists probably as a decamere (10X34 kD) and manifests maximal activity at weak alkaline pH range. The temperature optimum of the enzyme is --37 degrees C. DNAse appears to be Mg2+-dependent and has its maximal activity at 10 mM MgCl2. EDTA completely inhibits DNAse activity. The given DNAse has been determined to cleave a phosphodiether bond in 3'-position of deoxyribose and to have both exo- and endonuclease activity, since it has hydrolized both native linear doublestranded DNA and closed-circle plasmid DNA.

[Oligodeoxyribonucleotides complementary to sections of the mollicute ribosomal operon as transcription inhibitors in vitro]. / Oligodezoksiribonukleotidy, komplementarnye uchastkam ribosomal'nogo operona mollikutov, kak ingibitory transkriptsii in vitro.

Antisense oligodeoxyribonucleotides have been studied for their effect on the transcription in vitro in mollicutes. A synthetic fragment of DNA [symbol: see text] complementary to that part of DNA which codes the 1510-1521 area of the 3'-terminal sequence 16S-pRNA of all mollicutes was used in the study as well as its modifications by imidasophenasine derivatives: [symbol: see text], [symbol: see text] [symbol: see text]. Maximal inhibition of the mollicute transcription in vitro was observed with 100 nM oligonucleotide concentration. Lower or higher concentrations were less effective. Transcription initiated by RNA-polymerase of M. fermentans PG-18 (a mollicute strain referring to AIDS disease) proved to be the most sensitive to the effect of modified oligonucleotide: it was inhibited by 75-80%. It is concluded that modified oligonucleotides exert a dual effect on transcription: firstly, they participate in nonspecific interaction with RNA-polymerase which induces insignificant inhibition of transcription and, secondly, they complementary interact with homologous sections of one-stranded DNA-matrix and block the RNA synthesis. Binding of modified oligonucleotides with DNA is rather strong.

[A mechanism of transcription in prokaryotes: stages and place in the realization of genetic information]. / Mekhanizm transkriptsii u prokariot: étapy i mesto v realizatsii geneticheskoi informatsii.

Transcription, being the first stage on the way of genetic information realization, is the most important to regulate expression of genes. It is a complex process in procaryotes. At different stages it is controlled by definite systems of a cell, that in the end, provides a regulated information flow initiating all processes which proceed in the organisms. At present the problem of the gene activity regulation plays a key role in the molecular biology and intensively develops both in theoretical and applied aspects.

[Optimization of physico-chemical parameters of reaction media for determining the activity of RNA-polymerases of mollicutes]. / Optimizatsiia fiziko-khimicheskikh parametrov reaktsionnoi sredy dlia opredeleniia aktivnosti RNK-polimeraz mollikutov.

RNA-polymerases of mollicutes differ considerably from certain species of bacteria in the temperature optimum of the activity manifestation. The activity of mollicute enzymes is considerably higher in the presence of manganese ions than in the presence of magnesium ions. They differ from Escherichia coli transcriptase in this character but are similar to the RNA-polymerase of lactic bacteria, hypothetic ancestors of acholeplasm. Sensitivity of RNA-polymerases to metals may be one of arguments explaining phylogeny of mycoplasms. It is established that for studying mollicute transcriptase the reacting mixture for examining the enzymic activity besides the major components should have the following parameters: pH 8.0; the MnCl concentration--8-10 mM; for form II of the A. laidlawii subsp. granulum 118 enzyme--4.5 mM; ammonium sulphate concentration--40 mM; for form II of st. 118-20 mM; the reaction should be conducted at the temperature of 27 degrees C for 30 min.

[Increase in the in vitro activity of mollicute transcriptase using substances of an organic nature]. / Povyshenie in vitro aktivnosti transkriptaz mollikutov pri pomoshchi veshchestv organicheskoi prirody.

It is established that the use of DNA-dependent RNA-polymerases of mollicutes in the molecular-biological and gene-engineering works aimed to obtain a sufficient amount of the transcription products may promote a 1.5-2-fold increase of the yield by introducing such substances as glycerol, dimethylsulphoxide and spermidine (optimal concentrations: 10-12 vol. % and 12 mM, respectively) into the composition of the reacting mixture.

[The chromatographic properties of the DNA-dependent DNA polymerases from Acholeplasma laidlawii PG-8]. / Khromatograficheskie svoistva DNK-zavisimykh DNK-polimeraz iz Acholeplasma laidlawii PG-8.

The DNA-dependent DNA-polymerase (DNA polymerase I which is not sorbed on the column with DEAE-cellulose, and DNA-polymerase II, which is absorbed by this column and is eluted from it by 0.3 M of NaCl), have been isolated from Acholeplasma laidlawii PG-8. DNA-polymerase I in homogeneous state was obtained as a result of the stepwise treatment by heparin-sepharose (elution at 0.35 M of NaCl) and poly-U-sepharose (elution at 0.3 M of NaCl). It was presented on the electrophoregram by one polypeptide with molecular weight of 72 kDalton. The second form of DNA polymerase was also obtained in homogeneous state as a result of sequential treatment on heparin-sepharose (elution at 0.3 M of NaCl) and on poly-A-sepharose (elution at 0.25 M of NaCl): the protein which had manifested polymerase activity was a polypeptide with molecular weight of 45 kDalton.

[Inhibitors of nucleic acid synthesis as a means of identifying the forms of DNA-dependent DNA polymerases in Acholeplasma laidlawii PG-8 and of determining their functions]. / Ingibitory sinteza nukleinovykh kislot kak sredstvo identifikatsii form DNK-zavisimykh DNK-polimeraz Acholeplasma laidlawii PG-8 i opredeleniia ikh funktsii.

Antibiotics, inhibitors of nucleic acids' synthesis from the group of chromomycins (olivomycin of sodium salt), anthracyclines (carminomycin and doxorubicin) and streptonigrin (bruneomycin) have been studied for their effect on DNA synthesis in vitro performed by DNA polymerases (1st and 2nd forms) of Acholeplasma laidlawii PG-8. It has been stated that olivomycin inhibits the function of both the first and second forms of DNA polymerases in proportion to an increase of the antibiotic concentration in the medium. Carminomycin in the concentration of about 1 microgram/ml almost completely inhibited the activity of both DNA polymerases. However, doxorubicin also belonging to the group of anthracyclins completely inhibited the activity of the first form of DNA polymerase in the concentration of 1 microgram/ml and practically has no effect in the concentration up to 100 micrograms/ml on the activity of the second form possessing 3'-->5'-function. Streptonigrin also proved to be suitable for differentiate the forms of DNA polymerases and to determine their functions. The first form of DNA polymerase with 5'-->3'-polymerase and exonuclease functions was not sensitive by this antibiotic in the concentration of 1000 micrograms/ml, while the activity of the second form of DNA polymerase with 3'-->5'-exonuclease functions was fully inhibited by this concentration of the antibiotic in the medium. The combination of doxorhubicin and streptonigrin in the medium can be used to determine the form of DNA polymerases and to identify their 5'-->3'- or 3'-->5'-exonuclease function and for selectivity inhibition of the function of one or another DNA polymerase in the medium.

[The kinetic and functional characteristics of DNA-dependent DNA-polymerases in Acholeplasma laidlawii PG-8]. / Izuchenie kineticheskikh i funktsional'nykh kharakteristik DNK-zavisimykh DNK-polimeraz Acholeplasma laidlawii PG-8.

The kinetic and functional characteristics of I and II forms of DNA-dependent DNA-polymerases of Acholeplasma laidlawii PG-8 have been studied. It is stated that I form of DNA polymerase possesses 5'-3'-exonuclease activity and is a typical replicase; II form of DNA-polymerase possesses both 5'-3'-polymerase and 3'-5'-exonuclease activity and is, evidently, a reparase. Both forms of enzyme give preference to poly(U)- and poly(A)-matrices having extremely high activity on these polymers. The enzymatic reactions realized by both forms of DNA-polymerases are described by the first-order equation. The calculated Michaelis-Menten constants equaled 180 and 250 microM for I and II forms of polymerases, respectively. It indicates that affinity to substrate in II form of polymerase is one-third higher than in I form of enzyme.

[The isolation and purification of a nonspecific DNAse from Mycoplasma fermentans PG-18]. / Vydelenie i ochistka nespetsificheskoi DNK-azy Mycoplasma fermentans PG-18.

Nonspecific endogenic DNAase has been isolated from biomass of Mycoplasma fermentans PG-18 cells and purified to the homogeneous state. The scheme of isolation consists of purification stages on columns with phosphocellulose, DNA-cepharose CL-8B and phenylcepharose. DNAase was not bound to phosphocellulose, its volume was equal to zero. Then this DNAase was passed through column with DEA-cepharose CL-6B (elution by gradient KCl from 0.1 to 1.8M): enzyme was eluted at KCl concentration in the eluting buffer from 0.1 to 1.2 M. The enzyme was purified to the homogeneous state on column with phenylcepharose (elution by linear gradient of ethylene glycol from 30 to 80%): enzyme was eluted at the concentration of ethylene glycol in the eluting buffer from 43 to 80%. According to data obtained using gel-electrophoresis, under the denaturing conditions molecular mass of enzyme in acrylamide gel was 34 kDa.

[The effect of physicochemical factors on the activity of DNA-dependent DNA polymerases in Acholeplasma laidlawii PG-8]. / Vliianie nekotorykh fiziko-khimicheskikh faktorov na aktivnost' DNK-zavisimykh DNK-polimeraz Acholeplasma laidlawii PG-8.

The biological and physico-chemical properties of DNA-dependent DNA-polymerases of Acholeplasma laidlawii PG-8 have been studied. The optimal parameters of maximal enzymatic activity are determined. It is stated that N-ethylmaleimide in concentration of 1 mM activated DNA-polymerase I by 52%, whereas DNA-polymerase II with reagent concentration of 0.5 mM demonstrated the peak of activity exceeding the control only by 10%. Spermidine in concentration of 1.5 mM for the first form of DNA-polymerase and 0.15 mM-for the second one increased the ability of both forms of polymerases to synthesize DNA by 10%. Aphidicolin added to the reaction medium up to concentration of 10 mg/ml decreased activity of forms I and II of enzymes by 83 and 68%, respectively. The presence of 0.6 mM of EDTA in the medium also negatively affected the activity of polymerases inhibiting it by 83% in form I and by 77%-in form II.

[The isolation and partial purification of 2 DNA-dependent DNA polymerases from Acholeplasma laidlawii PG-8]. / Vydelenie i chastichnaia ochistka dvukh DNK-zavisimykh DNK-polimeraz Acholeplasma laidlawii PG-8.

Two forms of DNA-dependent DNA-polymerase have been isolated and partially purified from the limited amount of biomass of cells Acholeplasma laidlawii PG-8, a typical representative of genus Acholeplasmataceae, as a result of successive chromatography on the columns with DEAE-cellulose DE-52 and Green A-sepharose. The first form of DNA-polymerase is eluted from the ion-exchange column with NaCl concentration of 0.1 M from the column with Green A-sepharose of 0.27 M, while the second form-with NaCl concentrations of 0.6 and 0.4 M, respectively. The both enzymatic activities are able to implement DNA synthesis. The conditions of DNA-polymerase production proved to be rather convenient for isolation of the concentrated and highly active enzymes.

[The inhibitory action of 6-azacytidine on Mollicutes and its proposed mechanism]. / Ingibitornoe deistvie 6-azatsitidina na mollikuty i ego predpolagaemyi mekhanizm.

6-Azacytidine (6-AC) is shown to have an inhibitory effect on the Mollicutes of the different systematic position. The growth of type strains of Mollicutes (Acholeplasma laidlawii PG-8, Mycoplasma pneumoniae FH and M. fermentans PG-18) completely ceased in the nutrient medium at concentration of the above substance in it within the range of 125-250 micrograms/ml. 50% inhibiting concentration of 6-AC equaled: for M. fermentans PG-8: 23.43 micrograms/ml; M. pneumoniae FN: 46.8 micrograms/ml; Acholeplasma laidlawii PG-8: 62.5 micrograms/ml. 6-AC concentration 5 micrograms/ml decreased the process of DNA-dependent DNA synthesis in the in vitro system more than by 60%. 6-AC exerted less effect on the DNA-dependent RNA synthesis in the in vitro system: at different concentrations of 6-AC (up to 400 micrograms/ml) RNA synthesis decreased only by 20%. Translation on ribosomes of Mollicutes in the in vitro system completely ceased at 6-AC concentration 100 micrograms/ml. The results obtained indicate that for 6-AC in cells of Mollicutes and, possibly, for other microorganisms there are two targets: ribosomes and DNA-dependent DNA-polymerase. Total effect of blocking of the translation and replication processes by 6-azacytidine causes death of Mollicutes. Since 6-AC has no harmful effect on the human cells, it can be used as an efficient method for treatment of respiratory and urogenital diseases induced by Mollicutes.