[Mycoplasma restriction-modification system MunI and its possible role in pathogenesis processes]. / Mikoplazmennaia sistema restriktsii--modifikatsii MunI i ee vozmozhnaia rol' v protsessakh patogeneza.

The restriction-modification system, named RMMunI, has been purified and characterised from Friend murine erythroleukemia cells. The site-specific endonuclease recognizes and cleaves the 5'C1AATTG nucleotide sequence. RMunI is an isoschizomer of RMfeI from Mycoplasma fermentans. Site-specific methylase modifies the second adenine residue in the same sequence (5'Cam6ATTG). It was established that the discovered enzymatic system is from mycoplasma which contaminates cell lines. Mycoplasma's DNA hybridizes with species-specific DNA probed for Mycoplasma fermentans and Mycoplasma arginini. The possible role of mycoplasmic restriction-modification enzymes in the process of acquired immune deficiency syndrome are discussed.

[The results of clinical trials and the prospects of using the Soviet preparation of human recombinant interferon alpha-2 (reaferon) in medical practice]. / Rezul'taty klinicheskikh ispytanii i perspektivy primeneniia v meditsinskoi praktike otechestvennogo rekombinantnogo preparata chelovecheskogo interferona al'fa-2 (reaferona).

The results of the clinical trials of human recombinant interferon alpha-2 (reaferon) make it possible to come to the conclusion that the preparation is well-tolerated and produces a pronounced therapeutic effect in a number of viral and oncological diseases. The Pharmacological Committee of the USSR has recommended reaferon for use in acute hepatitis B, hairy cell leukemia, renal cancer at stage IV, disseminated sclerosis, ocular herpes. The use of reaferon has been found to be promising in the treatment of papillomatosis of the larynx, Kaposi's sarcoma, mycosis fungoides, chronic myeloleukemia.

[Site-specific endonucleases LplI and AagI]. / Sait-spetsificheskie éndonukleazy LplI i AagI.

New site-specific endonucleases LplI and AagI have been isolated from the Lactobacillus plantarum and Achromobacter agile cells, respectively. The enzymes' purification stages included treatment of cell-free extracts with polyethylenimine, fractionation in two-phase system by Albertsson's method, chromatography on blue Sepharose and DEAE-cellulose. The results of cleavage of a 5'-32P-labelled oligodeoxynucleotide duplex by restriction endonucleases LplI and AagI indicate that these enzymes recognize and cut the sequence AT decreases CGAT, being therefore true isoschizomers of the ClaI restriction endonuclease from Caryophanon latum. The L. plantarum strain has 400 fold endonuclease productivity as compared with the ClaI producent and is perspective for preparative isolation of LplI.

[Isolation and characteristics of new restriction endonucleases from Haemophilus influenzae]. / Vydelenie i kharakteristika novykh éndonukleaz restriktsii iz Haemophilus influenzae.

Various strains of Haemophilus influenzae have been examined for the presence of site-specific endonuclease activities, and eleven restriction endonucleases have been isolated from seven strains. For all the endonucleases recognition sequences were determined, for three of them cleavage sites being identified. The enzymes proved to be isoschizomers of known endonucleases, viz. Hin1 I, Hin8 I--Acy I; Hin1 II, Hin8 II--Nla III; Hin2 I, Hin5 I--Hpa II; Hin3 I--Cau II; Hin5 II--Asu I; Hin5 III--Hind III; Hin6 I, Hin7 I--Hha I. Restriction endonucleases Hin1 I, Hin1 II and Hin6 I recognize nucleotide [formula: see text] sequences 5'GRCGPYC, 5'CATG, 5'GCGC, respectively, and cleave them as indicated by arrows.

[Methyl-cytosine specific restriction in Escherichia coli K-12]. / Izuchenie metil-tsitozin spetsificheskoi restriktsii u Escherichia coli K-12.

Experiments on transformation of Escherichia coli K-12 cells by plasmids carrying RM systems with different recognition sites containing 5-methylcytosine have shown that the gene mcrB determines the function of restriction. The data obtained made it possible to believe that E. coli possesses no restriction system recognizing specifically cytosine methylated in position 4.

[B-A and B-Z transitions in deoxyoligoduplexes containing 4- and 5- methylcytosine]. / Perekhody B-A i B-Z dezoksioligodupleksakh, soderzhashchikh 4- i 5-metiltsitozin.

Self-complementary oligodeoxynucleotides: GGACCCGGGTCC, GGA4mCCCGGGTCC, GGA5mCCCGGGTCC, CGCGCGCG, CG4mCGCGCG, CG5mCGCGCG were synthetized to study the contribution of methyl groups into the energetics of the three known cooperative transitions in DNA: helix-coil, B-A and B-Z With the use of circular dichroism and absorbtion methods the profiles of the above transitions were obtained by variation of temperature (helix-coil), trifluoroethanol fraction (B-A), NaCl and trifluorethanol contents (B-Z). On the basis of the transition widths and shifts of the transition points due to the methylations the energetics of the methyl groups was estimated. 5mC stabilizes the B form relatively the A form by 0.33 kcal/mol; while 4mC by 0.5 kcal/mol. In the B-Z transition 5 mC stabilizes the Z form by 0.28 kcal/mol relatively the B form; 4mC stabilizes also the Z form although by 0.14 kcal/mol only. Thus, these naturally occurring modifications could modulate substantially the ability of a DNA piece to shift into the A or Z form.

[Cloning of the gene of Saccharomyces cerevisiae yeasts that determines resistance to the toxic action of cadmium ions]. / Klonirovanie gena drozhzhei Saccharomyces cerevisiae, opredeliaiushchego rezistentnost' k toksicheskomu deistviiu ionov kadmiia.

A gene conferring resistance to cadmium in Saccharomyces cerevisiae was isolated from a yeast gene library created on the basis of the pL3 vector. The phenotype of resistance is only expressed in the yeast cells with cloned DNA inserted into a multicopy plasmid. Integration of the plasmid into chromosome or introduction of the centromeric region into the plasmid decreases the level of cadmium resistance. The cloned Sau3A I fragment of the yeast chromosome is 3.5 kbp in size. Restriction analysis and subcloning experiments showed the gene to be located within 1.6 kbp of the XhoI-Sau3A I fragment of DNA. Instability was observed in the vicinity of the XhoI-Sau3A I fragment of the yeast DNA in Escherichia coli.

[Effect of N4-methylcytosine and 5-methylcytosine on the stability of the DNA helix]. / Vliianie N4-metiltsitozina i 5-metiltsitozina na stabil'nost' spirali DNK.

The thermodynamic parameters (delta H, delta S) of the helix-coil transition of self-complementary oligonucleotides d(CGCGCGCG), d(CG5mCGCGCG), d(CG4mCGCGCG), d(GGACCCGGGTCC), d(GGA5mCCCGGGTCC), and d(GGA4mCCCGGGTCC) were determined. The substitution of 4mC for C was found to decrease the melting temperature of the oligonucleotides. The destabilization effect of the two substitutions is equivalent to the change of A.T for G.C pair. The free energy decrease of the helix-coil transition due to the introduction of two 4mC into an octanucleotide was estimated to be 1,24 kcal/mol.

[Cloning of the restriction-modification genes of Bacillus centrosporus in Escherichia coli]. / Klonirovanie genov restriktsii-modifikatsii Bacillus centrosporus v Escherichia coli.

A genomic library of Bacillus centrosporus was obtained using pBR327 as a vector. The total plasmid DNA of the library was cleaved by the BcnI restriction endonuclease and then transformed in Escherichia coli RR1. Two clones possessing restriction and DNA modification profiles of BcnI were identified among the transformants. Their respective plasmids were 13.3 and 9.05 kbp in size. Restriction mapping of both plasmids showed each of them to contain two sites for HindIII and one for both Eco31I and Eco47III, located at the same distance. This was assumed to be the location region of the BcnI restriction-modification genes. Confirmation of the assumption was obtained by deletion mapping of the recombinant plasmids. Special features concerning cloning of the restriction-modification genes are discussed on the basis of the results obtained.

[Construction of derivatives of the diphtheria toxin gene and their expression in Escherichia coli cells]. / Konstruirovanie proizvodnykh gena difteriinogo toksina i izuchenie ikh ékspressii v kletkakh Escherichia coli.

A fragment of diphtheria toxin (tox) gene from beta 45 phage DNA was cloned on pUC19 plasmid in E. coli cells. The fragment is coding for toxA fragment of the toxin and contains the control region of the tox gene. The tox gene promoter is active in E. coli. The toxA protein is found mainly in periplasm of E. coli cells. The protein is enzymatically active in ADP-ribosilation of elongation factor 2 from eucaryotic cells. An in frame toxA-lacZ' fusion was constructed on pUC8 plasmid. The hybrid protein expresses both toxA and lacZ' activities. Two or seven base pairs were deleted from the central part of toxA gene by means of S1 nuclease digestion. Translation of hybrid toxA-lacZ' mRNA should be terminated downward the delections due to the frameshifts caused by them. Nevertheless, a functionally active alpha-peptide of beta-galactosidase is expressed by both the deletion fusions. The existence of another translational start site functioning in E. coli and located inside 3'-end region of toxA mRNA is suggested.

[Site of integration of IS1-element responsible for expression of the yeast ADE1 gene in Escherichia coli]. / Issledovanie mesta integratsii IS1-élementa, obespechivaiushchego ékspressiiu gena ADE1 drozhzhei v kishechnoi palochke.

Cloned yeast ADE1 gene is expressed in Escherichia coli, due to integration of the IS1 bacterial element into the non-coding 5' region. Primary structure analysis of the integration site of IS1 element demonstrated that this site is situated in a region flanked by two inverted repeats of yeast DNA which are homologous to the right end of the IS1 element. The region up-stream (5') to the left inverted repeat is AT-rich.

[Cloning of the ADE2 gene of Saccharomyces cerevisiae and localization of the ARS-sequence]. / Klonirovanie ADE2-gena Saccharomyces cerevisiae i lokalizatsiia ARS-posledovatel'nosti.

Mutational changes in ADE2 result in the accumulation of red pigment in cells, which serves as an indicator for the selection of mutants. This easily detectable phenotype of red-coloured colonies can account for the wide use of ade2 mutants in yeast genetics. ADE2 gene was cloned in a shuttle vector by complementing the ade2 mutation in the yeast. It was shown that the 2.2 kbp HindIII fragment of yeast DNA contains structural sequences of the ADE2 gene as well as the ARS sequence. Deletion analysis of the 5' end of the ADE2 gene showed the ARS sequence to be situated at the distal end of the 1 kbp HindIII fragment. Removal of the ARS sequence does not influence ADE2 gene complementation ability. Transformants containing the ADE2 gene comprised in their plasmids form white colonies. Loss of the plasmids results in colour change of colonies.

[Specificity of action of DNA methylases BcnI, CfrI and Cfr10I]. / Spetsifichnost' deistviia DNK-metilaz BcnI, CfrI i Cfr10I.

The site specificity of three DNA methylases BcnI, CfrI and Cfr10I was determined to be 5'Cm4C(C/G)GG, 5'PyGGm5CCPu and 5'Pum5CCGGPy, respectively. Using the modification methylases under investigation with known restriction endonucleases, fourteen new DNA cleavage specificities can be created. Some aspects of the use of restriction endonucleases in DNA methylation analysis are discussed.

[Nucleotide sequence of the ADE 1 gene of the yeast Saccharomyces cerevisiae]. / Nukleotidnaia posledovatel'nost' gena ADE 1 drozhzhei Saccharomyces cerevisiae.

The yeast ADE 1 gene has been cloned and sequenced. The primary structure deduced from the nucleotide sequence demonstrated that phosphoribosylaminoimidazole-succinocarboxamide synthetase is a protein with molecular weight of 34 500 D.

[Cloning and physical mapping of the ADE1 gene of the yeast Saccharomyces cerevisiae]. / Klonirovanie i fizicheskoe kartirovanie gena ADE1 drozhzhei Saccharomyces cerevisiae.

ADE1 gene of Saccharomyces cerevisiae codes for the primary structure of SAICAR-synthetase. Mutational changes of ADE1 gene result in the accumulation of red pigment in cells. Colour differences, thus, serve as a basis for the selection of mutants or transformants. ADE1 gene was cloned as a 4.0 kb HindIII fragment of yeast DNA in a shuttle vector by complementing the ade1 mutation in yeast. The study of ADE1 gene expression in Escherichia coli showed that the 4.0 kb fragment containing the ADE1 gene does not complement purC mutations in E. coli. However, prototrophic colonies appeared at a frequency of 10(-7)-10(-8) after incubating clones bearing the recombinant plasmid with ADE1 gene on selective media. The plasmid DNA isolated from such clones complements the purC mutation in E. coli and the ade1 mutation in S. cerevisiae. Structural analysis of the plasmid demonstrated that the cloned DNA fragment contained an additional insertion of the bacterial origin. Further restriction enzyme analysis proved the insertion to be the bacterial element IS1. Expression of the cloned ADE1 gene in S. cerevisiae is controlled by its own promoter, whereas in E. coli it is controlled by the IS1 bacterial element.

[Substrate specificity of restriction endonucleases Eco471 and Eco521]. / Opredelenie substratnoi spetsifichnosti endonukleaz restriktsii Eco471 i Eco521.

Cleavage sites for Eco47I and Eco52I restriction endonucleases, which are isoschizomers of Ava II and Xma III, respectively, have been structurally elucidated.

[Specificity of new restrictases and methylases. Unusual modification of cytosine at position 4]. / Izuchenie spetsifichnosti novykh restriktaz i metilaz. Neobychnaia modifikatsiia tsitozina po 4-mu polozheniiu.

Fourteen restriction endonucleases and 4 methylases were isolated and purified from 14 strains of Citrobacter freundii and Escherichia coli, which were isolated from natural sources. To determine the nucleotide sequence recognized by the endonucleases a comparison of DNA cleavage patterns, the evaluation of the cleavage frequency of some DNA with known recognition sequences and mapping was used. It was determined that Cfr101 is a new enzyme recognizing 5'PuCCGGPy. Other restriction enzymes isolated were isoschizomers of: Cfr5I, Cfr11I, Eco60I, Eco61I--EcoRII; Cfr4I, Cfr8I, Cfr13I--Sau96I; Cfr6I--PvuII, Cfr9I--SmaI, Eco26I--HgiJII; Eco32I--EcoRV; Eco52I--XmaIII; Eco56I--NaeI. Some of the enzymes in C. freundii and E. coli were found for the first time. The methylases MCfrI; MCfr6I, MCfr9I and MCfr10I recognize the same nucleotide sequence as specific endonucleases isolated from the same strain. DNA modification in vitro by MCfrI and MCfr10I yields 5-methylcytosine and 4-methylcytosine by MCfr6I and MCfr9I.