[Structural instability of co-integrates formed during interaction of plasmid R57 with pB322 and RP1. Possible role of IS1 element in the degradation of co-integrates]. / Strukturnaia nestabil'nost' kointegratov, obrazuiushchiksia pri vzaimodeistvii plazmidy R57 s pBR322 i RP1. Vozmozhnoe uchastie élementa IS1 v degradatsii kointegratov.

The conjugative plasmid R57 determines resistance to ampicillin and chloramphenicol. Earlier it was shown that R57 encodes site-specific recA-independent recombinase, which acts in cis and resolves IS1-mediated cointegrates arising in the Escherichia coli recA cells between R57 and pBR322. In the present work the properties of the cointegrates between R57 and pBR322 or RP1 arising in the E. coli rec+ strains were studied. It was found that the cointegrates between R57 and pBR322, obtained by mating of the respective biplasmid donors of E. coli rec+ and the rec+ recipients, lost as a result of deletion a large DNA segment of R57 containing determinant Cmr. The resulting hybrid replicons preserved determinants Apr and Tcr of pBR322 and the R57 conjugative properties and were structurally identical. By using plasmid RP1ts12, which is temperature-sensitive in replication, it was demonstrated that in cells rec+ the cointegrates between R57 and RP1 are extremely unstable. On storage they undergo structural degradation mainly affecting the RP1 replicon. The degradation products of the hydrid complex had lost their RP1 genes but preserved the R57 functional determinants. For elucidation of the observed phenomena the properties of the IS1-mediated cointegrates between pBR322:Tn9 and plasmid pBR3.1--deletion derivative of RP1 were studied. It was found that insertion of IS1 sometimes resulted in formation of unstable cointegrates capable of resolving and loosing determinant Cmr with a high frequency. It was suggested that IS1 encodes the site-specific recombinase responsible for resolution of the IS1-mediated cointegrates and deletion generation. Expression of this recombinase appears to be dependent on structure of the insertion sites. The possible role of IS1 and recombinase encoded by it in resolution and structural instability of the cointegrates between R57 and pBR322 or RP1 is discussed.

[Immunity to repeated transposition of the insertion sequence IS21]. / Immunitet k povtornomu vstraivaniiu insertsionnoi posledovatel'nosti IS21.

The ability of pBR325 derivatives carrying a copy of IS21-element to accept the second copy of this element from plasmid pRP19.6, a temperature-sensitive for replication mutant of RPI containing the duplicated IS21 was studied. It was shown that the frequency of IS21 transposition into plasmids pBR32S::IS21 differing by localization IS21 was lower by two orders of magnitude as compared to that of pBR325. The restriction endonuclease analysis revealed that the insertion of the second copy of IS21 resulted in the formation of pBR325 derivatives carrying the tandem repeated copies of IS21. It was also shown that the plasmids pBR325::IS21 were capable of increasing the frequency of pRP19.6 insertion into the bacterial chromosome from 3-9 to 200-300 times depending on IS21 localization. On the basis of the results obtained and literature data the possible mechanism of the transposition immunity is discussed.

[Interaction (integration and excision) of the pRP19.6 plasmid, a derivative of the RP1 plasmid containing duplicated sequence IS21, with the chromosome of Escherichia coli K12]. / Vzaimodeistvie (vstraivanie i iskliuchenie) plazmidy pRP19.6-proizvodnoi RP1, soderzhashchei duplitsirovannuiu posledovatel'nost' IS21, s khromosomoi Escherichia coli K12.

A pRP19.6 plasmid is the derivative of the temperature sensitive RPlts12 plasmid and contains a duplicated IS21 (IS8) element. Using temperature sensitive pRP19.6 replication, Hfr strains have been obtained by integration of the plasmid into the chromosome of E. coli rec+ and recA- cells and their properties were studied. According to the results obtained, pRP19.6 insertion into the genome of the rec+ bacteria IS reversible, and its integration into the chromosome of the recA- bacteria produced the stable Hfr strains. To elucidate the mechanism of pRP19.6 excision from the bacterial chromosome, plasmids of R+ transconjugates generated with a low frequency in the crosses between the stable Hfr strains and the rec+ recipients were analyzed. It was shown that the stable Hfr clones might produce stable R1 plasmids as well as a family of deletion KmsTra- derivatives of the pRP19.6. The structure of the KmsTra- was investigated and the mechanism of their formation was proposed. In the light of the data obtained, prospects of pRP19.6 practical application are discussed.

[Isolation and characteristics of a temperature-sensitive plasmid pRP19.6--an RP1 derivative containing the duplicated IS21 sequence]. / Poluchenie i kharakteristika termochuvstvitel'noi plazmidy pRP19.6--proizvodnoi RP1, soderzhashchei duplitsirovannuiu posledovatel'nost' IS21.

When analysing the antibiotic resistant, temperature-independent derivatives of Proteus mirabilis cells, carrying the plasmid RP1ts12, a derivative of the latter (pRP19.6) with an elevated frequency of integration into E. coli K12 chromosome, has been isolated. The structure and properties of pRP19.6 was studied. As revealed from the data of structural and genetic analyses pRP19.6 is identical to the factor R68.45 described earlier by Haas and Holloway. Similarly to R68.45, the plasmid under study contains two copies of IS21 sequence and mobilises nonconjugative plasmid pBR325 with high efficiency. Using the temperature sensitive replication of pRP19.6, frequency of it's integration into the chromosomes of E. coli rec+ and recA- stains is determined. It is demonstrated that the clones carrying the plasmid in integrated state are Hfr-strains. The possibilities to use the temperature sensitive R68.45 like plasmid for isolation of Hfr-strains in the broad range of gram-negative bacteria and for insertional inactivation of chromosomal genes are discussed.

[Deletions of plasmid pRP3.1ts12 derived from RP1 leading to the suppression of the thermosensitive ts12 mutation and mucoid phenotype induction in Escherichia coli K-12]. / Deletsii plazmidy pRP3.1ts12--proizvodnoi RP1, privodiashchie k supressii termochuvstvitel'noi mutatsii ts12 i induktsii mukoidnogo fenotipa u Escherichia coli K-12.

Properties of a temperature-sensitive in replication mutant pRP3.1ts12 derived from the broad host range RP1 plasmid have been studied. pRP3.1ts12 is a shortened variant of the temperature-sensitive RP1ts12 mutant carrying a deletion in a region from 2.3 to 7.6 MD. In contrast to RP1ts12, the plasmid pRP3.1ts12 is a leaky ts mutant and is characterized by an elevated frequency of reversions to the temperature-independent phenotype. Temperature-independent derivatives of pRP3.1ts12 were studied. Approx. 15% of these were found to induce mucoid growth of the host cells. As revealed from restriction endonuclease analysis, most of the latter derivatives contain deletions of small DNA segments in the region 0.56 to 2.3 MD of the RP1 map. The possible nature of the gene(s), whose deletions suppress the temperature-sensitive ts12 mutation and results in superproduction of Escherichia coli capsular poly-saccharide is discussed.

[Comparison of endogenous nuclear RNA-polymerase II at different stages of mycoplasmodial growth]. / Sravnenie éndogennoi RNK-polimerazy II v iadrakh Physarum polycephalum na raznykh stadiiakh rosta mikroplazmodiia.

The nuclei of Physarum polycephalum isolated from the 48- and 96-hour-old growing microplasmodium differ 7-8 fold by the rates of [3H]UTP incorporation in vitro accompanied by a slight (approximately 1.6 fold) change in concentration of endogenous RNA-polymerase II. Using mild fragmentation of chromatin nuclei by micrococcal nuclease and DEAE-Sephadex chromatography the bound enzyme was shown to consist of two forms differing in the degree of their binding to the template and in functional significance. Transcription in the Physarum polycephalum nuclei can be regulated by changes in the correlation of these forms.

[Comparative enzymatic study on the structure of chromatin in the nuclei of the growing microplasmodium of Physarum polycephalum]. / Sravnitel'noe fermentativnoe issledovanie struktury khromatina iader rastushchego mikroplasmodiia Physarum polycephalum.

The structure of chromatin in the growing myxomycetes P. polycephalum can be interpreted on the basis of three types of substructure: (i) soluble in a low ionic strength buffer and 2 mM EDTA (I), (ii) soluble in 0.6 M NaCl (II) and (iii) non-soluble and nuclease-insensitive (III). The ratio of these structures in the nuclear pellet obtained after autolysis was found to be 71 : 0, 4 : 12 for the young and 7.7 : 62 : 20.6 for the old cultures, respectively. The ageing of the culture involves transition of substructure I to substructure II. The substructures differ in the nuclease sensitivity. The specific features of enzyme fragmentation of chromatin from P. polycephalum microplasmodium nuclei are discussed.

[UV-light-tested protein--DNA interactions in the nuclei during the cell cycle in Physarum polycephalum]. / Testiruemye uf-svetom belok--DNK-kontakty v iadrakh na raznykh stadiiakh kletochnogo tsikla Physarum polysephalum.

The release of DNAs from nuclear preparations isolated at different steps of the cell cycle of the synchronous culture of Physarum polycephalum and irradiated with UV-light for 5 min (lambda 253.7 A, i = 1.8 . 10(2) j . m-2 . min-1) into 1% Na-DS - 1.5 M NaCl solution was studied. The value obtained correlated well with the degree of binding of interacting proteins with DNA in chromatin and was identical for all the preparations tested with the only exception of the nuclei isolated at the end of the S-phase, when a slight but significant (15 +/- 10%) increase in DNA release was observed. The results obtained are discussed in terms of structural transitions and function of chromatin during the cell cycle of the myxomycete Physarum polycephalum.

[Dependence of RNA synthesis in the cell of Physarum polycephalum on concentration and activity of RNA-polymerase II]. / Sintez RNK v kletochnom tsikle Physarum polycephalum v zavisimosti kontsentratsii i udel'noi aktivnosti RNK-polimerazy II.

The RNA synthesis detected by incorporation of [H3]uridine chase label proceeds in macroplasmodium and in isolated nuclei throughout the cell cycle of a synchronous myxomycetes culture of Physarum polycephalum, producing two well-shaped peaks at the postmitotic and premitotic steps. Such RNA synthesis is due to the activity of alpha-amanitin-sensitive RNA-polymerase II. Concentrations of RNA-polymerase II at various steps of the cell cycle were determined by alpha-amanitin titration; the specific activity of the enzyme was assayed. The increase in the rate of mRNA synthesis was accompanied by a rise in the concentration and specific activity of the enzyme: for the postmitotic peak, for instance, the corresponding values were about 2-2.5 and 2-4 times as high as minimum values revealed during the cycle. The results obtained are discussed in terms of transcription control in eukaryotic systems.