[Biochemical bases for the effect of combining kanamycin and nitrofuran resistance genes in Escherichia coli cells]. / Biokhimicheskie osnovy éffekta sovmeshcheniia genov ustoichivosti k kanamitsinu i nitrofuranam v kletkakh Escherichia coli.

The fact of a significant increase in resistance to aminoglycosides when nfr genes with chromosomal or plasmid localization are combined with the plasmid genes coding for kanamycin-transferase in E. coli cells is confirmed. Gel-filtration of homogenates of the cells with and without pLD105 plasmid carrying nfr gene and of the cells with a chromosomal nfr gene revealed a 10 kD polypeptide when the plasmid is present. Relying on these results, it is concluded that the discovered polypeptide fulfils two roles: inhibiting of specific nitrofuran-reductase, which leads to nitrofurans resistance and a drop of transmembrane electric potential contributing to the increase of resistance to aminoglycosides (kanamycin) in strains with the plasmid nfr gene. Absence of the 10 kD polypeptide in the cells with a chromosomal nfr gene and other data are indicative of a possible existence of a different mechanism of resistance to nitrofurans and an increase of resistance to aminoglycosides in the strains with a chromosomal nfr mutation.

[Synthesis of an artificial gene coding for thymosin alpha1 and its expression in Escherichia coli as a hybrid with human tumor necrosis factor]. / Sintez iskusstvennogo gena, kodiruiushchego timozin alpha1, i ego ékspressiia v Escherichia coli v sostave gibridov s faktorom nekroza opukholei cheloveka.

Chemical-enzymatic synthesis and cloning in Escherichia coli of an artificial gene encoding the immunoactive peptide thymosin alpha 1 have been carried out. Recombinant plasmids were constructed which contain fusion genes coding for hybrids of human tumour necrosis factor (TNF) and thymosin alpha 1 as N- or C-terminal part of the hybrid protein. In the C-terminal hybrid protein, TNF and thymosin alpha 1 are linked through a methionine residue, thus allowing for thymosin alpha 1 to be cleaved off the rest of the hybrid protein with cyanogen bromide. In case of the N-terminal hybrid protein, the linker between the thymosin alpha 1 and TNF sequences is the acid-labile dipeptide Asp-Pro. Expression of the hybrid genes in E. coli and properties of the recombinant proteins were studied. The N-terminal hybrid protein was secreted into periplasmic space, in contrast with the C-terminal hybrid protein, which formed insoluble aggregates inside bacterial cells. Procedures for the isolation of both hybrid proteins were developed. The N-terminal hybrid protein displayed full biological activity in the cytotoxic assay on the mouse fibroblast L-929 whereas the C-terminal hybrid protein proved to be much less active. Treatment of the hybrid protein TNF-thymosin alpha 1 with cyanogen bromide lead to a mixture of two polypeptides, from which thymosin alpha 1 was purified to homogeneity by simple chromatographic procedures.

[Synthesis, secretion, and proteolytic degradation of diphtheria toxin in Escherichia coli]. / Sintez, sekretsiia i proteoliticheskaia degradatsiia difteriinogo toksina v Escherichia coli.

The recombinant plasmids have been constructed encoding the synthesis of a full-sized diphtheria toxin from its own or PR, PL-promoters of bacteriophage lambda in Escherichia coli cells. The high level constitutive synthesis of toxin results in slow cell growth and plasmid elimination. The toxin was mainly detected in the periplasm, partially in the membrane and to a less extent in the cytoplasm and culturing medium. The dimeric form of toxin was found in the cytoplasm. Participation of toxin B-subunit in secreting of the toxin into culturing medium is discussed. Proteolytic degradation of the synthesized toxin in different Escherichia coli strains was demonstrated. The process takes place in cytoplasm and periplasm mainly. The main enzyme participating in the process is a La-protease. The data on proteolysis obtained by immunoprecipitation immunoblotting, affinity chromatography and in mini-cells of Escherichia coli are summarized.

[Specific proteolysis by fibrinolysin-coagulase from Yersinia pestis of Yersinia pseudotuberculosis outer membrane proteins coded by the Ca(2+)-dependence plasmid]. / Spetsificheskii proteoliz fibrinolizinokoagulazoi Yersinia pestis belkov vneshnei membrany, kodiruemykh plazmidoi Ca(2+)-zavisimosti v Yersinia pseudotuberculosis.

The pesticinogenicity 9.5 kb plasmid from Yersinia pestis strain EV76 has been marked by the kanamycin phosphotransferase gene inserted into PstI site and designated pP3. The obtained plasmid pP3 determines the synthesis of 45 kd pesticin, alpha and beta-forms of fibrinolysin coagulase (37 and 35 kd) and the 29, 19 and 13 kd proteins in Escherichia coli mini cells. When transferred into Yersinia pseudotuberculosis strain 6933 the plasmid causes the proteolysis of outer membrane proteins. The 150 kd protein is reduced to 138 kd, the 48.5 kd protein is reduced to 45 kd. The proteins secreted into the cultural medium (51 and 38 kd) are also cleaved. The proteolysis of the 150 kd protein was found to occur at the stage of secretion via the inner membrane. The purified fibrinolysin coagulase from Escherichia coli strain JM83 harbouring the plasmid pP3 induces the proteolysis in vitro of the isolated membrane proteins from Yersinia pseudotuberculosis strain 6953 similar to the proteolysis registered in vivo.

[The heterogeneity of collection strains of Yersinia pseudotuberculosis and their molecular biological properties]. / Geterogennost' muzeinykh shtammov Yersinia pseudotuberculosis i ikh molekuliarno-biologicheskie svoistva.

The capacity of Y. pseudotuberculosis strains for disassociation with the appearance of S- and P-forms has been studied. Strains 852 and 9547 show high stability in S-forms, their conversion into R-forms occurring at 40-42 degrees C. Strain 6953 shows pronounced polymorphism and instability of its associations at different growth temperatures. Strain 9532 exists in S- and R-forms which retain their stability during numerous subculturings at different growth temperatures and prolonged storage. This strain has plasmids of 130, 72.2, 5.7 kb. All plasmids are retained in S- and R-forms, i. e. the dissociation of the strain is not accompanied by the loss of plasmids. The conversion of the strain from the S-form into the R-form leads to changes in the structure of lipopolysaccharide and the composition of low-molecular (less than 23 kD) proteins in the outer and inner membranes. In tests on guinea pigs the LD50 of the R-form of the strain is tenfold greater than that of its S-form. The dissociants of strain 9532 are transformed by plasmid DNA with equal efficiency and equally inherit them without selective pressure.

[Cloning of Yersinia pseudotuberculosis calcium-dependence plasmid pYV6953 BamHI fragments and their analysis in Escherichia coli mini-cells]. / Klonirovanie BamHI-fragmentov plazmidy kal'tsiizavisimosti pYV 6953 Yersinia pseudotuberculosis i ikh analiz v mini-kletkakh Escherichia coli.

Calcium dependence plasmid pYV6953 (70.4 kb) in Yersinia pseudotuberculosis cells codes for the major quantities synthesis of 150; 48.5; 19.4 Kd outer membrane proteins and the 51, 38, 27 Kd proteins secreted into the culturing medium. These outer membrane and secreted proteins are synthesized in considerable amounts in Yersinia pseudotuberculosis strains 6953 and 9547 at 37 degrees C and in the absence of calcium ions in the culturing medium. BamHI fragments of the plasmid pYV6953 as components of the recombinant plasmids code for the synthesis of 150; 66.6; 51; 48.5; 47; 38 and 21.5 Kd proteins in Escherichia coli mini cells. The synthesis of 150 and 48.5 Kd proteins is determined by the BamHI fragment 9 of the plasmid pYV6953 (3.3 kb). Addition of up to 8% of ethanol inhibiting the protein synthesis eliminates the 150 Kd protein but not the 48.5 Kd synthesis. The 48.5 Kd protein is concluded to be a subunit of the 150 Kd protein. The plasmid pYV6953 is different from the known plasmids pIB1 and pCD1 plasmids as far as the outer membrane and secreted proteins coded by the plasmids are concerned.