[Directed modification of the Tcr gene region of the plasmid pBR322 using complementary single-stranded DNA fragments carrying alkylating groups]. / Napravlennaia modifikatsiia uchastka gena Tcr plazmidy pBR322 s pomoshch'iu komplementarnykh odnotsepochechnykh fragmentov DNK, nesushchikh alkiliruiushchie gruppy.

A method is suggested for chemical modification of preselected regions of plasmid DNA by complementary single-stranded restriction fragments of DNA (srf DNA), carrying alkylating reagents. The gene coding for tetracycline resistance of plasmid pBR322 was used as a target. Srf DNA was prepared by a partial digestion of a double-stranded EcoRI-BamHI restriction fragment (377 base pairs) from Tcr by E. coli exonuclease III. The residues of an alkylating reagent N,N,N'-tri(beta-chlorethyl)-N'-(p-formylphenyl) propylenediamine 1,3 (TFP) were attached covalently to 4-5% of sfr DNA bases. The alkylating derivative of the sfr DNA was hybridized with supercoiled pBR322 plasmid DNA. The hybridization conditions (37 degrees C, 40% formamide, 0,2 M NaCl, 0,1 M Tris-HCl pH 7,5, 0,001 M EDTA) under which the bases carrying TFP residues are not eliminated from the sfr DNA, and transforming activity of pBR322 DNA does not decrease were established. It was shown that about 20% of plasmid pBR322 molecules form D-loops with alkylating sfr DNA under these conditions. It was shown that sfr DNA, carrying TFP can alkylate the complementary region of plasmid DNA, forming cross-linked D-loops. A method for the site-directed mutagenesis of switching off the preselected genes or non-transcribed DNA functional regions (promotors, introns etc) integrated into plasmids of other vectors is suggested.

[Selective binding of oligodeoxyribonucleotides with Escherichia coli RNA-polymerase and their effect on DNA-dependent RNA synthesis]. / Izbiratel'noe sviazyvanie oligodezoksiribonukleotidov RNK-polimerazoi Escherichia coli i ikh vliianie na DNK-zavisimyi sintez RNK.

It was shown previously, that E. coli RNA polymerase selectively binds certain fractions of oligoribonucleotides with the length greater than or equal to 5 nucleotides from the mixtures of random oligonucleotides of definite length. The data presented demonstrate, that E. coli RNA polymerase from the mixtures of random oligodeoxynucleotides of various length selectively binds oligodeoxynucleotides with the length greater than or equal to 9. The activity of the enzyme correlates with its ability to bind oligodeoxynucleotides. The enzyme which has selectively bound oligodeoxynucleotides, manifests sedimentation position characteristic for E. coli RNA polymerase engaged in transcription. The oligodeoxynucleotides with high affinity to the enzyme act as competitive inhibitors of transcription catalyzed by E. coli RNA polymerase. The data suggest that E. coli RNA polymerase bound oligodeoxynucleotides mimic the nucleotide sequences of the promoter responsible for the binding of the enzyme. It was found that selectively bound oligoribo - and oligodeoxynucleotides do not compete for the site on the enzyme. This property of E. coli RNA polymerase is assumed to play a certain role in the regulation of transcription.

Biochemical cascade mediating the estradiol action.

The data suggest that estradiol enhances the formation of histamine in rat uterus by induction of histidine decarboxylase; histamine activates adenylate cyclase providing accumulation of cyclic 3',5'-AMP, which, probably, induces glycolytic enzymes via phosphorylation of chromatin proteins, and mediates other estradiol effects. The chain of successively acting enzymes and mediators constitutes, obviously, a cascade amplifying the estradiol action. Since histamine is known to act as an intercellular mediator, attempts were made to find out the distribution of estradiol, histamine, and cyclic 3',5'-AMP among uterine cells. Autoradiography has shown that 3H-estradiol is bound by the nuclei of myometrium cells, 3H-histamine was found in the cytoplasm of these cells, 3H-cyclic 3',5'-AMP is selectively bound by the cells of capillary endothelium of the uterus. The estradiol mediators seem to spread their effect on different types of cells which form together a kind of a multicellular functional system.