[Creation of a hybrid protein gene based on Bacillus thuringiensis delta endotoxins CryIIIA and CrIA(a) and expression of its derivatives in Escherichia coli]. / Sozdanie gena gibridnogo belka na osnove delta-éndotoksinov Bacillus thuringiensis CryIIIA i CryIA(a) i ékspressiia ego proizvodnykh v Escherichia coli.

The 5'-terminal fragment (containing 1-565th codons) of Bacillus thuringiensis var. tenebrionis gene for the Coleoptera-specific delta-endotoxin CryIIIA was cloned. This sequence was extended with either only a homologous fragment of CryIA(a) or that one together with in-frame NPTII or GUS coding sequences. The obtained gene derivatives were expressed in E. coli. The analysis of hybrid polypeptides confirmed the enzymatic activities of bifunctional proteins and showed toxic properties of "insectotoxin-NPTII" against Colorado potato beetle (Leptinotarsa decemlineata).

[Dependence of the activity of phi X 174 B-promoter in the expression of Escherichia coli gal-operon on the number of its copies and their orientation]. / Zavisimost' aktivnosti B-promotora bakteriofaga phiX174 v ékspressii gal-operona Escherichia coli ot chisla ego kopii i ikh orientatsii.

Promoter activities of different restriction fragments of the R8 DNA region of phage phi X 174 were compared. The studied DNA fragments included HindII fragment R8 (B-promoter), its left portion 49 nucleotide long, and the central segment containing 113 nucleotides generated by AluI. The promoter activity of these fragments was quantitated by the appearance of uridyltransferase and galactokinase activities in Escherichia coli clones carrying plasmids pHD68-17. The gal-promoters of these plasmids was substituted for the three aforementioned restriction fragments. The R8 region and its central part (BII-promoter) had comparable promoter activities while the left part containing the putative BI-promoter, did not induce clones with the expressed gal-operon. Clones containing 1, 2, 3 copies of the promoter fragment R8 were selected. No clones were revealed with more copies. All selected di- and tri-promoter clusters in plasmids had the same correct orientation of all inserted promoters with respect to the gal-operon. The expression of the gal-operon in E. coli was nearly directly proportional to the number of the phi X 174 B-promoters inserted before the operon.

[Discontinuous transfer of phage T7 DNA molecules into Escherichia coli cells during infection]. / Poetapnoe proniknovenie molekuly DNK faga T7 v kletku Escherichia coli pri infektsii..

HpaI restriction analysis of the part of T7 DNA molecule which comes off from E. coli after ultrasonic desorption of virion had been carried out. In such a way it was possible to follow the transfer of labelled T7 DNA into the host cell after the phage adsorption under different conditions. It was established that in the presence of chloramphenicol the left 60% of T7 chromosome is gradually (during 20 min) transferred into the cell and further transport is stopped. This suggests that some T7 gene(s) of I or (and) II class(es) is (are) necessary to transfer the last 40% of T7 DNA molecule containing the genes encoding capsid proteins. Also some new results are obtained which support thr idea about the tight coupling of the processes of T7 DNA transport and its transcription, and about the possibility for RNA polymerase to carry a mechanical function as well. All these results suggest a rather complicated mechanism of the process of T7 DNA transfer into the host cell consisting of at least three stages tightly connected with T7 gene expression temporal control. Some probable consequences of this model as well as its agreement with functional structure of T7 chromosome and with T7 development are discussed.

[Kinetic determination of the non-specific constants for the binding of Escherichia coli RNA polymerase to T7 phage DNA]. / Kineticheskoe opredelenie konstant nespetsificheskogo sviazyvaniia RNK-polimerazy Escherichia coli s DNK faga T7.

The kinetics of promoter complex formation of E. coli RNA polymerase with T7 phage DNA was studied under different ionic strengths at DNA concentrations 0.15, 1.5 and 20 microns/ml. Based on the results obtained the non-specific binding constants, Keq, and the promoter complex formation rate constants, kp, were estimated. Within ionic strength range 0 + 125 mM NaCl the Keq and kp vary from 5.10(6) to 9.10(5) M-1 and from 0.8 x 0.26 x 10(9) M-1 S-1, respectively. The results are discussed from the view point of the one-dimensional diffusion sliding of the enzyme along DNA in the process of phomoter site selection.

[Protection of segments of replicative form I of phiX174 phage DNA recognized by HindII, BspRI, and AluI restrictases by Escherichia coli RNA-polymerase]. / Zashchita RNK-polimerazoi Escherichia coli uchastkov replikativnoi formy I DNK faga phiX174, uznavaemykh restriktazami HindII, BspRI i AluI.

The preincubation of a DNA with E. coli RNA polymerase provides its partial protection against the HindII, BspRI and AluI cleavage giving possibility to determine the location of RNA polymerase tight-binding sites. Using this approach about 16 RNA polymerase tight-binding sites were detected on replicative form of phiX174 phage DNA. The protection degree of each of these sites depended on the preincubation conditions. Some of the protected sites hit the known phiX174 promoters and rho-dependent terminators and the other were distributed along the whole phiX174 DNA molecule. Many of them could be considered as potential promoters because they contain all the necessary elements specifying the real promoter sequences. At least some of the intrinsic promoter elements could be observed next to the rest of protected sites. One of the protected sites (R6b/l) is located in phiX174 DNA region which is very similar to the cAMP-CRP-controlled promoter sequences. It was confirmed that phiX174 DNA has two B promoters positioned by Sanger on the phiX174 nucleotide map, according to our data obtained by RNA polymerase protection experiments along with RNA product analysis of the R8 DNA fragment transcription in vitro.

[Kinetics of DNA-dependent synthesis of RNA: correlation between abortive and productive initiations]. / Kinetika DNK-zavisimogo sinteza RNK: sootnoshenie reaktsii abortivnoi i produktivnoi initsiatsii.

A general equation of abortive initiation kinetics is inferred at its coupling to a productive initiation. It describes the abortive initiation dependence upon time and concentrations of the initiating nucleotide and the following nucleosidetriphosphate (NTP). The equation analysis revealed the abortive initiation dependence upon NTP's concentration in a characteristic manner possessing a maximum at the time it comes to saturation. The kinetic constants were estimated experimentally from the plot when the extent of pApU synthesis was plotted first against time in presence of a complete set of NTP's and second, against AMP and UTP concentration in the absence of the other three NTPs. The estimated values of the constants were introduced into the equation to analyze the dependence of pApU synthesis upon NTPs concentration ([ATP]=0.2 [UTP]=[GTP]=[CTP]=[NTP]). The theoretically calculated curve was compared to the experimentally obtained. Good coincidence of these curves supports the correctness of the general equation. The general equation of abortive initiation gives possibility to infer an equation of transcription involving the initiation and the elongation studies.

[Participation of various adenosine and guanosine derivatives in the abortive RNA synthesis initiation reaction: effect of Mg2+, Mn2+, and rifampicin]. / Uchastie razlichnykh proizvodnykh adenozina i guanozina v reaktsii abortivnoi initsiatsii sinteza RNK: vliianie ionov Mg2+, Mn2+ i rifampitsina.

Adenosine, TMP, ADP, ATP and UpA along with guanosine and tis analogous derivatives have different reactivity towards [alpha-32P]UTP in abortive initiation reactions catalyzed by E. coli RNA polymerase on T2 DNA in the presence of Mg2+ or Mn2+. Rifampicin moderately inhibited almost all of the above mentioned reactions, except the ATP and the GTP which were even 2.5 times more reactive in the presence of this antibiotic.

[Gradual penetration of T7 phage DNA molecules into Escherichia coli cells during infection in the presence of chloramphenicol]. / Postepennoe proniknovenie molekuly DNK faga T7 v kletku Escherichi coli pri infektsii v prisutstvii khloramfenikola.

The cell-attached T7 phage DNA has been analyzed when E. coli was infected in the presence of chloramphenicol or rifampicin followed by sonication to provide phage desorption. Sucrose gradient sedimentation of the cell T7-DNA followed by HpaI digestion and agarose gel electrophoresis were performed. The results obtained suggest a gradual entrance of the T7-DNA molecule into the host cell starting on its left end bearing early genes. These data support the conception that the T7-DNA entrance into host cell is directly coupled with its transcription by RNA polymerase. At the same time one more HpaI fragment was found even in the cells, infected with the phage in the process of rifampicin. It may be that this fragment corresponds to the right end of the T7 chromosome, thus suggesting that short fragment of the T7-DNA right end can also enter the host cell early in infection.

[Transcription initiation: influence of ionic strength and actinomycin D on the kinetics of the open promoter complex formation between Escherichia coli RNA-polymerase and T7 DNA]. / Initsiatsiia transkriptsii: vliianie ionnoi sily i aktinomitsina D na kinetiku formirovaniia otkrytykh promotornykh kompleksov RNK-polimerazy Escherichia coli s DNK faga T7.

A membrane filter assay has been devised to study the influence of ionic strength (0--150 mM NaCl) and actinomycin D on the kinetics of the open promoter complex formation between E. coli RNA-polymerase and [3H]DNA of T7 phage. The dependence of the complex formation rate upon the ionic strength is non-monotoneus with a maximum at 75--100 mM. The addition of one actinomycin molecule per 200 base pairs decreases the open promoter complex formation rate at ionic strength less than 100 mM. A promoter site selection model including liner diffusional selection is proposed which is in a good agreement with the experimental results obtained.

[Isolation and properties of B. subtilis DNA-binding proteins inhibiting ATP-dependent deoxyribonuclease]. / Vydelenie i svoistva DNK-sviazyvaiushchikh belkov Bactillus subtilis, ingibiruiushchikh ATP-zavisimuiu dezoksiribonukleazu.

The fraction inhibiting ATP-dependent DNAase and some other enzyme activities was found in B. subtilis cell extracts. Two methods of its isolation were elaborated. It is established that the inhibiting activity fraction represents a set of some positively charged thermostable proteins of low molecular weight (M 9000--25 000). The inhibiting effect of the proteins in question may be attributed to their ability to form a complex with DNA. The complex is formed in low ionic strength conditions. The elevation of NaCl concentration to 0,3 M removes some proteins from the complex and causes the complete loss of inhibiting activity. At 0,5 M NaCl DNA-protein complex is completely dissociated. The discovered proteins seems to be localized in DNA-membrane cell fraction. It is supposed that these proteins (or some of them) are the structural ones of the bacterial nucleoid.