[RNA interference in Escherichia coli cells: the expression of molecules that are complementary to the lon gene mRNA in parallel orientation]. / RNK-interferentsiia v kletkakh Escherichia coli pri ékspressii molekul, komplementarnykh v parallel'nom napravlenii mRNK gena lon.

To study the effect of RNA interference (RNAi) on the activity of gene lon in Escherichia coli, genetic constructs were used that could express RNA molecules complementary to the 5' region of lon mRNA in the same direction. These RNAs were termed parallel RNAs (pRNAs). Two approaches were used to control expression. In one approach, lon gene activity was estimated genetically, based on the effect of the Lon protease on bioluminescence determined by the Vibrio fischeri lux regulon. The other approach was direct testing of ATP-dependent proteolysis in vitro. It was found that pRNA considerably suppressed lon expression. The antiparallel RNA (apRNA) was a less effective suppressor of this gene. The specific RNAi was found to decay gradually by the 40th generation. The data obtained indicate that Eubacterium cells have mechanisms for specific regulation of gene activity that are sensitive to the formation of both parallel and antiparallel RNA duplexes involving mRNA of the given gene.

[Integration of IS186 element into the -10-region of the promoter of heat shock lon-gene in Escherichia coli]. / Integratsiia élementa IS186 v -10-oblast' promotora teplovogo shoka lon-gena Escherichiia coli.

The insertion element IS186 was found to be incorporated into the -10 region of the promoter of the E. coli lon gene. The integration represses lon gene expression. Homology between the integration sites of IS186, the -10 region of E. coli heat shock promoters, and the terminal inverted repeat of IS186 was revealed.

[Use of thermostable DNA polymerase from Thermus thermophilus KTP in a combined reverse transcription and amplification reaction of detecting interleukin 2alpha RNA and determining expression of the multidrug resistance gene (MDR-1)]. / Ispol'zovanie termostabil'noi DNK-polimerazy iz Thermus thermophilus STP v sovmeshchennoi reaktsii obratnoi transkriptsii i amplifikatsii dlia detektsii RNK interleikina 2alpha i opredelenie ékspressii gena mnozhestvennoi lekarstvennoi ustichivosti (MDR-1).

According to our data native Tth DNA polymerase displays higher reverse transcription activity than Taq DNA polymerase. This allows one to use Tth DNA polymerase in the complete reaction of reverse transcription and amplification (RT/PCR). We used this enzyme to synthesize the interleukine (IL-2 alpha) RNA template synthesized by the RT/PCR method in vitro. The conditions for RNA IL-2 alpha detection were optimized. The maximum yield of the specific product was obtained at pH 8.5-9.0. The influence of bivalent cations on the efficiency of RT reaction of coupled RT/PCR can be expressed as: Mn2+ > or = Cu2+ > Mg2+ > Cd2+ >> Co2+. The optimal ratio is 1.25-1.88 for Mn2+/dNTPs and 1.88-2.5 for Cu2+/dNTPs and Cd2+/dNTPs. The maximum yield of the RT/PCR product is found at Mg2+/dNTPs = 3.75. When Mn2+ is used instead of Mg2+ in the PCR reaction the efficiency of RT/PCR decreases. The RT/PCR method embracing thermostable Tth DNA-polymerase provides detection of 10(3) copies of RNA IL-2 alpha. An efficient method of the express-diagnostics of MDR-1 gene expression by coupled RT/PCR using Tth DNA polymerase is described.

[The demonstration of the genes controlling enterotoxigenicity in Staphylococcus aureus strains by using the polymerase chain reaction]. / Vyiavlenie genov, kontroliruiushchikh énterotoksigennost' u shtammov Staphylococcus aureus, s pomoshch'iu polimeraznoi tsepnoi reaktsii.

The selection and subsequent synthesis, according to the nucleotide sequences of S. aureus genes responsible for the expression of enterotoxins A and B, of highly specific primers for polymerase chain reaction Pcr were carried out with the use of the program "Primer". The optimum temperature conditions of polymerase chain reaction for all pairs of primers were selected. The method for the rapid determination of the enterotoxigenic properties of S. aureus strains by means of Pcr was proposed. The enterotoxigenic properties of several S. aureus strains were determined, which revealed that 3 clinical isolates had the gene of enterotoxin A in their genome, while laboratory strain FRI-722(H) carried the genes of enterotoxins A and B.

[Cloning the Staphylococcus aureus enterotoxin B gene, obtained by polymerase chain reaction, and its expression in Escherichia coli cells]. / Klonirovanie gena énterotoksina B Staphylococcus aureus, poluchennogo metodom polimeraznoi tsepnoi reaktsii, i ego ékspressiia v kletkakh Escherichia coli.

To determine the Staphylococcus aureus enterotoxigenicity, we have developed an approach based on polymerase chain reaction (PCR). Using this method several S. aureus strains have been screened for the presence of the enterotoxin B gene. A DNA fragment of the selected strain (FRI 722H) containing enterotoxin B gene has been obtained by the PCR method and cloned in the pUC19 vector. It is shown that enterotoxin B with the leader peptide forms insoluble complexes in E. coli cells, whereas the mature toxin is present in cytoplasmic fraction in a soluble form. The recombinant toxin made up for 1.7% of the total cellular protein in E. coli JM 109 cells.

[Cloning, structure and expression of the full-size lon gene in Escherichia coli coding for ATP-dependent La-proteinase]. / Klonirovanie, struktura i ékspressiia polnorazmernogo gena lon Escherichia coli, kodiruiushchego ATP-zavisimuiu La-proteinazu.

Assembly of the full Escherichia coli K-12 lon gene from the EcoRI--SphI fragment of the bacterial DNA ("modified" gene) cloned and sequenced earlier and the PstI fragment of the same DNA containing 3'-terminal region of the lon gene has been performed. Both "modified" and full genes showed all phenotype properties of lon gene. The complete nucleotide sequence of the gene (2770 bp) coding for the 784 amino acid sequence of protease La was determined. Location of catalytically active serine, histidine and aspartic acid residues was suggested, and ATP-binding site found. The lon gene and protease La structures we found are compared with those described independently and differences observed are discussed.

[Expression of Staphylococcus aureus enterotoxin A gene in heterologous systems]. / Ekspressiia gena énterotoksiba A Staphylococcus aureus v geterologichnykh sistemakh.

The genomic library of Staphylococcus aureus genes on the plasmid vector pSL5 has been constructed. The library contains a 2.5 kb HindIII DNA fragment including the gene for enterotoxin A. The entA gene on the high copy number plasmids in the Escherichia coli cells deficient in proteolysis determines the synthesis of enterotoxin A in the amounts comparable to the ones in the parent strain Staphylococcus aureus FRI 722(H).

[Regulation of proteolytic processes using antisense RNA genes htpR and lon of Escherichia coli in hetero- and homologous genetic systems]. / Reguliatsiia proteoliticheskikh protsessov s pomoshch'iu antismyslovykh RNK genov htpR i lon Escherichia coli v getero- i gomologichnykh genetichestkikh sistemakh.

Possibility of correction of proteolytic processes in cells of Escherichia coli and Pseudomonas aeruginosa has been studied. For this purpose recombinant plasmids directing the synthesis of antisense RNAs were constructed. In Ps. aeruginosa the synthesis of htpR antisense RNA resulted in 2.5-fold reduction of the intensity of degradation of 3H-puromycin polypeptides under heat shock conditions. An antisense RNA complementary to the 5'- end of E. coli lon gene decreased the same index to the level observed in lon- mutants. Genes homologous to htpR and lon genes of E. coli were found in Pseudomonas: bacteria in hybridisation experiments. This finding suggests that the genetic system of heat shock in these microorganisms is organized in a similar manner.

[Mechanism of the metabolic death of Escherichia coli B cells following freezing and thawing]. / Mekhanizm metabolicheskoi gibeli kletok Escherichia coli B posle zamorazhivaniia-ottaivaniia.

The state of E. coli B genome after low-temperature freezing and subsequent thawing (F--T) has been studied. The study has shown that in the process of F--T the DNA of the cells remains intact. But after thawing the incubation of the cells at 37 degrees C leads to the accumulation of ruptures in their DNA and its low-molecular decomposition products. At the same time the processes of the synthesis of DNA and proteins are suppressed to a considerable extent. Repeated F--T cycles enhance, on one hand, the inhibition of the processes of synthesis and, on the other hand, the activation of DNA metabolic decomposition. Such unbalance of the competitive enzymatic systems governing the synthesis and decomposition of DNA, which appears in the process of the revival of the cell from anabiosis, leads supposedly to the accumulation of ruptures and "gaps" in its DNA and, as a result, to the fragmentation of the genome and the death of the cell.