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E A Skripkin

Publications and source records attributed to E A Skripkin.

At least 19 recordsLinked to original sources
PubMed1994

[Kinetics of luciferase gene expression from fireflies Photinus pyralis and Luciola mingrelica in Escherichia coli cells. I. Kinetics of the transformation of recombinant luciferase into enzymatically-active conformers].

The Photinus pyralis and Luciola mingrelica luciferases genes expression has been studied on the pME61 or pJG lambda plasmids with a thermoinducible lambda Pr promoter in the E. coli strain CA using three independent methods: SDS gel electrophoresis to quantify the synthesized luciferase protein, EIA to quantify the enzyme native conformer and activity measurements. The cultures were incubated by the temperature schemes 28 degrees-42 degrees-21 degrees C and 28 degrees-21 degrees C. The maximal amount of the Ph. pyralis protein reached 4.5%, while that of L. mingrelica luciferase was 4.1% of the total amount of the cellular proteins after 10-hr incubation. The amount of the native conformer and the luciferase activity began to grow after a long induction period, reaching the maximal level by hour 50-60 after the thermoinduction. The increase in the enzyme activity correlated well with the increase in the ATP content in the cell. The observed "low-temperature induction" of the enzyme activity is interpreted as a protein transition to an active conformation. This transformation is considerably behind the protein biosynthesis process. Intracellular metabolic reactions have been shown to play an active part in these conformational changes.

Animals↗
PubMed1993

A two-dimensional model at the nucleotide level for the central hairpin of coliphage Q beta RNA.

We describe the construction and testing of a structural model at the nucleotide level for conformation CH of the central hairpin of genomic RNA from coliphage Q beta. The model was developed with the computer program MFOLD using both optimal and suboptimal predictions. Structural information obtained by electron microscopic analysis of Kleinschmidt spreadings of Q beta RNA was used to guide the modeling. The model was tested in solution with three enzymatic probes: RNase T1, RNase T2, and RNase V1, as well as four chemical probes: dimethylsulfate, diethylpyrocarbonate, kethoxal and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMCT). The structural analyses in solution are consistent with the predicted structural model. The model is also supported by comparative structural analysis with the related coliphage SP. The model provides a structural basis for published biochemical and genetic studies implicating large, long-range structural features in the co-regulation of viral coat and replicase expression. In addition, we show that the read-through region of the viral protein A1 forms a separate structural domain, and we suggest that it functions as a nucleation site that participates in the folding and refolding of the molecule during replication and translation. In addition to the central hairpin, we have analyzed the structure of the viral coat initiation region. Our studies show that the entire region consists of small local hairpins and that 26 nucleotides immediately surrounding the coat initiation codon are single-stranded.

Base Sequence↗
PubMed1993

[mRNA with an extended Shine-Dalgarno sequence is translated independently of ribosomal protein S1].

The role of the ribosomal protein S1 in translation of a mRNA containing an extended Shine-Dalgarno sequence has been investigated. Using the toe printing technique, the formation of a ternary initiation complex with both S1-depleted 30S subunits and subunits treated with anti-S1 antibodies and mini-mRNA containing an lambda-cro-mRNA translational initiation region with a very long (9 nucleotides) Shine-Dalgarno sequence, has been demonstrated. It is concluded that initiation of translation on mRNA with extended SD-sequence is S1-independent. By means of an E. coli cell-free system of translation (S-100 extract), the translation of mini-cro-mRNA by S1-depleted ribosomes has been shown. In contrast with mini-cro-mRNA, the 30S subunits without protein S1 are inactive both in the ternary initiation complex formation and in cell-free translation with MS2 or fr phage RNAs and RNA protein III of phage fd.

Base Sequence↗
PubMed1992

Unusual ribosome binding properties of mRNA encoding bacteriophage lambda repressor.

The mRNA encoding repressor cI of phage lambda is the only known E. coli message which starts directly with the initiation AUG codon. The ability of in vitro synthesized cI mRNA fragments (150 or 400 nts) to form ternary initiation complexes has been studied using the toeprint method. In the presence of tRNA(Met)f, these fragments are capable of forming the ternary complexes at the 5'-terminal AUG codon not only with 30S subunits but also with undissociated 70S ribosomes (70S tight couples). In the latter case, no binding at other positions of cI mRNA can be detected at all. The starting region of cI mRNA has a single stranded conformation and is highly enriched in A-residues. This feature of cI mRNA RBS is suggested to be the main factor which allows cI mRNA to form the initiation complex with the ribosome. Unlike 30S subunits, the binding to 70S tight couples is not affected by any of the initiation factors, although it is as efficient as that to 30S subunits supplemented with the factors. 30S subunits prefer to associate with the internal RBSs of the preformed mRNA molecules, provided that they are not sequestered by the secondary structure. In contrast, 70S tight couples tend to avoid extra sequences upstream of the codon directed to the P site and occupy a position as close as possible to the 5'-end of the message. This has been found to be the case both for tRNA(Met)f and for elongator tRNA(Glu)2. The structural features of mRNA RBSs which influence their different binding for 30S subunits and 70S ribosomes are discussed.

Bacteriophage lambda↗
PubMed1992

Identification of the Escherichia coli 30S ribosomal subunit protein neighboring mRNA during initiation of translation.

To identify the proteins of the 30S ribosomal subunit of E coli that neighbor mRNA in the ternary initiation complex (mRNA*30S subunit*tRNA(fMet), we used an affinity cross-linking approach in which photoactivated groups were attached to different positions along the mRNA chain. A series of mini-genes originating from the 5'-end region of the cro gene of lambda bacteriophage were constructed as templates for mini-mRNA synthesis. Two strategies were used to introduce photo-reactive agents into the message. According to the first, two transcripts were isolated from E coli and chemically derivatized at their 5'-ends with a photoinducible diaziril group. One of these messages allowed for localization of the 5'-end of the Shine-Dalgarno sequence while the other one allowed for labeling of the ribosome at the 5'-end side of the initiation AUG codon in the P site. According to the second approach, 5-azidouridine (5N3U) was randomly incorporated into mRNA transcripts during a T7 RNA polymerase catalyzed reaction by using a mixture of 5N3UTP and UTP. A message that had U residues at either -4, -3, -1, +2 and +14, +19, +20 positions was used (A from cro AUG is +1). Whereas cross-links with the 5N3U transcripts were essentially 'zero-length', the 5'-derivatized transcripts were covalently attached to ribosomal components about 14 A from the 5'-end. We found that proteins S1, S7, S5, S3 and S4 compose, or were close to, the ribosomal mRNA-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1992

mRNA containing an extended Shine-Dalgarno sequence is translated independently of ribosomal protein S1.

The role of ribosomal protein S1 in the translation of mRNA containing an extended Shine-Dalgarno sequence was investigated. Using the toeprinting technique, formation of the ternary initiation complex between 30S subunits, both S1-depleted or treated with anti-S1 antibodies, and mini-mRNA containing the 9 nucleotide-long Shine-Dalgarno sequence was studied. It was concluded that the initiation of translation on mRNA with an extended Shine-Dalgarno sequence is S1-independent. It was demonstrated that S1-depleted ribosomes effectively translate the cro-mini-mRNA in a cell-free system. In contrast to cro-mini-mRNA, 30S subunits without protein S1 are inactive in ternary initiation complex formation with, and cell-free translation of, MS2 or fr phage RNAs and RNA protein III of phage fd.

Base Sequence↗
PubMed1991

Chemical ligation of DNA: the first non-enzymatic assembly of a biologically active gene.

An artificial gene comprising 183 base pairs has been assembled by template-directed condensation of 35- to 53-membered oligodeoxyribo nucleotides with cyanogen bromide as a condensing agent. The reaction is complete within several minutes at 0 degrees C in buffer. The resulting mini-gene was cloned and expressed in vivo and in vitro. We have also found that the polymerase inhibition technique (toe-printing) is a good way to ascertain that translation initiation complexes form in the case of single-stranded DNAs as well. Thus, along with the fully chemical assembly of synthetic genes, a rapid and sufficiently reliable method for determining their ribosome-binding properties was developed.

Base Sequence↗
PubMed1990

Secondary structure of the central region of bacteriophage MS2 RNA. Conservation and biological significance.

The RNA of the Escherichia coli RNA phages is highly structured with 75% of the nucleotides estimated to take part in base-pairing. We have used enzymatic and chemical sensitivity of nucleotides, phylogenetic sequence comparison and the phenotypes of constructed mutants to develop a secondary structure model for the central region (900 nucleotides) of the group I phage MS2. The RNA folds into a number of, mostly irregular, helices and is further condensed by several long-distance interactions. There is substantial conservation of helices between the related groups I and II, attesting to the relevance of discrete RNA folding. In general, the secondary structure is thought to be needed to prevent annealing of plus and minus strand and to confer protection against RNase. Superimposed, however, are features required to regulate translation and replication. The MS2 RNA section studied here contains three translational start sites, as well as the binding sites for the coat protein and the replicase enzyme. Considering the density of helices along the RNA, it is not unexpected to find that all these sites lie in helical regions. This fact, however, does not mean that these sites are recognized as secondary structure elements by their interaction partners. This holds true only for the coat protein binding site. The other four sites function in the unfolded state and the stability of the helix in which they are contained serves to negatively control their accessibility. Mutations that stabilize helices containing ribosomal binding sites reduce their efficiency and vice versa. Comparison of homologous helices in different phage RNAs indicates that base substitutions have occurred in such a way that the thermodynamic stability of the helix is maintained. The evolution of individual helices shows several distinct size-reduction patterns. We have observed codon deletions from loop areas and shortening of hairpins by base-pair deletions from either the bottom, the middle or the top of stem structures. Evidence for the coaxial stacking of some helical segments is discussed.

Base Composition↗
PubMed1990

[Design of new oligonucleotide derivatives resistant to cell nucleases].

A new type of modified antisense oligodeoxyribonucleotides is suggested, containing an intercalating drug (e.g., daunomycin (Dnm-NH2)) at one end of the oligonuleotide, and a compound, improving the oligonucleotide transport into the cell, e.g., polymyxin B1 (PmH) at its other end. Schemes for joining these antibiotics to the terminal groups of oligonucleotides have been developed. From undecanucleotide pTGTAAAACGACp (I); the corresponding derivative (Pm)pTGTAAAACGACp (NH-Dnm) (II) was obtained with 20% yield. It was found that joining of daunomycin significantly lowers the rate of the oligonucleotide's exonuclease degradation, whereas joining of polymyxin blocks the degradation completely. Derivative (II) is much more stable to hydrolysis by cell enzymes that the unmodified oligonucleotide (I): upon incubation with E. coli cells (I) is quantitatively hydrolyzed with in 40 minutes while (II) is hydrolyzed by 30% with in 24 h. Derivative (II) is also better adsorbed by E. coli cells. This sort of derivative of antisense oligonucleotides may prove to be efficient inhibitors of the gene expression.

Chemical Phenomena↗
PubMed1989

Synthesis and pathway of Luciola mingrelica firefly luciferase in Xenopus laevis frog oocytes and in cell-free systems.

Poly (A+)RNA was isolated from the lanterns of adult fireflies of L. mingrelica. The poly (A+)RNA was translated in a cell-free translation mixture from rabbit reticulocyte and from Krebs II mouse ascites cells and in Xenopus laevis frog oocytes. The synthesis of the enzymatically active firefly luciferase was demonstrated in all systems. In the cell-free systems a maximal quantity of luciferase is synthesized during the first 1-2 h, then the decreasing activity of luciferase is observed. The optimal concentration of mRNA for translation in the mouse ascites cell extract was found. The kinetics of the synthesis of the luciferase, its pathway and stability in X. laevis oocytes was studied. It was observed that luciferase is secreted from oocytes into the incubation medium.

Animals↗
PubMed1986

[Isolation of a restriction endonuclease with HindIII-specificity from Bordetella bronchiseptica].

Site-specific restriction endonuclease BbrI has been found in bacteriophage resistant strain B. bronchioseptica 4994. The technique was elaborated for purification of BbrI to the stage free of nuclease and phosphatase contamination. The yield of purified enzyme is 6000-20 000 units per 10 g of biomass. BbrI recognises and cleaves the same DNA sequence as HindIII with the formation of four-nucleotide cohesive ends. The simplicity of cultivation, security for human, presence of the single restriction endonuclease and the high level of its production make B. bronchioseptica 4994 a promising producer of BbrI restriction endonuclease, isoshizomeric to HindIII, for use in experimental practice in industry.

Bordetella↗
PubMed1983

The primary and secondary structure of the 5'-end region of encephalomyocarditis virus RNA. A novel approach to sequencing long RNA molecules.

The primary structure of a 149-nucleotide fragment of encephalomyocarditis (EMC) virus RNA from the 5'-terminus of the genome up to the poly(C) tract (S fragment) has been determined. For isolation of the S fragment, site-directed fragmentation of the viral RNA with RNase H and poly(dG) was employed. For sequencing the S fragment, a novel approach has been developed, which can be used for primary structure determination of long RNA molecules. A model of the secondary structure of the S fragment is proposed, according to which this region of RNA is highly structured. The role of complementary oligonucleotide stretches near both termini of the RNA molecule is discussed.

Base Sequence↗
PubMed1983

[Synthesis and expression of the DNA fragment coding the antigenic determinant of the surface antigen protein of hepatitis B virus].

With the use of chemical-enzymatic synthesis, a polynucleotide (I) has been obtained which codes the amino acid sequence 93-109 of the hepatitis B surface antigen (HBsAg). The plasmid pHS12, constructed by insertion of (I) into pBR325 between EcoRI and BamHI sites, transformed E. coli HB101 cells. The plasmid expression in cell-free system produced a chimeric protein, its molecular weight corresponding to that a polypeptide containing the heptadecapeptide from HBsAg. An approach is discussed for creating a new type of vaccines on the basis of chimeric proteins containing the antigenic determinants of virus proteins.

Amino Acid Sequence↗