Protonated pyrimidine-purine-purine triplex.

We have studied a protonated pyrimidine-purine-purine (Py-Pu-Pu) triplex, which is formed between the d(C)nd(G)n duplex and the d(AG)m oligonucleotide as the third strand and carries the CG*A+ protonated base-triads. We have observed such an intermolecular complex between a plasmid carrying the d(C)18 d(G)18 insert and the d(AG)5 oligonucleotide without bivalent cations in 200 mM of Na+ at pH4.0. Bivalent cations additionally stabilize the complex. We propose the structures for nearly isomorphous base-triads TA*A, CG*G and CG*A+. To identify the H-DNA-like structure, which includes the triplex between d(C)n d(G)n duplex and the AG-strand, we have cloned in a superhelical plasmid the insert: G10TTAA(AG)5. The data on photofootprinting and chemical modification with diethyl pyrocarbonate, potassium permanganate and dimethyl sulfate demonstrate that the H-like structure with triplex carrying CG*G and CG*A+ base triads is actually formed under acid conditions. In the course of this study we have come across unexpected results on probing of Py-Pu-Pu triplexes by dimethyl sulfate (DMS): the protection effect is observed not only for guanines entering the duplex but also for guanines in the third strand lying in the major groove. We have demonstrated this effect not only for the case the novel protonated Py-Pu-Pu triplex but also for the traditional non-protonated Py-Pu-Pu intramolecular triplex (H*-DNA) formed by the d(C)37 d(G)37 insert in supercoiled plasmid in the presence of Mg2+ ions.

[Isolation of recombinant interleukin-3 produced by E. coli]. / Vydelenie rekombinantnogo interleikina-3, produtsiruemogo E. coli.

A synthetic gene coding for human interleukin-3 (hIL3) was cloned in the plasmid pTE2IL3, the gene expression being controlled by the phage fd PVIII promotor and the phage T7 gene 10 translational enhancer. Under constitutive biosynthesis conditions in E. coli, the accumulation of recombinant hIL3 (in the inclusion bodies) was up to 30-40% of the total cell protein. An effective procedure of the hIL3 isolation is suggested. The hIL3 was solubilized in 5 M guanidinium chloride, renaturated and purified to homogeneity by a single chromatographic step. The protein's yield was 34 mg/g wet cells. The isolated hIL3 showed a specific biological activity.

RNA--protein interactions within the internal translation initiation region of encephalomyocarditis virus RNA.

Various derivatives of the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) RNA have been used to analyze by UV-cross-linking its interaction with mRNA binding proteins from ascites carcinoma Krebs-2 cells. A doublet of proteins with Mr 58 and 60 kD bound to two regions of the IRES. One site is centered at nt 420-421 of EMCV RNA whereas the other is located between nt 315-377. Both sites form hairpin structures, the loops of which contain UCUUU motif, conserved among cardio- and aphthoviruses. The interaction of p58 and p60 with IRES is affected by the integrity of the stem-loop structure proximal to the start AUG codon (nts 680-787), although, under similar conditions, cross-linking of these proteins to this region was not detected. Deletions in the main recognition site of p58 strongly reduce the initiation activity of the IRES in vitro. However, elimination of p58 (p60) binding by these mutations does not completely abolish the ability of the IRES to direct polypeptide synthesis starting from the authentic AUG codon. The IRES can be assembled in vitro from two covalently unlinked transcripts, one containing the target site for p58 and the other encompassing the remaining part of the IRES fused to a reporter gene, resulting in considerable restoration of its activity. Implications of these findings for the mechanism of initiation resulting from internal entry of ribosomes are discussed.

[Isolation of the porcine alpha-interferon gene using simultaneous directed multipoint mutagenesis]. / Poluchenie gena alpha-interferona svin'i metodom odnovremennogo napravlennogo mnogotochechnogo mutageneza.

A method of obtaining the pig alpha-interferon gene by means of simultaneous multidirected mutagenesis of the human alpha 2-interferon gene is presented. Nucleotide homology between these genes is 80.4%. Fourteen synthetic oligonucleotides forming a pig alpha-interferon gene's strand were ligated on a single-stranded template, carrying cDNA of the human alpha 2-interferon gene. The obtained DNA fragment was cloned in the single-stranded or double-stranded form. It was found that the method does not affect the cloning efficiency. The primary structure of the gene was confirmed by sequencing.

Insertion of short hepatitis virus A amino acid sequences into poliovirus antigenic determinants results in viable progeny.

In an infectious poliovirus cDNA construct, the determinant encoding antigenic epitope N-Ag1 (in a loop located between two beta-strands in poly-peptide VP1) was altered by site-directed mutagenesis, to be partially similar with the determinants for presumptive epitopes in polypeptides VP1 or VP3 of hepatitis A virus (HAV). The modified constructs proved to be infectious. However, another construct, in which the same locus encoded a 'nonsense' and a relatively hydrophobic amino acid sequence, exhibited no infectivity. These data showed the feasibility of the insertion of foreign sequences in a specific antigenically active locus of the poliovirus icosahedron, and suggest some limitations with respect to the sequences to be 'transplanted'.

Effective method for obtaining long nucleotide chains on partially complementary templates. Processed bovine gamma-interferon gene obtained from human gamma-interferon gene.

The method of obtaining the bovine gamma-interferon gene by means of simultaneous multidirected mutagenesis of the human gamma-interferon gene is presented. The first strand of the bovine gamma-interferon gene was obtained by ligation of synthetic oligonucleotides, using the cDNA of human gamma-interferon, cloned in the single-stranded phage M13mp19 as a template. The second strand was synthesized using a large fragment of E. coli DNA-polymerase I. The double-stranded gene was then treated by restriction nucleases and cloned in a pUC-18 derived vector. The primary structure was confirmed by sequencing.

[Genes coding for RNA polymerase in bacteria. III. The use of modified Sanger's method for sequencing the C-terminal region of rpoB gene, N-terminal region of rpoC gene and intercistron region of RNA polymerase in Pseudomonas putida]. / Geny, kodiruiushchie RNK-polimerazu bakterii. III. Ispol'zovanie modifitsirovannogo metoda Sengera dlia opredeleniia pervichnoi struktury C-kontsevoi oblasti gena rpoB, N-kontsevoi oblasti gena rpoC i mezhtsistronnogo uchastka RNK-polimerazy Pseudomonas putida.

The Sanger method was modified and the primary structure of the SalI-C fragment of the Pseudomonas putida rpoBC operon was elucidated.

trp operon induction during the expression in E. coli of two IFN-gamma sequences.

Two nucleotide sequences coding for mature human immune interferon (IFN-gamma) and differing from each other by nine N-terminal nucleotides were expressed in E. coli under the control of a trp promoter. The longer gene variant after the ATG initiatory codon contained a TGT TAC TGC sequence, which was absent in the shorter gene. When expressed in E. coli under the direction of identical transcription and translation regulatory elements, these genes showed different susceptibility to induction.

[Genes encoding the beta-subunit of bacterial RNA-polymerases. I. Primary structure of the EcoRI-C fragment of the Salmonella typhimurium gene rpoB]. / Geny, kodiruiushchie beta-sub''edinitsu RNK-polimeraz bakterii. I. Pervichnaia struktura EcoRI-C-fragmenta gena rpoB Salmonella typhimurium.

BglII fragment of S. typhimurium DNA, containing rpoB gene coding for the RNA polymerase beta-subunit, was cloned. The nucleotide sequence of the rpoB gene EcoRI-C fragment (2873 b.p.) was determined.