[Localization of an antigenic determinant of recombinant interleukin-2, recognized by monoclonal antibody 13B1]. / Lokalizatsiia antigennoi determinanty rekombinantnogo interleikina-2, uznavaemoi monoklonal'nymi antitelami 13B1.

We suggest a simple approach to localization of antigenic determinants for the monoclonal antibody 13B1 raised against recombinant human interleukin-2. The approach is based on the limited trypsin proteolysis, peptide separation by the O'Farrell method and identification of the peptides, interacting with monoclonal antibodies, and comparison of the charge and length of these peptides with the corresponding values of theoretically possible peptides.

[Cloning and nucleotide sequence of the 5'-flanking area of the human interleukin-2 gene]. / Klonirovanie i nukleotidnaia posledovatel'nost' 5'-flankiruiushchei oblasti gena interleikin-2 cheloveka.

We have cloned human interleukin-2 gene and sequenced its 1'-flanking region (-1940 to -936). The region contains promoter-like structures having a high degree of homology with the real promoter.

[Inhibition of replication of human hepatitis B virus]. / Ingibirovanie replikatsii virusa gepatita v cheloveka.

Several nucleoside 5'-triphosphate analogs were investigated as inhibitors of human hepatitis B virus replication. Different analogs inhibited DNA synthesis differently, 3'-azido-2',3'-dideoxythymidine 5'triphosphate being the most active compound. This inhibitor blocked DNA synthesis by 50% at inhibitor: substrate molar ratio 1:8, and by 80% - at 1:1. The hypothesis is formulated that 3'-azido-2',3'-dideoxythymidine 5'-triphosphate inhibits RNA directed viral DNA replication due to incorporation of this compound into 3'-termini of newly synthesized DNA chains. The phenomenon observed opens new possibilities for chemotherapy of acute and chronic human hepatitis B.

[Synthesis and functional activity of initiation regions of mRNA translation. II. RNA-ligase synthesis of hepta- and decaribonucleotides--components of 20-base polyribonucleotide templates]. / Sintez i funktsional'naia aktivnost' uchastkov initsiatsii transliatsii mRNR. II. RNK-ligaznyi sintez gepta- i dekaribonukleotidov--komponentov 20-chlennykh poliribonukleotidnykh matrits.

Preparative RNA-ligase synthesis of decaribonucleotides, the 5'-and 3'-constituent parts to be used for the synthesis of 20-base polyribonucleotides] simulating minimal translation initiation regions for phage RNA was carried out. The decamers were obtained via appropriate heptamers also by RNA-ligase catalyzed synthesis. Apart from decamers used to prepare the functionally active 20-base polyribonucleotide, the minimal translation initiation region of the replicase gene (R) in MS2 and fr phage--sequence R(-17----3) and two its variants, decanucleotides for other template modification were also synthesized. Three 5'-terminal decamers were isolated and identified including the natural decamer ApApApCpApUpGpApGpG (-17----(-)8) and those with G(-9)----A(-9) and U(-12)----C(-12) nucleotide substitutions, as well as three 3'-terminal products differing from the natural region ApUpUpCpCpCpApUpG (-7----3) in MS2 RNA by U(-6)----A(-6), U(-6)U(-5)----A(-6)A(-5) and CCC----UUU (-3----(-)1) substitutions.

[Study of the mRNA-binding region of ribosomes at different steps of translation. II. Affinity modification of Escherichia coli ribosomes by benzylidene derivative of AUGU6 in the 70S initiation complex]. / Issledovanie mRNK-sviazyvaiushchei ioblasti ribosomy na raznykh étapakh transliatsii. II. Affinnaia modifikatsiia ribosom Escherichia coli benzilidenovym proizvodnym AUGU6 v 70S komplekse initsiatsii.

2',3'-O-(4-[N-(2-chloroethyl)-N-methylamino]) benzylidene derivative of AUGU6 was used for identification of the proteins in the region of the mRNA-binding centre of E. coli ribosomes. This derivative alkylated ribosomes (preferentially 30S ribosomal) with high efficiency within the 70S initiation complex. In both 30S and 50S ribosomal subunits proteins and rRNA were modified. Specificity of the alkylation of ribosomal proteins and rRNA with the reagent was proved by the inhibitory action of AUGU6. Using the method of two-dimensional electrophoresis in polyacrylamide gel the proteins S4, S12, S13, S14, S15, S18, S19 and S20/L26 which are labelled by the analog of mRNA were identified.

[Study of the ribosome mRNA-binding region during different stages of translation. I. Functional activity of mRNA analogs, AUGU6 and its benzylidene derivatives, in ribosome-dependent protein synthesis]. / Issledovanie mRNK-sviazyvaiushchei oblasti ribosomy na raznykh étapakh transliatsii. I. Funktsional'naia aktivnost' analogov mRNK--AUGU6 i ego benzilidenovykh proizvodnykh--v ribosomnozavisimom belkovom sinteze.

Hexaribouridylic acid, prepared by digestion of poly(U) with cobra venom endonuclease, and trinucleotide AUG synthesized chemically by triester approach were joined by RNA-ligase to yield a nonaribonucleotide AUGU6 bearing the initiation codon at its 5'-terminus. 2',3'-O-(4-[N-(2-chloro(or hydroxy) ethyl-N-Methylamino])- benzylidene residues were introduced at the 3'-terminus of oligonucleotide AUGU6 and its benzylidene derivatives AUGU6CHRCl or AUGU6CHROH were obtained. The mRNA analogs synthesized were tested for their template activity in the formation of 70S initiation complex. AUGU6, AUGU6CHRC1 and AUGU6CHROH were shown to stimulate factor-dependent binding of fMet-tRNA to ribosomes. The effect of benzylidene fragment on the template activity of AUGU6CHROH in the course of translation process was studied. It was shown that AUGU6CHROH stimulates synthesis of di- and tripeptides with the same efficiency as AUGU6.

[Subjection of tetracycline resistance genes to cat promotor gene in plasmid pBR325]. / Podstanovka genov, opredeliaiushchikh ustoichivost' k tetratsiklinu, pod promotor gena cat v plazmide pBR325.

A series of plasmids with tetracycline resistance genes (Tcr-operon) subjected to transcription from chloramphenicol acetyl transferase promoter (Cmr-promoter) have been constructed on the basis of plasmid pBR325, AprCmrTcr. For this purpose, a 0.8 Md fragment in pBR325 DNA bordered by unique EcoRI and HindIII restriction sites was cut out and structural genes of Tcr-operon were fused to the cat gene nucleotides corresponding to Cmr-promoter and first 72 amino acids of cat (alton, Vapnek, 1979; Marcoli et al., 1980). These plasmids with molecular weight amounting to 3 Md confer AprTcr phenotype to host cells. Tetracycline resistance can be eliminated completely by the deletion of a) Cmr-promoter; b) part of the first Tcr-operon gene.

[Preparation of RNAase-free E. coli ribosomes active in initiation of protein biosynthesis]. / Vydelenie aktivnykh v initsiatsii biosinteza belka ribosom E. coli, svobodnykh ot primesei RNKaz.

The level of contaminating RNAases in the main components of the protein biosynthesis initiation system, the initiation factors and ribosomes of E. coli, was studied. It was shown that the ribosomes are the major source of contaminating RNAases. A simple procedure for purification of ribosomes active in initiation including Sephadex G-200 gel-filtration of unwashed ribosomes in a 1.5 M NH4Cl-containing buffer was developed.

[Recognition of messenger RNAs upon initiation of translation in prokaryotes]. / Uznavanie matrichnykh RNK pri initsiatsii transliatsii u prokariot.

Structural aspects of ribosomal recognition of initiation sites on messenger RNA in procaryotes are considered. Location of initiation sites on mRNA, their length, typical nucleotide sequences that build up the initiation signal, and also the influence of the macrostructure of mRNA on protein synthesis are discussed. Determined nucleotide sequences of mRNA and DNA flanking the origin of different phage and bacterial genes are given.

[Bacteriophage MS2 mutants with disrupted phage replicase repressor activity]. / Mutanty bakteriofaga MS2 a narusheniem repressornoi aktivnosti fagovoi replicazy.

A number of ts-mutants with altered characteristics of RNA synthesis in non-suppressor cells at elevated temperatures have been obtained after HNO2-treatment of phage MS2am623 (amber mutation in site 50). The mutant ts130 is studied in detail: its peak of RNA synthesis is displaced 10 min. later and the total amount of RNA is reduced in 5-10 times. No RNA synthesis is observed in ts130-infected cell at 46 degrees C. Changes in the character of RNA synthesis are accompanied by the over-production of phage subunit of replicase: at 42 degrees C the replicase/RNA ratio in ts130 is 20 times higher than that of the original phage MS2am623. It is assumed that there is a decrease in the activity of replicase--RNA repressor complex in ts130 resulting in the loss of replicase control over its own synthesis while the control of coat protein synthesis remains unaffected.

[Regulatory region of the MS2 phage RNA replicase cistron. Accesibility of MS2 RNA specific fragment to T1 RNAse digestion and chemical modification with kethoxal]. / Reguliatornyi uchastok tsistrona replikazy RNK faga MS2. Dostupnost' Spetsificheskogo fragmenta RNK faga MS2 deistviiu RNKazy T1 i khimicheskoi.

Partial digestion with T1 RNAase and chemical modification with kethoxal were used to study stability of two hairpin in the proposed secondary structure of the functionally active MS2 RNA fragment MS2 R(--53 leads to 6), containing the regulatory region of the phage replicase cistron. Analysis of the products obtained after the above treatments showed that T1 RNAase and kethoxal attacked predominantly the guanosine residues in the hairpin b of the MS2 R(--53 leads to 6). This implies that in contrast to the structurally stable hairpin a of the polynucleotide, hairpin b appears to be more labile and may exist under the present experimental conditions in equilibrium with its open form. The data of the competition experiments demonstrated that the kethoxal modified MS2 R(-53 leads to 6) and shorter polynucleotide MS2 R(-53 leads to-11) obtained from MS2 R(-53 leads to 6) after T1 RNAase digestion failed to bind with MS2 coat protein. The relatively unstable hairpin b region in the polynucleotide MS2 R(-53 leads to 6) is suggested to play essential role in the complex formation.

[Regulation of RNA replication in RNA-containing bacteriphages. RNA synthesis in coat protein polar mutants]. / Reguliatsiia replikatsii RNK u RNK-soderzhashchikh bakteriofagov. Sintez RNK u poliarnykh mutantov po belku obolochki.

The synthesis of RNA by polar coat protein mutants f2sus3 and Qbetaam12 under suppressor (Escherichia coli S26R1E, Su+-1; H12R8a Su+-3) and non-suppressor (E. coli AB259; S26) conditions was examined. It was demonstrated that the synthesis of viral RNA under non-suppressor conditions in the presence of rifamycin produced the same gaussian pattern of rates as the synthesis of RNA by wild type phage or non-polar coat protein mutants. However, the total amount of RNA was decreased approximately 10-fold and the peak of RNA synthesis was displaced 7--10 min later. The number of infective centers was reduced also 10-fold indicating that a certain time-lapse was required to overcome the polarity of the parental RNA, this process being of single occurrence, exclusively on the parental RNA, but not on the progeny strains. As a consequence, it was concluded that the initiation of translation at the replicase cistron starts on the nascent RNA chains within the replicative complexes and not on the fully-synthesized templates with their complete secondary structure. The data obtained are not in contradiction with the hypothesis concerning the role of the repressor complex II (replicase-RNA) to slow down the synthesis of replicase and RNA in the coat protein mutants. The polarity can not be responsible probably for the blocking of the replicase cistron on the nascent chain following the block of coat protein cistron. Therefore, it appears appropriate to assume the existence of two binding sites for the replicase as repressor which is in keeping with the conclusions of Weissmann and co-workers.