[Elimination of introns from the interleukin 1 beta genome by DNA amplification and expression of interleukin 1 beta in Escherichia coli]. / Udalenie intronov metodom amplifikatsii DNK iz genomnogo gena interleikina 1 beta i ékspressiia gena interleikina 1 beta v Escherichia coli]

The use of the polymerase chain reaction was proposed for intron excision from genomic genes with known nucleotide sequences. Three exons (5, 6 and 7) of genomic interleukin 1 beta gene were amplified by means of thermostable DNA polymerase TthI from Thermus thermophilus on the base of cloned in M13 phage human genomic interleukin 1 beta gene. Synthetic oligonucleotides complementary to sequences flanking exons were used as primers. The fragments obtained by exon DNA amplification were joined in the correct order due to reciprocal complementation of end sequences, that was foreseen during synthesis of oligonucleotide primers followed by amplification of the enlarged fragments. As a result the structural interleukin-1 beta gene consisting of three exons was assembled. DNA sequences carrying the ATG initiation codon and XbaI recognition site at the 5'-end, and PstI recognition site at the 3'-end (essential for insertion into the expression vector) were formed by the additional end sequences of primers. The nucleotide sequence analysis of the obtained structural gene revealed its complete identity with natural interleukin 1 beta human gene. We created the expression vector pPR114 with phage lambda promoter PR thermo-inducible in case of the cIts857 repressor presence in cells. It was used for expression of the present gene. The interleukin 1 beta synthesized in E. coli had biological activity.

[Bank of sequences specific for human genome. I. The structure of one of the repeats in the Alu family]. / Poluchenie banka posledovatel'nostei, spetsifichnykh dlia genoma cheloveka. I. Struktura odnogo iz povtorov Alu-semeistva.

A bank enriched in sequences specific for the human genome was obtained. In course of the analysis, a clone containing an Alu family repeat was identified and its primary structure determined.

[Primary structure of the alpha subunit of Na+,K+-ATPase. II. Isolation, reverse transcription and cloning of messenger RNA]. / Pervichnaia struktura alpha-sub''edinitsy Na+,K+-ATP-azy. II. Vydelenie, obratnaia transkriptsiia i klonirovanie matrichnoi RNK.

Messenger RNA, coding for the alpha-subunit of the Na+, K+-ATPase, was isolated from outer medulla of pig kidney. Within 25S-26S region the mRNA yields a band of specific hybridization with three oligonucleotide probes synthesized according to data on structures of three peptides isolated from the tryptic hydrolysate of the protein. Translation of the enriched poly(A+)-fraction of RNA in Xenopus laevis oocytes followed by the immunochemical identification of the products confirmed the presence of RNA coding for the desired protein. This RNA preparation was used for synthesis and cloning of double stranded cDNA.

[Problems associated with the use of highly degenerated oligonucleotide probes. Identification of mRNA and cDNA clones corresponding to the gene for the alpha subunit of Na+,K+-ATPase]. / Problemy, sviazannye s ispol'zovaniem oligonukleotidnykh zondov s bol'shoi stepen'iu vyrozhdennosti. Identifikatsiia mRNK i klonov kDNK, sootvetstvuiushchikh genu alpha-sub''edinitsy Na+,K+-ATP-azy.

Oligonucleotides deduced from the amino acid sequence of a hexapeptide Lys-Asp-Phe-Ala-Glu-Asn were synthesized and used as probes to screen a pig kidney cDNA library for a specific DNA sequence coding for the alpha-subunit of Na+, K+-ATPase. It was shown that the mixed oligoprobe, consisting of 64 heptadecamers, could be only suitable for mRNA blot analysis. To identify the clones with specific cDNA inserts, mixed oligoprobes were fractionated by HPLC technique. For the same purpose a new set of oligonucleotides, synthesized as four groups of 16 different heptadecamers each, was used.

[An experimental study of human recombinant gamma interferon with a proteolytically reduced C-end area of the protein]. / Eksperimental'noe izuchenie rekombinantnogo chelovecheskogo gamma-interferona s proteoliticheski redutsirovannym C-kontsevym uchastkom belka.

The work presents the results of experimental study of gamma interferon obtained by gene engineering techniques on the basis of Escherichia coli producer strains. The study has revealed that gamma interferon, whose molecular weight is 15 KD, due to intracellular proteolytic degradation shows the absence of some amino acids at the C-end of protein and is electrophoretically homogeneous, while its antiviral, antiproliferative and immunomodulating effects are less pronounced than those of gamma interferon with a molecular weight of 18 KD.

[Simple method for DNA fraction enrichment with unique genes]. / Prostoi sposob obog a shcheniia praktsii DNK po soderzhaniiu unikal'nykh genov.

The conditions for RNA--DNA molecular hybridization in 15% phenolic suspension, allowing to extend the temperature range of the R-loop formation and render the method insensitive to the nucleotide composition of RNA are proposed. The method described results in a 100-fold enrichment of the globin-gene content in a few days. The method was used for enrichment of rat DNA preparations with globin genes.

[Enzymatic synthesis and properties of DNA, complementary to foot and mouth disease virus RNA]. / Fermentativnyi sintez i svoistva DNK, komplementarnoi RNK virusa iashchura.

The RNAs of the aphthosa virus, serotypes A and O were isolated and characterized. The complementary DNAs used in experiments on molecular hybridization with matrix RNAs were synthesized on virion RNAs by means of reverse transcriptase.