[The immunogenic properties of a recombinant vaccinia virus with an incorporated DNA copy of the 26S RNA of the Venezuelan equine encephalomyelitis virus]. / Immunogennye svoistva rekombinantnogo virusa ospovaktsiny so vstroennoi DNK-kopiei 26S RNK virusa venesuél'skogo éntsefalomielita loshadei.

A recombinant strain of vaccinia virus (VR26) containing a DNA-copy of the subgenomic 26S RNA of Venezuelan equine encephalomyelitis virus (VEE) inserted into the coding region of thymidine kinase (TK) gene was produced. This subgenomic RNA contained the genes for all structural proteins of the VEE virus, the strain Trinidad donkey (TRD). VR26 effectively expressed VEE virus glycoproteins on the membranes of the infected cells. Blood sera of VR26-immunized animals were found to contain VEE virus-specific antibodies. VR26-immunized mice and rabbits showed a high level of resistance to subcutaneous inoculation with the pathogenic TRD strain of VEE virus. VR26 also provided a high level of protection in animals against aerogenic infection. The absence of virus-neutralizing antibodies in most VR26-immunized animals resistant to inoculation with high doses of VEE suggests the dominant role of the cell component in the immune response. The immune response induced by the recombinant VR26 strain was stable as demonstrated by the resistance of the animals to a challenge with VEE virus 7 months after immunization. The experimental results suggest that this recombinant strain may be considered as a candidate for vaccine preparation.

[Effective synthesis and cloning of the human interleukin-2 gene and its analog: expression of the interleukin-2 gene in E. coli cells]. / Effektivnyi sintez i klonirovanie gena interleikina-2 cheloveka i egoanaloga: ékspressiia v kletkakh E. coli gena interleikina-2.

Artificial DNA fragments encoding human interleukin-2 (133 a.a.) and its analogue (deletion of 14 C-terminal a.a.) were prepared by means of the DNA polymerase I mediated extension of synthetic polynucleotides having short overlapping sequences at their 3'-ends. The fragments were cloned in specially designed pFH-type plasmids and then excised by the FokI and other restriction endonucleases to yield the subfragments with the structurally predetermined 5'-unique cohesive ends. The complete synthetic gene was constructed by one or two-step ligation. The expressed IL-2 was tested by analysing the T-cell proliferation activity of E.coli crude lisates containing the pEXIL2 expression plasmid.

[Design of recombinant-stable plasmids of the pFH series]. / Konstruirovanie rekombinantno-ustoichivykh plazmid serii pFH.

By cloning synthetic oligonucleotides into pUC18 plasmid, pFH123--pFH127 plasmids have been constructed. Their polylinker area, along with sites of widely used restriction endonucleases, contains two pairs each of FokI and HgaI sites in the opposite orientation to provide subfragment with unique predetermined 5'-ends. Comparative stability of the new plasmids and their derivatives has been studied and compared with that of the earlier constructed pMB plasmids.

[Plasmids pMB123 and pMB124--vectors for obtaining subfragments of DNA with random "sticky" ends]. / Plazmidy pMB123 i pMB124--vektory dlia polucheniia subfragmentov DNK s prizvol'nymi "lipkimi" kontsami.

For preparing a DNA fragment with unique protruding ends, plasmid vectors pMB123 and pMB124 were constructed by inserting a synthetic polylinker into plasmid pUR222 at the EcoRI-PstI sites. The polylinker contains two FokI and HgaI sites at its ends in opposite orientation flanking a combination of SalGI, AccI, HindII, HindIII (the latter site is absent from pMB124) and BamHI sites. DNA fragment cloned at the SalGI and BamHI sites can be regenerated by either FokI or HgaI treatment, the SalGI and BamHI sites being deleted from the cloned sequence. Fragments coding for parts of human interleukin-2 were cloned in these vectors.

[Identification in vivo of promoter activity in the left site of the att region of bacteriophage lambda DNA]. / Identifikatsiia in vivo promotornoi aktivnosti v levom raione uchastka att DNK bakteriofaga lambda.

The promoter-probing vector (pSK plasmid) was explored for cloning of the fragments from lambda cI857 and lambda b2 DNAs containing different regions of the att site. We have constructed all-tet fusions where the fusions are: 1) HindIII/BamHI-491 base pairs (b. p.) fragment of lambda cI857 DNA containing POP' site (plasmid pSK-PP'); 2) AluI-242 b. p. fragment of lambda cI857 DNA containing the left arm of the POP' site (plasmid pSK-P); 3) AluI-242 b. p. fragment of lambda cI857 DNA with opposite orientation (plasmid pSK-P); 4) EcoRI/BamHI-750 b. p. fragment of lambda b2 DNA containing the right arm of the POP' site (plasmid pSK-P'). These fusions permit us to analyse the effect of various pieces of the attachment site on the expression tet gene as the result of reparation of this gene promoter. We find that expression of tet (tetracycline resistant phenotype) takes place in the pSK-PP' and pSK-P but not in the pSK-P' and pSK-P. These facts permit us to conclude that the left arm of the att site contains a rightward promoter functioning in vivo. We postulate that this promoter activity might correspond to the promoter patt, which was described in previous experiments in vitro.

[Effect of promoter duplication in the synthetic interferon gene on the level of its expression]. / Vliianie duplikatsii promotorov v iskusstvennom gene interferona na uroven' ego ékspressii.

The expression of the artificial gene for the human leukocyte interferon (IFN) synthesized by the chemo-enzymic method has been studied in Escherichia coli cells. The genetic constructions in which the IFN gene transcription is carried out from A2 and B promoters of T7 phage or from E. coli lacZ UV5 gene promoter have been obtained, based on pEMB101 plasmid. In addition, the level of interferon production, depending on the promoter used, has been studied. It has been shown that the tandemly located promoters increase efficiency of the IFN gene expression.

[Plasmid vector for cloning, determination of nucleotide sequence and directed assembly of DNA fragments]. / Plazmidnyi vektor dlia klonirovaniia, opredeleniia nukleotidnoi posledovatel'nosti i napravlennoi sborki fragmentov DNK.

A plasmid vector pNIMB has been constructed (starting) from the pUR222 plasmid as a result of substitution of the polylinker containing restriction sites: PstI, SalGI, AccI, HindII, BamHI EcoRI and by other synthetic linkers with additional sites for HindIII and HgaI. Plasmid pNIMB does not differ from the parent one phenotypically. Compared to pUR222 the vector contains an additional site for cloning HindIII fragments of DNA and allows to clone SalGI/BamHI- and PstI/SalGI-fragments. Cloning of DNA fragments in all seven unique sites of pNiMB gives the possibility for sequencing the fragments avoiding their isolation from the gel. Moreover, this vector may be useful for cloning and directed assembly of chemically synthesised DNA fragments when the endonuclease HgaI sites are used.

[Synthesis and cloning of a DNA fragment containing a probable site for eukaryotic mRNA binding to ribosome]. / Sintez i klonirovanie fragmenta DNK, soderzhashchego predpolagaemyi sait sviazyvaniia éukarioticheskoi mRNK s ribosomoU

A series of oligonucleotides, including two polynucleotides of 33 bases long, were synthesized by a solid-phase phosphotriester method. Potassium salt of 3-nitro-1,2,4-triazole in the presence of 18-crown-6 ether was used as nucleophilic catalyst. The partly complementary polynucleotides were elongated by DNA-polymerase I (Klenow fragment) to the full duplex, which was digested with SalGI and was inserted into a plasmid pUR222. Phe synthesized DNA fragment precedes the gene of human gamma-interferon in the chromosome and contains the site for mRNA binding to ribosome.

[Isolation and characterization of a phage T7 DNA fragment containing promoter B active in vivo and in vitro]. / Vydelenie i kharakterizatsiia fragmenta faga T7, soderzhashchego aktivnyi v usloviiakh in vivo i in vitro promotor B.

The fragment of 124 base pairs (Alu-124) has been isolated from the phage T7 DNA and cloned in the plasmid pSK. The sequence of the fragment was determined and position of this fragment on the physical map of phage DNA was found. It has been found that Alu-124 fragment contained an active in vivo and in vitro promoter for E. coli RNA-polymerase. In vitro transcription from this promoter was initiated with GTP. The start of the transcription was localised at about 104 or 105 nucleotide. The data obtained indicate that the Alu-124 fragment of the phage T7 promoter B, may be involved in the control of the transcription of the phage T7 early stage development.

[Synthesis of a 33-member polynucleotide containing the "core" att site of phage lambda DNA and its cloning]. / Sintez 33-chlennogo polinukleotida, soderzhashchego "core" att sait DNK bakteriofaga lambda, i ego klonirovanie.

Two polynucleotides containing 33 monomeric units were synthesized by a solid-phase phosphotriester method. These polynucleotides form a duplex with protruding 5'-ends, which allows to clone the duplex in EcoRI site of a cloning vehicle. Each polynucleotide was purified by electrophoresis in polyacrylamide gel, and the duplex obtained was cloned in EcoRI site of pUR 222 plasmid DNA. The structure of the cloned duplex containing the "core" att site of phage lambda was confirmed by sequencing.

[Integrase of lambda phage inhibits the activity of promotor Patt under in vivo and in vitro conditions]. / Integraza faga lamb'da ingibiruet aktivnost' promotora Patt v usloviiakh in vivo i in vitro.

In vitro and in vitro transcription of the region of lambda phage DNA containing promoter Patt has been studied. Active transcription was observed in the vicinity of the att site and at the promoter Patt in vivo during the first minutes after infection. This transcription considerably decreases at the 1th minute, and stops completely at the 15th minute after infection. Maximum synthesis of integrase also takes place at the 15th minute after infection. In vitro the partially purified int protein (integrase) completely inhibited transcription from the promoter Patt. Hence, integrase is most probably a repressor of Patt in vivo and in vitro.