[Synthesis of an artificial gene coding for thymosin alpha1 and its expression in Escherichia coli as a hybrid with human tumor necrosis factor]. / Sintez iskusstvennogo gena, kodiruiushchego timozin alpha1, i ego ékspressiia v Escherichia coli v sostave gibridov s faktorom nekroza opukholei cheloveka.

Chemical-enzymatic synthesis and cloning in Escherichia coli of an artificial gene encoding the immunoactive peptide thymosin alpha 1 have been carried out. Recombinant plasmids were constructed which contain fusion genes coding for hybrids of human tumour necrosis factor (TNF) and thymosin alpha 1 as N- or C-terminal part of the hybrid protein. In the C-terminal hybrid protein, TNF and thymosin alpha 1 are linked through a methionine residue, thus allowing for thymosin alpha 1 to be cleaved off the rest of the hybrid protein with cyanogen bromide. In case of the N-terminal hybrid protein, the linker between the thymosin alpha 1 and TNF sequences is the acid-labile dipeptide Asp-Pro. Expression of the hybrid genes in E. coli and properties of the recombinant proteins were studied. The N-terminal hybrid protein was secreted into periplasmic space, in contrast with the C-terminal hybrid protein, which formed insoluble aggregates inside bacterial cells. Procedures for the isolation of both hybrid proteins were developed. The N-terminal hybrid protein displayed full biological activity in the cytotoxic assay on the mouse fibroblast L-929 whereas the C-terminal hybrid protein proved to be much less active. Treatment of the hybrid protein TNF-thymosin alpha 1 with cyanogen bromide lead to a mixture of two polypeptides, from which thymosin alpha 1 was purified to homogeneity by simple chromatographic procedures.

[Cloning and expression of the gene for Escherichia coli thermostable enterotoxin]. / Klonirovanie i ékspressiia gena termostabil'nogo énterotoksina Escherichia coli.

Gene estA coding for thermostable enterotoxin of Escherichia coli has been cloned. It is shown that in the E. coli strain SA162 this gene is located on the chromosome. Using polymerase chain reaction a site-directed mutagenesis of the cloned gene has been carried out, resulted in a recombinant strain--producer of the thermostable enterotoxin.

[Construction of a gene coding for the precursor of human tumor necrosis factor]. / Konstruirovanie gena, kodiruiushchego predshestvennik faktora nekroza opukholei cheloveka.

The cDNA sequence for human tumor necrosis factor (hTNF) was reconstructed in vitro from genomic sequence. Using the oligonucleotide directed mutagenesis, a site for restriction endonuclease ClaI was introduced into the end of the first exon. The nucleotide sequence representing the second and third exons flanked with restriction sites ClaI and XhoI was obtained by means of chemical enzymatic synthesis. Assembly of the total gene coding for precursor of hTNF was accomplished in pTNF33 plasmid containing semisynthetic gene for mature hTNF with appropriate restriction sites.

[Synthesis, cloning, and expression of artificial genes, coding antigenic determinants of the foot-and-mouth virus substrain A22]. / Sintez, klonirovanie i ékspressiia iskusstvennykh genov, kodiruiushchikh antigennye determinanty virusa iashchura podtipa A22.

Chemical-enzymatic synthesis and cloning in Escherichia coli of double-stranded DNAs, coding for simple and complex antigenic determinants of foot-and-mouth disease virus (FMDV) strain A22, have been carried out. The simple antigenic determinants are a part of the viral coat protein VP1 (amino acid sequence 131-152 or 131-160) whereas the complex antigenic determinants comprise additionally the amino acid sequence 200-213 of VP1 linked to N-terminus of simple antigenic determinants through a tetrapeptide spacer Pro-Pro-Ser-Pro. Recombinant DNAs containing genes for antigenic determinants of FMDV fused with C-terminus of gene for human tumor necrosis factor (hrTNF) have been constructed. Expression of the hybrid genes and properties of the proteins coded were studied. All recombinant proteins were shown to interact specifically with polyclonal antibodies both against hrTNF and FMDV strain A22. The recombinant proteins produced by bacteria are perspective for study as a vaccine against FMDV.

[Hybrid human lymphokine genes. I. Design of recombinant plasmids coding hybrids of immune interferon and human tumor necrosis factor]. / Geny gibridnykh limfokinov cheloveka. I. Konstruirovanie rekombinantnykh plazmid, kodiruiushchikh gibridy immunnogo interferona i faktorov nekroza opukholei cheloveka.

Recombinant plasmids coding for hybrid proteins between human interferon gamma and human tumour necrosis factor alpha or beta have been constructed using site-directed mutagenesis. The genes were fused via a synthetic oligonucleotide linker coding for tetrapeptide Pro-Val-Gly-Pro. The fused genes were expressed in Escherichia coli under control of early promoters of bacteriophage T7. E. coli cells harbouring the plasmids with the hybrid genes gave rather high level of the fused proteins biosynthesis. The hybrid recombinant proteins proved to be unstable in E. coli cells.

[Enzymatic ligation of DNA fragments containing phosphoamide modification of the internucleotide bonds]. / Enzimaticheskoe ligirovanie fragmentov DNK, soderzhashchikh fosfamidnuiu modifikatsiiu mezhnukleotidnykh sviazei.

DNA fragments with the point amidophosphate (cyclohexylamido- or morpholido-) modification in the sugar-phosphate backbone were synthesized and separated into individual diastereoisomer. The isomers were separated by the reversed-phase HPLC (RPC), and chirality at phosphorus was assigned by a stereochemical correlation scheme using phosphorothioate standards. The RPC-retention time values for Rp-isomers were found to be lower than for Sp-analogues. Amidophosphate DNA fragments were used as P- and OH-components in the T4 DNA-ligation. The enzyme does not ligate amidated fragments with modified internucleotide linkage near 5'- or 3'-end, independently of the amidophosphate chirality. When an unmodified phosphodiester linkage separates the amidophosphate group from 3'-end in O-component, the ligation occurs only with Sp-isomer, whereas Rp-analogue does not give the ligation product. In the P-component of the ligation, configuration of the modified linkage separated from 5'-phosphate by an unmodified linkage does not affect the result of the enzymatic reaction: both Sp-and Rp-stereomers do take part in the ligation. As a result of the ligation of the modified fragments on unmodified templates a set of 31-mers was obtained. They contain FokI and EcoRI recognition sites with the cleavage points of both endonucleases coinciding and being amidated. Upon treatment of duplex DNA consisted of unmodified and amidated strands with these endonucleases Sp-configuration did not hinder the cleavage of the unmodified strand, whereas Rp-configuration inhibited the EcoRI and did not affect the FokI cleavage.

Construction and properties of fusion proteins between human interferon-gamma and human tumour necrosis factors alpha and beta.

A number of recombinant plasmids coding for fusion proteins between human interferon-gamma (IFN-gamma) and human tumour necrosis factor alpha (TNF alpha) or beta (TNF beta) were constructed by using site-directed mutagenesis and ligation of the respective genes. In these proteins the whole IFN-gamma sequence of the molecule is linked at the N terminus via a short polypeptide linker to the TNF alpha sequence lacking two N-terminal amino acid residues or to the whole TNF beta sequence. A series of mutants with deletions in the interferon part of the fusion proteins were also produced. All the fusion genes obtained were efficiently expressed in Escherichia coli under the control of early promoters of bacteriophage T7. The recombinant fusion proteins were found to be unstable inside bacterial cells. Bacterial cell lysates expressing these fusion genes or their deletion mutants showed both biological activities in vitro: the antiviral activity of IFN-gamma and the cytotoxic activity of TNF.

[Model polycistron operon for studying conjugated translation in E. coli cells. The role of a stream of ribosomes]. / Model'nyi politsistronnyi operon dlia izucheniia sopriazhennoi transliatsii v kletkakh E. coli. Rol' potoka ribosom.

The role of "stream" of ribosomes upon translation of polycistronic mRNAs has been studied using an artificial polycistron. It has been found that the levels of activation of cistron Ci + 1 out of two adjacent cistrons (Ci and Ci + 1) depends, in addition to earlier described effects of mutual arrangement of initiation and termination signals, also on efficiency of translation of the foregoing cistron Ci. The results obtained lead to the conclusion that in polycistronic systems the levels of translation of cistron Ci + 1 can be regulated by "stream" of ribosomes resulted from translation of the proximal cistron Ci.

Formation of intramolecular triplex in homopurine-homopyrimidine mirror repeats with point substitutions.

We have used two-dimensional gel electrophoresis to study the structural transition to the triplex H form of sequences 5'-AAGGGAGAAXGGGGTATAGGGGYAAGAGGGAA-3' where X and Y are any DNA bases. The transition was observed at acid pH under superhelical stress. For X = Y = A or X = Y = G the sequences corresponded to homopurine-homopyrimidine mirror repeats (H-palindrome) which are known to adopt the H form under acid pH and superhelical stress. We have shown that the H form is actually formed for all X and Y, though in cases other than X = Y = A and X = Y = G the transition requires larger negative superhelical stress. Different substitutions require different superhelicity levels for the transition to occur. Theoretical analysis of the data obtained made it possible to estimate the energy cost of triplex formation due to all possible mismatched base triads.

Critical role of the C-terminus in the biological activities of human tumour necrosis factor-alpha.

Alterations of the C-terminal amino acid sequence of human tumour necrosis factor-alpha (hTNF-alpha) caused significant changes in its biological activity. Thus shortening of the C-terminus by removal of two or three amino acids led to a very marked loss of cytotoxic activity. Other, more subtle changes introduced by site-directed mutagenesis resulted in a less drastic reduction in cytotoxicity. The mitogenic activity towards human fibroblasts of the hTNF-alpha was reduced in parallel with the loss of cytotoxicity. These results suggest that the C-terminal amino acids of hTNF-alpha are critical for its biological actions and that they may be part of the receptor-binding site.

[Solid phase ligation of synthetic DNA fragments]. / Tverdofaznoe ligirovanie sinteticheskikh fragmentov DNK.

A synthetic oligodeoxyribonucleotide (oligo) covalently bound by an internucleotide linkage to the succinylated Sephacryl S-500 support through 1.9-diaminononane spacer was used as starting compound to assemble the E. coli rec A promoter DNA fragment from synthetic oligos by means of T4 DNA ligase. The solid-phase assembly of the designed DNA was performed by two ways: stepwise ligation of two pairs of oligos (2 dyads) or simultaneous ligation of four oligos (tetrad). Both ways gave equal results with some preference in the tetrad case. The reliability of E. coli promoter DNA fragment assembly was demonstrated by cloning it in a plasmid vector and sequencing the cloned DNA by the solid-phase Maxam--Gilbert technique.

[Chemical-enzymatic synthesis and cloning of DNA, coding the signal for secretion of proteins in gram-negative bacteria]. / Khimiko-fermantativnyi sintez i klonirovanie DNK, kodiruiushchei signal dlia sekretsii belkov v gramootritsatel'nykh bakteriaiakh.

Chemical-enzymatic synthesis of an artificial gene encoding leader peptide and 22 N-terminal amino acids of mature carboxypeptidase G2 from Pseudomonas sp. followed by enterokinase signal sequence (Asp4Lys) has been accomplished. The resulted DNA was fused with semi-synthetic gene coding for polypeptide 4-157 of mature human tumour necrosis factor (TNF) and then placed under control of early promoters of T7 bacteriophage. The expression products of the construct obtained was analysed using anti-TNF anti-serum. In E.coli leader peptide was cleaved off during translocation through inner membrane and the resultant product was found in membrane fraction.

An unusual DNA structure detected in a telomeric sequence under superhelical stress and at low pH.

Telomeric sequences of DNA, which are found at the ends of linear chromosomes, have been attracting attention as potential sites for the formation of unusual DNA structures. They consist of (GnTm) or (GnATm) motifs (n greater than or equal to m) and, in the single-stranded state, form hairpins stabilized by non-canonical G.G pairs. In the duplex state and under superhelical stress they exhibit hypersensitivity to SI nuclease which by analogy with homopurine-homopyrimidine sequences may reflect the formation of an unusual structure. To determine whether this is the case we have inserted into a plasmid the Tetrahymena telomeric motif (G4T2).(A2C4) and probed it by two-dimensional gel electrophoresis, chemical modification and oligonucleotide binding. Our data demonstrate that, under superhelical stress and at low pH, the insert does indeed adopt a novel DNA conformation. We have concluded that in this structure the C-rich strand forms a hairpin stabilized by non-Watson-Crick base pairs C.C+ and A.A+, whereas the G-rich strand remains unstructured. We term this new DNA structure the (C,A)-hairpin.

[Synthesis of 5'-phosphorylated oligodeoxyribonucleotides by the H-phosphonate method]. / Sintez 5'-fosforilirovannykh oligodezoksiribonukleotidov H-fosfonatnym metodom.

N,O-protected 2'-deoxyribonucleotide-3'-H-phosphonates and 2-(p-nitrophenylethyl)-H-phosphonate were prepared and used in solid-phase synthesis of 5'-phosphorylated DNA. H-Phosphonate condensation is performed with 1:2 ratio of P-component to activator (pivaloyl chloride). Protection groups were removed either by sequential treatment with conc. NH3 and DBU or by combination of these reagents.

[Ethylation of internucleotide phosphite groups by the oligodeoxyribonucleotide method of oxidative phosphorylation]. / Etilirovanie mezhnukleotidnoi fosfitnoi gruppy oligodezoksiribonukleotidov metodom okislitel'nogo fosforilirovaniia.

A method, based on solid-phase technology and oxidative phosphorylation, has been developed for preparation of modified oligodeoxyribonucleotides containing internucleotide ethylphosphate group.

[Location of the initiating codon AUG in relation to the 5'-end of mRNA mediates the effectiveness of translation in E. coli cells]. / Polozhenie initsiiruiushchego kodona AUG otnositel'no 5'-kontsa mRNK vliiaet na éffektivnost' translitsii v kletkakh E. coli.

A semisynthetic gene for beta-galactosidase (lacZ) and a synthetic DNA fragment containing the "ideal" promoter sequence were used for construction of an artificial operon including translation initiation codon ATG and no SD sequence. Cloning this artificial operon into pBR322 vector resulted in a number of pV plasmids; ATG positions were varied by insertions of synthetic oligonucleotides between lacZ coding sequence and starting point of transcription. It was found that efficiency of beta-galactosidase synthesis in E. coli cells harbouring pV plasmids strongly depended on the relative position of AUG and mRNA 5'-end. High level of the synthesis was provided by translation of mRNA with AUG codon in 5'-terminal position. Amounts of synthesized beta-galactosidase diminished with increase of the distance (2, 4, and 5 nucleotides) between 5'-end of lacZ mRNA and AUG codon.

DNA H form requires a homopurine-homopyrimidine mirror repeat.

Regular homopurine-homopyrimidine tracts, (dG-dA)n(dT-dC)n and (dG)n(dC)n, undergo a superhelix-induced, strongly pH-dependent, structural transition into a novel DNA conformation, the H form. We have suggested that the H form can arise in any homopurine-homopyrimidine mirror repeat (H palindrome). We have now tested this prediction using a tailored series of plasmids carrying the inserts AAGGGAGAAXGGGGTATAGGGGYAAGAGGGAA, where X and Y may be either A or G, and subject them to two-dimensional gel electrophoresis. In support of our hypothesis, the inserts exhibited facile transitions into the H form for X = Y = G, or X = Y = A, whereas the transition was much more difficult or impossible for the two non-palindromes (X = A, Y = G or X = G, Y = A). We present evidence that the H form is the structural basis for S1-nuclease hypersensitivity.

[The vector containing a signal for specific degradation of chimeric proteins. Synthesis of [Leu5]enkephalin using enteropeptidase]. / Vektor, soderzhashchii signal dlia spetsificheskogo rasshchepleniia khimernykh belkov. Poluchenie [Leu5]énkefalina s pomoshch'iu énteropeptidazy.

A plasmid vector (pEK1) coding, in framework of beta-galactosidase gene, for the amino acid sequence (Asp)4Lys which is recognized by bovine enteropeptidase has been constructed. Using this vector and chemically synthesized DNA coding for the [Leu5]-enkephalin, a plasmid (pEK-ENK) has been obtained in which the beta-galactosidase gene is fused, through the enteropeptidase linker, with the gene for [Leu5]enkephalin. The chimeric protein produced by expression of this plasmid has been isolated and then cleaved by the enteropeptidase to give [Leu5]enkephalin with the yield 74%.