[Design of the human tumor-associated VNTR(Muc1) antigen gene, fused with streptavidin, its expression in Escherichia coli. Study of properties of the hybrid protein]. / Konstruirovanie gena opukholeassotsiirovannogo antigena VNTR(Muc1) cheloveka, slitogo so streptavidinom, ego ékspressiia v Escherichia coli. Izuchenie svoistv gibridnogo belka.

A gene of human tumor-associated antigen VNTR(MUC1) bound to streptavidin, an expression plasmid, and a highly effective hybrid protein-producing strain were constructed. It was shown that the streptavidin leader peptide ensures an effective secretion of the hybrid protein into the periplasmic space of Escherichia coli cells. The hybrid protein was isolated in a homogeneous state and its immunogenic properties were studied.

[Study of the role of histidine residues in the function of uridine phosphorylase from Escherichia coli K-12 by protein engineering]. / Issledovanie roli ostatkov gistidina v funktsionirovanii uridinfosforilazy iz Escherichia coli K-12 metodami belkovoi inzhenerii.

Using site-directed mutagenesis, mutant genes of the E.coli UDPase that coded proteins with point substitutions of histidine residues (i.e., H8N, H47N, H101N, H122N, H152N, H179N, and H240N) were constructed. Study of the enzymatic activity of mutant UDPases showed that histidine-asparagine substitutions at the positions 47, 101, 152, 179, and 240 do not affect protein functioning. Whereas H122N and H8N substitutions inhibit the activity of UDPase by 60 and 100%, respectively. This evidences the important functional role of the His122 and His8 residues for the formation of the active site fo the enzyme.

Identical amino acid sequence of the aroA(G) gene products of Bacillus subtilis 168 and B. subtilis Marburg strain.

A DNA fragment containing the aroA(G) gene of Bacillus subtilis 168, encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase-chorismate mutase, was cloned and sequenced. The N-terminus of the protein encoded by aroA(G) showed homology with chorismate mutase encoded by aroH of B. subtilis and with the chorismate mutase parts of proteins encoded by the pheA and tyrA genes of Escherichia coli. The C-terminus of the aroA(G) product has sequence similarity with 3-deoxy-D-manno-octulosonate 8-phosphate synthase of E. coli. It was shown that the proteins encoded by the aroA(G) gene of B. subtilis 168 and the aroA gene of B. subtilis ATCC 6051 Marburg strain are identical, so the observed differences in DAHP synthase activity from these two strains must result from other changes.

[Chemical synthesis of a proteinase inhibitor (eglin C) gene without use of T4-DNA-ligase and its expression in E. coli]. / Khimicheskii sintez gena ingibitora proteinaz (églin C) bez ispol'zovaniia T4-DNK-ligazy i ego ékspressiia v E. coli.

A synthetic gene for a proteinase inhibitor (eglin C) that was obtained by direct amplification with oligonucleotides without using DNA ligase and polynucleotide kinase of T4 phage was cloned into expression vectors. A high yield of the polypeptide (110-130 mg/l) was attained in E. coli strains.

[Directed introduction of amino groups by internucleotide phosphate group during solid-phase synthesis of oligodeoxyribonucleotides. Preparation of biotinylated oligonucleotide probes]. / Napravlennoe vvedenie aminogrupp po mezhnukleotidnoi fosfatnoi gruppe pri tverdofaznom sinteze oligodezoksiribonukleotidov. Poluchenie biotinilirovannykh oligonukleotidnykh zondov.

An efficient procedure for solid-phase synthesis of amino-oligonucleotides has been developed, leading to direct introduction of an aliphatic primary amino group at the internucleotide phosphate. Amino-oligonucleotides were successfully used for preparation of biotinylated oligonucleotides.

[Site-directed mutagenesis with the use of addressed fragmentation of single-stranded DNA for obtaining hybrids of homologous proteins]. / Sait-napravlennyi mutagenez s ispol'zovaniem adresovannogo fragmentirovaniia odnonitevoi DNK dlia polucheniia gibridov gomologichnykh belkov.

Site-directed multiple mutagenesis to obtain hybrids of homologous proteins was carried out by means of the oligonucleotide-targeting digestion of ss DNA. The procedure is more convenient, rapid and simple than the step-by-step approach. To demonstrate different approaches to targeting digestion of ss DNA, NcoI, HinfI, FokI endonucleases were used for DNA cleavage. A hydride of the human and porcine IFN-alpha-2-genes has been constructed.

[Site-directed mutagenesis in the uracil-repair system. Preparation of mutated forms of human alpha 2 interferon]. / Sait-napravlennyi mutagenez v uratsil-reparatsionnoi sisteme. Poluchenie mutantnykh form interferon alpha2 cheloveka.

The mutant forms of human IFN-alpha 2 gene are obtained by oligonucleotide-directed mutagenesis with the use of uracil-repair system. To intensify the process the procedure of the uracil-containing DNA template preparation is modified. It was determined that when mutagenesis is performed in the uracil-repair system the yield of the process depends on the mutant DNA-strand in vitro synthesis efficiency. It is shown that the stability of the 5'-end primer-template complex and the level of the endogenic primers elongation are the basis factors, that determine induction mutations.

[Study of the role of the insA open reading frame in the IS1 insertion element structure during transposition and resolution of the IS1 mediated cointegrates]. / Izuchenie roli ramki schityvaniia insA v strukture insertsionnogo élementa IS1 v protsessakh transpozitsii i razresheniia oposredovannykh IS1 kointegratov.

In order to elucidate the function of the IS1 insA gene derivatives of plasmid pUC19::Tn9' with insertions of synthetic oligonucleotides were obtained. The latter are equal or multiple of 9 b.p. in length and are located in the Pst1 site within each of the two IS1 copies of the Tn9' transposon. The insertions of the nine base oligonucleotides code for the neutral amino acids and do not shift the reading frame. One of the mutant transposon obtained - Tn9'/X was studied on the ability to form simple insertions and plasmid cointegrates. For this purpose the pUC19 derivatives carrying the wild type and mutant transposon were mobilized by conjugative plasmid pRP3.1. It was found that the damage of the insA gene does not influence the ability of transposon to form simple insertions and plasmid cointegrates in both recA - and rec+ cells of E. coli. However, the frequency of the cointegrate formation in the subsequent transposition of the mutant transposon from pRP3.1::Tn9'/X to pBR322 was by 10-20 times lower in comparison to the wild type transposon. Instable (dissociating) Tn9'/X-mediated plasmid cointegrates formed by interaction pUC19::Tn9'/X and pRP3.1 were obtained. It was shown that in the E. coli recA-cells such cointegrates dissociate, as a rule, "correctly", i.e. they segregate mainly plasmids of types pUC19::Tn9'/X and pUC19::IS1/X. The data obtained correspond with the notion that the gene insA product is not essential for transposition, but is, possibly, involved in the formation of the IS1-generated deletions.

TGATG vector: a new expression system for cloned foreign genes in Escherichia coli cells.

A TGATG vector system was developed that allows for the construction of hybrid operons with partially overlapping genes, employing the effects of translational coupling to optimize expression of cloned cistrons in Escherichia coli. In this vector system (plasmid pPR-TGATG-1), the coding region of a foreign gene is attached to the ATG codon situated on the vector, to form the hybrid operon transcribed from the phage lambda PR promoter. The cloned gene is the distal cistron of this hybrid operon ('overlappon'). The efficiently translated cro'-cat'-'trpE hybrid cistron is proximal to the promoter. The coding region of this artificial fused cistron [the length of the corresponding open reading frame is about 120 amino acids (aa)] includes the following: the N-terminal portions of phage lambda Cro protein (20 aa), the CAT protein of E. coli (72 aa) and 3' C-terminal codons of the E. coli trpE gene product. At the 3'-end of the cro'-cat'-'trpE fused cistron there is a region for efficient translation reinitiation: a Shine-Dalgarno sequence of the E. coli trpD gene and the overlapping stop and start codons (TGATG). In this sequence, the last G is the first nucleotide of the unique SacI-recognition site (GAGCT decreases C) and so integration of the structural part of the foreign gene into the vector plasmid may be performed using blunt-end DNA linking after the treatment of pPR-TGATG-1 with SacI and E. coli DNA polymerase I or its Klenow fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

[Structural-functional analysis of the par-region of ColE1 plasmid]. / Strukturno-funktsional'nyi analiz par-uchastka plazmidy ColE1.

The DNA fragment identical to the right shoulder of the inverted repeat from the par-region of ColE1 plasmid has been synthesized chemically. It is shown to participate in the plasmid multimers resolution and to define the stable inheritance of the plasmid pKS1 containing the fragment in Escherichia coli C600 cells as well as in the multirecombinogenic strain Escherichia coli JC8679. The efficiency of the fragments functioning in Escherichia coli JC8679 is not enough for resolution of all forms of oligomeric pKS1 DNA. The site for recombinase action is found to be located in the synthesized oligonucleotide. However, some extra sequences of DNA located within the region of inverted repeat are necessary for maximally efficient functioning of the recombinase, the enzyme participating in plasmid multimers resolution.

[Chemico-enzymatic synthesis of an oligonucleotide template for an analog of ACTH (4-10) fragment]. / Khimiko-fermentativnyi sintez oligonucleotidnoi matritsy analoga fragmenta 4-10 adrenokortikotropnogo gormona.

Six oligodeoxyribonucleotides ranging from 9-mer to 13-mer were synthesized in solution by the phosphotriester approach and enzymatically joined by T4 DNA ligase. The obtained double-stranded DNA (32 b.p.) with protruding 5'-ends corresponding to the recognition sites for restrictases EcoRI and BamHI represents an oligonucleotide template coding for the modified amino acid sequence 4-10 of the adrenocorticotropic hormone, [Pro8,Gly9,Pro10]ACTH-(4-10).

Reverse transcription of phage RNA and its fragment directed by synthetic heteropolymeric primers.

DNA synthesis catalysed by RNA-directed DNA-polymerase (reverse transcriptase) was found to proceed on the RNA template of an MS2 phage in the presence of heteropolymeric synthetic octa- and nonadeoxyribonucleotide primers complementary to the intercistronic region (coat protein binding site) and the region of the coat protein cistron, respectively. The product of synthesis consists of discrete DNA fractions of different length, including transcripts longer than 1,000 nucleotides. The coat protein inhibits DNA synthesis if it is initiated at its binding site, but has no effect on DNA synthesis initiated at the coat protein cistron. It has been suggested that, in this system, the initiation of DNA synthesis by synthetic primers is topographically specific. The MS2 coat protein binding site (an RNA fragment of 59 nucleotides) serves as a template for polydeoxyribonucleotide synthesis in the presence of octanucleotide primer and reverse transcriptase. The product of synthesis is homogenous and its length corresponds to the length of the template. The effective and complete copying of the fragment having a distinct secondary structure proves that the secondary structure does not interfere, in principle, with RNA being a template in the system of reverse transcription.