Construction of a generalized consensus matrix for recognition of vertebrate pre-mRNA 3'-terminal processing sites.

Using a set of sequences of 63 cleavage/polyadenylation sites of vertebrate pre-mRNA, a generalized consensus matrix was constructed. The elements of the matrix were the absolute frequencies of oligonucleotides of length l at the ith position of sites. The cleavage point of each site was assigned the same position number. To recognize a polyadenylation site in a nucleotide sequence, a multiplicative measure was obtained using the elements of the generalized consensus matrix as weight factors. For any omega-long fragment of a nucleotide sequence, the estimated value of the functional mu was compared with the threshold value mu*. Based on the results obtained, we determined whether or not the given fragment is a processing site.

[Design of the generalized consensus matrix for recognizing sites of 3'-terminal processing of vertebrate pre-mRNA]. / Postroenie matritsy obobshchennogo konsensusa dlia raspoznavaniia saitov 3'-kontsevogo protsessinga pre-mRNK pozvonochnykh.

Using a set of sequences of 63 cleavage/polyadenylation sites of vertebrate pre-mRNA, a generalized consensus matrix was constructed. The elements of the matrix were the absolute frequencies of oligonucleotides of length l at the i-th position of the sites (the cleavage point of each site was assigned the same position number). To recognize a polyadenylation site in a nucleotide sequence, a multiplicative measure was obtained using the elements of the generalized consensus matrix as weighting factors. For any omega-long fragment of a nucleotide sequence, the estimated value of the functional mu was compared with the threshold value mu. Based on the results obtained, we determined whether or not the given fragment is a processing site.

Artificial protein vaccines with predetermined tertiary structure: application to anti-HIV-1 vaccine design.

A successful approach to the development of a safe and effective synthetic vaccine requires that different B and T cell epitopes of the infectious agent be included in the vaccine construction. In this paper we suggest a new approach to vaccine design in the form of an artificial protein with a predetermined tertiary structure (PTS vaccines). Based on B and T cell epitope properties, we substantiate the possible use for vaccine construction of one well-known protein spatial motif--the four-alpha-helix bundle. Antigenic determinants of cellular immunity (amphipathic alpha-helices) and humoral immunity (flexible hydrophilic loop regions) are used as blocks for vaccine design. General principles of PTS vaccine construction have been applied to anti-HIV-1 vaccine design.

[Design of four-helix protein--a possible vaccine against human immunodeficiency virus (HIV-1)]. / Konstruirovanie chetyrekhspiral'nogo belka--vozmozhnoi vaktsiny protiv virusa immunodefitsita cheloveka (HIV-1).

Successful approach to the development of safe and effective synthetic vaccines requires that different B- and T-cell epitopes of the infectious agent be included into the vaccine construction. It is suggested that vaccines should be constructed as proteins with both optimal epitope composition and predetermined tertiary structure. Based on analysis of B-cell and T-cell epitope properties, a possibility to use one well-known protein spatial motif--four-alpha-helix bundle--for vaccine construction is substantiated. Antigenic determinants of cellular immunity (amphipathic alpha-helices) and humoral immunity (flexible hydrophilic loop regions) can be used as blocks for vaccine design. Nonloop B-epitopes and nonhelical T-epitopes may be introduced in the protein N- and C-terminal regions. General principles of PTS-vaccine construction have been applied to anti-HIV-1 vaccine design. Experimentally studied T- and neutralizing B-cell epitopes from HIV-1 proteins were analyzed. The sequence of one possible four-alpha-helix protein vaccine has been constructed. Predicted secondary structure and T- and B-cell epitopes of this protein coincided with the planned ones. The amino acid composition of the protein was found to be consistent with the composition of globular water-soluble proteins. The gene of the protein with codon composition optimal for expression in E. coli has been synthesized. The advantages and limitations of this approach to vaccine design are discussed.

[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.

[Effectiveness of distal gene translation in polycistrons depends upon the arrangement of regulatory signals on a template]. / Effektivnost' transliatsii distal'nogo gena v politsistronakh zavisit ot vzaimnogo raspolozheniia reguliatornykh signalov na matritse.

The role of the translational terminator and initiator signals arrangement for two adjacent genes in polycistronic mRNA has been studied. Semisynthetic beta-galactosidase gene (lacZ) of E. coli and fragment of phage M13 DNA (with promoter PVIII, gene IX, and part of gene VIII) were used for constructing of the IX-VIII-lacZ artificial polycistronic operon. Cloning of the constructs into pBR322 vector resulted in a number of pLZ381N plasmids differing by the mutual arrangement of gene VIII translation terminator codon and SD site and initiator codon (SD-ATG-region) of lacZ gene. The mutual arrangement of gene VIII terminator codon and SDlacZ-ATG region has been altered by means of deletions and insertions that have not affected lacZ translation initiation signals. The beta-galactosidase (beta-Gal) synthesis in E. coli harbouring different types of pLZ381N plasmids has been found to depend on type of cistron coupling (gene VIII and lacZ). The overlapping of terminator and initiator codons (ATGA) for genes VIII and lacZ (type I of polycistrons) provide approximately equal translational level for both cistrons. On the other side, levels of beta-Gal synthesis in case of polycistrons type II (gene VIII stop-codon position at the beginning of SDlacZ or 10 nucleotides upstream) were 20-30 times as high as for type I. Differences in beta-Gal levels have also been found for variants of VIII-lacZ coupling in types IV and III polycistrons (the SDlacZ-ATG region in 27-50 nucleotides downstream from the proximal cistron VIII stop-codon, which, in turn, is 41 nucleotides upstream this terminator). These data cannot be explained on the basis of possible secondary structure including the SDlacZ-ATG region and other parts of polycistronic mRNA. In all these cases similarly stable stem-loop structures have been found. Therefore, the arrangement of the translation termination and initiation signals for two adjacent genes in essential for distal gene translation efficiency. One can imagine that ribosome or its 30S subpartical, stalling on the proximal gene terminator codon, affects the distal gene translation initiation.

[Formation and properties of artificial polycistrons containing truncated genes for E. coli tryptophan operon and phage M13 envelope protein]. / Konstruirovanie i svoistva iskusstvennykh politsistronov na osnove ukorochennykh genov triptofanovogo operona E. coli i belka obolochki faga M13.

Using gene fragments encoding the leader peptide of E. coli tryptophane operon (as duplicated fragment HhaI-140) or M13 phage coat protein (as TaqI-381 or HaeIII-1623 fragments) and basing on pDS1 family of plasmids, expression vectors have been constructed which contained transcription promoters Ptrp, PVIII, and Pv + PVIII, respectively. An artificial gene for human leukocyte interferon alpha 2 (ifn-alpha 2) has been cloned into these plasmids, so that its transcription was a part of polycistronic mRNA and preceding translation was terminated upstream to the ribosome binding site and starting codon of the interferon gene. E. coli cells harbouring these recombinant plasmids provided high level of the interferon biosynthesis. The effect of the mRNA length on the amount of protein synthesised under control of the M13 coat protein transcription-translation signals has been found.

[Duplication of a synthetic gene for human leukocyte interferon and its expression in polycistron mRNA with coupled translation system]. / Duplikatsiia sinteticheskogo gena leikotsitarnogo interferona cheloveka i ego ékspressiia v sostave politsistronnykh mRNK s sopriazhennoi sistemoi transliatsii.

Using a chemically synthesised adapter, the coding part of an artificial gene for human leukocyte alpha 2 interferon (ifn-alpha 2) has been duplicated. The adapter contained a termination signal of the first gene (TAA) within the Shine-Dalgarno sequence of the second gene (TAAGGA), distance between the terminating codon and starting codon of the second gene being 11 nucleotides. In another case this distance was 69 nucleotides, with the same SD sequence. The expression of the tandems as a part of polycistrons has been studied under control of promoters Plac, (Ptrp)2 of E. coli, and PVIII of M13 phage. It was found that tandems of ifn-alpha 2 genes in polycistronic structures trp L-ifn-ifn and IX-VIII-ifn-ifn under control of promoters (Ptrp)2 and PVIII, respectively, provided high level of the interferon biosynthesis, thus differing from the tandem under Plac promoter control, which had only ifn-ifn translation coupling.