Biomedical subjects
Publications and source records attributed to V V Kravchenko.
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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.
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A plasmid expression vector (pCEQ3), using temperature-regulated transcription from the p'R promoter of bacteriophage lambda, has been constructed. The vector is derived from pBR327 in which the EcoRI-ClaI fragment of plasmid DNA is replaced with a 2.2-kb DNA module cI857-pR-Q-p'R-qut-t'R, consisting of two regions of the lambda genome. The first region contains the repressor gene cI857 and promoter pR; the second one contains gene Q and the late promoter p'R. When the repressor protein, product of the cI857 gene, becomes temperature-inactivated, it allows the promoter pR to initiate the transcription of the Q gene. The product of the Q gene, in turn, acts as a positive regulator of transcription from promoter p'R. The promoter activity of pR is fully repressed at a low temperature (30 degrees C) and transcription from p'R is terminated in the absence of Q gene product, but the shift of temperature up to 37 degrees C is sufficient to make the transcription from the p'R promoter highly active. Foreign genes can be inserted into the single EcoRI site downstream from the p'R promoter. The resultant constructions express extremely high levels of the cloned gene product in Escherichia coli.
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The variants of expression in Escherichia coli of artificial DNA coding for human tumor necrosis factor, an important immune modulator with selective cytotoxic action on a number of transformed cell lines have been described. The DNA was placed under control of either phage M13 promoter of gene for main coat protein or tandem of pair of E. coli tryptophane promoters. It has been shown that E. coli cells harbouring plasmids described with artificial TNF gene provide good level of protein biosynthesis. The protein has been purified by anion exchange chromatography near to homogeneity and used for preparation of monoclonals. As result three hybridomas effectively produced high affinity monoclonal anti-TNF antibodies have been obtained and characterized.
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.
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.
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Plasmid expression vector using the temperature-regulated promoter P'R of bacteriophage lambda is described. The vector carries a combination of two regions of lambda cI857indgenome, that contain: 1) gene cI and promoter PR, and 2) gene Q and promoter P'R. Transcription or gene Q is initiated at promoter PR, which is controlled by cI857 repressor. The Q gene product acts as a positive regulator of RNA synthesis from P'R. At 37 degrees C, sufficient amounts of protein Q are synthesised to initiate the expression of the cloned gene from P'R. Inactivation of Q gene (by elimination of a single NcoI site) results in the loss of P'R expression activity in the vector. E. coli beta-galactosidase gene (lacZ) and human leukocyte interferon alpha 2 gene (ifn alpha 2) were cloned into a single EcoRI cleavage site under the control of P'R. These constructs express high levels of beta-galactosidase and interferon alpha 2 in E. coli at 37 degrees C.
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.
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.
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A simplified technique for DNA sequence analysis has been developed, based on modification of a previous method [(1980) Methods Enzymol. 65, 499-560]. It employs an adsorptive immobilization of terminally labelled DNA on DEAE paper followed by G, A+G, C+T and C specific modification and cleavage reactions. This solid-phase technique is faster and more convenient than the original method. The efficiency is comparable. The total processing time taken to produce cleaved fragments loaded on a gel is less than 2 h.
A 169 b.p. fragment including the bla gene promoter p3 has been removed from pBR327 plasmid, and the deleted plasmid used for cloning the TaqI/BglII-fragment of the lambda c1857ind- DNA containing promoter pR and gene cI to obtain plasmid pCE119. Cells containing pCE119 produced a high level of beta-lactamase at 42 degrees C, the yield at 42 degrees C being 100 times higher than at 32 degrees C. For cloning and functional assays a pCEZ12 plasmid was constructed, in which promoter pR and repressor cI of lambda phage control the expression of the semi-synthetic beta-galactosidase gene. Yield of beta-galactosidase produced by pCEZ12 at 42 degrees C was ca. 300 times higher than at 32 degrees C.
The binding has been studied of Escherichia coli RNA-polymerase to the fragments of lambda DNA obtained by digestion with restriction endonucleases BsuI, HindIII, BamHI, EcoRI and HindII + III. There are at least six sites of RNA-polymerase binding in the b2-region. In vitro transcription of those BsuI-fragments of the b2-region which contain six binding sites is rightward. Therefore, the fragments contain promoters rather than mere RNA-polymerase binding sites. One of the promoters of the b2 region named patt was calculated to be about 50 bp to the left of the att site. We postulate that this promoter might correspond to the hef-target which was described as important for the site-specific recombination.