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A new method of optimization of foreign gene expression in E. coli, based on the construction of hybrid operons with partially overlapping genes is described. The partial overlapping of the translation termination and initiation sites in the formed operon must provide translational coupling of appropriate gene product synthesis. Such an approach has provided the synthesis of human interferon alpha F in E. coli cells under the control of the lacUV5-promotor up to about (3-4).10(7) units per liter of bacterial culture. The reinitiation of the distal gene translation is shown to take place in the intercistronic region. Substitution of the lacUV5 promotor by the more efficient tac one allowed to increase the synthesis level of interferon alpha F to (1-2).10(8) units per liter. The conclusion is made about the equimolarity of distal and proximal to the promotor genes products syntheses when the intercistronic region of E. coli trpE-trpD genes are used for translational coupling.

The trpOP, lacUV5, tacOP, PR and PL-promotors were cloned in the previously obtained pML4 vector plasmid. The expression of structural gene cat was studied by the chloramphenicolacetyltransferase determination in cell extracts. The level of protein synthesis by appropriate recombinant plasmids was analysed in vivo and in vitro. It was shown that the efficiency of the gene expression is determined by both the "strength" of the promotors and mRNA translation specificity. The obtained collection of the plasmids might be used for the determination of the promotor strength by the hybridization of pulse-labeled mRNA with DNA and an effective expression of the genes by means of "hybrid protein gene" and "hybrid operon" constructions.

The expression of human leucocyte interferon alpha F gene in plasmid pLM-IFN alpha F-273 is controlled by a hybrid tac (trp-lac) promoter. A structural gene for interferon alpha F is a component of the hybrid operon lacZ'-IFN alpha F-TcR, that contains an E. coli trp-operon intercystronic region. Plasmid pLM IFN alpha F-273--directed interferon synthesis allows to obtain about 10(7) IU/l. This plasmid was cloned in broad-host-range vector plasmid pAYC31. The hybrid bi-repliconed plasmid containing interferon gene as well as its single-repliconed deletion derivatives obtained by the in vivo recombination, were introduced into obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida PpG6. Methylotrophic strain and Pseudomonas were able to transcribe the interferon gene from E. coli tac promoter, the yield of interferon being 2-4-fold higher as compared with the one in the initial host.

The possibility of creating artificial overlappons was studied on the model of two genes, that coding for the N-terminal part of lambda cro protein and the cat of E. coli. To test the dependence of translational coupling efficiency on the intercistronic region a series of recombinant DNA molecules carrying different hybrid operons with partially overlapping genes was constructed. The translational efficiency of the distal to the promoter gene was shown to depend on the intercistronic region structure: overlapping of the AUG codon with the terminating one of the proximal gene in the UGAUG manner is optimal for the translational coupling, and the displacement of AUG at several nucleotides in both directions decreases the translational reinitiation efficiency for the ribosomes, that have synthesised the first gene product.

The basic results of the studies on expression of the genes of human alpha F- and beta 1-interferons in E. coli cells are presented. To synthesize the fibroblast interferon, the respective fragment of the human chromosome was cloned, the complete nucleotide sequence of the structural moiety of mature beta-interferon was determined and the genes of "hybrid (interferon-like) proteins" and "hybrid sites of ribosome binding" were constructed with control of the beta-interferon gene by the prokaryotic regulatory areas. Synthesis of beta-interferon was achieved (1.10(7)-5.10(7) IU per 1 l of the bacterial culture) with the use of the tryptophan operon promoter. A new procedure for optimization of allogenic genetic information in E. coli cells: constructing of "hybrid operons with partially overlapping genes" or artificial "overlappons" was developed following the example of the alpha F-interferon gene cloned in the Laboratory headed by E. D. Sverdlov at the Institute of Bioorganic Chemistry of the USSR Academy of Sciences. The use of this procedure enabled production of up to 5.10(7) IU/l of alpha F-interferon under the control of the lacUV5-promoter. On the basis of the newly constructed vector molecules expression of the genes of alpha F- and beta 1-interferons was amplified with the "overlappon" procedure.

The study on the rate of initiation of model gene cat transcription under the control of E. coli (Plac UV5, Ptrp, Pcat, Ptac), phage lambda (PL, PR), phi X174 (PD) promotors was carried out by means of hybridization of pulse labelled in vivo mRNA with the DNA coding parts. The presence of gene bla(Apr) with its own constitutive promoter in all the recombinants permitted to use the level of appropriate mRNA in the cell as an internal standard. This method allowed to evaluate the true efficiency of the promoters in question. The strength of the promoters studied is shown to be equal within the limit of one order value.

A fragment of Escherichia coli chromosome containing the intact threonine operon or its distinct genes has been cloned on the pBR322 plasmid. This fragment has been mapped using some restriction endonucleases. Cloning results in an increased level of appropriate enzyme activity in cells containing hybrid plasmids. Those carrying the complete threonine operon are capable of accumulating threonine up to 5 g/l in culture medium during 48 h. When multi-copy plasmids are used for gene cloning, interpretation of experiments aimed at transformation of auxotrophic bacterial strains, might be complicated. For example, transformation of appropriate threonine auxotrophs by a hybrid plasmid carrying mutation in the threonine gene, might result in prototrophic phenotype. It is possible that the great amount of mutant enzyme molecules compensated their low activity. On the contrary, the presence of a gene within the plasmid, as shown by restriction and biochemical analysis, did not always ensure the growth on a minimal medium of auxotrophs transformed by this plasmid.