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Escherichia coli open reading frames ydiO and ydiQRST were identified as genes encoding components of the acyl-CoA dehydrogenase complex of anaerobic fatty acid β-oxidation. Individual or concomitant inactivation of fadE gene, encoding known aerobic acyl-CoA dehydrogenase, and ydiO and/or ydiQRST genes did not affect cellular growth on glucose as a sole carbon source. Aerobic growth on sodium oleate was observed only for the cells with intact fadE gene. With an alternative electron acceptor, the cells possessing intact fadE gene demonstrated anaerobic growth on sodium oleate irrespective of the presence or absence of ydiO and ydiQRST genes. For the fadE-deficient mutants, anaerobic growth on sodium oleate was observed only for cells with intact ydiO and ydiQRST genes, while the fadE/ydiO and fadE/ydiQRST mutants failed to grow under the similar conditions.

Microorganism producer strains are the basis of industrial biotechnology. Their properties determine the economical parameters of the production. Methods of rational design (metabolic engineering) and combinatorial methods of mutagenesis and selection (laboratory evolution, adaptive evolution, protein and genomic shuffling) are used for the construction of microorganism strains. Combination of these methods is frequently used. Modern strains usually do not contain plasmids and markers of drug resistance. All changes are introduced into the chromosome by the methods of homologous and site-specific recombination. The sum of such approaches is called recombineering. Gene expression is carried out at the optimal level under the control of promoters of a certain power (frequently regulated). Knowledge of a complete genomic sequence is almost a mandatory condition for the use of methods of metabolic engineering. Bioinformatics significantly assists in the selection of enzymes and the search for necessary genes and metabolic reactions. Measurement of metabolic fluxes largely assists in the construction of strains. The current level of science makes it possible to construct metabolic pathways de novo in strains for the production of chemicals and biofuel. Carbon dioxide has potential as a raw material for microbiological industry; therefore, the study of CO2 fixation by acetogens and electrogens is a promising direction of studies.

In the present work it was shown that biosynthesis of silver sulfide nanoparticles from silver nitrate and sodium thiosulfate solutions of millimolar concentration occurs efficiently by living Shewanella oneidensis MR-1 cells, as well as by ultrasonically-disrupted cells and by the membrane fraction of the cells. The size of nanoparticles synthesized in the presence of living cells was 7.8 ± 1.5 nm, while in the presence of ultrasonically-disrupted cells--it was 6.52 nm. The shape of nanoparticles in both cases was close to spherical. It was also shown, that synthesis of nanoparticles occurs in a cell-free solution of sodium thiosulfate that has been incubated with cells previously and to which then a silver nitrate solution was added. In this case the nanoparticles were of elongated shape and their size was (11 ± 4) x (24 ± 6) nm. In the control experiment, when only silver nitrate and sodium thiosulfate solutions not incubated with cells were used, the nanoparticles were not detected. It was shown that biosynthesis of nanoparticles occurs both in aerobic and anaerobic conditions. Nanoparticles are not formed by using thermally inactivated cells as it was shown by us previously. The results show the important role of the native structures of cells for the nanoparticles formation.

In this study, we constructed and characterized Escherichia coli strains deficient for mixed acid fermentation pathways, which are capable of rapid aerobic growth on glucose without pronounced bacterial Crabtree effect. The main pathways of production of acetic and lactic acids and ethanol in these strains were inactivated by a deletion of the ackA, pta, poxB, IdhA, and adhEgenes. The phosphoenolpyruvate-dependent phosphotransferase system of glucose transport and phosphorylation was inactivated in the strains by a deletion of the ptsG gene. The possibility of alternative transport and phosphorylation of the carbohydrate substrate was ensured in recombinants by constitutive expression of the galP and glk genes, which encode the low-affinity H+-symporter of D-galactose and glucokinase, respectively. SGMI.0DeltaptsG PtacgalP and SG M1.0DeltaptsG PIglk PtacgalP strains were capable of rapid aerobic growth in a minimal medium containing 2.0 and 10.0 g/l of glucose and secreted only small amounts of acetic acid and trace amounts of pyruvic acid.

Anaerobic biosynthesis of 1-butanol from glucose is investigated in recombinant Escherichia coli strains which form butyryl-CoA using the heterologous enzyme complex of clostridia or as a result of a reversal in the action of native enzymes of the fatty acid beta-oxidation pathway. It was revealed that when the basic pathways of acetic and lactic acid formation are inactivated due to deletions in the ackA, pta, poxB, and ldhA genes, the efficiency of butyryl-CoA biosynthesis and its reduced product, i.e., 1-butanol, by two types of recombinant stains is comparable. The limiting factor for 1-butanol production by the obtained strains is the low substrate specificity of the basic CoA-dependent alcohol/aldehyde AdhE dehydrogenase from E. coli to butyryl-CoA. It was concluded that, in order to construct an efficient 1-butanol producer based on a model strain synthesizing butyryl-CoA as a result of a reversal in fatty acid beta-oxidation enzymes, it is necessary to provide intensive formation of acetyl-CoA and enhanced activity of alternative alcohol and aldehyde dehydrogenases in the cells of a strain.

Effect of constitutive expression of the aceEF-lpdA operon genes coding for the enzymes of NAD+ reducing pyruvate dehydrogenase complex on the anaerobic production of succinic acids from glucose by recombinant Escherichia coli strains was studied. Basic producer strains were obtained by inactivation of the main pathways for synthesis of acetic and lactic acids by deletion of the genes ackA, pta, poxB, and ldhA (SGMO.1) in E. coli strain MG 1655 cells and additional introduction of the Bacillus subtilis pyruvate carboxylase (SG M0.1 [pPYC]). A constitutive expression of the genes aceEF-lpdA in derivatives of the basic strains SGM0.1 PL-aceEF-lpdA and SGM0.1 PL-aceEF-lpdA [pPYC] was provided by replacing the native regulatory region of the operon with the lambda phage PL promoter. Molar yields of succinic acid in anaerobic glucose fermentation by strains SGM0.1 P(L)-aceEF-lpdA and SGM0.1 PL-aceEF-lpdA [pPYC] exceeded the corresponding yields displayed by several control strains (exceeded considerably in the case of the strains with a pyruvate carboxylase activity). It is concluded that an increase in the succinic acid production by strain SGM0.1 PL-aceEF-lpdA [pPYC] as compared with the strains SGM0.1 and SGM0.1 [pPYC], which synthesize this substance in the reductive tricarboxylic acid cycle, is determined by activation of the glyoxylate shunt.

A study has been conducted on the morphology of artificial spider silk fibers, prepared from recombinant analogues of spiridons 1 and 2. It has been shown that by stretching out the "as spun" fiber, a reorganization of its spongy matrix occurs, which leads to the formation of microfibrills, followed by a reduction of the diameter of the fiber. The durability of an artificial fiber depends on the degree of stretching and on the substructure of the microfibrills. The model process of artificial fibers preparation reproduces to the great detail the natural process of spider web spinning. Future applications of this model include production of biomaterials with unique properties.

The distribution of secondary structures along the polypeptide chains of spider proteins spidroins 1 and 2 and their recombinant analogs has been studied by statistical methods. It was found that these proteins in the monomolecular form contain only trace amounts of beta-structures. At the same time, the regions of the sequence including Ala and Gly are predicted as helical-containing (with alpha-helices and left-helices of polyproline II type). An analysis of literature data and our data obtained in this study shows that the main conformation of the polypeptide chain solutions of spidroins 1 and 2 and their recombinant analogs in water solutions is the left-helix of polyproline II type with some contaminations of alpha-helices and a very small share of beta-structures. The transition to the state with extended conformations, which are peculiar to mature filaments of spider webs, requires the dehydration of the polypeptide chain backbone. Thus, the genesis of beta-structure in spider web proteins is determined by the conditions of conformation transitions between the main regular conformations of the polypeptide chain backbone.

Over the last ten years, the recently discovered process of direct electron transfer from anaerobically grown microorganisms to an electrode of a fuel cell has been the object of intense study. The microorganisms responsible for such electron transport were termed electrogenic; the devices using them to generate electric current, microbial fuel cells (MFCS). The review discussed the molecular mechanisms of electron transfer to the environment in the case of the two best studied microorganisms, Shewanella oneidensis and Geobacter sulfurreducens. The discovery of bacterial conducting pili (nanowires) used for electron transfer to the electrode and between bacterial cells was sensational. In the real MFCS, which use complex substrates (industrial liquid waste), microbial associations are active, often as biofilms. The progress in MFCS design and the prospects of their practical application are considered.

The C825T polymorphism in the gene encoding the G protein beta 3 subunit (GNB3) causes enhanced G protein activation and the increased in vitro cell proliferation. We investigated the association of gene GNB3 C825T polymorphism with coronary artery disease (CAD) in the Russian population. A total of 313 patients with CAD diagnosed on the basis of clinical studies and coronary angyography were examined. The control group included 132 individuals that lacked clinical CAD symptoms and had matching profile of coronary artery disease risk factors. Blood pressure was measured using standard protocols. Increased levels of diastolic and systolic pressure was observed in both groups. The allele and genotype frequencies of this polimorphic marker were significantly higher in the CAD patients than in control. We found that the frequency of allele C and gen-. otype CC was significantly higher in the CAD patients (OR = 1.55; P = 0.0079; OR = 1.63; P = 0.0215, respectively), which suggests higher risk of this pathology in carriers of allele C and genotype CC. Thus, in the Russian population coronary artery disease is associated with GNB3 allele C and genotype CC.

Alleles and genotypes of polymorphic markers of paraoxonase 1 and paraoxonase 2 genes (PON1 and PON2) encoding enzymes of the body antioxidative defense were compared in type 1 diabetes mellitus patients with or without diabetic nephropathy. The patients with nonoverlapping ("polar") phenotypes constituted different groups. The first group contained patients with diabetic nephropathy (DN+, n = 62), clinical proteinuria (albuminuria above 300 mg per day), and at least 15-year disease duration. In control group, the patients had no diabetic nephropathy (DN-, n = 68), their albuminuria was below 200 mg per day, and disease duration was at least 20 years. Comparative analysis with exact Fisher's test revealed no significant differences in frequencies of alleles and genotypes of the PON1 gene polymorphic marker Gln192Arg and of PON2 gene polymorphic markers Ala148Gly and Cys311Ser. Our results suggest that the polymorphic markers studied are not associated with diabetic nephropathy among Russian patients in Moscow.

Sequences of spidroin 1 and 2 from spiders belonging to various species were analyzed by Fourier analysis. Specific periodical patterns were found in various segments of the sequences. These characteristic periodicities vary within the same proteins as well as between spidroin 1 and spidroin 2 sequences. It is possible that alterations in periodicity help to recognize contact sites between the molecules. Spidroins of 2 type have similar sequence structure consisting of four parts with a particular periodical pattern. These parts are a constant C-terminal part, a long-periodical part, a short-periodical part, and a constant N-terminal part.

Many species of Bacteria and Archaea posses a regularly structured surface layers (S-layers) as outermost cell envelope component. S-layers composed of a single protein or glycoprotein species. The individual subunits of S-layers interact with each other and with the supporting bacterial envelope component through non-covalent forces. Pores in the crystalline protein network are with mean diameter of 2-6 nm, the thickness of S-layer is 5-10 nm. The isolated S-layer subunits reassemble into two-dimensional crystalline arrays in solution, on solid supports, on planar lipid films. These unique features of S-layers have led to a broad spectrum applications. This review focuses on the structural properties S-layers and S-proteins and their applications with accent to using this structures in nanobiotechnology.

To obtain transgenic tobacco plants expressing recombinant analogs of spider dragline silk spidroin 1, artificial 1f5 and 1f9 coding for spidroin 1 analogs were 3'-fused in-frame with the reporter lichenase gene. The Tr2' weak constitutive promoter of Agrobacterium tumefaciens T-DNA and the strong constitutive promoter of the cauliflower mosaic virus 35S RNA gene were used as regulatory elements. The expression cassettes were used to transform agrobacteria and then introduced in tobacco leaf disks. On evidence of Southern hybridization, transgenic plants each carried a single copy of a hybrid gene, which corresponded in size to the constructed one. Zymography and Western blotting revealed full-length hybrid proteins in leaf extracts of transgenic plants. The results testified that plants can maintain and express synthetic genes for spider silks and, consequently, may be used as a convenient producer of recombinant silk analogs.

Polymorphism A1166C of the AT1R gene encoding angiotensin vascular receptor [replacement of C (cytosine) for A (adenine)) at position 1166] was compared in patients with insulin-dependent diabetes mellitus (IDDM) complicated by diabetic nephropathy (DN) and in noncomplicated patients (n = 27 and n = 41, respectively) and also in patients with IDDM complicated by diabetic retinopathy (DR) and in correspondent noncomplicated individuals (n = 30 and n = 44, respectively). The frequency of AT1R gene alleles and genotypes in patients with IDDM complicated by DN did not differ significantly from that observed in patients with noncomplicated IDDM. In contrast, in patients with IDDM complicated by retinopathy, a significant decrease in the content of A allele (68.3% against 82.6%) and a significant increase in the content of C allele (31.7% against 17.4%) was found as compared with the control group. Thus, in the Moscow population, A1166C polymorphism of the AT1R gene is not associated with diabetic renal complications but indeed associated with diabetic retinal complications. C allele is a risk factor of DR (the relative risk, RR, is equal to 2.17), and A allele is, in contrast, a protective factor against early retinopathy development (RR is equal to 0.49).

A representative genomic library of the Corynebacterium glutamicum ATCC 13032 genes in a cosmid vector Lorist6 was created. The cosmids contain inserts of bacterial DNA obtained by partial digestion with the Sau3A I restrictase. Five hundred and thirty individual primary recombinant clones were transferred into the wells of microtiter plates, where they are now being preserved. The average size of the bacterial DNA inserts determined via a sum of restriction fragment sizes of recombinant molecules is about 38 kb. The capacity of the obtained gene library is 8.4 equivalents of the C. glutamicum genome, i.e., every fragment of the genome is on average represented by eight clones and is presented in at least one clone with the probability > 99%. Clone grids (sets of recombinant clones located on the hybridization membrane in regular and reproducible order) were created. Specificity of the created clone library and its representativeness were confirmed experimentally by hybridization of clone grids with DNA probes corresponding to unique regions of the Corynebacterium genome. A plasmid containing the pheA prephenate dehydratase gene, olygonucleotide corresponding to the lysC gene, and the 21 RNA probe obtained from the insert ends in different cosmids were used as probes. The created set of clones allows the construction of a cosmid contig overlapping the C. glutamicum genome and a physical genetic map on its base.

The use of the polymerase chain reaction was proposed for intron excision from genomic genes with known nucleotide sequences. Three exons (5, 6 and 7) of genomic interleukin 1 beta gene were amplified by means of thermostable DNA polymerase TthI from Thermus thermophilus on the base of cloned in M13 phage human genomic interleukin 1 beta gene. Synthetic oligonucleotides complementary to sequences flanking exons were used as primers. The fragments obtained by exon DNA amplification were joined in the correct order due to reciprocal complementation of end sequences, that was foreseen during synthesis of oligonucleotide primers followed by amplification of the enlarged fragments. As a result the structural interleukin-1 beta gene consisting of three exons was assembled. DNA sequences carrying the ATG initiation codon and XbaI recognition site at the 5'-end, and PstI recognition site at the 3'-end (essential for insertion into the expression vector) were formed by the additional end sequences of primers. The nucleotide sequence analysis of the obtained structural gene revealed its complete identity with natural interleukin 1 beta human gene. We created the expression vector pPR114 with phage lambda promoter PR thermo-inducible in case of the cIts857 repressor presence in cells. It was used for expression of the present gene. The interleukin 1 beta synthesized in E. coli had biological activity.

Class II HLA molecules are the most useful markers for susceptibility to different autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM) and rheumatoid arthritis (RA). Polymerase chain reaction and hybridization with a set of allele-specific oligonucleotide have been used for analysis of allelic sequence variation. The analysis of frequencies of HLA-DQA1 alleles among 10 patients of the russian population revealed a uneven distribution. We have developed a method for preparing non-radioactive oligonucleotide probes with terminal deoxynucleotidyl transferase and Bio-11-dUTP. Comparison of biotinylated and 32P-labeled hybridization probes gave the same sensitivity for HLA-DQA1 typing of amplified DNA. Amplification of the HLA-DQA1 gene has been successful on 10 pg of total DNA. This amount of DNA is close to the amount of DNA in a single cell. Alternatively, HLA-DQA1 typing could be based on the analysis of buccal cells of saliva that would avoid the problem of individuals who object to giving blood samples.

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.

Novel cloning vectors for glutamic acid producing bacteria have been constructed. The cryptic plasmid pBO1 (4.4 kb) from Brevibacterium sp. recombined with the plasmid pACYC184 (4.0 kb) from Escherichia coli was used to produce composite plasmid named pKA1. The plasmid could propagate and express the Cm-r phenotype in E. coli and coryneform glutamic acid producing bacteria Br. flavum, C. glutamicum, Br. lactofermentum. The pKA1 plasmid and its variants deleted within non-essential plasmid regions with unique restriction sites HindIII, SalGI, SphI were used in cloning experiments. The genes coding for threonine biosynthesis of C. glutamicum and Br. flavum were subcloned into shuttle vectors in C. glutamicum cells. Recombinant plasmids were introduced into protoplasts by polyethylenglycol-mediated transformation of plasmid DNAs. It was shown that the presence of plasmids containing the Br. flavum thrA2 gene in C. glutamicum (thrB) caused 10-fold increase in homoserine dehydrogenase activity, as compared to that of wild type strain, and in homoserine production.

Upon a 50% isopropanol treatment of phage fl in a 1 M NaCl solution protein A (gene 3 product)--DNA complex is precipitated while protein B (gene 8 product) was still solubilized. After such a treatment the DNA--protein complex containing 10--40% of protein A and less than 0.0025% of protein B was obtained. Evidence was obtained that there was no non-specific rearrangement of protein A during the isolation procedure. The complex was treated with endonuclease R.HAC III, followed by electrophoresis of the resulted fragments and estimation of the [14C] protein A (labeled with [14C]histidine) throughout the gel. The maximal radioactivity coincided with the DNA bands, being proportional to the DNA content in the respective bands. The data obtained indicate that protein A is iniformly arranged along the DNA molecule.

The influence of phage f1 gene 5 protein on melting of the synthetic polynucleotides has been investigated, using UV-spectroscopy. In our experiments we have varied the proteins concentration. It has been shown, that the protein lowers the melting temperature of the studied polynucleotides (d/A--Tn dAndTn, rAndTn, rAn.r n, dAn.rn). The melting temperatures and the shapes of melting curves of various polynucleotides differ when the same protein concentrations are used. We have shown that the protein binds to the double-stranded polynucleotides, containing ribo-ribo-, deoxyribo-ribo-chains. The difference in melting temperatures and shapes of melting curves was explained using the data about the differences in the secondary structure of these polynucleotides. Only for d/A-Tn renaturation was observed after sample cooling. It may reflect the single-stranded hairpin structure of this polynucleotide.

The stability of Escherichia coli strains carrying hybrid plasmids which contain ColE1-like replicon and threonine operon genes was studied. It was shown that the main reason for instability is the loss of a plasmid. The second reason for instability is the rec-dependent recombination that leads to formation of new plasmids. All experiments where instability of strains was observed, can be quantitatively described by the model that presumes a random loss of plasmids in cell population with a frequency of about 7.10(-4), which results in diappearance of plasmid-borne cells due to their low growth rate. Instability increases during the stationary phase but it is not easy to quantitatively estimate this process.

Cloning of genes for threonine and lysine biosynthesis from Corynebacterium glutamicum was performed in Escherichia coli cells using the plasmid vector lambda pSL5. The cloned genes are identified via complementation of thrB and lysA mutations. The gene complementing thrB of E. coli is located within a 4.1 kb EcoRI fragment of C. glutamicum chromosomal DNA. All the recombinant phasmids complementing the lysA gene of E. coli contain common 2.2 kb and 3.3 kb EcoRI C. glutamicum DNA fragments. The cloned DNA fragments hybridize with identical EcoRI fragments of C. glutamicum chromosomal DNA.

The number of EcoRI restriction sites in DNAs of several lombdoid phages varies from 2 to 7 and the size of fragments ranges from 0.6 . 10(6) to 25 . 10(6) daltons. Effectiveness of restriction determined in vivo weakly correlates with a number of restriction sites, suggesting different sensitivity of different sites to EcoRI action. No correlation between molecular weights of DNA of different phages and the rate of heat inactivation of the phage has been found. The variability of number of the restriction sites and of the molecular weights of the fragments permits to conclude that: 1) closely-related organisms can be possibly used as convenient sources of certain genes (if a construction of new vector lambdoid phages is essential); 2) there is an opportunity to find phages with different distribution of the restriction sites to other restrictases within a group of phages with non-identical EcoRI restriction sites.

Centrifugation of Bacillus thuringiensis 351 DNA in CsCl-ethidium bromide density gradient has revealed, besides the main band of chromosomal DNA (p = 1.56 g/cm2), some additional bands with higher density, which usually correspond cicle superhelix DNAs, are revealed. 6 discrete bands are observed under electrophoresis of total fraction of minor DNA bands, which suggests, that the preparation contains cicle DNAs of different size. The treatment with DNAse results in the appearance of 3 bands corresponding to opened cicle DNA forms, their molecular weight being 12-10(6)--4-10(6) daltons. Cicle super-helix and opened cicle DNA forms are found in minor fraction by means of electrone microscopy. Statistical analysis has revealed the presence of at least three types of cicle molecules of different size. The variant 351-10, free of extrachromosomal elements, is isolated after ethidium bromide treatment at high temperature. Possible mechanisms of cicle DNAs formation are discussed. Biological function of DNA extrachromosomes fragments in Bacillus thuringiensis is still obscure.

It has been shown that the protein product of T4 bacteriophage gene 32 (protein 32) completely prevents RNA and poly(A) synthesis on the denatured DNA as a template at the protein/DNA ratio about 13:1. Under the same conditions protein 32 has no effect on these syntheses on the native DNA and inhibit poly(A) synthesis with oligo(dT)9 and oligo(dT)12 as a template, preventing binding of enzyme with oligonucleotide. It is possible that the unwinded regions of double-stranded DNA within transcription complexes are too small to allow cooperative binding of protein 32 or are unavailable for this protein. We suppose that the protein 32 and other "unwinding" protein plays not only "protective" and "structural" roles but also participates in the regulation of template activity of single-stranded DNA regions in the replicative forks.

We studied Bacillus thuringiensis var galleriae, strain 612 plasmids. B. thuringiensis cells contain double-stranded plasmid DNA molecules (ranging of about 12% from total DNA content) with buoyant density 1.59 g/cm3. Plasmid DNA content was constant during the exponential and stationary phases of bacterial growth. The plasmid fractions consist of DNA molecules with molecular weights of 5.9 x 10(6), 10.0 x 10(6), and 110.9 x 10(6) daltons (pVD1, pVD2 pVD3, respectively). Endonuclease EcoRI cuts the plasmids pVD2 and pVD3 into two and four fragments, respectivelyy, but pVDI seemed to be resistent to EcoRI treatment. We found that pVD2 and pVD3 plasmids contain a common DNA fragment with the molecular weight of 6.7 x 10(6) dalton as it was shown by restriction analysis. In contrast, the same plasmids contain the common fragment with molecular weight of 7.5 x 10(6) dalton as shown by heteroduplex analysis. Plasmid pVD3 has a transposon-like structure.

Influence of the protein product of F1 phage gene 5 (protein 5) on the synthesis RNA and poly(A) in vitro was studied. It has been shown, that protein 5 has no effect on the transcription of the native DNA by E. coli RNA-polymerase, but completely prevents RNA and poly(A) synthesis on the denatured or single-stranded DNA at the protein/DNA ratio 10:1. Protein 5 inhibits poly(A) synthesis with oligo(dT)9 and oligo(dT)12 as a template, preventing binding of enzyme to the oligonucleotide. After the initiation of the poly(A) synthesis the inhibition becomes considerably wearer. The biological function of the inhibition of the transcription by "unwinding" proteins is discussed.

A simple method for isolation of three DNA-polymerases from E. coli is developed; it is based on properties of the enzymes to bind with single-stranded DNA, fixed on cellulose. DNA-polymerases I, II and III were identified by their relation to templates, ionic strength, heat denaturation and inhibition of the activity with agents which blocked sulfhydryl groups. After purification which included chromatography on DNA-cellulose as well as fractionation with phosphocellulose and gel-filtration through Sephadex G-100, the enzymes were free the endonucleases contamination.

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 transforming activity of DNA from Bacillus subtilis 168, Bac. subtilis W23, Bac. subtilis NRS and Bac. aterrimus after the EcoRI restrictase treatment was studied. The auxotrophic strains of Bac. subtilis 168 were used as recipients in bacterial transformation. The transforming activity for different markers decreased up to 0.001--6 per cent from the initial level for different Bacilli DNAs. In some cases the sensitivity of the same marker from different Bacilli differed in more than 50 times. Bac. subtilis and Bac. aterrimus have demonstrated the maximal differences. Such differences can be used for the identification of close related Bacilli.

The possibility of entomocyde crystal protein synthesis was studied using a heterological cell-free system with Bacillus thuringiensis plasmid DNA as template. The high level of template activity is usual for Bac. thuringiensis plasmid DNA. Immunochemical studies of the in vitro synthesized polypeptides showed that Bac. thuringiensis plasmid DNA does not direct crystal protein synthesis.

Escherichia coli DNA with molecular weight of 20 - 10(6) daltons was digested by restriction endonuclease EcoR1, and the transforming activity of resctricts was studied. The transforming activity of restricts for two markers (Leu and Arg) was 2-5 fold increased, for two other markers (Thr and His) was not changed, and for one marker (Pro) was completely absent. The molecular weight of E. coli DNA restricts was 7,5-10(6) daltons. An increase and a decrease of the transforming activity for different markers appeared to be the result of two effects: 1) more efficient uptake of low molecular weight DNA into the cell and 2) the inactivation of markers as a result of location close to EcoR1 induced break.

A novel branch of national economy--biotechnology is being developed, based on genetic engineering. The construction of strains using the methods of molecular cloning has led to-date to creation of new biotechnological processes. Further advance in biotechnology would be mainly promoted by the possibilities of application of gene engineering to reorganization of industrially important microorganisms. These are bacilli employed for production of vitamins, enzymes, insecticides; streptomycetes--the producers of antibiotics; yeasts applied in bakery industry, in production of fodder proteins; pseudomonads which will be helpful in development of effective biological means for protection of environment, etc. So, vector molecules based on plasmids and phages have been constructed for best-studied representatives of industrial microorganisms, the methods of introduction into the cell of hybrid DNA molecules worked out, the problems of optimization of foreign gene expression being currently solved.