[Construction of the new Escherichia coli K-12 wild-type strain with improved growth characteristics for application in metabolic engineering].

MG1655 of Escherichia coli K-12 is frequently used in metabolic engineering as the wild-type strain. However, its two mutations, ilvG and rph-1 provide a negative effect on culture growth. The "polar effect" of rph-1 decreases the level of pyrE expression, causing partial auxotrophy for pyrimidines. Mutation ilvG leading to the appearance of Val(S) phenotype causes retardation of cell growth rate on media containing amino acids. In this work, the substitution of two loci in the genome of MG1655 with the recovery of the wild-type phenotype was accomplished. Gene rph(wt) from the chromosome of E. coli TG1 was marked via Red-dependent integration of DNA fragment carrying lambda attL-Cm(R)-lambda attR and transduced with phage P1 into MG1655; later, the Cm(R) marker was removed with the use of lambda Xis/Int recombinase. Parallel to this procedure, a spontaneous Val(R) mutant of E. coli MG1655 yielding colonies of maximal size on M9 medium with glucose in the presence of Val (50 microg/ml) was isolated. It was shown that a nucleotide deletion in the isolated Val(R) strain had been generated in the region of the identified E. coli K-12 ilvG mutation, which led to the recovery of the reading frame and active protein synthesis. This mutation named ilvG-15, which is the only reason for the Val(R) phenotype in the obtained strain, was transferred to MG1655-rph(wt) using cotransduction, by analogy to the transfer of rph(wt). Evaluation of rates of aerobically growing cells (microm, hour(-1)) on M9 medium with glucose produced the following values: 0.56, 0.69, and 0.73 for strains MG1655, MG1655-rph(wt), and MG1655-(rph(wt), ilvG-15), respectively.

[New method of construction of artificial translational-coupled operons in bacterial chromosome].

The new method of translational-coupled operons construction in bacterial chromosome has been developed on the basis of recombineering approach. It includes construction in vitro of the artificial operon with efficiently translated proximal cistron followed by its insertion E. coli chromosome, modification of the operon due to Red-driven insertion of the special "Junction" with excisable selective marker in the intercistronic region of the initial operon and excising the marker. The structure of this Junction has been designed and tested in the present investigation. It consists of: 1) E. coli rplC-rplD intercistronic region for placing the TAA-codon of the proximal operon's gene in the SD-sequence (TAAGGAG) of rplD; 2) Cm(R)-gene flanked by lambdaattL/R-sites in such a fashion that after lambdaInt/Xis-driven excision of the marker the residual lambdaattB-site would not contain the termination codons in frame with ATG of rplD; 3) E. coli trpE-trpD intercistronic region for location of ATG of trpD at the position of initiation codon of the distal gene of original operon. The general design of desired construction provides the conversion of the original two-cistronic operon into three-cistronic operon with translational-coupled genes, where the coupling of the artificial ORF (rplD'-lambdaattB-'trpE) with the proximal gene is occurred due to rplC-rplD intercistronic region and the coupling of this ORF with the distal gene--due to trpE-trpD. The experimental implementation of the described strategy was showed by construction of artificial operon P(tac-aroG4-serA5, where expression optimization of the distal serA5 gene was achieved via construction of three-cistronic operon with translational-coupled genes.

[Tuning of expression level of the genes of interest located in the bacterial chromosome].

The new method of construction of the set of E. coli clones, differing in the promoter strength upstream the gene of interest, has been developed and tested using native E. coli MG 1655 lacZ gene as the reporter. This method includes the construction of the promoter-carrying DNA fragment obtained by PCR with consensus P(tac) as a template and the primers that lead to randomization of 4 central nucleotides in the promoter "-35"-region, linking the obtained fragments with the selective marker (Cm(R)) followed by Red-driven integration of the resulted DNA fragments directly in E. coli MG1655 chromosome instead the native lacI-gene and promoter/operator region of lac-operon. Due to direct determination of LacZ-activity in the independently obtained clones-integrants, we have found 14 new promoters (from 44 = 256 possible variants) that differ in their strength up to 100 fold (LacZ-activity in the corresponding strains smoothly varies from 10(2) for the weakest tested promoter up to 10(4) Miller U detected for the initial P(tac)). Sequencing of obtained promoters revealed that randomization of three positions in the "-35"-region is sufficient to obtain representative promoter library that would decrease the total number of potential promoter variants from 256 up to 64. It seems probable that exploiting of the developed method leading to one-step construction the library of clones with varied expression of gene/operon of interest could be useful tool in the modem metabolic engineering for optimization of genes expression.

[Light effect at various wavelengths on gametogenesis of the Black Sea sea urchin (Strongylocentrotus nudus)]. / Vozdeistvie sveeta s razlichnoi dlinoi volny na gametogenez chernogo morskogo ezha (Strongylocentrotus nudus).

Comparative study of the effect of light at wavelengths of 720 and 520 nm on the reproductive process in black sea urchin Strongylocentrotus nudus was performed in artificial conditions. The results obtained not only suggest the distinct influence of light on the reproductive process in sea urchin, but also indicate that its effects on oogenesis and spermatogenesis are different. Light at the wavelength of 720 nm was found to activate gonad development, while at the wavelength of 520 nm it had a suppressive effect by decreasing the oogonial and spermatogonial content without disturbing their cellular structure. It is proposed that the gametes which are formed in sea urchins that are exposed to the light with differing wavelengths may possess different capacities for reproduction, thus influencing the viability of the young.

[The role of P-element sequences in the transcription activation]. / Rol' posledovatel'nostei P-élementa v aktivatsii transkriptsii.

A study was made of mutations resulting from insertion of the P element in the regulatory region of the yellow gene. Excision of the P element enhanced expression of yellow. Molecular analysis implicated P-element terminal sequences, which remained in the locus after the element was excised, in transcription activation of the yellow gene.

[Mechanisms of excising the P-element in a model system at the yellow locus of Drosophila melanogaster]. / Izuchenie mekhanizmov vyrezaniia P-élementa na model'noi v lokuse yellow u Drosophila melanogaster]

Patterns of excision of a single P element were studied in a model system of the yellow locus. The data obtained were in good agreement with the generally accepted SDSA (synthesis-dependent strand annealing) model. Specific features of P element excision in the presence of two tandemly repeated copies are presented. The pattern of P element excision depended on the sequences surrounding the insertion site and on the number of its additional copies present in the genome.

[Determinants of resistance to chlortetracycline and other antibiotics in chlortetracycline-producing strain of Streptomyces aureofaciens]. / Determinanty ustoichivosti k khlortetratsiklinu i drugim antibiotikam u shtamma Streptomyces aureofaciens--produtsenta khlortetratsiklina.

Data are presented on resistance of Streptomyces aureofaciens strain TB-633 FU--the producer of chlortetracycline (CTC) to autogenous antibiotics and a number of other antibiotics. It is demonstrated that resistance to CTC is specified by ctr genes of constitutive expression as well as by inducible genes. CTC and ethidium bromide may serve as efficient inductors of inducible ctr genes. The induction process is accompanied by increase in antibiotic biosynthesis level. Genes responsible for strain resistance to a number of macrolide antibiotics and thiostrepton are inducible and only function in the presence of appropriate antibiotics in the medium. The action of inducible mtr gene(s) is described in detail. The gene(s) simultaneously ensure increase in resistance to CTC and a number of macrolide antibiotics in the presence of exogenous inductors in media, such as both CTC and macrolide antibiotics. Mutants have been isolated which provide constitutive level of resistance to these antibiotics. A series of ctr and mtr mutants have increased CTC biosynthesis as compared to the initial level. Data on comparative analysis of the results obtained from hybridization of fragments of S. aureofaciens and S. rimosus DNAs to actI and actIII genes, responsible for polyketide synthases' synthesis, demonstrate that genes for CTC and OTC biosynthesis are situated on DNA fragments of similar size. This determines the strategy for cloning ctr and mtr genes as well as genes for CTC biosynthesis from S. aureofaciens.

[DNA cloning in actinomycetes: the creation of vector systems]. / Klonirovanie DNK u aktinomitsetov: sozdanie vektornykh sistem.

A new vector type was constructed on the basis of SLP 1.2 plasmid and the Kanr determinant of S. rimosus P3. It was shown that the determinant was capable of amplifying in the chromosomes of S. rimosus during its improvement for increasing the level of resistance to kanamycin. Cloning of the Kanr determinant was performed on the Pst I-A fragment of the SLP 1.2 plasmid in S. lividans 66. The Kanr determinant was expressed in the resulting hybrid plasmids, e. g. pSU 3, thus providing resistance of S. lividans to 20 micrograms/ml of kanamycin. As a result of repeated passages variants capable of growing in the presence of 50 000 micrograms/ml of the antibiotic were selected. The electrophoretic analysis of the total DNA fragments obtained after exposure to different endonucleases showed that they were identical to the respective fragments of the hybrid pSU 3 plasmid and amounted to approximately 40 per cent of the total DNA. The presence of the unique sites for the Bam HI, ClaI and SacI restriction endonucleases on the pSU 3 plasmid in an insignificant area and the relative stability (30-100 per cent) of the amplified variants allowed using it for cloning and amplification of DNA in Streptomyces. Plasmids were identified with the genetic and physical methods in 13 strains of the blue systematic group among the 72 strains studied. A multicopy plasmid with a molecular weight of 5.6 MD designated as pSB 24.1 was identified in the family of the plasmids of S. cyanogenus. The deletion and insertion variants of the pSB 24.1 plasmid were obtained. The comparative study of the properties of these plasmids revealed the areas insignificant for replication and maintenance of the pSB 24.1 plasmid and the area determining the Ltz+-phenotype. It was suggested that formation of the deletion and insertion variants of the pSB 24.1 plasmid was provided by the site specific recombination mechanisms. The presence of the unique sites for a number of the restriction endonucleases located in the insignificant area, the presence of the selective marker and the high transformation frequency allowed one to consider the multicopy pSB 24.1 plasmid an acceptable vector for cloning of DNA in Streptomyces.

[Genetic instability of the production of antibiotic substances controlled by the SCP2 plasmid of Streptomyces coelicolor A3(2)]. / Geneticheskaia nestabil'nost' produktsii antibioticheskikh veshchestv, kontroliruemoi SCP2-plazmidoi Streptomyces coelicolor A3(2).

Streptomyces coelicolor A3(2) strain bearing a variant of SCP2 plasmid produces three antibiotic substances: amromycin and streptocins A and B. After growth at elevated temperature (37 degrees C) of S. coelicolor A3(2), mutants which are not capable of producing amromycin and streptocins A and B (ant- mutants) were formed at high frequency (50%). These mutants retained SCP2 plasmid. Genetic analysis demonstrates that Ant phenotype is caused by a mutation in SCP2 plasmid. Genetic analysis demonstrates that Ant phenotype is caused by a mutation in SCP2. Heteroduplex and electrophoretic analysis of SCP2 DNAs from mutant variants indicates that these plasmids contained the identical deletion of 800 +/- 100 base pairs, as compared to SCP2 DNA of the initial strain. Ant variants revert to the initial phenotype at a frequency of 1.10(-2) to 1.10(-3). In turn, revertants from Ant variants a high frequency, similar to the initial strain. It is suggested that reversible Ant+ in equilibrium or formed from Ant- transitions might be the result of transposition of a genetic element in SCP2 controlling production of amromycin and streptocins A and B.

[Genetic control of pathways of pyrimidine metabolism in Aspergillus nidulans. 1. Isolation and genetic analysis of 6-azauracil resistant mutants]. / Geneticheskii kontrol' putei metabolizma pirimidinov u Aspergillus nidulans. Soobshchenie I. Vydelenie i geneticheskii analiz mutantov, ustoichivykh k 6-azauritsila.

147 mutants exhibiting the resistance to toxic effect of 6 azauracil have been isolated by nitrosoguanidine treatment from the wild strain of Aspergillus nidulans. This mutants have been divided into 11 phenotypic groups according to its cross-resistance to 5-fluoroderivatives of uracil, uridine and deoxyuridine. The genetic analysis has shown that all mutations of resistance are of nuclear origin and dominant nature, and they are distributed on six loci of the chromosome VIII.

[Metabolic pathways of exogenous pyrimidines in Aspergillus nidulans]. / Puti metabolizma ékzogennykh pirimidinov u Aspergillus nidulans.

Studies on the susceptibility of the wild-type strain of Aspergillus nidulans to 6-azauracil suggest that it synthesizes pyrimidines using a by-pass pathway which is induced with 6-azauracil. The effect of a series of pyrimidines on the toxic action of 5-fluorouracil, 5-fluorouidine and 5-fluorodeoxyuridine has been investigated. As the result of these studies, a scheme is proposed for the metabolism of some pyrimidine bases and nucleosides in Aso. nidulans.