A reporter system for prokaryotic and eukaryotic cells based on the thermostable lichenase from Clostridium thermocellum.

The coding region of the licB gene from Clostridium thermocellum was truncated at the 3' end. The modified lichenase encoded by the construct (LicBM2) retained the most important properties of the enzyme - its high activity and thermostability. LicBM2 consists of the catalytic domain and part of the Pro-Thr-box. We demonstrated the application of the licBM2 gene as a reporter system for prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells by expressing it either as a transcriptional fusion with selected promoters or as a translational fusion with the E. coli uidA gene. The assays available for LicB activity are sensitive, accurate and simple, and can be used for the analysis of various gene fusion systems or for screening of transformants.

Preparation and properties of Clostridium thermocellum lichenase deletion variants and their use for construction of bifunctional hybrid proteins.

Major properties (pH and temperature optimum, stability) of lichenase (beta-1,3-1,4-glucanase) deletion variants from Clostridium thermocellum were comparatively studied. The deletion variant LicBM2 was used to create hybrid bifunctional proteins by fusion with sequences of the green fluorescent protein (GFP) from Aequorea victoria. The data show that in hybrid proteins both GFP and lichenase retain their major properties, namely, GFP remains a fluorescent protein and the lichenase retains activity and high thermostability. Based on the results of this investigation and results that have been obtained earlier, the use of the deletion variants of lichenase and the bifunctional hybrid proteins as reporter proteins is suggested.

Identification of nah-1 genes of the Pseudomonas putida naphthalene-degrading NPL-41 plasmid operon.

Pseudomonas putida BS202 degrades naphthalene via a plasmid-encoded catabolic pathway. The nucleotide sequence of the nahC gene encoding one of this pathway enzymes, 1,2-dihydroxynaphthalene dioxygenase, has been determined. Analysis of nucleotide sequence of its flanking regions identified partially the nahF and putative nahQ genes. Comparison of these three genes with corresponding ones in the NAH7 plasmid and DOX operon showed a high degree of homology.

The use of a thermostable beta-glucanase gene from Clostridium thermocellum as a reporter gene in plants.

In order to take advantage of the high thermostability of its product, beta-1,3;1,4-glucanase (lichenase), we used a modified version of the licB gene from Clostridium thermocellum as a reporter gene for the analysis of gene expression in transformed plants. The coding region of the licB gene was truncated at both ends. The truncated enzyme retained its activity and thermostability. The modified gene (m-licB), with and without a plant leader peptide-encoding sequence, was expressed in tobacco plants under control of either the Agrobacterium octopine TR-DNA 2' gene promoter or the promoter of the gene for the small subunit of ribulose-1,5-bisphosphate carboxylase. Expression of licB can be measured quantitatively and accurately, the assay is sensitive and simple enough to be used for analysis of various gene fusion systems or for screening of transformants. The enzyme is very stable and remains active in tissue extracts even after storage for 1 year and survives many thawing-freezing cycles. The lichenase-encoding gene was expressed at high levels in transformed tobacco plants without any apparent detrimental effects on vegetative growth or flowering.

[Isolation of a spontaneous mutant of Escherichia coli superproducing proline and resistant to elevated concentrations of NaCl]. / Poluchenie spontannogo mutanta Escherichia coli, sposobnogo k sverkhsintezu prolina i ustoichivogo k povyshennoi kontsentratsii NaCl.

We obtained a spontaneous mutant of Escherichia coli that was characterized by both proline superproduction and the resistance to osmotic stress. The selection of mutants was carried out among 2.5.10(5) clones survived upon plating strain SU1604 containing the sex factor F104, with a chromosome fragment carrying genes proB and proA, on solid modium with the proline analogue L-azetidine-2-carboxylate (AzT). The obtained mutant AztR clones were used as donors in replica crossing with a pro- recipient, followed by subsequent selection of AztR Pro+ exconjugants. Analysis of growth of 456 exconjugants in liquid minimal medium with NaCl at a concentration of 0.6 M helped to identify 9 mutants with increased salt tolerance, as compared with control. From these, we selected one mutant (denoted as SU1604/F'104S) which demonstrated the highest salt tolerance correlating with higher production of proline. Analysis of the mutant's properties suggests that it belongs to the group of Osm mutants.

[The study of binding of the virG gene product with the virD gene promotor region of the Agrobacterium tumefaciens Ti-plasmid C58]. / Izuchenie sviazyvaniia produkta gena virG s promotornoi oblast'iu gena virD Ti-plazmidy Agrobacterium tumefaciens C58.

The 1.5 kb EcoRI--HindIII fragment of the pTiC58 containing the virD regulatory sequence demonstrates a constitutive promoter activity in E. coli background and an inducible one in agrobacterium. The virG gene was cloned in pTZ19R plasmid. To reveal the virG product--virD regulatory sequence interaction a few protein fractions of E. coli harbouring the obtained recombinant plasmid pTZ19G lysate were used. PAGE-retardation assay revealed the specific binding between the 1.5 kb DNA fragment containing 5'-end of virD and a separate protein fraction of the bacterial lysate.

The use of bacterial genes encoding herbicide tolerance in constructing transgenic plants.

The modes of action of some of the best-studied and widespread herbicides are briefly reviewed. Particular attention is given to those herbicide-inhibited processes that bacteria and plants have in common. We describe bacterial mutant genes of herbicide resistance, peculiarities of their introduction into plants, and success in the construction of transgenic resistant plants.

[Transfer of hybrid plasmids pRP1.2::MINI-Mu into Agrobacterium and Rhizobium cells]. / Perenos gibridnykh plazmid pRP1.2::MINI-Mu v kletki Agrobacterium i Rhizobium.

The study is devoted to determination of bacteriophage Mu genome regions responsible for transfer limitation and instability of the plasmids in cells of strains of practically important microorganisms. With this aim in view, we determined the frequency of transfer into Agrobacterium tumefaciens and Rhizobium meliloti cells of plasmids with mini-Mu phages carrying previously constructed deletions of various lengths. Sharp decrease has been noted in the frequency of transfer into A. tumefaciens strain PG2592 of all the plasmids used, as compared with the initial plasmid pRP1.2 with no dependence on the availability of mini-Mu killing functions. This gives evidence that deletions in the mini-Mu utilized do not include the sites affected by the recipient' restriction system. As regards R. meliloti L5-30-M27, it appeared that the transfer of pRM30 plasmid carrying mini-Mu 5 with conserved killing functions (the ability for autonomous transposition) is of the same frequency as the transfer of pRP1.2. In this mini-Mu, the region between the extreme HpAI sites in the right end is missing, this region being probably responsible for such low frequency of transfer into Rhizobium cells of Mu-containing plasmid.

[Effect of mutations of Escherichia coli K-12 chromosome on the integration of bacteriophage Mu introduced into cells by infection or conjugation]. / Vliianie mutatsii Escherichia coli K-12 na vnedrenie v khromosomu genoma bakteriofaga Mu, vnesennogo v kletki pri infektsii ili kon''iugatsii.

We present the detailed research on the previously described Escherichia coli K-12 Mud- mutants with impaired development of bacteriophage Mu. The ability of Mu phage DNA to penetrate into mutant cells on infection was shown. If introduced into the cells or combined with mud mutation by recombination, the prophage may be induced, which results in phage Mu lythic development and phage burst from mutant cells. In the course of conjugative transfer into the mutant cells, within a DNA fragment of the lysogenic donor chromosome, MupAp1 prophage is not inherited by recombinants. At the same time, Mu prophage deficient in genes A and B, whose products are required for transposition, is inherited by the mutant with the usual frequency. These data enable us to conclude that the mud mutations disturb the stage of conservative transposition which is connected with the insertion of the Mu prophage into the chromosome, after excision from the linear DNA introduced into the cells via infection or conjugation.

[Genetic analysis of Escherichia coli min81 mutation blocking the development of bacteriophage Mu]. / Geneticheskoe izuchenie mutatsii min81 Escherichia coli, blokiruiushchei razvitie bakteriofaga Mu.

Data characterizing mim81 mutation obtained by the method for direct selection of transposition mutations are presented. The development of Mu is shown to be dramatically suppressed in the mutant strain both upon infection and after induction from the lysogenic state. Frequencies of lysogenization and mini-Mu-dependent formation of cointegrates in the mutant strain are comparable with those in the wild-type strain. Mu development prohibition is removed if expression of early Mu gene is provided from the modified Pe promoter. The results obtained make us believe that the mechanism of mim81 mutation action involves reduction of early gene expression to the level that is sufficient for Mu DNA integration into the chromosome during infection and for single replicative events, but insufficient for vegetative development of bacteriophage Mu.

[Transposition immunity in bacteriophage Mu. The effect of a mutation at the kil gene on the establishment of immunity]. / Immunnost' transpozitsii bakteriofaga Mu. Vliianie mutatsii po genu kil na ustanovlenie immunnosti.

Bacteriophage Mu is characterized by a phenomenon similar to the transposition immunity of TnA: the frequency of transposition of Mu or mini-Mu into plasmids containing certain phage sequences is reduced by two orders of magnitude. In order to lend transposition immunity to Mu, the recipient replicon must contain a sequence of phage DNA including a 5.1 kb early region from the c-end of Mu. The product of the kil (or cim) gene takes part in establishing the immunity. The transposition immunity of Mu is connected with the disturbance of cointegrate formation.

[Genetic analysis of the Mud mutants of Escherichia coli K-12 with an impaired ability to maintain bacteriophage Mu development]. / Geneticheskii analiz Mud-mutantov Escherichia coli K-12 s narushennoi sposobnost'iu podderzhivat' razvitie bakteriofaga Mu.

Properties of mud mutations affecting intracellular development of bacteriophage Mu have been studied. We demonstrate pleiotropy of these mutations as well as their effect on the development of Mu-like heteroimmune D108 phage and P1 phage, on the ability to inherit Mu prophage within the chromosome during conjugation, and the abortive inheritance of chromosomal markers during P1 transduction. Experimental evidence is given to support the concept that the effect of the mutations has no relation to the mutants' impaired ability to adsorb the phages. Genetic analysis which involved conjugational crossings using different Hfr donors and F' plasmids made it possible to localize the mud-1 mutation between ilv and metE genes in the region of 84-85 min of the Escherichia coli K-12 genetic map. The nature of mutations and a possible functional role of the mud-impaired gene is discussed.

[Effect of the polymerase activity of DNA polymerase I on the development of the temperate bacteriophages Mu, lambda red- and lambda red-gam-. I. The effect of the polymerase activity of DNA polymerase I on the development of bacteriophages lambda red- and lambda red-gam-]. / Vliianie polimeraznoi aktivnosti DNK-polimerazy I na razvitie umerennykh bakteriofagov Mu, lambda red-, lambda red-gam-. Soobshchenie I. Vliianie polimeraznoi aktivnosti DNK-polimerazy I na razvitie bakteriofagov lambda red-, lambda red-gam-.

Phages lambda c1857 red3 and lambda bio10 in Escherichia coli K-12 cells with impaired function of DNA-polymerase I polymerizing activity are shown to restore their normal development level when exonucleases V and I are removed from cells. In other words, an indirect involvement of DNA-polymerase I in the development of phages lambda defective in general recombination systems and gene gam functions has been established. A probability of DNA-polymerase I participation in the recombination stage of phage lambda development in E. coli K-12 cells is discussed.

[Effect of the polymerase activity of DNA polymerase I on the development of moderate bacteriophages Mu, lambda red- and lambda red-gam-. II. The effect of the polymerase activity of DNA polymerase I on the development of bacteriophage Mu]. / Vliianie polimeraznoi aktivnosti DNK-polimerazy I na razvitie umerennykh bakteriofagov Mu, lambda red-, lambda red-gam-. Soobshchenie II. Vliianie polimeraznoi aktivnosti DNK-polimerazy I na razvitie bakteriofaga Mu.

The paper reports on the influence of polymerizing activity of DNA-polymerase I on different developmental stages of temperate bacteriophage Mu in Escherichia coli K-12 cells. This activity is shown to be necessary for optimization of phage Mu primary integration into cell chromosomes. The relative frequency of Mu integration into bacterial chromosomes is 5-6 times lower in polA cells than in isogenic polA+ control strains, the phage yield from cells being delayed during the phage infectious development, but not in the course of induction from the prophage state. Data have been obtained that show the process of phage Mu DNA integration into the plasmid pRP1 .2 and the process of Mu transposition from the cell chromosome into the plasmid to be independent of the polymerizing activity of DNA-polymerase I.

[Integration of the mini-Mu phage into multicopy plasmids]. / Integratsiia mini-Mu-faga v mul'tikopiinye plazmidy.

Preparation of mini-Mu4 phage introduced into the pRP1.2 plasmid and carrying a 18 kb deletion of the central EcoRI fragment is described. The ability of the mini-Mu4 for independent transposition is demonstrated in experiments conducted for cointegrate formation (replicon fusion). The system developed is applicable to studying the mechanism of transposition, since the frequency of cointegrate formation may be descriptive of the transposition process. Frequency changes due to some factors may point to a possible influence of these factors on the mini-Mu transcription. The described mini-Mu integration into small multicopy plasmids of Col type makes it possible to conduct a simple physical analysis of structures formed in the course of transposition.

[Effect of amino acid substitutions in the polypeptide chain of DNA-polymerase on manifestation of the mutator effect]. / Vliianie aminokislotnykh zamen v polipeptidnoi tsepi DNK-polimerazy na proiavlenie mutatornogo éffekta.

Thin map of gene 43, controlling the synthesis of T4 DNA polymerase, is obtained by mapping experiments performed with 39 amber mutants, and is used for analysis of the sites of DNA polymerase gene from the point of view of displaying the mutator effect. The mutant sites studied possessed different reaction on amino acid substitutions in the polypeptide chain of the enzyme. Most of sites of the DNA polymerase gene, with the exception of two "supersensitive", responsed only on the apparent type of the amino acid substitutions: the mutator effect of amber mutations, which are located at these sites, was exhibited only in the case of insertion of the definite amino acid in the respective point of polypeptide chain. The proposed system of amber mutations for studying the mutator effect, allowed the authors to obtain the data on the effect of concrete alterations in the polypeptide chain of the enzyme on the development of its mutator properties.

[Mutator effect of suppressed amber-alleles of early genes of bacteriophage T4]. / Izuchenie mutatornogo éffekta u supressirovannykh amber-allelei rannikh genov bakteriofagov T4

The mutator effect of amber alleles of three early genes (43, 32, 47), which were suppressed by the bacterial suppressor gene, was studied. There are some advantages in using the suppressed amber alleles instead of ts, because in this case definite amino acid substitutions take place in the protein due to certain suppressor gene effect. For example, in Escherichia coli CR63(Su+-1) the replacement of the original amino acid by serine takes place. Studying the mutator effect of 43 alleles of DNA polymerase gene of phage T4 with tester mutant r131 showed that in condition of suppression by the gene Su+ -1 only 13,9% of alleles possessed the mutator activity. In the same experiments with mutants of genes 32 and 47 the mutator effect was not observed.

[Allele specificity of the mutator action of bacteriophage T4 genes 43 and 32]. / Allel'-spetsifichnost' mutatornogo deistiviia genov 43 i 32 bakteriofaga T4

The substitution of tester mutant ri31 (fs-type) for transition mutants rUV30 and rUV48 allowed to reveal new mutator alleles of DNA polymerase gene of phage T4. In these conditions about 70% alleles of this gene were found to be mutators. Unlike experiments carried out with mutant-tester ri31, in experiments with mutants rUV30 and rUV48 the presence of mutator alleles in gene 32 was demonstrated. The transition pathway AT leads to GC is the preferential direction of the mutation alteration under the action of suppressed amber alleles of genes 43 and 32. The data obtained suggest allele specificity of the mutator effect in bacteriophage T4.