[Cloning of genes, purification and properties investigation of recombinant DNA ligases from the thermophilic archaeon Pyrococcus abyssi and Methanobacterium thermoautotrophicum].

The genes encoding of DNA ligases from the thermophilic archaeon Pyrococcus abyssi (PabDNA ligase) and Methanobacterium thermoautotrophicum (MthDNA ligase) were cloned and expressed in Escherichia coli. The activity of purified enzymes was studied by ligation of two oligonucleotides, one of which had preformed hairpin structure. In the used system the maximal output of reaction products for both DNA ligases was observed near 70 degrees C that is explained by substrate thermostability. At stoichiometric ratio of enzymes and substrate the output of a product reaches of plateau at 70-75% of theoretical ones. Investigated DNA ligases showed different thermostability. The half-time life of PabDNA ligase was about 60 min at 90 degrees C. MthDNA ligase was completely inactivated at this temperature during 10 min. Recombinant DNA ligases from P. abyssi and M. thermoautotrophicum possessed high stability during a storage at 4 degrees C.

Increase of tyrosine hydroxylase and dopamine transporter mRNA levels in ventral tegmental area of male mice under influence of repeated aggression experience.

Tyrosine hydroxylase (TH) and dopamine transporter (DAT) mRNA levels in the ventral tegmental area (VTA) of midbrain were measured by multiplex RT-PCR in male mice with repeated experience of social victories (winners) and social defeats (losers) in 10 daily agonistic confrontations. Two independent experiments revealed enhanced TH and DAT mRNA levels in VTA of the winners in comparison with the losers and controls (animals after 5 days of individual housing). A positive correlation between DAT and TH mRNA levels was shown.

[Effective expression of genes for interleukin-2 and its mutant analogs in E. coli cells]. / Effektivnaia ékspressiia v kletkakh E. coli genov interleikina-2 cheloveka i ego mutantnykh analogov.

Recombinant plasmids were constructed for the efficient expression in E. coli cells of the human interleukin-2 (HIL-2) gene and two its mutant analogues obtained by of chemical-enzymic synthesis and polymerase chain reaction (deletion of 14 C-terminal amino acids and a change of the codon for Trp121 to Phe). The recombinant HIL-2 but not the mutant analogues were shown to be biologically active. Both analogues obtained were weak antagonists to HIL-2.

[Cloning of bacteriophage T5 DNA fragments in the plasmid pBR322. Analysis of recombinant plasmids by the method of bonding with RNA-polymerase from Escherichia coli on nitrocellulose filters]. / Klonirovanie fragmentov DNK bakteriofaga T5 v plazmide pBR322. Analiz rekombinantnykh plazmid metodom sviazyvaniia s RNK-polimerazoi Escherichia coli na nitrotselliuloznykh fil'trakh.

DNA of bacteriophage T5 was hydrolyzed with restriction endonucleases HindIII and BamHI, and subjected to the combined hydrolysis with BamHI+EcoRI and BamHI+ +HindIII. Fragments obtained were cloned in the plasmid pBR322. About 17% of T5 genome were recovered in recombinant plasmids. Cloned fragments were localized on the physical map of the phage by restriction analysis and Southern hybridization. With the aim of direct cloning of T5 promoters, PstI/HindIII fragments were inserted into pBR322 followed by selection of recombinants on ApsTCr phenotype. Binding of BsuRI and AluI fragments of hybrid plasmids with E. coli RNA polymerase was studied by nitrocellulose filter assay. The fragments, which were capable to form heparin resistant complexes were identified.

Cloning of genes for bacteriophage T5 stable RNAs.

One EcoRI-generated fragment (440 basepairs) and two EcoRI/HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage lambda XIII and the plasmid pBR322 as vectors. Recombinant DNA molecules were studied by hybridization with in vivo 32P-labeled T5 4-5 S RNAs on nitrocellulose filters. Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments. For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed. It was shown that the EcoRI 440 fragment contains the gene for tRNA 10 (tRNAAsp), the EcoRI/HindIII 220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNAHis), and the EcoRI/HindIII 960 fragment contains only a part of the gene for tRNA 9 (tRNAGln). The arrangement of these genes on the physical map of T5 phage was as follows: -tRNAGln-tRNAHis-RNA III-RNA I-...-tRNAAsp.

Cloning of bacteriophage T5 promoters.

Bacteriophage T5 was subjected to combined hydrolysis with the restriction endonucleases PstI and HindIII and the resulting fragments were inserted into the plasmid pBR322. Selection of transformants for Aps-Tcr-phenotype made it possible to screen the hybrid plasmids that contained promoter sequences in the cloned fragments. Two PstI/HindIII fragments, 720 bp (51% of the T5 DNA length) and 1,200 bp (70%) were cloned in this study. Tcr levels for these plasmids were as high as 18 micrograms/ml and 75 micrograms/ml, respectively. The presence of Escherichia coli RNA polymerase binding sites on both fragments was shown using the nitrocellulose filter assay. These binding sites are situated between 35 bp and 95 bp from the HindIII cleavage site on the 1,200 bp fragment; and within 420 bp from the HindIII site on the 720 bp fragment.

[Isolation and characteristics of highly purified S1-nuclease from amylorizin P10X]. / Vydelenie i kharakteristika vysokoochishchennoi S1-nukleazy iz amilorizina P10X.

S1-nuclease was purified from the Soviet commercial enzyme amylorizin P10x prepared from the surface culture of Aspergillus oryzae. The enzyme yield was 33% of total activity. The molecular weight of the enzyme measured by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was equal to 30,000. The enzyme showed high specificity to single-stranded DNA.

Isolation of transposon TnA from plasmid RP4 carrying two copies of this element.

Employing heteroduplex and restriction analyses, two inverted copies of a 3.2.10(6) dalton transposable sequence, TnA, were found in RP4::TnA, a spontaneously arisen derivative of the plasmid RP4. Integration of the second copy of TnA causes loss of the conjugative properties of RP4. Both TnA sequences in RP4::TnA were localized and found to have opposite orientations. The DNA fragment corresponding to the individual transposon TnA was isolated after the endonuclease S1 digestion of RP4::TnA molecules annealed under conditions favoring intramolecular renaturation. The attempts to transform the cells of Escherichia coli QD5003, HB101[pCRI] and JC7623 with the isolated transposon were unsuccessful.