[Dependence of aminoglycoside 3'-phosphotransferase VIII activity on protein serine/threonine kinases in Streptomyces rimosus].

In Streptomyces rimosus, selection with aminoglycoside kanamycin triggers "silent" aminoglycoside 3'-phosphotransferase (aph) VIII gene. Expression of aphVIII was accompanied by amplification of a chromosomal DNA fragment, which contained aphVIII. Earlier, S. rimosus aphVIII gene was isolated, sequenced, and deduced APHVIII protein sequence was reported. Using in vitro labeling and immunoprecipitation with anti-APHVIII antibody, we demonstrate that one of the abundant proteins phosphorylated by endogenous protein kinases (PKs) in extracts of S. rimosus strain S683 is APHVIII. Phosphoamino acid assay has shown phosphorylation of two seryl residues in APH molecule. The amount of phosphate incorporated into APHVIII in the presence of Ca2+ was 1.84-fold as much as that detected without Ca2+. As shown by in the gel self-phosphorylation and in the substrate-containing gel phosphorylation analyses, two serine PKs with molecular masses of 74 kDa and 55 kDa were active against APHVIII. The 55-kDa PK showed a clear Ca2+ and calmodulin dependency in activity. The specific kanamycin phosphotransferase activity of exhaustedly phosphorylated APHVIII was 3.72-fold as much as that detected in the preparation of nonphosphorylated enzyme. These results suggest involvement of PKs under study in the modulation of APHVIII aminoglycoside phosphorylating activity and in the generation of kanamycin resistance in S. rimosus.

[Identification of Rad51 protein from Chlamydomonas reinhardtii: recombinational characteristics].

The unicellular green microalga Chlamydomonas reinhardtii is a perspective model object for basic and applied research. However, its homologous recombination (HR) system which lies in the basis of double-strand DNA break repair have still not been studied. Last years the program of C. reinhardtii nuclear genome sequence is realized and different nucleotide repeats in the genome structure have been revealed that can explain a low level of HR relative to nonhomologous recombination events. Analyses of the C. reinhardtii EST (Expressed Sequence Tag)--and genome libraries permitted us to reconstruct and clone cDNA of the RAD51 gene. In present work, the cDNA was expressed, its product was purified and some basal biochemical activities were studied. The results show that Rad51C protein from lower eukaryote C. reinhardtii is identified as typical representative of the Rad51C-like subfamily of higher eukaryotes.

[Streptomyces rimosus aminoglycoside-3'-phosphotransferase VIII: comparisons with aminoglycoside-3'-phosphotransferases of aminoglycoside-producing strains with eukaryotic protein kinases]. / Aminoglikozid-3'-fosfotransferaza VIII iz Streptomyces rimosus: sravnenie s aminoglikozid-3'-fosfotransferazami iz shtammov-produtsentov aminoglikozidov i s éukarioticheskimi proteinkinazami.

The nucleotide sequence was established for the aphVIII aminoglycoside phosphotransferase gene of an oxytetracycline-producing Streptomyces rimosus strain. The gene is 804 bp in size and possibly codes for APHVIII of 267 residues. Heterologous expression of aphVIII was studied in Escherichia coli and Chlamydomonas reinhardtii. The deduced APHVIII sequence was compared with known sequences of aminoglycoside phosphotransferases of aminoglycoside-producing actinomycete strains and of eukaryotic protein kinases. A local homology of 38 residues was found between APHVIII and actinomycete serine-threonine protein kinases in the conserved region possibly involved in ATP binding. APHVIII differed from aminoglycoside 3'-phosphotransferases of aminoglycoside-producing actinomycete strains and of clinical isolates, and can be classed to a separate group.

[Determination of the nucleotide sequence of the silent aminoglycoside phosphotransferase gene in Streptomyces rimosus P3]. / Opredelenie nukleotidnoi posledovatel'nosti "molchashchego" gena aminoglikozidfosfotransferazy Streptomyces rimosus P3.

The nucleotide sequence BgIII-Pstl of the DNA fragment containing the aph gene cloned from Streptomyces rimosus P3 not producing aminoglycoside antibiotics was determined. The aph gene was shown to encode neomycin phosphotransferase differing by the substrate specificity from the enzyme encoded by the aph genes from the organisms producing aminoglycoside antibiotics. Comparison of the cloned aph VIII gene and its product with the aph genes and their products from the antibiotic-producing actinomycetes and clinical microbial strains permitted to consider it as a representative of the third group genes which are likely widely distributed in soil microorganisms not producing aminoglycoside antibiotics. The gene length was 777 nucleotide pairs at the average GC content of 67 per cent. Comparison of the nucleotide and protein sequences of the S.rimosus gene with those of the genes cloned from the aminoglycoside-producing organisms (S.fradiae and Micromonospora chalcea) revealed high homology in the 3'-end region of the genes. However, the homology percentage by DNA for the S.rimosus gene was lower than that for the genes from the other organisms on their comparison with each other: 56-67 and 82-86 per cent respectively. Comparison of the profiles of the protein hydrophilic properties revealed differences in the region of the first 110 amino acids of the gene under the investigation. The amino acid sequence contained all the highly conservative regions characteristics of aminoglycoside phosphotransferases but had several amino acid substitutes detected for the first time including those in the region responsible for the antibiotic binding.

[Determination of the nucleotide sequence and characterization of the novel aminoglycoside phosphotransferase aphVIII gene from the Streptomyces rimosus strain]. / Opredelenie nukleotidnoi posledovatel'nosti i kharakteristika novogo aminoglikozidfosfotransferaznogo gena aphVIII ia shtamma Streptomyces rimosus]

The nucleotide sequence of a BglII-PstI DNA fragment that contains the cloned aphVIII gene from the Streptomyces rimosus P3 strain, the producer of oxytetracycline, was determined. It was established that the aph gene encodes neomycin phosphotransferase that differs by substrate specificity from neomycin phosphotransferases encoded by aph genes in producers of aminoglycoside antibiotics and clinical bacterial strains. The gene was shown to be 777 bp in length with the mean GC content equal to 67%. The amino acid sequence possesses all highly conserved regions typical for aminoglycoside phosphotransferases; however, this sequence contained several amino acid substitutions that have been detected for the first time, including those in the domain responsible for the association with antibiotics.

Stable nuclear transformation of Chlamydomonas reinhardtii with a Streptomyces rimosus gene as the selective marker.

The aminoglycoside 3'-phosphotransferase type VIII (APHVIII) encoding gene (aphVIII) from Streptomyces rimosus was introduced by glass-bead high-efficiency transformation into the nuclear genome of green unicellular alga Chlamydomonas reinhardtii for induction of transformants resistant to aminoglycoside antibiotics. The aphVIII structural sequence was flanked by S. rimosus regulatory sequences which failed to direct expression in C. reinhardtii. The pSU937 plasmid containing these sequences was able to transform C. reinhardtii strain cw15 arg7-8 mt+ for paromomycin resistance (PmR) at a frequency (1.3-1.9) x 10(-7), probably as a result of in vivo gene fusion and expression of the aphVIII gene from regulatory elements of nuclear DNA. Evidence for the real C. reinhardtii transformation includes blot-hybridization with a probe specific for aphVIII and demonstration of APHVIII enzyme activity in crude cell extracts of transformants. Integrated Streptomyces DNA sequences, APHVIII enzyme activity and the aminoglycoside-resistance phenotype were stable through mitosis in the presence and absence of selection. The PmR phenotype was inherited by the meiotic progeny of transformants from crosses with wild-type strains.

[Analysis of a chromosomal DNA fragment isolated from a hybrid autonomous plasmid of Chlamydomonas reinhardtii]. / Analiz fragmenta khromosomnoi DNK, izolirovannogo iz gibridnoi avtonomnoi plazmidy Chlamydomonas reinhardtii.

The ability of a previously cloned fragment of nuclear DNA of Chlamydomonas reinhardtii (a 2.4-kb BamH 1-EcoR I fragment) to function as the origin of replication of plasmid pARG7.8.2.4, carrying the homologous arg7 marker gene of prototrophy or arginine in cells of C. reinhardtii transformants, was studied. As shown by Southern blotting, plasmid pARG7.8.2.4 integrates at several sites of chromosomal DNA of chlamydomonas without autonomous maintenance in cells. Results obtained using Southern blotting and plasmid rescue techniques demonstrated that plasmid pARG7.8, containing only the arg7 gene, is able both to integrate into the chromosome and to be maintained, in some cases, in the autonomous state in Arg+ transformants of alga. Analysis of the nucleotide sequence of the DNA fragment under study revealed several types of GC-enriched repeats that manifested homology with the nucleotide sequence of the arg7 gene in introns 10 and 12.

[Kinetics of DNA-distaxin complex-formation]. / Kinetika kompleksoobrazovaniia DNK s distaktsinami.

Kinetics of DNA--distaxines complexes formation was studied spectrophotometrically by means of the stop--flow method. It has been found that increase of number of bulky pyrrolcarboxamide group in the row of distaxines: -0, -1, -2, - slows down the binding reactions and increases the number of their stages.

[Kinetics of the formation of AT-specific DNA-distamin complex]. / Kinetika obrazovaniia AT-spetsificheskogo kompleksa DNK-distamin.

Kinetics of DNA--distamin complex formation was studied by means of the stop-flow method. It has been found that the binding reaction is much faster than in the case with distamycin A and has only two stages. The differences in kinetic properties of the complexes of distamin and distamycin A with DNA has been interpreted as resulting from the formation of different bonds by protonated positively charged N-containing groups of ligands with negatively charged DNA phosphates.

[Action of levorin on amino acid transport in the rat small intestine]. / Deistvie levorina na transport aminokislot v tonkoi kishke krysy.

Levorin, a polyenic antibiotic, in concentration of 2.10(-6) M markedly inhibited the active transport of amino acids in the cells of rat thin intestine, thus decreasing the oxygen dependent transfer of 14C-glycine and 14C-leucine and antigradient accumulation of glycine in the enterocytes. The effect increases with an increase in the period of the intestine mucosa contact with the antibiotic. Introduction of levorin from th serous side had no effect on the glycine transport. Inhibition of the transmembrane transport of glycine by levorin is due to its effect on transfer and accumulation of amino acids in the cells.

[Action of levorin on glucose transport in the rat small intestine]. / Deistvie levorina na transport gliukozy v tonkoi kishke krysy.

The technique of accumulating preparation of the mucosa and "turned out sac" was used to show that levorin, a polyenic antibiotic in a concentration of 10(-6) M, lowered the transport rate and accumulation of glucose by the epithelial cells of the rat thin intestine under conditions of oxygenation. Suppression of the glucose transport in the first stages resulted in partial inhibition of the transmembrane transfer. It is suggested that levorin suppression of the glucose transport through the erythrocyte apical membrane in the thin intestine is associated with a decrease in the electrochemical gradient of Na+.

[Doses levorin display an antiandrogenic action?]. / Okazyvaet li levorin antiandrogennoe deistvie?

The antiandrogenic effect of levorin on immature castrated rats treated with exogenic testosterone was studied. In a dose of 200 mg/kg levorin lowered the cholesterin blood levels in the rats, inhibited the testosterone-induced increase in RNA concentration in the ventral and dorsal prostate and the seminal vesicles and to a less extent suppressed the growth of the accessory sexual glands. However, the antiandrogenic effect was observed with the use of levorin in the dose producing a pronounced toxic action evident from death of a part of the animals and a marked decrease in the animal body weight. This fact casts doubt on specificity of the levorin effect. Apparently, in high doses levorin impairs metabolism as a whole which cannot but affect the response of the sexual glands to administration of testosterone.