[MAST2-like protein kinase from grape vine Vitis vinifera: cloning of catalytic domain cDNA].

The aim of our work is the identification of protein kinases phosphorylating microtubule proteins in plant cells. Using bioinformatic approach, we found genes of putative homologues of microtubule-associated mammalian protein kinase MAST2 in higher plant genomes. The gene of closest MAST2 homologue, putative protein, named GMLK (Grape MAST2-Like Kinase, A7NTE9_VITVI), was found in grape Vitis vinifera. We report here the cloning of cDNA of GMLK (A7NTE9) from Pinot Noir grape vine leaves.

[Nuclease S1 for DNA molecular weight determination in multiplasmid Escherichia coli strains]. / Primenenie nukleazy S1 dlia opredeleniia molekuliarnoi massy DNK v mul'tiplazmidnykh shtammakh Escherhicia coli.

DNase treatment used to convert supercoiled DNAs to the open form is suitable for molecular weight determination by electron microscopy or agarose gel electrophoresis. The comparative experiments showed that S1-nuclease has advantages over DNase for molecular weight determination in preparations containing many species of plasmids of significantly differing sizes, isolated from multiplasmid E. coli strains.

[Covalent bond between RNA and protein in encephalomyocarditis virus]. / Kovalentnoe soedinenie RNK i belka v viruse zntsefalomnokardita.

Deproteinized encephalomyocarditis [32P]RNA, after digestion with a mixture of RNases A, T1 and T2, yields mononucleotides and a labelled compound, which is positively charged at pH 3.5. This product can be digested with pronase and has a close electrophoretic mobility to a protein with a molecular weight of 7000-8000. Covalently bound nucleotide-peptides were isolated from this compound after treatment with RNases and pronase. It was shown that the 5'-terminal uridylic acid is covalently linked with peptides. The phosphodiester bond between uridylic acid and peptides is discussed.

[Non-radioactive diagnosis of viral infections using "DNA-peroxidase" probes]. / Neradioaktivnaia diagnostika virusnykh infektsii zondami "DNA-peroksidaza".

DNA-peroxidase probes were synthesized according to a modified method (Renzetal) for the detection of lambda phage DNA (model system), polio, potato X and M, tobacco mosaic viral RNAs by spot hybridization onto nitrocellulose membranes. cDNAs (300-1400 bases) complementary to the viral RNAs were cloned in M13 phage DNA or pTZ19. Efficacy of each step of the probe construction and the diagnostic procedure were thoroughly examined. Peroxidase activity manifested with non-toxic stain (NTS) was 3-5 fold more sensitive in comparison with alpha-Cl-naphthol or bisanisidine. It was found that HRP became much more stable to heat in diluted samples and was 2-3 fold more active after coupling with polyethylene imine spacer. Also, sodium borohydride reduction of the cDNA and PEI-HRP adduct crosslinked by the glutardialdehyde resulted in the stabilization of the probes. Target nucleic acids or diagnostic samples were efficiently fixed onto nitrocellulose membranes by a short-time UV irradiation. Diagnostics of cellular extracts with the preliminary prepared probes takes 4-5 hours due to express hybridization (1 hr) with 100-200 ng/ml of specific nucleotide sequence. Up to 20 pg (less than 10(-17) M) of the purified viral nucleic acids and 30-50 pg of them in the total fraction of the cellular nucleic acids isolated from the infected cells were identified with the probes. 50-10000 fold diluted lysate of the HeLa cells infected with poliovirus (PV1) and both crude extracts of potato tuber or potato and tobacco leaf tissues infected with PVX, PVM or TMV displayed specific signals with the respective DNA-HRP probes.

[The type of covalent bond in a natural complex of the bacteriophage phi X 174 protein A* and nucleotides]. / Izuchenie tipa kovalentnoi sviazi v prirodnom soedinenii belka A* bakteriofaga phi X 174 s nukleotidami.

The 32P-labelled A* protein has been isolated from E. coli cells infected by phage phi X174 in the presence of [32P]orthophosphate. The snake venom phosphodiesterase treatment of the [32P]peptides obtained by the pronase digestion of the protein has revealed a phosphodiester bond between the protein and a nucleotide material of A, G base composition. The hydrolysis of nucleotide-peptides with a mixture of concentrated HCl and CF3COOH has yielded 4'O-phosphotyrosine.

[The nature of a covalent bond between the relaxation protein and ColE1-DNA in the relaxation complex]. / Priroda kovalentnoi sviazi relaksiruiushchego belka i ColE1-DNK v relaksatsionnom komplekse.

O-[32P]phosphoserine was found to be the only phosphoamino acid in the acid hydrolysate of the [32P]ColE1 DNA-peptide produced by action of proteases on the ColE1 DNA relaxation complex. This finding suggests that the relaxation protein is bound to ColE1 DNA in the relaxation complex via a phosphodiester linkage between a serine hydroxyl of the protein and the 5'-phosphate of the terminal deoxycytidine residue of the DNA.

[Replication characteristics of colicinogenic plasmid ColK in Escherichia coli cells]. / Osobennosti replikatsii kolitsinogennoi plazmidy ColK v kletkakh Escherichia coli.

A fraction of plasmid DNA from K and X colicins producing Escherichia coli K235 cells was studied. Cells of this strain are shown to contain four types of plasmids with molecular weights of 21.6.10(6), 38.8.10(6) daltons. Transformation of E. coli C600 by a total plasmid DNA yielded clones containing a single plasmid - colicinogenic K factor (ColK, a mol wt 4.4.10(6)). ColK DNA is present in cells in a large number of copies, replicates in the presence of chloramphenicol, requires DNA polymerase I. Two fragments with mol wts 3.4 and 1.1.10(6) are formed when ColK DNA is treated with EcoRI enzyme. After circularization using phage T4 DNA ligase, the 3.4.10(6) fragment was capable of autonomous replication and stable maintenance in E. coli cells, replicated in the presence of chloramphenicol and though unable to synthesize colicin, confered upon cells resistance to colicin K. The mode of ColK DNA replication is studied in mutants temperature-sensitive for the replication of chromosomal DNA. ColK DNA replication is shown to be virtually independent of the dnaA gene product and only slightly dependent on that of the dnaC gene. No replication occurs in the dnaB, dnaF and dnaG mutants at non-permissive temperature.

[Characteristics of an enzyme hydrolyzing the covalent bond between RNA and protein VPg of the encephalomyocarditis virus]. / Kharakteristika fermenta, gidrolizuiushchego prirodnuiu kovalentnuiu sviaz' mezhdu RNK i belkom VPg virusa entsefalomiokardita.

The enzyme termed by us as uridilylpolynucleotide-(5'P----O)-tyrosine phosphodiesterase (Y-pUpN PDE) was isolated from mouse ascites Krebs II cells by ion-exchange and affinity chromatography. The enzyme was found to specifically split the natural covalent bond between VPg and EMC or polio viral RNAs. The enzyme is completely inactivated at 55 degrees C and partially by EDTA. The enzyme preparation isolated by the above-mentioned procedure is not homogeneous and contains inhibiting admixture(s). Possible role of the enzyme in living cells is discussed.

[Detection of a protein firmly bound to the RNA of radish mosaic virus]. / Obnaruzhenie belka, prochno sviazannogo s RNK virusa mozaiki redisa.

The firmly bound protein VPg was found to be linked to the phenol-deproteinized virion RNAs of radish mosaic virus. This protein was specifically labelled with the [125I]-Bolton-Hunter reagent in vitro. The molecular weight of VPg as determined by SDS-PAAG electrophoresis is equal to 4000. It was assumed that VPg is covalently linked to the 5'-end of radish mosaic virus RNAs.

[An enzyme hydrolyzing the covalent bond between RNA and VPg of picornaviruses. Substrates and methods of analysis of activity]. / Ferment, gidroliziruiushchii kovalentnuiu sviaz' mezhdu RNK i VPg pikornavirusov. Substraty i metody analiza aktivnosti.

Some new substrates--RNA-VPg, RNA-peptide and a small fragment of RNA peptide--labelled at the peptide component with [125I]Bolton-Hunter reagent have been proposed for use in the isolation and characterization of the enzyme hydrolyzing the phosphodiester bond between the VPg protein and encephalomyocarditis virus RNA. A novel procedure for the analysis of the specific enzyme activity is based on thin-layer chromatography of hydrolytic products on silicagel or polyethylenimine cellulose. The molecular mass of the enzyme isolated by a modified procedure involving FPLC is 90-95 kD.

[Natural substrates of uridylylpolynucleotide-(5'P--->O)-tyrosine phosphodiesterase--an enzyme, hydrolyzing the covalent bond between RNA and picornaviral VPg and a synthetic model of them]. / Prirodnye substraty uridililpolinukleotid-(5'P--->O)-tirozin fosfodiésterazy--fermenta, gidrolizuiushchego kovalentnuiu sviaz' mezhdu RNK i VPg pikornavirusov, i ikh sinteticheskie modeli.

A phosphodiester derivative of 3,5-[125I]diiodotyrosine and 5'-[32P]oligodeoxynucleotide mimicking a natural compound--the picornaviral RNA Pg complex--was obtained by the method of active N-hydroxybenzotriazole phosphodiesters. Using the isotope iodine ion exchange reaction in the tyrosine residue, the specific activity of the model compound could be increased 50 times. It has been found that the unlinking enzyme does not hydrolyze the phosphodiester bond between the tyrosine residue and the oligodeoxynucleotide. Treatment of viral RNA-VPg by nuclease S1 gave a 5'-terminal fragment of RNA linked with the K-peptide. This fragment was shown to be split at a higher rate in comparison with the original compound, RNA-VPg. A two-step procedure for obtaining picornaviral RNA-KpA-Kp and RNA-VPg compounds with higher specific activity selectivity labelled at the protein fragment with 125I was developed. At the first stage, aliphatic amino groups of the polypeptide within the RNA-VPg or RNA-Kp complex were selectively acylated by the activated ester of hydroxyphenylpropionic acid, whose residue was iodinated at the second stage by radioactive NaI in the presence of chloramine T. The specific radioactivity of the thus labelled compounds exceeded 15 to 30 times the specific activity upon selective introduction of the label into the protein with the help of the Bolton-Hunter reagent. The method can be used for highly effective and selective introduction of the label into the protein fragment of other nucleoproteins simultaneously at the amino groups of the protein and at tyrosine residue.