Immunocytochemical study of Bcr and Bcr-Abl localization in K562 cells.

AIM: To obtain polyclonal antibodies against recombinant proteins recognizing Bcr domain and fusion region of Bcr-Abl and analyze the patterns of intracellular distribution of Bcr and Bcr-Abl proteins in K562 cells of chronic myelogenous leukemia. METHODS: The coding sequences of DH and PH domains of Bcr-Abl were cloned, and the recombinant proteins were expressed in E. coli. The rabbit polyclonal antibodies were produced and used for immunocytochemical study of Bcr and Bcr-Abl localization in K562 cells. RESULTS: The gene constructs containing sequences coding for DH and PH domains of Bcr-Abl have been obtained. The antibodies with relative specificity to corresponding recombinant proteins differ by the patterns of their intracellular reactivity with Bcr- and Bcr-Abl related structures. While Bcr protein is located predominantly perinuclearly, antibody against hybrid Bcr-Abl protein is reacted with the structures in cell periphery, namely on cell membranes. CONCLUSION: Antibodies against DH and PH domains of Bcr-Abl react with proteins located differently in chronic myelogenous leukemia cells. The difference in intracellular localization of Bcr and Bcr-Abl may be attributable to the different domains interacting with different multiprotein complexes.

[Analysis of GEF activity of Bcr protein DH domain].

Dbl protein and DH (Dbl homology) domains are key regulators of RhoGTPases and promote GDP release from the complex with GTPase. About 70 DH-containing proteins are found. DH domain is localized in tandem with PH (pleckstrin homology) domain in many proteins. Bcr protein is a partner of Abl in reciprocal translocation t(9;22) which leads to Philadelphia chromosome formation. In the present study we have cloned Bcr DH and PH domains into the vector for mammalian expression. GEF activity of Bcr DH domain was studied alone and together with PH domain. Our data suggest, that Bcr DH domains does not reveal GEF activity against RhoGTPases RhoA, Cdc42 and Racl subfamilies in vivo.

Genetic diversity in populations of Gentoo penguins (Pygoscelis papua).

RAPD analysis was used to examine the extent of genetic polymorphism in two populations of Gentoo penguin (Pygoscelis papua) from Antarctic Islands (Petermann and Livingston). The chosen two of three 10 mer oligonucleotide primers accordingly to preliminary results showed different levels of polymorphism in Gentoo penguins at Petermann Island (from 23.53 to 42.86%) and Livingston Island (from 52.94 to 57.14%). Nei's similarity coefficients were in range from 0.5606 (when Gentoo genome profiles were compared with RAPD profiles of two related penguin species: Pygoscelis adeliae (Adelie) and Pygoscelis antarctica (Chinstrep)) to 0.9281 among observed Gentoo penguin populations. Nei's distances values ranged from 0.0746 to 0.5787 among the populations and species. The obtained results will be used for further estimation of genetic diversity of Gentoo penguins and determination of their taxonomic status.

[Role of Ku protein in normal and cancer cells].

Ku protein is a multifunctional heterodimer of eukaryotes that consists of two subunits: Ku70 and Ku80. It plays a critical role in the regulation of many cellular processes such as: non-homologous end-joining, V(D)J-recombination of immunoglobulin genes and genes of T-cellular receptors, transcription regulation, DNA replication. Now the existence of relation between Ku protein functioning and processes that lead to transformation of normal cells into cancerous ones is determined, in Ph-positive leukemias and multiple myelomas, in particular. Nevertheless, the machinery of this processes is not still completely understood and needs further researches.

[The molecular biology diagnosis of neoplastic blood diseases]. / Molekuliano-biolohichna diahnostyka neoplastychnykh zakhvoriuvan' krovi.

Molecular-biological approaches was applied for detection of Philadelphia chromosome in patients with chronic myeloid and acute lymphoblastic leukemias. It includes bcr-genomic probes and polymerase chain reaction with specific bcr/abl primers. The latter is more preferable for clinic use. Different diagnostic methods are compared and leukemias etiology problems are discussed.

[The DNA polymorphism in the inhabitants of Ukraine detected with an M13 phage-based probe]. / Izuchenie polimorfizma DNK zhitelei Ukrainy, vyiavliaemogo zondom na osnove faga M13.

BspRI and HinfI polymorphism of DNA in the inhabitants of Ukraine was analyzed with probe based on M13 phage. In the region 3 - 8 kb, the mean probabilities of coincidence of to marker bands comprised 0.22 for BspRI polymorphism and 0.34 for HinfI polymorphism. The indexes of similarity by Lee between the parents comprised 0.44 for BspRI polymorphism and 0.45 for HinfI polymorphism. The characteristics obtained may be used for the correct description of DNA fingerprints in the inhabitants of Ukraine.

Activities and regulation of the enzymes involved in the first and the third steps of the aspartate biosynthetic pathway in Enterococcus faecium.

The enzymes aspartokinase and homoserine dehydrogenase catalyze the reaction at key branching points in the aspartate pathway of amino acid biosynthesis. Enterococcus faecium has been found to contain two distinct aspartokinases and a single homoserine dehydrogenase. Aspartokinase isozymes eluted on gel filtration chromatography at molecular weights greater than 250,000 and about 125,000. The molecular weight of homoserine dehydrogenase was determined to be 220,000. One aspartokinase isozyme was slightly inhibited by meso-diaminopimelic acid. Another aspartokinase was repressed and inhibited by lysine. Although the level of diaminopimelate-sensitive (DAPs) enzyme was not much affected by growth conditions, the activity of lysine-sensitive (Lyss) aspartokinase disappeared rapidly during the stationary phase and was depressed in rich media. The synthesis of homoserine dehydrogenase was controlled by threonine and methionine. Threonine also inhibited the specific activity of this enzyme. The regulatory properties of aspartokinase isozymes and homoserine dehydrogenase from E. faecium are discussed and compared with those from Bacillus subtilis.

[Analysis of key enzyme activities involved in aspartate amino acid biosynthesis in Streptococcus bovis]. / Analiz kliuchevykh fermentativnykh aktivnostei biosinteza aminokislot aspartatnogo semeistva Streptococcus bovis.

The first and the third steps in the aspartate biosynthesis pathway in Streptococcus bovis are catalyzed by two different forms of aspartokinase and a single homoserine dehydrogenase, respectively. These enzymes can be separated by ammonium sulfate fractionation and gel filtration on Sephadex G-200. The two aspartokinase isozymes differ in molecular weights and are subject to differential regulation. The aspartokinase system of S. bovis is characterized by the absence of specific negative allosteric effectors among the end products of the synthesis of amino acids of the aspartic family. Homoserine dehydrogenase, which catalyzes the third step of the aspartic family amino acid synthesis, also has such negative effectors as threonine and methionine. The aspartokinase isozymes do not form multienzyme complexes with homoserine hydrogenase in S. bovis.

[Regulation of enzymes of the first and last stage of lysine biosynthesis in Streptococcus bovis and Enterococcus faecium]. / Reguliatsiia fermentov pervoi i poslednei stadii biosinteza lizina u Streptococcus bovis i Enterococcus faecium.

Regulation of aspartate kinase and diaminopimelate decarboxylase activities in Streptococcus bovis and Enterococcus faecium cell-free extracts was studied. The levels of synthesis of aspartate kinase and diaminopimelate decarboxylase in both microorganisms are growth-dependent. The synthesis of these enzymes is depressed by lysine, but the activity of aspartate kinase is induced by addition of this amino acid and threonine to the reaction system. Meso-diaminopimelate dehydrogenase activity was not found in the extracts of Streptococcus bovis and Enterococcus faecium. The data excludes the possibility of lysine formation via six enzyme reactions.

[The characteristics of the regulation of lysine biosynthesis in Streptococcus bovis]. / Nekotorye osobennosti reguliatsii biosinteza lizina u Streptococcus bovis.

The effect of threonine and methionine on the growth of and lysine synthesis in Streptococcus bovis st. A024/85 was studied. The character and the degree of manifestation of growth and regulation effects of these amino acids depend on their contents in the fermentation medium. The target in the regulation of lysine biosynthesis is aspartate kinase, whose synthesis is controlled by threonine and methionine. Lysine excretion was stimulated by the addition of a low concentration of dimethylsulfoxide in the medium during fermentation.

[Integration of the plasmid-cloned lysA gene of Bacillus subtilis into the chromosome of this microorganism]. / Integratsiia klonirovannogo na plazmide lysA-gena Bacillus subtilis v khromosomy étogo mikroorganizma.

Integration of the Bacillus subtilis lysA gene cloned on pLP1 plasmid, into the 250 degrees region of the chromosome of this microorganism was performed. Significant differences in the level and character of their expression were shown between lysA gene integrated into the chromosome and plasmid-borne genes. It is suggested that the expression of the lysA gene could be under control of a certain cis-acting factor which is able to promote transcription of the lysA gene and mediate gene specific regulation.

[Cloning and regulation of the expression of the lysA gene from Bacillus subtilis]. / Klonirovanie i reguliatsiia ékspressii lysA-gena Bacillus subtilis.

Cloning of Bacillus subtilis DNA fragment with the lysA gene encoding diaminopimelatecarboxylase (EC 4.1.1.20) was done. The cloned gene in poorly expressed both in Escherichia coli and in Bacillus subtilis. Some DNA sequence distant from the lysA gene seems to be necessary for full gene expression, this sequence having been not cloned together with the lysA. The sequence in needed for regulation of the expression as well.

[Diaminopimelate pathway for lysine synthesis in Escherichia coli and bacilli]. / Diaminopimelinovyi put' sinteza lizina u Escherichia coli i batsill.

The diaminopimmelate (DAP) pathway for lysine biosynthesis in Escherichia coli and some species of Bacillus are presented in the review. It was shown that the major variations of the DAP pathway of Bacillus subtilis from that described and extensively studied in Escherichia coli exist.

Purification of a DNA methyltransferase from Bacillus natto B3364.

An S-adenosyl-L-methionine:DNA-methyltransferase, termed M.BnaI, was purified from Bacillus natto B3364 strain by successive column chromatography. The molecular weight determined by gel filtration was 37 kDa for M.BnaI. Analysis of methyltransferase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed correspondence of the M.BnaI activity with one protein band at a molecular weight of 35 kDa. Sequencing of pUC19 DNA methylated with M.BnaI showed the cytosine-5 methylation in the BnaI recognition sequence GGAT decreases CC at the position indicated by the arrow.

A new isoschizomer, BnaI, of the BamHI restriction endonuclease.

By assaying the yield of phage SPO1 we have identified a new restriction-modification activity in the Bacillus natto B3364 strain. A class II restriction endonuclease, BnaI, isolated from the crude extract of B3364 cells was shown to be a true isoschizomer of the BamHI endonuclease. The Mr, stability and optimal conditions required for DNA digestion were determined for BnaI. Although both enzymes show the same specificity, BnaI and BamHI differ from each other in all the properties specified above.

[The effect of the oligomerization of pBR322 on the level of transformation in Escherichia coli]. / Vliianie oligomerizatsii pBR322 na uroven' transformatsii Escherichia coli.

The ability of the known Escherichia coli strain JC3881 recB recC recF sbc15 to produce oligomeric and multimeric forms of pBR322 underlies the study presented. The individual oligomeric forms of pBR322 were isolated from the agarose gel. The plasmid forms were used for electron microscopic control and also introduced into the system of E. coli competent cells. The E. coli transformation level of different forms of plasmid DNA rose from monomers to pentamers. CCC forms of the plasmid possessed high efficiency of the E. coli cell transformation. The systems of the host recombination are to be significant in the process of plasmid oligomerization.

[The induction of auxotrophic mutations for riboflavin by the integration of plasmid pLRS33 into the chromosome of Bacillus subtilis]. / Induktsiia mutatsii auksotrofnosti po riboflavinu pri integratsii plazmidy pLRS33 v khromosomu Bacillus subtilis.

The transformation of Bacillus subtilis Lys- strains with plasmid pLRS33 containing pBR322 and the Bac. subtilis chromosomal fragment carrying the genes for lysin biosynthesis and the riboflavin operon regulatory operator region (ribO) leads to the appearance of Rib- mutants. It was shown that these mutants contained long deletions covering a great portion of the riboflavin operon.

[Analysis of mutants of Bacillus subtilis, auxotrophic for lysine]. / Analiz mutantov Bacillus subtilis, auksotrofnykh po lizinu.

A number of B. subtilis mutants auxotrophic for lysine has been isolated and mapped in relation to the flanking ser2 marker. One of the mutants has been found to have a mutation in lys A gene coding for DAP-decarboxylase activity. The expression of DAP-decarboxylase is dependent on the product of lys R gene that is not linked with lysine genes cluster.