[The status of nucleolus organizing regions in hybrids of pluripotent and somatic mouse cells cultured under different conditions].

In the present work we examined the status of nucleolus organizing regions of mitotic chromosomes (NOR) in hybrid cells obtained by fusion of the mouse teratocarcinoma cells PCC4aza1 and adult mouse spleenocytes upon cultivation of hybrid cells under different conditions. We have shown that extended cultivation of hybrid cells in medium supplemented with HAT (hypoxanthine, aminopterin, thymidine) promotes the maintenance of NO-chromosomes, whereas under nonselective conditions elimination of NO-chromosome occurs. In nonselective medium the number of active, i. e. Ag-positive, NORs has been augmented comparatively to that observed under selective conditions. This observation directly indicates that reprogramming of the parental cell genomes in hybrid cells includes changes in the status of chromosomal NORs. The number of active NORs depends on conditions of hybrid cells culturing and may be changed by either of the two major ways--by elimination of NO-chromosomes (under nonselective conditions) or by inactivation of some NORs, when the general number of NO-chromosomes remains unaltered (under selective conditions).

[Growth and differentiation of cell hybrids obtained by fusing mouse PCC4azal teratocarcinoma cells and mouse spleen cells under different in vitro culture conditions].

Cell hybrids obtained by fusing mouse PCC4azal teratocarcinoma cells and spleen cells induced to proliferation and treated with the demethylating agent 5-azacytidine prior to fusion are described. The obtained hybrids demonstrated no expression of T lymphocyte marker genes CD11 and CD45, which indicates possible somatic nucleus reprogramming by factors present in teratocarcinoma cells. Irrespective of culture conditions, cell hybrids demonstrated a relatively stable chromosome number: they lost on average no more than four chromosomes after 30 passages. Culturing in medium containing hypoxanthine, aminopterin, and thymidine (selective conditions) decreased the differentiation capacity of cell hybrids compared to nonselective conditions, which is likely due to the inhibition of their metabolism. For the first time, teratocarcinoma cell hybrid differentiation into cardiomyocytes under the influence of DMSO has been demonstrated in vitro.

[Expression of human interferon beta in the mammary gland of transgenic rabbits].

A biotechnological system for the production of human beta interferon was developed on the basis of a hybrid gene constructed from the coding sequence of the beta interferon gene inserted into the first exon of the sheep beta lactoglobulin gene. It is intended for the expression of human beta interferon in mammary glands of transgenic animals. Two lines of transgenic rabbits were obtained using the hybrid gene. The tissue specificity of the expression of the transgene and the frequency of its inheritance in the first and second generations were studied. The activity of interferon was 2.2 x 10(4)-7.2 x 10(4) IU per milliliter of milk of transgenic female rabbits. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http:// www.maik.ru.

[Activation of nucleolar organizers during in vitro cultivation of mouse R1 embryonic stem cells].

We studies the activities of ribosomal genes (nucleolus forming regions of chromosomes) at successive stages of cultivation of the mouse R1 embryonic stem cells. The total number and number of active nucleolar organizers were estimated by means of in situ hybridization with mouse rDNA probes and argentophilic staining of nucleolus forming chromosomes regions from the 16th until the 32nd passages. The data we obtained suggest that the total number of nucleolar organizers per metaphase plate was constant (as a rule, eight), while the mean number of active nucleolar organizers progressively increased from the early (16th) to the late (32nd) passages: 5.2 +/- 0.4 versus 7.4 +/- 0.9 argentophilic organizers per cell. Cell heterogeneity by the number of active nucleolar organizers also increased during the late passages. Taken together, these data suggest activation of DNA transcription and synthesis of ribosomes during cultivation of mouse R1 embryonic stem cells. Based on the experimental and published data, it has been proposed that activation of ribosomal genes correlates in time with a decreased capacity of embryonic stem cells for pluripotent differentiation.

Efficient human IFN-gamma expression in the mammary gland of transgenic mice.

Two hybrid genes (BLG-HuIFN-gamma2 and BLG-HuIFN-gamma3) were constructed on the basis of sheep beta-lactoglobulin (BLG) and human interferon-gamma (HuIFN-gamma) gene sequences. They were used to direct HuIFN-gamma synthesis in the mammary gland of transgenic mice. HuIFN-gamma was efficiently produced in the mammary gland of transgenic mice. BLG-HuIFN-gamma2 transgenic females expressed HuIFN-gamma in the milk at concentrations up to 570 mg/ml, and BLG-HuIFN-gamma3 transgenic females expressed up to 350 mg/ml. All females carrying the BLG-HuIFN-gamma3 gene expressed HuIFN-gamma in their milk. No significant changes were observed in the HuIFN-gamma expression level during the lactation period. Using RT-PCR analysis, ectopic expression for both hybrid genes was found in transgenic mice. Despite ectopic expression of HuIFN-gamma in transgenic mice, their development and pregnancy were normal. The heritability of the HuIFN-gamma expression level in milk was demonstrated up to the F2 generation. This work demonstrates that hybrid genes have the potential to develop in transgenic domestic animals producing HuIFN-gamma in milk.

N-glycosylation of recombinant human interferon-gamma produced in different animal expression systems.

Recombinant human interferon-gamma (IFN-gamma) was expressed in Chinese hamster ovary cells, baculovirus-infected Sf9 insect cells and the mammary gland of transgenic mice. The N-linked carbohydrate populations associated with both Asn25 and Asn97 glycosylation sites were characterized by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. A site-specific analysis of dual (2N) and single (1N) site-occupancy variants of IFN-gamma derived from Chinese hamster ovary cells showed that N-glycans were predominantly of the complex bi- and triantennary type. Although Asn25-linked glycans were substituted with a core fucose residue, Asn97 N-glycans were predominantly non-fucosylated, and truncated complex and high-mannose oligosaccharide chains were also evident. Transgenic mouse derived IFN-gamma exhibited considerable site-specific variation in N-glycan structures. Asn25-linked carbohydrates were of the complex, core fucosylated type, Asn97-linked carbohydrates were mainly of the oligomannose type, with smaller proportions of hybrid and complex N-glycans. Carbohydrates associated with both glycosylation sites of IFN-gamma from Sf9 insect cells were mainly tri-mannosyl core structures, with fucosylation confined to the Asn25 site. These data demonstrate the profound influence of host cell type and protein structure on the N-glycosylation of recombinant proteins.

Human gamma-interferon expression in the mammary gland of transgenic mice.

Transgenic mice carrying a hybrid gene consisting of ovine beta-lactoglobulin gene sequences and human gamma-interferon (hIFN-g) cDNA were produced. hIFN-g expression in the mammary gland of two lactating transgenic founder females was found. The concentration of active hIFN-g in the milk was estimated as being ca. 1800 IU/ml. The hIFN-g ability to express in the mammary gland was found in the progeny of transgenic founder male.

[Directed mutagenesis]. / Napravlennyi mutagenez.

Methods for generating mutations in preselected genes of prokaryotes and eukaryotes are reviewed. Directed mutagenesis is based on mutagenic treatment of genome fragments in vitro instead of a whole genome in vivo. The cloning methods make it possible to perform directed mutagenesis even using conventional nonspecific mutagens. Methods have been elaborated for selective modification of specific sequences in cloned genes, based on the specific DNA recognition properties of restriction endonucleases and complementary poly- and oligonucleotides. Chemically synthesized oligonucleotides may be used for constructing mutant genes containing any base substitutions, small insertions and deletions.

Cold-sensitive mutations in beta and beta' subunit gene affecting the interaction of RNA polymerase with promoters.

RNA polymerases with a cold-sensitive activity were purified from seven mutants of Escherichia coli. Subunit reconstitution experiments have shown that RNA polymerases from three mutants (Rpob262, RpoB264, and RpoB265) owed their cold sensitivity to alterations in the beta subunit. Three mutants (RpoC3, RpoC263, and RpoC267) were shown to be defective in the beta' subunit and one (RpoBC266) in both beta and beta' subunits. Two mutations (rpoC3 and rpoC263) reduced the level of RNA polymerase reconstitution. RNA polymerases from RpoC3 and RpoBC266 mutants are defective in RNA chain elongation at 6 degree C, while all the other mutants are defective in RNA polymerase-promoter interaction. most mutant RNA polymerases differ from the wild-type enzyme in transcription selectivity. The results obtained in this study indicate that both beta and beta' subunits are involved in RNA chain elongation and promoter binding and selection.

[Effect of DNA supercoiling on transcription performed by normal and mutant Escherichia coli RNA-polymerases]. / Vliianie sverkhspiralizatsii DNK na Transkriptsiiu, Osuchchestvliaemuiu, Normal'noi i mutantnymi RNK-polimerazami Escherichia coli.

A specific DNA-gyrase, inhibitor, coumermycin A1, causes differential changes in the spectrum of proteins synthesized in E. coli wild types cells, while protein spectrum in the mutant cells with coumermycin-resistant DNA-gyrase is left unaffected. The mutation of RNA-polymerase RpoB265 lowers the sensitivity of bacteria to coumermycin A1, whereas the RpoC3 enhances it. The differences between the normal and mutant RpoB265 RNA polymerases on interaction in vitro with ColE1 DNA plasmid depend on its supercoiling. No dependences of this kind were detected when comparing the normal and RpoC3-RNA polymerase. The obtained data indicate that the template supercoiling may significantly affect the transcription in vivo and that the properties of RNA polymerase may in some cases define this influence.

[Cold-sensitive mutation in the beta-subunit of Escherichia coli RNA polymerase affecting the opening of promoters]. / Knolodochuvstvitel'naia mutatsiia V beta-subdedinntse RNK-polimerazy Escherichia coli, vliiaiushchaia na otkryvanie promotorov.

A cold-sensitive mutation in Escherichia coli, affecting the beta-subunit of RNA polymerase and causing an increase in the temperature of promoter opening on T2 phage DNA, was obtained. The mutation also affects the stages preceding promoter opening by increasing the dissociation rate of RNA polymerase--DNA closed complexes. The affinity of RNA polymerase to T2- and lambda-DNA is differently changed by the mutation. The relative efficiency of transcription of these two templates is also changed. These results suggest a participation of the beta-subunit of RNA polymerase in the interaction with promoters.

A cold-sensitive beta subunit mutant RNA polymerase from Escherichia coli with defects in promoter opening in vitro.

A cold-sensitive mutation in the rpoB gene for the RNA polymerase beta subunit increasing the temperature of promoter opening on T2 phage DNA was obtained in Escherichia coli. The mutation also affects the stages preceding promoter opening by increasing the dissociation rate of RNA polymerase--DNA closed complexes. The affinity of RNA polymerase to T2 and lambda DNA is differentially changed by the mutation. The relative efficiency of transcription of these two templates is also changed. These results suggest a participation of the RNA polymerase beta subunit in the interaction with promoters.

DNA supercoiling and transcription in Escherichia coli: influence of RNA polymerase mutations.

Coumermycin A1, a specific inhibitor of DNA gyrase, differentially changes the spectrum of proteins synthesized in wild type E. coli cells but has no effect on the protein spectrum in mutant cells with coumermycin-resistant DNA gyrase. The rpoB265 mutation affecting RNA polymerase decreases the coumermycin A1-sensitivity of bacteria while the rpoC3 mutation increases it. The interaction of wild type and mutant RpoB265 RNA polymerases with ColEl plasmid DNA in vitro is differently affected by DNA supercoiling. No such differences are observed in the case of RpoC3 RNA polymerase. The results suggest that template supercoiling may have a substantial effect on transcription in vivo, an effect which, in some cases, depends on the properties of RNA polymerase.