[SUBUNITS OF MAMMAL DNA DEPENDENT RNA POLYMERASE III MODIFIED IN VIVO].

In subfractions of DNA-dependent RNA polymerase isolated from nuclei of 3T3 mouse fibroblasts, A431 epidermoid carcinoma cells and mature human placenta, we have identified three subunits modified by both phosphorylation and O-N-acetylglucosamine glucosilation. The subfractions of the human enzyme contained modified subunits with molecular weight of 60 and 45 kDa and a subunit of 52 kDa probably belonging to one of the basal factors of RNA Polymerase III transcription. The subfractions of 3T3 mouse cells RNA Polymerase III consisted of modificated subunits with molecular weight 49, 45 and 42 kDa, however, the 45 kDa subunit might be a component of the basal transcription factor of RNA Polymerase III, since it was not identified in mouse enzyme composition.

[Dynamic phosphorylation of RNA polymerase III holoenzyme subunits from human epidermoid carcinoma cells A431 cultivated under different conditions].

Dynamic phosphorylation in vivo of individual subunits of holoenzyme RNA polymerase III subfractions IIIa and IIIb on serine/threonine and tyrosine aminoacid residues has been shown in human epidermoid carcinoma cells A431. Cells cultivation under starvation on embryonic serum caused a prolongation of the cell cycle, while cultivation at low concentration of epidermal growth factor (EGF) activated cell proliferation. Subunit 45 kDa is phosphorylated on serine/threonine residues in subfraction IIIa only in starving cells. This subunit of subfraction IIIb is unphosphorylated. Phosphorylation of this particular subunit is restored under induction by EGF at low concentration (0.1 ng/ml). The level of phosphorylation on tyrosine aminoacid residues of subunits of holoenzyme subfractions IIIa and IIIb is high in cells cultivated under starvation. Subunit 60 kDa has a higher level of phosphorylation as compared with subunits 45 and 38 kDa. Induction by EGF at low concentrations increases the level of phosphorylation of subunits 60 and 38 kDa in both subfractions of holoenzyme.

[Subunits of human holoenzyme of DNA dependent RNA polymerase III phosphorylated in vivo].

Phosphorylation of human holoenzyme of DNA dependent RNA polymerase III subunits in vivo has been investigated. RNA polymerase III from human placenta nuclei and epidermoid carcinoma cells A431 was isolated as two subfractions (IIIa and IIIb) distinguished in the order of elution from DEAE Sephadex A-25 column chromatography and buoyant density at glycerol gradient centrifugation. The subfractions of RNA polymerase III holoenzyme consists of four subunits with molecular masses 60, 52, 45 and 38 kDa, respectively, phosphorylated in vivo. The subunits with molecular masses 60 and 45 kDa are phosphorylated both on tyrosine and serine/threonine residues. All these subunits belong to subunits of the molecules of RNA polymerase III proper. RNA polymerase III and RNA polymerase I have the 38 kDa subunit in common. The subunit with molecular mass 52 kDa is phosphorylated on serine/threonine residues and may be related to some basal transcription factors of RNA polymerase III.

Small stable RNA level depends on the physiological state of the cell.

The level of 5S rRNA and tRNAi(Met)1 synthesized by RNA polymerase III was investigated in human epidermoid carcinoma cells A431 at different physiological states: low and high proliferation and apoptosis. The real-time RT-PCR method using SYBR Green I was applied to measure certain RNA species in total cellular RNA. The share of 5S rRNA was practically the same in slowly and actively proliferating A431 cells, but increased about 2.5-fold in apoptotic cells. The share of initiator tRNAi(Met)1 in actively proliferating and apoptotic cells was 1.5-2.0 times higher than in slowly proliferating cells. Our results suggest a possible existence of special mechanisms regulating RNA polymerase III-directed transcription from different type promoters in accordance with the physiological state of the cell.

[Use of the real-time RT-PCR method for investigation of small stable RNA expression level in human epidermoid carcinoma cells A431]. / Ispol'zovanie metoda RT-PCR v real'nom vremeni dlia izucheniia urovnia ékspressii malykh stabil'nykh RNK v kletkakh épidermoidnoi kartsinomy cheloveka A431.

Real-time RT-PCR using fluorescence dyes (e.g. SYBR Green I) is currently the most sensitive and precise method for investigation of RNA level and has long been widely used for absolute and relative quantification of mRNA in the cell. This highly sensitive method allows measurement of different type RNA level in the cell based on the kinetics of the corresponding double-stranded cDNA amplification. Upon its binding to the minor groove of double-stranded DNA, SYBR Green I dye increases its fluorescence about 100-fold, and this increase can be recorded even at early cycles of amplification. During the real-time RT-PCR procedure the level of amplified DNA is measured after every cycle of amplification, which permits to perform quantification at the cycles when amplification curve has not yet reached the "plateau" range and corresponds to the range of exponential increase in DNA amount. This approach makes it possible to avoid misinterpretation of data typical of conventional PCR methods "in the end point" and caused by a deficiency of one or more reaction components at the late PCR cycles. We applied for the first time real-time RT-PCR using SYBR Green I for the measurement of the class III genes RNA-product level, that is, small stable non-translated RNAs--ribosomal 5S rRNA, initiator transfer RNAiMet1, and Alu-RNA, synthesized by DNA-dependent RNA polymerase III. We investigated the level of 5S rRNA-, tRNA- and Alu-gene expression in the cell being in different states: with prolonged generation period, activated to proliferation, and apoptotic. The expression level was judged from the content of corresponding RNA-products in the total cellular RNA. The used approach enabled us to find out the specific RNA share in the total cell RNA. Human epidermoid carcinoma cells A431 were used as a model for investigating class III gene expression level in vivo. These cells expose on their surface an abnormally large amount of receptors to epidermoid growth factor (EGF), and the result of EGF action on A431 cells depends on the growth factor concentration. Low concentrations of EGF (0.1 ng/ml) cause active proliferation of A431 cells, but its high concentrations (10-100 ng/ml) cause apoptosis in these cells. Besides, upon growing in serum-free media, A431 cells continue to proliferate, but by this extending the generation period to 48 h, against 30 h on growing in serum-containing media. Hence, A431 cells can serve as a useful model for investigation of specific gene expression level in cells being in different physiological states, in both slowly and actively proliferating cells, and in apoptotic cells. For successful use of real-time RT-PCR in 5S rRNA, tRNAi(Met)1 and Alu-RNA level quantification, we optimized the amplification reaction conditions. We took into account that the share of each particular RNA in the cell may vary--the share of ribosomal RNA is high, tRNAi(Met)1--low, and Alu-RNA--very low. Moreover, the level of some small RNAs (e.g. Alu-RNA) can vary significantly in cells of different lines. This explains why the amount of cDNA, gained by reverse transcription of total cellular RNA, and the concentration of specific primers used for PCR were different in each case. We showed that the expression of different class III genes--5S rRNA-, tRNA- and Alu-genes, was not similarly regulated in response to external stimuli, causing prolongation of generation period, activation of proliferation and apoptosis. 5S rRNA level was practically the same in A431 cells both having prolonged generation period and being activated by EGF in low concentration, but in apoptotic cells this level dramatically fell about 8-fold. Alu-RNA level was equal in cells with prolonged generation period and in apoptotic cells, and increased about 2-fold in cells activated by EGF in low concentration. The initiator tRNAi(Met)1 level in cells activated by EGF in low concentration and in apoptotic cells was by almost two times higher than in cells with prolonged generation period. The data obtained testify that the real-time RT-PCR method using SYBR Green I yields highly reliable and reproducible quantification for the level of class III gene RNA-products--small stable RNAs (5S rRNA, tRNA and Alu-RNA). Examination of each specific RNA level requires individual selection for the amplification reaction conditions: the amount of cDNA and primer concentration in the sample. This is primarily caused by different expression levels in some particular class III genes within the frames of the cells, and by different levels of some small stable RNAs (e. g. Alu-RNA) in different cell lines. Special attention must be paid to the internal control for discriminating between specific RNA levels in proliferating and apoptotic cells, as in the late apoptosis RNAs of most types are degraded (for example, mRNA of "house-keeping" gene for RPLP0 protein, used as a possible internal control in our experiments). As far as the applied approach allows estimation of a specific RNA share in the total cellular RNA, we propose to chose as internal control mRNA, whose share doesn't change during the total RNA degradation in apoptosis and thus, mRNA degradation is not selective (in relation to other type RNAs). In that way, the real-time RT-PCR method, which is currently the most sensitive and precise method for quantification of RNA in the cell, holds much promise for the investigation of not only different mRNAs, but also small stable RNAs, synthesized by RNA polymerase III.

[Phosphorylation and dephosphorylation of rna polymerase III holoenzyme are modifications regulating the level of transcription in vitro]. / Fosforilirovanie-defosforilirovanie kholoénzima RNK-polimerazy III--modifikatsii, reguliruiushchie uroven' transkriptsii in vitro.

Two subforms of RNA polymerase III-IIIa and IIIb--were identified in human placenta nuclei. These subforms differed in molecular weight of one subunit, and in buoyant density in glycerol concentration gradient. Protein kinase activity, which phosphorylates at least four subunits of RNA polymerase IIIa and three subunits of RNA polymerase IIIb in vitro, was copurified with both the subforms. Protein kinase activity was inhibited by wortmannin, a specific PI3-kinase inhibitor. RNA polymerase III dephosphorylation by alkaline phosphatase in vitro decrease the transcription level on specific Alu-template. The associated protein kinase was not able to phosphorylate dephosphorylated RNA polymerase IIIa and to restore the transcription level up to the control one.

[DNA-dependent RNA polymerase I from human placental nuclei]. / DNK-zavisimaia RNK-polimeraza I iz iader platsenty cheloveka.

This paper describes a large-scale method for solubilisation and purification of DNA-dependent RNA-Polymerase I from mature human placenta. The solubilisation method involves homogenization of the whole human placenta, isolation of cell nuclei, sonication of separated nuclei at high ionic strength and ammonium sulfate precipitation. The purification method consists of chromatography of RNA-Polymerase I activity on DEAE-Sephadex A-25 and Phosphocellulose P-11, and glycerol-density gradient centrifugation. In result, RNA-Polymerase I of human placenta nuclei has been shown to be completely resistant to alpha-amanitin. Besides dependence of RNA-Polymerase I on different Mg2+ and Mn2+ concentrations, glycerol concentration and ionic strength was studied. Using our results, an optimal RNA-Polymerase I assay mixture was developed. The subunit composition of RNA-Polymerase I was investigated by dodecylsulfate-gel electrophoresis. The RNA-Polymerase I molecule of human placenta consists of 13-14 polypeptides.

[The small Alu-like RNA from the A-431 cell line specifically regulates the activity of the RNA polymerase III from human placental nuclei]. / Malaia Alu-podobnaia RNK iz kletok linii A-431 spetsificheski reguliruet aktivnost' RNK-polimerazy III iz iader platsenty cheloveka.

The influence of small Alu-like RNA, isolated from specific RNP complexes (alpha-RNP), on the activity of RNA polymerase III in cell-free system has been studied. The RNAs transcribed in vitro from Alu-DNA template (BLUR, 8) were isolated and subjected to polyacrylamide gel electrophoresis. A specific stimulation of RNA polymerase III activity by alpha-RNA was demonstrated.

[DNA-dependent RNA-polymerase II from human placental nuclei]. / DNK-zavisimaia RNK-polimeraza II iz iader platsenty cheloveka.

The procedure for isolation and purification of RNA polymerase II from human placenta nuclei is described. The sensitivity of the enzyme to alpha-amanitin, bivalent cations, ionic strength and glycerol concentration in vitro has been studied. Eleven subunits of the RNA polymerase II molecule have been identified by gel electrophoresis. An optimal RNA polymerase II assay mixture has been developed.

[Inhibition of the reactions of DNA hydrolysis and RNA synthesis by the alkaloid sanguinarine]. / Izuchenie ingibirovaniia reakstii gidroliza DNK i sinteza RNK alkaloidom sangvinarinom.

The increase of the contour length of the low molecular linear duplex DNA in the complex with an alkaloid sanguinarine has been evidenced by the viscometric method. The enzymatic hydrolysis of modified DNA by pancreatic deoxyribonuclease I and RNA synthesis of DNA by rat liver nuclear RNA polymerase were studied. Sanguinarine has been shown to inhibit the first stages of DNA hydrolysis. This alkaloid is a weaker inhibitor than ethidium bromide, a more potent inhibitor than actinomycin D and exerts an inhibiting effect similar to that of distamycin A. Sanguinarine also decreases the rate of the labelled precursor incorporation into the acid-insoluble fractions by nuclear RNA polymerase from rat liver. A 50% inhibition by sanguinarine was observed at the same alkaloid concentration as that of ethidium bromide.