Proteins associated with heterogeneous nuclear RNA in eukaryotic cells

When HeLa cell nuclei axe mechanically disrupted in either hypotonic or isotonic buffers, heterogeneous nuclear RNA is recovered from the post-nucleolar fraction in the form of EDTA-resistant ribonucleoprotein particles, which sediment between 40 S and 250 S in sucrose gradients containing 0.01 m or 0.15 m-NaCl. That the RNA in these particles is HnRNA² is indicated by its heterodisperse sedimentation (20 to 80 S) and its continued synthesis in concentrations of actinomycin D that selectively inhibit the synthesis of ribosomal RNA. The specificity of the HnRNA-protein complexes is evidenced by the failure of deliberate attempts to generate artificial RNP by the addition of deproteinized HnRNA to intact or disrupted nuclei at low ionic strength.The proteins bound to HnRNA are complex. In HeLa cells, HnRNP particles contain proteins with molecular weights from 39,000 to approximately 180,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points between 4.9 and 8.3 (analytical isoelectric focusing). They are readily distinguishable from proteins in other cell fractions, including those in chromatin.Exposure of HeLa HnRNP particles to 0.5 m-NaCl reduces their average sedimentation velocity by approximately 30%. CsCl density-gradient analysis reveals that this is accompanied by the loss of a major portion of the proteins. However, a significant fraction of the HnRNP (25 to 30%) is resistant to high salt concentrations and continues to band at the same density as native HnRNP (1.43 g/cm³). This is true even after prolonged exposure (24 h) to high salt. The salt-resistant HnRNP is enriched for proteins above 60,000 molecular weight. In at least these two respects, this sub-class of HnRNP resembles “messenger RNP” prepared from cytoplasmic polyribosomes, which is also salt-stable and contains relatively high molecular weight proteins.HnRNP particles can also be recovered from HeLa cell nuclei lysed in high salt but these contain many extra proteins, notably histones, and sediment much faster in sucrose gradients than particles prepared as above. HnRNP is not liberated by extracting HeLa nuclei in 0.14 m-NaCl, pH 8.0 (Samarina et al., 1967) unless the temperature is 20 °C or higher. In this case the particles are converted to 45 S structures, which contain partially degraded HnRNA. 45 S particles can also be produced by subjecting 40 to 250 S HnRNP to a very limited digestion with pancreatic ribonuclease (1 to 2 hits/molecule).HnRNP particles have similar sedimentation velocities (40 to 300 S) when isolated under physiological ionic conditions from a variety of mammalian cells, including WI38 human diploid fibroblasts, mouse L-cells, monkey kidney cells and rat liver. However, electrophoresis reveals a distinct pattern of HnRNP proteins for each cell type. It is proposed that this cell-specificity reflects a situation in which HnRNA molecules that differ in nucleotide sequence are complexed with different sets of proteins, so that the resulting HnRNP particles are biochemically distinct at each genetic locus. This hypothesis is discussed in relation to the cytology of lampbrush and polytene chromosomes.     

Characterization of proteins associated with nuclear ribonucleoprotein particles by two-dimensional polyacrylamide gel electrophoresis.

Rat liver nuclear ribonucleoprotein particles were prepared by two different methods and defined as 40S ribonucleoprotein (40S RNP) and heterogeneous nuclear ribonucleoprotein (HnRNP) particles. The RNP particles were either solubilized in 8 M urea--6 mM 2-mercaptoethanol--20 mM glycine--20 mM Tris--HCl (pH 8.4) or subjected to removal of RNA by phenol extraction prior to solubilizing the proteins in the urea buffer. The proteins associated with 40S RNP and HnRNP were heterogeneous and very similar in their electrophoretic patterns when analyzed by two-dimensional PAGE, except a protein with molecular weight of 62 000 and an isoelectric point (pI) of 6.2 was present only in HnRNP particles. At least 12 major and 22 minor components could be identified in both preparations. The major proteins were found at pI values varying from 6.0 to 8.5 and with molecular weights from 32 000 to 42 000, and a group of proteins with molecular weight approximately 65 000 were more prominent in HnRNP than in 40S RNP. The other components were found mainly at pI ranges from 5.0 to 6.5 with molecular weights from 43 000 to 65 000. The phenol method extracted essentially all proteins associated with either 40S RNP and HnRNP, but was less effective in extracting a group of proteins with pI values from 5.0 to 5.5 and more efficient for proteins with pI values from 7.5 to 8.5. When chromatin proteins isolated by phenol extraction were compared with HnRNP particle proteins isolated by the same method, the electrophoretic mobilities of the HnRNP particle proteins were found to be identical with a fraction nonhistone chromatin proteins. The 40S RNP particles were further purified by metrizamide isopycnic density gradient centrifugation. The electrophoretic patterns of these proteins were very similar to those prepared by sucrose density gradient centrifugation. Therefore, we concluded that the proteins of RNP particles constituted part of the chromatin proteins.     

Distribution of snRNP complexes in rat liver nuclear extracts: Biochemical and immunochemical analysis

Summary Rat liver nuclei were extracted with 0.14 M NaCl and the extracts submitted to sucrose gradient fractionation. Aliquots of the nuclear residue remaining after the 0.14 M NaCl extraction were also extracted either with 0.3 M NaCl or 1 M urea, and the extracts similarly submitted to sucrose gradient fractionation. Thereafter, both the presence and relative distribution of individual U-snRNA (U1–U6) species was followed. Results showed an extensive association of all U-snRNAs to RNP structures of 40 S. However, characteristic differences in the association of mostly U1 and U5 — which were the major identifiable species in the extracts — to these structures were observed. Only a small fraction of U1 appeared complexed to 40 S RNP structures, while most of it sedimenting in the > 20 S region of the gradient. In contrast, U5-snRNA had a tight and almost exclusive association to 40 S RNP structures. No pool of 10–12 S U5-snRNP complexes was detected.Combined immunoprecipitation and immunoblotting experiments on nuclear 0.14 M NaCl extracts using anti-Sm and/or anti-RNP antisera showed that all snRNA species, whether recovered as 10–12 S complexes or segregated with 40 S RNP components, existed as snRNP structures bearing at least their Sm-antigenic polypeptides.These and our previous results [Guialis, A., Arvanitopoulou, A, Patrinou-Georgoula, M. and Sekeris, C.E. (1983) FEBS Lett. 151, 127–133], support the existence of snRNP-enriched RNP structures of 40 S. In such structures the core polypeptides (Mr = 32000–45000) of 40 S monoparticles are not obligatory components.     

Nuclear and polyribosomal ribonucleoprotein particles containing poly(A) in rat liver cells

Poly(A) containing ribonucleoprotein particles were prepared from rat liver nuclei and polyribosomes. The particles have sedimentation coefficients of 14 S and 9 S, respectively. In Cs₂SO₄ density gradients the particles banded at densities of 1.28–1.29 g cm^-3. Both nuclear and polyribosomal poly(A)-RNP contain in addition to some minor polypeptides, two main polypeptides having molecular weights of 63 000 and 90 000 dalton, respectively indistinguishable from each other according to their electrophoretic mobilities.Abbreviations STKM 0.25 M sucrose, 0.05 M Tris-HCl, pH 7.2, 0.025 M KCl, 0.005 M MgCl₂ - TKM 0.05 M Tris-HCl, pH 7.5, 0.025 M KCl, 0.005 M MgCl₂ - STM II 0.1 M NaCl, 0.01 M Tris-HCl, pH 8, 0.001 M MgCl₂ - DTT dithiothreitol - SDS sodium dodecylsulphate     

Ultraviolet light-induced crosslinking of HnRNA to informofer proteins in vitro

RNA-protein interaction in the 30S subunits of rat liver hnRNP has been studied by crosslinking of informofer proteins to hnRNA induced by UV irradiation.Irradiation of 30S particles with 254 nm UV light in doses of 1×10⁵ erg/mm² leads to the extensive crosslinking hnRNA to informofer proteins. The crosslinked material was analyzed either by resedimentation in a 15–30% sucrose gradient in the presence of 3 M guanidine-HCl and 1 M NaCl or by centrifugation in a Cs₂SO₄ density gradient containing guanidine-HCl and sarkosyl. The crosslinked complexes sedimented at about 25S in the sucrose gradient and proved to be heterogeneous in isopycnic centrifugation experiments. The proteins of the crosslinked complexes were analyzed by polyacrylamide gel electrophoresis. Proteins with M_r values of 70 000, 58 000, 43 000 and 40 000 appeared to be crosslinked with hnRNAs of the 30S particles.In the unirradiated 30S particles after centrifugation in the Cs₂SO₄ density gradient containing guanidine-HCl and sarkosyl two minor proteins were observed with M_r values of 70 000 and 58 000, banded in density zones characteristic for free RNA.     

Isolation and characterization of nuclear particles containing rapidly labelled hnRNA and snRNA in combination with a distinct set of polypeptides of Mr 74000 and 72000

A heterogeneous RNP structure has been isolated from rat liver nuclei by a method previously used for the isolation of 30S RNP complexes carrying heterogeneous RNA (hnRNA) [1]. The RNP sediments in sucrose gradients with s-values of 70-110S. Formaldehyde-fixed preparations band at Q = 1.40 in isopycnic CsCl gradients. The RNP structure is composed of a heterogeneous population of polypeptides, prominent among which are two proteins with Mr 74000 and 72000. It contains both rapidly labelled RNA as well as several species of snRNA, as demonstrated by double-labelling experiments and gel electrophoresis. Treatment of rats with alpha-amanitin leads to a significant decrease in the amount of recovered RNP. In the presence of 0.7 M NaCl the s-value of the complex changes from 70-110S to 40-80S. The RNP structure is stable to mild RNase A or micrococcal nuclease digestion. Transmission electron microscopy reveals the presence of a heterogeneous population of particles with a mean diameter of 300-360 A. The isolated RNP structure differs completely from the well-known monoparticle or polyparticle hnRNP complexes and from the 30S or smaller snRNP particles but could be similar to or identical with the heterogeneous complex described by Jacob et al. [29].     

ADP-ribosylation of proteins associated with heterogeneous nuclear RNA in rat liver nuclei

Purified rat liver nuclei were incubated in vitro in the presence of [adenylate-32P]nicotinamide adenine dinucleotide. The label was rapidly incorporated into trichloroacetic acid-insoluble material and also detected in particles carrying heterogeneous nuclear RNA. The particles were isolated by density gradient centrifugation, and their size determined to be 30-40 S from parallel experiments using nuclei labelled with [3H]uridine 5'-triphosphate under similar conditions. Treatment of the 30-40 S-particles with enzymes of different specificities showed that the label was tightly bound to proteins, not incorporated into nuclei acids and not utilized in phosphorylation of proteins. The label was detached by phosphodiesterase I from snake venom and identified as ADP-ribose and adenosine 5'-phosphate present at a ratio of 7.5 to 1 using thin layer chromatography on poly(ethyleneimine)-cellulose. Radioactively labelled (ADP-ribosylated) proteins were visualized by autoradiography following SDS-polyacrylamide gel electrophoresis. They included several major species of the ribonucleoprotein with molecular weights of 36000, 39000 and 42000, and a limited number of high molecular weight polypeptides.     

A comparative study on the two classes of heterogeneous nuclear ribonucleoprotein particles separated in metrizamide density gradient,by electrophoresis of proteins and chase experiments. Evidence for two distinct subfractions of HnRNP in mammalian nuclei

Heterogeneous nuclear ribonucleoprotein particles (HnRNP) were separated in metrizamide density gradients, into two fractions migrating to 1.31 g ml^-1 and 1.18 g ml^-1, respectively. Proteins associated with each of these fractions were analysed by SDS-acrylamide gel electrophoresis. It is shown that the whole proteins extracted from these two metrizamide fractions exhibit clearly different electrophoretic patterns: 1.31 HnRNP particles contain as major polypeptide chains molecules with molecular weights ranging from 40 000 to 65 000, while major polypeptides of 1.18 HnRNP are banding in the 30 000–40 000 molecular weight region of the gels. Both fractions contain numerous other associated polypeptide chains whose molecular weights are above 65 000. A possible kinetic relationship between these two HnRNP classes was investigatedin vivo by performing chase experiments. No clear evidence for a precursor-product relationship was found. Implications arising from these structural and kinetic observations, and problems relating to nuclear maturation of pre-messenger RNA, are discussed.     

The effect of inhibition of RNA synthesis by actinomycin D on the population of basic polypeptides of the 30S unclear ribonucleoprotein particles

In rats injected intraperitoneally with actinomycin D (2 mg/kg body weight) 12 h earlier, the yield of the 30S ribonucleoprotein particles isolated from liver nuclei by extraction with 0.1 M NaCl at pH 8.0 decreased by 60 per cent. The protein-to-RNA ratio of these particles increased to 32:1 from the ratio 4.4:1 found in the same particles isolated from the nuclei of liver of control rats. The particles isolated from the liver nuclei of rats injected with actinomycin D were depleted of all charge isomers of the two most prominent polypeptides (33,000 and 39,000 daltons) present in the particles of liver of control animals. The most abundant protein in these particles was a 43,000 dalton polypeptide. This polypeptide is the least prominent of the 3 major polypeptides present in the control particles. The same charge isomers of the 43,000 dalton polypeptide were present in the nuclear ribonucleoprotein particles isolated from the liver of control animals and from the liver of animals treated with actinomycin D 12 h earlier. In control animals the nuclear ribonucleoprotein monoparticles isolated from kidney contained 3 major polypeptides of the same molecular weight with the same distribution of their charge isomers as were present in the particles isolated from liver nuclei. The injection of actinomycin D 12 h earlier was without any effect on the protein composition of the 30S nuclear ribonucleoprotein particles of rat kidney.     

Isolation and characterization of nuclear ribonucleoprotein complexes from Drosophila melanogaster

The isolation of total nuclear ribonucleoprotein particles fromDrosophila melanogaster embryos, using a pH 8.0, 01 M NaCl extraction of purified nuclei, is described. When the extract is fractionated on isokinetic sucrose gradients, at least six major classes of nuclear ribonucleoprotein complexes, differing in RNA and protein content as well as sedimentation behavior, are observed. The two largest complexes are preribosomal complexes. The remaining four major classes of RNPs sediment at roughly 6S, 8S, 12S and 30S. A minor class at 17S is also observed. The 30S fraction is 200–250 Å in width and appears to be analogous to the mammalian monoparticle. It is composed primarily of polypeptides at about 36 000 and 37 000 daltons, along with 1–2 kilobase RNA fragments. The 6S, 8S and 12S complexes contain a few discrete small nuclear RNAs from 80–600 bases in length, along with a small number of polypeptides, about 50 000, 52 000, 56 000 and 75 000 daltons. These novel complexes are of the order of a 100 Å in width (60–120 Å range).     

Composition of hnRNA-associated proteins in rat liver is specifically altered after cycloheximide treatment

Rats were treated with non-lethal doses of -amanitin or cycloheximide. Nuclei were prepared and the particles carrying heterogeneous nuclear RNA (30–40 S-particles) isolated by density gradient centrifugation. In the case of -amanitin the yield of particles was reduced to about 45%. Cycloheximide affected the composition of proteins associated with the nuclear RNA. In particular, the concentration of a 110 000 molecular weight protein as determined by sodium dodecylsulfate gel electrophoresis was reduced to 20–30% after 2 h and then increased to 150–180% of the control before it approached the normal level after 30 h. Possible mechanisms underlying these changes are discussed.Abbreviations hnRNA heterogeneous nuclear RNA - hnRNP ribonucleoprotein which contains hnRNA     

Characterization of ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two different in vitro systems.

The ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two in vitro systems were studied and the following results were obtained. 1. When the isolated nuclei of regenerating rat liver labeled in vivo with [14C] orotic acid were incubated in medium containing ATP and an energy-regenerating system (medium I) release of labeled 40-S particles was observed. Analysis of these 40-S particles showed that they contained heterogeneous RNA but no 18 S or 28 S ribosomal RNAs and their buoyant density in CsCl was 1.42-1.45 g/cm3, suggesting that they were nuclear informosome-like particles released during incubation. 2. When the same nuclei were incubated in the same medium fortified with dialyzed cytosol, spermidine and yeast RNA (medium II), release of labeled 60-S and 40-S particles was observed. Using CsCl buoyant density gradient centrifugation, two components were found in the labeled ribonucleoprotein particles released from nuclei in this medium. The labeled 60-S particles were found to contain 28-S RNA as the main component and their buoyant density in CsCl was 1.61 g/cm3, suggesting that they were labeled large ribosomal subunits. The labeled 40-S particles contained both 18 S RNA and heterogeneous RNA and they formed two discrete bands in CsCl, at 1.40 and 1.56 g/cm3, suggesting that they contained small ribosomal subunits and nuclear informosome-like particles. 3. These results clearly indicate that addition of dialyzed cytosol, spermidine and low molecular yeast RNA to medium I causes the release of ribosomal subunits or their precursors from isolated nuclei in the in vitro system.     

Distribution of selected phospholipid modifying enzymes in rat brain microsomal subfractions prepared by density gradient zonal rotor centrifugation

A rat brain P₃ fraction enriched in ER derived microsomes was centrifuged through a 20–40% linear sucrose gradient in a Beckman Ti-14 Zonal rotor and 11 fractions were obtained. The distribution of marker enzyme activities and protein were determined in these 11 subfractions. NADPH-Cytochrome C reductase, choline phosphotransferase were employed for endoplasmic reticulum, Na⁺, K⁺-ATPase, 5-nucleotidase, and acetylcholinesterase were employed for plasma membrane, 2, 3-cyclic nucleotide phosphohydrolase was employed for myelin. The bulk of the protein was recovered in the 24–34% sucrose fractions, Na⁺, K⁺-ATPase, 5-nucleotidase, and acetylcholinesterase were in the 22–38% sucrose fractions while NADPH-cytochrome C reductase and CNPase were enriched in the 20–22% sucrose fractions. The ethanolamine and the serine base exchange activities had a bimodal distribution, with highest specific activities in sucrose fractions 32–34% and 20–24%. Choline base exchange activity was nearly undetectable in all the fractions. The specific activities of CDP-choline phosphotransferase, and phospholipid-N-methyltransferase were highest in the 20–22% sucrose fraction. Phospholipid-N-methyltransferase activity was significantly stimulated in the presence of exogenous phospholipid acceptors as phosphatidylethanolamine or phosphatidylmonomethylethanolamine or phosphatidyldimethylethanolamine, however, the greatest response was with phosphatidylmonomethylethanolamine. The rat brain P₃ fraction yielded a population of a membrane at the light end of the sucrose gradient which has a buoyant density similar to myelin but seemed to be enriched with NADPN cytochrome C reductase and phospholipid modifying enzymes. This is in contrast to liver microsomes submitted to a similar fractionation.     

Detection of beta-globin mRNA precursor in heterogeneous nuclear ribonucleoprotein associated with U1-RNP by using anti-RNP antibody

Heterogeneous nuclear RNA-ribonucleoprotein (hnRNP) fractions were isolated from Friend erythroleukemia cells and separated by 15-45% sucrose gradient centrifugation. The distribution of small nuclear RNAs (snRNAs) in hnRNP fractions indicated that the snRNAs are associated with hnRNP particles. HnRNP fractions were incubated with normal IgG or anti-U1 RNP IgG, and the resulting immunocomplexes were isolated by binding to a protein A-Sepharose column. HnRNP was found in bound fractions only when anti-U1 RNP IgG was used. By Northern hybridization of RNA extracted from the immunocomplexes with a beta-globin genomic DNA probe, 15S beta-globin mRNA precursors and 10S mature mRNA were detected. These findings suggest the existence of a complex of U1 RNP particles and hnRNP particles containing beta-globin pre-mRNA.     

STUDIES ON ISOLATED CELL COMPONENTS : XVII. The Distribution of Cytochrome Oxidase Activity in Rat Liver Brei Fractionated in the Zonal Ultracentrifuge

The zonal ultracentrifuge was used to separate the subcellular components of rat liver brei into soluble phase, microsomal, mitochondrial, membranous fragments, and nuclear fractions during a single centrifugation. The centrifuge was run at 10,000 to 30,000 RPM for 15 to 240 minutes, and the rotor contained a 1200 ml sucrose gradient, varying linearly with radius from 17 to 55 per cent sucrose with a "cushion" of 66 per cent sucrose at the rotor edge. The distribution of the mitochondria was determined using cytochrome oxidase as the marker enzyme. An automated assay system for cytochrome oxidase was developed utilizing reduced cytochrome c as substrate, modules of the Technicon Autoanalyzer, and the Beckman DB Spectrophotometer. All of the cytochrome oxidase activity was restricted to a single peak in the gradient, and no activity could be detected in the zones occupied by the microsomes and nuclei. The mitochondrial fraction was isolated from rat liver brei in 0.25 M sucrose by differential centrifugation, and then run in the zonal ultracentrifuge.This fraction behaved in the zonal ultracentrifuge in the same way as mitochondria separated directly from intact brei. Observations of the isolated fractions in the phase contrast microscope indicated that a wide variety of granules was present in the mitochondrial zone in addition to the true mitochondria. Under the conditions employed, the mitochondria were sedimented essentially to their isopycnic position in the gradient at approximately 43.8 per cent sucrose, density 1.20 gm/cc.     

Distribution of NG, NG-dimethylarginine in nuclear protein fractions

Nuclear proteins have been fractionated into five distinct classes according to their extractability from rat liver nuclei at different pH and salt concentrations. The fractions have been analyzed for their amino acid composition which shows the presence of N^G, N^G-dimethylarginine, in sizable amount, in non-histone nuclear proteins (NHNP). This modification is most prominent in proteins which are found associated with rapidly-labeled heterogeneous RNA (HnRNP proteins).     

Analysis of storage proteins in normal and aborted seeds from embryo-lethal mutants of Arabidopsis thaliana

The major storage proteins isolated from wild-type seeds of Arabidopsis thaliana (L.) Heynh., strain Columbia, were studied by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both the hypocotyl and cotyledons of mature embryos contained abundant 12 S (cruciferin) and 2 S (arabin) proteins that appeared similar in size and subunit composition to the cruciferin (12 S) and napin (1.7 S) seed-storage proteins of Brassica napus. The 12 S protein from Arabidopsis was resolved by SDS-PAGE into two groups of subunits with approximate relative molecular weights of 22–23 kDa (kilodalton) and 30–34 kDa. These polypeptides accumulated late in embryo development, disappeared early in germination, and were not detected in other vegetative or reproductive tissues. Accumulation of the 12 S proteins in aborted seeds from nine embryo-lethal mutants with different patterns of abnormal development was studied to determine the extent of cellular differentiation in arrested embryos from each mutant line. Abundant 12 S proteins were found in arrested embryos from two mutants with late lethal phases, but not in seven other mutants with lethal phases ranging from the globular to the cotyledon stages of embryo development. These results indicate that the accumulation of seed-storage proteins in wild-type embryos of Arabidopsis is closely tied to morphogenetic changes that occur during embryo development. Embryo-lethal mutants may therefore be useful in future studies on the developmental regulation of storage-protein synthesis.Abbreviations kDa kilodalton - M_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Is there anionic ATPase in the nuclei of animal cells?

The nuclei of the rat liver, heart, thymus and of the mouse liver isolated in sucrose gradient reveal ATPase sensitive to bicarbonate, sulfite, azide and thiocyanate. The admixture of mitochondria and submitochondrial particles in the nuclear preparation was found negligible, which could not contribute to the anion ATPase in the nuclei. This was demonstrated by the calculation and by the introducing of mitochondria into the nuclear preparations.     

Calcium-binding protein regucalcin inhibits deoxyribonucleic acid synthesis in the nuclei of regenerating rat liver

The effect of regucalcin, a Ca²⁺-binding protein isolated from rat liver cytosol, on deoxyribonucleic acid (DNA) synthesis in the nuclei of regenerating rat liver was investigated. At 1 day after partial hepatectomy, the liver weight was increased about 50% of that of sham-operated rats, and it reached to the same levels as sham operation at 3 days after hepatectomy. Nuclear DNA synthesis was markedly increased at 1 day after hepatectomy, and this increase was also seen at 3 days. Nuclear DNA synthesis was clearly enhanced in the presence of EGTA (0.4 mM) in the incubation mixture. The presence of Ca²⁺ ( 1.0–25 M) caused a significant decrease in the nuclear DNA synthesis of normal rat liver. Regucalcin (0.25 and 0.5 M) clearly inhibited the nuclear DNA synthesis of normal rat liver. This inhibition was also seen in the presence of Ca²⁺ (1.0 M). Moreover, in the liver nuclei obtained at 1 day after partial hepatectomy, the presence of regucalcin (0.05–0.5 M) caused a remarkable inhibition of nuclear DNA synthesis. This effect was also revealed in the presence of EGTA (0.4 mM). Thus, the inhibitory effect of regucalcin was remarkable in regenerating rat liver nuclei in comparison with that of normal rat liver. The present results demonstrate that regucalcin can suppress nuclear DNA synthesis in regenerating rat liver. We suppose that regucalcin may have a role in the regulation of nuclear DNA synthesis in liver cell proliferation.     

The effect of cytisine on mRNA transport in the cytoplasm of eukaryotic cells (plants and animals)

The action of alkaloid cytisine on protein biosynthesis in eukaryotic cells was studied. It was shown that the alkaloid had no effect on mRNA translation. Cytisine inhibited the release of mRNP particles from rat liver and wheat embryos nuclei. Sedimentation properties and distribution in CsCl gradient of the material extracted from alkaloid-treated nuclei did not differ from control one and were similar to informosomes from animal and plant cells described earlier. The main part of mRNA with sedimentation coefficients 14-18S, capable for translation in the cytoplasm is retained in alkaloid-treated nuclei.