Characterization of Novikoff hepatoma small RNAs homologous to repetitive DNAs

Three minor small RNA species from Novikoff hepatoma cells, with homology to repetitive DNA sequences, have been identified and characterized. These small RNAs, designated 5.1S, 6S and T3 RNAs, show homology to Alu 1, Alu 2, and Alu 3 sequences, respectively. 6S and T3 RNAs were found both in the nucleus and cytoplasm, whereas 5.1S RNA was not found in the nucleus. Neural tissues were found to contain a 6S-sized BC1 RNA with homology to I.D. sequences [19]; in contrast, the current study shows that Novikoff hepatoma cells contain a 75–80 nucleotide long (T3) RNA, homologous to I.D. sequences. These data suggest that BCl and T3 small RNAs, homologous to I.D. sequences, are expressed in a tissue-specific manner. These results also show that in addition to the abundant 7SL, 4.5S and 4.5S1 RNAs having homology to repetitive DNA, Novikoff hepatoma cells also contain several minor small RNAs with homology to repetitive sequences.     

Structural organization of ribosomal RNAs from Novikoff hepatoma. II. Characterization of possible binding sites of 5 S rRNA and 5.8 S rRNA to 28 S rRNA

Interrelationships among 5 S, 5.8 S, and 28 S rRNA were probed by methods employed in the accompanying report (Choi, Y. C. (1985) J. Biol. Chem. 260, 12769-12772). Two complexes were isolated from 20 S ribonucleoprotein (RNP) fraction and 60 S subunit. The 20 S RNP fraction was found to contain the 3'-340 nucleotide fragment (domain VII) in association with 5 S rRNA. The 60 S subunit contained a stable complex consisting of the 5'-upstream portion (4220-4462, domain VI and VII), the 3'-downstream portion (4463-4802, domain VII) of 3'-583 nucleotides fragment, and 5.8 S rRNA. By computer analysis and hybridization, the 5'-upstream portion was found to contain the 5.8 S rRNA contact site. By affinity chromatography, the 3'-downstream portion was found to contain the 5 S rRNA association site. Furthermore, by comparison with the secondary structure of 28 S rRNA proposed by Hadjiolov et al. (Hadjiolov, A. A., Georgiev, O. I., Nosikov, V. V., and Yavachev, L. P. (1984) Nucleic Acids Res. 12, 3677-3693), it was found that domain VII is capable of binding 5.8 S rRNA and 5 S rRNA juxtaposed to each other. Accordingly, a model was proposed to indicate that a possible contact site for 5.8 S rRNA is within the region surrounding the alpha-sarcin site (4333-4350) and is a possible association site of 5 S rRNA within the 3'-downstream portion (4463-4802) of the 3'-583 nucleotide fragment (4220-4802).     

Primary and secondary structure of 7-3 (K) RNA of Novikoff hepatoma

7-3 RNA (also known as K-RNA and 7SK-RNA) is a distinct small RNA found in insect to mammalian cells. Previous studies showed that this RNA is not capped, contains no modified nucleotides, is conserved through evolution, is synthesized by RNA polymerase III, and, in part, is associated by polyribosomes. In this study, the complete nucleotide sequence of 7-3 RNA was determined by RNA-sequencing methods, and the sequence is compared with several small RNAs and repetitive DNA sequences for homology. This 330-nucleotide-long RNA contained pppGp as its 5' terminus and exhibited heterogeneity with respect to the 3'-terminal AoH. The nucleotide sequence is: (sequence in text) The RNA is G-C rich, and evidence is presented that 7-3 RNA is in a ribonucleoprotein particle in the cytoplasm.     

Subcellular localization of ribonucleotide reductase in Novikoff hepatoma and regenerating rat liver

The subcellular localization of ribonucleotide reductase was ascertained in Novikoff heptoma and normal and regenerating rat tissue. Over 90% of the cellular ribonucleotide reductase is found to be associated with a membrane fraction derived from the postmicrosomal supernatant after centrifugation at 78,000g for 18 hr which bands at 1.3 m sucrose in a discontinuous sucrose gradient. The properties of this particular ribonucleotide reductase are similar to those reported for mammalian ribonucleotide reductase. This membrane fraction, which contains ribonucleotide reductase, had been previously shown to contain a DNA polymerase whose activity is related to cell proliferation. The association of these two enzymes involved in DNA synthesis leads to the suggestion that there may exist a complex of enzymes involved in deoxynucleotide and DNA synthesis in this membrane fraction.     

Characterization of the glucocorticoid-receptor proteins in the Novikoff hepatoma.

The response of brown adipose and liver mitochondria from the cold-acclimated hamster (Mesocricetus auratus) to the synthetic uncoupler 2-azido-4-nitrophenol has been measured. Brown adipose mitochondria are more readily uncoupled than liver mitochondria. Binding of 2-azido-4-nitrophenol to either kind of mitochondria is competitively inhibited by 2,4-dinitrophenol, by palmitic acid, and by the trifluoromethylphenylhydrazone of carbonyl cyanide. Separate experiments indicated that the number of high-affinity binding sites is approximately the same for either kind of mitochondria; hence it was concluded that observed differences in binding are due to dissimilar dissociation constants of the uncoupler. Brown adipose mitochondria bind 2-azido-4-nitrophenol less tightly than liver mitochondria, but this difference is probably due to the effect of residual long-chain fatty acids which cannot readily be removed. A convenient synthesis of 2-azido-4-nitrophenol is described, along with a method for tritiation of the reagent.     

A yeast endoribonuclease stimulated by Novikoff hepatoma small nuclear RNAs U1 and U2

Using [³H]m⁷Gppp[¹⁴C]RNA-poly(A) from yeast as a substrate, an endoribonuclease has been detected in enzyme fractions derived from a high salt wash of ribonucleoprotein particles of $\text{Saccharomyces}\text{cerevisiae}$. The [³H]m⁷Gppp[¹⁴C]RNA-poly(A) seems to be a preferred substrate since other polyribonucleotides are hydrolyzed more slowly, if at all. The enzyme is inhibited by ethidium bromide, but fully double-stranded polyribonucleotides are not hydrolyzed. The hydrolysis of [³H]m⁷Gppp[¹⁴C]RNA-poly(A) is stimulated about 2.5-fold by the addition of small nuclear RNAs U1 and U2 of Novikoff hepatoma cells. Results show that the stimulation involves an interaction of the labeled RNA with the small nuclear RNA.     

Cell growth-dependent subcellular localization of p8

p8 is a stress-induced protein, biochemically related to the architectural factor HMG-I/Y, overexpressed in many cancers and required for tumor expansion. The molecular mechanisms by which p8 may exert its effect in aspects of growth is unknown. Using immunocytochemistry, we found that p8 presents nuclear localization in sub-confluent cells, but it localizes throughout the whole cell in high density grown cells. Cells arrested in Go/G1, either by serum deprivation or by hydroxyurea treatment, show a nucleo-cytoplasmic localization of p8, whether in the rest of the cell cycle stages of actively dividing cells the localization is nuclear. A comparison of p8 sequences from human to fly predicts a conserved bipartite nuclear localization sequence (NLS). The putative NLS has been demonstrated to be functional, since nuclear import is energy dependent (inhibited by sodium azide plus 2-deoxyglucose), and fusion proteins GFP-p8 and GFP-NLSp8 localize to the nucleus, whereas GFP-p8NLSmut in which with Lys 65, 69, 76, and 77 mutated to Ala localized to the whole cell. p8 localization does not involve the CRM1 transporter, since it is insensitive to leptomycin B. Inhibitors of MAPK pathways did not affect p8 subcellular localization. The inhibition of deacetylation with Trichostatin A promotes cytoplasmic accumulation of p8. The results suggest that p8 growth stage-dependent localization is regulated by acetylation, that p8 is not free within the cell but forming part of a complex and that it may exert a role in both subcellular localizations.     

Osmotic permeability of Novikoff hepatoma cells

The osmotic permeability coefficient (P_f) for water movement across Novikoff hepatoma cells was found to be 82 ± 3 (S.E.) · 10^?5 cm · s^?1 at 20°C. The corresponding diffusional permeability coefficient for ³HHO (P_d) was 97 ± 10 (S.E.) · 10^?5 cm · s^?1, therefore the ratio $\text{P}{}_{\mathrm{<mtext>f</mtext>}}\text{P}{}_{\mathrm{<mtext>d</mtext>}}$ is close to unity. The apparent activation energy for water filtration was 10.4 ± 0.4 (S.E.) kcal · mol^?1. This value is significantly greater than the activation energy for the self diffusion of water. The product of the hydraulic permeability coefficient and the viscosity coefficient for water was temperature-dependent. However, the product of the hydraulic permeability coefficient and the viscosity coefficient for membrane lipid did not vary with temperature. These data are interpreted as evidence for water movement across a lipid membrane barrier rather than through aqueous channels.     

Characterization and subcellular localization of chlorophyllase from Ginkgo biloba

Chlorophyllase (Chlase) catalyzes the initial step of chlorophyll (Chl)-degradation, but the physiological significance of this reaction is still ambiguous. Common understanding of its role is that Chlase is involved in de-greening processes such as fruit ripening, leaf senescence, and flowering. But there is a possibility that Chlase is also involved in turnover and homeostasis of Chls. Among the de-greening processes, autumnal coloration is one of the most striking natural phenomena, but the involvement of Chlase during autumnal coloration is not clear. Previously, it was shown that Chlase activity and expression level of the Chlase gene were not increased during autumnal coloration in Ginkgo biloba, indicating that Chlase does not work specially in the de-greening processes in G. biloba. In this study, we characterized the recombinant Chlase and analyzed its subcellular localization to understand the role of the cloned Chlase of G. biloba (GbCLH). GbCLH exhibited its highest activity at pH 7.5, 40 degrees C. Kinetic analysis revealed that GbCLH hydrolyzes pheophytin (Pheo) a and Chl a more rapidly than Pheo b and Chl b. Transient expression analysis of 40 N-terminus amino acids of GbCLH fused with GFP (green fluorescent protein) and subcellular fractionation showed that GbCLH localizes within chloroplasts. Together with our previous results, property of GbCLH and its location within the chloroplasts suggest that GbCLH plays a role in the turnover and homeostasis of Chls in green leaves of G. biloba.     

Phosphorylation of purified Novikoff hepatoma topoisomerase I

The purified Novikoff hepatoma nuclear phosphoprotein with a molecular weight of 110 kdalton and pI 8.4, was found to be a type I topoisomerase. When isolated from 32P-labeled Novikoff ascites cells or incubated in vitro with protein kinase, phosphoserine was found to be its major phosphorylated amino acid. The enzymatic activity of topoisomerase I was altered by changes in phosphorylation. Its activity was increased by protein kinase and it was decreased by alkaline phosphatase.     

Characterization of Novikoff hepatoma mRNA methylation and heterogeneity in the methylated 5' terminus.

KOH digestion of methyl-labeled poly(A)+ mRNA purified by (dT)-cellulose chromatography produced mononucleotide and multiple peaks of a large oligonucleotide (-6 to -8 charge) when separated on the basis of charge by Pellionex-WAX high-speed liquid chromatography in 7 M urea. Heat denaturation of the RNA before application to (dT)-cellulose was required to release contaminants (mostly 18S rRNA) that persisted even after repeated binding to (dT)-cellulose at room temperature. Analysis of the purified poly(A)+ mRNA by enzyme digestion, acid hydrolysis, and a variety of chromatographic techniques has shown that the monucleotide (53%) is due entirely to N6-methyladenosine. The large oligonucleotides (47%) were found to contain 7-methylguanosine and the 2'-0-methyl derivatives of all four nucleosides. No radioactivity was found associated with the poly(A) segment. Periodate oxidation of the mRNA followed by beta elimination released only labeled 7-methylguanine consistent with a blocked 5' terminus containing an unusual 5'-5' bond. Alkaline phosphatase treatment of intact mRNA had no effect on the migration of the KOH produced oligonucleotides on Pellionex-WAX. When RNA from which 7-methylguanine was removed by beta elimination was used for the phosphatase treatment, distinct dinucleotides (NmpNp) and trinucleotides (NmpNmpNp) occurred after KOH hydrolysis and Pellionex-WAX chromatography. Thus Novikoff hepatoma poly(A)+ mRNA molecules can contain either one or two 2'-0-methylnucleotides linked by a 5'-5' bond to a terminal 7-methylguanosine and the 2'-0-methylation can occur with any of the four nucleotides. The 5' terminus may be represented by m7G5'ppp5' (Nmp)lor2Np, a general structure proposed earlier as a possible 5' terminus for all eucaryotic mRNA molecules (Rottman, F., Shatkin, A., and Perry, R. (1974), Cell 3, 197). The composition analyses indicate that there are 3.0 N6-methyladenosine residues, 1.0 7-methylguanosine residue, and 1.7 2'-0-methylnucleoside residues per average mRNA molecule.     

Cross-linking of Novikoff ascites hepatoma cytokeratin filaments

We have investigated the structure of solubilized cytokeratins from Novikoff ascites hepatoma using the cleavable cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) in the presence of 6 M urea to effect partial complex melting. By two-dimensional gel electrophoresis, in which the protein cross-links were broken in the second dimension, we have identified two major complexes as a p39-p56 dimer and a (p39-p56)2 tetramer, p39 and p56 being two of the major cytokeratins in Novikoff ascites hepatoma. Experiments investigating possible relationships between the dimer and tetramer employed immunoblots and two monoclonal antibodies which recognized either p56 or p39 cytokeratins. When very low protein concentrations were cross-linked, the dimer was the predominant product. As protein concentration increased, we noted a decrease in dimers and a corresponding increase in tetramers, suggesting that the dimer may be a precursor to the tetramer. In support of the cross-linking experiments, two-dimensional gel electrophoresis using 4 M urea in the first dimension indicated a predominant association of p56 and p39 in the Novikoff ascites hepatoma cytokeratin complexes.