The in vivo equivalence of a cell-free system for RNA processing and transport

The equivalence of messenger RNA released (transported) from isolated rat liver nuclei to three selected media, with messenger RNA normally released to liver cytoplasm $\text{in vivo}$, has been evaluated by competitive DNA: RNA hybridization. Near normal nuclear restriction was exhibited by nuclei in media fortified with ATP, salts, spermidine and dialyzed cytosol. The RNA transport in the latter system was markedly inhibited by colchicine as was also the transport of RNA $\text{in vivo}$. Both nuclear restriction and sensitivity of the RNA transport to colchicine in media lacking spermidine and cytosol deviated significantly from the $\text{in vivo}$ norm. The results emphasize the importance of establishing the $\text{in vivo}$ equivalence in cell-free systems designed to study RNA synthesis, processing and transport.     

Induction of single-strand regions in nuclear DNA by adriamycin.

Incubation of adriamycin with isolated nuclei converts nuclear DNA to a form which is susceptible to hydrolysis by $\text{Neurospora}$$\text{crassa}$ nuclease an enzyme highly specific for the cleavage of single-stranded DNA. The effect of adriamycin on nuclear DNA incubated in the presence of the nuclease can be determined by measuring the release of acid-soluble nucleotides or by analyzing the DNA after centrifugation in neutral sucrose gradients. Similar changes in chromatin structure are not observed during incubation of nuclei with adriamycin alone. In addition to adriamycin, daunomycin and ethidium bromide are also active in inducing the formation of DNA structures which are susceptible to the $\text{Neurospora}$$\text{crassa}$ nuclease. The results suggest that certain antitumor agents can induce the formation of single-strand regions in nuclear DNA and that these sites probably occur as a result of a DNA strand separating event.     

An enzymic analysis of a nuclear envelope fraction

A rat liver nuclear envelope fraction isolated essentially by the technique of Monneron et al. (J. Cell Biol. 55, 104–125 (1972)) is characterized by high levels of glucose-6-phosphatase and 5′-nucleotidase. A broadly specific nucleoside triphosphatase activity is present. Cytochromes $\text{b}{}_5$ and $\text{P-450}$ as well as NADPH- and NADH-cytochrome $\text{c}$ reductase activities are present but at lower levels than found in microsomes. Cytochrome $\text{c}$ oxidase activity is low. RNA polymerase activity is absent from the nuclear envelope fraction. Cytochemistry shows that glucose-6-phosphatase activity is strong and restricted to the nuclear envelope of nuclei. 5′-Nucleotidase shows weak reaction deposit in whole nuclei but in contrast gives clear reaction deposit in isolated nuclear envelopes. Cytochemical reaction deposit due to nucleoside trisphosphatase activity is not restricted to the nuclear envelope but is found to a larger extent within the nucleus.     

Phospholipid interactions in rat liver nuclear matrix

Rat liver nuclear matrix has been isolated by salt extraction and nuclease digestion of nuclei. Under the electron microscope, the matrix appears as a spongelike network joined by thinner fibrils. Biochemical analysis shows a high protein content and low amounts of nucleic acid and phospholipid. Treatment of the matrix with phospholipase C results in a release of most of the nucleic acid, and a disappearance of the fibrils, however the appearance of the matrix is largely unaffected. It seems likely that phospholipids are responsible for the hydrophobic interactions between nucleic acids and matrix fibrils. From $\text{in vitro}$ labelling studies the released DNA is more recently synthesised than the bulk material, however the matrix bound RNA appears to label less rapidly than total nuclear RNA.     

Capsaicin and potassium evoked substance P release from the nucleus tractus solitarius and spinal trigeminal nucleus invitro

The nucleus tractus solitarius and the spinal trigeminal nucleus receive peripheral sensory input from substance P containing afferent nerves. This study demonstrates that $\text{in}$$\text{vitro}$ depolarization of these nuclei in tissue slices evokes a calcium-dependent efflux of substance P immunoreactivity. Capsaicin (33μM) also elicits substance P release from the nucleus tractus solitarius and spinal trigeminal nucleus but not from the hypothalamus. The occurrence of potassium-stimulated SP release from the two medullary nuclei fulfills one of the criteria for neurotransmitter status. The capsaicin data support the contention that this agent elicits release of substance P from nuclear regions receiving peripheral afferent information in substance P nerves independent of the particular sensory modality served but is ineffective in nonsensory areas.     

Increased levels of rat hepatic nuclear free and engaged RNA polymerase activities during liver regeneration.

Rat liver nuclei, seventeen hours after partial hepatectomy, showed a two to three-fold increase in total RNA synthesis $\text{in vitro}$ over the sham operated controls. When tested with exogenous synthetic template, this was found to be mainly a reflection of increased levels of both the nuclear free and engaged RNA polymerase activities $\text{per se}$. It was also observed that there was a greater stimulation of the species of RNA polymerase that are α-amanitin resistant than sensitive (3.2 μg/ml). This observation was further confirmed by DEAE-Sephadex column chromatography of the solubilized nuclear free and engaged RNA polymerases and found RNA polymerase I and IIIa were the major species greatly stimulated during this period of liver regeneration. These data suggest not only that there exists a sensitive equilibrium between the nuclear free and engaged RNA polymerases; they also suggest the possibility that RNA polymerase itself may play a positive role in the regulation of gene expression.     

A new eukaryotic RNA polymerase factor: a factor from chicken myeloblastosis cells which stimulates transcription of denatured DNA

A heat-stable protein factor, capable of stimulating RNA synthesis by nuclear RNA polymerase II, was found in isolated nuclei of chicken myeloblastosis cells. It is adsorbed to a DEAE-Sephadex column used for RNA polymerase purification and then is eluted with 0.1 M ammonium sulfate. This factor appears to differ from previously reported eukaryotic RNA polymerase factors in its property of stimulating the activity of denatured (or single-stranded) DNA template. When heated, this factor contains no detectable endonuclease or exonuclease activity. The degree of stimulation is greater with chicken myeloblastosis RNA polymerase IIb than IIa and is most efficient when homologous DNA is used as template. This factor causes no stimulation of $\text{E. coli}$ RNA polymerase.     

Sub-nuclear fractionation. I. Procedure and characterization of fractions

A procedure for fractionation of nuclei from rat liver, Xenopus liver and Xenopus erythrocytes is described. It is based on mild sonication of isolated nuclei for 7–12 sec in a nearly isotonic medium, separation of nuclear sap and centrifugation on a discontinuous sucrose density gradient containing Na and K citrate. Nuclei are thus separated in a single operation into 8 fractions representing nucleoplasm, euchromatin, nucleoli, heterochromatin and nuclear membranes. The sub-nuclear fractions were characterized by chemical composition (DNA, protein, RNA and phospholipid), electron microscopy, thermal denaturation properties of chromatin, relative binding of ${}^3\text{H-actinomycin D}$, polyacrylamide gel electrophoresis of nuclear proteins and titration of membranes against Triton X-100. Approx. 10% of total DNA was recovered as heterochromatin associated with membranes but the bulk of nuclear membranes co-sedimented with the major euchromatin zones. Subnuclear fractions prepared in this way retain virtually all the RNA polymerase activity bound to chromatin [41].     

An improved method for the isolation of yeast nuclei active in RNA synthesis in vitro

An improved method has been developed for the isolation of nuclei from $\text{Saccharomyces cerevisiae}$ for the study of RNA synthesis $\text{in vitro}$. Utilization of Ficoll in the isolation procedure greatly increases the activity of RNA polymerase in isolated nuclei. Nuclei prepared by this procedure are essentially free of mitochondrial DNA.     

Studies on isolated cell components III. A cytological study of the effects of heparin on isolated nuclei

The gel released from isolated rat liver nuclei in response to heparin treatment has been found to stain with methylene blue, azure A, and methyl green when the dyes were added to the salt-sucrose nuclear isolation medium.Azure A and methylene blue caused rapid nuclear shrinkage to as little as $\text{1}\text{4}$ the original nuclear volume. Subsequent treatment with heparin caused the nuclei to fade rapidly and swell to approximately $\text{5}\text{4}$ of the original volume. With methylene blue stained nuclei heparin caused the extrusion of deeply stained, slightly birefringent rods through apertures on the nuclear surface. Methyl green also caused nuclear shrinkage, but to a lesser degree.Studies with the Feulgen reaction demonstrated structural damage in isolated rat liver nuclei as a result of heparin action. The viscous material released by heparin was shown to be Feulgen positive by resort to hydrolysis without prior fixation, since after customary fixatives the presence of a Feulgen positive reaction outside the nucleus could not be clearly demonstrated. The possibility is suggested that the Feulgen reaction following the customary fixatives depends in part on the manner in which the DNA is bound.The nuclei of leucocytes with visually intact cell membranes included in the nuclear preparations failed to show structural damage due to heparin and it is suggested that either the cell membrane provides some protection against heparin action or that damaged cells are more susceptible to this action.Observations made provide additional basis for the conclusion that heparin replaces DNA in the nucleo-histone of the nucleus, resulting in the structural damage observed, and releasing DNA in the form of a soluble viscous protein containing complex.     

Nuclear thyroid hormone receptors: evidence for association with nucleolar chromatin.

When hypothyroid rat liver nuclei labeled $\text{in vivo}$ with [125 I]L-triiodothyronine are incubated with micrococcal nuclease, the nuclear chromatin is digested and chromatin particles are released into the medium. The nuclease-treated nuclei contain intact nucleoli and a residual chromatin fraction. When this residual chromatin is purified, it contains only a small percentage of the initial nuclear DNA but is strikingly enriched in [125 I]L-triiodothyronine. This chromatin fraction has many of the characteristics of nucleolar chromatin including a high protein to DNA ratio, an abundance of nonhistone proteins, and a relatively high RNA to DNA ratio. An association of thyroid hormone receptors with a nucleolar component implicates this organelle in the early events of thyroid hormone action.     

In vitro synthesis and stability of RNA in isolated nuclei from bovine lymphocytes

Nuclei from Concanavalin A-stimulated lymphocytes (30 hr after Con A addition) incorporate up to 5 times more (3-H)UTP into RNA than nuclei from resting lymphocytes. The incorporation kinetics is linear for almost 60 min. 14–20% of the $\text{in vitro}$ labeled RNA is polyadenylated. Poly(A) (?)RNA from both types of nuclei sediments from 4–5S up to more than 30S on sucrose gradients. Nuclei from stimulated cells synthesize about double the amount of RNA larger than 18S than nuclei from resting cells. The same holds for poly(A) (+)RNA. Poly(A) (?) RNA labeled during 10 min in both types of nuclei is stable during a 30 min chase. Under the same conditions poly(A) (+)RNA in nuclei from resting cells is degraded to about 50% during the chase whereas it is stable in nuclei from stimulated cells.     

N-6-methyl-adenosine in adenovirus type 2 nuclear RNA is conserved in the formation of messenger RNA

In the biogenesis of adenovirus type 2 messenger RNAs, methylation occurs at the 5′ end (cap) and to internal adenosine residues to yield N⁶-methyl-adenosine (m⁶A) (Sommer et al., 1976; Moss &; Koczot, 1976; Wold et al., 1976). The kinetics of accumulation of ³H from methyl-labeled methionine and ¹⁴C from uridine into Ad-2²-specific RNA was measured late in Ad-2 infection. As reported previously (Nevins &; Darnell, 1978a), the rate of accumulation of [¹⁴C]uridine label in nuclear RNA was approximately four- to fivefold faster than in the cytoplasmic RNA, indicating a conservation of about 20% for the total RNA. The initial rates of [³H]methyl label in m⁶A in nuclear RNA and in the cytoplasmic RNA were approximately equal, suggesting a complete (or nearly complete) conservation of m⁶A.In accord with the accumulation kinetics, the ratio of ³H to ¹⁴C was higher in cytoplasmic RNA than in nuclear RNA that hybridized to equivalent regions of the Ad-2 DNA.A mathematical model was designed to evaluate the accumulation of methyl label in m⁶A, taking into consideration the three major parameters that affect the accumulation curves: equilibration of the S-adenosyl-methionine pool, the nuclear dwell time of sequences destined to be mRNA, and the cytoplasmic stability of mRNA. The half-time ($\text{t}\text{1}\text{2}$) for pool equilibration was determined experimentally to be 22 minutes and the nuclear dwell time and the mean life-time of cytoplasmic mRNA were estimated from ¹⁴C label to be about 30 and 70 minutes, respectively.The model gave an excellent fit to the data when the $\text{t}\text{1}\text{2}$ for pool equilibration time of 24 ± 2 minutes, a nuclear dwell time of 25 ± 10 minutes, and a mean cytoplasmic mRNA life-time of 75 ± 30 minutes were used to evaluate accumulation curves. Even when data from a restricted region of the genome, $\text{40.5–52.6}$, which encodes the main portion of at least five 3′ co-terminal mRNAs whose spliced junction with the tripartite leader sequence varies from $\text{38, 40, 43, 45}$, and $\text{48}$ was analyzed, it appeared that m⁶A was conserved.Finally, m⁶A was found to be added in a brief label (3.5 min) mainly to nuclear molecules that were longer than any cytoplasmic RNA. The conservation of m⁶A and its addition prior to splicing raise the possibility that internal methylations are involved, in the formation of mRNA.