SYNTHESIS OF NUCLEAR RNA IN NERVE AND GLIAL CELLS

—Tritium-labelled RNA precursors were injected at 30 min intervals into the fourth ventricle of rats or rabbits. After 4 h the nuclei from neurones, astrocytes, and other glial cells were isolated and RNA extracted. Investigations were performed in order to establish optimum conditions for RNA extraction from this particular material. The sedimentation patterns obtained in sucrose gradients were similar to those of nuclear RNA from other mammalian tissues and showed the presence of RNA species with high specific activities in the region of the gradient between 10S and 16S and above 28S. All three types of nuclei contained a 45S and a 38S RNA. Moreover, a 32S component could be identified in astrocytic nuclei, a 35S fraction in neuronal nuclei, and both a 32S and 35S RNA in nuclei from glial cells. The nuclei from the various cell types also differ with respect to the rate of incorporation of the label into the nuclear RNA, being four times higher in astrocytic and neuronal nuclei than in those derived from the other glial cells.     

RNA synthesis in isolated bovine thyroid nuclei and nucleoli. alpha-Amanitin effect, a hint to the existence of a specific regulatory system

DNA dependent RNA polymerase activities in isolated bovine thyroid nuclei and nucleoli have been studied. They retain their RNA synthetic activity for an extended period of time. This RNA synthetic activity is sensitive to actinomycin D and requires the presence of all four ribonucleoside triphosphates. The optimal conditions have been determined. Polyacrylamide gel electrophoresis reveals that the RNA synthesized has a size distribution ranging from 34S to 4S. The production of 18S-8S RNA is very sensitive to low concentrations of alpha-amanitin. However, in isolated bovine thyroid nuclei (not in nucleoli) this drug displays an effect on all RNA classes produced. The alpha-amanitin induced drastic decrease of [3H]-UMP incorporation in RNA of all sizes synthesized by isolated bovine thyroid nuclei is discussed.     

A polymerase activity forming 5S and pre-4S RNA in isolated HeLa cell nuclei

Isolated HeLa cell nuclei are capable of synthesizing 5S and pre-4S RNA. The labeling of these low molecular weight species has been compared with the labeling of nucleolar RNA and nuclear heterogeneous RNA. The 5S and pre-4S RNA molecules made in vitro were identified by their mobility on SDS acrylamide gels and by the sensitivity of pre-4S RNA to enzymes which cleave it in vitro to 4S RNA. Their mobilities and cleavage properties are similar to the RNA made in vivo. Unlike the nuclear heterogeneous RNA, the synthesis of the two small molecular weight RNAs is resistant to α-amanitin.A large proportion of 4S RNA labeled in vitro appears to be formed de novo. The ratio of the terminal uridine to the internal uridine 3′-monophosphate remains constant with time, even though there is linear incorporation into the pre-4S RNA in the isolated nuclei.Production of the nucleolar RNA and pre-4S RNA has been compared in the presence of various ions. The pre-4S RNA synthesis has a sharper maximum for (NH₄)SO₄ and MgCl₂ than does the synthesis of nucleolar RNA. The in vitro synthesis of pre-4S is more sensitive to ellipticine and pCMB than the production of nucleolar RNA. These differences between the production of pre-4S RNA and nucleolar RNA are discussed with respect to in vitro reinitiation and the possibility that different polymerases are involved in their synthesis.     

Transcription in Isolated Wheat Nuclei: I. ISOLATION OF NUCLEI AND ELIMINATION OF ENDOGENOUS RIBONUCLEASE ACTIVITY

Nuclei isolated from embryos of wheat (var. Yamhill) incorporated [(3)H]UTP into a trichloroacetic acid-insoluble product linearly for 60 minutes. When the RNA synthesized in vitro was analyzed on a sucrose gradient, the amount of RNA in the 4S region increased with longer incubation times. These data and the absence of higher molecular weight RNA of specific size classes in our work (and previously published reports) suggested that nuclear fractions from plant tissue contained active nucleases. This was confirmed when wheat nuclei were mixed with [(3)H]yeast RNA (4, 18, 26S). All of the radioactive yeast RNA was degraded within 30 minutes to species sedimenting between 4 and 10S. The inclusion of high salt (125 millimolar (NH(4))(2)SO(4), 100 millimolar KCl), EGTA, and exogenous RNA or DNA reduced but did not eliminate endogenous RNase activity. Wheat embryo nuclei were further purified by centrifugation on a gradient of a polyvinylpyrrolidone-coated colloidal silica suspension (Percoll). These nuclei were ellipsoidal, free of cytoplasmic material, and lacked endogenous nuclease activity when assayed with [(3)H]yeast RNA. Sucrose gradients were not as effective as Percoll gradients in purifying nuclei free of RNase activity. The Percoll method of isolating nuclei and the RNase assay reported here will be useful in isolating plant nuclei that are capable of synthesizing distinct RNA species in vitro.     

Processing of RNA by cytoplasmic extract

Nuclei are isolated from HeLa cells by a low speed centrifugation procedure. These nuclei can carry out RNA synthesis at 30°. The effect of cytoplasmic extract on the transport and processing of RNA is measured. In the presence of cytoplasmic extract the precursor to 4S RNA is processed to 4S RNA and an RNA species of 7–9S is transported outside the nuclei.Abbreviations HEPES N-2-Hydroxymethylpiperazine-N'-2'-ethanesulfonic acid - DTT dithiothreitol - cDNA complementary DNA     

Direct and reverse transport of RNA in a system of isolated HeLa cell nuclei

In a system containing isolated HeLa cell nuclei the release of RNA from the nuclei may be paralleled with the antagonistic process, i. e., RNA translocation into the nuclei. The RNA release from the nuclei depends on incubation time, pH, Mg2+ and nucleoside triphosphate concentration. The rate of reverse transport depends on pH, size of RNA to be translocated and the physiological state of the nuclear membrane. Low molecular weight RNAs (less than 10 S) are translocated into the nuclei most effectively. The nuclei of synchronized HeLa cells in the G1-period are more "permeable" for translocated RNA as compared with the S-phase HeLa cell nuclei.     

In Vitro RNA Synthesis by Rat Liver and I Leukemic Nuclei. Isolation of Undegraded RNA

Incubation of nuclei from rat liver or human leukemic cells in the presence of ³H-UTP² and other factors results in th incorporation of label into a material precipitable by acid, alcohol or ether. This materials is isolated by phenolsds extraction, is sensititve to ribonuclease digestion and presumed to be RNA.

The addition of Cu++ to the incubation system is necessary to inhibit RNA breakdown and allows the isolation of undegraded RNA without interefering with th incorporation of radiosactivity. The time patterns of labl incorporation by the two nuclei preparations are different. Whereas label incorporation by th two nuclei preparations are different. Whereas labelincorporation by liver nuclei continues to increase up to 60 minutes, incorporation by th leukemic nuclei is high during the first 10 minutes and continues at a slower rate up to 45 minutes of incubation. further, th two nuclei preparations also synthesize diferent RNA species. While liver nuclei synthesize RNA sedimenting at 4.5S and 7S to 13S, leukemic nuclei synthesize a heterogeneous, polydisperse type of RNA.     

Cytologische Lokalisation von 5S und 18/25S RNA Genorten in Mitose-Chromosomen von Vicia faba

Homologous in situ hybridization with tritiated 4S, 5S and 18/25S RNA from root tip meristems of Vicia faba has been used to study the pattern of distribution of DNA sequences coding for these RNAs in the diploid nuclei. 5S RNA hybridizes to two regions of the satellites of the pair of satellited chromosomes. The sites differ in the level of in situ hybridization implicating different degrees of redundancy. 18/25S RNA hybridization is concentrated to the secondary constriction of these satellite chromosomes. Both, 5S and 18/25S ribosomal RNA gene sites are located on the same pair of chromosomes, but obviously the sequences are not contiguous. An association of 5S RNA cistrons with heterochromatin is assumed. Additional RNA gene sites as well as 4S RNA gene sites are not detectable.     

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.     

Intercellular information transfer in the immunogenesis process. VIII. Analytical separation on agar and polyacrylamide gel of RNA fractions from the spleens of immunized and intact rats

RNA isolated from the spleens of intact rats and from rats with immunized sheep red cells was fractionated through three steps: 1 - extraction from phenol nuclei at 50-55 degrees and 65-75 degrees C, 2 - calcium-phosphate chromatography, 3 - agar electrophoresis. Eight agar fractions were obtained of the spleens of immunized rats, an increased RNA content was manifested in at least three agar fractions: the first (4 S), the third (21 S) and the eighth (26 S) ones. The first and the eighth immune RNA fractions, as it was shown earlier, induce the synthesis of antibodies in the rat transplantable lymphosarcoma cell. The first agar fraction of nuclear RNA from the spleens of immunized and intact rats were additionally separated using PAAG electrophoresis. The 4 S agar RNA fraction appears to be rather heterogeneous. It contains 4 S, 4.5 S, 5 S, 5.8 S, U1, U2 and 8 SII fractions, which are low-molecular nuclear RNAs, the 4 S subfraction prevailing. It is suggested that the 4 S PAAG subfraction is most active in the synthesis of antibodies induced by the heterogeneous agar 4 S RNA.     

A new species of virus-coded low molecular weight RNA from cells infected with adenovirus type 2

A virus-coded low molecular weight RNA (5.2S), which migrates slightly faster on polyacrylamide gels than the well characterized adenovirus-specific 5.5S RNA, has been isolated from cells infected with adenovirus type 2. Hybridization-competition experiments and RNA fingerprints indicate that the two virus-associated (VA) RNAs differ in their primary structures. The gene for 5.2S RNA is located to the right of the gene for 5.5S RNA, on the I strand of a DNA segment which extends between positions 30.3 and 32.2 on the map of adenovirus type 2 DNA.Both 5.5S and 5.2S RNA can be detected early after infection and also in the presence of cytosine-arabinoside or cycloheximide. After the onset of viral DNA replication, the synthesis of 5.2S RNA levels off, whereas 5.5S RNA is synthesized in increasing amounts. Both 5.2S and 5.5S RNAs are synthesized in isolated nuclei by an enzyme which resembles RNA polymerase III in its sensitivity to α-amanitin. In isolated nuclei, both RNA species are labeled with β-³²P-labeled GTP, which suggests that they are initiated at separate promoter sites.     

A cell-free assay system for the analysis of changes in RNA synthesis during the development of Xenopus laevis.

Conditions were established for the maximal synthesis of RNA by Xenopus cultured cell nuclei. These differed from those for mammalian nuclei in having a lower K⁺ optimum. The Xenopus nuclei showed all three RNA polymerase activities and processed rRNA to 28 S and 18 S species. Extracts of full-grown oocytes stimulated the rate of RNA synthesis 2.5-fold and caused it to continue linearly for at least 6 hr. This full effect could be produced by preincubation of the nuclei with oocyte extract, followed by their reisolation and assay under standard conditions, provided that the four ribonucleotide triphosphates were present during the preincubation. The stimulatory factor(s) were mainly present in the cytoplasm of the oocyte. They produced quantitatively identical stimulations of RNA synthesis in hamster nuclei. The overall stimulatory effect of cell extracts disappears in the egg, remains absent through cleavage, but reappears at the late blastula stage. This corresponds to the changes in RNA synthesis believed to occur in early development. The extracts affect polymerases I and III, but not II to a significant extent. They also stimulate the incorporation of [γ-³²P]ATP and GTP into RNA, though to a lesser extent than the incorporation of [³H]UTP. The egg extract inhibits γ-³²P incorporation. There therefore seems to be some effect on the initiation of new chain synthesis, but its magnitude is uncertain, and the effect could be indirect.     

A new class of small nuclear RNA molecules synthesized by a type I RNA polymerase in HeLa cells

A new class of previously undetected small RNA molecules with a range of discrete sizes between 6S and 10S has been identified in HeLa cell nuclei. They differ in size and location from the previously described small nuclear RNA species (snRNA). These RNA molecules were initially found by selective RNA labeling in vitro in isolated nuclei. The in vitro products migrate in gel electrophoresis in the region from 6–10S with predominant components between 8S and 10S. They are labeled in the presence of very high concentrations of α-amanitin (150–400 μg/ml), suggesting they are synthesized by a type I polymerase. Unlike the major polymerase I product, ribosomal precursor RNA, however, these molecules are found in the nucleoplasm and their labeling is not affected by pretreatment of cells with low concentrations of actinomycin D (0.04 μg/ml). Their formation by a presumptive polymerase I type of enzyme is the basis of their tentative designation as small nuclear polymerase I (snPI) RNAs.The snPI RNA molecules appear to be associated with chromatin and the nuclear matrix. They can be selectively eluted from nuclei leaving most of hnRNA behind. This association is used as the basis of fractionation procedures which separate these molecules from hnRNA and permit the demonstration of the synthesis of at least the most predominant of these RNA molecules in vivo. w     

Synthesis of messenger RNA-like molecules in isolated myeloma nuclei.

Nuclei isolated from mouse myeloma cells grown in tissue culture are capable of synthesizing RNA for prolonged periods of time. Addition of cytoplasmic extracts to the system stimulates slightly the rate and prolongs the time of synthesis. As judges by sedimentation in SDS and in formamide gradients, the size of the RNA synthesized is heterogeneous from smaller than 10S to larger than 45S, thus resembling in vivo made RNA. The characteristics of some of the RNA are in keeping with those expected to be for mRNA. Fifty percent of the RNA synthesis is sensitive to alpha-amanitin. After an incubation of two hours in the absence of alpha-amanitin about 10 percent of the newly synthesized RNA is found outside of the nuclei; it sediments with a broad distribution at 18S. A considerable fraction of the RNA that is released from nuclei in vitro can promote the formation of polyribosomes, and contains molecules that are polyadenylated and "capped".