Stability of rapidly labelled messenger ribonucleic acid in Bacillus amyloliquefaciens during the phases of minimum and maximum extracellular enzyme formation.

The stability of rapidly labelled hybridizable messenger RNA in both exponential and post-exponential phase cells of Bacillus amyloliquefaciens was measured in terms of the rate of loss of its radioactivity. In the exponential phase, where 96% of the mRNA was specific for cell proteins and only 4% was exoprotein mRNA, the label was lost exponentially from the rapidly labelled hybridizable mRNA fraction with a half-life of six minutes at 30 °C. The antibiotic rifampicin, at a concentration of 10 μg/ml, had no effect on the characteristics of decay of this exponential-phase mRNA. In the post-exponential phase, where there were equal amounts of cell protein and exoprotein-specific mRNA, rapidly labelled hybridizable mRNA decayed exponentially in the presence of rifampicin (10 μg/ml), with a half-life of six minutes at 30 °C. In the absence of rifampicin the characteristics of decay were more complex. The evidence available suggested that this was due to the superimposition of a component attributable to reincorporation of degradation products of radioactive RNA on the characteristic exponential decay pattern of the post-exponential mRNA.Measurement of the stability of active mRNA, by studying the loss of ability to incorporate l-[¹⁴C]leucine into protein in the presence of rifampicin (10 μg/ml), gave half-lives of 4.5 minutes and six minutes, respectively, for exponential and post-exponential material.     

The incorporation of radioactive fatty acids into the phospholipids of nerve-cell-body membranes in vivo. Evidence for highly labelled neuronal nuclei

1. Nerve cell bodies were isolated in bulk from cerebral cortices of 15 day-old rabbits after intrathecal injections of [³H]plamitate, [³H]oleate or [³H]arachidonate and [¹⁴C]glycerol. 2. Nuclear, microsomal and two mitochondrial fractions were isolated from homogenates of the radioactively labelled nerve cell bodies by using differential and discontinuous-gradient centrifugation. 3. After 7.5min in vivo, a high percentage (>80%) of the total ³H-labelled fatty acid radioactivity was found in the membrane fractions of the nerve cell bodies, whereas after 60min in vivo 50% of the total [¹⁴C]glycerol radioactivity was found in the high-speed supernatant. 4. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, and the radioactivity in neutral lipid and non-esterified fatty acid fractions were determined in the four subfractions, as were the distributions of several marker enzymes and nucleates. 5. With respect of ³H-labelled fatty acid, the phospholipids of the nuclear fraction had the highest specific radioactivities of the four subfractions. However, for [¹⁴C]glycerol labelling, generally the ¹⁴C specific radioactivities for individual phospholipids were comparable in the four subfractions. This latter observation suggests transport of phospholipids synthesized de novo between membranes of the nerve cell body. 6. Double-labelling experiments demonstrated that individual phospholipids and the combined neutral lipids of the nuclear fraction had higher labelling ratios of ³H-labelled fatty acid/[¹⁴C]glycerol than did the corresponding lipids of the microsomal or mitochondrial fractions. 7. On the basis of the labelling results and the marker studies, it is proposed that it is indeed the nuclei of the nuclear fraction that have these lipids highly labelled with ³H-labelled fatty acid, and the existence of nuclear acyl transferases that are responsible for this fatty acid incorporation is suggested.     

NEWLY SYNTHESIZED RNA IN NUCLEI ISOLATED FROM NERVE AND GLIAL CELLS

—Nuclear RNA from neurones, astrocytes and other glial cells was pulse-labelled in vivo with [³H]uridine and analysed by sucrose density-gradient centrifugation after various periods of incorporation. Thirty min after the injection of the isotope, rapidly-labelled RNA appeared in all three cell types, a heterogeneous fraction sedimenting above 30S, the others at 25 and 12S. The transformation rate of the two latter components was equally rapid in all three types of nuclei studied. These components are assumed to be of messenger nature. The heavy fractions underwent transformations which in other cells have been described to lead to rRNA formation. The temporal pattern as well as the sequence of changes were similar in nuclei from neurones and astrocytes, the only difference being that a 35S intermediate was found in the former and a 32S in the latter. In non-astrocytic glial nuclei, synthesis and transformation of the 45S component were delayed as compared to the other cell types and the processing of this component may involve both a 32S and a 35S intermediate. Moreover, the radioactivity incorporated in all the nuclear RNA species was always lower in these cells.     

Nucleo-cytoplasmic relationships of high-molecular-weight ribonucleic acid, including polyadenylated species, in the developing rat brain.

The metabolism of high-molecular-weight RNA in the nuclear and cytoplasmic fractions of newborn and adult rat brain was investigated after the intracranial administration of [32P]Pi. In young brain, a considerable proportion of the newly synthesized radioactive RNA is transferred to the cytoplasm, in contrast with the adult brain, where there appears to be a high intranuclear turnover. Electrophoretic analysis of the newly synthesized RNA showed that processing of the rRNA precursor to yield the 28S and 18S rRNA may be more rapid in the adult than in the young, although most of the adult rRNA in the nucleus is not transferred to the cytoplasm. In young brain, processing is probably tightly coupled to transport of rRNA into the cytoplasm, so that 28S and 18S rRNA are not subjected to possible degradation within the nucleus. Polyadenylated RNA turns over in concert with high-molecular-weight RNA in the nuclei of the adult rat brain. In the cytoplasm the polyadenylated RNA has a higher turnover rate relative to rRNA. In the young brain the polyadenylated RNA is transferred to the cytoplasm along with rRNA, although polyadenylated RNA is transported into the cytoplasm at a faster rate. The nuclear and cytoplasmic polyadenylated RNA species of young brain are larger than their corresponding adult counterparts. These results suggest that there are considerable changes in the regulation of the nucleo-cytoplasmic relationship of rRNA and polyadenylated RNA during the transition of the brain from a developing replicative phase to an adult differentiated and non-dividing state.     

Histone-acetylating enzyme of brain

1. Acetylation of histones by an enzyme system derived from rat brain and liver (histone acetylase) was studied by using [1-(14)C]acetyl-CoA as the acetyl group donor. 2. The activity of this enzyme was largely confined to the nucleus. 3. Histone-acetylating activity of cerebral nuclei purified by centrifugation through 1.9m-sucrose was not altered by the presence of the cytoplasmic fraction. 4. Cerebral nuclei from adult rats exhibited greater histone-acetylating activity than did the corresponding preparation from newborn animals. 5. Nuclear acetylating activity was higher in brain than in liver of adult rats but not in newborn animals. 6. The partially purified enzyme from cerebral nuclei, prepared by ammonium sulphate fractionation of an acetone-dried powder, specifically catalysed histone acetylation. 7. Polylysine, protamine, serum albumin and gamma-globulin were not enzymically acetylated by this preparation. 8. Soluble acetylating preparations from both brain and liver nuclei were more active towards arginine-rich F3 and slightly lysine-rich F2a and F2b histone fractions than towards the lysine-rich F1 fraction. 9. Enzymic acetylation of chromatin-bound proteins was much less extensive than that of free histones. 10. The high histone acetylase activity in mature brain may reflect the importance of this process in the genetic control of cerebral function.     

The properties and function of rapidly-labelled nuclear RNA

Summary Nuclei were isolated from cultured cells of Acer pseudoplatanus L. previously pulse-labelled with [5-³H]uridine or [³²P]phosphate and the properties of the rapidly-labelled RNA were studied. Polyacrylamide gel electrophoresis showed ribosomal RNA precursors and processing intermediates with molecular weights of 3.4, 2.5, 1.4 and 1×10⁶ daltons, together with polydisperse RNA. The relative proportions of ribosomal RNA precursors and polydisperse RNA varied according to the length of the labelling period, but after 30 min approximately 90% of the radioactive RNA was polydisperse. The relationship between this polydisperse RNA and messenger RNA was investigated. The percentage of total nuclear RNA retained by chromatography on oligodeoxythymidylic acid-cellulose columns varied from 6% to 16% depending on the length of the labelling period. This RNA fraction, which has an adenylic acid content of approximately 45%, is assumed to represent RNA with polyadenylic acid sequences attached. A larger proportion of the nuclear polydisperse RNA lacked polyadenylic acid. Both types of polydisperse RNA were similar in size and during polyacrylamide gel electrophoresis migrated as broad peaks with an average molecular weight of approximately 10⁶ daltons. The polydisperse nuclear RNA that lacks polyadenylic acid was found to be similar in nucleotide composition to ribosomal RNA and is assumed to represent growing chains of ribosomal precursor RNA. After short labelling times the majority of the radioactivity incorporated into nuclear RNA is present in molecules of this type. This suggests that the designation of pulse-labelled polydisperse RNA as messenger RNA or precursor to messenger RNA solely on the basis of rapid labelling and size heterogeneity is unsound. The average molecular weight of the polyadenylic acid-containing messenger RNA from the cytoplasm was less than that of the corresponding nuclear RNA (6 and 9×10⁵ daltons respectively). This suggest either that the majority of the nuclear polyadenylic acid-containing RNA does not enter the cytoplasm, or if it does, that it first undergoes a reduction in size.Abbreviations rRNA ribosomal RNA - mRNA messenger - RNA poly(A), polyadenylic acid, poly(A) and poly(A) - RNA RNA with and without poly(A) sequences attached - poly(U) polyuridylic acid - oligo (dT)-cellulose cellulose with oligo deoxythymidylic acid covalently attached - C cytidylic acid - A adenylic acid - G guanylic acid - U uridylic acid     

Effect of undernutrition on DNA and RNA synthesis in subcellular fractions from different regions of the developing rat brain

The effect of undernutrition on the incorporation of [methyl-³H]thymidine into DNA and of 5-[³H]uridine into RNA of cerebral hemispheres, cerebellum, and brain stem was studied in vivo and in vitro in rats. The labeling of DNA from nuclei and mitochondria and of RNA from nuclei, mitochondria, microsomes, and soluble fractions, was also measured in vitro. The results demonstrate that nucleic acid synthesis is impaired and delayed during undernutrition. Specific effects were observed for the different brain regions and subcellular fractions: at 10 days nuclear and mitochondrial DNA and RNA synthesis was impaired, whereas at 30 days only the mitochondrial nucleic acid synthesis was affected.The delay of DNA and RNA labeling, caused by undernutrition, was most evident in the cerebellum, probably due to its intense cell proliferation during postnatal development. The specific sensitivity of mitochondria as compared to other subcellular fractions, may be due to the intense biogenesis and/or turnover of nucleic acids in brain mitochondria not only during postnatal development, but also in the adult animal.     

Evidence for Two Metabolically Distinct Types of Ribonucleic Acid in Chromatin and Nucleoli

Patterns of radioisotope incorporation are useful characteristics in describing cellular RNA fractions, and have indicated a distinctive "nuclear" RNA. In order to characterize the RNA fractions of the two nuclear components, nucleoli and chromatin, and to determine thereby the precise localization of the RNA typical of isolated nuclei, time-courses of P³² incorporation into nucleolar, chromosomal, and cytoplasmic RNA of Drosophila salivary glands have been determined from autoradiograms. Two experiments are reported which cover 12 and 18 hour periods, including an initial 2 hour feeding on P³². Concentrations of RNA-P³² (identified by ribonuclease digestion) were determined by grain counts. After 1 hour only the nucleolar RNA is labelled. Activity is detectible in chromosomal and cytoplasmic RNA after the 2nd hour. The nucleolar fraction reaches its maximum activity shortly after transfer of the larvae to non-radioactive food, the other fractions several hours later. Maximum activities persist in the chromosomal and cytoplasmic fractions; nucleolar activity decreases after the 9th hour. The observed differences in times at which incorporation begins and maximum activities are reached, and in maintenance of maximum activities indicate that chromosomal and nucleolar RNA are distinct fractions. The metabolic characteristics which have been ascribed to "nuclear" RNA apply only to the nucleolar fraction.     

Mitochondrial DNA,RNA, and protein synthesis in different regions of developing rat brain

In vivo and in vitro (tissue slices) incorporation of labeled precursors into DNA, RNA, and proteins was measured in mitochondria obtained from cerebral hemispheres, cerebellum, and brain stem of rats at different days of postnatal development. To compare the synthesis of macromolecules in mitochondria with that in other subcellular fractions, the incorporation of labeled precursors into DNA, RNA, and proteins extracted from nuclei and into RNA and proteins extracted from microsomes and cytoplasmic soluble fractions was also measured.The results obtained showed that the incorporation of [³H]thymidine into DNA and of [¹⁴C]leucine into proteins of nuclei and mitochondria from the various brain regions examined decreased during postnatal development, however, at 30 days of age the specific radioactivity of mitochondrial DNA was higher than that of nuclear DNA. [³H]Uridine incorporation into RNA decreased from 10 to 30 days of age in nuclei while in mitochondria it was quite similar at both ages. This result may be due to a faster turnover of mitochondrial RNA compared to that of mitochondrial DNA and proteins. The results obtained suggest an active biosynthesis of macromolecules in brain mitochondria and might indicate an intense biogenesis of these organelles in rat brain during postnatal development.Preliminary reports of these results were presented at the XI FEBS Meeting, Copenhagen, August 14–19, 1977, Poster number A2-2-155-3, and at III Meeting of Italian Biochem. Soc., Siena, October 3–5, 1977, Abstract C6.     

The messenger ribonucleic acid content of Bacillus subtilis 168

Bacillus subtilis 168 messenger RNA was determined by DNA-RNA hybridization techniques, with denatured DNA immobilized upon cellulose nitrate membrane filters. The following results were obtained. (1) Cultures of B. subtilis, growing exponentially in enriched glucose-salts medium at 37 degrees , incorporated [5-(3)H]uracil into both ribosomal and messenger RNA fractions without the kinetic delay expected from the presence of the intracellular nucleotide pools. (2) However short the time of labelling with exogenous labelled uracil (down to 7sec.), 32-36% of the rapidly labelled RNA was messenger RNA and 68-64% was an RNA with the hybridization characteristics of ribosomal RNA. Analysis of the apparent nucleotide base composition of total (32)P-labelled rapidly labelled RNA and the two RNA fractions separated by hybridization at a DNA/RNA ratio 5:1 confirmed this finding. Of the rapidly labelled RNA, 31% readily hybridized with DNA at low DNA/RNA ratios and had an apparent base composition like that of the DNA, whereas 69% was hybridized only at low efficiency at low DNA/RNA ratios and had a composition identical with that of ribosomal RNA. (3) In cultures dividing every 48min. at 37 degrees , kinetic analysis of RNA labelled over a 20min. period showed that the average life-time of messenger RNA was 2.7-3.0min. and that its amount was 3.0% of the total RNA. (4) The hybridization of (3)H-labelled randomly labelled RNA with DNA at a DNA/RNA ratio 5:1 showed that 2.9% of the randomly labelled RNA had the characteristics of messenger RNA. (5) Experiments carried out as described by Pigott & Midgley (1968) indicated that hybridization at low DNA/RNA ratios (5:1) effectively accounted for all the messenger RNA in a given specimen. The efficiency coefficient of RNA hybridization lay within the range of 90-95% input, if an excess of DNA sites was offered for RNA binding. (6) These measurements are compared with other results obtained by different methods, and reasons for any major disagreement are suggested.     

In vitro transcription of calliphorin genes in isolated nuclei of fat body from larvae of Calliphora vicina

Nuclei from fat body of different developmental stages of Calliphora vicina were isolated. They appear to be polyploid and show polytene chromosomes. The isolated nuclei were incubated with [³²P]GTP and the RNA transcribed in vitro was hybridized with a DNA fragment encoding a polypeptide subunit of calliphorin. The isolated nuclei transcribe the calliphorin-mRNA correctly and with the same stage specificity as observed in vivo.     

DIFFERENTIAL METABOLISM OF RNA IN NEURONAL-ENRICHED AND GLIAL-ENRICHED FRACTIONS OF RAT CEREBRUM

Cerebral tissue of rat, disrupted by passage through a custom-designed tissue press, was diluted to a 10% (w/v) cellular suspension in 10% (w/v) Ficoll and was then fractionated in the Spinco Model L ultracentrifuge into: (1) two enriched cellular layers (alpha and beta) in an isopycnic Ficoll gradient in the Spinco 40 angular rotor; or (2) into four layers (A, B, C, and D) in a discontinuous Ficoll gradient in the Spinco SW 39 swinging bucket rotor. The cellular composition of these layers was identified microscopically and enzymically as either glial-enriched (alpha and B layers) or neuronal-enriched (beta and C layers) fractions of cerebral cortex. Portions of the neuronal and glial fractions were used for determinations of total nitrogen, and for colorimetric determinations of carbonic anhydrase activity (a glial cell marker). These data established that glial contamination of the neuronal-enriched layer averaged 6·5 per cent. The data also indicated glial enrichment of Layer B, although no quantitative assessment of the amount of neuronal contamination was possible. The kinetics of metabolism of RNA in the glial-enriched and neuronal-enriched fractions were studied from 0·5 to 16 h after-intracisternal injection of either [³H]cytidine or [³H]orotic acid. In addition, the incorporation of [³H]cytidine into crude nuclear and cytoplasmic components of the layers was studied by the use of 1 h pulses. Our findings indicated greater incorporation of [³H]cytidine into nuclear fractions than into cytoplasmic fractions at 1 h and greater incorporation of both precursors into neuronal-enriched fractions than into glial-enriched fractions at all pulse times.     

Preparation and characterization of chick erythrocyte nuclei from heterokaryons

A method for the isolation of reactivated chick erythrocyte nuclei from heterokaryons was developed. The heterokaryons were produced by fusing chick erythrocytes with HeLa or L cells in the presence of inactivated Sendai virus. At various time intervals after fusion nuclei were isolated directly from the monolayer by treatment with an acidic detergent solution. Chick erythrocyte nuclei were then separated from other nuclei (HeLa or L cell) by centrifugation on sucrose gradients. The purified preparation of reactivated chick erythrocyte nuclei was shown to be free from other nuclei and cytoplasmic contamination. By using L cells which had been labelled with ³H-leucine before fusion or heterokaryons labelled after fusion it was demonstrated that labelled mouse proteins migrate from the cytoplasm of the heterokaryons into the reactivating chick erythrocyte nuclei. ³H-uridine labelling of heterokaryons made by fusing UV-irradiated chick erythrocytes with L cells failed to reveal any significant migration of mouse RNA into the chick erythrocyte nuclei.     

PERSISTENCE OF ALTERED RNA SYNTHESIS IN RAT CEREBRAL CORTEX 12h AFTER A SINGLE ELECTROCONVULSIVE SHOCK

The effect of electroshock (ECS) on RNA synthesis in nuclei and cytoplasm of rat cerebral cortex was examined using a double label technique by intraventricular injection of [³H] and [¹⁴C]orotate. At t h after ECS, the incorporation into nuclear RNA was 80% of the control rate and the appearance of newly synthesized RNA in the cytoplasm was only 27.6%. Analysis on composite polyacrylamide-agarose gels of purified RNA showed that the ³H/¹⁴C ratio of each gel slice slowly increased with decreasing M.W. of the RNA. This has been interpreted as an inhibition in the rate of processing of nuclear RNA. When the nuclear RNA was subjected to denaturation with 50% dimethyl sulphoxide (DMSO) this effect was enhanced. In a similar experiment, rats were injected, treated to ECS and killed 12 h later. The overall incorporation into nuclear and cytoplasmic RNA was increased to 174%, and 137.5% respectively. Analysis on gels showed very little variation in the ³H/¹⁴C ratio of the steady state levels of nuclear RNA. They compared well with a control experiment where rats were injected with [³H] and [¹⁴C]orotate as described above but no ECS was applied to the [¹⁴C] labelled animals. However a 1 h pulse label given 11 h after ECS treatment revealed that the rate of incorporation into nuclear RNA still showed a decrease of 81% of the control. The nuclear RYA fractionated on gels clearly showed that the inhibition of the processing rate of nuclear RNA was still occurring. This effect was again magnified on denaturation of the RNA with DMSO. This suggests that ECS may disturb RNA metabolism in nervous tissue for much longer periods than previously realised.