Characterisation of RNA Synthesis in vitro in Neurone‐Rich, Oligodendroglial and Liver Nuclei

Abstract

1. In neurone-rich nuclei isolated from the adult guinea-pig cerebral cortex, DNA-dependent RNA polymerase B reaction is several times more active than in liver nuclei. This may be attributable to the unusual wealth of euchromatin in mature neuronal nuclei.2. Nuclear RNA labelled in vitro was hydrolysed by alkali, and the number as well as the average length of nascent RNA chains were determined by measuring [3H]uridine and [3H]UMP/[3H]uridine ratio, respectively. In neurone-rich nuclei, the number of growing RNA chains is greater, and the average chain length longer than in liver nuclei. In oligodendroglial nuclei which exhibit RNA polymerase B reaction of intermediate activity, the number of growing RNA chains is found comparable to, but the average chain length longer, than that in liver nuclei. Thus, the presence of a greater number of growing RNA chains appears to be a unique feature of neuronal nuclei.3. In neuronal chromatin, mean residue ellipticity in circular dichroism spectrum in the wavelength range of 260 -300 nm is significantly higher than in liver and oligodendroglial chromatins, and this is in apparent correlation with the number, rather than the average length, of growing RNA chains.4. Digestion of chromatin with trypsin leads to reversion of the circular dichroism spectrum toward that of free DNA, and completely abolishes the difference in ellipticity pattern that is observed among native neurone-rich, oligodendroglial and liver chromatins.5. In liver nuclei, RNA polymerase B reaction is greatly enhanced with trypsin, which is primarily attributable to the facilitated elongation of RNA chains that is otherwise restricted. In neuronal nuclei where the steric restriction imposed on RNA synthesis is thought to be intrinsically weakened, digestion with trypsin is more than necessary in disclosing the RNA polymerase B activity.6. RNA synthesis in neuronal and oligodendroglial nuclei in vitro is discussed in correlation with that reported in vivo.

Transcription and Motoneuron Size

“…The mean counts per minute per nucleus within individually counted nuclei increased nearly linearly with time ( Fig . 1A), with per nucleus values at 1 h falling well within the range of nucleotide triphosphate incorporation rates reported for isolated neuronal (Thompson, 1973 ;Mizobe et al ., 1974) and other (McKnight and Palmiter, 1979) nuclei under comparable conditions . Similarly, incorporation increased in a linear fashion when the concentration of labeled UTP was varied between 0.55 and 2.75 pM UTP (Fig .…”

“…Until now, this question has not been tested directly, although there is evidence suggesting a positive relationship between nuclear size and total transcription in neurons . For instance, nuclei from neuron-enriched brain fractions are larger and show higher labeling of RNA per unit DNA than do nuclei from glia or hepatocytes (Kato and Kurokawa, 1970;Lovtrup-Rein, 1970;Giuditta et al ., 1972;Thompson, 1973;Mizobe et al, 1974;Pearson et al ., 1984) . Neurons also possess conspicuous amounts of dispersed chromatin Peters et al, 1976) .…”

Effect of Ischemia on the Activity of DNA‐Dependent RNA Polymerase and DNA Polymerase

“…Neuronal and glial enriched nuclear fractions from the isolated spinal cord nuclei were separated by sucrose density centrifugation (Mizobe et al, 1974). The spinal cord had been rapidly removed, freed of meninges and chopped into small pieces in an ice-cold solution containing 2 molllitre sucrose, 1 mmoUlitre MgCI2 and 1 mmolllitre K2HP04, adjusted to pH 6.5.…”

Enhancement of Hydroxyindole‐O‐Methyltransferase and DNA‐Dependent RNA Polymerase Activities Induced by Oestradiol in Rat Pineals in Culture

“…5 x (w/v) perchloric acid. Labelled nucleosides and nucleotides in the mixture were adsorbed on charcoal, extracted and identified by thin-layer chromatography, and respective radioactivities were measured as described elsewhere [28]. Approx.…”