Asen A. Hadjiolov

Ribosome Biogenesis and Nucleolar Ultrastructure in Neuronal and Oligodendroglial Rat Brain Cells

Abstract: The absolute amounts of precursor to ribosomal RNA (pre‐rRNA) and ribosomal RNA (rRNA) in isolated rat brain neuronal and oligodendroglial nuclei were determined. The amount of the major pre‐rRNA and rRNA species in neuronal nuclei was about twofold higher than in oligodendroglial nuclei. The relative rate of pre‐rRNA synthesis in vivo was 2.3‐ to 2.7‐fold higher in neuronal as compared with oligodendroglial nuclei. This corresponds to a 2.7‐fold higher activity of the “template‐bound” RNA polymerase I in isolated neuronal nuclei, whereas the activity of the “free” enzyme in both neuronal and glial nuclei was almost identical. The higher transcription rates of rRNA genes correlated with the markedly more prominent fibrillar component in neuronal nucleoli. The turnover times of the major pre‐rRNA and rRNA species in neuronal and oligodendroglial nuclei were similar, except for 45S pre‐rRNA, which turned over at an 1.5‐fold slower rate in neuronal nuclei. The relative rates of processing of pre‐rRNA and of nucleocytoplasmic transport of rRNA in neuronal cells were 2.7‐fold higher than in oligodendroglial cells and corresponded to the differences in rRNA gene transcription rates. The established ribosome formation features correlated with an abundant (neurons) or exceedingly scarce (oligodendrocytes) nucleolar granular component. The turnover rate of cytoplasmic ribosomes in rat brain neurons was twofold slower than in oligodendrocytes, largely because of the about fivefold higher amount of ribosomes in the cytoplasm of neurons. We conclude that ribosome formation and turnover in neuronal and oligodendroglial cells are adapted to the protein synthetic levels in these two types of brain cells.

Inhibition of ribonucleic acid biosynthesis in mice liver by the exotoxin of Bacillus thuringiensis

The exotoxin of Bacillus thuringiensis, a structural analogue of ATP, is a strong inhibitor of in vivo RNA synthesis in mice liver. Administration of low doses of exotoxin causes the following changes in the labelling of liver RNA species. 1. 1. The synthesis of nuclear rRNA, including the 45-S precursor rRNA, is inhibited almost completely, while the labelling of nuclear heterogeneous “DNA-like” RNA is inhibited only to about 50 % of the level in control mice. 2. 2. The labelling of cytoplasmic 28-S and 18-S rRNA is inhibited to about 10 % of control levels. The same degree of inhibition is observed with rRNA extracted from isolated microsomes, ribosomes or postmicrosomal ribonucleoproteins. Cytoplasmic mRNA from microsomes and postmicrosomal ribonucleoproteins is inhibited to the same extent as nuclear and cytoplasmic rRNA. 3. 3. The labelling of 5-S rRNA extracted from ribosomes is decreased to about 10 % of control levels, identical to the inhibition of the other rRNA fractions studied. On the other hand, the synthesis of tRNA is relatively resistant to exotoxin, showing only about 50 % inhibition. The results are discussed and it is suggested that 5-S rRNA may be important in the nucleoplasmic control of rRNA synthesis and ribosome maturation.

Degradation of ribosomal precursor and polyadenylic acid-containing ribonucleic acids in Saccharomyces cerevisiae caused by actinomycin D

Abstract The synthesis of RNA in the osmotic-sensitive yeast mutant VY 1160 is inhibited by actinomycin D at concentrations which are without effect on the parental wild-type strain. Protein synthesis is not affected initially even at doses which block RNA synthesis completely. Lower concentrations of actinomycin D inhibit preferentially the synthesis of rRNA as compared to that of poly(A)+-RNA. Unspecific degradation of prelabeled precursors of rRNAs (pre-rRNA) and poly(A)+-RNA is caused by actinomycin D at doses which block new RNA synthesis. This degradation could not be correlated with changes in the nuclease activity of yeast cell lysates. It appears that the post-transcriptional degradation of pre-rRNA and poly(A)+-RNA caused by actinomycin D is not connected with the action of the drug on transcription.

The action of snake venom phosphodiesterase on liver ribosomal ribonucleic acids

Abstract The stepwise hydrolysis of rat-liver ribosomal RNAs (rRNA) with snake venom phosphodiesterase (EC 3.1.4.1) has been studied. The following results have been obtained: 1. 1. Hydrolysis of poly (U), poly (A) and equimolar mixtures of them proceeds to completion and yields pU and pA as the only major products of the reaction. Secondary structure in poly (U) · poly (A) has no detectable influence on the rate and the extent of hydrolysis. The substrate (Ap)nCp is resistant to the action of this enzyme, while (Ap)nC is rapidly degraded to give pA and pC. 2. 2. As shown by agar-gel electrophoresis, the liver rRNA studied is constituted of homogeneous 28-S and 18-S RNA molecules. They display secondary structure transitions typical for native rRNAs. The preparations studied are virtually free from endonucleolytic contaminants. 3. 3. Hydrolysis of rRNAs with venom phosphodiesterase goes to completion and yields pG, pC, pA and pU as the only major products. Analysis by determination of the nucleosides originating from chain terminals during hydrolysis, shows that the endonuclease contaminants in this system may account for no more than 1 to 2 internal breaks per RNA molecule. 4. 4. The molar ratio of the mononucleotides liberated on stepwise hydrolysis of liver rRNAs with venom phosphodiesterase varies with progress of enzymatic degradation. The product obtained at early stages of enzyme action has a higher content of pA and pU and a lower content of pG and pC. The same correlation is observed with 18-S rRNA and partly with 28-S rRNA. It is suggested that in rRNA the segment near the 3′-end of the chain contains more pA and pU and less pG and pC as compared with the segment near the 5′-end of the chain. 5. 5. Labeling in vivo of free uridine 5′-phosphates and cytidine 5′-phosphates with [6-14C]orotic acid shows with both nucleotides a maximum incorporation at 40 min. Ribosomal RNAs labeled for 90 min in vivo with [6-14C]orotic acid were analysed by partial degradation with venom phosphodiesterase and determination of the radioactivity of uridylic acid residues. The labeling of 18-S RNA is higher than of 28-S RNA. Enzymatic liberation of labeled uridylic acid from 18-S and 28-S RNA is non-uniform. With both 18-S and 28-S RNA, uridylic acid liberated at the initial stages of enzyme action is more highly labeled than uridylic acid from the remaining RNA fragments.

Changes in a nuclear matrix antigen during the cell cycle: interphase and mitotic cells

We studied the behaviour in interphase and mitotic human cells of a 125 kDa (pI 6.5) antigen, associated with the nuclear matrix and detected in proliferating cells. Indirect immunofluorescence with a specific monoclonal antibody reveals that during interphase in WISH and Namalwa cells, as well as phytohaemagglutinin—stimulated lymphocytes, the antigen displays a speckled distribution in the nucleoplasm of all cells. At early prophase the fluorescence intensity of the coalesced speckles increases markedly. During metaphase and anaphase the antigen gives maximal fluorescence distributed diffusely in the nucleoplasm, while chromosomes remain negative. At anaphase and cytokinesis the antigen is still cytoplasmic, but fluorescence intensity decreases. Two‐dimensional gel electrophoresis and immunoblotting reveal that the p125/6.5 antigen displays a net increase in isolated mitotic cells as compared to interphase cells. These results suggest that the p125/6.5 protein participates in late G2 phase and G2/M transition events preparing the cell for mitosis.

A comparison of nuclear and nucleolar matrix proteins from rat liver

The comparison of the gel electrophoresis patterns of nuclear and nucleolar matrix proteins reveals marked differences between these structures. The nucleolar matrix contains 5 prominent protein bands ( mol.wt.: 8.2; 7.0; 5.6; 4.0 and 3.0 x 10 4 ) which are not found in nuclei. It is suggested that the nucleolar matrix has a distinct structure participating in selective interactions.

Effect of cycloheximide on the in vivo and in vitro synthesis of ribosomal RNA in rat liver

The action of low (5 mg/kg body wt;) and high (20 mg/kg body wt.) doses of cycloheximide, both causing a rapid and almost complete inhibition of protein synthesis in rat liver is investigated. Short-term (15 min) [14C]orotate incorporation into nucleolar rRNA in vivo is inhibited only by the high dose acting for periods longer than 1 h. The effect may be correlated with a strongly reduced labelling of the cellular pool of free uridine nucleotides. These results indicate that in vivo transcription of rRNA genes may not be under stringent control. The activity of template-bound RNA polymerase A in nuclei isolated from animals treated with both doses of cycloheximide is reduced within 1 h to about 50% of controls reaching nearly plateau levels at longer times of action of the drug. The differential effect of cycloheximide inhibition of protein synthesis on in vivo and in vitro rRNA synthesis suggests the existence of elongation control protein(s) characterized by a rapid turnover and a loose association with the nucleus.

Toyocamycin inhibition of ribosomal ribonucleic acid processing in an osmotic-sensitive adenosine-utilizing Saccharomyces cerevisiae mutant

An adenosine-utilizing mutant of Saccharomyces cerevisiae (SY 15 ado) is isolated after remutagenesis of an osmotic-sensitive strain, auxotrophic for adenine, with ethyl methanesulfonate. It is shown that the SY15ado mutant can be used to achieve experimental conditions under which cell growth and RNA Synthesis are directly dependent on exogenous adenosine. After starvation for adenosine, toyocamycin is incorporated into pre-rRNA chains of SY15ado cells replacing adenosine residues. The extent of this replacement depends on the concentration of added toyocamycin. Lower doses slow down processing of pre-rRNA into mature rRNA with an accumulation of 27 S and 20 S pre-rRNA. At higher concentrations toyocamycin blocks the last steps of pre-rRNA processing i.e. the conversions 27 S pre-rRNA leads to 25 S rRNA and 20 S pre-rRNA leads to 18 S rRNA. It appears that the main site of toyocamycin action is at the last steps of ribosome formation, while transcription and the early stages of pre-RNA processing are less affected.

Pattern of [32P]orthophosphate incorporation in vitro into ribonucleic acid nucleotides of Ehrlich ascites tumor cells.

1. 1. Rapidly labelled RNA from whole Ehrlich ascites tumor cells had the same mononucleotide composition irrespective of conditions used for [32P]orthophosphate incorporation (in vivo or in vitro). It corresponded in each case to 30–40 % d-RNA (RNA similar in mononucleotide composition to DNA) and 60–70 % r-RNA (RNA similar in mononucleotide composition to ribosomal RNA). 2. RNA extracted with phenol-sodium dodecyl sulfate at 65° from nuclei of isolated ascites tumor cells showed three main peaks on agar gel electrophoresis, corresponding to ribosomal RNA (2 peaks) and soluble RNA. Polyvinyl sulfate was necessary to avoid partial degradation of RNA during its isolation and storage. 3. Nuclear RNA isolated from tumor cells labelled in vitro (30 min) with [32P]orthophosphate was hydrolyzed with 0.5 N KOH or with snake-venom phosphodiesterase (EC 3.1.4.1). The specific activities of the four nucleoside 2′(3′)-monophosphates were of the same order. The specific activity of GMP was on the average twelve-fold lower than that of the other three nucleoside 5′-monophosphates. Nucleotide incorporation into rapidly labelled nuclear RNA was statistically random. The mononucleotide composition of this RNA corresponds to 50 % d-RNA and 50 % r-RNA. 4. During the step-wise degradation of rapidly labelled nuclear RNA with snake-venom phosphodiesterase the specific activity of CMP increased, while that of AMP and UMP decreased.

Isolation and initial characterization of nucleolar fibrillar remnants from the liver of rats treated with D-galactosamine.

Abstract Administration of d -galactosamine (250 mg/kg body weight) to rats causes a rapid and complete block of transcription. Within 4 h the granular components of nucleoli disappear, while the fibrillar components are condensed and converted into fibrillar remnants. The amount of precursors to rRNA (pre-rRNA) and rRNA components (mostly 28S rRNA) is reduced to less than 10% of that in control nucleoli. Electron microscopic radioautography shows also that the nucleolar fibrillar remnants do not contain pre-rRNA in growth-arrested transcription complexes. The Ag-staining reaction, adapted for electron microscopy, is positive with the fibrillar components of nucleoli. After d -galactosamine, the positive Ag staining is associated exclusively with the nucleolar fibrillar remnants. The positive Ag staining and the presence of rRNA genes, as revealed by rRNA:DNA hybridization, show that the nucleolar fibrillar remnants contain nucleolus organizer components. The fraction of nucleolar fibrillar remnants, isolated from nuclei of d -galactosamine-treated rats, preserves its ultrastructural characteristics observed in situ. It contains mainly DNA and proteins, including histones and nucleolar matrix proteins. The implications of these results for the elucidation of structural-biochemical correlations in the nucleolus are discussed.