Synthesis and Processing of Ribosomal RNA in Cultured Plant Cells

Cox, B. J.; Turnock, G.

doi:10.1111/j.1432-1033.1973.tb02996.x

Cited by 40 publications

(19 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The incorporation of [3H]UTP in nuclei in vitro is thought to represent the additional elongation of RNA chains that were growing in vivo, but became arrested during the isolation of nuclei [14,16]. In our present experiment, this is indicated by the fact that while the average length of nascent portions of RNA chains in terms of [3H]UMP/[3H]uridine ratio ranges between 50 and 135 nucleotides, estimation of the average molecular weight of labelled RNA suggests the longer length of nucleotide chains (Fig.2).…”

Section: Discussionmentioning

confidence: 99%

“…RNA was extracted from nuclei and precipitated by cetyltrimethylammonium bromide [ 151, essentially as described by Cox [16]. RNA polymerase B reaction was allowed to proceed for 40min in the medium scaled up to 2.0 ml, and terminated by freezing the mixture at -80 "C. After the addition of 100 pg of deoxyribonuclease I and 40 pl of 250 mM MgClz, the mix was thawed, kept at 0-4 "C for 5 min, and adjusted to 0.1 % with respect to sodium dodecylsulphate.…”

Section: Isolation Of Rnafrom Nucleimentioning

confidence: 99%

“…Gels for calibration were scanned at 260 nm in a Joyce Loebl Chromoscan. The average molecular weight of labelled RNA was then calculated as described by Cox [16].…”

Section: Electrophoresis Of R N a On Polyacrylamide Gelsmentioning

confidence: 99%

See 2 more Smart Citations

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

Mizobe¹,

Tashiro²,

Kurokawa³

1974

European Journal of Biochemistry

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Isolation Of Rnafrom Nucleimentioning

confidence: 99%

See 1 more Smart Citation

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

Mizobe¹,

Tashiro²,

Kurokawa³

1974

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…These interactions may result in activation or inhibition of enzyme activity [36]. For example, heparin inhibits the activity of some enzymes such as lysosomal hydrolases [36], DNA polymerase [37], RNA polymerase [38], ribonuclease [39], ornithine decarboxylase [40] and casein kinase I1 [41,421. Other enzymes are activated by heparin, such as lipoprotein lipase [43], tyrosine hydroxylase [44], rabbit skeletal muscle and rat liver phosphorylase kinase [45,461 and the p68 kinase.…”

Section: The Signgicance Of' the P68 Kinase Activation By Heparinmentioning

confidence: 99%

The double‐stranded RNA‐dependent protein kinase is also activated by heparin

Hovanessian

Galabru

1987

European Journal of Biochemistry

114

View full text Add to dashboard Cite

The double-stranded(ds)-RNA dependent protein kinase from human cells is a M , 68 000 protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by dsRNA, the p68 kinase becomes autophosphorylated. The phosphorylated p68 kinase then can catalyze the phosphorylation of exogenous substrates, such as eIF2 and histone. The second phosphorylation step can take place in the absence of dsRNA. Here we show that, besides dsRNA other polyanions, especially heparin, can also activate the p68 kinase for the autophosphorylation reaction. Heparin activation of the p68 kinase is reversible since it can be prevented by addition of antithrombin 111, heparin-binding protein. However, when antithrombin I11 is added after autophosphorylation of the p68 kinase then phosphorylation of histone is not affected. The p68 kinase binds to heparin-Sepharose. Further evidence that the p68 kinase can be activated by heparin was provided by photoaffinity labeling with 8-a~ido-[a-~~P]ATP. This ATP analog can bind to the p68 kinase only in the presence of heparin or dsRNA. Thus suggesting that the activation of the p68 kinase triggers a conformational modification allowing the binding of ATP. Basic proteins, histone and protamine, prevent the activation process induced by heparin. This is probably due to binding of these basic proteins to heparin and thus sequestering the activator of the protein kinase.A protein kinase activity dependent on double-stranded (ds)RNA is enhanced in mouse and human cells following treatment with interferon [l -41. The protein kinase from human cells is a Mr-68000 protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon [5]. The p68 kinase is characterized by two distinct protein kinase activities [6]. The first one is functional for its phosphorylation whereas the second one is responsible for the phosphorylation of exogenous substrates such as calf thymus histones and the CI subunit of protein synthesis initiation factor eIF2. When activated by dsRNA in the presence of MnZ+ and ATP, p68 kinase is autophosphorylated. This phosphorylated p68 kinase is then capable of catalyzing phosphorylation of eIF2. Phosphorylation of exogenous substrates (histone, eIF2) is not dependent on dsRNA [7]. It might occur as long as p68 kinase is phosphorylated, i.e. there is as strong correlation between the degree of phosphate content of p68 kinase and its kinase activity on exogenous substrates [7, 81. The protein kinase activities mediating autophosphorylation of p68 kinase and phosphorylation of exogenous substrates are independent of cyclic AMP or cyclic GMP and are markedly stimulated by Mn2 +. Previously we have suggested that the dsRNA-dependent protein kinase is composed of two subunits; a M , 48000 protein (p48), which phosphorylates a Mr-68000 protein (p68; [7]). However, these results now have been shown to be artifactual owing to partial proteolysis of p68 kinase during preparation of cell extracts. Under ex- Abbreviations....

show abstract

“…Certainly, a significant proportion of this increased synthesis of rRNA in response to auxin relates to increased levels of RNA polymerase I enzyme and to increased activity of those RNA polymerase I molecules (1 1). Since mature rRNAs (1 8S and 25S or 0.7 and 1.3 x 106 D, respectively) are initially transcribed as a large precursor molecule of about 2.8 x 105 D in some plants (8,17,25) and about 2.3 x 106 in others (6, 28) including soybean (15), the rate of processing of these large transcripts could be involved in establishing the rate of accumulation of new ribosomes. Melanson and Ingle (23) in fact have reported that precursor processing, including a large increase in the rate of processing of the 1.4 x 106 precursor into the mature 1.3 x 106 (25S) rRNA, accounts for a part of the auxin-enhanced synthesis and accumulaton of ribosomes in artichoke tissue.…”

Section: Discussionmentioning

confidence: 99%

Fractionation of Nucleoli from Auxin-Treated Soybean Hypocotyl into Nucleolar Chromatin and Preribosomal Particles

Chen

Huang²,

Lin³

et al. 1983

Plant Physiol.

View full text Add to dashboard Cite

ABSTRACrNucleoli from auxin-treated tissues (Glycine max L. var Wayne or Kaoshiung No. 3) were isolated and purified by Percoll density gradient centrifugation. There was a 2.1-fold increase in RNA and a 2.8fold increase in protein after a 24-h auxin treatment per unit nucleolar DNA. More than 150 acid-soluble protein spots were associated with the auxintreated nucleoli on two dimensional (2-D) gel electropherograms.Nucleoli from auxin-treated tissue were fractionated by suspension in 20 millimolar dithiothreitol at room temperature for 20 minutes into two distinct fractions referred to as the nucleolar chromatin and preribosomal particle fractions. The DNA:RNA:protein ratio ofthe chromatin fraction was 1:2.5:14. Most of RNA polymerase 1 activity and nucleolar DNA recovered in this fraction. The acid-soluble proteins in the chromatin were resolved into 32 protein spots on 2-D gel electropherogram. The most abundant spots were identified as histones.The nucleolar preribosomal particle fraction had a DNA:RNA:protein ratio of 1:24:102 and contained only trace amounts of RNA polymerase 1 activity and only 10 per cent of the nucleolar DNA. Acid-soluble proteins associated with these particles were resolved into 78 protein spots; 72 of these (acid-soluble) protein spots corresponded in 2-D gel electrophoresis to 80S cytoplasmic ribosomal proteins. Some 15 protein spots found in 80S ribosomal proteins were absent in the preribosomal particles. It seems reasonable, based on these data, that the enlargement of nucleoli after auxin treatment is primarily due to the large increase in ribosomal proteins and rRNA which accumulate and assemble in the nucleoli in the form of preribosomal particles.A substantial body of literature has established that the synthesis and processing of pre-rRNA occurs in the nucleolus of eukaryotic organisms (2,22,29,31). The nucleolar organizer region (15) contains several hundred to several thousand copies of rRNA genes which are associated with proteins in the form of nucleolar chromatin. During the transcription of pre-rRNA, several ribosomal proteins become associated with the nascent RNA; this leads to further processing and packaging of the prerRNA in the nucleolus to form ribonucleoprotein complexes termed preribosomal particles or preribosomes (29).The nucleolar volume as a fraction of total nuclear volume varies considerably as tissues undergo altered rates of ribosome production. For example, partial hepatectomy or injection of thioacetamide into rats results in an increase in relative nucleolar

show abstract

Synthesis and Processing of Ribosomal RNA in Cultured Plant Cells

Cited by 40 publications

References 29 publications

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

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

The double‐stranded RNA‐dependent protein kinase is also activated by heparin

Fractionation of Nucleoli from Auxin-Treated Soybean Hypocotyl into Nucleolar Chromatin and Preribosomal Particles

Contact Info

Product

Resources

About