Differential thermal stability of old and new ribosomal RNA of rat liver

Kokileva, Ludmila; Mladenova, I.; Tsanev, Roumen

doi:10.1016/0014-5793(71)80003-0

Cited by 21 publications

(4 citation statements)

References 9 publications

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“…As to the origin of the minor fragments of 26-S rRNA, our data suggest that the specific nicks are introduced secondarily depending on preparation conditions, presumably by an endogenous endonuclease, as has been reported for insect rRNA by Ishikawa [31,32]. This is in contrast to reports of other authors in insect and mammalian cells [23,33,341, who described similar minor fragments which were found especially in 'old' rRNA and were assumed to be degradation intermediates in vivo of the large rRNA.…”

Section: Discussioncontrasting

confidence: 46%

“…Besides these 'primary nicks' (cf. [32,32]), additional hidden breaks leading to minor fragments upon denaturation have been observed to varying extent in the large rRNA of insects [23,31,32] as well as in mammalian cells such as rat liver [33,34]. In the present paper we describe the pattern and the sequence of introduction of hidden breaks in rRNA and their precursor molecules as well as the iiitracellular location of such events in Tetrahyniena.…”

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confidence: 85%

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Introduction of Hidden Breaks during rRNA Maturation and Ageing in Tetrahymena pyriformis

Eckert

Kaffenberger

Krohne

et al. 1978

European Journal of Biochemistry

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The stability of Tetralzymena pyriformis cytoplasmic rRNAs and nuclear rRNA precursors has been studied by polyacrylamide gel electrophoresis under partly and completely denaturing conditions. Cytoplasmic 17-S rRNA ( M , = 0.66 x lo6) consists of a continuous polynucleotide chain throughout its lifetime, whereas the bulk of 26-S rRNA ( M , = 1.27 x lo6) dissociates upon denaturation. Two large fragments (Fl, F2) of somewhat different molecular weights ( M , 0.63 x lo6 and 0.58 x lo6) and the small 5.8-S rRNA fragment ( M , about 50000) are regularly observed. Some additional distinct minor fragments (F3 -F6) are noted under certain preparative conditions, suggestive of artifactual origin. The following conclusions were made from the data obtained. (a) Newly synthesized 26-S rRNA molecules do not contain the 'central' hidden break (separating Fl and F,) until about 15 min after their appearance in the cytoplasm; however, they release during denaturation the 5.8-S and/or a short-lived 7-S fragment ( M , about 75 000) which might represent a direct precursor to the 5.8-S rRNA. (b) The immediate nuclear precursor to the 26-S rRNA ( M , 1.39 x lo6) releases a small fragment of similar size (7 S). (c) The largest stable transcription product of the rDNA (prerRNA) does not contain any hidden break.The macronucleus of the ciliated protozoan Tetrahymens pyriformis contains numerous nucleoli, including extra-chromosomal, amplified rDNA units (cf. [I -4]), and shows high rates of pre-rRNA synthesis and ribosome formation during logarithmic growth of the cells (e.g. [5 -101). The general pathway of pre-rRNA processing in this organism has been found to be relatively simple [7,8,10,11]. Based on polyacrylamide/agarose gel electrophoretic analyses under non-denaturing conditions, we have shown that the largest stable rRNA precursor found under logarithmic growth conditions has an apparent molecular weight of about 2.2-2.3 x lo6 and seems to be rapidly cleaved, via two intermediates, into the mature 26-S and 1 7 4 rRNAs [9,10]. The final processing Ahhveyiuriom. rDNA, fraction of nuclear DNA containing the sequences for the rRNA precursor (prc-rRNA) plus spacer aequences; NaCLCit, standard saline citrate = 0.15 M NaC1, 0.015 M sodium citrate, pH 7.0. All other abbreviations for nucleotides and nucleic acids are those proposed by the IUPAC-IUB commission on Biochemical Nomenclature, see Eur. J. Biocher)~. 15, 203-208 (1970).Enz,ymc.s. Deoxyribonuclease I (EC 3.1.4.5); proteinase K (EC 3,4.-,-); ribonuclease A (EC 3.1.4.22). steps, however, including those that lead to the production of the mature large rRNA together with the noncovalently bound, heat-dissociable 5.8-S rRNA, are unknown in this organism (for recent findings in other lower and higher organisms see for example [ 12 -161). Moreover, in Tetrahymena, as in various other protozoa and protostomian animals, at least one additional hidden break is introduced near or at the center of the large rRNA resulting in the formation of discrete fragments upon denaturation [17...

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Section: Discussioncontrasting

confidence: 46%

mentioning

confidence: 85%

Introduction of Hidden Breaks during rRNA Maturation and Ageing in Tetrahymena pyriformis

Eckert

Kaffenberger

Krohne

et al. 1978

European Journal of Biochemistry

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“…The conclusion from these experiments is that the big difference in the 18-Sj28-S ratio of 40-"C and of 5 5°C fractions could be hardly due to thermal dissociation of the nucIear 28-S RNA molecules into fragments. This is supported by the fact that the presence of hidden breaks has not been proved in the nuclear 2 8 3 RNA [56] and that in the cytoplasm they are present not in the newly synthesized but in the "old" 28-5 RNA [55].…”

Section: Profiles At 15-min Extraction Timesupporting

confidence: 48%

Characteristics of Nuclear‐Ribosomal and DNA‐Like Ribonucleic Acids Differentially Extracted by Hot‐Phenol Fractionation

Марков

Arion²

1973

European Journal of Biochemistry

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The purpose of the present study was to define more exactly the conditions of the method of hot-phenol fractionation of nuclear RNA (Georgiev and associates). The thermal RNA fractions obtained were characterized by agar-gel electrophoresis, determination of the specific radioactivity and base composition analysis, both of the total and newly synthesized RNA.It was found that the fraction extracted at 0-4 "C (considered as cytoplasmic RNA in the literature) contained some nuclear ribosomal precursor RNAs, expecially 3 2 4 and 23-S RNAs.The composition of the different thermal RNA fractions strongly depended on the extraction conditions (time, pH, volume ratios etc.). Conditions were well-defhed (15-min extraction a t each temperature, pH 6.2) for obtaining, as separate fractions, the nuclear RNA species of ribosomal type (40-"C RNA, i.e. that obtained a t 40°C) and two classes of DNA-like RNA (63-"C and 85-"C RNA). The 55-"C RNA fraction was found to be of mixed character (rRNA + DNA-like RNA) regardless of the extraction conditions. The 85-"C RNA contained the "giant" RNA molecules, had the highest specifh radioactivity and a (G + C)/(A + U) ratio of about 0.80. These findings are in agreement with the view that 85-"C RNA is the very fist product of transcription. 63-"C RNA was also DNA-like but different from 85-"C RNA with respect to electrophoretic prose, specific radioactivity and base composition. A high content of adenylic acid (above 30°/,) was found in the region 8 to 18 S of 63-"C RNA. It is suggested that poly(A) segments are attached at the 18-S stage of the processing of 63-"C RNA as a precursor to the cytoplasmic mRNA.The results presented demonstrate that under well-defined conditions the method of the hot-phenol fractionation of Georgiev may be used as a reproducible procedure for obtaining different types of nuclear RNA without preliminary isolation of nuclei and nucleoli.The nuclei of eukaryotic cells contain a complex set of ribonucleic acids [1-201. The study of their physical-chemical properties, turnover and functions is hampered by difficulties encountered in the complete extraction and good separation of the different types of nuclear RNAs in an undegraded state.One of the approaches followed is the isolation of sufficiently pure nuclei, their fractionation into nucleoli, nucleoplasm, chromatin etc. and extraction of RNA from these nuclear components [ll, [19][20][21][22].Abbreviations. rRNA, ribosomal RNA; dRNA, RNA with DNA-like base composition; nrRNA, nuclear RNA of ribosomal type; ndRNA, nuclear DNA-like RNA; mRNA, messenger RNA.Enzyme. DNAase, DNAase I, deoxyribonucleate oligonucleotidohydrolase (EC 3.1.4.5).Definition. An Azao unit is the quantity of material contained in 1 ml of a solution which haa an absorbance of 1 at 260 nm when measured i n a 1-cm path-length cell.However, these procedures often lead to degradation [23] or loss of some nuclear RNAs [24]. On the other hand, isolation of pure intact nuclei from many cell types (lymphocytes, tumour cells, endocrine organs etc.)...

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“…Some of the difference in observed turnover rates of 28S and 18S RNA, but not in conservation of rRNA, might be from progressive nicking of RNA in functional 60S ribosomal subunits (5,15,17,20,26). 1 If this is so, 60S and 40S ribosomal subunits should turn over at the same rate, but the nicked RNA extracted from the 60S subunits should sediment slower than normal 28S rRNA.…”

Section: Nicks In 28s Rrnamentioning

confidence: 97%

Conservation of ribosomal RNA during compensatory renal hypertrophy. A major mechanism in RNA accretion.

Melvin¹,

Kumar²,

Malt³

1976

The Journal of Cell Biology

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After removal of one mouse kidney, compensatory hypertrophy in the remaining kidney is marked in 2 days by a 20% average increase in ribosomal RNA (rRNA) per cell. Both 28S and 18S RNA are conserved during the initial stages of compensatory renal hypertrophy to an extent sufficient to account for the rest of the observed accumulation of rRNA. Like some cultured cells, the kidney conserves rRNA during physiological growth.2 days after unilateral nephrectomy in rats and mice, the amount of RNA per cell in the remaining kidney is increased 20-40% (6,8,9,19,22,32). Cell division and DNA synthesis are not pronounced, nor are they necessary for compensatory renal hypertrophy.Increases in total renal RNA during compensatory hypertrophy are likely to be due to an increase in the quantity of ribosomal RNA (rRNA), since rRNA accounts for at least 85% of cellular RNA (I 3). However, an increased rate of rRNA synthesis could not actually be demonstrated (11). An alternative explanation for the accumulation of RNA is that rRNA is degraded slower than normal while its rate of synthesis continues unchanged. Although the turnover rate of rRNA in mouse kidneys several days after the onset of renal hypertrophy is the same as that in kidneys of sham nephrectomized mice (3, 22), few measurements of the degradation rate of rRNA have been made during the first stages of growth, when the rate of accretion of RNA is most pronounced (10).We show that the rate of degradation of both 28S and 18S RNA during the first 4 days of compensatory renal growth is distinctly slower than in nongrowing kidney. MATERIALS AND METHODS Animals 40-day old male Charles River mice (Charles RiverBreeding Laboratories, Wilmington, Mass.) were used for unilateral nephrectomy and controls as described (23). Injections of radiochemicals were intraperitoneal. Isolation of Ribosmes and RNATwo kidneys were homogenized in 10 ml of ice-cold RSB buffer with 10 strokes of the tight pestle of a Dounce homogenizer (Kontes Glass Co., Vineland, N. J.). After removal of 1 ml of the homogenate for estimation of RNA and DNA, the remainder was centrifuged at 10,000 g for 10 rain. MgCI~ was added to the supernate to a final concentration of 70 mM, and the preparation was left for 45 min in ice. After centrifugation at 10,000 g for 10 rain. the ribosomal pellet was resuspended.For preparation of polyribosomes the cell homogenate was centrifuged at 10,000 g for 12 min. The sapernate was layered on a discontinuous sucrose gradient (1.5 ml of 1.5 M sucrose over 2.5 ml of 2.0 M sucrose in 0.25 M NaCI, 0.05 M MgCI~, and 0.01 M Tris, pH 7.4 at 20~ and centrifuged at 50,000 rpm in a Spinco type 65 rotor (Beckman Instruments, Inc., Spinco Div., Palo Alto, Calif.) for 4 h to deposit the polysomes.Polysome and ribosome preparations were dissolved in 548

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Differential thermal stability of old and new ribosomal RNA of rat liver

Cited by 21 publications

References 9 publications

Introduction of Hidden Breaks during rRNA Maturation and Ageing in Tetrahymena pyriformis

Introduction of Hidden Breaks during rRNA Maturation and Ageing in Tetrahymena pyriformis

Characteristics of Nuclear‐Ribosomal and DNA‐Like Ribonucleic Acids Differentially Extracted by Hot‐Phenol Fractionation

Conservation of ribosomal RNA during compensatory renal hypertrophy. A major mechanism in RNA accretion.

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