Some factors affecting the fractionation and mobility of RNA in agar gel electrophoresis

Tsanev, Roumen; Staynov, D. Z.; Kokileva, Ludmila; Mladenova, I.

doi:10.1016/0003-2697(69)90374-1

Cited by 20 publications

(5 citation statements)

References 24 publications

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“…Such a second centrifugation gives better resolution of the 40S fraction from the soluble region, which inevitably merges into the 40S region to some extent during zonal centrifugation of post-mitochondrial supernatant. Agar-gel electrophoresis (Tsanev et al, 1969;Hinton & Dessev, 1973) of RNA from different regions on the slowand fast-sedimentation sides of the peak showed that the change in radioactivity across the peak was due to change in the specific radioactivity of 18 S RNA, little or no RNA of other sizes being detectable. However, some of this approximately 18 S RNA might be mRNA.…”

Section: Resultsmentioning

confidence: 99%

Determination of the amount of small messenger ribonucleic acid-containing particles free in liver cytoplasm

Mullock

Hinton

1975

Biochemical Journal

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To assess the contribution made by mRNA-containing particles to the heterogeneity previously observed among rat liver 40S ribonucleoprotein particles, the amount of poly(A)-containing RNA in subribosomal particles was determined. RNA was labelled with orotate in vivo for 24h and then for 50min. Poly(A)-containing RNA was trapped on filters impregnated with poly(U). Very little poly(A)-containing RNA was found in conventionally prepared ribonucleoprotein particles after fractionation in sucrose. However, after preparation of ribonucleoprotein particles by sedimentation through 1 M-sucrose in the presence of 0.15M-KCl or by precipitation with Mg2+ as described by Leitin & Lerman [(1969) Biokhimiya 34, 839-849], amounts of poly(A)-containing RNA were similar to amounts of mRNA found by other workers in total ribonucleoprotein particles. Even in such preparations, less than 5% of the total rapidly labelled RNA in native subribosomal-particle fractions was mRNA. It seems that mRNA-containing particles make up only a very small part of the population of subribosomal particles in liver.

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Section: Resultsmentioning

confidence: 99%

Determination of the amount of small messenger ribonucleic acid-containing particles free in liver cytoplasm

Mullock

Hinton

1975

Biochemical Journal

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“…They found that the mobility was independent of size for DNA molecules larger than ∼400 base pairs (bp) 5, and varied with ionic strength 3, 5 and the identity and valence of the cation in the background electrolyte 2, 3. At about the same time, inspired by the separation of proteins in synthetic gel matrices 6–10, other investigators began to use similar matrices to separate RNA 11–19 and DNA molecules 15, 20–24 by molecular mass. The separation matrices included agar 11, 18, 20, 21, agarose 22, 23, polyacrylamide 13, 14, 19, 24–28 and composite agarose‐acrylamide 16, 17 gels.…”

Section: Historical Overviewmentioning

confidence: 99%

“…At about the same time, inspired by the separation of proteins in synthetic gel matrices 6–10, other investigators began to use similar matrices to separate RNA 11–19 and DNA molecules 15, 20–24 by molecular mass. The separation matrices included agar 11, 18, 20, 21, agarose 22, 23, polyacrylamide 13, 14, 19, 24–28 and composite agarose‐acrylamide 16, 17 gels. As electrophoretic methods were improved by the purification of agarose 29–31 and the use of slab gels instead of tube gels 25, 32, and as the discovery of restriction enzymes allowed the preparation of monodisperse DNA fragments of known size 33, 34, it became apparent that the separation of DNA fragments by molecular mass depended on the gel matrix in which the separation was carried out 33, 35.…”

Section: Historical Overviewmentioning

confidence: 99%

Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution

2009

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This review describes the electrophoresis of curved and normal DNA molecules in agarose gels, polyacrylamide gels and in free solution. These studies were undertaken to clarify why curved DNA molecules migrate anomalously slowly in polyacrylamide gels but not in agarose gels. Two milestone papers are cited, in which Ferguson plots were used to estimate the effective pore size of agarose and polyacrylamide gels. Subsequent studies on the effect of the electric field on agarose and polyacrylamide gel matrices, DNA interactions with the two gel matrices, and the effect of curvature on the free solution mobility of DNA are also described. The combined results suggest that the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are due primarily to preferential interactions of curved DNAs with the polyacrylamide gel matrix; the restrictive pore size of the matrix is of lesser importance. In free solution, DNA mobilities increase with increasing molecular mass until leveling off at a plateau value of (3.17 ± 0.01) × 10 -4 cm 2 /Vs in 40 mM Tris-acetate-EDTA buffer at 20°C. Curved DNA molecules migrate anomalously slowly in free solution as well as in polyacrylamide gels, explaining why the Ferguson plots of curved and normal DNAs containing the same number of base pairs extrapolate to different mobilities at zero gel concentration.Keywords agarose gels; capillary electrophoresis; DNA; free solution mobility; polyacrylamide gels Historical overviewThe study of DNA electrophoresis began in 1964, when three groups of investigators [1][2][3][4][5] measured the mobility in free solution using moving boundary methods. They found that the mobility was independent of size for DNA molecules larger than ∼400 base pairs (bp) [5], and varied with ionic strength [3,5] and the identity and valence of the cation in the background electrolyte [2,3]. At about the same time, inspired by the separation of proteins in synthetic gel matrices [6][7][8][9][10], other investigators began to use similar matrices to separate RNA [11][12][13][14][15][16][17][18][19] and DNA molecules [15,[20][21][22][23][24] by molecular mass. The separation matrices included agar [11,18,20,21], agarose [22,23], polyacrylamide [13,14,19,[24][25][26][27][28] and composite agarose-acrylamide [16,17] gels. As electrophoretic methods were improved by the purification of agarose [29][30][31] and the use of slab gels instead of tube gels [25,32], and as the discovery of restriction enzymes allowed the preparation of monodisperse DNA fragments of known size [33,34], it became apparent that the separation of DNA fragments by molecular mass depended on the gel matrix in which the separation was carried out Correspondence: Dr. Nancy C. Stellwagen, Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA, nancystellwagen@uiowa.edu, Fax: +319-335-9570. The author has declared no conflict of interest. NIH Public Access Author ManuscriptElectrophoresis. Author manuscript; available in PMC 2010 June 1. [33,35]. Electro...

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“…The subunits were obtained by centrifugation in a sucrose-density gradient (8-240/,) after dissociation by 0.02 mM EDTA in 10 mM Tris buffer pH 7.5 [14]. Ribosomal protein was obtained by means of extraction with LiC1-urea solution [16]. The free RNAs were separated by agar-gel electrophoresis [ 161. tRNA was prepared from liver [17].…”

Section: Methodsmentioning

confidence: 99%

The Temperature‐Dependent Enzymatic Breakdown of rRNA of Reticulocytes

Prehn

Rosenthal

Rapoport

1972

European Journal of Biochemistry

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The influence of temperature on the enzymatic breakdown of rRNA and ribosomal particles from rabbit reticulocytes by ribosomal ribonuclease and phosphatase was studied. Nucleosides were formed after the initial attack of ribonuclease on single-stranded regions of the RNA.The following conclusions were reached for free RNA: U and C were distributed in a random manner in the single-stranded loops a t 20 "C. Short (A-U)-rich sequences were liberated at 37 "C, the relative share of which appeared to be higher in the 28-5 RNA. It contained correspondingly longer G-C sequences. Clusters of A-U sequences of greater length than a t 37 "C were melted a t 50 "C. It was proved that only -U-U-U-sequences were split, by analysis of of the 3' and 5' terminal nucleotides of the primary split products of ribonuclease.The behaviour of the particle-bound RNA was modi6ed considerably by interactions with proteins. Some of the cytosines appear to be protected against enzymatic attack a t 20 "C. The double-stranded sections apparently exhibit variation both with regard to the number of bases in the sequence, which ranges from 4 to 10, and their chemical nature. There is extensive clustering of identical bases. The main method for the study of the ribosome has been the analysis of the temperature profiles and titrations, which lends itself only to statistical conclusions.Enzynzes. Ribonuclease (EC 2.7.7.16); Phosphatase (EC 3.1.3.1) ; Phosphodiesterase (spleen) (EC 3.1.4.1).Previously we reported temperature-dependent changes in the specifity of enzymatic degradation of free rRNA [12]. The U-rich double-stranded sections were melted between 30 "C and 50 "C and were subject to preferential enzymatic breakdown. The results agree with the model of Cox [ill. The purpose of the present study was to gain additional information from a systematic study of the temperaturedependent enzymatic breakdown of both free rRNA and ribosomal particles. MATERIALS AND METHODSThe ribosomes from reticulocytes were prepared according to a published procedure . To determine the nucleotide composition the tRNA was hydrolyzed for I8 h in 0.3 M KOH a t 25 "C. After neutralization with HC10, and removal of the KClO, by centrifugation the nucleotides were fractionated on a Dowex column (1x8, 100 to 200 mesh, formate form) by elution with formic acid/ sodium formate. They were assayed spectrophotometrically.

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Some factors affecting the fractionation and mobility of RNA in agar gel electrophoresis

Cited by 20 publications

References 24 publications

Determination of the amount of small messenger ribonucleic acid-containing particles free in liver cytoplasm

Determination of the amount of small messenger ribonucleic acid-containing particles free in liver cytoplasm

Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution

The Temperature‐Dependent Enzymatic Breakdown of rRNA of Reticulocytes

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