Separation of Microsomal RNA into Five Bands during Agar Electrophoresis

Bachvaroff, R; McMaster, Philip R. B.

doi:10.1126/science.143.3611.1177

Cited by 34 publications

(9 citation statements)

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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%

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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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Section: Historical Overviewmentioning

confidence: 99%

Section: Historical Overviewmentioning

confidence: 99%

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

2009

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“…We have used the technique of agar-gel electrophoresis that was further developed as an analytical method for RNA fractionation by Tsanev & Staynov (1964). This method was shown to have a better resolving power than sucrose-densitygradient centrifugation (Bachvaroff & McMaster, 1964;Tsanev, 1965a,b). It is possible to study by radioautography the incorporation of labelled precursors into different electrophoretic fractions by using dry agar electrophoretograms.…”

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confidence: 99%

Incorporation of labelled precursors into the electrophoretic fractions of rat-liver ribonucleic acid

Tsanev¹,

Марков²,

Dessev³

1966

Biochemical Journal

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1. Incorporation of [(32)P]orthophosphate and of [2-(14)C]orotic acid into rat-liver RNA was studied by agar-gel electrophoresis by using u.v.-densitometry and radioautography of dried agar electrophoretograms. 2. During the electrophoresis some low-molecular-weight contaminants, including inorganic phosphate present in the RNA preparations, were separated from the RNA fractions. Since nucleoside mono-, di- and tri-phosphates still interfered, the RNA preparations had to be subjected to a purification procedure [Sephadex G-25 or Dowex 1 (X8)]. 3. In RNA extracted from cytoplasm, isolated microsomes or ribosomes, whatever variations were made in the phenol procedure no special rapidly labelled RNA fraction was detected other than ;soluble' RNA and the ribosomal RNA components. 4. When the whole homogenate or cytoplasmic fraction was treated only with phenol (pH6) a considerable part of the cytoplasmic RNA was not extracted. The treatment of the cytoplasmic fraction with sodium dodecyl sulphate before the addition of phenol increased the yield of the high-molecular-weight RNA and at the same time a higher specific activity was found for the faster ribosomal RNA component. 5. The presence of four distinct rapidly labelled RNA fractions was established in the RNA not extracted by phenol, and they moved slower than the ribosomal RNA. They were extracted only with the use of phenol-sodium dodecyl sulphate at an elevated temperature.

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“…For fractionation of RNA species, a modification of the electrophoretic method on agarose-polyacrylamide gels described by Peacock and Dingman (1968) was used. Electrophoretic separation of RNA was first described with agar as gel substance (Bachvaroff and McMaster 1964), and there has been many indications that gel techniques are capable of higher resolution than the more commonly used techniques of zone sedimentation and column chromatography. Agar gel electrophoresis seems to be most valuable in fractionation of high molecular weight RNA.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Ovarian RNA

Ahrén

Hamberger

Jarlstedt

et al. 1970

Acta Physiologica Scandinavica

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Ovaries were obtained from 24–25 day old Sprague‐Dawley rats. Groups of 20 ovaries were collected corresponding to a wet weight of 100–120 mg, and incubated for 20, 30, 60 and 240 min at 37° C in Krebs bicarbonate buffer containing 5.5 mM glucose and uridine‐3H. Following incubation, ovarian RNA was extracted with a modified phenol extraction method described in detail. Fractionation was made by composite agarose‐polyacrylamide gel electrophoresis which gave good separation and high resolution of the various RN.4 fractions. Ovarian RNA separated in four well‐defined fractions classified as 28S, 18S, 5s and 4s. This classification was based upon comparisons with simultaneously run liver RNA. In addition to the four main fractions. a weak band just before 28S and two smaller bands between 28S and 18S were consistently observed. Labelling of ovarian RNA with uridine‐3H showed a polydisperse pattern after the shorter incubation periods with most of the radioactivity concentrated to RNA fractions of molecular weights higher than 28S, but also clear labelling of the 5S—4S region. After 60 min, radioactivity peaks were also associated with the 28S and 18s fractions and with fractions between those, while after 240 min incubation, the radioactivity was concentrated to the four main fractions. The incorporation of radioactivity was found to be markedly decreased in presence of actinomycin D.

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Separation of Microsomal RNA into Five Bands during Agar Electrophoresis

Cited by 34 publications

References 4 publications

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

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

Incorporation of labelled precursors into the electrophoretic fractions of rat-liver ribonucleic acid

Characterization of Ovarian RNA

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