Molecular‐Weight Determination of Animal‐Cell RNA by Electrophoresis in Formamide under Fully Denaturing Conditions on Exponential Polyacrylamide Gels

Abstract

A method for electrophoretic analysis of RNA under fully denaturing conditions on exponential gradient polyacrylamide gels is described. Full denaturation, and strand separation of DNA . RNA hybrids and double-stranded RNA is obtained in dry formamide only if electrophoresis is carried out at 45 O and 55 "C, respectively.In such conditions, the effects of secondary structure of RNA, important in aqueous medium, are suppressed and a linear correlation is obtained between the logarithm of the molecular weight of an RNA and its final position in the gel over the entire molecular weight range of lo4-lo7.Based on absolute molecular weight standards, obtained from sequenced rRNA of Escherichia coli and tRNA and extrapolating to higher molecular weights, the size of animal cell RNA was reexamined. Precursor rRNA (45 S) from HeLa cells migrates according to a molecular weight of 4.1 x lo6. Nascent precursor mRNA has molecular weights of up to 5 x lo6 in the case of duck erythroblasts and of up to lo7 in HeLa cells. This seems to represent the largest size of non-viral animal-cell RNA molecules.Polyacrylamide gel electrophoresis has developed into a widely applied tool for the evaluation of the molecular weight of RNA. The M , values obtained by this method for known RNA species, such as ribosomal RNAs from eukaryotes or prokaryotes [l], are consistent with length measurements in electron micrographs [2] or with size evaluations obtained from sedimentation rates [3,4]. The introduction of exponential gel electrophoresis [ 5 ] enlarged the scope of the method: it made possible the simultaneous analysis of all types of animal cell RNA on one gel in a MI-range from lo4 to lo7 and, furthermore, it made the final resolution independent of loading volume and time of electrophoresis.However, in the course of parallel analysis of nuclear RNA by electrophoretic migration and by sedimentation on sucrose gradients considerable discrepancies appeared : the amount of high-MI relative to low-M, pre-mRNA molecules (precursor messenger RNA, CJ [6]) was larger on gels than on gradients, as judged by their position relative to rRNA. Discrepan- cies in molecular weight values obtained by sedimentation analysis and electrophoresis were reported also for yeast mitochondria1 RNA [7].These differences were attributed to the effects of RNA secondary and tertiary structure on sedimentation and on electrophoretic mobility [8-141. In fact, large sequences of high secondary structure increase the apparent viscosity of the nucleic acids and, decreasing the mobility, lead to opposite conclusions in either analytical system. Furthermore, the hybridisation of complementary sequences, or random hydrogen bond formation between molecules, may lead to an overestimation of the M , of nuclear RNA and to the contamination of high-MI RNA with messenger RNA. Thus, we decided to further develop our analytical methods by suppressing the secondary structure of RNA during its analysis. This may be done by using a denaturing solvent, such as dimethylsulfoxide or for...

Size Distribution of Rat Liver Nuclear RNA Containing mRNA Sequences

“…Although in 75 "/, formamide in the absence of salt poly(rG) . poly(rC) would be totally devoid of secondary structure at our preheating temperature of 65 "C [47], we cannot exclude all RNA reaggregation at our sedimentation temperature of 22 "C [42].…”

“…Nuclear RNA has a high tendency to form aggregates and to remain aggregated after being nicked if rigorous denaturing conditions are not employed [14,36,37]. Careful studies by Ts'o and his coworkers [38,39] and recently by Pinder et al [40,41] and Spohr et al [42] have shown that formamide used under the proper conditions is an effective denaturant of RNA. We chose to fractionate nuclear RNA according to size by sedimentation at 22 "C in sucrose gradients containing 75 % formamide and no salt.…”

Characterization of the Different Electrophoretic Forms of the Cadang-Cadang Viroid

“…Sizes of the four ccRNAs were estimated by electrophoresis in 6~ polyacrylamide gel slabs (40 x 20 x 0.05 cm) containing 98~ formamide (Maniatis & Efstradiatis, 1980 (Spohr et al, 1976); alfalfa mosaic virus (AMV) RNA 4, 881 nucleotides (Brederode et al, 1980); yeast 5-8S RNA, 158 nucleotides (Rubin, 1973); yeast 5S RNA, 121 nucleotides (Miyazaki, 1974), Escherichia coli phenylalanine tRNA, 76 nucleotides (Barrell & Sanger, 1969).…”

Characterisation of a Chromatin Fraction Bearing Pulse-Labelled RNA. 1. Nascent RNA, RNA Polymerase B and Transcribed DNA Sequence Content

“…The RNA size distribution was not significantly changed by tlic prescncc of rat liver RNase inhibitor [27] during chromatin prcparation and we found that the reduction in mean size could reasonably be ascribed to breakagc of nascent RNA during were derived by electrophoresis of RNA in denaturing polyacrylamide gels (98 : ( formamide, 45 "C) [28]. RNA polyinerase B can be assayed quantitatively in cell and tissue homogenates [20,29] and chromatin fractions [30] using labelled amatoxins, which bind to the polymerase in a specific and stoichiometric fashion.…”