Abstract
A new method for mapping RNA initiation, termination, and splice sites was developed. The method involves: 1) hybridization of RNA to end-labeled single-stranded DNA; 2) mild digestion of the hybrid with a single strand specific nuclease; 3) high resolution gel electrophoresis and autoradiography. The regions of the labeled probe which are resistant to nuclease digestion are mapped by measuring the distance from the labeled end. The sequence can be determined by running the end-labeled probe sequenced according to the protocol of Maxam and Gilbert (Maxam, A.M., and Gilbert, W. (1980) Methods Enzymol. 65, 499-560) at the same time. The feasibility of this method was tested with adult chicken beta-globin mRNA, and we found that the transcription initiation, termination, and RNA splice sites can be determined to within a few bases of the known sites. Using this method we also found that ribosomes bind to beta-globin mRNA from 22 bases upstream from the translation start codon (AUG) to 15 bases past the stop codon (UAA).
Highlights
W. (1980)Methods Emymol. 65,499-560)at the same time. The feasibility of this method was tested with adult chicken /?-globin mRNA, and we found that the transcriptioninitiation,termination, and RNA
Acrylamide Gel Electrophoresis-Sequencing gels (0.3, 330, and 400 mm) were prepared asdescribed by Maxam and Gilbert [11].The gel was exposed to Kodak XAR-5 x-ray film with a DuPont Cronex intensifying screen at -70 "C
The basic strategy used in mapping RNA is similar to that of the DNase I footprinting method of Galas and Schmitz [5]
Summary
Isolation of Polysomes-Erythroid cells were obtained from white leghorn chickens after phenylhydrazine treatment and polysomes were isolated as described [7]. Preparation of Polysomal and Protected Polysomal RNA-To prepare undegraded total polysomal RNA the polysomal pellet was suspended in 1%SDS,’10m~ Tris, pH7.5,1 mM EDTA, 0.15 M NaCl. To prepare micrococcal nuclease-protected polysomal RNA the polysomal pellet was taken up in 5%glycerol, 10 mM Tris, pH 7.5, 0.2 mMMgC12, 1 mM CaClz, and insoluble material was removed by a short centrifugation (15 min, 12,000 X g, 4 “C). After digestion the solution was made 1 mM EDTA, 1% SDS andtreated with proteinase K (50 pg/ml), in the presence of vadanyl-adenosine complex for 2 h at 37 “C. Cbning-The chicken @-globingene fragments that will be described under “Results” were inserted into M13mp7RF by established methods and with observation of current NIH guidelines for recombinant DNA research. The abbreviations used are: SDS, sodium dodecylsulfate; P-RNA, nuclease-protected polysomal RNA; M13mp7RF, replicative form of M13mp DNA
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