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Electron microscopic mapping of RNA transcribed from the late region of polyoma virus DNA

Manor, Haim and Wu, Madeline and Baran, Nava and Davidson, Norman (1979) Electron microscopic mapping of RNA transcribed from the late region of polyoma virus DNA. Journal of Virology, 32 (1). pp. 293-303. ISSN 0022-538X. http://resolver.caltech.edu/CaltechAUTHORS:MANjvir79

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Abstract

The polyoma virus (Py) RNA species transcribed from the L DNA strand of the "late" region of the Py genome in Py-infected mouse cells have been mapped by hybridization with specific fragments of Py DNA followed by electron microscopic visualization of the hybrids. Total cellular polyadenylated Py-specific RNA molecules having an S value in the range of 16S to 20S were purified by oligodeoxythymidylic acidcellulose column chromatography, preparative hybridization with Py DNA, and sucrose gradient centrifugation. Cytoplasmic Py-specific RNA was similarily purified, except that it was not fractionated by sucrose gradient centrifugation. Hybrids of these RNA molecules and Py DNA fragments were spread for electron microscopy by either the cytochrome c technique or the bacteriophage T4 gene 32 protein method. The polyadenylic acid at the 3'-end of the RNA in the hybrids was identified by labeling with simian virus 40 DNA circles to which polybromodeoxyuridylic acid tails had been covalently attached. These experiments revealed the presence of three L DNA strand transcripts in both RNA preparations. Two of these RNA molecules were found to be spliced from chains transcribed from two noncontiguous parts of the late region. The third molecule either is a continuous transcript of the entire late region or contains a splicing feature which is too small to be reliably observed by the electron microscope methods used. The 5'-ends of the three RNA species map within a region extending from 68 to 70 map units on the Py restriction endonuclease map. Each of the two spliced molecules contains a 5'-terminal leader sequence transcribed from a DNA segment with an estimated length of 60 to 110 nuvleotides. The 3'-ends of the leaders map at 66.7 +/- 1.0 and 66.4 +/- 0.50 map units. In these molecules the 5'-ends of the other part (the main body) map at 59.4 +/- 0.90 and 49.4 +/- 2.0 map units, respectively. The 3'-termini of all three RNA species map at 24 to 25 map units.


Item Type:Article
Additional Information:Copyright © 1979 by the American Society for Microbiology. Received for publication 8 January 1979. This research was supported by grants from the United States-Israel Binational Science Foundation, Jersusalem, Israel, and the Leukemia Research Foundation, and by Public Health Service grant GM 20927 from the National Institutes of Health. H.M. was supported by an Eleanor Roosevelt-International Cancer Fellowship during the course of this work. We thank Yaffa Bot for able technical assistance.
Record Number:CaltechAUTHORS:MANjvir79
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:MANjvir79
Alternative URL:http://jvi.asm.org/cgi/content/abstract/32/1/293
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:4550
Collection:CaltechAUTHORS
Deposited By: Lindsay Cleary
Deposited On:28 Aug 2006
Last Modified:26 Dec 2012 09:00

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