This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lebowitz, P Articles by Weissman, S M Search for Related Content PubMed PubMed Citation Articles by Lebowitz, P Articles by Weissman, S M

 Previous Article  |  Next Article 

J Virol. 1977 May; 22(2): 430-445

Specificity of initiation of transcription of simian virus 40 DNA I by Escherichia coli RNA polymerase: identification and localization of five sites for initiation with [gamma-32P]ATP.

ABSTRACT

Simian virus 40 (SV40) DNA I was transcribed with Escherichia coli RNA polymerase in the presence of gamma-32P-labeled ribonucleoside triphosphates in order to investigate the specificity of initiation of in vitro transcription. ATP and GTP served as predominant initiating nucleotides, the former being incorporated about twice as much as the latter. Cleavage of [gamma-32P]ATP-labeled SV40 complementary RNA (cRNA) with T1 RNase followed by homochromatographic analysis of the resultant 5' initiation fragments revealed the presence of four specific initiation fragments 6 to 9 nucleotides in length, designated AI, AII, AIIIa, and AIIIb. By means of hybridization of [gamma-32P]ATP-labeled SV40 cRNA to DNA from specific adenovirus 2-SV40 hybrids and specific restriction endonuclease fragments of SV40 DNA before chromatographic analysis, it was possible to identify and determine approximate localizations of five [gamma-32P]ATP initiation sites on the SV40 genome: one in Hin-G close to the Hin-G-B junction, giving rise to the AII fragment, two in the overalpping fragment Hin-A-Hae-A,giving rise to AI and AIII fragments, and two in the fragment Hin-A-Hae-E, also giving rise to AI and AIII fragments. All five sites either fall within or lie near regions of the genome that are cleaved by S1 nuclease and subject to partial alkaline denaturation. These five sites lie on the minus strand of SV40 DNA and initiate RNAs that are copied in a leftward direction. Cleavage of [gamma-32P]GTP-labeled cRNA with pancreatic RNase liberated three major 5' initiation fragments of short length, GI, GII, and GIII, suggesting the presence of three principal GTP initiation sites.