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Ordered transcription of RNA tumor virus genomes

Haseltine, William A. and Kleid, Dennis G. and Panet, Amos and Rothenberg, Ellen V. and Baltimore, David (1976) Ordered transcription of RNA tumor virus genomes. Journal of Molecular Biology, 106 (1). pp. 109-131. ISSN 0022-2836. https://resolver.caltech.edu/CaltechAUTHORS:20170206-144004711

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Abstract

Recent improvements in the reaction conditions have allowed a detailed characterization of the DNA products synthesized in vitro by the reverse transcriptase of RNA tumor viruses from the 70 S genome. We have investigated the size distribution of DNA products synthesized in vitro under different reaction conditions using both Rous sarcoma virus (RSV) and Moloney murine leukemia virus (M-MuLV). Analysis of the reaction products on polyacrylamide gels shows that the majority of DNA synthesized, especially at high concentrations of dNTPs, comprised a set of DNA chains of discrete length the longest of which cover a major fraction of the genome. The effect of varying the time of incubation was to increase the fraction of the total DNA in longer chains. However, small DNA chains less than 200 nucleotides long were still abundant after 500 minutes. By varying which of the four dNTPs was at low concentration we revealed two classes of DNA fragments of discrete length, those present regardless of which dNTP was at low concentration (structural stops) and those which were made only when a specific dNTP was at a low concentration (sequence stops). A strong structural stop which gave a fragment 135 nucleotides long (M-MuLV) or 100 nucleotides long (RSV) comprised more than half of the population of DNA molecules even at the highest dNTP concentrations. The DNA products of the endogenous and exogenous (reconstructed) reactions were identical. The DNA chains of discrete length were initiated with a tRNA (tRNA^(Trp) RSV, tRNA^(Pro) M-MuLV). The DNA fragments of both RSV and M-MuLV have a common initiation sequence. We have sequenced the first 18 nucleotides of M-MuLV. The initial sequences of RSV (AATGAAGC) and M-MuLV (AATGAAAGA) are remarkably similar suggesting a common evolutionary ancestry. We analyzed the pyrimidine tracts of DNA chains of increasing length by electrophoresis followed by homochromatography. The pyrimidine tracts of the shorter pieces form a subset of the pyrimidine tracts of the longer pieces. These data demonstrate that the in vitro products of discrete length were initiated with a tRNA primer at a unique site along the 35 S genome and grew by linear extension.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/0022-2836(76)90303-XDOIArticle
http://www.sciencedirect.com/science/article/pii/002228367690303XPublisherArticle
ORCID:
AuthorORCID
Rothenberg, Ellen V.0000-0002-3901-347X
Baltimore, David0000-0001-8723-8190
Additional Information:© 1976 Elsevier Ltd. Received 16 February 1976. This work was supported by a contract from the Virus Cancer Program of the National Cancer Institute and grant no. CA-14051 from the National Cancer Institute, One of us (W. H.) was a postdoctoral fellow of the Helen Hay Whitney Foundation. Another author (D. K.) was a postdoctoral fellow of the National Institutes of Health in the laboratory of Dr Mark Ptashne. A third author (E. R.) was a predoctoral fellow of the National Science Foundation. A fourth author (D. B.) was a Research Professor of the American Cancer Society.
Funders:
Funding AgencyGrant Number
NIHCA14051
Helen Hay Whitney FoundationUNSPECIFIED
National Cancer InstituteUNSPECIFIED
NSF Predoctoral FellowshipUNSPECIFIED
NIH Postdoctoral FellowshipUNSPECIFIED
American Cancer SocietyUNSPECIFIED
Issue or Number:1
Record Number:CaltechAUTHORS:20170206-144004711
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170206-144004711
Official Citation:Wllliam A. Haseltine, Dennis G. Kleid, Amos Panet, Ellen Rothenberg, David Baltimore, Ordered transcription of RNA tumor virus genomes, Journal of Molecular Biology, Volume 106, Issue 1, 5 September 1976, Pages 109-131, ISSN 0022-2836, http://dx.doi.org/10.1016/0022-2836(76)90303-X. (//www.sciencedirect.com/science/article/pii/002228367690303X)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:74096
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:07 Feb 2017 00:06
Last Modified:03 Oct 2019 16:34

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