Transcription of Simian virus 40. I. Separation of the strands of SV40 DNA and hybridization of the separated strands to RNA extracted from lytically infected and transformed cells |
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| Authors: | J Sambrook P A Sharp W Keller |
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| Affiliation: | 2. Clayton Livestock Research Center, New Mexico State University, Clayton 88415;1. Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan;2. Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan;3. R&D Department, Hoya Technosurgical Co., 1-1-11, Tsutsujigaoka, Akishima-shi, Tokyo 196-0012, Japan;4. Laboratory of Biotechnology, Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529, Japan |
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| Abstract: | Asymmetric RNA was synthesized in vitro from SV40 component I DNA using Escherichia coli DNA-dependent RNA polymerase. When denatured, unit-length, single-stranded SV40 DNA was incubated in the presence of 6- to 20-fold excess of asymmetric RNA, about 50% of the DNA (E-DNA) formed DNA-RNA hybrids. The unhybridized DNA (L-DNA) was separated from the DNA-RNA hybrids by chromatography on hydroxyapatite. E-DNA and L-DNA were shown to be the complementary strands of SV40 DNA. After further purification and shearing, the separated strands were hybridized to RNA extracted at different stages of lytic infection and to RNA from transformed cells. “Early” RNA contained sequences complementary to 30% of E-strand DNA; “late” RNA bound to 30 to 35% of Estrand DNA and to 70% of L-strand DNA. RNA from SV3T3 cells hybridized with 50% of E-strand DNA and 15 to 20% of L-strand DNA. |
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