Analysis of early adenovirus 2 RNA using Eco R-R1 viral DNA fragments
Analysis of early adenovirus 2 RNA using Eco R-R1 viral DNA fragments
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使用 Eco R-R1 病毒 DNA 片段分析早期腺病毒 2 RNA
DOI：
10.1128/jvi.15.5.1202-1213.1975
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发表时间：
1975
影响因子：
5.4
通讯作者：
H. Raskas
中科院分区：
文献类型：
--
作者：
E. Craig;S. Zimmer;H. Raskas
通过RNA-DNA杂交分析腺病毒2在生产性感染早期合成的RNA。用腺病毒2型DNA进行杂交实验，用限制性内切酶EcoR. R1消化产生6个腺病毒2型DNA片段。用S（1）核酸酶消化法测定RNA和~（32）P标记的病毒DNA之间的双链体形成。来自细胞质的RNA退火了12%的总病毒DNA和以下百分比的每个R.R1片段：6%的R1-A、24%的R1-B、0%的R1-F、40%的R1-D、13%的R1-E和22%的R1-C。早期细胞质RNA由两种序列类别组成：I类，在感染后期（18 h）以大大减少的量存在，II类，在18 h保持高浓度。在杂交抑制实验中，II类RNA的杂交被晚期细胞质RNA抑制，而I类RNA的杂交不被晚期细胞质RNA阻断（J. J. Lucas和H. S. Ginsberg，1971; E.A.克雷格和H. J. Raskas，1971）。为了确定I类和II类序列在基因组上的位置，在杂交抑制实验中使用膜结合的DNA片段。这些研究表明，R1-D的早期细胞质转录本属于II类，而R1-C转录本属于I类序列。从片段A和B转录的细胞质RNA含有I类和II类序列。对按大小分级的细胞质RNA的分析表明，I类序列包括从R1-B转录的19 S RNA，II类序列包括从R1-D衍生的20 S RNA。从感染早期的培养物中纯化的核RNA与~（32）P标记的R1片段退火。所有六个片段的核RNA退火的DNA一样多或更多的细胞质RNA。Eco R1-F与早期核RNA至少退火25%，而在早期胞质RNA中未检测到与R1-F同源的序列。当感染后2至6小时标记培养物时，至少5%的-3H标记的早期核病毒RNA退火至Eco R1-F。来自R1-F的这些核转录物中的一些似乎与从基因组的连续区域（Eco R1-B）转录的序列共价连接。8.4通过R1-F杂交选择的RNA中有10%与R1-B再退火，而不超过1.5%与R1片段A、D、E或C再退火。
Adenovirus 2 RNA synthesized early in productive infection was analyzed by RNA-DNA hybridization. Hybridization experiments were performed with adenovirus 2 DNA and wit, the six adenovirus 2 DNA fragments generated 0y digestion with the restriction endonuclease Eco R.R1. Duplex formation between RNA and -32P-labeled viral DNA was assayed by S(1) nuclease digestion. RNA from the cytoplasm annealed 12 percent of the total viral DNA and the following percentage of each of the R.R1 fragments: 6 percent of R1-A, 24 percent of R1-B, 0 percent of R1-F, 40 percent of R1-D, 13 percent of R1-E, and 22 percent of R1-C. The early cytoplasmic RNA is composed of two sequence classes: class I, present in greatly reduced quantities at late times in infection (18 h), and class II, which remains at high concentrations at 18 h. In hybridization-inhibition experiments, hybridization of class II RNA is inhibited by late cytoplasmic RNA, whereas hybridization of class I RNA is not blocked by late cytoplasmic RNA (J. J. Lucas and H. S. Ginsberg, 1971; E.A. Craig and H. J. Raskas, 1971). To determine the location of class I and II sequences on the genome, membrane bound DNA fragments were used in hybridization-inhibition experiments. These studies demonstrated that the early cytoplasmic transcripts of R1-D belong to class II, whereas R1-C transcripts are class I sequences. The cytoplasmic RNAs transcribed from fragments A and B contain both class I and class II sequences. Analysis of cytoplasmic RNA fractionated by size demonstrated that the class I sequences include a 19 S RNA transcribed from R1-B and class II sequences include a 20S RNA derived from R1-D. Nuclear RNA purified from cultures early in infection was annealed with -32P-labeled R1 fragments. With all six fragments the nuclear RNA annealed as much or more of the DNA than did cytoplasmic RNA. Eco R1-F annealed at least 25 percent with early nuclear RNA, whereas no sequences homologous to R1-F were detected in early cytoplasmic RNA. When cultures were labeled from 2 to 6 h after infection, at least 5 percent of the -3H-labeled early nuclear viral RNA annealed to Eco R1-F. Some of these nuclear transcripts from R1-F appear to be covalently linked to sequences transcribed from a contiguous region of the genome (Eco R1-B). 8.4 percent of the RNA selected by hybridization of R1-F reannealed to R1-B, whereas no more than 1.5 percent reannealed to R1 fragments A, D, E, or C.