SIZING AND MAPPING OF EARLY ADENOVIRUS MESSENGER-RNAS BY GEL-ELECTROPHORESIS OF S1 ENDONUCLEASE-DIGESTED HYBRIDS
SIZING AND MAPPING OF EARLY ADENOVIRUS MESSENGER-RNAS BY GEL-ELECTROPHORESIS OF S1 ENDONUCLEASE-DIGESTED HYBRIDS
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DOI：
10.1016/0092-8674(77)90272-0
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发表时间：
1977-01-01
期刊：
影响因子：
64.5
通讯作者：
SHARP, PA
中科院分区：
文献类型：
--
作者：
BERK, AJ;SHARP, PA
开发了一种简单而灵敏的方法，用于对病毒或克隆 DNA 中的 RNA 转录本进行检测、大小测定和绘图。该技术用于检查 [人宫颈癌] HeLa 细胞在腺病毒 2 (Ad2) 感染的早期阶段产生的细胞质转录本。在高于 DNA 双链体 Tm（熔点）但低于 RNA-DNA 杂交双链体 Tm 的条件下，未标记的总细胞质或寡聚 (dT) 选择的细胞质 RNA 与 32P 标记的病毒 DNA 的限制性片段在 80% 甲酰胺中杂交。 DNA 与杂交 RNA 的长度精确互补，是通过在不向杂交双链体中引入链断裂的条件下用单链特异性 S1 核酸内切酶处理来生成的。然后通过碱性琼脂糖凝胶电泳测定 S1 抗性单链 DNA 的大小。终止于编码mRNA的区域内的限制性片段产生与从该限制性片段转录的mRNA部分大小相等的条带。这允许相对于限制性核酸内切酶切割位点的编码区的独特映射。所有早期 Ad2 转录物都聚集在病毒 DNA 的 4 个区域中。至少 5 个早期稳定的细胞质共线转录物从病毒基因组的左端转录到右侧，该区域是哺乳动物细胞转化所必需的基因组编码功能的区域。 650、350 和 1750 个核苷酸的转录本来自 1.7 .+-。 0.5-3.6.+-. 0.5, 3.6.+-. 0.2-4.6.+-. 0.4 和 4.7.+-。 0.3-9.5.+-.分别为0.5个单位，并且有两个450个核苷酸的转录物初步定位在3.0和11.0个单位的区域中。具有相同的3''末端和5''末端替换100个核苷酸的1600和1700个核苷酸的两个重叠转录物从66.2.+-转录到左侧。 0.3-61.6.+-. 0.2 和 66.5 .+-。 0.3-61.6.+-.分别为 0.2 个单位。其他七个不同的早期稳定细胞质转录本也位于基因组上。
A simple and sensitive method was developed for detecting, sizing and mapping RNA transcripts from viral or cloned DNA. This technique was used to examine the cytoplasmic transcripts produced during the early phase of adenovirus 2 (Ad2) infection of [human cervical carcinoma] HeLa cells. Unlabeled total cytoplasmic or oligo (dT)-selected cytoplasmic RNA is hybridized to restriction fragments of 32P-labeled viral DNA in 80% formamide under conditions above the Tm [melting point] of the DNA duplex, but below the Tm of the RNA-DNA hybrid duplex. DNA complements precisely the length of the hybridized RNA are generated by treating with single-strand-specific S1 endonuclease under conditions which do not introduce strand breaks into hybrid duplex. The sizes of the S1-resistant single-stranded DNA are then determined by alkaline agarose gel electrophoresis. A restriction fragment which terminates within a region coding for an mRNA yields a band equal in size to the portion of the mRNA transcribed from that restriction fragment. This allows unique mapping of coding regions relative to restriction endonuclease cleavage sites. All early Ad2 transcripts are clustered in 4 regions of the viral DNA. At least 5 early stable cytoplasmic colinear transcripts are transcribed to the right from the left end of the viral genome, the region of the genome coding functions necessary for transformation of mammalian cells. Transcripts of 650, 350 and 1750 nucleotides map from 1.7 .+-. 0.5-3.6 .+-. 0.5, 3.6 .+-. 0.2-4.6 .+-. 0.4, and 4.7 .+-. 0.3-9.5 .+-. 0.5 units, respectively, and there are two 450 nucleotide transcripts tentatively mapped in the region of 3.0 and 11.0 units. Two overlapping transcripts of 1600 and 1700 nucleotides having the same 3'' terminus and 5'' termini displaced by 100 nucleotides are transcribed to the left from 66.2 .+-. 0.3-61.6 .+-. 0.2 and 66.5 .+-. 0.3-61.6 .+-. 0.2 units, respectively. Seven other distinct early stable cytoplasmic transcripts were also positioned on the genome.