PURIFICATION AND TRANSLATION OF MURINE MAMMARY-TUMOR VIRUS MESSENGER-RNAS
PURIFICATION AND TRANSLATION OF MURINE MAMMARY-TUMOR VIRUS MESSENGER-RNAS
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DOI：
10.1128/jvi.39.1.207-218.1981
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发表时间：
1981-01-01
影响因子：
5.4
通讯作者：
VARMUS, HE
中科院分区：
文献类型：
--
作者：
DUDLEY, JP;VARMUS, HE
通过体外纯化和翻译研究了小鼠乳腺肿瘤病毒（MMTV）胞内RNA的功能。通过全细胞或多聚腺苷酸化 RNA 与共价结合到化学活化纸盘（重氮苯甲氧基甲基纸）的克隆 MMTV DNA 的制备性杂交，从感染 MMTV 的大鼠肉瘤（XC）细胞和培养的小鼠乳腺肿瘤细胞中分离出两个主要大小类别的 MMTV RNA，即 35 和 24S RNA。通过与明显缺乏与 24S RNA 同源序列的 4 千碱基 PstI 片段退火，制备不含 24S RNA 的基因组长度 (35S) RNA；通过蔗糖梯度中的速率区带沉降将 24S 物质与 35S RNA 分离。使用[3H]尿苷标记的细胞RNA进行的实验表明，制备型退火方法具有高度特异性，能够实现病毒RNA富集300倍；回收的 RNA 在变性条件下似乎是完整的，并在体外定向合成全长 gag 和 env 多肽。通过凝胶迁移率、针对gag和env产物的兔抗血清的免疫沉淀试验以及用葡萄球菌V8蛋白酶的部分消化来鉴定体外翻译的产物。 35S RNA 种类指导多种 gag 相关多肽的合成，其中包括之前在感染细胞提取物中报道的 3 种； 24S RNA 指导合成与来自感染细胞的 env 蛋白密切相关的 2 种多肽。因此35S RNA包括gag和gag-pol的mRNA； 24S RNA 是 env 的 mRNA。这些结果有助于确定 env 在 MMTV 基因组物理图谱上的位置，并影响基因组的编码潜力。
The functions of the intracellular RNA of mouse mammary tumor virus (MMTV) were studied by purification and translation in vitro. Two major size classes of MMTV RNA, 35 and 24S RNA, were isolated from MMTV-infected rat [sarcoma] (XC) cells and cultured [mouse] mammary tumor cells by preparative hybridization of whole cell or polyadenylated RNA to cloned MMTV DNA covalently bound to chemically activated paper disks (diazobenzyloxymethyl paper). Genomic-length (35S) RNA was prepared free of 24S RNA by annealing to a 4-kilobase PstI fragment apparently deficient in sequences homologous to 24S RNA; the 24S species was separated from 35S RNA by rate zonal sedimentation in sucrose gradients. Experiments using [3H]uridine-labeled cellular RNA indicated that the preparative annealing method was highly specific and capable of effecting a 300-fold enrichment for viral RNA; the recovered RNA appeared to be intact under denaturing conditions and directed synthesis of full-length gag and env polypeptides in vitro. The products of in vitro translation were identified by gel mobility, immunoprecipitation tests with rabbit antisera against gag and env products and partial digestion with Staphylococcus V8 protease. The 35S RNA species directed synthesis of several gag-related polypeptides, including 3 previously reported in extracts of infected cells; 24S RNA directed synthesis of 2 polypeptides closely related to env proteins from infected cells. Thus 35S RNA includes mRNA for gag and gag-pol; 24S RNA is the mRNA for env. These results help establish the position of env on the physical map of the MMTV genome and bear upon the coding potential of the genome.