Accurate transcription of cloned Xenopus rRNA genes by RNA polymerase I: demonstration by S1 nuclease mapping.
Accurate transcription of cloned Xenopus rRNA genes by RNA polymerase I: demonstration by S1 nuclease mapping.
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RNA 聚合酶 I 准确转录克隆的非洲爪蟾 rRNA 基因：通过 S1 核酸酶作图证明。
DOI：
10.1093/nar/10.11.3391
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发表时间：
1982
影响因子：
14.9
通讯作者：
McKnight,SL
中科院分区：
文献类型：
--
作者：
Sollner-Webb,B;McKnight,SL
我们已经证明了克隆的XenopusrRNA基因在注射到Xenopus卵母细胞后能够忠实地转录启动。这一观察是通过使用一种既灵敏又定量的S1核酸酶分析来实现的。为了检测注射模板中rRF4A的合成，利用了两种Xenopus种之间核糖体DNA序列的差异，以检测在卵母细胞中存在的rRNA的大背景之上。将克隆的莱氏X.laevis核糖体DNA注射到X.borealis卵母细胞的核中。然后分离出总的卵母细胞RUA，并将其与重叠在X的5‘端的放射性DNA探针杂交。北极血吸虫卵母细胞的内源rRNA不与探针杂交。RNA/DNA杂交体用S1核酸酶处理，用聚丙烯酰胺凝胶电泳法测定保护片段的大小。由注射的rDNA制成的RNA保护与realX相同的探针区域。Laevis前体rRNA。因此，转录似乎在克隆的、微注射的X上启动。在体内使用的相同位置上的LaevisrDNA。这种合成不会被联合注射足以抑制RNA聚合酶II和III的n-a所破坏，因此反应是由RNA聚合酶I介导的。转录的数量可以重复地定量，我们改变了一些参数，以便最大限度地提高注射的rDNA的转录表达。8个独立分离的X。LevisrDNA克隆以及这些基因的几个亚克隆起始区都以大致相同的效率被准确转录。这一分析方法应该有助于分析rRNA转录的几个方面，包括取消XenopusRNA聚合酶I启动子的位置。
We have demonstrated faithful transcriptional initiation of clonedXenopusrRNA genes upon injection intoXenopusoocytes. This observation has been made possible by the use of an S1 nuclease assay that is both sensitive and quantitative. In order to detect rRf4A synthesis from the injected tem plate above the large background of rRNA endogenously present in oocytes, the divergence of ribosomal DNA sequences between twoXenopusspecies was utilized. Cloned X. laevis ribosomal DNA was injected into the nuclei of X. borealis oocytes. Total oocyte RUA was then isolated and hybridized to a radioactive DNA probe that overlaps the 5′ end ofX. laevisrRNA; endogenous rRNA of the X. borealis oocytes does not hybridize to the probe. RNA/DNA hybrids were treated with S1 nuclease and protected fragments were sized by polyacrylamide gel electrophoresis. RNA made from the injected rDNA protects the same region of probe as does authenticX. laevisprecursor rRNA. Thus, transcription appears to initiate on the cloned, microinjectedX. laevisrDNA at the same site as is usedin vivo. This synthesis is not impaired by coinjectjon of an amount of n-a sufficient to inhibit RNA polymerase II and III; therefore the reaction is mediated by RNA polymerase I. The amount of transcription may be reproducibly quantitated and we have varied a number of parameters in order to maximize transcriptional expression of the injected rDNA. Eight independently isolatedX. laevisrDNA clones as well as several subcloned initiation regions of these genes are all accurately transcribed at approximately equal efficiency. This assay should facilitate analysis of several aspects of rRNA transcription, including deleniation of theXenopusRNA polymerase I promoter location.