Enhanced transcription of fibroin gene in vitro on covalently closed circular templates.
Enhanced transcription of fibroin gene in vitro on covalently closed circular templates.
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在共价闭合圆形模板上增强丝心蛋白基因的体外转录。
DOI：
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发表时间：
1985
影响因子：
4.8
通讯作者：
Y. Suzuki
中科院分区：
文献类型：
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作者：
S. Hirose;M. Tsuda;Y. Suzuki
本文提出了一种改进的S1核酸酶测定方法，用于在未标记的同源RNA存在下定量测定放射性转录物的量。利用这种方法，我们检测了丝素基因在环状和线状模板上的体外转录。在后部丝腺细胞提取物中，共价闭合环状（ccc）DNA形成超螺旋状态，并且支持比切口环状或线性DNA多3至10倍的转录。在中部丝腺细胞提取物中也观察到ccc模板上的有效转录，但在HeLa细胞提取物中从未观察到。当后部丝腺细胞提取物首先与pBR 322 DNA孵育，然后激发ccc DNA上的丝素基因转录时，ccc形式的pBR 322 DNA而不是线性形式的pBR 322 DNA抑制反应。一个缺失突变体保留了ccc模板的优先性，但在TATA盒和-20区的7个单点突变体则使其丧失了优先性，这表明丝腺提取物中的某些成分在缺少上游增强序列的情况下，能使ccc DNA超螺旋并刺激丝素基因的转录。
A modified method for S1 nuclease assay has been developed to quantitate an amount of radioactive transcripts in the presence of unlabeled homologous RNA. Using this method, we have assayed in vitro transcription of fibroin gene on circular and linear templates. In a posterior silk gland cell extract, covalently closed circular (ccc) DNA forms the superhelical state and supports three to 10 times more transcription than nicked circular or linear DNA does. The efficient transcription on ccc template is also observed in a middle silk gland cell extract but never seen in a HeLa cell extract. When the posterior silk gland cell extract is first incubated with pBR322 DNA and then challenged for the fibroin gene transcription on ccc DNA, the pBR322 DNA of the ccc form, but not of the linear one, inhibits the reaction. A deletion mutant carrying only 44 base pairs of the 5'-flanking sequence reserves the preference of the ccc template, but seven single-point mutants at the TATA box and -20 region abolish it. These results suggest that some components in the silk gland extract supercoil ccc DNA and stimulate the fibroin gene transcription in the absence of the upstream enhancing sequence.