Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas
Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas
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DOI：
10.1046/j.1365-313x.2002.01371.x
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发表时间：
2002-08-01
期刊：
影响因子：
7.2
通讯作者：
Vallon, O
中科院分区：
文献类型：
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作者：
Schroda, M;Beck, CF;Vallon, O
我们以前已经证明，当HSP70A(A)启动子融合在其他启动子的上游时，它们在驱动衣藻转基因表达方面的表现显著提高。在这里，我们利用细菌耐药基因ble，由RBCS2(R)启动子或AR启动子融合来确定A启动子可能通过什么机制发挥增强作用(S)。我们观察到AR-ble结构的转化率显著高于R-ble结构。然而，由两种构建类型产生的转化子池中的ble mRNA水平是相同的。共转化实验表明，R-ble转基因在80%的转化子中被沉默，而在含有AR-ble转基因的转化子中，这一比例降低到36%。我们得出结论，A启动子通过降低转基因转录沉默的概率而起作用。我们在A启动子中定位了两个负责这一效应的元件。第一个元件的核心似乎位于相对于HSP70A转录起始点的-7和+67核苷酸之间。其活性强烈依赖于其相对于R启动子的空间设置，并由上游序列(-196至-8)增强。第二个元素独立于第一个元素，位于-754到-197之间的区域。它的活性与第一个元素的间距无关，并且是相加的。
We have shown previously that the HSP70A (A) promoter, when fused upstream of other promoters, significantly improves their performance in driving transgene expression in Chlamydomonas. Here, we employed the bacterial resistance gene ble, driven by the RBCS2 (R) promoter or an AR promoter fusion, to determine, by which mechanism(s) the A promoter may exert its enhancing effect. We observed that transformation rates of AR-ble constructs were significantly higher than those of R-ble constructs. However, ble mRNA levels in pools of transformants generated with either construct type were the same. Co-transformation experiments revealed that the R-ble transgene was silenced in 80% of the transformants, whereas this fraction was reduced to 36% in transformants harbouring the AR-ble transgene. We conclude that the A promoter acts by decreasing the probability that a transgene becomes transcriptionally silenced. We mapped two elements within the A promoter that are responsible for this effect. The core of the first element appears to be located between nucleotides -7 and +67 relative to the HSP70A transcriptional start site. Its activity is strongly dependent on its spatial setting with respect to the R promoter and is increased by upstream sequences (- 196 to -8). The second element is independent of the first and is located to the region from - 754 to - 197. Its activity is spacing-independent and additive to the first element.