Nodal基因的第一内含子中存在着顺式调控增强子元件

A Cis-regulatory Enhancer Element of the nodal Gene is Present in Its First Intron

小鼠ｎｏｄａｌ基因是在胚胎发育时期表达的一个基因。它在原肠期起着重要作用，并且参与左一右体轴的决定［１，２］。４１３－ｄ小鼠胚胎于细胞系３５６３在ｎｏｄａｌ基因的第一内含子中含有单拷贝逆转录病毒的插入［３］。采用定量 ＲＮａｓｅ保护法比较了该细胞系及其母源 ＥＳ细胞 ＣＣＥ中ｎｏｄａｌ基因的表达量，ＣＣＥ细胞中ｎｏｄａｌ ｍＲＮＡ的含量是３５６３ＥＳ细胞含量的２．３倍。这一结果提示原病毒在ｎｏｄａｌ基因第一内含子中的整合降低了ｎｏｄａｌ ｍＲＮＡ的表达，其原因可能是病毒的插入破坏了ｎｏｄａｌ基因第一内含子中整合位点上的或其附近的顺式调控元件。为了验证这一推论，构建了一系列包含３５６３细胞系ｎｏｄａｌ基因的完整第一内含子及其部分片段的荧光素酶报告质粒，并以瞬时转化的方法测定了它们对报告基因转录活性的影响。该内含子５’端 ２．４ｋｂ ＤＮＡ片段亦足以使报告基因的转录活性提高 ８倍。这一结果表明：在 ｎｏｄａｌ基因的第一内含子中存在着控制ｎｏｄａｌ基因表达的顺式增强子元件。

The mouse nodal gene is expressed during early embryogenesis and plays an important role during gastrulation and the determintion of the left-right body symmetry/axis formation. The embryonic stem (ES) cell line 413-d 3563 contains a single copy of a retrovirus insertion in the first inton of the nodal gene. Using a quanhtahve RNase protechon assay, the level of the nodal gene expression in aams cell line was compared to that of the parental CCE cell line. A 2.3 fold down regulation of the nodal transcript in the 3563 ES cell line suggested that the proviral insertion might decrease the nodal mRNA expression by disrupting the potential cis regulatory elements present at the insertion site in the first intron of the nodal gene. Luciferase reporter constructs containing the entire first intron fragment or part of it were tested by a transient transfection assay for their potential transcriptional regulatory activity. A 2.4 kb DNA fragment from the 5' end of the intron was sufficient to increase the expression level of the reporter gene & fold, suggesting that an important cis acting enhancer element for the nodal gene expression is localized within the first intron.