Regulatory elements from the related spec genes of Strongylocentrotus purpuratus yield different spatial patterns with a lacZ reporter gene.
Regulatory elements from the related spec genes of Strongylocentrotus purpuratus yield different spatial patterns with a lacZ reporter gene.
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来自紫圆甲藻相关规格基因的调节元件与 lacZ 报告基因产生不同的空间模式。
DOI：
10.1016/0012-1606(90)90355-m
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发表时间：
1990
影响因子：
2.7
通讯作者：
Klein,WH
中科院分区：
文献类型：
--
作者：
Gan,L;Wessel,GM;Klein,WH
Spec 1和Spec 2基因与反口外胚层的分化密切相关。为了研究Spec基因空间表达的调控元件，我们将含有核靶向信号的大肠杆菌lacZ基因与来自Spec 1、Spec 2a和Spec 2c的5′侧翼DNA加5′非翻译前导序列融合。这三个基因在其上游区域都含有700 bp的高度保守的DNA，但在Spec 1和Spec 2c中，大的插入中断了保守区域。将Spec-lacZ报告基因质粒显微注射入鸡卵中。purpuratus、Lytechinus variegatus和L.用X-gal染色法原位测定β-半乳糖苷酶活性。Spec 2a-lacZ融合基因5′端侧翼序列为1516 bp，5′端非翻译前导序列为18 bp，该基因在3种动物的离口外胚层细胞中均优先表达。Spec 1-lacZfusion基因以一种截然不同的方式表达，优先在初级和次级间充质细胞中表达，偶尔在反口外胚层细胞中表达，而在口外胚层和内胚层细胞中表达较少。无论是同源或异源胚胎的染色模式是相同的。Spec 2c-lacZ融合基因，像Spec 2a-lacZ，优先表达在离口外胚层，但染色的其他细胞类型经常观察到。为了进一步描述正确空间表达所需的序列，我们从Spec 2a-lacZfusion基因中删除了800 bp的5′侧翼DNA，产生了仅包含保守DNA区域的Δ Spec 2a-lacZfusion基因。该基因融合体在无口外胚层细胞中优先表达。然而，细胞类型特异性不如亲本Spec 2a-lacZ质粒那么大。这些实验表明，与所有检测的Spec基因相关的保守DNA区域不足以形成完全的反口外胚层特异性，并表明Spec 2a的5′侧翼DNA中存在一个位于−1516和−697 bp之间的空间阻遏元件。
The Spec1 and Spec2 genes ofStrongylocentrotus purpuratusare closely associated with the differentiation of aboral ectoderm. To examinecis-regulatory elements involved in the spatial expression of the Spec genes, we fused theEscherichia coli lacZgene containing a nuclear targeting signal to 5′ flanking DNA plus 5′ untranslated leader sequences from Spec1, Spec2a, and Spec2c. All three genes contain 700 bp of highly conserved DNA in their upstream regions, but in Spec1 and Spec2c large insertions interrupt the conserved regions. The Spec-lacZreporter gene plasmids were microinjected into eggs ofS. purpuratus, Lytechinus variegatus, andL. pictus, and β-galactosidase activity was determinedin situby X-gal staining. The Spec2a-lacZfusion gene, which contained 1516 bp of 5′ flanking DNA and 18 bp of 5′ untranslated leader sequence, was preferentially expressed in aboral ectoderm cells in all three species. The Spec1-lacZfusion gene was expressed in a strikingly different fashion—preferentially in primary and secondary mesenchyme cells, occasionally in aboral ectoderm cells, and less often in oral ectoderm and endoderm cells. The staining pattern was the same in either homologous or heterologous embryos. The Spec2c-lacZfusion gene, like Spec2a-lacZ, was preferentially expressed in aboral ectoderm, but staining of other cell types was frequently observed. To further delineate sequences required for correct spatial expression, we deleted 800 bp of 5′ flanking DNA from the Spec2a-lacZfusion gene, resulting in a ΔSpec2a-lacZfusion gene that contained only the conserved DNA region. This gene fusion showed preferential expression in aboral ectoderm cells. However, the cell type specificity was not as great as with the parental Spec2a-lacZplasmid. These experiments implied that the conserved DNA region, associated with all Spec genes examined, was insufficient for complete aboral ectoderm specificity, and suggested that a spatial repressor element existed between −1516 and −697 bp in the 5′ flanking DNA of Spec2a.