A SPATIAL GRADIENT OF EXPRESSION OF A CAMP-REGULATED PRESPORE CELL-TYPE-SPECIFIC GENE IN DICTYOSTELIUM
A SPATIAL GRADIENT OF EXPRESSION OF A CAMP-REGULATED PRESPORE CELL-TYPE-SPECIFIC GENE IN DICTYOSTELIUM
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DOI：
10.1101/gad.4.4.596
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发表时间：
1990-04-01
影响因子：
10.5
通讯作者：
FIRTEL, RA
中科院分区：
文献类型：
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作者：
HABERSTROH, L;FIRTEL, RA
以前，我们确定了一类基因在Dictyosteoblasts是前孢子细胞类型的具体表达在多细胞聚集体，是indicible的cAMP通过细胞表面cAMP受体的作用。在本文中，我们报告的控制这样一个基因，编码孢子外壳蛋白，SP 60的表达的调控区域的克隆和分析。通过使用萤火虫荧光素酶基因和大肠杆菌lacZ的融合体[表达β-半乳糖苷酶（β- gal）]，我们已经鉴定了在多细胞聚集体中适当的空间和时间表达以及在振荡细胞培养物中cAMP诱导所需的顺式作用区域。缺失分析表明，一个富含CA的元件（CAE）和周围的序列存在三次内的5“侧翼序列需要适当的调节。包括所有这三个区域的SP 60-lacZ融合体仅在后部表达lacZ。85%的迁移蛞蝓（孢子前区）。研究表明，SP 60在中晚期聚集期间表达，并且SP 60-lacZ阳性细胞在空间上定位为形成聚集体内的甜甜圈形环。在围绕聚集体的裙部内并且仍然迁移到聚集体中的细胞不染色。连续的5“缺失的CAE和周围区域影响响应于发育信号和cAMP的SP 60-荧光素酶的表达水平，以及SP 60-lacZ的空间模式。这些区域中的第一个（最5“）的缺失将SP 60-lacZ融合体的空间表达限制在前孢子区的前部。当第一和第二区域都被去除时，表达水平下降，并且染色被限制在前孢子/前柄边界。此外，这两个缺失所见的染色模式在前孢子区中呈现为从前到后的梯度。所有三个区域的缺失导致cAMP和发育诱导的表达两者的损失。这些结果表明，存在一个梯度内的前孢子区，差异影响的活动，启动子含有不同数量的响应元件。
Previously, we identified a class of genes in Dictyostelium that are prespore cell-type specific in their expression in the multicellular aggregate and are indicible by cAMP acting through cell-surface cAMP receptors. In this paper, we report the cloning and analysis of the regulatory regions controlling the expression of one such gene that encodes a spore coat protein, SP60. By use of a fusion of the firefly luciferase gene and Escherichia coli lacZ [expresses .beta.-galactosidase (.beta.-gal)], we have identified cis-acting regions required for proper spatial and temporal expression in multicellular aggregates and for cAMP induction in shaking cell culture. Deletion analysis suggests that a CA-rich element (CAE) and surrounding sequences present three times within the 5''flanking sequence are required for proper regulation. SP60-lacZ fusions that include all three of these regions express lacZ only in the posterior .apprx. 85% of migrating slugs (prespore zone). Studies show that SP60 is expressed during mid to late aggregation, and SP60-lacZ-positive cells are spatially localized as a doughnut-shaped ring within the forming aggregate. Cells within the skirt that surrounds the aggregate and that are still migrating into the aggregate do not stain. Sequential 5'' deletions of CAEs and surrounding regions affect the expression level of SP60-luciferase in response to developmental signals and cAMP, as well as the spatial pattern of SP60-lacZ. Deletion of the first (most 5'') of these regions restricts the spatial expression of SP60-lacZ fusions to the anterior of the prespore zone. When both the first and second regions are removed, the expression level drops, and the staining is restricted to the prespore/prestalk boundary. Furthermore, the staining pattern that is seen with these two deletions is present as a gradient from anterior to posterior within the prespore zone. Deletion of all three regions results in a loss of both cAMP and developmentally induced expression. These results suggest the presence of a gradient within the prespore zone that differentially affects the activity of promoters containing different numbers of response elements.