Datta S, Gomer R H, Firtel R A
Mol Cell Biol. 1986 Mar;6(3):811-20. doi: 10.1128/mcb.6.3.811-820.1986.
We analyzed a developmentally regulated prestalk-specific gene from Dictyostelium discoideum encoding a cathepsin-like protease. A hybrid gene was constructed by fusing 2.5 kilobases of 5' flanking sequences and part of the coding region of the gene in-frame to the Escherichia coli beta-glucuronidase gene and was transformed into D. discoideum cells. In cells transformed with this vector, the gene fusion showed the same temporal regulation as the endogenous gene during multicellular development and, like endogenous prestalk genes, was highly inducible by cyclic AMP in in vitro cell cultures. Moreover, immunofluorescence studies showed that the fusion protein had the same spatial distribution within the migrating pseudoplasmodium as the endogenous gene. The results indicate that the regions of the D. discoideum prestalk-specific cathepsin gene contain all the necessary information for proper temporal, spatial, and cyclic AMP regulation of a prestalk cell-type gene in D. discoideum transformants and leads the way for experiments to identify the cell-type-specific regulatory elements.
我们分析了一种来自盘基网柄菌的发育调控的前柄特异性基因，该基因编码一种组织蛋白酶样蛋白酶。通过将该基因2.5千碱基的5'侧翼序列和部分编码区与大肠杆菌β-葡萄糖醛酸酶基因读框融合构建了一个杂交基因，并将其转化到盘基网柄菌细胞中。在用该载体转化的细胞中，基因融合在多细胞发育过程中表现出与内源基因相同的时间调控，并且与内源前柄基因一样，在体外细胞培养中可被环磷酸腺苷高度诱导。此外，免疫荧光研究表明，融合蛋白在迁移的伪原质团中的空间分布与内源基因相同。结果表明，盘基网柄菌前柄特异性组织蛋白酶基因的区域包含了盘基网柄菌转化体中前柄细胞类型基因进行适当的时间、空间和环磷酸腺苷调控所需的所有必要信息，并为鉴定细胞类型特异性调控元件的实验铺平了道路。
