Kruse F, Komro C T, Michnoff C H, MacDonald R J
Department of Biochemistry, University of Texas Health Science Center, Dallas 75235.
Mol Cell Biol. 1988 Feb;8(2):893-902. doi: 10.1128/mcb.8.2.893-902.1988.
Two separate domains within the 134-base-pair rat elastase I enhancer and a third domain at the enhancer-promoter boundary are required for selective expression in pancreatic acinar cells. The domains were detected by a series of 10-base-pair substitution mutations across the elastase I gene regulatory region from positions -200 to -61. The effect of each mutant on the pancreas-specific expression of a linked chloramphenicol acetyltransferase gene was assayed by transfection into pancreatic 266-6 acinar cells and control NIH/3T3 cells. The two enhancer domains are nonredundant, because mutations in either eliminated (greater than 100-fold reduction) expression in 266-6 cells. DNase I protection studies of the elastase I enhancer-promoter region with partially purified nuclear extracts from pancreatic tissue and 266-6 cells revealed nine discrete protected regions (footprints) on both DNA strands. One of three footprints that lie within the two functional domains of the enhancer contained a sequence, conserved among several pancreas-specific genes, which when mutated decreased linked chloramphenicol acetyltransferase expression up to 170-fold in 266-6 cells. This footprint may represent a binding site for one or more pancreas-specific regulatory proteins.
在134个碱基对的大鼠弹性蛋白酶I增强子内的两个独立结构域以及增强子 - 启动子边界处的第三个结构域是胰腺腺泡细胞中选择性表达所必需的。这些结构域是通过对弹性蛋白酶I基因调控区域（从 - 200到 - 61位）进行一系列10个碱基对的取代突变检测到的。通过转染到胰腺266 - 6腺泡细胞和对照NIH/3T3细胞中，检测每个突变体对连接的氯霉素乙酰转移酶基因胰腺特异性表达的影响。这两个增强子结构域是非冗余的，因为其中任何一个结构域发生突变都会消除（大于100倍降低）266 - 6细胞中的表达。用来自胰腺组织和266 - 6细胞的部分纯化核提取物对弹性蛋白酶I增强子 - 启动子区域进行DNase I保护研究，发现在两条DNA链上有九个离散的保护区域（足迹）。位于增强子两个功能结构域内的三个足迹之一包含一个在几个胰腺特异性基因中保守的序列，当该序列发生突变时，在266 - 6细胞中连接的氯霉素乙酰转移酶表达降低高达170倍。这个足迹可能代表一个或多个胰腺特异性调节蛋白的结合位点。
