Hayashi S, Goto K, Okada T S, Kondoh H
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Genes Dev. 1987 Oct;1(8):818-28. doi: 10.1101/gad.1.8.818.
We have previously shown that the tissue specificity determinant of the chicken delta 1-crystallin gene lies 3' of position -100 (Hayashi et al. 1985). Since the promoter of the gene (delta 1-crystallin promoter) did not show any tissue specificity, we examined various segments of the delta 1-crystallin gene for a tissue-specific enhancer activity by placing each segment downstream of a heterologous transcriptional unit coding for chloramphenicol acetyltransferase (CAT) and by transfecting chicken tissues in primary culture. We found that a segment spanning the third intron bears a strong lens-specific enhancer activity. This "delta 1-crystallin enhancer" activates transcription from the delta 1-crystallin promoter 20- to 40-fold in lens cells and to various degrees with other promoters. Deletion analysis of the enhancer region indicated that it covered nearly 1 kb but did not indicate clear-cut boundaries. For its enhancer effect the core region of 120 bp and associations with certain adjoining regions were required. Removal of the enhancer from the gene totally abolished delta 1-crystallin expression, and reinsertion of the enhancer in either upstream, internal, or downstream positions restored expression. We conclude that the delta 1-crystallin enhancer is an essential and major determinant for lens-specificity of delta 1-crystallin expression.
我们之前已经表明，鸡δ1-晶状体蛋白基因的组织特异性决定因素位于-100位点的3'端（Hayashi等人，1985年）。由于该基因的启动子（δ1-晶状体蛋白启动子）未显示出任何组织特异性，我们通过将δ1-晶状体蛋白基因的各个片段置于编码氯霉素乙酰转移酶（CAT）的异源转录单元下游，并转染原代培养的鸡组织，来检测这些片段是否具有组织特异性增强子活性。我们发现，跨越第三个内含子的一个片段具有很强的晶状体特异性增强子活性。这种“δ1-晶状体蛋白增强子”在晶状体细胞中可使δ1-晶状体蛋白启动子的转录激活20至40倍，对其他启动子也有不同程度的激活作用。对增强子区域的缺失分析表明，它覆盖了近1 kb，但没有明确的边界。其增强子效应需要120 bp的核心区域以及与某些相邻区域的协同作用。从基因中去除增强子会完全消除δ1-晶状体蛋白的表达，而将增强子重新插入上游、内部或下游位置均可恢复表达。我们得出结论，δ1-晶状体蛋白增强子是δ1-晶状体蛋白表达的晶状体特异性的重要和主要决定因素。
