Dedrick R L, Jones P P
Department of Biological Sciences, Stanford University, California 94305.
Mol Cell Biol. 1990 Feb;10(2):593-604. doi: 10.1128/mcb.10.2.593-604.1990.
We have examined the sequence elements and corresponding DNA-binding factors required for transient expression of the A alpha d promoter fused to the bacterial chloramphenicol acetyltransferase reporter gene in a variety of cultured cell lines. Deletion analysis demonstrated that only about 110 nucleotides of sequence 5' of the transcription start site are required for constitutive expression in the murine B-lymphoma cell line A20 or for gamma interferon-induced expression in the murine monocytic cell line WEHI-3. Linker-scanner mutation of this region indicated that at least three sequence elements are required for promoter activity. These elements correspond to the conserved sequence elements found in other human and mouse class II genes, the X box, the Y box, and the H box. Analysis of DNA-binding activity showed that the three most predominant factors present in extracts from WEHI-3, A20, or L cells (which do not express the class II genes) are actually a family of factors that bind to a fourth sequence element, overlapping the 3' end of the X-box sequence, that is homologous to the cyclic AMP-responsive enhancer element. A single common factor that binds to the Y box was detected in extracts from all cells tested, as has been seen with the Y-box elements of other class II genes. Another common factor was found that binds to the more conserved 5' region of the X-box element, although A20 extracts contained a second, distinct binding activity for this region. A common binding factor for the H-box element was detected in extracts from WEHI-3 and L cells. However, this activity was absent in A20 cell extracts. Instead, two different H-box-binding activities were detected, suggesting that different components are involved in class II gene expression in B cells and macrophages. Finally, gamma interferon treatment did not significantly alter the DNA-binding activity in WEHI-3 cells for any of the sequence elements shown to be required for induced chloramphenicol acetyltransferase expression.
我们已经研究了与细菌氯霉素乙酰转移酶报告基因融合的Aαd启动子在多种培养细胞系中瞬时表达所需的序列元件和相应的DNA结合因子。缺失分析表明，转录起始位点5'端仅约110个核苷酸的序列对于鼠B淋巴瘤细胞系A20中的组成型表达或鼠单核细胞系WEHI-3中γ干扰素诱导的表达是必需的。该区域的接头扫描突变表明，启动子活性至少需要三个序列元件。这些元件对应于在其他人类和小鼠II类基因中发现的保守序列元件，即X盒、Y盒和H盒。DNA结合活性分析表明，WEHI-3、A20或L细胞（不表达II类基因）提取物中存在的三种最主要的因子实际上是一个因子家族，它们与第四个序列元件结合，该元件与X盒序列的3'端重叠，与环磷酸腺苷反应增强元件同源。在所有测试细胞的提取物中都检测到了一种与Y盒结合的单一共同因子，其他II类基因的Y盒元件也是如此。还发现了另一种共同因子，它与X盒元件更保守的5'区域结合，尽管A20提取物中该区域存在第二种不同的结合活性。在WEHI-3和L细胞的提取物中检测到了H盒元件的共同结合因子。然而，这种活性在A20细胞提取物中不存在。相反，检测到了两种不同的H盒结合活性，这表明B细胞和巨噬细胞中II类基因表达涉及不同的成分。最后,γ干扰素处理并未显著改变WEHI-3细胞中任何所示诱导氯霉素乙酰转移酶表达所需序列元件的DNA结合活性。
