CHARACTERIZATION OF PROMOTER ELEMENTS OF AN INTERFERON-INDUCIBLE LY-6E/A DIFFERENTIATION ANTIGEN, WHICH IS EXPRESSED ON ACTIVATED T-CELLS AND HEMATOPOIETIC STEM-CELLS
CHARACTERIZATION OF PROMOTER ELEMENTS OF AN INTERFERON-INDUCIBLE LY-6E/A DIFFERENTIATION ANTIGEN, WHICH IS EXPRESSED ON ACTIVATED T-CELLS AND HEMATOPOIETIC STEM-CELLS
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DOI：
10.1128/mcb.10.10.5150
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发表时间：
1990-10-01
影响因子：
5.3
通讯作者：
BOTHWELL, ALM
中科院分区：
文献类型：
--
作者：
KHAN, KD;LINDWALL, G;BOTHWELL, ALM
Ly-6E/A 抗原在活化的鼠 T 细胞上表达。使用由先前表征的 cDNA 制成的探针，我们分离了编码 Ly-6A 抗原的基因组 DNA 克隆。我们确定了基因组克隆的 DNA 序列，并对启动子区域进行了功能分析。转染该基因组克隆的小鼠成纤维细胞 BALB/3T3 细胞在其细胞表面组成型表达 Ly-6A 抗原。该表达可由α/β和γ干扰素诱导。通过成纤维细胞中的氯霉素乙酰转移酶测定来分析 Ly-6E 5''侧翼区域的顺式作用元件。发现 L 细胞中至少需要两个阳性元件才能发挥最大的组成型启动子活性。电泳迁移率变动分析和足迹分析表明，其中一个阳性元件与粗核提取物中的 CCAAT 盒结合蛋白特异性结合。另一个包含 GGAAA 基序并与各种已知增强子具有同源性的元件也显示出特异性结合活性。当多聚化时，第二个积极元素成为非常强大的增强元素。干扰素处理可以增强稳定转染的 BALB/3T3 细胞中与 Ly-6E 5''侧翼区域融合的氯霉素乙酰转移酶基因的表达。负责这种增强的元件至少部分位于该基因的-1760 和-900 位之间。令人惊讶的是，Ly-6E 的该区域与已建立的干扰素刺激反应元件共有序列之间没有序列同源性，该元件已被证明对所有先前表征的 α/β 干扰素诱导型启动子具有重要的功能。 Ly-6E 基因可能被证明是用于研究干扰素诱导的新系统。
The Ly-6E/A antigen is expressed on activated murine T cells. Using probes made from the previously characterized cDNA, we have isolated a genomic DNA clone encoding the Ly-6A antigen. We determined the DNA sequence of the genomic clone and conducted a functional analysis of the promoter region. Mouse fibroblast BALB/3T3 cells transfected with this genomic clone constitutively expressed Ly-6A antigen on their cell surface. This expression was inducible by alpha/beta and gamma interferons. The Ly-6E 5''-flanking region was analyzed by chloramphenicol acetyltransferase assays in fibroblast cells for cis-acting elements. At least two positive elements were found to be needed for maximum constitutive promoter activity in L cells. One of the positive elements was specifically bound by a CCAAT box-binding protein from crude nuclear extract, as shown by electrophoretic mobility shift assays and footprinting. The other element, which contains a GGAAA motif and has homology to various known enhancers, also showed a specific binding activity. This second positive element when multimerized became a very powerful enhancing element. Interferon treatment could enhance expression of the chloramphenicol acetyltransferase gene fused to the Ly-6E 5''-flanking region in stably transfected BALB/3T3 cells. The elements responsible for this enhancement lie, at least in part, between positions -1760 and -900 of the gene. Surprisingly, there is no sequence homology between this region of Ly-6E and the established consensus for the interferon-stimulated response element, which has been shown functionally important to all previously characterized alpha/beta interferon-inducible promoters. The Ly-6E gene may prove to be a novel system for the study of interferon induction.