Javaux F, Donda A, Vassart G, Christophe D
IRIBHN, Université Libre de Bruxelles, Belgium.
Nucleic Acids Res. 1991 Mar 11;19(5):1121-7. doi: 10.1093/nar/19.5.1121.
A cDNA that encodes a transcription factor able to recognize the thyroglobulin gene promoter in vitro was isolated from a dog thyroid cDNA expression library in lambda gt11. The library was screened with a multimerized 20 bp-oligonucleotide probe corresponding to the -126 to -107 bp region of the bovine thyroglobulin gene promoter. The specificity of DNA sequence recognition was demonstrated by DNA binding experiments realized with beta-galactosidase-fusion protein immobilized on nitrocellulose filters and various unlabelled multimerized competing DNA fragments. The encoded protein, TFE, appears to be the canine counterpart of a recently cloned human transcription factor, ITF-2, that binds to the mu E5 kappa E2 motif found in both immunoglobulin heavy and light chains genes enhancers and belongs to the basic-Helix-Loop-Helix family of transcription factors. When TFE protein was produced in a rabbit reticulocyte lysate, it displayed the same specificity of DNA sequence recognition as the beta-galactosidase fusion protein and immobilization of the translation product on nitrocellulose still appeared to be essential for detecting in vitro DNA binding activity. Functional data failed to assign a role for TFE in the control of thyroglobulin gene transcription in vitro, suggesting that the selection of TFE clone resulted from the fortuitous presence of a high affinity binding site in the probe used for screening the expression library.
从λgt11载体的犬甲状腺cDNA表达文库中分离出一个能在体外识别甲状腺球蛋白基因启动子的转录因子的cDNA。用与牛甲状腺球蛋白基因启动子-126至-107 bp区域对应的多聚化20 bp寡核苷酸探针筛选该文库。通过用固定在硝酸纤维素滤膜上的β-半乳糖苷酶融合蛋白与各种未标记的多聚化竞争性DNA片段进行DNA结合实验，证明了DNA序列识别的特异性。编码的蛋白TFE似乎是最近克隆的人类转录因子ITF-2的犬类对应物，ITF-2可结合免疫球蛋白重链和轻链基因增强子中发现的μE5κE2基序，属于转录因子的碱性-螺旋-环-螺旋家族。当TFE蛋白在兔网织红细胞裂解物中产生时，它表现出与β-半乳糖苷酶融合蛋白相同的DNA序列识别特异性，并且将翻译产物固定在硝酸纤维素滤膜上对于检测体外DNA结合活性似乎仍然至关重要。功能数据未能确定TFE在体外甲状腺球蛋白基因转录调控中的作用，这表明TFE克隆的选择是由于用于筛选表达文库的探针中偶然存在高亲和力结合位点。
