Wrighton N, Grosveld F
Laboratory of Gene Structure and Expression, National Institute for Medical Research, Mill Hill, London, United Kingdom.
Mol Cell Biol. 1988 Jan;8(1):130-7. doi: 10.1128/mcb.8.1.130-137.1988.
We report the development of a novel in vivo transcription assay for trans-acting factors regulating the human gamma- and beta-globin genes. A cDNA coding for the human tissue-type plasminogen activator (t-PA) was inserted into the globin genes. Simian virus 40 small T-antigen splice and polyadenylation signals were included to produce a mature transcript coding for t-PA, whose activity can be detected in single cells by a fibrin-agarose plaque assay. Stable murine L-cell transfectants of the gamma.t-PA and beta.t-PA hybrid genes were fused to various cell lines to show that t-PA expression is increased specifically by erythroid MEL, HEL, and K562 cell fusion. The analogous H-2Kb.t-PA construct was not inducible under the same conditions. Interestingly, uninduced MEL cells increased beta.t-PA expression to the same extent as induced MEL cells. Chemiosmotic permeabilization of the beta-globin tester cell line and exposure to nuclear extracts were used to assay for trans-acting factors capable of stimulating beta.t-PA expression. Such factors were shown to be present in the nuclei of uninduced MEL cells.
我们报告了一种用于调控人类γ和β珠蛋白基因的反式作用因子的新型体内转录测定法的开发。将编码人组织型纤溶酶原激活剂（t-PA）的cDNA插入珠蛋白基因中。包含猿猴病毒40小T抗原剪接和聚腺苷酸化信号以产生编码t-PA的成熟转录本，其活性可通过纤维蛋白-琼脂糖噬菌斑测定法在单细胞中检测到。γ.t-PA和β.t-PA杂交基因的稳定鼠L细胞转染子与各种细胞系融合，以表明t-PA表达通过红系MEL、HEL和K562细胞融合特异性增加。类似的H-2Kb.t-PA构建体在相同条件下不可诱导。有趣的是，未诱导的MEL细胞将β.t-PA表达增加到与诱导的MEL细胞相同的程度。使用β珠蛋白测试细胞系的化学渗透通透化并暴露于核提取物来测定能够刺激β.t-PA表达的反式作用因子。已证明此类因子存在于未诱导的MEL细胞的细胞核中。
