摘要
借助于5'和3'末端删切后重建的IL-2R a链基因调控区次级克隆,在体外合成有放射性同位素参入的反意义RNA探针与总RNA进行液相杂交,结果表明TPA或PHA分别活化的T细胞在IL-2R a链表达过程中都在不同程度上有选择地利用了调控区内分别为-58(5')和+1(3')位两个转录起始点中3'转录起始点。热休克使PHA活化细胞更明显地利用+1位点。PHA诱导Jurkat细胞表达IL-2RamRNA斑点杂交证实,Jurkat细胞在活化16小时表达IL-2Ra基本达到高峰,至24小时已明显下降。根据这一规律提取PHA诱导活化15小时的Jurkat细胞S100和NE,进行有关结合蛋白的研究,初步结果显示磷酸纤维素柱的KCI洗脱组分中存在着DNA结合蛋白,有关结合蛋白性质的研究正在进行中。
High affinity receptor for IL2 is a heterodimer consisting of an a and a β chain, α chain can be induced by mitogen, antigen or anti-clonal typic antibodies, while β chain is considered to be expressed constitutively on certain types of lymphocytes . Thus the regulation of IL2R a gene expression became the most important event in elucidating the mechanism of transit expression of high affinity IL2 receptor.Using IL2 receptor a chain gene subclones with 5' and 3' deletions in the regulatory region, we synthesized 32P labeled antisense riboprobes in vitro. The ribo-probes hybridized with total RNA of T lymphocytes activated by either PHA or TPA for 10 hours. Results showed that two transcription initiation sites at -58 (5') and + 1 (3') on the 5' upstream of ILgR a gene are involved in both cases The 3' site is slightly predominant over the 5' initiation site, particularly in the case of TPA activation. Heat shock may strengthen the preference of +1 site in PHA activated cells. Based on our findings that IL2R a gene transcripts reach a peak level at around 16 h after the activation of PHA, we isolated the S100 fraction and nuclear extract from properly activated Jurkat cells and further fractionated both fractions on phospho-cellulose column. Preliminary results showed the existance of specific DNA binding proteins for IL-2R α5' regulatory sequences in the fractions eluted with 0.3 and 1.0 moI/L KCI.Further investigations on the specificity of the binding sequence, properties of the binding protein are now underway.
关键词
结合蛋白
白细胞介素2
受体
α链基因
IL-2R a chain gene, Gene transcription, Gene expression, Initiation site, DNA binding protein
