摘要
为了研究人淋巴毒素(LT)基因5’侧翼顺式作用片段在基因表达调控中的功能,首先从人LT基因组DNA分离到转录起始点上游704bp(-573～+131)片段并将其与指示基因—氯霉素酰基转移酶基因(cat)嵌合而构建出pLT704CAT质粒,继而用Bal-31核酸外切酶对704bp片段进行缺失并通过DNA序列分析确定缺失区段。将pLT704 CAT及各缺失突变子转染L929,Jurkat,Raji及Cos-1等哺乳动物细胞,通过瞬时表达CAT试验分析了704bp片段及各突变顺序对异源性基因转录表达的影响及其在不同细胞中对各种有丝分裂原刺激的反应性。实验结果表明,人LT基因启动子大致位于转录起始位点上游-78～-12bp之间,在启动子上游存在负调节顺序。LT基因转录调控区对异源性基因表达的调节具有明显的组织特异性。在Raji细胞中呈组成性促转录活性,在Jurkat细胞中抑制顺序的作用可以被PMA,PHA及ConA等诱导解除。当缺失突变破坏了kB样顺序时,PMA等的诱导效应消失,提示NF-kB可能参与人LT基因的正调控。实验结果还表明,人LT基因的组织特异性表达与该基因在转录水平的调节密切相关。
To investigate the role of the cis-acting elements of human lymphoto-xin (LT) gene in the process of gene expression control, a 704bp fragment from the 5' flanking sequences of human LT genomic DNA is isolated and linked to the reporter chloramphenicol acetyl transferase (CAT) gene, and thus constructed the plasmid pLT 704CAT. the 704bp fragment was subsequently und-er progressive deletion mutation with Bal-31 nucleasc, and the exact region of deletion was determined by DNA sequencing. pLT704CAT and its scries deletion mutants were transiently transected into L929, Jur-kat, Raji and Cos-1 cell lines. The effects of 704bp fragment and its deletion mutants on the instruction of foreign gene expression and the reactivity to various mitogens were analysed with CAT assay. The results suggest that the transcription promoter of huLT gene is located at -78 -12bp upstream the transcription initiaion site. There are negative sequences upstream the promoter element. A strict tissue specificity was demonstrated in the instruction of heterogenic gene expression by 5' flanking sequences of huLT gene. CAT was constilutively expressed in Raji cells and can be induced by PMA- PHA and ConA in Jurkat cell lines. When a ~KB like motif at -101 -9lbp was destroyed by deletion, the induction effect of the mitogens was disappeared, which indicats NF-~KB may take part in the positive regulation of LT gene expression. The results also suggests that the tissue specificity of huLT gene expression is closely related with its gene control mechanism at the transcription level.
出处
《免疫学杂志》
CAS
CSCD
北大核心
1992年第1期10-14,共5页
Immunological Journal
关键词
表达调控
启动子
基因
淋巴毒素
Lymphotoxin gene, Expreession regulation, Cis-acting elements, Promoter
