人淋巴毒素基因5′端上游NF－KB类因子特异性结合位点的研究

Study on the Specific Binding Site of NF-K B-like Factor at the 5'Upstream Region of Human Lymphotoxin Gene

淋巴毒素（Ｌｙｍｐｈｏｔｏｘｉｎ，ＬＴ）是由活化的Ｔ淋巴细胞分泌的一种糖蛋白。ＬＴ基因的表达调控主要是在转录水平上。ＰＨＡ＋ＰＭＡ诱导的Ｊｕｒｋａｔ细胞总ＲＮＡ点渍杂交发现，Ｊｕｒｋａｔ细胞的内源性ＬＴｍＲＮＡ的数量随诱导时间的延长而增加。凝胶迟滞电泳分析发现，ＰＨＡ＋ＰＭＡ诱导４小时的Ｊｕｒｋａｔ细胞核抽提物形成多种复合物，且在诱导不同时间后有明显变化。系列缺失片段的竞争反应表明，这些复合物的形成与ＬＴ基因５＇端上游──１２８ｂｐ──９３ｂｐ左右的序列相关。ＤＮａｓｅｌ足迹法及其竞争实验发现，ＬＴ基因５＇端上游─—１０４ｂｐ──８３ｂｐ（共２２ｂｐ）序列具有明显的蛋白质保护足迹。同源性分析发现此２２ｂｐ的序列中存在一个ＫＢ样结合位点：─１００ｂｐ──９０ｂｐ（５’－ＧＧＧＧＧＣＴＴＣＣＣ－３’）。ＮＰ－ＫＢ类蛋白质因子参与了此序列的ＤＮＡ－蛋白质的特异性结合，可能存在多种类型的ＮＰ－ＫＢ类蛋白因子参与了ＬＴ基因的表达调控。

Lymphotoxin (LT) is a glycoprotein secreted by activated T lymphocytes. The expression of LT gene is primarily controlled at the level of transcription. By using human LT DNA as a probe. we have carried out a RNA dot blotting test and found that the longer the Jurkat cells exposed to the PMA and PHA, the more was the endogeneous LT mRNA. Results of gel retardation assay showed that nuclear extract from Jurkat cells treated with PMA and PHA formed different complexes. Changes in the complex patterns were observed in different time of PMA and PHA induction.A specific protein-binding site was mapped to a 22bp sequence at the 5' upstream region of human LT gene by DNase I footprinting analysis. This region is similar to the sequence recognized by the proteins of NF-KB family. The results of fragment competition test and homology analysis indicated that the 22-bp sequence contains a KB-like motif only, which located at base pairs-100 to-90 (5'-GGGGGGCTTCCC-3'). Thus the NF-KB-like factors were involved in the protein-DNA interaction. Futhermore, there were more than one retarded bands appearing in the gel retardation assay. It suggested that there may be various kinds of NF-KB-like factors involved in the regulation of LT gene transcription at the same site.Supported by State Education Commission Research Foundation for Doctoral Disciplines and National Natural Science Foundation of China