Identification of protein-binding sites in the hepatitis B virus enhancer and core promoter domains.

We have investigated the role of liver-specific trans-acting factor(s) in the regulation of hepatitis B virus (HBV) gene expression. A recorder plasmid (pEcoAluCAT; HBV nucleotides 1 through 1878) was constructed containing the HBV enhancer and the promoter region of the pregenomic RNA, which was ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene. Upon transfecting this plasmid into various cell lines, the CAT gene was expressed only in cells of liver origin. Moreover, competition cotransfections with pEcoAluCAT and plasmids containing HBV enhancer sequences in human hepatoblastoma-derived HepG2 cells indicated the presence of titratable trans-acting factor(s) in these cells. Gel mobility shift assays using HBV enhancer and core promoter domains confirmed the existence of sequence-specific DNA-binding proteins in liver cell nuclear extract which bound to these regions. These binding sites encompass 17- and 12-nucleotide palindromes in the HBV enhancer and core promoter domains, respectively, when mapped by the methylation interference assay.     

A novel promoter sequence is involved in the oxidative stress-induced expression of the adult T-cell leukemia-derived factor (ADF)/human thioredoxin (Trx) gene.

Adult T cell leukemia-derived factor (ADF) is a human thioredoxin (Trx) and is a disulfide reducing protein with various biological functions. We found that expression of the ADF/Trx gene was increased by oxidative agents such as hydrogen peroxide, diamide and menadione in Jurkat cells. Analysis using a CAT expression vector plasmid under the control of the ADF/Trx gene promoter revealed that CAT gene expression in Jurkat cells was increased after exposure to oxidative agents. A series of deletion analyses showed that a region from -976 to -890 of the 5' flanking sequence was required for enhancement of ADF/Trx promoter activity against the oxidative agents. Gel mobility shift assay revealed the specific DNA binding activities to the sequences from -953 to -930 in the nuclear extracts from the Jurkat cells. The sequences in this region showed no homology with any known consensus sequences for DNA binding factors. It is suggested that ADF/Trx gene expression is enhanced through a novel cis-acting regulatory element responsive for the oxidative stress and a new factor(s) is involved in this oxidative stress responsive element.     

人apoA-I分泌型表达调控细胞模型构建

分别从pMD18-T质粒和人基因组DNA扩增出人apoA-I CDS区序列和apoA-I启动子(702bp片段),与pEGFP-N1重组,构成受apoA-I启动子调控的pEGFP-N1质粒和融合蛋白表达质粒,分别转染人肝癌HepG2细胞,以绿色荧光为标志筛选稳定转染系列克隆。用RT-PCR、荧光显微镜、免疫荧光术等鉴定其中一个克隆融合蛋白的表达;分别以胰岛素和葡萄糖刺激物鉴定该克隆的外源apoA-I启动子调控。结果表明:人apoA-I分泌型表达调控肝细胞模型初步建成。