REGULATION MECHANISM BY HORMONES OF THE TRANSCRIPTION OF RAT SERINE:PYRUVATE AMINOTRANSFERASE GENE

1. SPT基因上游区激素反应元件的分析（1）SPT基因上游区的限制性内切酶酶切图谱和序列分析--确定了包含10 Kb大鼠SPT基因的54 Kb区的限制性内切酶酶切位点。测定了-1.3Kb ~+1的核苷酸序列，并寻找了各种顺式元件的共有序列。(2)用重组质粒转染培养细胞的条件--除CAT（氯霉素乙酰转移酶）基因外，我还使用荧光素酶基因作为报告基因。通过共转染的pCH 110中编码的β-半乳糖苷酶的活性校正转染效率。通过磷酸钙法进行转染，并使细胞与磷酸钙-DNA复合物保持接触18小时。转染后48小时收获细胞并制备细胞提取物。我用 ...更多信息 人肝癌细胞系HepG 2作为受体细胞。(3)重组质粒的构建- I构建了SPT基因上游区（-5.5Kb至+1）与报告基因连接的重组克隆。现在我正在准备具有各种删除的构造。2.大鼠SPT基因有两个转录起始位点。由于下游转录起始位点周围没有TATA盒，因此我将研究重点放在下游启动子的分析上。将-493 ~+106的DNA片段与报告基因连接构建的重组质粒作为未诱导的阳性对照。通过删除+21至+37部分，报告基因的活性降低至20%，通过删除+37至+64部分，报告基因的活性进一步降低至4%。这表明在正常大鼠肝脏中SPT基因的转录主要是下游位点的转录，在+21和+37之间以及+37和+64之间存在两个功能性顺式元件。少

1. Analysis of the hormone responsive elements located in the upstream region of SPT (Serine:pyruvate aminotransferase) gene (1) Restriction enzyme mapping and sequencing analyses of the upstream region of SPT gene - The restriction enzyme sites, covering the 54 Kb region which contained 10 Kb of rat SPT gene, was determined. The nucleotide sequence from -1.3 Kb to +1 was also determined and the consensus sequences of various cis elements were searched. (2) Conditions to transfect the cultured cells with recombinant plasmids - I use luciferase gene as a reporter gene in addition to CAT (chloramphenicol acetyltransferase) gene. The efficiency of the transfection is corrected by the activity of beta-galactosidase encoded in the co-transfected pCH110. The transfection is performed by the calcium phosphate method and the cell is kept in contact with the calcium phosphate-DNA complex for 18 h. The cells are harvested 48 h after the transfection and the cell extracts are prepared. I use the … More human hepatoma cell line, HepG2, as the recipient cells. (3) Construction of the recombinant plasmid - I constructed the recombinant clone where the upstream region of SPT gene (-5.5 Kb to +1) was connected to the reporter gene. Now I am preparing the constructs having the various deletions.2. Analysis of the mechanism that determines the transcriptional initiationThere are two initiation sites of transcription in rat SPT gene. Because there is no TATA box around the initiation site of downstream transcription, I focused my study on the analysis of the downstream promoter. I used the recombinant plasmid constructed by connecting the DNA fragment of -493 to +106 with reporter gene as an uninduced positive control. The activity of the reporter gene decreased to 20% by deleting the +21 to +37 portion and further decreased to 4% by deleting the +37 to +64 portion. This indicates that the transcription from the downstream site mainly contributes in the transcription of SPT gene in normal rat liver and that two functional cis elements exist between +21 and +37, and +37 and +64. Less