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

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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