|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1997 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1996 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Deoxyribonucleosidetriphosphate (dNTP) pools in a human salivary gland adenocarcinoma cell line (HSG) which characterization was relatively well elucidated, was measured on high pressure liquid chromatography (HPLC) system. HPLC analysis were performed on a Waters 510 pump with a LC spectrophotometer (using A254 nm). Chromatography on Partisyl-10 SAX (4.6x250 mm) was done and eluted with 0.4M ammonium phosphate (pH 2.65) at a flow rate of 2 ml/min. HSG cells were seeded 100 mm plastic Petri dishes and grown in 6 ml of Dulbecco Is modified Eagle Is medium supplemented with 10% newborn calf serum in the presence of 5% CO2 in an incubator. We investigated about the method of dNTPs extraction from HSG cells, periodate-methylamine degradation of rNTPs and partial purification and concentration by solid phase Sep-Pak cartridge column. From these results, 12% TCA treatment for preparation of cell extracts and GARRETT Is method for periodate-oxidation treatment were more effective than the other methods we investigated. And partial purification by Sep-Pak cartridge was very effective if a sample dose not include TCA.We prepared cell extract of HSG cells which were cultured over 62 hours, by addition of TCA and then dNTP pools were measured. Thus, the amounts of dNTP per 1x106 cells were dCTP,5.3 (]SY.+-。[) 2.9 pmol ; dATP,13.7 (]SY.+-。[) 5.9 pmol ; dTTP,26.3 (]SY.+-。[) 11.5 pmol ; dGTP,6.2 (]SY.+-。[) 4.4 pmol on several stages of cell cycles. These results suspect that dNTP pools change followed by cell cycle, but the details were must be invetigated in detail. Degradation of rNTP must performed carefully to reduce by-products, because rNTP pools (CTP,ATP,UTP,GTP were 11,83,36,21 nmol/106 cells, respectively) were about 3000 times larger than dNTP pools. Additionally, concentration of dNTPs were very low and crude cell extracted sample injected to HPLC system, reproducibility of HPLC analysis were much more improved.