A PITFALL OF LOW SPECIFIC ACTIVITY RADIOACTIVE THYMIDINE.

Abstract

The action of x radiation on DNA synthesis in HeLa 53 and Chinese hamster is such that a plot of the doseresponse yields a two-component curve. In order to determine if the steep component was due to pool dilution, the effect of thymidine specific activity was investigated. Tissue cultures were irradiated with x rays and incubated with the tracer isotopes. Nucleic acids were extracted and the contents estimated by absorbancy measurements at 267 m mu . Radioactivity was determined in a spectrometer. Results show that incorporation of tritiated thymidine into DNA cultures incubated with higher concentrations (lower specific activity) of thymidine was less suppressed by the irradiation than cultures incubated with high specific activity material. Further work using granine labeled with carbon-14 failed to confirm the results. Therefore, a double labeling experiment was performed in which cells were incubated with both C/sup 14/-labeled granine and tritiated thymidine. The C/sup 14/-granine results paralleled the tritiated thymidine results. However in cultures where 4 mg/ml thymidine was used in conjunction with C/sup 14/-guanine instead of tracer thymidine, C/sup 14/ specific activity was half. It is apparent that this effect of thymidine is such that it masks the effects of x radiation on themore » DNA synthesizing system by detracting from the part of rate depression contributed by the steep component of the x-ray dose-response curve. Guanine apparently does not affect DNA synthesis rate. (H.M.G.)« less