By J. Kiefer (auth.), Professor Dr. Jürgen Kiefer (eds.)
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Extra resources for Radiation and Cellular Control Processes
A method for the determination of dATP and dTTP in picomole amounts. Analyt. Biochem. : Characteristics of deoxyribonucleic acid polymerase activity in nuclear and supernatant fractions of cultured mouse cells. Biochem. J. : Stimulation of DNA polymerase by factors isolated from Novikoff Hepatoma. Proc. Nat. Acad. Sci. : Thymidine kinase induction correlated with synchronous mitosis in Physarum polycephalum. Abstr. 4th FEBS Meeting, Oslo, p. : A permeable cell system for studying DNA replication in synchronized HeLa cells.
1974; Eckstein and Ahnefeld, in preparation). Estimation of DNA polymerase activity is based on the assay described by Lindberg and Skoog (1970) for deoxynucleotide estimation. This assay has proved useful also for analysis of DNA precursor contents in extracts from synchronized growing yeast cells. As far as dATP and dTTP are concerned, the values correlate with the amount of "concentrate" per assay, and with cell growth. Difficulties arise, however, in the estimation of dCTP. As shown in Figure 1, the incorporation of radioactivity from labelled dGTP decreases sharply with increasing amounts of yeast concentrate in the assay.
These conditions require (1) that harvest of cells and spheroplast preparation are carried out at room temperature and (2) that nuclei are isolated from spheroplasts as rapidly as possible, avoiding lengthy centrifugation, which are necessary to obtain optimal yields of pure nuclei. In other words, it was necessary to find a compromise between reasonable purity and active DNA synthesizing capacity of the isolated nuclei. This is not surprising in view of the fact that many components of the replication machinery rapidly leak out of the nuclei during an extensive purification.
Radiation and Cellular Control Processes by J. Kiefer (auth.), Professor Dr. Jürgen Kiefer (eds.)