In Vitro Deoxyribonucleic-acid-Replicating System Solubilized from Isolated Nuclei of Saccharomyces cerevisiae

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概要

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• An in vitro DNA replication system having a high specific activity could be prepared as a clear solution from isolated nuclei of Saccharomyces cerevisiae as described below. The isolated nuclei were treated with 0.4% Nonidet P-40 and precipitated by centrifugation in 20% sucrose. From the precipitate thus obtained, DNA-replicating activity was extracted with 0.5 m KC1. The extract has the ability to incorporate 3H-dTMP into the acid-insoluble fraction (DNA)in the presence of a template DNA, such as pJDB 219 or YRp 7. After 10 min incorporation reaction, in the template DNA, formation of a "replication eye" could be demonstrated under an electron microscope. The sum of the lengths of the eyes was found to be equivalent to the amount of the synthesized DNA calculated from incorporated 3H-dTMP.The specific activity (4.9 nmol dTMP incorporation/mg protein of solubilized fraction/60 min, at 28°C) of the DNA-replicating reaction measured with the present system was approximately 60-fold higher than the highest one previously reported.1, 2)

• 社団法人 日本農芸化学会の論文

著者

• Fukui Sakuzo

  Department Of Bioengineering Faculty Of Engineering Fukuyama University

• Tsuchiya Eiko

  Department Of Fermentation Technology Faculty Of Engineering Hiroshima University

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