Abstract
In yeast and other fungi the absence of thymidine kinase makes the specific labelling of DNA difficult. Until now, specific labellings of DNA have resulted in very low incorporation. On the other hand, after non-specific labelling of nucleic acids, DNA assay by modifications of Schmidt and Thannhauser's technique have provided a precision of only ± 30 %. We present a modification of this technique, which we have extensively used and which permits an estimate of the activity incorporated into DNA with an error of ± 2.5 %. All critical steps are described, including labelling of the culture. This method allows DNA of high specific activity to be obtained, and at the same time avoids the cell selection and DNA fragilization due to autoirradiation. The kinetics of incorporation of label into RNA and DNA have been studied. RNA, which represents 96–98 % of the total nucleic acids in yeast is labelled more rapidly than DNA. DNA specific activity increases considerably during the first generation in the labelled medium, before reaching an equilibrium. This situation makes obtaining DNA pulse labelling of high specific activity difficult. A method of “pseudo-pulse” experiment has been devised by comparing two cultures with different specific activities of neosynthesized DNA.
Published Version
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