A simple method is described for detecting 3H in polyacrylamide gels by scintillation autography (fluorography) using X-ray film. The gel is dehydrated in dimethyl sulphoxide, soaked in a solution of 2,5-diphenyloxazole (PPO) in dimethylsulphoxide, dried and exposed to R P Royal "X-Omat" film at -70 "C. Optimal conditions for each step are described. ,&particles from 3H interact with the 2,5-diphenyloxazole emitting light which causes local blackening of an X-ray film. The image produced resembles that obtained by conventional autoradiography of isotopes with higher emission energies such as 14C. 3000 dis. 3H/min in a band in a gel can be detected in a 24-h exposure. Similarly 500 dis./min can be detected in one week.When applied to the detection of s5S and l4C in polyacrylamide gels, this method is ten times more sensitive than conventional autoradiography. 130 dis. 3% or 14C/min in a band in a gel can be detected in 24 h.Autoradiography has been widely used to dettec labelled compounds after electrophoresis in polyacrylamide gels. It is simple, rapid and does not involve destruction of the sample, but its use has been restricted to such isotopes as 14C, 35S, 32P, l25I and 1311. In spite of its lower emission energy, 3H remains the most satisfactory isotope for a wide range of experiments because a wide range of 3H-labelled precursors is available at high specific radioactivity. Hitherto efficient detection of 3H in gels has required scintillation counting of dissolved gel slices, a timeconsuming procedure which affords limited resolution and destroys the sample.Wilson [l] showed that the detection efficiency for 3H on paper chromatograms can be increased by impregnating the chromatogram with a scintillator before applying film. Thus the film is not exposed by /%particles themselves, but by light generated by interaction of the b-particles with 2,5-diphenyloxazole (PPO). Randerath [2] has studied the factors which contribute to the efficiency of this process (fluorography or scintillation autography). He has shown that treatment of thin-layer chromatograms with a solution of PPO in ether permits efficient detection of 3H-labelled nucleotides by medical X-ray film. Until

Methods which use the scintillator PPO to record film images of 'H in chromatograms and polyacrylamide gels (fluorography) have been described elsewhere. This paper demonstrates that pre-exposure of the film to a brief flash of light greatly increases the sensitivity of fluorography. Pre-exposure also permits quantitative interpretation of the film image, because it corrects the non-linear relationship between radioactivity of the sample and absorbance of the film image. Therefore the distribution of radioactivity in the sample is accurately represented by microdensitometry of the image obtained on pre-exposed film.Using pre-exposed film 300 dis. 3H/min or 30 dis. 14C/min can be detected in a band in a gel in a 24-h exposure.The Appendix describes revisions and extensions of existing fluorographic procedures, including application to agarose gels and a rapid procedure for recovering PPO for re-use.Methods have been described for fluorographic detection in situ of molecules labelled with tritium in chromatograms [l-51 and polyacrylamide gels [6]. The more recent of these methods [3-61 use the organic scintillator 2,5-diphenyloxazole (PPO) to convert the energy of the P-particle to visible light, which forms an image on a blue-sensitive X-ray film. Thus absorption of P-particles within the sample is overcome by the increased penetration of light. In the case of polyacrylamide gels the same procedure increases the efficiency of detection of I4C and 35S ten-fold above the levels obtained with autoradiogIn this paper we investigate the quantitative relationship between the absorbance of the fluorographic film image and the distribution of radioactivity in the sample. The relationship is complex and is not linear with the amount of radioactivity or with time. Seriously misleading and incorrect results are obtained if the absorbance profile of the image is assumed to represent the distribution of radioactivity in the sample. This discrepancy may be explained in terms of the theory of the photographic process, and a single-step method is described for correcting it. raPhY [61. MATERIALS AND METHODS MaterialsThe materials used were those described previously [6]. Preparation of Isotopically Labelled ProteinsIsotopically labelled proteins were prepared by incubating oocytes of Xenopus laevis in saline containing individual radioactive amino acids exactly as described previously [6]. 'H-labelled haemoglobin was obtained by translating rabbit haemoglobin mRNA in Xenopus oocytes incubated in saline containing 0.5 mCi/ml ~-[2,5-'H]histidine (55 Ci/mmol) [7]. Other 3H-labelled proteins were prepared by translating encephalomyocarditis (EMC) viral RNA in oocytes [8] incubated in 0.5 mCi/ml ~-[4,5-~H]leucine (58 Ci/mmol). Preparation of Polyacrylamide Gels Containing Labelled ProteinsTwo types of gel sample were used to test the film response. In the first, haemoglobin labelled in oocytes with [3H]histidine was electrophoresed as described [8] in 20% acrylamide slab gels containing 0.065% bisacrylamide and 0.1 % sodium dodecylsu...

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The nucleosome subunits of chromatin are assembled from histones and DNA by an acidic protein which binds histones. The nucleosome assembly protein has been identified and purified from eggs of Xenopus laevis.

In eukaryotes the entire genome is replicated precisely once in each cell cycle. No DNA is re-replicated until passage through mitosis into the next S-phase. We have used a cell-free DNA replication system from Xenopus eggs to determine which mitotic changes permit DNA to re-replicate. The system efficiently replicates sperm chromatin, but no DNA is re-replicated in a single incubation. This letter shows that nuclei replicated in vitro are unable to re-replicate in fresh replication extract until they have passed through mitosis. However, the only mitotic change which is required to permit re-replication is nuclear envelope permeabilization. This suggests a simple model for the control of DNA replication in the cell cycle, whereby an essential replication factor is unable to cross the nuclear envelope but can only gain access to DNA when the nuclear envelope breaks down at mitosis.