Physarum polycephalum histones have been analysed by acrylamide gel electrophoresis. Two of the five major bands had electrophoretic mobilities identical with the mobilities of two bands from calf thymus histone. The P. polycephalum pattern is qualitatively the same at all stages of the synchronous mitotic cycle. Quantitative changes in the relative proportion and relative mobility of the very-lysine-rich histone are reported. I n particular, a dramatic increase in phosphate content of this histone occurred in late G2 phase with a peak where chromosome condensation is seen to be occurring in the phase contrast microscope. Phosphate content is low during S phase and the peaks of RNA synthesis.Thc presence of phosphorous, as 0-phospho-Lserine, has been clearly demonstrated in rat histone fraction FI and Langan [l] has isolated a phosphokinase and phosphatase specific for these histones. Phosphorylation can be stimulated by adenosine cyclic 3' : 5'-monophosphate and hormones. It has been suggested that histones and their chemical modifications have roles in the control of RNA synthesis, in DNA synthesis and in chromatin structure (for a review, see 121). The results presented here lead to the conclusions that phosphorylation of very-lysine-rich histonc is not directly linked with quantitative control of RNA synthesis during the mitotic cycle, nor with DNA synthesis, as has been previously suggested, but that it may be involved in the structural changes occurring during chromosome condensation.Physarum polycephalum has been used as a model of eukaryote chromosome structure for these studies because its naturally synchronous mitotic cycle gives very well synchronised mitoses without the need for interrupting growth or synchronising in any other way.Three criteria for a model of eukaryote chromosome structure have been proposed by Crick (unpublished communication) : the presence of a large amount of DNA, of histones in equal proportions and of heterogeneous nuclear DNA. P. polycephalum contains an appropriate amount of DNA, 1 pg per diploid nucleus [4] with a complexity typical of higher organisms [5,6]. Histones are present in the usual proportions [6-81. The presence of heterogeneous nuclear RNA has not been unequivocally demon-I)* strated due to practical difficulties although a highmolecular-weight ribosomal RNA precursor is present [9,10]. An additional important factor is the ability to carry out genetic analysis [3] and this is being undertaken with P. polycephalum [ll, 121 (and Haugli, unpublished results).I n the mitotic cycle of P. polycephalum DNA synthesis ( S phase) immediately follows mitosis and takes 3 to 4 h. The subsequent G2 phase takes about 5 h. RNA synthesis occurs during the S phase and in most of G2 phase with minimum values a t mitosis and the middle of interphase [4] (Fig. 8). Histone synthesis occurs during S phase [13-151. MATERIALS AND METHODS Preparation of HistonesPhysarum polycephalum was maintained in submerged shaken cultures [ 161. Synchronous surface plasmodia were grown on filter p...

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Summary As part of an 'enzyme-directed' approach to bioreductive drug development, we have measured the activity of NADH:cytochrome b5 reductase (B5R) in human cancer cell lines in order to assess the role of this enzyme in activating bioreductive drugs, and thus in influencing the cytotoxicity of these compounds. At present, there is no validated assay reported in the literature for measuring the activity of B5R in tumour cells, and current measurements have assumed that the enzyme activity can be measured either as the NADHdependent reduction of cytochrome c or as the non-dicoumarol-inhibitable activity in the DT-diaphorase assay. Using p-hydroxymercuribenzoate (pHMB) as an inhibitor of B5R, we have quantified the contribution of B5R to the NADH-dependent reduction of cytochrome c and to the overall reduction of cytochrome c in the DTdiaphorase assay. In the former we found that residual uninhibited activity remained in the presence of pHMB, in some cases accounting for up to 60% of the total reduction of cytochrome c. Thus, simply measuring the NADH-dependent reduction of cytochrome c consistently overestimated B5R activity. We also found that the non-dicoumarol-inhibitable activity in the DT-diaphorase assay underestimated B5R activity, especially in cell lines with high DT-diaphorase activity. Therefore, we have developed a spectrophotometric assay for measuring B5R activity as the pHMB-inhibitable NADH-dependent reduction of cytochrome c. This has been used to measure the B5R activity of a panel of 22 human tumour cell lines, in which we found 7-fold and 3-fold variations in activity expressed per cell or per mg protein respectively.