Analysis of X-Ray-Induced Chromosomal Translocations in Human and Marmoset Spermatogonial Stem Cells (Nature, Vol 253)

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We therefore characterised, using SDS-gel electrophoresis. the phosphoproteins produced in vitro by native particles to the corresponding densitograms of stained gels (Fig. 2a and c). The protein pattern of native nuclear particles (Fig. Zrr) is similar to those previously reported'.*, whereas that of the protein species of about 40.000 daltons in agreement with depleted in this protein species'. The distribution of '*P radioactivity shows two discrete peaks corresponding to molecular weights of about 85,000 and 40,000 for both native particles and 'particles wash'. It has consistently been observed that the 85,000-dalton species is preferentially phosphorylated in the wash fraction while the reverse is true in 'native particles'. A closer association of the kinase with the 40,000-dalton species Fig. 2 Sodium dodecyl sulphate-acrylamide gel electrophoresis in the presence of y3'P-ATP as described in the legend to Fig. I . Denaturation of the samples, electrophoresis on 10% acrylamide gels containing 0.1 % sodium dodecyl sulphate, staining of the gels and densitometer tracing were carried out as previously described.I0 Gels were then cut into 1 mm slices and counted directly in a Packard-Tricarb scintillation spectrometer wing Cerenkov radiation. Molecular werghts were estimated Nature Voi. 253 Fe Frs r : 6 lated species of molecular weight 26.000 has oc-nallv particle preparations of an adventitiously bex up roe possibly a histone, from another nuclear cornS Dent. 73 point is currently being investigated. The main proteins released by washing the m c l e s 9 0.5 M NaCl have molecular weights of about M.-XX(Fit I and ref. 2). We have recently isolated, using an 3Far~c h r o c gels is close to the above valuelo. Prelimin= tx?erime=! indicate that this protein fraction contains b e actk: I Laboratoire de Biofogie Mal6rribire, FacrcltP des Sciences. 1 0.5 M KCI. A few discrete protein species sec :o Received August 6; revised December IO. 1974. spermatogonial stem cells II translocations in human and marmoset . ONEof the major classes of genetic damage produced by ionish radiations is heritable reciprocal translocation. In a serk that each of several mammalian species had its own uniqu ' sensitivity to translocation induction in peripheral leukocyte and that human and marmoset leukocytes, although approLL mately equal in sensitivity, were twice as sensitix-e ;ts m o w leukocytes. Brewen and Preston4 also reported that in Chinese hamster and the mouse, the frequency of the tram- , location type analysed in the peripheral leukocytes w a s three 19 ; four times as high as the frequency of heritable translocatiom produced in spermatogonial stem cells and recovered in prirnar! ' spermatocytes. As the mouse is the mammal currently used ai the model for estimating man's genetic risk from mutagenic exposure, Brewen and Preston's earlier observations on gem cells should be extended to include a primate and man if possible Here we present o n extension of rhe interspecific a qd wr at ii Human Subjects Project

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' la?1 iature Vol. 253 Februarv 6 1975 !,lateral castration was performed, and preparations were .,Jde by the method of Evans et aLs, as modified by Chandley nine men, six of whom .had received testicular X-ray ;,posures of 78,200, or 600rad. Details of the irradiation regime ,re given by Heller and Rowley6. The interval between exposure ,,,d sampling varied among the individuals. because the :;rr each species in order that somatic against germ cell The data indicate that, as in the case of the peripheral dnsitive to reciprocal translocation induction in spermatogonial ,;em cells as the mouse. The ratio of somatic cell translocations ,that is, dicentrics) to germ cell translocations is approximately ::I in man and marmoset at X-ray doses of 100 rad and lower. t-his variation from the mouse data might be explained by the 4:sLiter efficacy in observing reciprocal translocations in the ;mates because the increased chiasmata frequency in primates Fig. 1 Examples of diplotene-diakinesis figure in marmoset and IV human; A ring-of-VI human. :resence of multivalent configurations (Fig. 1 ) which were the : < j U l t of reciprocal translocations induced in spermatogonial The results of this meiotic analysis are presented in Table 1. previously published data' from mousestudies are also presented Table 1 for the sake of comparison. Yields of dicentric Table 1, Frequencies of reciprocal translocations in meiotic cells and diccntrics in peripheral lcukocvtes following X-ray exposure 10 man, marmoset, and mouse Dose 7.; locations % locations % locations The number of cells analysed at 78, 200, and 600 rad were 371, t A total of 600 cells were analysed from four testes at each dose, $ 600 cells were analysed at each dose. except for 200 rad where 200 from two testes were analyscd. will give a higher probability of a multivalent association being teristics of translocation induction in the germ cells of man and marmoset are similar to those of the mouse. For all three, a peak yield is reached with an apparent decrease at higher doses. This peak yield, however, occurs at approximately 100 rad in the primates whereas it occurs at 500-600 rad in the m o w . Using the translocation frequencies observed at doses of 100 rad and lower, the mean rate of reciprocal translocation induction in man and marmoset is 7.7 x lo+ translocations per cell per rad. Using the data of Ford et d . 1 8 on the rate of transmission of cytologically scored translocations to Fl offspring of irradiated male mice a& the lower limit of transmission (that is 1:8), and the expected frequency of transmission of I :4 as the upper limit, a calculation of transmissable reciprocal translocations per gamete per rad can be made. This value is 0 . 9 6 ~10+-1.93 x lo+ translocations per gamete per rad for acute X-ray exposures. Jacobs9 and HarnertonlO have summarised the data on the frequency of translocations in live births. The figures are: 0.06% reciprocal translocations, 0.08% Robertsonian translocation, and 0.03% unbalanced translocations. Jacobs et al." have estimated that the spontaneous rate of viable translocations per gamete per generation is about 4 x lo-', of which 2 . 8 ~lo-' are balanced and 1.2 x IO-' are unbalanced. This estimate is a minimum as not all translocations can be detected by somatic cell chromosome analysis. So, if it is assumed that the measured spontaneous rate is between 0.25 and 1.00 of the true spontaneous rate, the estimated doubling dose for the induction of reciprocal translocations by acute X rays is 2-16 rad. In estimating a doubling dose for protracted (that is, chronic) exposures, one precaution must be taken. The experiments to date, using low dose rate irradiation, have been done with large total doses where the dose rate effect is maximised. In making risk estimates the principle concern is for low dose levels where, according to theory and experimental evidence, the dose rate effect for low LET radiations is minimal because of the small contribution by the two-track component.,Thus a conservative estimate of a doubling dose for low-dose chronic X-ray exposure might be expected to be the same as for acute low doses with a maximum of 4-30 rad. This research was sponsored by the United States Atomic Energy Commission under contract with the Union Carbide for help in obtaining biopsy material from the testicularly irradiated male volunteers. Dr Gengozian was supported by

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Oak Ridge Associated Unirrrsities, Oak Ridge, Tennessee 37830 Estimation of accessibility of DNA in chromatin from fluorescence measurements of electronic excitation energy transfer EUKARYOTICDNA is complexed with proteins which influence its conformation, stability and function1**.Attempts to correlate the chemical and biological properties of chromatin have As much as 50% of the DNA in chromatin is accessible to precipitants (such as basic dyes or or to limiting DNA accessibility in chromatin resembling a frayed telephone wire. Digestion extensively alters chromatin structure, however, and the results ofexperimentsinvolvingprecipitation or digestion may be influenced by processes associated with the aggregation phenomena serving as experimental endpoints. We have used an alternative approach to characterise DNA accessibility in chromatin using the transfer of electronic excitation energy between pairs of fluorescent dyes bound to chromatin at low ratios of dye to phosphate. The results suggest that at least one-third of the DNA in chromatin retains a high affinity for basic dyes. The exclusion of dyes or other moleculess from portions of chromatin may, however, depend on the conformation of chromatin as well as on the protein-DNA stoichiometry. The dyes quinacrine, acridine orange and 33258 Hoechst were chosen as energy donors. All three bind tightly to DNA and chromatin, exhibiting a fluorescence emission spectrum that extensively overlaps the absorption spectrum of the energy acceptor used, ethidium bromide. Following excitationof the donor, energy will be transferred with a high probability to ethidium molecules bound within a critical distance (approximately 25-35 A) of the donor. Energy transfer efficiency decreases sharply with donor-acceptor separations above this value. The critical donor-acceptor distance for 50%eflicient energy transfer, Ro, determined by individual spectroscopic parameters and relative dye orientations'O*ll, differs little between DNA and chromatin complexes with any particular donor-acceptor pair, although it varies somewhat between the three donor-acceptor pairs. The relative efficiency of. energy transfer between fixed amounts of donor and acceptor bound to either DNA or chromatin depends o n the probability that an acceptor molecule will bind within R, of the donor and thus, to a first approximation, this energy transfer varies inversely with the acceptor binding space. Hence it provides an estimate of DNA accessibility in chromatin. Energy transfer in any particular region of chromatin (of length & R,) should be proporrional to the product of the binding affinities of the donor and acceptor to this segment and will thus reflect the correlation between donor and acceptor both bind to DNA by intercalation in a mutually CQ. If ethidium bromide is added to a quinacrine-DNA quinacrine fluorescence is quenched by energy tri nearby ethidium molecules, which concomitantl! sensitised fluorescence. At low donor and acceptor sal under conditions such that virtually all dye molecules a. the donor fluorescence intensity (F) varies inversel) amount of added acceptor (Fig. 1). The amount oi bromide necessary for a 50% reduction in donor flu (ethidium bromide),, increases in proportion to accessible DNA phosphate concentration (Fig. 2). Q of the fluorescence of quinacrine bound to chromatin about one-third of the amount of added ethidium needed for a comparable effect on quinacrine-DNA c (Fig. 2). Direct measurements of quinacrine binding; that calf thymus chromatin also possesses about onenumber of quinacrine binding sites found in an equal ii DNA, 'consistent with the exktance of an extensh between quinacrine and ethidium bromide binding Fig. 1 Energy-transfer quenching of quinacrine Rut by ethidium in complexes with calf thymus DNA. Thi complexed with 2 x IO-' M calf thymus DNA (in0.01 additions of a I x ,IO-' solution of ethidium bromide. E was at 420 nm. Quinacrine fluorescence (f),measured a was corrected for dilution by the ethidium bromide SOIL for a very small amount of quenching due to the t m i a of fluorescence and reabsorption. fois the initial fluore the absence of added ethidium. The ratio of F,,F 1% against the amount of added ethidium bromtdc Estimates of DNA accessibility in chromatin range with acridine orange as the donor to 609& with the t dazole dye 33258 Hoechst (Table 1). This range n variations both in the correlation of donor an( binding distributions and in the quantum yield molecules bound to different DNA sequences. Differ< mode of binding of donor molecules might also be Quinacrine and acridine orange intercalate bet\\ bases", while 33258 Hoechst, which is a large bisben dye, may occupy one of the grooves of the DNA doc Preliminary measurements indicate that approxima! the sites in DNA for 33258 Hoechst binding are avaii thymus chromatin. If some of these sites are inac