HIGHER PLANT NUCLEOSOMES. A MICROMETHOD FOR ISOLATING AND DISPERSING CHROMATIN FIBRES

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1 New Phytol., (1978) 81, HIGHER PLANT NUCLEOSOMES. A MICROMETHOD FOR ISOLATING AND DISPERSING CHROMATIN FIBRES By M. L. MORENO, J. M. SOGO and C. DE LA TORRE Instituto debiologia Celular, C.S.LC. Velazquez, 144. Madrid-6. Spain {Received 31 January 1978) SUMMARY A one-step method to isolate and disperse chromatin fibres from unfixed plant tissues is described. Nucleosomes as units of chromatin organization in Allium cepa L. root meristems are demonstrated by this simple technique. INTRODUCTION The isolation of chromatin fibre had been used to study the pattern of DNA replication in plant meristems (Van't Hof, 1975, 1976). It involves isolation and lysis of nuclei in single root tips using modification of the methods of McLeish (1963) and Lark, Consigli and Toliver(1971). We have tried to test the dispersion ofthe fibres obtained by these methods. At the same time we have developed a simple direct method to effect, simultaneously, the isolation and dispersion of chromatin fibres, dispersion which is being evaluated by the conventional techniques to visualize unfixed living macromolecules. METHODS AND RESULTS Allium cepa L. roots growing at 25 C in the darkness in tap water and under continuous bubbling air were used. The tap water was renewed every 24 h. The roots were studied when they reached 3-5 cm of length and only the terminal 2 mm of the root, i.e. the region enclosing a nearly pure meristematic zone, were employed. Originally root meristems were either fixed in formaldehyde (0.01-1% w/v) at 0 C for 5 min, or unfixed and the squashed in M phosphate (TP) buffer ph 6.8. The drop of liquid which formed after squeezing the terminal zone between two slides at 0 C contained a suspension of nuclei. The nuclear membrane was broken by sonication, 30 s at 20 kilocycle/sg, at 0 C. A 1:5 dispersion of nuclear content in a 10 mm borate buffer, ph 9.2, was then carried out for 30 min. Centrifugation ofthe dispersed material was done on an electron microscope grid placed in a special chamber at 3200 rpm at 10 C for 10 min, following Miller and Bakken (1972). It was found that the method of getting nuclei by squeezing roots between two cold slides only worked when the salt concentration ofthe TP buffer was over 50 mm. However, nuclei were obtained independently of their previous fixation. The ultrastructural image of X/78/ $ Blackwell Scientific Publications 681

2 682 M. L. MORENO et al. such meristem material, after its 1:5 dispersion in 10 mm borate buffer ph 9.2 was not satisfactory, in the sense that there were hardly any areas on the grid where fibres whose size corresponds to the individual chromatin fibre (10-30 nm diameter, according to the preparative method) were found. Apparently dispersion of chromatin fibres was improved by lowering fixation and avoiding it as well as by lowering the molarity ofthe extracting buffer. Accordingly, we developed a simple method which is displayed in Fig. 1. The root is transversely sectioned with a blade in the middle of the meristem zone, about 1.5 mm from the tip. To demonstrate the extraction of nuclei, the freshly cut surfaces were directly exposed to 50 /nl of 50 mm TP buffer ph 6.8. We could see how, after 1-2 min, a number of nuclei was present in suspension. To SECTION WMm. 1,5 mm CUT ROOT 50;j lomm Borate buffer ph9 2 30min, 10'C DISPERSION Fig. 1. Scheme used for simultaneous extraction and dispersion of chromatin fibres in nonfixed root meristem. get nuclear dispersion simultaneously with nuclear extraction we exposed the freshly cut meristem section to 50 /il of the dispersion buffer (10 mm borate buffer ph 9.2), for 1-2 min. Afterwards, without any other step (sonication for instance) we centrifuged the dispersed material according to Miller and Bakken (1972). We could then observe a good degree of nuclear dispersion. Many fibres in the size of individual chromatin ones were found. Nucleosomes were easily recognized (Plate 1). We studied their size and the modal value was 16 ± 3.2 nm which corresponds to the nucleosome size when processed by similar techniques in other materials (Franke et al., 1976). DISCUSSION One advantage of the isolation method is that it is quite useful for plant cells whose size invalidates the use of micromanipulation. The cellular remnants accompanying the chromatin fibres were not very abundant. The advantage of the method developed is that it allows chromatin extraction at the molar concentration that we wanted, and makes it comparable to other methods involving micromanipulation of non-fixed cells (Franke et al, 1976).

3 T IE NEW PHYTOLOGIST, 81, 3 PLATE 1 Chromatin from root meristem dispersed according to the described method. The chromatin appears packed in nucleosomes with the known appearance of beads on a string. After centrifugation, the specimen was stained with 0.5 mm uranyl-acetate and then shadowed with platinum. The micrographs were taken at x primary magnification on a Jeol 100 B. -. MORENO ET AL. ISOLATION AND DISPERSION OF NUCLEOSOMES (facing p. 682)

4 Isolation and dispersion of nucleosomes 683 The universality of nucleosomes as conformation of chromatin presumed to be transcriptionally inactive though they may be capable of replicating (McKnight and Miller, 1977), appears to be clear now that it has been shown both in mammalian (Olins and Olins, 1974; Oudet, Gross-Bellard and Chambon, 1975), and unicellular organisms (Woodcock, Safer and Stanchfield, 1976) as well as in proliferating plant meristems. They are also present in mitotic chromosomes (Compton et al., 1976). Many authors have shown that the dispersion conditions to visualize nucleosomes are the same as those to visualize both nucleolar transcriptional complexes (the familiar Christmas trees) and the non-nucleolar transcriptional complexes (Franke et al., 1976). We have failed to find such complexes in the root meristem so far. However, their rarity in mammalian proliferating tissues also should be recalled (Puvion-Dutilleul et al., 1977). We intend to expand our method to quantify the frequencies of the different chromatin fibre configurations in normal proliferating plant tissues. ACKNOWLEDGMENTS We appreciate the receipt of grants from the Comision Asesora para la Investigaci6n Cienti'fica y T^cnica, Spain. REFERENCES COMPTON, J. L., HANCOCK, R., OUDET, P. & CHAMBON, P. (1976). Eur. J. Biochem., 70, 55. FRANKE, W. W., SCHEER, U., TRENDELENBURG, M. F., SPRING, H. & ZENTGRAF, H. (1976). Cytobiologie, 13,401. LARK, K. G., CONSIGLI, R. & TOLIVER, A. (1971). / MoL Biol, 58, 873. MCKNIGHT, S. L. & MILLER, O. L. Jr. (1977). Cell, 12, 795. McLEISH, H. (1963)./>oc roy. soc. B., 158, 261. MILLER, O. L. & BAKKEN, A. H. (1972). Acta EnocrinoL SuppL, 168, 155. OLINS, A. L. & OLINS, D. E. (1974). Science, 183, 330. OUDET, P., GROSS-BELLARD, M. & CHAMBON, P. (1975). Cell, 4, 281. PUVION-DUTILLEUL, F., BERNADAC, A., PUVION, E. & BERNHARD, W. (1977)7. Ultrast. Res., 58, 108. VANT HOF, J. (1975). Exp. Cell Res., 93,95. VANT HOF, J. (1916). Exp. Cell Res., 103, 395. WOODCOCK, C. L. F., SAFER, J. P. & STANCHFIELD, J. P. (1976). Exp. Cell Res., 97, 101.

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