By this method chromosomes reveal their entire substructure made up of fibres of about 250 ~ in diameter. The DNA double helix is thought to be tightly packed as a coiled snpercoiled nucleohistone fibril within these chromosome fibres [5, 6].
Nucleosome-DNA-Coil in Fibres of a Human Metaphase Chromosome as Demonstrated by Whole-Moun! Electron Microscopy F. Lampert Kinderpoliklinik der Universit~it, D-6300 Giegen Nucleosomes from interphase eukaryotic nuclei have been described as spherical bodies of about 70 A in diameter connected by thin filaments like 'particles on a string' [1]. It has been suggested that these particles consist of a loop of DNA gently coiled around a histone core [2]. This histone protein core would hold 140 basepairs of DNA in a single leRhanded, nouinterwound DNA supercoil approximately 95 base pairs in circumference [3]. I wish to present an electron micrograph of a human metaphase chromosome prepared by surface spreading and criticalpoint drying where amid the typical tangled and folded chromosome fibres a stretched subfibril with coils at rather uniform intervals was found. Human chromosomes prepared for wholemount electron microscopy were derived from an EB3-Burkitt lymphoma cell line. Mitosis was blocked by a final incubation with 0.1 pg colcemid/ml for 2-4 h. After centrifugation at 600 g for 5 min, cells were treated in a hypotonic medium (2.8 g KCI+ 1.6 ml heparin and 500ml H20) for 10 rain, washed once in distilled water and pelleted. One drop of the cell sediment Was then carefully applied to the clean surface of a trough containing distilled water at pH 6.4. The floating interphase nuclei and chromosome groups were picked up by touching the surface with formvar carboncoated grids. The grids were immediately mounted in a plastic grid holder under
598
30% ethanol, dehydrated through ascending concentrations to absolute ethanol, and transferred to amyl acetate. To preserve the three-dimensional structure as well as possible, the grids were dried by the cri;ical-point method of Anderson [4]. No stains were used.
These tightly folded nucleoprotein fibres of about 230-250 A in diameter can be seen in Figure 1 where part of a chromatid telomer of a well preserved chromosome of the human 6-X-12 group is shown. By a rare coincidence this intact chromosome fibre has been partially disintegrated revealing a stretched thin subfibril of about 35 A in diameter. At regular intervals (arrows), about 1100 1300A apart, this straight filament folds to form more or less hollow knobs of about 100 l l 0 A diameter. It is suggested that these structures are the loops of the naked or proteincoated DNA-filament around the histone cores of the nucleosome particles. Supported by the Deutsche Forschungsgemeinschaft. I thank Prof. Dr. O. Goetz, Virus Laboratory of the Children's University Hospital, Munich, for providing the cell culture and Prof. Dr. D. Hnhn, Institute of Hematology, Munich, for letting me use his electron microscope.
Received August 12, 1977
Fig. 1. Electron micrograph of the telomer of a water-spread and critical-point-dried, unstained human metaphase chromosome (EB3 ceil line). Amid the typical folded fibres of about 230 250/~_ in diameter, a straight subfibril can be seen which seems to be coiled (arrows) at regular intervals (x 105000)
1. Olins, A.L., Olins, D.E.: Science 183, 330 (1974) 2. Langmore, J.P., Wooley, J.C. : Proc. Nat. A_cad. Sci. USA 72, 2691 (1975) 3. Weintraub, H., Worcel, A., Alberts, B. : Cell 9, 409 (1976) 4. Anderson, T.F.: Trans. N.Y. Acad. Sci., Series II 13, 130 (1951) 5. Lampert, F.: Nature New Biol. 234, 187 (1971) 6. Finch, J.T., Klug, A.: Proc. Nat. Acad. Sci. USA 73, 1897 (1976)
Naturwissenschaften 64 (1977)
9 by Springer-Verlag 1977
Nucleosome-DNA-Coil in Fibres of a Human Metaphase Chromosome as Demonstrated by Whole-Moun! Electron Microscopy F. Lampert Kinderpoliklinik der Universit~it, D-6300 Giegen Nucleosomes from interphase eukaryotic nuclei have been described as spherical bodies of about 70 A in diameter connected by thin filaments like 'particles on a string' [1]. It has been suggested that these particles consist of a loop of DNA gently coiled around a histone core [2]. This histone protein core would hold 140 basepairs of DNA in a single leRhanded, nouinterwound DNA supercoil approximately 95 base pairs in circumference [3]. I wish to present an electron micrograph of a human metaphase chromosome prepared by surface spreading and criticalpoint drying where amid the typical tangled and folded chromosome fibres a stretched subfibril with coils at rather uniform intervals was found. Human chromosomes prepared for wholemount electron microscopy were derived from an EB3-Burkitt lymphoma cell line. Mitosis was blocked by a final incubation with 0.1 pg colcemid/ml for 2-4 h. After centrifugation at 600 g for 5 min, cells were treated in a hypotonic medium (2.8 g KCI+ 1.6 ml heparin and 500ml H20) for 10 rain, washed once in distilled water and pelleted. One drop of the cell sediment Was then carefully applied to the clean surface of a trough containing distilled water at pH 6.4. The floating interphase nuclei and chromosome groups were picked up by touching the surface with formvar carboncoated grids. The grids were immediately mounted in a plastic grid holder under
598
30% ethanol, dehydrated through ascending concentrations to absolute ethanol, and transferred to amyl acetate. To preserve the three-dimensional structure as well as possible, the grids were dried by the cri;ical-point method of Anderson [4]. No stains were used.
These tightly folded nucleoprotein fibres of about 230-250 A in diameter can be seen in Figure 1 where part of a chromatid telomer of a well preserved chromosome of the human 6-X-12 group is shown. By a rare coincidence this intact chromosome fibre has been partially disintegrated revealing a stretched thin subfibril of about 35 A in diameter. At regular intervals (arrows), about 1100 1300A apart, this straight filament folds to form more or less hollow knobs of about 100 l l 0 A diameter. It is suggested that these structures are the loops of the naked or proteincoated DNA-filament around the histone cores of the nucleosome particles. Supported by the Deutsche Forschungsgemeinschaft. I thank Prof. Dr. O. Goetz, Virus Laboratory of the Children's University Hospital, Munich, for providing the cell culture and Prof. Dr. D. Hnhn, Institute of Hematology, Munich, for letting me use his electron microscope.
Received August 12, 1977
Fig. 1. Electron micrograph of the telomer of a water-spread and critical-point-dried, unstained human metaphase chromosome (EB3 ceil line). Amid the typical folded fibres of about 230 250/~_ in diameter, a straight subfibril can be seen which seems to be coiled (arrows) at regular intervals (x 105000)
1. Olins, A.L., Olins, D.E.: Science 183, 330 (1974) 2. Langmore, J.P., Wooley, J.C. : Proc. Nat. A_cad. Sci. USA 72, 2691 (1975) 3. Weintraub, H., Worcel, A., Alberts, B. : Cell 9, 409 (1976) 4. Anderson, T.F.: Trans. N.Y. Acad. Sci., Series II 13, 130 (1951) 5. Lampert, F.: Nature New Biol. 234, 187 (1971) 6. Finch, J.T., Klug, A.: Proc. Nat. Acad. Sci. USA 73, 1897 (1976)
Naturwissenschaften 64 (1977)
9 by Springer-Verlag 1977
