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Electron microscopic visualization of nucleosomal organization in B12 starved and control Euglena chromatin
Cell Biology
International
ELECTRON MICROSCOPIC
Reports,
Vol. 6, No. 2, February
VISUALIZATION
197
1982
OF NUCLEOSOMAL ORGANIZATION
IN
B12 STARVED AND CONTROL EUGLENA CHROMATIN. DELPECH S.l,
BRE M.H.2,
MAZEN A.3,
de WRCIA
G.4,
CHAMPAGNE M.5;
LEFORT-TRAN 1~6 *. 1,2,6 3,4,5
: Laboratoire 91190 Gif : I.B.M.C.,
de Cytophysiologie de la Photosynth&e, sur Yvette, France. 15 Rue Descartes, 67084 Strasbourg-Cedex,
x : to whom correspondence
should
C.N.R.S France;
be addressed.
ABSTRACT Euglena chromatin, spread according to the Dubochet technique, displays the nucleosomal organization with the typical zig-zag conformation following short periods of digestion with micrococcal nuclease. After comparative kinetic experiments on vitamin 612 starved and control Euglena nuclei, a larger percentage of monosomes appears after 2 min. in the starved chromatin (86%) compared to the control (59%). No diff erence is found for longer period of incubation. INTRODUCTION In the absence of vitamin B12, Euqlena cell divisions stop at the end of S phase. In these blocked cells, the DNA amount is less than doubled while RNA and protein syntheses continue (Care11 et al., 1970; Bertaux et Valencia, 1975) at the same rate as in 812 control cells, at least in the first days of blockage (Bertaux et a1.,1978). The nuclear size increases as the cell volume and a progressive reduction of staining and size of chromatin clumps is observed with the progress of starvation (Bertaux et al., 1978). An extraction and electrophoresis of histones from normal and starved Euqlena cell nuclei allow us to show a full complement of histones in the two cases but a lower degree of acetylation appears in H4 histone from starved cell. The average DNA repeat length (225 bp) in starved Euglena cell nuclei (Bre et al., 1980) is the same as in the normal cell nuclei (Bre et al., 1980; Magnaval et a1.,1980);this length is among the largest found in eukaryotes. The DNA length of the core particle is 145 base pairs. In order to visualize the nucleosomal organization of Eu lena chromatin and to get a better understanding of its apparent +--aspersion in the nucleus during vitamin B12 starvation, electron microscope preparations on spread chromatin were examined according to Miller and Bakken (1972) and Dubochet et al., (1971). Nuclease digestion for a short time shows a greater liberation of mononucleosomes in the solubilized starved chromatin compared to the control. The significance of such differences between the two kinds 0309-l
651/82/020197-O7l$O2.OOlO
@ 1982 Academic
Press
Inc. (London)
Ltd.
198
Cell Biolog y International
of chromatin
is
Reports,
vol. 6, No. 2, February
1982
discussed.
MATERIAL AND METHODS Cells of Euqlena gracilis strain Z (Cambridge culture collection no 1224-5 D) were grown as previously described (Bre and Lefort-Tran, 1978), for the control and according to Br6 et al., 1975, for the starved cells. Nuclei were isolated according to Jardine and Leaver (1977) however, the cell breakage buffer added with 15 n+l Tris HCl was adjusted to pH 7.4 ; nuclei washing buffers were used according to the method of Hewish and Burgoyne (1973). Isolated control nuclei were lysed and spread on grids according to Miller and Bakken (1972). Micrococcal nuclease digestions were performed on washed nuclei from normal and starved Euglena using exactly the same conditions for the digestion. The nuclei were suspended in 5 nW Tris pH 7.4, 10 mM atmwnium acetate,0.2 mM EDTA and 2 IM B-mercaptoethanol at a concentration of 200 pg of DNA per ml (determined before digestion). After preincubation at 37"C, the suspension was made
Cell Biology
fig.
International
1 - Spread Miller
Reports,
Vol. 6, No. 2, February
chromatin from control and Bakken (1972).
Euglena
199
1982
nuclei
according
to
130 f 10 i, they are attached together with a thin DNA filament (fig. 2a and 2d).In these cases the zig-zag organization appears (arrows fig. 2d). To determine the extent of digestion of the chromatin following tt-eatment with endonuclease the percentage of nucleosornes contained in vh rious length chromatin fragments was determined by making visual counts on an EM grid. Table I first gives the percentage of nucleosomes of from one to six as compared to those fragments containing greater than six repeat unit lengths. The table I then gives the percentage of monomers, dimers, trimers and tetramers within the 6-orless repeat unit lengths. A preliminary test was performed to compare the results of this tnethod with those obtained using gel electrophoresis to separate a mixture of nuclease digested erythrocyte chromatin, The range of the results obtained upon repeat analysis for each method was found to be less than 10%. The results are shown in Fig. 3. The data obtained with the two methods are in close agreement, except for the monomeric particles. It was found that the adsorption of the mononucleosomes on the charged grid is a little faster than for larger fragments. Nevertheless, the statistical method can be considered as valuable in comparative analysis..According to Table 1, from the percentage of oligonucleosomes more than 6, it seems that the plateau is not reached for the two kinds of chromatin. When the micrococcal nuclease digestion advances the average length of the oligonucleosomes is reduced. After 16 min. and 30 min. of digestion the repartition in different oligomeric forms remains the same for the control and the starved chromatin. However, when the results concerning 2 min. of digestion are examined, striking differences appear between the two kinds of chromatin. The percentage of oligo-
200
Cell Biology
International
Reports,
Vol. 6, No. 2, February
1982
Fig. 2- Control (a,b,c) and starved (d,e,f) Euglena chromatin digested with micrococcal nuclease and spread according to Dubochet et al, 1971, after 2 min.(a,d), 16 min.(b,e) and 30 min.(c,f).(bar = 0.1~)
Cell Biology
International
Oligomeric forms 2 min.
Reports,
>6
Vol. S, No. 2, February
1
4~6
201
1982
2
3
4
17
C
79
21
S
45
55
ii!
7
10 3
6 1
16 min.
S C
El:
2
;5
19 21
1:
z
30 min.
sC
40 47
2:
64 62
20 22
i
;
I - Oligomeric 1 to 6 nucleosomes forms > 6 : more chain than; C6 : nucleosomes control ; S per : starved chain; per chromatjn. The results are expressed in percentage of the total nucleosomes counted (at least 800 for each treatment). The mono- ditritetrameric forms are expressed in percentage of nucleosomes in 6 association.
mrom Table
Fig. 3 - Comparison of electron microscopic results with gel electrophoresis results. The dotted line histogram represents the relative percentage of nucleosomes found associated with chromatin fragments of repeat lengths of from one t0 six as counted on an EM grid. The solid line histogram represents the relative percent of area under the peak for each of the separated chromatin fragments found after a scan of a gel electrophoresis of the same nuclease in digested chromatin. nucleosomes less than 6, in the starved chromatin (55 than the control (21 %). In this class (< 6) a large somes (87 %) is observed compared with only 59 % for This difference is no more found for respectively 16
%) is higher excess of monothe control. and 30 min.
DISCUSSION Using the Miller technique, we have shown that Euglena chromatin organization is clearly of the "nucleosomal type" as was previously supposed (Haapala and Soyer, 1975) and reported (Bre et al., 1979). With the Dubochet spreading technique after micrococcal digestion we have been able to avoid the stretching of the chromatin filaments and to preserve the zig-zag configuration. This conformation is good evidence for the presence of Hl histone in the nucleosome (Thoma et al., 1979). It was previously shown (Bre et al., 1980) that the nucleosomal repeat length (225 base pairs) was identical for control and vitamin B12 starved Euolena chromatin. For a better understanding of the differences observed in situ by other methods (Bertaux et al., 1978) between the two kindsnomatin, the electron microscope study of
202
Cell Biology
International
Reports,
Vol. 6, No. 2, February
1982
chromatin fragments after sequential micrococcal nuclease digestion appears interesting. Four times were chosen (30 sec.,2 min., 16 min.,30 min.).lhe preparations after 30 sec. of nuclease digestion display aolarge contamination with smaller particles than nucleosomes (% 90 A) which are present in the starved as in the control chromatin and might be assimilated to RNP particles. Because this contamination and numerous unfolded clusters of nucleosomes in starved chromatin, the 30 sec. digestion tiatments were not taken into account for the statistical analyses. The most striking differences in sensibility to the nuclease between the control and starved chromatin are observed after 2 min. (Table I). Percentage of oligonucleosomes of 1 up to 6 particles are significantly higher in starved than in control chromatin. This higher percentage results from a greater proportion (87 %) of triand tetramere associations are lesmonosomes, whereas the di-, ser than in the control. This observation is in agreement with the kinetics of chromatin nuclease digestion from B12 deprived cells which shows a marked increase of the initial rate of digestion. (Magnaval, 1981). After 2 min. of nuclease digestion, the percentage of "solubilized chromatin" is less than 10 % for the control and the starved chromatin. Indeed, under low ionic strength, the very low solubility of Euglena chromatin seems to be comparable with those of plants studied so far (Muller et al., 1980). However, in the low percentage of "solubilized chromatin", the repartition in different species of oligonucleosomes from 1 to 6 appears comparable in the pellet and in the supernatant of the solubilized chromatin (Muller et al., 1980). Therefore , it could be considered that the chromatin fraction examined in electron microscopy is representative of the nuclease digestion. Indeed, the significant increase of mononucleosomes with starved chromatin (87 %) compared to the control (59 %) might be considered as the evidence of a different sensibility which should be a consequence of a different molecular organization of the two kinds of chromatin. In consequence, one might think about a greater heterogeneity in the starved chromatin than in the control one. ACKNOWLEDGEMENTS Technical assistance from is gratefully acknowledged. help in Miller technique.
C. Bunoust, D. Bouvier
J. Dunand and M. Montaggioni is thanked for his valuable
REFERENCES Bertaux, 0. and leculaires throne. Les protistes. Lefort-Tran,
Valencia, R. (1975) Aspects structuraux et macromode l'avitaminose B12 chez Euglena en culture syncycles cellulaires et leur blocage chez plusieurs International Colloquium of CNRS no 240. Ed. M. and Valencia, R.
Cell Biology Bertaux,
International
Vol. 6, No. 2, February
1982
203
Moyne, G., Lafarge-Frayssinet, C. and Valencia, R. The nucleus of Euglena. II Ultrastructural modifications of the nucleus of B12-deprived Euglena gracilis Z. Journal of Ultrastructure Research, 62, n"3, 251-269. Bre, M.H., Delpech, S., Champagne, M., Mazen, A. et Lefort-Tran, M. (1980) Analyses des histones et de 1'ADN nucleosomal de l'euglene normale et carencee en vitamine 812. Comptes rendus de l'Academie des Sciences de Paris, 290, Serie D, 93-96. Bre, M.H., Delpech, S., El Ferjani, E., Champagne, M., Mazen, A. et Lefort-Tran, M. (1979) Organisation de la chromatine dans les noyaux temoins et carences en vitamine B12 chez l'euglene. Biologie Cellulaire, 35, 30 a. Bre, M.H., Diamond, J., Jacques, R. (1975) Factors mediating the vitamin 812 requirement-of Euglena. Journal of Protozoology, 22, (3), 432-434. Bre, M.H: et Lefort-Tran, M. (1978) Induction et reversibilite des evenements cuticulaires par carence et realimentation en vitamine B12 chez Euglena gracilis. Journal of Ultrastructure Research, 64, 362-376. Carell, E.F., Johnston, P-L., Christopher, A.R. (1970) Vitamin 812 and the macromolecular composition of Euglena. Journal of Cell Biology, 47, 525-530. Dubochet, J., Ducommun, M., Zollinger, M. and Kellenberger, E. (1971) A new preparation method for dark-field electron microscopy of biomacromolecules. Journal of Ultrastructure Research, 35, 147,167. Haapala, O.K. and Soyer, M-0. (1975) Organisation of chromosome fibrils in Euqlena gracilis. hereditas, 80, 185-194. L.A. (1973) Chromatin sub-structure. The Hewish, D.R. and Burgoyne, digestion of chromatin DNA at regularly spaced sites by a nuclear deoxyribonuclease. Biochemical and Biophysical Research Communications, 52, 504-510. Jardine, N.J. and Leaver, J.L. (1977) The isolation of nuclei and histones from Euglena gracilis. Experimental Cell Research, 106, 423-426. Magnaval, R., Valencia, R. and Paoletti, J. (1980) Subunit organiBiochemical and Biophysical Rezation of Euglena chromatin. search Communications, 92, no 4, 1415-1421. Magnaval, R. These Doctorat d'Etat, Orsay, 1981. Miller, O.L. Jr. and Bakken, A.H. (1972) Morphological studies of transcription. Acta Endocrinologica. Supplement 168, 155-177. G., and Gigot, C. (1980) Properties of condenMuller, A., Philipps, sed chromatin in barley nuclei. Planta, 149, 69-77. M. and Daune, M. (1978) Superstrucde Murcia, G., Das, G.C., Erard, ture and CD spectrum as probes of chromatin integrity. Nucleic Acids Research, 5, 523-535. Thoma, F., Koller, T.H. and Klug, A. (1979) Involvement of histone HI in the organization of the nucleosome and of the saltdependent superstructures of chromatin. Journal of Cell Biology 83, 403-426. Revised version accepted: 10th November 1981 27th July 1981 Received:
(1978)
O.,
Reports,
International
ELECTRON MICROSCOPIC
Reports,
Vol. 6, No. 2, February
VISUALIZATION
197
1982
OF NUCLEOSOMAL ORGANIZATION
IN
B12 STARVED AND CONTROL EUGLENA CHROMATIN. DELPECH S.l,
BRE M.H.2,
MAZEN A.3,
de WRCIA
G.4,
CHAMPAGNE M.5;
LEFORT-TRAN 1~6 *. 1,2,6 3,4,5
: Laboratoire 91190 Gif : I.B.M.C.,
de Cytophysiologie de la Photosynth&e, sur Yvette, France. 15 Rue Descartes, 67084 Strasbourg-Cedex,
x : to whom correspondence
should
C.N.R.S France;
be addressed.
ABSTRACT Euglena chromatin, spread according to the Dubochet technique, displays the nucleosomal organization with the typical zig-zag conformation following short periods of digestion with micrococcal nuclease. After comparative kinetic experiments on vitamin 612 starved and control Euglena nuclei, a larger percentage of monosomes appears after 2 min. in the starved chromatin (86%) compared to the control (59%). No diff erence is found for longer period of incubation. INTRODUCTION In the absence of vitamin B12, Euqlena cell divisions stop at the end of S phase. In these blocked cells, the DNA amount is less than doubled while RNA and protein syntheses continue (Care11 et al., 1970; Bertaux et Valencia, 1975) at the same rate as in 812 control cells, at least in the first days of blockage (Bertaux et a1.,1978). The nuclear size increases as the cell volume and a progressive reduction of staining and size of chromatin clumps is observed with the progress of starvation (Bertaux et al., 1978). An extraction and electrophoresis of histones from normal and starved Euqlena cell nuclei allow us to show a full complement of histones in the two cases but a lower degree of acetylation appears in H4 histone from starved cell. The average DNA repeat length (225 bp) in starved Euglena cell nuclei (Bre et al., 1980) is the same as in the normal cell nuclei (Bre et al., 1980; Magnaval et a1.,1980);this length is among the largest found in eukaryotes. The DNA length of the core particle is 145 base pairs. In order to visualize the nucleosomal organization of Eu lena chromatin and to get a better understanding of its apparent +--aspersion in the nucleus during vitamin B12 starvation, electron microscope preparations on spread chromatin were examined according to Miller and Bakken (1972) and Dubochet et al., (1971). Nuclease digestion for a short time shows a greater liberation of mononucleosomes in the solubilized starved chromatin compared to the control. The significance of such differences between the two kinds 0309-l
651/82/020197-O7l$O2.OOlO
@ 1982 Academic
Press
Inc. (London)
Ltd.
198
Cell Biolog y International
of chromatin
is
Reports,
vol. 6, No. 2, February
1982
discussed.
MATERIAL AND METHODS Cells of Euqlena gracilis strain Z (Cambridge culture collection no 1224-5 D) were grown as previously described (Bre and Lefort-Tran, 1978), for the control and according to Br6 et al., 1975, for the starved cells. Nuclei were isolated according to Jardine and Leaver (1977) however, the cell breakage buffer added with 15 n+l Tris HCl was adjusted to pH 7.4 ; nuclei washing buffers were used according to the method of Hewish and Burgoyne (1973). Isolated control nuclei were lysed and spread on grids according to Miller and Bakken (1972). Micrococcal nuclease digestions were performed on washed nuclei from normal and starved Euglena using exactly the same conditions for the digestion. The nuclei were suspended in 5 nW Tris pH 7.4, 10 mM atmwnium acetate,0.2 mM EDTA and 2 IM B-mercaptoethanol at a concentration of 200 pg of DNA per ml (determined before digestion). After preincubation at 37"C, the suspension was made
Cell Biology
fig.
International
1 - Spread Miller
Reports,
Vol. 6, No. 2, February
chromatin from control and Bakken (1972).
Euglena
199
1982
nuclei
according
to
130 f 10 i, they are attached together with a thin DNA filament (fig. 2a and 2d).In these cases the zig-zag organization appears (arrows fig. 2d). To determine the extent of digestion of the chromatin following tt-eatment with endonuclease the percentage of nucleosornes contained in vh rious length chromatin fragments was determined by making visual counts on an EM grid. Table I first gives the percentage of nucleosomes of from one to six as compared to those fragments containing greater than six repeat unit lengths. The table I then gives the percentage of monomers, dimers, trimers and tetramers within the 6-orless repeat unit lengths. A preliminary test was performed to compare the results of this tnethod with those obtained using gel electrophoresis to separate a mixture of nuclease digested erythrocyte chromatin, The range of the results obtained upon repeat analysis for each method was found to be less than 10%. The results are shown in Fig. 3. The data obtained with the two methods are in close agreement, except for the monomeric particles. It was found that the adsorption of the mononucleosomes on the charged grid is a little faster than for larger fragments. Nevertheless, the statistical method can be considered as valuable in comparative analysis..According to Table 1, from the percentage of oligonucleosomes more than 6, it seems that the plateau is not reached for the two kinds of chromatin. When the micrococcal nuclease digestion advances the average length of the oligonucleosomes is reduced. After 16 min. and 30 min. of digestion the repartition in different oligomeric forms remains the same for the control and the starved chromatin. However, when the results concerning 2 min. of digestion are examined, striking differences appear between the two kinds of chromatin. The percentage of oligo-
200
Cell Biology
International
Reports,
Vol. 6, No. 2, February
1982
Fig. 2- Control (a,b,c) and starved (d,e,f) Euglena chromatin digested with micrococcal nuclease and spread according to Dubochet et al, 1971, after 2 min.(a,d), 16 min.(b,e) and 30 min.(c,f).(bar = 0.1~)
Cell Biology
International
Oligomeric forms 2 min.
Reports,
>6
Vol. S, No. 2, February
1
4~6
201
1982
2
3
4
17
C
79
21
S
45
55
ii!
7
10 3
6 1
16 min.
S C
El:
2
;5
19 21
1:
z
30 min.
sC
40 47
2:
64 62
20 22
i
;
I - Oligomeric 1 to 6 nucleosomes forms > 6 : more chain than; C6 : nucleosomes control ; S per : starved chain; per chromatjn. The results are expressed in percentage of the total nucleosomes counted (at least 800 for each treatment). The mono- ditritetrameric forms are expressed in percentage of nucleosomes in 6 association.
mrom Table
Fig. 3 - Comparison of electron microscopic results with gel electrophoresis results. The dotted line histogram represents the relative percentage of nucleosomes found associated with chromatin fragments of repeat lengths of from one t0 six as counted on an EM grid. The solid line histogram represents the relative percent of area under the peak for each of the separated chromatin fragments found after a scan of a gel electrophoresis of the same nuclease in digested chromatin. nucleosomes less than 6, in the starved chromatin (55 than the control (21 %). In this class (< 6) a large somes (87 %) is observed compared with only 59 % for This difference is no more found for respectively 16
%) is higher excess of monothe control. and 30 min.
DISCUSSION Using the Miller technique, we have shown that Euglena chromatin organization is clearly of the "nucleosomal type" as was previously supposed (Haapala and Soyer, 1975) and reported (Bre et al., 1979). With the Dubochet spreading technique after micrococcal digestion we have been able to avoid the stretching of the chromatin filaments and to preserve the zig-zag configuration. This conformation is good evidence for the presence of Hl histone in the nucleosome (Thoma et al., 1979). It was previously shown (Bre et al., 1980) that the nucleosomal repeat length (225 base pairs) was identical for control and vitamin B12 starved Euolena chromatin. For a better understanding of the differences observed in situ by other methods (Bertaux et al., 1978) between the two kindsnomatin, the electron microscope study of
202
Cell Biology
International
Reports,
Vol. 6, No. 2, February
1982
chromatin fragments after sequential micrococcal nuclease digestion appears interesting. Four times were chosen (30 sec.,2 min., 16 min.,30 min.).lhe preparations after 30 sec. of nuclease digestion display aolarge contamination with smaller particles than nucleosomes (% 90 A) which are present in the starved as in the control chromatin and might be assimilated to RNP particles. Because this contamination and numerous unfolded clusters of nucleosomes in starved chromatin, the 30 sec. digestion tiatments were not taken into account for the statistical analyses. The most striking differences in sensibility to the nuclease between the control and starved chromatin are observed after 2 min. (Table I). Percentage of oligonucleosomes of 1 up to 6 particles are significantly higher in starved than in control chromatin. This higher percentage results from a greater proportion (87 %) of triand tetramere associations are lesmonosomes, whereas the di-, ser than in the control. This observation is in agreement with the kinetics of chromatin nuclease digestion from B12 deprived cells which shows a marked increase of the initial rate of digestion. (Magnaval, 1981). After 2 min. of nuclease digestion, the percentage of "solubilized chromatin" is less than 10 % for the control and the starved chromatin. Indeed, under low ionic strength, the very low solubility of Euglena chromatin seems to be comparable with those of plants studied so far (Muller et al., 1980). However, in the low percentage of "solubilized chromatin", the repartition in different species of oligonucleosomes from 1 to 6 appears comparable in the pellet and in the supernatant of the solubilized chromatin (Muller et al., 1980). Therefore , it could be considered that the chromatin fraction examined in electron microscopy is representative of the nuclease digestion. Indeed, the significant increase of mononucleosomes with starved chromatin (87 %) compared to the control (59 %) might be considered as the evidence of a different sensibility which should be a consequence of a different molecular organization of the two kinds of chromatin. In consequence, one might think about a greater heterogeneity in the starved chromatin than in the control one. ACKNOWLEDGEMENTS Technical assistance from is gratefully acknowledged. help in Miller technique.
C. Bunoust, D. Bouvier
J. Dunand and M. Montaggioni is thanked for his valuable
REFERENCES Bertaux, 0. and leculaires throne. Les protistes. Lefort-Tran,
Valencia, R. (1975) Aspects structuraux et macromode l'avitaminose B12 chez Euglena en culture syncycles cellulaires et leur blocage chez plusieurs International Colloquium of CNRS no 240. Ed. M. and Valencia, R.
Cell Biology Bertaux,
International
Vol. 6, No. 2, February
1982
203
Moyne, G., Lafarge-Frayssinet, C. and Valencia, R. The nucleus of Euglena. II Ultrastructural modifications of the nucleus of B12-deprived Euglena gracilis Z. Journal of Ultrastructure Research, 62, n"3, 251-269. Bre, M.H., Delpech, S., Champagne, M., Mazen, A. et Lefort-Tran, M. (1980) Analyses des histones et de 1'ADN nucleosomal de l'euglene normale et carencee en vitamine 812. Comptes rendus de l'Academie des Sciences de Paris, 290, Serie D, 93-96. Bre, M.H., Delpech, S., El Ferjani, E., Champagne, M., Mazen, A. et Lefort-Tran, M. (1979) Organisation de la chromatine dans les noyaux temoins et carences en vitamine B12 chez l'euglene. Biologie Cellulaire, 35, 30 a. Bre, M.H., Diamond, J., Jacques, R. (1975) Factors mediating the vitamin 812 requirement-of Euglena. Journal of Protozoology, 22, (3), 432-434. Bre, M.H: et Lefort-Tran, M. (1978) Induction et reversibilite des evenements cuticulaires par carence et realimentation en vitamine B12 chez Euglena gracilis. Journal of Ultrastructure Research, 64, 362-376. Carell, E.F., Johnston, P-L., Christopher, A.R. (1970) Vitamin 812 and the macromolecular composition of Euglena. Journal of Cell Biology, 47, 525-530. Dubochet, J., Ducommun, M., Zollinger, M. and Kellenberger, E. (1971) A new preparation method for dark-field electron microscopy of biomacromolecules. Journal of Ultrastructure Research, 35, 147,167. Haapala, O.K. and Soyer, M-0. (1975) Organisation of chromosome fibrils in Euqlena gracilis. hereditas, 80, 185-194. L.A. (1973) Chromatin sub-structure. The Hewish, D.R. and Burgoyne, digestion of chromatin DNA at regularly spaced sites by a nuclear deoxyribonuclease. Biochemical and Biophysical Research Communications, 52, 504-510. Jardine, N.J. and Leaver, J.L. (1977) The isolation of nuclei and histones from Euglena gracilis. Experimental Cell Research, 106, 423-426. Magnaval, R., Valencia, R. and Paoletti, J. (1980) Subunit organiBiochemical and Biophysical Rezation of Euglena chromatin. search Communications, 92, no 4, 1415-1421. Magnaval, R. These Doctorat d'Etat, Orsay, 1981. Miller, O.L. Jr. and Bakken, A.H. (1972) Morphological studies of transcription. Acta Endocrinologica. Supplement 168, 155-177. G., and Gigot, C. (1980) Properties of condenMuller, A., Philipps, sed chromatin in barley nuclei. Planta, 149, 69-77. M. and Daune, M. (1978) Superstrucde Murcia, G., Das, G.C., Erard, ture and CD spectrum as probes of chromatin integrity. Nucleic Acids Research, 5, 523-535. Thoma, F., Koller, T.H. and Klug, A. (1979) Involvement of histone HI in the organization of the nucleosome and of the saltdependent superstructures of chromatin. Journal of Cell Biology 83, 403-426. Revised version accepted: 10th November 1981 27th July 1981 Received:
(1978)
O.,
Reports,