Experimental changes in the width of the chromatin fibers from chicken erythrocytes

Experimental

EXPERIMENTAL

CHANGES FIBERS

FROM

Cell Research 67 (1971) 161-170

IN THE WIDTH CHICKEN

OF THE CHROMATIN

ERYTHROCYTES

A. J. SOLAR1 Centro

de Investigaciones

sobre Reproduccion,

Facultad

de Medicina,

Buenos

Aires,

Argentina

SUMMARY Widths of chromatin fibers prepared by spreading erythrocyte chromatin on water have been measured in different experimental conditions. Chromatin fibers from hemoglobin-free. EDTApretreated isolated nuclei, prepared by direct negative staining with man;1 acetate show an average width of 37 A with a standard deviation of 13 A. The same chromatin fibers. when previously treated with ethanol show a change in the average width from 37 to 138 A. The same chromatin, when floated on a hemoglobin solution and then treated with ethanol shows a further enlargement of its average width, from 37 to 313 A. These changes were compared with the measurements of chromatin fibers from whole erythrocytes spread on water. The average width of these fibers after ethanol treatment is 244 A. These results show that the 240-250 A chromatin fibers are the result of conformational changes of a thinner elementary fibril 30-40 8, wide, which are mainly dependent on the action of ethanol on this fibril and on the presence of additional proteins like hemoglobin.

The ultrastructure of nucleated erythrocyte chromatin from amphibia and birds has been the subject of several studies either with spreading techniques [8, 12, 241 or with the standard sectioning techniques [7]. As nucleated erythrocytes constitute a population of uniform cells in which the chromatin is fully repressed [2] chromatin from this source will probably be a uniform material for morphological measurements.In the previous studies on the ultrastructure of erythrocyte chromatin, fibers ranging from 200 to 250 A in width have been described [8, 12, 221. In other cell types the chromatin has been repeatedly described as formed by similar 250 A wide fibers, when it was processed by spreading techniques and the critical point method of drying [5, 14, 231. However, using a different technique (direct negative staining of the spread chromatin) Solari [ 15, 17, 181described thinner ll-

711811

elementary fibrils, about 30 A wide, in the chromatin of sea urchin sperm and in other cell types. This observation has been generalized to several kinds of eukaryotic cells [19]. Thus, the question is raised about the relationship between the 250 A wide fibers observed after the critical point method and the 30 A wide fibrils observed with direct negative staining. The aim of this paper is to show that the chromatin from erythrocytes may appear as 37 A wide fibrils or as wider fibers (138, 244 or 3 13 8, wide) depending on the presence of hemoglobin and on the effect of ethanol on the chromatin during the preparation procedures. MATERIALS

AND METHODS

Blood from chicken or from adult hens was collected on heparinized syringes. Phytohemagglutinin (Difco Laboratories, Detroit, Mich.), 0.2 ml per 10 ml of ExptI

Cell Res 67

162

A. J. Solari

blood, was added to the whole blood and then ervthrocvtes were sevarated bv low sveed centrifuaation (10 min at 80 g). Erythrocytes were resuspended in saline-EDTA (9 % NaCl. 1 mM EDTA) and sedimented as above. Then 9 vol of 1 mM EDTA were added to vroduce hemolvsis. After velletine the hemolyzed ‘erythrocytes, this stage was repeated twice. Then a solution of 0.5 % Triton X-100 (Mann Research Lab, New York) and 1 mM EDTA in water was added to the pellet, resuspended and sedimented. This stage was repeated until the nuclear pellet was white. Pellets were kept at 4°C in salineEDTA. Procedures chromatin

for spreading

and drying

the

The procedures were basically two: (1) the direct negative staining method, (2) the alcohol, amylacetate drying method. In both procedures the chromatin is first spread on a clean water surface which is touched with a carbon-coated, Formvar film covering the grids to pick up the chromatin with a drop of water. In the direct negative staining method [15, 171 the grids are then floated on a saturated solution of uranyl acetate in water for 30 set and touched on one side by filter paper to leave only a thin film of liquid which is rapidly air dried. In the alcohol, amyl-acetate drying method [12] the grids are immersed in ethanol for 5 min and then passed without drying to amyl acetate for 5 min and air dried; then these grids were floated on the uranyl acetate solution for negative staining as described above, or alternatively, they were examined directly or after shadowing with platinum. Experimental variations in this schedule are described in Results. Measurements Micrographs were taken at magnifications of x 40 000 and x 60 000 in a Siemens Elmiskop IA electron microscope. Actual magnification was calculated in each series of micrographs from a micrograph of a diffraction grating replica. After photographic enlargement, prints were made at magnifications of x 120 000, x 300 000 and x 450 000. Measurements of fiber widths in the prints were made with a x 6 magnifier carrying a scale with 0.1 mm units. The widths were measured at 2-3 mm intervals along the length of the isolated fibers.

RESULTS The chromatin direct negative

of isolated staining

nuclei after

When isolated nuclei are spread on water and the chromatin picked on grids is negatively stained with uranyl acetate, the morphology of the fibers is very similar to that of the sea urchin sperm chromatin [ 181(figs Exptl

Cell Res 67

Table 1. Method of drying

Source

Isolated nuclei without hemoglobin Isolated nuclei without hemo. globin Isolated nuclei, floated on hemoglobin Whole erythrocytes

Mean diameter m

Direct negative staining Alcohol-amyl acetate Alcohol-amyl acetate Alcohol-amyl acetate

31.3

SD. A

13.2

138.2

48

313.5

64

244.6

52.4

1, 9). Thin fibrils, about 30 8, wide, form the chromatin in well spread regions. Aggregated fibrils are present in the central regions. The width of these fibrils has an average of 37 A with a standard deviation of 13 A (fig. 5, table 1). The measurements show that the width is rather irregular and that the thinnest points are about 20 A wide. The chromatin of isolated nuclei after ethanol and amyl-acetate drying

The chromatin of isolated nuclei spread on water, floated on ethanol and dried from amyl acetate forms threads of an average width of 138 A with a standard deviation of 48 A (figs 2, 6, table 1). These threads show a substructure formed by granular-like images and sometimes rodlike images, about 20-30 A wide. Along the threads some thinner regions are frequent, where the width becomes40-80 A. When the preparations were directly examined or shadowed with platinum instead of negatively stained with uranyl acetate, the same morphology was observed. The effect of floating hemoglobin solution

the chromatin

on a

If the chromatin of isolated nuclei is spread on water and picked on grids and then the

Changes in the width of chromatin fibers

163

1. Chromatin from isolated nuclei prepared with direct negative staining with uranyl acetate. The average width of these fibrils is 37 A. x 120 000. (Figs 14 have identical magnifications). Fig. 2. Chromatin from isolated nuclei, passed by ethanol and amyl acetate and air-dried. The average width of these fibers is 138 A. This preparation is negatively stained with uranyl acetate after drying from amyl acetate. x 120 000. Fig.

Exptl

Cell Res 67

164 A. J. Solari

Fig. 3. Chromatin ethanol and amyl Fig. 4. Chromatin The average width Exptl

Cell Res 67

from isolated nuclei, floated on a hemoglobin solution after spreading and then passed by acetate and air-dried. The average width of these fibers is 313 A. x 120 000. from whole erythrocytes spread on water, passed by ethanol and amyl acetate and air-dried. of these fibers is 244 .& x 120 000.

Changes in the width of chromatin fibers

165

-r r

r

5-8. A/xissa: width of fibrils (A); ordinate: number of fibrils of a given width. 5. Histogram compiled from measurements of chromatin fibrils of isolated nuclei prepared with direct negative staining. Fig. 6. Histogram compiled from measurements of chromatin fibers of isolated nuclei passed by ethanol and amyl acetate and air-dried. Fia. 7. Histoaram comoiled from measurements of chromatin fibers of isolated nuclei floated on a hemoalobin soiution and-passed by ethanol and amyl acetate and air-dried. Fig. 8. Histogram compiled from measurements of chromatin fibers of whole erythrocytes spread on water, passed by ethanol and amyl acetate and air-dried. Figs Fig.

grids are floated on a hemoglobin solution (hemoglobin from the hemolysis of hen erythrocytes, dialysed against 1 mM EDTA in water and diluted to a concentration of 1 g per 100 ml of 1 mM EDTA in water) for 10 min and then passed through ethanol and dried from amyl acetate, the chromatin fibers are much wider (fig. 3). The average width of these fibers is 313 8, with a standard deviation of 64 8, (fig. 7, table 1).

It must be remarked that although the width of the fibers is enlarged, the pattern of the fibers is much the same and the degree of confluence of the fibers is not very different.

The chromatin from whole erythrocytes after ethanol and amyl-acetate drying As the presence of hemoglobin produced a large effect on the morphology of chromatin fibers, the spreading of whole erythrocytes Exptl

Cell Res 67

166

A. J. Solari

Fig. 9. Low-magnification electron micrograph thin, straight fibrils. x 12 000.

Exptl

CeN Res 67

of an isolated nucleus with direct negative staining, showing the

Changes in the width of chromatin fibers

167

Fig. 10. Chromatin from a whole erythrocyte spread on water, passed by ethanol and amyl acetate and air dried. The chromatin fibers are thicker and flexuous. x 50 000.

Exptl

Cell Res 67

168 A. J. Solari was performed as a control. The spreading of whole erythrocytes from heparinized blood, picked on grids and passed through ethanol and dried from amyl acetate showed fibers having an average width of 244 A with a standard deviation of 52 A (figs 4, 8, 10, table 1). DISCUSSION The morphology of chromatin fibers from whole erythrocytes after alcohol treatment and the critical point method of drying

In the chromatin from newt erythrocytes, Wolfe [22] observed a mean fiber diameter of 227 A with a standard deviation of 30.9 A when the chromatin was processed with the critical point method of drying. Ris [12] observed similar 200 A fibers after air drying of the chromatin from amyl acetate. Thus, air drying from amyl acetate does not change considerably the width of the fibers when compared with the critical point method. Wolfe’s observations [23] on the fibers dried from amyl acetate also show that the mean diameter is similar in the fibers treated with either the critical point or drying from amyl acetate, although in the latter method an enlarged frequency of branching of the fibers was observed [24]. The present results on the spreading of whole chicken erythrocytes show that the width of the chromatin fibers from these preparations (244 A) is very similar to that found in the above cited papers. Wolfe [24] has made systematic observations that show that the morphology of the chromatin fibers does not depend on the forces developed during spreading in the airwater interface. This author also observed [24] that the 250 A fibers obtained with the spreading method had the same width when embedded in epoxy resins and sectioned. Furthermore, it was shown [24] that the procedures made after the alcohol treatment Exptl

Cell Res 67

in the critical point method, did not introduce differences in width between the fibers directly embedded and those dried in wholemounts. However, this author did not analyse the action of alcohol on the wet chromatin, as all of his preparations passed through alcohol, either before embedding and sectioning, or after spreading and before drying. Wolfe’s observations have dealt on the changes between the 100 A fibers found in undamaged nuclei and the 250 A fibers found after spreading. These changes are mainly due to the interaction of chromatin with divalent cations [15, 171 and with additional protein ([17] and present results). However, the gap between the 30 a nucleohistone [15, 251 and the 100 A and the 250 A fibers was not studied by that author. The morphology of chromatin after direct negative staining and the action of ethanol

When chromatin from various sources is spread on water, but instead of being passed through alcohol is directly passed through uranyl acetate and dried (negative stain) fibrils 30-40 A in width have been observed [15, 181. In the present results, the spreading of isolated nuclei and the negative staining of the chromatin shows fibrils 37 A wide which are similar to those previously described. Wolfe [22] observed that the treatment of the spread chromatin with uranyl acetate solutions previous to the drying with the critical point method, does not affect the observed width (250 A) of the fibers. Furthermore, negative staining of viral nucleoproteins [20] seems to give a morphological picture very similar to that of the living state. Thus, a specific thinning of the fibers by the action of uranyl acetate seems to be improbable. Thus, it seems that the main cause of the differences in width between the fibrils ob-

Changes in the width of chromatin fibers

served with negative staining and those observed with other drying procedures is the action of ethanol on the wet chromatin. The aggregating action of ethanol on DNA molecules has been described [9]. A collapse of DNA molecules on themselves has also been observed by the action of ethanol [9]. The action of ethanol on the nucleohistone in chicken erythrocyte nuclei has been described as a coarse coagulation [3]. From these considerations and from the previous data on the plastic behaviour of chromatin fibrils [17, 181 it is concluded that the morphological picture of the chromatin fibers after any method that includes a passage of unfixed, wet chromatin in ethanol will not show the molecular units of the chromatin in their native configuration. The morphology of the chromatin in the presence of hemoglobin

Variations in the width of the chromatin fibers processed with the same technique but from different sources have been repeatedly reported. Thus, Gall [8] reported that the chromatin fibers from grasshopper spermatocytes were thinner than those of nucleated erythrocytes. Wolfe [22] observed that the chromatin fibers from barley leaflets have a mean width of 187 A, and those from barley root tips have a mean width of 170 A. However, in the milky endosperm, in which the quantity of contaminating protein would be greater, the chromatin fibers had a mean diameter of 244 A [22]. The effect of prefixation on the width of the chromatin fibers was also studied by Wolfe [22]. Prefixation in 2% formalin in 0.05 M phosphate buffer resulted in a reduced width of the fibers, about 12&130 A. Changes in the width of the chromatin fibers when the chromatin is in the presence of varying quantities of contaminating protein have been observed also with negative

169

staining [17, 181. All these observations suggest that the cellular or intercellular materials which are spread at the same time as the chromatin, do influence the width of the chromatin fibers. The present results on the effect of hemoglobin are very clear in this respect. Isolated nuclei, deprived from hemoglobin show chromatin fibers thinner (mean width, 137 A) than those from whole erythrocytes (244 A). This difference may be due to hemoglobin or to other substances that exist in much smaller amounts in whole erythrocytes. This idea was tested by the action of hemoglobin solutions on the wet chromatin, which resulted in enlarged widths, larger than those from whole erythrocytes. This further enlargement may be attributed to the larger availability of hemoglobin in the case of the chromatin floated on hemoglobin solutions compared with the hemoglobin of whole erythrocytes. In the case of the prefixation effects [22] it can be suggested that prefixation will diminish the interaction of chromatin with contaminating proteins [17, 221. However, it has been also suggested [13] that a chelating action of some buffers may result in a thinning of the fibers. As divalent cations have been shown to influence the degree of packing of the chromatin fibrils [15, 171 both mechanisms probably contribute to the final width of the fibers. The effect of hemoglobin on the fiber width was previously suggested [15]. The structure fibers

of the 250 A wide chromatin

The 250 8, chromatin fibers obtained with spreading methods have been interpreted in either of the following ways: (1) as a DNA longitudinal core surrounded by a matrix, probably proteinaceous [4, 211; (2) as a small number (two or four) of parallel DNAprotein fibrils, 100-80 8, wide [ll, 121; (3) as a supercoiled [5, 6, lo] or an irregularly Exptl

Cell Res 67

170 A. J. Solari folded smaller fibril, 30 ?, wide [ 15, 17, 181. The first interpretation cannot agree with the lengths of DNA present in most nuclei, which are under the form of very long molecules [15, 161 and which are above one order of magnitude longer than the fiber lengths [6]. The second view is not in agreement with most of the recent observations on the fine structure of the chromatin fibers [l, 15, 17, 231, its enzymatic digestion [15, 181 and the characteristics of its DNA [16]; furthermore, it also cannot account for the DNA. The third interpretation is the most likely. More recently DuPraw [6] has reported the existence of a different kind of chromatin fiber (“A fiber”) which seems to be homologous to the 30-40 A fibril previously described with negative staining methods [15, 17, 181. This work was supported by a grant (TW-00317) of the Public Health Service. The author is member of the Scientific Career, CNICT. Support from Professor R. E. Mancini is gratefully acknowledged. Additional support from the Population Council is thanked. Note added in proof: In a very recent paper Bram Ris (J mol biol 55 (1971) 325) have found that the thinner fibers from calf thymus chromatin are 25 8, wide. In agreement with p&vious observations on sea urchin chromatin [15, 17, 181 and with the present paper, these authors suggest that the chromatin fibers are formed by one DNA molecule irregularly sypercoiled or folded to make a thicker fiber 80-120 .. A wlae.

Exptl

CeN Res 67

REFERENCES 1. Barnicot, N A, J cell biol 32 (1967) 585. 2. Cameron,I L & Prescott,D M, Exptl cell res 30 (1963) 609. 3. Davies, H G & Spencer, M, J cell biol 14 (1962) 4. %+a,, E J, Proc natl acad sci US 53 (1965) 161. 5. - Nature 206 (1965) 338. 6. DuPraw, E J & Bahr, G F, Acta cytol 13 (1969) 188. 7. Everid, A C, Small, J V & Davies, H G, J cell sci 7 (1970) 35. 8. Gall, J G, Chromosoma 20 (1966) 221. Lang, E, J mol bio146 (1969) 209. 1:: Pardon, J F, Wilkins, M H F & Richards, B M, Nature 215 (1967) 508. 11. Ris, H, The interpretation of ultrastructure (ed R J C Harris) p. 69. Academic Press, New York (1962). 12. - Proc roy sot B, 164 (1966) 246. 13. - J cell biol 89 (1968) 158a. 14. Ris, H, & Chandler, B L, Cold Spring Harbor symp quant biol 28 (1963) 1. 15. Solari, A J, Proc natl acad sci US 53 (1965) 503. 16. - J ultrastruct res 17 (1967) 421. 17. - Exptl cell res 53 (1968) 553. 18. - Ibid 53 (1968) 567. 19. - Unpublished data. 20. Valentine, R C & Home, R W, The interpretation of ultrastructure (ed R J C Harris) p. 263. Academic Press. New York (1962‘1. 21. Wolfe, S L, J &rastruct res i2 (&5) 104. 22. - J cell biol 37 (1968) 610. 23. - The biological basis of medicine (ed E Bittar) p. 3, Academic Press, New York (1969). 24. Wolfe S L & Grim, N J, J ultrastruct res 19 (1967) 382. 25. Zubay, G & Doty, P, J mol biol 1 (1959) 1. Received December 9, 1970 Revised version received March 16, 1971