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X-ray diffraction from isolated metaphase chromosomes
J. Mol. Biol. (1973) 76, 267-270
X-ray Diffraction
from Isolated Metaphase Chromosomes
J. F. PAuuoNt
AND B. M. RICHARDS
Searle Research Laboratories, Lane End Road High Wycombe, Bucks, England AND
L. G. SKINNER AND C. H. OCKEY Paterson Laboratories, Christie Hospital and Holt Radium Institute Withington, Manchester, England (Received 22 December 1972) Metaphase chromosomes were isolated from Chinese hamster fibroblasts. They were found to give the same characteristic diffrmtion pattern in the wet state as isolated nucleohistone but when dry did not give the reflections normally associated with dry nucleohistone. No higher structural order than that associated with the nucleohistone supercoil was detected.
1. Introduction Electron micrographs obtained from metaphase chromosomes show that the basic structural element of the chromosome is a unit thread of diameter within the range 170 to 300 A (Davies, 1968; DuPraw, 1966; Lampert, 1971; Ris & Kubai, 1970;
Wolfe, 1965; Ockey, 1973). The macromolecular structure of the unit thread cannot be established from the electron micrographs. X-ray diffraction techniques have been used to study the structure of nucleohistone which is likely to represent the most important
structural
entity
within
the unit chromosomal
thread.
These studies
have now been extended to include the structure of the metaphase chromosome. The X-ray diffraction patterns obtained from DNH$ include a series of low angle rings, some slightly meridionally oriented, with spacings 110,55,37,27,22 and 18 A, and poorly oriented diffraction from the DNA (Luzzatti & Nicolaieff, 1959,1963; Wilkins et al., 1959). A super-coil model with pitch 120 A and radius 50 A has been proposed to explain the diffraction data (Pardon et al., 1967; Pardon & Wilkins, 1972). We have studied the structure of metaphase chromosomes isolated from Chinese hamster fibroblasts and shown that the diffraction patterns obtained from hydrated samples include the low angle diffraction maxima and are essentially the same as those from gels of DNH. When water is removed from specimens of nucleohistone a different diffraction pattern is obtained with low angle maxima at 76 A and 38 A. With metaphase chromosomes the patterns also change with dehydration ; as with DNH the changes are reversible, but with chromosome samples the 76 A maximum is not recorded. t To whom reprint requests should be addressed. 2 Abbreviation used: DNH, nucleohistone. 267
8
268
J. F. PARDON
ET
AL.
2. Materials and Methods (a) Preparation
and composition of metaphaae chromosomes
A Chinese hamster fibroblast (CHO) cell strain grown in McCoy’s 6a medium (McCoy et al., 1959) supplemented with 10% foetal calf serum was used for the extraction procedures. Cell culture ctnd harvesting of met&phase chromosomes were carried out as described by Skinner & Ockey (1971). Cell membranes were broken and chromosomes liberated by pressure homogenization in a medium containing O-1 M-sodium acetate, 0.1 MHCI, 0.001 M-MgCl,, 0.001 M-C&&, 0.1 M-sucrose, pH 3.2, and the chromosomes isolated and purified as described by the authors. For these studies, the final 900 g chromosome pellet was redispersed in a medium containing O-02 M-Tris (pH 7.01, O-002 M-CaCI,, 0.05% saponin, collected in the centrifuge at 900 g for 15 min and washed 3 times by centrifugation in this medium. Chemical analysis for DNA, RNA and protein were carried out as previously described (Skinner & Ockey, 1971). The isolated chromosome preparations were found to have the chemical composition shown in Table 1.
TABLE
1
Chemical composition of isolated metaphuse chromosomes o/o total DNA
of dry weight RNA
30.0*1G3
13.4Al.l
Each value represents preparations.
the mean with
S.E.
(b) X-ray
Protein
o!0 of total protein soluble in 0.2 M-HCl
66.7+2.7
62.1+3*4
of duplicate
determinations
on each of seven separate
techniques
diffraction
A pellet of the chromosomes was obtained by centrifugation at 51,000 g for 1 11. Part of this pellet W&S inserted into a thin-walled capillary tube of internal diameter I.0 mm (Pantak, Windsor) and the ends of the tube sealed with Araldite adhesive. With some samples a quantity of distilled water was introduced into the wide end of the capillary tube to prevent the specimen from drying during the exposure to the X-ray beam. Some
samples were maintained at a given relative humidity by placing a saturated solution of the appropriate salt in the wide end of the capillary away from the sample, or by placing the specimen in an open-ended capillary tube inside a chamber maintained at a constant relative humidity. X-ray diffraction patterns were obtained using a Searle X-ray diffraction camera (Baird & Tatlock, London) with either Elliott toroidal mirror or Franks double mirror focussing optics and specimen to flm distance 7.5 cm. The cameras were mounted on a Hilger and Watts Y33 generator (Rank Precision Industries, London) using a 0.1 mm x 6 mm line focus filament. The X-ray tube was operated at 50 kV, 2 mA/tube current and exposures were within the range 16 to 20 h. X-ray films (Kodirex) were developed in Kodak DX-80 developer and fixed in Kodak Unifix.
3. Results X-ray
diffraction
patterns obtained from the hydrated
pellets of chromosomes
further drying are shown in Plate I. The low angle pattern consists of a scatter profile with an inflexion at 93 A, a shoulder at 55 8, rings with spacings of 36 A, 27 A and 21 A and a trace of a ring at 18 A in some patterns. Diffuse difikaction rings from the DNA double helix are present at 12 A and 8 8. No attempt was
without
made
tation
to align
the specimen
was observed
within
within
the capillary
the diffraction
rings.
tube
and as a consequence
no orien-
X-RAY
STUDIES
OF METAPHASE
CHROMOSOMES
269
On drying the specimen to 95% relative humidity the pattern remained unchanged, but specimens dried to 84% relative humidity or lower relative humidities failed to produce the series of low angle maxima, Patterns obtained from specimens rehydrated after drying below 95% relative humidity included the low angle maxima. Diffuse rings at 38 A and 10 A were recorded from a sample maintained at 44% relative humidity; there was diffuse central scatter but no 76 A maximum.
4. Discussion Specimens of nucleohistone in the concentration range 0.4 < C < 0.6 (where C is the mass ratio of solute to solution) produce a well-defined series of diffraction rings at 100 A, 55 A, 37 A,27 A,22 A and 18 A upon a weak background intensity profile. mess concentrated gels produce the same diffraction maxima but with the lower DNH concentrations the background scatter profile is more intense (Pooley, Pardon & Richards, unpublished observations). For a DNH gel at concentration C = 0.3 the central scatter profile is intense, the 110 A and 55 A maxima are not resolved as discrete rings but as an inflexion in the region 90 A to 100 A and a shoulder at 55 A. In dehydrated specimens (C > O-7) the 110 A maxima and others in the series are replaced by two new maxima at 76 A and 38 A. The diffraction patterns obtained from the moist pellet of metaphase chromosomes are very similar to those obtained from gels of DNH in the concentration range 0.2 < C < 0.3. There were no maxima present in addition to those normally observed from DNH and the intensity distribution was essentially the same. On removing water by equilibrating the centrifuge pellet at 98 or 95% relative humidity the pattern changed very little; there was more central scatter in the region 110 A than is normally present from DNH at these humidities when the 110 A ring is no longer present. Specimens equilibrated at 84% relative humidity or lower relative humidities did not produce the 110 A series of maxima or the maxima at 76 A. The 38 A ring obtained from a sample maintained at 44% relative humidity was similar in appearance to the 32 A ring previously observed in samples of isolated histone (&bay $ Wilkins, 1962) and the 35 A ring observed from deoxyribonuclease-treated DNH (Garrett, 1968) and probably arises from the histone molecules. The X-ray diffraction patterns cannot be quantified but the results described here show that roughly the same proportion of the DNA in wet chromosome preparations exists in the same regular configuration as that in isolated nucleohistone. The most reasonable model to explain the low angle diffraction is the regular super helix proposed by Wilkins (Pardon & Wilkins, 1972). As with DNH, this regular tertiary structure is lost on removing water molecules but can be re-formed on rehydration. Unlike DNH, however, there are no features present in the diffraction from dried metaphase chromosomes to indicate the presence of any regular tertiary structure in the dry state. It may be that the 76 A maximum observed from specimens of DNH arises from a new configuration brought about as the result of a regular collapse of the super-coiled molecules. With metaphase chromosomes the presence of nonhistone proteins and RNA may prevent this regular collapse, especially if it involves interdigitation of neighbouring super helices or a re-arrangement of histone bridges. The diameter of the unit chromosomal thread found for Chinese hamster &romosomes isolated at metaphase and examined by electron microscopy was found to be 270 A to 280 A (Ockey, 1973). This diameter is greater than that of the nucleohistone
270
J. F. PARDON
ET
AL.
supercoil even though water and maybe other molecules have been removed during specimen preparation for electron microscopy. A comparative study of the structure of nucleohistone by electron microscopy and X-ray diffraction (Pooley, Pardon & Richards, unpublished observations) leads to the conclusion that removing water causes the supercoil to “collapse ” to produce an electron dense thread of greater diameter. In conclusion we can say that our results do not confirm or deny the existence in metaphase chromosomes of higher orders of structure over and above that of the proposed tertiary supercoil configuration for nucleohistone. If the regular shape of the metaphase chromosomes, seen in the light of results of microscopic studies, results from some kind of coiling or folding of the chromosome libril, this is not demonstrable by the X-ray studies reported here. REFERENCES Davies, H. G. (1968). J. CeZZ Sci. 3, 129. DuPraw, E. J. (1966). Nature, 209, 577. Garrett, R. A. (1968). Ph.D. thesis, London University. Lampert, F. (1971). Nature New Biol. 234, 187. Luzzatti, V. & Nicolaieff, A. (1959). J. Mol. Biol. 1, 127. Luzzatti, V. & Nicolaieff, A. (1963). J. Mol. Biol. 7, 142. McCoy, T. A., Maxwell, M. & Krnse, P. F. (1959). PTOC.Sot. Eqrx BioZ. Med. 100, 115. Ockey, C. H. (1973). PTOC. Nobel Symposium, no. 23. In the press. Almquist and Wiksell, Uppsala, Sweden. Pardon, J. F., Wilkins, M. H. F. & Richards, B. M. (1967). Nature, 215, 508. Pardon, J. F. & Wilkins, M. H. F. (1972). J. Mol. BioZ. 68, 115. Ris, H. & Kubai, D. F. (1970). An/n. Rev. Genet. 4, 263. Skinner, L. G. & Ockey, C. H. (1971). Chromosoma, 35, 125. Wilkins, M. H. F., Zubay, G. & Wilson, H. R. (1959). J. Mol. BioZ. 1, 179. Wolfe, S. L. (1965). J. Ultraetruct. Res. 12, 104. Zubay, G. & Wilkins, M. H. F. (1962). J. Mol. BioZ. 4, 444.
X-ray Diffraction
from Isolated Metaphase Chromosomes
J. F. PAuuoNt
AND B. M. RICHARDS
Searle Research Laboratories, Lane End Road High Wycombe, Bucks, England AND
L. G. SKINNER AND C. H. OCKEY Paterson Laboratories, Christie Hospital and Holt Radium Institute Withington, Manchester, England (Received 22 December 1972) Metaphase chromosomes were isolated from Chinese hamster fibroblasts. They were found to give the same characteristic diffrmtion pattern in the wet state as isolated nucleohistone but when dry did not give the reflections normally associated with dry nucleohistone. No higher structural order than that associated with the nucleohistone supercoil was detected.
1. Introduction Electron micrographs obtained from metaphase chromosomes show that the basic structural element of the chromosome is a unit thread of diameter within the range 170 to 300 A (Davies, 1968; DuPraw, 1966; Lampert, 1971; Ris & Kubai, 1970;
Wolfe, 1965; Ockey, 1973). The macromolecular structure of the unit thread cannot be established from the electron micrographs. X-ray diffraction techniques have been used to study the structure of nucleohistone which is likely to represent the most important
structural
entity
within
the unit chromosomal
thread.
These studies
have now been extended to include the structure of the metaphase chromosome. The X-ray diffraction patterns obtained from DNH$ include a series of low angle rings, some slightly meridionally oriented, with spacings 110,55,37,27,22 and 18 A, and poorly oriented diffraction from the DNA (Luzzatti & Nicolaieff, 1959,1963; Wilkins et al., 1959). A super-coil model with pitch 120 A and radius 50 A has been proposed to explain the diffraction data (Pardon et al., 1967; Pardon & Wilkins, 1972). We have studied the structure of metaphase chromosomes isolated from Chinese hamster fibroblasts and shown that the diffraction patterns obtained from hydrated samples include the low angle diffraction maxima and are essentially the same as those from gels of DNH. When water is removed from specimens of nucleohistone a different diffraction pattern is obtained with low angle maxima at 76 A and 38 A. With metaphase chromosomes the patterns also change with dehydration ; as with DNH the changes are reversible, but with chromosome samples the 76 A maximum is not recorded. t To whom reprint requests should be addressed. 2 Abbreviation used: DNH, nucleohistone. 267
8
268
J. F. PARDON
ET
AL.
2. Materials and Methods (a) Preparation
and composition of metaphaae chromosomes
A Chinese hamster fibroblast (CHO) cell strain grown in McCoy’s 6a medium (McCoy et al., 1959) supplemented with 10% foetal calf serum was used for the extraction procedures. Cell culture ctnd harvesting of met&phase chromosomes were carried out as described by Skinner & Ockey (1971). Cell membranes were broken and chromosomes liberated by pressure homogenization in a medium containing O-1 M-sodium acetate, 0.1 MHCI, 0.001 M-MgCl,, 0.001 M-C&&, 0.1 M-sucrose, pH 3.2, and the chromosomes isolated and purified as described by the authors. For these studies, the final 900 g chromosome pellet was redispersed in a medium containing O-02 M-Tris (pH 7.01, O-002 M-CaCI,, 0.05% saponin, collected in the centrifuge at 900 g for 15 min and washed 3 times by centrifugation in this medium. Chemical analysis for DNA, RNA and protein were carried out as previously described (Skinner & Ockey, 1971). The isolated chromosome preparations were found to have the chemical composition shown in Table 1.
TABLE
1
Chemical composition of isolated metaphuse chromosomes o/o total DNA
of dry weight RNA
30.0*1G3
13.4Al.l
Each value represents preparations.
the mean with
S.E.
(b) X-ray
Protein
o!0 of total protein soluble in 0.2 M-HCl
66.7+2.7
62.1+3*4
of duplicate
determinations
on each of seven separate
techniques
diffraction
A pellet of the chromosomes was obtained by centrifugation at 51,000 g for 1 11. Part of this pellet W&S inserted into a thin-walled capillary tube of internal diameter I.0 mm (Pantak, Windsor) and the ends of the tube sealed with Araldite adhesive. With some samples a quantity of distilled water was introduced into the wide end of the capillary tube to prevent the specimen from drying during the exposure to the X-ray beam. Some
samples were maintained at a given relative humidity by placing a saturated solution of the appropriate salt in the wide end of the capillary away from the sample, or by placing the specimen in an open-ended capillary tube inside a chamber maintained at a constant relative humidity. X-ray diffraction patterns were obtained using a Searle X-ray diffraction camera (Baird & Tatlock, London) with either Elliott toroidal mirror or Franks double mirror focussing optics and specimen to flm distance 7.5 cm. The cameras were mounted on a Hilger and Watts Y33 generator (Rank Precision Industries, London) using a 0.1 mm x 6 mm line focus filament. The X-ray tube was operated at 50 kV, 2 mA/tube current and exposures were within the range 16 to 20 h. X-ray films (Kodirex) were developed in Kodak DX-80 developer and fixed in Kodak Unifix.
3. Results X-ray
diffraction
patterns obtained from the hydrated
pellets of chromosomes
further drying are shown in Plate I. The low angle pattern consists of a scatter profile with an inflexion at 93 A, a shoulder at 55 8, rings with spacings of 36 A, 27 A and 21 A and a trace of a ring at 18 A in some patterns. Diffuse difikaction rings from the DNA double helix are present at 12 A and 8 8. No attempt was
without
made
tation
to align
the specimen
was observed
within
within
the capillary
the diffraction
rings.
tube
and as a consequence
no orien-
X-RAY
STUDIES
OF METAPHASE
CHROMOSOMES
269
On drying the specimen to 95% relative humidity the pattern remained unchanged, but specimens dried to 84% relative humidity or lower relative humidities failed to produce the series of low angle maxima, Patterns obtained from specimens rehydrated after drying below 95% relative humidity included the low angle maxima. Diffuse rings at 38 A and 10 A were recorded from a sample maintained at 44% relative humidity; there was diffuse central scatter but no 76 A maximum.
4. Discussion Specimens of nucleohistone in the concentration range 0.4 < C < 0.6 (where C is the mass ratio of solute to solution) produce a well-defined series of diffraction rings at 100 A, 55 A, 37 A,27 A,22 A and 18 A upon a weak background intensity profile. mess concentrated gels produce the same diffraction maxima but with the lower DNH concentrations the background scatter profile is more intense (Pooley, Pardon & Richards, unpublished observations). For a DNH gel at concentration C = 0.3 the central scatter profile is intense, the 110 A and 55 A maxima are not resolved as discrete rings but as an inflexion in the region 90 A to 100 A and a shoulder at 55 A. In dehydrated specimens (C > O-7) the 110 A maxima and others in the series are replaced by two new maxima at 76 A and 38 A. The diffraction patterns obtained from the moist pellet of metaphase chromosomes are very similar to those obtained from gels of DNH in the concentration range 0.2 < C < 0.3. There were no maxima present in addition to those normally observed from DNH and the intensity distribution was essentially the same. On removing water by equilibrating the centrifuge pellet at 98 or 95% relative humidity the pattern changed very little; there was more central scatter in the region 110 A than is normally present from DNH at these humidities when the 110 A ring is no longer present. Specimens equilibrated at 84% relative humidity or lower relative humidities did not produce the 110 A series of maxima or the maxima at 76 A. The 38 A ring obtained from a sample maintained at 44% relative humidity was similar in appearance to the 32 A ring previously observed in samples of isolated histone (&bay $ Wilkins, 1962) and the 35 A ring observed from deoxyribonuclease-treated DNH (Garrett, 1968) and probably arises from the histone molecules. The X-ray diffraction patterns cannot be quantified but the results described here show that roughly the same proportion of the DNA in wet chromosome preparations exists in the same regular configuration as that in isolated nucleohistone. The most reasonable model to explain the low angle diffraction is the regular super helix proposed by Wilkins (Pardon & Wilkins, 1972). As with DNH, this regular tertiary structure is lost on removing water molecules but can be re-formed on rehydration. Unlike DNH, however, there are no features present in the diffraction from dried metaphase chromosomes to indicate the presence of any regular tertiary structure in the dry state. It may be that the 76 A maximum observed from specimens of DNH arises from a new configuration brought about as the result of a regular collapse of the super-coiled molecules. With metaphase chromosomes the presence of nonhistone proteins and RNA may prevent this regular collapse, especially if it involves interdigitation of neighbouring super helices or a re-arrangement of histone bridges. The diameter of the unit chromosomal thread found for Chinese hamster &romosomes isolated at metaphase and examined by electron microscopy was found to be 270 A to 280 A (Ockey, 1973). This diameter is greater than that of the nucleohistone
270
J. F. PARDON
ET
AL.
supercoil even though water and maybe other molecules have been removed during specimen preparation for electron microscopy. A comparative study of the structure of nucleohistone by electron microscopy and X-ray diffraction (Pooley, Pardon & Richards, unpublished observations) leads to the conclusion that removing water causes the supercoil to “collapse ” to produce an electron dense thread of greater diameter. In conclusion we can say that our results do not confirm or deny the existence in metaphase chromosomes of higher orders of structure over and above that of the proposed tertiary supercoil configuration for nucleohistone. If the regular shape of the metaphase chromosomes, seen in the light of results of microscopic studies, results from some kind of coiling or folding of the chromosome libril, this is not demonstrable by the X-ray studies reported here. REFERENCES Davies, H. G. (1968). J. CeZZ Sci. 3, 129. DuPraw, E. J. (1966). Nature, 209, 577. Garrett, R. A. (1968). Ph.D. thesis, London University. Lampert, F. (1971). Nature New Biol. 234, 187. Luzzatti, V. & Nicolaieff, A. (1959). J. Mol. Biol. 1, 127. Luzzatti, V. & Nicolaieff, A. (1963). J. Mol. Biol. 7, 142. McCoy, T. A., Maxwell, M. & Krnse, P. F. (1959). PTOC.Sot. Eqrx BioZ. Med. 100, 115. Ockey, C. H. (1973). PTOC. Nobel Symposium, no. 23. In the press. Almquist and Wiksell, Uppsala, Sweden. Pardon, J. F., Wilkins, M. H. F. & Richards, B. M. (1967). Nature, 215, 508. Pardon, J. F. & Wilkins, M. H. F. (1972). J. Mol. BioZ. 68, 115. Ris, H. & Kubai, D. F. (1970). An/n. Rev. Genet. 4, 263. Skinner, L. G. & Ockey, C. H. (1971). Chromosoma, 35, 125. Wilkins, M. H. F., Zubay, G. & Wilson, H. R. (1959). J. Mol. BioZ. 1, 179. Wolfe, S. L. (1965). J. Ultraetruct. Res. 12, 104. Zubay, G. & Wilkins, M. H. F. (1962). J. Mol. BioZ. 4, 444.