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Early and late histones during sea urchin development
J. ,%fol. Biol. (1971.) 57, 355-358
arly and Late Histones during Sea Urchin DeveIopmen During cleavage of sea urchin embryos, replication
of the chromosomes occurs
at about hourly intervals whereas at later stages of development the cells divide a6 a much slower rate. A single slightly lysine-rich histone predominates in the rapidly dividing chromosomes. When cell division slows, lysine-rich and argininerich histones become the major components.
ephcation of the chromosomesduring early cleavage of the seaurchin embryo is an extremely rapid event. Hinegardiner, Rao & Feldman (1964) reported that the S period lasted about 13 minutes at 15°C and that the equivalent t,ime at 37°C woukl be three minutes. This period must be considered very short when compared to mammalian systems where the S period lasts six to eight hours (Defendi & M.anson, 1963). Since the S period and histone synthesis are closely timed events (Prescott, 1966; Robbins & Borun, 1967), it was questioned whether the major histone fractions esent at later stagesof development (Marushige & Qzaki, 1967; Vorobyev, Cineitis Vinogradova, 1969) are present during rapid replication of the chromosomes. Sea urchins, Lytechinus variegatus, were obtained from the Bermuda Biological Station for Research, Inc. Embryos were grown at 22 to 23°C in glasstrays at a concentration of approximately 2000 embryos/ml. of filtered sea water containing 100 units of penicillin/ml. When embryos reached the 32-cell stage, the mesenchyrne blastula stage, and the prism gastrula stage they were harvested, washed once with sea water, once with 0.45 M-NaCl, 0.05 M-KCl, 0.01 r+r-Tris (pH 7~3)~095 X-EDT& and then rapidly with 0.075 M-xaC1, 0.025 M-EDTA. The embryos were lysed in a ground-glass homogenizer with five volumes of the 6nal wash solution. Chrometin was isolated using a modification of the method of Huang & Bonner (1965). The total cell homogenate of the 32-cell stage was layered on 1.6 M-sucrose, 0.01 M-‘&is (pB 703)~ 0.001 M-EDTA, the top third of the tube gently mixed, and the chromatin collected as a pellet after centrifugation for two hours at 25,000 rev./min in a Spinco SW25.1 rotor. The chromatin was washed once in dilute saline-EDTA before freezing a.t -20°C. At the two later stages,the chromatin and unbroken nuclei were first isolated by centrifugation at lOOOg,washed once in 0.01 i?r-Tris (pN 8.0), 0.001 N-EDTB and resuspendedin the samesolution before centrifugation through sucrose. Chromatin was prepared from washed sperm by extracting the sperm twice with 0.075 M-NaCI, O-025M-EDTA and twice with 0.04 xl-Tris (pH 89), 0.0011~-EDTA. The final gel was stored at -20°C prior to acid-extraction of histones. Hi&ones were extracted in 0.4 N-H&SO, at 0°C for one hour. Acid-soluble proteins were precipitated with three volumes of 95% ethanol at -20°C for 18 hours. The precipitated material was washed once in cold ethanol before drying under vacuum. ,The samples were dissolved in freshly deionized 8 M-urea containing 0.01 IA:2mercaptoethanol and incubated at room temperature 30 minutes before gel electrooresis. This incubation was found to be necessary to prevent a,ggregation of the @nine-rich histones (Panyim & Chalkley, 19696). Studies with a variety of plant and animal material have indicated five main histone fractions with only minimal tissue and speciesspecificities (Nekon & Yunis, 11969;Neidle & Waelsch, 1964; Panyim & ChalMey, 196%; Hnilica, Edwards & 356
366
L.
C.
BENTTINEN
AND
D.
G.
COMB
1966; Fambrough, Fujimara & Bonner, 1968). Isolation and fractionation procedures demonstrate the close chemical similarity of corresponding fractions from different sources, and amino-acid analyses reveal only slight variations between these fractions (Hnilica, 1967; DeLange, Fambrough, Smith & Bonner, 1969a,b). Figure 1 shows the polyacrylamide gel patterns of histones characteristic of the three stages of development. The patterns obtained in five separate preparations of each developmental stage were essentially identical to those shown in Figure 1. The 2
FIG. 1. Histone patterns of the developing embryo. Tracing (a), 32-cell, and (c), prism gastrula are of 1Opg samples applied to the double gel pictured on the left in Plate I. Tracing (b) is that of a 25-pg sample of mesenchyme blastula run on a single gel (not pictured). Tracings were done with a Joyce-Loebl microdensitometer. In histone 5, we have labelled the slower moving band as 5’. This is the reverse of Panyim & Chalkley’s (1969a) nomenclature.
bands are numbered 1 through 5 in accordance with the nomenclature of Panyim & Chalkley (1969a), who designate these bands in order of increasing mobility as : (1) lysine-rich; (2) arginine-rich; (3) slightly lysine-rich; (4) slightly lysine-rich; and (5) arginine-rich. They correspond to fractions I, III, IIa, IIb and IV, respectively, of Fambrough et al. (1968) and fractions Fl, F3, F2b, F2a2 and F2al of Johns (1967). At the 32-cell stage when rapid cell division is in progress, a slightly lysine-rich fraction (band 3) is the major histone fraction of the chromosomes. Sample A in Plate I shows this more clearly. The lysine-rich histone (band l), which stains blue-black, is present in much smaller amounts during cleavage than at later stages of development. An increase in this histone during development in the sea urchin has also been reported by Vorobyev et al. (1969) and in maturing pea seedlings by Fambrough et al. (1968). Band 5 is a single component at the 32-cell stage, but at later stagesis resolved into two components 5 and 5’. The appearance of this second component may represent the appearance of a new histone or it may represent somechemical modification of the parent molecule such as acetylation or methylation. Vidali, Gershey & Allfrey (1.968)reported that fraction 5 is acetylated in vitro, and Panyim & Chalkley (1969a) claim that such a modification could be resolved by gel electrophoresis. DeLange et al. (1969a,b) noted that such modifications are the major differences between immature pea histone IV and calf histone IV.
8
D
I2-----345--
PLATE I. Sea urchin histones. & 32.cell histones; B, mesenchyme blastula histones;C, prism gastrulahistones; D, spermhistones. Acrylamide (10%) gels containing 8.0 w-urea were prepared following the method of Riesfeld, Lewis & Williams (1962) as modified by Leboy, Cox & Flaks (1964). Double gels were prepared by inserting 2 pieces of plastic tubing (i.d. 2 mm) into the large pore gel before polymerization. The gels measured 0.6 cm x 7 cm. Electrophoresis at 20°C was carried out for 2.6 to 3 hr at 4 m.k/gel. Gels were stained for 1 hr in 1 y0 amido black, 7 y0 acetic acid, and were destained with 7 y0 acetic acid. The sperm histone which has the same electrophoretie mobility as histone 1 stains light blue, has a very high serine content, and is, therefore, not considered to be a corresponding histone.
LETTERS
TO
THE
357
EDITOR
At later stages of development (Fig. 1, (lo) and (c) or Plate I, B and C) when cell division has slowed considerably, band 3 represents only a minor component; and band 2, an arginine-rich histone, has increased dramatically relative to the other histone components. Band 4 is resolved into two components at the prism gastrula stage. The histone patterns obtained in these studies were highly reproducible and demonstrate clearly that the relative proportions of the various histone components are different at early and late stages of development. The amount of the lysine-rich fracfion increasesabout 50% from the 32-cell stage to the mesenchymeblastula stage. This is a period when the nuclei decreasein volume and increasein density (Benttinen & Comb, unpublished results) and is of interest in view of the observations of Mirsky, urdick, Davidson & Littau (1968) that lysine-rich histones condense cbromatin. ming this sameperiod we have found that much more arginine-rich histone (band 2) becomes associatedwith the chromosomes,and this is also a time when the rate of nuclear RNA synthesis decreases(Kijima & Wilt, 1969). Plate I (B and D) showsthe pattern of spermhistonesrun on a gel with mesenebyme blastula histones. Of the five distinct sperm bands, only two (bands 3 and 5) are comparable to embryonic histones. Since histones 3 and 5 are also major compoaents of cleavage chromatin, several experiments were performed to determine whetber they represent a pool introduced by the sperm at fertilization or contamination by excesssperm. Fertilized eggs, in amount comparable to the above studies were harvested and treated in a manner identical to that used for cleavage histones. Bands were not detected in the histone region of the gel when the sampleused was twice that of the cleavage sample. It is, therefore, most unlikely that bands 3 and 5 are the result of contamination by excess sperm. The possibility cannot be ruled out that the entering sperm does in fact contribute these two fractions, although these studi.es show that such a contribution could not suffice for more than three or four cleavages. To determine if the unfertilized egg contains a reservoir of histones for use during rapid replication of the chromosomes,the basic proteins from the soluble fraction of the cell (after removal of nuclei and ribosomes by centrifugation) were concentrated by adsorption and elution from carboxymethyl-cellulose and run on a double pol.yacrylamide gel with sperm histonesthat had been treated in an identical manner. The results showed that a protein with an electrophoretic mobility on IO% gels identical to bistone 5 was present in the soluble fraction of the unfertilized egg. Basic proteins with electrophoretic mobilities similar to the other four embryonic histones were not detected. In view of the report of Kedes $ Gross (1969) that histone synthesis occurs throughout early development, it would appear that at least four out of the five histone fractions are synthesized, and that the lysine-rich and arginine-rich histones become more important as development proceeds. This work was aided by grant no. United States Public Health Service Foundation. Department of Biological Chemistry Harvard Medical School Boston, Mass. 02115, U.S.A.
Received 27
July
1970
12632 and
from grant
the National no. GB7703
Instit,utes from the
of Health National
LEE DONALD
of ihe Science
C.%XKlTINEN ~.COMB
358
L. 6.
BENTTINEN
AND
I).
0.
COMB
REFERENCES Defendi, V. & Manson, L. A. (1963). Nature, 198, 359. DeLange, R. J., Fambrough, D. M., Smith, E. L. & Bonner, J. (1969a). J. BioZ. Chem. 244, 319. DeLange, R. J., Fambrough, D. M., Smith, E. L. & Bonner, J. (19695). J. BioZ. Chem. 244, 6669. Fambrough, D. M., Fujimara, F. & Bonner, J. (1968). Biochemistry, 7, 575. Hinegardiner, R. T., Rao, B. & Feldman, D. F. (1964). Eq. Cell Res. 36, 53. Hnilica, L. (1967). In Progress in Nucleic Acid Research and MoZecular Biology, ed. by J. N. Davidson & W. E. Cohn, vol. 7, p. 25. New York: Academic Press. Hnilioa, L., Edwards, L. J. & Hey, A. E. (1966). Biochim. biophys. Acta, 124, 109. Huang, R. C. C. & Bonner, J. (1965). Proc. Nat. Acad. Sci., Wash. 54, 960. Johns, E. W. (1967). Biochem. J. 104, 78. Kedes, L. H. & Gross, P. R. (1969). Nature, 233, 1335. Kijima, S. & Wilt, F. H. (1969). J. Mol. BioZ. 40, 235, Leboy, P. S., Cox, E. 6. & Flaks, J. G. (1964). Proc. Nat. Acad. Sci., Wash. 52, 1367. Marushige, K. & Ozaki, H. (1967). Devel. Biol. 16, 474. Mix-sky, A. E., Burdick, C. J., Davidson, E. H. & Littau, V. C. (1968). Proc. Nat. Acad. Sci., Wash. 61, 592. Neidle, A. & Waelsoh, H. (1964). Science, 145, 1059. Nelson, R. D. & Yunis, J. J. (1969). Eq. Cell Res. 57, 311. Panyim, S. & Chalkley, R. (1969a). Arch. Biochem. Biophys. 130, 337. Panyim, S. & Chalkley, R. (1969b). Biochemistry, 8, 3972. Prescott, D. M. (1966). J. Cell BioZ. 31, 1. Riesfeld, R. A., Lewis, V. J. & Williams, D. E. (1962). Nature, 195, 281. Robbins, E. & Borun, T. W. (1967). Proc. Nat. Acad. Sci., Wash. 57, 409. Vidali, G., Gershey, E. L. & Allfrey, V. G. (1968). J. BioZ. Chem. 243, 6361. Vorobyev, V. I., Gineitis, A. A. & Vinogradova, I. A. (1969). Exp. CeZZRes. 57, 1.
arly and Late Histones during Sea Urchin DeveIopmen During cleavage of sea urchin embryos, replication
of the chromosomes occurs
at about hourly intervals whereas at later stages of development the cells divide a6 a much slower rate. A single slightly lysine-rich histone predominates in the rapidly dividing chromosomes. When cell division slows, lysine-rich and argininerich histones become the major components.
ephcation of the chromosomesduring early cleavage of the seaurchin embryo is an extremely rapid event. Hinegardiner, Rao & Feldman (1964) reported that the S period lasted about 13 minutes at 15°C and that the equivalent t,ime at 37°C woukl be three minutes. This period must be considered very short when compared to mammalian systems where the S period lasts six to eight hours (Defendi & M.anson, 1963). Since the S period and histone synthesis are closely timed events (Prescott, 1966; Robbins & Borun, 1967), it was questioned whether the major histone fractions esent at later stagesof development (Marushige & Qzaki, 1967; Vorobyev, Cineitis Vinogradova, 1969) are present during rapid replication of the chromosomes. Sea urchins, Lytechinus variegatus, were obtained from the Bermuda Biological Station for Research, Inc. Embryos were grown at 22 to 23°C in glasstrays at a concentration of approximately 2000 embryos/ml. of filtered sea water containing 100 units of penicillin/ml. When embryos reached the 32-cell stage, the mesenchyrne blastula stage, and the prism gastrula stage they were harvested, washed once with sea water, once with 0.45 M-NaCl, 0.05 M-KCl, 0.01 r+r-Tris (pH 7~3)~095 X-EDT& and then rapidly with 0.075 M-xaC1, 0.025 M-EDTA. The embryos were lysed in a ground-glass homogenizer with five volumes of the 6nal wash solution. Chrometin was isolated using a modification of the method of Huang & Bonner (1965). The total cell homogenate of the 32-cell stage was layered on 1.6 M-sucrose, 0.01 M-‘&is (pB 703)~ 0.001 M-EDTA, the top third of the tube gently mixed, and the chromatin collected as a pellet after centrifugation for two hours at 25,000 rev./min in a Spinco SW25.1 rotor. The chromatin was washed once in dilute saline-EDTA before freezing a.t -20°C. At the two later stages,the chromatin and unbroken nuclei were first isolated by centrifugation at lOOOg,washed once in 0.01 i?r-Tris (pN 8.0), 0.001 N-EDTB and resuspendedin the samesolution before centrifugation through sucrose. Chromatin was prepared from washed sperm by extracting the sperm twice with 0.075 M-NaCI, O-025M-EDTA and twice with 0.04 xl-Tris (pH 89), 0.0011~-EDTA. The final gel was stored at -20°C prior to acid-extraction of histones. Hi&ones were extracted in 0.4 N-H&SO, at 0°C for one hour. Acid-soluble proteins were precipitated with three volumes of 95% ethanol at -20°C for 18 hours. The precipitated material was washed once in cold ethanol before drying under vacuum. ,The samples were dissolved in freshly deionized 8 M-urea containing 0.01 IA:2mercaptoethanol and incubated at room temperature 30 minutes before gel electrooresis. This incubation was found to be necessary to prevent a,ggregation of the @nine-rich histones (Panyim & Chalkley, 19696). Studies with a variety of plant and animal material have indicated five main histone fractions with only minimal tissue and speciesspecificities (Nekon & Yunis, 11969;Neidle & Waelsch, 1964; Panyim & ChalMey, 196%; Hnilica, Edwards & 356
366
L.
C.
BENTTINEN
AND
D.
G.
COMB
1966; Fambrough, Fujimara & Bonner, 1968). Isolation and fractionation procedures demonstrate the close chemical similarity of corresponding fractions from different sources, and amino-acid analyses reveal only slight variations between these fractions (Hnilica, 1967; DeLange, Fambrough, Smith & Bonner, 1969a,b). Figure 1 shows the polyacrylamide gel patterns of histones characteristic of the three stages of development. The patterns obtained in five separate preparations of each developmental stage were essentially identical to those shown in Figure 1. The 2
FIG. 1. Histone patterns of the developing embryo. Tracing (a), 32-cell, and (c), prism gastrula are of 1Opg samples applied to the double gel pictured on the left in Plate I. Tracing (b) is that of a 25-pg sample of mesenchyme blastula run on a single gel (not pictured). Tracings were done with a Joyce-Loebl microdensitometer. In histone 5, we have labelled the slower moving band as 5’. This is the reverse of Panyim & Chalkley’s (1969a) nomenclature.
bands are numbered 1 through 5 in accordance with the nomenclature of Panyim & Chalkley (1969a), who designate these bands in order of increasing mobility as : (1) lysine-rich; (2) arginine-rich; (3) slightly lysine-rich; (4) slightly lysine-rich; and (5) arginine-rich. They correspond to fractions I, III, IIa, IIb and IV, respectively, of Fambrough et al. (1968) and fractions Fl, F3, F2b, F2a2 and F2al of Johns (1967). At the 32-cell stage when rapid cell division is in progress, a slightly lysine-rich fraction (band 3) is the major histone fraction of the chromosomes. Sample A in Plate I shows this more clearly. The lysine-rich histone (band l), which stains blue-black, is present in much smaller amounts during cleavage than at later stages of development. An increase in this histone during development in the sea urchin has also been reported by Vorobyev et al. (1969) and in maturing pea seedlings by Fambrough et al. (1968). Band 5 is a single component at the 32-cell stage, but at later stagesis resolved into two components 5 and 5’. The appearance of this second component may represent the appearance of a new histone or it may represent somechemical modification of the parent molecule such as acetylation or methylation. Vidali, Gershey & Allfrey (1.968)reported that fraction 5 is acetylated in vitro, and Panyim & Chalkley (1969a) claim that such a modification could be resolved by gel electrophoresis. DeLange et al. (1969a,b) noted that such modifications are the major differences between immature pea histone IV and calf histone IV.
8
D
I2-----345--
PLATE I. Sea urchin histones. & 32.cell histones; B, mesenchyme blastula histones;C, prism gastrulahistones; D, spermhistones. Acrylamide (10%) gels containing 8.0 w-urea were prepared following the method of Riesfeld, Lewis & Williams (1962) as modified by Leboy, Cox & Flaks (1964). Double gels were prepared by inserting 2 pieces of plastic tubing (i.d. 2 mm) into the large pore gel before polymerization. The gels measured 0.6 cm x 7 cm. Electrophoresis at 20°C was carried out for 2.6 to 3 hr at 4 m.k/gel. Gels were stained for 1 hr in 1 y0 amido black, 7 y0 acetic acid, and were destained with 7 y0 acetic acid. The sperm histone which has the same electrophoretie mobility as histone 1 stains light blue, has a very high serine content, and is, therefore, not considered to be a corresponding histone.
LETTERS
TO
THE
357
EDITOR
At later stages of development (Fig. 1, (lo) and (c) or Plate I, B and C) when cell division has slowed considerably, band 3 represents only a minor component; and band 2, an arginine-rich histone, has increased dramatically relative to the other histone components. Band 4 is resolved into two components at the prism gastrula stage. The histone patterns obtained in these studies were highly reproducible and demonstrate clearly that the relative proportions of the various histone components are different at early and late stages of development. The amount of the lysine-rich fracfion increasesabout 50% from the 32-cell stage to the mesenchymeblastula stage. This is a period when the nuclei decreasein volume and increasein density (Benttinen & Comb, unpublished results) and is of interest in view of the observations of Mirsky, urdick, Davidson & Littau (1968) that lysine-rich histones condense cbromatin. ming this sameperiod we have found that much more arginine-rich histone (band 2) becomes associatedwith the chromosomes,and this is also a time when the rate of nuclear RNA synthesis decreases(Kijima & Wilt, 1969). Plate I (B and D) showsthe pattern of spermhistonesrun on a gel with mesenebyme blastula histones. Of the five distinct sperm bands, only two (bands 3 and 5) are comparable to embryonic histones. Since histones 3 and 5 are also major compoaents of cleavage chromatin, several experiments were performed to determine whetber they represent a pool introduced by the sperm at fertilization or contamination by excesssperm. Fertilized eggs, in amount comparable to the above studies were harvested and treated in a manner identical to that used for cleavage histones. Bands were not detected in the histone region of the gel when the sampleused was twice that of the cleavage sample. It is, therefore, most unlikely that bands 3 and 5 are the result of contamination by excess sperm. The possibility cannot be ruled out that the entering sperm does in fact contribute these two fractions, although these studi.es show that such a contribution could not suffice for more than three or four cleavages. To determine if the unfertilized egg contains a reservoir of histones for use during rapid replication of the chromosomes,the basic proteins from the soluble fraction of the cell (after removal of nuclei and ribosomes by centrifugation) were concentrated by adsorption and elution from carboxymethyl-cellulose and run on a double pol.yacrylamide gel with sperm histonesthat had been treated in an identical manner. The results showed that a protein with an electrophoretic mobility on IO% gels identical to bistone 5 was present in the soluble fraction of the unfertilized egg. Basic proteins with electrophoretic mobilities similar to the other four embryonic histones were not detected. In view of the report of Kedes $ Gross (1969) that histone synthesis occurs throughout early development, it would appear that at least four out of the five histone fractions are synthesized, and that the lysine-rich and arginine-rich histones become more important as development proceeds. This work was aided by grant no. United States Public Health Service Foundation. Department of Biological Chemistry Harvard Medical School Boston, Mass. 02115, U.S.A.
Received 27
July
1970
12632 and
from grant
the National no. GB7703
Instit,utes from the
of Health National
LEE DONALD
of ihe Science
C.%XKlTINEN ~.COMB
358
L. 6.
BENTTINEN
AND
I).
0.
COMB
REFERENCES Defendi, V. & Manson, L. A. (1963). Nature, 198, 359. DeLange, R. J., Fambrough, D. M., Smith, E. L. & Bonner, J. (1969a). J. BioZ. Chem. 244, 319. DeLange, R. J., Fambrough, D. M., Smith, E. L. & Bonner, J. (19695). J. BioZ. Chem. 244, 6669. Fambrough, D. M., Fujimara, F. & Bonner, J. (1968). Biochemistry, 7, 575. Hinegardiner, R. T., Rao, B. & Feldman, D. F. (1964). Eq. Cell Res. 36, 53. Hnilica, L. (1967). In Progress in Nucleic Acid Research and MoZecular Biology, ed. by J. N. Davidson & W. E. Cohn, vol. 7, p. 25. New York: Academic Press. Hnilioa, L., Edwards, L. J. & Hey, A. E. (1966). Biochim. biophys. Acta, 124, 109. Huang, R. C. C. & Bonner, J. (1965). Proc. Nat. Acad. Sci., Wash. 54, 960. Johns, E. W. (1967). Biochem. J. 104, 78. Kedes, L. H. & Gross, P. R. (1969). Nature, 233, 1335. Kijima, S. & Wilt, F. H. (1969). J. Mol. BioZ. 40, 235, Leboy, P. S., Cox, E. 6. & Flaks, J. G. (1964). Proc. Nat. Acad. Sci., Wash. 52, 1367. Marushige, K. & Ozaki, H. (1967). Devel. Biol. 16, 474. Mix-sky, A. E., Burdick, C. J., Davidson, E. H. & Littau, V. C. (1968). Proc. Nat. Acad. Sci., Wash. 61, 592. Neidle, A. & Waelsoh, H. (1964). Science, 145, 1059. Nelson, R. D. & Yunis, J. J. (1969). Eq. Cell Res. 57, 311. Panyim, S. & Chalkley, R. (1969a). Arch. Biochem. Biophys. 130, 337. Panyim, S. & Chalkley, R. (1969b). Biochemistry, 8, 3972. Prescott, D. M. (1966). J. Cell BioZ. 31, 1. Riesfeld, R. A., Lewis, V. J. & Williams, D. E. (1962). Nature, 195, 281. Robbins, E. & Borun, T. W. (1967). Proc. Nat. Acad. Sci., Wash. 57, 409. Vidali, G., Gershey, E. L. & Allfrey, V. G. (1968). J. BioZ. Chem. 243, 6361. Vorobyev, V. I., Gineitis, A. A. & Vinogradova, I. A. (1969). Exp. CeZZRes. 57, 1.