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Differential kinetics of histone H1o accumulation in neuronal and glial cells from rat cerebral cortex during postnatal development
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 123, No. 2, 1984 September
Pages 697-702
17, 1984
DIFFERENTIAL KINETICS OF HISTONE Hl' ACCUMULATION IN NEURONAL AND GLIAL CELLS FROM RAT CEREBRAL CORTEX DURING POSTNATAL DEVELOPMENT B. Piiia,
P. Martinez,
Departamento de Bioqufmica, Aut6noma de Barcelona.
Received
August
L. Simdn and P. Suau+ Facultad de Ciencias, Bellaterra, Barcelona,
Universidad Spain
6, 1984
The accumulation of histone H1° has been studied in neuronal and glial nuclei from rat cerebral cortex during postnatal development. In neurons Hl" represents -2% of the Hl content at birth and remains unchanged until day 8. Beyond this point H1° accumulates rapidly until day 18, where it levels off at 16% of Hl. The midpoint of the transition is at day 14. In glial cells Hl" represents -2.5% of the Hl at birth. It starts to accumulate between days 18 and 21; its concentration raises rapidly up to day 30 slowing down from then on. At day 300 (the farthest point examined) it represents 21% of Hl. These results are discussed in relation to the events of the postnatal development of the cerebral cortex in the rat. It is concluded that H1° probably does not suppress cell proliferation.
species
The Hl class of histones contains even within the same cellular type
fractions, HlO, is predominantly found or no cellular proliferation (2-5). It
several molecular (1). One of the sub-
in tissues
showing
little
accumulates after birth at various stages of development in mouse liver, brain, kidney, pancreas and retina (6). It has been shown to decrease in regenerating rat pancreas (7) and liver (4) and to be hormonally regulated in several gland tissues (6). H1° can be chemically induced -in vitro in several cell lines such as Friend erythroleukemia (8-lo), murine neuroblastoma (11) and Chinese hamster suggested that Hl" plays a role in the DNA replication
(2,4)
or in cell
ovary arrest
differentiation
(12). It of cell
has been division and
(11).
We have performed a detailed analysis of the accumulation of H1° in neuronal and glial nuclei from rat cerebral cortex during postnatal development. Both cellular types show distinct patterns of H1° accumulation that can be correlated with cell matu+To whom correspondence ABBREVIATIONS;
should
be addressed.
SDS; sodium didecyl
sulfate. 0006-291X/84 $1.50 697
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 123, No. 2, 1984
ration during HI0 reflects cated in cell
development. at the level maturation
MATERIALS
AND METHODS
Fractionation
of cortex
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The results suggest that accumulation of of histone composition the events impliand terminal differentiation.
nuclei
Cerebral cortices from male Sprage-Dawley rats were homogenized by hand with 30 up-and-down strokes of a Dounce homogenizer in 1M sucrose, 1mM sodium cacodylate, 5mM MgC12 and 1mM dithiothreitol, pH 6.5. Cortex nuclei were fractionated by the method of Thompson (13), except that a third layer of sucrose (2.6M) was added to improve the purity of the glial fraction. Neuronal nuclei were obtained from the interphase of the 2.4M and 1.8M sucrose layers, whereas glial nuclei were recovered as a pellet at the bottom of the 2.6M sucrose cushion. All operations were performed at 2'C and phenyl methyl sulphonyl fluoride (0.1 mM) was used throughout to inhibit proteolitic activity. Gel
electrophoresis
Purified nuclei were pelleted and resuspended in 1OmM EDTA, pH 7.4. After sonication proteins were extracted four times with HC10f.(5% v(v). Acid extracts were dialysed against 5% acetic acid and lophylized. When proteins from whole nuclei were to be analized by SDS-polyacrylamide gel electrophoresis the latter were dispersed by sonication in 1OmM EDTA, pH 7.4. Proteins were analyzed by electrophoresis in SDS-polyacrylamide gels (15%), essentially as described by Laemmli (14), and in urea-acetic acid-polyacrylamide gels by the method of Panyim and Chalkley (15). Slab gels were fixed and stained in methanol : water : acetic acid (5:5:1) containing 0.25% Coomassie Brillant Blue R-250 and destained in water : acetic acid : methanol : ethanol (7:l:l:l). After destaining the gels Were soaked in dimethyl sulfoxide for 2 minutes to remove all the stain background between bands and then resuspended in 10% acetic acid as described (16). Band intensities were quantified by gel scanning at 540 nm using a Beckman DU-8B spectrophotometer. Measure
of
the
chromatin
repeat
unit
Micrococcalnuclease digestion of nuclei and DNA extraction were performed according to Thomas and Thompson (17). DNA fragments were analyzed on 1% agarose slab gels and sized with a Hae III digest of @X 174 RF DNA (Bio Labs). RESULTS Neuronal and glial nuclear fractions were isolated from rat cerebral cortex. The deqree of cross-contamination of each nuclear type with the other was of the order of 10% for either nuclear fraction, as judged by phase contrast microscopy. To ascertain the purity of the nuclear fractions we made use of the known fact 698
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 123, No. 2, 1984
Hla+b, Hl”--
Figure
of proteins form neuronal 1. Gel electrophoresis and glial nuclei from rat cerebral cortex SDSpolyacrylamide gel electrophoresis of proteins. A, from neuronal and glial nuclei. Nuclei were from the same group of rats of 21 days age.
(Lane
1) Whole
Perchloric (Lane
neuronal
acid extract
3) Whole
glial
extract
the chromatin
from
nuclei.
neuronal
repeat
unit
nuclei.
(Lane
2)
from neuronal nuclei.
acid extract from glial acid gel electrophoresis
that
-
(Lane
4)
Perchloric
nuclei. B, urea-acetic of a prechloric acid nuclei.
of neuronal
nuclei
contains
160-170
base pairs as compared to 200 base pairs for nonastrocytic glial nuclei (17). Values of 16826 and 19626 base pairs were found for neuronal shown).
and glial
fractions,
Accumulation
respectively,
of H1° in neuronal
mined between 0 and 300 days after its mobility on SDS and urea-acetic
in adult
and glial
rats
nuclei
(not
was exa-
birth. H1° was identified acid-polyacrylamide gels
by (Figu-
Hl* to ordinary Hl (Hla+Hlb) and total Hl (Hla+ re 1). The ratios Hlb+Hl') to core histones (H2a+H2b+H3+H4) were obtained from electrophoretic analysis on SDS-polyacrylamide gels of whole purified nuclei or HC104 (5% v/v) extractable proteins. Both methods gave
the
same
values
within
the
stadistical
error.
Figure 2 shows examples of gel scans of Hl subfractions from specific times of the postnatal development. Kinetics of accumulation of H1° in neuronal and glial nuclei are shown in Figure 3. In neurons Hl“ represents "2% of H1 at birth and remains at the same level until day 8. Beyond this point H1° accumulates 699
BIOCHEMICAL
Vol. 123, No. 2, 1984
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
7 DAYS
12 DAYS
Figure
rapidly until point of the to core
lk h
21 DAYS
2. SDS-polyacrylamide gel electrophoresis profiles of neuronal nuclei Hl subfraction at times 0, 12 and 21 days of postnatal development. A, total nuclei., B, perchloric acid extracts. day 18, transition
histones
7,
where it levels off at -16% of Hl. The midis at day 14. The amount of Hl" relative
increases
I I I I I I 0 20 50
about
seven-fold
I
I 100
I 200
in ten days.
I
I 300
I
DAYS
Figure
nuclei from rat 3. Accumulation of H1° in purified cerebral cortex during postnatal development. (0) Neuronal nuclei. (A) Glial nuclei. Each point is the average of six determinations from three different samples. 700
BIOCHEMICAL
Vol. 123, No. 2, 1984
In glial
H1° represents
cells
starting
to accumulate
increases 300 (the
steadily farthest
between
days
2-3% of Hl at birth,
18 and 21. Its
concentration
up to day 30 slowing down from then on. At day point examined) it represents "21% of Hl.
The amount of total within the standard
unchanged
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Hl relative deviation
to core histones remains (210%) during postnatal
development in both neurons and glial cells. It would be interesting to know whether H1° accumulates or substitutes for Hla and/or Hlb. Unfortunately, the standard deviation of the ratio total Hl/ core
histones
histones
(210%) is higher
than
the highest
ratio
Hl'/core
(2.5-3%).
DISCUSSION In this of accumulation bral
work we have characterized of histone
the different
H1° in neurons
and glia
modes
from rat
cere-
cortex. We have shown that
in neurons
the level
seven-fold during a period of ten days centered ning constant at 16% of Hl thereafter. In glial to accumulate rapidly until
represents is delayed
21% of Hl. Thus, by approximately
increases much more
accumulation nine days with
to neurons. It
is
firmly
established
mammalian forebrain do not tion begins long after the precursor
around day 14 remaicells H1° starts
between days 18 and 21. Its concentration day 30 and then continues to accumulate
slowly; at day 300 it of H1° in glial cells respect
of H1° increases
cells.
divide arrest
The existence
between the arrest of cell accumulation is inconsistent
that
neurons
after birth. of neuronal
of a lag period
proliferation with the
in the
cortex
of
Thus, H1° accumulaproliferation from of
and the involvement
several
days
onset of H1° of Hl'in the
arrest of neuronal proliferation. Instead, the period of accumulation of Hl' coincides with that of terminal differentiation of neurons, which is characterized by the growth and ramification of cell processes. By the age of 18 days, when accumulation of H1° is completed, the cerebral cortex has assumed most of the features characteristic of the adult (18). Proliferation
of glial
cells
in rat
cortex
is
largely
postnatal. It reaches a peak from 2-3 to 7-8 days after birth and is almost complete by the end of the second week after birth (19). The increase of the Hl" level in glial cells takes place 701
Vol. 123, No. 2, 1984
between is also tion tiation.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
days 18 and 21. Thus, as in neurons, accumulation clearly out of phase with cell proliferation.
Our results are consistent with of H1° is part of the events implicated
the
idea that in terminal
of H1° accumuladifferen-
ACKNOWLEDGMENTS We thank Dr. L. Cornudella for reading the manuscript and for suggestions. This work was supported by grants from the Comisidn Asesora de Investigaci6n Cientffica y T&nica of M.E.C. (1715/ Sanitarias de la Seguridad Social and 821, Fondo de Investigaciones CIRIT de la Generalitat de Catalunya. B. Piiia was supported by a fellowship from the Ministerio de EducaciBn y Ciencia (Spain). REFERENCES l23456789 10 11 12 13 14 15 16 17 -
Cole, R.D. (1977) in "Molecular Biology of the Mammalian Genetic Apparatus" (Ts'o, P., ed.) p. 93. Elsevier/North Holland, Amsterdam. Panyim, S. and Chalkley, R. (1969) Biophys. Biochem. Res. Commun. 2, 1042-1049. Balhorn, R., Chalkley, R. and Granner, D. (1972) Biochemistry, 11, 1094-1098. P.J. (1973) Fed. Proc. 32, Marsh, W.H. and Fitzerald, 2119-212s. Harris, M.R., Harbone, N., Smith, B.J. and Allan, J. (1982) Biophys. Biochem. Res. Commun. 109, 78-82. Gjerset, R., Gorka, L., HarthorK S., Lawrence, J.J. and Eisen, H. (1982) Proc. Natl. Acad. Sci. USA 79, 2333-2337. Benjamin, W.B. (1971) Nature (London) 234, 1%19. B. and Eisen, H. (lm) Proc. Natl. Acad. Keppel, F., Allet, Sci. USA 74, 653-656. Keppel, F., Allet, B. and Eisen, H. (1979) Eur. J. Biochem. 96, 477-482. flatanova, J., Oberhummer, K. and Swetly, P. (1980) in "In vivo and in vitro Erytropoiesis: The Friend System” (Rossi, Elsevier/North Holland, Amsterdam. G.B., ea.1 PP. 297-307, Pieler, C., Adolf, G.R. and Swetly, P, (1981) Eur. J. Biochem. 115, 329-333. Anna, J.A., Tobey, R.A. and Gurley, L.R. (1980) Biochemistry, 19, 2656-2671. Thompson, R.J. (1973) J. Neurochem. 21, 19-40. Laemmli, U.K. (1970) Nature 227, 680-D5. Panyim, S. and Chalkley, R. m69) Arch. Biochem. Biophys. 130, 337-346. Etstra, A. (1982) J. Biol. Chem. 257, 13088-13094. Thomas, J-0. and Thompson, R-J. (1977) Cell, -10, 633-640.
702
Vol. 123, No. 2, 1984 September
Pages 697-702
17, 1984
DIFFERENTIAL KINETICS OF HISTONE Hl' ACCUMULATION IN NEURONAL AND GLIAL CELLS FROM RAT CEREBRAL CORTEX DURING POSTNATAL DEVELOPMENT B. Piiia,
P. Martinez,
Departamento de Bioqufmica, Aut6noma de Barcelona.
Received
August
L. Simdn and P. Suau+ Facultad de Ciencias, Bellaterra, Barcelona,
Universidad Spain
6, 1984
The accumulation of histone H1° has been studied in neuronal and glial nuclei from rat cerebral cortex during postnatal development. In neurons Hl" represents -2% of the Hl content at birth and remains unchanged until day 8. Beyond this point H1° accumulates rapidly until day 18, where it levels off at 16% of Hl. The midpoint of the transition is at day 14. In glial cells Hl" represents -2.5% of the Hl at birth. It starts to accumulate between days 18 and 21; its concentration raises rapidly up to day 30 slowing down from then on. At day 300 (the farthest point examined) it represents 21% of Hl. These results are discussed in relation to the events of the postnatal development of the cerebral cortex in the rat. It is concluded that H1° probably does not suppress cell proliferation.
species
The Hl class of histones contains even within the same cellular type
fractions, HlO, is predominantly found or no cellular proliferation (2-5). It
several molecular (1). One of the sub-
in tissues
showing
little
accumulates after birth at various stages of development in mouse liver, brain, kidney, pancreas and retina (6). It has been shown to decrease in regenerating rat pancreas (7) and liver (4) and to be hormonally regulated in several gland tissues (6). H1° can be chemically induced -in vitro in several cell lines such as Friend erythroleukemia (8-lo), murine neuroblastoma (11) and Chinese hamster suggested that Hl" plays a role in the DNA replication
(2,4)
or in cell
ovary arrest
differentiation
(12). It of cell
has been division and
(11).
We have performed a detailed analysis of the accumulation of H1° in neuronal and glial nuclei from rat cerebral cortex during postnatal development. Both cellular types show distinct patterns of H1° accumulation that can be correlated with cell matu+To whom correspondence ABBREVIATIONS;
should
be addressed.
SDS; sodium didecyl
sulfate. 0006-291X/84 $1.50 697
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 123, No. 2, 1984
ration during HI0 reflects cated in cell
development. at the level maturation
MATERIALS
AND METHODS
Fractionation
of cortex
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The results suggest that accumulation of of histone composition the events impliand terminal differentiation.
nuclei
Cerebral cortices from male Sprage-Dawley rats were homogenized by hand with 30 up-and-down strokes of a Dounce homogenizer in 1M sucrose, 1mM sodium cacodylate, 5mM MgC12 and 1mM dithiothreitol, pH 6.5. Cortex nuclei were fractionated by the method of Thompson (13), except that a third layer of sucrose (2.6M) was added to improve the purity of the glial fraction. Neuronal nuclei were obtained from the interphase of the 2.4M and 1.8M sucrose layers, whereas glial nuclei were recovered as a pellet at the bottom of the 2.6M sucrose cushion. All operations were performed at 2'C and phenyl methyl sulphonyl fluoride (0.1 mM) was used throughout to inhibit proteolitic activity. Gel
electrophoresis
Purified nuclei were pelleted and resuspended in 1OmM EDTA, pH 7.4. After sonication proteins were extracted four times with HC10f.(5% v(v). Acid extracts were dialysed against 5% acetic acid and lophylized. When proteins from whole nuclei were to be analized by SDS-polyacrylamide gel electrophoresis the latter were dispersed by sonication in 1OmM EDTA, pH 7.4. Proteins were analyzed by electrophoresis in SDS-polyacrylamide gels (15%), essentially as described by Laemmli (14), and in urea-acetic acid-polyacrylamide gels by the method of Panyim and Chalkley (15). Slab gels were fixed and stained in methanol : water : acetic acid (5:5:1) containing 0.25% Coomassie Brillant Blue R-250 and destained in water : acetic acid : methanol : ethanol (7:l:l:l). After destaining the gels Were soaked in dimethyl sulfoxide for 2 minutes to remove all the stain background between bands and then resuspended in 10% acetic acid as described (16). Band intensities were quantified by gel scanning at 540 nm using a Beckman DU-8B spectrophotometer. Measure
of
the
chromatin
repeat
unit
Micrococcalnuclease digestion of nuclei and DNA extraction were performed according to Thomas and Thompson (17). DNA fragments were analyzed on 1% agarose slab gels and sized with a Hae III digest of @X 174 RF DNA (Bio Labs). RESULTS Neuronal and glial nuclear fractions were isolated from rat cerebral cortex. The deqree of cross-contamination of each nuclear type with the other was of the order of 10% for either nuclear fraction, as judged by phase contrast microscopy. To ascertain the purity of the nuclear fractions we made use of the known fact 698
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 123, No. 2, 1984
Hla+b, Hl”--
Figure
of proteins form neuronal 1. Gel electrophoresis and glial nuclei from rat cerebral cortex SDSpolyacrylamide gel electrophoresis of proteins. A, from neuronal and glial nuclei. Nuclei were from the same group of rats of 21 days age.
(Lane
1) Whole
Perchloric (Lane
neuronal
acid extract
3) Whole
glial
extract
the chromatin
from
nuclei.
neuronal
repeat
unit
nuclei.
(Lane
2)
from neuronal nuclei.
acid extract from glial acid gel electrophoresis
that
-
(Lane
4)
Perchloric
nuclei. B, urea-acetic of a prechloric acid nuclei.
of neuronal
nuclei
contains
160-170
base pairs as compared to 200 base pairs for nonastrocytic glial nuclei (17). Values of 16826 and 19626 base pairs were found for neuronal shown).
and glial
fractions,
Accumulation
respectively,
of H1° in neuronal
mined between 0 and 300 days after its mobility on SDS and urea-acetic
in adult
and glial
rats
nuclei
(not
was exa-
birth. H1° was identified acid-polyacrylamide gels
by (Figu-
Hl* to ordinary Hl (Hla+Hlb) and total Hl (Hla+ re 1). The ratios Hlb+Hl') to core histones (H2a+H2b+H3+H4) were obtained from electrophoretic analysis on SDS-polyacrylamide gels of whole purified nuclei or HC104 (5% v/v) extractable proteins. Both methods gave
the
same
values
within
the
stadistical
error.
Figure 2 shows examples of gel scans of Hl subfractions from specific times of the postnatal development. Kinetics of accumulation of H1° in neuronal and glial nuclei are shown in Figure 3. In neurons Hl“ represents "2% of H1 at birth and remains at the same level until day 8. Beyond this point H1° accumulates 699
BIOCHEMICAL
Vol. 123, No. 2, 1984
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
7 DAYS
12 DAYS
Figure
rapidly until point of the to core
lk h
21 DAYS
2. SDS-polyacrylamide gel electrophoresis profiles of neuronal nuclei Hl subfraction at times 0, 12 and 21 days of postnatal development. A, total nuclei., B, perchloric acid extracts. day 18, transition
histones
7,
where it levels off at -16% of Hl. The midis at day 14. The amount of Hl" relative
increases
I I I I I I 0 20 50
about
seven-fold
I
I 100
I 200
in ten days.
I
I 300
I
DAYS
Figure
nuclei from rat 3. Accumulation of H1° in purified cerebral cortex during postnatal development. (0) Neuronal nuclei. (A) Glial nuclei. Each point is the average of six determinations from three different samples. 700
BIOCHEMICAL
Vol. 123, No. 2, 1984
In glial
H1° represents
cells
starting
to accumulate
increases 300 (the
steadily farthest
between
days
2-3% of Hl at birth,
18 and 21. Its
concentration
up to day 30 slowing down from then on. At day point examined) it represents "21% of Hl.
The amount of total within the standard
unchanged
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Hl relative deviation
to core histones remains (210%) during postnatal
development in both neurons and glial cells. It would be interesting to know whether H1° accumulates or substitutes for Hla and/or Hlb. Unfortunately, the standard deviation of the ratio total Hl/ core
histones
histones
(210%) is higher
than
the highest
ratio
Hl'/core
(2.5-3%).
DISCUSSION In this of accumulation bral
work we have characterized of histone
the different
H1° in neurons
and glia
modes
from rat
cere-
cortex. We have shown that
in neurons
the level
seven-fold during a period of ten days centered ning constant at 16% of Hl thereafter. In glial to accumulate rapidly until
represents is delayed
21% of Hl. Thus, by approximately
increases much more
accumulation nine days with
to neurons. It
is
firmly
established
mammalian forebrain do not tion begins long after the precursor
around day 14 remaicells H1° starts
between days 18 and 21. Its concentration day 30 and then continues to accumulate
slowly; at day 300 it of H1° in glial cells respect
of H1° increases
cells.
divide arrest
The existence
between the arrest of cell accumulation is inconsistent
that
neurons
after birth. of neuronal
of a lag period
proliferation with the
in the
cortex
of
Thus, H1° accumulaproliferation from of
and the involvement
several
days
onset of H1° of Hl'in the
arrest of neuronal proliferation. Instead, the period of accumulation of Hl' coincides with that of terminal differentiation of neurons, which is characterized by the growth and ramification of cell processes. By the age of 18 days, when accumulation of H1° is completed, the cerebral cortex has assumed most of the features characteristic of the adult (18). Proliferation
of glial
cells
in rat
cortex
is
largely
postnatal. It reaches a peak from 2-3 to 7-8 days after birth and is almost complete by the end of the second week after birth (19). The increase of the Hl" level in glial cells takes place 701
Vol. 123, No. 2, 1984
between is also tion tiation.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
days 18 and 21. Thus, as in neurons, accumulation clearly out of phase with cell proliferation.
Our results are consistent with of H1° is part of the events implicated
the
idea that in terminal
of H1° accumuladifferen-
ACKNOWLEDGMENTS We thank Dr. L. Cornudella for reading the manuscript and for suggestions. This work was supported by grants from the Comisidn Asesora de Investigaci6n Cientffica y T&nica of M.E.C. (1715/ Sanitarias de la Seguridad Social and 821, Fondo de Investigaciones CIRIT de la Generalitat de Catalunya. B. Piiia was supported by a fellowship from the Ministerio de EducaciBn y Ciencia (Spain). REFERENCES l23456789 10 11 12 13 14 15 16 17 -
Cole, R.D. (1977) in "Molecular Biology of the Mammalian Genetic Apparatus" (Ts'o, P., ed.) p. 93. Elsevier/North Holland, Amsterdam. Panyim, S. and Chalkley, R. (1969) Biophys. Biochem. Res. Commun. 2, 1042-1049. Balhorn, R., Chalkley, R. and Granner, D. (1972) Biochemistry, 11, 1094-1098. P.J. (1973) Fed. Proc. 32, Marsh, W.H. and Fitzerald, 2119-212s. Harris, M.R., Harbone, N., Smith, B.J. and Allan, J. (1982) Biophys. Biochem. Res. Commun. 109, 78-82. Gjerset, R., Gorka, L., HarthorK S., Lawrence, J.J. and Eisen, H. (1982) Proc. Natl. Acad. Sci. USA 79, 2333-2337. Benjamin, W.B. (1971) Nature (London) 234, 1%19. B. and Eisen, H. (lm) Proc. Natl. Acad. Keppel, F., Allet, Sci. USA 74, 653-656. Keppel, F., Allet, B. and Eisen, H. (1979) Eur. J. Biochem. 96, 477-482. flatanova, J., Oberhummer, K. and Swetly, P. (1980) in "In vivo and in vitro Erytropoiesis: The Friend System” (Rossi, Elsevier/North Holland, Amsterdam. G.B., ea.1 PP. 297-307, Pieler, C., Adolf, G.R. and Swetly, P, (1981) Eur. J. Biochem. 115, 329-333. Anna, J.A., Tobey, R.A. and Gurley, L.R. (1980) Biochemistry, 19, 2656-2671. Thompson, R.J. (1973) J. Neurochem. 21, 19-40. Laemmli, U.K. (1970) Nature 227, 680-D5. Panyim, S. and Chalkley, R. m69) Arch. Biochem. Biophys. 130, 337-346. Etstra, A. (1982) J. Biol. Chem. 257, 13088-13094. Thomas, J-0. and Thompson, R-J. (1977) Cell, -10, 633-640.
702