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DNA replication during muscle cell differentiation: identification of multiple DNA-dependent DNA polymerases
Cell Differentiation, 6 ( 1977 ) 9--16 © Elsevier/North-Holland Scientific Publishers Ltd.
9
DNA REPLICATION DURING MUSCLE CELL DIFFERENTIATION: IDENTIFICATION OF MULTIPLE DNA-DEPENDENT D N A POLYIV.:ERASES
Pasquale G R I P P O i, Giulio C O S S U 2 and Mario M O L I N A R O
2
C.N.R. Laborm¢ory of Molecular Embryology. Areo Feliee, Naples, Italy,and 2 Instituteof Histology and General Embryology, University of Rome, Italy Accepted 1 February, 1977
DNA-dependent DNA polymerases have been studied during chick embryo muscle differentiation in vitro. The total activity, extracted at both low and high ionic strengths, does no~ change throughout the differentiative process, although DNA synthesis stops at the moment of fusion. Analyses by glycerol gr~:dient centrifugation of the extracts at low and high ionic strengths show two major DNA polymerase forms, one sedimenting at 7.5 S and another at 3--4 S. Both enzymes are present in similar amqunts in duplicating myoblasts and in post-mitotic myotubes. These data suggest that the arrest of DNA synthesis which accompanies myoblast differentiation is not dependent on the disappearance or decrease of the major DNA polymerase activities described.
INTRODUCTION D N A synthesis is irreversibly b l o c k e d during m y o g e n e s i s s h o r t l y p r i o r t o m y o b l a s t fusion ( H o l t z e r , 1 9 7 0 ; B i s c h o f f et al., 1965). A l t h o u g h the m e c h a n i s m which regulates t h e w i t h d r a w a l o f m y o b l a s t s f r o m t h e cell cycle is still u n k n o w n , it c a n n o t be ascribed t o the c o n t a c t - i n h i b i t i o n m e c h a n i s m s which o c c u r in c o n f l u e n t cultures (Martz et al., 1972). In fact, m y o b l a s t s p r e v e n t e d f r o m fusing b y decreasing the Ca ~+ c o n c e n t r a t i o n o f the culture m e d i u m s t o p D N A synthesis a f t e r a few cycles, long b e f o r e t h e y reach confluence ( A d a m o et al., 1976). It has been r e p o r t e d b y O'Neill et al. ( 1 9 6 9 ) tha:~ a decrease in soluble D N A p o l y m e r a s e activity occurs in c u l t u r e d m y o blasts in c o n c o m i t a n c e with the arrest o f D N A replication. This suggests t h a t this e n z y m e p l a y s a regulative role in D N A synthesis. Since t w o f o r m s o f D N A p o l y m e r a s e have b e e n described in the chick e m b r y o ( B r u n e t al., 1 9 7 4 ) ( o n e s e d i m e n t i n g at 7.5 S and a n o t h e r at 3 S), we have investigated w h e t h e r the decrease in D N A p o l y m e r a s e activity, described b y these authors, could be ascribed to the d i s a p p e a r a n c e o f o n e or b o t h o f these e n z y m e species. H o w e v e r , in o u r c o n d i t i o n s , no qualitative or q u a n t i t a t i v e changes in over-all D N A - p o l y m e r a s e activity have been d e t e c t e d .
10
i;
MATERIALSAND METHODS
Culture conditions Muscle cell suspensions, obtained by trypsin treatment (Difco Trypsin 0.05%, 10 rain at 37°C) from breast muscle c.f ll-day-old chick embryos, were seeded in collagen.:coated plastic Petri dishes (Falcon Plastics) and cultured as previously described (Mclinaro et al., 1973).
Preparation o f cells At various times of culture, cells were incubated with 0.05% EDTA in Dulbecco's Phosphate Buffered Saline Ca2÷, Mg2+-free (CMF) for 10 min at 37°C, then gently scraped off until completely detached and centrifuged. After centrffugation, the pellet was suspend~l in 50 mM Tris-HC1 (pH 7.6), 5 mM MgC12, 2 mM 2-mereaptoethanol, 25 mH KC1, and homogenized in a motor-driven P o t ~ r homogenizer. In the expeJdments described in Table III, the pellet was also extracted in CMF (see above)and in a balanced salt solution (BSS) according to O'Neill et al. (1970). The homogenate was centrifuged at 10,000 rpm for 15 rain, the supernatant was collected and assayed for DNA polyraerase activity (low ionic strength so~!uble D:NA polymerase activity) or used for gradient analysis. The pellet was again extracted in 50 mM Tris-HC1 (pH 7.6), 5 mM MgC12, 2 mM 2-mercaptoethanol, 1 M KC1 and centrifuged at .~[0,000 rpm for 15 rain; the supernatant was a~ayed for DNA polymerase activity (high ionic strength soluble DNA polyraerase activity) or used for gradient analysis.
DNA polymerase assay DNA polymerase activity was assayed in a reaction mixture containing in a total volume of 0.3 ml: 50 mM potassium phosphate buffer (pH 7.4), 10 mM MgC1, 3.3 mM 2-mercaptoethanol, 30 ILg of activa~d salmon sperm DNA, 0.165 mM of each dCTP, dGTP, dATP (Boehtdnger) and tritiumlabelled dTTP (NEN) (specific activity of 1.5 :~ 10 s cpm/nmol). Assays were performed as previously described (Grippo et al., 1~75)o
Glycerol gradients sedimentation analysis 100 #1 of h!igb and low ionic strength extrac~ were :layered on l~near (10--30%) glycerol ~adients in 0.2 M potasshnn phosphate buffer {pH 7A), 0.2 M KC1, 2 ram 2-mercaptoethanol, 0.5 ram EDTA and centrifuged in a SW 50.1 rotor at 49,000 rpm for 14 h. Fractions of :[50 ~ul were collected and assayed as described above.
Yeast alcohol dehydrogenase (ADH) a~e~y The enzyme was assayed according go Vallee et al. (1955).
11. Protein and D N A measurements
An aliquot of the cell extract was used for determination of the protein content by Lowry's method. Bovine serum albumin (Sigma) was used as a standard. D N A c o n t e n t was m e a s u r e d b y t h e d i p h e n y l a m i n e reaction according to B u r t o n (1956). Calf t h y m u s D N A was used as a standard (Calbiochem). RESULTS
Chick embryo myoblasts in cultu:.~estart fusing after a few cell cycles, about 48 h after plating. The D N A content of these ceils increases during the duplicative phase, approaching a plateau after 72 h when fusion is complete (l~ig. I). D N A polymerase activity per plate, extracted with both low and high ionic strength buffers, rises during fusion, while the specific activity of the enzyme does not significantly change until at least 24 h after fusion (Table I). It should be pointed out that, in our conditions, about 30% of mononucleated cells (fibroblasts and non-fused myoblasts) axe stillpresent in culture after 72 h. In order to evaluate the contribution of these cells to t h e t o t a l D N A p o l y m e r a s e activil~y d e t e r m i n e d at this time, we m e a s u r e d their e n z y m a t i c activity a f t e r removal o f t h e m y o t u b e s b y filtration o f t h e cell suspension. Table II shows t h a t ~he activity o f t h e m o n o n u c l e a t e d cells a m o u n t s t o a b o u t 30% o f t o t a l activity, thus indicating t h a t their D N A polymerase activity is c o m p a r a b l e t o t h a t o f m y o t u b e s . These results are at vaxiance with t h o s e o f O'Neill et al. (1969), w h o have described a steep decline in t h e solube D N A p o l y m e r a s e activity in p r i m a r y
/el ///I*~S/S/ oj ~
o
~A
lO
~e of cuIturelhr)
Fig. !. DNA cot~tent and fusion in muscle cell cultures. Percentage of fusion was deterrnh~ed by counting 20 randomly selected fields of stained cultures. DNA content was me~ured ~ de~ribed in Materials and Methods.
12 TABLE I DNA polymerase specific activity at various times of culture. Age of culture
pmoles 3H TMP incorporated/p~ DNA
(h)
24 36 48 72 96
Low ionic strength soluble activity
High ionic strength Soluble activity
4.0 4.3 4.5 4.3 4.3
2.4 2.6 2.8 2.7 2.5
Extracts were prepared and DNA polymerase assays were carried out as described in Materials and Methods.
cultures o f c h i c k e m b r y o m y o b l a s t s a t t h e time: o f fusion. T h e possibility t h a t ~he differences m i g h t b e d u e t o d i f f e r e n t e x t r a c t i o n p r o c e d u r e s seems t o b e ruled o u t b y t h e d a t a p r e s e n t e d in T a b l e I I I . T h e d a t a s h o w n o differe n c e in t h e D N A p o l y m e r a s e activity w h e n m y o b l a s t s and fused m y o t u b e s w e r e e x t r a c t e d with e i t h e r BSS b u f f e r ( t h e m e d i u m used b y O'Neill et al.,
1970) or with CMF buffer (see Methods). In o r d e r t o a n s w e r t h e q u e s t i o n w h e t h e r changes in o n e o f t h e m u l t i p l e f o r m s described f o r c h i c k e m b r y o D N A c o u l d b e responsible f o r t h e arrest o f D N A synthesis, e x t r a c t s o f d u p l i c a t i n g cells a n d o f m y o t u b e s w e r e analysed b y c e n t r i f u g a t i o n in glycerol gradients. Figs. 2 a n d 3 s h o w glycerol gradients o f l o w a n d high ionic s t r e n ~ h e x t r a c t s o f d u p l i c a t i n g cells at 36 h {Fig. 2) a n d
TABLE H DNA polymerase content of post-mitotic non-fused cells.
pg DNA/phte % of fused nuclei m g of protein pmoles !H T M P incorporated plate pmoles 3H TMP incorporated pg D N A
Mononucleated cells
Mononucleated cells + myotubes
9.2 0 1.25 42
27.3 67 3.80 125
d.5
4.6
Non-fund ceiis-i7-2~.-of cuiture)-wereseparated f r o m myotubes by fiitra-~m-n-throu~g~a layer of cheese-cloth and collected by centrifugation. Microscope observation indicated tha~ no myotubes were present after filtration. Extraction procedures, DNA polymeric assays, DNA and protein measurements were carried out as described in Materials and Methods.
13 TABLE I I I DNA polymera.~-., specific ~ , i v i t y extracted with different buffers,
Age of vulture(h)
36 72
pmol~s 3H TMP incorporatedlpgD N A Tris-HCl buffer
CMF
BSS
4.5 4.3
5.8 5.2
5.0 4.9
Extracts were prepared and DNA polymerase assays were castled out as described in Materials and Methods.
o.f fused rnyotubes at 72 h (Fig. 3). In the low ionic strength extracts, of both duplicating myoblasts and mlfotubes, a main component sedimenting at 7 ~5 S and a small amount of low .~edimentingcomponent (3--4 S) have been d e t e c t e d . I n the h i g h i o n i c s t r e n g i ~ e x t r a c t s , t h e s i t u a t i o n is r e v e r s e d : b o t h in duplicating rnyoblas~ ~d
~ n y ( t u b e s t h e 3 - - 4 S s p e c i e s is t h e m a j o r c o r n -
ADH
R
z,,,,
o
-~
6
~E l--=z= 7
2
i, fraction number
Fig. 2. SedL~nentation pattern of duplicating rnyoblasts (36 h). 100 ~l samples were layered on linear (10--30%) glycerol gradients and sedimentation analysis performed as described in Materia~ and Methods. 25 ~l samples of each fraction were used for DNA polyrner~ ~y (see Methods). Yeast alcohol dehydrogenase (7.4 S) was used as a marker. Migration was from right to left. ~ . . . . . -e low ionic strength extracts; m. . . . . . m high ionic s t r e n ~ h extracts.
ADH
f;
~5 ft. ii Q. I"'-
/
2
''
-
I ~
;
.
/ 10
• 2O
fraction
30
number
,
F!~g. 3. S e d i m e n t a t i o n p a t t e r n o f m y o t u b e s (72 h). 1 0 0 pl samples were layered o n linear ( 1 0 - - 3 0 % ) glycerol gradients a n d sedimev .ation analysis p e r f o r m e d ~.9 descrlbed in Materials a n d M e t h o d s . 25 M! samples o f each f r a c t i o n were used for D N A p o l y m e r a s e assay (see M e t h o d s ) . Yeast alcohol d e h y d r o g e n a s e (7.4 S) was used as a m a r k e r . Migration was f r o m right to left. ®. . . . . -~ low ionic s t r e n g t h e x t r a c t s ; R- . . . . -m high ionic s t r e n g t h
extracts.
ponent, while the 7.5 S form is p r e ~ n t in a small amount. Furthermore, the relative proportion of the various DNA polymerase species does n o t show any significant difference between myoblasts and myotubes. DISCUSSION The results described in this paper show no changes in either the total DNA polymerase activity or in that of any one i ~ multiple forms during rayoblast differentiation at the time when DNA replication is halted. The two DNA polymerase components found in our experimental conditions have the same sedimentation coefficient and similar I,rL,perties to those already described in chick embryo extracts (Brun et al., 1974). However, it cannot be ruled out that, during myogenesis, DNA repJicat~on is under the control t,f either some minor form of DNA polymerase, or of factor: which interact i'n vivo with the enzyme(s) and which are lost during extraction. On the other hand, regulation could be achieved by the depletion of the nueleotide pool (Berlin et al., 1975). In this connection, our finding (Molinaro et
al., in p ~ p a r a t i o n ) o f a decrease o f t h y m k t i n e u p t a k e at the time o f fusion is suggestive o f an alternat.ive f o r m o f control. O'Neill et al. ( 1 9 6 9 ) have described a decrease in D N A r ~lymerase activity occurring at fusion. B u t it should be n o t e d t h a t t h e y have s u b m i t t e d their cultures t o very f r e q u e n t serum changes, which are k n o w n t o stimulate cell division in s t a t i o n a r y cultures ar, d t o s y n c h r o n i z e t h e cell p o p u l a t i o n (Griffiths, 1971). As a m a t t e r o f facL in O'Neill e t al.'s cultures t h e D N A c o n t e n t p e r plate still increased steadily after fusion, w h e n m o r e t h a n 80% o f t h e nuclei should be present in t h e m y o t u b e s and c o n s e q u e n t l y should have s t o p p e d D N A synthesis. T h e r e f o r e , the change observed b y t h e m should p r o b a b l y be ascribed t o changes in D N A p o l y m e r a s e activity occurring during t h e cell cycle (Bollum, 1975). In conclusion, it seems likely t h a t the arrest o f D N A replication in m y o genesis is n o t regulated t h r o u g h changes in D N A p o l y m e r a s e activity, at least o f t h e f o r m s which have been d e t e c t e d b y o u r analytical procedures, On t h e o t h e r hand, o u r results are in a g r e e m e n t w~th t h e lack o f correlation, which has b e e n r e p o r t e d in o t h e r biological systems, b e t w e e n the ability o f a cell t o u n d e r g o D N A replication and t h e presence o f D N A p o l y m e r a s e activity. F o r example, all t h e m a j o r D N A p o l y m e r a s e species which are also d e t e c t a b l e a f t e r fertilization and in e m b r y o s up to at least t h e gastrula stage are also p r e s e n t in unfertilized eggs o f X e n o p u s laevis ( G r i p p o e t al., 1976). ACKNOWLEDGEMENTS We .wish to thank Professors A. Monroy and J. Braehet for reading this mav.useript, Dr. B. Zani for helpful discussion and Mr. G. Locorotondo for excellent technical assistance. This work has been in part suppc,rted by grants from C.N.R. and from the Muscular Dystrophy A~ociation Inc. of America. REFERENCES Adamo, S., B. ,'ani, G. Siracusa and M. Molinaro: Cell Diff. 5, 53--76 (1976). Berlin, R.D. ann J.M. Olivier: In: International Review of Cytology, eds. G.H. Bourne and J.F. Jan;. ill, Vol. 42 (Academic Press, New York) pp. 287--338 (1975). B~ehoff, R. and H. Holtzer: J. Cell Biol. 41, 188--195 (1965). Bollum, F.J.: In: Progess in Nucleic Acid Research and Molecular Biology, ed. W.E. Cohn, Vol. 15, (Academic Press, New York) pp. 109--144 (1975). Brun, G., F. Rougaon, M. Lauber and F. Chape,~iI|e: Eur. J. Biochem. 41,241--251 (1974). Burton, K.: B~uehem. J. 62,315--319 (195~!3). Griffiths, J.B~: J. Cell Sci. 8, 43--.52 (1971). Grippo, P., G. Locorotondo and A. Caruso: FEBS Le~t. 51,137--142 (1975). Grippo, P., A. Caruso, G. Locorot~ndo and T. Labella: Cell Diff. 5,121--128 {1976). Holtzer, H.: In: Cell Differentiation, eds. I).A. S~:hijeide and J. De Vellis (Van Nostrand Relnhold Co., New York) pp. 476--503 c11970j. Lowry, O., Rosebrough, A. Farr and R. Randall: J. Biol. Chem. 193,265--275 (1951). MartT,, E. and M.S. Steinberg: J. Cell Physiol. 79:189--210 (1972). Molinaro, M. and M. Martinozzi: Exp. Cell Res. 78,329--334 (1973).
10 Molinaro, M., G. C o . u , B. Zani and M. Pacific'i: (in vreparat|0n). O'Neill, M. and R.C. Strohman: J. Cell Physiol. 73, 61--67 (1969)~ O'Neill, M. ~nd R.C. Strohman" Biochemistry 9, 2832--2838 (19"*.~'s. Vallee, B.L. m d F.L. Hoch: Proc. Natl. Acad. Sci. US 41, ~27--3~8 (1955).
9
DNA REPLICATION DURING MUSCLE CELL DIFFERENTIATION: IDENTIFICATION OF MULTIPLE DNA-DEPENDENT D N A POLYIV.:ERASES
Pasquale G R I P P O i, Giulio C O S S U 2 and Mario M O L I N A R O
2
C.N.R. Laborm¢ory of Molecular Embryology. Areo Feliee, Naples, Italy,and 2 Instituteof Histology and General Embryology, University of Rome, Italy Accepted 1 February, 1977
DNA-dependent DNA polymerases have been studied during chick embryo muscle differentiation in vitro. The total activity, extracted at both low and high ionic strengths, does no~ change throughout the differentiative process, although DNA synthesis stops at the moment of fusion. Analyses by glycerol gr~:dient centrifugation of the extracts at low and high ionic strengths show two major DNA polymerase forms, one sedimenting at 7.5 S and another at 3--4 S. Both enzymes are present in similar amqunts in duplicating myoblasts and in post-mitotic myotubes. These data suggest that the arrest of DNA synthesis which accompanies myoblast differentiation is not dependent on the disappearance or decrease of the major DNA polymerase activities described.
INTRODUCTION D N A synthesis is irreversibly b l o c k e d during m y o g e n e s i s s h o r t l y p r i o r t o m y o b l a s t fusion ( H o l t z e r , 1 9 7 0 ; B i s c h o f f et al., 1965). A l t h o u g h the m e c h a n i s m which regulates t h e w i t h d r a w a l o f m y o b l a s t s f r o m t h e cell cycle is still u n k n o w n , it c a n n o t be ascribed t o the c o n t a c t - i n h i b i t i o n m e c h a n i s m s which o c c u r in c o n f l u e n t cultures (Martz et al., 1972). In fact, m y o b l a s t s p r e v e n t e d f r o m fusing b y decreasing the Ca ~+ c o n c e n t r a t i o n o f the culture m e d i u m s t o p D N A synthesis a f t e r a few cycles, long b e f o r e t h e y reach confluence ( A d a m o et al., 1976). It has been r e p o r t e d b y O'Neill et al. ( 1 9 6 9 ) tha:~ a decrease in soluble D N A p o l y m e r a s e activity occurs in c u l t u r e d m y o blasts in c o n c o m i t a n c e with the arrest o f D N A replication. This suggests t h a t this e n z y m e p l a y s a regulative role in D N A synthesis. Since t w o f o r m s o f D N A p o l y m e r a s e have b e e n described in the chick e m b r y o ( B r u n e t al., 1 9 7 4 ) ( o n e s e d i m e n t i n g at 7.5 S and a n o t h e r at 3 S), we have investigated w h e t h e r the decrease in D N A p o l y m e r a s e activity, described b y these authors, could be ascribed to the d i s a p p e a r a n c e o f o n e or b o t h o f these e n z y m e species. H o w e v e r , in o u r c o n d i t i o n s , no qualitative or q u a n t i t a t i v e changes in over-all D N A - p o l y m e r a s e activity have been d e t e c t e d .
10
i;
MATERIALSAND METHODS
Culture conditions Muscle cell suspensions, obtained by trypsin treatment (Difco Trypsin 0.05%, 10 rain at 37°C) from breast muscle c.f ll-day-old chick embryos, were seeded in collagen.:coated plastic Petri dishes (Falcon Plastics) and cultured as previously described (Mclinaro et al., 1973).
Preparation o f cells At various times of culture, cells were incubated with 0.05% EDTA in Dulbecco's Phosphate Buffered Saline Ca2÷, Mg2+-free (CMF) for 10 min at 37°C, then gently scraped off until completely detached and centrifuged. After centrffugation, the pellet was suspend~l in 50 mM Tris-HC1 (pH 7.6), 5 mM MgC12, 2 mM 2-mereaptoethanol, 25 mH KC1, and homogenized in a motor-driven P o t ~ r homogenizer. In the expeJdments described in Table III, the pellet was also extracted in CMF (see above)and in a balanced salt solution (BSS) according to O'Neill et al. (1970). The homogenate was centrifuged at 10,000 rpm for 15 rain, the supernatant was collected and assayed for DNA polyraerase activity (low ionic strength so~!uble D:NA polymerase activity) or used for gradient analysis. The pellet was again extracted in 50 mM Tris-HC1 (pH 7.6), 5 mM MgC12, 2 mM 2-mercaptoethanol, 1 M KC1 and centrifuged at .~[0,000 rpm for 15 rain; the supernatant was a~ayed for DNA polymerase activity (high ionic strength soluble DNA polyraerase activity) or used for gradient analysis.
DNA polymerase assay DNA polymerase activity was assayed in a reaction mixture containing in a total volume of 0.3 ml: 50 mM potassium phosphate buffer (pH 7.4), 10 mM MgC1, 3.3 mM 2-mercaptoethanol, 30 ILg of activa~d salmon sperm DNA, 0.165 mM of each dCTP, dGTP, dATP (Boehtdnger) and tritiumlabelled dTTP (NEN) (specific activity of 1.5 :~ 10 s cpm/nmol). Assays were performed as previously described (Grippo et al., 1~75)o
Glycerol gradients sedimentation analysis 100 #1 of h!igb and low ionic strength extrac~ were :layered on l~near (10--30%) glycerol ~adients in 0.2 M potasshnn phosphate buffer {pH 7A), 0.2 M KC1, 2 ram 2-mercaptoethanol, 0.5 ram EDTA and centrifuged in a SW 50.1 rotor at 49,000 rpm for 14 h. Fractions of :[50 ~ul were collected and assayed as described above.
Yeast alcohol dehydrogenase (ADH) a~e~y The enzyme was assayed according go Vallee et al. (1955).
11. Protein and D N A measurements
An aliquot of the cell extract was used for determination of the protein content by Lowry's method. Bovine serum albumin (Sigma) was used as a standard. D N A c o n t e n t was m e a s u r e d b y t h e d i p h e n y l a m i n e reaction according to B u r t o n (1956). Calf t h y m u s D N A was used as a standard (Calbiochem). RESULTS
Chick embryo myoblasts in cultu:.~estart fusing after a few cell cycles, about 48 h after plating. The D N A content of these ceils increases during the duplicative phase, approaching a plateau after 72 h when fusion is complete (l~ig. I). D N A polymerase activity per plate, extracted with both low and high ionic strength buffers, rises during fusion, while the specific activity of the enzyme does not significantly change until at least 24 h after fusion (Table I). It should be pointed out that, in our conditions, about 30% of mononucleated cells (fibroblasts and non-fused myoblasts) axe stillpresent in culture after 72 h. In order to evaluate the contribution of these cells to t h e t o t a l D N A p o l y m e r a s e activil~y d e t e r m i n e d at this time, we m e a s u r e d their e n z y m a t i c activity a f t e r removal o f t h e m y o t u b e s b y filtration o f t h e cell suspension. Table II shows t h a t ~he activity o f t h e m o n o n u c l e a t e d cells a m o u n t s t o a b o u t 30% o f t o t a l activity, thus indicating t h a t their D N A polymerase activity is c o m p a r a b l e t o t h a t o f m y o t u b e s . These results are at vaxiance with t h o s e o f O'Neill et al. (1969), w h o have described a steep decline in t h e solube D N A p o l y m e r a s e activity in p r i m a r y
/el ///I*~S/S/ oj ~
o
~A
lO
~e of cuIturelhr)
Fig. !. DNA cot~tent and fusion in muscle cell cultures. Percentage of fusion was deterrnh~ed by counting 20 randomly selected fields of stained cultures. DNA content was me~ured ~ de~ribed in Materials and Methods.
12 TABLE I DNA polymerase specific activity at various times of culture. Age of culture
pmoles 3H TMP incorporated/p~ DNA
(h)
24 36 48 72 96
Low ionic strength soluble activity
High ionic strength Soluble activity
4.0 4.3 4.5 4.3 4.3
2.4 2.6 2.8 2.7 2.5
Extracts were prepared and DNA polymerase assays were carried out as described in Materials and Methods.
cultures o f c h i c k e m b r y o m y o b l a s t s a t t h e time: o f fusion. T h e possibility t h a t ~he differences m i g h t b e d u e t o d i f f e r e n t e x t r a c t i o n p r o c e d u r e s seems t o b e ruled o u t b y t h e d a t a p r e s e n t e d in T a b l e I I I . T h e d a t a s h o w n o differe n c e in t h e D N A p o l y m e r a s e activity w h e n m y o b l a s t s and fused m y o t u b e s w e r e e x t r a c t e d with e i t h e r BSS b u f f e r ( t h e m e d i u m used b y O'Neill et al.,
1970) or with CMF buffer (see Methods). In o r d e r t o a n s w e r t h e q u e s t i o n w h e t h e r changes in o n e o f t h e m u l t i p l e f o r m s described f o r c h i c k e m b r y o D N A c o u l d b e responsible f o r t h e arrest o f D N A synthesis, e x t r a c t s o f d u p l i c a t i n g cells a n d o f m y o t u b e s w e r e analysed b y c e n t r i f u g a t i o n in glycerol gradients. Figs. 2 a n d 3 s h o w glycerol gradients o f l o w a n d high ionic s t r e n ~ h e x t r a c t s o f d u p l i c a t i n g cells at 36 h {Fig. 2) a n d
TABLE H DNA polymerase content of post-mitotic non-fused cells.
pg DNA/phte % of fused nuclei m g of protein pmoles !H T M P incorporated plate pmoles 3H TMP incorporated pg D N A
Mononucleated cells
Mononucleated cells + myotubes
9.2 0 1.25 42
27.3 67 3.80 125
d.5
4.6
Non-fund ceiis-i7-2~.-of cuiture)-wereseparated f r o m myotubes by fiitra-~m-n-throu~g~a layer of cheese-cloth and collected by centrifugation. Microscope observation indicated tha~ no myotubes were present after filtration. Extraction procedures, DNA polymeric assays, DNA and protein measurements were carried out as described in Materials and Methods.
13 TABLE I I I DNA polymera.~-., specific ~ , i v i t y extracted with different buffers,
Age of vulture(h)
36 72
pmol~s 3H TMP incorporatedlpgD N A Tris-HCl buffer
CMF
BSS
4.5 4.3
5.8 5.2
5.0 4.9
Extracts were prepared and DNA polymerase assays were castled out as described in Materials and Methods.
o.f fused rnyotubes at 72 h (Fig. 3). In the low ionic strength extracts, of both duplicating myoblasts and mlfotubes, a main component sedimenting at 7 ~5 S and a small amount of low .~edimentingcomponent (3--4 S) have been d e t e c t e d . I n the h i g h i o n i c s t r e n g i ~ e x t r a c t s , t h e s i t u a t i o n is r e v e r s e d : b o t h in duplicating rnyoblas~ ~d
~ n y ( t u b e s t h e 3 - - 4 S s p e c i e s is t h e m a j o r c o r n -
ADH
R
z,,,,
o
-~
6
~E l--=z= 7
2
i, fraction number
Fig. 2. SedL~nentation pattern of duplicating rnyoblasts (36 h). 100 ~l samples were layered on linear (10--30%) glycerol gradients and sedimentation analysis performed as described in Materia~ and Methods. 25 ~l samples of each fraction were used for DNA polyrner~ ~y (see Methods). Yeast alcohol dehydrogenase (7.4 S) was used as a marker. Migration was from right to left. ~ . . . . . -e low ionic strength extracts; m. . . . . . m high ionic s t r e n ~ h extracts.
ADH
f;
~5 ft. ii Q. I"'-
/
2
''
-
I ~
;
.
/ 10
• 2O
fraction
30
number
,
F!~g. 3. S e d i m e n t a t i o n p a t t e r n o f m y o t u b e s (72 h). 1 0 0 pl samples were layered o n linear ( 1 0 - - 3 0 % ) glycerol gradients a n d sedimev .ation analysis p e r f o r m e d ~.9 descrlbed in Materials a n d M e t h o d s . 25 M! samples o f each f r a c t i o n were used for D N A p o l y m e r a s e assay (see M e t h o d s ) . Yeast alcohol d e h y d r o g e n a s e (7.4 S) was used as a m a r k e r . Migration was f r o m right to left. ®. . . . . -~ low ionic s t r e n g t h e x t r a c t s ; R- . . . . -m high ionic s t r e n g t h
extracts.
ponent, while the 7.5 S form is p r e ~ n t in a small amount. Furthermore, the relative proportion of the various DNA polymerase species does n o t show any significant difference between myoblasts and myotubes. DISCUSSION The results described in this paper show no changes in either the total DNA polymerase activity or in that of any one i ~ multiple forms during rayoblast differentiation at the time when DNA replication is halted. The two DNA polymerase components found in our experimental conditions have the same sedimentation coefficient and similar I,rL,perties to those already described in chick embryo extracts (Brun et al., 1974). However, it cannot be ruled out that, during myogenesis, DNA repJicat~on is under the control t,f either some minor form of DNA polymerase, or of factor: which interact i'n vivo with the enzyme(s) and which are lost during extraction. On the other hand, regulation could be achieved by the depletion of the nueleotide pool (Berlin et al., 1975). In this connection, our finding (Molinaro et
al., in p ~ p a r a t i o n ) o f a decrease o f t h y m k t i n e u p t a k e at the time o f fusion is suggestive o f an alternat.ive f o r m o f control. O'Neill et al. ( 1 9 6 9 ) have described a decrease in D N A r ~lymerase activity occurring at fusion. B u t it should be n o t e d t h a t t h e y have s u b m i t t e d their cultures t o very f r e q u e n t serum changes, which are k n o w n t o stimulate cell division in s t a t i o n a r y cultures ar, d t o s y n c h r o n i z e t h e cell p o p u l a t i o n (Griffiths, 1971). As a m a t t e r o f facL in O'Neill e t al.'s cultures t h e D N A c o n t e n t p e r plate still increased steadily after fusion, w h e n m o r e t h a n 80% o f t h e nuclei should be present in t h e m y o t u b e s and c o n s e q u e n t l y should have s t o p p e d D N A synthesis. T h e r e f o r e , the change observed b y t h e m should p r o b a b l y be ascribed t o changes in D N A p o l y m e r a s e activity occurring during t h e cell cycle (Bollum, 1975). In conclusion, it seems likely t h a t the arrest o f D N A replication in m y o genesis is n o t regulated t h r o u g h changes in D N A p o l y m e r a s e activity, at least o f t h e f o r m s which have been d e t e c t e d b y o u r analytical procedures, On t h e o t h e r hand, o u r results are in a g r e e m e n t w~th t h e lack o f correlation, which has b e e n r e p o r t e d in o t h e r biological systems, b e t w e e n the ability o f a cell t o u n d e r g o D N A replication and t h e presence o f D N A p o l y m e r a s e activity. F o r example, all t h e m a j o r D N A p o l y m e r a s e species which are also d e t e c t a b l e a f t e r fertilization and in e m b r y o s up to at least t h e gastrula stage are also p r e s e n t in unfertilized eggs o f X e n o p u s laevis ( G r i p p o e t al., 1976). ACKNOWLEDGEMENTS We .wish to thank Professors A. Monroy and J. Braehet for reading this mav.useript, Dr. B. Zani for helpful discussion and Mr. G. Locorotondo for excellent technical assistance. This work has been in part suppc,rted by grants from C.N.R. and from the Muscular Dystrophy A~ociation Inc. of America. REFERENCES Adamo, S., B. ,'ani, G. Siracusa and M. Molinaro: Cell Diff. 5, 53--76 (1976). Berlin, R.D. ann J.M. Olivier: In: International Review of Cytology, eds. G.H. Bourne and J.F. Jan;. ill, Vol. 42 (Academic Press, New York) pp. 287--338 (1975). B~ehoff, R. and H. Holtzer: J. Cell Biol. 41, 188--195 (1965). Bollum, F.J.: In: Progess in Nucleic Acid Research and Molecular Biology, ed. W.E. Cohn, Vol. 15, (Academic Press, New York) pp. 109--144 (1975). Brun, G., F. Rougaon, M. Lauber and F. Chape,~iI|e: Eur. J. Biochem. 41,241--251 (1974). Burton, K.: B~uehem. J. 62,315--319 (195~!3). Griffiths, J.B~: J. Cell Sci. 8, 43--.52 (1971). Grippo, P., G. Locorotondo and A. Caruso: FEBS Le~t. 51,137--142 (1975). Grippo, P., A. Caruso, G. Locorot~ndo and T. Labella: Cell Diff. 5,121--128 {1976). Holtzer, H.: In: Cell Differentiation, eds. I).A. S~:hijeide and J. De Vellis (Van Nostrand Relnhold Co., New York) pp. 476--503 c11970j. Lowry, O., Rosebrough, A. Farr and R. Randall: J. Biol. Chem. 193,265--275 (1951). MartT,, E. and M.S. Steinberg: J. Cell Physiol. 79:189--210 (1972). Molinaro, M. and M. Martinozzi: Exp. Cell Res. 78,329--334 (1973).
10 Molinaro, M., G. C o . u , B. Zani and M. Pacific'i: (in vreparat|0n). O'Neill, M. and R.C. Strohman: J. Cell Physiol. 73, 61--67 (1969)~ O'Neill, M. ~nd R.C. Strohman" Biochemistry 9, 2832--2838 (19"*.~'s. Vallee, B.L. m d F.L. Hoch: Proc. Natl. Acad. Sci. US 41, ~27--3~8 (1955).