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Selective inhibition of the semiconservative replication of mouse satellite DNA
PRELIMINARY
273
NOTES
BBA 9q264
Selective inhibition of the semiconservotive replicotion of mouse sotellite DNA When the nuclear DNA of the mouse (Mus musculus) is centrifuged to equilibrium in a CsC1 density gradient two bands are formed 1,*. The minor band has a buoyant density of 1.691 g" cm -~ and constitutes IO % of the total mass of nuclear DNA while the other band, found at a density of 1.7o2 g . c m -3, comprises the remaining 9 ° °/o1,~. The former, referred to as satellite DNA, has been isolated in a high state of purity ~ from a variety of mouse tissues; its chemical and physical properties have been extensively studied 4-1°. Although the DNA of the satellite has been shown to differ dramatically from the bulk of DNA present in the main band fraction, no differences have been observed relative to its rate of replication 5,6 or turnover 5,~ when compared to that oi the major band. The satellite is an integral part of the mouse genome s,x° and is, presumably, covalently attached to nucleotide sequences which comprise the main band 6,8. Nevertheless, we show, in this report, that exposure of mouse lymphoma cells (Fisher L5178Y) to high concentrations of sulfur mustard causes the uncoupling of the synthesis of satellite DNA from that of its main band. TABLE EXTENT
I OF ALKYLATION
OF MAIN BAND
AND SATELLITE
DNA's
M o u s e l y m p h o m a c e l l s ( L - 5 1 7 8 Y ) w e r e g r o w n i n s u s p e n s i o n c u l t u r e t o a c o n c e n t r a t i o n o f 3" lO6 e e l l s / m l ; o. 4 / ~ m o l e o f [ 8 5 S ] m u s t a r d g a s (63 ° m C / m m o l e ) w a s a d d e d t o 3 o o m l o f s u s p e n s i o n c u l t u r e . A f t e r 3 ° r a i n , D N A w a s e x t r a c t e d a n d f r a c t i o n a t e d o n p r e p a r a t i v e CsC1 d e n s i t y g r a d i e n t s as described elsewhere n. A and B are duplicate experiments.
Fxpt.
DNA
Counts per rain per ttg
Base pairs per alkyl group
G-C pairs per alkyl group
A
Main band Satellite
22. 9 19.o
I.O. io~ 1.2. lO5
0 . 4 2 . lO 5 0 . 4 2 - lO b
B
Main band Satellite
22. 7 18.o
1 . o . lOS 1. 3 • lO 5
0 . 4 2 . lO 5 o . 4 4 . io~
Treatment of lymphoma cells (grown in suspension culture, 3" lO5 cells/ml) with concentrations of [~sSlsulfur mustard (i/~M) that are IO times higher than the mean lethal dose results in the alkylation of about one DNA nucleotide per I . lO5 nucleotide pairs. The satellite and main band fractions are alkylated (Table I) to about the same extent. What small difference exists can be accounted for on the basis of differing G + C contents. Nevertheless, semiconservative replication of the satellite is more strongly inhibited than is the replication of the main band (Fig. I). To determine this we employed one of the standard methods used to demonstrate semiconservative replication in mammalian cells4. This involves adding relatively large quantities of 5-bromodeoxyuridine to the culture medium so that the halogenated pyrimidine is incorporated into new DNA instead of thymine. Because of the higher density imparted to the DNA by the bromine atom, the new DNA is Biochim. Biophys. Acta, 195 (1969) 2 7 3 - 2 7 5
274
PRELIMINARY NOTES
/¢/
(O]
__;'= J'L i
I
J
1.739
I
I
,,, I;l i ',~91
691
(b)
I I
i i
i 1.715
ft')
J
I t J t F
x__
1.717
Fig. I. Microdensitometer tracings of mouse D N A centrifuged to equilibrium in CsCI density gradients. I n a and b, l y m p h o m a cells were g r o w n for 4 h in the presence of 5 # g of 5-bromodeoxyuridine per ml culture m e d i u m , in c-f, cells were g r o w n for the same period in 13/~g 5-bromode°xY uridine per ml. ~SS]mustard gas (0.4/,mole) was added to 300 ml of culture m e d i u m (3' IO~ cells/ ml) 3 ° rain prior to the addition of 5-bromodeoxyuridine /a, c, e). The D N A ' s s h o w n in e and f r e p r e s e n t the h e a v y density material of c and d, respectively, and were obtained b y fractionation of c and d D N A ' s on p r e p a r a t i v e CsC1 density gradients TM. The satellite h e a v y h y b r i d was observed on the film to (d) and (f) at a density position of approx. 1.755 g. cm -8 b u t was n o t resolved b y the densitometer.
made more dense and thus becomes distinguishable from the original material. (The new DNA is called a hybrid duplex because it consists of the one heavy strand containing 5-bromodeoxyuridine and one light, pre-existing strand.) The extent to which the buoyant density of the hybrid is increased depends upon: (I) the thymine content of the DNA and (2) the extent to which 5-bromodeoxyuridine has replaced thymine in the newly replicated strand (this being dependent upon the relative concentrations of 5-bromodeoxyuridine and thymine). Hence, we find that the buoyant density of replicated DNA is higher when the concentration of 5-bromodeoxyuridine in the growth media is increased from 5 to 13 Fg/ml (Fig. I, a, b compared to c, d). With either concentration, however, it is evident that the small hybrid band at 1.715 g. cm -3 in (b) and 1.717 g. cm -3 (d) is exhibited only b y those cells which have not been exposed to mustard gas (Fig. I, b and d). This band is, in fact, a product of the semiconservative replication of mouse satellite DNA 4. It should be recognized, however, that unlike the major band which produces a single hybrid band following one generation in 5-bromodeoxyuridine, the satellite DNA produces two such bands following its replication 4. The reason for this is that one of the two strands of the satellite duplex contains over twice as m a n y thymine residues (H strand) as its complement (L strand) 7. Biochim. Biophys. Acta, 195 (1969) 273-275
275
PRELIMINARY NOTES
Hence, a hybrid consisting of a pre-existing H strand and a newly synthesized L strand contains less 5-bromodeoxyuridine and bands at a lower buoyant density than does the other possibility (the duplex consisting of a newly synthesized H strand and pre-existing L strand). The heavy hybrid is not readily visable from the tracings of the controls (Fig. I, b and d) because the extent to which 5-bromodeoxyuridine has replaced thymine was insufficient to provide the necessary resolution. (A heavy hybrid band could be discerned on the ultraviolet film to tracing (d) and (f) however.) In the event the light satellite hybrid was obscured by an overloading or spreading of the original main band (c), hybrid density material was prefractionated on preparative CsC1 gradients 3 and then recycled as shown in (e) (and (f) from (d)). A small amount of contamination from the nonreplicated main band is seen in both tracings (e and f) though only the untreated control shows any evidence of a satellite hybrid (f). Using the above method, the concentration of sulfur mustard required to depress by 5 ° % the replication of the satellite relative to that of the main band is approx, o.i #M (data not shown). The authors are grateful to Drs. Falk, Fishbein and Brubaker for their interest and advice.
National Institute o/Environmental Health Sciences, National Institutes o/ Health, Public Health Service and Department o] Health, Education, and Wel/are, Research Triangle Park, N.C. 27 709 (U.S.A.) I 2 3 4 5 6 7 8 9 IO ti
W. G. FLAMM N. J. BERNnEIM J. SPALDING
S. KIT, J. Mol. Biol., 3 (1961) 711. S. KIT, Nature, 193 (1962) 274. W. G. FLAMM, H. E. BOND AND H. E. BURR, Biochim. Biophys. Acta, 129 (1966) 31o. E. H. CHUN AND J. W. LIT'rLEFIELD, J. Mol. Biol., 7 (1963) 245" W. G. FLAMM, I-I. E. BOND, H. E. BURR AND G. B. BOND, Biochim. Biophys. Acta, 129 (1966) 652. H. E. BOND, W. G. FLAMM, H. E. BURR AND S. B. BOND, J. Mol. Biol., 27 (1967) 289. W. G. FLAMM, M. MCCALLUM AND P. M. B. WALKER, Proc. Natl. Acad. Sci. U.S., 57 (1967) 1729. W. G. FLAMM, P. M. B. WALKER AND M. MCCALLUM, J. Mol. Biol., 4 ° (1969) 423 . C. L. SCHILDKRAUT AND J. J. MAID, Biochim. Biophys. Acta, 161 (1968) 76. J. J. ~]~AIO AND C. L. SCHILDKRAUT,J. Mol. Biol., 4 ° (I969) 203. W. G. FLAMM, J. L. BIRNSTIEL AND P. M. B. WALKER, in G. D. BIRNIE AND S. M. F o x , Subcellular Components: Preparation and Fractionation, B u t t e r w o r t h s , London, 1969, p. 125-155.
Received August Ist, 1969 Biochim. Biophys. Acta, 195 (1969) 273-275
273
NOTES
BBA 9q264
Selective inhibition of the semiconservotive replicotion of mouse sotellite DNA When the nuclear DNA of the mouse (Mus musculus) is centrifuged to equilibrium in a CsC1 density gradient two bands are formed 1,*. The minor band has a buoyant density of 1.691 g" cm -~ and constitutes IO % of the total mass of nuclear DNA while the other band, found at a density of 1.7o2 g . c m -3, comprises the remaining 9 ° °/o1,~. The former, referred to as satellite DNA, has been isolated in a high state of purity ~ from a variety of mouse tissues; its chemical and physical properties have been extensively studied 4-1°. Although the DNA of the satellite has been shown to differ dramatically from the bulk of DNA present in the main band fraction, no differences have been observed relative to its rate of replication 5,6 or turnover 5,~ when compared to that oi the major band. The satellite is an integral part of the mouse genome s,x° and is, presumably, covalently attached to nucleotide sequences which comprise the main band 6,8. Nevertheless, we show, in this report, that exposure of mouse lymphoma cells (Fisher L5178Y) to high concentrations of sulfur mustard causes the uncoupling of the synthesis of satellite DNA from that of its main band. TABLE EXTENT
I OF ALKYLATION
OF MAIN BAND
AND SATELLITE
DNA's
M o u s e l y m p h o m a c e l l s ( L - 5 1 7 8 Y ) w e r e g r o w n i n s u s p e n s i o n c u l t u r e t o a c o n c e n t r a t i o n o f 3" lO6 e e l l s / m l ; o. 4 / ~ m o l e o f [ 8 5 S ] m u s t a r d g a s (63 ° m C / m m o l e ) w a s a d d e d t o 3 o o m l o f s u s p e n s i o n c u l t u r e . A f t e r 3 ° r a i n , D N A w a s e x t r a c t e d a n d f r a c t i o n a t e d o n p r e p a r a t i v e CsC1 d e n s i t y g r a d i e n t s as described elsewhere n. A and B are duplicate experiments.
Fxpt.
DNA
Counts per rain per ttg
Base pairs per alkyl group
G-C pairs per alkyl group
A
Main band Satellite
22. 9 19.o
I.O. io~ 1.2. lO5
0 . 4 2 . lO 5 0 . 4 2 - lO b
B
Main band Satellite
22. 7 18.o
1 . o . lOS 1. 3 • lO 5
0 . 4 2 . lO 5 o . 4 4 . io~
Treatment of lymphoma cells (grown in suspension culture, 3" lO5 cells/ml) with concentrations of [~sSlsulfur mustard (i/~M) that are IO times higher than the mean lethal dose results in the alkylation of about one DNA nucleotide per I . lO5 nucleotide pairs. The satellite and main band fractions are alkylated (Table I) to about the same extent. What small difference exists can be accounted for on the basis of differing G + C contents. Nevertheless, semiconservative replication of the satellite is more strongly inhibited than is the replication of the main band (Fig. I). To determine this we employed one of the standard methods used to demonstrate semiconservative replication in mammalian cells4. This involves adding relatively large quantities of 5-bromodeoxyuridine to the culture medium so that the halogenated pyrimidine is incorporated into new DNA instead of thymine. Because of the higher density imparted to the DNA by the bromine atom, the new DNA is Biochim. Biophys. Acta, 195 (1969) 2 7 3 - 2 7 5
274
PRELIMINARY NOTES
/¢/
(O]
__;'= J'L i
I
J
1.739
I
I
,,, I;l i ',~91
691
(b)
I I
i i
i 1.715
ft')
J
I t J t F
x__
1.717
Fig. I. Microdensitometer tracings of mouse D N A centrifuged to equilibrium in CsCI density gradients. I n a and b, l y m p h o m a cells were g r o w n for 4 h in the presence of 5 # g of 5-bromodeoxyuridine per ml culture m e d i u m , in c-f, cells were g r o w n for the same period in 13/~g 5-bromode°xY uridine per ml. ~SS]mustard gas (0.4/,mole) was added to 300 ml of culture m e d i u m (3' IO~ cells/ ml) 3 ° rain prior to the addition of 5-bromodeoxyuridine /a, c, e). The D N A ' s s h o w n in e and f r e p r e s e n t the h e a v y density material of c and d, respectively, and were obtained b y fractionation of c and d D N A ' s on p r e p a r a t i v e CsC1 density gradients TM. The satellite h e a v y h y b r i d was observed on the film to (d) and (f) at a density position of approx. 1.755 g. cm -8 b u t was n o t resolved b y the densitometer.
made more dense and thus becomes distinguishable from the original material. (The new DNA is called a hybrid duplex because it consists of the one heavy strand containing 5-bromodeoxyuridine and one light, pre-existing strand.) The extent to which the buoyant density of the hybrid is increased depends upon: (I) the thymine content of the DNA and (2) the extent to which 5-bromodeoxyuridine has replaced thymine in the newly replicated strand (this being dependent upon the relative concentrations of 5-bromodeoxyuridine and thymine). Hence, we find that the buoyant density of replicated DNA is higher when the concentration of 5-bromodeoxyuridine in the growth media is increased from 5 to 13 Fg/ml (Fig. I, a, b compared to c, d). With either concentration, however, it is evident that the small hybrid band at 1.715 g. cm -3 in (b) and 1.717 g. cm -3 (d) is exhibited only b y those cells which have not been exposed to mustard gas (Fig. I, b and d). This band is, in fact, a product of the semiconservative replication of mouse satellite DNA 4. It should be recognized, however, that unlike the major band which produces a single hybrid band following one generation in 5-bromodeoxyuridine, the satellite DNA produces two such bands following its replication 4. The reason for this is that one of the two strands of the satellite duplex contains over twice as m a n y thymine residues (H strand) as its complement (L strand) 7. Biochim. Biophys. Acta, 195 (1969) 273-275
275
PRELIMINARY NOTES
Hence, a hybrid consisting of a pre-existing H strand and a newly synthesized L strand contains less 5-bromodeoxyuridine and bands at a lower buoyant density than does the other possibility (the duplex consisting of a newly synthesized H strand and pre-existing L strand). The heavy hybrid is not readily visable from the tracings of the controls (Fig. I, b and d) because the extent to which 5-bromodeoxyuridine has replaced thymine was insufficient to provide the necessary resolution. (A heavy hybrid band could be discerned on the ultraviolet film to tracing (d) and (f) however.) In the event the light satellite hybrid was obscured by an overloading or spreading of the original main band (c), hybrid density material was prefractionated on preparative CsC1 gradients 3 and then recycled as shown in (e) (and (f) from (d)). A small amount of contamination from the nonreplicated main band is seen in both tracings (e and f) though only the untreated control shows any evidence of a satellite hybrid (f). Using the above method, the concentration of sulfur mustard required to depress by 5 ° % the replication of the satellite relative to that of the main band is approx, o.i #M (data not shown). The authors are grateful to Drs. Falk, Fishbein and Brubaker for their interest and advice.
National Institute o/Environmental Health Sciences, National Institutes o/ Health, Public Health Service and Department o] Health, Education, and Wel/are, Research Triangle Park, N.C. 27 709 (U.S.A.) I 2 3 4 5 6 7 8 9 IO ti
W. G. FLAMM N. J. BERNnEIM J. SPALDING
S. KIT, J. Mol. Biol., 3 (1961) 711. S. KIT, Nature, 193 (1962) 274. W. G. FLAMM, H. E. BOND AND H. E. BURR, Biochim. Biophys. Acta, 129 (1966) 31o. E. H. CHUN AND J. W. LIT'rLEFIELD, J. Mol. Biol., 7 (1963) 245" W. G. FLAMM, I-I. E. BOND, H. E. BURR AND G. B. BOND, Biochim. Biophys. Acta, 129 (1966) 652. H. E. BOND, W. G. FLAMM, H. E. BURR AND S. B. BOND, J. Mol. Biol., 27 (1967) 289. W. G. FLAMM, M. MCCALLUM AND P. M. B. WALKER, Proc. Natl. Acad. Sci. U.S., 57 (1967) 1729. W. G. FLAMM, P. M. B. WALKER AND M. MCCALLUM, J. Mol. Biol., 4 ° (1969) 423 . C. L. SCHILDKRAUT AND J. J. MAID, Biochim. Biophys. Acta, 161 (1968) 76. J. J. ~]~AIO AND C. L. SCHILDKRAUT,J. Mol. Biol., 4 ° (I969) 203. W. G. FLAMM, J. L. BIRNSTIEL AND P. M. B. WALKER, in G. D. BIRNIE AND S. M. F o x , Subcellular Components: Preparation and Fractionation, B u t t e r w o r t h s , London, 1969, p. 125-155.
Received August Ist, 1969 Biochim. Biophys. Acta, 195 (1969) 273-275