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Density labelling in vivo of replicating DNA in a rat
173
Biochimica et Biophysica Acta, 349 (1974) 173--177 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA
97993
DENSITY L A B E L L I N G IN VIVO OF REPLICATING DNA IN A RAT
H.J. D A N P U R E
and M.G. O R M E R O D
Chester Beatty Research Institute,Clifton Avenue Belmont, Sutton, Surrey SM2-5PX (Great Britain) (Received December 3rd, 1973)
Summary A m e t h o d for labelling newly replicated DNA in tissues of a rat is described. Rats were infused with a mixture of bromodeoxyuridine and [3 H] thymidine. After the animal had been killed and the different tissues extracted, the heavy newly replicated DNA could be separated from the light parental DNA on a density gradient of CsC1.
Introduction If cells are incubated in the presence of 5'-bromodeoxyuridine, this compound is incorporated into the cellular DNA in place of thymidine. The resulting DNA has a higher density than normal DNA and can be separated isopycnicaUy on CsC1 gradients. This technique has been used to study the replication of normal DNA [1] and to study the "repair replication" of damaged DNA [2] in cells grown in tissue culture. We wanted to evaluate the possibility of studying repair replication in the intact animal. To this end, we have develped a m e t h o d of density labelling newly replicated DNA in the rat. Since this technique may be of interest to workers in other fields we have described it in this paper. Experimental m e t h o d s Rats
Male Wistar rats of average weight about 320 g were used. Cannulation procedure. The animal was anaesthetised with ether before making an incision alongside one of the lateral tail veins. The exposed vein was carefully dissected o u t from the surrounding connective tissue, ligated with sterile thread and a small incision made on the posterior vena cava side of the ligature. A sterile intravenous cannula of outside diameter 0.63 mm, to which a syringe of heparinised isotonic saline (100 units of heparin per ml of saline) had
174
previously been attached, was gently inserted into the vein until 3--4 cm were in the animal. It was then tied to the vein and the heparinised saline was slowly forced into the vein to check t h a t the cannula was firmly in position and was not leaking. The skin on the tail was sewn up and the cannula was taped to the tail. The animal was then placed in a restraining cage which prevented it from moving except in a backward or forward direction. The syringe of heparinised saline was exchanged for one containing isotonic saline alone and attached to a slow infusion pump, which was set in motion to prevent blocking of the cannula. When the animal had recovered from the anaesthetic (usually about 15 min), the syringe of saline was removed and replaced by one containing unlabeUed DNA precursors. In some experiments, we cannulated the femoral vein. The cannulation procedure was the same as before but extreme care had to be taken when dissecting out the vein as it is closely aligned to the femoral artery and nerve. After the vein had been cannulated, the body wall was sewn up and the cannula was taped to the rat's thigh. Preparation of tissues. After the rat had been labelled with DNA precursors as described in Results and Discussion, the animal was removed from the restraining cage, killed with ether and the different organs removed. The spleen was rinsed in sterile TC199 tissue culture medium (Wellcome Reagents Ltd.) and cut longitudinally in half. The cells were forced out of the capsule by gently squeezing the spleen with a pair of forceps. The capsule was discarded, the cells were pelleted at 500 × g for 2 min and the pellet was taken up in 15 times its volume of TC199 medium. The t h y m u s was rinsed in TC199 medium and a cell suspension prepared by forcing the tissue through a coarse wire mesh. The cells were pelleted and the pellet suspended in 20 times its volume. Bone marrow cells were extracted from the two femurs by dissecting out the bones, cutting off the epiphyses and flushing out the cells with a syringe of TC199 medium. The cells were pelleted and resuspended in 10 .times the volume of the cell pellet. Epithelial cells from the inner wall of the small intestine were prepared by dissecting out a portion of the small intestine adjacent to the d u o d e n u m . The food was flushed out using a large syringe filled with 0.25 M sucrose. The instestine was then cut longitudinally and the mucosal epithelial cells were scraped off the inner wall with a scalpel. The cells were suspended in TC199 medium and pelleted. The liver was thoroughly rinsed in ice-cold 0.25 M sucrose and chopped up. All the connective tissue was discarded and an approx. 25% (w/v) suspension of liver in sucrose was homogenised using three strokes of a Potter-Elvejheim homogeniser. The cell suspension was centrifuged at 1000 × g for 10 min at 4°C to pellet the nuclei and unbroken cells. 0.5 ml of the pellet was added to 1.0 ml sucrose.
Extraction of DNA This was a modification of 0.3 ml aliquots of the different carbonate centrifuge tubes (MSE sulphate, 0.005 M Na4 EDTA and
the m e t h o d described by Flamm et al. [3]. cell suspensions were put into 10-ml polyLtd.) containing 0.2 ml 2% sodium dodecylsheared once through a 19 G syringe needle.
175 4 ml of alkaline CsC1 (refractive index 1.4070 and density 1.786 g/ml) containing 0.02 M EDTA, pH 12.75, were added to each sample and sheared again. Centrifugation at 12000 rev./min for 20 min in the MSE 10 ml X 10 ml titanium angle head rotor on the MSE super speed 65 centrifuge gave a solid pellicle of precipitated protein, which floated on top of the CsC1 solution. 4 ml of the clear liquid from below the peUicle were removed with a syringe and 0.1 ml of the remainder was put onto a filter paper disc to measure the total radioactivity on the gradient. The tube was washed out, dried and the 4 ml of CsC1--DNA solution were returned. 0.02 ml 32 P-labelled T2DNA was added as a marker. Solid CsC1 was then added to bring the refractive index to 1.4055 (density = 1.77 g/ml) as measured on an Abb~ refractometer. The solution was then overlaid with liquid paraffin, the tubes were capped and centrifuged in the 10 ml X 10 ml titanium angle head rotor at 50000 rev./min at 20°C overnight. After centrifugation, the polypropylene tubes were punctured using an MSE tube piercer and 6-drop fractions were collected directly onto 2.5 cm 3-MM Whatman filter paper discs. The discs were washed in ice-cold 10% trichloroacetic acid, rinsed twice in absolute ethanol and dried in acetone before counting in a Packard liquid scintillation counter in vials containing 4 ml of a suitable scintillant. Results and Discussion The DNA of the rats was labelled with a mixture of tritiated thymidine and bromodeoxyuridine. The reason we chose this mixture rather than using tritiated b r o m o d e o x y u r i d i n e was for reasons of economy. Thymidine and bromodeoxyuridine have a short half-life in the animal [4]; injections of these compounds would result in varying amounts of the compounds being incorporated into the DNA of the animal during the time course of the experiment. We, therefore, chose to continually infuse the labelling compounds. In order to achieve a complete separation on a density gradient between newly replicated and bulk DNA, it was necessary to ensure that no tritiated t h y m i d i n e was incorporated into light DNA. So we labelled the animal with b r o m o d e o x y u r i d i n e solution, then with tritiated thymidine plus bromodeoxyuridine, and finally with bromodeoxyuridine alone. The heavy tritiated DNA was thus sandwiched between two lengths of heavy unlabelled DNA. To ensure that sufficient bromodeoxyuridine was incorporated into the replicating DNA, it was necessary to maintain a certain level of bromodeoxyuridine in the animal t h r o u g h o u t the time course of the experiment. There were two limitations on the a m o u n t of bromodeoxyuridine that could be infused into a rat in a given period of time: (1) the solubility of the bromodeoxyuridine and (2) the a m o u n t of liquid which could be tolerated by the rat within a given period of time. We used a 32 mM solution of bromodeoxyuridine which was close to saturation. The total volume injected in a 2.5 h period was 20 ml, which is approximately equal to the total blood volume. This is obviously an extreme burden to the animal and any greater volume was likely to lead to severe oedema and heart failure. After trial and error, we established a protocol which appeared to give the best separation between tritiated heavy DNA and unlabelled light DNA. This
176 TABLE
I
OPTIMUM
CONDITIONS
FOR
DENSITY
LABELLING
REPLICATING
DNA
IN A W I S T A R
Label
T i m e of i n f u s i o n (h)
Infusion v o l u m e (ml)
C o m p o n e n t s infused
Pre-label
0.5
4
Radioactive label
1.0
8
Post-label
1.0
8
3 1 1 6 1 6 2
ml ml ml ml ml ml ml
RAT
32 m M b r o m o d e o x y u r i d i n e saline [3H] thymidine (i mCi) 32 m M bromodeoxyuridine saline 32 m M b r o m o d e o x y u r i d i n e saline
protocol is shown in Table I. The results obtained on the CsC1 density gradients are shown in Fig. 1; it can be seen that in all cases, a clear-cut separation between the newly replicated D N A and the light D N A marker was obtained. We also attempted to label the D N A with Na3 3 2 PO4. Over a 1 h labelling period, little radioactivity was incorporated into the D N A of the tissues studied, possibly because there was insufficient time for the radioactive mate-
a
-4
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.2 ~5-
.5.
o '
'
b
o~
b
)(
0
~
>( E
e
E cL
.4~
2.5.
3" ft.
-2 0
0 c
J
10.
-4
.10
-2
0
|
0
10
20 FractrOn
,
3o
i
Io
0
o
|
~b
|
~
.
3o
FractiOn
Fig. 1. T h e in vivo d e n s i t y labelling o f r e p l i c a t i n g D N A in a Wlstar r a t . T h e labelling c o n d i t i o n s a r e s u m m a r l s e d in T a b l e I. T h e p a n e l s s h o w CsCI g r a d i e n t s o f [ 3 H I D N A f r o m (a) liver, (b) s m a l l i n t e s t i n e , (c) t h y m u s , (d) s p l e e n a n d (e) b o n e m a r r o w . T h e l o w f r a c t i o n n u m b e r s r e p r e s e n t t h e d e n s e s t p a r t o f t h e g r a d i e n t . 3 2 p - l a b e U e d T 2 p h a g e D N A (e~..e) w a s u s e d as a " l i g h t " D N A m a r k e r .
rial to dilute o u t the existing pools. If we used longer labelling times, we observed a satisfactory incorporation of radioactive label, b u t insufficient b r o m o d e o x y u r i d i n e was incorporated to give a good density shift. Our objective was to establish conditions under which newly replicated DNA could be separated from the bulk of the DNA in the different tissues of a rat; our primary interest was not to maximise the amount of radioactive label incorporated. Obviously, the presence of bromodeoxyuridine reduced the amount of tritiated thymidine incorporated. The specific activity of the labelled DNA could have been increased by either using tritiated deoxycytidine or tritiated bromodeoxyuridine. We did n o t use either of these compounds in this series of experiments because of their high cost. References 1 Painter, R.B., J e r m a n y , D.A. and Rasmussens, R.E. (1966) J. Mol. Biol. 17, 47--56 2 Painter, R.B. and Cleaver, J.E., (1969) Radiat. Res. 3 7 , 4 5 1 - - 4 6 6 3 Flamm, W.G., Birnstiel, M.L. and Walker, P.M.B. (1969) in Subcellular Components, Preparation and Fractionation (Birnie, G.D. and Fox, S.M., eds), pp. 125--155, Butterworth, L o n d o n 4 Chang, L.O. and Looney, W.B. (1965) Cancer Res. 25, 1 8 1 5 - - 1 8 2 2
Biochimica et Biophysica Acta, 349 (1974) 173--177 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA
97993
DENSITY L A B E L L I N G IN VIVO OF REPLICATING DNA IN A RAT
H.J. D A N P U R E
and M.G. O R M E R O D
Chester Beatty Research Institute,Clifton Avenue Belmont, Sutton, Surrey SM2-5PX (Great Britain) (Received December 3rd, 1973)
Summary A m e t h o d for labelling newly replicated DNA in tissues of a rat is described. Rats were infused with a mixture of bromodeoxyuridine and [3 H] thymidine. After the animal had been killed and the different tissues extracted, the heavy newly replicated DNA could be separated from the light parental DNA on a density gradient of CsC1.
Introduction If cells are incubated in the presence of 5'-bromodeoxyuridine, this compound is incorporated into the cellular DNA in place of thymidine. The resulting DNA has a higher density than normal DNA and can be separated isopycnicaUy on CsC1 gradients. This technique has been used to study the replication of normal DNA [1] and to study the "repair replication" of damaged DNA [2] in cells grown in tissue culture. We wanted to evaluate the possibility of studying repair replication in the intact animal. To this end, we have develped a m e t h o d of density labelling newly replicated DNA in the rat. Since this technique may be of interest to workers in other fields we have described it in this paper. Experimental m e t h o d s Rats
Male Wistar rats of average weight about 320 g were used. Cannulation procedure. The animal was anaesthetised with ether before making an incision alongside one of the lateral tail veins. The exposed vein was carefully dissected o u t from the surrounding connective tissue, ligated with sterile thread and a small incision made on the posterior vena cava side of the ligature. A sterile intravenous cannula of outside diameter 0.63 mm, to which a syringe of heparinised isotonic saline (100 units of heparin per ml of saline) had
174
previously been attached, was gently inserted into the vein until 3--4 cm were in the animal. It was then tied to the vein and the heparinised saline was slowly forced into the vein to check t h a t the cannula was firmly in position and was not leaking. The skin on the tail was sewn up and the cannula was taped to the tail. The animal was then placed in a restraining cage which prevented it from moving except in a backward or forward direction. The syringe of heparinised saline was exchanged for one containing isotonic saline alone and attached to a slow infusion pump, which was set in motion to prevent blocking of the cannula. When the animal had recovered from the anaesthetic (usually about 15 min), the syringe of saline was removed and replaced by one containing unlabeUed DNA precursors. In some experiments, we cannulated the femoral vein. The cannulation procedure was the same as before but extreme care had to be taken when dissecting out the vein as it is closely aligned to the femoral artery and nerve. After the vein had been cannulated, the body wall was sewn up and the cannula was taped to the rat's thigh. Preparation of tissues. After the rat had been labelled with DNA precursors as described in Results and Discussion, the animal was removed from the restraining cage, killed with ether and the different organs removed. The spleen was rinsed in sterile TC199 tissue culture medium (Wellcome Reagents Ltd.) and cut longitudinally in half. The cells were forced out of the capsule by gently squeezing the spleen with a pair of forceps. The capsule was discarded, the cells were pelleted at 500 × g for 2 min and the pellet was taken up in 15 times its volume of TC199 medium. The t h y m u s was rinsed in TC199 medium and a cell suspension prepared by forcing the tissue through a coarse wire mesh. The cells were pelleted and the pellet suspended in 20 times its volume. Bone marrow cells were extracted from the two femurs by dissecting out the bones, cutting off the epiphyses and flushing out the cells with a syringe of TC199 medium. The cells were pelleted and resuspended in 10 .times the volume of the cell pellet. Epithelial cells from the inner wall of the small intestine were prepared by dissecting out a portion of the small intestine adjacent to the d u o d e n u m . The food was flushed out using a large syringe filled with 0.25 M sucrose. The instestine was then cut longitudinally and the mucosal epithelial cells were scraped off the inner wall with a scalpel. The cells were suspended in TC199 medium and pelleted. The liver was thoroughly rinsed in ice-cold 0.25 M sucrose and chopped up. All the connective tissue was discarded and an approx. 25% (w/v) suspension of liver in sucrose was homogenised using three strokes of a Potter-Elvejheim homogeniser. The cell suspension was centrifuged at 1000 × g for 10 min at 4°C to pellet the nuclei and unbroken cells. 0.5 ml of the pellet was added to 1.0 ml sucrose.
Extraction of DNA This was a modification of 0.3 ml aliquots of the different carbonate centrifuge tubes (MSE sulphate, 0.005 M Na4 EDTA and
the m e t h o d described by Flamm et al. [3]. cell suspensions were put into 10-ml polyLtd.) containing 0.2 ml 2% sodium dodecylsheared once through a 19 G syringe needle.
175 4 ml of alkaline CsC1 (refractive index 1.4070 and density 1.786 g/ml) containing 0.02 M EDTA, pH 12.75, were added to each sample and sheared again. Centrifugation at 12000 rev./min for 20 min in the MSE 10 ml X 10 ml titanium angle head rotor on the MSE super speed 65 centrifuge gave a solid pellicle of precipitated protein, which floated on top of the CsC1 solution. 4 ml of the clear liquid from below the peUicle were removed with a syringe and 0.1 ml of the remainder was put onto a filter paper disc to measure the total radioactivity on the gradient. The tube was washed out, dried and the 4 ml of CsC1--DNA solution were returned. 0.02 ml 32 P-labelled T2DNA was added as a marker. Solid CsC1 was then added to bring the refractive index to 1.4055 (density = 1.77 g/ml) as measured on an Abb~ refractometer. The solution was then overlaid with liquid paraffin, the tubes were capped and centrifuged in the 10 ml X 10 ml titanium angle head rotor at 50000 rev./min at 20°C overnight. After centrifugation, the polypropylene tubes were punctured using an MSE tube piercer and 6-drop fractions were collected directly onto 2.5 cm 3-MM Whatman filter paper discs. The discs were washed in ice-cold 10% trichloroacetic acid, rinsed twice in absolute ethanol and dried in acetone before counting in a Packard liquid scintillation counter in vials containing 4 ml of a suitable scintillant. Results and Discussion The DNA of the rats was labelled with a mixture of tritiated thymidine and bromodeoxyuridine. The reason we chose this mixture rather than using tritiated b r o m o d e o x y u r i d i n e was for reasons of economy. Thymidine and bromodeoxyuridine have a short half-life in the animal [4]; injections of these compounds would result in varying amounts of the compounds being incorporated into the DNA of the animal during the time course of the experiment. We, therefore, chose to continually infuse the labelling compounds. In order to achieve a complete separation on a density gradient between newly replicated and bulk DNA, it was necessary to ensure that no tritiated t h y m i d i n e was incorporated into light DNA. So we labelled the animal with b r o m o d e o x y u r i d i n e solution, then with tritiated thymidine plus bromodeoxyuridine, and finally with bromodeoxyuridine alone. The heavy tritiated DNA was thus sandwiched between two lengths of heavy unlabelled DNA. To ensure that sufficient bromodeoxyuridine was incorporated into the replicating DNA, it was necessary to maintain a certain level of bromodeoxyuridine in the animal t h r o u g h o u t the time course of the experiment. There were two limitations on the a m o u n t of bromodeoxyuridine that could be infused into a rat in a given period of time: (1) the solubility of the bromodeoxyuridine and (2) the a m o u n t of liquid which could be tolerated by the rat within a given period of time. We used a 32 mM solution of bromodeoxyuridine which was close to saturation. The total volume injected in a 2.5 h period was 20 ml, which is approximately equal to the total blood volume. This is obviously an extreme burden to the animal and any greater volume was likely to lead to severe oedema and heart failure. After trial and error, we established a protocol which appeared to give the best separation between tritiated heavy DNA and unlabelled light DNA. This
176 TABLE
I
OPTIMUM
CONDITIONS
FOR
DENSITY
LABELLING
REPLICATING
DNA
IN A W I S T A R
Label
T i m e of i n f u s i o n (h)
Infusion v o l u m e (ml)
C o m p o n e n t s infused
Pre-label
0.5
4
Radioactive label
1.0
8
Post-label
1.0
8
3 1 1 6 1 6 2
ml ml ml ml ml ml ml
RAT
32 m M b r o m o d e o x y u r i d i n e saline [3H] thymidine (i mCi) 32 m M bromodeoxyuridine saline 32 m M b r o m o d e o x y u r i d i n e saline
protocol is shown in Table I. The results obtained on the CsC1 density gradients are shown in Fig. 1; it can be seen that in all cases, a clear-cut separation between the newly replicated D N A and the light D N A marker was obtained. We also attempted to label the D N A with Na3 3 2 PO4. Over a 1 h labelling period, little radioactivity was incorporated into the D N A of the tissues studied, possibly because there was insufficient time for the radioactive mate-
a
-4
.10
.2 ~5-
.5.
o '
'
b
o~
b
)(
0
~
>( E
e
E cL
.4~
2.5.
3" ft.
-2 0
0 c
J
10.
-4
.10
-2
0
|
0
10
20 FractrOn
,
3o
i
Io
0
o
|
~b
|
~
.
3o
FractiOn
Fig. 1. T h e in vivo d e n s i t y labelling o f r e p l i c a t i n g D N A in a Wlstar r a t . T h e labelling c o n d i t i o n s a r e s u m m a r l s e d in T a b l e I. T h e p a n e l s s h o w CsCI g r a d i e n t s o f [ 3 H I D N A f r o m (a) liver, (b) s m a l l i n t e s t i n e , (c) t h y m u s , (d) s p l e e n a n d (e) b o n e m a r r o w . T h e l o w f r a c t i o n n u m b e r s r e p r e s e n t t h e d e n s e s t p a r t o f t h e g r a d i e n t . 3 2 p - l a b e U e d T 2 p h a g e D N A (e~..e) w a s u s e d as a " l i g h t " D N A m a r k e r .
rial to dilute o u t the existing pools. If we used longer labelling times, we observed a satisfactory incorporation of radioactive label, b u t insufficient b r o m o d e o x y u r i d i n e was incorporated to give a good density shift. Our objective was to establish conditions under which newly replicated DNA could be separated from the bulk of the DNA in the different tissues of a rat; our primary interest was not to maximise the amount of radioactive label incorporated. Obviously, the presence of bromodeoxyuridine reduced the amount of tritiated thymidine incorporated. The specific activity of the labelled DNA could have been increased by either using tritiated deoxycytidine or tritiated bromodeoxyuridine. We did n o t use either of these compounds in this series of experiments because of their high cost. References 1 Painter, R.B., J e r m a n y , D.A. and Rasmussens, R.E. (1966) J. Mol. Biol. 17, 47--56 2 Painter, R.B. and Cleaver, J.E., (1969) Radiat. Res. 3 7 , 4 5 1 - - 4 6 6 3 Flamm, W.G., Birnstiel, M.L. and Walker, P.M.B. (1969) in Subcellular Components, Preparation and Fractionation (Birnie, G.D. and Fox, S.M., eds), pp. 125--155, Butterworth, L o n d o n 4 Chang, L.O. and Looney, W.B. (1965) Cancer Res. 25, 1 8 1 5 - - 1 8 2 2