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A comparison of 5-bromodeoxyuridine and thymidine incorporation into fertilized sea urchin eggs
Printed in Sweden Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved
Experimental Cell Research 84 (1974) 395-398
A COMPARISON
OF 5BROMODEOXYURIDINE
INCORPORATION
INTO
J. L. GRAINGER
FERTILIZED
AND THYMIDINE
SEA URCHIN
EGGS
and R. T. HINEGARDNER
Division of Natural Sciences, University of California at Santa Cruz, Santa Cruz, Calif. 95064, USA
SUMMARY SH-Thymidine (BH-TdR) and *H-5-bromo-2-deoxyuridine CH-BUdR) are first incorporated into newly fertilized Strongyfocentrotus purpuratus eggs 30 min after fertilization, which is about the time of pronuclear fusion. An initial incorporation of 8H-BUdR immediately following fertilization has been previously reported. This was found to be due to a radioactive impurity present in samples of SH-BUdR stored in aqueous solutions. 8H-TdR stored in water, or *HBUdR stored in 50 % ethanol, did not contain this impurity.
Using 3H-thymidine CH-TdR) as the radioactive tracer, the initiation of the first DNA synthesis period following fertilization of the eggs of the sea urchin, Strongylocentrotus purpuratus, occurred at about 30 min (at 15’C) [l]. This is approximately the time of pronuclear fusion. Recently, the relationship between DNA synthesis and pronuclear fusion has been examined more closely [2], and it has been demonstrated that in Arbacia punctulatu DNA synthesis begins just after fusion is complete. By contrast, Zeitz et al. [3] found that if 5-bromodeoxyuridine (BUdR) was used as the radioactive nucleoside, there was significant incorporation of radioactivity into an acid-insoluble component of the egg immediately following fertilization. Their autoradiographic evidence indicated that the 3H-BUdR may have been incorporated into mitochondrial DNA and that the reason others had not detected this early synthesis was because thymidine was not phosphorylated, and thus utilized,
by the egg until about the time of pronuclear fusion. We have repeated this work using somewhat different techniques and different controls and we will show in this paper that even though there is radioactive incorporation at fertilization, the compound being incorporated is not 3H-BUdR but some contamination that is present in samples of 3H-BUdR that are stored as aqueous solutions. MATERIALS AND METHODS Eggs of the sea urchin Strongylocentrotus purpuratus were used in these experiments and handled by the methods described in Hinegardner et al. [l], except that the radioactivity was determined with a Beckman liquid scintillation counter using PPO in toluene (5 g PPO per liter toluene) as scintillation fluid. The radioactive contaminant in the JH-BUdR preparations was separated by paper chromatography using Whatman No. 1. paper and 95 % alcohol, 0.1 M ammonium acetate (7 : 1) (PH 6.0) as the solvent. The chromatograms were dried in air and the ammonium acetate allowed to sublimate off. The portion of the paper with the contaminant spot was then cut out and Exptl Cell Res 84 (1974)
396
Grainger and Hinegardner ham-Searle. SH-BUdR in 50% ethanol (15.3 Ci/ mmole) and aH-methyl-thymidine in water (6 Ci/ mmole) were purchased from Schwarz BioResearch.
RESULTS
0
0
10
20
30
40
50
60
70
Fig. 1. Abscissa: time (min) after fertilization; ordinate: cpm x lOa. Incorporation of l , SH-BUdR; A, SH-TdR; 0, SH-BUdR + 5 x non-radioactive BUdR; A, *HBUdR + 5 x non-radioactive thymidine at 15°C. The specific activities and concentrations of 8HBUdR and “H-TdR used in the experiments were the same. placed into the egg suspension. The aH-BUdR spot was treated similarly. The addition of the chromatogram had no effect on the rate of cleavage or proportion of surviving embryos. DNAase treatment was carried out as in Hinegardner et al. [l]. sH-5-Bromo-2-deoxyuridine-6 in aqueous solution (2 Ci/mmole, Batch 24) was purchased from Amers-
We can show by four different methods that fertilized sea urchin eggs incorporate neither BUdR nor TdR in any significant amount until nuclear fusion. (I) If two batches of eggs were raised, one in the presence of 3H-BUdR and the other in an equal amount of 3H-BUdR diluted with 5 x as much non-radioactive BUdR, the level of incorporated radioactivity up to about 20 min was essentially the same in both cases (fig. 1). After 20 min, the radioactive incorporation is reduced in proportion to the amount of cold BUdR added. The presence of non-radioactive thymidine also had no effect on the initial incorporation. (2) DNAase does not remove the prefusion radioactivity, but does eliminate 92 % of the post-fusion counts. It has been postulated that the initial incorporation of BUdR was into replicating mitochondria where the DNA was protected [3]. (3) When the aqueous samples of 3HBUdR that show prefusion incorporation were chromatogrammed, the radioactivity
150 -
125 -
100 -
75 -
Fig. 2. Abscissa: fraction no. ordinate: cpm x 108. Chromatogram of 8H-BUdR stored in aqueous solution. Region I: impurity; Region 2: Inseparable impurity, probably 3HBU; Region 3: Uncontaminated aHBUdR.
50 -
25 -
0
5
IO
15
ExptI Cell Res 84 (1974)
20
25
30
35
40
45
Incorporation of BUdR and TdR into fertilized eggs 391
separated into 3 components (fig. 2). The bulk of the activity was associated with the BUdR. When this portion of the chromatogram was added to the eggs, incorporation followed the time course for 3H-TdR (fig. 3). There is no appreciable pre-fusion incorporation of radioactivity. A small amount of a second component, which ran just behind the BUdR, was in the same position as known samples of bromouridine, but the peak did not separate well enough to be isolated. A third component, constituting about 1.6 % of the total radioactivity, ran slower than the other two and could be cut out and added to the eggs. This contained the component responsible for pre-fusion incorporation (fig. 3). (4) Not all tritiated nucleoside preparations gave pre-fusion incorporation. Fig. 4 is a plot of samples from two different suppliers. Those that were sold in 50% ethanol yield little incorporation, whereas aqueous samples contained much more contaminant. Also, no impurity peak was discernible on a chromatogram of 3H-BUdR stored in the ethanol.
0
IO
20
30
40
50
60
70
Fig. 3. Abscissa: time (min) after fertilization;
ordi-
nate: cnm x 102. Incorporation of l , 8H-BUdR impurity; 0, uncontaminated SH-BUdR at 15°C. SH-BUdR impurity was eluted from Region I and SH-BUdR from Region 3 of the chromatogram (fig. 2).
0
0
IO
20
30
40
50
60
70
Fig. 4. Abscissa: time (min) after fertilization; ordinate:
cpm x lOa. Incorporation of 0, 8H-BUdR stored in aqueous solution; 0, SH-BUdR stored in 50 % ethanol at 15°C.
DISCUSSION These results clearly show that BUdR incorporation into fertilized sea urchin eggs follows close to the same time course as that of thymidine. Radioactive incorporation prior to nuclear fusion is due to a contaminant and not to BUdR. Figs 1 and 3 both demonstrate that almost no BUdR is taken up at that time. It has been demonstrated that 3H-TdR is taken into Arbacia eggs and phosphorylated to 3H-thymidine triphosphate (3H-dTTP) between 5-10 min after fertilization [2]. Incorporation into an acid-insoluble fraction does not appear until pronuclear fusion, or 15-20 min post-fertilization. Apparently, a large pool of pre-formed dTTP which could inhibit the phosphorylation of TdR by thymidine kinase until 20 min after fertilization, does not exist. Wand et al. [4], studying the incorporation of 3H-TdR and its self-radiolytic impurities in Tetrahymena, found that the impurities and not the pure 3H-TdR were incorporated into the cytoplasm. The impurities could not be removed from their autoradiographs, or from an acid-insoluble Exptl Cell Res 84 (1974)
398 Grainger and Hinegardner
fraction, by DNAase or RNAase. The incorporation rate of the impurity did not follow that of pure 3H-TdR. The impurity incorporated immediately after addition to the medium and gradually leveled off, while the incorporation of 3H-TdR gradually increased, stopped, and then increased again, following the pattern of cell division. The rate of initial incorporation of our 3HBUdR impurity into the sea urchin eggs resembled that of the 3H-TdR impurity. The 3H-BUdR impurity also could not be removed by DNAase. Therefore, the cytoplasmic incorporation that has been called the incorporation of 3H-BUdR into mitochondrial DNA [3] seemsto be, in fact, the incorporation of an impurity, possibly selfradiolytic, that is present in aqueous 3HBUdR solutions. Shapiro & Kang [5] have shown that the uncatalysed hydrolysis of
Exptl Ceil Res 84 (1974)
BUdR occurs much more readily in aqueous solution than in ethanol. Our results indicate that BUdR is a good analogue to TdR. It is incorporated at about the same level as TdR and follows essentially the same time course. Like TdR [6-g], it does not appear to enter the egg in significant amounts until after fertilization. REFERENCES 1. Hineaardner. R T. Rao. B & Feldman. D E. Expticell res 36 (1964) 53. 2. Lonao. F J & Phmkett. W. Dev biol30 (1973) 56. 3. Zeiti, L, Garfinkel, E & Ferguson, R, dytobfos 4 (1971) 129. 4. Wand, M, Zeuthen, E & Evans, E A, Science 157 (1967) 436. Shaniro. R & Kang. S, Biochemistry 8 (1969) 1806. 2: Nemer,‘M, J biol &em 237 (1962)~143: ’ . 7. Czihak. G & Pohl._ E., Z Naturforsch 25b (1970) 1047. ’ 8. Ledebur-Villiger, M, Exptl cell res 72 (1972) 285.
ReceivedJune25 1g73 Revised version r&eived November 5, 1973
Experimental Cell Research 84 (1974) 395-398
A COMPARISON
OF 5BROMODEOXYURIDINE
INCORPORATION
INTO
J. L. GRAINGER
FERTILIZED
AND THYMIDINE
SEA URCHIN
EGGS
and R. T. HINEGARDNER
Division of Natural Sciences, University of California at Santa Cruz, Santa Cruz, Calif. 95064, USA
SUMMARY SH-Thymidine (BH-TdR) and *H-5-bromo-2-deoxyuridine CH-BUdR) are first incorporated into newly fertilized Strongyfocentrotus purpuratus eggs 30 min after fertilization, which is about the time of pronuclear fusion. An initial incorporation of 8H-BUdR immediately following fertilization has been previously reported. This was found to be due to a radioactive impurity present in samples of SH-BUdR stored in aqueous solutions. 8H-TdR stored in water, or *HBUdR stored in 50 % ethanol, did not contain this impurity.
Using 3H-thymidine CH-TdR) as the radioactive tracer, the initiation of the first DNA synthesis period following fertilization of the eggs of the sea urchin, Strongylocentrotus purpuratus, occurred at about 30 min (at 15’C) [l]. This is approximately the time of pronuclear fusion. Recently, the relationship between DNA synthesis and pronuclear fusion has been examined more closely [2], and it has been demonstrated that in Arbacia punctulatu DNA synthesis begins just after fusion is complete. By contrast, Zeitz et al. [3] found that if 5-bromodeoxyuridine (BUdR) was used as the radioactive nucleoside, there was significant incorporation of radioactivity into an acid-insoluble component of the egg immediately following fertilization. Their autoradiographic evidence indicated that the 3H-BUdR may have been incorporated into mitochondrial DNA and that the reason others had not detected this early synthesis was because thymidine was not phosphorylated, and thus utilized,
by the egg until about the time of pronuclear fusion. We have repeated this work using somewhat different techniques and different controls and we will show in this paper that even though there is radioactive incorporation at fertilization, the compound being incorporated is not 3H-BUdR but some contamination that is present in samples of 3H-BUdR that are stored as aqueous solutions. MATERIALS AND METHODS Eggs of the sea urchin Strongylocentrotus purpuratus were used in these experiments and handled by the methods described in Hinegardner et al. [l], except that the radioactivity was determined with a Beckman liquid scintillation counter using PPO in toluene (5 g PPO per liter toluene) as scintillation fluid. The radioactive contaminant in the JH-BUdR preparations was separated by paper chromatography using Whatman No. 1. paper and 95 % alcohol, 0.1 M ammonium acetate (7 : 1) (PH 6.0) as the solvent. The chromatograms were dried in air and the ammonium acetate allowed to sublimate off. The portion of the paper with the contaminant spot was then cut out and Exptl Cell Res 84 (1974)
396
Grainger and Hinegardner ham-Searle. SH-BUdR in 50% ethanol (15.3 Ci/ mmole) and aH-methyl-thymidine in water (6 Ci/ mmole) were purchased from Schwarz BioResearch.
RESULTS
0
0
10
20
30
40
50
60
70
Fig. 1. Abscissa: time (min) after fertilization; ordinate: cpm x lOa. Incorporation of l , SH-BUdR; A, SH-TdR; 0, SH-BUdR + 5 x non-radioactive BUdR; A, *HBUdR + 5 x non-radioactive thymidine at 15°C. The specific activities and concentrations of 8HBUdR and “H-TdR used in the experiments were the same. placed into the egg suspension. The aH-BUdR spot was treated similarly. The addition of the chromatogram had no effect on the rate of cleavage or proportion of surviving embryos. DNAase treatment was carried out as in Hinegardner et al. [l]. sH-5-Bromo-2-deoxyuridine-6 in aqueous solution (2 Ci/mmole, Batch 24) was purchased from Amers-
We can show by four different methods that fertilized sea urchin eggs incorporate neither BUdR nor TdR in any significant amount until nuclear fusion. (I) If two batches of eggs were raised, one in the presence of 3H-BUdR and the other in an equal amount of 3H-BUdR diluted with 5 x as much non-radioactive BUdR, the level of incorporated radioactivity up to about 20 min was essentially the same in both cases (fig. 1). After 20 min, the radioactive incorporation is reduced in proportion to the amount of cold BUdR added. The presence of non-radioactive thymidine also had no effect on the initial incorporation. (2) DNAase does not remove the prefusion radioactivity, but does eliminate 92 % of the post-fusion counts. It has been postulated that the initial incorporation of BUdR was into replicating mitochondria where the DNA was protected [3]. (3) When the aqueous samples of 3HBUdR that show prefusion incorporation were chromatogrammed, the radioactivity
150 -
125 -
100 -
75 -
Fig. 2. Abscissa: fraction no. ordinate: cpm x 108. Chromatogram of 8H-BUdR stored in aqueous solution. Region I: impurity; Region 2: Inseparable impurity, probably 3HBU; Region 3: Uncontaminated aHBUdR.
50 -
25 -
0
5
IO
15
ExptI Cell Res 84 (1974)
20
25
30
35
40
45
Incorporation of BUdR and TdR into fertilized eggs 391
separated into 3 components (fig. 2). The bulk of the activity was associated with the BUdR. When this portion of the chromatogram was added to the eggs, incorporation followed the time course for 3H-TdR (fig. 3). There is no appreciable pre-fusion incorporation of radioactivity. A small amount of a second component, which ran just behind the BUdR, was in the same position as known samples of bromouridine, but the peak did not separate well enough to be isolated. A third component, constituting about 1.6 % of the total radioactivity, ran slower than the other two and could be cut out and added to the eggs. This contained the component responsible for pre-fusion incorporation (fig. 3). (4) Not all tritiated nucleoside preparations gave pre-fusion incorporation. Fig. 4 is a plot of samples from two different suppliers. Those that were sold in 50% ethanol yield little incorporation, whereas aqueous samples contained much more contaminant. Also, no impurity peak was discernible on a chromatogram of 3H-BUdR stored in the ethanol.
0
IO
20
30
40
50
60
70
Fig. 3. Abscissa: time (min) after fertilization;
ordi-
nate: cnm x 102. Incorporation of l , 8H-BUdR impurity; 0, uncontaminated SH-BUdR at 15°C. SH-BUdR impurity was eluted from Region I and SH-BUdR from Region 3 of the chromatogram (fig. 2).
0
0
IO
20
30
40
50
60
70
Fig. 4. Abscissa: time (min) after fertilization; ordinate:
cpm x lOa. Incorporation of 0, 8H-BUdR stored in aqueous solution; 0, SH-BUdR stored in 50 % ethanol at 15°C.
DISCUSSION These results clearly show that BUdR incorporation into fertilized sea urchin eggs follows close to the same time course as that of thymidine. Radioactive incorporation prior to nuclear fusion is due to a contaminant and not to BUdR. Figs 1 and 3 both demonstrate that almost no BUdR is taken up at that time. It has been demonstrated that 3H-TdR is taken into Arbacia eggs and phosphorylated to 3H-thymidine triphosphate (3H-dTTP) between 5-10 min after fertilization [2]. Incorporation into an acid-insoluble fraction does not appear until pronuclear fusion, or 15-20 min post-fertilization. Apparently, a large pool of pre-formed dTTP which could inhibit the phosphorylation of TdR by thymidine kinase until 20 min after fertilization, does not exist. Wand et al. [4], studying the incorporation of 3H-TdR and its self-radiolytic impurities in Tetrahymena, found that the impurities and not the pure 3H-TdR were incorporated into the cytoplasm. The impurities could not be removed from their autoradiographs, or from an acid-insoluble Exptl Cell Res 84 (1974)
398 Grainger and Hinegardner
fraction, by DNAase or RNAase. The incorporation rate of the impurity did not follow that of pure 3H-TdR. The impurity incorporated immediately after addition to the medium and gradually leveled off, while the incorporation of 3H-TdR gradually increased, stopped, and then increased again, following the pattern of cell division. The rate of initial incorporation of our 3HBUdR impurity into the sea urchin eggs resembled that of the 3H-TdR impurity. The 3H-BUdR impurity also could not be removed by DNAase. Therefore, the cytoplasmic incorporation that has been called the incorporation of 3H-BUdR into mitochondrial DNA [3] seemsto be, in fact, the incorporation of an impurity, possibly selfradiolytic, that is present in aqueous 3HBUdR solutions. Shapiro & Kang [5] have shown that the uncatalysed hydrolysis of
Exptl Ceil Res 84 (1974)
BUdR occurs much more readily in aqueous solution than in ethanol. Our results indicate that BUdR is a good analogue to TdR. It is incorporated at about the same level as TdR and follows essentially the same time course. Like TdR [6-g], it does not appear to enter the egg in significant amounts until after fertilization. REFERENCES 1. Hineaardner. R T. Rao. B & Feldman. D E. Expticell res 36 (1964) 53. 2. Lonao. F J & Phmkett. W. Dev biol30 (1973) 56. 3. Zeiti, L, Garfinkel, E & Ferguson, R, dytobfos 4 (1971) 129. 4. Wand, M, Zeuthen, E & Evans, E A, Science 157 (1967) 436. Shaniro. R & Kang. S, Biochemistry 8 (1969) 1806. 2: Nemer,‘M, J biol &em 237 (1962)~143: ’ . 7. Czihak. G & Pohl._ E., Z Naturforsch 25b (1970) 1047. ’ 8. Ledebur-Villiger, M, Exptl cell res 72 (1972) 285.
ReceivedJune25 1g73 Revised version r&eived November 5, 1973