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A radiometric method for developing the alkaline sucrose gradient sedimentation patterns of DNA from nondividing cells
ANALYTICAL
BIOCHEMISTRY
536-542
86,
(1978)
A Radiometric Method for Developing the Alkaline Sucrose Gradient Sedimentation Patterns of DNA from Nondividing Cells M. V. SKLOBOVSKAJA,
A. S. SAENKO, Ju. A. SIOMIN, AND A. M. POVERENNY
Research
of Medical
Institute
Radiology, Ohninsk, Kaluga
Received A radiometric tion
method
patterns
of
method is the of the binding The procedure filtration
December
of the
DNA
for from
30,
Academy Region,
1976:
of Medical USSR
accepted
developing
the
nonlabeled
cells
alkaline
October sucrose
is described.
Sciences
17,
1977
gradient The
of USSR,
sedimenta-
principle
of the
labeling of the DNA contained in the gradient fractions by means of a labeled amino acid to DNA in the presence of formaldehyde. involves incubation of the fractions with the labeling reagent, incubation
of the filters. The DNA concentration sufficient to escape labeled described
mammalian and those
in vivo
are
mixtures
through
nitrocellulose
filters.
relationship between the radioactivity on the in the sample is linear; the DNA detection overloading of the gradients. It was shown cell DNA of DNA
sedimentation from the
same
patterns cells
developed labeled with
and
radiometry
filters and the sensitivity is that the nonby the method [3H]thymidine
identical.
DNA from nondividing and slowly dividing cells cannot be radioactively labeled in viva and as a consequence cannot be characterized with the help of McGrath and Williams’ method in its original version (I). To develop the sedimentation patterns of those DNAs, an analytical method is required sensitive enough to detect submicrogram quantities of nonlabeled DNA in gradient fractions. At the present time the most commonly adopted approach to the problem utilizes some modifications of the Kissane and Robins’ fluorometric procedure for the determination of DNA (2-5). As a rule, the fluorometric method is used to determine high molecular weight mammalian DNA in gradients of large volume (30 to 36 ml), the load being l-2. lo6 cells and the fraction number 15- 18. The procedure consists of 12-14 steps. In this communication, we present an alternative radiometric method for determining DNA in gradient fractions. The method is more sensitive and less time-consuming than fluorometric one. We render DNA in the fractions radioactive using the interaction between DNA and aminomethylolic compounds, the reaction having been described previously 0003-2697/78/0862-0536$02.00/O Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
536
DNA
DETECTION
IN SUCROSE
GRADIENTS
537
(6-9). In this case, an aminomethylolic compound is formed from formaldehyde and a labeled amino acid. The procedure involves incubation of the fractions with a labeling reagent which contains labeled amino acid, formaldehyde and buffer, filtration of the incubation mixtures through nitrocellulose filters, and radiometry of the filters. Using this method, the alkaline sucrose gradient sedimentation profiles of DNA from Ehrlich ascitic carcinoma cells and mouse thymocytes have been obtained. The gradient volumes were 4.8, 13, and 22 ml, the loads were 5. 104-5. lo5 cells, and fraction numbers were 24-30. MATERIALS
AND METHODS
Materials. All the solutions were filtered through dense glass or nitrocellulose filters. Five- and twenty-percent sucrose solutions were made in 0.2 N NaOH, 0.8 N NaCl, and0.01 M Na,EDTA, pH 12.5. Alkaline lysing agents were 0.5 N NaOH, 0.5 N NaCl, 0.01 M Na,EDTA (LA-l), and 0.5 N NaOH, 0.1 M Na,EDTA (LA-2). The detergents used were: sodium dodecyl sulfate, Serwa, Sweden; and Sarkosyl. Schuchardt, BDR. Formaldehyde was from Merck. Medium 199 with 5% mouse or calf fetal serum was used for the cell suspension preparation. Calf thymus or chicken erythrocyte DNA was used in the model experiments. [3H]Thymidine was obtained from Isotop, USSR. Labeled amino acids were: [r4C]glycine, 51 mCi/mmol, Isotop, USSR; [3H]lysine 20 Ci/mmol, Amersham, England; and [3H]glycine, 0.6-2.5 Ciimmol, Isotop, USSR. Nitrocellulose filters, 0.45- 1.5 pm (Synpor nn 6-3) (diam., 24 mm), were obtained from Chemapol, Czechoslovakia. Labeling of ascitic Ehrlich carcinoma (AEC) cell DNA. AEC cell DNA was labeled by injecting 400 &i of [3H]thymidine ip into animal bearing 2- to 3-day tumor (100 PCi at 24, 20, 16. and 2 hr before decapitation). Cell suspensions: tion. The labeled
Preparation,
irradiation,
and postirradiation
incuba-
and nonlabeled ascitic fluids from the decapitated animals were isolated and washed twice with 0.9% NaCl. Aliquots of the cell suspension in 0.9% NaCl at a concentration of approximately 5. IO5 cells/ml were irradiated in ice by y-rays in a Gammacellat a dose rate of 17 rads/sec. then spun down, resuspended in the fresh 0.9% NaCl, and incubated at 37°C. Mouse thymus was removed from a decapitated animal, squashed gently in a manual Teflon homogenizer with two or three strokes of the pestle, and then sieved through a layer of the cotton in a 20-m] syringe without a needle. Irradiation and incubation of these cells was done in medium 199. Cell vitality was tested by means of erythrosine. The colored thymocyte fraction did not exceed 10% after postirradiation incubation. Cell lysis. When 4.8-m] gradients were used, 0.2 ml of lysing agent and 0.1 ml of the cell suspension were layered over the sucrose surface,
538
SKLOBOVSKAJA
ET AL.
while with 13-ml gradients, 0.5 ml of lysing agent and 0.5 ml of the suspension were used. The load of gradient, the type of lysing agent, and the lysis time and temperature are detailed in the legend to the Fig. 2. Preparation of the labeled aminomethylolic reagent (LAR). The LAR components were a labeled amino acid, formaldehyde, and phosphate buffer, pH 6.8. The concentrations of the components were such as to give a final concentration of formaldehyde in the incubation mixture (the probe plus LAR aliquot) of about 2% and that of the buffer, about 0.2 M. The labeled amino acid content was calculated to obtain the required sensitivity of DNA determination (see Results and Discussion) and as a rule 5-10 $Zi per sample in our experiments. First the amino acid was added to the buffer and then formaldehyde. LAR may polymerize in 20-24 hr. so it should be prepared on the day of use and filtered through membrane filter No. 5 or 6. Incubation andfiltration of the incubation mixtures. The DNA solution probes or gradient fractions of 0.2- I .O ml were incubated with 0.2 ml of LAR 16-20 hr at 18-22°C. Then the samples were diluted approximately 1:3 with 0.1 N NaCl and filtered through the nitrocellulose filters under a slight vacuum. After the sample had been sieved, the filter was washed with 0.1 N NaCI, 5% TCA, and 0. I N NaCl once more, then dried with ethanol and inserted into a scintillation vial. In some cases, e.g., if sodium dodecyl sulfate is present in a probe, it is preferable to precipitate the incubation mixture with cold 5% TCA and then filter as mentioned above. Linear gradients. Linear gradients were made using a three-channel mixing chamber, MSE, and three-channel micropump, Multiperpex 2115, LKB, Sweden. Ultracentrifkgation. The gradients were spun at 20°C in the Beckman SW-39 rotor of the Spinco Model L-50 ultracentrifuge or in MSE rotors 3 x 25 or 6 x 16.5 of the MSE Superspeedultracentrifuge. The ultracentrifugation time and speed are detailed in the legend to the Fig. 2. Gradientfractionation. This was also performed using the micropump, from the bottom of the gradient. The fractions of gradients of labeled AEC cell DNA were collected either on filter paper disks or strips of adequate size or into test tubes and then filtered, after TCA precipitation, through the membrane filters. The gradients which contained nonlabeled DNA from AEC cells or thymocytes were fractionated into the test tubes, and the fractions were then incubated with LAR and filtered as described above. Instead of test tubes, the holes of polystyrolic plates commonly used in the immunologic studies are convenient for use in fractionation and incubation. Measurement ofradioactivity. The filter radioactivity was determined in a liquid scintillation spectrometer (Ansitron, Picker, USA; or SL-30 Intertechnique, France) using the toluene scintillation fluid.
DNA
DETECTION
IN
RESULTS
SUCROSE
539
GRADIENTS
AND DISCUSSION
In this work we applied the initial step of the interaction between DNA and the aminothylolic compound, the binding of the amino acid to DNA in the presence of formaldehyde. All the steps and conditions of the reaction were described in detail earlier (6-9). We have chosen as our method a combination of reaction conditions such that some variations of the parameters (duration, 16-20 hr; temperature, 18-22°C; pH in the incubation mixture, 8-10) have little effect on the results. DNA is not a specific substrate for the aminothylolic compounds. These substances also react with RNA, proteins, and various amines. However, the alkaline sucrose gradient ultracentrifugation method per se provides satisfactory conditions for the selective detection of DNA: RNA is destroyed in the alkaline gradient and remains at the top of it along with the proteins and the compounds of low molecular weight. For the sake of specificity enhancement, we have introduced a filtration step into
I
2 DNA
FIG. 1. The relationship concentration in the incubation composition. 12% sucrose,
concentration,
,q
per ml
between the radioactivity retained on the filter mixture. LAR volume, 0.2 ml; probe volume. 0.2 N NaOH. 0.8 N NaCI. 0.01 M Na,EDTA.
present and the labeled amino acid preparation and detergent, [3H]lysine. 20 Ci/mmol, 3.5 &i/sample; 2.5 Ci/mmol, IO &i/sample: (c) no detergent, [RH]glycine,
and the DNA 0.2 ml; probe The detergent
quantity were as follows: (a) (b) 0.1% Sarkosyl, [3H]glycine, 2.5 Ci/mmol. 36 yCi/sample.
no
540
SKLOBOVSKAJA
/Y\ ‘\’\ \ 2 \\ A’/’ \
2,000
1,000 0k
ET AL.
&
b
i ._ D 5 s h .Z .; 1,000 :: 2 BL.
c
4,000 3,000 d 2,000 1,000
5
IO
15
20 fraction
25 number
FIG. 2. The alkaline sucrose gradient sedimentation patterns of DNA from labeled and nonlabeled in viva cells. Details for each gradient are as follows: cell type, [3H]TdR label in rive, if any; irradiation dose and postirradiation time of incubation at 37°C; the gradient volume and load; the lysing agent type and, the lysis time and temperature; the ultracentrifugation time and speed; the labeled amino acid preparation, if needed, and its quantity per fraction: (a) curve I: ascite, [3H]TdR; 3 kR; 4.8 ml, 5.104 cells; LA-l, I8 hr. 22°C; 90 min. 30,000 rpm; none; (a) curve 2: ascite, no label; as in curve 1 except for amino acid preparation, [3H]glycine, 20 &I; (b) curve 1: ascite, [3H]TdR; 3 kR; 13 ml, 1.5. IO5 cells: LA-l, I8 hr, 22°C; 5 hr, 24.000 rpm; none: (b) curve 2: ascite, no label; as in curve I except for amine acid preparation, [3H]lysine, 3 &i; (c) curve 1: ascite, no label; 2 hr. 29°C and 16 hr. 0°C; I65 min. 24,000 rpm; I kR; I3 ml, 1.5, IO5 cells; LA-2, dose. 5 kR; (c) curve [3H]glycine. 20 &i; (c) curve 2: as in curve I except for irradiation
DNA
DETECTION
IN
SUCROSE
GRADIENTS
541
the procedure; the nitrocellulose membrane filters are known to sorb the denatured DNA selectively (IO). Model experiments were undertaken to evaluate the additional radioactivity on the membrane filter resulting from the binding of labeled amino acid to RNA and protein presented in the DNA sample. The probes were alkaline sucrose solutions of DNA, RNA, and bovine serum albumin (BSA) in various combinations and concentrations: DNA, 0.1-2.0 pg; RNA, up to 10 pg: BSA, up to 20 Fg in a 0.2-ml volume. It was shown, by use of the procedure described above, that the presence of 10 pg of RNA in the probe gives no additional radioactivity; the presence of protein gives some but very little additional radioactivity (-5% of that from the same quantity of DNA). So use of a nonspecific reaction of aminomethylolic compounds with DNA for the DNA determination under the present conditions (ultracentrifugation at alkaline pH and filtration through membrane filters) provided little interference from RNA and proteins. The method to be applied for developing the DNA sedimentation patterns must satisfy two additional requirements: a linear relationship between a registered parameter and the DNA concentration, and a high sensitivity of the DNA determination, adequate to the permissible load of the gradients. Such a load in the case of the intact mammalian cells must not exceed 5. IO4 cells on the 4.8-ml gradient (11); i.e., approximately 0.5 pg of DNA per gradient or 0.05 pg of DNA in the peak fraction. The sensitivity of the method must be high enough for the reliable determination of hundreths of a microgram of DNA. These characteristics were also evaluated in model experiments. Micro- and submicrogram quantities of DNA have been determined by the method described in alkaline sucrose solutions containing NaCl. EDTA, detergents, and the other compounds used in cell lysis and gradient centrifugation. Several labeled amino acids were used for DNA determination: [3H]glycine. [‘*C]glycine, and [“Hllysine. A relationship between the observed count rate from the radioactivity precipitated on the filter and the DNA content in a probe was linear in all cases; some examples are given in Fig. 1. It was also shown that the quantity of DNA-bound amino acid depends linearly on the amino acid content in the sample and is far from saturated under the conditions used. Hence the sensitivity may be varied by means of varying the quantity of amino acid per sample (for comparison. see the standard curves b and c, Fig. 1) and amounted to the desired values (the slope of line c, Fig. I. is approximately 500 cpm/O.Ol pg of DNA in a sample of 0.2 ml). One can also increase the fraction number without a loss of sensitivity 3: as in curve no label: not
I except irradiated:
for
irradiation 13 ml.
8.10’
dose, 5 kR and cells; LA-Z.
I .5 hr at 37°C: (d) curve 2 hr. 29°C and 16 hr.
I: thymocytes, 0°C: I65 min.
24.000 rpm: [“Hlglycine. IO &i: (d) curve 2: as in curve I except for gradient lead, 2.105 cells. and amino acid preparation. [“Clglycine. IO PCi: (d) curve 3: as in curve I except for irradiation dose, IO kR, and amino acid preparation [“Hlglycine. IO yCi; (d) curve 4: as in curve I except for irradiation dose, IO kR and I hr at 37°C. and amino acid preparation, [“HJglycine, IO &i.
542
SKLOBOVSKAJA
ET AL.
at the expense of label quantity. Amino acid and Isotope natures as well as individual features of the commercial preparations may have some influence on the binding of amino acid to DNA and on the nonspecific sorption on the filter. It is therefore advisable to check any new labeled preparation for its properties. It is enough to obtain two points of a standard curve for some label quantity (lo-20 @/sample): one for a blank probe and one for a probe with a known DNA quantity (0.2-0.5 pg). With the data obtained one can evaluate the optimal label quantity per fraction, taking into consideration the required sensitivity of the DNA determination and the linear nature of the two concentration relationships. The above-mentioned characteristics of the method have allowed us to use the procedure for developing the sedimentation patterns of DNA from cells nonlabeled in rsivo. A comparison of the DNA sedimentation profiles for labeled and nonlabeled cells has been made using AEC cells. The data in Fig. 2a and b show that the correspondent profiles are similar. Figure 2 also presents the sedimentation patterns of DNA from nonlabeled AEC cells given y-doses of 1 and 5 kR in vitro and subjected to the incubation at 37°C (Fig. 2c) and those of DNA from nonlabeled thymocytes, intact, given 10 kR and incubated at 37°C after the dose (Fig. 2d). The results shown (DNA molecular weight range, the effective process of the single breaks rejoining in AEC cells in vitro, and the slow one in thymocytes in vitro) are in accordance with the data obtained for the same cell types using the original McGrath and Williams method or its fluorometric modification (4,12). The method presented for radiometric development of the DNA sedimentation patterns by postfractionation labeling of DNA can be applied under generally accepted gradient ultracentrifugation conditions to nondividing and slowly dividing cells and is believed to be useful in sedimentation studies of the mammalian cell DNA damage and repair. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. IO. I I. I?.
McGrath, R. A., and Williams. R. W. (1966) Nature (London) 209, 49. Karran. P.. and Ormerod, M. G. (1973) Biochim. Biophps. Acta 299, 54. Ono. T., and Okada, S. (1973) J. Radiat. Res. 14, 204. Ono, T.. and Okada. S. (1974) Int. J. Radiuf. Biol. 25, 291. Zubroff. J.. and Sarma, D. S. R. (1976) Anal. Biochem. 70, 387. Siomin. Ju. A.. Kolomiytseva. E. N., and Poverenny, A. M. (1974) Molecu/crrnuju Biologia 8, 276; 220 (in English). Poverenny. A. M., Siomin. Ju. A., Saenko, A. S., and Sinzinis, B. I. (1975) Mutar. Res. 27, 123. Siomin, Ju. A.. Simonov. V. V., and Poverenny, A. M. (1973) Biochim. Biophys. Actu 33, 27. Simonov, V. V.. Siomin. Ju. A., Suminov. S. I., and Poverenny. A. M. (1974) Biokhirniu 39, 517. Nygaard, A. P., and Hall, B. D. (1963) Biochem. Biophys. Res. Cornmun. 12, 98. Len. J. T.. Caldwell, J., Dean, C. J.. and Alexander, P. (1967) Nnture (London) 204,790. Moroson, H.. and Furlan. M. (1970) Radint. Res. 44, 713.
BIOCHEMISTRY
536-542
86,
(1978)
A Radiometric Method for Developing the Alkaline Sucrose Gradient Sedimentation Patterns of DNA from Nondividing Cells M. V. SKLOBOVSKAJA,
A. S. SAENKO, Ju. A. SIOMIN, AND A. M. POVERENNY
Research
of Medical
Institute
Radiology, Ohninsk, Kaluga
Received A radiometric tion
method
patterns
of
method is the of the binding The procedure filtration
December
of the
DNA
for from
30,
Academy Region,
1976:
of Medical USSR
accepted
developing
the
nonlabeled
cells
alkaline
October sucrose
is described.
Sciences
17,
1977
gradient The
of USSR,
sedimenta-
principle
of the
labeling of the DNA contained in the gradient fractions by means of a labeled amino acid to DNA in the presence of formaldehyde. involves incubation of the fractions with the labeling reagent, incubation
of the filters. The DNA concentration sufficient to escape labeled described
mammalian and those
in vivo
are
mixtures
through
nitrocellulose
filters.
relationship between the radioactivity on the in the sample is linear; the DNA detection overloading of the gradients. It was shown cell DNA of DNA
sedimentation from the
same
patterns cells
developed labeled with
and
radiometry
filters and the sensitivity is that the nonby the method [3H]thymidine
identical.
DNA from nondividing and slowly dividing cells cannot be radioactively labeled in viva and as a consequence cannot be characterized with the help of McGrath and Williams’ method in its original version (I). To develop the sedimentation patterns of those DNAs, an analytical method is required sensitive enough to detect submicrogram quantities of nonlabeled DNA in gradient fractions. At the present time the most commonly adopted approach to the problem utilizes some modifications of the Kissane and Robins’ fluorometric procedure for the determination of DNA (2-5). As a rule, the fluorometric method is used to determine high molecular weight mammalian DNA in gradients of large volume (30 to 36 ml), the load being l-2. lo6 cells and the fraction number 15- 18. The procedure consists of 12-14 steps. In this communication, we present an alternative radiometric method for determining DNA in gradient fractions. The method is more sensitive and less time-consuming than fluorometric one. We render DNA in the fractions radioactive using the interaction between DNA and aminomethylolic compounds, the reaction having been described previously 0003-2697/78/0862-0536$02.00/O Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
536
DNA
DETECTION
IN SUCROSE
GRADIENTS
537
(6-9). In this case, an aminomethylolic compound is formed from formaldehyde and a labeled amino acid. The procedure involves incubation of the fractions with a labeling reagent which contains labeled amino acid, formaldehyde and buffer, filtration of the incubation mixtures through nitrocellulose filters, and radiometry of the filters. Using this method, the alkaline sucrose gradient sedimentation profiles of DNA from Ehrlich ascitic carcinoma cells and mouse thymocytes have been obtained. The gradient volumes were 4.8, 13, and 22 ml, the loads were 5. 104-5. lo5 cells, and fraction numbers were 24-30. MATERIALS
AND METHODS
Materials. All the solutions were filtered through dense glass or nitrocellulose filters. Five- and twenty-percent sucrose solutions were made in 0.2 N NaOH, 0.8 N NaCl, and0.01 M Na,EDTA, pH 12.5. Alkaline lysing agents were 0.5 N NaOH, 0.5 N NaCl, 0.01 M Na,EDTA (LA-l), and 0.5 N NaOH, 0.1 M Na,EDTA (LA-2). The detergents used were: sodium dodecyl sulfate, Serwa, Sweden; and Sarkosyl. Schuchardt, BDR. Formaldehyde was from Merck. Medium 199 with 5% mouse or calf fetal serum was used for the cell suspension preparation. Calf thymus or chicken erythrocyte DNA was used in the model experiments. [3H]Thymidine was obtained from Isotop, USSR. Labeled amino acids were: [r4C]glycine, 51 mCi/mmol, Isotop, USSR; [3H]lysine 20 Ci/mmol, Amersham, England; and [3H]glycine, 0.6-2.5 Ciimmol, Isotop, USSR. Nitrocellulose filters, 0.45- 1.5 pm (Synpor nn 6-3) (diam., 24 mm), were obtained from Chemapol, Czechoslovakia. Labeling of ascitic Ehrlich carcinoma (AEC) cell DNA. AEC cell DNA was labeled by injecting 400 &i of [3H]thymidine ip into animal bearing 2- to 3-day tumor (100 PCi at 24, 20, 16. and 2 hr before decapitation). Cell suspensions: tion. The labeled
Preparation,
irradiation,
and postirradiation
incuba-
and nonlabeled ascitic fluids from the decapitated animals were isolated and washed twice with 0.9% NaCl. Aliquots of the cell suspension in 0.9% NaCl at a concentration of approximately 5. IO5 cells/ml were irradiated in ice by y-rays in a Gammacellat a dose rate of 17 rads/sec. then spun down, resuspended in the fresh 0.9% NaCl, and incubated at 37°C. Mouse thymus was removed from a decapitated animal, squashed gently in a manual Teflon homogenizer with two or three strokes of the pestle, and then sieved through a layer of the cotton in a 20-m] syringe without a needle. Irradiation and incubation of these cells was done in medium 199. Cell vitality was tested by means of erythrosine. The colored thymocyte fraction did not exceed 10% after postirradiation incubation. Cell lysis. When 4.8-m] gradients were used, 0.2 ml of lysing agent and 0.1 ml of the cell suspension were layered over the sucrose surface,
538
SKLOBOVSKAJA
ET AL.
while with 13-ml gradients, 0.5 ml of lysing agent and 0.5 ml of the suspension were used. The load of gradient, the type of lysing agent, and the lysis time and temperature are detailed in the legend to the Fig. 2. Preparation of the labeled aminomethylolic reagent (LAR). The LAR components were a labeled amino acid, formaldehyde, and phosphate buffer, pH 6.8. The concentrations of the components were such as to give a final concentration of formaldehyde in the incubation mixture (the probe plus LAR aliquot) of about 2% and that of the buffer, about 0.2 M. The labeled amino acid content was calculated to obtain the required sensitivity of DNA determination (see Results and Discussion) and as a rule 5-10 $Zi per sample in our experiments. First the amino acid was added to the buffer and then formaldehyde. LAR may polymerize in 20-24 hr. so it should be prepared on the day of use and filtered through membrane filter No. 5 or 6. Incubation andfiltration of the incubation mixtures. The DNA solution probes or gradient fractions of 0.2- I .O ml were incubated with 0.2 ml of LAR 16-20 hr at 18-22°C. Then the samples were diluted approximately 1:3 with 0.1 N NaCl and filtered through the nitrocellulose filters under a slight vacuum. After the sample had been sieved, the filter was washed with 0.1 N NaCI, 5% TCA, and 0. I N NaCl once more, then dried with ethanol and inserted into a scintillation vial. In some cases, e.g., if sodium dodecyl sulfate is present in a probe, it is preferable to precipitate the incubation mixture with cold 5% TCA and then filter as mentioned above. Linear gradients. Linear gradients were made using a three-channel mixing chamber, MSE, and three-channel micropump, Multiperpex 2115, LKB, Sweden. Ultracentrifkgation. The gradients were spun at 20°C in the Beckman SW-39 rotor of the Spinco Model L-50 ultracentrifuge or in MSE rotors 3 x 25 or 6 x 16.5 of the MSE Superspeedultracentrifuge. The ultracentrifugation time and speed are detailed in the legend to the Fig. 2. Gradientfractionation. This was also performed using the micropump, from the bottom of the gradient. The fractions of gradients of labeled AEC cell DNA were collected either on filter paper disks or strips of adequate size or into test tubes and then filtered, after TCA precipitation, through the membrane filters. The gradients which contained nonlabeled DNA from AEC cells or thymocytes were fractionated into the test tubes, and the fractions were then incubated with LAR and filtered as described above. Instead of test tubes, the holes of polystyrolic plates commonly used in the immunologic studies are convenient for use in fractionation and incubation. Measurement ofradioactivity. The filter radioactivity was determined in a liquid scintillation spectrometer (Ansitron, Picker, USA; or SL-30 Intertechnique, France) using the toluene scintillation fluid.
DNA
DETECTION
IN
RESULTS
SUCROSE
539
GRADIENTS
AND DISCUSSION
In this work we applied the initial step of the interaction between DNA and the aminothylolic compound, the binding of the amino acid to DNA in the presence of formaldehyde. All the steps and conditions of the reaction were described in detail earlier (6-9). We have chosen as our method a combination of reaction conditions such that some variations of the parameters (duration, 16-20 hr; temperature, 18-22°C; pH in the incubation mixture, 8-10) have little effect on the results. DNA is not a specific substrate for the aminothylolic compounds. These substances also react with RNA, proteins, and various amines. However, the alkaline sucrose gradient ultracentrifugation method per se provides satisfactory conditions for the selective detection of DNA: RNA is destroyed in the alkaline gradient and remains at the top of it along with the proteins and the compounds of low molecular weight. For the sake of specificity enhancement, we have introduced a filtration step into
I
2 DNA
FIG. 1. The relationship concentration in the incubation composition. 12% sucrose,
concentration,
,q
per ml
between the radioactivity retained on the filter mixture. LAR volume, 0.2 ml; probe volume. 0.2 N NaOH. 0.8 N NaCI. 0.01 M Na,EDTA.
present and the labeled amino acid preparation and detergent, [3H]lysine. 20 Ci/mmol, 3.5 &i/sample; 2.5 Ci/mmol, IO &i/sample: (c) no detergent, [RH]glycine,
and the DNA 0.2 ml; probe The detergent
quantity were as follows: (a) (b) 0.1% Sarkosyl, [3H]glycine, 2.5 Ci/mmol. 36 yCi/sample.
no
540
SKLOBOVSKAJA
/Y\ ‘\’\ \ 2 \\ A’/’ \
2,000
1,000 0k
ET AL.
&
b
i ._ D 5 s h .Z .; 1,000 :: 2 BL.
c
4,000 3,000 d 2,000 1,000
5
IO
15
20 fraction
25 number
FIG. 2. The alkaline sucrose gradient sedimentation patterns of DNA from labeled and nonlabeled in viva cells. Details for each gradient are as follows: cell type, [3H]TdR label in rive, if any; irradiation dose and postirradiation time of incubation at 37°C; the gradient volume and load; the lysing agent type and, the lysis time and temperature; the ultracentrifugation time and speed; the labeled amino acid preparation, if needed, and its quantity per fraction: (a) curve I: ascite, [3H]TdR; 3 kR; 4.8 ml, 5.104 cells; LA-l, I8 hr. 22°C; 90 min. 30,000 rpm; none; (a) curve 2: ascite, no label; as in curve 1 except for amino acid preparation, [3H]glycine, 20 &I; (b) curve 1: ascite, [3H]TdR; 3 kR; 13 ml, 1.5. IO5 cells: LA-l, I8 hr, 22°C; 5 hr, 24.000 rpm; none: (b) curve 2: ascite, no label; as in curve I except for amine acid preparation, [3H]lysine, 3 &i; (c) curve 1: ascite, no label; 2 hr. 29°C and 16 hr. 0°C; I65 min. 24,000 rpm; I kR; I3 ml, 1.5, IO5 cells; LA-2, dose. 5 kR; (c) curve [3H]glycine. 20 &i; (c) curve 2: as in curve I except for irradiation
DNA
DETECTION
IN
SUCROSE
GRADIENTS
541
the procedure; the nitrocellulose membrane filters are known to sorb the denatured DNA selectively (IO). Model experiments were undertaken to evaluate the additional radioactivity on the membrane filter resulting from the binding of labeled amino acid to RNA and protein presented in the DNA sample. The probes were alkaline sucrose solutions of DNA, RNA, and bovine serum albumin (BSA) in various combinations and concentrations: DNA, 0.1-2.0 pg; RNA, up to 10 pg: BSA, up to 20 Fg in a 0.2-ml volume. It was shown, by use of the procedure described above, that the presence of 10 pg of RNA in the probe gives no additional radioactivity; the presence of protein gives some but very little additional radioactivity (-5% of that from the same quantity of DNA). So use of a nonspecific reaction of aminomethylolic compounds with DNA for the DNA determination under the present conditions (ultracentrifugation at alkaline pH and filtration through membrane filters) provided little interference from RNA and proteins. The method to be applied for developing the DNA sedimentation patterns must satisfy two additional requirements: a linear relationship between a registered parameter and the DNA concentration, and a high sensitivity of the DNA determination, adequate to the permissible load of the gradients. Such a load in the case of the intact mammalian cells must not exceed 5. IO4 cells on the 4.8-ml gradient (11); i.e., approximately 0.5 pg of DNA per gradient or 0.05 pg of DNA in the peak fraction. The sensitivity of the method must be high enough for the reliable determination of hundreths of a microgram of DNA. These characteristics were also evaluated in model experiments. Micro- and submicrogram quantities of DNA have been determined by the method described in alkaline sucrose solutions containing NaCl. EDTA, detergents, and the other compounds used in cell lysis and gradient centrifugation. Several labeled amino acids were used for DNA determination: [3H]glycine. [‘*C]glycine, and [“Hllysine. A relationship between the observed count rate from the radioactivity precipitated on the filter and the DNA content in a probe was linear in all cases; some examples are given in Fig. 1. It was also shown that the quantity of DNA-bound amino acid depends linearly on the amino acid content in the sample and is far from saturated under the conditions used. Hence the sensitivity may be varied by means of varying the quantity of amino acid per sample (for comparison. see the standard curves b and c, Fig. 1) and amounted to the desired values (the slope of line c, Fig. I. is approximately 500 cpm/O.Ol pg of DNA in a sample of 0.2 ml). One can also increase the fraction number without a loss of sensitivity 3: as in curve no label: not
I except irradiated:
for
irradiation 13 ml.
8.10’
dose, 5 kR and cells; LA-Z.
I .5 hr at 37°C: (d) curve 2 hr. 29°C and 16 hr.
I: thymocytes, 0°C: I65 min.
24.000 rpm: [“Hlglycine. IO &i: (d) curve 2: as in curve I except for gradient lead, 2.105 cells. and amino acid preparation. [“Clglycine. IO PCi: (d) curve 3: as in curve I except for irradiation dose, IO kR, and amino acid preparation [“Hlglycine. IO yCi; (d) curve 4: as in curve I except for irradiation dose, IO kR and I hr at 37°C. and amino acid preparation, [“HJglycine, IO &i.
542
SKLOBOVSKAJA
ET AL.
at the expense of label quantity. Amino acid and Isotope natures as well as individual features of the commercial preparations may have some influence on the binding of amino acid to DNA and on the nonspecific sorption on the filter. It is therefore advisable to check any new labeled preparation for its properties. It is enough to obtain two points of a standard curve for some label quantity (lo-20 @/sample): one for a blank probe and one for a probe with a known DNA quantity (0.2-0.5 pg). With the data obtained one can evaluate the optimal label quantity per fraction, taking into consideration the required sensitivity of the DNA determination and the linear nature of the two concentration relationships. The above-mentioned characteristics of the method have allowed us to use the procedure for developing the sedimentation patterns of DNA from cells nonlabeled in rsivo. A comparison of the DNA sedimentation profiles for labeled and nonlabeled cells has been made using AEC cells. The data in Fig. 2a and b show that the correspondent profiles are similar. Figure 2 also presents the sedimentation patterns of DNA from nonlabeled AEC cells given y-doses of 1 and 5 kR in vitro and subjected to the incubation at 37°C (Fig. 2c) and those of DNA from nonlabeled thymocytes, intact, given 10 kR and incubated at 37°C after the dose (Fig. 2d). The results shown (DNA molecular weight range, the effective process of the single breaks rejoining in AEC cells in vitro, and the slow one in thymocytes in vitro) are in accordance with the data obtained for the same cell types using the original McGrath and Williams method or its fluorometric modification (4,12). The method presented for radiometric development of the DNA sedimentation patterns by postfractionation labeling of DNA can be applied under generally accepted gradient ultracentrifugation conditions to nondividing and slowly dividing cells and is believed to be useful in sedimentation studies of the mammalian cell DNA damage and repair. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. IO. I I. I?.
McGrath, R. A., and Williams. R. W. (1966) Nature (London) 209, 49. Karran. P.. and Ormerod, M. G. (1973) Biochim. Biophps. Acta 299, 54. Ono. T., and Okada, S. (1973) J. Radiat. Res. 14, 204. Ono, T.. and Okada. S. (1974) Int. J. Radiuf. Biol. 25, 291. Zubroff. J.. and Sarma, D. S. R. (1976) Anal. Biochem. 70, 387. Siomin. Ju. A.. Kolomiytseva. E. N., and Poverenny, A. M. (1974) Molecu/crrnuju Biologia 8, 276; 220 (in English). Poverenny. A. M., Siomin. Ju. A., Saenko, A. S., and Sinzinis, B. I. (1975) Mutar. Res. 27, 123. Siomin, Ju. A.. Simonov. V. V., and Poverenny, A. M. (1973) Biochim. Biophys. Actu 33, 27. Simonov, V. V.. Siomin. Ju. A., Suminov. S. I., and Poverenny. A. M. (1974) Biokhirniu 39, 517. Nygaard, A. P., and Hall, B. D. (1963) Biochem. Biophys. Res. Cornmun. 12, 98. Len. J. T.. Caldwell, J., Dean, C. J.. and Alexander, P. (1967) Nnture (London) 204,790. Moroson, H.. and Furlan. M. (1970) Radint. Res. 44, 713.