- Email: [email protected]
Studies on the action and chemical nature of the nuclear factor promoting degradation of chromatin DNA
Printed in Sweden Copyright 0 1974 by Academic Press, Inc. AN rights of reproduction in any form reserved
Experimental Cell Research 84 (1974) 388-394
STUDIES ON THE ACTION AND CHEMICAL FACTOR PROMOTING DEGRADATION K. M. JANDIERI,
NATURE OF THE NUCLEAR OF CHROMATIN DNA
G. D. TUMANISHVILI, N. G. AVALISHVILI, N. D. MGVDELADZE
D. V. DZIDZIGURI
AND
Institute of Experimental Morphology, Academy of Sciences of the Georgian SSR, Digomi 380059, Tbilisi, Georgian SSR
SUMMARY Chromatin DNA of liver and kidney, obtained by the method of Dingman & Sporn, is inaccessible in 0.14 M NaCl to pancreatic DNase and cytoplasmic DNase. Under the combined action of DNase and nuclear extract (NE) (extraction with 0.14 M NaCI) on chromatin, the DNA of the latter is intensively degraded. The action of NE is tissue-specific-liver NE has almost no effect on kidney chromatin DNA degradation. The removal of protein or RNA from NE deprives it of its ability to accelerate chromatin DNA degradation by DNases. It is assumed that the active part of NE is a complex of a protein and RNA. Here, tissue specificity is determined by both components of this complex. The biological role of the nuclear factor promoting chromatin DNA degradation is not known at present.
Recently in experiments with chick embryos jetted to the action of nuclear extracts, in vivo, data have been obtained indicating thus providing a system more suitable for the the existence of a factor in hen liver nuclei analysis than that used in the previous causing DNA degradation in nuclei of the work. chick embryo liver [16, 171. Subsequent experiments carried out on MATERIALS AND METHODS homogenates of rat liver and kidney have Wistar albino rats, weighing 20&250 g, were shown that nuclei of liver and kidney actually decapitated to obtain chromatin and nuclear contain the factor promoting DNA degrada- extract from liver and kidney using the technique of Dingman & Sporn [2]. To extract chromatin, a tion in the nuclei. The action of the factor 0.2 mM solution of EDTA, was used at 1 ml/g of was found to be organ-specific. The sub- the initial tissue weight. It is known that under urevailina in this study nearly all the stance promoting DNA degradation is ex- conditions chromatin present goes into solution [2]. tracted from nuclei by 0.14 M NaCl and is To obtain nuclear extract (NE) of the liver or nuclei were isolated using the method of evidently one of the components of the kidney, Berthet & de Duve [l], namely, 20 % tissue nuclear sap [4]. homogenate in 0.25 M solution of sucrose, was at 600 g for 10 min and the precipitate The present paper studies the action of the centrifuged washed each time with sucrose solution. The nuclear factor promoting DNA degradation sunernatant obtained after the first centrifugation the cytoplasmic fraction. The nuclear fraction in chromatin of rat liver and kidney in order was was incubated for 1 h in 0.14 M solution of NaCl to obtain information on the mechanism at + 4°C the volume ratio being 1: 2, then the mixture of action of the nature. Chromatin
factor and its chemical preparations were sub-
Exptl CeN Res 84 (1974)
was centrifuged for 10 min. The precipitate was not used in the experiments and the supernatant represented the nuclear extract.
The nuclear factor promoting DNA degradation
389
Table 1. Change in pH of the incubation medium and the rate of liver chromatin DNA degradation pH value
Incubation medium Chromatin + cytoplasmic fraction + 0.14 M NaCl Chromatin + cytoplasmic fraction + NE Chromatin + DNase + 0.14 M NaCl Chromatin + DNase + NE Chromatin + DNase +0.14 M NaCl (pH previously brought to pH 7.15)
To study the chemical nature of the nuclear factor, the NE was heated (45”, 50” and 55”), digested with crystalline trypsin produced by SPOFA (Czechoslovakia) (2 mg/ml of the extract, incubation for 1 h at 37°C) and with the pancreatic RNase (from Reanal, Budapest, Hungary) with an activity of 40 Kunitz units (10 pg/ml of the extract, incubation for 15 min at 37°C). Trypsinization was stopped by trypsin soy bean inhibitor (Reanal), while RNase was removed with askangel-a kind of bentonite received from the Institute of Physical and Organic Chemistry (Academy of Sciences of the Georgian SSR), and treated at the Institute of Plant Biochemistry (Academy of Sciences of the Georgian SSR) [5]. To study the action of NE on the rate of chromatin DNA degradation, the latter was incubated in the following way: either 0.5 ml of cytoplasmic fraction of liver or kidney, or 0.5 ml of DNase solution (25 pg pancreatic DNase; Sigma, activity 2 000 Kunitz units, in 0.14 M solution of NaCl) was added to 0.5 ml chromatin. 0.5 ml of NE of either liver or kidney was added to chromatin with either of the media. In the control experiments 0.5 ml of 0.14 M NaCl or (in a number of experiments) 0.25 M sucrose solution was added to the incubation mixture. Thus, the volume of the incubation mixture was always 1.5 ml, except in those cases when the ratio of the mixture components was changed with a special aim (see below). The incubation proceeded for 1 h at 37°C. After incubation the extent of chromatin DNA degradation was estimated by increasing the concentration of acid-soluble nucleotides or by decreasing the DNA concentration in the incubation medium. The concentration of acid-soluble nucleotides was determined spectrophotometrically according to Spirin [14] and that of DNA according to Tsanev & Markov [15] or Dische [3]. Since the results of the determinations of the extent of DNA degradation by the different methods were in good agreement, the amount of acid-soluble nucleotides alone was determined in most cases. Determinations were made before and after cbromatin incubation in different media. A decrease of DNA content was
Before incubation
After incubation
Increase in content of free nucleotides (%I
6.60 6.80 6.67 7.15
6.67 6.75 6.65 7.15
3-2 56-5 3-l 43-3
7.15
7.15
4-2
expressed as a percentage of its initial concentration. The increment in free nucleotide concentration was expressed as a percentage, as the ratio of difference in extinctions, corresponding to the nucleotide concentration in the medium at the beginning of the experiment and at its end, to the extinction corresponding to the DNA amount in the solution. To characterize the obtained chromatin and NE, their content of DNA [3], RNA [3] and protein [9] was determined. On average, 1 ml of chromatin contains 147+21 pg of DNA, 23)4,ug of RNA and 165 - 12 ,ug of protein. The protein/DNA ratio was l&1.2. The content of RNA and protein in NE of liver and kidney is given in table 2. DNA was not found in NE.
RESULTS Degradation of chromatin DNA of rat liver and kidney in presence and absence of NE
When chromatin is incubated with cytoplasmic fraction or DNase only a negligible part of chromatin DNA is degraded. The content of free nucleotides in the medium is increased by 3 %, on average, and DNA decreasesrespectively by 3-5 % (fig. 1). It is seen in the figure that when NE is absent, the amount of chromatin DNA of liver and kidney degraded during 1 h is the same. Approximately the same amount of chromatin DNA is degraded when the latter is incubated with the respective NE (fig. 1) in the absence of cytoplasmic fraction or DNase. Exptl Cell Res 84 (1974)
Jandieri et al.
1
7c 60
The amount of degraded DNA and thereby the concentration of free nucleotides is sharply increased when nuclear extract of an organ is added to the chromatin incubated with the cytoplasmic fraction of the same organ or DNase (fig. 1). The increase of the rate of chromatin DNA degradation was more then IO-fold. Addition of MgCl, to the incubation medium to compensate Mg ions bound by EDTA (0.8 mM of MgCl, to 0.2 mM EDTA) did not affect the experimental results. Under these conditions not more then 5 % of the chromatin DNA was degraded, as occurs in the controls.
5c
Effect of pH on chromatin DNA degradation 40 30 20 10 0 6
6.5
7.0
7.5
8
3 Fig. 1. Degradation of chromatin DNA of rat liver and kidney in the presence and absence of NE. Abscissa: I, liver chromatin + liver cytoplasmic fraction + NaClO.14 M; II, liver chromatin + DNase + NaCl 0.14 M or 0.25 M sucrose solution; III, liver chromatin +liver NE +NaCl 0.14 M; IV, kidney chromatin +DNase +NaCl 0.14 M; V, liver chromatin + liver cytoplasmic fraction + liver NE; VI, liver chromatin + DNase + liver NE; VII, kidney chromatin + DNase + kidney NE; ordinate: increase in content of acid-soluble nucleotides. Fig. 2. Abscissa: pH values; ordinate: increase in content of acid soluble nucleotides. Effect of pH on the extent of degradation of chromatin DNA. Exptl Cell Res 84 (1974)
In most of our experiments we did not add any buffer to incubation media for fear that its presencein the cytoplasmic fraction might upset the conditions required for the observed processes. Degradation of chromatin DNA proceeded in both a slightly acid and slightly alkaline medium (table 1). When the cytoplasmic fraction was used, the pH of the medium, whether with or without NE, is almost the same and in this case the acceleration of chromatin DNA degradation appears not to depend on the pH value. With DNase present, the reaction in the presence and absence of NE proceeds at different pH values (6.8-7.15 respectively). Such a change of pH could enhance chromatin DNA degradation. However, the previous increase of pH in the absence of
Fig. 3. Effect of NE and cytoplasmic fractions of heterologous tissues on degradation of chromatin DNA. Abscissa: I, liver chromatin +liver cytoplasmic fraction + kidney NE; II, liver chromatin + DNase + kidney NE; III, kidney chromatin + DNase -Iliver Ne; IV, liver chromatin + DNase + kidney chromatin (1.Oml); V, liver chromatin + kidney cytoplasmic fraction + liver NE; VI, kidney chromatin + liver cytoplasmic fraction + kidney NE; ordinate: increase in content of acid-soluble nucleotides.
The nuclear factor promoting DNA degradation
391
NE to 7.15 did not cause any degradation of chromatin DNA (table 1). We were able to establish that the NE activity is exerted at pH 6.8-7.15. At pH values higher and lower than those, NE does not enhance the chromatin DNA degradation (fig. 2). The indicated range is rather narrower than the pH values within which the activity of DNase is exerted.
60
Degradation of chromatin DNA under the influence of NE and cytoplasmic fraction of heterologous tissues
50
40
30 25' 5
10
15
20
2.5
50
50 40
* 3?
30
T
25 20 10 0 I
II
*ml-h 111
IV
v
VI
wt
VIII
whh IX
x-
XI
Fig. 4. Abscissa: amount of DNase &g); ordinate: increase in content of acid-soluble nucleotides. 0, without NE; x , with NE. Extent of chromatin DNA degradation under the action of DNAase in different concentration. Fig. 5. Abscissa: amount of DNase (up); ordinate: increase in content of acid-soluble nucleotides. 0, without; x, with NE. Effect of NE on digestion of DNA by DNase. Fig. 6. Effects of heating, trypsin, and DNase treatment on NE activity. Abscissa: I, liver chromatin + DNase + liver NE, heated (45°C); II, liver cbromatin + DNase + liver NE, heated (50%); III, liver chromatin + DNase -t liver NE (55°C); IV, liver chromatin + DNase + liver NE treated with
If kidney NE is added to the liver chromatin, chromatin DNA degradation is not accelerated in the presence of the cytoplasmic fraction of liver or DNase; Even in the case when 1.0 ml kidney NE was added instead of 0.5 ml, the rate of liver chromatin DNA degradation by DNase was not increased (fig. 3). Similarly, when liver NE is added to kidney chromatin in the presenceof kidney cytoplasmic fraction or DNase the degradation of kidney chromatin DNA does not accelerate (fig. 3). At the same time, when kidney cytoplasmic fraction is used instead of liver cytoplasmic fraction, liver chromatin DNA is degraded in the presenceof liver NE at the same rate as in the liver cytoplasmic fraction (fig. 3). In experiments with kidney chromatin it was also shown that the action of the cytoplasmic fraction is not tissue-specific (fig. 3). trypsin; V, liver chromatin + DNase + liver NE treated with RNase; VI, liver chromatin + DNase + liver NE, heated (55°C) +liver NE treated with RNase; VII, liver chromatin + DNase + liver NE treated with trypsin + liver NE treated with RNase; VIII, liver chromatin + DNase + liver NE, heated (55°C) +kidney NE treated with RNase; IX, liver chromatin + DNase + liver NE treated with RNase + kidney NE, heated (SST); X, liver chromatin+ DNase + liver NE treated with trypsin + kidney NE treated with RNase; XI, Liver chromatin-tDNase +liver NE treated with RNase + kidney NE treated with trypsin. ordinate: increase in content of acid-soluble nucleotides. Exptl Cell Res 84 (1974)
392
Jandieri et al.
Thus, relative acceleration of chromatin DNA degradation occurs only in the presence of NE from a homologous tissue, while the substitution of homologous by heterologous cytoplasmic fraction does not affect degradation.
Effects of heating, trypsin and DNase treatment on NE activity
If NE is heated to 55°C and maintained at this temperature for 5 min, its addition to the cytoplasmic fraction or to DNase solution does not cause chromatin DNA degradation (fig. 6). Heating to 45 and 50°C does not abolish its activity. Thus, Extent of chromatin DNA degradation 55°C is the threshold temperature for NE by DNase in various concentrations inactivation. As shown above, a chosen concentration of Incubation of NE to 55°C causesdenaturaDNase does not cause chromatin DNA tion of only part of the protein present in degradation in the absence of NE. As it the solution. When the precipitate is removed turned out, DNase, even in higher conby centrifugation (600 g, for 10 min) almost centrations, does not digest chromatin half of the initial protein is still found in the DNA when incubated in the absence of solution (table 2). NE (fig. 4). In the case of liver chromatin NE also lost its activity if treated previincubation in the presence of liver NE, the ously with trypsin (see Materials and Methrate of chromatin DNA degradation depends ods), the amount of protein in NE being on the amount of DNase (fig. 4). An increase also decreased (table 2). in liver NE up to 1.0 ml in the presence NE loses its activity also following treatof 25 ,ug DNase in the incubation medium ment with RNase (fig. 6). Since the extract does not alter the result (44 + 2.0 %). had been treated with askangel to remove RNase, it was necessary to make sure that DNase activity of NE and the effect of askangel itself did not cause NE inactivation. NE on DNase activity It was found that askangel did not deprive DNase activity of NE was determined by NE of the ability to accelerate chromatin DNA degradation in the presence of DNase the extent of degradation of pure DNA (fig. 6). (erythrocyte DNA; Sigma). 0.5 ml DNA Treatment with RNase reduced to less (20 pg) were placed in a phosphate buffer (0.1 M) pH -7.2, containing 0.01 M MgCl,. than half the initial RNA amount in the 0.5 ml of liver NE was added to the solution extract (table 2). Treatment with askangel and incubated for 1 h at 37°C. Under such only slightly reduced the concentrations of conditions the appearance of acid-soluble RNA and protein in NE. It follows from nucleotides was not observed (2.0 t- 1.O%), these experiments that partial removal of which shows the absence of DNase activity both protein and RNA from NE deprived it of its ability to accelerate chromatin DNA in NE. To determine the effect of NE on the degradation by DNase. If NE inactivated by heating or trypsin DNase activity, we carried out a series of experiments with increasing amounts of was combined with an equal volume of NE DNase in the absence and presence of the inactivated by RNase, the activity of NE liver NE. The intensity of pure DNA degrada- was restored and it was again able to accetion did not depend on the presence of NE lerate chromatin DNA degradation in the presence of DNase (fig. 6). However, when in the incubation medium (fig. 5). Exptl CeN Res 84 (1974)
The nuclear factor promoting DNA degradation
393
the experiments of Marushige & Bonner [I 11, little or no chromosomal DNA becomes acid-soluble by treatment with Protein RNA DNase, some degree of digestion of DNA per 1 ml per 1 ml was detected when applying other criteria to of NE of NE Kind of treatment of NE (md tug) their results. Recently it has been found that in 0.14 M 7.6kO.3 210& 17 Without treatment NaCl the activity of DNase may be 170+12 4.2kO.2 Heating (SYC) 3.1kO.5 182+10 Trypsin reduced by almost 50 % [13]. However, this 86&8 6.6kO.2 RNase was not observed in our experiments, where 186&10 7.0+0.3 Askangel the addition of 0.25 M sucrose solution, instead of 0.14 M NaCl, in a number of control experiments did not affect the two portions of NE treated in different ways result. were combined, the total amount of NE was The absence of chromatin DNA degradafound to be twice as much as before (1 ml), tion may be due to the binding of Mg which might cause the apparent recovery of ions by EDTA, while an increase in its activity. Therefore in the control experi- DNase activity on addition of NE could be ments, 1 ml of NE treated by heating, a result of addition of Mg2+ ions contained trypsin, or RNase, was added to 0.5 ml of in the extract. However, such an assumpchromatin solution. The increase in the tion has been excluded by experiments with amount of extract did not affect the results addition of an excessive amount of MgCl, of the experiments. in the absence of NE, which did not cause When liver NE treated with heat or any increase in chromatin DNA degradatrypsin was combined with kidney NE tion. treated with RNase or vice versa, the Enhancement of chromatin DNA degradaactivity of NE was not restored (fig. 6). tion cannot be attributed to a small shift of pH caused by the addition of the nuclear extract to the medium. Neither the rise in DISCUSSION pH to 7.15 nor the incubation of chromatin The experiments have confirmed our assump- in the phosphate buffer causes enhancement tion [4] (that there is a nuclear factor of chromatin DNA degradation. Especially promoting chromatin DNA degradation by when cytoplasmic fraction is used instead of DNase). It seems surprising that under the DNase, the above-mentioned shift was hardly action of DNase such a small part of chroma- noticeable. tin DNA is degraded. Actually, a more The optimum values of pH, determined complete digestion of the chromatin DNA for the action of NE, are within 6.8-7.15. by DNase has been reparted (e.g. [13]). This fact, evidently, should be taken into However, the high reproducibility of our account while planning experiments and results rules out all possible doubt in this interpreting the data obtained. respect. It may be that the discrepancy in The loss of NE activity after heating and the results depends on the method of treatment shows the protein nature of chromatin extraction and the kind of tissue the nuclear factor promoting chromatin DNA used. It must be noted that although in degradation. Table 2. Effect of heat, trypsin and RNase on the amount of protein and RNA in NE
Exptl Cell Res 84 (1974)
394
Jandieri et al.
The ability of the nuclear extract to promote chromatin DNA degradation is related to its other component as wellRNA. RNA, though not degraded either by heating to 55°C or trypsin, is sufficiently well removed from the extract treated with RNase. As askangel does not cause any decrease in NE activity, the inactivation of the nuclear extract after treatment with RNase must be due to RNA degradation and not to the absorption by some other substances by askangel. The evidence that the nuclear factor promoting chromatin DNA degradation consists of at least two components, is supplied by experiments with combinations of two portions of the nuclear extract, one of which was previously treated by heating or trypsin and the other by RNase. After these two portions had been reunited, the extract was found to be active. A striking feature, hard to explain is the fact that the tissue specificity of NE is determined by both of its components, protein and RNA. We cannot say at present whether we are dealing with some peculiar class of RNA or with some other mechanism of this phenomenon. This problem is under investigation. Probably, the factor found is identical with that extracted from Rana pipiens liver, which when injected into the fertilized egg of the same species causes chromosome distortions leading to the cessation of the embryonic development at the stage of the early gastrula [lo, 181.As was found, this factor may be identified as an acid protein [12], though the authors themselves do not think that the data obtained by them are sufficiently clear-cut. In a number of her papers, Ermolaeva has described a substance which is not one of the known DNases, but participates in
Exptl Cell Res 84 (1974)
the chromatin DNA degradation [6, 7, 81. She considers the nuclear factor to be an enzyme, the action of which in her opinion has no tissue specificity. Ermolaeva’s conclusions have not been confirmed by our data either with respect to the specificity or to the chemical nature of NE. The biological role of the nuclear factor promoting chromatin DNA destruction is unknown. Most probably it is the same factor which causes DNA degradation in the experiments in vivo mentioned previously. REFERENCES 1. Berthet, J & de Duve, C D, Biochem j 50 (1951) 174. 2. Dingman, M B & Sporn, M B, J biol them 239 (1964) 3483. 3. Dische, Z., The nucleic acids (ed E Chargaff & J N Davtdson) p. 426. Academic Press, New York (1955). 4. Jandieri, K’M, Tumanishvili G D, Vopr biofisiki i teoreticheskoi biol. vol. 3._ D. _ 45. Tbilisi TGU (1971). 5. Jokhadse, D I, Bull Georgian acad sci 12 (1966) 621. Ermolaeva, N V, Biokhimia 31 (1966) 860. 7”: - Ibid 32 (1967) 774. - Ibid 35 (1970) 17. i: Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1953) 265. 10. Forkert, C L & Ursprung, H, Dev biol 7 (1963) 11. Marushiae. K & Bonner. J. Proc natl acad sci US 68 (1971)2941. ’ . Melton. Ch G. J exotl zoo1 164 (1967) 325. ::: Mirskyj A E, ‘Proc-natl acad si‘ US -68 (1971) 2945. Spirin, A S, Biokhimia 23 (1958) 656. ::: Tsanev, R G & Markov, G G, Biokhimia 25 (1960) 151. 16. Tumanishvili. G D & Salamatina. N V, Vopr biofiziki i teoreticheskoi biol, vol. 2, p. 89. Tbilisi TGU (1969). 17. Tumanishvili, G D, Vepkhvadze, L K, Salamatina, N V, Arm embryo1 morphogen, suppl. 1 (1969) 275. 18. Ursprung, H & Markert, C L, Dev biol 8 (1963) 309. Received September 8, 1973 Revised version received October 29, 1973
Experimental Cell Research 84 (1974) 388-394
STUDIES ON THE ACTION AND CHEMICAL FACTOR PROMOTING DEGRADATION K. M. JANDIERI,
NATURE OF THE NUCLEAR OF CHROMATIN DNA
G. D. TUMANISHVILI, N. G. AVALISHVILI, N. D. MGVDELADZE
D. V. DZIDZIGURI
AND
Institute of Experimental Morphology, Academy of Sciences of the Georgian SSR, Digomi 380059, Tbilisi, Georgian SSR
SUMMARY Chromatin DNA of liver and kidney, obtained by the method of Dingman & Sporn, is inaccessible in 0.14 M NaCl to pancreatic DNase and cytoplasmic DNase. Under the combined action of DNase and nuclear extract (NE) (extraction with 0.14 M NaCI) on chromatin, the DNA of the latter is intensively degraded. The action of NE is tissue-specific-liver NE has almost no effect on kidney chromatin DNA degradation. The removal of protein or RNA from NE deprives it of its ability to accelerate chromatin DNA degradation by DNases. It is assumed that the active part of NE is a complex of a protein and RNA. Here, tissue specificity is determined by both components of this complex. The biological role of the nuclear factor promoting chromatin DNA degradation is not known at present.
Recently in experiments with chick embryos jetted to the action of nuclear extracts, in vivo, data have been obtained indicating thus providing a system more suitable for the the existence of a factor in hen liver nuclei analysis than that used in the previous causing DNA degradation in nuclei of the work. chick embryo liver [16, 171. Subsequent experiments carried out on MATERIALS AND METHODS homogenates of rat liver and kidney have Wistar albino rats, weighing 20&250 g, were shown that nuclei of liver and kidney actually decapitated to obtain chromatin and nuclear contain the factor promoting DNA degrada- extract from liver and kidney using the technique of Dingman & Sporn [2]. To extract chromatin, a tion in the nuclei. The action of the factor 0.2 mM solution of EDTA, was used at 1 ml/g of was found to be organ-specific. The sub- the initial tissue weight. It is known that under urevailina in this study nearly all the stance promoting DNA degradation is ex- conditions chromatin present goes into solution [2]. tracted from nuclei by 0.14 M NaCl and is To obtain nuclear extract (NE) of the liver or nuclei were isolated using the method of evidently one of the components of the kidney, Berthet & de Duve [l], namely, 20 % tissue nuclear sap [4]. homogenate in 0.25 M solution of sucrose, was at 600 g for 10 min and the precipitate The present paper studies the action of the centrifuged washed each time with sucrose solution. The nuclear factor promoting DNA degradation sunernatant obtained after the first centrifugation the cytoplasmic fraction. The nuclear fraction in chromatin of rat liver and kidney in order was was incubated for 1 h in 0.14 M solution of NaCl to obtain information on the mechanism at + 4°C the volume ratio being 1: 2, then the mixture of action of the nature. Chromatin
factor and its chemical preparations were sub-
Exptl CeN Res 84 (1974)
was centrifuged for 10 min. The precipitate was not used in the experiments and the supernatant represented the nuclear extract.
The nuclear factor promoting DNA degradation
389
Table 1. Change in pH of the incubation medium and the rate of liver chromatin DNA degradation pH value
Incubation medium Chromatin + cytoplasmic fraction + 0.14 M NaCl Chromatin + cytoplasmic fraction + NE Chromatin + DNase + 0.14 M NaCl Chromatin + DNase + NE Chromatin + DNase +0.14 M NaCl (pH previously brought to pH 7.15)
To study the chemical nature of the nuclear factor, the NE was heated (45”, 50” and 55”), digested with crystalline trypsin produced by SPOFA (Czechoslovakia) (2 mg/ml of the extract, incubation for 1 h at 37°C) and with the pancreatic RNase (from Reanal, Budapest, Hungary) with an activity of 40 Kunitz units (10 pg/ml of the extract, incubation for 15 min at 37°C). Trypsinization was stopped by trypsin soy bean inhibitor (Reanal), while RNase was removed with askangel-a kind of bentonite received from the Institute of Physical and Organic Chemistry (Academy of Sciences of the Georgian SSR), and treated at the Institute of Plant Biochemistry (Academy of Sciences of the Georgian SSR) [5]. To study the action of NE on the rate of chromatin DNA degradation, the latter was incubated in the following way: either 0.5 ml of cytoplasmic fraction of liver or kidney, or 0.5 ml of DNase solution (25 pg pancreatic DNase; Sigma, activity 2 000 Kunitz units, in 0.14 M solution of NaCl) was added to 0.5 ml chromatin. 0.5 ml of NE of either liver or kidney was added to chromatin with either of the media. In the control experiments 0.5 ml of 0.14 M NaCl or (in a number of experiments) 0.25 M sucrose solution was added to the incubation mixture. Thus, the volume of the incubation mixture was always 1.5 ml, except in those cases when the ratio of the mixture components was changed with a special aim (see below). The incubation proceeded for 1 h at 37°C. After incubation the extent of chromatin DNA degradation was estimated by increasing the concentration of acid-soluble nucleotides or by decreasing the DNA concentration in the incubation medium. The concentration of acid-soluble nucleotides was determined spectrophotometrically according to Spirin [14] and that of DNA according to Tsanev & Markov [15] or Dische [3]. Since the results of the determinations of the extent of DNA degradation by the different methods were in good agreement, the amount of acid-soluble nucleotides alone was determined in most cases. Determinations were made before and after cbromatin incubation in different media. A decrease of DNA content was
Before incubation
After incubation
Increase in content of free nucleotides (%I
6.60 6.80 6.67 7.15
6.67 6.75 6.65 7.15
3-2 56-5 3-l 43-3
7.15
7.15
4-2
expressed as a percentage of its initial concentration. The increment in free nucleotide concentration was expressed as a percentage, as the ratio of difference in extinctions, corresponding to the nucleotide concentration in the medium at the beginning of the experiment and at its end, to the extinction corresponding to the DNA amount in the solution. To characterize the obtained chromatin and NE, their content of DNA [3], RNA [3] and protein [9] was determined. On average, 1 ml of chromatin contains 147+21 pg of DNA, 23)4,ug of RNA and 165 - 12 ,ug of protein. The protein/DNA ratio was l&1.2. The content of RNA and protein in NE of liver and kidney is given in table 2. DNA was not found in NE.
RESULTS Degradation of chromatin DNA of rat liver and kidney in presence and absence of NE
When chromatin is incubated with cytoplasmic fraction or DNase only a negligible part of chromatin DNA is degraded. The content of free nucleotides in the medium is increased by 3 %, on average, and DNA decreasesrespectively by 3-5 % (fig. 1). It is seen in the figure that when NE is absent, the amount of chromatin DNA of liver and kidney degraded during 1 h is the same. Approximately the same amount of chromatin DNA is degraded when the latter is incubated with the respective NE (fig. 1) in the absence of cytoplasmic fraction or DNase. Exptl Cell Res 84 (1974)
Jandieri et al.
1
7c 60
The amount of degraded DNA and thereby the concentration of free nucleotides is sharply increased when nuclear extract of an organ is added to the chromatin incubated with the cytoplasmic fraction of the same organ or DNase (fig. 1). The increase of the rate of chromatin DNA degradation was more then IO-fold. Addition of MgCl, to the incubation medium to compensate Mg ions bound by EDTA (0.8 mM of MgCl, to 0.2 mM EDTA) did not affect the experimental results. Under these conditions not more then 5 % of the chromatin DNA was degraded, as occurs in the controls.
5c
Effect of pH on chromatin DNA degradation 40 30 20 10 0 6
6.5
7.0
7.5
8
3 Fig. 1. Degradation of chromatin DNA of rat liver and kidney in the presence and absence of NE. Abscissa: I, liver chromatin + liver cytoplasmic fraction + NaClO.14 M; II, liver chromatin + DNase + NaCl 0.14 M or 0.25 M sucrose solution; III, liver chromatin +liver NE +NaCl 0.14 M; IV, kidney chromatin +DNase +NaCl 0.14 M; V, liver chromatin + liver cytoplasmic fraction + liver NE; VI, liver chromatin + DNase + liver NE; VII, kidney chromatin + DNase + kidney NE; ordinate: increase in content of acid-soluble nucleotides. Fig. 2. Abscissa: pH values; ordinate: increase in content of acid soluble nucleotides. Effect of pH on the extent of degradation of chromatin DNA. Exptl Cell Res 84 (1974)
In most of our experiments we did not add any buffer to incubation media for fear that its presencein the cytoplasmic fraction might upset the conditions required for the observed processes. Degradation of chromatin DNA proceeded in both a slightly acid and slightly alkaline medium (table 1). When the cytoplasmic fraction was used, the pH of the medium, whether with or without NE, is almost the same and in this case the acceleration of chromatin DNA degradation appears not to depend on the pH value. With DNase present, the reaction in the presence and absence of NE proceeds at different pH values (6.8-7.15 respectively). Such a change of pH could enhance chromatin DNA degradation. However, the previous increase of pH in the absence of
Fig. 3. Effect of NE and cytoplasmic fractions of heterologous tissues on degradation of chromatin DNA. Abscissa: I, liver chromatin +liver cytoplasmic fraction + kidney NE; II, liver chromatin + DNase + kidney NE; III, kidney chromatin + DNase -Iliver Ne; IV, liver chromatin + DNase + kidney chromatin (1.Oml); V, liver chromatin + kidney cytoplasmic fraction + liver NE; VI, kidney chromatin + liver cytoplasmic fraction + kidney NE; ordinate: increase in content of acid-soluble nucleotides.
The nuclear factor promoting DNA degradation
391
NE to 7.15 did not cause any degradation of chromatin DNA (table 1). We were able to establish that the NE activity is exerted at pH 6.8-7.15. At pH values higher and lower than those, NE does not enhance the chromatin DNA degradation (fig. 2). The indicated range is rather narrower than the pH values within which the activity of DNase is exerted.
60
Degradation of chromatin DNA under the influence of NE and cytoplasmic fraction of heterologous tissues
50
40
30 25' 5
10
15
20
2.5
50
50 40
* 3?
30
T
25 20 10 0 I
II
*ml-h 111
IV
v
VI
wt
VIII
whh IX
x-
XI
Fig. 4. Abscissa: amount of DNase &g); ordinate: increase in content of acid-soluble nucleotides. 0, without NE; x , with NE. Extent of chromatin DNA degradation under the action of DNAase in different concentration. Fig. 5. Abscissa: amount of DNase (up); ordinate: increase in content of acid-soluble nucleotides. 0, without; x, with NE. Effect of NE on digestion of DNA by DNase. Fig. 6. Effects of heating, trypsin, and DNase treatment on NE activity. Abscissa: I, liver chromatin + DNase + liver NE, heated (45°C); II, liver cbromatin + DNase + liver NE, heated (50%); III, liver chromatin + DNase -t liver NE (55°C); IV, liver chromatin + DNase + liver NE treated with
If kidney NE is added to the liver chromatin, chromatin DNA degradation is not accelerated in the presence of the cytoplasmic fraction of liver or DNase; Even in the case when 1.0 ml kidney NE was added instead of 0.5 ml, the rate of liver chromatin DNA degradation by DNase was not increased (fig. 3). Similarly, when liver NE is added to kidney chromatin in the presenceof kidney cytoplasmic fraction or DNase the degradation of kidney chromatin DNA does not accelerate (fig. 3). At the same time, when kidney cytoplasmic fraction is used instead of liver cytoplasmic fraction, liver chromatin DNA is degraded in the presenceof liver NE at the same rate as in the liver cytoplasmic fraction (fig. 3). In experiments with kidney chromatin it was also shown that the action of the cytoplasmic fraction is not tissue-specific (fig. 3). trypsin; V, liver chromatin + DNase + liver NE treated with RNase; VI, liver chromatin + DNase + liver NE, heated (55°C) +liver NE treated with RNase; VII, liver chromatin + DNase + liver NE treated with trypsin + liver NE treated with RNase; VIII, liver chromatin + DNase + liver NE, heated (55°C) +kidney NE treated with RNase; IX, liver chromatin + DNase + liver NE treated with RNase + kidney NE, heated (SST); X, liver chromatin+ DNase + liver NE treated with trypsin + kidney NE treated with RNase; XI, Liver chromatin-tDNase +liver NE treated with RNase + kidney NE treated with trypsin. ordinate: increase in content of acid-soluble nucleotides. Exptl Cell Res 84 (1974)
392
Jandieri et al.
Thus, relative acceleration of chromatin DNA degradation occurs only in the presence of NE from a homologous tissue, while the substitution of homologous by heterologous cytoplasmic fraction does not affect degradation.
Effects of heating, trypsin and DNase treatment on NE activity
If NE is heated to 55°C and maintained at this temperature for 5 min, its addition to the cytoplasmic fraction or to DNase solution does not cause chromatin DNA degradation (fig. 6). Heating to 45 and 50°C does not abolish its activity. Thus, Extent of chromatin DNA degradation 55°C is the threshold temperature for NE by DNase in various concentrations inactivation. As shown above, a chosen concentration of Incubation of NE to 55°C causesdenaturaDNase does not cause chromatin DNA tion of only part of the protein present in degradation in the absence of NE. As it the solution. When the precipitate is removed turned out, DNase, even in higher conby centrifugation (600 g, for 10 min) almost centrations, does not digest chromatin half of the initial protein is still found in the DNA when incubated in the absence of solution (table 2). NE (fig. 4). In the case of liver chromatin NE also lost its activity if treated previincubation in the presence of liver NE, the ously with trypsin (see Materials and Methrate of chromatin DNA degradation depends ods), the amount of protein in NE being on the amount of DNase (fig. 4). An increase also decreased (table 2). in liver NE up to 1.0 ml in the presence NE loses its activity also following treatof 25 ,ug DNase in the incubation medium ment with RNase (fig. 6). Since the extract does not alter the result (44 + 2.0 %). had been treated with askangel to remove RNase, it was necessary to make sure that DNase activity of NE and the effect of askangel itself did not cause NE inactivation. NE on DNase activity It was found that askangel did not deprive DNase activity of NE was determined by NE of the ability to accelerate chromatin DNA degradation in the presence of DNase the extent of degradation of pure DNA (fig. 6). (erythrocyte DNA; Sigma). 0.5 ml DNA Treatment with RNase reduced to less (20 pg) were placed in a phosphate buffer (0.1 M) pH -7.2, containing 0.01 M MgCl,. than half the initial RNA amount in the 0.5 ml of liver NE was added to the solution extract (table 2). Treatment with askangel and incubated for 1 h at 37°C. Under such only slightly reduced the concentrations of conditions the appearance of acid-soluble RNA and protein in NE. It follows from nucleotides was not observed (2.0 t- 1.O%), these experiments that partial removal of which shows the absence of DNase activity both protein and RNA from NE deprived it of its ability to accelerate chromatin DNA in NE. To determine the effect of NE on the degradation by DNase. If NE inactivated by heating or trypsin DNase activity, we carried out a series of experiments with increasing amounts of was combined with an equal volume of NE DNase in the absence and presence of the inactivated by RNase, the activity of NE liver NE. The intensity of pure DNA degrada- was restored and it was again able to accetion did not depend on the presence of NE lerate chromatin DNA degradation in the presence of DNase (fig. 6). However, when in the incubation medium (fig. 5). Exptl CeN Res 84 (1974)
The nuclear factor promoting DNA degradation
393
the experiments of Marushige & Bonner [I 11, little or no chromosomal DNA becomes acid-soluble by treatment with Protein RNA DNase, some degree of digestion of DNA per 1 ml per 1 ml was detected when applying other criteria to of NE of NE Kind of treatment of NE (md tug) their results. Recently it has been found that in 0.14 M 7.6kO.3 210& 17 Without treatment NaCl the activity of DNase may be 170+12 4.2kO.2 Heating (SYC) 3.1kO.5 182+10 Trypsin reduced by almost 50 % [13]. However, this 86&8 6.6kO.2 RNase was not observed in our experiments, where 186&10 7.0+0.3 Askangel the addition of 0.25 M sucrose solution, instead of 0.14 M NaCl, in a number of control experiments did not affect the two portions of NE treated in different ways result. were combined, the total amount of NE was The absence of chromatin DNA degradafound to be twice as much as before (1 ml), tion may be due to the binding of Mg which might cause the apparent recovery of ions by EDTA, while an increase in its activity. Therefore in the control experi- DNase activity on addition of NE could be ments, 1 ml of NE treated by heating, a result of addition of Mg2+ ions contained trypsin, or RNase, was added to 0.5 ml of in the extract. However, such an assumpchromatin solution. The increase in the tion has been excluded by experiments with amount of extract did not affect the results addition of an excessive amount of MgCl, of the experiments. in the absence of NE, which did not cause When liver NE treated with heat or any increase in chromatin DNA degradatrypsin was combined with kidney NE tion. treated with RNase or vice versa, the Enhancement of chromatin DNA degradaactivity of NE was not restored (fig. 6). tion cannot be attributed to a small shift of pH caused by the addition of the nuclear extract to the medium. Neither the rise in DISCUSSION pH to 7.15 nor the incubation of chromatin The experiments have confirmed our assump- in the phosphate buffer causes enhancement tion [4] (that there is a nuclear factor of chromatin DNA degradation. Especially promoting chromatin DNA degradation by when cytoplasmic fraction is used instead of DNase). It seems surprising that under the DNase, the above-mentioned shift was hardly action of DNase such a small part of chroma- noticeable. tin DNA is degraded. Actually, a more The optimum values of pH, determined complete digestion of the chromatin DNA for the action of NE, are within 6.8-7.15. by DNase has been reparted (e.g. [13]). This fact, evidently, should be taken into However, the high reproducibility of our account while planning experiments and results rules out all possible doubt in this interpreting the data obtained. respect. It may be that the discrepancy in The loss of NE activity after heating and the results depends on the method of treatment shows the protein nature of chromatin extraction and the kind of tissue the nuclear factor promoting chromatin DNA used. It must be noted that although in degradation. Table 2. Effect of heat, trypsin and RNase on the amount of protein and RNA in NE
Exptl Cell Res 84 (1974)
394
Jandieri et al.
The ability of the nuclear extract to promote chromatin DNA degradation is related to its other component as wellRNA. RNA, though not degraded either by heating to 55°C or trypsin, is sufficiently well removed from the extract treated with RNase. As askangel does not cause any decrease in NE activity, the inactivation of the nuclear extract after treatment with RNase must be due to RNA degradation and not to the absorption by some other substances by askangel. The evidence that the nuclear factor promoting chromatin DNA degradation consists of at least two components, is supplied by experiments with combinations of two portions of the nuclear extract, one of which was previously treated by heating or trypsin and the other by RNase. After these two portions had been reunited, the extract was found to be active. A striking feature, hard to explain is the fact that the tissue specificity of NE is determined by both of its components, protein and RNA. We cannot say at present whether we are dealing with some peculiar class of RNA or with some other mechanism of this phenomenon. This problem is under investigation. Probably, the factor found is identical with that extracted from Rana pipiens liver, which when injected into the fertilized egg of the same species causes chromosome distortions leading to the cessation of the embryonic development at the stage of the early gastrula [lo, 181.As was found, this factor may be identified as an acid protein [12], though the authors themselves do not think that the data obtained by them are sufficiently clear-cut. In a number of her papers, Ermolaeva has described a substance which is not one of the known DNases, but participates in
Exptl Cell Res 84 (1974)
the chromatin DNA degradation [6, 7, 81. She considers the nuclear factor to be an enzyme, the action of which in her opinion has no tissue specificity. Ermolaeva’s conclusions have not been confirmed by our data either with respect to the specificity or to the chemical nature of NE. The biological role of the nuclear factor promoting chromatin DNA destruction is unknown. Most probably it is the same factor which causes DNA degradation in the experiments in vivo mentioned previously. REFERENCES 1. Berthet, J & de Duve, C D, Biochem j 50 (1951) 174. 2. Dingman, M B & Sporn, M B, J biol them 239 (1964) 3483. 3. Dische, Z., The nucleic acids (ed E Chargaff & J N Davtdson) p. 426. Academic Press, New York (1955). 4. Jandieri, K’M, Tumanishvili G D, Vopr biofisiki i teoreticheskoi biol. vol. 3._ D. _ 45. Tbilisi TGU (1971). 5. Jokhadse, D I, Bull Georgian acad sci 12 (1966) 621. Ermolaeva, N V, Biokhimia 31 (1966) 860. 7”: - Ibid 32 (1967) 774. - Ibid 35 (1970) 17. i: Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1953) 265. 10. Forkert, C L & Ursprung, H, Dev biol 7 (1963) 11. Marushiae. K & Bonner. J. Proc natl acad sci US 68 (1971)2941. ’ . Melton. Ch G. J exotl zoo1 164 (1967) 325. ::: Mirskyj A E, ‘Proc-natl acad si‘ US -68 (1971) 2945. Spirin, A S, Biokhimia 23 (1958) 656. ::: Tsanev, R G & Markov, G G, Biokhimia 25 (1960) 151. 16. Tumanishvili. G D & Salamatina. N V, Vopr biofiziki i teoreticheskoi biol, vol. 2, p. 89. Tbilisi TGU (1969). 17. Tumanishvili, G D, Vepkhvadze, L K, Salamatina, N V, Arm embryo1 morphogen, suppl. 1 (1969) 275. 18. Ursprung, H & Markert, C L, Dev biol 8 (1963) 309. Received September 8, 1973 Revised version received October 29, 1973