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The measurement of nucleic acid phosphorus turnover in rat liver by the constant exposure technique
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
67, 59-73 (1957)
The Measurement of Nucleic Acid Phosphorus Turnover in Rat Liver by the Constant Exposure Technique’*” Robert W. Swick and Arthur From the Division
of Biological Laboratory, Received
L. Koch3
and Medical Research, Lemont, Illinois
drgonne
Xational
June 15, 1956
In a previous communication (1) we have indicated the difficulties attendant on the int,erpretation of turnover experiments in which the animal receives only a single short exposure to the isotope. It was pointed out that these difficulties may, in part, be eliminated or compensated for by use of a continuous exposure technique. With this method of administration and using U40n as the t’racer it, was found possible to estimate the rates of purine synthesis and degradation in ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) in rat liver. In t,he present paper we will describe the extension of this technique to the measurement of the rate of phosphorus incorporation into, and release from, RNA and DNA. There are, however, two important differences between Cl4 and P32 as isotopic tracers in experiments of this type. First, Peeis a short-lived isotope whose half-life is of the order of the duration of the experiments. Hence the question arises whether the phosphorus should be administered in such a way that t,he specific activity is maintained constant or should be allowed to decay exponentially as t,he isotope decays with appropriate correction back to some arbitrary time, e.g., the beginning of the experiment. It can be shown t,hat the latter procedure is not only simpler but yields results that are more easily interpreted. Let us assume that X is t,he specific activity of component z at any 1 Work performed under the auspices of the U. S. Atomic Energy Commission. 2 A preliminary report was presented at the meetings of the American Society of Biological Chemists, Atlantic Cit.y, April 16-20, 1956. 3 Present address: Department of Biochemistry, College of Medicine, University of Florida, Gainesville, Florida. 59
60
ROBERT
IV.
SWICK
.4ND
.4RTHUR.
L.
KOCH
t.ime; that Y is the specific activity at any time of a component present in a constant amount y; and that y is formed from x at a rate V.4 If a long-lived isotope is used in any particular experiment and if X = F(t) where F(t) can be any function of time, t,hen
-dY = $(X) dt
After multiplication by an integration factor, integrated [see, for example, Ref. (5)] to yield
Nom if a short-lived becomes
(1)
F(Y). this equation
may be
isotope with a decay constant X is used, Eq. (1)
g = ;(x) - (5 + A) Y. Let us assume momentarily
t,hat X = e-“F(t); e"t'UF(t)&
_
(3)
then, Eq. (2) becomes Ce-"t/,
.
(4
4 The theory of isotope kinetics in biological systems is reviewed in Refs. (23). The basic equation as exemplified by Eqs. (I), (3), (i), and (11) of the present paper states that the rate of change of total isotope content of any component is equal to the difference in the rate at which isotopic molecules enter the pool and the rate at which they leave. In our nomenclature wherein the specific activity of the component is expressed as a capital letter and the amount of the constituent by the corresponding lower case letter, the total activity is given by their product. Thus the rate of change of the total isotope content of component Y is given bJ d( Yy)/dt, which becomes y X dY/dt when the component is in dynamic equilibrium (the amount y is constant). The rate at which isotope molecules enter a pool is simply the product of the reaction velocity with which molecules enter the pool times the specific activity of the source, if there is only one source. The rate at which isotopic molecules leave the pool is the product of the reaction velocity with which molecules leave times the specific activity of the pool. Hence the net rate of increase in isotope content is the difference of these two products. The validity of this relationship will be apparent when it is remembered that a specific activity is equivalent to a knowledge of the fraction of molecules that are isotopic, and consequently the product of reaction velocities, V, times the specific activity of the appropriate pool is the number of isotopic molecules introduced or removed per unit time.
NUCLEIC
ACID
TURNOVER
61
Hence it is apparent that if the specific activity of any component is e-At times the value expected had the experiment been conducted with a long-lived isotope, then any other component formed from this component mill have e-“’ times the specific act#ivity predicted from the longlived experiment. Therefore, in an experiment in which the rate of administration of the radioactive compound is constant and in which the specific activity of this material is decreasing exponentially, the effects of isotopic decay on the specific activity of any isolated fraction may be eliminated by simply correcting for decay, no matter when the component was isolated. The second difference between the previous experiments with CY402 and the present studies with P32 is the great disparity in size of t’he metabolic pools under study. In the former case, the fraction of acidsoluble purine in rat liver is very small compared to the amount of nucleic acid purine, whereas in the second case the amount of acidsoluble phosphorus is roughly equal to the amount of nucleic acid phosphorus. In addition, purines are synthesized intracellularly, so that events in one cell have little effect upon events in another, whereas the phosphorus metabolism of cells and bones will have a marked effect on the specific activity of the phosphorus in a liver cell. Thus the specific activity of the intracellular phosphorus pool could not be inferred simply from a knowledge of the specific activity of the dietary phosphorus and the turnover rates of a particular tissue. It was therefore necessary actually to measure the specific activity of the intracellular acid-soluble pool. There are several points which may be considered to justify this procedure. First,, it will be shown that in liver there is no significant difference between the specific activities of the two readily accessible acid-soluble phosphorus fractions (inorganic and organic P) after only 4 hr. of continuous P32 administration. Secondly, the existent phosphatases and phosphorylases could be expected to equilibrate rapidly the phosphorus in various pools. Thirdly, the rate of equilibration which must exist in order that no significant error in calculations be introduced is much less for the continuous exposure type of experiment than for a study made with an acute exposure. In addition to these considerations, the experimental data obtained are consistent with t’he hypothesis that the nucleic acids are synthesized at any time from material having the specific activity of the measured acid-soluble pool. Consider the following scheme which suggests itself from general considerations and from our previous results (1).
62
ROBERT
W.
SWICK
AND
ARTHUR
L.
I-
gut e
VI
KOCH
bone, etc.
I1 vz
intracellular poolRN-~ Va (Xl , DN.4 I 1 V4 excretion
If at time t = 0, P3*is introduced and maintained at a specific activity of 100 (after correction for decay), then the specific activity of any component within the rat, and in particular the intracellular phosphorus pool of the liver, may be represented (after correction for decay) as 100 minus a sum of exponential terms x = 100 - Z&?-k’t
(5)
where Ai are constants such that 2,4; = 1OO.6For the purpose of calculating the activities of the RNA and DNA, two exponential terms are more than adequate. Thus x = l(-J()- Jle-“‘t
_ &-“2t
(6)
It was shown previously (1) that even in the growing animal t,he rate at which activity is introduced into the RNA is given by dR = V,(X vz
- R)
(7)
where R is the corrected specific activity of RNA and T is the amount of RNA present. Combining and solving the resultant differential equation, and assuming that r is approximately constant, we obtain R = 100 - 3'v Ale-w - ;
--r
2
kl
A2e-“2’ I' 2
-r
k2
(8) 2.1
-
;A2
100 - + -- 2 T
k1
-Gy -LT
-v*c/r e
I
6 It is easily seen that Eq. (5) satisfies the given boundary conditions. At t = 0, e--k; = 1 and therefore X = 0; at t = m, e- X* = 0 and therefore X = 100; i.e., the acid-soluble phosphorus fraction is in equilibrium with the dietary phosphorus.
NUCLEIC
ACID
63
TURNOVER
There are several special cases which are pertinent where kz = m and X = -4 r(1 - e -klt),
-V*llr
here. In Case I,
--t1t
(9)
In Case II, where Ai = 0, A2 = 100, and X = lOO(1 - es”*“), it is use: ful to reintroduce this expression for X into Eq. (8). One obtains
R = l(-jo(l - e-v2t’r) For the incorporation into DNA, From the scheme above,
(10)
only the second case is of interest.
d(Dd) ___ = v*x dt
(11)
where D is the corrected specific activity of the DNA and d is the amount of DNA present. There is no expression for the degradation of DNA for reasons discussed in Ref. (1). Therefore,
02 4
(12)
where d, is the amount of DNA present at time T, the conclusion of the experiment. Here, as in the previous work with C 14,the interest lies in the mean rate of DNA synthesis, V4/& where a is the average amount of DNA present. If L is the ratio of the final weight to initial weight of the liver, and the amount of DNA per gram of liver is considered to be constant,B 6 It will be shown below that this assumption is not valid for our animals. The amount of DNA per gram of liver may decrease by as much as 3074,.It may readily be shown that this has little effect on Eq. (13). In an experiment wherein the liver weight doubled but the DNA concentration decreased by 3OoJc,the true value for V,/d would still be 88% of that obtained from Eq. (13).
64
ROBERT IV’. SU’ICK AND ARTHUR L. KOCH
then we may express dj in terms of d and L and obtain ___D - 2L (13) ,-xXL+l k2 If the specific activity of the acid-soluble pool rose abruptly to 100% at the beginning of the exposure, then in Eq. (13) the term X/k, would be dropped from the denominator. Thus X/k2 is t,he correction term for the changing specific activity of the acid-soluble pool and has the units of days. An independent estimate of V4/d is given by the average growth rate: v, L-l 2 -z-z (14) d izTXT I74 := d
Thus we have developed a set of equations by which the data obtained by the continuous administration of isotopic phosphorus may be used to calculate two quantities of nucleic acid turnover: V2/r and V4,/d, which may then be compared with values obtained using 04 as the tracer. MATERIALS
AND METHODS
Young Sprague-Dawley male rats were fed at hourly intervals (6) a purified diet composed of cerelose, 72%; easein, 18%; corn oil, 5%; salt mixture, 4%; vitamin mixture, O.S%, for periods ranging from 4 hr. to 16 days. Initially, P3* as Na*HPOl was added at a level of 1.3-1.6 pt./g. of raGon, which led, after several days, to severe radiation damage in the intestinal tract as manifested by anorexia and melena. Subsequently, the isotope was incorporated at a level of 0.5 pt./g. without evidence of injury to the young rats. Thus the level of isotopic phosphorus in the animal’s tissues would be expected to rise in time as a result of the continuous ingestion, exchange, and excretion of phosphorus-containing compounds, until t.he specific activity of the internal phosphorus equals that of the diet, and it would then remain constant. The nucleic acids were isolated by the procedure of Tyner et al. (7) with only slight modifications (1). In order to reduce the possibility of contamination, care was taken to add acid to the various fractions only when the solutions were at 0”. The acid-soluble phosphorus fraction was divided by precipitating the inorganic phosphorus compounds with &1g++ and NH4OH (8). That the RNA was essentially free of DNA was indicated by a negative Dische test sensitive to 2y0 contamination. Purification of the DNA fraction was completed with a second hydrolysis at pH 13 followed by acidification to pH 2.’ Pzawas counted in solution 7 It is inherent in the technique of isotope administration used here that the effect of any possible contamination of one fraction by another will be small. Where single-exposure experiments usually result in fractions differing in isotope
WJCLEIC
ACID
TURNOVER
65
with a Geiger-Mueller tube with an accuracy of &57c and phosphorus was determined by the Fiske-SubbaRow met’hod (8). The results are expressed as relative specific activity in cps./pmole of P where the specific activity of the diet arbitrarily equals 109. The initial weight of the livers of the rats used in the experiments was calculated from the body weight and the regression of liver weight on body weight obtained by sacrificing a number of rats of similar body weight and history. In order to determine whether the concentration of DNA in the livers of our animals remained constant during the experimental period, groups of five young rats were sacrificed at intervals, and the DNA content of the liver was determined with diphenylamine according to the procedure of Schneider (9). RESULTS
These studies were performed under several different physiological conditions which profoundly affected the time course of the incorporation of radioactivity into the acid-soluble fraction, but had very little effect on the uptake of P32into the nucleic acid fractions. Short-Term Imorporation
into Slowly Growing Rais
In order to establish the validity of the constant-exposure technique as applied to P35,a number of 150-g. rats were fed a radioactive diet for 4, 8, 16, 24, 36, and 48 hr. The results of this experiment are given in Fig. 1. It will be seen that in the 48-hr. time period, the specific activity of both inorganic and organic acid-soluble phosphorus appears to approach a liiit that is considerably less than 100. Therefore, these data may be best expressed in terms of Case I above: X = Al(l
- e”l’)
(15)
where -41 may be estimated from the relationship (10) Al==
x1 2x1 - x2
(16)
where X1 is the value of X at some particular time and Xz is the value at twice that time. Then values of A1 - X may be plotted on semilogarithmic paper and kl may be estimated. The average specific-activity data for acid-soluble phosphorus gives the following equation: X = 53.3(1 - e-‘.r2”)
(17)
where t is measured in days; this equation is plotted as a smooth curve content by orders of magnitude, the radioactivity of the various fractions obtained in the present experiments usually were of the same order of magnitude.
ROBERT
W.
SWICK
AND
ARTHUR
L.
KOCH
a
Acid- Soluble
1 0
4.0
8.0
16.0
24.0 TIME
36.0
L
2
48.0 '16 days
(hours)
FIG. 1. The incorporation of P** into the acid-soluble P and RNA P of 150-g. rats, short-term experiments. 0 = acid-soluble, inorganic P; LII = acid-soluble, organic P; X = RNA P. The relative specific activity of the diet is taken as 100. The smooth curve through the acid-soluble P data satisfiedX = 53.3 (1 - e-l.lzt). The curve through the RNA data satisfies R = 53.3 (1 - 1.23 e-OJI1 + 0.23 e--OJzL). Each point along the abscissa represents one animal.
in Fig. 1. An estimate of VJr may be obtained from the equation
dR -=dt
Vz r @ - RI
(18)
and, from the last four pairs of determinations, one obtains an average of 0.21 for Vz/r. The line through the RNA data is calculated from Eq. (9) with this value of VJr. The agreement between the observed values and the predicted values is excellent in experiments of 16 hr. duration or longer. However, after shorter periods of administration, the predicted value is lower than that actually found, suggesting that the specific activity of the precursor pool
MJCLEIC
ACID
TURNOVER
67
is somewhat higher than the value assumed. The reason for this may lie in the following. At these early times, the smooth curve falls below the data for inorganic phosphorus; had the data for inorganic phosphorus been fitted, rather than the average of the organic and inorganic fractions, the expected values for the RNA would have agreed with those found. Thus even at times as short as 4 hr., there is agreement with t,he hypothesis that inorganic, acid-soluble phosphorus has the same specific activity as the immediate precursor of RNA phosphorus. The hypothesis is unassailable at longer times, viz., in the remainder of t,he experiment,s presented belolv. Long-Tom
Incorporation
of Px2 into Rapidly Growing Rats
In Fig. 2 are presented the data for the incorporation of P32 into the phosphorus fractions of young, rapidly growing rats (MM5 g.). The specific activities of the acid-soluble fractions are easily fitted to a singleterm exponential S = loot1 - c-o.afj
iw
Insertion of this value for liz (0.4) and of an assumed value for V2/r of O.l7/day into Eq. (10) gives a smooth curve through the RNA points. The data for the specific activity of DNA P may be corrected for the effect of the changing specific activity of the acid-soluble phosphorus by use of Eq. (13). The corrections are of the order of 2.3-2.5 days out of t,he 8-15 days duration of the experiment. These corrected data are tabulated in Table I together mit,h rate of liver growth estimated from liver weight. The Effect of High Phosphorus Diet In four experiments, the phosphorus level of the diet was increased threefold by the addition of Ca3(P0& in an attempt to increase the turnover of the acid-soluble phosphorus pool. The results are shown in Fig. 3. Although this treat#ment failed to affect the rate of incorporation into the acid-soluble pool, it did modify the ultimate value attained in that the specific activity was greater than 100%. From the data, the limiting value appears to be 113% of the diet. This suggests that there was incomplet,e solubilization of the Caa(POJs and therefore incomplete equilibration of NazHP3”04 with the Ca salt prior to absorption. As a result the specific activity of the phosphorus absorbed was 1.13 times
68
ROBERT
0
2.0
W.
4.0
SWICK
.4ND
6.0 TIME
8.0
ARHTUR
IO.0
L.
KOCH
12.0 14.0
16.0
(days)
FIG. 2. The incorporation of P32 into acid-soluble P, RNA P, and DNA P of rapidly growing weanling rats. 0 = acid-soluble P; X = RNA P; and q = DNA P. The relative specific activity of the diet is taken as 100. The curve through the acid-soluble P values is given by X = 100 (1 - e-O.4Oand that through the values for RNA P by R = 174 (1 - e-0.171) - 74X. The numbers above the points for DNA P are the ratios of final liver weight to initial liver weight as estimated from the sacrifice of a number of comparable rats. Each point along the abscissa represents one rat. the average specific activity of the phosphorus in the diet. It should be noted that the rats did not thrive on the high phosphorus diet and that the growth rate was markedly reduced. An estimate of VJr (026/day) and values for V,/d (Table I) were obtained when the dat#a were treated mathematically as in the previous sections.
NUCLEIC
ACID
TURNOVER
TABLE The Growth Diet
Normnl
High
phosphorus
phosphorus
I
Rate of Liver,
Length of CCpX.“E dUYS
8 8 13 15 7 15
T’Jd
Estimated liver growth per day Lix;e;wdeaiyght, DN.4 Pa*, per day
0.037 0.085 0.037 0.035 0.058 0.025
i?)
0.064 0.056 0.063 0.054 0.056 0.027
Changes in DNA4 Conknt with Inxrea8ing Body Weight Examination of the DNA content of the livers of young rats whose body weight (and presumably age) were similar to those used in the tracer experiments showed that t.here was a decrease in t,he concentration of this fraction during this stage of growth (Fig. 4). This decrease, which amounted bo 25 % in one case and 27 % in another, occurred during the period when the animals were doubling their init,ial body weight of 40 g. After this time the concentration of DK;A remained essentially constant. Although the magnitude of the decrease in DNA was similar in the two experiments, there was about a 25% difference in the absolute amounts of DNA found, which may be partly explained by improved technique with each experiment. Thus the growth rate measured in terms of liver weights can differ from that determined from the DNA content, and the discrepancy can be large. If in an experiment where there is a 25 % decrease in the DNA concentrat,ion during the time that the liver weight doubles, use of Eq. (14) would result in a “weight” growth rate twice t.he “DNA” growth rate. This fact resolves the observed discrepancy between the “weight” growth rates and the “DNA P3*” growth rates (Table I). If both quantities could be corrected for the change in the DNA concentratSion, the “weight” growt#h rate would be halved, whereas there would be little change in the values estimated from the isotope data.6 DISCUSSION
The difference in the rate of incorporation of radioactivity into the acid-soluble phosphorus fraction between the 40-g. weanling and the larger, adult animal is marked and may be ascribed to the difference in the food requirement of the two animals. While t,he weanling consumed
70
ROBERT
W.
SWICK
AND
ARTHUR
L.
KOCH
110 100 90
w >
40
c a 2
30
I-
E 20 IO 0
I
I
I
I.0
3.0
7.0
c
1%
TIME (days) 3. The incorporation of P*2 into rapidly growing weanling rats fed a diet high in phosphorus. See legend, Fig. 2. The curve through the acid-soluble phosphorus data is given by X = 113 (1 - e- 0.321)and that through the values for RNA P by R = 603 (1 - e-o.*al) - 4.36X. Each point along the abscissa represents one animal. FIG.
0.12-0.16 g. diet/day/g. body weight, the larger rat ate only one-third to one-half as much. Although the rapid growth of the young rat releases proportionately more bone phosphorus into the system than the simple turnover of bone in the adult, it is estimated that the weanling rat derives some 90% of his acid-soluble phosphorus from the diet while in the older animal only about 50 % of this pool was so derived. The rate of nucleic acid synthesis, however, is much less affected by
NUCLEIC
6.0
I
I
ACID
I
71
TURNOVER
I
I
I
I
I
a W > 5
5.0 - % 4-o -0
P-
P
\
I
4
3.0 -
B
2.0 -
1.0 40
I 50
I 70
I 60 BODY
I 80 WEIGHT
I
I
I
90
100
110
I .
120
(grams)
FIG. 4. The concentration of DNA in the livers of rapidly growing rats. a, Expt. 1; a, Expt. 2; 0, Expt. 3. Each point represents the average of five animals with its standard deviation.
these factors, since the rate constant for RNA synthesis obtained in these experiments is 0.2l/day for the 150-g. animal, O.l7/day for the weanling rat, and 0.26/day for the weanling given the high phosphorus diet. In the previous paper, the estimate for young animals was 0.20/day for RNA adenine and 0.24/day for the RNA guanine (1). It appears clear, then, t,hat the values obtained with isotopic carbon or phosphorus are similar. One may conclude that either method may be used to give a valid measure of RNA turnover. It may also be concluded that the molecule is renewed as an entity. For reasons which have been discussed at length in the previous paper (l), the expression of these results in t,he usual t,erms of the concept of “half-life” would be inappropriate. Let us consider next the DNA. The values for the rate constant of formation of DNA as measured with phosphorus are significantly smaller than those obtained from the liver weight data. This is also the observation reported previously when radioactive carbon was used as the tracer substance, and it is clearly explained by a decrease in the DNA per gram of tissue as the animal matures. It is also clear that Cl4 and P32as tracers
72
ROBERT
W.
SWICK
AND
ARTHUR
L.
KOCH
give similar estimates and that both are equally valid measures of DNA formation. It may be concluded that this molecule, like that of RNA, is synthesized as a unit. It also is apparent that DNA4 is formed at a rate determined by the growth of the animal, and, as has been done previously (l), it may be shown that the existing DNA is retained during the processes of cell division and probably for the life of the cell. Other workers have noted differences in the incorporation of various isotopes into the two types of nucleic acid. It appears possible to hope that eventually all of the experimental results obtained with the single dose technique may be reconciled with the hypothesis that the various moieties of the nucleic acids are incorporated into the macromolecule at the same rate. It would appear that the differences that have been seen (11, 12) are reflections of (a) variations in the time curves of the immediate precursors of the nucleic acids, (b) variations in t,he permeability of various cell types present in the tissue to the several tracers, (c) variation in the specific activity of the precursor pools in various portions of the cell, and (d) the possible effects of the movement of polynucleotides from one part of the cell to another. h?,KNOWLEDGMENTS
The authors gratefully acknowledge the valuable technical assistance of Mrs. Dorothy Handa and Mr. Samuel S. Thomas. We are indebted to Dr. Peter D. Klein for his interest and many helpful suggestions, and especially to Dr. J. Z. Hearon, Naval Research Medical Institute, National Institutes of Health, Bethesda, Maryland, for guidance and discussion on the theoretical aspects of tracer kinetics. SUMMARY
Using the continuous exposure technique, we have compared measurements of nucleic acid turnover obtained with phosphate-P3’ with previous observations in which U402 was used as a tracer. When proper correction was made for the precursor pool, the rate constant for P39 incorporation into the RNA was found to be similar to that previously observed for CY incorporation into the RNA purines. It is t,herefore concluded that RNA is synthesized and degraded as a unit. In young growing rats the rate of growth, estimated from the increase in liver weight, was compared with the increase in DNA estimated from the incorporation of P32. The latter values were lower than the former as was observed previously with CY. This discrepancy may be attributed to a decreasing concentration of DNA in the livers during the experi-
NUCLEIC ACID TURNOVER
73
mental period chosen. From the similarity in the rates of incorporation of CL4 and of P32 into the DNA, it appears t,hat DNA is likewise synthesized as a unit. REFERENCES 1. SWICH, R. W., KOCH, A. L., AND HANDA, D. T., Arch. Biochem. and Biophys. 63, 226 (1956). 2. SIRI, w. E., “Isotopic Tracers and Nuclear Radiations xvit.h Applications t,o Biology and Medicine,” Chap. 15, pp. 388402. McGraw Hill, New York, 1949. 3. SOLOMON, A. K., in “Advances in Biological and hledical Physics” (J. H. Lawrence and C. 9. Tobias, eds.), Vol. III, pp. 62-99. Academic Press, New York, 1953. 4. REINER, J. ill., Arch. Biochem. and Biophys. 46, 53, 80 (1953). 5. hIARGENAU, H., AND hkJRPHY, G. hl., “The Mathematics of Physics and Chemistry,” 1st ed., Eqs. 2-6. Van Nostrand, New York, 1943. 6. SWICR, R. W., AND HANDA, D. T., J. Biol. Chem. 288, 517 (1956). 7. TYNER, E. P., HEIDELBERGER, C., AND I.EP?LGE, G. A., Cancer Research 13, 186 (1953). 8. ALBERT,S.,JOHNSON, R.M.,AND C~~~~,hLS.,cuncer Research 11,772 (1951). 9. SCHNEIDER, W. C., J. Biol. Chew 161, 293 (1945). 10. WOOD, H. G., in “A Symposium on the Use of Isotopes in Biology and Medicine,” p. 219. University of Wisconsin Press, Madison, 1948. 11. BROWN, G. B., AND ROLL, P. hl., in “Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. II, p. 341. Academic Press, New York, 1955. 12. SMELLIE, R. M. S., in “Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), p. 393. Academic Press, New York, 1955.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
67, 59-73 (1957)
The Measurement of Nucleic Acid Phosphorus Turnover in Rat Liver by the Constant Exposure Technique’*” Robert W. Swick and Arthur From the Division
of Biological Laboratory, Received
L. Koch3
and Medical Research, Lemont, Illinois
drgonne
Xational
June 15, 1956
In a previous communication (1) we have indicated the difficulties attendant on the int,erpretation of turnover experiments in which the animal receives only a single short exposure to the isotope. It was pointed out that these difficulties may, in part, be eliminated or compensated for by use of a continuous exposure technique. With this method of administration and using U40n as the t’racer it, was found possible to estimate the rates of purine synthesis and degradation in ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) in rat liver. In t,he present paper we will describe the extension of this technique to the measurement of the rate of phosphorus incorporation into, and release from, RNA and DNA. There are, however, two important differences between Cl4 and P32 as isotopic tracers in experiments of this type. First, Peeis a short-lived isotope whose half-life is of the order of the duration of the experiments. Hence the question arises whether the phosphorus should be administered in such a way that t,he specific activity is maintained constant or should be allowed to decay exponentially as t,he isotope decays with appropriate correction back to some arbitrary time, e.g., the beginning of the experiment. It can be shown t,hat the latter procedure is not only simpler but yields results that are more easily interpreted. Let us assume that X is t,he specific activity of component z at any 1 Work performed under the auspices of the U. S. Atomic Energy Commission. 2 A preliminary report was presented at the meetings of the American Society of Biological Chemists, Atlantic Cit.y, April 16-20, 1956. 3 Present address: Department of Biochemistry, College of Medicine, University of Florida, Gainesville, Florida. 59
60
ROBERT
IV.
SWICK
.4ND
.4RTHUR.
L.
KOCH
t.ime; that Y is the specific activity at any time of a component present in a constant amount y; and that y is formed from x at a rate V.4 If a long-lived isotope is used in any particular experiment and if X = F(t) where F(t) can be any function of time, t,hen
-dY = $(X) dt
After multiplication by an integration factor, integrated [see, for example, Ref. (5)] to yield
Nom if a short-lived becomes
(1)
F(Y). this equation
may be
isotope with a decay constant X is used, Eq. (1)
g = ;(x) - (5 + A) Y. Let us assume momentarily
t,hat X = e-“F(t); e"t'UF(t)&
_
(3)
then, Eq. (2) becomes Ce-"t/,
.
(4
4 The theory of isotope kinetics in biological systems is reviewed in Refs. (23). The basic equation as exemplified by Eqs. (I), (3), (i), and (11) of the present paper states that the rate of change of total isotope content of any component is equal to the difference in the rate at which isotopic molecules enter the pool and the rate at which they leave. In our nomenclature wherein the specific activity of the component is expressed as a capital letter and the amount of the constituent by the corresponding lower case letter, the total activity is given by their product. Thus the rate of change of the total isotope content of component Y is given bJ d( Yy)/dt, which becomes y X dY/dt when the component is in dynamic equilibrium (the amount y is constant). The rate at which isotope molecules enter a pool is simply the product of the reaction velocity with which molecules enter the pool times the specific activity of the source, if there is only one source. The rate at which isotopic molecules leave the pool is the product of the reaction velocity with which molecules leave times the specific activity of the pool. Hence the net rate of increase in isotope content is the difference of these two products. The validity of this relationship will be apparent when it is remembered that a specific activity is equivalent to a knowledge of the fraction of molecules that are isotopic, and consequently the product of reaction velocities, V, times the specific activity of the appropriate pool is the number of isotopic molecules introduced or removed per unit time.
NUCLEIC
ACID
TURNOVER
61
Hence it is apparent that if the specific activity of any component is e-At times the value expected had the experiment been conducted with a long-lived isotope, then any other component formed from this component mill have e-“’ times the specific act#ivity predicted from the longlived experiment. Therefore, in an experiment in which the rate of administration of the radioactive compound is constant and in which the specific activity of this material is decreasing exponentially, the effects of isotopic decay on the specific activity of any isolated fraction may be eliminated by simply correcting for decay, no matter when the component was isolated. The second difference between the previous experiments with CY402 and the present studies with P32 is the great disparity in size of t’he metabolic pools under study. In the former case, the fraction of acidsoluble purine in rat liver is very small compared to the amount of nucleic acid purine, whereas in the second case the amount of acidsoluble phosphorus is roughly equal to the amount of nucleic acid phosphorus. In addition, purines are synthesized intracellularly, so that events in one cell have little effect upon events in another, whereas the phosphorus metabolism of cells and bones will have a marked effect on the specific activity of the phosphorus in a liver cell. Thus the specific activity of the intracellular phosphorus pool could not be inferred simply from a knowledge of the specific activity of the dietary phosphorus and the turnover rates of a particular tissue. It was therefore necessary actually to measure the specific activity of the intracellular acid-soluble pool. There are several points which may be considered to justify this procedure. First,, it will be shown that in liver there is no significant difference between the specific activities of the two readily accessible acid-soluble phosphorus fractions (inorganic and organic P) after only 4 hr. of continuous P32 administration. Secondly, the existent phosphatases and phosphorylases could be expected to equilibrate rapidly the phosphorus in various pools. Thirdly, the rate of equilibration which must exist in order that no significant error in calculations be introduced is much less for the continuous exposure type of experiment than for a study made with an acute exposure. In addition to these considerations, the experimental data obtained are consistent with t’he hypothesis that the nucleic acids are synthesized at any time from material having the specific activity of the measured acid-soluble pool. Consider the following scheme which suggests itself from general considerations and from our previous results (1).
62
ROBERT
W.
SWICK
AND
ARTHUR
L.
I-
gut e
VI
KOCH
bone, etc.
I1 vz
intracellular poolRN-~ Va (Xl , DN.4 I 1 V4 excretion
If at time t = 0, P3*is introduced and maintained at a specific activity of 100 (after correction for decay), then the specific activity of any component within the rat, and in particular the intracellular phosphorus pool of the liver, may be represented (after correction for decay) as 100 minus a sum of exponential terms x = 100 - Z&?-k’t
(5)
where Ai are constants such that 2,4; = 1OO.6For the purpose of calculating the activities of the RNA and DNA, two exponential terms are more than adequate. Thus x = l(-J()- Jle-“‘t
_ &-“2t
(6)
It was shown previously (1) that even in the growing animal t,he rate at which activity is introduced into the RNA is given by dR = V,(X vz
- R)
(7)
where R is the corrected specific activity of RNA and T is the amount of RNA present. Combining and solving the resultant differential equation, and assuming that r is approximately constant, we obtain R = 100 - 3'v Ale-w - ;
--r
2
kl
A2e-“2’ I' 2
-r
k2
(8) 2.1
-
;A2
100 - + -- 2 T
k1
-Gy -LT
-v*c/r e
I
6 It is easily seen that Eq. (5) satisfies the given boundary conditions. At t = 0, e--k; = 1 and therefore X = 0; at t = m, e- X* = 0 and therefore X = 100; i.e., the acid-soluble phosphorus fraction is in equilibrium with the dietary phosphorus.
NUCLEIC
ACID
63
TURNOVER
There are several special cases which are pertinent where kz = m and X = -4 r(1 - e -klt),
-V*llr
here. In Case I,
--t1t
(9)
In Case II, where Ai = 0, A2 = 100, and X = lOO(1 - es”*“), it is use: ful to reintroduce this expression for X into Eq. (8). One obtains
R = l(-jo(l - e-v2t’r) For the incorporation into DNA, From the scheme above,
(10)
only the second case is of interest.
d(Dd) ___ = v*x dt
(11)
where D is the corrected specific activity of the DNA and d is the amount of DNA present. There is no expression for the degradation of DNA for reasons discussed in Ref. (1). Therefore,
02 4
(12)
where d, is the amount of DNA present at time T, the conclusion of the experiment. Here, as in the previous work with C 14,the interest lies in the mean rate of DNA synthesis, V4/& where a is the average amount of DNA present. If L is the ratio of the final weight to initial weight of the liver, and the amount of DNA per gram of liver is considered to be constant,B 6 It will be shown below that this assumption is not valid for our animals. The amount of DNA per gram of liver may decrease by as much as 3074,.It may readily be shown that this has little effect on Eq. (13). In an experiment wherein the liver weight doubled but the DNA concentration decreased by 3OoJc,the true value for V,/d would still be 88% of that obtained from Eq. (13).
64
ROBERT IV’. SU’ICK AND ARTHUR L. KOCH
then we may express dj in terms of d and L and obtain ___D - 2L (13) ,-xXL+l k2 If the specific activity of the acid-soluble pool rose abruptly to 100% at the beginning of the exposure, then in Eq. (13) the term X/k, would be dropped from the denominator. Thus X/k2 is t,he correction term for the changing specific activity of the acid-soluble pool and has the units of days. An independent estimate of V4/d is given by the average growth rate: v, L-l 2 -z-z (14) d izTXT I74 := d
Thus we have developed a set of equations by which the data obtained by the continuous administration of isotopic phosphorus may be used to calculate two quantities of nucleic acid turnover: V2/r and V4,/d, which may then be compared with values obtained using 04 as the tracer. MATERIALS
AND METHODS
Young Sprague-Dawley male rats were fed at hourly intervals (6) a purified diet composed of cerelose, 72%; easein, 18%; corn oil, 5%; salt mixture, 4%; vitamin mixture, O.S%, for periods ranging from 4 hr. to 16 days. Initially, P3* as Na*HPOl was added at a level of 1.3-1.6 pt./g. of raGon, which led, after several days, to severe radiation damage in the intestinal tract as manifested by anorexia and melena. Subsequently, the isotope was incorporated at a level of 0.5 pt./g. without evidence of injury to the young rats. Thus the level of isotopic phosphorus in the animal’s tissues would be expected to rise in time as a result of the continuous ingestion, exchange, and excretion of phosphorus-containing compounds, until t.he specific activity of the internal phosphorus equals that of the diet, and it would then remain constant. The nucleic acids were isolated by the procedure of Tyner et al. (7) with only slight modifications (1). In order to reduce the possibility of contamination, care was taken to add acid to the various fractions only when the solutions were at 0”. The acid-soluble phosphorus fraction was divided by precipitating the inorganic phosphorus compounds with &1g++ and NH4OH (8). That the RNA was essentially free of DNA was indicated by a negative Dische test sensitive to 2y0 contamination. Purification of the DNA fraction was completed with a second hydrolysis at pH 13 followed by acidification to pH 2.’ Pzawas counted in solution 7 It is inherent in the technique of isotope administration used here that the effect of any possible contamination of one fraction by another will be small. Where single-exposure experiments usually result in fractions differing in isotope
WJCLEIC
ACID
TURNOVER
65
with a Geiger-Mueller tube with an accuracy of &57c and phosphorus was determined by the Fiske-SubbaRow met’hod (8). The results are expressed as relative specific activity in cps./pmole of P where the specific activity of the diet arbitrarily equals 109. The initial weight of the livers of the rats used in the experiments was calculated from the body weight and the regression of liver weight on body weight obtained by sacrificing a number of rats of similar body weight and history. In order to determine whether the concentration of DNA in the livers of our animals remained constant during the experimental period, groups of five young rats were sacrificed at intervals, and the DNA content of the liver was determined with diphenylamine according to the procedure of Schneider (9). RESULTS
These studies were performed under several different physiological conditions which profoundly affected the time course of the incorporation of radioactivity into the acid-soluble fraction, but had very little effect on the uptake of P32into the nucleic acid fractions. Short-Term Imorporation
into Slowly Growing Rais
In order to establish the validity of the constant-exposure technique as applied to P35,a number of 150-g. rats were fed a radioactive diet for 4, 8, 16, 24, 36, and 48 hr. The results of this experiment are given in Fig. 1. It will be seen that in the 48-hr. time period, the specific activity of both inorganic and organic acid-soluble phosphorus appears to approach a liiit that is considerably less than 100. Therefore, these data may be best expressed in terms of Case I above: X = Al(l
- e”l’)
(15)
where -41 may be estimated from the relationship (10) Al==
x1 2x1 - x2
(16)
where X1 is the value of X at some particular time and Xz is the value at twice that time. Then values of A1 - X may be plotted on semilogarithmic paper and kl may be estimated. The average specific-activity data for acid-soluble phosphorus gives the following equation: X = 53.3(1 - e-‘.r2”)
(17)
where t is measured in days; this equation is plotted as a smooth curve content by orders of magnitude, the radioactivity of the various fractions obtained in the present experiments usually were of the same order of magnitude.
ROBERT
W.
SWICK
AND
ARTHUR
L.
KOCH
a
Acid- Soluble
1 0
4.0
8.0
16.0
24.0 TIME
36.0
L
2
48.0 '16 days
(hours)
FIG. 1. The incorporation of P** into the acid-soluble P and RNA P of 150-g. rats, short-term experiments. 0 = acid-soluble, inorganic P; LII = acid-soluble, organic P; X = RNA P. The relative specific activity of the diet is taken as 100. The smooth curve through the acid-soluble P data satisfiedX = 53.3 (1 - e-l.lzt). The curve through the RNA data satisfies R = 53.3 (1 - 1.23 e-OJI1 + 0.23 e--OJzL). Each point along the abscissa represents one animal.
in Fig. 1. An estimate of VJr may be obtained from the equation
dR -=dt
Vz r @ - RI
(18)
and, from the last four pairs of determinations, one obtains an average of 0.21 for Vz/r. The line through the RNA data is calculated from Eq. (9) with this value of VJr. The agreement between the observed values and the predicted values is excellent in experiments of 16 hr. duration or longer. However, after shorter periods of administration, the predicted value is lower than that actually found, suggesting that the specific activity of the precursor pool
MJCLEIC
ACID
TURNOVER
67
is somewhat higher than the value assumed. The reason for this may lie in the following. At these early times, the smooth curve falls below the data for inorganic phosphorus; had the data for inorganic phosphorus been fitted, rather than the average of the organic and inorganic fractions, the expected values for the RNA would have agreed with those found. Thus even at times as short as 4 hr., there is agreement with t,he hypothesis that inorganic, acid-soluble phosphorus has the same specific activity as the immediate precursor of RNA phosphorus. The hypothesis is unassailable at longer times, viz., in the remainder of t,he experiment,s presented belolv. Long-Tom
Incorporation
of Px2 into Rapidly Growing Rats
In Fig. 2 are presented the data for the incorporation of P32 into the phosphorus fractions of young, rapidly growing rats (MM5 g.). The specific activities of the acid-soluble fractions are easily fitted to a singleterm exponential S = loot1 - c-o.afj
iw
Insertion of this value for liz (0.4) and of an assumed value for V2/r of O.l7/day into Eq. (10) gives a smooth curve through the RNA points. The data for the specific activity of DNA P may be corrected for the effect of the changing specific activity of the acid-soluble phosphorus by use of Eq. (13). The corrections are of the order of 2.3-2.5 days out of t,he 8-15 days duration of the experiment. These corrected data are tabulated in Table I together mit,h rate of liver growth estimated from liver weight. The Effect of High Phosphorus Diet In four experiments, the phosphorus level of the diet was increased threefold by the addition of Ca3(P0& in an attempt to increase the turnover of the acid-soluble phosphorus pool. The results are shown in Fig. 3. Although this treat#ment failed to affect the rate of incorporation into the acid-soluble pool, it did modify the ultimate value attained in that the specific activity was greater than 100%. From the data, the limiting value appears to be 113% of the diet. This suggests that there was incomplet,e solubilization of the Caa(POJs and therefore incomplete equilibration of NazHP3”04 with the Ca salt prior to absorption. As a result the specific activity of the phosphorus absorbed was 1.13 times
68
ROBERT
0
2.0
W.
4.0
SWICK
.4ND
6.0 TIME
8.0
ARHTUR
IO.0
L.
KOCH
12.0 14.0
16.0
(days)
FIG. 2. The incorporation of P32 into acid-soluble P, RNA P, and DNA P of rapidly growing weanling rats. 0 = acid-soluble P; X = RNA P; and q = DNA P. The relative specific activity of the diet is taken as 100. The curve through the acid-soluble P values is given by X = 100 (1 - e-O.4Oand that through the values for RNA P by R = 174 (1 - e-0.171) - 74X. The numbers above the points for DNA P are the ratios of final liver weight to initial liver weight as estimated from the sacrifice of a number of comparable rats. Each point along the abscissa represents one rat. the average specific activity of the phosphorus in the diet. It should be noted that the rats did not thrive on the high phosphorus diet and that the growth rate was markedly reduced. An estimate of VJr (026/day) and values for V,/d (Table I) were obtained when the dat#a were treated mathematically as in the previous sections.
NUCLEIC
ACID
TURNOVER
TABLE The Growth Diet
Normnl
High
phosphorus
phosphorus
I
Rate of Liver,
Length of CCpX.“E dUYS
8 8 13 15 7 15
T’Jd
Estimated liver growth per day Lix;e;wdeaiyght, DN.4 Pa*, per day
0.037 0.085 0.037 0.035 0.058 0.025
i?)
0.064 0.056 0.063 0.054 0.056 0.027
Changes in DNA4 Conknt with Inxrea8ing Body Weight Examination of the DNA content of the livers of young rats whose body weight (and presumably age) were similar to those used in the tracer experiments showed that t.here was a decrease in t,he concentration of this fraction during this stage of growth (Fig. 4). This decrease, which amounted bo 25 % in one case and 27 % in another, occurred during the period when the animals were doubling their init,ial body weight of 40 g. After this time the concentration of DK;A remained essentially constant. Although the magnitude of the decrease in DNA was similar in the two experiments, there was about a 25% difference in the absolute amounts of DNA found, which may be partly explained by improved technique with each experiment. Thus the growth rate measured in terms of liver weights can differ from that determined from the DNA content, and the discrepancy can be large. If in an experiment where there is a 25 % decrease in the DNA concentrat,ion during the time that the liver weight doubles, use of Eq. (14) would result in a “weight” growth rate twice t.he “DNA” growth rate. This fact resolves the observed discrepancy between the “weight” growth rates and the “DNA P3*” growth rates (Table I). If both quantities could be corrected for the change in the DNA concentratSion, the “weight” growt#h rate would be halved, whereas there would be little change in the values estimated from the isotope data.6 DISCUSSION
The difference in the rate of incorporation of radioactivity into the acid-soluble phosphorus fraction between the 40-g. weanling and the larger, adult animal is marked and may be ascribed to the difference in the food requirement of the two animals. While t,he weanling consumed
70
ROBERT
W.
SWICK
AND
ARTHUR
L.
KOCH
110 100 90
w >
40
c a 2
30
I-
E 20 IO 0
I
I
I
I.0
3.0
7.0
c
1%
TIME (days) 3. The incorporation of P*2 into rapidly growing weanling rats fed a diet high in phosphorus. See legend, Fig. 2. The curve through the acid-soluble phosphorus data is given by X = 113 (1 - e- 0.321)and that through the values for RNA P by R = 603 (1 - e-o.*al) - 4.36X. Each point along the abscissa represents one animal. FIG.
0.12-0.16 g. diet/day/g. body weight, the larger rat ate only one-third to one-half as much. Although the rapid growth of the young rat releases proportionately more bone phosphorus into the system than the simple turnover of bone in the adult, it is estimated that the weanling rat derives some 90% of his acid-soluble phosphorus from the diet while in the older animal only about 50 % of this pool was so derived. The rate of nucleic acid synthesis, however, is much less affected by
NUCLEIC
6.0
I
I
ACID
I
71
TURNOVER
I
I
I
I
I
a W > 5
5.0 - % 4-o -0
P-
P
\
I
4
3.0 -
B
2.0 -
1.0 40
I 50
I 70
I 60 BODY
I 80 WEIGHT
I
I
I
90
100
110
I .
120
(grams)
FIG. 4. The concentration of DNA in the livers of rapidly growing rats. a, Expt. 1; a, Expt. 2; 0, Expt. 3. Each point represents the average of five animals with its standard deviation.
these factors, since the rate constant for RNA synthesis obtained in these experiments is 0.2l/day for the 150-g. animal, O.l7/day for the weanling rat, and 0.26/day for the weanling given the high phosphorus diet. In the previous paper, the estimate for young animals was 0.20/day for RNA adenine and 0.24/day for the RNA guanine (1). It appears clear, then, t,hat the values obtained with isotopic carbon or phosphorus are similar. One may conclude that either method may be used to give a valid measure of RNA turnover. It may also be concluded that the molecule is renewed as an entity. For reasons which have been discussed at length in the previous paper (l), the expression of these results in t,he usual t,erms of the concept of “half-life” would be inappropriate. Let us consider next the DNA. The values for the rate constant of formation of DNA as measured with phosphorus are significantly smaller than those obtained from the liver weight data. This is also the observation reported previously when radioactive carbon was used as the tracer substance, and it is clearly explained by a decrease in the DNA per gram of tissue as the animal matures. It is also clear that Cl4 and P32as tracers
72
ROBERT
W.
SWICK
AND
ARTHUR
L.
KOCH
give similar estimates and that both are equally valid measures of DNA formation. It may be concluded that this molecule, like that of RNA, is synthesized as a unit. It also is apparent that DNA4 is formed at a rate determined by the growth of the animal, and, as has been done previously (l), it may be shown that the existing DNA is retained during the processes of cell division and probably for the life of the cell. Other workers have noted differences in the incorporation of various isotopes into the two types of nucleic acid. It appears possible to hope that eventually all of the experimental results obtained with the single dose technique may be reconciled with the hypothesis that the various moieties of the nucleic acids are incorporated into the macromolecule at the same rate. It would appear that the differences that have been seen (11, 12) are reflections of (a) variations in the time curves of the immediate precursors of the nucleic acids, (b) variations in t,he permeability of various cell types present in the tissue to the several tracers, (c) variation in the specific activity of the precursor pools in various portions of the cell, and (d) the possible effects of the movement of polynucleotides from one part of the cell to another. h?,KNOWLEDGMENTS
The authors gratefully acknowledge the valuable technical assistance of Mrs. Dorothy Handa and Mr. Samuel S. Thomas. We are indebted to Dr. Peter D. Klein for his interest and many helpful suggestions, and especially to Dr. J. Z. Hearon, Naval Research Medical Institute, National Institutes of Health, Bethesda, Maryland, for guidance and discussion on the theoretical aspects of tracer kinetics. SUMMARY
Using the continuous exposure technique, we have compared measurements of nucleic acid turnover obtained with phosphate-P3’ with previous observations in which U402 was used as a tracer. When proper correction was made for the precursor pool, the rate constant for P39 incorporation into the RNA was found to be similar to that previously observed for CY incorporation into the RNA purines. It is t,herefore concluded that RNA is synthesized and degraded as a unit. In young growing rats the rate of growth, estimated from the increase in liver weight, was compared with the increase in DNA estimated from the incorporation of P32. The latter values were lower than the former as was observed previously with CY. This discrepancy may be attributed to a decreasing concentration of DNA in the livers during the experi-
NUCLEIC ACID TURNOVER
73
mental period chosen. From the similarity in the rates of incorporation of CL4 and of P32 into the DNA, it appears t,hat DNA is likewise synthesized as a unit. REFERENCES 1. SWICH, R. W., KOCH, A. L., AND HANDA, D. T., Arch. Biochem. and Biophys. 63, 226 (1956). 2. SIRI, w. E., “Isotopic Tracers and Nuclear Radiations xvit.h Applications t,o Biology and Medicine,” Chap. 15, pp. 388402. McGraw Hill, New York, 1949. 3. SOLOMON, A. K., in “Advances in Biological and hledical Physics” (J. H. Lawrence and C. 9. Tobias, eds.), Vol. III, pp. 62-99. Academic Press, New York, 1953. 4. REINER, J. ill., Arch. Biochem. and Biophys. 46, 53, 80 (1953). 5. hIARGENAU, H., AND hkJRPHY, G. hl., “The Mathematics of Physics and Chemistry,” 1st ed., Eqs. 2-6. Van Nostrand, New York, 1943. 6. SWICR, R. W., AND HANDA, D. T., J. Biol. Chem. 288, 517 (1956). 7. TYNER, E. P., HEIDELBERGER, C., AND I.EP?LGE, G. A., Cancer Research 13, 186 (1953). 8. ALBERT,S.,JOHNSON, R.M.,AND C~~~~,hLS.,cuncer Research 11,772 (1951). 9. SCHNEIDER, W. C., J. Biol. Chew 161, 293 (1945). 10. WOOD, H. G., in “A Symposium on the Use of Isotopes in Biology and Medicine,” p. 219. University of Wisconsin Press, Madison, 1948. 11. BROWN, G. B., AND ROLL, P. hl., in “Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. II, p. 341. Academic Press, New York, 1955. 12. SMELLIE, R. M. S., in “Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), p. 393. Academic Press, New York, 1955.