DETERMINATION OF NUCLEIC ACIDS IN ANIMAL TISSUES

RY GIOVANNI CERIOTTI*

(From the Division of Experimental Chemotherapy, Sloan-Kettering Institute for Cancer Research, New York, New York)

(Received for publication, September 21, 1954)

As reported in a previous paper (l), a new microchemical method for the determination of deoxyribonucleic acid (DNA) has been devised which permits the evaluation of DNA in amounts as small as 2.5 y per ml. The method is particularly useful with very small quantities of tissue. For this reason a study was undertaken to learn the best conditions for the application of the method to the determination of DNA in animal tissues. The reaction of DNA with indole was compared with the diphenylamine reaction according to Dische (2). At the same time, a modification of the Barrenscheen and Peham (3) method for ribose determination with orcinol was worked out in order to determine pentose nucleic acid (PNA) in the same organ extracts. Total nucleic acid P of tissues was calculated from the PNA and DNA contents, found by means of color reactions. These values were compared to those obtained by a direct chemical method. Readings were also taken at 260 and 268 rnp and their values compared with those calculated from the results of the color reactions.

EXPERIMENTAL

DNA Reaction with Indole-Since DNA was to be determined in tissue extracts, it was necessary to test the effect of the usual nucleic acid extrac- tion media (trichloroacetic acid, HC104, NaOH) on the indole reaction.

The standard curve was made with a sample of DNA from calf thymus, prepared according to the Hammarsten method and having the following characteristics: N 13.4, P 8.0, adenine 10.0, guanine 7.4, cytosine 4.7, thymine 8.4 per cent, N:P 1.68 (from Dr. L. Cavalieri).

The DNA reaction with indole’ was performed under several different conditions. Previous boiling of the DNA solution in 0.1 N NaOH for half an hour did not affect the results. This was also true when the solution in N KOH was incubated for 24 hours at 37”. The small amount of alkali introduced into the reaction did not cause any variation in the results; however, when used for tissue extraction, alkaline treatment presented

* Present address, Centro Tumori di Busto Arsizio (Italy), Repnrto Ricerche Scientifiche.

1 The indole was purified by distillnt.ion under reduced pressure.

59

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60 DETERMINATION OF NCCLEIC .4CIDR

several disadvantages. With organs rich in lipides or connective tissue, the extracts are never completely clear; furthermore, consistent,ly lower values were obtained for PNA determinations than with the perchloric acid procedure.

Trichloroacetic acid, in the amounts usually employed for deproteiniza- tion, reacts with indole, giving a pink, cloudy color that is extracted com- pletely by chloroform. At the same time it inhibits the reaction between indole and DNA.

Perchloric acid tested in a final concentration of 3.3 per cent, which is about 3 times that usually employed in the test, did not enhance or de- crease the intensity of the color formed during the reaction.

Pentose Reaction with Orcinol-In 1941 Barrenscheen and Peham (3) re- ported that the Bial reaction can be improved by substituting cupric chlo- ride for ferric chloride. They emphasized the fact that the boiling time should be exactly 10 minutes, because longer boiling increases the intensity of the color and causes a precipitation of the colored compound even at relatively low concentrations; they pointed out also the different slopes of the absorption curves given by arabinose, xylose, adenosine, and adenosine- triphosphoric acid under the same experimental conditions. As Albaum and Umbreit (4) have demonstrated, the different slopes of these curves are due to the different rates of furfural formation from the compounds under consideration. For adenosinetriphosphoric acid, the furfural yield is completed in 10 minutes of hydrolysis; for the other compounds more time is required. After 40 minutes of boiling the pentose and pentose purine nucleosides and nucleotides give all the furfural accounted for by their ribose content; no furfural is formed from pyrimidine nucleotides. Also with PNA the color intensity increases until 40 minutes hydrolysis and then remains almost constant.

At the low concentrations used (2 to 20 y of ribose per ml.), and after prolonged hydrolysis, formation of precipitates has never been observed. Precipitates form at higher concentrations and eventually, when trichloro- acetic acid is present, in rather large amounts. After 10 minutes of hy- drolysis, the color formed is blue; it then turns green. After 40 minutes of hydrolysis the color intensity remains constant or decreases only slightly. The color is easily extracted without any change by either amyl or isoamyl alcohol. By this means the color can be concentrated and the sensitivity of the reaction thus increased. Also when the color is precipitated, it dis- solves completely in the alcohol.

The reaction is performed according to the following procedure. Reapnts--0.004 M CuC12.H~0 in concentrated HCl (density 1.19); con-

centrated HCl, c.p. (density 1.19) ; orcinol freshly recrystallized (m.p. 106”). Solution to be tested, at concentrations of ribose ranging from 2 to 20 y

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G. CERIO’ITI 61

per ml. Isoamyl alcohol (b.p. 128.5’), freshly redistilled and filtered after shaking with Darco G-60.

200 mg. of orcinol are dissolved in concentrated HCl; 10 ml. of the CuC12*H20 solution are added, and the volume is made up to 100 ml. with concentrated HCl. To 5 ml. of the solution to be tested, 5 ml. of the re- agent are added. The test-tubes are shaken thoroughly and immersed in a

1.5 --

0.1 __

400 500 600 700 mp WAVE LENGTH

FIG. 1. Absorption spectrum of the color formed by the reaction of ribose and PNA with orcinol.

boiling water bath for 40 minutes; then they are cooled under running water.

The color is extracted with 5 ml. of isoamyl alcohol, and, after centrifuga- tion, is read on a Beckman spectrophotometer at 675 rnp against a blank treated in the same manner (see Fig. 1).

Lambert-Beer’s law is followed at concentrations ranging between 2 and 15 y of ribose per ml. The results are constant and reproducible, and the color is stable. The optical density for 1 y of ribose per ml. is 0.0715 (Fig. 2).

The results given by PNA were especially studied. The PNA used presented the following characteristics: N 14.5, P 8.3 per

cent, N:P 1.74; content of purine and pyrimidine bases in millimoles per

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62 DETERMINSTION OF NUCLEIC ACIDS

gm., guanine 0.86, adenine 0.64, cytidine 0.56, uridine 0.50, ribose 2.56 gm. (38.4 per cent). The purine-bound ribose corresponds to 57.6 per cent of the total ribose. From several tests at different concentrations of PNA, the ribose hydrolyzed was found to be 59.87 per cent of the total,

1.1 _-

1.0 __

0.9 __

0.8 __

0.7 __

0.6 __

0.5 __

0.4 ._

c

8 0.3 __ n

2 0.2 u i= “0 0.1

o 5 10 15 20 25 30 35 40 8/ml.

Fro. 2. Absorption curves of the orcinol reaction. 0, ribose; fl, PNA (P con- tent 8.3 per cent; purine-bound ribose 57.6 per cent). The straight line indicates the theoretical absorption for a PNA containing 9.5 per cent P.

with a deviation in the different tests of 11~1.94. These differences are within the limits of error of the method and therefore are not significant.

Practically, only the purine-bound ribose was hydrolyzed. On the basis of the data reported above, ‘the average ratio between the optical density for 1 y of ribose and 1 y of PNA is 3.76.

This fart)or ran be used to determine the PNA content of an unknown sample on t,he basis of the curve for ribose. When the PNA was dissolved in 0.1 N NaOH solution and boiled for half an hour before performing t’he reaction, the dat,a obtained were the same as mit,h a neutral unboiled solu-

tion. DNA solutions at a concentration of 2 mg. per ml., when submitted

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G. CERIOTTI 63

to the orcinol test, gave a color corresponding to 4.5 y of ribose per ml.; that is, to 17.0 y of PNA. This represents an interference of 0.85 per cent.

Determinations by Ultraviolet Absorption-These were carried out both at 260 and 268 mp. In fact, as Ogur and Rosen (5) have pointed out, the absorption maximum for PNR is at 260 rnp, for DNA at 268 rnp, when the readings are performed in perchloric acid solution. The optical density for PNA was 0.316 at 260 rnp and 0.298 at 268 rnp per y of P (Table I). The optical density for PNA appeared to be the same when the readings were made in 0.1 N NaOH as those in 10 per cent perchloric acid solution. On the contrary, the DNA absorption decreased in 0.1 N NaOH solution compared with that in 10 per cent perchloric acid.

TABLE I

Optical Densities of PNA and DNA with Different Reactions

The optical densities per microgram of nucleic acid have been calculated for DNA and PNA corresponding to the classical tetranucleotide formula; therefore P = 9.89 and 9.5 per cent, respectively.

I DNA PNA

Optical density Indole Diphenyl-

reaction am’?e reaction

Ultra- Ultra- violet’ violet*

at260 m,, at268 mp

Per y I’. 0.228 0.0265 0.274 0.286 0.200 0.316 0.295 C‘ ” nucleic acid.. 0.0225 0.00263 0.0270 0.0284 0.019 0.0300 0.0283

* In 10 per cent perchloric acid solution.

Phosphorus Determination-The P was determined, after incineration with 0.2 ml. of perchloric acid at 250”, by the amidol (2,4-diaminophenol dihydrochloride) reagent (6). The weights of the different organs ranged usually between 0.5 and 2.0 mg. (dry weight) according to their nucleic acid content. Samples of 0.4 to 1.5 ml. of the tissue extracts were made up to 2 ml. with 10 per cent perchloric acid and then incinerated.

Preparation of Organ Extracts-Extraction of organs is based on the tech- nique described by Ogur and Rosen (5). Immediately after the animal was sacrificed, the organs were minced, weighed, and extracted twice with absolute alcohol and twice with ether. After drying, the residue was ex- tracted with cold 2 per cent perchloric acid in the refrigerator for 20 min- utes and then centrifuged in the cold. The supernatant fluid was dis- carded. The procedure was repeated. The residue was then extracted with 10 per cent perchloric acid at 70” for 20 minutes, centrifuged, the su- pernatant fluid saved, and the residue extracted again. The combined supernatant solutions were diluted to provide an estimated concentration

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64 DETERMINATION OF NUCLEIC ACIDS

ranging between 10 and 15 y of DNA per ml. and about 25 to 35 y of PNA per ml.

An alternative technique was often used. The organs were left in abso- lute alcohol for a few hours, and then the alcohol was changed. They could be stored in alcohol, then in alcohol-ether and ether, for a few weeks, and subsequently dried completely in a desiccator without any noticeable variation in DNA and PNA content. After determination of the dry weight, the perchloric acid extractions were performed as described above.

The amount of tissues used and the final dilution varied according to the estimated difference in nucleic acid content. Usually the extraction was performed with 1 ml. of 10 per cent perchloric acid per 3 to 4 mg. for liver and kidney, 1.5 to 2 mg. for spleen, lung, and pancreas, and 0.5 mg. for thymus. The perchloric acid extracts were further diluted 6 to 12 times with distilled water for the indole reaction. The final perchloric acid con-

TABLE II

Recovery of DNA from Spleen Extract by Indole Reaction

DNA of spleen extracts, 31.7 y per ml.

DNA added Total DNA

y per ml. y per ml.

17.2 48.9 34.4 66.1 51.6 83.3

Recovery

y per ml.

48.2 64.2 81.9

Per cent recovery

98.5 97.0 98.0

tent was thus lower than that used for testing its eventual influence on the reaction.

For the orcinol reaction, dilution with distilled water ranged between 3 and 10 times. For ultraviolet readings, the perchloric acid extracts were diluted 15 to 20 times with 10 per cent perchloric acid. Phosphorus de- terminations and the diphenylamine reaction were performed on the undi- luted extracts. 3 ml. were used for the DNA and 5 ml. for the PNA de- terminations. The tests were always run in duplicate.

The DNA determination with diphenylamine was also made. All the extraction procedure was performed in the same centrifuge tube in order to avoid any loss and to permit the use of very small samples of tissues.

The indole reaction makes it possible to work with samples as small as 25 mg. of wet tissue (sarcoma 180) ; when thymus is employed, the amounts may be much smaller. Some recovery experiments were conducted by adding different amounts of pure DNA to the organ extracts. The re- covery was complete within the limits of error of the method (f4 per cent) (Table II).

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G. CERIOTTI 65

Calculations-In order to compare the results obtained by t,he various methods, calculations were made according to the following outline. The DNA and PNA contents for 100 y of dry tissue were calculated from the results of the absorption given by the different reactions on the basis of their specific optical densities (Table I). The per cent of DNA and PNA, multiplied by 0.0989 and by 0.095, respectively, gave the theoretical phos- phorus content required by the color reactions for theoretical DNA and PNA. The theoretical absorption in the ultraviolet was also calculated by multiplying the per cent values by the optical densities for 1 y of DNA or PNA, both at 260 and at 268 ml.r.

The sum of the values of PNA and DNA phosphorus and that of PNA and DNA absorption in the ultraviolet, calculated on the basis of the color reactions, were compared with the percentages of phosphorus and of the optical densities found by direct determination.

Materials-Several organs of C57 black mice and of albino rats were studied: liver, kidney, spleen, lung, thymus, and pancreas. The average weight of the animals was 19.5 f 0.5 gm. for mice and 150 f 5 gm. for rats. Several determinations were also made with sarcoma 180. The values reported are the mean of at least five determinations. All the tests were run in duplicate.

Results

The results obtained are reported in Tables III, IV, and V. All the values refer to the dry weight of the defatted organs. For comparison with the data of the literature, which are often given as per cent of the wet weight, the average per cent of dry weight of the organs studied is also reported.

DISCUSSION

As may be seen from Table III, the values by the indole reaction com- pare favorably with those by the diphenylamine reaction within the range of the experimental error for both methods. The standard deviation, which is the result of both the individual variations and the experimental error, is sometimes larger with one method, sometimes with the other. The range of error of the two procedures is almost the same, notwith- standing the much smaller amounts of tissue used for the indole reaction.

Some organs, especially spleen, show very important individual vari- ations. This fact is confirmed by data in the literature (7) and has to be kept in mind when comparisons are made between different groups of animals. It may be easily correlated with the very variable histological picture of this organ, sometimes very rich and sometimes poor in germi- native centers; furthermore, it is difficult to free the organ from blood to

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66 DETERMIN.4TION OF NUCLEIC ACIDS

the same extent in every animal. Thymus also shows rather large indi- vidual variations. Smaller variations are found in some other organs, for

TABLE III

Comparison of Determination of DNA and PNA Content of Mouse and Rat Organs

Data expressed as per cent of the dry weight. The standard deviations are the results both of individual variation and error of the methods used.

Mouse liver

“ kidney

I‘ spleen

“ lung

“ thymus

‘L pancreas

ltut liver

“ kidney

I‘ spleen

“ lung

“ thymus

“ pancreas

Sarcoma 180

DV veight,

Per cent of

wet weight

24.0

20.0

20.8

20.2

20.8

22.8

23.9

20.0

21.7

17.2

17.6

23.5

15.4

1.66 f 0.218 1.71 f 0.282 4.55 f 0.298 1.34- 1.95

2.76 f 0.155 2.53 f 0.293 3.01 f 0.154 2.60- 2.98

8.32 f 1.02 8.46 f 0.488 5.72 f 0.210 7.30- 9.70

3.73 f 0.202 3.69 f 0.311 2.71 f 0.126 3.44- 3.90

4.80 f 1.74 13.80 f 1.74 5.97 f 0.22212.0 - 16.0

2.42 f 0.336 2.37 f 0.26213.95 f 0.680 1.93- 2.66

1.15 f 0.124 1.20 f 0.136 4.16 + 0.358 1.02- 1.31

2.05 f 0.232 2.12 f 0.173 2.61 f 0.082 1.74- 2.35

3.34 f 1.08 3.58 f 1.01 3.66 f 0.290 2.06 3.68

3.66 f 0.164 3.88 f 0.241 2.75 f 0.293 3.45- 3.85

2.12 f 1.88 12.96 f 1.02 5.70 f 0.175 9.30- 13.8(

1.99 f 0.175 2.02 A 0.16310.64 f 0.923 1.72- 2.15

4.78 f 0.231 7.75 f 0.309 4.55-

DNA, per cent of dry weight

Indole Diphenylamine

Extreme values

PNA

1

3

) 5.25

4.05- 4.84

2.86- 3.25

5.50- 5.98

2.55- 2.86

5.70- 6.24

3.4 - 14.8

3.84- 4.77

2.55- 2.71

2.81- 4.03

2.46- 3.20

5.60- 5.90

9.45- 12.00

7.20- 8.05

instance kidney and lung. This holds true also for sarcoma 180 when healthy parts of tumors of the same age are chosen.

When comparing the DNA content of organs from mice and rats, one notices a more or less higher content for the former, except for lung. Also PNA is higher in mouse than in rat spleen and pancreas. The data in the literature are very scarce for the mouse; those for the rat are much more abundant. The present data compare favorably with most of the

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G. CERIOTTI 67

published data (7). Considerable differences may be found, depend- ing upon the strain, age, and sex of the animals used (7). Therefore ani- mals of the same sex and of similar weight and, for mice, of an inbred strain (C57 black) have been chosen.

Special consideration must be given to comparison among the data ob- tained from color reactions (indole and orcinol), phosphorus determi- nation, and ultraviolet readings. In Table IV, one observes some differ- ences between the phosphorus calculated on the basis of the color reactions and that determined directly. These are especially marked for lung and

TABLE IV

DNA P and PNA P of Mouse and Rat Organs

Comparison between the dry weight percentages calculated on the basis of the indole and the orcinol reactions with the values found experimentally.

nlouseliver ............... “ kidney. ............ ‘L spleen ............. “ lung. .............. ‘C thymus ............ “ pancreas. ..........

Rat liver .................. “ kidney. ............. “ spleen ............... (‘ lung .................. “ thymus ............... “ pancreas ..............

Sarcoma 180. .............

-

NAP er ent of #B ry

weight (indole)

0.164 0.273 0.824 0.369 1.462 0.239 0.114 0.203 0.330 0.362 1.200 0.197 0.474

‘NA P, Per :ent of dry

weight

DNA P + PNA P, per cent of dry weight PNA P

DNA P lalculated Found

0.433 0.597 0.626 f 0.055 2.64 0.286 0.559 0.537 f 0.026 1.05 0.543 1.367 1.385 f 0.077 0.66 0.25s 0.627 0.582 f 0.063 0.70

0.567 2.029 1.836 f 0.274 0.39

1.325 1.564 1.740 f 0.090 5.54 0.395 0.509 0.506 f 0.052 3.46 0.258 0.461 0.434 f 0.025 1.27 0.348 0.678 0.623 f 0.132 1.05 0.261 0.623 0.551 f 0.050 0.72 0.541 1.741 1.600 f 0.180 0.45 1.010 1.207 1.240 f 0.039 5.13 0.718 1.190 1.140 f 0.053 1.52

-

thymus, both of mouse and rat, and for rat spleen. The phosphorus con- tent of these organs is lower than expected; on the contrary, for mouse pancreas it is higher.

Considering the values calculated and those found for ultraviolet ab- sorption, practically the only important difference to be observed is in mouse pancreas, in which the absorption is higher than expected (Table V).

There is greater agreement between the results of the color reactions and the ultraviolet readings than between these determinations and those of phosphorus.

The different ratio of purines to pyrimidines possibly present in the PNA of various organs may explain the lack of agreement when the phos-

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68 DETERMINATION OF NUCLEIC ACIDS

phorus and ultraviolet data compare favorably. Only PNA has to be taken into consideration, because DNA has been shown to be very con- stant in its composition, at least in the same species (8, 9); on the con- trary, in PNA the ratio between purines and pyrimidines may vary con- siderably in different organs (9, 10).

Since the orcinol reaction reveals only the purine-bound ribose, a lower content of purines may demonstrate a lower PNA content than is ac- counted for by P determination and ultraviolet absorption. On the other

26Ol44

Calculated, indole + orcino’

Mouse liver. ‘C kidney.... “ spleen. .‘ lung. . . “ thymus...... I‘ pancreas.

Rat liver. . . . “ kidney. “ spleen. “ lung. “ thymus, “ pancreas......

Sarcoma 180.

0.181 0.165 0.400 0.188 0.580 0.484 0.154 0.134 0.200 0.182 0.502 0.373 0.355

-

Ultraviolet absorption per 100 y. dry weight

T 268 nw 7

Calculated, ndole + orcinol Found Found

0.187 f 0.013 0.160 f 0.015 0.410 f 0.023 0.183 f 0.014 0.590 f 0.059 0.557 f 0.045 0.152 f 0.010 0.125 f 0.013 0.217 f 0.025 0.173 f 0.015 0.486 f 0.049 0.389 f 0.031 0.350 f 0.016

0.186 0.187 f 0.013 0.164 0.163 f 0.015 0.406 0.426 f 0.028 0.184 0.189 f 0.015 0.590 0.620 f 0.067 0.465 0.554 f 0.045 0.149 0.151 f 0.009 0.132 0.131 ztr 0.013 0.199 0.215 f 0.029 0.173 0.176 f 0.014 0.505 0.495 i 0.051 0.359 0.384 f 0.033

TABLE V

Ultraviolet Absorption Values of Perchloric Acid Extracts of Mouse and Rat Organs

Comparison between the ultraviolet absorption values found experimentally and those calculated from the results of the indole and the orcinol reactions.

I -

hand, if the purine content is higher, the PNA may appear to be higher than that expected from P determination and ultraviolet absorption.

However, when the ultraviolet absorption and the results of the color reactions are in agreement and the P values are lower than expected, the possibility exists, as pointed out for PNA by Gulland (ll), that 1 P residue may be bound to 3 instead of to 2 nucleosides. This could account for a lack of as much as one-fourth of the PNA P. This possibility could exist also for DNA, but it has not been proved.

To explain the present results in another way, one should admit the presence in the extracts of some interfering substance (or substances) giving both the color reactions and absorption in the ultraviolet. A sub- stance which interferes with the reaction for DNA with indole has been

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G. CERIO!l’TI 69

found in serum extracts, but it does not show any specific absorption in the ultraviolet. The unknown substance gives a yellow color with indole, which behaves in the same manner that the DNA color does towards chloroform extraction. However, the color produced by serum presents a maximum of absorption at 460 rnp, and the color formed by DNA pos- sesses a maximum at 490 mp. In addition, the color formed by DNA is completely adsorbed by alumina and that formed by the unknown sub- stance is not, under the selected conditions. Absorption curves of the organ extracts after indole reaction did not differ from the absorption curve given by pure DNA. For this reason the reacting substance of serum does not constitute a limitation for the method of DNA determi- nation in animal tissues. The reacting substance of serum could possibly correspond to the perchloric acid-soluble mucoprotein, which also gives the diphenylamine and the tryptophan perchlaric acid reaction (12-16).

The indole reaction for DNA, in the present form, has some limitations, but it is useful when very small quantities of tissue are available.

Comparison among different methods of nucleic acid determination, based on their different components, shows that both sugars and phospho- rus determinations yield only relative values, at least as far as PNA is concerned. They may be used alone for comparison of the same organ or tissue. Otherwise, simultaneous study of the ultraviolet absorption is also advisable. The latter seems, at present, to be the best approach to the real value. It is, however, to be noted that this method may produce large variations jvith small changes in the experimental conditions, as, for instance, pH or heating, and that a variation in the proportion of the different bases could possibly induce variation in the absorption values.

SUMMARY

The application of the color reaction of DNA with indole to the determi- nation of this substance in animal tissues has been studied under different conditions.

An improved method far PNA determination with orcinol and its appli- cation to the tissues is also described.

Comparison has been made between the DNA reaction with indole and with diphenylamine.

The total nucleic acid values obtained by the methods proposed have been compared with those from nucleic acid phosphorus determinations and from the ultraviolet absorption.

The limitations of the methods and their validity for direct determi- nation of nucleic acids have been discussed.

I wish to express my appreciation to Dr. C. Chester Stock for his con- tinued interest and aid in the present work, to Dr. L. Cavalieri for kindly

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70 DETERMINATION OF SUCLEIC ACIDS

supplying the PNA and DNA samples, and to Miss Mary Tompkins and Miss Emma Berti for their technical assistance.

BIBLIOGRAPHY

1. Ceriotti, G., J. Biol. Che~m., 198, 297 (1952). 2. Dische, Z., Mikrochemie, 8, 4 (1930). 3. Barrenscheen, H. K., and Peham, A., Z. physiol. Chem., 272, 81 (194132). 4. Albaum, H. G., and Umbreit, W. W., J. Bio2. Chem., 167, 369 (1947). 5. Ogur, M., and Rosen, G., Arch. Biochem., 26, 262 (1950). 6. Miiller, E., Z. physiol. Chem., 237, 35 (1935). 7. Davidson, J. N., Cold Spring Harbor Symposia &ant. Biol., 12, 51 (1947). 8. Chargaff, E:., Ezperientia, 6, 201 (1950). 9. Chargaff, E., J. Cell. and Comp. Physiol., 38, 41 (1951).

10. Magasanik, Is., Vischer, E., Doniger, It., Elson, D., and Chargaff, E., J. Biol. Chem., 186, 37 (1950).

11. Gulland, J. M., Symposia Sot. Exp. Biol., 1, 1 (1947). 12. Seibert, F. Is., Pfaff, M. L., and Seibert, M. V., Arch. Biochem., 18, 279 (1948). 13. Petermann, M. L., and Hogness, K. R., Cancer, 1, 100 (1948). 14. Weimer, H. E., Mehl, J. W., and Winzier, R. J., J. Biol. Chem., 186, 561 (1950). 15. Butler, L. O., Brit. J. Cancer, 6, 225 (1951). 16. Coburn, A. J., and Haninger, J., J. Exp. Med., 99, 1 (1954).

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Giovanni CeriottiIN ANIMAL TISSUES

DETERMINATION OF NUCLEIC ACIDS

1955, 214:59-70.J. Biol. Chem. 

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