BIOCHIMICA ET BIOPHYSICA ACTA

953

DISTRIBUTION OF 5-METHYLCYTOSINE
IN P Y R I M I D I N E SEQUENCES OF DEOXYRIBONUCLEIC ACIDS
J. D O S K O ~ I L AND F. ~ORM

Institute o/Organic Chemistry and Biochemistry, Czechoslovak Academy o/Sciences,

Praha (Czechoslovakia)
(Received October 6th, 1961)

SUMMARY

Several samples of DNA from different mammalian organs and from wheat germ were
degraded with Burton reagent and the location of derivatives of 5-methylcytosine in
separate fractions of the hydrolysate was investigated. In mammalian DNA's 5-methylcytosine was found to occur almost exclusively in the fraction of solitary pyrimidine nucleotides and in the terminal groups of polypyrimidine series carrying monoesterified phosphorus on C-3' of deoxyribose. The ratio of 5-methylcytosine to cytosine
was nearly identical in these positions. In the DNA from wheat germ the highest degree of replacement occurred in similar positions, but a fairly high amount of 5-methylcytosine was found in the sequence MpT. The relation of these findings to earlier
evidence obtained by enzymic degradation of DNA is discussed. It is concluded that
the replacement of cytosine by 5-methylcytosine is determined by the nature of the
nucleotide attached to C-3' of deoxycytidine. The replacement occurs with highest
probability in the sequence CpG; somewhat less probable is the replacement in the
sequence CpT, whereas in the sequences CpM, CpC and CpA the substitution takes
place only rarely, even in preparations with high overall content of 5-methylcytosine.

INTRODUCTION

DNA from several plant and animal sources contains small quantities of 5-methylcytosineL 2. According to the Watson-Crick double-helix model of DNA, 5-methylcytosine should be freely exchangeable with cytosine. However, when determining the
content of 5-methylcytosine in fractions3 and degradation products 4,5of DNA, significant deviations from random distribution and free exchangeability were found.
SINSHEIMER4 demonstrated that in the hydrolysate of calf-thymus DNA, large quantities of the dinucleotide pMpG occur, whereas other dinucleotides are completely free
of methylcytosine. SI~APII~OAND CHARGAFF 5, investigating the composition of the
acid hydrolysate of rye-germ DNA, found 5-methylcytosine to be accumulated in
the fraction of solitary nucleotides and in dipyrimidine fragments, thus showing some
similarity to the relative distribution of thymidylic acid.
Abbreviations: G, A, C, M. T, deoxyguanosine, deoxyadenosine, deoxycytidine, deoxy-5methylcytidine and thymidine, respectively; X, a n y of these nucleosides; Pu, purine deoxyribonucleosides; Py, pyrimidine deoxyribonucleosides; p represents orthophosphate esterified with these
nucleosides; for abbreviations of nucleotides see ref. 6.

Bioehim. Biophys. Acta, 55 (1962) 953-959

954

j. DOSKOCIL, F. SORM

In the present study the location of 5-methylcytidylic acid in polypyrimidine

sequences was investigated. Preparations of DNA from calf thymus, rat spleen, mouse
leukemic liver and wheat germ were hydrolysed with formic acid and diphenylamine 6.
Using this method of hydrolysis the purine deoxyribonucleotides are degraded to
purine bases and inorganic orthophosphate is formed from tile phosphodiester groups
joining two purine nucleosides; the pyrimidine nucleosides, however, are not degraded
and remain as nucleotides of the general structure PY.P.+v The fractions of n-nucleoside (n+I)-phosphates were degraded succesively with prostate phosphomonoesterase and with phosphodiesterase from calf spleen. With this type of degradation,
deoxycytidylic acid occuring in the sequence CpPy appears in the form of deoxycytidine 3'-phosphate, whereas that forming part of the sequence CpPu in the original
chain of DNA is recovered as deoxycytidine. The extent of replacement of cytosine
with 5-methylcytosine in these positions was then determined by analysis of the deoxyribonucleoside 3'-phosphate and deoxyribonucleoside fractions.
EXPERIMENTAL

DNA from rat spleen and mouse leukemic liver (strain Black C 57, transplantable
post-irradiation blastic leukemia, 20-30 % blastic infiltration) was prepared according
to KAY, SIMMONSAND DOUNCE7. DNA from calf thymus was isolated by the method
o f SCHWANDER AND SIGNER 8. The method of LIPSHITZ AND CHARGAFF 9 w a s used, to
prepare DNA from wheat germ except that RNA was removed by treatment with
ribonuclease instead of the absorption on charcoal. The analyses of the preparations
are given in Table I.
TABLE I
COMPOSITION O F THE SAMPLI~S O F D N A
Source

Calf t h y m u s
R a t spleen
Mouse leukemic liver
Wheat germ

P(%)

RIgA (%)

9.1
8. 7
8. 7
9.0

o.3
0. 3
o. 5
1.5

21.5
21. 3
21.6
22.6

28.3
28. 5
28.2
27. 3

2o.1
20.2
21.o
15.8

1.5
1. 3
1. 3
6. 7

28.6
28. 7
27. 9
27.6

The phosphomonoesterase was prepared from human hypertrophic prostatic

tissue according to DAVlDSON AND FISHMANlo. The activity was determined with
sodium glycerophosphate, using a o.25 % (w/v) solution in o.o5 M acetate buffer, pH
5.5. o.o5 ml of the solution of the enzyme (absorbancy at 28o m# equal to o.315) was
added/ml of the buffered solution of sodium glycerophosphate and the mixture was
incubated at 37 . i ml of the solution of the enzyme produced 39oo#g Pi/h under these
conditions.
The splenic phosphodiesterase was isolated from calf spleen by the method of
HILMOEn. A mixture of oligonucleotides from the hydrolysis of calf-thymus DNA with
splenic DNAase II was used as substrate for testing the phosphodiesterase activity.
The hydrolysate of DNA with DNAase II was ckromatographed on a column of
Dowex-I X2. The fraction eluted by 1.o-1.2 M ammonium formate (pH 4.65) was
evaporated to dryness and the formate was removed by vacuum sublimation at
Biochim.

Biophys.

Acta,

55 (1962) 953-959

DISTRIBUTION OF 5-METHYLCYTOSINE IN D N A

955

4 o. The residue, consisting mostly of tetranucleotides, was dissolved in an appropriate amount of 0.05 M acetate buffer, p H 6.5, to give a solution having A2eo equal to
5.0-5.5.0.025 ml of enzyme was added/ml of this solution and the mixture was incubated at 37 . The production of mononucleotides was followed by circular paper
chromatography in ethanol-I M ammonium acetate TM, p H 7-5 (75:30). With usual
preparations of the enzyme (absorbancy at 280 m# about IO) the splitting to mononucleotides was virtually complete in 3o min of incubation. The preparation contained
some phosphomonoester ase activity, but if care was taken to interrupt the incubation
just as the cleavage of phosphodiester bonds was complete, equal amounts Of nucleotides and nucleosides were always obtained in analyses of dinucleoside monophosphates.
The hydrolysis of DNA and the remova] of formic acid were carried out as described b y BURTON AND PETERSEN6. 400 mg of mammalian DNA (except in the case
of DNA fl:om mouse leukemic liver, where only 18o mg were available) and 200 mg of
wheat-germ DNA were used for one experiment. The hydrolysate was chromatographed on a column of Dowex-I X2 (20 I cm) with ammonium formate buffer, p H
4.65 4-0.0.5. A stock solution of formate buffer, 4 M in ammonium formate and approx.
I M in formic acid, was appropriately diluted with watel to obtain the indicated
concentration of formate. Exact control of p H of the buffer was essential, since even
slight deviations from the correct value caused incomplete separation of pCpCp from
pTp or from pTpCp and pCpTp. Two linear concentration gradients were used succesively to elute the n-nucleoside (n +I)-phosphates: 250 ml o. i M to 25oml 0.6 M and
250 ml o.6 M to 250 ml 1.2 M formate. About 9 % of the total ultraviolet-absorbing
material was eluted. The elution curve was registered b y means of a Uvicord, type

15

10
I

"7

o'.6

1'.2

=o~5

5
5

o.1

ab

o'.e

~:2

Conch. of formate (#4)

Fig. I. Elution curves of the hydrolysate of DNA with Burton reagent. The column of Dowex-I
X2 (z 20 cm) was charged with a hydrolysate of 4o0 mg DNA and eluted with two succesive
linear gradients of ammonium formate buffer, p H 4.65 (25o ml o.i M to 25o ml 0.6 M and 25 m l
o . 6 M to 25oml 1.2 M formate). The volume of the fractions collected was 7 ml. A, calf-thymus
DNA; B, wheat-germ DNA. Fraction I, pCp, pMp, 2, pTp; 3, pCpCp, pCpMp; 3a, pMpNIp, pCpMp,
pMpCp; 3b, pCpCp; 4, pCpTp, pTpCp, pTpMp; 4 a, pTpMp, pMpTp; 4b, pTpCp, pCpTp; 5, pTpTp,
pCpC]?Cp; 6, trinucleotides TCC; 7, trinucleotides TTC; 8, mostly tetranucleotides.

Biochim. Biophys. Acta, 55 (1962) 953-959

956

3. DOSKO~IL, F. ~ORM

47Ol A (LKB, Sweden). The fractions forming separate peaks were pooled and evaporated to dryness i n vacuo. Ammonium formate was removed b y sublimation
at 4 0 i n vacuo. The oligonucleotides were then dissolved in 2 ml of 0.05 M acetate buffer, p H 5.5. o.I ml of prostatic phosphomonoesterase was added and the solution incubated for 8 h at 37 . The p H of the mixture was brought to 6.5 b y addition of approx. 0.02 ml of 0.5 M ammonia and the phosphomonoesterase was inactivated by
heating the solution on a water bath at 8o for 15 min. After cooling 0.05 m l o f p h o s phodiesterase was added and the solution was incubated at 37 usually for 2 h. Since
the preparation of phosphodiesterase was not entirely free of phosphomonoesterase
activity, the incubation had to be interrupted as soon as the substrate was completely
decomposed to mononucleotides and nucleosides. For this purpose samples were
withdrawn from the reaction mixture in 3o-min intervals and analysed b y circular
paper chromatography on a disk of W h a t m a n No. 4 paper with ethanol-I M a m m o n i u m
acetate, p H 7.5 (75 : 30). As soon as no residual spot of oligonucleotides could be discerned on tile chromatogram, the reaction was interrupted and the samples were:
rapidly concentrated to small volume on a horizontal r o t a r y vacuum evaporator.
The mixture was then applied on a sheet of W h a t m a n No. 3 paper in a line IO cm of
length and subjected to descending chromatography with ethanol-ammonium acetate,
p H 7.5, for approx. 14 h. The zones of nucleotides and nucleosides were eluted with
water; the eluates were evaporated to dryness and hydrolysed with concentrated
formic acid according to VISCHER AND CHARGAFF13 using sealed capillary tubes
approx. I m m of inner diameter. The hydrolysates were evaporated to dryness i n
vacuo, dissolved in o.i N HCI and chromatographed on W h a t m a n No. 3 paper in a
system 1 isopropanol-HCl-water (17o :41:39). Usually satisfactory separation of 5methylcytosine and cytosine was achieved b y the first chromatography, so t h a t rechromatography 1 was not necessary, If, however, the eluted 5-methylcytosine was
not spectroscopically pure, rechromatography in the same solvent was used for further purification. If no distinct zone of 5-methylcytosine could be detected on the
chromatogram, the zone of cytosine was also rechromatographed to be sure that no
methylcytosine remained in the cytosine zone.
The zones of pyrimidines were cut out of tile chromatogram and eluted with 4 ml
o.I N HCI. Ultraviolet spectra were then recorded in the range 250-300 m# in 5-m#
intervals. The amounts of pyrimidines were calculated using the following values
of millimolar extinction coefficients14: lO. 5 at 275 # m for cytosine, 9.8 at 283 m# for
5-methylcytosine and 7.95 at 265 m# for thymine.
To check the reliability of the analytical method aliquots of fractions of the hydrolysate were analysed for 5-methylcytosine content without previous enzymic hydrolysis. The results obtained were then compared with the content of 5-methylcytosine calculated from the determinations after enzymic degradation. I t m a y be seen
from Table I I that the agreement is satisfactory. Similarly, good agreement was
obtained when calculating the content of 5-methylcytosine in whole DNA from the
analyses of separate fractions and comparing it with the data obtained b y direct analysis, indicating that the hydrolysis method using formic acid to determine nitrogenous constituents is trustworthy.
When analysing the mixed dipyrimidines additional information was obtained
giving the ratio of both sequential isomers, pCpTp and pTpCp, without their actual
separation. I t has been shown b y BURTON AND PETERSEN6,15, that this ratio is sigBiochim. Biophys. Acta, 55 (1962) 953-959

DISTRIBUTION OF 5-METHYLCYTOSINE IN D N A

957

nificantly different from unity in many preparations of DNA from different organisms. The ratios given in Table II are derived from the ratio of cytosine (plus 5methylcytosine) to thymine in the nucleoside fraction, which is equal to the reciprocal of this ratio in the 3'-phosphate fraction of the enzymic hydrolysate. This equality provides a test for the correctness of the degradative and analytical procedures
employed.
RESULTS

The experiments summarized in Table II show that in all three preparations of

mammalian DNA 5-methylcytosine occurs almost exclusively in the fraction pCp and
in the terminal nucleotide of polypyrimidine units bearing monoesterified phosphorus on the C-3' atom of deoxyribose. This fact indicates that methylcytosine
replaces cytosine only in the sequence PupCpPu (sohtary deoxycytidyhc acid) or
PypCpPu (3'-phosphate terminus of po]ypyrimidine sequences), but not in the sequence PupCpPy. E.g. in the isomeric mixed dipyrimidine nucleotides partial replacement of cytosine with 5-methylcytosine occurs in the less frequent pTpCp isomer, but not in the more frequent pCpTp isomer; the preponderance of the latter
sequential isomer observed by BURTON AND PETERSEN6 in calf-thymus DNA seems
to be quite common in preparations of DNA from mammalian tissues, since almost
equal ratios of both sequential isomers were observed with calf-thymus, rat-spleen
and mouse-leukemic-hver DNA's.
It can therefore be readily understood that the solitary cytosine is replaced
with 5-methylcytosine about twice as frequently as the total cytosine in the dipyrimidine sequences and that even less 5-methylcytosine is contained in higher polypyrimidine sequences TM. A similar type of distribution was suspected by SHAPIRO
AND CHARGAFF5 to exist in rye-germ DNA, accounting for the observed tendency of
5-methylcytosine to be accumulated in the fraction of solitary pyrimidine nucleotides and in lower polypyrimidine sequences.
The extent of actual replacement of exchangeable cytosine with 5-methylcytosine is nearly the same in all types of sequences, namely PupMpPu, CpMpPu, TpMpPu
as well as in terminal deoxycytidylic acid occuring at the 3'-phosphate end of tripyrimidine sequences. This fact indicates that, in mammalian preparations studied,
the replacement of cytosine with 5-methylcytosine is determined exclusively by the
nature of the nucleotide attached to 3'-carbon atom of deoxycytidylic acid, whereas
the nucle,otide on the 5'-carbon atom is irrelevant in this respect.
In the DNA from wheat germ the distribution of 5-methylcytosine is somewhat
different. The greater part of 5-methylcytosine occurs in the sequence MpPu, but a
definite, though smaller, amount of it is found in the sequence MpPy as well. Especially the sequence MpT is favored, being only slightly less frequent than TpM. It
cannot be decided by the present method, whether the II % Mp occuring in the hydrolysate of the dicytidyhc acid fraction comes from the sequence pMpCp or pMpMp;
the isolation of appreciable amount of pMpMp from acid hydrolysates of rye-germ
DNA by SHAPIRO AND CHARGAFF5 indicates that a part of the Mp from the dicytidyhc acid fraction was originally bound in the form of dideoxy-5-methylcytidylic
acid.
Another significant difference from mammalian preparations is apparent in
wheat-germ DNA. The degree of replacement of deoxycytidyhc acid occurring in the
Biochim. Biophys. dcta, 55 (I962) 953-959

958

j . DOSKOCIL, F. SORM

sequences CpPu is considerably

pyrimidine

higher in dipyrimidine

fragments

than in higher poly-

sequences.

TABLE II
DISTRIBUTION

OF 5-METHYLCYTOSINE IN THE HYDROLYSATES OF D N A WITH FORMIC ACID AND

DIPHENYLAMINE

The f r a c t i o n s of t h e h y d r o l y s a t e were d e g r a d e d w i t h p h o s p h o m o n o e s t e r a s e a n d s pl e ni c p h o s p h o diesterase.

5-Melkylcytosine as a percentage o] the sum cytosine + 5"methylcytosine
Composition oI maior
component in the ]raction

Call-thymus

In nucIeoside
3"-phosphates

In the whole/raction

In nucleosides

Found

Calculated

DNA

pep

--

xi

11.oo.8

pCpCp

<I

4.84-0-7

5.2

pCpTp 63.1%
p T p C p 36.9 %
TCC
36.9 %
63.1%
TTC
36.1%
63. 9 %

<I

13

5.70.7

5.2

<I

12

5.32,-0.3

5.2

<I

II

4.44-0.6

4.4

< i
<i

8
io
9

--

--

<i

io

--

--

<i

12

--

--

pCp
pCpCp

-< i

9
8

---

---

p C p T p 64. 3 %
p T p C p 35.7 %

<2

--

--

C,
Cp
C,
Cp

Rat-spleen

DNA

p Cp
pCp Cp
p C p T p 62.8 %
p T p C p 37.2 %
TCC
38.2 %
61.8 % Cp
TTC
3 6 . 1 % C,
63. 9 % Cp
Mouse-leukemic-liver

DNA

Wheat-germ DNA

pCp
p CpCp

-II

39
47

39
27

4- 2
+2

-29.0

p C p T p 49 %
pTpCp 51%

33

45

37

4-2

39.2

24

12

q-i

11.2

io

34

16

4-1

18.o

35

i6

q-i

15.o

TCC
TTC

36.80/1o C,
63.2 % Cp

33.2 % C,
66.8 ~o Cp
T e t r a n u c l e o t i d e s 27.3 % C,
7 2 . 7 % Cp

3.7

Biochim.

Biophys.

A c t a , 55 (1962) 9 5 3 - 9 5 9

DISTRIBUTION OF 5-METHYLCYTOSINE IN DNA

959

DISCUSSION

The experiments indicate that in mammalian preparations as well as in DNA from

wheat germ the highest degree of replacement of cytosine with 5-rnethylcytosine occurs in the sequence CpPu. The purine moiety of this sequence cannot be identified
b y the present method. Since in the hydrolysate of calf-thymus and wheat-germ DNA
with DNAase I, much of the dinucleotide pMpG but no pMpA was found, although
the dinudeotide pCpA was abundant 4, it is very probable that the purine nucleotide
attached 1:o the phosphorus of Mp is deoxyguanylic acid and not deoxyadenylic acid.
SHAPIRO AND CHARGAFF5 found a similar hypothesis to be consistent with the peculiar distribution of 5-methylcytosine in fractions of acid hydrolysate of rye-germ DNA.
F r o m our results, together with the observation of SINSHEIMER4, it follows that this
explanation is correct and that in mammalian DNA's the methylcytosine occurs exclusively in the sequence MpG. In DNA from wheat germ a similar location of 5-methylcytosiue is also the most frequent one; but, besides that, a fairly high amount of
deoxy-5-nlethylcytidylic acid is found in the sequence MpT and a small, but definite,
quantity of it occurs in MpC or MpM.
In spite of the apparent differences the sequential arrangement of deoxy-5methylcyfidylic acid seems to be determined b y a common principle in mammalian
as well as in wheat-germ DNA. Gradual differences in the probability of replacement
of cytosine with 5-methylcytosine are supposed to be characteristic for different sequences of the type CpX, the exchange taking place most probably in the sequence
CpG, with somewhat smal]er probability in CpT, and only rarely in the sequences
CpM, CpC and CpA. The actual degree of replacement in these sequences obviously
depends on the overall content of 5-rnethylcytosine in DNA. In mammalian samples
with low overall content of 5-methylcytosine only the most probable type of substitution in the sequence CpG takes place. In wheat-germ DNA, containing relatively
high percentage of 5-methylcytosine, other less probable types of substitution become
apparent, namely in the sequence CpT and, to a very small extent, also in CpC, CpM
and possibly also inCpA.
The limited experimental material with DNA from mouse leukemic liver seems
to indicate that the location of 5-methylcytosine in DI~A from neoplastic tissue follows the same general principles as in normal mammalian DNA. Further analysis
will be required before this question can be unequivocally answered.
REFERENCES
x G. R. WYATT, Biochem. J., 48 (1951) 584 .

2 G. BRAWERMAN AND E. CHARGAFF, J. Am. Chem. Soc., 73 (1951) 4052.

3 E. CHARGAFF, C. F. CRAMPTON AND R. LIPSHITZ, Nature, 172 (1953) 289.

* R. L. SINSHEIMER,J. Biol. Chem., 2I 5 (1955) 579.

H. S. SHAPIRO AND E. CHARGAFF, Biochim. Biophys. Acta, 39 (196o) 68.

s K. BURTON AND G. B. PETERSEN, Biochem. J., 75 (196o) 17.
* E. R. M. KAY, N. S. SIMMONS AND A. L. I)OUNCE, J. Am. Chem. Soc., 74

(1952) 17248 M. SCHWAI~DERANDR. SIGNER,Helv. Chim. Acta, 33 (195o) 1521.

R. LIPSHITZAND E. CHARGAFF,Bioehim. Biophys. Acta, I9 (I956) 256.
10 I~. IV[.DAVIDSONANDW. H. FISHMAN,J. Biol. Chem., 234 (1959) 526.
xl R. J. HILMOE,J. Biol. Chem., 235 (196o) 2117.
1~ F. FELIX, J. L. POTTERAND M. LASKOWSKI,J. Biol. Chem., 235 (196o) 115o.
18 E. VISCHE:R AND E. CHARGAFF, J. Biol. Chem., 176 (1948) 715.
1, G. R. WYATT, in E. CHARGAFFAND J. N. DAVlDSON,The Nucleic Acids, Vol. I, Academic Press
Inc., Ne~ York, 1955, p. 262.
1~ K. BURTON, Bioehem. J., 77 (196o) 547.
16 j. DOSKO~:ILANDF. ~ORM,Coll. Czeehoslov. Chem. Communs., 26 (1961) 2739.
Biochim. Biophys. Acta, 55 (1962) 953-959