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Methylation of DNA in developing sea urchin embryos
J. Mol. Biol.
(1968) 36, 195-208
Methylation
of DNA
in Developing
Sea Urchin
Embryos
P. GRIPPO, M. IACCARINO, E. PARISI AND E. SCARANO-~ International Laboratory of Geneticsand Biophysics, Naples and Chair of Molecular Biology, University of Palermo, Italy (Received 8 April
1968, and in revisedform 7 June 1968)
The methylation of DNA in developing sea urchin embryos has been studied. 5-methylcytosine is the only “minor” base found in DNA of sea urchin embryos at all stages of the early embryonic development. The occurrence of other methylated bases up to a level of l/20 of 5-methyloytosine can be excluded. The methyl group of methionine is transferred onto specific DNA cytosines. The distribution of 5-methylcytosine on DNA is non-random: about 60% of total 5methyloytosine was found in the monopyrimidine fraction of the DNA pyrimidine isostichs. In addition 90% of 5methylcytosine wasfound in the dinucleotide (CG) separated from a DNase digest. The data suggest that DNA 5-methylcytosine is synthesized at the polymer level in developing sea urchin embryos as it is known to occur in other organisms. Experiments which indicate an active transport for z-methionine and n-serine by developing sea urchin embryos are also reported.
1. Introduction The non-random distribution of 5-methylcytosine in the DNA’s from several organisms was demonstrated by Sinsheimer (1954), Shapiro & Chargaff (1960), Spencer & Chargaff (1963), DoskoEil & Sormova (1965a). The specific distribution of 5-methylcytosine on DNA cannot be explained by the DNA polymerase reaction mechanism, since the enzyme does not distinguish between dCTP and CH,-dCTP (Bessmanet al., 1958). Bacterial DNA methylating enzymes have been found which transfer in vitro the methyl group of S-adenosyl-methionine to bases contained in specific nucleotide sequencesof DNA (Gold, Hurwitz & Anders, 1963a,b; Gold & Hurwitz, 1964a,b; Fujimoto, Srinivasan & Borek, 1965). The enzymes do not methylate homologous DNA but do methylate DNA from other organismsand methyl-poor DNA from the same organism. Recently Sheid, Srinivasan & Borek (1967, 1968), and Burdon, Martin & La1 (1967) have described DNA methylating enzymes from mammalian tissues, The methylation of specific oytosines to 5-methylcytosines at the polymer level by specific enzymes accounts for the non-random distribution of DNA B-methylcytosines. Nevertheless, the biological meaning of DNA methylation is not clear. A possiblerole of DNA methylation in cell differentiation has been discussed(Scarano 1967f; Scarano, Iaccarino, Grippo & Parisi, 1967; Scarano & Augusti-Tocco, 1967). In a preliminary communication Scarano, Iaccarino, Grippo & Winckelmans (1965) reported on the methylation of DNA in developing sea urchin embryos. t Requests for reprints should Biophysics, Naples, Italy. $ Symposium lecture Regulation Interlaken, Sept. 1967.
be sent to me at the International of Protein
Sycnthis 195
in. Embryos,
Laboratory
8th Int.
of Genetics Cong.
Embryology,
and
196
P. GRIPPO
ET
AL.
This paper reports experiments on methionine uptake and on methylation of DNA in developing sea urchin embryos. The experiments on DNA methylation demonstrate that (1) 5-methylcytosine is the only minor base present in DNA of developing sea urchin embryos; (2) its methyl group originates from methionine; (3) the in vivo synthesized 5-methyloytosine is non-randomly distributed in DNA; (4) methylation of DNA occurs at all stagesof the early embryonic development.
2. Materials
and Methods
l-Methyladenine was purchased from Sigma Chemical Corp. (St. Louis, MO.); $-methyladenine, 6-dimethyladenine and 1-methylguanine were purchased from Fluka (Buchs, Switzerland) ; 5-methyladenine and 5methylcytosine were purchased from Calbiochem (Los Angeles, Calif.). We thank Dr G. H. Hitchings (Wellcome Research Laboratory, Tuckahoe, N.Y.) for samples of 2-methylguanine, 2-dimethylguanine, 1-methylguanine, 2-methyladenine, 6-methyladenine, and 6-dimethyladenine. 8-Methyladenine was synthesized according to Koppel & Robins (1958). The compound behaved as a pure substance on paper chromatography in the solvents 1 to 6 (see below), and its ultraviolet absorption spectrum was identical to that described by Koppel. Aminopterine was purchased from L. Light & Co. Ltd. (Colnbrook, Bucks., England). Highly polymerized DNA from calf thymus was obtained from Sigma Chemical Corp., (St. Louis, MO.). n-[methyZ-14C]methionine was obtained from the Radiochemical Centre (Amersham, England); the different samples used, had a specific activity in the range between 5 me/mmole and 29.5 ma/m-mole. n-[methyl-“Hlmethionine (110 ma/m-mole) and L-[14C]serine (120 ma/m-mole) were purchased from New England Nuclear Corp. (Boston, Mass.). Sterile 32P-labelled sodium phosphate was purchased from the Radiochemical Centre (Amersham, England), L-[methyZ-2H3]methionine (99% deuterium) was synthesized according to Rachele, Kuchinskas, Kratzer & du Vigneaud (1955) by New England Nuclear Corp. (575 Albany St., Boston, Mass.). Crystalline pancreatic deoxyribonuclease, snake venom phosphodiesterase, and Escherichia coli alkaline phosphatase were obtained from Worthington Biochemical Corp., (Freehold, N. J.). The phosphodiesterase was free of 5’ nucleotidase activity under the conditions of our experiments. Russel’s viper venom was obtained from the Haffkine Institute (Parel, Bombay). Charcoal was purchased from Barnebey-Cheney (Columbus 19, Ohio). Dimethyl POPOP, PPO, [14C]toluene, E3H]toluene, tritiated water, hydroxide of hyamine 10X and Cab-0-Sil were purchased from Packard Instrument Corp. (La Grange, Ill.). All other chemicals were reagent grade of the highest purity available. (a)
Sea
urchin
Paracentrotus livid~ and Sph.aerechinue granularis, were obtained from the Zoological Station, Naples. Eggs were collected by gently stirring the ovaries in filtered sea water, filtered through bolting silk, and washed twice with sea water by sedimentation. Fertilization was performed with the minimum amount of freshly diluted sperm which caused 100% fertilization in a control trial. After formation of the fertilization membrane, the fertilized eggs were washed twice with sea water and were kept at 18% with continuous gentle stirring at a concentration of 5000 eggs/ml. In most of the experiments the embryos were cultured in sea water containing 5 mg penicillin G/l. and 10 mg streptomycin sulphate/l. 32P-labelled bacteria were used to exclude possible bacterial contamination of the sea urchin embryos. P. lividzls embryos at the gastrula stage and S. granularis embryos at the swimming blastula stage were mixed with 32P-labelled bacteria. About 95% of the 32P was washed away during the centrifugation of the embryos. (b) an
Embryos were International
collected Centrifuge
Extraction
at 4°C by centrifugation PR2; were washed
of DNA at 800 rev./mm in the no. 259 head of twice with a solution containing 0.55
DNA M-NaCl above, sodium
METHYLATION
in 0.1 M-EDTA were extracted lauryl sulphate. (c)
IN
SEA
URCHIN
EMBRYOS
197
at pH 8. 10 to 15 ml. of embryos packed by centrifugation as with 50 ml. of 0.55 M-NaCl in O-1 M-EDTA, pH 8, and 4 ml. of 25% DNA was then purified according to the method of Marmur (1961).
Extraction
of DNA
nucleotides
and
DNA
nucleosides
About 10 ml. of packed embryos were treated twice for 30 min at 37’C with a mixture of absolute ethanol and ether (3:l v/v). The residue from the extraction was dried under vacuum over KOH. The dry powder was dissolved in 10 ml. of O-3 N-NaOH and kept at 37°C for 18 hr to hydrolyse the RNA. The NaOH hydrolysate was chilled at 0°C and acidtied with perchloric acid to a final concentration of 5%. The precipitate was collected by centrifugation, washed 4 times with cold 1 y. perchloric acid, and dissolved in water by addition of NaOH until pH 7 was obtained. Deoxynucleotides were obtained by enzymic hydrolysis with DNase and phosphodiesterase. When deoxynucleosides were prepared, snake venom hydrolysis followed DNase hydrolysis (see below). (d) (i)
DNA was dissolved 7 with 0.1 m-NaOH. 10 pg/ml., respectively. (ii)
hydrolysis
in water at a concentration of 20 mg/ml. and the pH MgSO* and DNase were added to a final concentration The mixture was incubated for 5 hr at 37°C.
was adjusted of 2 mM
to and
to pH 9.6 with 0.1 M-NaGH and glycine buffer, pH of 0.02 M. 100 pg/ml. of snake venom phosphodiesterase at 37°C for 5 hr.
9.6,
Phosphodiesterase
The DNase digest was added to a final were added and the (iii)
Enzymic
DNase
Snake
was adjusted concentration mixture kept
venom
Oligonucleotides at a concentration of about 2 mg/ml. in 0.02 na-glycine buffer, MgSO,, pH 9.6, were hydrolysed, at 37°C for 5 hr, by addition of 100 pg of snake ml. (iv)
2 mMvenom/
Phosphatase
Nucleotides phosphatase/ml.
at a concentration of about 2 mg/ml. were at 37OC for 4 hr. The pH was continuously (e)
Hydrolysis
to purines
and
hydrolysed adjusted
with 20 pg of alkaline to 9 with 0.1 M-NaOH.
pyrimidines
DNA, deoxyribonuoleotides, and deoxyribonucleosides were hydrolysed with 70% perchloric acid, according to Bendich (1955). The recovery after acid hydrolysis of standard methylated bases was measured. Recovery of 90% or more of &methylcytosine, 2methyladenine, 6-methyladenine, 6-dimethyladenine, 7-methyladenine, %methyladenine, I-methylguanine, 2-methylguanine, 2-dimethylguanine was found. (f)
Degradation
The methyl group of thymine Sprinson (1954). The crystallized its radioactivity measured. The standard of [l*C]toluene. (g)
of the methyl
group
Preparation
of DNA
isostichs
DNA was hydrolysed with 3% diphenylamine in formic The isostiohs were separated according to chain length using a linear gradient of triethylammonium bicarbonate. each isostich fraction by sublimation under vacuum. (h)
Preparation
of thymine
was converted to iodoform as described by Elwin & iodoform was dissolved in the scintillator solution and efficiency of counting was determined with an internal
of d&ucleot&
fractions
acid (Burton on a column The salt
from
Hydrolysis of DNA from sea urchin embryos with pancreatic dinucleotide fractions were performed according to methods Tocco, Carestia, Grippo, Parisi & Scarano, 1968).
a DNa.se
& Petersen, 1960). of DEAE cellulose was removed from digest
DNase, and separation of already published (Augusti-
198
ET
P. GRIPPO (i)
Desalting
on
AL.
charcoal columne (Cohn, 1963 PersMzal
cornmunicatiom)
Charcoal WM suspended in 0.2 N-HCl and washed with water to neutrality. It was then stirred in isopropanol-water-toluene (50: 50: 1: by vol.) for 30 min. The charcoal was sedimented and the supernatant was discarded. The reversibly adsorbed toluene was eluted from the charcoal by washing it on a column with 95% ethanol-water-conon NH,OH (2 :2:1 by vol.) until no material with optical density absorbance at 260 rnp appeared in the effluent. The sample of nucleotides or nucleosides, about 100 optical density units at 260 mp, was brought to pH 2 and adsorbed on a column of toluenetreated charcoal (0.6 cm x 2 cm). The column was then washed with water to neutrality and the sample eluted with 95% ethanol-water-concn NH,OH (2: 2: 1 by vol.). The yield of nucleotides and nucleosides was 90 to 100°&
(j) Paper chromatography
and thin-layer
chromatography
Sheets of 3 MM Whatman paper were washed with 200 ml. of 2 N-acetic acid and with glass distilled water in a ohromatographic tank. Thin-layer chromatographic separations were performed on micro-crystalline cellulose (Avicel) or DEAE-cellulose according to Grippo, Iaccarino, Rossi & Scarano (1965). The nucleic acid derivatives were detected by Mineralight ultraviolet lamp Products Inc. (San Gabriel, Calif.). The spots were eluted by water or dilute acid. The Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent
following chromatographic solvents were used : 1: isopropanol-HCl-water (65:16.7:18.3, by vol. (Wyatt, 1951)). 2: n-butanol-water (86:14, by vol. (Markham & Smith, 1949)). 3 : isobutyric acid-water-25% NHBOH (400 :208 :0*4, by vol. (Lijfgren, 1952)). 4: isopropanol-water-concn NH,OH (85: 15:1*3, by vol. (Hershey, Dixon & Chase, 1953)). 5: n-butanol-water-formic acid (77 :13:10, by vol. (Markham & Smith, 1949)). 6: saturated (NH&SO,-0.5M-sodium acetate-isoprop?fiol (80:18:2, by vol. (Markham & Smith, 1952)). 7: 95% ethanol-lM-ammonium acetate saturated with tetraborate (70: 30, by vol. (Plesner, 1955)). 8: n-butanol-water (86: 14, by vol.), with 5% by vol., of eoncn NH&OH on the bottom of chromatographic tank (Markham & Smith, 1949). 9: 5% aqueous ammonium bicarbonate (Broom, Townsend, Jones & Robins, 1964). 10: n-butanol-water-concn NH,OH (86: 13: 1, by vol. (Wacker & Ebert, 1959)). 11: isopropanol-5% aqueous ammonium sulphate (l:lQ, by vol. (Brookes & Lawley, 1960)). 12: ethanol-water (7 :3, by vol. (Broom et al., 1964)). 13: N-N-dimethylphormamide-isopropanol-concn NH,OH (25: 65: 10, by vol. (Broom et al., 1964)). 14: n-butanol-ethanol-water (50:15:35, by vol. (Wacker & Ebert, 1959)). 15: methanol-ooncn HCl-water (7: 2: 1, by vol. (Brookes & Lawley, 1960) ). 16:
n-butanol Lawley,
saturated 1960)).
with
(k) Electrophoretic Whatman 3 MM 4°C.
separations paper. The
High-voltage
NH40H
Spec6rophotometri.c
acids and their Quantitative
(100:
1, by
vol.
(Brookes
&
e~ectr~horesis
were performed according electrophoresis was performed (1)
The U.V. spectra of nucleic recording spectrophotometer. DU spectrophotometer.
water-concn
to Smith (1955) on a refrigerated
on plate
washed kept at
measurements
derivatives were determinations
taken were
with a Beckman DK2A made with a Beckman
DNA
METHYLATION
IN
SEA
(m) Radioactitity
URCHIN
EMBRYOS
199
mecwurerrmts
Measurements of radioactivity were performed with a Packard Tri-Curb 314-&X liquid-scintillation counting system. The following scintillators were used: (a) 1200 ml. of Cab-O-W were mixed with 700 ml. of 0.01% dimethyl-POPOP, 0.4% PPO in toluene, and 300 ml. of absolute methanol ; (b) 40 g of Cab-0-Sil were mixed with 500 ml. of dioxane and 500 ml. of xylene containing 80 g of naphthalene, 5 g of PPO and 05 g of dimethyl-POPOP. 20 ml. of this mixture can hold up to 0.2 ml. of aqueous solution. Tritium in this scintihator has an efficiency higher than in scintillator (a). Quenching corrections were made by internal standardization with standard labelled compounds. Radioactivity on paper or on thin layer ohromatograms was measured by adding the paper or the cellulose powder to a vial with 20 ml. of scintillator (a). 3zP was measured with a Nuclear-Chicago low background flow counter. (n)
Mass epectrometry
measurements
Mass speotrometry analysis was performed with a Consolidated Electrodynamics Co. double focusing mass spectrometer. Samples of about 60 pg were introduced asdry powder in the ionization chamber of the instrument. The following operating conditions were used : energy of ionizing electron beam 25 ev; temperature of the ionization chamber 70 to 120°c.
3. Results (a) Uptake of methionine Knowledge of the rate of uptake of methionine by embryos at different stages of development is a necessaryprerequisite to studies of in vivo methylation. Experiments on X. granularis and P. lividus embryos have revealed striking changesof the rate of uptake of r,-methionine during development. Figure 1 shows the uptake of 4.85 PM-methionine as a function of time by sea urchin embryos P. lividus at the 16 cells stage and at the gastrula stage. The methionine which disappears from the incubation medium is recovered in the embryos.
Time
(hr)
Fro. 1. Time course-of uptake of z-methionine by P. 1ividu.s embryos. Ordhate: radioa&ivity taken up by the embryos as a percentage of the total radioactivity. Absci88a: time in hours. --O--O-, Embryos at the gastrula stage; -A-A-, embryos at the 16.cells stage; --m-f-J--, embryos at the gastrula stage + 1 rnn-.z-serine; -e--a-, embryos at the gastrula stage + CrnX-L-serine; --w-m--, embryos at the gastrula stage + 4 mm-L-glycine. In all samples 4.85 pM-L-[~eth?/Z-‘*C]methionine (13.9 me/mole) was present. At the indicated times radioactivity was measured on 100 ~1. samples of sea water.
P. GRIPPO
200
ET
I
AL. I
I
60
90
too-
g
&O-
s0 “a 2
60-
?! ‘E 2
40-
ii 20-
0
30 Time
(mid
FIG. 2. Inhibition by n-serine of methionine uptake. S. granularis embryos at hatching blastula stage cultured in the presence of 28.2 PM-L-[methyl-‘W] methionine (5.45 ma/m-mole). Ordinate and abscissa as in Fig. 1. --O--O-, Embryos + [r*C]methionine; -A-A-, embryos + [Wlmethionine + 20 pML-serine; -I-+-~---, embryos + [W]methionine + 0.1 ma6--r;-serine; -•--•---, embryos + [14C]methionine + 1 mx-L-serine. At the indiosted times radioactivity was measured on 100 ~1. samples of filtered sea. water.
60 Time
(mid
FIG. 3. Time-course of uptake of L-serine and its inhibition by methionine. S. granularis embryos at the hatching blastula stage cultured in the presence of 0.33 ~M-L-[W] serine. Ordinate end ebscisse es in Fig. 1. -O-O--,Embryos + [%!]serine;--n-n-, embryos + [l%]serine + 1 mM-L-methionine. At the indicated times radioactivity was measured on 250 ~1. samples.
DNA
METHYLATION
IN
SEA
URCHIN
EMBRYOS
201
The uptake of methionine is inhibited to the same extent by 1 mM- and 4 mllr-serine. 4 mM-glycine causes 100% inhibition of the methionine uptake. Thymidine and 5.methyldeoxycytidine do not inhibit the uptake of methionine. Figure 2 shows the uptake of 28.2 +?f-methionine as a function of time by sea urchin embryos S. granularis at the swimming blastula stage. The uptake is inhibited by the of L-serine. presence of L-serine and the inhibition is proportional to the concentration The uptake of 0.33 PM-serine by S. granularis embryos at the swimming blastula stage is shown in Figure 3. Serine uptake is also inhibited by 1 mM-methionine. A similar curve of uptake has been obtained for P. lividus embryos.
P 3 Stoge of development
4. Rate of methionine uptake at different stages of development. S. grawularis embryos &t the indicated stages cultured in the presence of 19.6 pM-L-[methyl-14C]methionine (10.2 me/mmole) per 15 min. FIQ.
In Figure 4 the rate of methionine uptake by S. granularis embryos at several stages of development is reported. Unfertilized eggs under these conditions do not take up methionine even after a previous stirring for 16 hours in seawater. A sudden change in permeability to methionine occurs immediately after fertilization. The uptake becomeseasily measurable and increaseswith development. At the gastrula stage 567” of the methionine is taken up by the embryos in a 15-minute period in the incubation medium. In the experiments reported in Figures 1, 2, 3 and 4 almost all the radioactivity disappearing from the seawater was recovered in the embryos. A very small fraction of the methyl group of methionine was oxidized to carbon dioxide during incubation as demonstrated by the following experiment. 100 ml. of a suspensionof P. lividus embryos at gastrula stage were incubated fox 5.5 hours with 1.2 pmole L-[methyl-14C] methionine (13.9 me/m-mole). During the incubation the CO, produced by the embryos was collected as BaC03. Lessthan O-1y0 of the total radioactivity was found in BaCO,. (b) DNA
methylation
Incubation times of five hours or longer were chosento minimize the effects of the different rates both of the uptake of methionine and of the synthesis of DNA during the early embryonic development of the sea urchin (Elson, Gustafson & Chargaff, 1954). The methylation of DNA, which occurs during the chosenperiods of incubation with labelled L-methionine is of such an extent, at all stages of development, that it 14
202
P.
GRIPPO
ET
AL.
is possible to detect it even at the very early stages when there is a small uptake oi methionine and the total amount of DNA synthesized is small. (i) Search for DNA methylated bases 2.2 litres of a suspension of P. lividus embryos at the early pluteus stage were incubated for 12 hours with 25 FU of L-[methyL1*C]methionine. At the end of the incubation the embryos were collected, 7.5 mg of carrier DNA from calf thymus were added, and DNA was extracted. The purified DNA was hydrolysed with DNase and phosphodiesterase. The deoxymononucleotides obtained were desalted on charcoal and separated by high-voltage ionophoresis on paper. All the radioactivity was found in the four ultraviolet absorbing bands (Fig. 5). The bands were eluted and each
6
FIG.
5. Eleotrophorstio
212 6 lo 14 IB 22 26 cm from origin Start separation of the 4 deoxynuoleotides.
For
details
see text.
deoxynucleotide was chromatographed on paper with solvent 7 to purify it from possible contaminating 5’-ribonucleotides. Almost 100% of the radioactivity was recovered in the deoxynucleotide spots. Each deoxynucleotide was identified by its ultraviolet spectra. 2-l pmoles of dCMP, 3.9 pmoles of dTMP, 3.9 pmoles of dAMP and 2.4 pmoles of dGMP were recovered and the total radioactivity in each spot was 17,600; 43,600; 73,800 and 31,600 ctsjmin, respectively. The deoxynucleotides were hydrolysed with concentrated perchloric acid and the bases obtained were analysed in several chromatographic solvents with methylated bases as standards. All the radioactivity was recovered in the bases. This excludes the possibility that the radioactivity found in the nucleotides was in the sugar moiety. The bases were analysed as follows. Cytosine. Samples of the hydrolysed dCMP were analysed by uni-dimensional chromatography in solvents 1, 2, 6, 8 and by two-dimensional chromatography in solvents 1 and 2. All the radioactivity was found in the Et-methylcytosine spot. No radioactivity was detected in the cytosine and in carrier 6.methyladenine spots. Carrier 6.methyladenine was added, because in the electrophoretic separation 6methyl&WI? does not separate satisfactorily from dCMP. Thymine. Samples of the hydrolysed dTMP were chromatographed uni-dimensionally with solvents 1, 2, 3, 4, 5, 6, 8 and two-dimensionally (first direction: solvent 2; second direction: solvent 1). In every case all the radioactivity was found in the thymine spot. A sample of thymine containing 16,500 disint’egrations/min of l*C was hydrolysed by the method of Elwin & Sprinson (1954), 100°J of the radioactivity was recovered
DNA
METHYLATION
IN
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URCHIN
EMBRYOS
203
in iodoform, demonstrating that all the radioactivity of thymine was in the methyl group. Adenine. Samples of the hydrolysed dAMP were chromatographed with solvents 1,2,3,4,5,6 and 8. No radioaotivity was found in l-methyladenine, 2-methyladenine, 6-methyladenine, 6dimethyladenine, 7-methyladenine, and 8-methyladenine, added as carriers and separated from adenine in the indicated chromatographic solvents. In all the solvents, the radioactivity was found always in the adenine spot. Furthermore, the adenine sample was checked by chromatography with solvents 9,10, 11,12,13,14,15 and 16 under oonditions in which 10% of the total radioactivity should have been detected if separated from the main radioactive spot. In all the solvents one radioactive spot was found in coincidence with adenine. Finally, the purified adenine was crystallized several times from water and no change of its specific activity was found. Quanine. Samples of hydrolysed dGMP were chromatographed with solvents 1, 4 and 8. No methyl bases were found. Identical results for all four bases were obtained in two similar experiments performed with P. lividus embryos at the prism stage. (ii) Origin. of the methy.! group of DNA 5-methylcytosine (a) Experiments with doubly-labelledmethionine The origin of the methyl group of 5-methylcytosine and of the radioactivity in the thymine, adenine and guanine sampleswere studied by incubating the embryos in the presence of L-[methyl-14C]methionine and L-[methyl-3H]methionine. Direct transfer of the methyl group of methionine is suggested,when a basehas a ratio 3H/14Cequal to that of the added methionine. A ratio lower than the initial one would indicate oxidation of the methyl group of methionine and incorporation by the reaction of the C(1) pool metabolism, either in the methyl group of thymine or in the purine ring of adenine and guanine. TABLE
1
Ratio 14CJ3Hin basesof seaurchin embryo DNA Exp. 1 2 3 4
S-Methylcytosine 2.9 2.8 3.1 26
Thymine
Adenine
1.8 1.7 1.5 1.9
l-7 1.4 1.3 l-3
Guanine
0.4 0.5 0.6 0.6
Experiment 1. P. lividus embryos et the prism stage incubated for 13 hr with 25 po ofn-[m&yZrW]methionine and 80 PC of n-[~ethyPH]methionine. In experiments 2,3, end 4 8. granzcZa& embryos at the stages 8 to 16 cells, hatching bl&,ulee, end blastulae with mesenchyme respectively, were incub&sd for 14 hr with 50 po of r,-[w&hyZ-l4C] methionme end 50 pc of h-[methyZ-3H]methioaine. The specific activity of L-[methyZ-l4C]methionine end of r.-[6-&hyZ-3H]methionine was 295 me/m-mole end 100 me/m-mole, respectively. In all experiments the sH/W ratios were normuheed to the initial value in methionine t&ken equal to 3.
Table 1 lists the ratios 3H/14C of the DNA bases.The data show that the in&,] 3HI 1% ratio was found only in 5-methylcytosine. About one-third of the tritium was
204
P.
GRIPPO
ET
AL.
lr
HO 4
“L
I I 126 127 128 129
j
m/e
(a)
Fla. 6. Mass spectra (a) 5-methylcytosine from DNA incubated with [nzethyZ-aHs]methionine.
I
.
120 121 122 123 124 I!2! 126 127 I28 129 (b)
of 5methylcytosine from 124 m/e to 130 m/e. of P. lividus. (b) 5-methylcytosine from DNA For details see text.
of P. Zividua
lost in the case of thymine, and more than one-third in the case of the purines. (However, a definite conclusion cannot be reached on the basis of these experiments because of the possible occurrence of isotopic effects.) (b) Experiments
with L-[methyL2H3]methionine
To have direct evidence of the transfer of the methyl group of methionine to DNA cytosine, L-[methyL2H,]methionine was used in the following experiment. Four litres of a suspension of P. 1ividu-s embryos at the blastula stage were incubated with 20 mg
SO-
I% 0%
of total S-methylcytosine of total pyrimidine
FIN. 7. Percentage distribution of 5-methylcytosine and of total pyrimidine deoxynucleosides in DNA pyrimidine isostichs of 8. gralzulccris embryos. 8. granulcwis embryos at blastnla stage cultured in the presence of 1 mo of ~-[m&yZ-r~C] methionine (13.9 me/m-mole) for 7 hr. The embryo DNA was extracted and hydrolysed with 3% diphenylamine in formic acid. The pyrimidine isostiohs were separated and each isostich fraction was hydrolysed to deoxynucleosides. 5-Methyldeoxycytidine was purified by paper cbromatography in solvents 1, 7, and 4. The spots were eluted and the radioactivity measured. The percentage of total pyrimidine deoxynucleosides in each isostich fraction was measured by U.V. spectrophotometry.
DNA
METHYLATION
IN
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URCHIN
EMBRYOS
205
of deuterated L-methionine. After five hours the embryos were collected and DNA was extracted. The DNA was hydrolysed to bases with perchloric acid and the bases were separated and purified by paper chromatography in solvents 2 and 4. The spots of cytosine and 5methylcytosine were eluted together. After elution the samples were dried and used for mass spectrometry analysis. Figure 6 shows the mass-spectra of a standard 5-methylcytosine and of a Smethylcytosine from DNA of embryos grown in the presence of labelled methionine. The presence of [5-methyl-2H,]cytosine in the DNA of sea urchin embryos grown in the presence of n-[methyL2H,]methionine demonstrates the direct transfer of the methyl group of methionine to DNA oytosine. (iii) Non-randomness of DNA methylation. The data of Figure 7 demonstrate that the methylation of DNA cytosine in. viva is non-random. In fact, 60% of the synthesized 5-methylcytosine was recovered in the monopyrimidine fraction, although only 25% of pyrimidines occur in this fraction. The total counts of 5-methyldeoxycytidine of each isostich are proportional to the amounts of the deoxynucleoside because the specific activities of the Ei-methyldeoxyoytidine of all the isostich fractions are the same. This is demonstrated by the following experiment. Embryos were incubated with L-[methyL2H,]methionine and DNA was extracted as described above. The DNA pyrimidine isostichs were prepared and from each isostich fraction 5-methylcytosine was purified and analysed by mass spectrometry. The ratio of [5-methyL2H,]cytosine over 5-methylcytosine was the same in all fractions.
iooI 0
% of total % of total
rl
fl
dinucleotide dinucleotides
S-methylcytosine
SO-
0,
r
(CT) (CCKCC)(CA)(AA)
n (AC)
l-l n (AT) (CT)
(TT)
Fra. 8. Percentage distribution of 5-methylcytosine and of the 4 major bases in the dinucleotides from a DNase digest of 6’. granularis embryo DNA. S. granularis embryos at the blast& stage incubated for 7 hr with 1 mc of n-[methyVW]methionine (1 I.7 me/m-mole). DNA and dinucleotides were prepared and separated as described in Materials and Methods. Each dinucleotide was hydrolysed with snake venom, and deoxynucleosides were separated by paper chromatography in Solvent 1. 5-methyldeoxycytidine spots were eluted and the radioactivity measured. The percentage of each dinucleotide was measured by u.v. spectrophotometry. Dinuoleotide (GG) was not measurable.
Moreover, the non-random distribution of 5-methylcytosine was demonstrated by the analysis of dinucleotide fractions from the DNase digest of labelled DNA from 1.9. granularis embryos grown in the presence of L-[methyl-14C]methionine. J?igure 8 shows the distribution of 5-methylcytosine in the DNA dinucleotide fraction. (iv) ikfethylation of DNA during the early embryonic development The data of Table 2 demonstrate that methylation of DNA occurs at all stagesof development in P. lividus. No conclusion about quantitative differences of methylation at the different stagescan be drawn becauseno information was collected on the
P.
206
GRIPPO
ET
AL.
pool of methionine and on the rate of DNA synthesis during the period of incubation with labelled methionine. Similar results on methylation of DNA at all stages of development were reported previously (Scarano et al., 1965) for S. granularis embryos.
TABLE
2
S~eci$c activity of DNA 5-methylcytosilzefrom sea urchin embryos(P. Iividus) at different stagesof development Exp. 1 2 3 4 5 6
Stage
2 disintegrations/min
128 cells hatching hatching blastulae blast&e prisms
blastulae blastulae with mesenchyme with mesenohyme
14C X 10-6/~mole 6 2 3 6 9 1
At the indicated stages embryos were incubated for 5 hr with 150 PC/I. of n-[methyl-i4C]methionine (29 me/m-mole). At the end of a 5hr period the embryos were collected. DNA deoxynuoleosides were purified and the specific activity determined on deoxycytodine + 5methyldeoxycytidine. The deoxynucleosides were then hydrolysed to bases and analysed: all the radioactivity of the original deoxycytidine + 5methyldeoxycytidine sample was in 5-methylcytosine, while cytosine was not radioactive. The speciflo radioactivity of 5-methylcytosine was calculated from the known base composition of P. Zividua sperm DNA (Chargaff, Lipshitz & Green, 1952).
4. Discussion The experiments on the uptake of ammo acids reported in the present paper indicate the existence of an active transport mechanismfor L-methionme and rJ-serine in developing sea urchin embryos P. lividus and S. granularis. The uptake of Lmethionine is inhibited by the presenceof L-serine and vice zlersa,but is not affected by thymidine and 5-methyldeoxycytidine. These facts are in agreement with the tidings of Mitchison & Cummins (1966) on the active transport mechanismfor valine in developing seaurchin embryos. Unfertilized seaurchin eggsdo not take up methionine from sea water under our experimental conditions. The uptake begins soon after fertilization. The rate of the uptake increases, at least until the gastrula stage. The labelling of the methyl group of thymine and of the purine ring of adenine and guanine, which arises from the methyl group of L-methionine, indicates that a fraction of the methionine methyl group is oxidized to one carbon pool intermediates. By this pathway the C(2) and C(8) of the purine ring and the methyl group of thymine are labelled. Thus, the only valid criterion for DNA methylation is the isolation of a DNA methyl base labelled in the methyl group. The label of the methyl group of L-methionine does not enter the pyrimidine ring of cytosine, LLmethylcytosine and thymine. 5-Methylcytosine is the only minor methylated base in the DNA of developing sea urchin embryos. On the assumption that all DNA minor methyl baseswould be labelled with a specific activity equal to that of 5-methylcytosine, our experiments can exclude the presenceof other methylated basesup to a level of l/20 of B-methylcyto-
DNA
METHYLATION
IN
SEA
URCHIN
EMBRYOS
207
sine. However, we are investigating further the problem of the labelling of the methyl group of thymine on the assumption that a small fraction of the labelled thymine arises from DNA B-methylcytosine at the polymer level (Scarano, et al., 1967). It is interesting to notice that in several bacterial strains (Dosko&il & Sormova, 19653)and in the blue-green alga Plectonema boryanum (Kaye, Salomon & Fridlender, 1967) 6-methyladenine was found to occur in DNA in addition to 5-methylcytosine, while in higher organisms 5-methylcytosine is the only DNA minor base found. The experiments with doubly-labelled methionine and with [2H,]methyl-labelled methionine demonstrate the dire& transfer of the methyl group of methionine onto DNA cytosine. It is likely that methylation of DNA oytosine in seaurchin embryos occurs, as in all the other organisms,via X-adenosylmethionine. The non-random distribution of 5-methylcytosine in the sea urchin embryo DNA suggeststhat the methylation of DNA occurs at the polymer level as it is known to occur in the other organismsin which it has been studied (Gold et al., 1963a, b; Gold & Hurwitz, 1964a, b; Fujimoto et al., 1965). Bacterial contamination of the embryos cannot affect the results described in this paper becauseof the following facts. (1) 6-Methyladenine was never found in the embryo DNA; yet 6-methyladenine is a component of bacterial DNA (Dunn & Smith, 1958). (2) Most of the experiments were done in the presenceof penicillin G and streptomycin sulphate. (3) Unfertilized eggswere cultured for five hours in sea water containing labelled methionine; no radioactivity was taken up by the collected eggs, while under the same conditions sea urchin embryos take up lOOo/oof the added methionine. Further work is necessary to identify possible quantitative differences in DNA methylation and/or possible changes in the distribution of 5-methylcytosine in the DNA from embryos at different stagesof development. We want to emphasizethe possibleusefulnessof massspectrometry in determining quantitatively minor basesin nucleic acid fragments. We are attempting to develop methods for determining by massspectrometry the distribution of 5-methylcytosine in pyrimidine isostichs and in DNase digest. We thank Professor M. Ageno of the Istituto Superiore di &nit&, Rome, for his interest in this work and for advice in mass spectrometry analysis, as well as Mr A. Rosati, Mr M. Flamini and Mr A. Martinangelo for their technical assistance in the measurements. We are grateful to Mr A. Granieri and M.r G. Forlani for skilful technical assistance. This work was supported by Euratom-C.N.R.-C.N.E.N. contract no. 012-61-12 BIAI, and by research Grant no. HD-0126304 from the U.S. Public Health Service.
REFERENCES Augusti-Tocco,
G.,
Carestia,
C.,
Grippo,
P.,
Parisi,
E.
& Scarana,
E.
(1968).
Biochim.
biophys. Acta, 155, 8. Bendich, A. (1955). In MeMods in. EnzymoEogy, ed. by S. P. Colowick & N. 0. Kaplan, vol. 3, p. 715. New York: Academic Press. Bessman, M. S., Lehman, I. It., Adler, J., Zimmerman, S. B., Simms, E. S. & Kornberg, A. (1958). Proc. Nat. Acad. Sci., Wash. 44, 633. Brookes, P. & Lawley, P. D. (1960). J. Chem. Sot. 1, 539. Broom, A. D., Townsend, L. B., Jones, J. W. & Robins, R. K. (1964). Biochemistry, 3, 494. Burdon, R. H., Martin, B. T. & Lal, B. M. (1967). J. Mol. Biol. 28, 357. Burton, K. & Petersen, G. B. (1960). Biochem. J. 75, 17. Chargaff, E., Lipshitz, R. & Green C. (1952). J. Biol. Chem, 195, 155. Doskosil, J. & Sormova, Z. (1965a). Biochem. BiophyB. Res. Comm. 20, 334.
208
P.
GRIPPO
ET
AL.
Dosko&l, J. & SormovB, Z. (19656). Biochim. bio&ys. Acta, 95, 513. Dunn, D. B. & Smith, J. D. (1958). Biochem. J. 68, 627. Elson, D., Gustafson, T., & Chargaff, E. (1954). J. Biol. Chem. 209, 285. Elwin, D. & Sprinson, D. B. (1954). J. BioE. Chem. 207, 467. Fujimoto, D., Srinivasan, P. R. & Borek, E. (1965). Biochemistry, 4, 2849. Gold, M. & Hurwitz, J. (1964a). J. Biol. Chem. 239, 3858. Gold, M. & Hurwitz, J. (1964b). J. Biol. Chem. 239, 3866. Gold, M., Hurwitz, J. & Anders, M. (1963a). Biochem. Biophys. Res. Comm. 11, 107. Gold, RI., Hurwitz, J. & Anders, M. (19636). PPOC. Nat. Acad. Sci., Wash. 50, 164. Grippo, P., Iaccarino, M., Rossi, M. & Scarano, E. (1965). Biochim. biophys. Acta, 95, 1. Hershey, A. D., Dixon, J. & Chase, M. (1953). J. Gen. PhysioE. 36, 777. Kaye, A. M., Salomon, R. & Fridlender, B. (1967). J. Mol. BioZ. 24, 479. Koppel, H. C. & Robins, R. K. (1958). J. Organ. Chem. 23, 1457. LSfgren, N. (1952). Acta Chem. Stand. 6, 1030. Markham, R. & Smith, J. D. (1949). Biochem. J. 45, 294. Markham, R. & Smith, J. D. (1952). Biochem. J. 52, 558. Marmur, J. (1961). J. Mol. BioZ. 3, 208. Mitohison, J. H. & Cummins, J. E. (1966). J. Cell Science, 1, 35. Plesner, P. E. (1955). Acta Chem. Stand. 9, 197. Rachele, J. R., Kuchinskas, E. S., Kratzer, F. H. & du Vigneaud (1955). J. BioZ. Chem. 215, 593. Soarano, E. & Augusti-Tocco, G. (1967). In Comprehensive Biochemistry ed. by M. Florkin & E. H. Stotz, vol. 28, p. 55. Amsterdam: Elsevier Publishing Company. Scarano, E., Iaccarino, M., Grippo, P. & Parisi, E. (1967). Proc. Nat. Acad. Sci., Wash. 57, 1394. Scarano, E., Iaccarino, M., Grippo, P. & Winckelmans, D. (1965). J. MOE. BioZ. 14, 603. Shapiro, H. S. & Chargaff, E. (1960). Biochim. biophys. Acta, 39, 68. Sheid, B., Srinivasan, P. R. & Borek, E. (1967). Fed. Proc. 26, 775. Sheid, B., Srinivasan, P. R. & Borek, E. (1968). Biochemistry, 7, 281. Sinsheimer, R. L. (1954). J. BioZ. Chem. 208, 445. Smith, J. D. (1955). In Nucleic Acids, vol. 1, p. 267. New York: Academic Press. Spencer, J. H. & Chargaff, E. (1963). Biochim. biophys. Acta, 68, 9. Wacker, A. & Ebert, M. (1959). 2. Naturf. 14b, 709. Wyatt, G. R. (1951). Biochem. J. 48, 584.
(1968) 36, 195-208
Methylation
of DNA
in Developing
Sea Urchin
Embryos
P. GRIPPO, M. IACCARINO, E. PARISI AND E. SCARANO-~ International Laboratory of Geneticsand Biophysics, Naples and Chair of Molecular Biology, University of Palermo, Italy (Received 8 April
1968, and in revisedform 7 June 1968)
The methylation of DNA in developing sea urchin embryos has been studied. 5-methylcytosine is the only “minor” base found in DNA of sea urchin embryos at all stages of the early embryonic development. The occurrence of other methylated bases up to a level of l/20 of 5-methyloytosine can be excluded. The methyl group of methionine is transferred onto specific DNA cytosines. The distribution of 5-methylcytosine on DNA is non-random: about 60% of total 5methyloytosine was found in the monopyrimidine fraction of the DNA pyrimidine isostichs. In addition 90% of 5methylcytosine wasfound in the dinucleotide (CG) separated from a DNase digest. The data suggest that DNA 5-methylcytosine is synthesized at the polymer level in developing sea urchin embryos as it is known to occur in other organisms. Experiments which indicate an active transport for z-methionine and n-serine by developing sea urchin embryos are also reported.
1. Introduction The non-random distribution of 5-methylcytosine in the DNA’s from several organisms was demonstrated by Sinsheimer (1954), Shapiro & Chargaff (1960), Spencer & Chargaff (1963), DoskoEil & Sormova (1965a). The specific distribution of 5-methylcytosine on DNA cannot be explained by the DNA polymerase reaction mechanism, since the enzyme does not distinguish between dCTP and CH,-dCTP (Bessmanet al., 1958). Bacterial DNA methylating enzymes have been found which transfer in vitro the methyl group of S-adenosyl-methionine to bases contained in specific nucleotide sequencesof DNA (Gold, Hurwitz & Anders, 1963a,b; Gold & Hurwitz, 1964a,b; Fujimoto, Srinivasan & Borek, 1965). The enzymes do not methylate homologous DNA but do methylate DNA from other organismsand methyl-poor DNA from the same organism. Recently Sheid, Srinivasan & Borek (1967, 1968), and Burdon, Martin & La1 (1967) have described DNA methylating enzymes from mammalian tissues, The methylation of specific oytosines to 5-methylcytosines at the polymer level by specific enzymes accounts for the non-random distribution of DNA B-methylcytosines. Nevertheless, the biological meaning of DNA methylation is not clear. A possiblerole of DNA methylation in cell differentiation has been discussed(Scarano 1967f; Scarano, Iaccarino, Grippo & Parisi, 1967; Scarano & Augusti-Tocco, 1967). In a preliminary communication Scarano, Iaccarino, Grippo & Winckelmans (1965) reported on the methylation of DNA in developing sea urchin embryos. t Requests for reprints should Biophysics, Naples, Italy. $ Symposium lecture Regulation Interlaken, Sept. 1967.
be sent to me at the International of Protein
Sycnthis 195
in. Embryos,
Laboratory
8th Int.
of Genetics Cong.
Embryology,
and
196
P. GRIPPO
ET
AL.
This paper reports experiments on methionine uptake and on methylation of DNA in developing sea urchin embryos. The experiments on DNA methylation demonstrate that (1) 5-methylcytosine is the only minor base present in DNA of developing sea urchin embryos; (2) its methyl group originates from methionine; (3) the in vivo synthesized 5-methyloytosine is non-randomly distributed in DNA; (4) methylation of DNA occurs at all stagesof the early embryonic development.
2. Materials
and Methods
l-Methyladenine was purchased from Sigma Chemical Corp. (St. Louis, MO.); $-methyladenine, 6-dimethyladenine and 1-methylguanine were purchased from Fluka (Buchs, Switzerland) ; 5-methyladenine and 5methylcytosine were purchased from Calbiochem (Los Angeles, Calif.). We thank Dr G. H. Hitchings (Wellcome Research Laboratory, Tuckahoe, N.Y.) for samples of 2-methylguanine, 2-dimethylguanine, 1-methylguanine, 2-methyladenine, 6-methyladenine, and 6-dimethyladenine. 8-Methyladenine was synthesized according to Koppel & Robins (1958). The compound behaved as a pure substance on paper chromatography in the solvents 1 to 6 (see below), and its ultraviolet absorption spectrum was identical to that described by Koppel. Aminopterine was purchased from L. Light & Co. Ltd. (Colnbrook, Bucks., England). Highly polymerized DNA from calf thymus was obtained from Sigma Chemical Corp., (St. Louis, MO.). n-[methyZ-14C]methionine was obtained from the Radiochemical Centre (Amersham, England); the different samples used, had a specific activity in the range between 5 me/mmole and 29.5 ma/m-mole. n-[methyl-“Hlmethionine (110 ma/m-mole) and L-[14C]serine (120 ma/m-mole) were purchased from New England Nuclear Corp. (Boston, Mass.). Sterile 32P-labelled sodium phosphate was purchased from the Radiochemical Centre (Amersham, England), L-[methyZ-2H3]methionine (99% deuterium) was synthesized according to Rachele, Kuchinskas, Kratzer & du Vigneaud (1955) by New England Nuclear Corp. (575 Albany St., Boston, Mass.). Crystalline pancreatic deoxyribonuclease, snake venom phosphodiesterase, and Escherichia coli alkaline phosphatase were obtained from Worthington Biochemical Corp., (Freehold, N. J.). The phosphodiesterase was free of 5’ nucleotidase activity under the conditions of our experiments. Russel’s viper venom was obtained from the Haffkine Institute (Parel, Bombay). Charcoal was purchased from Barnebey-Cheney (Columbus 19, Ohio). Dimethyl POPOP, PPO, [14C]toluene, E3H]toluene, tritiated water, hydroxide of hyamine 10X and Cab-0-Sil were purchased from Packard Instrument Corp. (La Grange, Ill.). All other chemicals were reagent grade of the highest purity available. (a)
Sea
urchin
Paracentrotus livid~ and Sph.aerechinue granularis, were obtained from the Zoological Station, Naples. Eggs were collected by gently stirring the ovaries in filtered sea water, filtered through bolting silk, and washed twice with sea water by sedimentation. Fertilization was performed with the minimum amount of freshly diluted sperm which caused 100% fertilization in a control trial. After formation of the fertilization membrane, the fertilized eggs were washed twice with sea water and were kept at 18% with continuous gentle stirring at a concentration of 5000 eggs/ml. In most of the experiments the embryos were cultured in sea water containing 5 mg penicillin G/l. and 10 mg streptomycin sulphate/l. 32P-labelled bacteria were used to exclude possible bacterial contamination of the sea urchin embryos. P. lividzls embryos at the gastrula stage and S. granularis embryos at the swimming blastula stage were mixed with 32P-labelled bacteria. About 95% of the 32P was washed away during the centrifugation of the embryos. (b) an
Embryos were International
collected Centrifuge
Extraction
at 4°C by centrifugation PR2; were washed
of DNA at 800 rev./mm in the no. 259 head of twice with a solution containing 0.55
DNA M-NaCl above, sodium
METHYLATION
in 0.1 M-EDTA were extracted lauryl sulphate. (c)
IN
SEA
URCHIN
EMBRYOS
197
at pH 8. 10 to 15 ml. of embryos packed by centrifugation as with 50 ml. of 0.55 M-NaCl in O-1 M-EDTA, pH 8, and 4 ml. of 25% DNA was then purified according to the method of Marmur (1961).
Extraction
of DNA
nucleotides
and
DNA
nucleosides
About 10 ml. of packed embryos were treated twice for 30 min at 37’C with a mixture of absolute ethanol and ether (3:l v/v). The residue from the extraction was dried under vacuum over KOH. The dry powder was dissolved in 10 ml. of O-3 N-NaOH and kept at 37°C for 18 hr to hydrolyse the RNA. The NaOH hydrolysate was chilled at 0°C and acidtied with perchloric acid to a final concentration of 5%. The precipitate was collected by centrifugation, washed 4 times with cold 1 y. perchloric acid, and dissolved in water by addition of NaOH until pH 7 was obtained. Deoxynucleotides were obtained by enzymic hydrolysis with DNase and phosphodiesterase. When deoxynucleosides were prepared, snake venom hydrolysis followed DNase hydrolysis (see below). (d) (i)
DNA was dissolved 7 with 0.1 m-NaOH. 10 pg/ml., respectively. (ii)
hydrolysis
in water at a concentration of 20 mg/ml. and the pH MgSO* and DNase were added to a final concentration The mixture was incubated for 5 hr at 37°C.
was adjusted of 2 mM
to and
to pH 9.6 with 0.1 M-NaGH and glycine buffer, pH of 0.02 M. 100 pg/ml. of snake venom phosphodiesterase at 37°C for 5 hr.
9.6,
Phosphodiesterase
The DNase digest was added to a final were added and the (iii)
Enzymic
DNase
Snake
was adjusted concentration mixture kept
venom
Oligonucleotides at a concentration of about 2 mg/ml. in 0.02 na-glycine buffer, MgSO,, pH 9.6, were hydrolysed, at 37°C for 5 hr, by addition of 100 pg of snake ml. (iv)
2 mMvenom/
Phosphatase
Nucleotides phosphatase/ml.
at a concentration of about 2 mg/ml. were at 37OC for 4 hr. The pH was continuously (e)
Hydrolysis
to purines
and
hydrolysed adjusted
with 20 pg of alkaline to 9 with 0.1 M-NaOH.
pyrimidines
DNA, deoxyribonuoleotides, and deoxyribonucleosides were hydrolysed with 70% perchloric acid, according to Bendich (1955). The recovery after acid hydrolysis of standard methylated bases was measured. Recovery of 90% or more of &methylcytosine, 2methyladenine, 6-methyladenine, 6-dimethyladenine, 7-methyladenine, %methyladenine, I-methylguanine, 2-methylguanine, 2-dimethylguanine was found. (f)
Degradation
The methyl group of thymine Sprinson (1954). The crystallized its radioactivity measured. The standard of [l*C]toluene. (g)
of the methyl
group
Preparation
of DNA
isostichs
DNA was hydrolysed with 3% diphenylamine in formic The isostiohs were separated according to chain length using a linear gradient of triethylammonium bicarbonate. each isostich fraction by sublimation under vacuum. (h)
Preparation
of thymine
was converted to iodoform as described by Elwin & iodoform was dissolved in the scintillator solution and efficiency of counting was determined with an internal
of d&ucleot&
fractions
acid (Burton on a column The salt
from
Hydrolysis of DNA from sea urchin embryos with pancreatic dinucleotide fractions were performed according to methods Tocco, Carestia, Grippo, Parisi & Scarano, 1968).
a DNa.se
& Petersen, 1960). of DEAE cellulose was removed from digest
DNase, and separation of already published (Augusti-
198
ET
P. GRIPPO (i)
Desalting
on
AL.
charcoal columne (Cohn, 1963 PersMzal
cornmunicatiom)
Charcoal WM suspended in 0.2 N-HCl and washed with water to neutrality. It was then stirred in isopropanol-water-toluene (50: 50: 1: by vol.) for 30 min. The charcoal was sedimented and the supernatant was discarded. The reversibly adsorbed toluene was eluted from the charcoal by washing it on a column with 95% ethanol-water-conon NH,OH (2 :2:1 by vol.) until no material with optical density absorbance at 260 rnp appeared in the effluent. The sample of nucleotides or nucleosides, about 100 optical density units at 260 mp, was brought to pH 2 and adsorbed on a column of toluenetreated charcoal (0.6 cm x 2 cm). The column was then washed with water to neutrality and the sample eluted with 95% ethanol-water-concn NH,OH (2: 2: 1 by vol.). The yield of nucleotides and nucleosides was 90 to 100°&
(j) Paper chromatography
and thin-layer
chromatography
Sheets of 3 MM Whatman paper were washed with 200 ml. of 2 N-acetic acid and with glass distilled water in a ohromatographic tank. Thin-layer chromatographic separations were performed on micro-crystalline cellulose (Avicel) or DEAE-cellulose according to Grippo, Iaccarino, Rossi & Scarano (1965). The nucleic acid derivatives were detected by Mineralight ultraviolet lamp Products Inc. (San Gabriel, Calif.). The spots were eluted by water or dilute acid. The Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent Solvent
following chromatographic solvents were used : 1: isopropanol-HCl-water (65:16.7:18.3, by vol. (Wyatt, 1951)). 2: n-butanol-water (86:14, by vol. (Markham & Smith, 1949)). 3 : isobutyric acid-water-25% NHBOH (400 :208 :0*4, by vol. (Lijfgren, 1952)). 4: isopropanol-water-concn NH,OH (85: 15:1*3, by vol. (Hershey, Dixon & Chase, 1953)). 5: n-butanol-water-formic acid (77 :13:10, by vol. (Markham & Smith, 1949)). 6: saturated (NH&SO,-0.5M-sodium acetate-isoprop?fiol (80:18:2, by vol. (Markham & Smith, 1952)). 7: 95% ethanol-lM-ammonium acetate saturated with tetraborate (70: 30, by vol. (Plesner, 1955)). 8: n-butanol-water (86: 14, by vol.), with 5% by vol., of eoncn NH&OH on the bottom of chromatographic tank (Markham & Smith, 1949). 9: 5% aqueous ammonium bicarbonate (Broom, Townsend, Jones & Robins, 1964). 10: n-butanol-water-concn NH,OH (86: 13: 1, by vol. (Wacker & Ebert, 1959)). 11: isopropanol-5% aqueous ammonium sulphate (l:lQ, by vol. (Brookes & Lawley, 1960)). 12: ethanol-water (7 :3, by vol. (Broom et al., 1964)). 13: N-N-dimethylphormamide-isopropanol-concn NH,OH (25: 65: 10, by vol. (Broom et al., 1964)). 14: n-butanol-ethanol-water (50:15:35, by vol. (Wacker & Ebert, 1959)). 15: methanol-ooncn HCl-water (7: 2: 1, by vol. (Brookes & Lawley, 1960) ). 16:
n-butanol Lawley,
saturated 1960)).
with
(k) Electrophoretic Whatman 3 MM 4°C.
separations paper. The
High-voltage
NH40H
Spec6rophotometri.c
acids and their Quantitative
(100:
1, by
vol.
(Brookes
&
e~ectr~horesis
were performed according electrophoresis was performed (1)
The U.V. spectra of nucleic recording spectrophotometer. DU spectrophotometer.
water-concn
to Smith (1955) on a refrigerated
on plate
washed kept at
measurements
derivatives were determinations
taken were
with a Beckman DK2A made with a Beckman
DNA
METHYLATION
IN
SEA
(m) Radioactitity
URCHIN
EMBRYOS
199
mecwurerrmts
Measurements of radioactivity were performed with a Packard Tri-Curb 314-&X liquid-scintillation counting system. The following scintillators were used: (a) 1200 ml. of Cab-O-W were mixed with 700 ml. of 0.01% dimethyl-POPOP, 0.4% PPO in toluene, and 300 ml. of absolute methanol ; (b) 40 g of Cab-0-Sil were mixed with 500 ml. of dioxane and 500 ml. of xylene containing 80 g of naphthalene, 5 g of PPO and 05 g of dimethyl-POPOP. 20 ml. of this mixture can hold up to 0.2 ml. of aqueous solution. Tritium in this scintihator has an efficiency higher than in scintillator (a). Quenching corrections were made by internal standardization with standard labelled compounds. Radioactivity on paper or on thin layer ohromatograms was measured by adding the paper or the cellulose powder to a vial with 20 ml. of scintillator (a). 3zP was measured with a Nuclear-Chicago low background flow counter. (n)
Mass epectrometry
measurements
Mass speotrometry analysis was performed with a Consolidated Electrodynamics Co. double focusing mass spectrometer. Samples of about 60 pg were introduced asdry powder in the ionization chamber of the instrument. The following operating conditions were used : energy of ionizing electron beam 25 ev; temperature of the ionization chamber 70 to 120°c.
3. Results (a) Uptake of methionine Knowledge of the rate of uptake of methionine by embryos at different stages of development is a necessaryprerequisite to studies of in vivo methylation. Experiments on X. granularis and P. lividus embryos have revealed striking changesof the rate of uptake of r,-methionine during development. Figure 1 shows the uptake of 4.85 PM-methionine as a function of time by sea urchin embryos P. lividus at the 16 cells stage and at the gastrula stage. The methionine which disappears from the incubation medium is recovered in the embryos.
Time
(hr)
Fro. 1. Time course-of uptake of z-methionine by P. 1ividu.s embryos. Ordhate: radioa&ivity taken up by the embryos as a percentage of the total radioactivity. Absci88a: time in hours. --O--O-, Embryos at the gastrula stage; -A-A-, embryos at the 16.cells stage; --m-f-J--, embryos at the gastrula stage + 1 rnn-.z-serine; -e--a-, embryos at the gastrula stage + CrnX-L-serine; --w-m--, embryos at the gastrula stage + 4 mm-L-glycine. In all samples 4.85 pM-L-[~eth?/Z-‘*C]methionine (13.9 me/mole) was present. At the indicated times radioactivity was measured on 100 ~1. samples of sea water.
P. GRIPPO
200
ET
I
AL. I
I
60
90
too-
g
&O-
s0 “a 2
60-
?! ‘E 2
40-
ii 20-
0
30 Time
(mid
FIG. 2. Inhibition by n-serine of methionine uptake. S. granularis embryos at hatching blastula stage cultured in the presence of 28.2 PM-L-[methyl-‘W] methionine (5.45 ma/m-mole). Ordinate and abscissa as in Fig. 1. --O--O-, Embryos + [r*C]methionine; -A-A-, embryos + [Wlmethionine + 20 pML-serine; -I-+-~---, embryos + [W]methionine + 0.1 ma6--r;-serine; -•--•---, embryos + [14C]methionine + 1 mx-L-serine. At the indiosted times radioactivity was measured on 100 ~1. samples of filtered sea. water.
60 Time
(mid
FIG. 3. Time-course of uptake of L-serine and its inhibition by methionine. S. granularis embryos at the hatching blastula stage cultured in the presence of 0.33 ~M-L-[W] serine. Ordinate end ebscisse es in Fig. 1. -O-O--,Embryos + [%!]serine;--n-n-, embryos + [l%]serine + 1 mM-L-methionine. At the indicated times radioactivity was measured on 250 ~1. samples.
DNA
METHYLATION
IN
SEA
URCHIN
EMBRYOS
201
The uptake of methionine is inhibited to the same extent by 1 mM- and 4 mllr-serine. 4 mM-glycine causes 100% inhibition of the methionine uptake. Thymidine and 5.methyldeoxycytidine do not inhibit the uptake of methionine. Figure 2 shows the uptake of 28.2 +?f-methionine as a function of time by sea urchin embryos S. granularis at the swimming blastula stage. The uptake is inhibited by the of L-serine. presence of L-serine and the inhibition is proportional to the concentration The uptake of 0.33 PM-serine by S. granularis embryos at the swimming blastula stage is shown in Figure 3. Serine uptake is also inhibited by 1 mM-methionine. A similar curve of uptake has been obtained for P. lividus embryos.
P 3 Stoge of development
4. Rate of methionine uptake at different stages of development. S. grawularis embryos &t the indicated stages cultured in the presence of 19.6 pM-L-[methyl-14C]methionine (10.2 me/mmole) per 15 min. FIQ.
In Figure 4 the rate of methionine uptake by S. granularis embryos at several stages of development is reported. Unfertilized eggs under these conditions do not take up methionine even after a previous stirring for 16 hours in seawater. A sudden change in permeability to methionine occurs immediately after fertilization. The uptake becomeseasily measurable and increaseswith development. At the gastrula stage 567” of the methionine is taken up by the embryos in a 15-minute period in the incubation medium. In the experiments reported in Figures 1, 2, 3 and 4 almost all the radioactivity disappearing from the seawater was recovered in the embryos. A very small fraction of the methyl group of methionine was oxidized to carbon dioxide during incubation as demonstrated by the following experiment. 100 ml. of a suspensionof P. lividus embryos at gastrula stage were incubated fox 5.5 hours with 1.2 pmole L-[methyl-14C] methionine (13.9 me/m-mole). During the incubation the CO, produced by the embryos was collected as BaC03. Lessthan O-1y0 of the total radioactivity was found in BaCO,. (b) DNA
methylation
Incubation times of five hours or longer were chosento minimize the effects of the different rates both of the uptake of methionine and of the synthesis of DNA during the early embryonic development of the sea urchin (Elson, Gustafson & Chargaff, 1954). The methylation of DNA, which occurs during the chosenperiods of incubation with labelled L-methionine is of such an extent, at all stages of development, that it 14
202
P.
GRIPPO
ET
AL.
is possible to detect it even at the very early stages when there is a small uptake oi methionine and the total amount of DNA synthesized is small. (i) Search for DNA methylated bases 2.2 litres of a suspension of P. lividus embryos at the early pluteus stage were incubated for 12 hours with 25 FU of L-[methyL1*C]methionine. At the end of the incubation the embryos were collected, 7.5 mg of carrier DNA from calf thymus were added, and DNA was extracted. The purified DNA was hydrolysed with DNase and phosphodiesterase. The deoxymononucleotides obtained were desalted on charcoal and separated by high-voltage ionophoresis on paper. All the radioactivity was found in the four ultraviolet absorbing bands (Fig. 5). The bands were eluted and each
6
FIG.
5. Eleotrophorstio
212 6 lo 14 IB 22 26 cm from origin Start separation of the 4 deoxynuoleotides.
For
details
see text.
deoxynucleotide was chromatographed on paper with solvent 7 to purify it from possible contaminating 5’-ribonucleotides. Almost 100% of the radioactivity was recovered in the deoxynucleotide spots. Each deoxynucleotide was identified by its ultraviolet spectra. 2-l pmoles of dCMP, 3.9 pmoles of dTMP, 3.9 pmoles of dAMP and 2.4 pmoles of dGMP were recovered and the total radioactivity in each spot was 17,600; 43,600; 73,800 and 31,600 ctsjmin, respectively. The deoxynucleotides were hydrolysed with concentrated perchloric acid and the bases obtained were analysed in several chromatographic solvents with methylated bases as standards. All the radioactivity was recovered in the bases. This excludes the possibility that the radioactivity found in the nucleotides was in the sugar moiety. The bases were analysed as follows. Cytosine. Samples of the hydrolysed dCMP were analysed by uni-dimensional chromatography in solvents 1, 2, 6, 8 and by two-dimensional chromatography in solvents 1 and 2. All the radioactivity was found in the Et-methylcytosine spot. No radioactivity was detected in the cytosine and in carrier 6.methyladenine spots. Carrier 6.methyladenine was added, because in the electrophoretic separation 6methyl&WI? does not separate satisfactorily from dCMP. Thymine. Samples of the hydrolysed dTMP were chromatographed uni-dimensionally with solvents 1, 2, 3, 4, 5, 6, 8 and two-dimensionally (first direction: solvent 2; second direction: solvent 1). In every case all the radioactivity was found in the thymine spot. A sample of thymine containing 16,500 disint’egrations/min of l*C was hydrolysed by the method of Elwin & Sprinson (1954), 100°J of the radioactivity was recovered
DNA
METHYLATION
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in iodoform, demonstrating that all the radioactivity of thymine was in the methyl group. Adenine. Samples of the hydrolysed dAMP were chromatographed with solvents 1,2,3,4,5,6 and 8. No radioaotivity was found in l-methyladenine, 2-methyladenine, 6-methyladenine, 6dimethyladenine, 7-methyladenine, and 8-methyladenine, added as carriers and separated from adenine in the indicated chromatographic solvents. In all the solvents, the radioactivity was found always in the adenine spot. Furthermore, the adenine sample was checked by chromatography with solvents 9,10, 11,12,13,14,15 and 16 under oonditions in which 10% of the total radioactivity should have been detected if separated from the main radioactive spot. In all the solvents one radioactive spot was found in coincidence with adenine. Finally, the purified adenine was crystallized several times from water and no change of its specific activity was found. Quanine. Samples of hydrolysed dGMP were chromatographed with solvents 1, 4 and 8. No methyl bases were found. Identical results for all four bases were obtained in two similar experiments performed with P. lividus embryos at the prism stage. (ii) Origin. of the methy.! group of DNA 5-methylcytosine (a) Experiments with doubly-labelledmethionine The origin of the methyl group of 5-methylcytosine and of the radioactivity in the thymine, adenine and guanine sampleswere studied by incubating the embryos in the presence of L-[methyl-14C]methionine and L-[methyl-3H]methionine. Direct transfer of the methyl group of methionine is suggested,when a basehas a ratio 3H/14Cequal to that of the added methionine. A ratio lower than the initial one would indicate oxidation of the methyl group of methionine and incorporation by the reaction of the C(1) pool metabolism, either in the methyl group of thymine or in the purine ring of adenine and guanine. TABLE
1
Ratio 14CJ3Hin basesof seaurchin embryo DNA Exp. 1 2 3 4
S-Methylcytosine 2.9 2.8 3.1 26
Thymine
Adenine
1.8 1.7 1.5 1.9
l-7 1.4 1.3 l-3
Guanine
0.4 0.5 0.6 0.6
Experiment 1. P. lividus embryos et the prism stage incubated for 13 hr with 25 po ofn-[m&yZrW]methionine and 80 PC of n-[~ethyPH]methionine. In experiments 2,3, end 4 8. granzcZa& embryos at the stages 8 to 16 cells, hatching bl&,ulee, end blastulae with mesenchyme respectively, were incub&sd for 14 hr with 50 po of r,-[w&hyZ-l4C] methionme end 50 pc of h-[methyZ-3H]methioaine. The specific activity of L-[methyZ-l4C]methionine end of r.-[6-&hyZ-3H]methionine was 295 me/m-mole end 100 me/m-mole, respectively. In all experiments the sH/W ratios were normuheed to the initial value in methionine t&ken equal to 3.
Table 1 lists the ratios 3H/14C of the DNA bases.The data show that the in&,] 3HI 1% ratio was found only in 5-methylcytosine. About one-third of the tritium was
204
P.
GRIPPO
ET
AL.
lr
HO 4
“L
I I 126 127 128 129
j
m/e
(a)
Fla. 6. Mass spectra (a) 5-methylcytosine from DNA incubated with [nzethyZ-aHs]methionine.
I
.
120 121 122 123 124 I!2! 126 127 I28 129 (b)
of 5methylcytosine from 124 m/e to 130 m/e. of P. lividus. (b) 5-methylcytosine from DNA For details see text.
of P. Zividua
lost in the case of thymine, and more than one-third in the case of the purines. (However, a definite conclusion cannot be reached on the basis of these experiments because of the possible occurrence of isotopic effects.) (b) Experiments
with L-[methyL2H3]methionine
To have direct evidence of the transfer of the methyl group of methionine to DNA cytosine, L-[methyL2H,]methionine was used in the following experiment. Four litres of a suspension of P. 1ividu-s embryos at the blastula stage were incubated with 20 mg
SO-
I% 0%
of total S-methylcytosine of total pyrimidine
FIN. 7. Percentage distribution of 5-methylcytosine and of total pyrimidine deoxynucleosides in DNA pyrimidine isostichs of 8. gralzulccris embryos. 8. granulcwis embryos at blastnla stage cultured in the presence of 1 mo of ~-[m&yZ-r~C] methionine (13.9 me/m-mole) for 7 hr. The embryo DNA was extracted and hydrolysed with 3% diphenylamine in formic acid. The pyrimidine isostiohs were separated and each isostich fraction was hydrolysed to deoxynucleosides. 5-Methyldeoxycytidine was purified by paper cbromatography in solvents 1, 7, and 4. The spots were eluted and the radioactivity measured. The percentage of total pyrimidine deoxynucleosides in each isostich fraction was measured by U.V. spectrophotometry.
DNA
METHYLATION
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of deuterated L-methionine. After five hours the embryos were collected and DNA was extracted. The DNA was hydrolysed to bases with perchloric acid and the bases were separated and purified by paper chromatography in solvents 2 and 4. The spots of cytosine and 5methylcytosine were eluted together. After elution the samples were dried and used for mass spectrometry analysis. Figure 6 shows the mass-spectra of a standard 5-methylcytosine and of a Smethylcytosine from DNA of embryos grown in the presence of labelled methionine. The presence of [5-methyl-2H,]cytosine in the DNA of sea urchin embryos grown in the presence of n-[methyL2H,]methionine demonstrates the direct transfer of the methyl group of methionine to DNA oytosine. (iii) Non-randomness of DNA methylation. The data of Figure 7 demonstrate that the methylation of DNA cytosine in. viva is non-random. In fact, 60% of the synthesized 5-methylcytosine was recovered in the monopyrimidine fraction, although only 25% of pyrimidines occur in this fraction. The total counts of 5-methyldeoxycytidine of each isostich are proportional to the amounts of the deoxynucleoside because the specific activities of the Ei-methyldeoxyoytidine of all the isostich fractions are the same. This is demonstrated by the following experiment. Embryos were incubated with L-[methyL2H,]methionine and DNA was extracted as described above. The DNA pyrimidine isostichs were prepared and from each isostich fraction 5-methylcytosine was purified and analysed by mass spectrometry. The ratio of [5-methyL2H,]cytosine over 5-methylcytosine was the same in all fractions.
iooI 0
% of total % of total
rl
fl
dinucleotide dinucleotides
S-methylcytosine
SO-
0,
r
(CT) (CCKCC)(CA)(AA)
n (AC)
l-l n (AT) (CT)
(TT)
Fra. 8. Percentage distribution of 5-methylcytosine and of the 4 major bases in the dinucleotides from a DNase digest of 6’. granularis embryo DNA. S. granularis embryos at the blast& stage incubated for 7 hr with 1 mc of n-[methyVW]methionine (1 I.7 me/m-mole). DNA and dinucleotides were prepared and separated as described in Materials and Methods. Each dinucleotide was hydrolysed with snake venom, and deoxynucleosides were separated by paper chromatography in Solvent 1. 5-methyldeoxycytidine spots were eluted and the radioactivity measured. The percentage of each dinucleotide was measured by u.v. spectrophotometry. Dinuoleotide (GG) was not measurable.
Moreover, the non-random distribution of 5-methylcytosine was demonstrated by the analysis of dinucleotide fractions from the DNase digest of labelled DNA from 1.9. granularis embryos grown in the presence of L-[methyl-14C]methionine. J?igure 8 shows the distribution of 5-methylcytosine in the DNA dinucleotide fraction. (iv) ikfethylation of DNA during the early embryonic development The data of Table 2 demonstrate that methylation of DNA occurs at all stagesof development in P. lividus. No conclusion about quantitative differences of methylation at the different stagescan be drawn becauseno information was collected on the
P.
206
GRIPPO
ET
AL.
pool of methionine and on the rate of DNA synthesis during the period of incubation with labelled methionine. Similar results on methylation of DNA at all stages of development were reported previously (Scarano et al., 1965) for S. granularis embryos.
TABLE
2
S~eci$c activity of DNA 5-methylcytosilzefrom sea urchin embryos(P. Iividus) at different stagesof development Exp. 1 2 3 4 5 6
Stage
2 disintegrations/min
128 cells hatching hatching blastulae blast&e prisms
blastulae blastulae with mesenchyme with mesenohyme
14C X 10-6/~mole 6 2 3 6 9 1
At the indicated stages embryos were incubated for 5 hr with 150 PC/I. of n-[methyl-i4C]methionine (29 me/m-mole). At the end of a 5hr period the embryos were collected. DNA deoxynuoleosides were purified and the specific activity determined on deoxycytodine + 5methyldeoxycytidine. The deoxynucleosides were then hydrolysed to bases and analysed: all the radioactivity of the original deoxycytidine + 5methyldeoxycytidine sample was in 5-methylcytosine, while cytosine was not radioactive. The speciflo radioactivity of 5-methylcytosine was calculated from the known base composition of P. Zividua sperm DNA (Chargaff, Lipshitz & Green, 1952).
4. Discussion The experiments on the uptake of ammo acids reported in the present paper indicate the existence of an active transport mechanismfor L-methionme and rJ-serine in developing sea urchin embryos P. lividus and S. granularis. The uptake of Lmethionine is inhibited by the presenceof L-serine and vice zlersa,but is not affected by thymidine and 5-methyldeoxycytidine. These facts are in agreement with the tidings of Mitchison & Cummins (1966) on the active transport mechanismfor valine in developing seaurchin embryos. Unfertilized seaurchin eggsdo not take up methionine from sea water under our experimental conditions. The uptake begins soon after fertilization. The rate of the uptake increases, at least until the gastrula stage. The labelling of the methyl group of thymine and of the purine ring of adenine and guanine, which arises from the methyl group of L-methionine, indicates that a fraction of the methionine methyl group is oxidized to one carbon pool intermediates. By this pathway the C(2) and C(8) of the purine ring and the methyl group of thymine are labelled. Thus, the only valid criterion for DNA methylation is the isolation of a DNA methyl base labelled in the methyl group. The label of the methyl group of L-methionine does not enter the pyrimidine ring of cytosine, LLmethylcytosine and thymine. 5-Methylcytosine is the only minor methylated base in the DNA of developing sea urchin embryos. On the assumption that all DNA minor methyl baseswould be labelled with a specific activity equal to that of 5-methylcytosine, our experiments can exclude the presenceof other methylated basesup to a level of l/20 of B-methylcyto-
DNA
METHYLATION
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URCHIN
EMBRYOS
207
sine. However, we are investigating further the problem of the labelling of the methyl group of thymine on the assumption that a small fraction of the labelled thymine arises from DNA B-methylcytosine at the polymer level (Scarano, et al., 1967). It is interesting to notice that in several bacterial strains (Dosko&il & Sormova, 19653)and in the blue-green alga Plectonema boryanum (Kaye, Salomon & Fridlender, 1967) 6-methyladenine was found to occur in DNA in addition to 5-methylcytosine, while in higher organisms 5-methylcytosine is the only DNA minor base found. The experiments with doubly-labelled methionine and with [2H,]methyl-labelled methionine demonstrate the dire& transfer of the methyl group of methionine onto DNA cytosine. It is likely that methylation of DNA oytosine in seaurchin embryos occurs, as in all the other organisms,via X-adenosylmethionine. The non-random distribution of 5-methylcytosine in the sea urchin embryo DNA suggeststhat the methylation of DNA occurs at the polymer level as it is known to occur in the other organismsin which it has been studied (Gold et al., 1963a, b; Gold & Hurwitz, 1964a, b; Fujimoto et al., 1965). Bacterial contamination of the embryos cannot affect the results described in this paper becauseof the following facts. (1) 6-Methyladenine was never found in the embryo DNA; yet 6-methyladenine is a component of bacterial DNA (Dunn & Smith, 1958). (2) Most of the experiments were done in the presenceof penicillin G and streptomycin sulphate. (3) Unfertilized eggswere cultured for five hours in sea water containing labelled methionine; no radioactivity was taken up by the collected eggs, while under the same conditions sea urchin embryos take up lOOo/oof the added methionine. Further work is necessary to identify possible quantitative differences in DNA methylation and/or possible changes in the distribution of 5-methylcytosine in the DNA from embryos at different stagesof development. We want to emphasizethe possibleusefulnessof massspectrometry in determining quantitatively minor basesin nucleic acid fragments. We are attempting to develop methods for determining by massspectrometry the distribution of 5-methylcytosine in pyrimidine isostichs and in DNase digest. We thank Professor M. Ageno of the Istituto Superiore di &nit&, Rome, for his interest in this work and for advice in mass spectrometry analysis, as well as Mr A. Rosati, Mr M. Flamini and Mr A. Martinangelo for their technical assistance in the measurements. We are grateful to Mr A. Granieri and M.r G. Forlani for skilful technical assistance. This work was supported by Euratom-C.N.R.-C.N.E.N. contract no. 012-61-12 BIAI, and by research Grant no. HD-0126304 from the U.S. Public Health Service.
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