Proc. Nati. Acad. Sci. USAVol. 76, No. 11, pp. 5794-5798, November 1979Genetics

Methylation of chloroplast DNAs in the life cycle of Chlamydomonas(maternal inheritance/DNA methylation/restriction enzyme analysis)

HANS-DIETER ROYER AND RUTH SAGER*Sidney Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115

Contributed by Ruth Sager, August 10, 1979

ABSTRACT Methylation patterns of Chlamydomonaschlor-oplast DNAs (chIDNAs) were examined in the vegetative,gametic, and zygotic stages of the life cycle. Restriction endo-nuclease fragment patterns produced by EcoRI, BamHI, HpaII, and Msp I were compared; the last two cleave DNA at thesequence CC-C-C, but Hpa II is blocked by prior methylationof the internal cytidine whereas Msp I is not. chlDNAs fromvegetative cells of both mating types showed no evidence ofmethylation at C-C-CG-. Gametic mt+ chlDNA was heavilymethylated at C-C-C-, whereas the homologous chlDNA frommt- gametes showed very slight methylation at C-C-C-G.Methylation of additional sites in chlDNA from mt+ gametesbut not from mt- gametes was shown by blockage of someEcoRI and BamHI sites that were cleaved in the chlDNA fromvegetative cells. chlDNA from 6-hr zygotes was much moremethylated than gametic mt+ DNA, as shown by its almost totalresistance to cleavage by all four restriction enzymes. Thesefindings support and extend previous evidence that chlDNA ofmt+ cells is methylated during gametogenesis and that furthermethylation occurs after gametic fusion in the young zy-gotes.

The methylation and restriction of chloroplast DNA (chlDNA)has been shown (1-4) to play a decisive role in the regulationof maternal inheritance of the chloroplast genome inChlamydomonas. In this paper, the methylation patterns ofchIDNAs purified from vegetative cells and gametes of bothmating types and from zygotes have been compared with theuse of four restriction endonucleases: Hpa II, Msp I, EcoRI, andBamHI.Hpa II and Msp I both cleave DNA at the C-C-G-G se-

quence, but Hpa II is blocked by prior methylation of the in-ternal cytidine (5, 6) whereas Msp I is not (7). Thus, comparisonof fragment patterns produced by digestion of the same DNAwith each of these enzymes provides a simple way to detect invivo methylation (7, 8). EcoRI and BamHI patterns were alsoexamined, and evidence is presented that restriction sites ofthese enzymes can be blocked by methylation. The EcoRI andBamHI restriction fragment patterns of chlDNAs from vege-tative cells of our strains, 21gr and 5177D, were compared withthose of the related strain CW15 (9) and no differences weredetected.We report here that the gametic chlDNA from mating type

plus (mt+ ) cells is strongly methylated at C-C-G-G sites,whereas the homologous chlDNA from mating type minus(mt-) cells is only slightly methylated at these sites and not atthe other sites. Furthermore, the chlDNA from zygotes is soheavily methylated that it is almost fully resistant to cleavageby each of the enzymes, including Msp I. These findings sup-port and extend previous evidence based upon the density shiftseen in zygotic chlDNA (1, 3) and upon radioisotope labeling(3, 4) that mt+ gametic chlDNA is methylated and that furthermethylation occurs after zygote formation.

MATERIALS AND METHODSCells and Culture Conditions. Chlamydomonas reinhardi

strains 21gr (maternal, mt+) and 5177D (paternal, mt-) wereused. The strains and their growth conditions and methods forgametogenesis and zygote formation have been described (10).Gametes were induced by nitrogen starvation. Zygotes wereprepared by mixing equal numbers of mt+ and mt- gametes.Mating and zygote formation were monitored in the micro-scope. The yield of viable zygotes and the frequency of ma-ternal and exceptional zygotes was determined as described(10). The frequency of exceptional zygotes did not exceed 1%and the frequency of viable zygotes was >90%.

Isolation of chlDNA. chlDNA was isolated by publishedprocedures (3). Vegetative cells, gametes, and zygotes werelysed with 2% sodium dodecyl sulfate/2% Sarkosyl after the cellwall was broken in a French pressure cell. The lysates weretreated with Pronase (600 ,g/ml) at 50'C overnight and thenextracted twice with freshly distilled phenol. The aqueous phasewas extracted with chloroform/isoamyl alcohol, 24:1 (vol/vol),followed by three ether extractions to remove residual phenol.The aqueous phase was dialyzed against 150 mM NaCI/15 mMNa citrate; RNase T1 (20 units/ml) and pancreatic RNase (200sg/ml) were added and the mixture was incubated at 370Covernight. The mixture was again phenol extracted as describedabove and the DNA was spooled on a glass rod after the additionof 2 vol of ethanol. The chlDNA was purified by two cycles ofCsCl density gradient centrifugation. chlDNA was stored in 10mM Tris/1 mM EDTA, pH 7.5, at 40C.

Endonuclease Digestion and Gel Electrophoresis. chlDNAwas digested with the endonucleases under conditions suggestedby the distributors. Restriction fragments were separated onhorizontal 0.8% agarose gels and the bands were visualized byethidium bromide fluorescence and photographed accordingto published procedures (11, 12). EcoRI, BamHI, and Msp Iwere from New England Biolabs; Hpa II was from BethesdaResearch Laboratories. Control OX174 replicative form II DNAwas from New England Biolabs and plasmid pLJ3 was a giftfrom B. Royer-Pokora.

RESULTSHpa II and Msp I Fragment Patterns. A comparison of

restriction fragment patterns produced by digestion of vege-tative, gamete, and zygote chlDNAs with Hpa II and Msp I isshown in Fig. 1. The chlDNA from mt + vegetative cells ap-peared to be identical whether digested with Hpa II (lane a)or Msp I (lane b). The same results were observed with mt-vegetative cells (not shown). This result demonstrates thatchlDNA is not methylated at the C-C-G-G sequence in vege-tative cells. The Hpa II digest revealed 28 bands, some of whichare present in twice-molar amount.

chlDNA restriction fragments from gametes of mt- cells

Abbreviations: mC, 5-methylcytidine; chlDNA, chloroplast DNA.* To whom reprint requests should be addressed.

5794

The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

Proc. Nati. Acad. Sci. USA 76 (1979) 5795

a b

-1-2-3-4

-5

-67-8-9

-1011-12-13

-14-15-16m17+18-19-20

-21-22-23

-2425-26-27

f h

-89-10-12-13

-14-15-16717+18-19-20

-2122

-23

24-25

-26

-27

-28 -28 .. --

FIG. 1. Hpa II and Msp I digestion and agarose gel analysis of Chlamydomonas chlDNAs. Lanes: a, Hpa II digest of mt+ vegetative chlDNA;b, Msp I digest of mt+ chlDNA; c, Hpa II digest of mt- gamete DNA; d, Msp I digest of mt- gamete chlDNA; e, Msp I digest of mt+ gametechlDNA; f, Hpa II digest ofmt+ gamete chlDNA; g, Hpa II digest of mt+ gamete chlDNA plus OX174 DNA; h, kX174 DNA without endonuclease;i, Hpa II digest of zygote chlDNA plus OX174 DNA; j, Hpa II digest of zygote chlDNA; k, Msp I digest of zygote chlDNA. The small OX174 HpaII/Msp I fragments have been electrophoresed out of the gel under the conditions of electrophoresis\

A

are shown in lanes c (Hpa II) and d (Msp I) of Fig. 1. The MspI pattern is almost identical with that of the vegetative cells, butthe Hpa II pattern shows some differences. Bands 9, 14, 15, and27 are absent; bands 19, 20, 21, and 22 are almost undetectable.New bands, presumably the result of cleavage blocked bymethylation, are seen between bands 4 and 5, 5 and 6, and 13and 14. This result shows that some methylation occurred at theC-C-G-G sequence.

Digestion of chlDNA from gametes of mt + cells showed amuch more dramatic difference, compared to the mt- gam-etes, between the patterns of DNA digested with Hpa II (lanef) and Msp I (lane e). The chlDNA from mt+ gametes wasalmost totally resistant to Hpa II digestion-only a few bandsare visible in the low molecular weight range (bands 18, 24, 25,26, and 28). This result shows that the C-C-G-G sequence isalmost completely methylated in the chlDNA of the mt+gamete. The Msp I digest of mt+ gametic chlDNA was similarto the pattern of vegetative chlDNA but showed some differ-

ences: band 13 was decreased; extra bands were seen above 8and 14 and between 15 and 16; the double band 17,18 was de-creased; band 19 was absent; an additional diffuse fluorescencewas seen between 23 and 24; and band 27 was decreased. Theoccurrence of a smear in the high molecular weight range in-dicates the presence of partially cleaved fragments. BecauseMsp I evidently did not cleave all of its recognition sites in thisDNA, we conclude that the C-C-G-G sequence is furthermodified or else that the chlDNA of mt + gametes has un-dergone some other change that makes certain of these sitesunavailable to the enzyme. Controls (lanes g and h) showed thatthe enzyme Hpa II was not inhibited by the gametic chlDNAto which OX174 DNA was added; the undigested OX174 DNAis shown in lane h. A similar lack of inhibition was seen with theMsp I digest of chlDNA from mt+ gametes (lane e); the mix-ture containing qX174 is not shown.

Zygote DNA isolated 6 hr after gamete fusion was completelyresistant to digestion by Hpa II (lane j). Msp I digestions of this

Genetics: Royer and Sager

5796 Genetics: Royer and Sager

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01-02-

0304-05-

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b c dProc. Natl. Acad. Sci. USA 76 (1979)

chlDNA of mt + gametes. As a control on the extent of diges-tion, kX174 DNA was included in the Hpa II digestion of zy-gote DNA (lane i) and was totally cleaved. In all digestions weused at least a 5-fold excess of enzyme.EcoRI Restriction Fragment Patterns. The EcoRI restric-

tion fragment patterns obtained from chlDNA of our strains(21gr, mt + and 5177D, mt-) did not differ from those pub-lished (9) for strain CW 15, except that we could separatefragments R 11 and R 12 which comigrated in the publishedreport. Similarly, we found no differences in BamHI fragmentsbetween our strains and CW 15. Therefore, we follow the no-menclature and restriction map of Rochaix (9) in this study.

Fig. 2 shows a comparison of the EcoRI restriction fragments.Gamete DNA of mt- cells (lane b) had basically the same re-striction fragment pattern as vegetative DNA (lane a); however,R 20 was absent. In addition, a smear in the low molecularweight range was visible, indicated by diffuse fluorescence, andthe high molecular weight bands above R 21 to R 26 were lessintense than expected. These observations suggest that degra-dation of the mt- DNA is already beginning in the gamete.The mt + gamete DNA (lane c) had a similar restriction

pattern as the mt- gamete DNA with some changes-namely,bands appeared to be less fluorescent or were absent and newbands could be recognized. R 04 and R 05 were missing, R3 andR 5 were decreased, and R 7 was decreased whereas R 10 wasabsent, R 11 and R 13 were decreased and R 20 could hardlybe recognized. R 20 was also absent in mt- gamete DNA. Onthe other hand new bands, which we consider to be fusedfragments due to modified EcoRI sites, were seen between R2 and R 3, between R 22 and R 23, and between R 24 and R 25;another band was visible above R 26.The zygote DNA (lane d), isolated -6 hr after gamete fusion,

was almost completely resistant to digestion with EcoRI; onlya few faint bands in the high molecular weight range were seen.The DNA preparation was not inhibiting the enzyme because

a b c d

FIG. 2. Agarose gel analysis of Chlamydomonas chlDNAs di-gested with EcoRI. Lanes: a, vegetative chlDNA; b, mt- gametechlDNA; c, mt+ gamete chlDNA; d, zygote chlDNA isolated 6 hr aftergamete fusion. The bands are numbered as described by Rochaix(9).

DNA yielded only a few faint bands in the high molecularweight range (lane k), indicating an increased resistance todigestion of zygote DNA with this enzyme compared to mt+gametic DNA (lane e). Thus, zygote DNA appears to be furthermodified at or near the C-C-G-G sequence, compared to the

FIG. 3. Degradation of mt- chlDNA in gametes and zygotes.Same gel as in Fig. 2 but overexposed in order to increase the fluo-rescence in the low molecular weight range, to show the start of deg-radation of mt- chlDNA. Lanes: a, vegetative chlDNA; b, mt-gamete chlDNA; c, mt+ gamete chlDNA; d, zygote chlDNA.

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Proc. Natl. Acad. Sci. USA 76 (1979) 5797

a b c d

-15-14

13 12-11

-10

-7

-654- 4

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-(2)

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FIG. 4. Agarose gel analysis of Chlamydomonas chIDNA digestedwith BamHI. Lanes: a, vegetative chlDNA; b, mt- chlDNA; c, mt+chlDNA; d, zygote chlDNA isolated 6 hr after gamete fusion. Thebands are numbered as described (9); for bands in parentheses, 1.1and 1.2 are nuclear rDNA and 2 and 8 are mitochondrial DNA.

plasmid pLJ3 DNA, when included in the mixture, was com-

pletely digested (not shown). A smear in the low molecularweight range probably resulted from degradation of chlDNAof mt- origin (2-4). Fig. 3 shows an overexposed gel in whichthe smear in the low molecular weight range, indicating thebeginning of DNA degradation, was clearly visible.BamHI Restriction of chIDNA. Fig. 4 presents the BamHI

restriction fragment patterns of chlDNA. When the BamHIsite G-G-A-T-C-C (13) is modified at the internal cytosine bythe homologous methylase (14) to G-G-A-T-mC-C, this sitebecomes resistant to digestion. In the vegetative DNA and inthe mt- gamete DNA, this site was not detectably methylated;the restriction fragment patterns of both DNAs were identical.In the mt + gamete this site was partially methylated: some

bands were absent and fusion fragments could be seen. Bands5 and 6 were absent and in the higher molecular weight rangethe background fluorescence was relatively high, indicating theappearance of some partially digested fragments due to themodification of some BamHI sites. In the zygote all BamHI sitesseemed to be methylated; this DNA was almost totally resistantto digestion with BamHI.

DISCUSSIONMethylation of chlDNA from gametes and zygotes ofChlamydomonas serves to differentiate gametic DNAs of thetwo mating types in the zygote and to protect the chlDNA ofmt + gametes and zygotes from restriction and degradation.Maternal inheritance of the chlDNA genome is determined bythis methylation-restriction system (1-4) which closely re-sembles the bacterial modification-restriction systems thatprotect bacterial cells against foreign DNA (15).

In this study, evidence of methylation has been based uponthe comparison of restriction fragment patterns obtained afterdigestion of vegetative, gametic, and zygotic chlDNAs with therestriction enzymes Hpa II, Msp I, EcoRI, and BamHI. HpaII and Msp I both recognize the sequence C-C-G-G, but onlyMsp I can cleave the methylated sequence C-mC-G-G. Thus,comparison of the restriction fragment patterns generated bythese two enzymes acting on the same DNA permits easy de-tection of methylation at C-C-G-G sites. chlDNA from vege-tative cells is not methylated at C-C-G-G; and gametic chlDNAfrom mt + cells is heavily methylated at C-C-G-G, whereasgametic chlDNA from mt- cells is methylated at very fewC-C-G-G sites. Comparisons of vegetative and gameticchlDNAs cleaved by EcoRI and BamHI have shown that someEcoRI and BamHI sites in mt+ gametic DNA, but not in mt-gametic DNA, have become resistant to digestion by theseenzymes as well as to digestion by Hpa II. Zygotic chlDNA isheavily methylated not only at C-C-G-G but also at other sites,such that it was almost totally resistant to attack by all fourenzymes used in this study.The EcoRI restriction site is G-A-A-T-T-C (16), and the

bacterial EcoRI methylase modifies the internal adenosine(G-A-mA-T-T-C) and renders the site resistant to digestion (17).Methylation of adenine has not been detected in Chlamydo-monas (3), and thus the resistance of gametic and zygoticchIDNAs to EcoRI digestion may be the result of methylationof the terminal cytidine of the restriction site, but this point hasnot been established. Decreased cleavage resulting frommethylation of Ranking sequences could also account for thedisappearance of particular bands in EcoRI digests (18).A site-specific methylase from Chlamydomonas has been

isolated and characterized (19). The methylated sequence hasbeen identified as T-mC-A, T-mC-G, or T-mC-C on the basis ofthe nearest-neighbor analysis. These sequences are partiallycontained within the EcoRI (G-A-A-T-T-C) and BamHI (G-G-A-T-C-C) recognition sites, and thus the EcoRI and BamHIsites may be methylated by this enzyme. On the other hand, thisenzyme does not methylate C-C-G-G sequences, and thus atleast two methylases must be present. It is also possible that othermodifications in addition to methylation may occur but havenot as yet been analyzed.A two-step methylation process would be consistent with our

data. The first step, occurring in mt + gametes, would resultin methylation of C-C-G-G sites by an unknown methylase aswell as a small amount of methylation by the previously iden-tified methylase. The second step, occurring after zygote for-mation, could be performed by the methylase isolated by Sanoand Sager (19). The occurrence of a second round of methyl-ation after zygote formation has been documented both in thispaper and in previous work (3) in which radioisotope incor-poration revealed additional methylation after zygote forma-tion. In eukaryotes the occurrence of mc is almost exclusivelyseen in the dinucleotide CpG (20). In the chlDNA of mt+ cellsduring gametogenesis this dinucleotide seems to be primarilymethylated at the Hpa II site but in zygotes additional recog-nition sites are presumably methylated. We have not testedother C-G enzymes (21) as yet.

Genetics: Royer and Sager

5798 Genetics: Royer and Sager

In summary, these results confirm and extend a previousreport (3) that mt+ gametic chlDNA is methylated and thatfurther extensive methylation of this DNA occurs after zygoteformation. Previous studies (1, 3) also showed that mt- gameticDNA is degraded after zygote formation -and disappears fromdensity gradients. In the present work we have found evidenceof degradation of mt- chIDNA in the form of a low molecularweight smear in both mt- gametic and zygotic DNA prepa-rations. No comparable evidence of degradation has been seenin preparations of mt+ gametic or vegetative chlDNAs.ChIDNA of maternal (mt+ ) origin is protected against diges-tion in the zygote by extensive methylation, seen in these ex-periments as resistance to digestion by four different en-zymes.Two kinds of methylation systems have been described: those

involving methylation-restriction in which methylation protectsthe DNA against cleavage by a restriction enzyme with ho-mologous site specificity (22, 23); and those in which methyl-ation probably has a. regulatory role (24-34). Methylation-restriction systems have been documented in prokaryotes (22,23) and proposed in eukaryotes (2, 35, 36).

We thank Constance Grabowy for invaluable assistance. The re-search was supported by Research Grant GM 22874 from the NationalInstitutes of Health and a fellowship to H.-D.R. from the Max-PlanckSociety.

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