Development 1990 Supplement, 15-20Printed in Great Britain © The Company of Biologists Limited 1990

15

Variation and inheritance of cytosine methyiation patterns in wheat at the

high molecular weight giutenin and ribosomai RNA gene loci

R. B. FLAVELL and M. O'DELL

John limes Institute, John Innes Centre for Plant Science Research, Colney Lane, Norwich NR4 7UH, UK

Summary

Chromosome marking by cytosine methyiation has beenexamined in two gene systems in wheat - at the lociencoding high molecular weight (HMW) giutenin sub-units (seed proteins) and ribosomai RNA. Variation incytosine methyiation occurs between progeny in highlyinbred lines around the HMW giutenin locus. Thevariation is inherited through meiosis to F, and F2

generations but occasionally a new variant arises.Specific cytosine residues lose their methyl group in theseed, the organ where the genes are expressed.

Within the multigene family of ribosomai RNA genes,several subsets of genes can be defined based upon thecytosine methyiation patterns. High activity of aribosomai RNA gene locus is correlated with loss ofmethyiation at specific cytosine residues, especially in

the promoter and upstream regulatory regions. A modelis described in which the subset of genes selected to beused are those to which specific regulatory proteins andtranscription complexes bind most favourably. Bindingof such proteins inhibits cytosine methyiation and somarks the subset of genes for expression in subsequentcell generations. Examples are described where newtypes of RNA genes are introduced via sexual crossesthat result in changes to the methyiation patterns of theribosomai RNA genes. The processes determining thechanges begin, it is believed, in the fertilised egg.

Key words: wheat, cytosine methyiation, rRNA.inheritance, gene expression.

Introduction

There is now substantial evidence that the activity ofchromosome segments is correlated with modificationsof specific bases, most commonly cytosine.5-methylcytosine occurs especially frequently in plantgenomes in the symmetrical dinucleotide CG or in thetrinucleotide CXG (see Table 1). In plant species, from12 to 33 % of the cytosine residues are methylated(Wagner and Cespius, 1981). It is likely that themodification of cytosine provides information affectingmany kinds of nuclear processes. The best documentedis in mammalian genomes where the stable, inheritedmodification of DNA sequences around a gene caninfluence the binding of transcription and other factorsto the gene and hence influence its expression (Cedar,1988). Genes which contain a high proportion ofmethylated cytosines are usually inactive and may beinaccessible to the transcription machinery, while thosewhich are active or potentially active are not methyl-ated at critical cytosine residues. Results for plantspecies consistent with this general conclusion havebeen published for Agrobacterium T-DNA genes(Hepburn et al. 1983; Gelvin et al. 1983; van Slogteren etal. 1984 and Peerbolte et al. 1986), for rRNA genes(Blundyera/. 1987; Watson et al. 1987 and Flavell et al.1988), for maize zein storage protein genes (Bianchi

and Viotti, 1988) and for maize transposable elements(Chomet et al. 1987; Chandler and Walbot, 1986;Chandler et al. 1988; Schwartz and Dennis, 1986;Fedoroff, 1989 and Martienssen et al. 1990).

The studies on transposable elements (Fedoroff,1989; Martienssen et al. 1990) are particularly interest-ing, because they reveal not only the correlationbetween activity and the presence of specific unmethy-lated cytosines around the start sites of transcription,but also the activation and demethylation of partiallymethylated copies in a specific developmental patternand by the presence of additional active copies of theelement in the genome. The altered methyiationpattern can be inherited giving the element a differentactivity potential in the next generation. Whether theloss of methyl groups is due to active transcriptioninterfering with the methyiation process or whether it isthe consequence of regulatory proteins binding to theDNA before transcription and thus interfering withmethyiation is often unclear. However, several studieshave shown that methylated DNA is not transcribedwhen introduced into cells (Cedar, 1988).

Specific methyltransferases which use S-adenosyl-methionine as the donor of methyl groups are presentto modify the cytosine residues (Kirnos et al. 1981).Few detailed studies of the processes involved andtheir control have been carried out in plants, but

16 R. B. Flavell and M. O'Dell

Kirnos and collaborators have provided some datawhich show that newly replicated DNA fragments areundermethylated (Vanyushin and Kirnos, 1988). Muchof the methylation occurs after replication by a processthat does not involve DNA repair. The methylasespresumably recognise the hemimethylated symmetricalCG or CXG motifs and methylate the cytosine in thenew strand, unless something such as a DNA-boundprotein interferes with the process. The methylationprocess must be very efficient to ensure faithfulpropagation of the cytosine methylation pattern.

Estimates of the error frequency in the methylationprocess have not been made in plants. However, in amajor survey of maize plants regenerated from tissueculture, many changes in cytosine methylation wererecorded especially in a particular genotype (Brown,1989). These changes appear to be stably transmittedfrom one generation to the next. While phenotypicchanges are frequent in these plants propagatedthrough tissue culture, there is no evidence yet to provethat this is due to altered gene expression as a result offailure to methylate a specific cytosine residue.

Recently we have initiated studies to investigatevariation in cytosine methylation in hexaploid bread-wheat at specific loci that encode high molecular weightglutenin subunits which are major seed proteins. Wehave also studied the inheritance of this variation. Thisseemed a worthwhile study because if methylation ofcytosine residues is an important feature of the controlof the activity of chromosome segments, then a betterunderstanding of the control and fidelity of themethylation process in plants possessing high levels ofcytosine methylation is desirable.

Another way of examining the control of cytosinemethylation is to study a large number of copies of thesame gene. The ribosomal RNA genes constitute a verylarge multigene family in wheat, with between 8000 and15 000 members. Members of the family can becompared within and between genotypes to examinevariation in cytosine methylation.

In this paper we summarise results of these studieswhich demonstrate a strong correlation between thestatus of cytosine methylation at specific sites and geneexpression, although cytosine methylation at other sitesappear not to show such correlation. Variation incytosine methylation at specific sites was uncoveredbetween inbred plants and between members of the

Table 1. Nearest-neighbour analysis of m'C indifferent methvlated sequences present in plant DNA

DNA sequence

C-GC-AC-TC-CC-A-GC-T-GC-A-T

Data are from Gruenbaum et al

% methvlation

8219197

>S0

>so< 4

(19S1).

rRNA multigene family. Some of the methylationmodifications are inherited through meiosis.

Variation in cytosine methylation at the highmolecular weight glutenin loci in leaf tissue

The genes for high molecular weight (HMW) gluteninsubunits are important because they are major con-tributors to the visco-elastic properties of dough madefrom flour (Payne et al. 1981; Flavell et al. 1989). Thereare three pairs of HMW glutenin genes in hexaploidwheat, one pair on each of the closely related group 1chromosomes, 1A, IB and ID. When nuclear DNAfrom the variety Chinese Spring is restricted withBamH\ and probed with the cDNA encoding a HMWglutenin, eight fragments ranging from 2.0 to 10.2 kbpare detected (Fig. 1A). From studies on DNAs fromaneuploid lines lacking chromosomes 1 A. 1B or ID thechromosomal origin of each of the fragments has beendetermined. When DNAs from different seedlings ofChinese Spring, regenerated from callus tissue initiatedfrom scutellum, were treated with BamHl and Hha\ (anenzyme which does not cleave GCGC sites when theinternal cytosine is methylated) then several differenthybridisation patterns were obtained. Out of a sampleof 36 seedlings studied, 7 variant patterns were seen.Some are shown in Fig. 1A. The variation is due tovariation in cytosine methylation at one or more of theHha\ sites on the BamW\ fragments containing theHMW glutenin genes. For example, in one plant (E2a,Fig. 1A) the 10.2 kbp fragment of the locus onchromosome ID is cleaved to give a 5.1 kbp fragment,while in another (Ela) it is cleaved to give a 2.9 kbpfragment (Fig. IB). Occasionally both kinds of frag-ment are visible (E3a, Fig. 1A). Such variation wasunexpected given that Chinese Spring seeds are highlyinbred. Seedlings grown directly from inbred seed werealso examined. Similar variation was seen. Theseresults show that variation in cytosine methylationarises during plant life cycles and probably also duringtissue culture.

The variation is stably inherited in most cases. Thiswas established by making crosses between individualswhose methylation patterns in leaf DNAs are different.The F| progeny had the additive pattern of hybridisingfragments expected if homozygous chromosomal pat-terns were transmitted through meiosis, the zygote andperpetuated somatically. The methylation patterns ofF, plants were unaffected by which plant donated theegg or the pollen. This implies that the single pollen andegg cells had the same methylation patterns as the leafand stem cells. A few F2 progeny have also beenexamined. Parental and heterozygous patterns weredetected endorsing once again the stability of methyl-ation patterns during meiosis. In addition some newmethylation patterns have been detected in F2 progeny.The time in development when the variation arose isnot known. It could have occurred in the F| plantsbefore meiosis. during meiosis, post meiosis duringzygote formation or during development and growth of

E1aE3a E7a E12a _ ,

E2a E4a E10a E13aVariation of cytosine methylation in wheat 17

not surprising, given that 3 out of our original sample of19 Chinese Spring seeds were variants.

«• * • * '

— 9.7

— 5.1

— 3.5

_ 2.9

— 2.3

— 2.0

Methylation changes in the seed

The HMW glutenin genes are expressed only in theseed. Therefore it was interesting to study themethylation patterns in and around the loci in DNAisolated from the developing seeds and compare themwith those of other organs. After treatment withBamHl and Hha\, similar patterns of fragments wereobserved in the endosperm and leaf samples. However,some additional 1.4 and 1.6 kbp fragments wereproduced in the endosperm and these must be due tocleavage of Hhal sites close to or within a HMWglutenin gene. These sites are not cleaved in leaf DNAof the same plant. Also, the sites giving rise to the2.9 kbp fragments (see Fig. IB) were unmethylated inseed of the plant where they were methylated in the leafcells. These discoveries imply that the undermethyla-tion of these specific sites in the seed, most of which isthe endosperm tissue, is associated with gene ex-pression.

BamHI

BamH\/Hha\

CS tissue cultured plants

' Clu-ID-1BtmiHl

Hhal

_JL_Hhal Hhal

BamHl

10.9 -

8.1

• 5 . 1

• 2 . 9 -Sizes in kb pairs

Fig. 1. (A) Variation at the HMW glutenin subunit locuscontrolling cytosine methylation at the Hhal sites. Plants(Ela to EJ3a) were regenerated from scutellum tissueculture. DNA was extracted, treated with BamHl andHhal as shown, fractionated in agarose by electrophoresis,transferred to nitrocellulose (Southern, 1975) andhybridised with a 32P-labelled cDNA clone encoding aHMW glutenin subunit. The fragments from the Glu-1D-1locus are JO.2 kbp (BamHl), 5.J and 2.9 kbp(BamHl-Hhal). The other fragments are from the Glu-1D-2, Glu-lA-1, Glu-lA-2, Glu-1B-1 and Glu-lB-2 loci.Size markers record kilobase pairs. (B) Map of the Hhalsites in the BamHl fragment from the Glu-Dl-1 locus. TheHhal sites give rise to the 5.1 and 2.9kbp fragments visiblein (A).

the F2 plant. The methylation change did not occur inall copies of the glutenin gene subunits which implies itmight have arisen late during seedling development.Alternatively, the change may have occurred in onlyone of the two alleles. The discovery of new variation is

Cytosine methylation and expression ofmembers of the ribosomal RNA gene family

In the second system we examined the heterogeneityamongst the members of the large family of ribosomalRNA genes in wheat. Cytological studies at the lightand electron microscope level as well as studies onstocks carrying major ribosomal DNA deletions haveshown that there is an excess number of genes and notall are required (Flavell el al. 1988). Indeed perhapsfewer than 20% are used. What determines how manyand which subset of the genes are used? Are the choicesmade during every cell generation? Are the activegenes marked differentially from the inactive set?

The rRNA genes are organised in long tandem arrayscalled nucleolus organisers. The plethora of rRNAgenes are remarkably uniform in sequence due torecombination and gene conversion events whichhomogenise the sequences over time within andbetween the complex loci (Flavell, 1985). However,there is considerable heterogeneity within the genefamily with respect to the patterns of cytosine methyl-ation. Many of the genes are methylated at most or allof the CG residues assayed by restriction enzymes.Another subset of the genes contains one or a fewunmethylated cytosines but many different sites areinvolved. The third class carries an unmethylatedCCGG site at 165 base pairs upstream from the start oftranscription.

The number of genes which carry one or moremethylated CCGG sites was studied in related plantsthat differ greatly in the total number of rRNA genes(Flavell et al. 1988). As the total number of rRNA genesincreases, the proportion containing one or moreunmethylated CCGG sites decreases. In addition.

18 R. B. Flavell and M. O'Dell

S Kb

I I I I I I If

I I III I IfiDO

i i iD D D n

OD

25S 16 S

Fig. 2. Map of intergenic region of a ribosomal RNA gene from wheat. Bottom line shows twelve A (135bp), two C. twoD. and three B repeats, recognised from the complete nucleotide sequence (Barker et al. 1988). that lies between the 25Sand 18S coding sequences. These latter sequences are shown hatched. Transcription is initiated between D and C repeats(Vincentz and Flavell. 1989). The upper two lines indicate some of the restriction sites and the lengths of DNA involved.See Barker et al. (1988) for details.

amongst the genes that contain unmethylated CCGGsites, fewer genes contain more than one unmethylatedsite. This relationship suggested that the total numberof unmethylated CCGG sites in rDNA might berelatively constant between genotypes, the sites beingdistributed (but not at random) among the availablerRNA genes. The extent to which the number ofunmethylated cytosines is controlled is difficult todefine with precision but it is clear that the cytosinemethylation patterns in rDNA are highly regulated.Furthermore, these different patterns of methylationcorrelate with nucleolus activity as determined cytologi-cally (Flavell et al. 1988) and described below.

In hexaploid wheat there are two major nucleolarorganiser loci, on chromosomes IB and 6B respect-ively. Each ribosomal RNA gene of the array isassociated with an intergenic region. This regioncontains amongst other features an array of 135 bprepeats as shown in Fig. 2 (Barker et al. 1988). In somevarieties the genes at the IB locus produce a moreactive nucleolus than those at the 6B locus while inother varieties it is the reverse. The genes at the two lociare often distinguishable because in the intergenicregion there are different numbers of 135 bp sub-repeats which are responsible for the production ofrestriction fragments of different lengths when DNA istreated with an appropriate restriction endonuclease. Ithas thus been possible by combining cytological assaysof nucleolus volume with the methylation status ofspecific restriction fragments to examine the relation-ship between nucleolus volume, gene number andmethylation status of the genes in specific IB and 6Bloci present together in the same cell. (Flavell et al.1988; Sardana and Flavell, unpublished).

The loci which give the larger nucleolus in a cellinvariably have a larger number of rRNA genes withouta methylated cytosine at the -165 CCGG site.Conversely, an inactive or weakly active nucleolusorganiser has a much higher proportion of its rRNAgenes methylated at all the CCGG and GCGC sitesassayed. These results imply a correlation between theextent of cytosine methylation and locus activity; anactive locus is associated with non-methvlation of

cytosine residues. The genes at a more active locus alsohave more 135 bp repeats in the intergenic region. Thiscorrelation suggests that 135 bp repeats may act asenhancers of gene activity, as the equivalent repeatshave been shown to do in Xenopus (Reeder, 1984). The135 bp repeats also have an interesting pattern ofcytosine methylation. In almost all those genes with the-165 CCGG site unmethylated, one or more of the135bp repeats has a GCGC site unmethylated. Theobservation that only one or a few of the 135 bp repeatsin such genes are unmethylated is especially interestingbecause the primary sequences of all the 135 bp repeatsare essentially identical. Furthermore, the distributionof unmethylated 135 bp repeats in the tandem array isnot random. The probability of a 135 bp repeatcontaining an unmethylated GCGC site is much greaterin the 3' half of the array. The more active nucleoli aretherefore characterised by having more genes withunmethylated 135 bp repeats close to the promoter.How is this achieved? How is this subset of genesdistinguished by unmethylated sites selected?

A model to account for the rRNA genemethylation and activity patterns

A model and working hypothesis to relate genemethylation and expression has been presented else-where (Flavell et al. 1986, 1989). We have proposed thatspecific proteins in limiting concentrations can bind co-operatively to sequences in the promoter and the 135 bprepeats when certain cytosines are not methylated. Wehave recently recognised a protein species in cellextracts that can bind to sequences in these regions (S.Jackson and R. B. Flavell, unpublished observations).When the proteins are bound, the DNA is preventedfrom being methylated in these regions, which in turnprevents it being wrapped up in heterochromatin andthereby becoming inaccessible to transcription com-plexes. The model further postulates that these proteinsfacilitate the binding of transcription complexes andthus play an important role in the control of transcrip-tion. The co-operative binding of such proteins andtranscription processes in a competitive manner be-

Variation of cytosine methylation in wheat 19

tween the rRNA gene variants available would lead to asituation where sufficient genes with a greater capacityto bind transcription complex proteins are distinguishedfrom the total set of rRNA genes by lacking methylatedcytosines at specific sites. Because the work on rRNAgenes and the studies on the HMW glutenin regionpresented earlier show that methylation patterns areinherited somatically and even through meiosis, then itis reasonable to propose that the marking of the genesby de-methylation ensures that the same subset, by andlarge, is used from one cell generation to the next andthe competition process between genes for specificprotein binding does not involve all the genes in everycell cycle.

However, when new rRNA genes are introduced viaa sexual cross, the situation changes because genes of adifferent competitive ability may be added. This isfrequently the case when wide crosses are made.Suppression of the nucleolar organisers of one speciesin the presence of those of another is common in plantsand animals (Flavell el al. 1986). We have described thesituation in wheat-rye hybrids (Flavell, 1989) and alsoin the wheat lines in which the nucleolar organiserbearing chromosome from a wild relative, Aegilopsumbellulata, has been added (Martini et al. 1982). Thewheat nucleolus organisers are suppressed while theextra one of the Aegilops species is very active.Consistent with the results presented above, manymore of the wheat rRNA genes are methylated at allsites assayed in the presence of the Aegilops umbellulatachromosome, while many of the Aegilops umbellulatarRNA genes have unmethylated sites in the intergenicregulatory regions (Flavell et al. 1988).

A similar situation occurs in wide crosses betweenHordeum species. Cytological analyses of the fertilisedegg cells and cells of subsequent generations (R. Finch,M. D. Bennett and R. B. Flavell, unpublishedobservations) have shown that while nucleolar orga-nisers of both parents are active in the fertilised egg cell,only those of one parent can be observed after 2 or 3 cellcycles. We presume that these cytological changes areaccompanied by the molecular changes in methylation,and therefore the restructuring of the rRNA gene locito mark the set of genes potentially most useful to thecell occurs as soon as the new genotype is established,i.e. in the fertilised egg. This subset, we predict, isperpetuated by somatic inheritance of the methylationpattern.

Concluding remarks

The analyses of both sorts of loci described here areconsistent with the conclusion that methylation ofcytosine at CG dinucleotides is very common in plantsand that loss of the methyl group from specific sites iscorrelated with gene expression or the potential for thegene to be expressed.

As we have described for the rRNA genes, thisspecific interference with the methylation process islikely to involve the binding of specific regulatory

proteins and possibly transcription complexes.Although it can be argued whether the initial eventinterfering with methylation comes before or aftertranscription, the inheritance of a methylation patternraises the important point that once the pattern isestablished in cells of a tissue or physiological state, it isperpetuated through cell division to predispose thegene in derived cells to be accessible to regulatoryproteins and transcription complexes. Thus, the choiceof which gene template to use need not be made denovo in each daughter cell after cell division. Rather, itis established for a particular cell lineage and stablyinherited through cell division. Chromosome or genelocus marking associated with the modification of basesmay therefore play a very significant role in develop-ment. This theme is elaborated in the discoveries ofgenomic imprinting in mammals in which cytosinemethylation patterns are determined by whether thegene was inherited from the father or the mother(Sapienzaefa/. 1987; Reik etal. 1987). It is also relevantto the studies on plant transposable elements whosemethylation state varies during development, alteringthe potential activity of the element (Federoff, 1989;Martienssen et al. 1990). The activity of the elementitself or another element in the genome is involved inthe methylation change, suggesting that the transposaseproduct may interact with the element's DNA andinterfere with the methylation process. Because inplants the egg and pollen cells are formed from somaticlineages, changes such as these in somatic cells can beinherited meiotically to alter the potential activity of anelement in the next generation. The extent to whichinherited epigenetic changes are a source of geneticvariation in evolution needs to be assessed. Methylatedcytosine residues have higher mutation rates than non-methylated residues (Anitiquera and Bird, 1988).Therefore, active genes may be shielded from mutationcompared with inactive ones.

While some cytosine methylation changes are associ-ated with developmental changes in gene expression itis obvious that a change in the DNA template isinsufficient for gene expression - the appropriatetranscription factors are also essential. Thus errors inthe methylation process would not lead inherently toerrors in transcription, although in certain situationsthis could occur. In this paper we have describedmethylation variants at specific sites around the HMWglutenin loci and within the large rRNA multigenefamily of a specific plant. These may result from 'errors'and have no consequences in leaf cells either becausethe transcription factors are not present (gluteninsubunit genes) or because the particular sites necessaryfor transcription are not unmethylated. If several sitesmust be unmethylated to facilitate access to transcrip-tion factors, then the probability of all these beingunmethylated by error is very low, and biologicalaberrations will be similarly low. Therefore theinherited variation that we have revealed around theHMW glutenin locus may be genomic noise of littlebiological significance. No variation in the levels ofHMW glutenins accumulated in seeds has been found

20 R. B. Flavell and M. O'Dell

to be associated with this variation (J. Rogers,unpublished observations). However, such variationprovides a useful marker to study the inheritance ofspecific chromosome fragments. This is especiallyvaluable in a highly inbred organism where RFLPvariation is not as extensive as in other species.Exploitation of cytosine methylation variation inpedigree analysis has already been described for humanfamilies (Silva and White, 1988).

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