An Inverted Repeat Triggers Cytosine Methylation of Identical Sequences in Arabidopsis

Molecular Cell, Vol. 3, 505–511, April, 1999, Copyright 1999 by Cell Press

An Inverted Repeat Triggers Cytosine Methylation of Identical Sequences in Arabidopsis Bradley Luff, Laura Pawlowski, and Judith Bender* Department of Biochemistry Johns Hopkins University School of Public Health Baltimore, Maryland 21205

Summary The Wassilewskija (WS) strain of Arabidopsis has four PAI genes at three sites: an inverted repeat at one locus plus singlet genes at two unlinked loci. These four genes are methylated over their regions of DNA identity. In contrast, the Columbia (Col) strain has three singlet PAI genes with no methylation. To test the hypothesis that the WS inverted repeat locus triggers methylation of unlinked identical sequences, we introduced this locus into the Col background by genetic crosses. The inverted repeat induced de novo methylation of all three unmethylated Col PAI genes, with methylation efficiency varying with the position of the target locus. These results, plus results with inverted repeat transgenes, show that methylation is communicated by a DNA/DNA pairing mechanism. Introduction Many species of animals, plants, and fungi modify their genomic DNA by methylation of the 5 position of cytosine (5-Me-C). Eukaryotic cytosine methylation is found predominantly on repetitive sequences (Yoder et al., 1997; Finnegan et al., 1998). However, within methylated regions of the genome, there is little sequence specificity that determines which cytosines become methylated: in mammals, cytosines in the minimal symmetric context 59-CG-39 are methylated (Bestor and Tycho, 1996), while in fungi (Selker et al., 1993; Goyon et al., 1994) and higher plants (Martienssen and Baron, 1994; Meyer et al., 1994; Jacobsen and Meyerowitz, 1997; Jeddeloh et al., 1998), both symmetrically and asymmetrically disposed cytosines are methylated. Cytosine methylation usually correlates with a loss of gene expression. For example, methylation in mammalian genomes is involved in inactivation of one of the two X chromosomes in females (Panning and Jaenisch, 1998) and with imprinting of certain genomic loci where one allele is methylated and silenced whereas the other allele is hypomethylated and expressed (Jaenisch, 1997). Inappropriate methylation of tumor suppressor genes has also been implicated in cancer (Baylin et al., 1998). Several examples of methylation-correlated silencing have been documented in plants, affecting endogenous genes (Das and Messing, 1994; Bender and Fink, 1995; Jacobsen and Meyerowitz, 1997), transposable elements (Banks et al., 1988; Brutnell and Dellaporta, 1994; Martienssen and Baron, 1994), and * To whom correspondence should be addressed (e-mail: jbender@ welchlink.welch.jhu.edu)

transgene sequences (Meyer et al., 1993; Matzke et al., 1994; Ye and Signer, 1996; Davies et al., 1997; Mittelsten Scheid et al., 1998). In petunia and Arabidopsis thaliana, transgenes methylated in their promoter regions have been shown by nuclear run-on analysis to have impaired transcription initiation (Meyer et al., 1993; Ye and Signer, 1996). In the fungi Neurospora crassa and Ascobolus immersus, methylation in the body of the gene has been shown to impede transcription elongation (Barry et al., 1993; Rountree and Selker, 1997). The phenotypes of methylation-impaired mutants suggest that genomic cytosine methylation plays a critical role in the biology of organisms that have evolved with methylation systems. For example, mouse mutants deficient in a cytosine methyltransferase gene die during embryogenesis (Li et al., 1992). Methylation-impaired strains of Arabidopsis display progressive developmental defects (Finnegan et al., 1996; Kakutani et al., 1996; Ronemus et al., 1996). An Ascobolus deletion mutant in a gene with methyltransferase homology is sterile in homozygous matings (Malagnac et al., 1997). The underlying mechanisms of these abnormalities remain to be determined, but it has been speculated that they reflect, either directly or indirectly, inappropriate expression of methylation-controlled genes. Little is known about the mechanisms that establish methylation patterns on particular regions of the eukaryotic genome. In Neurospora and Ascobolus, duplicated sequences are methylated de novo specifically in premeiotic cells (Selker, 1990, 1997). A number of lines of evidence suggest that duplications in these fungi are detected as substrates for methylation by a DNA/DNA pairing mechanism (see Discussion). In plants, repeated sequences generated by genomic rearrangements or transformation are also frequently methylated de novo (Matzke et al., 1996; Finnegan et al., 1998). Several models have been proposed to account for plant methylation phenomena including sensing of repeated DNA by a DNA/DNA pairing mechanism, sensing of repeated DNA by overexpression of transcripts that participate in RNA/ DNA pairing, perception of repeated DNA as foreign, and perception of repeated DNA as transposon-like (Matzke and Matzke, 1995; Matzke et al., 1996). However, a detailed mechanism for de novo methylation has not been determined for any plant system. Similarly, mechanisms of de novo methylation have not yet been elucidated in animal systems (Bestor and Tycho, 1996). We previously found that a naturally occurring rearrangement in an endogenous Arabidopsis gene family, the PAI genes, is associated with methylation of the gene family members. The PAI genes encode enzymes that catalyze the third step of the tryptophan biosynthetic pathway. In the standard strain Columbia (Col), there are three nearly identical PAI genes at three unlinked genomic sites, and these genes have no methylation (Bender and Fink, 1995). However, in the Wassilewskija (WS) strain there is a duplication at one of three PAI loci to yield a tail-to-tail inverted repeat of two PAI

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Figure 1. PAI Gene Structure in Col and WS The MspI restriction maps of the WS PAI1–PAI4 locus, the Col PAI1 locus, and the PAI2 and PAI3 loci (which are identical between WS and Col) are shown. Sites internal to the PAI-identical sequences and the next closest flanking sites are indicated by M. Black arrows indicate PAI genes, with PAI3 hatched to represent reduced identity between this gene and its sister genes. Gray arrows indicate 2.9 kb direct repeat sequences that flank the WS PAI1–PAI4 genes and the Col PAI1 gene. The direct repeat downstream of Col PAI1 is partially deleted. The region covered by the PAI probe used in Southern blot analyses is indicated. The white arrowheads in the PAI4 gene and the pai4 59 partial duplication indicate the orientation and extent of the duplicated sequences. The white box indicates a patch of heterologous sequence upstream of Col PAI1.

genes (Figure 1), and in this strain there is dense methylation of all four PAI sequences. One of the PAI genes in the inverted repeat is expressed at a high level despite being methylated and confers a normal plant phenotype (Bender and Fink, 1995; J. B., unpublished data). In this report, we dissect the signals that direct methylation to particular sequences using the Arabidopsis PAI genes as a model system. We show that the inverted repeat PAI locus can trigger methylation of identical sister gene sequences elsewhere in the genome. The patterns of methylation, and dependence of methylation efficiency on the relative genomic positions of the inverted repeat initiator locus and target unmethylated loci, suggest that de novo methylation is established by DNA/DNA pairing. Our results offer insights into why certain complex transgenes trigger methylation and silencing in higher eukaryotes, and suggest strategies to design transgenes that either avoid or induce methylation of identical resident sequences. Results WS PAI Methylation Is Dense, Asymmetric, and Coextensive with PAI Sequence Identity The WS strain of Arabidopsis carries four PAI genes at three unlinked sites (Bender and Fink, 1995). At one locus on the upper arm of chromosome 1, there is a tail-to-tail inverted repeat of almost identical genes, PAI1–PAI4, flanked by 2.9 kb of perfect direct repeat sequences (Figure 1). Immediately beyond the PAI1proximal direct repeat, there is a partial duplication, pai4 59. At a second locus on the upper arm of chromosome 5, there is an almost identical singlet gene, PAI2. At a third locus in the middle of chromosome 1, there is a divergent singlet gene with approximately 90% identity to its sisters, PAI3. All four full-length PAI genes were previously found to be heavily methylated over their

regions of DNA sequence identity by Southern blot analysis (Bender and Fink, 1995). To obtain a detailed picture of PAI methylation patterns, we used a bisulfite mutagenesis genomic sequencing method that monitors the methylation status of every cytosine in a sequenced region (Frommer et al., 1992). PAI1 and PAI2 promoter top and bottom strands were PCR amplified, cloned, and sequenced from bisulfite-mutagenized WS genomic DNA. This analysis revealed that PAI methylation could occur at any cytosine residue, regardless of whether it was in a symmetric context (59-CG-39 or 59-CNG-39) or in an asymmetric context (Figure 2). However, the dense methylation of PAI1 and PAI2 ended at or near the boundary of identity, which is shared with PAI4 and pai4 59. In over half of the molecules sequenced, methylation stopped at this boundary, and in the molecules where methylation continued beyond this region, it became less dense. The promoter of the PAI3 gene, which lacks the upstream identical region, was also sequenced. The dense methylation in the body of PAI3 ended at the breakpoint of identity with the other genes (Figure 2). In addition, the downstream identity boundary of the partial pai4 59 duplication was sequenced to determine whether this segment, which has only 731 bp of identity to its sister PAI genes, is methylated. The segment was as densely methylated as the full-length WS PAI genes, and the dense methylation became reduced beyond the point in the second intron sequence where the duplication terminates (data not shown). These sequencing data are consistent with the model that nucleic acid identity determines cytosine methylation patterns. The PAI1–PAI4 Inverted Repeat Triggers De Novo Methylation We used genetic crosses between WS and Col to test the hypothesis that PAI methylation in WS is signaled by the PAI1–PAI4 inverted repeat locus. We found that when the WS inverted repeat was combined with unmethylated Col PAI1, PAI2, or PAI3 genes, the unmethylated Col genes were methylated de novo within a few generations of inbreeding (see below). These results suggest that the WS inverted repeat or a tightly linked locus provides the primary signal for methylation of PAIidentical sequences elsewhere in the genome. WS and Col were crossed, and F2 progeny were screened for PAI genotypes with linked polymorphic markers. Several independent hybrid lines were identified that were homozygous for the WS PAI1–PAI4 inverted repeat locus and homozygous for the Col PAI2 and/or PAI3 genes (Hybs 1–3, Figure 3). DNA was prepared from F3 progeny plants of each F2 hybrid individual for Southern blot analysis of methylation. Methylation patterns in hybrid lines were followed with a standard HpaII/MspI restriction digest assay that monitors methylation of an internal site for each PAI locus (Figure 1). The methylation of HpaII/MspI sites parallels the methylation observed at several other sites across each WS PAI gene and the methylation patterns determined by sequencing, and it thus serves as a diagnostic marker for the overall methylation status of each PAI locus (Bender and Fink, 1995; Jeddeloh et al., 1998). F3 Hyb1–3 lines displayed substantial de novo methylation

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Figure 2. Methylation Patterns in PAI Promoter Regions Bisulfite genomic sequencing of methylation patterns was performed for the top and bottom strands of the indicated PAI genes, with eight independent molecules sequenced for each strand. Vertical lines indicate the positions of cytosines, with the height of each line representing how many sequenced molecules had 5-Me-C at that position. Black indicates cytosines in the context 59-CG-39, blue indicates cytosines in the context 59-CNG-39, and red indicates cytosines in an asymmetric context. Asterisks indicate sites where none of the sequenced molecules had a 5-Me-C. The black horizontal line indicates the region of PAI identity, and the gray horizontal line indicates flanking upstream heterologous sequence unique to each gene. For each PAI gene, the transcription start site lies just interior to the point where PAI3 identity to PAI1 and PAI2 begins.

of Col PAI2 but no methylation of Col PAI3 (Figure 3). In representative inbred lines, PAI2 became as densely methylated as in WS by the F4 generation. PAI3 was methylated more stochastically between the F4 and F6 generations, with only some lines achieving full methylation. The effect of the WS PAI1–PAI4 locus on the allelic Col singlet PAI1 gene was tested by combining the two alleles in a heterozygote. DNA prepared from F1 heterozygotes showed a small amount of a methylated Col PAI1 species when WS but not Col was the female parent in the cross (data not shown). Therefore, de novo Col PAI1 methylation can occur at a low efficiency during the F1 generation, but factors that mediate this process behave differently when passed through the female versus the male gametes. Methylation of Col PAI1 in heterozygous plants was also monitored beyond the F1 generation using F2 plants heterozygous for WS PAI1–PAI4/Col PAI1 but homozygous for Col PAI2 and Col PAI3 (four independent P1Hyb lines). F3 progeny of each line were

screened to identify segregating individuals homozygous for Col PAI1. DNA prepared from F4 progeny of each homozygous segregant line displayed substantial methylation of PAI1 (Table 1). Progressive methylation changes in P1Hyb lines were determined by inbreeding heterozygous siblings of each line so that the PAI1–PAI4 locus was present in heterozygous form for three, four, or five generations before being segregated away (F5, F6, and F7 columns, respectively, in Table 1). PAI2 became methylated by the fourth generation, but PAI3 was not methylated even after five generations. PAI Methylation Is Maintained in the Absence of the PAI1–PAI4 Inverted Repeat To determine whether the inverted repeat locus is required for maintenance of sister gene methylation, we examined PAI methylation in progeny of WS 3 Col crosses where the WS PAI1–PAI4 inverted repeat was segregated away but the methylated WS PAI2 and/or PAI3 genes remained (Hybs 4–6, Figure 3). PAI methylation was maintained for at least five generations, but Figure 3. De Novo Methylation in WS 3 Col Hybrids

(A) F2 progeny of WS 3 Col crosses were screened for individuals homozygous for all permutations of the WS and Col PAI loci (7 Hyb1, 2 Hyb2, 4 Hyb3, 4 Hyb4, 4 Hyb5, and 1 Hyb6). DNA prepared from F3 progeny of individual hybrids was tested for PAI methylation by HpaII(H)/MspI(M) Southern blot with an internal PAI probe. (B) The same hybrid lines illustrated in (A) were inbred for three more generations, and DNA prepared from F6 progeny was tested for PAI methylation. Black indicates WS DNA, and gray indicates Col DNA. Arrows indicate PAI genes. The arrow corresponding to PAI3 is hatched to represent the reduced identity between this gene and its sister genes. Boxes around arrows indicate cytosine methylation, with the thickness of the line corresponding to the density of methylation observed. The upper chromosome corresponds to chromosome 1 carrying the PAI1–PAI4 locus and the unlinked PAI3 gene. The lower chromosome corresponds to chromosome 5 carrying the PAI2 gene. Asterisks indicate the positions of bands diagnostic of methylation.

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Table 1. Methylation Patterns in Inbred Hybrid Lines Segregated from a PAI1–PAI4/PAI1 Heterozygote PAI1 Methylation

PAI2 Methylation

PAI3 Methylation

P1Hyb

F4

F5

F6

F7

F4

F5

F6

F7

F4

F5

F6

F7

Line Line Line Line

1a 1 1 1

1 1 1 1

1 1 1 1

1 1 1 1

1 2 2 2

1 2 2 2

1 1 1 1

1 1 1 1

2b 2 2 2

2 2 2 2

2 2 2 2

2 2 2 2

a b

1 2 3 4

A plus sign denotes detectable cytosine methylation as determined by a HpaII/MspI Southern blot assay. A minus sign denotes no detectable cytosine methylation as determined by a HpaII/MspI Southern blot assay.

the density of methylation was reduced. Sequenced methylation patterns for PAI2 from a representative hybrid line showed that the residual methylation was almost entirely on symmetric cytosines (Figure 2). Neither a methylated PAI2 nor a methylated PAI3 singlet locus triggered de novo methylation of unmethylated PAI sequences (Figure 3). Similarly, homozygous P1Hyb lines with a methylated Col PAI1 singlet gene (the F4 generation lines tested in Table 1) showed no methylation changes upon inbreeding (data not shown). To determine whether a methylated singlet can trigger the methylation of an allelic singlet, we identified four F2 plants homozygous for Col PAI1 and PAI3, but heterozygous for WS PAI2/Col PAI2. The heterozygous form was inbred for two or three generations before segregating away the WS PAI2 allele, and homozygous Col PAI2 progeny were tested for methylation. No de novo PAI methylation was detected (data not shown).

A Promoterless pai1–pai4 Transgene Triggers De Novo Methylation To more precisely map the determinants at the PAI1– PAI4 locus that trigger methylation, we constructed a promoterless inverted repeat pai1–pai4 transgene and introduced it as a single-copy insert in the Arabidopsis genome. In each of five independent random insertion lines examined—3 in Col and 2 in a WS derivative, Rev1, where the endogenous PAI1–PAI4 locus is deleted and the remaining singlet genes are unmethylated (Bender and Fink, 1995)—the homozygous pai1–pai4 construct displayed de novo methylation over the symmetrical portions of the sequence within two generations of inbreeding. The methylation became progressively more dense in subsequent generations without a spread to flanking regions (Figure 4). No fortuitous transgene RNA expression was detected with an RT-PCR assay. These results show that the inverted repeat structure per se rather than an RNA or protein product of the endogenous locus provides a methylation signal. In no single-copy line did the promoterless transgene trigger methylation of endogenous PAI genes. However, in some multicopy lines both transgenes and endogenous PAI genes were methylated by the second homozygous generation. Therefore, it is likely that the promoterless transgene carries all the sequence determinants for self- and trans-methylation, but when present in single copy it might require several additional generations of inbreeding and/or an appropriate insertion site to trigger trans-methylation.

Discussion Our results show that a locus carrying a methylated inverted repeat arrangement of PAI genes triggers de novo cytosine methylation of three target PAI sequences elsewhere in the Arabidopsis genome. In contrast, methylated singlet PAI genes do not promote methylation of either unlinked or allelic unmethylated singlet PAI targets. Three additional lines of evidence indicate that the inverted repeat PAI gene structure per se rather than a gene product or tightly linked locus causes the effect. First, a single-copy promoterless pai1–pai4 inverted repeat transgene cassette triggers its own methylation (Figure 4) in either the WS or the Col background. Second, methylation is directed to PAI sequences specifically over the regions of shared identity with the inverted repeat genes (Figure 2). Third, in a survey of PAI structure and methylation in other Arabidopsis strains, only strains carrying an inverted repeat at the PAI1 locus display PAI methylation (J. B., unpublished data). Consistent with our observations, inverted repeats and/or complex tandem repetitive arrays have been correlated with cytosine methylation and gene silencing effects in a number of plant and animal systems (Dorer and

Figure 4. A Promoterless pai1–pai4 Inverted Repeat Transgene Becomes Methylated De Novo DNA was prepared from an inbred line carrying the homozygous pai1–pai4 transgene for two (2) or four (4) generations and analyzed by HpaII (H)/MspI (M) Southern blot with an internal PAI probe. Asterisks indicate the positions of bands diagnostic of methylation. The molecular weights of unmethylated and methylated transgene bands are indicated in the right margin. LB is the left border, RB is the right border, and kanR is the selectable marker on the transgene.

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Henikoff, 1994; Matzke et al., 1994; Kermicle et al., 1995; Ye and Signer, 1996; Davies et al., 1997; Stam et al., 1997; Chopra et al., 1998; Garrick et al., 1998; Mittelsten Scheid et al., 1998). Our experiments suggest that de novo PAI methylation is induced by DNA/DNA pairing. Strikingly, PAI methylation in the WS strain is coextensive with PAI DNA sequence identity, including 250 bp of upstream untranscribed sequences in the WS PAI1 and PAI2 genes, and intron sequences in all four WS PAI genes, without a significant spread into neighboring genes (Figure 2; Bender and Fink, 1995). Furthermore, PAI methylation is sensitive to the genomic positions of interacting segments. In crosses with PAI1–PAI4, the allelic identical PAI1 gene is methylated more rapidly than the unlinked identical PAI2 gene (Table 1). This observation suggests that during a DNA/DNA homology search, a PAI sequence at the allelic position has a higher probability of pairing with the PAI1–PAI4 locus than does the same sequence on a different chromosome. The reduced efficiency of PAI3 methylation (Figure 3; Table 1) suggests that this gene lies in an unfavorable genomic position for PAI interactions, although it might also reflect the reduced sequence identity between PAI3 and its sisters. Consistent with a DNA/DNA pairing model, a promoterless pai1–pai4 transgene triggers its own methylation over the regions of mirror image sequence within a few generations after insertion in the Arabidopsis genome (Figure 4), regardless of insertion site or strain background. This result argues strongly that an RNA molecule is not involved in PAI methylation, although RNAs have been proposed to trigger gene silencing and methylation in other plant systems, perhaps as a defense response to RNA viruses (Wassenegger et al., 1994; Matzke and Matzke, 1995; Jones et al., 1998; Mette et al., 1999). Based on our observations, we have proposed that the PAI1–PAI4 inverted repeat might form a hairpin or cruciform that serves as an ideal substrate for methylation just along the regions of PAI identity (Bender, 1998). This unusual structure might also trap unlinked identical sequences to promote their trans-methylation, whereas transiently paired singlet unlinked repeat sequences such as the PAI genes in Col or normally paired homologous chromosomes would not be efficiently recognized as methylation substrates. A number of lines of evidence suggest that repeatinduced methylation events in the fungi Neurospora and Ascobolus are triggered by DNA/DNA pairing. Methylation is coextensive with the duplicated sequences (Selker et al., 1993; Goyon et al., 1994), and methylation (or in Neurospora, methylation-associated mutations) is always observed on both copies of a duplication, suggesting that the methylation/mutation process is induced by interaction of the copies (Selker, 1997). Moreover, duplications that are genetically linked are detected and modified more efficiently than unlinked copies, suggesting that the ability of identical sequences to find each other during a genome-wide homology search influences the process (Selker, 1990; Goyon et al., 1996). Because methylation of PAI genes is also coextensive with sequence identity and sensitive to genomic position, the fungal and plant de novo methylation processes

might be mechanistically related. However, the methylation machineries in Ascobolus and Neurospora can efficiently detect two unlinked single-copy repeats whereas the unlinked PAI1, PAI2, and PAI3 genes in the Col strain of Arabidopsis do not serve as efficient methylation substrates. Therefore, the methylation activity that affects the PAI genes in Arabidopsis has different substrate requirements than the methylation activities in Neurospora and Ascobolus. Furthermore, repeat-induced fungal methylation occurs specifically in premeiotic cells, whereas de novo Arabidopsis PAI methylation can occur in postmeiotic cells: methylated Col PAI1 species are present during the F1 generation of plants made by crossing WS female and Col male gametes. The observation that PAI methylation primarily affects symmetrically disposed cytosines in the absence of the PAI1–PAI4 inverted repeat but affects both symmetric and asymmetric cytosines in the presence of the inverted repeat suggests that PAI genes in the two different contexts form different substrates for cytosine methylation (Figure 2; Jeddeloh et al., 1998). The PAI genes in the inverted repeat context might either stimulate the activity of additional methyltransferase enzymes or alter the activity of the enzyme(s) responsible for the basal level of symmetric methylation. The persistence of asymmetric PAI methylation in WS indicates that the PAI substrate formed in the inverted repeat context is continuously present, suggesting that methylation does not block PAI pairing. Consistent with this view, studies in Ascobolous suggest that methylation suppresses recombination at a step after the initial pairing of methylated sequences (Maloisel and Rossignol, 1998). Experimental Procedures Plant Growth, DNA Extraction, and Transgene Analysis Plants were grown under continuous illumination in Fafard Growing Mix #2 (Griffin Greenhouse Supplies). For experiments involving inbred lines, seeds were collected from a single plant in each generation. DNA was extracted from total plant tissue of approximately 20 4-week-old plants as described (Luschnig et al., 1998) and Southern blotted using standard methods (Jeddeloh et al., 1998). PAI genotypes were determined using linked polymorphisms, described in http://genome-www.stanford.edu/Arabidopsis/aboutcaps.html. The pai1–pai4 construct is an insertion of a 3.2 kb HincII fragment into the SmaI site of pBIN19 (Bevan, 1984). RT-PCR was performed with primers to PAI-identical exon sequences flanking a polymorphic second exon SacI site. WS PAI1 and PAI4 lack the site, but all other WS and Col PAI genes carry the site. Thus, RT-PCR products were cleaved with SacI to distinguish endogenous PAI transcripts (cut) from transgene transcripts (uncut). Bisulfite Genomic Sequencing of Methylation Patterns WS genomic DNA (10 mg) was cleaved with XhoI to separate the inverted repeat genes from each other, mutagenized with sodium bisulfite, and tested for efficiency of mutagenesis by analysis of the unmethylated ASA1 gene as described (Jeddeloh et al., 1998). PCR primers to amplify the PAI2 bottom strand were as previously described (Jeddeloh et al., 1998). Detailed information regarding the PAI2 top strand, PAI1 top and bottom strand, PAI3 top and bottom strand, and pai4 59 top and bottom strand primer pairs is available upon request from J. B. For each region analyzed, eight independent PCR products were cloned and sequenced. Acknowledgments The authors would like to thank Eric Selker and Cecile Pickart for critical comments on the manuscript. This work was supported by

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a March of Dimes Basil O’Connor Starter Scholar Award 5-FY980535 and a Searle Scholars Award 97-E-103 to J. B.

Jones, A.L., Thomas, C.L., and Maule, A.J. (1998). De novo methylation and co-suppression induced by a cytoplasmically replicating plant RNA virus. EMBO J. 17, 6385–6393.

Received November 13, 1998; revised January 28, 1999.

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