MOLECULAR AND CELLULAR BIOLOGY, Aug. 1992, p. 3372-3379
Vol. 12, No. 8
0270-7306/92/083372-08$02.00/0 Copyright X) 1992, American Society for Microbiology
Extrachromosomal Homologous DNA Recombination in Plant Cells Is Fast and Is Not Affected by CpG Methylation HOLGER PUCHTA,* SERGE KOCHER, AND BARBARA HOHN
Friedrich Miescher-Institut, P. O. Box 2543, CH-4002 Basel, Switzerland Received 9 March 1992/Accepted 1 May 1992
Using a sensitive transient assay, we investigated extrachromosomal homologous DNA recombination (ECR) in plant cells. As the plant genome is highly C methylated, we addressed the question of whether CpG methylation has an influence on DNA recombination efficiencies. Whereas the expression level of the fully CpG-methylated DNA molecules was reduced drastically, we found no significant changes in ECR efficiencies between two partly CpG-methylated plasmids or between one fully CpG-methylated and one nonmethylated plasmid. Using a modified polymerase chain reaction analysis, we were able to detect recombination between two fully CpG-methylated plasmids. Furthermore, we characterized the kinetics of the ECR reaction. Cotransfection of plasmids carrying truncated copies of the 1-glucuronidase (GUS) gene resulted in enzyme activity with a delay of only half an hour compared with that of the plasmid carrying the functional marker gene. This indicates that the ECR reaction itself requires no more than 30 min. By polymerase chain reaction, we were able to detect the recombined GUS gene as early as 2 h after transfection. This result and the time course of the transient GUS activity indicate that ECR occurs mainly early after transfection. The biological significance of this finding is discussed, and properties of ECR and intrachromosomal recombination are compared. The biological role of C methylation in eukaryotes has attracted wide interest in recent years (for reviews, see references 7, 8, 23, and 43). Plant genomes are especially highly methylated (32). C methylation of promoter regions in the plant genome could be correlated with gene inactivation (51), as is frequently found for animal systems. In addition, the expression of a gene transfected into tobacco protoplasts was inhibited by in vitro hemi-C (16, 50) or in vitro CpG methylation of the DNA (15). It has been shown that C methylation of specific sequence motifs present in different plant promoters leads to reduced binding of cognate transcription factors (18, 44). Methylation was also correlated with inhibition of transposition (14, 41) and with inhibition of expression of homologous genes in transgenic plants (19, 26, 31). In the present communication, we address the question of whether C (in this case, CpG) methylation influences recombination efficiencies. We cotransfected Nicotiana plumbaginifolia protoplasts with two nonreplicating plasmids carrying different deletions of the ,3-glucuronidase (GUS) gene. To detect recombination products, we used a transient extrachromosomal recombination (ECR) assay system (28, 38, 39) and a modified polymerase chain reaction (PCR) analysis (38). Applying the same techniques, we furthermore investigated the kinetics of the ECR reaction in plant cells. In plants, ECR occurs naturally in caulimoviruses (21) and geminiviruses (45) and between T-DNAs of Agrobacterium tumefaciens (33).
35S promoter and the nopaline synthetase terminator. pGUS 23N1 has a 187-bp deletion in the 5' part of the GUS gene and pGUS 23C1 has a 601-bp deletion in the 3' part of the gene. Plasmid DNA was prepared for the transfection experiments as described by using either Escherichia coli DH5at or dam mutant strain BZ 101. In vitro methylation of DNA. For in vitro methylation of supercoiled circular plasmid DNA, the enzymes M-SssI, M-HpaII, M-FnuDII, and M-HhaI were used at conditions recommended by the manufacturer (New England Biolabs, Schwalbach, Germany). For testing methylation efficiency, the methylated as well as nonmethylated plasmid DNAs were digested with the respective methyl-sensitive restriction nuclease (HhaI, HpaII, or BstUI; all from New England Biolabs) and were analyzed by gel electrophoresis. The methylation of the DNA was regarded as complete when no degradation of 1 ,ug of DNA by a methyl-sensitive enzyme was detectable by ethidium bromide staining. The methylated DNA was extracted with phenol, ethanol precipitated, and dissolved in sterile water, and its concentration was determined spectrophotometrically. For PCR analysis of recombination of M-SssI-methylated plasmid DNAs, the methylated molecules were first separated from trace amounts of nonmethylated molecules. The plasmids were digested with an excess of the restriction endonuclease HhaI, the digested samples were separated by agarose gel electrophoresis, and the respective supercoiled circular, monomeric plasmid bands were cut out of the gel. The DNA was eluted, extracted with phenol, precipitated with ethanol, and redissolved. The DNA concentration was determined spectrophotometrically. Isolation of protoplasts, transfection of DNA, and f8-glucuronidase assays. Mesophyll protoplasts of N. plumbaginifolia were enzymatically isolated from leaves of sterile shoot cultures. Polyethylene glycol-mediated transfections of protoplasts were done as previously described (38). For the analysis of the role of C methylation in recombination, transfections were performed with 1 p,g per plasmid and 9 p,g of carrier DNA per 0.6 x 106 protoplasts. Every experimen-
MATERIALS AND METHODS Cloning procedures. Standard cloning procedures were used (40). Construction of plasmids pGUS 23, pGUS 23C1, and pGUS 23N1 (Fig. 1) was described previously (38). The plasmids are pUC7 derivatives carrying 3-glucuronidase (GUS) sequences flanked by the cauliflower mosaic virus *
Corresponding author. 3372
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VOL. 12, 1992 35 S CpG-sites
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Kpnl for transfection of protoplasts. The plasmids are pUC7 derivatives carrying ,B-glucuronidase (GUS) of the used FIG. 1. Scheme plasmids sequences flanked by the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthetase terminator (Nos T). Plasmid pGUS 23N1 has a 187-bp deletion in the 5' part of the GUS gene and plasmid pGUS 23C1 has a 601-bp deletion in the 3' part of the gene (38). The GUS-specific overlap of pGUS 23N1 and pGUS 23C1 is hatched in pGUS 23. The number of methylated CpG sites in each region is indicated. The DNA was treated by M-HpaII, M-FnuDII, and M-HhaI (HFH) or M-SssI (S) methylase. The restriction sites for KpnI and SacI that were used to liberate the 2-kb-long recombined GUS gene are indicated with vertical open arrows. The recombined gene was detected by PCR with the primers pNl and pCl, both marked by horizontal black arrows.
tal series included transfections with 1 ,ug of nonmethylated as well as with methylated pGUS 23 (complemented by salmon sperm DNA to a total amount of 10 ,ug of DNA) as controls. An aliquot of protoplasts was mock transfected as a background control. Protoplasts were harvested 18 h after transfection. In addition to the described transfections (see Results) (Table 1), we also used in control experiments 5 ,ug or 250 ng of each plasmid, in the second case complemented with carrier DNA to 10 ,ug. In some experiments CsClpurified plasmid DNA instead of sheared salmon sperm
TABLE 1. Effects of CpG methylation on gene expression and ECR in protoplastsa Plasmid methylation status (no. of expts)
pGUS 23 pGUS 23HFH (3) pGUS 23s (4)
pGUS 23C1/pGUS 23N1 pGUS 23C1HF/pGUS 23NlHFH (5)
pGUS 23C1/pGUS 23N1s (4) pGUS 23C1S/pGUS 23N1 (5) pGUS 23C1S/pGUS 23N1s (5)
GUS activity
Recombination
efficiency"
1
1.30 ± 0.19 0.048 ± 0.028
1 1.19 ± 0.92 ± 0.052 ± 0.057 ±
0.68 0.30 0.049 0.022
1 0.92 0.92 NTC N'T
a The normalized average values of the indicated number of different cotransfection experiments (in parentheses) with standard deviations are given (normalizations in the upper section are to the value of pGUS 23, and those in the lower section are to that of pGUS 23C1/pGUS 23N1). GUS background of the plant cells, 0.014 ± 0.009 (4), is normalized to the value of pGUS 23C1/pGUS 23N1. HFH, DNA methylated by M-HpaII, M-FnuDII, and M-HhaI methylases; S, DNA methylated by M-SssI methylase. b Recombination efficiency is calculated as normalized GUS activity of the respective cotransfection relative to activities of the functional GUS gene with the same methylation status. c The recombination substrate pGUS 23C1 carries the promoter necessary for the expression of the recombined GUS gene. Because of our transient assay relying on the expression of the recombined gene, recombination efficiencies could only be calculated for the combinations in which this plasmid was transcriptionally active. Combinations including pGUS 23C1 M-SssI are not testable (NT) with this assay system. The measured low GUS activities indicate, however, that during recombination no extensive demethylation of the recombining molecules takes place.
DNA was used as a carrier. However, the results were similar to the data presented in Table 1. For the kinetics of the appearance of the GUS activity (see Fig. 3), multiples of 5 ,ug of pGUS 23C1 and pGUS 23N1 were used per 0.6 x 106 protoplasts. As a control, 0.025 to 0.25 ,ug of pGUS 23 supplemented to a total of 10 ,ug with carrier DNA (salmon sperm DNA sheared to 2 to 10 kb) per 0.6 x 106 protoplasts was transfected. Samples of 0.6 x 106 protoplasts were taken at different times after transfection, and the GUS activity of each was monitored. Soluble extracts of protoplasts were prepared and GUS activities were measured fluorometrically by using the substrate methylumbelliferyl-D-glucuronide (Sigma) as previously described (38), with the exception that 100 ,u1 instead of 20 ,ul of extract was used for the GUS assays to enhance sensitivity. The measured GUS activity was normalized to the determined protein content of the respective sample. Samples with an extremely poor protein extraction were discarded. In a second step, we normalized the results to the GUS activity of one representative plasmid cotransfection (see 7-h value in Fig. 3 and nonmethylated pGUS 23C1/pGUS 23N1 value in Table 1) and set this value arbitrarily at 1. Isolation of protoplast DNA and detection of the recombined GUS gene by PCR. Multiples of 5 ,ug of either pGUS 23N1 or pGUS 23C1 or both were transfected into batches of 0.6 x 106 protoplasts. At the appropriate time, cells were harvested by centrifugation and total DNA was isolated by the method of Burr and Burr (6). After extraction with phenol, the DNA was taken up in 50 mM Tris-HCI (pH 8.0)-20 mM EDTA. The sample was digested first with RNase A, next with Pronase and, after extraction with phenol, with the two restriction enzymes KpnI and SacI as previously described (38). After agarose gel electrophoresis, DNA of the expected size of the recombined GUS gene was cut out of the gel and eluted, and similar amounts of DNA per sample were used for PCR. PCR was done with Taq Polymerase (Perkin-Elmer Cetus) as described by the manufacturer by using the primers pCl and pNl (Fig. 1) (38). Next, 30 or 35 cycles with 1 min at 94°C, 1.5 min at 56°C, and 4 min at 72°C were performed.
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RESULTS To determine whether CpG methylation influences the efficiency of extrachromosomal homologous DNA recombination, we applied a transient assay by using the recombination substrates pGUS 23C1 and pGUS 23N1 (38). The two plasmids contain truncated copies of the ,B-glucuronidase (GUS) gene and have an overlap of 1,251 bp of GUS-specific sequences. Although both plasmids contain the cauliflower mosaic virus 35S promoter, neither the methylated nor the nonmethylated versions are able to express any GUS activity by themselves (Fig. 1). To characterize the influence of different methylation patterns and thus to discriminate between the influence of methylation on transcription and on recombination, the plasmid pGUS 23 carrying the functional GUS gene was used as a positive control. All three plasmids were CpG methylated with different commercially available methylases (Table 1 and Fig. 1). To exclude the possibility that dam methylation might influence the recombination efficiencies of plasmids, owing to propagation in dam' E. coli, pGUS 23N1 and pGUS 23C1 were also propagated in a dam mutant strain. Control experiments performed with the dam mutant plasmids or combinations of dam mutant and dam + plasmids revealed no significant differences compared with the data found with the dam' plasmids (data not shown). Recombination between two partly CpG-methylated plasmids. Supercoiled circular plasmids successively methylated with the three 4-bp sequence-specific methylases M-HpaII, M-FnuDII, and M-HhaI were used for transfection. Eighteen hours later, the GUS activity of methylated pGUS 23 was similar to the GUS activity of the nonmethylated plasmid DNA (Table 1). Thus, transcription was not inhibited by methylation of the specific recognition sites in the GUS gene. This pattern of methylation also had no influence on the recombination behavior of pGUS 23C1 and pGUS 23N1. The 16 methylated CpG pairs in the overlapping GUS sequence (Fig. 1) did not reduce ECR frequencies (Table 1). Recombination between one fully CpG-methylated and one nonmethylated plasmid. Plasmid DNA methylated at CpG sites by M-SssI was used for this experiment. The expression of the functional GUS gene (pGUS 23) was drastically reduced by this modification (Table 1), which methylates 19 CpG sites in the cauliflower mosaic virus 35S promoter and 86 in the homologous region (Fig. 1). As shown below, the remaining low GUS activity is due to incomplete methylation, a phenomenon that was described for this enzyme before (15). When combinations of methylated and nonmethylated recombination substrates were transfected into N. plumbaginifolia protoplasts, we found that the methylation status of pGUS 23C1 influenced the recombination-dependent transient GUS activities to the same extent that the methylation status of the intact gene affects expression (Table 1). This reduction in enzyme activity therefore is most likely due to inhibition of expression by the pGUS 23C1 part of the recombination product. This part includes the 35S promoter used for the expression of the recombined gene. The methylation status of the second recombination substrate pGUS 23N1 had no influence on the GUS expression rates. Recombination between two fully CpG-methylated plasmids. Since our enzyme assay relies on the expression of the recombined gene, we could not use it to address the question of whether recombination occurs between two fully CpGmethylated molecules. To solve this problem we used PCR
MOL. CELL. BIOL.
to detect the CpG-methylated recombination product directly. The prerequisite for such an analysis was that all partly or nonmethylated molecules were excluded from the M-SssImethylated plasmid preparation. These "impurities," which also seem to be responsible for the low GUS activities detected in (co-) transfections of M-SssI-methylated pGUS 23 or pGUS 23C1 (Table 1) (see below), would cause a positive PCR signal, even if recombination of most molecules would be inhibited by CpG methylation. To obtain a pure plasmid preparation, pGUS 23C1, pGUS 23N1, and pGUS 23 were M-SssI methylated and were then digested with an amount of a methyl-sensitive endonuclease that completely digested a severalfold excess of nonmethylated plasmid DNA in a control reaction. The digests were separated on agarose gels; the DNA bands representing the circular supercoiled, monomeric plasmid form were cut out, and the DNA was eluted. To test whether this procedure yielded fully CpG-methylated molecules only, we transfected under standard conditions M-SssI-treated preparations of pGUS 23 or pGUS 23C1 plus pGUS 23N1 into protoplasts. This time we were not able to detect any GUS activity above background (data not shown), demonstrating that our DNA preparations contained methylated molecules only. For the PCR analysis we transfected 1.2 x 106 protoplasts with a combination of the M-SssI-methylated or nonmethylated pGUS 23C1 plus pGUS 23N1. Simultaneously, a batch of protoplasts was transfected with pGUS 23C1 or with pGUS 23N1 alone. After 6 h, protoplasts were harvested and total DNA from the respective samples was isolated and digested with the restriction enzymes SacI and KpnI. The two recombination substrates pGUS 23C1 and pGUS 23N1 were linearized by one or the other enzyme at the promoter or the terminator of the GUS gene, respectively, to yield a 5-kb fragment in both cases (Fig. 1). In contrast, the same digestion should liberate a 2-kb GUS-specific DNA fragment in the recombined molecules. The DNA preparations were size fractionated by agarose gel electrophoresis, and the 2-kb fractions were eluted and used for PCR with the GUSspecific primers pNl and pCl (Fig. 1). Only recombined molecules should contain binding sites for both primers. The result of a PCR analysis after 35 cycles is given in Fig. 2. No band could be detected in a reconstitution experiment in which DNA samples originating from protoplasts transfected with either pGUS 23C1 or pGUS 23N1 alone were mixed (lane 2). The recombined GUS gene could be detected in the protoplasts transfected with pGUS 23C1 plus pGUS 23N1 (lane 3) and in the protoplasts transfected with a combination of the same plasmids in their M-SssI-methylated form (lane 4). The lack of PCR products in the reconstitution experiment clearly demonstrates that bands in lanes 3 and 4 are caused by the recombined GUS gene. Thus, ECR occurs regardless of the methylation status of the recombination partners. Time course of ECR. Our finding that CpG methylation is not influencing ECR in plant cells may indicate that the ECR reaction was faster than the binding of a putative methylCpG binding protein to the DNA. We wanted to test this possibility by determining when and for how long after transfection DNA molecules can undergo recombination. For this analysis we used two independent strategies, the transient expression assay and the PCR assay, both described above. Cotransfection of equal amounts of the two supercoiled, circular plasmids pGUS 23N1 and pGUS 23C1 resulted in
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FIG. 2. PCR analysis to detect the recombined GUS gene in cotransfections of fully CpG-methylated recombination substrates. Total DNA of the protoplasts was purified and size fractionated as described. PCR was performed with the two GUS-specific primers pNl and pCl (Fig. 1), and the products were separated on an agarose gel. Lanes: P, phage 4X174 DNA digested with HaeIII; 1, PCR analysis of pGUS 23 digested with Sacl and KpnI as a positive control; 2, PCR analysis of mixed DNA extracts of protoplasts transfected with either pGUS 23C1 or pGUS 23N1; 3, PCR analysis of DNA extracts of protoplasts cotransfected with pGUS 23C1 and pGUS 23N1; 4, PCR analysis of DNA extracts of protoplasts cotransfected with pGUS 23C1 M-SssI and pGUS 23N1 M-SssI; 5, PCR analysis of DNA extracts of mock-transfected protoplasts harvested 2 h after transfection.
GUS activity of 0.5 to 5% of that of the functional plasmid pGUS 23 (38). Therefore, in the control transfections, the amount of pGUS 23 plasmid DNA was decreased to a level that was expected to result in a GUS activity directly comparable to the one expected from the recombination partners.
After transfection of the same batch of protoplasts with either pGUS 23 or pGUS 23C1 plus pGUS 23N1, aliquots were taken at different times and GUS activities were monitored. The experiment was repeated several times. A representative time course of the appearance of GUS activities is shown in Fig. 3. In this particular experiment, the GUS activities 7 h after transfection of protoplasts with pGUS 23 (0.025 ,ug of plasmid per 0.6 x 106 protoplasts) and pGUS 23Cl/pGUS 23N1 (5 ,ug [each] of pGUS 23C1 and pGUS 23N1 per 0.6 x 106 protoplasts) were almost identical (1:1.09; 22-h value, 1:1.85). For both time courses the respective 7-h value was arbitrarily set as 1 and the GUS activities of the different times were normalized to it (Fig. 1). We could detect GUS activity in protoplasts transfected with pGUS 23 after 4 h, and in protoplasts cotransfected with pGUS 23C1/pGUS 23N1 after 4.5 h. Thus, there is only a 30-min delay in enzyme expression in the protoplasts that require recombination for GUS expression. Furthermore, comparison of the slopes of both curves indicates not only qualitatively that ECR is fast but also quantitatively that it occurs mainly early after transfection. This can be concluded because the rate of appearance of GUS activity in the
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FIG. 3. Time course of transient expression of j-glucuronidase in protoplasts transfected with either pGUS 23C1 and pGUS 23N1 (continuous line) or pGUS 23 (dotted line). For reasons of sensitivity, an amount (0.025 p.g complemented with carrier DNA to 10 ,ug) of pGUS 23 DNA that resulted in about the same GUS activity as the cotransfection of 5 p.g (each) of pGUS 23C1 and pGUS 23N1 per 0.6 x 106 protoplasts was used. The GUS activities of both transfections measured after 7 h were arbitrarily set as 1 (real values, 1:1.09).
protoplasts transfected with the recombination substrates did not differ markedly from the one in which all functional template molecules are available for transcription from the point of transfection on. If the number of recombined active genes were to have increased over time, the slope should also have increased compared with that of the control. Detection of the recombined GUS gene by PCR early after transfection. The kinetics of the appearance of the test enzyme in protoplasts cotransfected with the recombination substrates indicates that ECR takes place mainly within 30 min. To determine the time of appearance of the recombined GUS gene directly, we used the sensitive PCR procedure. A part of the same batch of protoplasts that was used for the kinetics in Fig. 3 was transfected with either pGUS 23C1 or pGUS 23N1 or was cotransfected with both plasmids simultaneously; 1.5 and 2 h after transfection, the protoplasts were harvested and total DNA from the respective samples was isolated. The DNA was prepared for PCR as described above. The result of a PCR analysis after 30 cycles is shown in Fig. 4. No band could be detected in the samples originating from protoplasts transfected with either pGUS 23C1 (lane 2) or pGUS 23N1 (lane 3) alone, both harvested 2 h after transfection. Also, no signal was visible from the reconstitution experiment in which these two samples were mixed prior to PCR (lane 4) or from protoplasts cotransfected with pGUS 23C1 plus pGUS 23N1 harvested 1.5 h after transfection (lane 5). However, the recombined GUS gene could be detected in the protoplasts transfected with pGUS 23C1 plus pGUS 23N1, which were harvested 2 h after transfection (lane 6). DISCUSSION
Methylation and gene activity. In vitro-methylated plasmid DNA carrying the functional GUS gene was tested for activity in plant protoplasts. When the gene was methylated with three different methylases only at some CpG sites (Fig. 1), gene activity was not reduced. This result clearly indicates that analysis of methylation of (trans)genes carried out
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FIG. 4. PCR analysis to detect the recombined GUS gene early after transfection. Protoplasts were transfected with pGUS 23C1, pGUS 23N1, or a combination of both. Protoplasts were harvested 1.5 or 2 h after transfection, and DNA was purified and size fractionated as previously described. PCR was performed with the two GUS-specific primers pNl and pCl (Fig. 1), and the products were separated on an agarose gel. Lanes: P, phage 4X174 DNA digested with HaeIII; 1, PCR analysis of pGUS 23 digested with Sacl and Kpinl as a positive control; 2, PCR analysis of DNA extracts of protoplasts transfected with pGUS 23C1, harvested 2 h after transfection; 3, PCR analysis of DNA extracts of protoplasts transfected with pGUS 23N1, harvested 2 h after transfection; 4, PCR analysis of mixed DNA extracts of protoplasts transfected with either pGUS 23C1 or pGUS 23N1, harvested 2 h after transfection; 5, PCR analysis of DNA extracts of protoplasts cotransfected with pGUS 23C1 and pGUS 23N1, harvested 1.5 h after transfection; 6, PCR analysis of DNA extracts of protoplasts cotransfected with pGUS 23C1 and pGUS 23N1, harvested 2 h after transfection; 7, PCR analysis of DNA extracts of mock-transfected protoplast, harvested 2 h after transfection.
by the enzyme pair HpaII-MspI, as is frequently done, may not be important for gene activity. The methylation status of the single HpaII site in the 35S promoter has indeed been shown to be unrelated to gene activity (26). However, when the gene was fully CpG methylated with M-SssI methylase, gene activity was essentially abolished (Table 1; Fig. 5, numbers 1 and 2). This confirms earlier results obtained with hemi-C (16, 50) or CpG-methylated DNA (15). Cotransfections of the GUS deletion mutants, however, provide additional information on the parts of the gene that are crucial for the correlation of CpG methylation and transcriptional inactivation. In these cotransfection experiments, only recombined molecules were able to express GUS activity (38). These molecules are expected to inherit the methylation patterns of their parents (for detailed discussion, see below). Apparently, methylation of the promoter as well as of the 5' end of the gene is sufficient to block transcription (Fig. 5, compare numbers 3 and 5). The C-terminal part of the gene and the terminator apparently play no role in transcriptional inhibition (Fig. 5, compare numbers 3 and 4). This finding is similar to what is observed in mammalian systems, in which methylation of the promoter sequences plays a crucial role in regulation of transcription (20). Methylation and recombination. There are three possible ways by which C methylation may inhibit recombination. (i) Methyl groups might inhibit the binding of the recombination
enzymes to the DNA. (ii) The binding of a general methylCpG binding protein might interfere with the recombination reaction. For mammalian cells, Boyes and Bird (5) demonstrated the presence of a protein (MeCO-1) able to inhibit transcription. (iii) The inhibition could be indirect through inhibition of transcription. It has been demonstrated for intrachromosomal recombination (ICR) that transcription enhances recombination efficiencies (48). Since CpG methylation is correlated with inhibition of transcription (Fig. 5, numbers 1 and 2), the recombination efficiency of a methylated gene in comparison to a nonmethylated one should be reduced, according to the third hypothesis. However, in our experiments we obtained no evidence that the methylation status of the plasmid has any influence on ECR efficiencies. Recombination between partially CpGmethylated substrates was as efficient as that between nonmethylated controls (Table 1). Recombination efficiency between the fully CpG-methylated 5'-deleted recombination partner pGUS 23N1 and pGUS 23C1 was not reduced compared with that of the two unmodified partners (Table 1; Fig. 5, compare numbers 3 and 4). As both recombination substrates carried the 35S promoter sequence, we can conclude that there is neither a direct nor an indirect (via inhibition of transcription of pGUS 23N1) influence of CpG methylation on extrachromosomal recombination efficiencies in these combinations. Thus, the binding of a hypothetical methyl-CpG binding protein would not block recombination in the same way as it may block transcription. Our results also clearly indicate that the recombination substrates are not extensively demethylated during the recombination reaction. If the substrates-including part of the promoters-were extensively demethylated during the recombination reaction, we would have detected higher GUS activities in our transient assay (Table 1, compare pGUS 23s with pGUS 23C1s/pGUS 23N1s or pGUS 23C1s/ pGUS 23N1). This confirms a report by Hershkovitz et al. (15), who showed that methylation patterns of nonreplicating plasmids are stably maintained in plant protoplasts. As stated above, CpG methylation of one plasmid (pGUS 23N1) is not influencing recombination rates (Fig. 5, number 4). Therefore, we have to assume that recombination also takes place in the combination pGUS 23C1 M-SssI plus pGUS 23N1 (Fig. 5, number 5), although we are not able to directly test this with the transient assay system. However, we were able to demonstrate via PCR that recombination occurs between the two completely CpG-methylated substrates (Fig. 5, number 6). In spite of being only semiquantitative, the PCR signals of the methylated and nonmethylated recombination substrates nevertheless indicate that in both cases the recombination efficiencies do not differ by orders of magnitudes. Genomic DNA of plants, in contrast to that of mammals, is also CpNpG methylated (32). As there is no easy method to obtain this kind of methylation in vitro, the role of CpNpG methylation on transcription and homologous recombination remains to be elucidated. The effect of CpG methylation on ICR in plant cells has not yet been investigated. Given that ECR is not inhibited by CpG methylation, it might be tempting to speculate that CpG methylation itself is not influencing the recombination efficiencies of ICR either. There is one reason to favor this argument; some members of repetitive, homologous sequence families in the plant genome, for example, the rDNA genes, are highly C methylated (10). In order to conserve homogeneity of these genes, homologous recombination and/or gene conversion events should be efficient.
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FIG. 5. Schematic outline of the influence of CpG methylation of the transfected plasmid substrates on recombination and transcription. The shaded bars reflect plasmids CpG methylated by M-SssI methylase. The outline is based on the results presented in Table 1. Abbreviations: enz. act., enzyme activity; transcr., transcription; recomb., recombination; n.t., not testable (see also Table 1); Nr., number.
However, an indirect effect of more densely packed, transcriptionally inactive DNA on recombination is likely to exist (43) and has been demonstrated for yeast cells (48). We could not detect such an indirect recombination-enhancing effect on ECR. Since in our transient assay one recombination partner (pGUS 23C1) was always transcribed, we were only able to demonstrate that transcription of the second plasmid (pGUS 23N1) had no influence on recombination efficiencies. A recently published study about the influence of CpG methylation on site-specific recombination [V(D)J immunoglobulin switching] in mammalian cells (17) is of significance for our findings. In this system, early after transfection the nonmethylated and fully CpG-methylated plasmid molecules showed no difference in their recombination behavior. At a later time-after replication of the plasmids had startedsite-specific recombination efficiencies of the fully CpGmethylated molecules was drastically reduced. The inhibition could be correlated with a dense protein-packing of the methylated in comparison to that in the nonmethylated plasmid molecules. Since we could not detect any influence of CpG methylation on recombination in our system, the questions of when after transfection and for how long ECR takes place in plant cells arose. Kinetics of ECR in plant cells. To answer this question the timing of the ECR reaction was determined by using a transient assay and PCR analysis. The rationale of our experiment was to compare the time and the rate of appearance of the transient
P-glucuronidase
activity of protoplasts requiring ECR for enzyme expression with those of protoplasts transfected with the complete gene. Efficiencies of uptake of DNA into the nucleus, transcription, and translation as well as degradation of DNA, RNA, and enzyme (37) should be identical in both protoplast populations. Differences in the appearance of the enzyme activity should directly reflect properties of the ECR reaction itself. The detected delay indicates that the time required for most ECR events appears to be no more than 30 min (Fig. 3). The time of ECR might even be less, as samples were taken in half-hour intervals. Within this short time period, a multitude of different processes has to take place: breaks have to be introduced in the DNA substrates, the recombination partners have to find each other and anneal, the gaps in the recombined DNA have to be repaired, and finally restoration of a functional DNA molecule by ligation has to take place. Whereas the time of GUS appearance gives us an answer to the question of how much time ECR needs, the shape of the curve gives us a quantitative indication of how long ECR can take place in the protoplast population. If ECR takes place only in a 30-min period, the two curves in Fig. 3 should have the same slope. If, on the other hand, new template molecules are made by ECR during the whole assay period, GUS expression should start very slowly and the curve should rise later in an exponential manner. In reality, the slope of the ECR-dependent curve in Fig. 3 is very similar to the one of the expression of the functional GUS gene. This indicates that most templates are made by ECR early after
3378
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TABLE 2. Comparison of general properties of ECR and ICR in higher eukaryotesa RE correlated with length of homology RE influenced by CpG methylation RE drastically reduced by mismatches RE dependent on the position in the genome Time requirement of recombination reaction Recombination reaction is conservative Putative mechanism of the recombination reaction Recombination frequency"
Reference(s)
ECR
Property
1, 38 This work 49
+ -
Vol. 12, No. 8
0270-7306/92/083372-08$02.00/0 Copyright X) 1992, American Society for Microbiology
Extrachromosomal Homologous DNA Recombination in Plant Cells Is Fast and Is Not Affected by CpG Methylation HOLGER PUCHTA,* SERGE KOCHER, AND BARBARA HOHN
Friedrich Miescher-Institut, P. O. Box 2543, CH-4002 Basel, Switzerland Received 9 March 1992/Accepted 1 May 1992
Using a sensitive transient assay, we investigated extrachromosomal homologous DNA recombination (ECR) in plant cells. As the plant genome is highly C methylated, we addressed the question of whether CpG methylation has an influence on DNA recombination efficiencies. Whereas the expression level of the fully CpG-methylated DNA molecules was reduced drastically, we found no significant changes in ECR efficiencies between two partly CpG-methylated plasmids or between one fully CpG-methylated and one nonmethylated plasmid. Using a modified polymerase chain reaction analysis, we were able to detect recombination between two fully CpG-methylated plasmids. Furthermore, we characterized the kinetics of the ECR reaction. Cotransfection of plasmids carrying truncated copies of the 1-glucuronidase (GUS) gene resulted in enzyme activity with a delay of only half an hour compared with that of the plasmid carrying the functional marker gene. This indicates that the ECR reaction itself requires no more than 30 min. By polymerase chain reaction, we were able to detect the recombined GUS gene as early as 2 h after transfection. This result and the time course of the transient GUS activity indicate that ECR occurs mainly early after transfection. The biological significance of this finding is discussed, and properties of ECR and intrachromosomal recombination are compared. The biological role of C methylation in eukaryotes has attracted wide interest in recent years (for reviews, see references 7, 8, 23, and 43). Plant genomes are especially highly methylated (32). C methylation of promoter regions in the plant genome could be correlated with gene inactivation (51), as is frequently found for animal systems. In addition, the expression of a gene transfected into tobacco protoplasts was inhibited by in vitro hemi-C (16, 50) or in vitro CpG methylation of the DNA (15). It has been shown that C methylation of specific sequence motifs present in different plant promoters leads to reduced binding of cognate transcription factors (18, 44). Methylation was also correlated with inhibition of transposition (14, 41) and with inhibition of expression of homologous genes in transgenic plants (19, 26, 31). In the present communication, we address the question of whether C (in this case, CpG) methylation influences recombination efficiencies. We cotransfected Nicotiana plumbaginifolia protoplasts with two nonreplicating plasmids carrying different deletions of the ,3-glucuronidase (GUS) gene. To detect recombination products, we used a transient extrachromosomal recombination (ECR) assay system (28, 38, 39) and a modified polymerase chain reaction (PCR) analysis (38). Applying the same techniques, we furthermore investigated the kinetics of the ECR reaction in plant cells. In plants, ECR occurs naturally in caulimoviruses (21) and geminiviruses (45) and between T-DNAs of Agrobacterium tumefaciens (33).
35S promoter and the nopaline synthetase terminator. pGUS 23N1 has a 187-bp deletion in the 5' part of the GUS gene and pGUS 23C1 has a 601-bp deletion in the 3' part of the gene. Plasmid DNA was prepared for the transfection experiments as described by using either Escherichia coli DH5at or dam mutant strain BZ 101. In vitro methylation of DNA. For in vitro methylation of supercoiled circular plasmid DNA, the enzymes M-SssI, M-HpaII, M-FnuDII, and M-HhaI were used at conditions recommended by the manufacturer (New England Biolabs, Schwalbach, Germany). For testing methylation efficiency, the methylated as well as nonmethylated plasmid DNAs were digested with the respective methyl-sensitive restriction nuclease (HhaI, HpaII, or BstUI; all from New England Biolabs) and were analyzed by gel electrophoresis. The methylation of the DNA was regarded as complete when no degradation of 1 ,ug of DNA by a methyl-sensitive enzyme was detectable by ethidium bromide staining. The methylated DNA was extracted with phenol, ethanol precipitated, and dissolved in sterile water, and its concentration was determined spectrophotometrically. For PCR analysis of recombination of M-SssI-methylated plasmid DNAs, the methylated molecules were first separated from trace amounts of nonmethylated molecules. The plasmids were digested with an excess of the restriction endonuclease HhaI, the digested samples were separated by agarose gel electrophoresis, and the respective supercoiled circular, monomeric plasmid bands were cut out of the gel. The DNA was eluted, extracted with phenol, precipitated with ethanol, and redissolved. The DNA concentration was determined spectrophotometrically. Isolation of protoplasts, transfection of DNA, and f8-glucuronidase assays. Mesophyll protoplasts of N. plumbaginifolia were enzymatically isolated from leaves of sterile shoot cultures. Polyethylene glycol-mediated transfections of protoplasts were done as previously described (38). For the analysis of the role of C methylation in recombination, transfections were performed with 1 p,g per plasmid and 9 p,g of carrier DNA per 0.6 x 106 protoplasts. Every experimen-
MATERIALS AND METHODS Cloning procedures. Standard cloning procedures were used (40). Construction of plasmids pGUS 23, pGUS 23C1, and pGUS 23N1 (Fig. 1) was described previously (38). The plasmids are pUC7 derivatives carrying 3-glucuronidase (GUS) sequences flanked by the cauliflower mosaic virus *
Corresponding author. 3372
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VOL. 12, 1992 35 S CpG-sites
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Kpnl for transfection of protoplasts. The plasmids are pUC7 derivatives carrying ,B-glucuronidase (GUS) of the used FIG. 1. Scheme plasmids sequences flanked by the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthetase terminator (Nos T). Plasmid pGUS 23N1 has a 187-bp deletion in the 5' part of the GUS gene and plasmid pGUS 23C1 has a 601-bp deletion in the 3' part of the gene (38). The GUS-specific overlap of pGUS 23N1 and pGUS 23C1 is hatched in pGUS 23. The number of methylated CpG sites in each region is indicated. The DNA was treated by M-HpaII, M-FnuDII, and M-HhaI (HFH) or M-SssI (S) methylase. The restriction sites for KpnI and SacI that were used to liberate the 2-kb-long recombined GUS gene are indicated with vertical open arrows. The recombined gene was detected by PCR with the primers pNl and pCl, both marked by horizontal black arrows.
tal series included transfections with 1 ,ug of nonmethylated as well as with methylated pGUS 23 (complemented by salmon sperm DNA to a total amount of 10 ,ug of DNA) as controls. An aliquot of protoplasts was mock transfected as a background control. Protoplasts were harvested 18 h after transfection. In addition to the described transfections (see Results) (Table 1), we also used in control experiments 5 ,ug or 250 ng of each plasmid, in the second case complemented with carrier DNA to 10 ,ug. In some experiments CsClpurified plasmid DNA instead of sheared salmon sperm
TABLE 1. Effects of CpG methylation on gene expression and ECR in protoplastsa Plasmid methylation status (no. of expts)
pGUS 23 pGUS 23HFH (3) pGUS 23s (4)
pGUS 23C1/pGUS 23N1 pGUS 23C1HF/pGUS 23NlHFH (5)
pGUS 23C1/pGUS 23N1s (4) pGUS 23C1S/pGUS 23N1 (5) pGUS 23C1S/pGUS 23N1s (5)
GUS activity
Recombination
efficiency"
1
1.30 ± 0.19 0.048 ± 0.028
1 1.19 ± 0.92 ± 0.052 ± 0.057 ±
0.68 0.30 0.049 0.022
1 0.92 0.92 NTC N'T
a The normalized average values of the indicated number of different cotransfection experiments (in parentheses) with standard deviations are given (normalizations in the upper section are to the value of pGUS 23, and those in the lower section are to that of pGUS 23C1/pGUS 23N1). GUS background of the plant cells, 0.014 ± 0.009 (4), is normalized to the value of pGUS 23C1/pGUS 23N1. HFH, DNA methylated by M-HpaII, M-FnuDII, and M-HhaI methylases; S, DNA methylated by M-SssI methylase. b Recombination efficiency is calculated as normalized GUS activity of the respective cotransfection relative to activities of the functional GUS gene with the same methylation status. c The recombination substrate pGUS 23C1 carries the promoter necessary for the expression of the recombined GUS gene. Because of our transient assay relying on the expression of the recombined gene, recombination efficiencies could only be calculated for the combinations in which this plasmid was transcriptionally active. Combinations including pGUS 23C1 M-SssI are not testable (NT) with this assay system. The measured low GUS activities indicate, however, that during recombination no extensive demethylation of the recombining molecules takes place.
DNA was used as a carrier. However, the results were similar to the data presented in Table 1. For the kinetics of the appearance of the GUS activity (see Fig. 3), multiples of 5 ,ug of pGUS 23C1 and pGUS 23N1 were used per 0.6 x 106 protoplasts. As a control, 0.025 to 0.25 ,ug of pGUS 23 supplemented to a total of 10 ,ug with carrier DNA (salmon sperm DNA sheared to 2 to 10 kb) per 0.6 x 106 protoplasts was transfected. Samples of 0.6 x 106 protoplasts were taken at different times after transfection, and the GUS activity of each was monitored. Soluble extracts of protoplasts were prepared and GUS activities were measured fluorometrically by using the substrate methylumbelliferyl-D-glucuronide (Sigma) as previously described (38), with the exception that 100 ,u1 instead of 20 ,ul of extract was used for the GUS assays to enhance sensitivity. The measured GUS activity was normalized to the determined protein content of the respective sample. Samples with an extremely poor protein extraction were discarded. In a second step, we normalized the results to the GUS activity of one representative plasmid cotransfection (see 7-h value in Fig. 3 and nonmethylated pGUS 23C1/pGUS 23N1 value in Table 1) and set this value arbitrarily at 1. Isolation of protoplast DNA and detection of the recombined GUS gene by PCR. Multiples of 5 ,ug of either pGUS 23N1 or pGUS 23C1 or both were transfected into batches of 0.6 x 106 protoplasts. At the appropriate time, cells were harvested by centrifugation and total DNA was isolated by the method of Burr and Burr (6). After extraction with phenol, the DNA was taken up in 50 mM Tris-HCI (pH 8.0)-20 mM EDTA. The sample was digested first with RNase A, next with Pronase and, after extraction with phenol, with the two restriction enzymes KpnI and SacI as previously described (38). After agarose gel electrophoresis, DNA of the expected size of the recombined GUS gene was cut out of the gel and eluted, and similar amounts of DNA per sample were used for PCR. PCR was done with Taq Polymerase (Perkin-Elmer Cetus) as described by the manufacturer by using the primers pCl and pNl (Fig. 1) (38). Next, 30 or 35 cycles with 1 min at 94°C, 1.5 min at 56°C, and 4 min at 72°C were performed.
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PUCHTA ET AL.
RESULTS To determine whether CpG methylation influences the efficiency of extrachromosomal homologous DNA recombination, we applied a transient assay by using the recombination substrates pGUS 23C1 and pGUS 23N1 (38). The two plasmids contain truncated copies of the ,B-glucuronidase (GUS) gene and have an overlap of 1,251 bp of GUS-specific sequences. Although both plasmids contain the cauliflower mosaic virus 35S promoter, neither the methylated nor the nonmethylated versions are able to express any GUS activity by themselves (Fig. 1). To characterize the influence of different methylation patterns and thus to discriminate between the influence of methylation on transcription and on recombination, the plasmid pGUS 23 carrying the functional GUS gene was used as a positive control. All three plasmids were CpG methylated with different commercially available methylases (Table 1 and Fig. 1). To exclude the possibility that dam methylation might influence the recombination efficiencies of plasmids, owing to propagation in dam' E. coli, pGUS 23N1 and pGUS 23C1 were also propagated in a dam mutant strain. Control experiments performed with the dam mutant plasmids or combinations of dam mutant and dam + plasmids revealed no significant differences compared with the data found with the dam' plasmids (data not shown). Recombination between two partly CpG-methylated plasmids. Supercoiled circular plasmids successively methylated with the three 4-bp sequence-specific methylases M-HpaII, M-FnuDII, and M-HhaI were used for transfection. Eighteen hours later, the GUS activity of methylated pGUS 23 was similar to the GUS activity of the nonmethylated plasmid DNA (Table 1). Thus, transcription was not inhibited by methylation of the specific recognition sites in the GUS gene. This pattern of methylation also had no influence on the recombination behavior of pGUS 23C1 and pGUS 23N1. The 16 methylated CpG pairs in the overlapping GUS sequence (Fig. 1) did not reduce ECR frequencies (Table 1). Recombination between one fully CpG-methylated and one nonmethylated plasmid. Plasmid DNA methylated at CpG sites by M-SssI was used for this experiment. The expression of the functional GUS gene (pGUS 23) was drastically reduced by this modification (Table 1), which methylates 19 CpG sites in the cauliflower mosaic virus 35S promoter and 86 in the homologous region (Fig. 1). As shown below, the remaining low GUS activity is due to incomplete methylation, a phenomenon that was described for this enzyme before (15). When combinations of methylated and nonmethylated recombination substrates were transfected into N. plumbaginifolia protoplasts, we found that the methylation status of pGUS 23C1 influenced the recombination-dependent transient GUS activities to the same extent that the methylation status of the intact gene affects expression (Table 1). This reduction in enzyme activity therefore is most likely due to inhibition of expression by the pGUS 23C1 part of the recombination product. This part includes the 35S promoter used for the expression of the recombined gene. The methylation status of the second recombination substrate pGUS 23N1 had no influence on the GUS expression rates. Recombination between two fully CpG-methylated plasmids. Since our enzyme assay relies on the expression of the recombined gene, we could not use it to address the question of whether recombination occurs between two fully CpGmethylated molecules. To solve this problem we used PCR
MOL. CELL. BIOL.
to detect the CpG-methylated recombination product directly. The prerequisite for such an analysis was that all partly or nonmethylated molecules were excluded from the M-SssImethylated plasmid preparation. These "impurities," which also seem to be responsible for the low GUS activities detected in (co-) transfections of M-SssI-methylated pGUS 23 or pGUS 23C1 (Table 1) (see below), would cause a positive PCR signal, even if recombination of most molecules would be inhibited by CpG methylation. To obtain a pure plasmid preparation, pGUS 23C1, pGUS 23N1, and pGUS 23 were M-SssI methylated and were then digested with an amount of a methyl-sensitive endonuclease that completely digested a severalfold excess of nonmethylated plasmid DNA in a control reaction. The digests were separated on agarose gels; the DNA bands representing the circular supercoiled, monomeric plasmid form were cut out, and the DNA was eluted. To test whether this procedure yielded fully CpG-methylated molecules only, we transfected under standard conditions M-SssI-treated preparations of pGUS 23 or pGUS 23C1 plus pGUS 23N1 into protoplasts. This time we were not able to detect any GUS activity above background (data not shown), demonstrating that our DNA preparations contained methylated molecules only. For the PCR analysis we transfected 1.2 x 106 protoplasts with a combination of the M-SssI-methylated or nonmethylated pGUS 23C1 plus pGUS 23N1. Simultaneously, a batch of protoplasts was transfected with pGUS 23C1 or with pGUS 23N1 alone. After 6 h, protoplasts were harvested and total DNA from the respective samples was isolated and digested with the restriction enzymes SacI and KpnI. The two recombination substrates pGUS 23C1 and pGUS 23N1 were linearized by one or the other enzyme at the promoter or the terminator of the GUS gene, respectively, to yield a 5-kb fragment in both cases (Fig. 1). In contrast, the same digestion should liberate a 2-kb GUS-specific DNA fragment in the recombined molecules. The DNA preparations were size fractionated by agarose gel electrophoresis, and the 2-kb fractions were eluted and used for PCR with the GUSspecific primers pNl and pCl (Fig. 1). Only recombined molecules should contain binding sites for both primers. The result of a PCR analysis after 35 cycles is given in Fig. 2. No band could be detected in a reconstitution experiment in which DNA samples originating from protoplasts transfected with either pGUS 23C1 or pGUS 23N1 alone were mixed (lane 2). The recombined GUS gene could be detected in the protoplasts transfected with pGUS 23C1 plus pGUS 23N1 (lane 3) and in the protoplasts transfected with a combination of the same plasmids in their M-SssI-methylated form (lane 4). The lack of PCR products in the reconstitution experiment clearly demonstrates that bands in lanes 3 and 4 are caused by the recombined GUS gene. Thus, ECR occurs regardless of the methylation status of the recombination partners. Time course of ECR. Our finding that CpG methylation is not influencing ECR in plant cells may indicate that the ECR reaction was faster than the binding of a putative methylCpG binding protein to the DNA. We wanted to test this possibility by determining when and for how long after transfection DNA molecules can undergo recombination. For this analysis we used two independent strategies, the transient expression assay and the PCR assay, both described above. Cotransfection of equal amounts of the two supercoiled, circular plasmids pGUS 23N1 and pGUS 23C1 resulted in
VOL. 12, 1992
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FIG. 2. PCR analysis to detect the recombined GUS gene in cotransfections of fully CpG-methylated recombination substrates. Total DNA of the protoplasts was purified and size fractionated as described. PCR was performed with the two GUS-specific primers pNl and pCl (Fig. 1), and the products were separated on an agarose gel. Lanes: P, phage 4X174 DNA digested with HaeIII; 1, PCR analysis of pGUS 23 digested with Sacl and KpnI as a positive control; 2, PCR analysis of mixed DNA extracts of protoplasts transfected with either pGUS 23C1 or pGUS 23N1; 3, PCR analysis of DNA extracts of protoplasts cotransfected with pGUS 23C1 and pGUS 23N1; 4, PCR analysis of DNA extracts of protoplasts cotransfected with pGUS 23C1 M-SssI and pGUS 23N1 M-SssI; 5, PCR analysis of DNA extracts of mock-transfected protoplasts harvested 2 h after transfection.
GUS activity of 0.5 to 5% of that of the functional plasmid pGUS 23 (38). Therefore, in the control transfections, the amount of pGUS 23 plasmid DNA was decreased to a level that was expected to result in a GUS activity directly comparable to the one expected from the recombination partners.
After transfection of the same batch of protoplasts with either pGUS 23 or pGUS 23C1 plus pGUS 23N1, aliquots were taken at different times and GUS activities were monitored. The experiment was repeated several times. A representative time course of the appearance of GUS activities is shown in Fig. 3. In this particular experiment, the GUS activities 7 h after transfection of protoplasts with pGUS 23 (0.025 ,ug of plasmid per 0.6 x 106 protoplasts) and pGUS 23Cl/pGUS 23N1 (5 ,ug [each] of pGUS 23C1 and pGUS 23N1 per 0.6 x 106 protoplasts) were almost identical (1:1.09; 22-h value, 1:1.85). For both time courses the respective 7-h value was arbitrarily set as 1 and the GUS activities of the different times were normalized to it (Fig. 1). We could detect GUS activity in protoplasts transfected with pGUS 23 after 4 h, and in protoplasts cotransfected with pGUS 23C1/pGUS 23N1 after 4.5 h. Thus, there is only a 30-min delay in enzyme expression in the protoplasts that require recombination for GUS expression. Furthermore, comparison of the slopes of both curves indicates not only qualitatively that ECR is fast but also quantitatively that it occurs mainly early after transfection. This can be concluded because the rate of appearance of GUS activity in the
5.5
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FIG. 3. Time course of transient expression of j-glucuronidase in protoplasts transfected with either pGUS 23C1 and pGUS 23N1 (continuous line) or pGUS 23 (dotted line). For reasons of sensitivity, an amount (0.025 p.g complemented with carrier DNA to 10 ,ug) of pGUS 23 DNA that resulted in about the same GUS activity as the cotransfection of 5 p.g (each) of pGUS 23C1 and pGUS 23N1 per 0.6 x 106 protoplasts was used. The GUS activities of both transfections measured after 7 h were arbitrarily set as 1 (real values, 1:1.09).
protoplasts transfected with the recombination substrates did not differ markedly from the one in which all functional template molecules are available for transcription from the point of transfection on. If the number of recombined active genes were to have increased over time, the slope should also have increased compared with that of the control. Detection of the recombined GUS gene by PCR early after transfection. The kinetics of the appearance of the test enzyme in protoplasts cotransfected with the recombination substrates indicates that ECR takes place mainly within 30 min. To determine the time of appearance of the recombined GUS gene directly, we used the sensitive PCR procedure. A part of the same batch of protoplasts that was used for the kinetics in Fig. 3 was transfected with either pGUS 23C1 or pGUS 23N1 or was cotransfected with both plasmids simultaneously; 1.5 and 2 h after transfection, the protoplasts were harvested and total DNA from the respective samples was isolated. The DNA was prepared for PCR as described above. The result of a PCR analysis after 30 cycles is shown in Fig. 4. No band could be detected in the samples originating from protoplasts transfected with either pGUS 23C1 (lane 2) or pGUS 23N1 (lane 3) alone, both harvested 2 h after transfection. Also, no signal was visible from the reconstitution experiment in which these two samples were mixed prior to PCR (lane 4) or from protoplasts cotransfected with pGUS 23C1 plus pGUS 23N1 harvested 1.5 h after transfection (lane 5). However, the recombined GUS gene could be detected in the protoplasts transfected with pGUS 23C1 plus pGUS 23N1, which were harvested 2 h after transfection (lane 6). DISCUSSION
Methylation and gene activity. In vitro-methylated plasmid DNA carrying the functional GUS gene was tested for activity in plant protoplasts. When the gene was methylated with three different methylases only at some CpG sites (Fig. 1), gene activity was not reduced. This result clearly indicates that analysis of methylation of (trans)genes carried out
3376 P
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FIG. 4. PCR analysis to detect the recombined GUS gene early after transfection. Protoplasts were transfected with pGUS 23C1, pGUS 23N1, or a combination of both. Protoplasts were harvested 1.5 or 2 h after transfection, and DNA was purified and size fractionated as previously described. PCR was performed with the two GUS-specific primers pNl and pCl (Fig. 1), and the products were separated on an agarose gel. Lanes: P, phage 4X174 DNA digested with HaeIII; 1, PCR analysis of pGUS 23 digested with Sacl and Kpinl as a positive control; 2, PCR analysis of DNA extracts of protoplasts transfected with pGUS 23C1, harvested 2 h after transfection; 3, PCR analysis of DNA extracts of protoplasts transfected with pGUS 23N1, harvested 2 h after transfection; 4, PCR analysis of mixed DNA extracts of protoplasts transfected with either pGUS 23C1 or pGUS 23N1, harvested 2 h after transfection; 5, PCR analysis of DNA extracts of protoplasts cotransfected with pGUS 23C1 and pGUS 23N1, harvested 1.5 h after transfection; 6, PCR analysis of DNA extracts of protoplasts cotransfected with pGUS 23C1 and pGUS 23N1, harvested 2 h after transfection; 7, PCR analysis of DNA extracts of mock-transfected protoplast, harvested 2 h after transfection.
by the enzyme pair HpaII-MspI, as is frequently done, may not be important for gene activity. The methylation status of the single HpaII site in the 35S promoter has indeed been shown to be unrelated to gene activity (26). However, when the gene was fully CpG methylated with M-SssI methylase, gene activity was essentially abolished (Table 1; Fig. 5, numbers 1 and 2). This confirms earlier results obtained with hemi-C (16, 50) or CpG-methylated DNA (15). Cotransfections of the GUS deletion mutants, however, provide additional information on the parts of the gene that are crucial for the correlation of CpG methylation and transcriptional inactivation. In these cotransfection experiments, only recombined molecules were able to express GUS activity (38). These molecules are expected to inherit the methylation patterns of their parents (for detailed discussion, see below). Apparently, methylation of the promoter as well as of the 5' end of the gene is sufficient to block transcription (Fig. 5, compare numbers 3 and 5). The C-terminal part of the gene and the terminator apparently play no role in transcriptional inhibition (Fig. 5, compare numbers 3 and 4). This finding is similar to what is observed in mammalian systems, in which methylation of the promoter sequences plays a crucial role in regulation of transcription (20). Methylation and recombination. There are three possible ways by which C methylation may inhibit recombination. (i) Methyl groups might inhibit the binding of the recombination
enzymes to the DNA. (ii) The binding of a general methylCpG binding protein might interfere with the recombination reaction. For mammalian cells, Boyes and Bird (5) demonstrated the presence of a protein (MeCO-1) able to inhibit transcription. (iii) The inhibition could be indirect through inhibition of transcription. It has been demonstrated for intrachromosomal recombination (ICR) that transcription enhances recombination efficiencies (48). Since CpG methylation is correlated with inhibition of transcription (Fig. 5, numbers 1 and 2), the recombination efficiency of a methylated gene in comparison to a nonmethylated one should be reduced, according to the third hypothesis. However, in our experiments we obtained no evidence that the methylation status of the plasmid has any influence on ECR efficiencies. Recombination between partially CpGmethylated substrates was as efficient as that between nonmethylated controls (Table 1). Recombination efficiency between the fully CpG-methylated 5'-deleted recombination partner pGUS 23N1 and pGUS 23C1 was not reduced compared with that of the two unmodified partners (Table 1; Fig. 5, compare numbers 3 and 4). As both recombination substrates carried the 35S promoter sequence, we can conclude that there is neither a direct nor an indirect (via inhibition of transcription of pGUS 23N1) influence of CpG methylation on extrachromosomal recombination efficiencies in these combinations. Thus, the binding of a hypothetical methyl-CpG binding protein would not block recombination in the same way as it may block transcription. Our results also clearly indicate that the recombination substrates are not extensively demethylated during the recombination reaction. If the substrates-including part of the promoters-were extensively demethylated during the recombination reaction, we would have detected higher GUS activities in our transient assay (Table 1, compare pGUS 23s with pGUS 23C1s/pGUS 23N1s or pGUS 23C1s/ pGUS 23N1). This confirms a report by Hershkovitz et al. (15), who showed that methylation patterns of nonreplicating plasmids are stably maintained in plant protoplasts. As stated above, CpG methylation of one plasmid (pGUS 23N1) is not influencing recombination rates (Fig. 5, number 4). Therefore, we have to assume that recombination also takes place in the combination pGUS 23C1 M-SssI plus pGUS 23N1 (Fig. 5, number 5), although we are not able to directly test this with the transient assay system. However, we were able to demonstrate via PCR that recombination occurs between the two completely CpG-methylated substrates (Fig. 5, number 6). In spite of being only semiquantitative, the PCR signals of the methylated and nonmethylated recombination substrates nevertheless indicate that in both cases the recombination efficiencies do not differ by orders of magnitudes. Genomic DNA of plants, in contrast to that of mammals, is also CpNpG methylated (32). As there is no easy method to obtain this kind of methylation in vitro, the role of CpNpG methylation on transcription and homologous recombination remains to be elucidated. The effect of CpG methylation on ICR in plant cells has not yet been investigated. Given that ECR is not inhibited by CpG methylation, it might be tempting to speculate that CpG methylation itself is not influencing the recombination efficiencies of ICR either. There is one reason to favor this argument; some members of repetitive, homologous sequence families in the plant genome, for example, the rDNA genes, are highly C methylated (10). In order to conserve homogeneity of these genes, homologous recombination and/or gene conversion events should be efficient.
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n t.
pGUS 23
pGUS 23C1 s
pGUS 23N1
FIG. 5. Schematic outline of the influence of CpG methylation of the transfected plasmid substrates on recombination and transcription. The shaded bars reflect plasmids CpG methylated by M-SssI methylase. The outline is based on the results presented in Table 1. Abbreviations: enz. act., enzyme activity; transcr., transcription; recomb., recombination; n.t., not testable (see also Table 1); Nr., number.
However, an indirect effect of more densely packed, transcriptionally inactive DNA on recombination is likely to exist (43) and has been demonstrated for yeast cells (48). We could not detect such an indirect recombination-enhancing effect on ECR. Since in our transient assay one recombination partner (pGUS 23C1) was always transcribed, we were only able to demonstrate that transcription of the second plasmid (pGUS 23N1) had no influence on recombination efficiencies. A recently published study about the influence of CpG methylation on site-specific recombination [V(D)J immunoglobulin switching] in mammalian cells (17) is of significance for our findings. In this system, early after transfection the nonmethylated and fully CpG-methylated plasmid molecules showed no difference in their recombination behavior. At a later time-after replication of the plasmids had startedsite-specific recombination efficiencies of the fully CpGmethylated molecules was drastically reduced. The inhibition could be correlated with a dense protein-packing of the methylated in comparison to that in the nonmethylated plasmid molecules. Since we could not detect any influence of CpG methylation on recombination in our system, the questions of when after transfection and for how long ECR takes place in plant cells arose. Kinetics of ECR in plant cells. To answer this question the timing of the ECR reaction was determined by using a transient assay and PCR analysis. The rationale of our experiment was to compare the time and the rate of appearance of the transient
P-glucuronidase
activity of protoplasts requiring ECR for enzyme expression with those of protoplasts transfected with the complete gene. Efficiencies of uptake of DNA into the nucleus, transcription, and translation as well as degradation of DNA, RNA, and enzyme (37) should be identical in both protoplast populations. Differences in the appearance of the enzyme activity should directly reflect properties of the ECR reaction itself. The detected delay indicates that the time required for most ECR events appears to be no more than 30 min (Fig. 3). The time of ECR might even be less, as samples were taken in half-hour intervals. Within this short time period, a multitude of different processes has to take place: breaks have to be introduced in the DNA substrates, the recombination partners have to find each other and anneal, the gaps in the recombined DNA have to be repaired, and finally restoration of a functional DNA molecule by ligation has to take place. Whereas the time of GUS appearance gives us an answer to the question of how much time ECR needs, the shape of the curve gives us a quantitative indication of how long ECR can take place in the protoplast population. If ECR takes place only in a 30-min period, the two curves in Fig. 3 should have the same slope. If, on the other hand, new template molecules are made by ECR during the whole assay period, GUS expression should start very slowly and the curve should rise later in an exponential manner. In reality, the slope of the ECR-dependent curve in Fig. 3 is very similar to the one of the expression of the functional GUS gene. This indicates that most templates are made by ECR early after
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PUCHTA ET AL.
TABLE 2. Comparison of general properties of ECR and ICR in higher eukaryotesa RE correlated with length of homology RE influenced by CpG methylation RE drastically reduced by mismatches RE dependent on the position in the genome Time requirement of recombination reaction Recombination reaction is conservative Putative mechanism of the recombination reaction Recombination frequency"
Reference(s)
ECR
Property
1, 38 This work 49
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