The remaining dna 25 in female gametes 50 in male

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Unformatted text preview: , 75% of DNA in egg cells versus 50% of DNA in sperm nuclei should be fully demethylated. The remaining DNA (25% in female gametes, 50% in male) should be hemimethylated (Fig. 3d). If, as suggested21, A. thaliana gametes undergo an additional round of DNA synthesis and fertilize in G2, demethylation ratios should be higher. We determined the actual frequency of demethylation and reactivation of the GUS locus in reciprocal backcrosses of F1 hybrids (genotype MET1+/– GUS+/–). The frequency of reactivation was significantly higher when met1 and GUS were maternally inherited (74.5% among backcrossed plants; 102 active and 35 silent, hypothesis 3:1; χ2 = 0.004; P > 0.99) than after paternal transmission (42% among backcrossed plants; 53 active and 72 silent, hypothesis 1:1; χ2 = 2.59; P > 0.10). nature genetics • volume 34 • may 2003 © 2003 Nature Publishing Group letter We confirmed this ‘epigenetic segregation’ using Southern blots for the methylation status of the GUS locus in backcrossed plants (Fig. 3b). The accurate match between observed segregation frequencies and expected parental effects support the hypothesis that MET1 is required during gametogenesis. We may also conclude that the residual hemimethylation provides necessary and sufficient information for remethylation of the second DNA strand as soon as MET1 is again provided in the heterozygous zygotes. This remethylation of hemimethylated templates probably occurs before the first S phase, that is, before zygotic DNA replication, as the backcross progeny included plants without detectable GUS activity in any tissue (indicating complete silencing and methylation of the gene; Fig. 3d). The observed reactivation ratios are consistent with fertilization occurring at G1 stage; however, carryover of MET1 activity into the first postmeiotic mitosis resulting in delayed demethylation cannot be excluded. Notably, we observed gametophytic effects only after total depletion of MET1 activity. Previous genetic analysis in F2 populations, using a partial loss-of-function allele of MET1 (met1-1, met1-2) that retains up to 30–50% of CpG methylation in early generations of homozygous mutant plants9, suggested a clearly recessive character of the mutation9,19. In contrast, comparable F2 examination of met1-3 or met1-4 crosses showed clear gametophytic effects (see Supplementary Table 1 online). Divergent methylation in met1 heterozygotes was not restricted to the transgenic GUS genes but was also shown for endogenous loci. Southern-blot analysis of the Rap2.1 promoter on chromosome I (ref. 22) and of the FWA promoter on chromosome IV (ref. 18), both known to be methylated in wild-type A. thaliana, showed that T2 plants heterozygous with respect to the met1 mutation inherited either one or no hypomethylated allele of each gene in a process resembling random segregation (Fig. 3c). These arbitrary combinations of methylated and unmethylated loci match the phenotypic variation observed among heterozygous met1 plants (Fig. 2) and reflect independent epigenetic segregation. It is well documented that maintenance of DNA methylatio...
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