Pl-Rhoades is more susceptible to DNase I cleavage than Pl-Blotched
--Hoekenga, OA and Cone, KC

Pl-Rhoades (Pl-Rh) and Pl-Blotched (Pl-Bh) are alleles with strikingly different phenotypes. Pl-Rh leads to uniform pigmentation in the plant and is silent in the kernel. Pl-Bh leads to variegated pigmentation in both the plant and the kernel. Pl-Rh and Pl-Bh have essentially identical DNA sequences, but Pl-Bh DNA is hypermethylated relative to Pl-Rh. The degree of methylation correlates with the phenotype (more methylated, less pigment) and with mRNA levels (more methylated, less mRNA) (Cocciolone and Cone, Genetics 135:575, 1992). In other systems, transcriptionally inactive genes are frequently methylated and located in tightly packed or condensed chromatin (Eden and Cedar, Curr Op Genet Dev 4:255, 1994). If this trend holds for the Pl gene, then the chromatin structure of Pl-Rh should be more "open" than that of Pl-Bh.

To address this prediction, we compared the chromatin structure of the two Pl alleles using a nuclease protection assay (Spiker, Murray and Thompson, PNAS USA 80:815, 1983). In this type of assay, "open" chromatin should be more accessible to nuclease digestion than tightly condensed chromatin. Nuclei from Pl-Rh and Pl-Bh husks were incubated with the nuclease DNase I. The DNA was purified, digested with restriction enzymes, and analyzed on Southern blots. The results indicated that both genes are susceptible to DNase I cleavage at the same sites within the coding region in 5' flanking sequences. However, Pl-Rh is more susceptible to DNase I digestion than Pl-Bh. These observations are consistent with our expectations. Pl-Rh and Pl-Bh were predicted to share the same DNase I sensitive sites, as an active Pl-Bh should be indistinguishable from Pl-Rh. Furthermore, the enhanced susceptibility of Pl-Rh correlates with its higher transcriptional activity and lower level of DNA methylation. Future experiments will be aimed at investigating chromatin structure of the two alleles in the kernel as a step in trying to explain the ectopic expression of Pl-Bh in the kernel. 

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