Last year we reported on a survey of the patterns of ribosomal DNA (rDNA) fragments in inbred lines of maize produced by digestion of leaf tissue DNA with the methylation-sensitive restriction enzyme HpaII. Inbred lines were heterogeneous with respect to the number of bands produced by HpaII digestion of nuclear DNA with either one, two, or three bands being present. R. Phillips et al. (Keystone Conference, 1985) have reported that DNA purified from leaf tissue of A188 has a single hypomethylated HpaII site, but DNA from endosperm tissue harvested at 17 days after pollination (DAP) displayed four bands indicating that additional HpaII sites were undermethylated.
We have initiated a survey of rDNA patterns from specific tissues of our previously assayed inbred lines to determine if there is a relationship between the heterogeneous methylation pattern in leaf tissue and the methylation pattern of other tissues. Figure 1 shows the result of HpaII digests of DNA isolated from the hybrid SX19 (B73 X Mo17). HpaII digestion of SX19 rDNA from leaf tissue produced a single 9.1 kbp band and left a significant uncut fraction. This result is identical to those we reported for the inbred parent lines in our communication last year. HpaII digestion of 13 DAP endosperm rDNA produced three distinct new bands at 5.2, 3.5, and 2.6 kbp. The 9.1 kbp band is no longer visible, and there is considerable background smear in this lane indicating that, in general, the rDNA is less methylated. However, a significant fraction of the rDNA still is not accessible to HpaII cleavage in the endosperm tissue. These results are similar to those previously reported by Phillips et al. in 17 DAP endosperm from A188. We are now examining rDNA hypomethylation in other tissues from this hybrid, in specific tissues from its progenitors, and from B37N.
Figure 1. SX19 DNA, isolated from seedling leaves or from endosperm collected at 13 DAP, was digested with HpaII, and fragments were separated by electrophoresis on 0.8% agarose gels. Fragments were transferred to nitrocellulose by Southern blotting and probed with a maize rDNA probe (pZmrl) containing the entire repeat unit (M.D. McMullen et al., Nucl. Ac. Res. 14:4953,1986). Fragment sizes were derived from lambda DNA cut with HindIII. We wish to thank Pioneer Hi-Bred International, Inc. for providing SX19 seeds, and M.D. McMullen for providing the plasmid pZmrl.
Vishal Sachdev, Eldon Jupe and Elizabeth Zimmer
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