In our last two communications to the MNL we have reported on rDNA methylation patterns in different inbred lines of maize. We have now initiated studies to examine rDNA methylation in Tripsacum. DNA from several species (T. australe, T. cundinamarce, T. dactyloides and T. laxum) and three accessions of a single species (T. dactyloides) were digested with the methylation-sensitive restriction enzyme HpaII and probed with pZmr1 to characterize their rDNA methylation patterns. These experiments showed that most of the rDNA was not accessible to HpaII cleavage indicating that the rDNA arrays were completely methylated. However, a significant fraction of the rDNA (15-25%) was digested by HpaII into fragments of repeat unit length (see Figure 1A; lanes 1-7). These HpalI single digest patterns are similar to those of maize shown in our previous work. Some species of Tripsacum are heterogeneous for rDNA length. In Figure 1A (lanes 1 and 4) we can see that rDNA repeat units of two distinct lengths have been released by Hpall digestion. In Figure 1B, we have mapped the distance from the conserved XbaI site (in the 18S gene) to the site of HpaII cleavage. The 8.6 kbp band released in a HpaII/XbaI double digest maps the site of Hpall cleavage to a location within the intergenic spacer of Tripsacum rDNA. Considering the difference in total repeat unit length between T. dactyloides (9.5 kbp) and inbred lines of maize (9.1 kbp), this site falls in a region very near that previously observed in inbred lines of maize (R. Phillips et al., Keystone Conference, 1985; E. Jupe and E. Zimmer, unpublished observations).
Figure 1. DNA purified from leaves of various species of Tripsacum was digested with appropriate restriction enzymes, electrophoresed on UK agarose gels, and transferred to Zeta bind membranes by Southern blotting. In (A) we have HpaII digests of DNA from five species of Tripsacum (including three accessions of T. dactyloides) probed with a full length maize rDNA probe (pZmr1). Samples loaded in lanes are as follows: (1) T. australe (2) T. dactyloides (acc. 68-50-5) (3) T. dactyloides (acc. 63-229) (4) T. dactyloides (acc. 63-39) (5) T. cundinamarce (6) T. laxum (7) T. peruvianum. In (B) we show preliminary mapping experiments for T. dactyloides (acc. 63-229). DNA was digested with XbaI (lane 1). HpaII (lane 2) and HpaII/XbaI (lane 3). A 400 base pair probe (pXBr1) adjacent to the conserved XbaI site was used to indirectly end-label the fragments produced in double digests. Fragment sizes were calculated from maize rDNA digested with BamHI and detected with the probe pZmr1 (size indicated by arrows on left) and lambda DNA digested with HindIII. We wish to thank Dr. M. D. McMullen for providing the plasmid pZmr1 and Dr. D. H. Timothy for Tripsacum seeds.
Vishal Sachdev, Eldon Jupe and Elizabeth Zimmer
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