Replication termini in the rDNA of synchronized pea root cells (Pisum sativum)

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In synchronized root cells of Pisum sativum (cv. Alaska) the joining of nascent replicons is delayed until cells reach the S–G2 boundary or early G2 phase. To determine if the delayed ligation of nascent chains occurs at specific termination sites, we mapped the location of arrested forks in the ribosomal DNA (rDNA) repeats from cells in late S and G2 phases. Two-dimensional (neutral-alkaline) agarose electrophoresis and Southern blot hybridization with specific rDNA sequences show that only cells located at the S–G2 boundary and early G2 phase produce alkali-released rDNA fragments of discrete size. The released fragments are from a particular restriction fragment, demonstrating that the replication forks stop non-randomly within the rDNA repeats. Indirect end-labeling with probes homologous to one or the other end of the fork-containing restriction fragment shows that there are two termination regions, T1 and T2, where forks stop. T1 is located in the non-transcribed spacer and T2 is at the junction between the non-transcribed spacer and the 18S gene. The two termini are separated by ˜1.3 kb. Replication forks stop at identical sites in both the 8.6- and 9.0-kb rDNA repeat size classes indicating that these sites are sequence determined.

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