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

Abstract

In synchronized root cells of Pisum sativum (cv. Alaska) the joining of nascent replicons is delayed until cells reach the S-G(2) boundary or early G(2) 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 G(2) phases. Two-dimensional (neutral-alkaline) agarose electrophoresis and Southern blot hybridization with specific rDNA sequences show that only cells located at the S-G(2) boundary and early G(2) 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, T(1) and T(2), where forks stop. T(1) is located in the non-transcribed spacer and T(2) 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.