Studies of mammalian chromosome replication. II. Evidence for the existence of defined chromosome replicating units.
Sister chromatids of metaphase chromosomes can be differentially stained if the cells have replicated their DNA semiconservatively for two cell cycles in a medium containing 5-bromodeoxyuridine (BrdU). When prematurely condensed chromosomes (PCC) are induced in cells during the second S phase after BrdU is added to the medium, the replicated chromosome segments show sister chromatid differential (SCD) staining. Employing this PCC-SCD system on synchronous and asynchronous Chinese hamster ovary (CHO) cells, we have demonstrated that the replication patterns of the CHO cells can be categorized into G1/S, early, early-mid, mid-late, and late S phase patterns according to the amount of replicated chromosomes. During the first 4 h of the S phase, the replication patterns show SCD staining in chains of small chromosome segments. The amount of replicated chromosomes increase during the mid-late and late S categories (last 4 h). Significantly, small SCD segments are also present during these later intervals of the S phase. Measurements of these replicated segments indicate the presence of characteristic chromosome fragment sizes between 0.2 to 1.2 micrometers in all S phase cells except those at G1/S which contain no SCD fragments. These small segments are operationally defined as chromosome replicating units of chromosomal replicons. They are interpreted to be composed of clusters of molecular DNA replicons. The larger SCD segments in the late S cells may arise by the joining of adjacent chromosomal replicons. Further application of this PCC-SCD method to study the chromosome replication process of two other rodents, Peromyscus eremicus and Microtus agrestis, with peculiar chromosomal locations of heterochromatin has demonstrated an ordered sequence of chromosome replication. The euchromatin and heterochromatin of the two species undergo two separate sequences of decondensation, replication, and condensation during the early-mid and mid-late intervals respectively of the S phase. Similar-sized chromosomal replicons are present in both types of chromatin. These data suggest that mammalian chromosomes are replicated in groups of replicating units, or chromosomal replicons, along their lengths. The organization and structure of these chromosomal replicons with respect to those of the interphase nucleus and metaphase chromosomes are discussed.
D000818:Animals; D003411:Arvicolinae / Q000235:genetics; D001973:Bromodeoxyuridine; D002460:Cell Line; D002842:Chromatids / Q000648:ultrastructure*; D002875:Chromosomes / Q000648:ultrastructure*; D006224:Cricetinae; D003412:Cricetulus; D003584:Cytological Techniques; D004261:DNA Replication; D005260:Female; D010053:Ovary; D010542:Peromyscus / Q000235:genetics; D013045:Species Specificity
D001973:Bromodeoxyuridine