Initiation of DNA synthesis by nuclei from scrape-ruptured quiescent mammalian cells in high-speed supernatants of Xenopus egg extracts.
Demembranated sperm heads, detergent-isolated somatic nuclei and even naked DNA are efficiently replicated in cytoplasmic extracts of activated amphibian eggs, but only after nuclear assembly and the formation of an intact nuclear envelope. DNA synthesis has not previously been shown to be initiated in high-speed (200,000 g) supernatants of egg cytoplasm because they are depleted of the vesicular material required to support nuclear envelope formation. Here we show that mammalian nuclei prepared by scrape-rupture are able to initiate DNA replication in such high-speed supernatants. These nuclei begin DNA synthesis asynchronously. This asynchrony cannot be attributed to differences in the time taken for nuclear assembly. Instead, we suggest that the asynchrony reflects intrinsic differences between nuclei and that these differences are a major cause of cell cycle variability. Our demonstration of initiation in high-speed supernatants now enables the initiation of eukaryotic DNA synthesis to be studied independently of nuclear assembly.
Hola
M
M
Division of Biomedical Sciences, King's College, Randall Institute, London, UK.
Castleden
S
S
Howard
M
M
Brooks
R F
RF
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis*; D004261:DNA Replication; D005260:Female; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D007399:Interphase; D008297:Male; D009685:Nuclear Envelope / Q000378:metabolism; D010063:Ovum / Q000378:metabolism* / Q000648:ultrastructure; D013077:Sperm Head / Q000378:metabolism; D014461:Ultracentrifugation; D014981:Xenopus
D004247:DNA