Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts.
Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define 'S-phase promoting factor' (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.
Strausfeld
U P
UP
Imperial Cancer Research Fund, Clare Hall Laboratories, Potters Bar, Herts, England.
Howell
M
M
Descombes
P
P
Chevalier
S
S
Rempel
R E
RE
Adamczewski
J
J
Maller
J L
JL
Hunt
T
T
Blow
J J
JJ
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D005314:Embryonic and Fetal Development; D005260:Female; D066298:In Vitro Techniques; D008938:Mitosis / Q000187:drug effects; D010063:Ovum / Q000166:cytology / Q000187:drug effects / Q000254:growth & development; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D016196:S Phase / Q000187:drug effects*; D014981:Xenopus
D016213:Cyclins; D011994:Recombinant Proteins; D016203:CDC2 Protein Kinase; D018844:Cyclin-Dependent Kinases