Overproduction of Rb protein after the G1/S boundary causes G2 arrest.

摘要: The Rb protein is known to exert its activity at decision points in the G1 phase of cell cycle. To investigate whether it may also play some role(s) later cycle, we used a system rapid inducible gene amplification conditionally overexpress during G2 phase. A line expressing temperature-sensitive simian virus 40 large T antigen (T-Ag) was stably transfected with plasmids containing cDNA linked origin replication: pRB-wt, pRB-fs, and pRB-Dra, carrying wild-type murine cDNA, frameshift mutation close beginning coding region, single-amino-acid deletion E1A/T-Ag binding pocket, respectively. Numerous independent lines were isolated nonpermissive temperature; displaying high level episomal an intact expression cassette following shiftdown permissive temperature chosen for further analysis. Plasmid pRB-fs did not express detectable antigen, while pRB-Dra expressed full-length protein. Dra has previously been shown abrogate phosphorylation as well T-Ag binding. Fluorescence-activated sorting (FACS) analysis revealed that cultures induced either or mutant proteins significantly enriched cells DNA content. Cultures amplified rearranged pRB-wt had normal cycle profiles. Double-label FACS showed overexpressing Rb-Dra uniformly accumulating G2, whereas endogenous levels found throughout These results indicate interacting component(s) cycle-regulatory machinery