Quinn LM , Herr A , McGarry TJ , Richardson H .

Figure 1. Drosophila Geminin and comparison withXenopus and human Geminin. (A) Drosophila(Dm) geminin gene structure showing location ofP-element alleles relative to the transcriptional unit. (B) Sequence comparison with Xenopus (Xl) and human (Hs) Geminin. The sequences were aligned using the GCG Gap program. Identical amino acids are marked with a vertical line and conservative changes are indicated (*). Differences between Xl H and L forms are indicated. The putative destruction box is in boldface type. The neuralization domain is underlined and the DNA replication inhibition domain is indicated by the broken underline. The double line above the sequence alignment shows the coiled-coil region. The degree of identity and similarity (in parentheses) between Xl Geminin L, Hs and Dm Geminin sequences is indicated for each domain.

Figure 2. Drosophila Geminin inhibits DNA replication in Xenopus extracts by preventing Mcm loading and interacts with Drosophila Cdt1(Dup) in vivo. (A)Drosophila (Dm) Geminin inhibits DNA replication inXenopus extracts. Purified Xl Geminin (⋄), GST-Dm Geminin (○), His-6 Dm Geminin (▵), or GST (□) were added at the concentrations shown to a Xenopus DNA replication in vitro assay system. Replication assays were carried out as described previously (McGarry and Kirschner 1998). (B) Dm Geminin inhibits Mcm loading. The indicated amounts of Dm Geminin were added to the Xenopus in vitro assay system and DNA replication measured (top) or the chromatin binding of xMcm3 determined (bottom). Concentrations of Dm Geminin that inhibit DNA replication (top) strongly inhibited xMcm3 chromatin binding. The GST control added at a concentration of 8 μg/mL had no effect on DNA replication or xMcm3 binding. In the control tracks (no sperm DNA or no calcium), DNA replication and xMcm3 binding were not observed. (C) Anti-Dm Geminin Western analysis. Dm Geminin bands are indicated (arrowheads). The faint band above 30 kD is a background band caused by the secondary antibody (data not shown). No Dm Geminin antibody reacting bands were detected elsewhere on the gel (data not shown). (WT) Wild type; (Hs-Gem) heat shocked hsp70–GAL4 UAS–Dm geminin embryos; (mutant 1) l(2)k14019/CyO; and (mutant 2)l(2)k03202/CyO. (+) Heat shocked. Equal amounts of protein were loaded per track. (D) Dm Dup(Cdt1) and Dm Geminin form a complex in Drosophila embryos. Dup or Dm Geminin were immunoprecipitated from embryonic extracts and the precipitate analyzed by immunoblotting with the Dup antibody (left) or Dm Geminin antibody (right). (Left) Dup bands (arrows); Dm Geminin bands (arrowheads); and IgG heavy chain (*) are indicated. Controls, (S) Supernatant; (PI) preimmune sera.

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