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Fig. 2. Characterization of B-type cyclins from U. maydis. (A) Scheme of Clb1 and Clb2 proteins. U. maydis cyclins contained the FLRRXSK motif (white boxes) and `destruction box' consensus sequences (gray boxes). (B) Dendrogram of selected B-type cyclins from fungi. U. maydis Clb1 groups with the S- and M-phase cyclins from S. pombe, while U. maydis Clb2 groups with a heterogeneous B-type cyclins. Accession numbers for the proteins are: ScClb5, P30283; CaCyb1, U40430; ScClb2, S14166; SpCig1, P24865; CaCyb4, AAC79857; ScClb3, A60048; UmClb2, AY260970; UmClb1, AY260969; SpCig2, P36630; SpCdc13, P10815. Bar: 0,05 substitutions per aa. (C) Clb1 and Clb2 are highly abundant in S- and G2/M-arrested cells, but present at low levels in G1 cells. Extracts from cells carrying an epitope-tagged copy of Clb1 and Clb2 were analyzed by immunoblotting. Samples are as follows: G1, culture enriched in G1 phase cells; Ben, cells arrested at G2/M transition after treatment with benomyl for 1 hour; HU, cells arrested in S phase by treatment with hydroxyurea for 90 minutes; As, asynchronous cells. The same filters were also probed with anti-PSTAIRE antibodies (bottom panel), showing that the abundance of Cdk1 varied less than twofold. (D) Clb1 associates with Cdk1. Lysates prepared from wild-type FB1 and Clb1-tagged UMP19 (clb1-1) cells were incubated with Suc1 beads to pull down Cdk1 (upper panel) or were immunoprecipitated with anti-VSV antibody to pull down Clb1 (lower panel). The whole cell lysates as well as the precipitates were separated by SDS-PAGE and immunoblotted with anti-PSTAIRE and anti-VSV to detect Cdk1 and Clb1 proteins respectively. (E) Clb2 associates with Cdk1. Lysates prepared from wild-type FB1 and Clb2-tagged UMP27 (clb2-1) cells were incubated with Suc1 beads to pull down Cdk1 (upper panel) or were immunoprecipitated with anti-MYC antibody to pull down Clb2 (lower panel). The whole cell lysates, as well as the precipitates, were separated by SDS-PAGE and immunoblotted with anti-PSTAIRE and anti-MYC to detect Cdk1 and Clb2 proteins respectively. The different bands with higher electrophoretic mobility detected in the anti-MYC IP are degradation products of Clb2.
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