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GST-T7-Cdc14 (RDB 872) and GST-Cdc14 (RDB 1183) were expressed in E. coli BL21(DE3) pLysS cells, and purified on glutathione-agarose beads (Sigma) as described previously [ 33 ] . His6-T7-Net1(1-341) (Net1N, RDB1153) [ 11 ] was expressed in E. coli BL21(DE3) pLysS cells, and purified on Ni-NTA agarose beads (Qiagen) as described previously for p28 Kix1 [ 34 ] . Purification of His6-Plx1, His6-Plx1(N172), and His6-HA-Cdc5 from baculovirus-infected insect cells followed an earlier protocol [ 24 ] . The concentrations of purified proteins were in the ranges of: Net1N~0.
The autogene pool was transformed into DH5Δlac pLysS cells by electroporation.
The wild type autogene thus obtained (pET/T7p/T7) was transformed into strain HMS174 pLysS (Novagen, Madison, WI), the same cell line Studier et al.
To test for overexpression of the mutant enzymes, E. coli BL21(DE3)pLysS cells were transformed with the mutated over-expression plasmids, and small scale growth cultures were used [ 68 ] . Large-scale (4 L) preparations of the L1 mutants were performed as described previously [ 36 ] . Protein purity was ascertained by SDS-PAGE.
E. coli strains DH5α and BL21(DE3) pLysS were obtained from Gibco BRL and Novagen, respectively.