Differential control of Eg5-dependent centrosome separation by Plk1 and Cdk1Mammalian centrosomes separate prior to mitosis in a process involving Cdk1 and the kinesin Eg5. Dissection by chemical genetics reveals a Cdk1-independent alternative mechanism, in which Plk1 counteracts both centrosomal cohesion factors and Eg5-opposing cytoskeletal forces.

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The cDNA of soybean agglutinin (SBA), a glycoprotein lectin, obtained from the mRNA of soybean seeds at mid‐maturation, was cloned in a λgt 10 phage and subcloned in a pUC‐8 plasmid. Probing with a fragment of the lectin gene [Vodkin, L. O., Rhodes, P. R. & Goldberg, R. B. (1983) Cell 34, 1023–1031] afforded a clone of 1012 nucleotides containing the complete coding region of 858 nucleotides for the precursor to soybean agglutinin. The deduced amino acid sequence contains the 253 residues of the mature lectin and an hydrophobic N‐terminal signal peptide of 32 amino acids. Expression in Escherichia coli of the cDNA coding for the precursor to the lectin or for the mature lectin led to the accumulation of large quantities of inclusion bodies, from which mature SBA was isolated in small yield (up to 1 mg/1). It was identical with the native lectin in the hemagglutinating activity and carbohydrate specificity, N‐terminal sequence and oligomeric structure, but, because it was not glycosylated, its subunit mass was lower by 2 kDa. Our findings show that pre‐SBA is processed into the mature form in the bacteria, and that, contrary to what has been suggested [Nagai, K. & Yamaguchi, H. (1993) J. Biochem. (Tokyo) 113, 123–125], glycosylation is not essential for the folding of the lectin, nor for its subunit assembly into a biologically active tetramer. To obtain recombinant SBA in secreted form, the pre‐SBA cDNA was subcloned in pTM1 vector and the construct inserted into vaccinia virus. When monkey BS‐C‐1 cells were infected by the virus, using a double expression protocol, recombinant lectin was secreted into the growth medium, from which it was isolated by immunoaffinity chromatography at a yield similar to that from the bacteria. Except for its lower hemagglutinating activity, the product was indistinguishable from native SBA in all properties tested. It was also susceptible to digestion by endo‐β‐N ‐acetylglucosaminidase H or N‐glycanase which caused a decrease of 2 kDa in its subunit mass and gave the same results on lectin blot analysis, indicating that it too is a glycoprotein with a single oligomannose unit.

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With a Hunsdiecker-Barton iododecarboxylation strategy, we converted the carboxylate group of the oseltamivir precursor into exemplary phosphonate monoesters. In all cases, K(i) values towards influenza virus sialidase remained in the sub-nanomolar range. We have thus made valuable structural space available for the design of novel oseltamivir-based tools for influenza virus research.