Cyclin‐dependent kinases (cdk) play an essential role in the intracellular control of the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. Enzymatic screening has recently led to the discovery of specific inhibitors of cyclin‐dependent kinases, such as butyrolactone I, flavopiridol and the purine olomoucine. Among a series of C2, N⁶, N9‐substituted ade‐nines tested on purified cdc2/cylin B, 2‐(1‐ethyl‐2‐hydroxyethylamino)‐6‐benzylamino‐9‐isopropylpu‐rine (roscovitine) displays high efficiency and high selectivity towards some cyclin‐dependent kinases. The kinase specificity of roscovitine was investigated with 25 highly purified kinases (including protein kinase A, G and C isoforms, myosin light‐chain kinase, casein kinase 2, insulin receptor tyrosine kinase, c‐src, v‐abl). Most kinases are not significantly inhibited by roscovitine. cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdkSlp35 only are substantially inhibited (IC₅₀values of 0.65, 0.7, 0.7 and 0.2 μM, respectively). cdk4/cyclin D1 and cdk6/cyclin D2 are very poorly inhibited by roscovitine (IC₅₀>100 μM). Extracellular regulated kinases erkl and erk2 are inhibited with an IC₅₀of 34 μM and 14 μM, respectively. Roscovitine reversibly arrests starfish oocytes and sea urchin embryos in late pro‐phase. Roscovitine inhibitsin vitroM‐phase‐promoting factor activity andin vitroDNA synthesis inXenopusegg extracts. It blocks progesterone‐induced oocyte maturation ofXenopusoocytes andin vivophosphorylation of the elongation factor eEF‐1. Roscovitine inhibits the proliferation of mammalian cell lines with an average IC₅₀] of 16 μM. In the presence of roscovitine L1210 cells arrest in G1 and accumulate in G2.In vivophosphorylation of vimentin on Ser55 by cdc2/cyclin B is inhibited by roscovitine. Through its unique selectivity for some cyclin‐dependent kinases, roscovitine provides a useful anti‐mitotic reagent for cell cycle studies and may prove interesting to control cells with deregulated cdc2, cdk2 or cdk5 kinase activities.

The minichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes. Thus far, all eukaryotes have been shown to contain six highly related MCMs that apparently function together in DNA replication. Sequencing of the entire genome of the thermophilic archaeon Methanobacterium thermoautotrophicum has allowed us to identify only a single MCM-like gene (ORF Mt1770). This gene is most similar to MCM4 in eukaryotic cells. Here we have expressed and purified the M. thermoautotrophicum MCM protein. The purified protein forms a complex that has a molecular mass of ≈850 kDa, consistent with formation of a double hexamer. The protein has an ATP-independent DNA-binding activity, a DNA-stimulated ATPase activity that discriminates between single- and double-stranded DNA, and a strand-displacement (helicase) activity that can unwind up to 500 base pairs. The 3′ to 5′ helicase activity requires both ATP hydrolysis and a functional nucleotide-binding site. Moreover, the double hexamer form is the active helicase. It is therefore likely that an MCM complex acts as the replicative DNA helicase in eukaryotes and archaea. The simplified replication machinery in archaea may provide a simplified model for assembly of the machinery required for initiation of eukaryotic DNA replication.

The replication licensing factor (RLF) is an essential initiation factor that is involved in preventing re-replication of chromosomal DNA in a single cell cycle. In Xenopus egg extracts, it can be separated into two components: RLF-M, a complex of MCM/P1 polypeptides, and RLF-B, which is currently unpurified. In this paper we investigate variations in RLF activity throughout the cell cycle. Total RLF activity is low in metaphase, due to a lack of RLF-B activity and the presence of an RLF inhibitor. RLF-B is rapidly activated on exit from metaphase, and then declines during interphase. The RLF inhibitor present in metaphase extracts is dependent on the activity of cyclin-dependent kinases (Cdks). Affinity depletion of Cdks from metaphase extracts removed the RLF inhibitor, while Cdc2/cyclin B directly inhibited RLF activity. In metaphase extracts treated with the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP), both cyclin B and the RLF inhibitor were stabilized although the extracts morphologically entered interphase. These results are consistent with studies in other organisms that invoke a key role for Cdks in preventing re-replication of DNA in a single cell cycle.

Methanococcus maripaludis has two surface appendages, namely flagella and pili. Flagella have been shown to be required for swimming, but no specific