Akihisa Nagata

Cdc25A is a novel phosphatase functioning early in the cell cycle.

The cdc25+ tyrosine phosphatase is a key mitotic inducer of the fission yeast Schizosaccharomyces pombe, controlling the timing of the initiation of mitosis. Mammals contain at least three cdc25+ homologues called cdc25A, cdc25B and cdc25C. In this study we investigate the biological function of cdc25A. Although very potent in rescuing the S.pombe cdc25 mutant, cdc25A is less structurally related to the S.pombe enzyme. Northern and Western blotting detection reveals that unlike cdc25B, cdc25C and cdc2, cdc25A is predominantly expressed in late G1. Moreover, immunodepletion of cdc25A in rat cells by microinjection of a specific antibody effectively blocks their cell cycle progression from G1 into the S phase, as determined by laser scanning single cell cytometry. These results indicate that cdc25A is not a mitotic regulator but a novel phosphatase that plays a crucial role in the start of the cell cycle. In view of its strong ability to activate cdc2 kinase and its specific expression in late G1, cdc2‐related kinases functioning early in the cell cycle may be targets for this phosphatase.

Dephosphorylation of human p34cdc2 kinase on both Thr-14 and Tyr-15 by human cdc25B phosphatase.

In mammalian cells, p34 cdc2 kinase undergoes phosphorylation at threonine‐14, tyrosine‐15 and threonine‐161 in the S and G2 phases of the cell cycle. At the onset of mitosis, the kinase becomes dephosphorylated at threonine‐14 and tyrosine‐15, resulting in activation. Cdc25 phosphatase has been shown to dephosphorylate tyrosine‐15 in vitro, but whether it also does at threonine‐14 remains unclear. In this study, we have found that human cdc25B phosphatase dephosphorylates both threonine‐14 and tyrosine‐15 but not threonine‐161.

Cell cycle control in fission yeast and mammals: identification of new regulatory mechanisms.

Publisher Summary This chapter discusses the recent rapid progress in understanding of the molecular mechanisms controlling the G1 and G2 phases of the cell cycle in fission yeast and mammals, focusing on the newly identified control genes and highly conserved control mechanisms between these two apparently remote organisms. The cells of the fission yeast, Schizosaccharomyces pombe, are rod shaped, grow in the longitudinal direction, and divide by septation and medial fission. The chapter describes the cell cycle starts control of fission yeast and mammals. The mitotic start control of fission yeast and mammals have also been described in the chapter. Cell cycle control is one of the most complex and fundamental cellular regulatory processes that eukaryotes possess. Cell cycle control is one of the most complex and fundamental cellular regulatory processes that eukaryotes possess. Once cells have committed to start the cell cycle, they are unable to differentiate until they return to G1. The next few years will be the period during which rapid progress continues to be made and will witness the discovery of new factors and new mechanisms and the resolution of some of these questions.

Isolation and characterization of nucleoid proteins from Escherichia coli

SummaryOf the molecular species of proteins associated with the nucleoids of Escherichia coli cells, those with relatively high affinity to bind to DNA were isolated and characterized. Seven classes of nucleoid proteins with molecular weights of 9,000, 17,000 (two molecular species), 22,000, 24,000, 27,000 and 28,000 were isolated at more than 90% purity or were partially purified. On the basis of its amino acid composition and other chemical properties, the 9,000 dalton protein was identified as HLP II (or HU protein or BH2) (Pettijohn 1982: Rouvière-Yaniv and Gros 1975; Varshavsky et al. 1978). The 17 K protein consisted of two molecular species and one of these, 17 K (a) protein, seemed to be identical with HLPI (or protein 1 or BH1) reported previously (Pettijohn 1982; Varshavsky et al. 1977; Varshavsky et al. 1978). The 26 K protein was identical to the 22 K protein (Kishi et al. 1982). The 27 K protein showed immunological cross-reactivity with the antibody for histone H2A and was thus identified as the H protein reported previously (Hübscher et al. 1980). Two basic proteins, 9 K and 17 K(a), showed relatively high binding affinities to DNA, while the 28 K protein showed moderate binding affinity. The biological significance of these nucleoid proteins, which constitute a family of proteins participating in formation of the nucleoid structure, is discussed.

In vivo enhancement of general and specific transcription in Escherichia coli by DNA gyrase activity

The effect of drugs which inhibit DNA gyrase, including nalidixic acid, oxolinic acid and coumerycin, on transcription of Escherichia coli bacteria, phage and plasmid genomes was studied. Quantitative estimates of the synthesis of RNA under drug-treatment conditions showed that synthesis of many RNA species, including trp mRNA, was subject to inhibiton by the drug. Transcription directed by the lambda promoter pR was selectively less sensitive to the drug action than transcription initiated at the lambda promoter pL. Evidence was obtained showing that diminished transcription resulted from less frequent RNA chain initiation rather than a premature arrest of the chain elongation. Inhibiton of transcription by these DNA gyrase inhibitors was observed even in the absence of DNA replication. The inhibition by oxolinic acid or coumerycin was not observed in an E. coli strain bearing a nalAr mutation or a cour mutation, respectively. The reduction of trp mRNA synthesis in oxolinic acid-treated cells cannot be attributed to the increase in the rate of nascent mRNA degradation. These results indicate that DNA gyrase is generally required for intracellular RNA synthesis, and suggest that the supercoiling of DNA by this winding enzyme enhances the initiation of transcription.

Dnacin A1 and dnacin B1 are antitumor antibiotics that inhibit cdc25B phosphatase activity

The p80cdc25 protein is a protein phosphatase directly involved in p34cdc2 protein kinase activation by dephosphorylation. The cdc25B gene is one of three human cdc25 homologs which can complement the temperature-sensitive cdc25 mutation of Schizosaccharomyces pombe, and is expressed a high levels in human cell lines, particularly in some cancer cells. A fusion protein of glutathione-S-transferase (GST) and the catalytic domain of cdc25B protein was constructed and found to retain phosphatase activity in the manner of a p80cdc25 phosphatase by using a chromogenic substrate, p-nitrophenylphosphate. Two benzoquinoid antitumor compounds, dnacin A1 and dnacin B1, inhibited phosphatase activity in a non-competitive manner.

Alteration of ribosomes and RNA polymerase in drug-resistant clinical isolates of Mycobacterium tuberculosis.

The biochemical mechanism of resistance to kanamycin, viomycin, and rifampin in five clinical isolates of Mycobacterium tuberculosis was studied. Resistance to viomycin and kanamycin was attributed to altered ribosomes, whereas resistance to rifampin was attributed to an alteration of RNA polymerase. Ribosomal resistance was, however, not the only way of expressing resistance to viomycin and kanamycin.

Cdc6 requires anchorage for its expression

Fibroblasts need anchorage to extracellular matrix to transit from G1 to S phase, but no longer after oncogenic transformation. Here we report that Cdc6 protein essential for the activation of replication origins requires anchorage or oncogenic stimulation for its execution. Upon anchorage loss, Cdc6 expression is shut off both transcriptionally and post-transcriptionally in a rat fibroblast despite enforced activation of E2F-dependent promoters. However, stimulation of this cell with oncogenic growth factors suppresses this shutoff and concurrently activates Cdk2 and Cdk6/4, thereby overriding the anchorage requirement for the G1-S transition and consequently enabling cells to perform anchorage-independent S phase entry. Analysis with enforced expression of Cdc6 indicates that the G1 cyclin-dependent kinases and Cdc6 constitute major cell cycle targets for the restriction of the G1-S transition by anchorage loss.

Analysis of the promoter region in the rRNA operon fromMycobacterium bovis BCG

At least two transcriptional initiation sites were observed in rRNA operon ofMycobacterium bovis BCG at approximately-187 and-265. The former transcriptional signal was recognized byEscherichia coli RNA polymerase, whereas the later was not.

Primary Structure of Rat Brain Prostaglandin D Synthetase Deduced from the cDNA Sequence

The amino acid sequence of rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi, O. (1985) J. Biol. Chem. 260, 12410-12415) was determined by a combination of cDNA and protein sequencing. cDNA clones specific for this enzyme were isolated from a lambda gt11 rat brain cDNA expression library. Nucleotide sequence analyses of cloned cDNA inserts revealed that this enzyme consisted of a 564- or 549-base pair open reading frame coding for a 188- or 183-amino acid polypeptide with a Mr of 21,232 or 20,749 starting at the first or second ATG. About 60% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of tryptic peptides of the purified enzyme. The recognition sequence for N-glycosylation was seen at two positions of amino acid residues 51-53 (-Asn-Ser-Ser-) and 78-80 (-Asn-Leu-Thr-) counted from the first Met. Both sites were considered to be glycosylated with carbohydrate chains of Mr 3,000, since two smaller proteins with Mr 23,000 and 20,000 were found during deglycosylation of the purified enzyme (Mr 26,000) with N-glycanase. The prostaglandin D synthetase activity was detected in fusion proteins obtained from lysogens with recombinants coding from 34 and 19 nucleotides upstream and 47 and 77 downstream from the first ATG, indicating that the glycosyl chain and about 20 amino acid residues of N terminus were not essential for the enzyme activity. The amino acid composition of the purified enzyme indicated that about 20 residues of hydrophobic amino acids of the N terminus are post-translationally deleted, probably as a signal peptide. These results, together with the immunocytochemical localization of this enzyme to rough-surfaced endoplasmic reticulum and other nuclear membrane of oligodendrocytes (Urade, Y., Fujimoto, N., Kaneko, T., Konishi, A., Mizuno, N., and Hayaishi, O. (1987) J. Biol. Chem. 262, 15132-15136) suggest that this enzyme is a membrane-associated protein.