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T Enoch

Publications and source records attributed to T Enoch.

27 records · Page 2Linked to original sources
PubMed1995

The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis.

Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired. Controls called 'checkpoints', mediate cell cycle arrest in response to unreplicated or damaged DNA. Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation. We have cloned and sequenced the hus5+ gene. It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs). To understand the role of hus5+ in cell cycle control we have characterized the phenotypes of the hus5 mutants and the hus5 gene disruption. We find that, whilst the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest. Thus, the hus5+ gene product is not directly involved in checkpoint control. However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants. In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation. We conclude that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation.

Amino Acid Sequence↗
PubMed1994

mik1+ encodes a tyrosine kinase that phosphorylates p34cdc2 on tyrosine 15.

mik1+ and wee1+ function to regulate the tyrosine phosphorylation of p34cdc2 in Schizosaccharomyces pombe (Lundgren, K., Walworth, N., Booher, R., Dembski, M., Kirschner, M., and Beach, D. (1991) Cell 64, 1111-1122). wee1+ encodes a tyrosine kinase that directly phosphorylates p34cdc2 on tyrosine 15, resulting in the inactivation of the cyclin B/p34cdc2 complex. We have overproduced the mik1+ gene product in insect cells and in S. pombe in order to characterize it biochemically. Immunoprecipitates of Mik1 from both sources catalyzed the phosphorylation of p34cdc2 on tyrosine 15 whereas immunoprecipitates of a kinase-deficient mutant of Mik1 were negative in this assay. Mik1 overproduced in insect cells was partially purified by column chromatography, and column fractions were assayed for their ability to phosphorylate p34cdc2 on tyrosine 15. Two major peaks of Mik1 protein were detected by gel filtration chromatography. One peak eluted in the void volume, and a second peak eluted with an apparent molecular mass expected for monomeric Mik1 (approximately 68 kDa). The tyrosine 15 kinase activity co-eluted with the 68 kDa form of Mik1. These results indicate that mik1+ encodes a tyrosine kinase that directly phosphorylates p34cdc2 on tyrosine 15.

Amino Acid Sequence↗
PubMed1993

Checkpoint check.

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Genes, Fungal↗
PubMed1992

Fission yeast genes involved in coupling mitosis to completion of DNA replication.

We have isolated fission yeast mutants that enter mitosis when DNA replication is blocked with hydroxyurea. The mutants define eight linkage groups, three of which consist of alleles of the rad1, rad3, and rad17 genes. Recently, these fission yeast genes have been shown to be required for radiation-induced cell cycle arrest, as is the budding yeast RAD9 gene. The other five genes are called hus (hydroxyurea sensitive) 1-5. We propose that these genes participate in an intracellular signal transduction pathway that monitors the completion of DNA replication and transmits information to the mitotic control protein cdc2. Mutations that bypass the requirement for cdc25 (an activator of the mitotic regulator cdc2) also uncouple mitosis from DNA replication. However, mitosis is blocked by inhibitors of DNA replication in strains in which the cdc25 gene has been deleted, indicating that although cdc25 influences the coupling of mitosis to the completion of DNA replication, it is not essential for this control.

Alleles↗
PubMed1991

p34cdc2 acts as a lamin kinase in fission yeast.

The nuclear lamina is an intermediate filament network that underlies the nuclear membrane in higher eukaryotic cells. During mitosis in higher eukaryotes, nuclear lamins are phosphorylated by a mitosis-specific kinase and this induces disassembly of the lamina structure. Recently, p34cdc2 protein kinase purified from starfish has been shown to induce phosphorylation of lamin proteins and disassembly of the nuclear lamina when incubated with isolated chick nuclei suggesting that p34cdc2 is likely to be the mitotic lamin kinase (Peter, M., J. Nakagawa, M. Dorée, J.C. Labbe, and E.A. Nigg. 1990b. Cell. 45:145-153). To confirm and extend these studies using genetic techniques, we have investigated the role of p34cdc2 in lamin phosphorylation in the fission yeast. As fission yeast lamins have not been identified, we have introduced a cDNA encoding the chicken lamin B2 protein into fission yeast. We report here that the chicken lamin B2 protein expressed in fission yeast is assembled into a structure that associates with the nucleus during interphase and becomes dispersed throughout the cytoplasm when cells enter mitosis. Mitotic reorganization correlates with phosphorylation of the chicken lamin B2 protein by a mitosis-specific yeast lamin kinase with similarities to the mitotic lamin kinase of higher eukaryotes. We show that a lamin kinase activity can be detected in cell-free yeast extracts and in p34cdc2 immunoprecipitates prepared from yeast cells arrested in mitosis. The fission yeast lamin kinase activity is temperature sensitive in extracts and immunoprecipitates prepared from strains bearing temperature-sensitive mutations in the cdc2 gene. These results in conjunction with the previously reported biochemical studies strongly suggest that disassembly of the nuclear lamina at mitosis in higher eukaryotic cells is a consequence of direct phosphorylation of nuclear lamins by p34cdc2.

Animals↗
PubMed1990

Mutation of fission yeast cell cycle control genes abolishes dependence of mitosis on DNA replication.

Entry into mitosis in fission yeast is controlled by the p34cdc2 protein kinase, which is activated by cdc25+ and inhibited by wee1+. In "wee" mutants one or the other of these controls is circumvented resulting in advancement of mitosis. We report that dependence of mitosis on DNA synthesis is lost in wee mutants in which cdc25+ control is circumvented either by mutations in cdc2+ or by overproduction of cdc25+. In contrast, dependence is maintained when the wee1+ control is bypassed. We propose that cdc25+ activity requires completion of earlier cell-cycle events such as DNA synthesis, and thus links p34cdc2 kinase activation to completion of these earlier events. Constitutive expression of cdc25+ homologs could explain why mitosis is not dependent on DNA replication in some early embryos.

CDC2 Protein Kinase↗
PubMed1986

Activation of the human beta-interferon gene requires an interferon-inducible factor.

beta-Interferon (beta-IFN) gene expression can be induced by poly(I)-poly(C) or virus, but there is considerable variation in the extent of induction between different cell lines. We characterized two poorly inducible human cell lines, HeLa and 143 thymidine kinase negative (143 tk-), to define cellular factors involved in the activation of the beta-IFN gene. We show that the deficiency in beta-IFN induction in these cells can be complemented by fusion to highly inducible mouse cells. We conclude that the human cells are deficient in a trans-acting factor required for B-IFN gene activation. The level of induction of the beta-IFN gene in HeLa and 143 tk- cells can also be increased by priming with IFN before induction. If IFN priming is carried out in the presence of cycloheximide, a approximately 200-fold increase in induction is observed. We conclude that activation of the beta-IFN gene requires an IFN-inducible factor that is only expressed at low levels in unprimed HeLa and 143 tk- cells.

Cell Fusion↗