Molecular and Cellular Biology

Hixon, Mary L.; Flores, Ana I.; Wagner, Mark W.; Gualberto, Antonio

doi: N/Apmid: 9774639

Primary human fibroblasts arrest growth in response to the inhibition of mitosis by mitotic spindle-depolymerizing drugs. We show that the mechanism of mitotic arrest is transient and implicates a decrease in the expression of cdc2/cdc28 kinase subunit Homo sapiens 1 (CKsHs1) and a delay in the metabolism of cyclin B. Primary human fibroblasts infected with a retroviral vector that drives the expression of a mutant p53 protein failed to downregulate CKsHs1 expression, degraded cyclin B despite the absence of chromosomal segregation, and underwent DNA endoreduplication. In addition, ectopic expression of CKsHs1 interfered with the control of cyclin B metabolism by the mitotic spindle cell cycle checkpoint and resulted in a higher tendency to undergo DNA endoreduplication. These results demonstrate that an altered regulation of CKsHs1 and cyclin B in cells that carry mutant p53 undermines the mitotic spindle cell cycle checkpoint and facilitates the development of aneuploidy. These data may contribute to the understanding of the origin of heteroploidy in mutant p53 cells.

Rahmatullah, Mohammed; Schroering, Allen; Rothblum, Katrina; Stahl, Richard C.; Urban, Bobbi; Carey, David J.

doi: N/Apmid: 9774641

A peptide corresponding to the epidermal growth factor homology domain of -heregulin stimulated autophosphorylation of the heregulin receptors erbB2 and erbB3 in Schwann cells and activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2. Heregulin-dependent activation of PAK65, a component of the stress-activated signaling pathway, ribosomal S6 kinase, and a cyclic AMP (cAMP) response element binding protein (CREB) kinase, identified as p95 RSK2 , was also observed. Receptor phosphorylation and activation of these kinases in response to heregulin occurred in the absence of forskolin stimulation and were not augmented in cells treated with forskolin, a direct activator of adenylyl cyclase. Schwann cell proliferation in response to heregulin was observed only when the cells were also exposed to an agent that elevates cAMP levels. In the absence of heregulin, elevation of cAMP levels failed to stimulate Schwann cell proliferation. Forskolin significantly enhanced heregulin-stimulated expression of cyclin D and phosphorylation of the retinoblastoma gene product. In cells treated with both heregulin and forskolin there was a sustained accumulation of phospho-CREB, which was not observed in cells treated with either agent alone. Heregulin and forskolin synergistically activated transcription of a cyclin D promoter construct. These results demonstrate that heregulin-stimulated activation of MAP kinase is not sufficient to induce maximal Schwann cell proliferation. Expression of critical cell cycle regulatory proteins and cell division require activation of both heregulin and cAMP-dependent processes.

Gwynn, Babette; Lueders, Kira; Sands, Mark S.; Birkenmeier, Edward H.

doi: N/Apmid: 9774663

The severity of human mucopolysaccharidosis type VII (MPS VII), or Sly syndrome, depends on the relative activity of the enzyme -glucuronidase. Loss of -glucuronidase activity can cause hydrops fetalis, with in utero or postnatal death of the patient. In this report, we show that -glucuronidase activity is not detectable by a standard fluorometric assay in C3H/HeOuJ (C3H) mice homozygous for a new mutation, gus mps2J . These gus mps2J /gus mps2J mice are born and survive much longer than the previously characterized -glucuronidase-null B6.C-H-2 bm1 /ByBir- gus mps ( gus mps /gus mps ) mice. Northern blot analysis of liver from gus mps2J /gus mps2J mice demonstrates a 750-bp reduction in size of -glucuronidase mRNA. A 5.4-kb insertion in the Gus-s h nucleotide sequence from these mice was localized by Southern blot analysis to intron 8. The ends of the inserted sequences were cloned by inverse PCR and revealed an intracisternal A-particle (IAP) element inserted near the 3' end of the intron. The sequence of the long terminal repeat (LTR) regions of the IAP most closely matches that of a composite LTR found in transposed IAPs previously identified in the C3H strain. The inserted IAP may contribute to diminished -glucuronidase activity either by interfering with transcription or by destabilizing the message. The resulting phenotype is much less severe than that previously described in the gus mps /gus mps mouse and provides an opportunity to study MPS VII on a genetic background that clearly modulates disease severity.

Espinoza, F. Hernán; Farrell, Alison; Nourse, Jamison L.; Chamberlin, Holly M.; Gileadi, Opher; Morgan, David O.

doi: N/Apmid: 9774652

Cak1 Is Required for Kin28 Phosphorylation and Activation In Vivo F. Hernán Espinoza 1 , Alison Farrell 1 , Jamison L. Nourse 1 , Holly M. Chamberlin 1 , Opher Gileadi 2 , and David O. Morgan 1 , * Departments of Physiology and Biochemistry & Biophysics, University of California, San Francisco, California 94143-0444, 1 and Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel 2 ABSTRACT Complete activation of most cyclin-dependent protein kinases (CDKs) requires phosphorylation by the CDK-activating kinase (CAK). In the budding yeast, Saccharomyces cerevisiae , the major CAK is a 44-kDa protein kinase known as Cak1. Cak1 is required for the phosphorylation and activation of Cdc28, a major CDK involved in cell cycle control. We addressed the possibility that Cak1 is also required for the activation of other yeast CDKs, such as Kin28, Pho85, and Srb10. We generated three new temperature-sensitive cak1 mutant strains, which arrested at the restrictive temperature with nonuniform budding morphology. All three cak1 mutants displayed significant synthetic interactions with loss-of-function mutations in CDC28 and KIN28 . Loss of Cak1 function reduced the phosphorylation and activity of both Cdc28 and Kin28 but did not affect the activity of Pho85 or Srb10. In the presence of the Kin28 regulatory subunits Ccl1 and Tfb3, Kin28 was phosphorylated and activated when coexpressed with Cak1 in insect cells. We conclude that Cak1 is required for the activating phosphorylation of Kin28 as well as that of Cdc28.

Chen, Wenliang; Jinks-Robertson, Sue

doi: N/Apmid: 9774668

Mismatch repair (MMR) proteins actively inhibit recombination between diverged sequences in both prokaryotes and eukaryotes. Although the molecular basis of the antirecombination activity exerted by MMR proteins is unclear, it presumably involves the recognition of mismatches present in heteroduplex recombination intermediates. This recognition could be exerted during the initial stage of strand exchange, during the extension of heteroduplex DNA, or during the resolution of recombination intermediates. We previously used an assay system based on 350-bp inverted-repeat substrates to demonstrate that MMR proteins strongly inhibit mitotic recombination between diverged sequences in Saccharomyces cerevisiae . The assay system detects only those events that reverse the orientation of the region between the recombination substrates, which can occur as a result of either intrachromatid crossover or sister chromatid conversion. In the present study we sequenced the products of mitotic recombination between 94%-identical substrates in order to map gene conversion tracts in wild-type versus MMR-defective yeast strains. The sequence data indicate that (i) most recombination occurs via sister chromatid conversion and (ii) gene conversion tracts in an MMR-defective strain are significantly longer than those in an isogenic wild-type strain. The shortening of conversion tracts observed in a wild-type strain relative to an MMR-defective strain suggests that at least part of the antirecombination activity of MMR proteins derives from the blockage of heteroduplex extension in the presence of mismatches.

Karpichev, Igor V.; Small, Gillian M.

doi: N/Apmid: 9774671

Two transcription factors, Oaf1p and Pip2p (Oaf2p), are key components in the pathway by which several Saccharomyces cerevisiae genes encoding peroxisomal proteins are activated in the presence of a fatty acid such as oleate. By searching the S. cerevisiae genomic database for the consensus sequence that acts as a target for these transcription factors, we identified 40 genes that contain a putative Oaf1p-Pip2p binding site in their promoter region. Quantitative Northern analysis confirmed that the expression of 22 of the genes identified is induced by oleate and that either one or both of these transcription factors are required for the activation. In addition to known peroxisomal proteins, the regulated genes encode novel peroxisomal proteins, a mitochondrial protein, and proteins of unknown location and function. We demonstrate that Oaf1p regulates certain genes in the absence of Pip2p and that both of these transcription factors play a role in maintaining the glucose-repressed state of one gene. Furthermore, we provide evidence that the defined consensus binding site is not required for the regulation of certain oleate-responsive genes.

Hofman, Edward R.; Boyanapalli, Madanamohan; Lindner, Daniel J.; Weihua, Xiao; Hassel, Bret A.; Jagus, Rosemary; Gutierrez, Peter L.; Kalvakolanu, Dhananjaya V.

doi: N/Apmid: 9774665

Interferons (IFNs) and retinoids are potent biological response modifiers. By using JAK-STAT pathways, IFNs regulate the expression of genes involved in antiviral, antitumor, and immunomodulatory actions. Retinoids exert their cell growth-regulatory effects via nuclear receptors, which also function as transcription factors. Although these ligands act through distinct mechanisms, several studies have shown that the combination of IFNs and retinoids synergistically inhibits cell growth. We have previously reported that IFN- -all- trans -retinoic acid (RA) combination is a more potent growth suppressor of human tumor xenografts in vivo than either agent alone. Furthermore, the IFN-RA combination causes cell death in several tumor cell lines in vitro. However, the molecular basis for these growth-suppressive actions is unknown. It has been suggested that certain gene products, which mediate the antiviral actions of IFNs, are also responsible for the antitumor actions of the IFN-RA combination. However, we did not find a correlation between their activities and cell death. Therefore, we have used an antisense knockout approach to directly identify the gene products that mediate cell death and have isolated several genes associated with retinoid-IFN-induced mortality (GRIM). In this investigation, we characterized one of the GRIM cDNAs, GRIM-12. Sequence analysis suggests that the GRIM-12 product is identical to human thioredoxin reductase (TR). TR is posttranscriptionally induced by the IFN-RA combination in human breast carcinoma cells. Overexpression of GRIM-12 causes a small amount of cell death and further enhances the susceptibility of cells to IFN-RA-induced death. Dominant negative inhibitors directed against TR inhibit its cell death-inducing functions. Interference with TR enzymatic activity led to growth promotion in the presence of the IFN-RA combination. Thus, these studies identify a novel function for TR in cell growth regulation.

Smider, Vaughn; Rathmell, W. Kimryn; Brown, Greg; Lewis, Susanna; Chu, Gilbert

doi: N/Apmid: 9774698

Failure of Hairpin-Ended and Nicked DNA To Activate DNA-Dependent Protein Kinase: Implications for V(D)J Recombination Vaughn Smider 1 , W. Kimryn Rathmell 1 , Greg Brown 2 , Susanna Lewis 2 , and Gilbert Chu 1 , * Departments of Medicine and Biochemistry, Stanford University Medical Center, Stanford, California 94305, 1 and Department of Immunology, University of Toronto, and Division of Immunology/Cancer, Hospital for Sick Children, Toronto, Ontario, Canada 2 ABSTRACT V(D)J recombination is initiated by a coordinated cleavage reaction that nicks DNA at two sites and then forms a hairpin coding end and blunt signal end at each site. Following cleavage, the DNA ends are joined by a process that is incompletely understood but nevertheless depends on DNA-dependent protein kinase (DNA-PK), which consists of Ku and a 460-kDa catalytic subunit (DNA-PK CS or p460). Ku directs DNA-PK CS to DNA ends to efficiently activate the kinase. In vivo, the mouse SCID mutation in DNA-PK CS disrupts joining of the hairpin coding ends but spares joining of the open signal ends. To better understand the mechanism of V(D)J recombination, we measured the activation of DNA-PK by the three DNA structures formed during the cleavage reaction: open ends, DNA nicks, and hairpin ends. Although open DNA ends strongly activated DNA-PK, nicked DNA substrates and hairpin-ended DNA did not. Therefore, even though efficient processing of hairpin coding ends requires DNA-PK CS , this may occur by activation of the kinase bound to the cogenerated open signal end rather than to the hairpin end itself.

Kobayashi, Masatomo; Aita, Noriko; Hayashi, Shigeo; Okada, Kohichi; Ohta, Tsutomu; Hirose, Susumu

doi: N/Apmid: 9774687

DNA Supercoiling Factor Localizes to Puffs on Polytene Chromosomes in Drosophila melanogaster Masatomo Kobayashi 1 , 2 , Noriko Aita 1 , 2 , Shigeo Hayashi 1 , 3 , Kohichi Okada 1 , 2 , Tsutomu Ohta 2 , † , and Susumu Hirose 1 , 2 , * The Graduate University for Advanced Studies, 1 Department of Developmental Genetics, 2 and Genetic Stock Research Center, 3 National Institute of Genetics, Mishima, Shizuoka-ken 411-8540, Japan ABSTRACT DNA supercoiling factor (SCF) was first identified in silkworm as a protein that generates negative supercoils in DNA in conjunction with eukaryotic topoisomerase II. To analyze the in vivo role of the factor, we cloned a cDNA encoding Drosophila melanogaster SCF. Northern analysis revealed 1.6- and 1.8-kb mRNAs throughout development. The longer mRNA contains an open reading frame that shares homology with mouse reticulocalbin whereas the shorter one encodes a truncated version lacking the N-terminal signal peptide-like sequence. An antibody against SCF detected a 45-kDa protein in the cytoplasmic fraction and a 30-kDa protein in the nuclear fraction of embryonic extracts. Immunoprecipitation suggests that the 30-kDa protein interacts with topoisomerase II in the nucleus, and hence that it is a functional form of SCF. Immunostaining of blastoderm embryos showed that SCF is present in nuclei during interphase but is excluded from mitotic chromosomes. In larvae, the antibody stained the nuclei of several tissues including a posterior part of the salivary gland. This latter staining was associated with natural or ecdysteroid-induced puffs on polytene chromosomes. Upon heat treatment of larvae, the staining on the endogenous puffs disappeared, and strong staining appeared on heat shock puffs. These results implicate SCF in gene expression.

Yamaguchi-Iwai, Yuko; Sonoda, Eiichiro; Buerstedde, Jean-Marie; Bezzubova, Olga; Morrison, Ciaran; Takata, Minoru; Shinohara, Akira; Takeda, Shunichi

doi: N/Apmid: 9774659

Homologous Recombination, but Not DNA Repair, Is Reduced in Vertebrate Cells Deficient in RAD52 Yuko Yamaguchi-Iwai 1 , Eiichiro Sonoda 1 , Jean-Marie Buerstedde 2 , † , Olga Bezzubova 2 , † , Ciaran Morrison 1 , Minoru Takata 1 , Akira Shinohara 3 , ‡ , and Shunichi Takeda 1 , * Bayer-chair Department of Molecular Immunology and Allergology, Faculty of Medicine, Kyoto University, Konoe Yoshida, Sakyo-ku, Kyoto 606-8501, 1 and Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560-0043, 3 Japan, and Basel Institute for Immunology, CH-4005 Basel, Switzerland 2 ABSTRACT Rad52 plays a pivotal role in double-strand break (DSB) repair and genetic recombination in Saccharomyces cerevisiae , where mutation of this gene leads to extreme X-ray sensitivity and defective recombination. Yeast Rad51 and Rad52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in vitro, suggesting that Rad51 and Rad52 act cooperatively. To define the role of Rad52 in vertebrates, we generated RAD52 −/− mutants of the chicken B-cell line DT40. Surprisingly, RAD52 −/− cells were not hypersensitive to DNA damages induced by γ-irradiation, methyl methanesulfonate, or cis -platinum(II)diammine dichloride (cisplatin). Intrachromosomal recombination, measured by immunoglobulin gene conversion, and radiation-induced Rad51 nuclear focus formation, which is a putative intermediate step during recombinational repair, occurred as frequently in RAD52 −/− cells as in wild-type cells. Targeted integration frequencies, however, were consistently reduced in RAD52 −/− cells, showing a clear role for Rad52 in genetic recombination. These findings reveal striking differences between S. cerevisiae and vertebrates in the functions of RAD51 and RAD52 .