RESUMEN
BACKGROUND: Transforming growth factor-beta (TGF-ß) induces the epithelial-to-mesenchymal transition (EMT) leading to increased cell plasticity at the onset of cancer cell invasion and metastasis. Mechanisms involved in TGF-ß-mediated EMT and cell motility are unclear. Recent studies showed that p53 affects TGF-ß/SMAD3-mediated signalling, cell migration, and tumorigenesis. We previously demonstrated that Nox4, a Nox family NADPH oxidase, is a TGF-ß/SMAD3-inducible source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. Our present study investigates the involvement of p53 in TGF-ß-regulated Nox4 expression and cell migration. METHODS: We investigated the effect of wild-type p53 (WT-p53) and mutant p53 proteins on TGF-ß-regulated Nox4 expression and cell migration. Nox4 mRNA and protein, ROS production, cell migration, and focal adhesion kinase (FAK) activation were examined in three different cell models based on their p53 mutational status. H1299, a p53-null lung epithelial cell line, was used for heterologous expression of WT-p53 or mutant p53. In contrast, functional studies using siRNA-mediated knockdown of endogenous p53 were conducted in MDA-MB-231 metastatic breast epithelial cells that express p53-R280K and MCF-10A normal breast cells that have WT-p53. RESULTS: We found that WT-p53 is a potent suppressor of TGF-ß-induced Nox4, ROS production, and cell migration in p53-null lung epithelial (H1299) cells. In contrast, tumour-associated mutant p53 proteins (R175H or R280K) caused enhanced Nox4 expression and cell migration in both TGF-ß-dependent and TGF-ß-independent pathways. Moreover, knockdown of endogenous mutant p53 (R280K) in TGF-ß-treated MDA-MB-231 metastatic breast epithelial cells resulted in decreased Nox4 protein and reduced phosphorylation of FAK, a key regulator of cell motility. Expression of WT-p53 or dominant-negative Nox4 decreased TGF-ß-mediated FAK phosphorylation, whereas mutant p53 (R280K) increased phospho-FAK. Furthermore, knockdown of WT-p53 in MCF-10A normal breast epithelial cells increased basal Nox4 expression, whereas p53-R280K could override endogenous WT-p53 repression of Nox4. Remarkably, immunofluorescence analysis revealed MCF-10A cells expressing p53-R280K mutant showed an upregulation of Nox4 in both confluent and migrating cells. CONCLUSIONS: Collectively, our findings define novel opposing functions for WT-p53 and mutant p53 proteins in regulating Nox4-dependent signalling in TGF-ß-mediated cell motility.
Asunto(s)
Neoplasias de la Mama/patología , Células Epiteliales/fisiología , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/patología , NADPH Oxidasas/biosíntesis , Proteínas de Neoplasias/fisiología , Proteína p53 Supresora de Tumor/fisiología , Mama/citología , Neoplasias de la Mama/metabolismo , Línea Celular Transformada , Línea Celular Tumoral , Movimiento Celular , Inducción Enzimática , Transición Epitelial-Mesenquimal , Femenino , Proteína-Tirosina Quinasas de Adhesión Focal/fisiología , Genes p53 , Humanos , Pulmón/citología , Neoplasias Pulmonares/metabolismo , Masculino , Mutación Missense , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Metástasis de la Neoplasia , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , ARN Interferente Pequeño/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transfección , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
The spindle checkpoint is an evolutionarily conserved mitotic regulatory mechanism that ensures that anaphase is not attempted until chromosomes are properly aligned on the spindle. Two different cell-cycle transitions must be inhibited by the spindle checkpoint to arrest cells at metaphase and prevent mitotic exit. The checkpoint proteins interact in ways that are more complex than was originally envisioned. This review summarizes the evidence for two pathways of spindle-checkpoint regulation in budding yeast. We describe how the proteins are involved in these pathways and discuss the ways in which the spindle checkpoint inhibits the cell-cycle machinery.
Asunto(s)
Proteínas de Ciclo Celular , Ciclo Celular/fisiología , Proteínas de Saccharomyces cerevisiae , Huso Acromático/fisiología , Animales , Proteínas Fúngicas/fisiología , Humanos , Proteínas Nucleares/fisiología , Saccharomycetales , Securina , Transducción de SeñalRESUMEN
We have investigated the function of p55CDC, a mammalian protein related to Cdc20 and Hct1/Cdh1 in Saccharomyces cerevisiae, and Fizzy and Fizzy-related in Drosophila. Immunofluorescence studies and expression of a p55CDC-GFP chimera demonstrate that p55CDC is concentrated at the kinetochores in M phase cells from late prophase to telophase. Some p55CDC is also associated with the spindle microtubules and spindle poles, and some is diffuse in the cytoplasm. At anaphase, the concentration of p55CDC at the kinetochores gradually diminishes, and is gone by late telophase. In extracts prepared from M phase, but not from interphase HeLa cells, p55CDC coimmunoprecipitates with three important elements of the M phase checkpoint machinery: Cdc27, Cdc16, and Mad2. p55CDC is required for binding Mad2 with the Cdc27 and Cdc16. Thus, it is likely that p55CDC mediates the association of Mad2 with the cyclosome/anaphase-promoting complex. Microinjection of anti-p55CDC antibody into mitotic mammalian cells induces arrest or delay at metaphase, and impairs progression of late mitotic events. These studies suggest that mammalian p55CDC may be part of a regulatory and targeting complex for the anaphase-promoting complex.
Asunto(s)
Proteínas de Unión al Calcio , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Ligasas/metabolismo , Mitosis/fisiología , Proteínas/metabolismo , Complejos de Ubiquitina-Proteína Ligasa , Anafase , Ciclosoma-Complejo Promotor de la Anafase , Animales , Anticuerpos/metabolismo , Proteínas Cdc20 , División Celular , Extractos Celulares , Cromosomas/metabolismo , Células HeLa , Humanos , Cinetocoros/metabolismo , Células LLC-PK1 , Proteínas Mad2 , Metafase , Microinyecciones , Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras , Intercambio de Cromátides Hermanas , Porcinos , Ubiquitina-Proteína LigasasRESUMEN
Cell viability requires accurate chromosome segregation at mitosis. The spindle checkpoint ensures that anaphase is not attempted until the sister chromatids of each chromosome are attached to spindle microtubules from opposite poles. The checkpoint mechanism involves a signal transduction cascade that is more complex than was originally envisioned.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Fúngicas/fisiología , Mitosis/fisiología , Proteínas de Saccharomyces cerevisiae , Animales , Genes cdc/fisiología , Humanos , Mitosis/genética , LevadurasRESUMEN
The spindle checkpoint regulates microtubule-based chromosome segregation and helps to maintain genomic stability [1,2]. Mutational inactivation of spindle checkpoint genes has been implicated in the progression of several types of human cancer. Recent evidence from budding yeast suggests that the spindle checkpoint is complex. Order-of-function experiments have defined two separable pathways within the checkpoint. One pathway, defined by MAD2, controls the metaphase-to-anaphase transition and the other, defined by BUB2, controls the exit from mitosis [3-6]. The relationships between the separate branches of the checkpoint, and especially the events that trigger the pathways, have not been defined. We localized a Bub2p-GFP fusion protein to the cytoplasmic side of the spindle pole body and used a kar9 mutant to show that cells with misoriented spindles are arrested in anaphase of mitosis. We used a kar9 bub2 double mutant to show that the arrest is BUB2 dependent. We conclude that the separate pathways of the spindle checkpoint respond to different classes of microtubules. The MAD2 branch of the pathway responds to kinetochore microtubule interactions and the BUB2 branch of the pathway operates within the cytoplasm, responding to spindle misorientation.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas Fúngicas/metabolismo , Genes cdc , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Huso Acromático/metabolismo , Proteínas de Unión al Calcio/metabolismo , Ciclo Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Proteínas Fúngicas/genética , Genes Reporteros , Proteínas Mad2 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Huso Acromático/ultraestructura , Tubulina (Proteína)/inmunología , Tubulina (Proteína)/metabolismoRESUMEN
Protein synthesis inhibitors have often been used to identify regulatory steps in cell division. We used cell division cycle mutants of the yeast Saccharomyces cerevisiae and two chemical inhibitors of translation to investigate the requirements for protein synthesis for completing landmark events after the G1 phase of the cell cycle. We show, using cdc2, cdc6, cdc7, cdc8, cdc17 (38 degrees C), and cdc21 (also named tmp1) mutants, that cells arrested in S phase complete DNA synthesis but cannot complete nuclear division if protein synthesis is inhibited. In contrast, we show, using cdc16, cdc17 (36 degrees C), cdc20, cdc23, and nocodazole treatment, that cells that arrest in the G2 stage complete nuclear division in the absence of protein synthesis. Protein synthesis is required late in the cell cycle to complete cytokinesis and cell separation. These studies show that there are requirements for protein synthesis in the cell cycle, after G1, that are restricted to two discrete intervals.
Asunto(s)
Núcleo Celular/ultraestructura , Cicloheximida/farmacología , Replicación del ADN/efectos de los fármacos , Proteínas Fúngicas/biosíntesis , Saccharomyces cerevisiae/crecimiento & desarrollo , Tricodermina/farmacología , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Genotipo , Fase S/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismoRESUMEN
Dicentric chromosomes are genetically unstable and depress the rate of cell division in Saccharomyces cerevisiae. We have characterized the effects of a conditionally dicentric chromosome on the cell division cycle by using microscopy, flow cytometry, and an assay for histone H1 kinase activity. Activating the dicentric chromosome induced a delay in the cell cycle after DNA replication and before anaphase. The delay occurred in the absence of RAD9, a gene required to arrest cell division in response to DNA damage. The rate of dicentric chromosome loss, however, was elevated in the rad9 mutant. A mutation in BUB2, a gene required for arrest of cell division in response to loss of microtubule function, diminished the delay. Both RAD9 and BUB2 appear to be involved in the cellular response to a dicentric chromosome, since the conditionally dicentric rad9 bub2 double mutant was highly inviable. We conclude that a dicentric chromosome results in chromosome breakage and spindle aberrations prior to nuclear division that normally activate mitotic checkpoints, thereby delaying the onset of anaphase.
Asunto(s)
Proteínas de Ciclo Celular , Ciclo Celular/genética , Cromosomas Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Diploidia , Citometría de Flujo , Proteínas Fúngicas/genética , Mitosis , Protamina Quinasa/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
Saccharomyces cerevisiae, like most eucaryotic cells, can prevent the onset of anaphase until chromosomes are properly aligned on the mitotic spindle. We determined that Cdc55p (regulatory B subunit of protein phosphatase 2A [PP2A]) is required for the kinetochore/spindle checkpoint regulatory pathway in yeast. ctf13 cdc55 double mutants could not maintain a ctf13-induced mitotic delay, as determined by antitubulin staining and levels of histone H1 kinase activity. In addition, cdc55::LEU2 mutants and tpd3::LEU2 mutants (regulatory A subunit of PP2A) were nocodazole sensitive and exhibited the phenotypes of previously identified kinetochore/spindle checkpoint mutants. Inactivating CDC55 did not simply bypass the arrest that results from inhibiting ubiquitin-dependent proteolysis because cdc16-1 cdc55::LEU2 and cdc23-1 cdc55::LEU2 double mutants arrested normally at elevated temperatures. CDC55 is specific for the kinetochore/spindle checkpoint because cdc55 mutants showed normal sensitivity to gamma radiation and hydroxyurea. The conditional lethality and the abnormal cellular morphogenesis of cdc55::LEU2 were suppressed by cdc28F19, suggesting that the cdc55 phenotypes are dependent on the phosphorylation state of Cdc28p. In contrast, the nocodazole sensitivity of cdc55::LEU2 was not suppressed by cdc28F19. Therefore, the mitotic checkpoint activity of CDC55 (and TPD3) is independent of regulated phosphorylation of Cdc28p. Finally, cdc55::LEU2 suppresses the temperature sensitivity of cdc20-1, suggesting additional roles for CDC55 in mitosis.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Cinetocoros/fisiología , Mitosis/fisiología , Fosfoproteínas Fosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , ADN de Hongos/biosíntesis , Proteínas Fúngicas/genética , Rayos gamma , Prueba de Complementación Genética , Hidroxiurea/farmacología , Microtúbulos/efectos de los fármacos , Mutación , Nocodazol/farmacología , Proteínas Nucleares/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fosfoproteínas Fosfatasas/genética , Fosforilación , Protamina Quinasa/metabolismo , Proteína Fosfatasa 2 , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/efectos de la radiación , Huso Acromático/fisiología , Esporas Fúngicas , Supresión Genética , TemperaturaRESUMEN
Inhibition of mitosis by antimitotic drugs is thought to occur by destruction of microtubules, causing cells to arrest through the action of one or more mitotic checkpoints. We have patterned experiments in the yeast Saccharomyces cerevisiae after recent studies in mammalian cells that demonstrate the effectiveness of antimitotic drugs at concentrations that maintain spindle structure. We show that low concentrations of nocodazole delay cell division under the control of the previously identified mitotic checkpoint genes BUB1, BUB3, MAD1, and MAD2 and independently of BUB2. The same genes mediate the cell cycle delay induced in ctf13 mutants, limited for an essential kinetochore component. Our data suggest that a low concentration of nocodazole induces a cell cycle delay through checkpoint control that is sensitive to impaired kinetochore function. The BUB2 gene may be part of a separate checkpoint that responds to abnormal spindle structure.
Asunto(s)
Genes Fúngicos/fisiología , Cinetocoros/fisiología , Nocodazol/farmacología , Saccharomyces cerevisiae/fisiología , División Celular/efectos de los fármacos , División Celular/genética , Genes Fúngicos/efectos de los fármacos , Genotipo , Cinética , Cinetocoros/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Microtúbulos/fisiología , Mitosis/efectos de los fármacos , Mutación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Tubulina (Proteína)/análisisRESUMEN
Checkpoints prevent inaccurate chromosome segregation by inhibiting cell division when errors in mitotic processes are encountered. We used a temperature-sensitive mutation, dbf4, to examine the requirement for DNA replication in establishing mitotic checkpoint arrest. We used gamma-irradiation to induce DNA damage and hydroxyurea to limit deoxyribonucleotides in cells deprived of DBF4 function to investigate the requirement for DNA replication in DNA-responsive checkpoints. In the absence of DNA replication, mitosis was not inhibited by these treatments, which normally activate the DNA damage and DNA replication checkpoints. Our results support a model that indicates that the assembly of replication structures is critical for cells to respond to defects in DNA metabolism. We show that activating the spindle checkpoint with nocodazole does not require prior progression through S phase but does require a stable kinetochore.
Asunto(s)
Proteínas de Ciclo Celular , Replicación del ADN , Mitosis/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Antineoplásicos/farmacología , División Celular , Cromátides/metabolismo , ADN/metabolismo , Daño del ADN , ADN de Hongos/metabolismo , Proteínas Fúngicas/metabolismo , Cinetocoros/metabolismo , Nocodazol/farmacología , Proteínas Nucleares/metabolismo , Fase S , Huso Acromático/metabolismoRESUMEN
Previous analysis of cdc20 mutants of the yeast Saccharomyces cerevisiae suggests that the CDC20 gene product (Cdc20p) is required for two microtubule-dependent processes, nuclear movements prior to anaphase and chromosome separation. Here we report that cdc20 mutants are defective for a third microtubule-mediated event, nuclear fusion during mating of G1 cells, but appear normal for a fourth microtubule-dependent process, nuclear migration after DNA replication. Therefore, Cdc20p is required for a subset of microtubule-dependent processes and functions at multiple stages in the life cycle. Consistent with this interpretation, we find that cdc20 cells arrested by alpha-factor or at the restrictive temperature accumulate anomalous microtubule structures, as detected by indirect immunofluorescence. The anomalous microtubule staining patterns are due to cdc20 because intragenic revertants that revert the temperature sensitivity have normal microtubule morphologies. cdc20 mutants have a sevenfold increase in the intensity of antitubulin fluorescence in intranuclear spindles compared with spindles from wild-type cells, yet the total amount of tubulin is indistinguishable by Western immunoblot analysis. This result suggests that Cdc20p modulates microtubule structure in wild-type cells either by promoting microtubule disassembly or by altering the surface of the microtubules. Finally, we cloned and sequenced CDC20 and show that it encodes a member of a family of proteins that share homology to the beta subunit of transducin.
Asunto(s)
Proteínas Fúngicas/genética , Genes Fúngicos , Microtúbulos/fisiología , Saccharomyces cerevisiae/genética , Transducina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cruzamientos Genéticos , Reparación del ADN , ADN de Hongos/genética , Técnica del Anticuerpo Fluorescente , Genotipo , Datos de Secuencia Molecular , Plásmidos , Mapeo Restrictivo , Saccharomyces cerevisiae/fisiología , Homología de Secuencia de Ácido Nucleico , Tubulina (Proteína)/análisis , Tubulina (Proteína)/genéticaRESUMEN
We determined the sequence of a Drosophila tRNA gene cluster containing a tRNAHis gene and a tRNAHis pseudogene in close proximity on the same DNA strand. The pseudogene contains eight consecutive base pairs different from the region of the bona fide gene which codes for the 3' portion of the anticodon stem of tRNAHis. The tRNAHis gene is transcribed efficiently in Drosophila Kc cell extract, whereas the pseudogene is not. The pseudogene is also a much poorer competitor than the real gene in a stable transcription complex formation assay, even though the sequence alteration in the pseudogene does not affect the sequence or spacing of the putative internal transcription control regions. Recombinant clones were constructed in which the 5'-flanking regions are exchanged. The transcription efficiencies and competitive abilities of the recombinant clones resemble those of the genes from which the 5' flank was derived; for example, the tRNAHis pseudogene with the 5'-flanking sequence of the tRNAHis gene is now efficiently transcribed. Deletion analysis of the pseudogene 5' flank failed to uncover an inhibitory element. Deletion analysis of the real gene showed very high dependence on the presence of the wild-type 5'-flanking sequence for factor binding to the internal control regions and stable complex formation. The 5'-flanking sequence of a Drosophila tRNAArg gene active in the Drosophila Kc cell extract does not restore transcriptional activity or stable complex formation. The tRNAHis gene and pseudogene behave atypically in HeLa cell extract. Both genes compete for HeLa transcription factors, but neither of them is efficiently transcribed. Removal of the 5'-flanking sequences of each gene and replacement with various sequences, including the tRNAArg gene 5' flank, does not allow increased transcription in HeLa cell extract.
Asunto(s)
Genes , Aminoacil-ARN de Transferencia/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Deleción Cromosómica , Drosophila , Células HeLa , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos , Aminoacil-ARN de Transferencia/análisis , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
The three-dimensional organization of mitotic microtubules in a mutant strain of Saccharomyces cerevisiae has been studied by computer-assisted serial reconstruction. At the nonpermissive temperature, cdc20 cells arrested with a spindle length of approximately 2.5 microns. These spindles contained a mean of 81 microtubules (range, 56-100) compared with 23 in wild-type spindles of comparable length. This increase in spindle microtubule number resulted in a total polymer length up to four times that of wild-type spindles. The spindle pole bodies in the cdc20 cells were approximately 2.3 times the size of wild-type, thereby accommodating the abnormally large number of spindle microtubules. The cdc20 spindles contained a large number of interpolar microtubules organized in a "core bundle." A neighbor density analysis of this bundle at the spindle midzone showed a preferred spacing of approximately 35 nm center-to-center between microtubules of opposite polarity. Although this is evidence of specific interaction between antiparallel microtubules, mutant spindles were less ordered than the spindle of wild-type cells. The number of noncore microtubules was significantly higher than that reported for wild-type, and these microtubules did not display a characteristic metaphase configuration. cdc20 spindles showed significantly more cross-bridges between spindle microtubules than were seen in the wild type. The cross-bridge density was highest between antiparallel microtubules. These data suggest that spindle microtubules are stabilized in cdc20 cells and that the CDC20 gene product may be involved in cell cycle processes that promote spindle microtubule disassembly.
Asunto(s)
Proteínas de Ciclo Celular/genética , Mutación , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Huso Acromático/química , Huso Acromático/ultraestructura , Proteínas Cdc20 , Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica , Modelos BiológicosRESUMEN
The functional interaction of Arg-, Ile-, Leu-, Lys- and Met-tRNA synthetases occurring within the same rat liver multienzyme complex are investigated by examining the enzymes catalytic activities and inactivation kinetics. The Michaelis constants for amino acids, ATP and tRNAs of the dissociated aminoacyl-tRNA synthetases are not significantly different from those of the high-Mr multienzyme complex, except in a few cases where the Km values of the dissociated enzymes are higher than those of the high-Mr form. The maximal aminoacylation velocities of the individual aminoacyl-tRNA synthetases are not affected by the presence of simultaneous aminoacylation by another synthetase occurring within the same multienzyme complex. Site-specific oxidative modification by ascorbate and nonspecific thermal inactivation of synthetases in the purified rat liver 18 S synthetase complex are examined. Lys- and Arg-tRNA synthetases show remarkably parallel time-courses in both inactivation processes. Leu- and Met-tRNA synthetases also show parallel kinetics in thermal inactivation and possibly oxidative inactivation. Ile-tRNA synthetase shows little inactivation in either process. The oxidative inactivation of Lys- and Arg-tRNA synthetases can be reversed by addition of dithiothreitol. These results suggest that synthetases within the same high-Mr complex catalyze aminoacylation reactions independently; however, the stabilities of some of the synthetases in the multienzyme complex are coupled. In particular, the stability of Arg-tRNA synthetase depends appreciably on its association with fully active Lys-tRNA synthetase.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Hígado/enzimología , Complejos Multienzimáticos/metabolismo , Aminoácidos/farmacología , Animales , Arginino-ARNt Ligasa/metabolismo , Ácido Ascórbico/farmacología , Ditiotreitol/farmacología , Calor , Isoleucina-ARNt Ligasa/metabolismo , Cinética , Leucina-ARNt Ligasa/metabolismo , Lisina-ARNt Ligasa/metabolismo , Metionina-ARNt Ligasa/metabolismo , Peso Molecular , RatasRESUMEN
DNA fragments corresponding to genes encoding the MSP of Caenorhabditis elegans sperm have been isolated by recombinant DNA techniques. Analyses of individual genomic clones suggest that there are multiple MSP genes that are dispersed in the genome. From restriction enzyme digests of genomic DNA fractionated and hybridized with an MSP complementary DNA probe, there appear to be more than 30 MSP genes in the genome. Despite the occurrence of this large dispersed multigene family, the MSP messenger RNA from both males and hermaphrodites is homogene in size. There are at least three different proteins of identical molecular weight but different isoelectric point that cross-react with anti-MSP antisera. Each protein is a primary translation product with no detectable post-translational modifications, suggesting that at least three of the MSP genes are expressed.
Asunto(s)
Caenorhabditis/genética , Genes , Proteínas/genética , Espermatozoides/análisis , Animales , ADN/análisis , Enzimas de Restricción del ADN , ADN Recombinante , Electroforesis en Gel de Agar , Glicoproteínas/análisis , Masculino , Hibridación de Ácido Nucleico , Fosfoproteínas/análisis , Biosíntesis de Proteínas , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
The major sperm proteins (MSPs) are a family of closely related, small, basic proteins comprising 15% of the protein in Caenorhabditis elegans sperm. They are encoded by a multigene family of more than 50 genes, including many pseudogenes. MSP gene transcription occurs only in late primary spermatocytes. In order to study the genomic organization of transcribed MSP genes, probes specific for the 3' untranslated regions of sequenced cDNA clones were used to isolate transcribed genes from genomic libraries. These and other clones of MSP genes were located in overlapping cosmid clones by DNA fingerprinting. These cosmids were aligned with the genetic map by overlap with known genes or in-situ hybridization to chromosomes. Of 40 MSP genes identified, 37, including all those known to be transcribed, are organized into six clusters composed of 3 to 13 genes each. Within each cluster, MSP genes are not in tandem but are separated by at least several thousand bases of DNA. Pseudogenes are interspersed among functional genes. Genes with similar 3' untranslated sequences are in the same cluster. The six MSP clusters are confined to only three chromosomal loci; one on the left arm of chromosome II and two near the middle of chromosome IV. Additional sperm-specific genes are located in one cluster of MSP genes on chromosome IV. The multiplicity of MSP genes appears to be a mechanism for enhancing MSP synthesis in spermatocytes, and the loose clustering of genes could be a result of the mechanism of gene duplication or could play a role in regulation.
Asunto(s)
Caenorhabditis/genética , Genes , Proteínas Nucleares/genética , Seudogenes , Animales , Secuencia de Bases , Mapeo Cromosómico , Cósmidos , ADN/genética , Datos de Secuencia Molecular , Hibridación de Ácido NucleicoRESUMEN
Previous experiments suggest that mitotic chromosome segregation in some fungi is a nonrandom process in which chromatids of the same replicative age are destined for cosegregation. We have investigated the pattern of chromatid segregation in Saccharomyces cerevisiae by labeling the DNA of a strain auxotrophic for thymidine with 5-bromodeoxyuridine. The fate of DNA strands was followed qualitatively by immunofluorescence microscopy and quantitatively by microphotometry using an anti-5-bromodeoxyuridine monoclonal antibody. Chromatids of the same replicative age were distributed randomly to daughter cells at mitosis. Quantitative measurements showed that the amount of fluorescence in the daughter nuclei derived from parents with hemilabeled chromosomes diminished in intensity by one half. The concentration of 5-bromodeoxyuridine used in the experiments had little effect on the frequency of either homologous or sister chromatid exchanges. We infer that the 5-bromodeoxyuridine was distributed randomly due to mitotic segregation of chromatids and not via sister chromatid exchanges.
Asunto(s)
Cromátides/fisiología , Mitosis , Saccharomyces cerevisiae/citología , Anticuerpos Monoclonales , Bromodesoxiuridina/metabolismo , División Celular , Replicación del ADN , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Microscopía Fluorescente , Fotometría , Recombinación Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Intercambio de Cromátides HermanasRESUMEN
The spindle checkpoint ensures accurate chromosome segregation by inhibiting anaphase onset in response to altered microtubule function and impaired kinetochore function. In this study, we report that the ability of the anti-microtubule drug nocodazole to inhibit cell cycle progression in Saccharomyces cerevisiae depends on the function of the kinetochore protein encoded by NDC10. We examined the role of the spindle checkpoint in the arrest in cdc20 mutants that arrest prior to anaphase with an aberrant spindle. The arrest in cdc20 defective cells is dependent on the BUB2 checkpoint and independent of the BUB1, BUB3, and MAD spindle checkpoint genes. We show that the lesion recognized by Bub2p is not excess microtubules, and the cdc20 arrest is independent of kinetochore function. We show that Cdc20p is not required for cyclin proteolysis at two points in the cell cycle, suggesting that CDC20 is distinct from genes encoding integral proteins of the anaphase promoting complex.
Asunto(s)
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Cinetocoros/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Huso Acromático/fisiología , Animales , Proteínas Cdc20 , Epistasis Genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Microtúbulos/fisiología , Ratas , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/fisiología , Transducción de SeñalRESUMEN
We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Asociadas a Microtúbulos , Proteínas de Saccharomyces cerevisiae , Transducción de Señal/fisiología , Huso Acromático/fisiología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Sitios de Unión , Cromatina/genética , Cromatina/fisiología , Proteínas Cromosómicas no Histona , Mapeo Cromosómico , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/genética , Glucógeno Sintasa Quinasa 3 , Cinetocoros , Mutagénesis , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologíaRESUMEN
Auto-processing of the non-structural polypeptide 3ABC of foot and mouth disease virus (FMDV) expressed in Escherichia coli-BL21-DE3 was prevented by mutating either four glutamic acid residues at the 3A/3B1, 3B1/2, 3B2/3 and 3B3/3C junctions (3ABCtet) or a single cysteine residue at position 383 within the 3C domain (3ABCm). Independent expression of 3ABC and 3ABCtet genes induced expression of chaperone DnaK and degradation of ribosomal S1 protein in E. coli. They also induced cleavage of nucleosomal histone H3 when transiently expressed in BHK21 cells. 3ABCtet, 3ABCm, 3AB and 3A proteins concentrated in the perinuclear region suggesting that peptide sequences within the 3A domain specify intracellular targeting of 3ABC in BHK-21 cells. We propose that 3ABC molecules localized in the nuclear periphery are a source of protease 3C activity and are responsible for histone H3 processing during FMDV infections.