RESUMEN
BACKGROUND: Colon cancer predisposition is associated with mutations in BRCA1. BRCA1 protein stability depends on binding to BARD1. In different cancers, expression of differentially spliced BARD1 isoforms is correlated with poor prognosis and decreased patient survival. We therefore suspected a role of BARD1 isoforms in colon cancer. METHODS: We performed immunohistochemistry in 168 colorectal cancers, using four antibodies directed against differentially expressed regions of BARD1. We determined structure and relative expression of BARD1 mRNA isoforms in 40 tumour and paired normal peri-tumour tissues. BARD1 expression was correlated with clinical outcome. RESULTS: BARD1 isoforms were expressed in 98% of cases and not correlated with BRCA1. BARD1 mRNA isoforms were upregulated in all tumours as compared with paired normal peri-tumour tissues. Non-correlated expression and localisation of different epitopes suggested insignificant expression of full-length (FL) BARD1. Expression of N- and C-terminal epitopes correlated with increased survival, but expression of epitopes mapping to the middle of BARD1 correlated with decreased survival. Middle epitopes are present in oncogenic BARD1 isoforms, which have pro-proliferative functions. Correlated upregulation of only N- and C-terminal epitopes reflects the expression of isoforms BARD1δ and BARD1φ. CONCLUSION: Our results suggest that BARD1 isoforms, but not FL BARD1, are expressed in colon cancer and affect its progression and clinical outcome.
Asunto(s)
Neoplasias del Colon/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Proteína BRCA1/metabolismo , Neoplasias del Colon/mortalidad , Neoplasias del Colon/patología , Neoplasias Colorrectales/metabolismo , Metilación de ADN , Progresión de la Enfermedad , Mapeo Epitopo , Estrógenos/farmacología , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Resultado del Tratamiento , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: Telomere length has been considered in many cross-sectional studies as a biomarker of aging. However the association between shorter telomeres with lower survival at advanced ages remains a controversial issue. This association could reflect the impact of other health conditions than a direct biological effect. OBJECTIVE: To test whether leukocyte telomere length is associated with 5-year survival beyond the impact of other risk factors of mortality like comorbidity, functional, nutritional and cognitive status. DESIGN: Prospective study. SETTING AND PARTICIPANTS: A population representative sample of 444 patients (mean age 85 years; 74% female) discharged from the acute geriatric hospital of Geneva University Hospitals (January-December 2004), since then 263 (59.2%) had died (December 2009). MEASUREMENTS: Telomere length in leukocytes by flow cytometry. RESULTS: In univariate model, telomere length at baseline and cognitive status were not significantly associated with mortality even when adjusting for age (R²=9.5%) and gender (R²=1.9%). The best prognostic predictor was the geriatric index of comorbidity (GIC) (R²=8.8%; HR=3.85) followed by more dependence in instrumental (R²=5.9%; HR=3.85) and based (R²=2.3%; HR=0.84) activities of daily living and lower albumin levels (R²=1.5%; HR=0.97). Obesity (BMI>30: R²=1.6%; HR=0.55) was significantly associated with a two-fold decrease in the risk of mortality compared to BMI between 20-25. When all independent variables were entered in a full multiple Cox regression model (R²=21.4%), the GIC was the strongest risk predictor followed by the nutritional and functional variables. CONCLUSION: Neither telomeres length nor the presence of dementia are predictors of survival whereas the weight of multiple comorbidity conditions, nutritional and functional impairment are significantly associated with 5-year mortality in the oldest old.
Asunto(s)
Envejecimiento/fisiología , Estado de Salud , Leucocitos/citología , Estado Nutricional , Alta del Paciente/estadística & datos numéricos , Homeostasis del Telómero , Anciano de 80 o más Años , Biomarcadores , Índice de Masa Corporal , Trastornos del Conocimiento/mortalidad , Comorbilidad , Femenino , Citometría de Flujo , Evaluación Geriátrica , Humanos , Masculino , Obesidad/mortalidad , Estudios Prospectivos , Análisis de Supervivencia , Telómero/ultraestructuraRESUMEN
p53 has been called the cellular gatekeeper of the genome because it can induce cell-cycle arrest in G1, apoptosis or affect DNA replication in response to DNA damage. As p53 has been observed in first-trimester cytotrophoblastic cells (CTB), but its expression in normal cells is generally not detectable because of its short half-life, p53 could play an important role in cellular differentiation and/or in the control of the invasion of trophoblastic cells; therefore, p53 status was investigated in these cells. Using different antibodies recognizing different epitopes of p53 protein, abundant p53 expression was observed both in nuclear and in cytoplasmic compartments of first-trimester CTB. Whereas p53 was detected in the nuclei of few trophoblastic cells with an antibody recognizing the N-terminal epitope of the protein, high expression level of p53 in the cytoplasm of CTB was detected with an antibody recognizing the middle part of p53. The lack of immunoreactivity of p53 with antibodies recognizing the epitopes located at the N-terminus of p53 and the high level of p53 protein observed in the cytoplasm of CTB suggest that the N-terminus of p53 is involved in the formation of complexes. These cytoplasmic complexes were detected under non-reducing conditions in western blot analysis and had apparent molecular weights (MW) of 195, 167 or 125 kDa. These complexes could prolong the half-life of p53 in the cytoplasm of CTBs. By contrast, in the nuclei of CTBs, p53 seems to be present as a tetramer.
Asunto(s)
Primer Trimestre del Embarazo/metabolismo , Trofoblastos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Células Cultivadas , Dimerización , Femenino , Humanos , Inmunohistoquímica , Embarazo , Isoformas de Proteínas , Estructura Terciaria de Proteína , Trofoblastos/citología , Proteína p53 Supresora de Tumor/químicaRESUMEN
The present review on aging research in Switzerland describes ongoing gerontological and geriatric research in the field of both basic science and clinical research. Although Switzerland is situated at the rear end of the scale in regard of size or number of inhabitants, the number of high quality research groups per inhabitant positions it amongst the leading countries in the Western world. Being a small country Switzerland counts only five universities with clinical affiliations. Aging research in Switzerland therefore does not cover all areas of this rapidly developing discipline but some of the scientific contributions are mirrored in highest scored journals or others focus on topics that clearly bridge geriatric research and research on cellular and molecular mechanisms of aging.
Asunto(s)
Envejecimiento , Envejecimiento/genética , Envejecimiento/fisiología , Enfermedad de Alzheimer/etiología , Animales , Vasos Sanguíneos/fisiología , Geriatría , Humanos , Ratones , Modelos Animales , Modelos Biológicos , Investigación/tendencias , SuizaRESUMEN
The BRCA1-associated protein BARD1 is a putative tumor suppressor. We suggest that BARD1 is a mediator of apoptosis since (1) cell death in vivo (ischemic stroke) and in vitro is accompanied by increased levels of BARD1 protein and mRNA; (2) overexpression of BARD1 induces cell death with all features of apoptosis; and (3) BARD1-repressed cells are defective for the apoptotic response to genotoxic stress. The proapoptotic activity of BARD1 involves binding to and elevations of p53. BRCA1 is not required for but partially counteracts apoptosis induction by BARD1. A tumor-associated mutation Q564H of BARD1 is defective in apoptosis induction, thus suggesting a role of BARD1 in tumor suppression by mediating the signaling from proapoptotic stress toward induction of apoptosis.
Asunto(s)
Apoptosis , Proteínas Portadoras/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Doxorrubicina/farmacología , Expresión Génica/efectos de los fármacos , Expresión Génica/efectos de la radiación , Genes Supresores de Tumor , Células HeLa , Humanos , Hipoxia Encefálica/genética , Hipoxia Encefálica/metabolismo , Hipoxia Encefálica/patología , Infarto de la Arteria Cerebral Media , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutágenos/farmacología , Mutación/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , Rayos Ultravioleta , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
We have investigated whether repression of the putative tumor suppressor gene BARD1 or expression of the Notch4(int-3) oncogene in non-tumorigenic mammary epithelial cells affects their in vitro morphogenetic properties. Bard1 (Brca1-associated ring domain) is a protein interacting with Brca1 and thought to be involved in Brca1-mediated tumor suppression. To investigate the potential role of Bard1 in mammary gland development, we repressed its expression in TAC-2 cells, a murine mammary epithelial cell line which, when grown in three-dimensional collagen gels, forms branching ducts in response to hepatocyte growth factor (HGF) and alveolar-like cysts in response to hydrocortisone. Whereas Bard1 repression did not markedly modify the tubulogenic response of TAC-2 cells to HGF, it dramatically altered cyst development, resulting in the formation of compact cell aggregates devoid of central lumen. In addition, when grown to post-confluence in two-dimensional cultures, Bard1-suppressed TAC-2 cells overcame contact-inhibition of cell proliferation and formed multiple cell layers. The Notch4(int-3) oncogene, which codes for a constitutively activated form of the Notch4 receptor, has been reported to induce undifferentiated carcinomas when expressed in the mammary gland. The potential effect of activated Notch4 on mammary gland morphogenesis was investigated by retroviral expression of the oncogene in TAC-2 cells. Notch4(int-3) expression was found to significantly reduce HGF-induced tubulogenesis and to markedly inhibit hydrocortisone-induced cyst formation. In addition, Notch4(int-3) expressing TAC-2 cells formed multilayers in post-confluent cultures and exhibited an invasive behavior when grown on the surface of collagen gels. Taken together, these results indicate that both repression of Bard1 and expression of Notch4(int-3) disrupt cyst morphogenesis and induce an invasive phenotype in TAC-2 mammary epithelial cells.
Asunto(s)
Mama , Proteínas Portadoras/fisiología , Proteínas Proto-Oncogénicas/fisiología , Receptores de Superficie Celular , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Mama/embriología , Mama/fisiología , Línea Celular , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes Supresores de Tumor , Humanos , Morfogénesis/fisiología , Receptor Notch4 , Receptores NotchRESUMEN
We have shown previously that rats can be cured from induced peritoneal colon carcinomatosis by injections of apoptotic bodies derived from tumor cells and interleukin 2. This curative treatment generated a tumor-specific cytotoxic T-cell response associated with a humoral response. Autoantibodies from sera of cured rats strongly recognized a Mr 67,000 protein from apoptotic bodies and weakly reacted with a protein of Mr approximately 97,000 in PROb parental cells. We now show that these autoantibodies are directed against BARD1, originally identified as a protein interacting with the product of the breast cancer gene 1, BRCA1. We demonstrate that the Mr 67,000 antigen is a cleaved form of BARD1 present in apoptotic bodies derived from rat and human colon and mammary carcinoma cell lines. Moreover, we show that the cleavage site of BARD1 is located NH2 terminally but downstream of the RING domain essential for BARD1 and BRCA1 protein interaction. In vitro studies using [35S]methionine-labeled human BARD1 and apoptotic cellular extracts derived from SW48 carcinoma cells indicate that BARD1 proteolysis occurs at an early stage of apoptosis and in a cell cycle-dependent manner. This hydrolysis is inhibited by EGTA, and the calpain inhibitor I, N-acetyl-leu-leu-norleucinal, but not by several caspases inhibitors, suggesting that BARD1 is hydrolyzed by the calcium-dependent cysteine proteases, calpains. Thus, the highly immunogenic form of cleaved BARD1 could contribute to the antitumoral response mediated by apoptotic bodies.
Asunto(s)
Apoptosis , Autoantígenos/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Autoantígenos/química , Proteína BRCA1/metabolismo , Western Blotting , Neoplasias de la Mama/metabolismo , Calpaína/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Ciclo Celular , Fraccionamiento Celular , Clonación Molecular , Neoplasias del Colon/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , ADN Complementario/metabolismo , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Biblioteca de Genes , Humanos , Leupeptinas/farmacología , Neoplasias Mamarias Animales/metabolismo , Ratones , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
About half of the familial breast cancer cases are found to bear mutations in the breast cancer susceptibility gene 1 (BRCA1). The majority of BRCA1 mutations produce a truncated protein and BRCA1-associated breast tumors exhibit a number of defined tumor phenotypes. The function of BRCA1 has been examined in gene knockout mice in which the nullizygous mice die early in utero, but this lethality can be partially rescued by a nullizygous p53 mutation. Wild-type BRCA1 protein binds to a number of cellular proteins, including DNA repair protein Rad51, tumor suppressor p53, RNA polymerase II holoenzyme, RNA helicase A, CtBP-interacting protein, c-myc, BRCA1-associated RING domain protein (BARD1), BRCA2 protein, etc. These proteins likely mediate the involvement of BRCA1 in DNA repair, transcriptional transactivation, and cell cycle control. Overall, BRCA1 protein may act as a converging vehicle for cell regulatory proteins to associate with. Therefore, mutations in BRCA1 may affect the composition of these complexes on which dysregulation of cellular functions with eventual development of malignancy is expected.
Asunto(s)
Proteína BRCA1/genética , Neoplasias de la Mama/genética , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Animales , Proteína BRCA1/metabolismo , Proteína BRCA1/fisiología , Proteínas Portadoras/metabolismo , Ciclo Celular , Reparación del ADN , Femenino , Predisposición Genética a la Enfermedad , Humanos , Ratones , Ratones Noqueados , Mutación , Fenotipo , Activación Transcripcional , Dedos de ZincRESUMEN
BRCA1-associated RING domain (BARD1) was identified as a protein interacting with the breast cancer gene product BRCA1. The identification of tumorigenic missense mutations within BRCA1 that impair the formation of BARD1-BRCA1 complexes, and of BARD1 mutations in breast carcinomas, sustain the view that BARD1 is involved in BRCA1-mediated tumor suppression. We have cloned the murine Bard1 gene and determined that its expression in different tissues correlates with the expression profile of Brca1. To investigate the function of Bard1, we have reduced Bard1 gene expression in TAC-2 cells, a murine mammary epithelial cell line that retains morphogenetic properties characteristic of normal breast epithelium. Partial repression of Bard1, achieved by the transfection of TAC-2 cells with plasmids constitutively expressing ribozymes or antisense RNAs, resulted in marked phenotypic changes, consisting of altered cell shape, increased cell size, high frequency of multinucleated cells, and aberrant cell cycle progression. Furthermore, Bard1-repressed cell clones overcame contact inhibition of cell proliferation when grown in monolayer cultures and lost the capacity to form luminal structures in three-dimensional collagen gels. These results demonstrate that Bard1 repression induces complex changes in mammary epithelial cell properties which are suggestive of a premalignant phenotype.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Animales , Secuencia de Bases , Neoplasias de la Mama/genética , Clonación Molecular , Inhibición de Contacto , ADN Complementario/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes Supresores de Tumor , Humanos , Glándulas Mamarias Animales/crecimiento & desarrollo , Neoplasias Mamarias Experimentales/genética , Ratones , Fenotipo , Lesiones Precancerosas/genética , Fase SRESUMEN
Phosphorylation, dimerization and binding to calmodulin have been reported to influence the microtubule assembly capacities of MAPs (microtubule-associated proteins). Here we report that the Drosophila 205K MAP is a phosphoprotein in vivo and can be phosphorylated by cdc2/p34 in vitro. Bacterially produced 205K MAP is competent of microtubule assembly and microtubule bundling and binds to immobilized calmodulin in a Ca2+-dependent way. EM rotary shadowing analyses suggest that 205K MAP consists of an amino-terminal flexible extended region and a carboxy-terminal globular domain. This carboxy-terminal region harbors the microtubule binding site and sequences required for dimerization, as confirmed by in vitro crosslinking experiments of truncated proteins.
Asunto(s)
Proteínas Asociadas a Microtúbulos/química , Microtúbulos/metabolismo , Animales , Reactivos de Enlaces Cruzados/química , Dimerización , Drosophila , Proteínas Asociadas a Microtúbulos/aislamiento & purificación , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Porcinos , Tubulina (Proteína)/metabolismoRESUMEN
Microtubule-associated proteins (MAPs) promote the assembly of microtubules from purified tubulin in vitro. In order to establish a model system for the investigation of the role of MAPs in microtubule assembly in vivo, we have generated Saccharomyces cerevisiae strains that permit the modulation of the expression levels of MHP1 (MAP-Homologous Protein 1) and of the alpha and beta-tubulin genes. Simultaneous overexpression of alpha and beta tubulin results in the accumulation of long aberrant microtubules in interphase, a similar phenotype as was observed in cells overexpressing MHP1. We demonstrate that overexpression of MHP1 in asynchronously growing yeast cultures leads to cell cycle arrest in G2. In cells that overexpress MHP1 and the tubulin genes, a suppression of both the MHP1 and the tubulin overexpression phenotypes can be observed. Progressive induction of alpha and beta tubulin overexpression and constitutive overexpression of MHP1 lead to the formation of long cytoplasmic microtubules more frequently than observed in cells overproducing tubulin or Mhplp individually and the increased microtubule polymerization could be correlated with the increase of a and beta tubulin expression. However, the overexpression of MHP1 did not alter the phenotypes of individual overexpression of a or beta-tubulin. These data indicate that Mhplp not only stabilizes microtubules but promotes microtubule assembly in vivo, and suggest that the role of other mammalian MAPs in the promotion of microtubule assembly could be tested in this yeast system.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/fisiología , Saccharomyces cerevisiae/citología , Western Blotting , Ciclo Celular/fisiología , Citometría de Flujo , Microscopía Fluorescente , Plásmidos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Tubulina (Proteína)/metabolismoRESUMEN
The gene for a microtubule-associated protein (MAP), termed MHP1 (MAP-Homologous Protein 1), was isolated from Saccharomyces cerevisiae by expression cloning using antibodies specific for the Drosophila 205K MAP. MHP1 encodes an essential protein of 1,398 amino acids that contains near its COOH-terminal end a sequence homologous to the microtubule-binding domain of MAP2, MAP4, and tau. While total disruptions are lethal, NH2-terminal deletion mutations of MHP1 are viable, and the expression of the COOH-terminal two-thirds of the protein is sufficient for vegetative growth. Nonviable deletion-disruption mutations of MHP1 can be partially complemented by the expression of the Drosophila 205K MAP. Mhp1p binds to microtubules in vitro, and it is the COOH-terminal region containing the tau-homologous motif that mediates microtubule binding. Antibodies directed against a COOH-terminal peptide of Mhp1p decorate cytoplasmic microtubules and mitotic spindles as revealed by immunofluorescence microscopy. The overexpression of an NH2-terminal deletion mutation of MHP1 results in an accumulation of large-budded cells with short spindles and disturbed nuclear migration. In asynchronously growing cells that overexpress MHP1 from a multicopy plasmid, the length and number of cytoplasmic microtubules is increased and the proportion of mitotic cells is decreased, while haploid cells in which the expression of MHP1 has been silenced exhibit few microtubules. These results suggest that MHP1 is essential for the formation and/or stabilization of microtubules.
Asunto(s)
Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/fisiología , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , División Celular , Clonación Molecular , Mapeo Epitopo , Proteínas Fúngicas/análisis , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Expresión Génica , Prueba de Complementación Genética , Sueros Inmunes , Interfase , Punto Isoeléctrico , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mitosis , Datos de Secuencia Molecular , Fenotipo , Fosforilación , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae , Análisis de SecuenciaRESUMEN
The Drosophila melanogaster (Dm) gene lethal(3)73Ah, essential at the late pupal stage, encodes a protein with a novel Cys-rich sequence motif, typical for ring-finger proteins. Amino-acid sequence comparison revealed a striking homology of the entire lethal(3)73Ah sequence to the gene products of the mammalian oncogenes, mel-18 and bmi-1, and to the zinc-finger-containing N-terminal region of the Dm proteins encoded by the Posterior sex combs and Suppressor two of zeste genes. The lethal(3)73Ah gene is located in a densely transcribed region sharing 3'-untranslated sequences with the adjacent sex-determining gene, transformer. Its transcription is temporally and spatially regulated with maximal expression in adult females. In all stages the mRNA can be localized to the fat body and, in addition, to the ovaries of adult females.
Asunto(s)
Proteínas de Unión al ADN/genética , Drosophila melanogaster/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Femenino , Expresión Génica , Ratones , Datos de Secuencia Molecular , Complejo Represivo Polycomb 1 , ARN Mensajero/análisis , Homología de Secuencia de AminoácidoRESUMEN
We have purified microtubule-associated proteins from Neurospora crassa on the basis of heat stability and affinity to calmodulin. Two proteins of molecular masses 170 kDa and 190 kDa have been partially purified. A third protein of 145 kDa was purified almost to homogeneity, and we present evidence that this protein is a specific substrate for a Ca2+/calmodulin-dependent protein kinase. The purified 170-, 190-, and 145-kDa proteins induce the assembly of microtubules from purified porcine brain tubulin. We demonstrate that all three proteins are microtubule-associated proteins on the basis of an in vitro microtubule-binding assay.
Asunto(s)
Calmodulina/metabolismo , Proteínas Asociadas a Microtúbulos/aislamiento & purificación , Neurospora crassa/química , Anticuerpos/inmunología , Especificidad de Anticuerpos , Western Blotting , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Cromatografía de Afinidad , Estabilidad de Medicamentos , Calor , Microscopía Electrónica , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Especificidad por SustratoRESUMEN
We have sequenced cDNA clones encoding the Drosophila 205K microtubule-associated protein (MAP), a protein that may be the species specific homologue of mammalian MAP4. The peptide sequence deduced from the longest open-reading frame reveals a hydrophilic protein, which has basic and acidic regions that are similar in organization to mammalian MAP2. Using truncated forms of the 205K MAP, a 232-amino acid region could be defined that is necessary for microtubule binding. The amino acid sequence of this region shares no similarity with the binding motif of MAP2 or tau. We also analyzed several embryonic cDNA clones, which show the existence of differentially spliced mRNAs. Finally, we identified several potential protein kinase target sequences. One of these is distal to the microtubule-binding site and fits the phosphorylation consensus sequence of proteins phosphorylated by the mitosis specific protein kinase cdc2. Our data suggest that the 205K MAP uses a microtubule-binding motif unlike that found in other MAPs, and also raise the possibility that the activities of the 205K MAP may be regulated by alternative splicing and phosphorylation.
Asunto(s)
Drosophila/genética , Proteínas Asociadas a Microtúbulos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Clonación Molecular , ADN/genética , Drosophila/metabolismo , Genes , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica , ARN/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
In Drosophila, the primary signal for sex determination is given by the ratio of X chromosomes to sets of autosomes (X:A). The primary signal is read by a key gene (Sxl) and transmitted down to the differentiation genes by the subordinate control genes tra, tra-2, ix and dsx. Mutations in tra transform chromosomal females (X/X; tra/tra) into sterile males (pseudomales). We have cloned the tra region by microdissection and chromosomal walking. We identified the gene using deficiency breakpoints, DNA aberrations in three different alleles of tra and by P-mediated transformation. A 3.8-kb fragment perfectly rescued the mutant phenotype of X/X; tra/tra flies, showing that it contained all the necessary information to restore female-specific functions in the mutant flies. We present evidence that most of the function of tra can be provided by a subsegment of 2 kb that is differentially transcribed or processed in males and females.