RESUMEN
The neural crest is a migratory cell population that gives rise to multiple cell types in the vertebrate embryo. The intrinsic determinants that segregate neural crest cells from multipotential dorsal progenitors within the neural tube are poorly defined. In this study, we show that the winged helix transcription factor Foxd3 is expressed in both premigratory and migratory neural crest cells. Foxd3 is genetically downstream of Pax3 and is not expressed in regions of Pax3 mutant mice that lack neural crest, implying that Foxd3 may regulate aspects of the neural crest differentiation program. We show that misexpression of Foxd3 in the chick neural tube promotes a neural crest-like phenotype and suppresses interneuron differentiation. Cells that ectopically express Foxd3 upregulate HNK1 and Cad7, delaminate and emigrate from the neural tube at multiple dorsoventral levels. Foxd3 does not induce Slug and RhoB, nor is its ability to promote a neural crest-like phenotype enhanced by co-expression of Slug. Together these results suggest Foxd3 can function independently of Slug and RhoB to promote the development of neural crest cells from neural tube progenitors.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Cresta Neural/citología , Neuronas/citología , Proteínas Represoras/genética , Animales , Proteínas Aviares , Biomarcadores , Antígenos CD57/genética , Cadherinas/genética , Diferenciación Celular/genética , Movimiento Celular , Embrión de Pollo , Proteínas de Unión al ADN/metabolismo , Inducción Embrionaria/genética , Factores de Transcripción Forkhead , Secuencias Hélice-Giro-Hélice , Ratones , Ratones Mutantes , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Proteínas Represoras/metabolismo , Factores de Transcripción de la Familia Snail , Médula Espinal/embriología , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína de Unión al GTP rhoB/genéticaRESUMEN
Interneurons in the ventral spinal cord are essential for coordinated locomotion in vertebrates. During embryogenesis, the V0 and V1 classes of ventral interneurons are defined by expression of the homeodomain transcription factors Evx1/2 and En1, respectively. In this study, we show that Evx1 V0 interneurons are locally projecting intersegmental commissural neurons. In Evx1 mutant embryos, the majority of V0 interneurons fail to extend commissural axons. Instead, they adopt an En1-like ipsilateral axonal projection and ectopically express En1, indicating that V0 interneurons are transfated to a V1 identity. Conversely, misexpression of Evx1 represses En1, suggesting that Evx1 may suppress the V1 interneuron differentiation program. Our findings demonstrate that Evx1 is a postmitotic determinant of V0 interneuron identity and reveal a critical postmitotic phase for neuronal determination in the developing spinal cord.
Asunto(s)
Células del Asta Anterior/metabolismo , Movimiento Celular/fisiología , Proteínas de Homeodominio/metabolismo , Interneuronas/metabolismo , Locomoción/fisiología , Alelos , Animales , Células del Asta Anterior/embriología , Axones/metabolismo , Embrión de Pollo , Femenino , Proteínas de Homeodominio/genética , Masculino , Ratones , Ratones Noqueados , Ratones Mutantes , Fenotipo , Médula Espinal/embriología , Médula Espinal/metabolismoRESUMEN
In mammalian embryos, myogenic precursor cells emigrate from the ventral lip of the dermomyotome and colonize the limbs, tongue and diaphragm where they differentiate and form skeletal muscle. Previous studies have shown that Pax3, together with the c-Met receptor tyrosine kinase and its ligand Scatter Factor (SF) are necessary for the migration of hypaxial muscle precursors in mice. Lbx1 and Pax3 are co-expressed in all migrating hypaxial muscle precursors, raising the possibility that Lbx1 regulates their migration. To examine the function of Lbx1 in muscle development, we inactivated the Lbx1 gene by homologous recombination. Mice lacking Lbx1 exhibit an extensive loss of limb muscles, although some forelimb and hindlimb muscles are still present. The pattern of muscle loss suggests that Lbx1 is not required for the specification of particular limb muscles, and the muscle defects that occur in Lbx1(-/-) mice can be solely attributed to changes in muscle precursor migration. c-Met is expressed in Lbx1 mutant mice and limb muscle precursors delaminate from the ventral dermomyotome but fail to migrate laterally into the limb. Muscle precursors still migrate ventrally and give rise to tongue, diaphragm and some limb muscles, demonstrating Lbx1 is necessary for the lateral, but not ventral, migration of hypaxial muscle precursors. These results suggest that Lbx1 regulates responsiveness to a lateral migration signal which emanates from the developing limb.
Asunto(s)
Extremidades/embriología , Proteínas Musculares/genética , Músculos/embriología , Células Madre/metabolismo , Factores de Transcripción , Animales , Animales Recién Nacidos , Movimiento Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Diafragma/embriología , Diafragma/crecimiento & desarrollo , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Ratones , Ratones Noqueados , Desarrollo de Músculos , Proteínas Musculares/metabolismo , Mutación , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Proteínas Proto-Oncogénicas c-met/genética , Transducción de Señal , Lengua/embriología , Lengua/crecimiento & desarrolloRESUMEN
The transcription factor Oct-4 is expressed specifically in the totipotent germline cycle of mice. Cells that lose Oct-4 differentiate along different paths to form embryonic and extraembryonic somatic tissue. Oct-4 may maintain the potency of stem and germline cells by preventing all other differentiation pathways. Oct-4 may also regulate the molecular differentiation of cells in the germ lineage as it progresses from the fertilized egg, through cleavage stage/morula blastomeres, blastocyst, inner cell mass, epiblast, germ cells, and gametes. The factors that regulate, and are regulated by, Oct-4 are reviewed with respect to the phenomena of cell potency and germ/soma segregation and differentiation.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Desarrollo Embrionario y Fetal/genética , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/citología , Factores de Transcripción/fisiología , Animales , Diferenciación Celular , Línea Celular , Linaje de la Célula , Proteínas de Unión al ADN/genética , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Femenino , Humanos , Masculino , Meiosis/genética , Ratones/embriología , Ratones/genética , Familia de Multigenes , Factor 3 de Transcripción de Unión a Octámeros , Especificidad de la Especie , Factores de Transcripción/genética , Vertebrados/anatomía & histología , Vertebrados/embriologíaRESUMEN
The POU transcription factor Oct-4 is expressed specifically in the germ line, pluripotent cells of the pregastrulation embryo and stem cell lines derived from the early embryo. Osteopontin (OPN) is a protein secreted by cells of the preimplantation embryo and contains a GRGDS motif that can bind to specific integrin subtypes and modulate cell adhesion/migration. We show that Oct-4 and OPN are coexpressed in the preimplantation mouse embryo and during differentiation of embryonal cell lines. Immunoprecipitation of the first intron of OPN (i-opn) from covalently fixed chromatin of embryonal stem cells by Oct-4-specific antibodies indicates that Oct-4 binds to this fragment in vivo. The i-opn fragment functions as an enhancer in cell lines that resemble cells of the preimplantation embryo. Furthermore, it contains a novel palindromic Oct factor recognition element (PORE) that is composed of an inverted pair of homeodomain-binding sites separated by exactly 5 bp (ATTTG +5 CAAAT). POU proteins can homo- and heterodimerize on the PORE in a configuration that has not been described previously. Strong transcriptional activation of the OPN element requires an intact PORE. In contrast, the canonical octamer overlapping with the downstream half of the PORE is not essential. Sox-2 is a transcription factor that contains an HMG box and is coexpressed with Oct-4 in the early mouse embryo. Sox-2 represses Oct-4 mediated activation of i-opn by way of a canonical Sox element that is located close to the PORE. Repression depends on a carboxy-terminal region of Sox-2 that is outside of the HMG box. Expression, DNA binding, and transactivation data are consistent with the hypothesis that OPN expression is regulated by Oct-4 and Sox-2 in preimplantation development.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Desarrollo Embrionario/genética , Elementos de Facilitación Genéticos , Proteínas Nucleares/fisiología , Sialoglicoproteínas/antagonistas & inhibidores , Sialoglicoproteínas/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Composición de Base , Secuencia de Bases , Sitios de Unión/genética , Blastocisto/metabolismo , Blastocisto/fisiología , Carcinoma Embrionario , Diferenciación Celular/genética , Cromatina/aislamiento & purificación , Cromatina/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Dimerización , Embrión de Mamíferos , Femenino , Proteínas HMGB , Intrones , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factor 3 de Transcripción de Unión a Octámeros , Osteopontina , Factores del Dominio POU , Embarazo , Conformación Proteica , Factores de Transcripción SOXB1 , Sialoglicoproteínas/biosíntesis , Factores de Transcripción/fisiología , Células Tumorales CultivadasRESUMEN
Controlled environment experiments were conducted to determine the influence of temperature and leaf wetness duration on infection of perennial ryegrass by Rhizoctonia solani. Infection of grass plants raised in pots and exposed to mycelium of R. solani was evaluated at various combinations of temperature and leaf wetness duration. Temperatures included 15, 18, 21, 24, and 27°C. Leaf wetness periods were 9, 12, 15, 18, and 24 h. Disease was most severe (more than 50 leaves with brown patch lesions per pot) when plants were in contact with the inoculum source for 24 h of leaf wetness at 24°C. The least amount of disease (0 leaves with lesions per pot) occurred at 15°C and a 9-h wet period. The data were subjected to analysis of variance with orthogonal polynomial contrasts. Significant effects were included in a regression model that described the response of infection to temperature and wetness duration. The polynomial model included linear and quadratic terms for temperature and wetness duration. The adjusted coefficient of determination for the fitted model was 0.93, indicating an excellent fit to the data. The model is intended for use in an improved brown patch warning system for perennial ryegrass in the midwestern United States.
RESUMEN
Pax3 RNA is expressed in neural crest when Schwann cell (SC) precursors migrate to the PNS. Pax3 RNA and SC markers were monitored in sciatic nerves of mice during development and nerve repair. An inverse correlation was observed between expression of Pax3 RNA and myelin basic protein (MBP). Inverse correlation was also observed in SC primary cultures. Treating cultures with forskolin, an adenylate cyclase agonist, repressed Pax3 RNA, GFAP, NGFR, N-CAM, and L1 and elevated MBP. Subsequent microinjection with Pax3 expression vector elevated Pax3 RNA, GFAP, NGFR, N-CAM, and L1 and repressed MBP. Thus, Pax3 is likely involved in the differentiation pathway to myelinating SCs. Pax3 repressed a 1.3 kb MBP promoter fragment in cotransfection assays, suggesting that it represses MBP transcription.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de Unión al ADN/genética , Vaina de Mielina/fisiología , Sistema Nervioso Periférico/fisiología , Factores de Transcripción , Animales , Axones/fisiología , Células Cultivadas , Colforsina/farmacología , Proteínas de Unión al ADN/farmacología , Regulación de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/metabolismo , Ratones , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Regiones Promotoras Genéticas , ARN/análisis , ARN/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Células de Schwann/metabolismo , Nervio Ciático/metabolismoRESUMEN
The mouse homeodomain protein Hoxa-7, expressed under the control of an inducible promoter, was able to inducibly activate reporter genes containing multimerized Hoxa-7 binding sites in Saccharomyces cerevisiae. This tight regulation was exploited in an attempt to screen for Hoxa-7 responsive elements. A reporter library consisting of a randomised 10 bp element inserted into the minimal gal1 promoter was constructed. In a surprisingly small screen, 24 reporters were isolated which had all of the transactivation characteristics expected for a Hoxa-7 binding site insertion. However, further characterisation revealed that the selected elements lacked homeodomain (HD) binding core motifs and were not bound by a purified Hoxa-7/beta-galactosidase fusion protein capable of binding known sites. The minimal promoter context contains 16 HD core motifs in 410 bp. Careful re-examination of basal levels revealed a low residual response of the gal1 minimal promoter to Hoxa-7. The 11 characterised 10 bp inserts amplified Hoxa-7 responsiveness in a manner correlated to increases in basal reporter activity. Thus, a quantitative range of Hox-responsiveness was produced by slight sequence alterations that did not change HD binding sites of their relative spacing in the promoter. These data suggest how, without altering resident HD base contact zones, mouse promoters could be optimised by natural selection to give appropriate quantitative outputs in each anatomical region defined by an assortment of Hox proteins. The selected elements were pyrimidine rich on the sense strand, containing (T)nC motifs, strikingly similar to sequences which enhance Hoxa-7 binding and activation from outside the HD contact zone. A search of defined sequence databases demonstrated that these elements were over-represented in promoters. Two elements altered the mobility shift patterns produced by cell extracts on minimal promoter fragments.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Oligodesoxirribonucleótidos/genética , Regiones Promotoras Genéticas/genética , Activación Transcripcional , beta-Galactosidasa/genética , Animales , Secuencia de Bases , Sitios de Unión , Genes Reporteros , Proteínas de Homeodominio/genética , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Selección Genética , Factores de TiempoRESUMEN
The murine developmental control gene product, Hoxa-7, was shown to function as a DNA-binding transactivator in Saccharomyces cerevisiae. The importance of the ATTA core, the preference for antp class flanking nucleotides, the importance of Asn-51 of the homeodomain (HD), and the synergism of multiple binding sites all reflect properties that have previously been described for HOM or Hox proteins in tissue culture systems. A comparison of contact positions among genes of paralog groups and classes of mammalian HDs points to a lack of diversity in positions that make base contact, suggesting that besides the combination of HD amino acid-base pair contacts, another means of recognizing differences between targets must exist if Hox genes select different targets. The HD of antennapedia is identical to the Hoxa-7 HD. The interaction of Hoxa-7 with the exact sequence used in the nuclear magnetic resonance three-dimensional structural analysis on the antennapedia HD was studied. Hoxa-7 binding and transactivation was influenced by sequences outside of the known base contact zone of this site. We conclude that Hoxa-7 protein has a second means to interact with DNA or/and that the sequences flanking the base contact zone influence HD interactions by distorting DNA within the contact zone (base or backbone). This result is discussed in terms of DNA flexure and two modes of transcription used in S. cerevisiae.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio , Saccharomyces cerevisiae/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , ADN Recombinante/genética , ADN Recombinante/metabolismo , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Proteínas de Unión al ADN/genética , Genes Fúngicos , Genes Reporteros , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Mutación Puntual , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Activación TranscripcionalRESUMEN
Bordetella pertussis, the pathogen responsible for whooping cough, produces a toxic calmodulin-sensitive adenylyl cyclase which enters animal cells and increases intracellular cAMP. A point mutant of B. pertussis with abolished adenylyl cyclase catalytic activity was over 1000-fold less pathogenic to newborn mice than wild-type bacteria, demonstrating the importance of the adenylyl cyclase for B. pertussis virulence (Gross et al.). The B. pertussis adenylyl cyclase is highly sensitive to calmodulin with an apparent Kd for calmodulin of approximately 1 nM. The importance of this high-affinity calmodulin binding for virulence in vivo was examined by the creation of a B. pertussis point mutant (Trp-242 to Glu-242) with 200-fold lower calmodulin affinity than the native enzyme. This mutant B. pertussis strain retained its virulence in a newborn mouse model of pertussis, but the time course for establishment of a lethal infection in vivo was significantly delayed for the mutant strain. These data illustrate that high-affinity calmodulin binding is not obligatory for the activity of this toxin but is important for the rate for establishment of a lethal infection.
Asunto(s)
Adenilil Ciclasas/metabolismo , Bordetella pertussis/enzimología , Bordetella pertussis/patogenicidad , Calmodulina/metabolismo , Adenilil Ciclasas/genética , Animales , Animales Recién Nacidos , Bordetella pertussis/genética , Ratones , Ratones Endogámicos BALB C , Mutación Puntual , Unión Proteica , VirulenciaRESUMEN
Bordetella pertussis produces a calmodulin-stimulated adenylyl cyclase that invades animal cells and raises intracellular cAMP levels [Confer, D. L., & Eaton, J. W. (1982) Science 217, 948-950; Shattuck, R. L., & Storm, D. R. (1985) Biochemistry 24, 6323-6328]. The mechanism for invasion of animal cells by this enzyme has not been defined, but there is considerable evidence that it does not enter by receptor-mediated endocytosis [Gordon, V. M., Leppla, S. H., & Hewlett, E. L. (1988) Infect. Immun. 56, 1066-1069; Donovan, M. G., & Storm, D. R. (1990) J. Cell. Physiol. 145, 444-449]. In this study, the importance of high-affinity calmodulin (CaM) binding for entry of the enzyme into neuroblastoma cells was evaluated using a mutant enzyme that has significantly lower affinity for calmodulin than the wild-type enzyme. Oligonucleotide-directed site-specific mutagenesis was used to create a point mutant at a critical tryptophan residue (Trp-242) within the proposed CaM binding domain of the B. pertussis adenylyl cyclase. Substitution of Trp-242 with Glu lowered the apparent affinity of the enzyme for calmodulin by 250-fold; however, the maximal enzyme activity in the presence of saturating calmodulin was equivalent to the wild-type enzyme. The Glu-242 mutant adenylyl cyclase was returned to B. pertussis by homologous recombination, and the enzyme produced by this strain was examined for invasion of neuroblastoma cells. Although the mutant enzyme stimulated the production of intracellular cAMP in neuroblastoma cells, the rate of cAMP accumulation was at least 10-fold lower than that caused by the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Adenilil Ciclasas/metabolismo , Bordetella pertussis/enzimología , Calmodulina/metabolismo , Adenilil Ciclasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Transporte Biológico , Bordetella pertussis/genética , Proteínas de Unión a Calmodulina/metabolismo , Activación Enzimática , Genes Bacterianos , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neuroblastoma , Oligodesoxirribonucleótidos/química , Mapeo Restrictivo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Bordetella pertussis, the causative agent of whooping cough, secretes several toxins implicated in this disease. One of these putative virulence factors is the adenylate cyclase (AC) toxin that elevates intracellular cAMP in eukaryotic cells to cytotoxic levels. This toxin is a bifunctional protein comprising both AC and hemolysin (HLY) enzymatic domains. The gene encoding the AC toxin (cyaA) is expressed as part of an operon that includes genes required for secretion or activation of the toxin. Because of this genetic organization, it is difficult to create B. pertussis mutants of cyaA that are ablations of a single enzyme function by conventional means, such as transposon mutagenesis. Therefore, to clarify the role of individual toxin functions in the virulence of B. pertussis, we have used site-directed or deletion mutagenesis and genetic recombination to specifically target the cyaA gene of B. pertussis to produce mutants that lack only the AC or HLY activity of this toxin. A point mutant of B. pertussis with abolished AC catalytic activity was greater than 1000 times less pathogenic to newborn mice than wild-type bacteria, directly demonstrating the importance of the AC toxin in pertussis virulence. Similarly, an in-frame deletion mutant of B. pertussis that lacks HLY is equally avirulent, supporting observations that the HLY domain plays a critical role in AC toxin entry into cells. Furthermore, the genetically inactivated AC toxin produced by the point mutant is antigenically similar to the native toxin, suggesting that this strain may be useful in the development of pertussis component vaccines.
Asunto(s)
Toxina de Adenilato Ciclasa , Bordetella pertussis/patogenicidad , Proteínas Hemolisinas/genética , Factores de Virulencia de Bordetella/toxicidad , Adenilil Ciclasas/química , Adenilil Ciclasas/genética , Animales , Bordetella pertussis/genética , Análisis Mutacional de ADN , Genes Bacterianos , Proteínas Hemolisinas/química , Ratones , Ratones Endogámicos BALB C , Mapeo Restrictivo , Relación Estructura-Actividad , Factores de Virulencia de Bordetella/químicaRESUMEN
African trypanosomes are protozoan parasites that evade the host immune system by varying their dense antigenic coat. The Variant Surface Glycoprotein (VSG) is expressed exclusively from telomere-linked expression sites that contain in addition to the VSG gene, a number of open reading frames termed Expression Site Associated Genes (ESAGs). Here we demonstrate by complementation of a yeast mutant deleted for adenylate cyclase (cyr-1), that an ESAG from Trypanosoma equiperdum encodes an adenylate cyclase. Furthermore, we report that adjacent to adenylate cyclase in the expression site, is a separate open reading frame that encodes a protein sequence motif similar to the leucine-rich repeat regulatory domain of Saccharomyces cerevisiae and Schizosaccharomyces pombe adenylate cyclases. The finding of two adjacent open reading frames homologous to a single enzyme in yeast suggests that the two expression site encoded proteins may interact to regulate adenylate cyclase activity during the course of an infection.
Asunto(s)
Adenilil Ciclasas/genética , Genes Reguladores , Leucina/genética , Trypanosoma/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Regulación Enzimológica de la Expresión Génica , Genes Fúngicos , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Plásmidos , ARN Protozoario/análisis , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoAsunto(s)
Adenilil Ciclasas/aislamiento & purificación , Espermatozoides/enzimología , Adenosina Trifosfato/metabolismo , Adenilil Ciclasas/análisis , Adenilil Ciclasas/metabolismo , Animales , Calmodulina/metabolismo , Calmodulina/farmacología , Cromatografía de Afinidad/métodos , Cromatografía en Gel/métodos , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/métodos , AMP Cíclico/metabolismo , Caballos , Indicadores y Reactivos , Cinética , Masculino , Radioisótopos de Fósforo , Técnica de Dilución de Radioisótopos , Termodinámica , TritioRESUMEN
The extracellular calmodulin-sensitive adenylate cyclase produced by Bordetella pertussis is synthesized as a 215-kDa precursor. This polypeptide is transported to the outer membrane of the bacteria where it is proteolytically processed to a 45-kDa catalytic subunit which is released into the culture supernatant [Masure, H.R., & Storm, D.R. (1989) biochemistry 28, 438-442]. The gene encoding this enzyme, cyaA, is part of the cya operon that also includes the genes cyaB, cyaD, and cyaE. A comparison of the predicted amino acid sequences encoded by cyaA, cyaB, and cyaD with the amino acid sequences encoded by hlyA, hlyB, and hlyD genes from the hemolysin (hly) operon from Escherichia coli shows a large degree of sequence similarity [Glaser, P., Sakamoto, H., Bellalou, J., Ullmann, A., & Danchin, A. (1988) EMBO J. 7, 3997-4004]. Complementation studies have shown that HlyB and HlyD are responsible for the secretion of HlyA (hemolysin) from E. coli. The signal sequence responsible for secretion of hemolysin has been shown to reside in its C-terminal 27 amino acids. Similarly, CyaB, CyaD, and CyaE are required for the secretion of CyaA from Bordetella pertussis. We placed the cyaA gene and a truncated cyaA gene that lacks the nucleotides that code for a putative C-terminal secretory signal sequence under the control of the lac promoter in the plasmid pUC-19. These plasmids were transformed into strains of E. coli which contained the hly operon. The truncated cyaA gene product, lacking the putative signal sequence, was not secreted but accumulated inside the cell.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Adenilil Ciclasas/genética , Bordetella pertussis/genética , Factores de Hemolisina/genética , Operón/fisiología , Plásmidos/genética , Adenilil Ciclasas/metabolismo , Bordetella pertussis/enzimología , Escherichia coli/enzimología , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Proteínas Hemolisinas/metabolismo , Operón Lac/fisiología , Regiones Promotoras GenéticasRESUMEN
The molecular basis for replication-dependent expression of thymidine kinase (TK) activity (EC 2.7.1.21) was investigated in mouse skeletal muscle cells transformed with multiple copies of the chicken TK gene. When shifted to mitogen-depleted medium, proliferating myoblasts irreversibly withdraw from the cell cycle and commit to terminal differentiation. Early after commitment, postreplicative myocytes maintain nearly proliferative levels of TK mRNA but have greatly reduced levels of TK activity. Metabolic labeling studies with [35S]methionine indicated that the decrease in TK activity was associated with a 10-fold reduction in the rate of TK protein synthesis. Commitment had little effect on the stability or catalytic efficiency of TK protein. The decrease in TK synthetic rate in the continued presence of TK mRNA indicated that translation of TK mRNA was repressed in committed cells. The distribution of TK mRNA between ribonucleoprotein particles and polysomes was determined. In both proliferative cells and committed cells, TK mRNA levels were maximal in polysomes containing five to seven ribosomes. Thus, the synthesis of TK protein in nonreplicating muscle cells was inhibited by a translational mechanism that did not alter the average number of ribosomes engaged by TK mRNA.
Asunto(s)
Músculos/enzimología , Polirribosomas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Timidina Quinasa/biosíntesis , Animales , Diferenciación Celular , División Celular , Células Cultivadas , Replicación del ADN , Cinética , Ratones , Músculos/citología , ARN Mensajero/metabolismo , Timidina Quinasa/genéticaRESUMEN
Replication-dependent changes in levels of enzymes involved in DNA precursor biosynthesis are accompanied frequently by changes in levels of cognate mRNA. We tested the common assumption that changes in mRNA levels are responsible for growth-dependent expression of these enzymes using a line of mouse muscle cells that irreversibly withdraws from the cell cycle as part of its terminal differentiation program. Thymidine kinase (TK) mRNA, activity, and protein levels were quantitated in cells transformed with multiple copies of the chicken TK gene. The decline in TK mRNA (both whole cell and cytoplasmic) during myogenesis was poor (2-fold average) and variable (1.2 to 8-fold). In contrast, TK activity always was regulated efficiently (20-fold), even in cells which regulated TK mRNA very poorly. Thus, regulation of TK activity was independent of TK mRNA regulation as myoblasts withdrew from the cell cycle. A TK/beta-galactosidase fusion protein was used to derive an antibody against chicken TK. Immunoblot and immunoprecipitation analyses demonstrated TK protein levels, like TK activity levels, declined to a greater extent than TK mRNA levels. Thus, TK activity likely was regulated by a mechanism involving either decreased translation of TK mRNA or increased degradation of TK protein in committed muscle cells.
Asunto(s)
Ciclo Celular , Músculos/enzimología , ARN Mensajero/fisiología , Timidina Quinasa/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Regulación de la Expresión Génica , Ratones , Músculos/metabolismo , Músculos/fisiología , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Timidina Quinasa/genética , Timidina Quinasa/aislamiento & purificación , Transformación GenéticaRESUMEN
TK mRNA levels were determined in mouse L cells transformed with intron deletion mutations of the chicken TK gene. Whether normalized per cell, per integrated gene, or per internal control signal, intron deletion did not diminish the efficiency of TK mRNA formation in transformed L cells. The results demonstrated that introns are not required for efficient biogenesis of cellular mRNA in transformed mouse L cells.
Asunto(s)
Intrones , ARN Mensajero/biosíntesis , Timidina Quinasa/genética , Animales , Pollos , Células L , Ratones , Mutación , Hibridación de Ácido Nucleico , Empalme del ARN , Ribonucleasas , Transcripción Genética , Transformación GenéticaRESUMEN
Thymidine kinase (TK) is representative of a class of enzymes involved in DNA precursor biosynthesis that declines as cells withdraw from the cell cycle. If TK activity is regulated exclusively by the availability of messenger RNA, changes in enzyme activity levels should not precede or excede changes in TK mRNA levels. This prediction was tested in several tissues during chicken embryogenesis and in differentiating muscle cells in culture. A sensitive method of determining absolute TK mRNA levels was developed. A synthetic complimentary RNA probe spanning an intron acceptor site in the chicken TK gene was hybridized with cellular RNA or synthetic colinear TK RNA of known concentration. After RNase digestion and gel electrophoresis, the intensity of the protected fragment was used to calculate absolute TK mRNA levels. As few as 0.02 molecules of TK mRNA per cell could be measured accurately. Depending on the tissue type, 8-day embryos contained between 3 and 12 TK mRNAs per cell. Proliferating mouse muscle cells transformed with the chicken TK gene contained between 30 and 150 TK mRNAs per cell. Both in vivo and in vitro, TK mRNA levels declined as cells withdrew from the cell cycle during differentiation. In vivo, the decline in TK activity never preceded or exceeded observed changes in TK mRNA. However, in the cell culture system, TK activity consistently declined to a greater extent than TK mRNA. Thus, a translational or a post-translational mechanism must also be operative in controlling TK activity levels. Estimation of transcription rates in nuclei isolated from proliferating and differentiated muscle cell transformants indicated that the TK gene was transcriptionally repressed in postreplicative cells.
Asunto(s)
Encéfalo/embriología , Diferenciación Celular , Genes , Corazón/embriología , Hígado/embriología , Músculos/citología , ARN Mensajero/genética , Timidina Quinasa/genética , Transcripción Genética , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Embrión de Pollo , Clonación Molecular , Represión Enzimática , Ratones , Músculos/enzimología , Timidina Quinasa/biosíntesisRESUMEN
Calmodulin (CaM), the calcium binding protein that modulates the activity of a number of key regulatory enzymes, is present at high levels in sperm. To determine whether CaM regulates adenylate cyclase in mammalian sperm, the actions of EGTA and selected CaM antagonists on a solubilized adenylate cyclase from mature equine sperm were examined. The activity of equine sperm adenylate cyclase was inhibited by EGTA in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 2 mM. Equine sperm adenylate cyclase was also inhibited in a concentration-dependent manner by the CaM antagonists chlorpromazine and calmidazolium (IC50 = 400 and 50 microM, respectively). The inhibition of enzyme activity by these agents correlated with their known potency and specificity as anti-CaM agents. The activity of the enzyme in the presence of 200 microM calmidazolium was restored by the addition of authentic CaM (EC50 = 15 microM); full activity was restored by the addition of 50 microM CaM. La3+, an ion that dissociates CaM from tightly bound CaM-enzyme systems, inhibited equine sperm adenylate cyclase (IC50 = 1 mM). Incubation of equine sperm adenylate cyclase with La3+ dissociated endogenous CaM from the enzyme so that most of the enzyme bound to a CaM-Sepharose column equilibrated with Ca2+. Specific elution of CaM-binding proteins from the CaM-Sepharose column with EGTA yielded a CaM-depleted adenylate cyclase fraction that was stimulated 2-fold by the addition of exogenous CaM.