RESUMEN

One of the most dominant influences in the patterning of multicellular embryos is exerted by the Hedgehog (Hh) family of secreted signaling proteins. Here, we identify a segment polarity gene in Drosophila melanogaster, skinny hedgehog (ski), and show that its product is required in Hh-expressing cells for production of appropriate signaling activity in embryos and in the imaginal precursors of adult tissues. The ski gene encodes an apparent acyltransferase, and we provide genetic and biochemical evidence that Hh proteins from ski mutant cells retain carboxyl-terminal cholesterol modification but lack amino-terminal palmitate modification. Our results suggest that ski encodes an enzyme that acts within the secretory pathway to catalyze amino-terminal palmitoylation of Hh, and further demonstrate that this lipid modification is required for the embryonic and larval patterning activities of the Hh signal.

Asunto(s)

Aciltransferasas/genética , Aciltransferasas/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas de Insectos/metabolismo , Ácido Palmítico/metabolismo , Transducción de Señal , Acilación , Aciltransferasas/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Tipificación del Cuerpo , Colesterol/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Expresión Génica , Genes de Insecto , Proteínas Hedgehog , Proteínas de Insectos/química , Proteínas de Insectos/genética , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Transgenes

RESUMEN

Mathematical descriptors, coupled with experimental observations, are used to quantify differential uptake of an experimental herbicide in Japonica and Indica rice (Oryza sativa, non-target) and barnyardgrass (Echinochloa crus-galli, target). Partitioning, degradation, plant uptake and metabolism are described using mass-balance conservation equations in the form of kinetic approximations. Estimated environmental concentrations, governed by the pesticide formulation, are described using superimposed analytical solutions for the one-dimensional diffusion equation in spherical coordinates and by a finite difference representation of the two-dimensional diffusion equation in Cartesian coordinates. Formulation attributes from granules include active ingredient release rates, particle sizes, pesticide loading, and granule spacing. The diffusion model for pesticide transport is coupled with the compartment model to follow the fate and transport of a pesticide from its initial application location to various environmental matrices of interest. Formulation effects, partitioning and degradation in the various environmental matrices, differential plant uptake and metabolism, and dose-response information for plants are accounted for. This novel model provides a mechanism for selecting formulation delivery systems that optimize specific attributes (such as weed control or the therapeutic index) for risk-assessment procedures. In this report we describe how this methodology was used to explore the factors affecting herbicide efficacy and to define an optimal release rate for a granule formulation.

Asunto(s)

Herbicidas/química , Oryza/efectos de los fármacos , Poaceae/efectos de los fármacos , Algoritmos , Transporte Biológico , Química Agrícola , Química Farmacéutica , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Análisis de Fourier , Herbicidas/metabolismo , Herbicidas/toxicidad , Cinética , Modelos Biológicos , Oryza/metabolismo , Tamaño de la Partícula , Hojas de la Planta/metabolismo , Poaceae/metabolismo , Medición de Riesgo , Distribución Tisular , Agua/metabolismo

RESUMEN

The total synthesis of the epidermal growth factor inhibitor reveromycin B (2) in 25 linear steps from chiral methylene pyran 13 is described. The key steps involved an inverse electron demand hetero-Diels-Alder reaction between dienophile 13 and diene 12 to construct the 6,6-spiroketal 11 which upon oxidation with dimethyldioxirane and acid catalyzed rearrangement gave the 5,6-spiroketal aldehyde 9. Lithium acetylide addition followed by oxidation/reduction and protective group manipulation provided the reveromycin B spiroketal core 8 which was converted into the reveromycin A (1) derivative 6 in order to confirm the stereochemistry of the spiroketal segment. Introduction of the C1-C10 side chain began with sequential Wittig reactions to form the C8-C9 and C7-C6 bonds, and a tin mediated asymmetric aldol reaction installed the C4 and C5 stereocenters. The final key steps to the target molecule 2 involved a Stille coupling to introduce the C21-C22 bond, succinoylation, selective deprotection, oxidation, and Wittig condensation to form the final C2-C3 bond. Deprotection was effected by TBAF in DMF to afford reveromycin B (2) in 72% yield.

Asunto(s)

Antibióticos Antineoplásicos/síntesis química , Piranos/síntesis química , Compuestos de Espiro/síntesis química , Antibióticos Antineoplásicos/farmacología , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Piranos/farmacología , Compuestos de Espiro/farmacología , Estereoisomerismo

RESUMEN

The demonstration over 30 years ago that inhibitors of cholesterol biosynthesis disrupt animal development suggested an intriguing connection between fundamental cellular metabolic processes and the more global processes of embryonic tissue patterning. Adding a new dimension to this relationship is the more recent finding that the Hedgehog family of tissue patterning factors are covalently modified by cholesterol. Here we review the mechanism of the Hedgehog autoprocessing reaction that results in this modification, and compare this reaction to that undergone by other autoprocessing proteins. We also discuss the biological consequences of cholesterol modification, in particular the use of cholesterol as a molecular handle in the spatial deployment of the protein signal in developing tissues. Finally, the developmental consequences of chemical and genetic disruption of cholesterol homeostasis are summarized, along with the potential importance of cholesterol-rich lipid rafts in production of and response to the Hh signal.

Asunto(s)

Colesterol/química , Colesterol/metabolismo , Proteínas/química , ATPasas de Translocación de Protón , Proteínas de Saccharomyces cerevisiae , Transactivadores , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Drosophila , Inducción Embrionaria , Endodesoxirribonucleasas/química , Proteínas Hedgehog , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Modelos Químicos , Datos de Secuencia Molecular , Nematodos , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Transducción de Señal , Teratógenos/farmacología , Alcaloides de Veratrum/farmacología

RESUMEN

Basal cell carcinoma, medulloblastoma, rhabdomyosarcoma and other human tumours are associated with mutations that activate the proto-oncogene Smoothened (SMO) or that inactivate the tumour suppressor Patched (PTCH). Smoothened and Patched mediate the cellular response to the Hedgehog (Hh) secreted protein signal, and oncogenic mutations affecting these proteins cause excess activity of the Hh response pathway. Here we show that the plant-derived teratogen cyclopamine, which inhibits the Hh response, is a potential 'mechanism-based' therapeutic agent for treatment of these tumours. We show that cyclopamine or synthetic derivatives with improved potency block activation of the Hh response pathway and abnormal cell growth associated with both types of oncogenic mutation. Our results also indicate that cyclopamine may act by influencing the balance between active and inactive forms of Smoothened.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Proteínas de Drosophila , Proteínas de la Membrana/genética , Proteínas/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Receptores Acoplados a Proteínas G , Transducción de Señal/efectos de los fármacos , Transactivadores , Alcaloides de Veratrum/farmacología , Células 3T3 , Animales , Síndrome del Nevo Basocelular/tratamiento farmacológico , Síndrome del Nevo Basocelular/genética , Síndrome del Nevo Basocelular/metabolismo , Línea Celular , Transformación Celular Neoplásica/efectos de los fármacos , Clonación Molecular , Drosophila , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Hedgehog , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/metabolismo , Ratones , Mutación , Oncogenes , Receptores Patched , Receptor Patched-1 , Proteínas/metabolismo , Proto-Oncogenes Mas , Receptores de Superficie Celular/metabolismo , Receptor Smoothened , Alcaloides de Veratrum/química

RESUMEN

[structure: see text] The total synthesis of the epidermal growth factor inhibitor reveromycin B (2) is described. A novel, convergent, and stereoselective reaction sequence was utilized to construct the 5,6-spiroketal system 10 which was converted into the natural product 2 by a 16-step sequence.

Asunto(s)

Antineoplásicos/síntesis química , Piranos/síntesis química , Compuestos de Espiro/síntesis química , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Estereoisomerismo

RESUMEN

Nucleosomes prevent the recognition of TATA promoter elements by the basal transcriptional machinery in the absence of induction. However, while Saccharomyces cerevisiae histones H3 and H4 contain N-terminal regions involved in the activation and repression of GAL1 and in the expression of heterochromatin-like regions, the sequences involved in repressing basal transcription have not yet been identified. Here, we describe the mapping of new N-terminal domains, in all four core histones (H2A, H2B, H3 and H4), required for the repression of basal, uninduced transcription. Basal transcription was monitored by the use of a GAL1 promoter-URA3 reporter construct whose uninduced activity can be detected through cellular sensitivity to the drug, 5-fluoroorotic acid. We have found for each histone that the N-terminal sequences repressing basal activity are in a short region adjacent to the structured alpha-helical core. Analysis of minichromosome DNA topology demonstrates that the basal domains are required for the proper folding of DNA around the chromosomal particle. Deletion of the basal domain at each histone significantly decreases plasmid superhelical density, which probably reflects a release of DNA from the constraints of the nucleosome into the linker region. This provides a means by which basal factors may recognize otherwise repressed regulatory elements.

Asunto(s)

Regulación Fúngica de la Expresión Génica , Histonas/genética , Saccharomyces cerevisiae/genética , Transcripción Genética , Acetilación , Secuencia de Bases , Cromatina/ultraestructura , Genes Reporteros , Datos de Secuencia Molecular , Nucleosomas/metabolismo , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Eliminación de Secuencia , TATA Box , Telómero/genética

RESUMEN

Effect of 1,25,50 and 75 nM of okadaic acid (OA) on human lymphocytes from healthy individuals in culture has been investigated. To our surprise, we observed induction of significantly high sister chromatid exchanges (SCEs) at concentrations known to inhibit both protein phosphatase-1 (PP-1) and protein phosphatase -2A (PP-2A). However, 1 nM okadaic acid, known to inhibit PP-2A alone, did not induce this cellular feature/phenotype. This novel preliminary observation lays foundation for investigating further the role of PP-1 inhibition in governing as yet unknown finer controls in the induction of high SCEs, the mechanism for which has eluded answers till date.

Asunto(s)

Inhibidores Enzimáticos/farmacología , Éteres Cíclicos/farmacología , Linfocitos/efectos de los fármacos , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Intercambio de Cromátides Hermanas/efectos de los fármacos , Células Cultivadas , Humanos , Ácido Ocadaico , Proteína Fosfatasa 1 , Proteína Fosfatasa 2 , Valores de Referencia

RESUMEN

Previous work has shown that N-terminal deletions of yeast histone H3 cause a 2- to 4-fold increase in the induction of GAL1 and a number of other genes involved in galactose metabolism. In contrast, deletions at the H4 N terminus cause a 10- to 20-fold decrease in the induction of these same GAL genes. However, H3 and H4 N-terminal deletions each decrease PHO5 induction only 2- to 4-fold. To define the GAL1 gene regulatory elements through which the histone N termini activate or repress transcription, fusions were made between GAL1 and PHO5 promoter elements attached to a beta-galactosidase reporter gene. We show here that GAL1 hyperactivation caused by the H3 N-terminal deletion delta 4-15 is linked to the upstream activation sequence. Conversely, the relative decrease in GAL1 induction caused by the H4N-terminal deletion delta 4-28 is linked to the downstream promoter which contains the TATA element. These data indicate that the H3 N terminus is required for the repression of the GAL1 upstream element, whereas the H4N terminus is required for the activation of the GAL1 downstream promoter element.

Asunto(s)

Histonas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Saccharomyces cerevisiae/genética , Transcripción Genética , Secuencia de Bases , Cartilla de ADN , Galactoquinasa/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Eliminación de Secuencia , TATA Box

RESUMEN

Yeast chromosomes may lack the linker histone H1 (normally required to compact 10 nm beads-on-a-string fiber into the 30 nm fiber) and there is no cytological evidence for higher order fiber structure but they do contain regions which correspond to euchromatin and heterochromatin of higher eukaryotes. Both euchromatin and heterochromatin contain nucleosomal particles (composed of two molecules each of H2A, H2B, H3 and H4), however histones have been shown to regulate genes in these regions in quite different ways. The mechanisms by which such regulation occurs are the topic of this paper.

Asunto(s)

Cromatina/fisiología , Regulación Fúngica de la Expresión Génica/genética , Heterocromatina/fisiología , Histonas/fisiología , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Eucromatina , Datos de Secuencia Molecular

RESUMEN

Recent work has shown that the yeast histone H4 N-terminus, while not essential for viability, is required for repression of the silent mating loci and activation of GAL1 and PHO5 promoters. Because histone H3 shares many structural features with histone H4 and is intimately associated with H4 in the assembled nucleosome, we asked whether H3 has similar functions. While the basic N-terminal domain of H3 is found to be non-essential (deletion of residues 4-40 of this 135 amino acid protein allows viability), its removal has only a minor effect on mating. Surprisingly, both deletions (of residues 4-15) and acetylation site substitutions (at residues 9, 14 and 18) within the N-terminus of H3 allow hyperactivation of the GAL1 promoter as well as a number of other GAL4-regulated genes including GAL2, GAL7 and GAL10. To a limited extent glucose repression is also alleviated by H3 N-terminal deletions. Expression of another inducible promoter, PHO5, is shown to be relatively unaffected. We conclude that the H3 and H4 N-termini have different functions in both the repression of the silent mating loci and in the regulation of GAL1.

Asunto(s)

Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Histonas/genética , Regiones Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción , Secuencia de Aminoácidos , División Celular/genética , Análisis Mutacional de ADN , Proteínas de Unión al ADN , Inducción Enzimática/efectos de los fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Galactosa/farmacología , Glucosa/farmacología , Datos de Secuencia Molecular , Mutagénesis , Nucleosomas/metabolismo , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Represoras/genética , beta-Galactosidasa/genética

RESUMEN

We have previously shown that nucleosome loss, obtained by repressing histone H4 mRNA synthesis, activates otherwise inactive PHO5, GAL1, and CYC1 gene promoters (fused to the bacterial beta-galactosidase [lacZ] reporter gene) to moderate levels of activity (approximately 2 to 15% of fully induced levels). We now report that nucleosome loss activates the expression of two additional promoters that are normally induced by independent mechanisms: CUP1 (induced by heavy-metal toxicity) and HIS3 (induced by amino acid starvation). Surprisingly, the level of CUP1-lacZ and HIS3-lacZ activation by nucleosome loss approximates fully induced levels of transcription. These CUP1 and HIS3 promoter activities are increased similarly from either episomal or genomic constructs. Our results emphasize the universality of the mechanism by which nucleosome loss activates yeast promoters. Moreover, a comparison of absolute levels of activation for different promoters suggests that activation by nucleosome loss results in a relatively constant level of activation, while levels obtained by normal induction vary considerably. These data argue that nucleosome loss may play a uniquely dominant role in the regulation of certain promoters.

Asunto(s)

Regulación Fúngica de la Expresión Génica , Nucleosomas , Regiones Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Aminoácidos/metabolismo , Inducción Enzimática , Histonas , Metales/metabolismo , Metales/toxicidad , Biosíntesis de Proteínas , Proteínas Recombinantes , Transcripción Genética

RESUMEN

To search for histone domains that may regulate transcription in vivo, we made deletions and amino acid substitutions in the histone N-termini of S. cerevisiae. Histone H4 N-terminal residues 4-23, which include the extremely conserved, reversibly acetylated lysines (at positions 5, 8, 12, and 16), were found to encompass a region required for the activation of the GAL1 promoter. Deletions in the H4 N-terminus reduce GAL1 activation 20-fold. This effect is specific to histone H4 in that large deletions in the N-termini of H2A, H2B, and H3 do not similarly decrease induction. Activation of the PHO5 promoter is reduced approximately 4- to 5-fold by these H4 deletions. Mutations in histone H4 acetylation sites and surrounding residues can cause comparable and, in some cases, even greater effects on induction of these two promoters. We postulate that the H4 N-terminus may interact with a component of the transcription initiation complex, allowing nucleosome unfolding and subsequent initiation.

Asunto(s)

Regulación Fúngica de la Expresión Génica , Histonas/fisiología , Nucleosomas/fisiología , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Transcripción Genética , Acetilación , Secuencia de Aminoácidos , Proteínas Fúngicas/genética , Histonas/química , Datos de Secuencia Molecular , Nucleosomas/ultraestructura , ARN de Hongos/biosíntesis , Relación Estructura-Actividad