Modelado dinámico y control de maniobras de dispositivos submarinos

Es bien conocido por todos que la Primera Revolución Industrial, que tuvo su inicio en la segunda mitad del Siglo XVIII, conllevó un aumento del uso de los recursos energéticos que no se ha detenido para llegar a los niveles de desarrollo tecnológico, industrial y de calidad de vida, de los que se dispone en la actualidad. A simple vista podría intuirse que para disponer de un mayor nivel tecnológico, industrial, de confort, etc. sea necesario un mayor consumo de energía primaria. La actual generación de energía está principalmente basada en el procesamiento de los diversos compuestos del carbono (hidrocarburos, gases y productos derivados del petróleo), que son contaminantes y además, se agotan. Desde hace unas pocas décadas, la humanidad ha sido consciente que es necesario generar energía a partir de fuentes de origen renovable, y que además resulten menos contaminantes. Así, en la actualidad, se ha llegado a un estado de desarrollo avanzado para la explotación de diversas fuentes de energías como la eólica, a la vez que se comienza a mirar con realismo la posibilidad de explotación de diversas energías de origen marino. Se considera que las energías renovables procedentes de los océanos que se encuentran más desarrolladas tecnológicamente hablando, sin tener en cuenta la energía eólica fuera costa (offshore), son la denominada energía undimotriz o de las olas y la energía de las corrientes marinas, no necesariamente en este orden. El trabajo propuesto en esta Tesis se centra en este último recurso energético y, aunque no se dispone todavía de ningún dispositivo en fase de explotación comercial, la concepción, diseño y desarrollo de dispositivos para la extracción de energía de las corrientes, y su evolución, han sido relativamente rápidos e importantes en estos últimos años. Existen ya diferentes dispositivos en fase de pruebas con resultados muy prometedores. Aunque los dispositivos actuales se encuentran limitados a la explotación energética en zonas de poca profundidad, los diferentes estudios del recurso indican la necesidad de explotar corrientes marinas a mayores profundidades, para lo que se están desarrollando actualmente dispositivos, cuya evolución en lo que a sistemas de fondeo se refiere, está siendo muy parecida a la que se ha producido en los parques eólicos fuera costa, similar a su vez, a la evolución llevada a cabo en las plataformas oceánicas para la explotación de recursos petrolíferos (denominados oil & gas) que se extraen de profundidades cada vez mayores. Las soluciones tecnológicas que resulten válidas han de ser también económicamente viables, y en la actualidad se requiere todavía reducir costos en todas las fases de instalación, explotación y mantenimiento de estos dispositivos, sea cual sea su profundidad de operación. Uno de los focos de estudio para abaratar los costes de explotación en general, pasa por abaratar y reducir los costes en las maniobras necesarias de inmersión (de la superficie del mar a la profundidad de operación) y emersión (de la profundidad de operación a la superficie del mar) de estos dispositivos, para llevar a cabo tareas de mantenimiento in situ, en el mar, y sin necesidad de buques especializados ni de su transporte a tierra. En esta Tesis se propone, en primer lugar, un método para evaluar el ciclo de vida de diversos dispositivos de aprovechamiento de las corrientes marinas. Se evidencia que el coste de la energía así generada sigue siendo no plenamente competitivo, por lo que se requiere avanzar en el abaratamiento de costes, principalmente en la instalación y en su mantenimiento. Para ello se propone como novedad principal, introducir sistemas de control en lazo cerrado para realizar maniobras de instalación y mantenimiento de forma automática. También se aporta un modelo dinámico original y muy sencillo para dispositivos bajo estos movimientos de emersión/inmersión, a partir del cual se han desarrollado los algoritmos de control para el propósito mencionado, que no es otro sino automatizar en todo lo posible las maniobras completas. Los algoritmos de control propuestos han sido validados mediante simulación. Se proponen trayectorias de referencia de movimiento suaves (smooth) similares a las utilizadas en robótica. Estos movimientos de cambios de profundidad en lazo cerrado, combinados con secuencias de movimientos en bucle abierto para cuando el dispositivo interacciona en la superficie libre, han dado lugar a nuevas maniobras completas de instalación y mantenimiento que se presentan en esta Tesis, diferentes a las actuales. Finalmente, y como justificación de la viabilidad económica del método novedoso aportado, se ha realizado un estudio comparativo de los costes de la tecnología propuesta, frente a la tecnología actual. Este nuevo sistema de maniobras automáticas implica un ciclo de vida diferente para los dispositivos de aprovechamiento de la energía de las corrientes, ciclo que se cuantifica a partir de un dispositivo base que ha sido modificado y adaptado para la nueva tecnología propuesta, demostrando su viabilidad tanto técnica como económica. ABSTRACT It’s well known that the First Industrial Revolution started in the second half of the eighteenth century, carried the increasing of the use of energy resource which have not been stopped until reach the present technology, industrial evolution and daily life quality. On the surface, it can be known intuitively that a higher consumption of primary energy resource is demanded for benefiting from a higher technological industrial and daily life level. Today, the generation of energy is mainly based in the processing of carbon products (hydrocarbons, gases and petroleum products) which are pollutants, and additionally, are depleted. From a few decades ago, the humanity is aware the energy should be obtained from renewable resources, which besides, should be cleaner. So, at the present, a technical develop has been gained to exploit several energy source, as wind energy, and, at the same time, the extraction of the marine energy starts to seem as a reality. The renewable marine energies considered more advanced and technically developed, without keeping in mind, the offshore wind energy, are the wave energy and the tidal current energy, not necessarily in that order. This Thesis is focused in this last energy resource, and, although, any device is under commercial operation, the concept, design and develop of this type of devices to extract the tidal current energy and their evolution has been comparatively fast and important the last years. There are several devices under test with promising results. Even through the current devices are limited to lower depth areas, the several studies of the tidal energy resource suggest the need to exploit the marine current at greater depths to what is being developed devices, where their evolution in the anchoring system is being very similar to the evolution performed in the offshore wind farms, which is at the same time, similar to the evolution in the oil and gas exploitation which are extracted to greatest depths. Viable technical solutions should be also viable economically and nowadays the cost in all phases of the project (installation, maintenance and operation) should be decreased whatever the operation depth is. One focus of study to lower the operation cost is the cost decreasing of immersion manoeuvring operations (from sea surface to the operation depth) and immersion manoeuvring operations (from operation depth to the sea surface), therefore the maintenance operations can be performed on – site, in the sea, and no specialized vessels are required to transport the devices from the sea to shore. In this dissertation, firstly is proposed a method to evaluate the life cycle of the tidal energy current devices. It is proved the energy generated by these devices is not fully competitive; therefore, the cost falling is mainly an objective in the installation and the maintenance operations. For that, it is proposed as main novelty, the using of closed loop control systems to perform the automatic installation and manoeuvring operations. It is also contributed with an original and simple dynamic model and for controlling the immersion/emersion movements of these devices, from which the control algorithms are developed in order to automate as much as possible the complete manoeuvring. The control algorithms proposed has been validated by simulations. Reference paths with smooth movements, similar which are used in robotics, are suggested. These movements to change the depth using closed loop control, combined with the sequences in open loop movements when the device is in free surface, have been development for a new complete manoeuvring to installation and maintenance operations which are advanced in this Thesis and they are different to the present manoeuvrings. Finally and as justification of the economic viability of this original method, a comparative cost study between the technology proposed and the current technology is performed. This new automatic manoeuvring system involves a different life cycle for the tidal energy current devices, cycle that is quantified from a base device which has been modified and adapted for the new proposed technology, showing the technical and economic viability.

Diseño y realización de una base de datos para el control de inventario del Museo Historia de la Informática

El objetivo principal de este proyecto es la realización de un sistema, que permita a través de alguna herramienta accesible a cualquier usuario, poder interactuar con una base de datos que contenga un catálogo bien documentado de los objetos disponibles en el Museo Histórico de la Informática (MHI) perteneciente a la Escuela Técnica Superior de Ingenieros Informáticos (ETSIINF) de la Universidad Politécnica de Madrid (UPM). Hasta el momento, no existía inventario alguno, por lo que la contribución del trabajo que aquí se presenta, supone un gran avance en la organización de los fondos del Museo. Ello contribuirá al desarrollo del objetivo principal del MHI, que es la difusión de la historia de la informática, mediante un medio de los más usados hoy en día, internet. El trabajo realizado se presenta a lo largo de 10 capítulos. En los que se muestran, el análisis del problema, los requisitos y las distintas alternativas posibles de solución, así como la solución adoptada y su desarrollo, tanto en el diseño de la base de datos como de sitio Web que hace posible la visualización e interacción de la información. En el primer capítulo, se puede encontrar una breve introducción del proyecto. Se indican los objetivos, la motivación y el alcance del mismo. En el segundo capítulo, se muestran los requisitos del problema, se analizan las tecnologías, herramientas y lenguajes disponibles para diseñar bases de datos, y se propone la elección de una de las tecnologías, teniendo en cuenta las limitaciones del entorno en el cual se va a implantar la solución. En el tercer capítulo, se diseña la solución propuesta para el sistema. Primero se muestra el diseño de bajo nivel, que serán los cimientos y posteriormente se explica el diseño de alto nivel. Finalmente, se introduce el conjunto de pruebas que el sistema tendrá que pasar para garantizar su correcto funcionamiento. El cuarto capítulo, muestra todas las tecnologías, herramientas, lenguajes y plantillas utilizadas para la implementación de la WEB. Mientras que en el capítulo cinco, se pueden ver los resultados de las pruebas realizadas. En el capítulo seis, se evalúan los costes económicos de realización de proyecto y se presenta la agenda de actividades y tareas llevadas a cabo para su desarrollo. El séptimo capítulo, resume las contribuciones técnicas del proyecto tratadas en los capítulos anteriores, así como las conclusiones personales. Mientras que, el capítulo ocho, apunta una serie de trabajos futuros que se podrían realizarse utilizando como base este proyecto. El capítulo nueve contiene las referencias de la información que se han consultado y que se citan en el texto, y el décimo complementa este proceso de información, incluyendo un glosario de términos técnicos. El contenido de la memoria concluye con el manual de usuario para la administración de la base de datos, que se incluye en forma de anexo.---ABSTRACT---The main goal of this project is the development of a system that would allow through some accessible tool for any user to interact with a database that contains a well-documented objects available in the Computer History Museum's (MHI) catalog, which belongs to the School of Computer Engineers (ETSIINF) of the Polytechnic University of Madrid (UPM). So far, there was no inventory, so the contribution of the work presented here, is a breakthrough in the organization of the Museum's collections. This will contribute to the development of the main goal of the MHI, which is the diffusion of computer history, by means of the most used today, internet. The work is presented along 10 chapters. Which show the analysis of the problem, requirements, the different possible solutions and the solution adopted and its development, both in the design of the database and Web site, which enables the visualization and interaction of the information. In the first chapter, a brief introduction of the project is found. Objectives, motivation and scope of the project are specified. In the second chapter, the requirements of the problem are shown. Technologies, tools and languages available to design databases are analysed, and the choice of a technology is proposed, taking into account the limitations of the environment in which it will to implement the solution. In the third chapter, the proposed system solution is designed. First, low-level design, which will be the foundation of the project, is shown, and then the high-level design is explained. Finally, test suite, which the system will have to past to ensure their proper functioning, are introduced. The fourth chapter shows all technologies, tools, languages and templates used to implement the WEB. While in chapter five, the results of the tests are shown. The economic costs of development the project are evaluated in chapter six, and the schedule of activities and tasks carried out for this development are shown. The seventh chapter summarizes the technical contributions of the project discussed in previous chapters, as well as personal conclusions. While the eighth chapter, suggests future works that could be made, based on this project. Ninth chapter contains references to information that have been consulted and cited in the text, and the tenth chapter includes a glossary of technical terms, to complement that process of information. Finally an annex includes a user manual for managing the database.

Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control

In fission yeast, the rad3 gene product plays a critical role in sensing DNA structure defects and activating damage response pathways. A structural homologue of rad3 in humans (ATR) has been identified based on sequence similarity in the protein kinase domain. General information regarding ATR expression, protein kinase activity, and cellular localization is known, but its function in human cells remains undetermined. In the current study, the ATR protein was examined by gel filtration of protein extracts and was found to exist predominantly as part of a large protein complex. A kinase-inactivated form of the ATR gene was prepared by site-directed mutagenesis and was used in transfection experiments to probe the function of this complex. Introduction of this kinase-dead ATR into a normal fibroblast cell line, an ATM-deficient fibroblast line derived from a patient with ataxia–telangiectasia, or a p53 mutant cell line all resulted in significant losses in cell viability. Clones expressing the kinase-dead ATR displayed increased sensitivity to x-rays and UV and a loss of checkpoint control. We conclude that ATR functions as a critical part of a protein complex that mediates responses to ionizing and UV radiation in human cells. These responses include effects on cell viability and cell cycle checkpoint control.

Use of phosphorous mapping and sea-level change data in assessing coastal activity zones and establishing site chronology : A case study from the Icelandic multi-period site of Vatnsfjörður

This paper forms part of Lukasz Mikolajczyk's PhD dissertation, which is supervised by Karen Milek

Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea

Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5′ upstream region of the grisea ORF. One mutation is a G → A transition at the 5′ splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA′-′lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Promoting detachment of neutrophils adherent to murine postcapillary venules to control inflammation: Effect of lipocortin 1

In this study we investigated, using intravital microscopy, how neutrophil extravasation across mouse mesenteric postcapillary venules is inhibited by the glucocorticoid-regulated protein lipocortin (LC; also termed annexin) 1. Intraperitoneal injection of 1 mg of zymosan into mice induced neutrophil rolling on the activated mesenteric endothelium followed by adhesion (maximal at 2 hr: 5–6 cells per 100-μm of vessel length) and emigration (maximal at 4 hr: 8–10 cells per high-powered field). Treatment of mice with human recombinant LC1 (2 mg/kg s.c.) or its mimetic peptide Ac2–26 (13 mg/kg s.c.) did not modify cell rolling but markedly reduced (≥50%) the degree of neutrophil adhesion and emigration (P < 0.05). Intravenous treatment with peptide Ac2–26 (13 mg/kg) or recombinant human LC1 (0.7–2 mg/kg) promoted detachment of neutrophils adherent to the endothelium 2 hr after zymosan administration, with adherent cells detaching within 4.12 ± 0.75 min and 2.36 ± 0.31 min, respectively (n = 20–25 cells). Recruitment of newly adherent cells to the endothelium was unaffected. The structurally related protein LC5 was inactive in this assay, whereas a chimeric molecule constructed from the N terminus of LC1 (49 aa) attached to the core region of LC5 produced cell detachment with kinetics similar to LC1. Removal of adherent neutrophils from activated postcapillary endothelium is a novel pharmacological action, and it is at this site where LC1 and its mimetics operate to down-regulate this aspect of the host inflammatory response.

The increased level of β1,4-galactosyltransferase required for lactose biosynthesis is achieved in part by translational control

β1,4-Galactosyltransferase (β4GalT-I) participates in both glycoconjugate biosynthesis (ubiquitous activity) and lactose biosynthesis (mammary gland-specific activity). In somatic tissues, transcription of the mammalian β4GalT-I gene results in a 4.1-kb mRNA and a 3.9-kb mRNA as a consequence of initiation at two start sites separated by ≈200 bp. In the mammary gland, coincident with the increased β4GalT-I enzyme level (≈50-fold) required for lactose biosynthesis, there is a switch from the 4.1-kb start site to the preferential use of the 3.9-kb start site, which is governed by a stronger tissue-restricted promoter. The use of the 3.9-kb start site results in a β4GalT-I transcript in which the 5′- untranslated region (UTR) has been truncated from ≈175 nt to ≈28 nt. The 5′-UTR of the 4.1-kb transcript [UTR(4.1)] is predicted to contain extensive secondary structure, a feature previously shown to reduce translational efficiency of an mRNA. In contrast, the 5′-UTR of the 3.9-kb mRNA [UTR(3.9)] lacks extensive secondary structure; thus, this transcript is predicted to be more efficiently translated relative to the 4.1-kb mRNA. To test this prediction, constructs were assembled in which the respective 5′-UTRs were fused to the luciferase-coding sequence and enzyme levels were determined after translation in vitro and in vivo. The luciferase mRNA containing the truncated UTR(3.9) was translated more efficiently both in vitro (≈14-fold) and in vivo (3- to 5-fold) relative to the luciferase mRNA containing the UTR(4.1). Consequently, in addition to control at the transcriptional level, β4GalT-I enzyme levels are further augmented in the lactating mammary gland as a result of translational control.

Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II

Griffonia simplicifolia leaf lectin II (GSII), a plant defense protein against certain insects, consists of an N-acetylglucosamine (GlcNAc)-binding large subunit with a small subunit having sequence homology to class III chitinases. Much of the insecticidal activity of GSII is attributable to the large lectin subunit, because bacterially expressed recombinant large subunit (rGSII) inhibited growth and development of the cowpea bruchid, Callosobruchus maculatus (F). Site-specific mutations were introduced into rGSII to generate proteins with altered GlcNAc binding, and the different rGSII proteins were evaluated for insecticidal activity when added to the diet of the cowpea bruchid. At pH 5.5, close to the physiological pH of the cowpea bruchid midgut lumen, rGSII recombinant proteins were categorized as having high (rGSII, rGSII-Y134F, and rGSII-N196D mutant proteins), low (rGSII-N136D), or no (rGSII-D88N, rGSII-Y134G, rGSII-Y134D, and rGSII-N136Q) GlcNAc-binding activity. Insecticidal activity of the recombinant proteins correlated with their GlcNAc-binding activity. Furthermore, insecticidal activity correlated with the resistance to proteolytic degradation by cowpea bruchid midgut extracts and with GlcNAc-specific binding to the insect digestive tract. Together, these results establish that insecticidal activity of GSII is functionally linked to carbohydrate binding, presumably to the midgut epithelium or the peritrophic matrix, and to biochemical stability of the protein to digestive proteolysis.

The AMPA receptor subunit GluR-B in its Q/R site-unedited form is not essential for brain development and function

Calcium permeability of l-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in excitatory neurons of the mammalian brain is prevented by coassembly of the GluR-B subunit, which carries an arginine (R) residue at a critical site of the channel pore. The codon for this arginine is created by site-selective adenosine deamination of an exonic glutamine (Q) codon at the pre-mRNA level. Thus, central neurons can potentially control the calcium permeability of AMPARs by the level of GluR-B gene expression as well as by the extent of Q/R-site editing, which in postnatal brain, positions the R codon into >99% of GluR-B mRNA. To study whether the small amount of unedited GluR-B is of functional relevance, we have generated mice carrying GluR-B alleles with an exonic arginine codon. We report that these mutants manifest no obvious deficiencies, indicating that AMPAR-mediated calcium influx into central neurons can be solely regulated by the levels of Q/R site-edited GluR-B relative to other AMPAR subunits. Notably, a targeted GluR-B gene mutant with 30% reduced GluR-B levels had 2-fold higher AMPAR-mediated calcium permeability in hippocampal pyramidal cells with no sign of cytotoxicity. This constitutes proof in vivo that elevated calcium influx through AMPARs need not generate pathophysiological consequences.

Control of alternative pre-mRNA splicing by distributed pentameric repeats

Multiple copies of the hexamer TGCATG have been shown to regulate fibronectin pre-mRNA alternative splicing. GCATG repeats also are clustered near the regulated calcitonin-specific 3′ splice site in the rat calcitonin/CGRP gene. Specific mutagenesis of these repeats in calcitonin/CGRP pre-mRNA resulted in the loss of calcitonin-specific splicing, suggesting that the native repeats act to enhance alternative exon inclusion. Mutation of subsets of these elements implies that alternative splicing requires a minimum of two repeats, and that the combination of one intronic and one exonic repeat is necessary for optimal cell-specific splicing. However, multimerized intronic repeats inhibited calcitonin-specific splicing in both the wild-type context and in a transcript lacking endogenous repeats. These results suggest that both the number and distribution of repeats may be important features for the regulation of tissue-specific alternative splicing. Further, RNA containing a single repeat bound cell-specific protein complexes, but tissue-specific differences in protein binding were not detected by using multimerized repeats. Together, these data support a novel model for alternative splicing regulation that requires the cell-specific recognition of multiple, distributed sequence elements.

Benthic–pelagic links and rocky intertidal communities: Bottom-up effects on top-down control?

Insight into the dependence of benthic communities on biological and physical processes in nearshore pelagic environments, long considered a “black box,” has eluded ecologists. In rocky intertidal communities at Oregon coastal sites 80 km apart, differences in abundance of sessile invertebrates, herbivores, carnivores, and macrophytes in the low zone were not readily explained by local scale differences in hydrodynamic or physical conditions (wave forces, surge flow, or air temperature during low tide). Field experiments employing predator and herbivore manipulations and prey transplants suggested top-down (predation, grazing) processes varied positively with bottom-up processes (growth of filter-feeders, prey recruitment), but the basis for these differences was unknown. Shore-based sampling revealed that between-site differences were associated with nearshore oceanographic conditions, including phytoplankton concentration and productivity, particulates, and water temperature during upwelling. Further, samples taken at 19 sites along 380 km of coastline suggested that the differences documented between two sites reflect broader scale gradients of phytoplankton concentration. Among several alternative explanations, a coastal hydrodynamics hypothesis, reflecting mesoscale (tens to hundreds of kilometers) variation in the interaction between offshore currents and winds and continental shelf bathymetry, was inferred to be the primary underlying cause. Satellite imagery and offshore chlorophyll-a samples are consistent with the postulated mechanism. Our results suggest that benthic community dynamics can be coupled to pelagic ecosystems by both trophic and transport linkages.

Spatio-temporally controlled site-specific somatic mutagenesis in the mouse

The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type will facilitate studies of gene function and the generation of animal models for human diseases. We have shown previously that conditional recombination–excision between two loxP sites can be achieved in mice by using the Cre recombinase fused to a mutated ligand binding domain of the human estrogen receptor (Cre-ERT), which binds tamoxifen but not estrogens. DNA excision was induced in a number of tissues after administration of tamoxifen to transgenic mice expressing Cre-ERT under the control of the cytomegalovirus promoter. However, the efficiency of excision varied between tissues, and the highest level (≈40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ERT in a given cell type, we have now crossed Cre-ERT-expressing mice with reporter mice in which expression of Escherichia coli β-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. We show that site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ERT. These results indicate that cell-specific expression of Cre-ERT in transgenic mice can be used for efficient tamoxifen-dependent, Cre-mediated recombination at loci containing loxP sites to generate site-specific somatic mutations in a spatio-temporally controlled manner.

Mapping of a Myosin-binding Domain and a Regulatory Phosphorylation Site in M-Protein, a Structural Protein of the Sarcomeric M Band

The myofibrils of cross-striated muscle fibers contain in their M bands cytoskeletal proteins whose main function seems to be the stabilization of the three-dimensional arrangement of thick filaments. We identified two immunoglobin domains (Mp2–Mp3) of M-protein as a site binding to the central region of light meromyosin. This binding is regulated in vitro by phosphorylation of a single serine residue (Ser76) in the immediately adjacent amino-terminal domain Mp1. M-protein phosphorylation by cAMP-dependent kinase A inhibits binding to myosin LMM. Transient transfection studies of cultured cells revealed that the myosin-binding site seems involved in the targeting of M-protein to its location in the myofibril. Using the same method, a second myofibril-binding site was uncovered in domains Mp9–Mp13. These results support the view that specific phosphorylation events could be also important for the control of sarcomeric M band formation and remodeling.

Recognition of Yeast mRNAs as “Nonsense Containing” Leads to Both Inhibition of mRNA Translation and mRNA Degradation: Implications for the Control of mRNA Decapping

A critical step in the degradation of many eukaryotic mRNAs is a decapping reaction that exposes the transcript to 5′ to 3′ exonucleolytic degradation. The dual role of the cap structure as a target of mRNA degradation and as the site of assembly of translation initiation factors has led to the hypothesis that the rate of decapping would be specified by the status of the cap binding complex. This model makes the prediction that signals that promote mRNA decapping should also alter translation. To test this hypothesis, we examined the decapping triggered by premature termination codons to determine whether there is a down-regulation of translation when mRNAs were recognized as “nonsense containing.” We constructed an mRNA containing a premature stop codon in which we could measure the levels of both the mRNA and the polypeptide encoded upstream of the premature stop codon. Using this system, we analyzed the effects of premature stop codons on the levels of protein being produced per mRNA. In addition, by using alterations either in cis or in trans that inactivate different steps in the recognition and degradation of nonsense-containing mRNAs, we demonstrated that the recognition of a nonsense codon led to a decrease in the translational efficiency of the mRNA. These observations argue that the signal from a premature termination codon impinges on the translation machinery and suggest that decapping is a consequence of the change in translational status of the mRNA.