Workflow-centric research objects: First class citizens in scholarly discourse.

A workflow-centric research object bundles a workflow, the provenance of the results obtained by its enactment, other digital objects that are relevant for the experiment (papers, datasets, etc.), and annotations that semantically describe all these objects. In this paper, we propose a model to specify workflow-centric research objects, and show how the model can be grounded using semantic technologies and existing vocabularies, in particular the Object Reuse and Exchange (ORE) model and the Annotation Ontology (AO).We describe the life-cycle of a research object, which resembles the life-cycle of a scienti?c experiment.

Improved heuristic drift elimination with magnetically-aided dominant directions (MiHDE) for pedestrian navigation in complex buildings

The main problem of pedestrian dead-reckoning (PDR) using only a body-attached inertial measurement unit is the accumulation of heading errors. The heading provided by magnetometers in indoor buildings is in general not reliable and therefore it is commonly not used. Recently, a new method was proposed called heuristic drift elimination (HDE) that minimises the heading error when navigating in buildings. It assumes that the majority of buildings have their corridors parallel to each other, or they intersect at right angles, and consequently most of the time the person walks along a straight path with a heading constrained to one of the four possible directions. In this article we study the performance of HDE-based methods in complex buildings, i.e. with pathways also oriented at 45°, long curved corridors, and wide areas where non-oriented motion is possible. We explain how the performance of the original HDE method can be deteriorated in complex buildings, and also, how severe errors can appear in the case of false matches with the building's dominant directions. Although magnetic compassing indoors has a chaotic behaviour, in this article we analyse large data-sets in order to study the potential use that magnetic compassing has to estimate the absolute yaw angle of a walking person. Apart from these analysis, this article also proposes an improved HDE method called Magnetically-aided Improved Heuristic Drift Elimination (MiHDE), that is implemented over a PDR framework that uses foot-mounted inertial navigation with an extended Kalman filter (EKF). The EKF is fed with the MiHDE-estimated orientation error, gyro bias corrections, as well as the confidence over that corrections. We experimentally evaluated the performance of the proposed MiHDE-based PDR method, comparing it with the original HDE implementation. Results show that both methods perform very well in ideal orthogonal narrow-corridor buildings, and MiHDE outperforms HDE for non-ideal trajectories (e.g. curved paths) and also makes it robust against potential false dominant direction matchings.

Mobile devices: dominant design as a goal

Since the beginning of the smartphones in the 80s, the mobile device market has grown and evolved towards devices connected everywhere, with hardware more and more close to computers and laptops than a classic mobile telephone. Nowadays, this market seems to be crowded and some companies seem not to know exactly which step is next. In this manner, a concept appears in the market as a solution or a difficulty to overcome: the dominant design. The thesis aims to establish an analysis and definition of what a dominant design is and how we should understand this concept: which are the costumers’ demands and needs? How can we relate this information with the dominant design? What is the strategy of the firm before designing a device? Do they use a concept similar to a dominant design?. The research base its analysis in a theoretical framework based in innovation and marketing literature, to then compare the model studied with data collected from surveys made to customers, interviews made to workers of the mobile device market, and different new projects on the market. The research finishes with a discussion about the theoretical and the empirical frameworks, and concludes replying the research questions, and defining a dominant design and its current situation in the market. RESUMEN. Desde la aparición de los Smartphones en los años 80, el mercado de los dispositivos móviles ha crecido y evolucionado hacia dispositivos cada vez más conectados, con hardware cada vez más cercano a los ordenadores de sobremesa y portátiles que al clásico teléfono móvil. A día de hoy, el mercado está saturado y algunas compañías parecen dubitativas ante el próximo paso a seguir. De esta manera, el concepto del diseño dominante aparece en el mercado como una solución a esta dificultad. El primer capítulo de este estudio se centra en establecer, a modo de introducción, los antecedentes al caso a estudiar, el objetivo de la tesis con sus limitaciones y delimitaciones, así como la metodología utilizada. También se plantean las preguntas principales (Research Questions) sobre el objetivo de la tesis, las cuales darán respuesta en la conclusión final al caso de estudio. Este proyecto tiene como objetivo establecer un análisis y definición sobre que es un diseño dominante y qué deberíamos entender como tal: ¿cuáles son las necesidades y las exigencias de los clientes? ¿Cómo se puede relacionar esta información con el diseño dominante en el sector tecnológico? ¿Cuáles son las estrategias de las empresas antes de diseñar un nuevo dispositivo? ¿Usan un concepto o modelos similares a un diseño dominante? Posteriormente, el segundo capítulo expone la bibliografía utilizada, y el enfoque analítico que se llevará a cabo con las 3 principales fuentes de datos. La investigación enfoca su análisis en un marco teórico, basado en publicaciones y bibliografía relacionadas con la innovación y el marketing, para luego comparar el modelo estudiado con un marco empírico: datos obtenidos de encuestas a consumidores, entrevistas a profesionales del sector de los dispositivos móviles, y diferentes prototipos y nuevos proyectos en este mercado. Entre esta literatura se encuentran manuales de marketing como “22 Immutable laws of Marketing” (de Al Ries & Jack Trout), publicaciones sobre el sector industrial de la tecnología y negocios: “Crossing the Chasm” de Geoffrey A. Moore y modelos de innovación entre otros como “Mastering the Dynamics of Innovation” de James M. Utterback. El tercer capítulo corresponde al estudio del marco teórico de la tesis, donde se analizará principalmente el modelo de innovación utilizado (el modelo cíclico de Utterback) y varios principios de marketing aplicados a este sector. Se plantean las bases de este modelo, la definición que el propio Utterback ofrece sobre el diseño dominante, y las 3 fases del proceso del mismo (Fluid Phase, Transitional Phase y Specific Phase), donde las empresas cambian de estrategia según las circunstancias evolutivas del dispositivo, su posición respecto el líder del mercado, o los procesos de estandarización y de costes. Por último se plantea la base para el desarrollo del diseño dominante en un ciclo evolutivo constante en el tiempo. Respecto a la parte más analítica de la tesis, el cuarto capítulo se desarrolla a partir de los datos obtenidos de las fuentes de información en el marco empírico de estudio. Se obtienen conclusiones sobre los datos realizados en ambas encuestas (en Español e Inglés) y sobre la relevancia de esta información; se estudian uno por uno hasta cuatro casos de nuevos dispositivos a corto-medio plazo en el mercado y se obtienen unas conclusiones globales sobre las entrevistas realizadas a los profesionales del sector y la relevancia de todas estas informaciones. En el quinto capítulo de la tesis se desarrolla la discusión en torno a los marcos teórico y empírico utilizados, para concluir respondiendo a las “Research Questions”, definiendo de esta manera el concepto de diseño dominante y comparando esta definición con la situación real del mercado. Se contrastan las bases del modelo de Utterback con los datos obtenidos en el capítulo cuarto, enfatizando la comparación entre las fases de este modelo con la realidad obtenida a través del estudio. Las encuestas realizadas a los consumidores se enmarcan en la segunda y tercera fase del ciclo, donde el desarrollo del diseño dominante ya está establecido y más desarrollado, mientras que las entrevistas unifican varios puntos clave a tener en cuenta en la primera y segunda fases, orientándose a las capas previas del proceso. Después se comparan uno a uno los 4 dispositivos analizados, a fin de establecer su jerarquía dentro del mercado, como posibles nuevos diseños dominantes o evoluciones especializadas de otros que ya aparecieron en el mercado con anterioridad. Así mismo, en esta parte final del estudio se comparan entre sí los resultados similares entre las tres fuentes de datos, y se analiza la veracidad de todas las fuentes consultadas. Finalmente, se han registrado en un sexto capítulo todas las referencias utilizadas en este proyecto, tanto publicaciones bibliográficas, entrevistas, citas de personajes relevantes del sector y enlaces en la red sobre noticias relevantes. En el apartado de apéndices se adjuntan tres anexos, donde se adjunta información utilizada en el caso de estudio, y la cual se ha obviado del texto principal con el objetivo de agilizar la lectura y la comprensión del mismo. Estos tres apéndices corresponden a las dos encuestas realizadas en ambos idiomas y la entrevista realizada a los profesionales del sector de los dispositivos móviles.

A Comparative Analysis of Hedging in a Corpus of Two Written Legal Discourse Genres

Esta tesis doctoral, que es la culminación de mis estudios de doctorado impartidos por el Departamento de Lingüística Aplicada a la Ciencia y a la Tecnología de la Universidad Politécnica de Madrid, aborda el análisis del uso de la matización (hedging) en el lenguaje legal inglés siguiendo los postulados y principios de la análisis crítica de género (Bhatia, 2004) y empleando las herramientas de análisis de córpora WordSmith Tools versión 6 (Scott, 2014). Como refleja el título, el estudio se centra en la descripción y en el análisis contrastivo de las variedades léxico-sintácticas de los matizadores del discurso (hedges) y las estrategias discursivas que con ellos se llevan a cabo, además de las funciones que éstas desempeñan en un corpus de sentencias del Tribunal Supremo de EE. UU., y de artículos jurídicos de investigación americanos, relacionando, en la medida posible, éstas con los rasgos determinantes de los dos géneros, desde una perspectiva socio-cognitiva. El elemento innovador que ofrece es que, a pesar de los numerosos estudios que se han podido realizar sobre los matizadores del discurso en el inglés general (Lakoff, 1973; Hübler, 1983; Clemen, 1997; Markkanen and Schröder, 1997; Mauranen, 1997; Fetzer 2010; y Finnegan, 2010 entre otros) académico (Crompton, 1997; Meyer, 1997; Skelton, 1997; Martín Butragueňo, 2003) científico (Hyland, 1996a, 1996c, 1998c, 2007; Grabe and Kaplan, 1997; Salager-Meyer, 1997 Varttala, 2001) médico (Prince, 1982; Salager-Meyer, 1994; Skelton, 1997), y, en menor medida el inglés legal (Toska, 2012), no existe ningún tipo de investigación que vincule los distintos usos de la matización a las características genéricas de las comunicaciones profesionales. Dentro del lenguaje legal, la matización confirma su dependencia tanto de las expectativas a macro-nivel de la comunidad de discurso, como de las intenciones a micro-nivel del escritor de la comunicación, variando en función de los propósitos comunicativos del género ya sean éstos educativos, pedagógicos, interpersonales u operativos. El estudio pone de relieve el uso predominante de los verbos modales epistémicos y de los verbos léxicos como matizadores del discurso, estos últimos divididos en cuatro tipos (Hyland 1998c; Palmer 1986, 1990, 2001) especulativos, citativos, deductivos y sensoriales. La realización léxico-sintáctica del matizador puede señalar una de cuatro estrategias discursivas particulares (Namsaraev, 1997; Salager-Meyer, 1994), la indeterminación, la despersonalización, la subjectivisación, o la matización camuflada (camouflage hedging), cuya incidencia y función varia según género. La identificación y cuantificación de los distintos matizadores y estrategias empleados en los diferentes géneros del discurso legal puede tener implicaciones pedagógicos para los estudiantes de derecho no nativos que tienen que demostrar una competencia adecuada en su uso y procesamiento. ABSTRACT This doctoral thesis, which represents the culmination of my doctoral studies undertaken in the Department of Linguistics Applied to Science and Technology of the Universidad Politécnica de Madrid, focusses on the analysis of hedging in legal English following the principles of Critical Genre Analysis (Bhatia, 2004), and using WordSmith Tools version 6 (Scott, 2014) corpus analysis tools. As the title suggests, this study centers on the description and contrastive analysis of lexico-grammatical realizations of hedges and the discourse strategies which they can indicate, as well as the functions they can carry out, in a corpus of U.S. Supreme Court opinions and American law review articles. The study relates realization, incidence and function of hedging to the predominant generic characteristics of the two genres from a socio-cognitive perspective. While there have been numerous studies on hedging in general English (Lakoff, 1973; Hübler, 1983; Clemen, 1997; Markkanen and Schröder, 1997; Mauranen, 1997; Fetzer 2010; and Finnegan, 2010 among others) academic English (Crompton, 1997; Meyer, 1997; Skelton, 1997; Martín Butragueňo, 2003) scientific English (Hyland, 1996a, 1996c, 1998c, 2007; Grabe and Kaplan, 1997; Salager-Meyer, 1997 Varttala, 2001) medical English (Prince, 1982; Salager-Meyer, 1994; Skelton, 1997), and, to a lesser degree, legal English (Toska, 2012), this study is innovative in that it links the different realizations and functions of hedging to the generic characteristics of a particular professional communication. Within legal English, hedging has been found to depend on not only the macro-level expectations of the discourse community for a specific genre, but also on the micro-level intentions of the author of a communication, varying according to the educational, pedagogical, interpersonal or operative purposes the genre may have. The study highlights the predominance of epistemic modal verbs and lexical verbs as hedges, dividing the latter into four types (Hyland, 1998c; Palmer, 1986, 1990, 2001): speculative, quotative, deductive and sensorial. Lexical-grammatical realizations of hedges can signal one of four discourse strategies (Namsaraev, 1997; Salager-Meyer, 1994), indetermination, depersonalization, subjectivization and camouflage hedging, as well as fulfill a variety of functions. The identification and quantification of the different hedges and hedging strategies and functions in the two genres may have pedagogical implications for non-native law students who must demonstrate adequate competence in the production and interpretation of hedged discourse.

El tiempo como sustancia de la forma : una aproximación al Museo de Arte Romano de Mérida desde los presupuestos del vitalismo

El modelo dominante durante la Era Moderna asume la presencia del hombre como sujeto dentro del gran engranaje mecánico del Cosmos. Asimismo, recoge una idea acerca del ser natural dentro de la tradición ontológica iniciada por el eleatismo presocrático que concibe a éste como lo inmutable y estático frente al cambio y al movimiento, los cuales se constituyen como meras apariencias. Durante el periodo anterior a la aparición de los grandes filósofos griegos se produce una transformación en donde, de la inicial cosmología vinculada a un tiempo primordial, se pasará a una visión del Universo como ente indestructible, atemporal, inmutable, perfecto, geométrico y espacial. Si en Demócrito se admite un universo sometido al azar y a la necesidad, en Platón el Universo sólo atiende a la necesidad. Este modelo ontológico se pone en entredicho cuando el hombre ya no es concebido como pieza de un sistema más amplio, sino como centro radical del pensamiento. La condición radical del hombre es entonces su propia vida, siendo éste el concepto troncal del denominado vitalismo cuyo más influyente representante en España es José Ortega y Gasset. El estatismo del ser –del hombre- pierde sentido; en palabras del propio Ortega, “no es un ser sino un estar siendo” lo que caracteriza a la vida humana. La razón cartesiana es ahora la razón vital y su objeto de estudio no es la naturaleza sino el propio devenir, es decir, el tiempo, la historia. Este planteamiento es fundamental para comprender el edificio que es objeto de este estudio, el Museo de Arte Romano de Mérida (1980-1985) de Rafael Moneo. Por ello el concepto de tiempo es utilizado como marco y estructura de la presente tesis, a sabiendas del notorio y muy significativo papel que este edificio desempeñó en la carrera de su autor y en el panorama nacional e internacional de la arquitectura y de la museología. Este proyecto nos permite acercarnos al pensamiento de su autor a través de un edificio que, aun habiendo sido ampliamente reconocido, no cuenta con un estudio suficientemente exhaustivo que recoja la amplitud y riqueza que encierra. Esta tesis no es un compendio de lo que ya se ha investigado sobre Mérida; es una aproximación global e interpretativa cuyo sentido sólo puede concebirse al vislumbrar la estructura completa de la misma en sintonía con el "lógos" vital, histórico y narrativo que el proyecto encierra. Se revisará la concepción histórica según la cual, la configuración espacial de la forma habría tenido primacía respecto a su configuración temporal, al remitir esta última a una condición espacializada y circunstancial. La componente vicaria de la circunstancia será elevada por Ortega a la categoría de esencial, visualizando así una paradoja cuya reformulación nos lleva a la concepción de un tiempo sustancial. El diccionario de la RAE, en su tercera acepción, define la sustancia como “aquello que permanece en algo que cambia”, lo cual nos remite al pensamiento antiguo. Se mostrará que lo que permanece no necesariamente implica una concepción estática y eleática de la forma, que la arquitectura esencial no es unívocamente la arquitectura atemporal del platonismo y que cabe concebir la "firmitas" desde la atención a la "durée" bergsoniana. Al asociar tradicionalmente la sustancia con el referido estatismo, se margina al tiempo y a la duración a lo no sustancial; por ello, se tratará de aproximar los términos de tiempo y sustancia para definir la forma. Ello conllevará al estudio de las notables patologías derivadas de la asunción de un tiempo cronológico en nuestra contemporaneidad frente a las cuales, las intuiciones contenidas en Mérida, se alinearán con la actual actitud revisionista en el ámbito del pensamiento filosófico y científico. En Mérida, la memoria recogerá los aspectos de la conciencia así como los aspectos vinculados a la experiencia íntima y colectiva como soporte para la consecución de un discurso. La dualidad entre intuición e inteligencia será recogida por Moneo con idea de trascender su incomunicabilidad, mediante una operación que consistirá en la reivindicación de una memoria irreductible, cuya morada estaría incardinada en el propio tiempo de la duración y de la vida y no en la espacialidad coextensiva del presente y de la acción funcional sobre la materia. Moneo asumirá el papel de la memoria como condición central de una forma que se encarnará al concebirse como un teatro. En la respuesta a la contradicción entre el hecho físico y el efecto psíquico de la experiencia humana residirá la pertinencia de un tiempo narrativo. Será entonces el lenguaje el encargado de aportar sentido a la obra mediante el recurso fundado en la dramatización de la experiencia, es decir, a través de una conexión entre la conciencia íntima y el carácter social y colectivo intrínseco en la arquitectura. ABSTRACT TIME AS A SUBSTANCE OF FORM. AN APPROACH TO THE ROMAN ART MUSEUM OF MÉRIDA FROM THE VIEWPOINT OF VITALISM. The dominant model during the Modern Era placed man as a subject inside the great mechanism of the cosmos. It is also based in an idea about natural being within the ontological tradition initiated by the pre-Socratic Eleatism that conceives it as something immutable and static in respect with change and movement, which are considered as mere appearances. Prior to the emergence of the great Greek philosophers occurred a transformation where concepts of cosmology linked to a primordial time, changed to a view of the universe as indestructible, timeless, unchanging, perfect, geometric and spatial. If Democritus accepted a universe subjected to randomness and necessity, Plato thought that the universe only worked by necessity. This ontological model is called into question when man is not conceived as a piece of a broader system, but as a radical center of thinking. The radical condition of man then is his own life. This is the core concept of so-called Vitalism, whose most influential representative in Spain was José Ortega y Gasset. The stillness of being – of man - loses its meaning; in the words of Ortega, “it is not being but being in progress” that characterizes human life. The Cartesian reason is now the vital reason and its subject of study is no longer nature but its own evolution, in other words, time and history. This approach is fundamental to understand the building which is the subject of this study, the Museum of Roman Art in Mérida (1980-1985) by Rafael Moneo. The concept of time is used as a framework and structure of this thesis, demonstrating the notorious and very significant role this building has implied in the career of its author and in the national and international panorama of architecture and museology. This project allow us to approach the thought of its author through a building that, even whilst widely recognized, does not yet have a sufficiently comprehensive study covering its breadth and richness. This thesis is not a compendium of what already has been researched on Merida; it is a global and interpretative approach whose meaning can only be conceived as a study of its complete structure in line with the vital, historical and narrative logos the project implies. We will review the historical idea where spatial configuration of the form would have had primacy with respect to temporary configuration, because the latter refers to a spatial and circumstantial condition. The vicarious nature of the circumstance will be elevated by Ortega to the category of essential, thus showing a paradox which reformulation leads us to the conception of a substantial time. The dictionary of the Spanish Royal Academy, in its third meaning, defines substance as "that which remains in something that changes". This is a reference to ancient thought. It will be shown that what remains does not necessarily imply a static and Eleatic conception of form. It will also be shown that the essential architecture is not uniquely the timeless architecture of Platonism and that it is possible to conceive the "firmitas" parallel to the "durée" of Henri Bergson. As a result of this traditional association between substance and stillness, it marginalizes the time and the duration to the non-substantial; for this reason, we will try to approach terms of time and substance to define the shape. This will involve studying significant pathologies resulting from an assumption of chronological time in our contemporary world against which, the insights contained in Merida, will be aligned with the current revisionist attitude in the fields of philosophical and scientific thought. In Merida, memory includes aspects of consciousness as well as aspects linked to the intimate and collective experience as a foundation for the achievement of discourse. The duality between intuition and intelligence is put forward by Moneo with the idea of transcending its lack of communication, by means of a resource consisting of the vindication of an irreducible memory, whose home would be embodied in the time of duration and life and not in the coextensive spatiality of the present and in the functional action on the matter. Moneo demonstrates the role of memory as a central condition of form as a theatre. In response to the contradiction between the physical fact and the psychological effect of human experience lies the relevance of narrative time. Language then assumes the responsibility of giving meaning to the work through the dramatization of experience, i.e., through a connection between the intimate consciousness and the intrinsic social and collective character of architecture.

Delineating the Boundaries for Religious Speech in Public Discourse in Kierkegaard and Habermas

How is it that one of the most famous Christian thinkers - Soren Kierkegaard -- and one of the most famous contemporary secular thinkers -- Jurgen Habermas - both agree: the religious has nothing to say in the public realm of social, ethical discourse.

Recessive and dominant mutations in the ethylene biosynthetic gene ACS5 of Arabidopsis confer cytokinin insensitivity and ethylene overproduction, respectively

We identified a set of cytokinin-insensitive mutants by using a screen based on the ethylene-mediated triple response observed after treatment with low levels of cytokinins. One group of these mutants disrupts ACS5, a member of the Arabidopsis gene family that encodes 1-aminocyclopropane-1-carboxylate synthase, the first enzyme in ethylene biosynthesis. The ACS5 isoform is mainly responsible for the sustained rise in ethylene biosynthesis observed in response to low levels of cytokinin and appears to be regulated primarily by a posttranscriptional mechanism. Furthermore, the dominant ethylene-overproducing mutant eto2 was found to be the result of an alteration of the carboxy terminus of ACS5, suggesting that this domain acts as a negative regulator of ACS5 function.

Retardation of skeletal development and cervical abnormalities in transgenic mice expressing a dominant-negative retinoic acid receptor in chondrogenic cells

Skeletal formation is a fundamental element of body patterning and is strictly regulated both temporally and spatially by a variety of molecules. Among these, retinoic acid (RA) has been shown to be involved in normal skeletal development. However, its pleiotropic effects have caused difficulty in identifying its crucial target cells and molecular mechanisms for each effect. Development of cartilage primordia is an important process in defining the skeletal structures. To address the role of RA in skeletal formation, we have generated mice expressing a dominant-negative retinoic acid receptor (RAR) in chondrogenic cells by using the type II collagen α1 promoter, and we have analyzed their phenotypes. These mice exhibited small cartilage primordia during development and retarded skeletal formation in both embryonic and postnatal periods. They also showed selective degeneration in their cervical vertebrae combined with homeotic transformations, but not in their extremities. The cervical phenotypes are reminiscent of phenotypes involving homeobox genes. We found that the expression of Hoxa-4 was indeed reduced in the cartilage primordia of cervical vertebrae of embryonic day 12.5 embryos. These observations demonstrate that endogenous RA acts directly on chondrogenic cells to promote skeletal growth in both embryonic and growing periods, and it regulates the proper formation of cervical vertebrae. Furthermore, RA apparently specifies the identities of the cervical vertebrae through the regulation of homeobox genes in the chondrogenic cells. Great similarities of the phenotypes between our mice and reported RAR knockout mice revealed that chondrogenic cells are a principal RA target during complex cascades of skeletal development.

Dominant transformation by mutated human ras genes in vitro requires more than 100 times higher expression than is observed in cancers

The gene-mutation-cancer hypothesis holds that mutated cellular protooncogenes, such as point-mutated proto-ras, “play a dominant part in cancer,” because they are sufficient to transform transfected mouse cell lines in vitro [Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. & Watson, J. D. (1994) Molecular Biology of the Cell (Garland, New York)]. However, in cells transformed in vitro mutated human ras genes are expressed more than 100-fold than in the cancers from which they are isolated. In view of the discrepancy between the very low levels of ras transcription in cancers and the very high levels in cells transformed in vitro, we have investigated the minimal level of human ras expression for transformation in vitro. Using point-mutated human ras genes recombined with different promoters from either human metallothionein-IIA or human fibronectin or from retroviruses we found dominant in vitro transformation of the mouse C3H cell line only with ras genes linked to viral promoters. These ras genes were expressed more than 120-fold higher than are native ras genes of C3H cells. The copy number of transfected ras genes ranged from 2–6 in our system. In addition, nondominant transformation was observed in a small percentage (2–7%) of C3H cells transfected with ras genes that are expressed less than 20 times higher than native C3H ras genes. Because over 90% of cells expressing ras at this moderately enhanced level were untransformed, transformation must follow either a nondominant ras mechanism or a non-ras mechanism. We conclude that the mutated, but normally expressed, ras genes found in human and animal cancers are not likely to “play a dominant part in cancer.” The conclusion that mutated ras genes are not sufficient or dominant for cancer is directly supported by recent discoveries of mutated ras in normal animals, and in benign human tissue, “which has little potential to progress” [Jen, J., Powell, S. M., Papadopoulos, N., Smith, K. J., Hamilton, S. R., Vogelstein, B. & Kinzler, K. W. (1994) Cancer Res. 54, 5523–5526]. Even the view that mutated ras is necessary for cancer is hard to reconcile with (i) otherwise indistinguishable cancers with and without ras mutations, (ii) metastases of the same human cancers with and without ras mutations, (iii) retroviral ras genes that are oncogenic without point mutations, and (iv) human tumor cells having spontaneously lost ras mutation but not tumorigencity.

A variant of DNA polymerase β acts as a dominant negative mutant

In eukaryotic cells, DNA polymerase β (polβ) carries out base-excision repair (BER) required for DNA maintenance, replication, recombination, and drug resistance. A specific deletion in one allele in the coding sequence of the polβ gene occurs in colorectal and breast carcinomas. The 87-bp deleted region encodes amino acid residues 208–236 in the catalytic domain of the enzyme. Here, we report evidence for expression of the wild-type (WT) and the truncated polβ proteins in colorectal tumors. To elucidate the potential functional consequences of polβ truncation, stable HeLa cell lines were established from cloned WT and variant polβΔ208–236. Cells expressing the variant protein exhibited substantially decreased BER activity. To test our hypothesis that truncated polβ may disrupt the function of the WT enzyme, we stably transfected mouse embryonic fibroblast 16.3 cells with polβΔ208–236 cDNA. Reverse transcription–PCR and Western blot analyses showed that the new cell line, 16.3ΔP, expresses the WT and the truncated polβ mRNA and proteins. BER and binding activities were undetectable in these cells. Furthermore, in vivo the 16.3ΔP cells were more sensitive to N-methyl-N′-nitro-N-nitrosoguanidine than the 16.3 cells. On adding increasing amounts of 16.3ΔP protein extracts, the BER and DNA binding activities of extracts of the parent 16.3 cell line progressively declined. These results strongly suggest that truncated polβ acts as a dominant negative mutant. The defective polβ may facilitate accumulation of mutations, leading to the expression of a mutator phenotype in tumor cells.

A dominant form of inherited retinal degeneration caused by a non-photoreceptor cell-specific mutation

We have isolated a dominant mutation, night blindness a (nba), that causes a slow retinal degeneration in zebrafish. Heterozygous nba fish have normal vision through 2–3 months of age but subsequently become night blind. By 9.5 months of age, visual sensitivity of affected fish may be decreased more than two log units, or 100-fold, as measured behaviorally. Electroretinographic (ERG) thresholds of mutant fish are also raised significantly, and the ERG b-wave shows a delayed implicit time. These defects are due primarily to a late-onset photoreceptor cell degeneration involving initially the rods but eventually the cones as well. Homozygous nba fish display an early-onset neuronal degeneration throughout the retina and elsewhere in the central nervous system. As a result, animals develop with small eyes and die by 4–5 days postfertilization (pf). These latter data indicate that the mutation affecting nba fish is not in a photoreceptor cell-specific gene.

Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel

ATP-sensitive K+ (KATP) channels are known to play important roles in various cellular functions, but the direct consequences of disruption of KATP channel function are largely unknown. We have generated transgenic mice expressing a dominant-negative form of the KATP channel subunit Kir6.2 (Kir6.2G132S, substitution of glycine with serine at position 132) in pancreatic beta cells. Kir6.2G132S transgenic mice develop hypoglycemia with hyperinsulinemia in neonates and hyperglycemia with hypoinsulinemia and decreased beta cell population in adults. KATP channel function is found to be impaired in the beta cells of transgenic mice with hyperglycemia. In addition, both resting membrane potential and basal calcium concentrations are shown to be significantly elevated in the beta cells of transgenic mice. We also found a high frequency of apoptotic beta cells before the appearance of hyperglycemia in the transgenic mice, suggesting that the KATP channel might play a significant role in beta cell survival in addition to its role in the regulation of insulin secretion.

Isolation and characterization of iron-independent positive dominant mutants of the diphtheria toxin repressor DtxR

It is well known that the functional activity of the diphtheria toxin repressor DtxR is controlled by iron, which serves as an essential cofactor necessary for activation of target DNA binding by this regulatory element. In this communication, we describe the isolation and characterization of a unique series of DtxR mutants that are constitutively active and repress the expression of β-galactosidase from a diphtheria tox promoter/operator–lacZ transcriptional fusion, even in the absence of iron. These self-activating mutants of DtxR (SAD) were isolated through the use of a positive selection system for the cloning of functional dtxR alleles and target DNA operator sites. Of the four independently isolated SAD mutants that were characterized, two (SAD2 and SAD11) were found to carry a single missense mutation (E175K) in their respective C-terminal SH3-like domains. In contrast, the mutant allele encoding SAD3 was found to carry a total of six missense mutations distributed throughout the N- and C-terminal domains of the repressor. Partial diploid analysis of strains carrying both native dtxR and alleles encoding either SAD2 or SAD3 demonstrate that these iron-independent mutants possess a positive dominant phenotype in the regulation of β-galactosidase expression from a diphtheria tox promoter/operator–lacZ transcriptional fusion.