The construction of sexpartite vaults in Europe

Sexpartite vaults constitute one of the most interesting chapters in European Gothic architecture. Originally, the use of the square cross-ribbed vault was limited to relatively small spaces, but when the need arose to cover spaces of considerable size, a new vault with very peculiar characteristics appeared. This new vault was a cross-ribbed vault that was reinforced in the centre by a rib that was parallel to the transverse ribs which effectively divided the vault in half. This configuration breaks the side arch into two fragments, creating a pair of windows on each side. The volumetrics of these vaults is extremely complex and the difficulties involved in their construction perhaps explain why they were abandoned in favour of the simple cross ribbed vault, now with rectangular sections. The existence of the sexpartite vault barely lasted more than fifty years, from the end of the XII century and the beginning of the XIII. Towards the end of the 19th century Viollet-le-Duc gave a succinct explanation of this type of vault. A. Choisy also, later, devotes some pages to the French sexpartite vault; since then, the subject has only been broached in a few references in later studies on Gothic architecture. However, despite its short period of existence, the sexpartite vault spread throughout Europe and was used to build important vaulting. Viollet-le-Duc's sexpartite vault could be considered to be the prototype of them all, while it is true that the studies that we have conducted so far lead us to affirm that there is a wide variety of vaults, with different volumetric spaces and different construction strategies. Therefore, we believe that this chapter of international Gothic deserves further study applying the knowledge and resources that are available today. This paper has been written to explore the most significant European sexpartite vaults. New measurement technology has led to a revolution in research into the history of construction, allowing studies to be conducted that were hitherto impossible. Thorough data collection using total station and photogrammetry has enabled us to identify the stereotomy of the voussoirs, tas-de-charges and keystones, as well as the bonding of the surfaces of the severies. A comparison of the construction techniques employed in the different vaults studied reveals common construction features and aspects that are specific to each country. Thus we are able to establish the relationship between sexpartite vaults in different European countries and their influence on each other.

La deformación del tipo Construcción de bóvedas no-canónicas en España (siglos XVI-XVIII)

Una bóveda no canónica es una bóveda que se adapta a una forma distinta de aquella para la que ha sido inicialmente concebida. Bóvedas raras, anormales, no convencionales, habitualmente consideradas excepciones o casos particulares, resultan ser más frecuentes de lo inicialmente esperado. El interés por este tipo de bóvedas surge a raíz de una investigación inicial sobre las bóvedas empleadas para cubrir espacios de planta anular, como en el caso de las girolas de las iglesias. Sin embargo, el problema de la bóveda anular no puede ser abordado directamente, sino como parte de una investigación más general sobre bóvedas que se deforman para adaptarse a una situación anómala. El análisis de las posibilidades que un determinado tipo de bóveda brinda para resolver el abovedamiento de espacios de planta irregular, trascendiendo el problema de la planta anular, es lo que da origen a esta investigación. La cuestión de las bóvedas deformadas forma parte de un contexto mayor, el de la deformación en arquitectura abovedada. Ante una contradicción, la deformación de la bóveda es sólo una de las posibles opciones que esta arquitectura ofrece para resolver un problema de deformación. La tesis se estructura en dos partes: en la primera parte se analizan los conceptos de forma y deformación en el contexto de la arquitectura abovedada con objeto de sentar las bases para una teoría de las bóvedas no canónicas. El objetivo es establecer un punto de partida para la investigación en un campo que todavía no había sido abordado. En la segunda parte se analizan tres tipos de bóveda desde la perspectiva de las bóvedas no canónicas, a partir de un estudio de casos de bóvedas en España entre los siglos XVI y XVIII. El estudio de la deformación en arquitectura abovedada se centra en el problema de la girola, por tratarse de un caso generalizado de deformación, directamente relacionado con el problema de las bóvedas irregulares y cuyo estudio, llamativamente, no había sido llevado a cabo hasta la fecha. Se propone una primera aproximación al problema de la girola, desde un punto de vista puramente morfológico, al margen de consideraciones históricas. En el caso de las bóvedas deformadas, el análisis se centra en tres tipos de bóveda: la bóveda de crucería, la bóveda de arista y la bóveda baída. Estos tres tipos de bóveda, aunque basadas en criterios formales distintos, están íntimamente relacionados entre sí. Por un lado permiten resolver el mismo problema –planta cuadrada delimitada por arcos–, por otro lado es posible establecer una relación formal entre la bóveda de arista y la bóveda baída a través de la bóveda de crucería. El estudio de casos recogido en la segunda parte de la tesis se fundamenta en dos líneas de investigación, la primera sobre soluciones teóricas de bóvedas no convencionales propuestas en los manuscritos y tratados de cantería, y la segunda sobre bóvedas efectivamente construidas, tratado de establecer una comparación entre teoría y práctica, confrontando el grado de relación entre ambas. Sin embargo este doble análisis sólo se ha podido llevar a cabo en contadas ocasiones. Constatamos que las bóvedas no canónicas reflejadas en los tratados son pocas y apenas se han llevado a la práctica, mientras que las soluciones construidas no responden a modelos teóricos propuestos, manifestando un divorcio entre teoría y práctica. El estudio de estas bóvedas permite poner en cuestión la definición tradicional que relaciona los conceptos de ‘bóveda’ y ‘superficie’. Al iniciar el trabajo nos encontramos con un modelo teórico extremadamente rígido que deja fuera un gran número de bóvedas, obligando a agruparlas bajo el término «no canónicas». El trabajo realizado pone en evidencia lo limitado del modelo. El problema no está en la presencia de bóvedas anómalas, que no se adaptan al modelo tradicionalmente propuesto, sino en la extrema rigidez del modelo. ABSTRACT A non canonical vault is a vault adapted to a different form from that for which was originally conceived. These rare, abnormal, unconventional vaults are usually considered as exceptions or special cases. However they prove to be more frequent than it was initially expected. Interest in this type of vaults arises from an initial research on the vaults used to roof annular spaces, such as ambulatories. Nevertheless, the annular vault question cannot be addressed directly, but as a part of a broader research on distorted vaults; a research on vaults deformed to conform an anomalous layout. The analysis of the possibilities that a particular type of vault provides to solve the vaulting of an irregular layout, beyond the problem of the annular plan is the origin of this research. The argument of deformed vaults is part of a greater context, the context of deformation in vaulted architecture. Facing a contradiction, deforming a vault is just one of the options that vaulted architecture offers to solve a problem of deformation. This dissertation is organised in two parts: in the first part we analyse the concepts of form and deformation in the context of vaulted architecture in order to lay the foundations for a non canonical vaults theory. The objective is to establish a starting point for future research in a field that has not been addressed yet. In the second part, we analyse three types of vault from the perspective of non canonical vaults, based on a case study of Spanish vaults between the 16th and 18th Centuries. The analysis of deformation in vaulted architecture focuses on the question of the ambulatory, because it is a generalized example of deformation, directly related to the problem of irregular vaults. Remarkably, the analysis of these spaces had not been conducted to date. We propose a first approach to the question of the ambulatory, from a purely morphological point of view, setting aside historical considerations. The analysis of deformed vaults focuses on three types of vault: the groin vault, the ribbed vault and the sail vault. These three types of vault, although based on different formal criteria, are closely related between them. On the one hand, they allow to solve the same problem –a square perimeter limited by arcs-; on the other hand, it is possible to establish a formal relationship between the groin vault and the sail vault through the ribbed vault. The case study presented in the second part of this dissertation is based on two research lines: theoretical non conventional vaults solutions proposed on stonecutting treatises; and currently built vaults. The aim of this double analysis was to establish a comparison between theory and practice, comparing the degree of relationship between them. Nevertheless, this double analysis has only been carried out on rare occasions. It is noted that non canonical vaults reflected in treaties are few and hardly been employed, while the built solutions do not meet proposed theoretical models, expressing a divorce between theory and practice. The analysis of these vaults allows us to question the traditional definition that connects the concepts of 'vault' and 'surface'. When we began this research, we found an extremely rigid theoretical model that leaved out many vaults, forcing to group them under the term of «non canonical vaults». This research evidences the limitations of the model. The problem is not the presence of abnormal vaults, which cannot adapt to the traditional model, but in the very high stiffness of the model.

Aspectos formales y constructivos en la obra de Guillem Sagrera: el uso de las plantillas

Los enjarjes, jarjas o jarjamentos conforman el comienzo de la bóveda de crucería, el lugar en el que todos los nervios nacen reunidos. Permiten la transición entre el muro y las dovelas que constituyen nervios, resolviendo un encuentro de gran importancia constructiva. Están compuestos por piezas de lechos horizontales que traban con el muro y que, además de conformar el arranque de los nervios, han de proporcionar apoyo a las primeras dovelas. Al construirse a la vez que el muro y antes que el resto de la bóveda, se podría decir que son los encargados de portar su ADN: en ellos queda registrado el número de nervios que tendrá la bóveda, su curvatura, desde qué altura arrancará cada uno, su perfil, etc. El objetivo de este trabajo se centra en estudio de los enjarjes en la obra de Guillem Sagrera abordando su dimensión tecnológica, constructiva y de diseño. Dos características los distinguen de otras soluciones y justifican su interés: los nervios surgen directamente del muro, sin la intermediación de capiteles, ménsulas o pilastras; y se diseñan con la intención de facilitar la aparición de intersecciones entre las molduras de sus perfiles, en algunos casos mediante el cruce de los ejes de los nervios. Sagrera nos acerca a una innovación arquitectónica en la que el esfuerzo no se centra en realizar bóvedas con muchos nervios o con trazados en planta complejos, sino en la cuidada resolución de este encuentro de nervios. En ella se adivina el trabajo minucioso con las plantillas que controlan el trazado del contorno de los lechos de las piezas y la hábil mano de los canteros que son capaces de tallar intersecciones de gran complejidad. Se ha realizado un repaso de los primeros experimentos relativos al modo de relacionarse los nervios entre sí para ilustrar el contexto y origen de las soluciones realizadas por Guillem Sagrera. Mostramos que ante ciertas dificultades, consecuencia de la reunión de nervios, los constructores fueron capaces de desarrollar nuevas soluciones, mediante la experimentación con un sistema constructivo que conocían y manejaban con destreza. Para acercarnos a la comprensión de las estrategias de diseño que permiten el proyecto de estos enjarjes y los procedimientos técnicos y constructivos necesarios para su ejecución, nos vimos en la necesidad de adentrarnos en la problemática general de los enjarjes de la bóveda de crucería. De este modo, lo que empezó siendo una introducción para poder contextualizar la obra del mallorquín acabó convirtiéndose en la primera parte de la tesis, cuyo volumen prácticamente equipara a la segunda. En ella presentamos el proceso de diseño, trazado y talla de los cuatro enjarjes llevados a cabo en el taller de cantería de la ETSAM, en los que hemos podido experimentar de manera práctica los aspectos teóricos desarrollados. Estos ensayos nos han permitido contrastar hipótesis y baremar la dificultad de ciertos procedimientos o procesos, así como acercarnos realmente al elemento constructivo. El trabajo práctico nos ha enseñado a no fiarnos siempre de las hipótesis que se desarrollan modelando con el ordenador o dibujando; a valorar el pensar con las manos. En relación con la obra de Sagrera, la presente investigación realiza aportaciones al conocimiento del cambio proyectual y constructivo llevado a cabo en los arranques de las bóvedas entre los siglos XIII y XV, cuando los nervios comienzan a surgir directamente de los soportes y se dan los primeros cruzamientos. Mostramos que ya no solamente se construyen enjarjes fruto directo de la geometría general de la bóveda, sino que se llevan a cabo cambios deliberados en relación a su resultado, en los que se advierten decisiones proyectuales que, por supuesto, no serían viables sin las posibilidades que ofrece el trabajo con plantillas. ABSTRACT The solid blocks commonly known as tas-de-charge (in Spanish enjarjes, jarjas or jarjamentos) constitute the beginning of the ribbed vault – the place from which all the ribs spring together. They facilitate a transition between the wall and the rib voussoirs, and thus solve a junction of utmost constructive importance. They consist of blocks set in horizontal courses which interlock with the wall and which, as well as constituting the springing of the ribs, serve as a support for their first voussoirs. The tas-de-charge are built simultaneously with the wall and well before the remainder of the vault – thus, they arguably carry its ‘DNA’, since they register how many ribs the vault will have as well as their curvature, their springing height or their profile. This work is focused on the study of the tas-de-charge in the works of Guillem Sagrera, and will address their technological, constructive and design aspects. Two characteristics set these apart from other solutions and justify their relevance: these are that the ribs spring directly from the wall without the mediation of capitals, corbels or pilasters; and that they are deliberately designed to force the intersection of their mouldings, in some cases by crossing the rib axes. Sagrera’s work tells a story of architectural innovation – one where the effort is not centred on creating vaults with numerous ribs or a sophisticated ground plan, but on carefully solving the rib unions, which evidence a meticulous use of templates to control the tracing of the pieces’ profiles as well as the skill of the stonemasons, able to carve highly complex intersections. An overview of the first experiments with rib relationships will illustrate the context and origin of Guillem Sagrera’s solutions. We show how, faced with difficulties arising from the convergence of ribs into the tas-de-charge, builders were able to develop new solutions by experimenting with a construction system that they were already familiar with and could control easily. In order to gain a better insight into the design strategies behind his tas-de-charge and the technical and constructive procedures required for their execution, we found ourselves facing the need to address the general subject of tas-de-charge in ribbed vaults. This, which began as an introduction meant as context for Sagrera’s work, took on a life of its own and became the first half of the thesis, with a volume practically equal to that of the second. We have devoted a chapter to experimental archaeology. It comprises the design, tracing and carving processes for the four tas-de-charge executed at the ETSAM Stonecutting Workshop, in which we have tested experimentally the theories studied in the previous chapters. These tests have allowed us to contrast hypotheses, assess the difficulty of certain procedures or processes and understand the built element as a real entity. The practical work has taught us not to always trust the hypotheses proposed through computer modelling or drawing – and to recognise the importance of coordinating the hands and the mind. After studying Sagrera’s work and contrasting it with other related or previous tas-de-charge, our research will seek to make a contribution to the study of the shift in the design and construction of vault springers that took place between the 13th and 14th centuries, when ribs began to spring directly from their support and moulding crossings began to appear. We show that, from then on, tas-de-charge would not only depend on the general vault geometry – deliberate modifications would be carried out in order to achieve the desired result. This reveals design decisions that would have been unworkable if not for the effective use of template strategies.

Bóvedas sexpartitas : los orígenes del gótico

Uno de los capítulos más interesantes del gótico europeo lo constituyen las bóvedas sexpartitas, sin lugar a dudas una de las bóvedas más singulares jamás creadas dentro de este estilo. Las primeras bóvedas góticas, en cruce de ojivas y de planta cuadrada, limitaban su uso a espacios relativamente pequeños, pero ante la necesidad de cubrir espacios de considerables dimensiones, apareció una nueva bóveda de características muy peculiares; la sexpartita. Esta bóveda en cruce de ojivas está reforzada por el centro con un arco paralelo a los arcos fajones que la divide por la mitad y que fragmenta el arco formero en dos, creando una pareja de ventanales en cada lado. La sencilla superficie en bóveda de arista, en el origen de las bóvedas de crucería, se complica extraordinariamente creando una volumetría de gran complejidad con seis cuarteles de plementería y con seis apoyos de distinto tamaño, cuatros esquineros y dos centrales más pequeños. Las dificultades que implica la construcción de este tipo de bóvedas explican quizás su abandono prematuro y la vuelta a la bóveda de crucería simple, ahora de tramos rectangulares. No obstante, a pesar de su corta existencia, la bóveda sexpartita fue la gran protagonista de los inicios del gótico y con ella se llevaron a cabo importantes abovedamientos, desde Inglaterra hasta Rumanía. La disciplina de la Historia de la Construcción se vio extraordinariamente favorecida por los estudios realizados en el siglo XIX, sin embargo su investigación se abandona durante el siglo XX para ser recuperada recientemente. Viollet-le-Duc, a finales del s. XIX, hace una sucinta explicación de este tipo de bóvedas. También Auguste Choisy, más tarde, dedica unas páginas a la bóveda sexpartita francesa; desde entonces, este tema, ha merecido escasísimas referencias en los estudios posteriores. Esta investigación se enmarca en este contexto y pretende poner de manifiesto los conocimientos geométricos y constructivos que hicieron posible la realización de las bóvedas sexpartitas europeas. Para ello se ha llevado a cabo la investigación de las principales bóvedas en Europa occidental; Francia, España, Inglaterra, Alemania, Suiza e Italia. Su estudio comparativo nos ha permitido poner de manifiesto sus características constructivas comunes y aquellos aspectos propios de cada país, así como algunos de los canales de comunicación que permitieron la expansión de esta arquitectura. Las nuevas tecnologías de medición, el escáner láser, la estación total, la fotogrametría, etc., han supuesto una revolución para la documentación y restauración del Patrimonio y un salto cualitativo formidable para el análisis de las bóvedas góticas, permitiendo estudios de la arquitectura histórica hasta ahora inabordables. Para realizar el análisis de las bóvedas sexpartitas europeas se ha llevado a cabo un levantamiento exhaustivo de las mismas, lo que ha permitido definir su despiece, obteniendo la forma de la talla de cada uno de los elementos constructivos que la componen; jarjas, dovelas, claves y plementería. La obtención de estos datos nos ha permitido abordar un profundo estudio de su estereotomía y construcción, aportando datos inéditos hasta el momento. Por otro lado se ha llevado a cabo la detección y catalogación de las principales bóvedas sexpartitas que aún se conservan en Europa. Los estudios realizados nos permiten afirmar que la bóveda sexpartita surge en Francia en la segunda mitad del siglo XII, utilizándose en las principales catedrales francesas, como Notre Dame de Paris, Bourges o Laon. A comienzos del siglo XIII cae en desuso en Francia y comienza su expansión por el resto de Europa, donde se abandona medio siglo después, desapareciendo definitivamente del gótico europeo. Mientras que los ejemplos que datan del siglo XII muestran soluciones escasamente desarrolladas y propias del románico, las bóvedas construidas en el siglo XIII muestran soluciones enormemente complejas, con grandes jarjamentos e inteligentes estrategias constructivas y geométricas que permiten la simplificación de sus estructuras auxiliares y una mayor libertad en su diseño. Estas bóvedas son el reflejo del desarrollo de la estereotomía gótica en sus comienzos por lo que su estudio nos ha permitido conocer el desarrollo y la evolución del gótico primitivo en Europa. ABSTRACT One of the most interesting chapters of European Gothic is the sexpartite vault, without doubt one of the most remarkable vaults ever created within this style. The first Gothic vaults, with crossed ribs on a square base, were restricted to relatively small areas, but a new vault, with very particular characteristics emerged to address the need to cover spaces of considerable size; the sexpartite vault. This cross-ribbed vault is reinforced in the centre by an arch that runs parallel to the transverse arches, divides the vault in half and splits the wall arch in two, creating a pair of windows, one on each side. The simple groin vault surface, the source of ribbed vaults, was greatly complicated creating a highly complex volume with six sections of severies and with six supports of different sizes, four on the corners and two smaller central ones. The construction difficulties involved in building this type of vault may explain its premature abandonment and a return to the simple cross-ribbed vault, now in rectangular sections. However, despite its brief existence, the sexpartite vault was the great protagonist of the beginnings of Gothic architecture and important vaulting was built using this system from England to Romania. Studies undertaken in the 19th century helped the History of Construction as a discipline tremendously. Research was abandoned during the twentieth century however, and has only recently been taken up again. Towards the end of the 19th century, Viollet-le-Duc gave a brief description of this type of vault. Later, Auguste de Choisy also devoted some pages to the French sexpartite vault; since then, later studies have made very few references to it. Against this background, this research now attempts to bring to light the knowledge of geometry and construction that made the construction of the European sexpartite vault possible. To this end, the main vaults in Western Europe - France, Spain, England, Germany, Switzerland and Italy, have been studied. By making a comparative study we have been able to reveal the common construction features and those that are specific to each country, as well as some of the channels of communication that enabled this architecture to spread. New measuring technologies, the laser scanner, total station, photogrammetry, etc., have given rise to a revolution in heritage documentation and restoration, as well as facilitating a huge qualitative leap for the analysis of Gothic vaults, enabling studies of historical architecture that until now were inaccessible. A comprehensive survey was carried out to be able to analyse European sexpartite vaults. We could thus create an exploded view, which enabled us to obtain the form of each of the elements; tas-de-charges, voussoirs, keystones and severies. The data gathered provided previously unknown facts that enabled us to make an in-depth study of stereotomy and construction. Furthermore, the main sexpartite vaults still preserved in Europe have been identified and categorised. The studies undertaken allowed us to affirm that the sexpartite vault appeared in France in the second half of the twelfth century, being used in the main French cathedrals, such as Notre Dame de Paris, Bourges or Laon. At the beginning of the thirteenth century it fell into disuse in France and began to expand throughout the rest of Europe, where it was abandoned half a century later, disappearing from European Gothic for good. While the examples dating back to the 12th century display poorly developed solutions more characteristic of the Romanesque, the vaults built in the 13th century reveal enormously complex solutions, with large tas-de-charges and intelligent construction and geometric strategies that allowed auxiliary support structures to be simplified, and gave more freedom to design. These vaults reflect the beginnings of Gothic stereotomy and by studying them we have been able to learn more about the development and evolution of Early Gothic architecture in Europe.

Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex

The Arp2/3 complex, a stable assembly of two actin-related proteins (Arp2 and Arp3) with five other subunits, caps the pointed end of actin filaments and nucleates actin polymerization with low efficiency. WASp and Scar are two similar proteins that bind the p21 subunit of the Arp2/3 complex, but their effect on the nucleation activity of the complex was not known. We report that full-length, recombinant human Scar protein, as well as N-terminally truncated Scar proteins, enhance nucleation by the Arp2/3 complex. By themselves, these proteins either have no effect or inhibit actin polymerization. The actin monomer-binding W domain and the p21-binding A domain from the C terminus of Scar are both required to activate Arp2/3 complex. A proline-rich domain in the middle of Scar enhances the activity of the W and A domains. Preincubating Scar and Arp2/3 complex with actin filaments overcomes the initial lag in polymerization, suggesting that efficient nucleation by the Arp2/3 complex requires assembly on the side of a preexisting filament—a dendritic nucleation mechanism. The Arp2/3 complex with full-length Scar, Scar containing P, W, and A domains, or Scar containing W and A domains overcomes inhibition of nucleation by the actin monomer-binding protein profilin, giving active nucleation over a low background of spontaneous nucleation. These results show that Scar and, likely, related proteins, such as the Cdc42 targets WASp and N-WASp, are endogenous activators of actin polymerization by the Arp2/3 complex.

The FEZ1 gene at chromosome 8p22 encodes a leucine-zipper protein, and its expression is altered in multiple human tumors

Alterations of human chromosome 8p occur frequently in many tumors. We identified a 1.5-Mb common region of allelic loss on 8p22 by allelotype analysis. cDNA selection allowed isolation of several genes, including FEZ1. The predicted Fez1 protein contained a leucine-zipper region with similarity to the DNA-binding domain of the cAMP-responsive activating-transcription factor 5. RNA blot analysis revealed that FEZ1 gene expression was undetectable in more than 60% of epithelial tumors. Mutations were found in primary esophageal cancers and in a prostate cancer cell line. Transcript analysis from several FEZ1-expressing tumors revealed truncated mRNAs, including a frameshift. Alteration and inactivation of the FEZ1 gene may play a role in various human tumors.

Unusual phenotypic alteration of β amyloid precursor protein (βAPP) maturation by a new Val-715 → Met βAPP-770 mutation responsible for probable early-onset Alzheimer’s disease

We have identified a novel β amyloid precursor protein (βAPP) mutation (V715M-βAPP770) that cosegregates with early-onset Alzheimer’s disease (AD) in a pedigree. Unlike other familial AD-linked βAPP mutations reported to date, overexpression of V715M-βAPP in human HEK293 cells and murine neurons reduces total Aβ production and increases the recovery of the physiologically secreted product, APPα. V715M-βAPP significantly reduces Aβ40 secretion without affecting Aβ42 production in HEK293 cells. However, a marked increase in N-terminally truncated Aβ ending at position 42 (x-42Aβ) is observed, whereas its counterpart x-40Aβ is not affected. These results suggest that, in some cases, familial AD may be associated with a reduction in the overall production of Aβ but may be caused by increased production of truncated forms of Aβ ending at the 42 position.

A stochastic approximation algorithm with Markov chain Monte-Carlo method for incomplete data estimation problems

We propose a general procedure for solving incomplete data estimation problems. The procedure can be used to find the maximum likelihood estimate or to solve estimating equations in difficult cases such as estimation with the censored or truncated regression model, the nonlinear structural measurement error model, and the random effects model. The procedure is based on the general principle of stochastic approximation and the Markov chain Monte-Carlo method. Applying the theory on adaptive algorithms, we derive conditions under which the proposed procedure converges. Simulation studies also indicate that the proposed procedure consistently converges to the maximum likelihood estimate for the structural measurement error logistic regression model.

Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association

Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its amino terminus. Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron. The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3′-truncated transcripts. We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3′-end formation a short distance downstream. Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element in vitro with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation in vivo. A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1. Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth. Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient. Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1. In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function.

Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members

The cell cycle inhibitor p21/WAF1/Cip1 is expressed in many cell types and is regulated by p53-dependent and p53-independent mechanisms. p21 is an important regulator of hepatocyte cell cycle, differentiation, and liver development, but little is known about the regulation of its synthesis in hepatocytes. We report herein that the p21 gene is constitutively expressed in human hepatoma HepG2 cells. Deletion analysis of the p21 promoter showed that it contains a distal (positions −2,300/−210) and a proximal (positions −124 to −61) region that act synergistically to achieve high levels of constitutive expression. The proximal region that consists of multiple Sp1 binding sites is essential for constitutive p21 promoter activity in hepatocytes. This region also mediates the transcriptional activation of the p21 promoter by members of the Smad family of proteins, which play important role in the transduction of extracellular signals such as transforming growth factor β, activin, etc. Constitutive expression of p21 was severely reduced by a C-terminally truncated form of Smad4 that was shown previously to block signaling through Smads. Smad3/4 and to a much lesser extent Smad2/4 caused high levels of transcriptional activation of the p21 promoter. Transactivation was compromised by N- or C-terminally truncated forms of Smad3. By using Gal4-Sp1 fusion proteins, we show that Smad proteins can activate gene transcription via functional interactions with the ubiquitous factor Sp1. These data demonstrate that Smad proteins and Sp1 participate in the constitutive or inducible expression of the p21 gene in hepatic cells.

MgATP activates the β cell KATP channel by interaction with its SUR1 subunit

ATP-sensitive potassium (KATP) channels in the pancreatic β cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca2+ influx and exocytosis. Metabolic regulation of KATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The β cell KATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate KATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg2+ caused an apparent reduction in the inhibitory action of ATP on wild-type KATP channels, and MgATP actually activated KATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates KATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg2+.

Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA

Integration of transgenic DNA into the plant genome was investigated in 13 transgenic oat (Avena sativa L.) lines produced using microprojectile bombardment with one or two cotransformed plasmids. In all transformation events, the transgenic DNA integrated into the plant genome consisted of intact transgene copies that were accompanied by multiple, rearranged, and/or truncated transgene fragments. All fragments of transgenic DNA cosegregated, indicating that they were integrated at single gene loci. Analysis of the structure of the transgenic loci indicated that the transgenic DNA was interspersed by the host genomic DNA. The number of insertions of transgenic DNA within the transgene loci varied from 2 to 12 among the 13 lines. Restriction endonucleases that do not cleave the introduced plasmids produced restriction fragments ranging from 3.6 to about 60 kb in length hybridizing to a probe comprising the introduced plasmids. Although the size of the interspersing host DNA within the transgene locus is unknown, the sizes of the transgene-hybridizing restriction fragments indicated that the entire transgene locus must be at least from 35–280 kb. The observation that all transgenic lines analyzed exhibited genomic interspersion of multiple clustered transgenes suggests a predominating integration mechanism. We propose that transgene integration at multiple clustered DNA replication forks could account for the observed interspersion of transgenic DNA with host genomic DNA within transgenic loci.

Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease α-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of α-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH2, RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.

Assembly of a catalytic unit for RNA microhelix aminoacylation using nonspecific RNA binding domains

An assembly of a catalytic unit for aminoacylation of an RNA microhelix is demonstrated here. This assembly may recapitulate a step in the historical development of tRNA synthetases. The class-defining domain of a tRNA synthetase is closely related to the primordial enzyme that catalyzed synthesis of aminoacyl adenylate. RNA binding elements are imagined to have been added so that early RNA substrates could be docked proximal to the activated amino acid. RNA microhelices that recapitulate the acceptor stem of modern tRNAs are potential examples of early substrates. In this work, we examined a fragment of Escherichia coli alanyl-tRNA synthetase, which catalyzes aminoacyl adenylate formation but is virtually inactive for catalysis of RNA microhelix aminoacylation. Fusion to the fragment of either of two unrelated nonspecific RNA binding domains activated microhelix aminoacylation. Although the fusion proteins lacked the RNA sequence specificity of the natural enzyme, their activity was within 1–2 kcal⋅mol−1 of a truncated alanyl-tRNA synthetase that has aminoacylation activity sufficient to sustain cell growth. These results show that, starting with an activity for adenylate synthesis, barriers are relatively low for building catalytic units for aminoacylation of RNA helices.

RNase E is required for the maturation of ssrA RNA and normal ssrA RNA peptide-tagging activity

During recent studies of ribonucleolytic “degradosome” complexes of Escherichia coli, we found that degradosomes contain certain RNAs as well as RNase E and other protein components. One of these RNAs is ssrA (for small stable RNA) RNA (also known as tm RNA or 10Sa RNA), which functions as both a tRNA and mRNA to tag the C-terminal ends of truncated proteins with a short peptide and target them for degradation. Here, we show that mature 363-nt ssrA RNA is generated by RNase E cleavage at the CCA-3′ terminus of a 457-nt ssrA RNA precursor and that interference with this cleavage in vivo leads to accumulation of the precursor and blockage of SsrA-mediated proteolysis. These results demonstrate that RNase E is required to produce mature ssrA RNA and for normal ssrA RNA peptide-tagging activity. Our findings indicate that RNase E, an enzyme already known to have a central role in RNA processing and decay in E. coli, also has the previously unsuspected ability to affect protein degradation through its role in maturation of the 3′ end of ssrA RNA.