Stable isotope, seismic sequence boundaries and bioevents in ODP Hole 166-1006A

During ODP Leg 166, the recovery of cores from a transect of drill sites across the Bahamas margin from marginal to deep basin environments was an essential requirement for the study of the response of the sedimentary systems to sea-level changes. A detailed biostratigraphy based on planktonic foraminifera was performed on ODP Hole 1006A for an accurate stratigraphic control. The investigated late middle Miocene-early Pliocene sequence spans the interval from about 12.5 Ma (Biozone N12) to approximately 4.5 Ma (Biozone N19). Several bioevents calibrated with the time scale of Berggren et al. (1995a,b) were identified. The ODP Site 1006 benthic oxygen isotope stratigraphy can be correlated to the corresponding deep-water benthic oxygen isotope curve from ODP Site 846 in the Eastern Equatorial Pacific (Shackleton et al., 1995. Proc. ODP Sci. Res. 138, 337-356), which was orbitally tuned for the entire Pliocene into the latest Miocene at 6.0 Ma. The approximate stratigraphic match of the isotopic signals from both records between 4.5 and 6.0 Ma implies that the paleoceanographic signal from the Bahamas is not simply a record of regional variations but, indeed, represents glacio-eustatic fluctuations. The ODP Site 1006 oxygen and carbon isotope record, based on benthic and planktonic foraminifera, was used to define paleoceanographic changes on the margin, which could be tied to lithostratigraphic events on the Bahamas carbonate platform using seismic sequence stratigraphy. The oxygen isotope values show a general cooling trend from the middle to late Miocene, which was interrupted by a significant trend towards warmer sea-surface temperatures (SST) and associated sea-level rise with decreased ice volume during the latest Miocene. This trend reached a maximum coincident with the Miocene/Pliocene boundary. An abrupt cooling in the early Pliocene then followed the warming which continued into the earliest Pliocene. The late Miocene paleoceanographic evolution along the Bahamas margin can be observed in the ODP Site 1006 delta13C values, which support other evidence for the beginning of the closure of the Panama gateway at 8 Ma followed by a reduced intermediate water supply of water from the Pacific into the Caribbean at about 5 Ma. A general correlation of lower sedimentation rates with the major seismic sequence boundaries (SSBs) was observed. Additionally, the SSBs are associated with transitions towards more positive oxygen isotope excursions. This observed correspondence implies that the presence of a SSB, representing a density impedance contrast in the sedimentary sequence, may reflect changes in the character of the deposited sediment during highstands versus those during lowstands. However, not all of the recorded oxygen isotope excursions correspond to SSBs. The absence of a SSB in association with an oxygen isotope excursion indicates that not all oxygen isotope sea-level events impact the carbonate margin to the same extent, or maybe even represent equivalent sea-level fluctuations. Thus, it can be tentatively concluded that SSBs produced on carbonate margins do record sea-level fluctuations but not every sea-level fluctuation is represented by a SSB in the sequence stratigraphic record.

Geometric characteristics of conics in Bézier form

Derive coordinate-free expressions for geometric characteristics of conics written in Bézier form in terms of their control points and weights.

Analysis of the geometric altimetry to support aircraft optimal profiles within future 4D trajectory management

The use of barometric altimetry is to some extent a limiting factor on safety, predictability and efficiency of aircraft operations, and reduces the potential of the trajectory based operations capabilities. However, geometric altimetry could be used to improve all of these aspects. Nowadays aircraft altitude is estimated by applying the International Standard Atmosphere which differs from real altitude. At different temperatures for an assigned barometric altitude, aerodynamic forces are different and this has a direct relationship with time, fuel consumption and range of the flight. The study explores the feasibility of using sensors providing geometric reference altitude, in particular, to supply capabilities for the optimization of vertical profiles and also, their impact on the vertical Air Traffic Management separation assurance processes. One of the aims of the thesis is to assess if geometric altitude fulfils the aeronautical requirements through existing sensors. Also the thesis will elaborate on the advantages of geometric altitude over the barometric altitude in terms of efficiency for vertical navigation. The evidence that geometric altitude is the best choice to improve the efficiency in vertical profile and aircraft capacity by reducing vertical uncertainties will also be shown. In this paper, an atmospheric study is presented, as well as the impact of temperature deviation from International Standard Atmosphere model is analyzed in order to obtain relationship between geometric and barometric altitude. Furthermore, an aircraft model to study aircraft vertical profile is provided to analyse trajectories based on geometric altitudes.

Geometric characteristics of conics in Bézier form

Synthetic derivation of closed formulae of the geometric characteristic of a conic given in Bézier form in terms of its control polygon, (P; Q; R) and weights, (1; w; 1g)

Transductive neurofuzzy-based torque control of a milling process: results of a case study

This paper presents the design and implementation of an intelligent control system based on local neurofuzzy models of the milling process relayed through an Ehternet-based application. Its purpose is to control the spindle torque of a milling process by using an internal model control paradigm to modify the feed rate in real time. The stabilization of cutting cutting torque is especially necessary in milling processes such as high-spedd roughing of steel moulds and dies tha present minor geometric uncertainties. Thus, maintenance of the curring torque increaes the material removal rate and reduces the risk of damage due to excessive spindle vibration, a very sensitive and expensive component in all high-speed milling machines. Torque control is therefore an interesting challenge from an industrial point of view.

A unified framework for linear function approximation of value functions in stochastic control

This paper contributes with a unified formulation that merges previ- ous analysis on the prediction of the performance ( value function ) of certain sequence of actions ( policy ) when an agent operates a Markov decision process with large state-space. When the states are represented by features and the value function is linearly approxi- mated, our analysis reveals a new relationship between two common cost functions used to obtain the optimal approximation. In addition, this analysis allows us to propose an efficient adaptive algorithm that provides an unbiased linear estimate. The performance of the pro- posed algorithm is illustrated by simulation, showing competitive results when compared with the state-of-the-art solutions.

Contributions to trajectory prediction theory and its application to arrival management for air traffic control

La gestión del tráfico aéreo (Air Traffic Management, ATM) está experimentando un cambio de paradigma hacia las denominadas operaciones basadas trayectoria. Bajo dicho paradigma se modifica el papel de los controladores de tráfico aéreo desde una operativa basada su intervención táctica continuada hacia una labor de supervisión a más largo plazo. Esto se apoya en la creciente confianza en las soluciones aportadas por las herramientas automatizadas de soporte a la decisión más modernas. Para dar soporte a este concepto, se precisa una importante inversión para el desarrollo, junto con la adquisición de nuevos equipos en tierra y embarcados, que permitan la sincronización precisa de la visión de la trayectoria, basada en el intercambio de información entre ambos actores. Durante los últimos 30 a 40 años las aerolíneas han generado uno de los menores retornos de la inversión de entre todas las industrias. Sin beneficios tangibles, la industria aérea tiene dificultades para atraer el capital requerido para su modernización, lo que retrasa la implantación de dichas mejoras. Esta tesis tiene como objetivo responder a la pregunta de si las capacidades actualmente instaladas en las aeronaves comerciales se pueden aplicar para lograr la sincronización de la trayectoria con el nivel de calidad requerido. Además, se analiza en ella si, conjuntamente con mejoras en las herramientas de predicción trayectorias instaladas en tierra en para facilitar la gestión de las arribadas, dichas capacidades permiten obtener los beneficios esperados en el marco de las operaciones basadas en trayectoria. Esto podría proporcionar un incentivo para futuras actualizaciones de la aviónica que podrían llevar a mejoras adicionales. El concepto operacional propuesto en esta tesis tiene como objetivo permitir que los aviones sean pilotados de una manera consistente con las técnicas actuales de vuelo optimizado. Se permite a las aeronaves que desciendan en el denominado “modo de ángulo de descenso gestionado” (path-managed mode), que es el preferido por la mayoría de las compañías aéreas, debido a que conlleva un reducido consumo de combustible. El problema de este modo es que en él no se controla de forma activa el tiempo de llegada al punto de interés. En nuestro concepto operacional, la incertidumbre temporal se gestiona en mediante de la medición del tiempo en puntos estratégicamente escogidos a lo largo de la trayectoria de la aeronave, y permitiendo la modificación por el control de tierra de la velocidad de la aeronave. Aunque la base del concepto es la gestión de las ordenes de velocidad que se proporcionan al piloto, para ser capaces de operar con los niveles de equipamiento típicos actualmente, dicho concepto también constituye un marco en el que la aviónica más avanzada (por ejemplo, que permita el control por el FMS del tiempo de llegada) puede integrarse de forma natural, una vez que esta tecnología este instalada. Además de gestionar la incertidumbre temporal a través de la medición en múltiples puntos, se intenta reducir dicha incertidumbre al mínimo mediante la mejora de las herramienta de predicción de la trayectoria en tierra. En esta tesis se presenta una novedosa descomposición del proceso de predicción de trayectorias en dos etapas. Dicha descomposición permite integrar adecuadamente los datos de la trayectoria de referencia calculada por el Flight Management System (FMS), disponibles usando Futuro Sistema de Navegación Aérea (FANS), en el sistema de predicción de trayectorias en tierra. FANS es un equipo presente en los aviones comerciales de fuselaje ancho actualmente en la producción, e incluso algunos aviones de fuselaje estrecho pueden tener instalada avionica FANS. Además de informar automáticamente de la posición de la aeronave, FANS permite proporcionar (parte de) la trayectoria de referencia en poder de los FMS, pero la explotación de esta capacidad para la mejora de la predicción de trayectorias no se ha estudiado en profundidad en el pasado. La predicción en dos etapas proporciona una solución adecuada al problema de sincronización de trayectorias aire-tierra dado que permite la sincronización de las dimensiones controladas por el sistema de guiado utilizando la información de la trayectoria de referencia proporcionada mediante FANS, y también facilita la mejora en la predicción de las dimensiones abiertas restantes usado un modelo del guiado que explota los modelos meteorológicos mejorados disponibles en tierra. Este proceso de predicción de la trayectoria de dos etapas se aplicó a una muestra de 438 vuelos reales que realizaron un descenso continuo (sin intervención del controlador) con destino Melbourne. Dichos vuelos son de aeronaves del modelo Boeing 737-800, si bien la metodología descrita es extrapolable a otros tipos de aeronave. El método propuesto de predicción de trayectorias permite una mejora en la desviación estándar del error de la estimación del tiempo de llegada al punto de interés, que es un 30% menor que la que obtiene el FMS. Dicha trayectoria prevista mejorada se puede utilizar para establecer la secuencia de arribadas y para la asignación de las franjas horarias para cada aterrizaje (slots). Sobre la base del slot asignado, se determina un perfil de velocidades que permita cumplir con dicho slot con un impacto mínimo en la eficiencia del vuelo. En la tesis se propone un nuevo algoritmo que determina las velocidades requeridas sin necesidad de un proceso iterativo de búsqueda sobre el sistema de predicción de trayectorias. El algoritmo se basa en una parametrización inteligente del proceso de predicción de la trayectoria, que permite relacionar el tiempo estimado de llegada con una función polinómica. Resolviendo dicho polinomio para el tiempo de llegada deseado, se obtiene de forma natural el perfil de velocidades optimo para cumplir con dicho tiempo de llegada sin comprometer la eficiencia. El diseño de los sistemas de gestión de arribadas propuesto en esta tesis aprovecha la aviónica y los sistemas de comunicación instalados de un modo mucho más eficiente, proporcionando valor añadido para la industria. Por tanto, la solución es compatible con la transición hacia los sistemas de aviónica avanzados que están desarrollándose actualmente. Los beneficios que se obtengan a lo largo de dicha transición son un incentivo para inversiones subsiguientes en la aviónica y en los sistemas de control de tráfico en tierra. ABSTRACT Air traffic management (ATM) is undergoing a paradigm shift towards trajectory based operations where the role of an air traffic controller evolves from that of continuous intervention towards supervision, as decision making is improved based on increased confidence in the solutions provided by advanced automation. To support this concept, significant investment for the development and acquisition of new equipment is required on the ground as well as in the air, to facilitate the high degree of trajectory synchronisation and information exchange required. Over the past 30-40 years the airline industry has generated one of the lowest returns on invested capital among all industries. Without tangible benefits realised, the airline industry may find it difficult to attract the required investment capital and delay acquiring equipment needed to realise the concept of trajectory based operations. In response to these challenges facing the modernisation of ATM, this thesis aims to answer the question whether existing aircraft capabilities can be applied to achieve sufficient trajectory synchronisation and improvements to ground-based trajectory prediction in support of the arrival management process, to realise some of the benefits envisioned under trajectory based operations, and to provide an incentive for further avionics upgrades. The proposed operational concept aims to permit aircraft to operate in a manner consistent with current optimal aircraft operating techniques. It allows aircraft to descend in the fuel efficient path managed mode as preferred by a majority of airlines, with arrival time not actively controlled by the airborne automation. The temporal uncertainty is managed through metering at strategically chosen points along the aircraft’s trajectory with primary use of speed advisories. While the focus is on speed advisories to support all aircraft and different levels of equipage, the concept also constitutes a framework in which advanced avionics as airborne time-of-arrival control can be integrated once this technology is widely available. In addition to managing temporal uncertainty through metering at multiple points, this temporal uncertainty is minimised by improving the supporting trajectory prediction capability. A novel two-stage trajectory prediction process is presented to adequately integrate aircraft trajectory data available through Future Air Navigation Systems (FANS) into the ground-based trajectory predictor. FANS is standard equipment on any wide-body aircraft in production today, and some single-aisle aircraft are easily capable of being fitted with FANS. In addition to automatic position reporting, FANS provides the ability to provide (part of) the reference trajectory held by the aircraft’s Flight Management System (FMS), but this capability has yet been widely overlooked. The two-stage process provides a ‘best of both world’s’ solution to the air-ground synchronisation problem by synchronising with the FMS reference trajectory those dimensions controlled by the guidance mode, and improving on the prediction of the remaining open dimensions by exploiting the high resolution meteorological forecast available to a ground-based system. The two-stage trajectory prediction process was applied to a sample of 438 FANS-equipped Boeing 737-800 flights into Melbourne conducting a continuous descent free from ATC intervention, and can be extrapolated to other types of aircraft. Trajectories predicted through the two-stage approach provided estimated time of arrivals with a 30% reduction in standard deviation of the error compared to estimated time of arrival calculated by the FMS. This improved predicted trajectory can subsequently be used to set the sequence and allocate landing slots. Based on the allocated landing slot, the proposed system calculates a speed schedule for the aircraft to meet this landing slot at minimal flight efficiency impact. A novel algorithm is presented that determines this speed schedule without requiring an iterative process in which multiple calls to a trajectory predictor need to be made. The algorithm is based on parameterisation of the trajectory prediction process, allowing the estimate time of arrival to be represented by a polynomial function of the speed schedule, providing an analytical solution to the speed schedule required to meet a set arrival time. The arrival management solution proposed in this thesis leverages the use of existing avionics and communications systems resulting in new value for industry for current investment. The solution therefore supports a transition concept from mixed equipage towards advanced avionics currently under development. Benefits realised under this transition may provide an incentive for ongoing investment in avionics.

Analysis of the geometric altimetry to support aircraft optimal trajectories within the future 4d trajectory management

Over the past few years, the common practice within air traffic management has been that commercial aircraft fly by following a set of predefined routes to reach their destination. Currently, aircraft operators are requesting more flexibility to fly according to their prefer- ences, in order to achieve their business objectives. Due to this reason, much research effort is being invested in developing different techniques which evaluate aircraft optimal trajectory and traffic synchronisation. Also, the inefficient use of the airspace using barometric altitude overall in the landing and takeoff phases or in Continuous Descent Approach (CDA) trajectories where currently it is necessary introduce the necessary reference setting (QNH or QFE). To solve this problem and to permit a better airspace management born the interest of this research. Where the main goals will be to evaluate the impact, weakness and strength of the use of geometrical altitude instead of the use of barometric altitude. Moreover, this dissertation propose the design a simplified trajectory simulator which is able to predict aircraft trajectories. The model is based on a three degrees of freedom aircraft point mass model that can adapt aircraft performance data from Base of Aircraft Data, and meteorological information. A feature of this trajectory simulator is to support the improvement of the strategic and pre-tactical trajectory planning in the future Air Traffic Management. To this end, the error of the tool (aircraft Trajectory Simulator) is measured by comparing its performance variables with actual flown trajectories obtained from Flight Data Recorder information. The trajectory simulator is validated by analysing the performance of different type of aircraft and considering different routes. A fuel consumption estimation error was identified and a correction is proposed for each type of aircraft model. In the future Air Traffic Management (ATM) system, the trajectory becomes the fundamental element of a new set of operating procedures collectively referred to as Trajectory-Based Operations (TBO). Thus, governmental institutions, academia, and industry have shown a renewed interest for the application of trajectory optimisation techniques in com- mercial aviation. The trajectory optimisation problem can be solved using optimal control methods. In this research we present and discuss the existing methods for solving optimal control problems focusing on direct collocation, which has received recent attention by the scientific community. In particular, two families of collocation methods are analysed, i.e., Hermite-Legendre-Gauss-Lobatto collocation and the pseudospectral collocation. They are first compared based on a benchmark case study: the minimum fuel trajectory problem with fixed arrival time. For the sake of scalability to more realistic problems, the different meth- ods are also tested based on a real Airbus 319 El Cairo-Madrid flight. Results show that pseudospectral collocation, which has shown to be numerically more accurate and computa- tionally much faster, is suitable for the type of problems arising in trajectory optimisation with application to ATM. Fast and accurate optimal trajectory can contribute properly to achieve the new challenges of the future ATM. As atmosphere uncertainties are one of the most important issues in the trajectory plan- ning, the final objective of this dissertation is to have a magnitude order of how different is the fuel consumption under different atmosphere condition. Is important to note that in the strategic phase planning the optimal trajectories are determined by meteorological predictions which differ from the moment of the flight. The optimal trajectories have shown savings of at least 500 [kg] in the majority of the atmosphere condition (different pressure, and temperature at Mean Sea Level, and different lapse rate temperature) with respect to the conventional procedure simulated at the same atmosphere condition.This results show that the implementation of optimal profiles are beneficial under the current Air traffic Management (ATM).

Global instability analysis and control of three-dimensional long open cavity flow

The three-dimensional wall-bounded open cavity may be considered as a simplified geometry found in industrial applications such as leading gear or slotted flats on the airplane. Understanding the three-dimensional complex flow structure that surrounds this particular geometry is therefore of major industrial interest. At the light of the remarkable former investigations in this kind of flows, enough evidences suggest that the lateral walls have a great influence on the flow features and hence on their instability modes. Nevertheless, even though there is a large body of literature on cavity flows, most of them are based on the assumption that the flow is two-dimensional and spanwise-periodic. The flow over realistic open cavity should be considered. This thesis presents an investigation of three-dimensional wall-bounded open cavity with geometric ratio 6:2:1. To this aim, three-dimensional Direct Numerical Simulation (DNS) and global linear instability have been performed. Linear instability analysis reveals that the onset of the first instability in this open cavity is around Recr 1080. The three-dimensional shear layer mode with a complex structure is shown to be the most unstable mode. I t is noteworthy that the flow pattern of this high-frequency shear layer mode is similar to the observed unstable oscillations in supercritical unstable case. DNS of the cavity flow carried out at different Reynolds number from steady state until a nonlinear saturated state is obtained. The comparison of time histories of kinetic energy presents a clearly dominant energetic mode which shifts between low-frequency and highfrequency oscillation. A complete flow patterns from subcritical cases to supercritical case has been put in evidence. The flow structure at the supercritical case Re=1100 resembles typical wake-shedding instability oscillations with a lateral motion existed in the subcritical cases. Also, This flow pattern is similar to the observations in experiments. In order to validate the linear instability analysis results, the topology of the composite flow fields reconstructed by linear superposition of a three-dimensional base flow and its leading three-dimensional global eigenmodes has been studied. The instantaneous wall streamlines of those composited flows display distinguish influence region of each eigenmode. Attention has been focused on the leading high-frequency shear layer mode; the composite flow fields have been fully recognized with respect to the downstream wave shedding. The three-dimensional shear layer mode is shown to give rise to a typical wake-shedding instability with a lateral motions occurring downstream which is in good agreement with the experiment results. Moreover, the spanwise-periodic, open cavity with the same length to depth ratio has been also studied. The most unstable linear mode is different from the real three-dimensional cavity flow, because of the existence of the side walls. Structure sensitivity of the unstable global mode is analyzed in the flow control context. The adjoint-based sensitivity analysis has been employed to localized the receptivity region, where the flow is more sensible to momentum forcing and mass injection. Because of the non-normality of the linearized Navier-Stokes equations, the direct and adjoint field has a large spatial separation. The strongest sensitivity region is locate in the upstream lip of the three-dimensional cavity. This numerical finding is in agreement with experimental observations. Finally, a prototype of passive flow control strategy is applied.

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.

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.

rRNA complementarity within mRNAs: A possible basis for mRNA-ribosome interactions and translational control

Our recent demonstration that many eukaryotic mRNAs contain sequences complementary to rRNA led to the hypothesis that these sequences might mediate specific interactions between mRNAs and ribosomes and thereby affect translation. In the present experiments, the ability of complementary sequences to bind to rRNA was investigated by using photochemical cross-linking. RNA probes with perfect complementarity to 18S or 28S rRNA were shown to cross-link specifically to the corresponding rRNA within intact ribosomal subunits. Similar results were obtained by using probes based on natural mRNA sequences with varying degrees of complementarity to the 18S rRNA. RNase H cleavage localized four such probes to complementary regions of the 18S rRNA. The effects of complementarity on translation were assessed by using the mRNA encoding ribosomal protein S15. This mRNA contains a sequence within its coding region that is complementary to the 18S rRNA at 20 of 22 nucleotides. RNA from an S15-luciferase fusion construct was translated in a cell-free lysate and compared with the translation of four related constructs that were mutated to decrease complementarity to the 18S rRNA. These mutations did not alter the amino acid sequence or the codon bias. A correlation between complementarity and translation was observed; constructs with less complementarity increased the amount of translation up to 54%. These findings raised the possibility that direct base-pairing of particular mRNAs to rRNAs within ribosomes may function as a mechanism of translational control.

Orientation-dependent and sequence-specific expansions of CTG/CAG trinucleotide repeats in Saccharomyces cerevisiae

A quantitative and selective genetic assay was developed to monitor expansions of trinucleotide repeats (TNRs) in yeast. A promoter containing 25 repeats allows expression of a URA3 reporter gene and yields sensitivity to the drug 5-fluoroorotic acid. Expansion of the TNR to 30 or more repeats turns off URA3 and provides drug resistance. When integrated at either of two chromosomal loci, expansion rates were 1 × 10−5 to 4 × 10−5 per generation if CTG repeats were replicated on the lagging daughter strand. PCR analysis indicated that 5–28 additional repeats were present in 95% of the expanded alleles. No significant changes in CTG expansion rates occurred in strains deficient in the mismatch repair gene MSH2 or the recombination gene RAD52. The frequent nature of CTG expansions suggests that the threshold number for this repeat is below 25 in this system. In contrast, expansions of the complementary repeat CAG occurred at 500- to 1,000-fold lower rates, similar to a randomized (C,A,G) control sequence. When the reporter plasmid was inverted within the chromosome, switching the leading and lagging strands of replication, frequent expansions were observed only when CTG repeats resided on the lagging daughter strand. Among the rare CAG expansions, the largest gain in tract size was 38 repeats. The control repeats CTA and TAG showed no detectable rate of expansions. The orientation-dependence and sequence-specificity data support the model that expansions of CTG and CAG tracts result from aberrant DNA replication via hairpin-containing Okazaki fragments.

Molecular keys to speciation: DNA polymorphism and the control of genetic exchange in enterobacteria

Speciation involves the establishment of genetic barriers between closely related organisms. The extent of genetic recombination is a key determinant and a measure of genetic isolation. The results reported here reveal that genetic barriers can be established, eliminated, or modified by manipulating two systems which control genetic recombination, SOS and mismatch repair. The extent of genetic isolation between enterobacteria is a simple mathematical function of DNA sequence divergence. The function does not depend on hybrid DNA stability, but rather on the number of blocks of sequences identical in the two mating partners and sufficiently large to allow the initiation of recombination. Further, there is no obvious discontinuity in the function that could be used to define a level of divergence for distinguishing species.

Endoplasmic reticulum quality control of asialoglycoprotein receptor H2a involves a determinant for retention and not retrieval

The human asialoglycoprotein receptor H2a subunit contains a charged pentapeptide, EGHRG, in its ectodomain that is the only sequence absent from the H2b alternatively spliced variant. H2b exits the endoplasmic reticulum (ER) even when singly expressed, whereas H2a gives rise to a cleaved soluble secreted ectodomain fragment; uncleaved membrane-bound H2a molecules are completely retained and degraded in the ER. We have inserted the H2a pentapeptide into the sequence of the H1 subunit (H1i5), which caused complete ER retention but, unexpectedly, no degradation. This suggests that the pentapeptide is a determinant for ER retention not colocalizing in H2a with the determinant for degradation. The state of sugar chain processing and the ER localization of H1i5, which was unchanged at 15°C or after treatment with nocodazole, indicate ER retention and not retrieval from the cis-Golgi or the intermediate compartment. H1i5 folded similarly to H1, and both associated to calnexin. However, whereas H1 dissociated with a half time of 45 min, H1i5 remained bound to the chaperone for prolonged periods. The correct global folding of H2a and H1i5 and of other normal precursors and unassembled proteins and the true ER retention, and not exit and retrieval, suggest a difference in their quality control mechanism compared with that of misfolded proteins, which does involve retrieval. However, both pathways may involve calnexin.