Tri a 14, wheat Lipid Transfer Protein, a marker of wheat respiratory allergy

Over 30 wheat allergens have been associated to baker’s asthma and much of them have been also implied in food allergy. Few of them have rendered as major allergens. Tri a 14, wheat LTP, has been associated to baker’s asthma as major allergen in patients that can consume peach and wheat derived foodstuffs. In Spanish baker’s asthma patients, 60% showed positive response to Tri a 14 and 45% to Pru p 3. However, the cross-reactivity between peach and wheat has been unusual in allergic population (1,8). Moreover, wheat allergy is not so often as should be attending to the high consume.

On the simulation of the UPMSAT-2 microsatellite power

Simulation of satellite subsystems behaviour is extramely important in the design at early stages. The subsystems are normally simulated in the both ways : isolated and as part of more complex simulation that takes into account imputs from other subsystems (concurrent design). In the present work, a simple concurrent simulation of the power subsystem of a microsatellite, UPMSat-2, is described. The aim of the work is to obtain the performance profile of the system (battery charging level, power consumption by the payloads, power supply from solar panels....). Different situations such as battery critical low or high level, effects of high current charging due to the low temperature of solar panels after eclipse,DoD margins..., were analysed, and different safety strategies studied using the developed tool (simulator) to fulfil the mission requirements. Also, failure cases were analysed in order to study the robustness of the system. The mentioned simulator has been programed taking into account the power consumption performances (average and maximum consumptions per orbit/day) of small part of the subsystem (SELEX GALILEO SPVS modular generators built with Azur Space solar cells, SAFT VES16 6P4S Li-ion battery, SSBV magnetometers, TECNOBIT and DATSI/UPM On Board Data Handling -OBDH-...). The developed tool is then intended to be a modular simulator, with the chance of use any other components implementing some standard data.

Multidisciplinary Design Optimization Application to Conceptual Design of University-class Microsatellite Projects

Esta tesis se ha realizado en el contexto del proyecto UPMSat-2, que es un microsatélite diseñado, construido y operado por el Instituto Universitario de Microgravedad "Ignacio Da Riva" (IDR / UPM) de la Universidad Politécnica de Madrid. Aplicación de la metodología Ingeniería Concurrente (Concurrent Engineering: CE) en el marco de la aplicación de diseño multidisciplinar (Multidisciplinary Design Optimization: MDO) es uno de los principales objetivos del presente trabajo. En los últimos años, ha habido un interés continuo en la participación de los grupos de investigación de las universidades en los estudios de la tecnología espacial a través de sus propios microsatélites. La participación en este tipo de proyectos tiene algunos desafíos inherentes, tales como presupuestos y servicios limitados. Además, debido al hecho de que el objetivo principal de estos proyectos es fundamentalmente educativo, por lo general hay incertidumbres en cuanto a su misión en órbita y cargas útiles en las primeras fases del proyecto. Por otro lado, existen limitaciones predeterminadas para sus presupuestos de masa, volumen y energía, debido al hecho de que la mayoría de ellos están considerados como una carga útil auxiliar para el lanzamiento. De este modo, el costo de lanzamiento se reduce considerablemente. En este contexto, el subsistema estructural del satélite es uno de los más afectados por las restricciones que impone el lanzador. Esto puede afectar a diferentes aspectos, incluyendo las dimensiones, la resistencia y los requisitos de frecuencia. En la primera parte de esta tesis, la atención se centra en el desarrollo de una herramienta de diseño del subsistema estructural que evalúa, no sólo las propiedades de la estructura primaria como variables, sino también algunas variables de nivel de sistema del satélite, como la masa de la carga útil y la masa y las dimensiones extremas de satélite. Este enfoque permite que el equipo de diseño obtenga una mejor visión del diseño en un espacio de diseño extendido. La herramienta de diseño estructural se basa en las fórmulas y los supuestos apropiados, incluyendo los modelos estáticos y dinámicos del satélite. Un algoritmo genético (Genetic Algorithm: GA) se aplica al espacio de diseño para optimizaciones de objetivo único y también multiobjetivo. El resultado de la optimización multiobjetivo es un Pareto-optimal basado en dos objetivo, la masa total de satélites mínimo y el máximo presupuesto de masa de carga útil. Por otro lado, la aplicación de los microsatélites en misiones espaciales es de interés por su menor coste y tiempo de desarrollo. La gran necesidad de las aplicaciones de teledetección es un fuerte impulsor de su popularidad en este tipo de misiones espaciales. Las misiones de tele-observación por satélite son esenciales para la investigación de los recursos de la tierra y el medio ambiente. En estas misiones existen interrelaciones estrechas entre diferentes requisitos como la altitud orbital, tiempo de revisita, el ciclo de vida y la resolución. Además, todos estos requisitos puede afectar a toda las características de diseño. Durante los últimos años la aplicación de CE en las misiones espaciales ha demostrado una gran ventaja para llegar al diseño óptimo, teniendo en cuenta tanto el rendimiento y el costo del proyecto. Un ejemplo bien conocido de la aplicación de CE es la CDF (Facilidad Diseño Concurrente) de la ESA (Agencia Espacial Europea). Está claro que para los proyectos de microsatélites universitarios tener o desarrollar una instalación de este tipo parece estar más allá de las capacidades del proyecto. Sin embargo, la práctica de la CE a cualquier escala puede ser beneficiosa para los microsatélites universitarios también. En la segunda parte de esta tesis, la atención se centra en el desarrollo de una estructura de optimización de diseño multidisciplinar (Multidisciplinary Design Optimization: MDO) aplicable a la fase de diseño conceptual de microsatélites de teledetección. Este enfoque permite que el equipo de diseño conozca la interacción entre las diferentes variables de diseño. El esquema MDO presentado no sólo incluye variables de nivel de sistema, tales como la masa total del satélite y la potencia total, sino también los requisitos de la misión como la resolución y tiempo de revisita. El proceso de diseño de microsatélites se divide en tres disciplinas; a) diseño de órbita, b) diseño de carga útil y c) diseño de plataforma. En primer lugar, se calculan diferentes parámetros de misión para un rango práctico de órbitas helio-síncronas (sun-synchronous orbits: SS-Os). Luego, según los parámetros orbitales y los datos de un instrumento como referencia, se calcula la masa y la potencia de la carga útil. El diseño de la plataforma del satélite se estima a partir de los datos de la masa y potencia de los diferentes subsistemas utilizando relaciones empíricas de diseño. El diseño del subsistema de potencia se realiza teniendo en cuenta variables de diseño más detalladas, como el escenario de la misión y diferentes tipos de células solares y baterías. El escenario se selecciona, de modo de obtener una banda de cobertura sobre la superficie terrestre paralelo al Ecuador después de cada intervalo de revisita. Con el objetivo de evaluar las interrelaciones entre las diferentes variables en el espacio de diseño, todas las disciplinas de diseño mencionados se combinan en un código unificado. Por último, una forma básica de MDO se ajusta a la herramienta de diseño de sistema de satélite. La optimización del diseño se realiza por medio de un GA con el único objetivo de minimizar la masa total de microsatélite. Según los resultados obtenidos de la aplicación del MDO, existen diferentes puntos de diseños óptimos, pero con diferentes variables de misión. Este análisis demuestra la aplicabilidad de MDO para los estudios de ingeniería de sistema en la fase de diseño conceptual en este tipo de proyectos. La principal conclusión de esta tesis, es que el diseño clásico de los satélites que por lo general comienza con la definición de la misión y la carga útil no es necesariamente la mejor metodología para todos los proyectos de satélites. Un microsatélite universitario, es un ejemplo de este tipo de proyectos. Por eso, se han desarrollado un conjunto de herramientas de diseño para encarar los estudios de la fase inicial de diseño. Este conjunto de herramientas incluye diferentes disciplinas de diseño centrados en el subsistema estructural y teniendo en cuenta una carga útil desconocida a priori. Los resultados demuestran que la mínima masa total del satélite y la máxima masa disponible para una carga útil desconocida a priori, son objetivos conflictivos. En este contexto para encontrar un Pareto-optimal se ha aplicado una optimización multiobjetivo. Según los resultados se concluye que la selección de la masa total por satélite en el rango de 40-60 kg puede considerarse como óptima para un proyecto de microsatélites universitario con carga útil desconocida a priori. También la metodología CE se ha aplicado al proceso de diseño conceptual de microsatélites de teledetección. Los resultados de la aplicación del CE proporcionan una clara comprensión de la interacción entre los requisitos de diseño de sistemas de satélites, tales como la masa total del microsatélite y la potencia y los requisitos de la misión como la resolución y el tiempo de revisita. La aplicación de MDO se hace con la minimización de la masa total de microsatélite. Los resultados de la aplicación de MDO aclaran la relación clara entre los diferentes requisitos de diseño del sistema y de misión, así como que permiten seleccionar las líneas de base para el diseño óptimo con el objetivo seleccionado en las primeras fase de diseño. ABSTRACT This thesis is done in the context of UPMSat-2 project, which is a microsatellite under design and manufacturing at the Instituto Universitario de Microgravedad “Ignacio Da Riva” (IDR/UPM) of the Universidad Politécnica de Madrid. Application of Concurrent Engineering (CE) methodology in the framework of Multidisciplinary Design application (MDO) is one of the main objectives of the present work. In recent years, there has been continuing interest in the participation of university research groups in space technology studies by means of their own microsatellites. The involvement in such projects has some inherent challenges, such as limited budget and facilities. Also, due to the fact that the main objective of these projects is for educational purposes, usually there are uncertainties regarding their in orbit mission and scientific payloads at the early phases of the project. On the other hand, there are predetermined limitations for their mass and volume budgets owing to the fact that most of them are launched as an auxiliary payload in which the launch cost is reduced considerably. The satellite structure subsystem is the one which is most affected by the launcher constraints. This can affect different aspects, including dimensions, strength and frequency requirements. In the first part of this thesis, the main focus is on developing a structural design sizing tool containing not only the primary structures properties as variables but also the satellite system level variables such as payload mass budget and satellite total mass and dimensions. This approach enables the design team to obtain better insight into the design in an extended design envelope. The structural design sizing tool is based on the analytical structural design formulas and appropriate assumptions including both static and dynamic models of the satellite. A Genetic Algorithm (GA) is applied to the design space for both single and multiobejective optimizations. The result of the multiobjective optimization is a Pareto-optimal based on two objectives, minimum satellite total mass and maximum payload mass budget. On the other hand, the application of the microsatellites is of interest for their less cost and response time. The high need for the remote sensing applications is a strong driver of their popularity in space missions. The satellite remote sensing missions are essential for long term research around the condition of the earth resources and environment. In remote sensing missions there are tight interrelations between different requirements such as orbital altitude, revisit time, mission cycle life and spatial resolution. Also, all of these requirements can affect the whole design characteristics. During the last years application of the CE in the space missions has demonstrated a great advantage to reach the optimum design base lines considering both the performance and the cost of the project. A well-known example of CE application is ESA (European Space Agency) CDF (Concurrent Design Facility). It is clear that for the university-class microsatellite projects having or developing such a facility seems beyond the project capabilities. Nevertheless practicing CE at any scale can be beneficiary for the university-class microsatellite projects. In the second part of this thesis, the main focus is on developing a MDO framework applicable to the conceptual design phase of the remote sensing microsatellites. This approach enables the design team to evaluate the interaction between the different system design variables. The presented MDO framework contains not only the system level variables such as the satellite total mass and total power, but also the mission requirements like the spatial resolution and the revisit time. The microsatellite sizing process is divided into the three major design disciplines; a) orbit design, b) payload sizing and c) bus sizing. First, different mission parameters for a practical range of sun-synchronous orbits (SS-Os) are calculated. Then, according to the orbital parameters and a reference remote sensing instrument, mass and power of the payload are calculated. Satellite bus sizing is done based on mass and power calculation of the different subsystems using design estimation relationships. In the satellite bus sizing, the power subsystem design is realized by considering more detailed design variables including a mission scenario and different types of solar cells and batteries. The mission scenario is selected in order to obtain a coverage belt on the earth surface parallel to the earth equatorial after each revisit time. In order to evaluate the interrelations between the different variables inside the design space all the mentioned design disciplines are combined in a unified code. The integrated satellite system sizing tool developed in this section is considered as an application of the CE to the conceptual design of the remote sensing microsatellite projects. Finally, in order to apply the MDO methodology to the design problem, a basic MDO framework is adjusted to the developed satellite system design tool. Design optimization is done by means of a GA single objective algorithm with the objective function as minimizing the microsatellite total mass. According to the results of MDO application, there exist different optimum design points all with the minimum satellite total mass but with different mission variables. This output demonstrates the successful applicability of MDO approach for system engineering trade-off studies at the conceptual design phase of the design in such projects. The main conclusion of this thesis is that the classical design approach for the satellite design which usually starts with the mission and payload definition is not necessarily the best approach for all of the satellite projects. The university-class microsatellite is an example for such projects. Due to this fact an integrated satellite sizing tool including different design disciplines focusing on the structural subsystem and considering unknown payload is developed. According to the results the satellite total mass and available mass for the unknown payload are conflictive objectives. In order to find the Pareto-optimal a multiobjective GA optimization is conducted. Based on the optimization results it is concluded that selecting the satellite total mass in the range of 40-60 kg can be considered as an optimum approach for a university-class microsatellite project with unknown payload(s). Also, the CE methodology is applied to the remote sensing microsatellites conceptual design process. The results of CE application provide a clear understanding of the interaction between satellite system design requirements such as satellite total mass and power and the satellite mission variables such as revisit time and spatial resolution. The MDO application is done with the total mass minimization of a remote sensing satellite. The results from the MDO application clarify the unclear relationship between different system and mission design variables as well as the optimum design base lines according to the selected objective during the initial design phases.

Mapping Genetic Diversity of Cherimoya (Annona cherimola Mill.): Application of Spatial Analysis for Conservation and Use of Plant Genetic Resources.

There is a growing call for inventories that evaluate geographic patterns in diversity of plant genetic resources maintained on farm and in species' natural populations in order to enhance their use and conservation. Such evaluations are relevant for useful tropical and subtropical tree species, as many of these species are still undomesticated, or in incipient stages of domestication and local populations can offer yet-unknown traits of high value to further domestication. For many outcrossing species, such as most trees, inbreeding depression can be an issue, and genetic diversity is important to sustain local production. Diversity is also crucial for species to adapt to environmental changes. This paper explores the possibilities of incorporating molecular marker data into Geographic Information Systems (GIS) to allow visualization and better understanding of spatial patterns of genetic diversity as a key input to optimize conservation and use of plant genetic resources, based on a case study of cherimoya (Annona cherimola Mill.), a Neotropical fruit tree species. We present spatial analyses to (1) improve the understanding of spatial distribution of genetic diversity of cherimoya natural stands and cultivated trees in Ecuador, Bolivia and Peru based on microsatellite molecular markers (SSRs); and (2) formulate optimal conservation strategies by revealing priority areas for in situ conservation, and identifying existing diversity gaps in ex situ collections. We found high levels of allelic richness, locally common alleles and expected heterozygosity in cherimoya's putative centre of origin, southern Ecuador and northern Peru, whereas levels of diversity in southern Peru and especially in Bolivia were significantly lower. The application of GIS on a large microsatellite dataset allows a more detailed prioritization of areas for in situ conservation and targeted collection across the Andean distribution range of cherimoya than previous studies could do, i.e. at province and department level in Ecuador and Peru, respectively.

Artificial analysis of molecular marker loci linked to tree resistance response by an artificial neural network

One of the biggest challenges that software developers face is to make an accurate estimate of the project effort. Radial basis function neural networks have been used to software effort estimation in this work using NASA dataset. This paper evaluates and compares radial basis function versus a regression model. The results show that radial basis function neural network have obtained less Mean Square Error than the regression method.

Analytical models for the power subsystem and the attitude control subsystem of a microsatellite

El trabajo realizado en la presente tesis doctoral se debe considerar parte del proyecto UPMSat-2, que se enmarca dentro del ámbito de la tecnología aeroespacial. El UPMSat-2 es un microsatélite (de bajo coste y pequeño tamaño) diseñado, construido, probado e integrado por la Universidad Politécnica de Madrid (España), para fines de demostración tecnológica y educación. El objetivo de la presente tesis doctoral es presentar nuevos modelos analíticos para estudiar la interdependencia energética entre los subsistemas de potencia y de control de actitud de un satélite. En primer lugar, se estudia la simulación del subsistema de potencia de un microsatélite, prestando especial atención a la simulación de la fuente de potencia, esto es, los paneles solares. En la tesis se presentan métodos sencillos pero precisos para simular la producción de energía de los paneles en condiciones ambientales variables a través de su circuito equivalente. Los métodos propuestos para el cálculo de los parámetros del circuito equivalente son explícitos (o al menos, con las variables desacopladas), no iterativos y directos; no se necesitan iteraciones o valores iniciales para calcular los parámetros. La precisión de este método se prueba y se compara con métodos similares de la literatura disponible, demostrando una precisión similar para mayor simplicidad. En segundo lugar, se presenta la simulación del subsistema de control de actitud de un microsatélite, prestando especial atención a la nueva ley de control propuesta. La tesis presenta un nuevo tipo de control magnético es aplicable a la órbita baja terrestre (LEO). La ley de control propuesta es capaz de ajustar la velocidad de rotación del satélite alrededor de su eje principal de inercia máximo o mínimo. Además, en el caso de órbitas de alta inclinación, la ley de control favorece la alineación del eje de rotación con la dirección normal al plano orbital. El algoritmo de control propuesto es simple, sólo se requieren magnetopares como actuadores; sólo se requieren magnetómetros como sensores; no hace falta estimar la velocidad angular; no incluye un modelo de campo magnético de la Tierra; no tiene por qué ser externamente activado con información sobre las características orbitales y permite el rearme automático después de un apagado total del subsistema de control de actitud. La viabilidad teórica de la citada ley de control se demuestra a través de análisis de Monte Carlo. Por último, en términos de producción de energía, se demuestra que la actitud propuesto (en eje principal perpendicular al plano de la órbita, y el satélite que gira alrededor de ella con una velocidad controlada) es muy adecuado para la misión UPMSat-2, ya que permite una área superior de los paneles apuntando hacia el sol cuando se compara con otras actitudes estudiadas. En comparación con el control de actitud anterior propuesto para el UPMSat-2 resulta en un incremento de 25% en la potencia disponible. Además, la actitud propuesto mostró mejoras significativas, en comparación con otros, en términos de control térmico, como la tasa de rotación angular por satélite puede seleccionarse para conseguir una homogeneización de la temperatura más alta que apunta satélite y la antena. ABSTRACT The work carried out in the present doctoral dissertation should be considered part of the UPMSat-2 project, falling within the scope of the aerospace technology. The UPMSat-2 is a microsatellite (low cost and small size) designed, constructed integrated and tested for educational and technology demonstration purposes at the Universidad Politécnica de Madrid (Spain). The aim of the present doctoral dissertation is to present new analytical models to study the energy interdependence between the power and the attitude control subsystems of a satellite. First, the simulation of the power subsystem of a microsatellite is studied, paying particular attention to the simulation of the power supply, i.e. the solar panels. Simple but accurate methods for simulate the power production under variable ambient conditions using its equivalent circuit are presented. The proposed methods for calculate the equivalent circuit parameters are explicit (or at least, with decoupled variables), non-iterative and straight forward; no iterations or initial values for the parameters are needed. The accuracy of this method is tested and compared with similar methods from the available literature demonstrating similar precision but higher simplicity. Second, the simulation of the control subsystem of a microsatellite is presented, paying particular attention to the new control law proposed. A new type of magnetic control applied to Low Earth Orbit (LEO) satellites has been presented. The proposed control law is able to set the satellite rotation speed around its maximum or minimum inertia principal axis. Besides, the proposed control law favors the alignment of this axis with the normal direction to the orbital plane for high inclination orbits. The proposed control algorithm is simples, only magnetorquers are required as actuators; only magnetometers are required as sensors; no estimation of the angular velocity is needed; it does not include an in-orbit Earth magnetic field model; it does not need to be externally activated with information about the orbital characteristics and it allows automatic reset after a total shutdown of attitude control subsystem. The theoretical viability of the control law is demonstrated through Monte Carlo analysis. Finally, in terms of power production, it is demonstrated that the proposed attitude (on principal axis perpendicular to the orbit plane, and the satellite rotating around it with a controlled rate) is quite suitable for the UPMSat-2 mission, as it allows a higher area of the panels pointing towards the sun when compared to other studied attitudes. Compared with the previous attitude control proposed for the UPMSat-2 it results in a 25% increment in available power. Besides, the proposed attitude showed significant improvements, when compared to others, in terms of thermal control, as the satellite angular rotation rate can be selected to achieve a higher temperature homogenization of the satellite and antenna pointing.

The location of Z- and W-linked marker genes and sequence on the homomorphic sex chromosomes of the ostrich and the emu

Perhaps the most striking fact about early Cenozoic avian history some 70 million years ago was the rapid radiation of large, flightless, ground-living birds. It has been suggested that, for a time, there was active competition between these large terrestrial birds and the early mammals. Probably reflecting the above noted early start of Ratitae of the infraclass Eoaves, the presumptive sex chromosomes of their present day survivors, such as the emu and the ostrich, largely remained homomorphic. The signs of genetic differentiation between their still-homomorphic Z and W chromosomes were tested by using two marker genes (Z-linked ZOV3 and the gene for the iron-responsive element-binding protein) and one marker sequence of a part of a presumptive pseudogene (W-linked EE0.6 of the chicken). Their homologues, maintaining 71–92% identities to the chicken counterparts, were found in both the emu (Dromaius novaehollandiae) and the ostrich (Struthio camelus). Their locations were visualized on chromosome preparations by fluorescence in situ hybridization. In the case of the emu, these three marker sequences were localized on both members of the fifth pair of a female, thus revealing no sign yet of genetic differentiation between the Z and the W. The finding was the same with regard to both members of the fourth pair of male ostriches. In the female ostrich, however, the sequence of the gene for the iron-responsive element-binding protein was missing from one of the pairs, thus revealing the differentiation by a small deletion of the W from the Z.

Computerized polymorphic marker identification: Experimental validation and a predicted human polymorphism catalog

A computational system for the prediction of polymorphic loci directly and efficiently from human genomic sequence was developed and verified. A suite of programs, collectively called pompous (polymorphic marker prediction of ubiquitous simple sequences) detects tandem repeats ranging from dinucleotides up to 250 mers, scores them according to predicted level of polymorphism, and designs appropriate flanking primers for PCR amplification. This approach was validated on an approximately 750-kilobase region of human chromosome 3p21.3, involved in lung and breast carcinoma homozygous deletions. Target DNA from 36 paired B lymphoblastoid and lung cancer lines was amplified and allelotyped for 33 loci predicted by pompous to be variable in repeat size. We found that among those 36 predominately Caucasian individuals 22 of the 33 (67%) predicted loci were polymorphic with an average heterozygosity of 0.42. Allele loss in this region was found in 27/36 (75%) of the tumor lines using these markers. pompous provides the genetic researcher with an additional tool for the rapid and efficient identification of polymorphic markers, and through a World Wide Web site, investigators can use pompous to identify polymorphic markers for their research. A catalog of 13,261 potential polymorphic markers and associated primer sets has been created from the analysis of 141,779,504 base pairs of human genomic sequence in GenBank. This data is available on our Web site (pompous.swmed.edu) and will be updated periodically as GenBank is expanded and algorithm accuracy is improved.

Mitochondrial localization of a NADR-dependent isocitrate dehydrogenase isoenzyme by using the green fluorescent protein as a marker

In this work, we describe the isolation of a new cDNA encoding an NADP-dependent isocitrate dehydrogenase (ICDH). The nucleotide sequence in its 5′ region gives a deduced amino acid sequence indicative of a targeting peptide. However, even if this cDNA clearly encodes a noncytosolic ICDH, it is not possible to say from the targeting peptide sequence to which subcellular compartment the protein is addressed. To respond to this question, we have transformed tobacco plants with a construct containing the entire targeting signal-encoding sequence in front of a modified green fluorescent protein (GFP) gene. This construct was placed under the control of the cauliflower mosaic virus 35S promoter, and transgenic tobacco plants were regenerated. At the same time, and as a control, we also have transformed tobacco plants with the same construct but lacking the nucleotide sequence corresponding to the ICDH-targeting peptide, in which the GFP is retained in the cytoplasm. By optical and confocal microscopy of leaf epiderm and Western blot analyses, we show that the putative-targeting sequence encoded by the cDNA addresses the GFP exclusively into the mitochondria of plant cells. Therefore, we conclude that this cDNA encodes a mitochondrial ICDH.

Induction of microsatellite instability by oxidative DNA damage

Instability of repetitive sequences, both in intronic sequences and within coding regions, has been demonstrated to be a hallmark of genomic instability in human cancer. Understanding how these mutational events arise may provide an opportunity for prevention or early intervention in cancer development. To study the source of this instability, we have identified a region of the β-lactamase gene that is tolerant to the insertion of fragments of exogenous DNA as large as 1,614 bp with minimal loss of enzyme activity, as determined by antibiotic resistance. Fragments inserted out-of-frame render Escherichia coli sensitive to antibiotic, and compensatory frameshift mutations that restore the reading frame of β-lactamase can be selected on the basis of antibiotic resistance. We have utilized this site to insert a synthetic microsatellite sequence within the β-lactamase gene and selected for mutations yielding frameshifts. This assay provides for detection of one frameshift mutation in a background of 106 wild-type sequences. Mismatch repair deficiency increased the observed frameshift frequency ≈300-fold. Exposure of plasmid containing microsatellite sequences to hydrogen peroxide resulted in frameshift mutations that were localized exclusively to the microsatellite sequences, whereas DNA damage by UV or N-methyl-N′-nitro-N-nitrosoguanidine did not result in enhanced mutagenesis. We postulate that in tumor cells, endogenous production of oxygen free radicals may be a major factor in promoting instability of microsatellite sequences. This β-lactamase assay may provide a sensitive methodology for the detection and quantitation of mutations associated with the development of cancer.

Epigenetic phenotypes distinguish microsatellite-stable and -unstable colorectal cancers

Aberrant DNA methylation is a common phenomenon in human cancer, but its patterns, causes, and consequences are poorly defined. Promoter methylation of the DNA mismatch repair gene MutL homologue (MLH1) has been implicated in the subset of colorectal cancers that shows microsatellite instability (MSI). The present analysis of four MspI/HpaII sites at the MLH1 promoter region in a series of 89 sporadic colorectal cancers revealed two main methylation patterns that closely correlated with the MSI status of the tumors. These sites were hypermethylated in tumor tissue relative to normal mucosa in most MSI(+) cases (31/51, 61%). By contrast, in the majority of MSI(−) cases (20/38, 53%) the same sites showed methylation in normal mucosa and hypomethylation in tumor tissue. Hypermethylation displayed a direct correlation with increasing age and proximal location in the bowel and was accompanied by immunohistochemically documented loss of MLH1 protein both in tumors and in normal tissue. Similar patterns of methylation were observed in the promoter region of the calcitonin gene that does not have a known functional role in tumorigenesis. We propose a model of carcinogenesis where different epigenetic phenotypes distinguish the colonic mucosa in individuals who develop MSI(+) and MSI(−) tumors. These phenotypes may underlie the different developmental pathways that are known to occur in these tumors.

Philopatry of male marine turtles inferred from mitochondrial DNA markers

Recent studies of mitochondrial DNA (mtDNA) variation among marine turtle populations are consistent with the hypothesis that females return to beaches in their natal region to nest as adults. In contrast, less is known about breeding migrations of male marine turtles and whether they too are philopatric to natal regions. Studies of geographic structuring of restriction fragment and microsatellite polymorphisms at anonymous nuclear loci in green turtle (Chelonia mydas) populations indicate that nuclear gene flow is higher than estimates from mtDNA analyses. Regional populations from the northern and southern Great Barrier Reef were distinct for mtDNA but indistinguishable at nuclear loci, whereas the Gulf of Carpentaria (northern Australia) population was distinct for both types of marker. To assess whether this result was due to reduced philopatry of males across the Great Barrier Reef, we determined the mtDNA haplotypes of breeding males at courtship areas for comparison with breeding females from the same three locations. We used a PCR-restriction fragment length polymorphism approach to determine control region haplotypes and designed mismatch primers for the identification of specific haplotypes. The mtDNA haplotype frequencies were not significantly different between males and females at any of the three areas and estimates of Fst among the regions were similar for males and females (Fst = 0.78 and 0.73, respectively). We conclude that breeding males, like females, are philopatric to courtship areas within their natal region. Nuclear gene flow between populations is most likely occurring through matings during migrations of both males and females through nonnatal courtship areas.

Phylogenetic assessment of length variation at a microsatellite locus

Sixty-six haplotypes at a locus containing a simple dinucleotide (CA)n microsatellite repeat were isolated by PCR–single-strand conformational polymorphism from populations of the horseshoe crab Limulus polyphemus. These haplotypes were sequenced to assess nucleotide variation directly. Thirty-four distinct sequences (alleles) were identified in a region 570 bp long that included the microsatellite motif. In the repeat region itself, CA-number varied in integer values from 5 to 11 across alleles, except that a (CA)8 class was not observed. Differences among alleles were due also to polymorphisms at 22 sites in regions immediately flanking the microsatellite repeats. Nucleotide substitutions in these regions were used to estimate phylogenetic relationships among alleles, and the gene phylogeny was used to trace the evolution of length variation and CA repeat numbers. A low correlation between size variation and genealogical relationships among alleles suggests that absolute fragment size (as normally scored in microsatellite assays) is an unreliable indicator of historical affinities among alleles. This finding on the molecular fine structure of microsatellite variation suggests the need for caution in the use of repeat counts at microsatellite loci as secure indicators of allelic relationships.

Long-range disruption of gene expression by a selectable marker cassette

Recent studies have suggested that the retention of selectable marker cassettes (like PGK–Neo, in which a hybrid gene consisting of the phosphoglycerate kinase I promoter drives the neomycin phosphotransferase gene) in targeted loci can cause unexpected phenotypes in “knockout” mice due to disruption of expression of neighboring genes within a locus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the β-like globin gene cluster. The insertion of PGK–Neo into the granzyme B gene, the most 5′ gene in the granzyme B gene cluster, severely reduced the normal expression of multiple genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK–Neo cassette into the β-globin locus control region (LCR) abrogates the expression of multiple globin genes downstream from the cassette. In contrast, a targeted mutation of the promyelocyte-specific cathepsin G gene (which lies just 3′ to the granzyme genes in the same cluster) had minimal effects on upstream granzyme gene expression. Although the mechanism of these long distance effects are unknown, the expression of PGK–Neo can be “captured” by the regulatory domain into which it is inserted. These results suggest that the PGK–Neo cassette can interact productively with locus control regions and thereby disrupt normal interactions between local and long-distance regulatory regions within a tissue-specific domain.

Complexities in the genetic structure of Anopheles gambiae populations in west Africa as revealed by microsatellite DNA analysis

Chromosomal forms of Anopheles gambiae, given the informal designations Bamako, Mopti, and Savannah, have been recognized by the presence or absence of four paracentric inversions on chromosome 2. Studies of karyotype frequencies at sites where the forms occur in sympatry have led to the suggestion that these forms represent species. We conducted a study of the genetic structure of populations of An. gambiae from two villages in Mali, west Africa. Populations at each site were composed of the Bamako and Mopti forms and the sibling species, Anopheles arabiensis. Karyotypes were determined for each individual mosquito and genotypes at 21 microsatellite loci determined. A number of the microsatellites have been physically mapped to polytene chromosomes, making it possible to select loci based on their position relative to the inversions used to define forms. We found that the chromosomal forms differ at all loci on chromosome 2, but there were few differences for loci on other chromosomes. Geographic variation was small. Gene flow appears to vary among different regions within the genome, being lowest on chromosome 2, probably due to hitchhiking with the inversions. We conclude that the majority of observed genetic divergence between chromosomal forms can be explained by forces that need not involve reproductive isolation, although reproductive isolation is not ruled out. We found low levels of gene flow between the sibling species Anopheles gambiae and Anopheles arabiensis, similar to estimates based on observed frequencies of hybrid karyotypes in natural populations.