Studies of subcellular localization and complex formation of the Agrobacterium tumefaciens transport ATPase VirB11

The VirB11 ATPase is an essential component of an Agrobacterium tumefaciens type IV bacterial secretion system that transfers oncogenic nucleoprotein complexes to susceptible plant cells. This dissertation investigates the subcellular localization and homo-oligomeric state of the VirB11 ATPase in order to provide insights about the assembly of the protein as a subunit of this membrane-associated transfer system. Subcellular fractionation studies and quantitative immunoblot analysis demonstrated that $\sim$30% of VirB11 partitioned as soluble protein and $\sim$70% was tightly associated with the bacterial cytoplasmic membrane. No differences were detected in VirB11 subcellular localization and membrane association in the presence or absence of other transport system components. Mutations in virB11 affecting protein function were mapped near the amino terminus, just upstream of a region encoding a Walker 'A' nucleotide-binding site, and within the Walker 'A' motif partitioned almost exclusively with the cytoplasmic membrane, suggesting that an activity associated with nucleotide binding could modulate the affinity of VirB11 for the cytoplasmic membrane. Merodiploid analysis of VirB11 mutant and truncation derivatives provided strong evidence that VirB11 functions as a homo- or heteromultimer and that the C-terminal half of VirB11 contains a protein interaction domain. A combination of biochemical and molecular genetic approaches suggested that VirB11 and the green fluorescence protein (GFP) formed a mixed multimer as demonstrated by immunoprecipitation experiments with anti-GFP antibodies. Second, a hybrid protein composed of VirB11 fused to the N-terminal DNA-binding domain of bacteriophage $\lambda$ cI repressor conferred immunity to $\lambda$ superinfection, demonstrating that VirB11 self-association promotes dimerization of the chimeric repressor. A conserved Walker 'A' motif, though required for VirB11 function in T-complex export, was not necessary for VirB11 self-association. Sequences in both the N- and the C-terminal halves of the protein were found to contribute to self-association of the full length protein. Chemical cross-linking experiments with His$\sb6$ tagged VirB11 suggested that VirB11 probably assembles into a higher order homo-oligomeric complex. ^

Flexibility Properties in Complex Analysis and Affine Algebraic Geometry

In the last decades affine algebraic varieties and Stein manifolds with big (infinite-dimensional) automorphism groups have been intensively studied. Several notions expressing that the automorphisms group is big have been proposed. All of them imply that the manifold in question is an Oka–Forstnerič manifold. This important notion has also recently merged from the intensive studies around the homotopy principle in Complex Analysis. This homotopy principle, which goes back to the 1930s, has had an enormous impact on the development of the area of Several Complex Variables and the number of its applications is constantly growing. In this overview chapter we present three classes of properties: (1) density property, (2) flexibility, and (3) Oka–Forstnerič. For each class we give the relevant definitions, its most significant features and explain the known implications between all these properties. Many difficult mathematical problems could be solved by applying the developed theory, we indicate some of the most spectacular ones.

Nonlinear tests for genetic studies of complex disease

Linkage and association studies are major analytical tools to search for susceptibility genes for complex diseases. With the availability of large collection of single nucleotide polymorphisms (SNPs) and the rapid progresses for high throughput genotyping technologies, together with the ambitious goals of the International HapMap Project, genetic markers covering the whole genome will be available for genome-wide linkage and association studies. In order not to inflate the type I error rate in performing genome-wide linkage and association studies, multiple adjustment for the significant level for each independent linkage and/or association test is required, and this has led to the suggestion of genome-wide significant cut-off as low as 5 × 10 −7. Almost no linkage and/or association study can meet such a stringent threshold by the standard statistical methods. Developing new statistics with high power is urgently needed to tackle this problem. This dissertation proposes and explores a class of novel test statistics that can be used in both population-based and family-based genetic data by employing a completely new strategy, which uses nonlinear transformation of the sample means to construct test statistics for linkage and association studies. Extensive simulation studies are used to illustrate the properties of the nonlinear test statistics. Power calculations are performed using both analytical and empirical methods. Finally, real data sets are analyzed with the nonlinear test statistics. Results show that the nonlinear test statistics have correct type I error rates, and most of the studied nonlinear test statistics have higher power than the standard chi-square test. This dissertation introduces a new idea to design novel test statistics with high power and might open new ways to mapping susceptibility genes for complex diseases. ^

A hardware in the loop design methodology for FPGA system and its application to complex functions

In this work, a unified algorithm-architecture-circuit co-design environment for complex FPGA system development is presented. The main objective is to find an efficient methodology for designing a configurable optimized FPGA system by using as few efforts as possible in verification stage, so as to speed up the development period. A proposed high performance FFT/iFFT processor for Multiband Orthogonal Frequency Division Multiplexing Ultra Wideband (MB-OFDM UWB) system design process is given as an example to demonstrate the proposed methodology. This efficient design methodology is tested and considered to be suitable for almost all types of complex FPGA system designs and verifications.

Wind tunnel analysis of the flow topology over complex topographies

El principal objetivo de este trabajo es aportar conocimiento para contestar la pregunta: ¿hasta que punto los ensayos en túnel aerodinámico pueden contribuir a determinar las características que afectan la respuesta dinámica de los aerogeneradores operando en terreno complejo?. Esta pregunta no es nueva, de hecho, el debate en la comunidad científica comenzó en el primer tercio del siglo pasado y aún está intensamente vivo. El método generalmente aceptado para enfrentar el mencionado problema consiste en analizar un caso de estudio determinado en el cual se aplican tanto ensayos a escala real como análisis computacionales y ensayos en túnel aerodinámico. Esto no es ni fácil ni barato. Esta es la razón por la cual desde el experimento de Askervein en 1988, los modelizadores del flujo atmosférico tuvieron que esperar hasta 2007 a que el experimento de Bolund fuese puesto en marcha con un despliegue de medios técnicos equivalentes (teniendo en cuenta la evolución de las tecnologías de sensores y computación). El problema contempla tantos aspectos que ambas experiencias fueron restringidas a condiciones de atmósfera neutra con efectos de Coriolis despreciables con objeto de reducir la complejidad. Este es el contexto en el que se ha desarrollado la presente tesis doctoral. La topología del flujo sobre la isla de Bolund ha sido estudiada mediante la reproducción del experimento de Bolund en los túneles aerodinámicos A9 y ACLA16 del IDR. Dos modelos de la isla de Bolund fueron fabricados a dos escalas, 1:230 y 1:115. El flujo de entrada en el túnel aerodinámico simulando la capa límite sin perturbar correspondía a régimen de transición (transitionally rough regime) y fue usado como situación de referencia. El modelo a escala 1:230 fue ensayado en el túnel A9 para determinar la presión sobre su superficie. La distribución del coeficiente de presión sobre la isla proporcionó una visualización y estimación de una región de desprendimiento sobre el pequeño acantilado situado al frente de la misma. Las medidas de presión instantánea con suficiente grado de resolución temporal pusieron de manifiesto la no estacionariedad en la región de desprendimiento. El modelo a escala 1:115 fue ensayado utilizando hilo caliente de tres componentes y un sistema de velocimetría por imágenes de partículas de dos componentes. El flujo fue caracterizado por el ratio de aceleración, el incremento normalizado de energía cinética turbulenta y los ángulos de inclinación y desviación horizontal. Los resultados a lo largo de la dirección 270°y alturas de 2 m y 5 m presentaron una gran similitud con los resultados a escala real del experimento de Bolund. Los perfiles verticales en las localizaciones de las torres meteorológicas mostraron un acuerdo significativo con los resultados a escala real. El análisis de los esfuerzos de Reynolds y el análisis espectral en las localizaciones de los mástiles meteorológicos presentaron niveles de acuerdo variados en ciertas posiciones, mientras que en otras presentaron claras diferencias. El mapeo horizontal del flujo, para una dirección de viento de 270°, permitió caracterizar el comportamiento de la burbuja intermitente de recirculación sobre el pequeño acantilado existente al frente de la isla así como de la región de relajación y de la capa de cortadura en la región corriente abajo de Bolund. Se realizaron medidas de velocidad con alta resolución espacial en planos perpendiculares a la dirección del flujo sin perturbar. Estas medidas permitieron detectar y caracterizar una estructura de flujo similar a un torbellino longitudinal con regiones con altos gradientes de velocidad y alta intensidad de turbulencia. Esta estructura de flujo es, sin duda, un reto para los modelos computacionales y puede considerarse un factor de riesgo para la operación de los aerogeneradores. Se obtuvieron y analizaron distribuciones espaciales de los esfuerzos de Reynolds mediante 3CHW y PIV. Este tipo de parámetros no constituyen parte de los resultados habituales en los ensayos en túnel sobre topografías y son muy útiles para los modelizadores que utilizan simulación de grades torbellinos (LES). Se proporciona una interpretación de los resultados obtenidos en el túnel aerodinámico en términos de utilidad para los diseñadores de parques eólicos. La evolución y variación de los parámetros del flujo a lo largo de líneas, planos y superficies han permitido identificar como estas propiedades del flujo podrían afectar la localización de los aerogeneradores y a la clasificación de emplazamientos. Los resultados presentados sugieren, bajo ciertas condiciones, la robustez de los ensayos en túnel para estudiar la topología sobre terreno complejo y su comparabilidad con otras técnicas de simulación, especialmente considerando el nivel de acuerdo del conjunto de resultados presentados con los resultados a escala real. De forma adicional, algunos de los parámetros del flujo obtenidos de las medidas en túnel son difícilmente determinables en ensayos a escala real o por medios computacionales, considerado el estado del arte. Este trabajo fue realizado como parte de las actividades subvencionadas por la Comisión Europea como dentro del proyecto FP7-PEOPLE-ITN-2008WAUDIT (Wind Resource Assessment Audit and Standardization) dentro de la FP7 Marie-Curie Initial Training Network y por el Ministerio Español de Economía y Competitividad dentro del proyecto ENE2012-36473, TURCO (Determinación en túnel aerodinámico de la distribución espacial de parámetros estadísticos de la turbulencia atmosférica sobre topografías complejas) del Plan Nacional de Investigación (Subprograma de investigación fundamental no orientada 2012). El informe se ha organizado en siete capítulos y un conjunto de anexos. En el primer capítulo se introduce el problema. En el capítulo dos se describen los medios experimentales utilizados. Seguidamente, en el capítulo tres, se analizan en detalle las condiciones de referencia del principal túnel aerodinámico utilizado en esta investigación. En el capítulo tres se presentan resultados de ensayos de presión superficial sobre un modelo de la isla. Los principales resultados del experimento de Bolund se reproducen en el capítulo cinco. En el capítulo seis se identifican diferentes estructuras del flujo sobre la isla y, finalmente, en el capitulo siete, se recogen las conclusiones y una propuesta de lineas de trabajo futuras. ABSTRACT The main objective of this work is to contribute to answer the question: to which extend can the wind tunnel testing contribute to determine the flow characteristics that affect the dynamic response of wind turbines operating in highly complex terrains?. This question is not new, indeed, the debate in the scientific community was opened in the first third of the past century and it is still intensely alive. The accepted approach to face this problem consists in analysing a given case study where full-scale tests, computational modelling and wind tunnel testing are applied to the same topography. This is neither easy nor cheap. This is is the reason why since the Askervein experience in 1988, the atmospheric flow modellers community had to wait till 2007 when the Bolund experiment was setup with a deployment of technical means equivalent (considering the evolution of the sensor and computing techniques). The problem is so manifold that both experiences were restricted to neutral conditions without Coriolis effects in order to reduce the complexity. This is the framework in which this PhD has been carried out. The flow topology over the Bolund Island has been studied by replicating the Bolund experiment in the IDR A9 and ACLA16 wind tunnels. Two mock-ups of the Bolund island were manufactured at two scales of 1:230 and 1:115. The in-flow in the empty wind tunnel simulating the incoming atmospheric boundary layer was in the transitionally rough regime and used as a reference case. The 1:230 model was tested in the A9 wind tunnel to measure surface pressure. The mapping of the pressure coefficient across the island gave a visualisation and estimation of a detachment region on the top of the escarpment in front of the island. Time resolved instantaneous pressure measurements illustrated the non-steadiness in the detachment region. The 1:115 model was tested using 3C hot-wires(HW) and 2C Particle Image Velocimetry(PIV). Measurements at met masts M3, M6, M7 and M8 and along Line 270°were taken to replicate the result of the Bolund experiment. The flow was characterised by the speed-up ratio, normalised increment of the turbulent kinetic energy, inclination angle and turning angle. Results along line 270°at heights of 2 m and 5 m compared very well with the full-scale results of the Bolund experiment. Vertical profiles at the met masts showed a significant agreement with the full-scale results. The analysis of the Reynolds stresses and the spectral analysis at the met mast locations gave a varied level of agreement at some locations while clear mismatch at others. The horizontal mapping of the flow field, for a 270°wind direction, allowed to characterise the behaviour of the intermittent recirculation bubble on top of the front escarpment followed by a relaxation region and the presence of a shear layer in the lee side of the island. Further detailed velocity measurements were taken at cross-flow planes over the island to study the flow structures on the island. A longitudinal vortex-like structure with high mean velocity gradients and high turbulent kinetic energy was characterised on the escarpment and evolving downstream. This flow structure is a challenge to the numerical models while posing a threat to wind farm designers when siting wind turbines. Spatial distribution of Reynold stresses were presented from 3C HW and PIV measurements. These values are not common results from usual wind tunnel measurements and very useful for modellers using large eddy simulation (LES). An interpretation of the wind tunnel results in terms of usefulness to wind farm designers is given. Evolution and variation of the flow parameters along measurement lines, planes and surfaces indicated how the flow field could affect wind turbine siting. Different flow properties were presented so compare the level of agreement to full-scale results and how this affected when characterising the site wind classes. The results presented suggest, under certain conditions, the robustness of the wind tunnel testing for studying flow topology over complex terrain and its capability to compare to other modelling techniques especially from the level of agreement between the different data sets presented. Additionally, some flow parameters obtained from wind tunnel measurements would have been quite difficult to be measured at full-scale or by computational means considering the state of the art. This work was carried out as a part of the activities supported by the EC as part of the FP7- PEOPLE-ITN-2008 WAUDIT project (Wind Resource Assessment Audit and Standardization) within the FP7 Marie-Curie Initial Training Network and by the Spanish Ministerio de Economía y Competitividad, within the framework of the ENE2012-36473, TURCO project (Determination of the Spatial Distribution of Statistic Parameters of Flow Turbulence over Complex Topographies in Wind Tunnel) belonging to the Spanish National Program of Research (Subprograma de investigación fundamental no orientada 2012). The report is organised in seven chapters and a collection of annexes. In chapter one, the problem is introduced. In chapter two the experimental setup is described. Following, in chapter three, the inflow conditions of the main wind tunnel used in this piece of research are analysed in detail. In chapter three, preliminary pressure tests results on a model of the island are presented. The main results from the Bolund experiment are replicated in chapter five. In chapter six, an identification of specific flow strutures over the island is presented and, finally, in chapter seven, conclusions and lines for future works related to the presented one are included.

Differential expression of major histocompatibility complex class II genes on murine macrophages associated with T cell cytokine profile and protective/suppressive effects

Protective/suppressive major histocompatibility complex (MHC) class II alleles have been identified in humans and mice where they exert a disease-protective and immunosuppressive effect. Various modes of action have been proposed, among them differential expression of MHC class II genes in different types of antigen-presenting cells impacting on the T helper type 1 (Th1)–Th2 balance. To test this possibility, the expression of H-2 molecules from the four haplotypes H-2b, H-2d, H-2k, and H-2q was determined on bone marrow-derived macrophages (BMDMs) and splenic B cells. The I-Ab and I-Ek molecules, both well characterized as protective/suppressive, are expressed at a high level on almost all CD11b+ BMDMs for 5–8 days, after which expression slowly declines. In contrast, I-Ad, I-Ak, and I-Aq expression is lower, peaks over a shorter period, and declines more rapidly. No differential expression could be detected on B cells. In addition, the differential MHC class II expression found on macrophages skews the cytokine response of T cells as shown by an in vitro restimulation assay with BMDMs as antigen-presenting cells. The results indicate that macrophages of the protective/suppressive haplotypes express MHC class II molecules at a high level and exert Th1 bias, whereas low-level expression favors a Th2 response. We suggest that the extent of expression of the class II gene gates the back signal from T cells and in this way controls the activity of macrophages. This effect mediated by polymorphic nonexon segments of MHC class II genes may play a role in determining disease susceptibility in humans and mice.

Functionality of major histocompatibility complex class II molecules in mice doubly deficient for invariant chain and H-2M complexes

By combining two previously generated null mutations, Ii° and M°, we produced mice lacking the invariant chain and H-2M complexes, both required for normal cell-surface expression of major histocompatibility complex class II molecules loaded with the usual diverse array of peptides. As expected, the maturation and transport of class II molecules, their expression at the cell surface, and their capacity to present antigens were quite similar for cells from Ii°M° double-mutant mice and from animals carrying just the Ii° mutation. More surprising were certain features of the CD4+ T cell repertoire selected in Ii°M° mice: many fewer cells were selected than in Ii+M° animals, and these had been purged of self-reactive specificities, unlike their counterparts in Ii+M° animals. These findings suggest (i) that the peptides carried by class II molecules on stromal cells lacking H-2M complexes may almost all derive from invariant chain and (ii) that H-2M complexes edit the peptide array displayed on thymic stromal cells in the absence of invariant chain, showing that it can edit, in vivo, peptides other than CLIP.

Role of the major histocompatibility complex class II Ea gene in lupus susceptibility in mice

The gene(s) encoded within major histocompatibility complex (MHC) act as one of the major genetic elements contributing to the susceptibility of murine systemic lupus erythematosus (SLE). We have recently demonstrated that lupus susceptibility is more closely linked to the I-E− H-2b haplotype than to the I-E+ H-2d haplotype in lupus-prone BXSB and (NZB × BXSB)F1 hybrid mice. To investigate whether the reduced susceptibility to SLE in H-2d mice is related to the expression of the MHC class II Ea gene (absent in H-2b mice), we determined the possible role of the Ea gene as a lupus protective gene in mice. Our results showed that (i) the development of SLE was almost completely prevented in BXSB (H-2b) mice expressing two copies of the Ead transgene at the homozygous level as well as in BXSB H-2k (I-E+) congenic mice as for H-2d BXSB mice, and (ii) the expression of two functional Ea (transgenic and endogenous) genes in either H-2d/b (NZB × BXSB)F1 or H-2k/b (MRL × BXSB)F1 mice provided protection from SLE at levels comparable to those conferred by the H-2d/d or H-2k/k haplotype. In addition, the level of the Ea gene-mediated protection appeared to be dependent on the genetic susceptibility to SLE in individual lupus-prone mice. Our results indicate that the reduced susceptibility associated with the I-E+ H-2d and H-2k haplotypes (versus the I-E− H-2b haplotype) is largely, if not all, contributed by the apparent autoimmune suppressive effect of the Ea gene, independently of the expression of the I-A or other MHC-linked genes.

DNA annealing by Rad52 Protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA–ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA–Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA–ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.

Chromatin-bound PCNA complex formation triggered by DNA damage occurs independent of the ATM gene product in human cells

Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases δ and ɛ, is involved in DNA replication as well as in diverse DNA repair pathways. In quiescent cells, UV light-induced bulky DNA damage triggers the transition of PCNA from a soluble to an insoluble chromatin-bound form, which is intimately associated with the repair synthesis by polymerases δ and ɛ. In this study, we investigated the efficiency of PCNA complex formation in response to ionizing radiation-induced DNA strand breaks in normal and radiation-sensitive Ataxia telangiectasia (AT) cells by immunofluorescence and western blot techniques. Exposure of normal cells to γ-rays rapidly triggered the formation of PCNA foci in a dose-dependent manner in the nuclei and the PCNA foci (40–45%) co-localized with sites of repair synthesis detected by bromodeoxyuridine labeling. The chromatin-bound PCNA gradually declined with increasing post-irradiation times and almost reached the level of unirradiated cells by 6 h. The PCNA foci formed after γ-irradiation was resistant to high salt extraction and the chromatin association of PCNA was lost after DNase I digestion. Interestingly, two radiosensitive primary fibroblast cell lines, derived from AT patients harboring homozygous mutations in the ATM gene, displayed an efficient PCNA redistribution after γ-irradiation. We also analyzed the PCNA complex induced by a radiomimetic agent, Bleomycin (BLM), which produces predominantly single- and double-strand DNA breaks. The efficiency and the time course of PCNA complex induced by BLM were identical in both normal and AT cells. Our study demonstrates for the first time that the ATM gene product is not required for PCNA complex assembly in response to DNA strand breaks. Additionally, we observed an increased interaction of PCNA with the Ku70 and Ku80 heterodimer after DNA damage, suggestive of a role for PCNA in the non-homologous end-joining repair pathway of DNA strand breaks.

Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium.

Conventional major histocompatibility complex (MHC) class I genes encode molecules that present intracellular peptide antigens to T cells. They are ubiquitously expressed and regulated by interferon gamma. Two highly divergent human MHC class I genes, MICA and MICB, are regulated by promoter heat shock elements similar to those of HSP70 genes. MICA encodes a cell surface glycoprotein, which is not associated with beta 2-microglobulin, is conformationally stable independent of conventional class I peptide ligands, and almost exclusively expressed in gastrointestinal epithelium. Thus, this MHC class I molecule may function as an indicator of cell stress and may be recognized by a subset of gut mucosal T cells in an unusual interaction.

Biosynthesis of major histocompatibility complex molecules and generation of T cells in Ii TAP1 double-mutant mice.

Major histocompatibility complex (MHC) class I and II molecules are loaded with peptides in distinct subcellular compartments. The transporter associated with antigen processing (TAP) is responsible for delivering peptides derived from cytosolic proteins to the endoplasmic reticulum, where they bind to class I molecules, while the invariant chain (Ii) directs class II molecules to endosomal compartments, where they bind peptides originating mostly from exogenous sources. Mice carrying null mutations of the TAP1 or Ii genes (TAP10) or Ii0, respectively) have been useful tools for elucidating the two MHC/peptide loading pathways. To evaluate to what extent these pathways functionally intersect, we have studied the biosynthesis of MHC molecules and the generation of T cells in Ii0TAP10 double-mutant mice. We find that the assembly and expression of class II molecules in Ii0 and Ii0TAP10 animals are indistinguishable and that formation and display of class I molecules is the same in TAP10 and Ii0TAP10 animals. Thymic selection in the double mutants is as expected, with reduced numbers of both CD4+ CD8- and CD4- CD8+ thymocyte compartments. Surprisingly, lymph node T-cell populations look almost normal; we propose that population expansion of peripheral T cells normalizes the numbers of CD4+ and CD8+ cells in Ii0TAP10 mice.

Complex flexibility of the transforming growth factor beta superfamily.

The transforming growth factors beta (TGF-beta s) are important modulators of growth and differentiation. They are intermolecular disulfide-bonded homodimeric molecules. The monomer fold has a conserved cystine knot and lacks a hydrophobic core. The biological specificity of a given member of the family is believed to be determined by the conformational flexibility of the variable loop regions of the monomer. The monomer subunit assembly in the dimer is stabilized mainly by hydrophobic contacts and a few hydrogen bonds. Since these interactions are nondirectional, we examined subunit assemblies of TGF-beta by using conformational analysis. The different subunit assemblies in TGF-beta 2 dimer were characterized in terms of the intersubunit disulfide torsion. Our analyses show that the subunit assemblies fall into two states: the crystallographically observed gauche+conformation and the previously not reported gauche--conformation, both having almost identical interaction energies. Furthermore, there is significant flexibility in the subunit assembly within the gauche+ and the gauche- states of the disulfide bond. The monomer subunit assembly is independent of the variations about the loop regions. The variations in the loop regions, coupled with flexibility in the monomer assembly, lead to a complex flexibility in the dimer of the TGF-beta superfamily. For the TGF-beta superfamily, the cystine knot acts as a scaffold and complex flexibility provides for biological selectivity. Complex flexibility might provide an explanation for the diverse range of biological activities that these important molecules display.

Use of space by domestic chicks housed in complex aviaries.

To improve the understanding of the development of locomotor capacity in layer hens, we measured how female laying hen chicks (n=120) of four different strains (LSL-lite, Hyline Brown, Dekalb White, Lohmann Brown; 3 groups of 10 chicks per line) utilized the ground, the air, elevated horizontal (platforms and perches) and inclined surfaces (ramps and ladders) in an aviary until 9 weeks of age. We used infra-red video recordings to perform all-occurrences sampling of locomotive behavioural and perching events that occurred on the ground—where bedding material, food and water were provided, in the air, and on elevated horizontal and inclined surfaces within weekly 30-min sampling periods. Chicks preferred level ground during the first week of life compared to weeks 5–9 (P<0.0001) and performed 52% of all behavioural events in this section. Elevated surface use began at 2 weeks of age and increased over time (P=0.003), where most behaviour was performed in S2 (45% of all events). Chicks preferred horizontal to inclined surfaces, which were used from weeks 2–5 with maximum use occurring during weeks 2 and 3. Lohmann LSL chicks used the space above the ground most frequently (P=0.05) and performed more aerial ascent/descent behaviour than other lines (P<0.0001). Overall activity levels declined with age (P<0.0001). In summary layer chicks almost exclusively locomoted on the ground but utilized elevated horizontal surfaces (perch, first platform) as early as 2 weeks. These results provide information for improving space use in rearing aviaries by introducing lower perches, platforms and ramps/ladders to accommodate age-dependent locomotor abilities.

Targeting a complex transcriptome: The construction of the mouse full-length cDNA encyclopedia

We report the construction of the mouse full-length cDNA encyclopedia, the most extensive view of a complex transcriptome, on the basis of preparing and sequencing 246 libraries. Before cloning, cDNAs were enriched in full-length by Cap-Trapper, and in most cases, aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads, which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU), which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC), which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large. numbers of clusters (and TUs) of this project, which also include non-protein-coding RNAs, and the lower gene number estimation of genome annotations. Altogether, S'-end clusters identify regions that are potential promoters for 8637 known genes and S'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.