Development of a fuzzy control based self-adaptive system for energy saving in constrained networks for the Internet of Things

Las redes del futuro, incluyendo las redes de próxima generación, tienen entre sus objetivos de diseño el control sobre el consumo de energía y la conectividad de la red. Estos objetivos cobran especial relevancia cuando hablamos de redes con capacidades limitadas, como es el caso de las redes de sensores inalámbricos (WSN por sus siglas en inglés). Estas redes se caracterizan por estar formadas por dispositivos de baja o muy baja capacidad de proceso y por depender de baterías para su alimentación. Por tanto la optimización de la energía consumida se hace muy importante. Son muchas las propuestas que se han realizado para optimizar el consumo de energía en este tipo de redes. Quizás las más conocidas son las que se basan en la planificación coordinada de periodos de actividad e inactividad, siendo una de las formas más eficaces para extender el tiempo de vida de las baterías. La propuesta que se presenta en este trabajo se basa en el control de la conectividad mediante una aproximación probabilística. La idea subyacente es que se puede esperar que una red mantenga la conectividad si todos sus nodos tienen al menos un número determinado de vecinos. Empleando algún mecanismo que mantenga ese número, se espera que se pueda mantener la conectividad con un consumo energético menor que si se empleara una potencia de transmisión fija que garantizara una conectividad similar. Para que el mecanismo sea eficiente debe tener la menor huella posible en los dispositivos donde se vaya a emplear. Por eso se propone el uso de un sistema auto-adaptativo basado en control mediante lógica borrosa. En este trabajo se ha diseñado e implementado el sistema descrito, y se ha probado en un despliegue real confirmando que efectivamente existen configuraciones posibles que permiten mantener la conectividad ahorrando energía con respecto al uso de una potencia de transmisión fija. ABSTRACT. Among the design goals for future networks, including next generation networks, we can find the energy consumption and the connectivity. These two goals are of special relevance when dealing with constrained networks. That is the case of Wireless Sensors Networks (WSN). These networks consist of devices with low or very low processing capabilities. They also depend on batteries for their operation. Thus energy optimization becomes a very important issue. Several proposals have been made for optimizing the energy consumption in this kind of networks. Perhaps the best known are those based on the coordinated planning of active and sleep intervals. They are indeed one of the most effective ways to extend the lifetime of the batteries. The proposal presented in this work uses a probabilistic approach to control the connectivity of a network. The underlying idea is that it is highly probable that the network will have a good connectivity if all the nodes have a minimum number of neighbors. By using some mechanism to reach that number, we hope that we can preserve the connectivity with a lower energy consumption compared to the required one if a fixed transmission power is used to achieve a similar connectivity. The mechanism must have the smallest footprint possible on the devices being used in order to be efficient. Therefore a fuzzy control based self-adaptive system is proposed. This work includes the design and implementation of the described system. It also has been validated in a real scenario deployment. We have obtained results supporting that there exist configurations where it is possible to get a good connectivity saving energy when compared to the use of a fixed transmission power for a similar connectivity.

Coherent structures in statistically-stationary homogeneous shear turbulence

La región cerca de la pared de flujos turbulentos de pared ya está bien conocido debido a su bajo número de Reynolds local y la separación escala estrecha. La región lejos de la pared (capa externa) no es tan interesante tampoco, ya que las estadísticas allí se escalan bien por las unidades exteriores. La región intermedia (capa logarítmica), sin embargo, ha estado recibiendo cada vez más atención debido a su propiedad auto-similares. Además, de acuerdo a Flores et al. (2007) y Flores & Jiménez (2010), la capa logarítmica es más o menos independiente de otras capas, lo que implica que podría ser inspeccionado mediante el aislamiento de otras dos capas, lo que reduciría significativamente los costes computacionales para la simulación de flujos turbulentos de pared. Algunos intentos se trataron después por Mizuno & Jiménez (2013), quien simulan la capa logarítmica sin la región cerca de la pared con estadísticas obtenidas de acuerdo razonablemente bien con los de las simulaciones completas. Lo que más, la capa logarítmica podría ser imitado por otra turbulencia sencillo de cizallamiento de motor. Por ejemplo, Pumir (1996) encontró que la turbulencia de cizallamiento homogéneo estadísticamente estacionario (SS-HST) también irrumpe, de una manera muy similar al proceso de auto-sostenible en flujos turbulentos de pared. Según los consideraciones arriba, esta tesis trata de desvelar en qué medida es la capa logarítmica de canales similares a la turbulencia de cizalla más sencillo, SS-HST, mediante la comparación de ambos cinemática y la dinámica de las estructuras coherentes en los dos flujos. Resultados sobre el canal se muestran mediante Lozano-Durán et al. (2012) y Lozano-Durán & Jiménez (2014b). La hoja de ruta de esta tarea se divide en tres etapas. En primer lugar, SS-HST es investigada por medio de un código nuevo de simulación numérica directa, espectral en las dos direcciones horizontales y compacto-diferencias finitas en la dirección de la cizalla. Sin utiliza remallado para imponer la condición de borde cortante periódica. La influencia de la geometría de la caja computacional se explora. Ya que el HST no tiene ninguna longitud característica externa y tiende a llenar el dominio computacional, las simulaciopnes a largo plazo del HST son ’mínimos’ en el sentido de que contiene sólo unas pocas estructuras media a gran escala. Se ha encontrado que el límite principal es el ancho de la caja de la envergadura, Lz, que establece las escalas de longitud y velocidad de la turbulencia, y que las otras dos dimensiones de la caja debe ser suficientemente grande (Lx > 2LZ, Ly > Lz) para evitar que otras direcciones estando limitado también. También se encontró que las cajas de gran longitud, Lx > 2Ly, par con el paso del tiempo la condición de borde cortante periódica, y desarrollar fuertes ráfagas linealizadas no físicos. Dentro de estos límites, el flujo muestra similitudes y diferencias interesantes con otros flujos de cizalla, y, en particular, con la capa logarítmica de flujos turbulentos de pared. Ellos son exploradas con cierto detalle. Incluyen un proceso autosostenido de rayas a gran escala y con una explosión cuasi-periódica. La escala de tiempo de ruptura es de aproximadamente universales, ~20S~l(S es la velocidad de cizallamiento media), y la disponibilidad de dos sistemas de ruptura diferentes permite el crecimiento de las ráfagas a estar relacionado con algo de confianza a la cizalladura de turbulencia inicialmente isotrópico. Se concluye que la SS-HST, llevado a cabo dentro de los parámetros de cílculo apropiados, es un sistema muy prometedor para estudiar la turbulencia de cizallamiento en general. En segundo lugar, las mismas estructuras coherentes como en los canales estudiados por Lozano-Durán et al. (2012), es decir, grupos de vórticidad (fuerte disipación) y Qs (fuerte tensión de Reynolds tangencial, -uv) tridimensionales, se estudia mediante simulación numérica directa de SS-HST con relaciones de aspecto de cuadro aceptables y número de Reynolds hasta Rex ~ 250 (basado en Taylor-microescala). Se discute la influencia de la intermitencia de umbral independiente del tiempo. Estas estructuras tienen alargamientos similares en la dirección sentido de la corriente a las familias separadas en los canales hasta que son de tamaño comparable a la caja. Sus dimensiones fractales, longitudes interior y exterior como una función del volumen concuerdan bien con sus homólogos de canales. El estudio sobre sus organizaciones espaciales encontró que Qs del mismo tipo están alineados aproximadamente en la dirección del vector de velocidad en el cuadrante al que pertenecen, mientras Qs de diferentes tipos están restringidos por el hecho de que no debe haber ningún choque de velocidad, lo que hace Q2s (eyecciones, u < 0,v > 0) y Q4s (sweeps, u > 0,v < 0) emparejado en la dirección de la envergadura. Esto se verifica mediante la inspección de estructuras de velocidad, otros cuadrantes como la uw y vw en SS-HST y las familias separadas en el canal. La alineación sentido de la corriente de Qs ligada a la pared con el mismo tipo en los canales se debe a la modulación de la pared. El campo de flujo medio condicionado a pares Q2-Q4 encontró que los grupos de vórticidad están en el medio de los dos, pero prefieren los dos cizalla capas alojamiento en la parte superior e inferior de Q2s y Q4s respectivamente, lo que hace que la vorticidad envergadura dentro de las grupos de vórticidad hace no cancele. La pared amplifica la diferencia entre los tamaños de baja- y alta-velocidad rayas asociados con parejas de Q2-Q4 se adjuntan como los pares alcanzan cerca de la pared, el cual es verificado por la correlación de la velocidad del sentido de la corriente condicionado a Q2s adjuntos y Q4s con diferentes alturas. Grupos de vórticidad en SS-HST asociados con Q2s o Q4s también están flanqueadas por un contador de rotación de los vórtices sentido de la corriente en la dirección de la envergadura como en el canal. La larga ’despertar’ cónica se origina a partir de los altos grupos de vórticidad ligada a la pared han encontrado los del Álamo et al. (2006) y Flores et al. (2007), que desaparece en SS-HST, sólo es cierto para altos grupos de vórticidad ligada a la pared asociados con Q2s pero no para aquellos asociados con Q4s, cuyo campo de flujo promedio es en realidad muy similar a la de SS-HST. En tercer lugar, las evoluciones temporales de Qs y grupos de vórticidad se estudian mediante el uso de la método inventado por Lozano-Durán & Jiménez (2014b). Las estructuras se clasifican en las ramas, que se organizan más en los gráficos. Ambas resoluciones espaciales y temporales se eligen para ser capaz de capturar el longitud y el tiempo de Kolmogorov puntual más probable en el momento más extrema. Debido al efecto caja mínima, sólo hay un gráfico principal consiste en casi todas las ramas, con su volumen y el número de estructuras instantáneo seguien la energía cinética y enstrofía intermitente. La vida de las ramas, lo que tiene más sentido para las ramas primarias, pierde su significado en el SS-HST debido a las aportaciones de ramas primarias al total de Reynolds estrés o enstrofía son casi insignificantes. Esto también es cierto en la capa exterior de los canales. En cambio, la vida de los gráficos en los canales se compara con el tiempo de ruptura en SS-HST. Grupos de vórticidad están asociados con casi el mismo cuadrante en términos de sus velocidades medias durante su tiempo de vida, especialmente para los relacionados con las eyecciones y sweeps. Al igual que en los canales, las eyecciones de SS-HST se mueven hacia arriba con una velocidad promedio vertical uT (velocidad de fricción) mientras que lo contrario es cierto para los barridos. Grupos de vórticidad, por otra parte, son casi inmóvil en la dirección vertical. En la dirección de sentido de la corriente, que están advección por la velocidad media local y por lo tanto deforman por la diferencia de velocidad media. Sweeps y eyecciones se mueven más rápido y más lento que la velocidad media, respectivamente, tanto por 1.5uT. Grupos de vórticidad se mueven con la misma velocidad que la velocidad media. Se verifica que las estructuras incoherentes cerca de la pared se debe a la pared en vez de pequeño tamaño. Los resultados sugieren fuertemente que las estructuras coherentes en canales no son especialmente asociado con la pared, o incluso con un perfil de cizalladura dado. ABSTRACT Since the wall-bounded turbulence was first recognized more than one century ago, its near wall region (buffer layer) has been studied extensively and becomes relatively well understood due to the low local Reynolds number and narrow scale separation. The region just above the buffer layer, i.e., the logarithmic layer, is receiving increasingly more attention nowadays due to its self-similar property. Flores et al. (20076) and Flores & Jim´enez (2010) show that the statistics of logarithmic layer is kind of independent of other layers, implying that it might be possible to study it separately, which would reduce significantly the computational costs for simulations of the logarithmic layer. Some attempts were tried later by Mizuno & Jimenez (2013), who simulated the logarithmic layer without the buffer layer with obtained statistics agree reasonably well with those of full simulations. Besides, the logarithmic layer might be mimicked by other simpler sheardriven turbulence. For example, Pumir (1996) found that the statistically-stationary homogeneous shear turbulence (SS-HST) also bursts, in a manner strikingly similar to the self-sustaining process in wall-bounded turbulence. Based on these considerations, this thesis tries to reveal to what extent is the logarithmic layer of channels similar to the simplest shear-driven turbulence, SS-HST, by comparing both kinematics and dynamics of coherent structures in the two flows. Results about the channel are shown by Lozano-Dur´an et al. (2012) and Lozano-Dur´an & Jim´enez (20146). The roadmap of this task is divided into three stages. First, SS-HST is investigated by means of a new direct numerical simulation code, spectral in the two horizontal directions and compact-finite-differences in the direction of the shear. No remeshing is used to impose the shear-periodic boundary condition. The influence of the geometry of the computational box is explored. Since HST has no characteristic outer length scale and tends to fill the computational domain, longterm simulations of HST are ‘minimal’ in the sense of containing on average only a few large-scale structures. It is found that the main limit is the spanwise box width, Lz, which sets the length and velocity scales of the turbulence, and that the two other box dimensions should be sufficiently large (Lx > 2LZ, Ly > Lz) to prevent other directions to be constrained as well. It is also found that very long boxes, Lx > 2Ly, couple with the passing period of the shear-periodic boundary condition, and develop strong unphysical linearized bursts. Within those limits, the flow shows interesting similarities and differences with other shear flows, and in particular with the logarithmic layer of wallbounded turbulence. They are explored in some detail. They include a self-sustaining process for large-scale streaks and quasi-periodic bursting. The bursting time scale is approximately universal, ~ 20S~l (S is the mean shear rate), and the availability of two different bursting systems allows the growth of the bursts to be related with some confidence to the shearing of initially isotropic turbulence. It is concluded that SS-HST, conducted within the proper computational parameters, is a very promising system to study shear turbulence in general. Second, the same coherent structures as in channels studied by Lozano-Dur´an et al. (2012), namely three-dimensional vortex clusters (strong dissipation) and Qs (strong tangential Reynolds stress, -uv), are studied by direct numerical simulation of SS-HST with acceptable box aspect ratios and Reynolds number up to Rex ~ 250 (based on Taylor-microscale). The influence of the intermittency to time-independent threshold is discussed. These structures have similar elongations in the streamwise direction to detached families in channels until they are of comparable size to the box. Their fractal dimensions, inner and outer lengths as a function of volume agree well with their counterparts in channels. The study about their spatial organizations found that Qs of the same type are aligned roughly in the direction of the velocity vector in the quadrant they belong to, while Qs of different types are restricted by the fact that there should be no velocity clash, which makes Q2s (ejections, u < 0, v > 0) and Q4s (sweeps, u > 0, v < 0) paired in the spanwise direction. This is verified by inspecting velocity structures, other quadrants such as u-w and v-w in SS-HST and also detached families in the channel. The streamwise alignment of attached Qs with the same type in channels is due to the modulation of the wall. The average flow field conditioned to Q2-Q4 pairs found that vortex clusters are in the middle of the pair, but prefer to the two shear layers lodging at the top and bottom of Q2s and Q4s respectively, which makes the spanwise vorticity inside vortex clusters does not cancel. The wall amplifies the difference between the sizes of low- and high-speed streaks associated with attached Q2-Q4 pairs as the pairs reach closer to the wall, which is verified by the correlation of streamwise velocity conditioned to attached Q2s and Q4s with different heights. Vortex clusters in SS-HST associated with Q2s or Q4s are also flanked by a counter rotating streamwise vortices in the spanwise direction as in the channel. The long conical ‘wake’ originates from tall attached vortex clusters found by del A´ lamo et al. (2006) and Flores et al. (2007b), which disappears in SS-HST, is only true for tall attached vortices associated with Q2s but not for those associated with Q4s, whose averaged flow field is actually quite similar to that in SS-HST. Third, the temporal evolutions of Qs and vortex clusters are studied by using the method invented by Lozano-Dur´an & Jim´enez (2014b). Structures are sorted into branches, which are further organized into graphs. Both spatial and temporal resolutions are chosen to be able to capture the most probable pointwise Kolmogorov length and time at the most extreme moment. Due to the minimal box effect, there is only one main graph consist by almost all the branches, with its instantaneous volume and number of structures follow the intermittent kinetic energy and enstrophy. The lifetime of branches, which makes more sense for primary branches, loses its meaning in SS-HST because the contributions of primary branches to total Reynolds stress or enstrophy are almost negligible. This is also true in the outer layer of channels. Instead, the lifetime of graphs in channels are compared with the bursting time in SS-HST. Vortex clusters are associated with almost the same quadrant in terms of their mean velocities during their life time, especially for those related with ejections and sweeps. As in channels, ejections in SS-HST move upwards with an average vertical velocity uτ (friction velocity) while the opposite is true for sweeps. Vortex clusters, on the other hand, are almost still in the vertical direction. In the streamwise direction, they are advected by the local mean velocity and thus deformed by the mean velocity difference. Sweeps and ejections move faster and slower than the mean velocity respectively, both by 1.5uτ . Vortex clusters move with the same speed as the mean velocity. It is verified that the incoherent structures near the wall is due to the wall instead of small size. The results suggest that coherent structures in channels are not particularly associated with the wall, or even with a given shear profile.

Ubiquitin-dependent degradation of IκBα is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1

Activation of the transcription factor nuclear factor kappa B (NF-κB) is controlled by proteolysis of its inhibitory subunit (IκB) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of IκBα by a large multisubunit complex containing IκB kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/βTrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with IκBα only when IκBα is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of IκBα in concert with IκB kinases, resulting in nuclear translocation of NF-κB. In addition, FWD1 strikingly evoked the ubiquitination of IκBα in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of IκBα. These results suggest that the substrate-specific degradation of IκBα is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated IκBα. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-κB through control of IκB protein stability.

The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA–RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5–200kD and U5–100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplices in vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5–200kD protein but lack U5–100kD, suggesting that the U5–200kD protein could mediate U4/U6 duplex unwinding. Finally, U5–200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5–200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.

Architectural specificity in chromatin structure at the TATA box in vivo: Nucleosome displacement upon β-phaseolin gene activation

Extensive studies of the β-phaseolin (phas) gene in transgenic tobacco have shown that it is highly active during seed embryogenesis but is completely silent in leaf and other vegetative tissues. In vivo footprinting revealed that the lack of even basal transcriptional activity in vegetative tissues is associated with the presence of a nucleosome that is rotationally positioned with base pair precision over three phased TATA boxes present in the phas promoter. Positioning is sequence-dependent because an identical rotational setting is obtained upon nucleosome reconstitution in vitro. A comparison of DNase I and dimethyl sulfate footprints in vivo and in vitro strongly suggests that this repressive chromatin architecture is remodeled concomitant with gene activation in the developing seed. This leads to the disruption of histone-mediated DNA wrapping and the assembly of the TATA boxes into a transcriptionally competent nucleoprotein complex.

An interplay between TATA box-binding protein and transcription factors IIE and IIA modulates DNA binding and transcription

The basal transcription factor IIE (TFIIE) is thought to be one of the last factors to be assembled into a preinitiation complex (PIC) at eukaryotic promoters after RNA polymerase II and TFIIF have been incorporated. It was shown that a primary function of TFIIE is to recruit and cooperate with TFIIH in promoter melting. Here, we show that the large subunit of TFIIE (E56) can directly stimulate TBP binding to the promoter in the absence of other basal factors. The zinc-finger domain of E56, required for transcriptional activity, is critical for this function. In addition, the small subunit of TFIIE (E34) directly contacts DNA and TFIIA and thus providing a second mechanism for TFIIE to help binding of a TBP/IIA complex to the promoter, the first critical step in the PIC assembly. These studies suggest an alternative PIC assembly pathway in which TFIIE affects both TBP and TFIIH functions during initiation of RNA synthesis.

Human CUL1 forms an evolutionarily conserved ubiquitin ligase complex (SCF) with SKP1 and an F-box protein

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by catalyzing ubiquitination of the S phase cyclin-dependent kinase inhibitor SIC1. SCF is composed of three proteins—ySKP1, CDC53 (Cullin), and the F-box protein CDC4—that are conserved from yeast to humans. As part of an effort to identify components and substrates of a putative human SCF complex, we isolated hSKP1 in a two-hybrid screen with hCUL1, the closest human homologue of CDC53. Here, we show that hCUL1 associates with hSKP1 in vivo and directly interacts with both hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-like particle. Moreover, hCUL1 complements the growth defect of yeast cdc53ts mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. Taken together, these data suggest that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. Further application of biochemical assays similar to those described here can now be used to identify regulators/components of hCUL1-based SCF complexes, to determine whether the hCUL2–hCUL5 proteins also are components of ubiquitin ligase complexes in human cells, and to screen for chemical compounds that modulate the activities of the hSKP1 and hCUL1 proteins.

RAP74 induces promoter contacts by RNA polymerase II upstream and downstream of a DNA bend centered on the TATA box

RAP74, the large subunit of transcription factor IIF, associates with a preinitiation complex containing RNA polymerase II (pol II) and other general initiation factors. We have mapped the location of RAP74 in close proximity to promoter DNA at similar distances both upstream and downstream of a DNA bend centered on the TATA box. Binding of RAP74 induces a conformational change that affects the position of pol II relative to that of the DNA. This reorganization of the preinitiation complex minimally requires the N-terminal region of RAP74 containing both its RAP30-binding domain and another region necessary for accurate transcription in vitro. We propose a role for RAP74 in controlling the topological organization of the pol II preinitiation complex.

Distal enhancer regulation by promoter derepression in topologically constrained DNA in vitro

Long-range promoter–enhancer interactions are a crucial regulatory feature of many eukaryotic genes yet little is known about the mechanisms involved. Using cloned chicken βA-globin genes, either individually or within the natural chromosomal locus, enhancer-dependent transcription is achieved in vitro at a distance of 2 kb with developmentally staged erythroid extracts. This occurs by promoter derepression and is critically dependent upon DNA topology. In the presence of the enhancer, genes must exist in a supercoiled conformation to be actively transcribed, whereas relaxed or linear templates are inactive. Distal protein–protein interactions in vitro may be favored on supercoiled DNA because of topological constraints. In this system, enhancers act primarily to increase the probability of rapid and efficient transcription complex formation and initiation. Repressor and activator proteins binding within the promoter, including erythroid-specific GATA-1, mediate this process.

A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2

The prolamin box (P-box) is a highly conserved 7-bp sequence element (5′-TGTAAAG-3′) found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys2-Cys2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.

The polo-box-dependent induction of ectopic septal structures by a mammalian polo kinase, Plk, in Saccharomyces cerevisiae

Members of the polo subfamily of protein kinases play pivotal roles in cell-cycle control and proliferation. In addition to a high degree of sequence similarity in the kinase domain, polo kinases contain a strikingly conserved motif termed “polo-box” in the noncatalytic C-terminal domain. We have previously shown that the mammalian polo-like kinase Plk is a functional homolog of Saccharomyces cerevisiae Cdc5. Here, we show that, in a polo-box- and kinase activity-dependent manner, ectopic expression of Plk in budding yeast can induce a class of cells with abnormally elongated buds. In addition to localization at spindle poles and cytokinetic neck filaments, Plk induces and localizes to ectopic septin ring structures within the elongated buds. In contrast, mutations in the polo-box abolish both localization to, and induction of, septal structures. Consistent with the polo-box-dependent subcellular localization, the C-terminal domain of Plk, but not its polo-box mutant, is sufficient for subcellular localization. Our data suggest that Plk may contribute a signal to initiate or promote cytokinetic event(s) and that an intact polo-box is required for regulation of these cellular processes.

Pontin52, an interaction partner of β-catenin, binds to the TATA box binding protein

β-catenin, the vertebrate homolog of the Drosophila Armadillo protein, has been shown to have dual cellular functions, as a component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway. At Wnt signaling, β-catenin becomes stabilized in the cytoplasm and subsequently available for interaction with transcription factors of the lymphocyte enhancer factor-1/T-cell factor family, resulting in a nuclear localization of β-catenin. Although β-catenin does not bind DNA directly, its carboxyl- and amino-terminal regions exhibit a transactivating activity still not well understood molecularly. Here we report the identification of an interaction partner of β-catenin, a nuclear protein designated Pontin52. Pontin52 binds β-catenin in the region of Armadillo repeats 2–5 and, more importantly, also binds the TATA box binding protein. We provide evidence for an in vivo multiprotein complex composed of Pontin52, β-catenin, and lymphocyte enhancer factor-1/T-cell factor. Our results suggest involvement of Pontin52 in the nuclear function of β-catenin.

Hydrophobic sequence minimization of the α-lactalbumin molten globule

The molten globule, a widespread protein-folding intermediate, can attain a native-like backbone topology, even in the apparent absence of rigid side-chain packing. Nonetheless, mutagenesis studies suggest that molten globules are stabilized by some degree of side-chain packing among specific hydrophobic residues. Here we investigate the importance of hydrophobic side-chain diversity in determining the overall fold of the α-lactalbumin molten globule. We have replaced all of the hydrophobic amino acids in the sequence of the helical domain with a representative amino acid, leucine. Remarkably, the minimized molecule forms a molten globule that retains many structural features characteristic of a native α-lactalbumin fold. Thus, nonspecific hydrophobic interactions may be sufficient to determine the global fold of a protein.

Role of the Box C/D Motif in Localization of Small Nucleolar RNAs to Coiled Bodies and Nucleoli

Small nucleolar RNAs (snoRNAs) are a large family of eukaryotic RNAs that function within the nucleolus in the biogenesis of ribosomes. One major class of snoRNAs is the box C/D snoRNAs named for their conserved box C and box D sequence elements. We have investigated the involvement of cis-acting sequences and intranuclear structures in the localization of box C/D snoRNAs to the nucleolus by assaying the intranuclear distribution of fluorescently labeled U3, U8, and U14 snoRNAs injected into Xenopus oocyte nuclei. Analysis of an extensive panel of U3 RNA variants showed that the box C/D motif, comprised of box C′, box D, and the 3′ terminal stem of U3, is necessary and sufficient for the nucleolar localization of U3 snoRNA. Disruption of the elements of the box C/D motif of U8 and U14 snoRNAs also prevented nucleolar localization, indicating that all box C/D snoRNAs use a common nucleolar-targeting mechanism. Finally, we found that wild-type box C/D snoRNAs transiently associate with coiled bodies before they localize to nucleoli and that variant RNAs that lack an intact box C/D motif are detained within coiled bodies. These results suggest that coiled bodies play a role in the biogenesis and/or intranuclear transport of box C/D snoRNAs.

Box H and Box ACA Are Nucleolar Localization Elements of U17 Small Nucleolar RNA

The nucleolar localization elements (NoLEs) of U17 small nucleolar RNA (snoRNA), which is essential for rRNA processing and belongs to the box H/ACA snoRNA family, were analyzed by fluorescence microscopy. Injection of mutant U17 transcripts into Xenopus laevis oocyte nuclei revealed that deletion of stems 1, 2, and 4 of U17 snoRNA reduced but did not prevent nucleolar localization. The deletion of stem 3 had no adverse effect. Therefore, the hairpins of the hairpin–hinge–hairpin–tail structure formed by these stems are not absolutely critical for nucleolar localization of U17, nor are sequences within stems 1, 3, and 4, which may tether U17 to the rRNA precursor by base pairing. In contrast, box H and box ACA are major NoLEs; their combined substitution or deletion abolished nucleolar localization of U17 snoRNA. Mutation of just box H or just the box ACA region alone did not fully abolish the nucleolar localization of U17. This indicates that the NoLEs of the box H/ACA snoRNA family function differently from the bipartite NoLEs (conserved boxes C and D) of box C/D snoRNAs, where mutation of either box alone prevents nucleolar localization.