Characterization of the human SCF ubiquitin ligases - structure, function, and regulation

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by cata lyzi ng ubiquitination of the S phase CDK inhibitor SIC1. SCF is composed of several evolutionarily conserved proteins, including ySKP1, CDC53 (Cullin), and the F-box protein CDC4. We isolated hSKP1 in a two-hybrid screen with hCUL1, the human homologue of CDC53. We showed that hCUL1 associates with hSKP1 in vivo and directly interacts with hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-Iike particle. Moreover, hCUL1 complements the growth defect of yeast CDC53^(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. These data demonstrated that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. However, purified human SCF complexes consisting of CUL1, SKP1, and SKP2 are inactive in vitro, suggesting that additional factors are required.

Subsequently, mammalian SCF ubiquitin ligases were shown to regulate various physiological processes by targeting important cellular regulators, like lĸBα, β-catenin, and p27, for ubiquitin-dependent proteolysis by the 26S proteasome. Little, however, is known about the regulation of various SCF complexes. By using sequential immunoaffinity purification and mass spectrometry, we identified proteins that interact with human SCF components SKP2 and CUL1 in vivo. Among them we identified two additional SCF subunits: HRT1, present in all SCF complexes, and CKS1, that binds to SKP2 and is likely to be a subunit of SCF5^(SKP2) complexes. Subsequent work by others demonstrated that these proteins are essential for SCF activity. We also discovered that COP9 Signalosome (CSN), previously described in plants as a suppressor of photomorphogenesis, associates with CUL1 and other SCF subunits in vivo. This interaction is evolutionarily conserved and is also observed with other Cullins, suggesting that all Cullin based ubiquitin ligases are regulated by CSN. CSN regulates Cullin Neddylation presumably through CSNS/JAB1, a stochiometric Signalosome subunit and a putative deneddylating enzyme. This work sheds light onto an intricate connection that exists between signal transduction pathways and protein degradation machinery inside the cell and sets stage for gaining further insights into regulation of protein degradation.

Sequence-function relationships in E. coli transcriptional regulation

Understanding how transcriptional regulatory sequence maps to regulatory function remains a difficult problem in regulatory biology. Given a particular DNA sequence for a bacterial promoter region, we would like to be able to say which transcription factors bind there, how strongly they bind, and whether they interact with each other and/or RNA polymerase, with the ultimate objective of integrating knowledge of these parameters into a prediction of gene expression levels. The theoretical framework of statistical thermodynamics provides a useful framework for doing so, enabling us to predict how gene expression levels depend on transcription factor binding energies and concentrations. We used thermodynamic models, coupled with models of the sequence-dependent binding energies of transcription factors and RNAP, to construct a genotype to phenotype map for the level of repression exhibited by the lac promoter, and tested it experimentally using a set of promoter variants from E. coli strains isolated from different natural environments. For this work, we sought to ``reverse engineer'' naturally occurring promoter sequences to understand how variations in promoter sequence affects gene expression. The natural inverse of this approach is to ``forward engineer'' promoter sequences to obtain targeted levels of gene expression. We used a high precision model of RNAP-DNA sequence dependent binding energy, coupled with a thermodynamic model relating binding energy to gene expression, to predictively design and verify a suite of synthetic E. coli promoters whose expression varied over nearly three orders of magnitude.

However, although thermodynamic models enable predictions of mean levels of gene expression, it has become evident that cell-to-cell variability or ``noise'' in gene expression can also play a biologically important role. In order to address this aspect of gene regulation, we developed models based on the chemical master equation framework and used them to explore the noise properties of a number of common E. coli regulatory motifs; these properties included the dependence of the noise on parameters such as transcription factor binding strength and copy number. We then performed experiments in which these parameters were systematically varied and measured the level of variability using mRNA FISH. The results showed a clear dependence of the noise on these parameters, in accord with model predictions.

Finally, one shortcoming of the preceding modeling frameworks is that their applicability is largely limited to systems that are already well-characterized, such as the lac promoter. Motivated by this fact, we used a high throughput promoter mutagenesis assay called Sort-Seq to explore the completely uncharacterized transcriptional regulatory DNA of the E. coli mechanosensitive channel of large conductance (MscL). We identified several candidate transcription factor binding sites, and work is continuing to identify the associated proteins.

Expanding the Toolkit for Synthetic Biology: Frameworks for Native-like Non-natural Gene Circuits

Synthetic biology combines biological parts from different sources in order to engineer non-native, functional systems. While there is a lot of potential for synthetic biology to revolutionize processes, such as the production of pharmaceuticals, engineering synthetic systems has been challenging. It is oftentimes necessary to explore a large design space to balance the levels of interacting components in the circuit. There are also times where it is desirable to incorporate enzymes that have non-biological functions into a synthetic circuit. Tuning the levels of different components, however, is often restricted to a fixed operating point, and this makes synthetic systems sensitive to changes in the environment. Natural systems are able to respond dynamically to a changing environment by obtaining information relevant to the function of the circuit. This work addresses these problems by establishing frameworks and mechanisms that allow synthetic circuits to communicate with the environment, maintain fixed ratios between components, and potentially add new parts that are outside the realm of current biological function. These frameworks provide a way for synthetic circuits to behave more like natural circuits by enabling a dynamic response, and provide a systematic and rational way to search design space to an experimentally tractable size where likely solutions exist. We hope that the contributions described below will aid in allowing synthetic biology to realize its potential.

A multiple scattering problem

The present work deals with the problem of the interaction of the electromagnetic radiation with a statistical distribution of nonmagnetic dielectric particles immersed in an infinite homogeneous isotropic, non-magnetic medium. The wavelength of the incident radiation can be less, equal or greater than the linear dimension of a particle. The distance between any two particles is several wavelengths. A single particle in the absence of the others is assumed to scatter like a Rayleigh-Gans particle, i.e. interaction between the volume elements (self-interaction) is neglected. The interaction of the particles is taken into account (multiple scattering) and conditions are set up for the case of a lossless medium which guarantee that the multiple scattering contribution is more important than the self-interaction one. These conditions relate the wavelength λ and the linear dimensions of a particle a and of the region occupied by the particles D. It is found that for constant λ/a, D is proportional to λ and that |Δχ|, where Δχ is the difference in the dielectric susceptibilities between particle and medium, has to lie within a certain range.

The total scattering field is obtained as a series the several terms of which represent the corresponding multiple scattering orders. The first term is a single scattering term. The ensemble average of the total scattering intensity is then obtained as a series which does not involve terms due to products between terms of different orders. Thus the waves corresponding to different orders are independent and their Stokes parameters add.

The second and third order intensity terms are explicitly computed. The method used suggests a general approach for computing any order. It is found that in general the first order scattering intensity pattern (or phase function) peaks in the forward direction Θ = 0. The second order tends to smooth out the pattern giving a maximum in the Θ = π/2 direction and minima in the Θ = 0 , Θ = π directions. This ceases to be true if ka (where k = 2π/λ) becomes large (> 20). For large ka the forward direction is further enhanced. Similar features are expected from the higher orders even though the critical value of ka may increase with the order.

The first order polarization of the scattered wave is determined. The ensemble average of the Stokes parameters of the scattered wave is explicitly computed for the second order. A similar method can be applied for any order. It is found that the polarization of the scattered wave depends on the polarization of the incident wave. If the latter is elliptically polarized then the first order scattered wave is elliptically polarized, but in the Θ = π/2 direction is linearly polarized. If the incident wave is circularly polarized the first order scattered wave is elliptically polarized except for the directions Θ = π/2 (linearly polarized) and Θ = 0, π (circularly polarized). The handedness of the Θ = 0 wave is the same as that of the incident whereas the handedness of the Θ = π wave is opposite. If the incident wave is linearly polarized the first order scattered wave is also linearly polarized. The second order makes the total scattered wave to be elliptically polarized for any Θ no matter what the incident wave is. However, the handedness of the total scattered wave is not altered by the second order. Higher orders have similar effects as the second order.

If the medium is lossy the general approach employed for the lossless case is still valid. Only the algebra increases in complexity. It is found that the results of the lossless case are insensitive in the first order of k_imD where k_im = imaginary part of the wave vector k and D a linear characteristic dimension of the region occupied by the particles. Thus moderately extended regions and small losses make (k_imD)² ≪ 1 and the lossy character of the medium does not alter the results of the lossless case. In general the presence of the losses tends to reduce the forward scattering.

Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning.

Small RNAs have several important biological functions. MicroRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) regulate mRNA stability and translation, and siRNAs cause post-transcriptional gene silencing of transposons, viruses and transgenes and are important in both the establishment and maintenance of cytosine DNA methylation. Here, we study the role of the four Arabidopsis thaliana DICER-LIKE genes (DCL1-DCL4) in these processes. Sequencing of small RNAs from a dcl2 dcl3 dcl4 triple mutant showed markedly reduced tasiRNA and siRNA production and indicated that DCL1, in addition to its role as the major enzyme for processing miRNAs, has a previously unknown role in the production of small RNAs from endogenous inverted repeats. DCL2, DCL3 and DCL4 showed functional redundancy in siRNA and tasiRNA production and in the establishment and maintenance of DNA methylation. Our studies also suggest that asymmetric DNA methylation can be maintained by pathways that do not require siRNAs.

The structure of turbulent stratified and premixed methane/air flames I: Non-swirling flows

A series of flames in a turbulent methane/air stratified swirl burner is presented. The degree of stratification and swirl are systematically varied to generate a matrix of experimental conditions, allowing their separate and combined effects to be investigated. Non-swirling flows are considered in the present paper, and the effects of swirl are considered in a companion paper (Part II). A mean equivalence ratio of φ=0.75 is used, with φ for the highest level of stratification spanning 0.375-1.125. The burner features a central bluff-body to aid flame stabilization, and the influence of the induced recirculation zone is also considered. The current work focuses on non-swirling flows where two-component particle image velocimetry (PIV) measurements are sufficient to characterize the main features of the flow field. Scalar data obtained from Rayleigh/Raman/CO laser induced fluorescence (CO-LIF) line measurements at 103μm resolution allow the behavior of key combustion species-CH 4, CO 2, CO, H 2, H 2O and O 2-to be probed within the instantaneous flame front. Simultaneous cross-planar OH-PLIF is used to determine the orientation of the instantaneous flame normal in the scalar measurement window, allowing gradients in temperature and progress variable to be angle corrected to their three dimensional values. The relationship between curvature and flame thickness is investigated using the OH-PLIF images, as well as the effect of stratification on curvature.The main findings are that the behavior of the key combustion species in temperature space is well captured on the mean by laminar flame calculations regardless of the level of stratification. H 2 and CO are significant exceptions, both appearing at elevated levels in the stratified flames. Values for surface density function and by extension thermal scalar dissipation rate are found to be substantially lower than laminar values, as the thickening of the flame due to turbulence dominates the effect of increased strain. These findings hold for both premixed and stratified flames. The current series of flames is proposed as an interesting if challenging set of test cases for existing and emerging turbulent flame models, and data are available on request. © 2012 The Combustion Institute.

The structure of turbulent stratified and premixed methane/air flames II: Swirling flows

Experimental results are presented from a series of turbulent methane/air stratified flames stabilized on a swirl burner. Nine operating conditions are considered, systematically varying the level of stratification and swirl while maintaining a lean global mean equivalence ratio of φ̄=0.75. Scalar data are obtained from Rayleigh/Raman/CO laser induced fluorescence (CO-LIF) line measurements at 103μm resolution, allowing the behavior of the major combustion species-CH 4, CO 2, CO, H 2, H 2O and O 2-to be probed within the instantaneous flame front. The corresponding three-dimensional surface density function and thermal scalar dissipation rate are investigated, along with geometric characteristics of the flame such as curvature and flame thickness. Hydrogen and carbon monoxide levels within the flame brush are raised by stratification, indicating models with laminar premixed flame chemistry may not be suitable for stratified flames. However, flame surface density, scalar dissipation and curvature all appear insensitive to the degree of stratification in the flames surveyed. © 2012 The Combustion Institute.

Random function priors for exchangeable arrays with applications to graphs and relational data

A fundamental problem in the analysis of structured relational data like graphs, networks, databases, and matrices is to extract a summary of the common structure underlying relations between individual entities. Relational data are typically encoded in the form of arrays; invariance to the ordering of rows and columns corresponds to exchangeable arrays. Results in probability theory due to Aldous, Hoover and Kallenberg show that exchangeable arrays can be represented in terms of a random measurable function which constitutes the natural model parameter in a Bayesian model. We obtain a flexible yet simple Bayesian nonparametric model by placing a Gaussian process prior on the parameter function. Efficient inference utilises elliptical slice sampling combined with a random sparse approximation to the Gaussian process. We demonstrate applications of the model to network data and clarify its relation to models in the literature, several of which emerge as special cases.

Application of scalar dissipation rate model to siemens DLE combustors

The standard design process for the Siemens Industrial Turbomachinery, Lincoln, Dry Low Emissions combustion systems has adopted the Eddy Dissipation Model with Finite Rate Chemistry for reacting computational fluid dynamics simulations. The major drawbacks of this model have been the over-prediction of temperature and lack of species data limiting the applicability of the model. A novel combustion model referred to as the Scalar Dissipation Rate Model has been developed recently based on a flamelet type assumption. Previous attempts to adopt the flamelet philosophy with alternative closure models have failed, with the prediction of unphysical phenomenon. The Scalar Dissipation Rate Model (SDRM) was developed from a physical understanding of scalar dissipation rate, signifying the rate of mixing of hot and cold fluids at scales relevant to sustain combustion, in flames and was validated using direct numerical simulations data and experimental measurements. This paper reports on the first industrial application of the SDRM to SITL DLE combustion system. Previous applications have considered ideally premixed laboratory scale flames. The industrial application differs significantly in the complexity of the geometry, unmixedness and operating pressures. The model was implemented into ANSYS-CFX using their inbuilt command language. Simulations were run transiently using Scale Adaptive Simulation turbulence model, which switches between Large Eddy Simulation and Unsteady Reynolds Averaged Navier Stokes using a blending function. The model was validated in a research SITL DLE combustion system prior to being applied to the actual industrial geometry at real operating conditions. This system consists of the SGT-100 burner with a glass square-sectioned combustor allowing for detailed diagnostics. This paper shows the successful validation of the SDRM against time averaged temperature and velocity within measurement errors. The successful validation allowed application of the SDRM to the SGT-100 twin shaft at the relevant full load conditions. Limited validation data was available due to the complexity of measurement in the real geometry. Comparison of surface temperatures and combustor exit temperature profiles showed an improvement compared to EDM/FRC model. Furthermore, no unphysical phenomena were predicted. This paper presents the successful application of the SDRM to the industrial combustion system. The model shows a marked improvement in the prediction of temperature over the EDM/FRC model previously used. This is of significant importance in the future applications of combustion CFD for understanding of hardware mechanical integrity, combustion emissions and dynamics of the flame. Copyright © 2012 by ASME.

Kelvin-Helmholtz-like instability of turbulent flows over riblets

The turbulent drag reduction due to riblets is a function of their size and, for different configurations, collapses well with a length scale l+g=(A+g)1/2, based in the groove cross-section Ag. The initially linear drag reduction breaks down for l+g≈11, which agrees in our DNS with the previously reported appearance of quasi-two-dimensional spanwise rollers immediately above the riblets. They are similar to those found over porous surfaces and plant canopies, and can be traced to a Kelvin-Helmholtz-like instability associated with the relaxation of the impermeability condition for the wall-normal velocity. The extra Reynolds stress associated with them accounts quantitatively for the drag degradation. An inviscid model for the instability confirms its nature, agreeing well with the observed perturbation wavelengths and shapes. The onset of the instability is determined by a length scale L+w that, for conventional riblet geometries, is proportional to l+g. The instability onset, L+w≥4, corresponds to the empirical breakdown point l+g≈11.

Toll-like receptor 3 regulates Mx expression in rare minnow Gobiocypris rarus after viral infection

Toll-like receptor 3 (TLR3) plays a key role in activating immune responses during viral infection. To study the genes involved in the regulatory function of TLR3 in the rare minnow Gobiocypris rarus after viral infection, a full-length cDNA of TLR3 (GrTLR3) with a splice variant (GrTLR3s) was identified by homologous cloning and RACE techniques. The antiviral effector molecule Mx gene was cloned and partially sequenced. The mRNA expression levels of GrTLR3, GrTLR3s, and Mx were studied in different tissues before and after virus infection by real-time quantitative RT-PCR. The transcripts of all three genes in liver were significantly increased following GCRV infection (P<0.05). The mRNA levels in liver were upregulated at 24 h post-injection for GrTLR3 and GrTLR3s, and at 12 h for Mx. The upregulated expression levels were several folds for GrTLR3s, tens of folds for GrTLR3, and hundreds of folds for Mx. By semi-quantitative RT-PCR, GrTLR3 and Mx expressed at all the developmental stages, whereas GrTLR3s could only be detected at later developmental stages. Using RNAi and transgenic techniques, GrTLR3 mediated Mx expression but GrTLR3s did not. The time-dependent upregulation of receptor and effector, and the Mx over-expression dependent on TLR3, indicated that GrTLR3 regulated Mx expression in viral infection through a configuration change in rare minnow, and its splice variant did not contribute to the process.

Energy spectra of two-electron two-dimensional quantum dots confined by elliptical and bowl-like potentials

The laterally confining potential of quantum dots (QDs) fabricated in semiconductor heterostructures is approximated by an elliptical two-dimensional harmonic-oscillator well or a bowl-like circular well. The energy spectrum of two interacting electrons in these potentials is calculated in the effective-mass approximation as a function of dot size and characteristic frequency of the confining potential by the exact diagonalization method. Energy level crossover is displayed according to the ratio of the characteristic frequencies of the elliptical confinement potential along the y axis and that along the x axis. Investigating the rovibrational spectrum with pair-correlation function and conditional probability distribution, we could see the violation of circular symmetry. However, there are still some symmetries left in the elliptical QDs. When the QDs are confined by a "bowl-like" potential, the removal of the degeneracy in the energy levels of QDs is found. The distribution of energy levels is different for the different heights of the barriers. (C) 2003 American Institute of Physics.

Coherent transport through a quantum dot embedded in a double-slit-like Aharonov-Bohm ring

Coherent transport through a quantum dot embedded in one arm of a double-slit-like Aharonov-Bohm (AB) ring is studied using the Green's function approach. We obtain experimental observations such as continuous phase shift along a single resonance peak and sharp inter-resonance phase drop. The AB oscillations of the differential conductance of the whole device are calculated by using the nonequilibrium Keldysh formalism. It is shown that the oscillating conductance has a continuous bias-voltage-dependent phase shift and is asymmetric in both linear and nonlinear response regimes.

Transmission through a Quantum Dot Embedded in a Double-slit-like Aharonov-Bohm Ring

We investigate the electron transport through a double-slit-like Aharonov-Bohm (AB) ring with a quantum dot (QD) embedded in one of its arms. Considering both the resonance of the dot and interference effect, the magnitude and phase of the transmission amplitude through the QD are calculated using Green's function approach. The numerical results are in good agreement with the experimental observations.