Hypersensitive site 4 of the human β-globin locus control region.

The Locus Control Region (LCR) of the human beta globin gene domain is defined by four erythroid-specific DNasel hypersensitive sites (HSS) located upstream of this multigene cluster. The LCR confers copy number dependent high levels of erythroid specific expression to a linked transgene, independent of the site of integration. To assess the role of the individual hypersensitive sites of the LCR, we have localized HSS4 to a 280bp fragment that is functional both in murine erythroleukaemia (MEL) cells and in transgenic mice. This fragment coincides with the major area of hypersensitivity 'in vivo' and contains a number of DNasel footprints. Bandshift analysis shows that these footprints correspond to binding sites for the erythroid specific proteins GATA1 and NF-E2 and a number of ubiquitous proteins, including jun/fos, Sp1 and TEF2

Transcriptional Activation by hypersensitive site three of the human β-globin Locus Control Region in murine erythroleukemia cells.

In this paper we describe a complete deletional analysis of hypersensitive site three (HS3) of the human beta-globin Locus Control Region (LCR). The previously defined core fragment consists of 6 footprinted regions, with multiple binding sites for the erythroid-specific factor GATA-1 and G-rich motifs that can interact with ubiquitous factors such as Sp1 and TEF-2. We show in this paper that the 5' half of this fragment is the most important for activity in murine erythroleukemia (MEL) cells. A fragment containing footprints 1-4 can stimulate transcription of a linked human beta-globin gene to levels of about 40% of that obtained with footprints 1-6. Constructs containing either footprints 1-3 or 3-6 cannot be distinguished from the beta-globin gene alone. We further show that binding sites for the erythroid-specific factor NF-E2 can co-operatively interact with parts of the HS3 core fragment, and that HS3 requires elements upstream from -103 in the human beta-globin promoter for full activity. The importance of these results is discussed in the context of the regulation of the genes in the human beta-globin cluster

Estrogen-inducible and liver-specific expression of the chicken very low density apolipoprotein II gene locus in transgenic mice.

We have examined the chicken Very Low Density Apolipoprotein II (apoVLDL II) gene locus in transgenic mice. A DNA fragment composed of the transcribed region, 16 kb of 5' flanking and 400 bp of 3' flanking sequences contained all the information sufficient for estrogen-inducible, liver-specific expression of the apoVLDL II gene. The far-upstream region contains a Negative Regulating Element coinciding with a DNaseI-hypersensitive site at -11 kb. In transgenic mice, the NRE at -11 kb is used for downregulating the expression to a lower maximum level. The NRE might be used for modulating apoVLDL II gene expression, and may be involved in the rapid shut-down of the expression after hormone removal

Staircase structures in fluid dynamical systems

Stratified turbulent fluids exhibit a wide variety of fascinating behaviours. One of the most interesting is the phenomenon of staircase formation, where the density field spontaneously evolves into a series of well-mixed layers separated by sharp interfaces with high gradients. Staircases appear in a wide range of contexts, from the geophysical examples of oceanic thermohaline staircases and atmospheric potential vorticity staircases, to the E×B staircase of plasma physics. In this thesis we present models for staircases in stirred stratified convection and double diffusive convection. We derive a one-dimensional horizontally-averaged mixing-length model from the Boussinesq equations, which we apply first to layering in stirred stratified convection, and then to double-diffusive layering. In stirred stratified convection, the model consists of equations for the buoyancy and kinetic energy, closed via a length scale parameterised in terms of the variables. We investigate the linear stability of the system, determining the effects of varying the viscous and molecular diffusivities. A novel choice of boundary conditions allows us to investigate the behaviour of numerical solutions to very late times. Staircase solutions undergo layer mergers, which we demonstrate occur on a logarithmic timescale, providing a link with other models of layering. We also present an experimental study to test predictions of the theory. For double-diffusive convection, the model consists of three equations, for temperature, salinity and energy. We present a linear stability analysis for a general three-component flux-law system, which we apply to our specific model. A suitable parameterisation for the length scale allows the model to produce staircases in salt fingering convection without the need for any external forcing. In diffusive convection, some energy source is required for layering. Temperature and salinity fluxes through staircases increase during layer mergers, accounting for heightened fluxes in observed staircases in comparison with non-layered states

The minimal requirements for activity in transgenic mice of hypersensitive site 3 of the beta globin locus control region.

Proper expression of the human beta-like globin genes is completely dependent on the presence of the locus control region or LCR, a region containing four DNase hypersensitive sites (HS1-4) situated 5' to the structural genes. Linkage of the LCR to a transgene results in copy number-dependent transcription, independent of the site of integration in the host genome. We have analysed a small region of the LCR (HS3) in transgenic animals to determine the minimal interactions that are required for this property. The results show that a specific combination of a G-rich sequence flanked on each side by one binding site for the transcription factor GATA1 is essential to obtain position-independent expression of a linked beta globin gene in erythroid cells. The overall transcriptional activity of HS3 is achieved through synergy with other combinations of similar binding sites

Salt fingering staircases and the three-component Phillips effect

Understanding the dynamics of staircases in salt fingering convection presents a long-standing theoretical challenge to fluid dynamicists. Although there has been significant progress, particularly through numerical simulations, there are a number of conflicting theoretical explanations as to the driving mechanism underlying staircase formation. The Phillips effect proposes that layering in stirred stratified flow is due to an antidiffusive process, and it has been suggested that this mechanism may also be responsible for salt fingering staircases. However, the details of this process, as well as mathematical models to predict the evolution and merger dynamics of staircases, have yet to be established. We generalise the theory of the Phillips effect to a three component system (e.g. temperature, salinity, energy) and demonstrate a regularised nonlinear model of layering based on mixing-length parameterisations. The model predicts both the inception of layering and its long-term evolution through mergers, while generalising, and remaining consistent with, previous results for double diffusive layering based on flux ratios. Our model of salt fingering is formulated using spatial averaging processes and closed by a mixing length parameterised in terms of the kinetic energy and the ratio of the temperature and salt gradients. The model predicts a layering instability for a bounded range of parameter values in the salt fingering regime. Nonlinear solutions show that an initially unstable linear buoyancy gradient develops into layers, which proceed to merge through a process of stronger interfaces growing at the expense of weaker ones. Our results indicate that these mergers are responsible for the characteristic increase of buoyancy flux through thermohaline staircases