The brown Dominant (bw D ) allele contains a large insertion of heterochromatin, which causes the locus to aberrantly associate with heterochromatin in interphase nuclei and silences the wild-type allele in heterozygotes. Transgenes placed near the bw ؉ locus, in trans to bw D , can also be silenced. The strength of silencing (called trans inactivation) varies with the regulatory sequences of the transgene and its distance away from the bw D insertion site in trans. In this study, we examine endogenous sequences in cis that influence susceptibility of a reporter gene to trans inactivation. Flanking deletions were induced in two parental lines containing P-element transgenes showing trans inactivation of the mini-white reporter. These new lines, which have mini-white under the influence of different endogenous sequence elements, now show varied ability to be silenced by bw D . Determination of the deleted regions and the levels of mini-white expression and trans inactivation has allowed us to explore the correlation between cis sequence elements and susceptibility to trans inactivation and to identify a 301-bp sequence that acts as an enhancer of trans inactivation. Intriguingly, this region encompasses the upstream regions of two divergently transcribed genes and contains a sequence motif that may bind BEAF, a protein involved in delimiting chromatin boundaries.Transcription of the eukaryotic genome is controlled at a number of levels. Not only must sequences near the coding region bind the basal transcription machinery and appropriate transcription factors, but also the surrounding chromatin must be arranged to promote access of these proteins to their binding sites. Recent evidence suggests yet another level of control: the positioning of a locus within the three-dimensional space of the interphase nucleus. In certain circumstances, it has been shown that loci that are being silenced move to a different region of the nucleus, where they associate with masses of highly condensed constitutive heterochromatin (for review, see reference 36). As the consequences of positioning in the nucleus have only recently begun to be explored, it is not surprising that the interplay among these levels of transcriptional regulation is poorly understood. This paper presents results that begin to address the interaction between cis-acting regulatory sequences, chromatin structure, and nuclear positioning.One of the first systems that identified a role for nuclear positioning in gene silencing involves an unusual allele of an eye-color locus in Drosophila melanogaster. The brown Dominant (bw D ) allele contains an insertion of approximately 1.6 Mbp of heterochromatin into the brown (bw) coding sequence near the distal end of the right arm of the second chromosome (27). Eyes of bw D /bw ϩ flies almost totally lack red eye pigment, except in a few small distinct spots. Silencing of the bw ϩ gene on the homologous chromosome, called trans inactivation, has similarities to classic cis-acting position effect variegation (PEV), where a ...
Abstrac tWe have developed an innovative ray-tracing simulation algorithm to describe Relativistic Effects in SpaceTime ("REST") . Our algorithm, called REST frame, models light rays that hav e assumed infinite speed in conventional ray-tracing to have a finite speed in spacetime, and uses the non-Newtonian Lorent z Transformation to relate measurements of a single event in different inertial coordinate systems (inertial frames) . Our earlie r work [5][6][7] explored the power of REST frame as an experimentation tool to study the rich visual properties in natura l world modeled by Special Relativity . Non-intuitive images o f the anisotropic deformation ("warping " ) of space, the intensity concentration/spreading of light sources in spacetime, and th e relativistic Doppler shift were visualized from our simulations .REST frame simulations are computationally expensive . Several hours of CPU time may be needed to generate one intricate image on a relatively powerful DECStation 3100 . Thi s high simulation cost of REST frame precludes its application i n interactive, real-time graphics environments .In this paper, we report a scanline based REST frame rendering method that provides a faster alternative to the original ray-tracing based RESTframe implementation . This ne w method operates in the spirit of the classical Z-buffer in computer graphics[2] and the inter-inertial frames point-mappin g method investigated in physics in the early 1960's [14][12], an d determines the visibility of points in spacetime by their spatial and temporal visibility . Specifically, all spacetime even t points that are potentially visible from the viewpoint at th e imaging time are geometrically projected in three dimensiona l (3D) space to the image plane pixel buffer . Multiple points with a same pixel affiliation are sorted by their time distance
Abstrac tWe have developed an innovative ray-tracing simulation algorithm to describe Relativistic Effects in SpaceTime ("REST") . Our algorithm, called REST frame, models light rays that hav e assumed infinite speed in conventional ray-tracing to have a finite speed in spacetime, and uses the non-Newtonian Lorent z Transformation to relate measurements of a single event in different inertial coordinate systems (inertial frames) . Our earlie r work [5] [6][7] explored the power of REST frame as an experimentation tool to study the rich visual properties in natura l world modeled by Special Relativity . Non-intuitive images o f the anisotropic deformation ("warping " ) of space, the intensity concentration/spreading of light sources in spacetime, and th e relativistic Doppler shift were visualized from our simulations .REST frame simulations are computationally expensive . Several hours of CPU time may be needed to generate one intricate image on a relatively powerful DECStation 3100 . Thi s high simulation cost of REST frame precludes its application i n interactive, real-time graphics environments .In this paper, we report a scanline based REST frame rendering method that provides a faster alternative to the original ray-tracing based RESTframe implementation . This ne w method operates in the spirit of the classical Z-buffer in computer graphics[2] and the inter-inertial frames point-mappin g method investigated in physics in the early 1960's [14][12], an d determines the visibility of points in spacetime by their spatial and temporal visibility . Specifically, all spacetime even t points that are potentially visible from the viewpoint at th e imaging time are geometrically projected in three dimensiona l (3D) space to the image plane pixel buffer . Multiple points with a same pixel affiliation are sorted by their time distance from the imaging time, and the most recent spacetime point i s displayed .This method, which we call 'Time-Buffer" or 'T-Buffer", offers a significant speed improvement over the original RESTframe in software, and permits a dedicated Z-buffer-type hardware implementation that promises interactive, real-time relativistic effects in simulations on a contemporary graphic workstation .Motion blur in real world images caused by the noninfinitesimal exposure time of image-taking can be simulated by "Stochastic T-Buffer", which perturbs the time componen t of the scan-converted spacetime events that are potentially visible . The classical A-Buffer technique[1] that models translucency also can be adapted easily in T-Buffer . The limitation of T-Buffer is its inability to model specular reflection and refraction in optics, ' which our original REST frame implementatio n simulates completely .
The brown Dominant (bw D ) allele of Drosophila contains a heterochromatic block that causes the locus to interact with centric heterochromatin. This association silences bw 1 in heterozygotes (trans-inactivation) and is dependent on nuclear organizational changes later in development, suggesting that transinactivation may not be possible until later in development. To study this, a P element containing an upstream activating sequence (UAS)-GFP reporter was inserted 5 kb from the bw D insertion site. Seven different GAL4 driver lines were used and GFP fluorescence was compared in the presence or the absence of bw D . We measured silencing in different tissues and stages of development and found variable silencing of GFP expression driven by the same driver. When UAS-GFP was not expressed until differentiation in the eye imaginal disc it was more easily trans-inactivated than when it was expressed earlier in undifferentiated cells. In contrast to some studies by other workers on silencing in cis, we did not find consistent correlation of silencing with level of expression or evidence of relaxation of silencing with terminal differentiation. We suggest that such contrasting results may be attributed to a potentially different role played by nuclear organization in cis and trans position-effect variegation. P OSITION-EFFECT variegation (PEV) is the silencing of gene expression by nearby heterochromatin. The phenomenon was first described in Drosophila and has long served as a model for the downregulation of expression by chromatin compaction and changes in higher-order chromatin organization. Classic cis-acting PEV results from a chromosomal break and rejoining that juxtaposes a euchromatic gene and a block of constitutive heterochromatin. In contrast to earlier ideas concerning the continuous, straightforward linear spreading of the silent state, a collection of recent studies have found that the phenomenon is more complex. Many factors influence the susceptibility or resistance of a gene to silencing by heterochromatin in cis, such as the size, composition, and distance of the heterochromatic block and, most importantly, the specifics of the expression properties of the gene being silenced (for review see Talbert and Henikoff 2006).While these studies addressed cis-acting PEV, there is a similar, related phenomenon in flies of silencing by heterochromatin in trans. In trans-acting PEV, or ''transinactivation,'' the block of heterochromatin and the silenced gene are not necessarily on the same DNA molecule, but are brought close to each other within the space of the interphase nucleus. It is probable that this type of PEV is similar to downregulation of a number of normal genes in higher eukaryotes, as increasing evidence finds developmentally silenced loci that move into association with large blocks of heterochromatin (for example, see Su et al. 2004). A well-studied example of trans-inactivation is the brown Dominant (bw D
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