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Nucleosomes are believed to inhibit DNA binding by transcription factors. Theoretical attempts to understand the significance of nucleosomes in gene expression and regulation are based upon this assumption. However, nucleosomal inhibition of transcription factor binding to DNA is not complete. Rather, access to nucleosomal DNA depends on a number of factors, including the stereochemistry of transcription factor-DNA interaction, the in vivo kinetics of thermal fluctuations in nucleosome structure, and the intracellular concentration of the transcription factor. In vitro binding studies must therefore be complemented with in vivo measurements. The inducible PHO5 promoter of yeast has played a prominent role in this discussion. It bears two binding sites for the transcriptional activator Pho4, which at the repressed promoter are positioned within a nucleosome and in the linker region between two nucleosomes, respectively. Earlier studies suggested that the nucleosomal binding site is inaccessible to Pho4 binding in the absence of chromatin remodeling. However, this notion has been challenged by several recent reports. We therefore have reanalyzed transcription factor binding to the PHO5 promoter in vivo, using 'chromatin endogenous cleavage' (ChEC). Our results unambiguously demonstrate that nucleosomes effectively interfere with the binding of Pho4 and other critical transcription factors to regulatory sequences of the PHO5 promoter. Our data furthermore suggest that Pho4 recruits the TATA box binding protein to the PHO5 promoter.
Citation: Mao C, Brown CR, Griesenbeck J, Boeger H (2011) Occlusion of Regulatory Sequences by Promoter Nucleosomes In Vivo. PLoS ONE6(3): e17521. https://doi.org/10.1371/journal.pone.0017521
Editor: Laszlo Tora, Institute of Genetics and Molecular and Cellular Biology, France
Received: November 29, 2010; Accepted: February 3, 2011; Published: March 3, 2011
Copyright: © 2011 Mao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: RB was supported by National Research Service Award (NRSA) grant F32GM087867 from the National Institutes of Health. JG was supported by two DFG travel grants, GR 1706/4-1 and GR 1706/5-1. HB acknowledges support from National Institutes of Health grant GM078111-01 and the Pew Scholars Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
In vitro studies indicated that the wrapping of DNA in nucleosomes limits the accessibility of core particle DNA to nucleases and transcription factors and interferes with the initiation of transcription [1], [2], [3]. This conclusion provides the basis for theories regarding the gene-regulatory function of chromatin structure, and has sparked great interest in the mechanism of nucleosome positioning [4], [5], [6], [7], and the kinetics of nucleosome transactions in vivo [8].
However, the occlusion of binding sites by nucleosomes is not complete. A small number of DNA binding proteins appears to bind wrapped nucleosomal DNA, albeit at reduced affinity, as long as their recognition sequence is rotationally properly positioned [9]. Most transcription factors probably depend on the spontaneous unwrapping of nucleosomal DNA to access interior sequences of the core particle [2]. This requires sufficiently high concentrations of the transcription factor to overcome the fast rewrapping kinetics of nucleosomal DNA [10]. Clustering of binding sites within the nucleosome core particle can lead to cooperative binding in the absence of direct interactions between the transcription factors (indirect cooperativity) [11]. Furthermore, it has been argued that histone modifications affect the extent of DNA wrapping about the histone octamer [12], [13].
Because of these and other mitigating factors, in vitro binding studies, which have mostly been performed on nucleosomes reconstituted in vitro on artificial DNA sequences, need to be complemented by in vivo experiments when considering the effect of specific nucleosomes on transcription factor binding [14]. In vivo binding studies have focused on a small number of biological models. The inducible PHO5 promoter of yeast (Saccharomyces cerevisiae) has served as a prominent paradigm in this discussion [15]. PHO5, which encodes a secreted acidic phosphatase, is induced in response to phosphate starvation. The PHO5 promoter contains three regulatory sequence elements, two upstream activation sequences, UASp1 and UASp2, and a TATA box. Under repressing conditions (high phosphate media), the promoter is characterized by nucleosomes in defined positions, with UASp1 exposed in the linker region between the two nucleosome core particles, N-2 and N-3, and UASp2 positioned close to the center of core particle N-2; the TATA box is wrapped in core particle N-1 [16]. Under activating conditions (media with little or no phosphate), the transcriptional activator Pho4, a helix-loop-helix DNA binding protein, enters the nucleus and binds together with the homeodomain factor Pho2 at both upstream activating sequences. The activation domain of Pho4 is required for the depletion of promoter nucleosomes and the activation of PHO5 transcription [8], [17].
Nucleosome N-2 isolated from native yeast chromatin was found to prevent the binding of Pho4 and Pho2 at UASp2 [18]. A classic experiment used dimethylsulfate (DMS) footprinting in vivo to show that Pho4, when deprived of its activation domain, binds at UASp1, but not UASp2 [19]. Consistently, UASp1 residues were methylated at a faster rate than UASp2 residues upon activation of PHO5 by Pho4 fused to a DNA methyltransferase [20]. Thus in vitro and in vivo binding experiments suggested occlusion of the Pho4 binding site at UASp2 by nucleosome N-2.
However, doubts remain. The pattern of a potential Pho4-DMS footprint on nucleosomal DNA is unknown. The absence of a recognizable pattern does therefore not exclude the possibility of Pho4 binding to the nucleosome. Free methyltransferase methylates nucleosomal DNA at a slower rate than naked DNA [20]. Slower methylation kinetics may therefore reflect the presence of a nucleosome rather than the inability of Pho4-DMS to access its binding site at UASp2. Furthermore, recent studies provided evidence, either by DMS footprinting or chromatin immunoprecipitation (ChIP), for binding of Pho4 at UASp2 in the absence of apparent chromatin remodeling, which was inhibited by deletion of the histone chaperone gene ASF1 [21], [22], [23]. These results suggested that Pho4 binding to DNA was uninhibited by nucleosome formation. On the basis of these conclusions, the binding of Pho4 and Pho2 at UASp2 has been construed as an in vivo example for indirect cooperative binding of transcription factors to nucleosomal DNA [24].
Because of its significance for theoretical attempts to understand the role of chromatin structure in gene expression and regulation [8], [25], we reanalyzed Pho4 binding, and investigated the binding of TBP and Pho2 at the PHO5 promoter in wild type cells and various mutants by 'chromatin endogenous cleavage' (ChEC). This approach allows one to monitor binding of transcription factors to their recognition elements by measuring the frequency of binding-site dependent DNA cleavage, after in vivo crosslinking, by micrococcal nuclease that was linked to the transcription factor [26]. This approach allowed for quantitative measurements of high molecular specificity, sufficient spatial resolution, as well as low background, but was superior to other previously used methods mostly because the absence of signal was more easily interpretable (see Discussion).
PHO5 promoter cleavage by Pho4-MNase
To analyze Pho4 binding at the PHO5 promoter, we generated strains that express micrococcal nuclease linked to the C-terminus of the transcription factor (Pho4-MNase). Phosphatase assays indicated that the fusion protein was equally effective in activating PHO5 as the wild type Pho4 protein (Fig. 1).
Acidic phosphatase activities were measured after 0, 1, 2, 3, and 4 hours of culturing cells in phosphate-free medium. Phosphatase activities for cells expressing the Pho4 wild type protein and Pho4-MNase are indicated by gray and black bars, respectively. Phosphatase activity is given in arbitrary units normalized to cell density.
Cells expressing Pho4-MNase were briefly treated with formaldehyde to cross-link promoter-bound proteins and DNA, either before or at different times after transfer into phosphate-free medium. Extracts prepared from cross-linked cells were incubated for various amounts of time in the presence of Ca2+ ions to activate micrococcal nuclease. To determine cleavage frequencies, isolated DNA was digested with restriction enzymes to release a 3 kb fragment encompassing the PHO5 gene, fractionated by gel electrophoresis, blotted and hybridized with a radiolabeled DNA probe that recognizes sequences upstream of the PHO5 promoter (Fig. 2). As expected, PHO5 DNA isolated from induced cells was cleaved at two sites, close to UASp1 and UASp2. Both sites most certainly represent a cluster of closely spaced cutting events, as micrococcal nuclease lacks sequence specificity. There was little or no cleavage of PHO5 DNA isolated from repressed cells. Cleavage frequencies for samples taken at 3, 4 and 6 hours after induction were virtually identical (Fig. 2), suggesting that Pho4 reached binding equilibrium at UASp1 between 2 and 3 hours after induction. The slow approach toward binding equilibrium may explain, in part, the slow kinetics of PHO5 induction and promoter nucleosome loss [27]. An even slower approach toward binding equilibrium was previously observed by ChIP [28]. The discrepancy may be attributable to the lack of resolution in the ChIP experiments, which did not allow for distinguishing between binding at UASp1 and UASp2.
ChEC analysis of Pho4 binding at the wild type PHO5 promoter. Cells were cultured in phosphate-free medium for 0, 2, 3, 4, and 6 hours before analysis. For each induction time point, aliquots of extract prepared after in vivo cross-linking were incubated in the presence of Ca2+ ions for 0, 5, 15, 30, and 60 minutes (triangles above autoradiographs). The DNA was isolated, digested with PstI and ApaI, fractionated by agarose gel electrophoresis, blotted and hybridized with a 32P-labeled DNA probe complementary to sequences upstream of the PHO5 promoter. Autoradiography of the Southern blot is shown on top. The graphic on the right shows the nucleosome configuration of the repressed PHO5 promoter [16], [29], where nucleosomes and regulatory sequence elements are indicated by gray ovals and small circles, respectively. The relative frequency of cleavage at UASp1, which provides a measure for Pho4 binding at UASp1, is indicated on the ordinate of the histogram (bottom). Different gray tones correspond to different induction times (see triangles above autoradiography). Numbers on the abscissa indicate incubation times (in minutes) in the presence of calcium ions. The similarity of cleavage kinetics for induction time points 3, 4, and 6 hours suggests that Pho4 reached its binding equilibrium at UASp1 between 2 and 3 hours after transfer of cells into phosphate-free medium. Marker bands (lane M) indicate (from top to bottom) restriction sites for PstI (3′-end of PHO5 gene), DraI (beginning of open reading frame), ClaI (UASp2), and BamHI (5′-edge of nucleosome N-3).
The restriction of cleavage to sites close to UASp1 and UASp2 suggested that cutting was due to sequence-specific binding of Pho4-MNase at the promoter. To test this conjecture, we mutated the Pho4 binding site at UASp2. The mutation selectively abolished cleavage at UASp2 but not UASp1 (Fig. 3), indicating that cleavage close to UASp2 was due to Pho4 binding at UASp2, and not due to Pho4 binding at UASp1 or cross-linking of Pho4-MNase that did not interact with the PHO5 promoter in a sequence-specific manner.
ChEC analysis of Pho4 binding at the PHO5 promoter. Cells were cultured in phosphate-free medium for 0, 2, 2.5, 3, and 4 hours before analysis. Cell extracts for each induction time point were incubated in the presence of Ca2+ ions for 0, 5, 15, 30, and 60 minutes (gray triangles above autoradiographs). The DNA was isolated, digested with PstI and ApaI, fractionated by agarose gel electrophoresis, blotted and hybridized with a radioactively labeled DNA probe spanning sequences upstream of the PHO5 promoter. A diagram of the repressed PHO5 promoter chromatin structure is shown to the right of the autoradiographs. Gray ovals represent nucleosome core particles. Positions of the TATA-box, UASp2, and UASp1 are indicated by a white, gray and black dot, respectively. A black bar indicates the position of the probe used for indirect end-labeling. Autoradiographs on the top, middle and bottom show Pho4 binding in PHO5 wild type cells, UASp2Δ cells, and UASp1Δ cells, respectively. Bands in the marker lane (M) indicate, from top to bottom, positions of PstI, DraI, ClaI and BamHI restriction sites, respectively. Pho4 binding at the PHO8 promoter was unimpaired in UASp1Δ cells (data not shown).
Mutation of UASp1 prevents Pho4 binding at UASp2
To analyze effect of UASp mutations on the chromatin remodeling on the PHO5 promoter, we employed strains that allow for the formation of PHO5 gene circles in vivo and subsequent analysis of chromatin remodeling by topology analysis [29]. We isolated gene circle topoisomers from UASp1 and UASp2 mutant strains, and resolved topoisomers by agarose gel electrophoresis. Consistent with earlier nuclease accessibility measurements at nucleosome N-2 [30], mutation of UASp1 completely abolished remodeling of PHO5 promoter chromatin, as indicated by virtually identical gene circle topoisomer distributions between induced and non-induced cells (Fig. 4A). In contrast, mutation of UASp2 allowed for chromatin remodeling, although remodeling was less effective than in promoter wild type cells (Fig. 4B).
Nucleosome loss at the PHO5 promoter was measured by topology analysis [29], in UASp1 and UASp2 deletion mutants. Gene circles were formed in vivo by site-specific recombination of the PHO5 locus [29]. Gene circle topoisomers were fractionated by chloroquine gel electrophoresis, blotted and hybridized with a 32P-labeled DNA probe spanning the PHO5 gene. (A) Topoisomer distributions of PHO80 (R) and pho80Δ (A) PHO5 gene circles isolated from UASp1Δ cells. Pho80 is an inhibitor of the PHO signaling pathway. In its absence Pho4 is constitutively nuclear. (B) Topoisomer distributions of PHO80 (R) and pho80Δ (A) PHO5 gene circles isolated from UASp2Δ cells. The intensity profiles of distributions are shown on the right next to the autoradiographs. Distribution centers are indicated by arrowheads. The center of the topoisomer distribution for the repressed gene is additionally marked by a dashed line in the intensity profiles. Numbers indicate the linking difference, ΔLk°, between distribution means (arrowheads), which corresponds to the average number of nucleosomes lost from the activated promoter [29]. Wild type PHO5 gene circles have a linking difference of 1.85 [8], [29]. Positions of nicked circles are indicated by (*).
To assess whether nucleosome N-2 interferes with Pho4 binding at UASp2, we repeated our ChEC analysis in a UASp1 mutant. If nucleosome N-2 does not interfere with Pho4 binding at UASp2, mutation of UASp1 should selectively abolish cleavage at UASp1, but not UASp2. In contrast, if Pho4 binding at UASp2 is inhibited by nucleosome N-2, mutation of UASp1 is expected to abolish cleavage at both UASp1 and UASp2. Our results bore out the latter expectation (Fig. 3). The simplest interpretation of this result is that UASp2 is inaccessible to Pho4, unless nucleosome N-2 is removed due to Pho4 binding at UASp1 and recruitment of chromatin remodeling or other activities.
Mutation of UASp1 prevents Pho2 binding at UASp2
Multiple Pho2-binding sites have been detected by DNase I footprinting in vitro at the PHO5 promoter, including one site juxtaposed to UASp1, and four sites occupied by nucleosome N-2 under repressing conditions [31]. A corresponding cleavage pattern of PHO5 DNA was observed for cells expressing Pho2-MNase after induction (Fig. 5). Mutation of UASp1 abolished cleavage by Pho2-MNase, except at UASp1, indicating that nucleosome N-2 interfered with Pho2 binding. In contrast, mutation of UASp2 allowed for Pho2 binding at N-2 sequences, albeit with reduced apparent affinity, consistent with the increased promoter nucleosome occupancy in the induced UASp2 mutant compared to wild type (Fig. 4B).
ChEC analysis of Pho4 binding at the PHO5 promoter. Cells were cultured in phosphate-free medium for 0, 2, 3, and 4 hours before analysis. Cell extracts for each induction time point were incubated in the presence of Ca2+ ions for 0, 5, 15, 30, and 60 minutes (gray triangles above autoradiographs). The DNA was isolated, digested with ScaI, fractionated by agarose gel electrophoresis, blotted and hybridized with a radioactively labeled DNA probe spanning sequences within the PHO5 open reading frame. A diagram of the repressed PHO5 promoter chromatin structure is shown to the right of the autoradiographs. Gray ovals represent nucleosome core particles. Positions of the TATA-box, UASp2, and UASp1 are indicated by a white, gray and black dot, respectively. White circles indicate positions of Pho2 binding sites mapped by DNase I footprinting in vitro and corresponding Pho2-MNase cleavage pattern (autoradiography). A black bar indicates the position of the probe used for indirect end-labeling. Autoradiographs on the top, middle and bottom show Pho2p binding in PHO5 wild type cells, UASp1Δ cells, and UASp2Δ cells, respectively. Bands in the marker lane (M) indicate, from top to bottom, positions of ScaI, BamHI, ClaI and DraI restriction sites, respectively. (The ScaI/ScaI fragments of the samples in the bottom autoradiograph migrated slower than the Sca/ScaI fragment of the marker, because the Pho2-MNase expressing strain was derived from a PHO5 gene circle strain, which contains an RS element between nucleosome N-3 and the 5′ ScaI site [29], whereas the marker was prepared from a strain lacking this insertion. This did not affect the positioning of Pho2-MNase cleavage sites relative to the ClaI and DraI marker bands.
TBP binding at the PHO5 promoter coincides with transcriptional activation of PHO5
Does nucleosome N-1 occlude the promoter's TATA box? To address this question, we investigated the cutting of PHO5 promoter DNA by micrococcal nuclease linked to the TATA box binding protein (TBP-MNase). Upon induction, a distinct cleavage pattern was observed, with strong cutting at the TATA box, and weaker cutting at UASp1 and UASp2 (Fig. 6). In contrast, little or no cleavage was observed under repressing conditions (Fig. 6), consistent with the notion that TBP binding at the PHO5 TATA box requires prior removal of nucleosome N-1.
ChEC analysis of TBP-MNase cleavage of PHO5 promoter DNA in the PHO5 wild type, and strains bearing mutations either in the TATA box or UASp1 of the PHO5 promoter. Autoradiographs show cleavage of PHO5 promoter DNA for PHO5 wild type (top), a TATA box mutant (middle), and a UASp1Δ mutant (bottom), respectively. Bands in the marker lane (M) indicate, from top to bottom, ScaI, BamHI, ClaI, and DraI cleavage, respectively.
Cleavage at all three promoter sites was abolished in a UASp1 mutant, indicating that cutting, including cuts at the non-nucleosomal UASp1, required binding of Pho4 to the promoter (Fig. 6). While cleavage at UASp2 and the TATA box might have been due to loss of nucleosomes from positions N-1 and N-2 and nonspecific DNA binding by TBP-MNase, Pho4-dependent cleavage at UASp1 either indicated recruitment of TBP-MNase by the promoter-bound activator, or interactions between the core promoter and upstream activating sequences. To distinguish between these two possibilities, we investigated cutting by TBP-MNase in a TATA box mutant. If cleavage at upstream activating sequences was entirely due to TBP-MNase binding at the TATA box and looping of DNA between the TATA box and activator binding sites, mutation of the TATA box should diminish cleavage at all three promoter sites. In contrast, we found that mutation of the TATA box diminished cleavage at the core promoter, but not at UASp1 and UASp2 (Fig. 6).
Pho4 and Pho2 bind cooperatively at the PHO5 promoter in vivo
In vitro binding studies showed that Pho4 binds cooperatively with Pho2 at UASp1 [31]. To determine the significance of this cooperativity for DNA binding in vivo, which depends on the intracellular concentration of both transcription factors, we investigated cleavage of PHO5 promoter DNA by Pho4-MNase and Pho2-MNase in pho2Δ and pho4 Δ strains, respectively.
Deletion of PHO2 abolished cleavage of the activated promoter DNA by Pho4-MNase (Fig. 7). This suggested that the cooperativity between Pho4 and Pho2 observed in vitro is essential for Pho4 binding at UASp1 in vivo, consistent with the absolute requirement of Pho2 for transcriptional activation of PHO5. Deletion of PHO4 abolished cleavage of the activated promoter by Pho2-MNase at N-2 sequences, as expected, and decreased cleavage at UASp1 (Fig. 8). The latter observation suggested that the direct interaction between Pho4 and Pho2 also stabilized Pho2 binding at UASp1. Consistent with an earlier finding that Pho2 recruits the histone acetyltransferase NuA4 to the repressed PHO5 promoter [32], our data suggested weak binding of Pho2 at UASp1 in the absence of Pho4.
ChEC analysis of Pho4 binding at PHO5 promoter in wild type and pho2Δ cells. PHO2 wild type (PHO2) and pho2Δ cells (pho2Δ) cells expressing Pho4-MNase were cultured in phosphate-free media for 0, 2, 3, and 4 hours. Cell extracts for each induction time point were incubated in the presence of Ca2+ ions for 0, 10, 30, and 60 minutes (triangles above autoradiograph). For marker bands see legend to Fig. 3.
ChEC analysis of Pho2 binding at the PHO5 promoter. Pho4 wild type (PHO4) and deletion (pho4Δ) cells of Pho2-MNase strains were treated as described in the legend to Fig. 5. Top autoradiograph (analysis of PHO4 wild type strain) is identical to the top autoradiograph of Fig. 5. Bottom autoradiograph shows PHO5 promoter cleavage by Pho2-MNase for pho4Δ cells.
Our data demonstrate that, in vivo, PHO5 promoter sequences occupied by nucleosomes remain largely inaccessible to the critical transcription factors Pho4, Pho2, and TBP, unless nucleosomal inhibition is relieved upon Pho4 binding at the linker-positioned UASp1 (Fig. 3, 5, 6).
Occlusion of UASp2 by nucleosome N-2 is consistent with the crystal structures of the nucleosome, and the basic helix-loop-helix domain of Pho4 bound at UASp2 [33], [34]. The Pho4 homodimer contacts its binding sequence in the major groove on two opposite faces of the DNA, with the helix-loop-helix domain on one face and the remainder of the protein, including its activation domain, on the other face of the DNA [34]. This stereochemistry precludes binding to DNA adhering to a surface. It must be assumed, therefore, that binding of Pho4 to UASp2 requires unwrapping of the DNA from the histone octamer of nucleosome N-2. Consistently, the DNA in the crystal structure of Pho4-UASp2 complex is unbent with a helical repeat of 10.77 base pairs per turn [34], close to that of free DNA under physiological conditions [35], but different from the average helical repeat of 10.2 base pairs per turn for the nucleosome core particle [9], [33].
Contrary to recent suggestions [21], [22], [23], our data indicate that the physiological concentration of Pho4 is insufficient to capture transiently unfolded states of the nucleosome due to either thermal fluctuations in DNA-histone interactions [2], or constitutive enzymatic remodeling. The effect of UASp1 deletion on PHO5 expression and chromatin remodeling can be suppressed by overexpression of Pho4 [19], [36]. Suppression may result from non-specific binding of Pho4 at linker DNA, or direct access to UASp2. The latter possibility does not contradict our conclusions, as Pho4 concentrations above the physiological level may allow for efficient capturing of short-lived unfolded nucleosome states [2].
Like Pho4, Pho2 and the TATA box binding protein are efficiently excluded from their binding sequences when the latter are wrapped in a nucleosome (Fig. 5, 6). Under repressing conditions Pho2 occupied its binding site close to UASp1, but no binding was detectable at N-2 sequences (Fig. 5). The latter cannot be explained by the assumption that Pho2 binding at N-2 sequences required Pho4 binding at UASp2, because Pho2 could access N-2 sequences under activating conditions in UASp2 mutant cells (Fig. 5). Although positioned close to the entry site to nucleosome core particle N-1 [29], [37], the TATA box was not measurably occupied by TBP under repressing conditions (Fig. 6), suggesting that nucleosome N-1 interfered with TBP binding at the TATA box. This conclusion is consistent with other experimental findings. Based on the assumption that transcriptional activation of PHO5 requires removal of nucleosome N-1, it has been possible to explain the quantitative relationship between expression level, promoter nucleosome occupancy, and the magnitude of steady-state fluctuations in gene expression at the single cell level [8]. Furthermore, depletion of nucleosomes in vivo promotes activator-independent transcription of PHO5 and initiates transcription from cryptic promoters [38], [39].
Most recently, it has been suggested that absence of Asf1 allows for binding of Pho4 to a partially unfolded core particle rather than a fully wrapped nucleosome [22], [23]. As pointed out above, for structural reasons it must be assumed that Pho4 binding at UASp2 requires at least partial unfolding of nucleosome N-2. The partially unfolded structure was assumed to be conducive to Pho4 binding at UASp2 and yet proved resistant to nuclease digestion [21], [23]. No attempt was made to explain the apparent contradiction. A first attempt to prove the existence of the altered nucleosome fell short because the resolution afforded by ChIP of sonicated chromatin was insufficient to distinguish between Pho4 binding at UASp1 and UASp2 [22]. Instead, it was argued that immunoprecipitation of PHO5 promoter DNA from chromatin preparations of asf1Δ cells with an anti-Pho4 antibody was mostly due to Pho4 binding at UASp2 rather than UASp1, since Pho4 binds (naked) UASp2 with higher affinity than UASp1 [22], [23]. However, this argument presupposed the validity of what the experiment needed to show, that Pho4 bound with equal affinity to naked and nucleosomal UASp2. In a second attempt, micrococcal nuclease-generated nucleosome core particles were sequentially immunoprecipitated, firstly with an anti-Pho4 antibody and secondly with an anti-histone H3 antibody [23]. Nucleosome N-2 DNA was pulled down more efficiently in chromatin preparations from asf1Δ cells than wild type cells, suggesting that Pho4 and histones bound to the same sequences simultaneously [23]. However, the difference in immunoprecipitation efficiency between wild type and asf1Δ cells may be attributable to the histone antibody alone. In chromatin preparation from wild type cells, Pho4-bound sequences must have been mostly naked and thus degraded by micrococcal nuclease, whereas UASp2 sequences that were precipitated non-specifically in the first immunoprecipitation step were mostly nucleosomal in preparations from asf1Δ cells, and thus efficiently precipitated by the histone antibody in the second step. These experiments did not, therefore, provide a compelling argument for simultaneous binding of histones and Pho4 to N-2 sequences. We have analyzed Pho4 binding at UASp2 in a UASp1 asf1Δ mutant to address the possibility that nucleosome N-2 is altered rather than removed in asf1Δ cells upon PHO5 induction. We could find no evidence for this possibility. Our results were indistinguishable from those obtained with ASF1 wild type cells that bore a mutated UASp1 (data not shown).
Two objections may be raised against the naïve interpretation of ChEC data. First, the fusion of Pho4 to micrococcal nuclease prevented binding of Pho4 to nucleosome N-2. This appears unlikely because micrococcal nuclease did not measurably interfere with PHO5 activation (Fig. 1). Second, N-2 may not have prevented Pho4 binding at UASp2, but cleavage of promoter DNA by micrococcal nuclease. The question of whether the absence of signal may be interpreted as absence of binding equally applies to other methods for detecting the binding of transcription factors to DNA (see Introduction). For ChEC, the question may be answered affirmatively. Binding of Pho4-MNase at the nucleosomal UASp2, close to the dyad axis of the N-2 nucleosome core particle, would position the nuclease in close proximity to the nucleosomal linkers. Since micrococcal nuclease does not rely on specific DNA sequences for cleavage and effectively cleaves linker DNA when tethered to nucleosomes by linkage to the C-terminus of histones [26], [40], it must be assumed that absence of cleavage indicates absence of transcription factor binding. This is not necessarily the case for DNA methylation and DMS footprinting assays, as both methods rely on modification of DNA occupied by core particle N-2 for detection of Pho4 binding at UASp2.
Different conclusions regarding the ability of Pho4 to access its binding site within nucleosome N-2 are most likely attributable to the different methods used to prevent the loss of promoter nucleosomes under inducing conditions - mutation of the Pho4 binding site at UASp1 in this and other studies [19], [41], and deletion of ASF1 in recent reports [21], [22], [23]. The effect of the UASp1 mutation on PHO5 regulation is undoubtedly direct. This may not be true for the asf1Δ mutant. Cells that lack Asf1 function exhibit a wide range of defects in gene expression, including the expression of histone genes [42], [43]. Furthermore, ASF1 was found to be required for PHO5 chromatin remodeling at intermediate but not low phosphate concentrations [44]. This may have rendered the outcome of induction experiments in asf1Δ cells susceptible to small irregularities in the phosphate concentration of the media [21], [22], [44].
Our ChEC results provided evidence for recruitment of TBP to the PHO5 promoter by Pho4 in vivo (Fig. 6). This result is consistent with earlier demonstrations of Pho4 and TBP interaction in vitro [45]. The in vivo interaction does not have to be direct but may be mediated by other factors such as TFIIB, which was also found to interact with Pho4 in vitro [46]. Cleavage of PHO5 promoter DNA by TBP-MNase at upstream activating sequences may be due to TBP-MNase bound at the TATA box and loop formation between the core promoter and upstream activating sequences, rather than recruitment by Pho4. Two observations argue against this possibility. Loop formation should promote cleavage of core promoter sequences by Pho4-MNase. No such cleavage was observed (Fig. 7). If cleavage at upstream activating sequences by TBP-MNase were due to loop formation, mutation of the TATA box should diminish the frequency of cleavage at the upstream activating sequences. However, cleavage was diminished only at the TATA box, and not at the upstream activating sequences (Fig. 6). Activator interactions with other proteins are promiscuous by nature. Their relevance for the regulatory mechanism is therefore uncertain. However, the suggested interaction between TBP and Pho4 may be physiologically significant. Analysis of the steady-state fluctuations in PHO5 promoter-controlled gene expression indicated that the Pho4 activator stimulates the rate of transcription machinery assembly after promoter nucleosome removal [8].
Materials and Methods
Plasmids and Strains
The PHO5 UASp1, UASp2 and TATA box mutations were described previously [8], [29]. Pho4 and Pho2 deletion plasmids pCM90.1 and pCM115.1 were constructed by replacing the PHO4 or PHO2 open reading frame with a 1.1 kb URA3 gene.
PHO4 and PHO2-MNase fusion strains YR22 and YR3 were derived from strains NOY505 and yM2.1 [29], respectively, as previously described [40]. Strain y1185, expressing TBP-MNase, was reported earlier [40]. PHO5Δ::URA3 strains yC01.2, yC11.3, and yC131.8 were constructed by replacing PHO5 in YR22, y1185, and YR3 using plasmid pM51.1 as described previously [29]. Strains yC03.1 and yC15.1, containing PHO5 UASp1 and UASp2 mutations in addition to Pho4-MNase, were derived from yC01.2 by homologous recombination with plasmids pCM46.9 and pCM65.1, respectively. Similarly, strain yC16.1, expressing TBP-MNase, was derived from yC11.3 with plasmid pCM46.9. Strains yC159.3, yC138.6, and yC139.5, expressing PHO2-MNase, were derived from yC131.8 with plasmids pM50.1, pCM46.9, and pCM47.8 bearing wild type PHO5, PHO5 UASp1 mutation, and PHO5[GC] UASp2 mutation. PHO5 TATA box mutation strain yC22.2, expressing TBP-MNase, was derived from yC11.3 with plasmid pCM74.3. Strain yC162.1, a PHO4Δ::URA3 strain expressing PHO2-MNase, was derived from yC159.3 with plasmid pCM90.1. Strain yC132.1, a PHO2Δ::URA3 strain expressing Pho4-MNase, was derived from YR22 with plasmid pCM115.1. A list of all strains used is provided in Table 1. All yeast transformations were performed using the lithium acetate method.
Chromatin Endogenous Cleavage (ChEC)
Micrococcal nuclease-tagged strains were cultured at 30°C in 260 ml of YPAD to a final density of 3–4×107 cells per ml. One hundred milliliters of culture were harvested for the zero hour time point. Cells from the remaining 155 ml of culture were washed with 20 ml of sterile water, resuspended in 420 ml of phosphate-free SCD medium and cultured at 30°C. ChEC was performed using cell extracts from 100 ml formaldehyde-cross-linked yeast cells as described previously [26] with the following exceptions: Formaldehyde was added to cultures at room temperature for 15 minutes. Cells were washed once with sterile water after harvesting. Protease inhibitors 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), Pepstatin A, and E-64 were added to all buffers at a final concentration of 2 µM. Complete Mini EDTA-free Protease Inhibitor Cocktail (Roche) was added prior to bead beating. The bead beating was performed on a MP FastPrep-24 machine (MPBio), set at 5.0 m/s for 2×20 seconds with a one minute break after the first 20 second beating.
After micrococcal nuclease cleavage reactions, samples were treated with RNase A at a final concentration of 100 mg/ml for 1 hour at 37°C. Incubation was continued for an additional hour after adding 1/40 volume of 10% SDS and 100 mg/ml Proteinase K. Reverse cross-linking was performed at 65°C overnight. One-third of the isolated DNA was digested with a restriction enzyme and separated in a 1.6% agarose gel in Tris-borate-EDTA at 4 V/cm for 4 hours and 30 min. Southern blotting and probe preparation was performed as previously described [29]. 32P-labeled PHO5 upstream and open reading frame probes were prepared by random priming using a 500 bp NotI/BamHI fragment and a 687 bp ScaI/SalI fragment as template, respectively.
Phosphatase Assay
Fifty microliters of cells were mixed with 250 µl 0.1 M pH 4.2 sodium acetate and 250 µl of 10 mg/ml 4-orthonitrophenylphosphate and incubated for 15 minutes at 37°C. Following incubation, 900 µl of 1.4 M sodium carbonate was added to the mixture and 800 µl of the final mixture was measured at optical density (OD) at 420 nm. The OD reading at 420 nm was divided by the OD at 600 nm to normalize for cell density.
Topology Analysis
Analysis of topoisomer distributions was performed as previously described [29].
Author Contributions
Conceived and designed the experiments: CM CB HB JG. Performed the experiments: CM CB HB. Analyzed the data: CM CB HB JG. Contributed reagents/materials/analysis tools: CM CB HB JG. Wrote the manuscript: CM CB HB JG.
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Tag Archives: orange pebbles
Irish Pebbles 1
Rusty Pebbles at Whiteford
Vast swathes of pebbles on the beach at Whiteford in Gower are coloured orange – or at least they were the last time I looked. (The beach sediments there are very mobile so it cannot be guaranteed that you will see exactly the same thing on each visit). These coloured pebbles are found in a band stretching from the base of the sand dunes at the eastern end of the beach towards the disused Victorian Whiteford Lighthouse.
The pebbles seem to be coated in rust rather than rusty because of their intrinsic composition. I guess the first couple of times that I noticed the orange pebbles I vaguely thought that they were stained by rust emanating from the decomposing remains of the old iron causeway that linked the lighthouse to the shore. You can often find pieces of the iron framework of the walkway – sometimes supports still in situ and other times single pieces of the structure lying free.
However, lately, I have been discovering more and more about the Quaternary geology of the Gower – a relatively recent geological period dating from about 2.5 million years ago to the present. This includes the Pleistocene with a variety of glacial, peri-glacial and inter-glacial deposits; and the recent Holocene (from 11,800 years ago) with peat and submerged forests, marsh, dune, beach and alluvial deposits. As I read more, I am gradually reaching something of an understanding about some of the natural phenomena that I observe and photograph on Gower beaches. So I now tentatively consider that the rusty pebbles are not related to the dilapidation of the old lighthouse but are the result of a much older natural geological process.
I have already mentioned in Jessica's Nature Blog the remains of the submerged forest at Broughton Bay which lies to the west and adjacent to Whiteford Sands. These ancient tree trunks are embedded in peat deposits. While I was reading George (2008), I learnt that the peat decomposes to form a hard ferruginous layer called an iron-pan or hardpan. This has led me to wonder if the iron compounds that coat the pebbles at Whiteford are derived from an iron pan layer.
Supporting evidence for this idea comes from the presence of ancient tree trunks emerging from black peat deposits close to the rusty pebbles – similar to those stumps found at Broughton. The old waterlogged wood is also stained with rust – as you will see from the photographs below. Additionally, slightly higher on the beach, closer to the dunes, the shore is strewn with pebbles around which orange-coloured watery 'tears' rise to the surface and weep across the surface of the sand – making me think they might originate from a concealed ferruginous hardpan below.
Then again, I suppose the rust could come from buried decomposing munitions as the beach was used for firing practice in the Second World War!
George, Gareth T. (2008) The Geology of South Wales – A Field Guide, G.T.George at firstname.lastname@example.com , ISBN 978-0-9559371-0-1, p 70.
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An odd ball from Ringstead Bay
A naturally-occurring pebble-studded ball of Kimmeridge Clay. It was found rolling around on the water's edge at Ringstead Bay in Dorset. I like the shape of this beachcombed object. It's quite decorative. The pattern of the bright orange and lighter-coloured stones looks attractive against the blue-grey clay.
The main body of the clay outcrops high on the seashore and has a very glutinous texture when wet. Sometimes pieces of this clay crack off in dry conditions or may be loosened by wave action. When wet, these lumps of clay stick to anything they come into contact with.
The seashore at this point is mostly flint shingle with smaller pebbles and finer gravel at low tide level. The strong currents and crashing waves have moved the clay around until it has become this interesting rounded shape and texture. It has acquired the pebble coating in much the same way that a soft sweet acquires its finish of "hundreds and thousands".
Revision of a post first published 23 June 2009
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Waves at Lyme Regis
How many ways are there for the waves to break on the seashore? Each beach, each tide, is different. Have you ever noticed how the circular motion of waves means that waves are retreating and advancing at the same time? No sooner has a wave crashed on the shore creating millions of salty bubbles, than it is being dragged back seawards under the sediments, while the next wave builds and advances above. The whole cyclical dynamic process generating hypnotic movement and sound accompanied by changing lacey patterns of seafoam on water and shingle.
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Pebbles from Corsica
Many years ago, I picked up these wonderfully patterned and coloured pebbles on a beach in Corsica. They remind me of the warm sunny day that I saw the fantastic sight of an entire seashore covered with pebbles just like them.
I have to confess that I know very little about them except that they are extremely beautiful and varied in many ways: geological origin and structure; pattern (plain, spotted, marbled and striped); texture and shape (smooth, rounded or flattened); and colour (yellow, orange, red, black and white). The rocks from which these stones were derived must to be volcanic or igneous in origin. I remember that in Corsica the whole mountainsides are pink and red.
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Some pebbles from Eype
Eype in Dorset has an interesting beach. Mostly it is shingle – orange and grey rounded pebbles of various sizes. However, there are large boulders of different geological types that have fallen from the cliff and are now scattered across the shore. When the tide comes in, it sometimes washes the pebbles high over the boulders so that, when the tide goes out, groups of small pebbles remain in nooks and crannies on the surface of the larger rocks – looking like odd eggs in a strange stony nest.
Click here for more PEBBLES.
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subset: commoncrawl


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Recently Netflix has made a channel available for download at the WiiShop channel to replace its streaming disc. I had always used the streaming disc which is on my wbfs hdd, but now they won't let you watch certain movies unless you load the Netflix channel directly from the Wii Menu. The error tells you to go to the WiiShop channel to download the Netflix Channel. When I go to WiiShop it tells me I needs first go to the Wii Settings and update my Wii and then come back and update my WiiShop channel. Am I able to safely update my Wii directly from my Wii Settings menu or no? Thanks for your time whoever helps!!!
Follow the shop channel guide in my signature.
Damn I suck!!!!


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Bliss and Bloom Ltd
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24 piece cutlery set. Beautiful pearlized cream/ivory coloured handles and stainless steel stems with detail in the middle. 6 knives, 6 forks, 6 dessert spoons & 6 tea spoons. Dishwasher safe more details
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They look gorgeous when the dining table is laid out for those special occasions.
The knife, fork and dessert spoon measure L 22cm. The tea spoon measures L 16cm
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United announce tour details
Manchester United have announced the kick-off times for their five pre-season friendlies in Thailand, Australia, Japan and Hong Kong.
United face a Singha All Star XI at the Rajamangala Stadium in Bangkok on July 13 at 8pm local time (2pm BST) and then face the A-League All-Stars at ANZ Stadium on July 20 at 7.30pm local time (10.30am BST).
They then head to Japan, first facing Yokohama Marinos on July 23 at Nissan Stadium at 7.20pm local time (11.20am BST) and then play Cerezo Osaka on July 26 at Osaka Nagai Stadium at 7pm local time (11am BST).
The tour concludes with a trip to Hong Kong on July 29 to face Kitchee FC at Hong Kong Stadium at 8pm local time (1pm BST).
Information from the Press Association was used in this report
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In The Heights: Indoor nationals marked Mantz's first trip to New York City. He was awed by the spectacle of the Armory, situated in the colorful Washington Heights section of Manhattan. Mantz's victory, in 14:24.33, was his first national title. The next day, he celebrated with Dunkin' Donuts for breakfast, sightseeing with his father and a long run in Central Park.
Surge to Victory: In the 5,000m, his first on the track, Mantz traded the lead with Aaron Templeton of Tennessee. Both Mantz and Templeton—as well as defending champion Elijah Armstrong of Idaho—were Foot Locker cross country finalists in December. Feeling strong from consistent winter training at 50 miles per week, and coming off of many great workouts, Mantz broke away with three laps to go, running his last 600 meters in 1:39 to triumph by 13 seconds.
Seeking Fast Times: Mantz's name was added to the meet's illustrious 5,000m victory list that includes all-time greats Lukas Verzbicas and Edward Cheserek. Mantz reassessed his spring goals after the win, and now he's looking for a sub-8:55 at Arcadia, which draws an elite field in the 3,200m. Last year at Arcadia, Mantz placed 21st (a record 16 boys broke 9:00) and set his PR, 9:04.05, one of the fastest sophomore times in the country in 2013.
Pulling An All-Nighter: Mantz, 17, hoped to break 9 minutes at the Simplot Games in February in Pocatello, Idaho. After the 3,200m trials on a Thursday, he realized he had a school project due on Friday and was up until 4 a.m., getting his work done. He felt wasted in the Saturday final, taking third in 9:12.99. Still, his time, at 4,500 feet altitude, was worth close to 9:00 at sea level according to altitude conversions.
Foot Locker Finals: After making Foot Locker finals as a sophomore and taking 12th place, Mantz felt much fitter last December in his junior season. With workouts like a hilly 5-mile tempo run at 5:12 pace, he hoped to contend for the victory. But Mantz couldn't put it together mentally, feeling "really nervous" before the race. He placed seventh, 16 seconds behind victorious Grant Fisher of Michigan.
Cross Times Two: With his early state meet in mid-October, Mantz had, in effect, two cross country seasons while pursuing national honors. After winning his state 4A title, Mantz ran the Nike Cross Nationals Southwest Regional in Tempe, Ariz., just to get in a race, and placed second. He never intended to run the NXN finals in Portland, Ore., because of the muddy conditions he'd heard about from Utah runners the previous year; instead Mantz ran the Foot Locker West in Walnut, Calif., taking fourth, to return to San Diego for Foot Locker nationals.
Working for More: "Conner's excellence comes from his relentless training," said Sky View coach Kyle Wright. "He runs in any weather when no one else is willing to go outside. He never allows himself to become stagnant."
Belly of the Beast: Last summer, Mantz trained daily. With the mountain trails of northern Utah practically at his doorstep, Mantz did one 10-miler that took him up to 10,000 feet altitude and another workout that his team calls the "beast": 10 sprints up a steep 300m hill. "I was heaving afterwards," said Mantz. "It's the most miserable workout, but it makes you faster." He also attended the Nike elite youth camp in Portland, Ore.
The Freshman Experience: As a freshman, Mantz got mixed up in one meet, ran off course and was disqualified, costing Sky View the team title. The week of the state meet, Mantz was distressed with a root canal. And in the Foot Locker West Regional, Mantz was dejected when he learned he'd missed an all-West medal by one place. Nevertheless, Mantz was a budding star. He was Sky View's top runner, placing third at state, and took 25th in the regional, the first freshman in the field.
Going the Distance: Mantz's freshman fitness was rooted in the long distance racing he'd done since age 12. When his father got into marathoning, Conner started running about 15 to 20 miles a week. Even on modest mileage, Conner entered half-marathons and 15Ks and found he could handle it. In 2011, at 14, he ran the Utah Valley half in 1:13:20, a 14-and-under course record. In 2012, after his freshman season, he ran the event in his 1:11:25 PR, a 15-to-18 course record. Taking fifth overall, Mantz won $75 in prize money but had to refuse it to maintain his amateur status. "Hopefully," he said, "I'll get more in college scholarship money."


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Devin Deckert has helped Temescal Canyon High's boys water polo team flirt with section titles each of the last two seasons. On Friday night, the Titans junior pushed the swimming program to the brink and beyond.
Deckert's 21.32-second split on the last leg of the 200-yard medley relay vaulted the Titans from third to first in that championship heat, and his two individual third-place finishes ---- in the 200 and 100 freestyle ---- helped Temescal Canyon hold off Beaumont for the program's first championship at the CIF Southern Section Division IV finals at Riverside City College.
"There was an expectation that we could do well," said Titans coach Damien Andrews, whose team scored 275 points to Beaumont's 255 a year after placing third at this meet. "We knew that we had to swim well to win. ... By no means were we thinking this could be a cakewalk."
Deckert certainly swam well, clocking in at 1 minute, 46.42 seconds in the 200 free and 47.74 seconds in the 100 free. He also swam the anchor leg on the boys' second-place 400 relay team (Kjell Hiniker, Naz Pounds and Jesse Hayward), which clocked in at 3:19.73. But Deckert's last-leg push for the 200 medley relay team (Pounds, Jake Gallagher and Hayward) clearly was the Titans' highlight of the meet.
"It was awesome," said Andrews, whose team edged Woodcrest Christian (1:43.05) with a time of 1:41.20 for the win. "It was a killer rush."
Deckert, of course, wasn't the only Titan scoring key points. The 200 free-relay team placed fifth (1:32.27), Pounds placed third in the 100 back (57.27), and Hiniker placed fifth in the 50 free (22.48). Also, Kelly Giordano and Jake Gallagher placed 10th in the 200 free and 100 breaststroke, respectively.
Calvary Murrieta independent swimmer David Riser placed fourth in the 500 free (4:46.82) and sixth in the 200 IM (2:00.99).
On the girls side, Temescal Canyon (143.5) finished eighth as a team and Hemet (135) finished 10th ---- well behind champion Pasadena La Salle (249.5).
Sydnee Inman and Sammi Murphy scored a pair of top-10 showings for the Titans, with Inman placing fifth in the 100 free (55.82) and sixth in the 50 free (25.55). Murphy, meanwhile, placed ninth in both the 200 free (2:04.33) and 100 back (1:06.49).
The Titans' 200 medley relay had an eighth-place showing (1:58.87) and the 200 free relay placed sixth (1:44.49).
Freshman McKenna Glasser had a remarkable finals debut for Hemet, placing second in the 200 IM (2:10.39) and third in the 100 butterfly (58.61) and helping the 200 free relay team to a third-place showing (1:43.47).


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I have been in the High-End Audio industry for almost 22 years now,I have had a small loudspeaker business for about that long,but for the last 6 or 7 years my intrest in computers and computer building has sparked my intrest in putting the two together,for years computers have been limited to business word processing and data storage,but with all the latest innovations in audio and video processing the future seems promising for high-end computing especially in the area of multi-media.I still love 2 channel audio,but all of this new computer stuff gets me excited too!!! Any Thoughts??
You are here
June 9, 2006 - 2:24pm#1
High-End Multimedia
June 19, 2006 - 12:27am#2
Re: High-End Multimedia
I think it will be some time before we can get real high end sound out of a computer system, but you can get decent sound.
There are some sound cards that get good sound and any amp speaker combo can be connected to it. You can also connect the computers sound card to an input on a preamp/integrated amp, etc.


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The Son That Former NFL Player Rae Carruth Tried To Kill Turns 18, Still Suffering From Permanent Brain Damage
The former wide receiver, who plotted the murder of his ex, Cherica Adams, is scheduled to be released from prison soon.
Can oral sex really cause throat cancer? What is throat cancer…and how is it related to HPV?
Cancer death rates continue to decline, according to the American Cancer Society. But cancer caused by the human papillomavirus (HPV), a sexually transmitted infection most well known for causing genital warts and cervical cancer, is still largely a cause for concern. Recent reports have shown a rise in both oral and anal cancer caused by HPV in both women and men.
Oral infections of human papillomavirus, or HPV, affect nearly 7 percent of Americans, affecting three times as many men (10 percent) as women (3.6 percent).
In fact, HPV is now a more common cause of throat and other oral cancers than tobacco.
What is HPV?
Genital human papillomavirus, or HPV, is one of the most common sexually transmitted infections (STIs). There are more than 40 types of HPV that can infect the genital areas of males and females. These HPV types can also infect the mouth and throat.
HPV can cause serious health problems, including genital warts and certain cancers, particularly cervical cancer and throat cancer. There is no certain way to tell who will develop health problems from HPV and who will not. In most cases HPV goes away by itself before it causes any health problems, and most people who become infected with HPV do not even know they have it.
Experts are now looking into determining if the newly available HPV vaccine is effective in males. The vaccine has been shown to be almost 100 percent effective for preventing cervical infection.
Read more about the link between HPV and oral cancer at BlackDoctor.Org.
BET Health News - We go beyond the music and entertainment world to bring you important medical information and health-related tips of special relevance to Blacks in the U.S. and around the world. Click here to subscribe to our newsletter.
Get ready for the BET Experience, featuring Beyoncé, Snoop Dogg, R. Kelly, Erykah Badu, Kendrick Lamar and many more. Go here for more details and info on how to buy tickets. Sign up now to attend free film screenings, celebrity basketball games and more.
(Photo: Roy McMahon/Getty Images)
The former wide receiver, who plotted the murder of his ex, Cherica Adams, is scheduled to be released from prison soon.
Each of the men could be served a life sentence in prison.
Presented by


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St. Charles Schools name Laura Abbey High School teacher of the year
Staff Report - May 11, 2006
Laura, a teacher at Hahnville High School, graduated from the University of Louisiana-Monroe with a Bachelor of Business Administration Degree, Alternative Teaching Certification, and a Masters of Education Degree.
She is currently pursuing Gifted Certification at the University of New Orleans. She is a supervisor of student teachers for Southeastern Louisiana University, the co-chair for the Health and Human Services Smaller Learning Community, and the sponsor of the HHS Cheerleaders. Laura has been named to Who's Who Among American High School Teachers annually since 1998.
Read More Stories at HeraldGuide.com!
|heraldguide.com is a supplement to St. Charles Herald Guide.
Copyright © 2001 - 2017 St. Charles Herald Guide, Inc. All rights reserved.
Please contact our WebMaster if you experience problems with the website.


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WALNUT CREEK — The plan for a major Safeway development at the corner of Ygnacio Valley and Oak Grove Roads is continuing to slowly move forward.
The City Council will hold yet another study session on the Shadelands Gateway Specific Plan during its meeting 7 p.m. Tuesday, 1666 N. Main St. The plan will eventually lay out the zoning of what will be allowed on a 25-acre Safeway-owned parcel. At the same time, Safeway is already proposing what they want to build — a 219,000-square-foot development called The Orchards at the entrance to the Shadelands Business Park, that would include senior housing.
The preliminary plan calls for not only a 55,000-square-foot grocery store — double the size of the Safeway store across the street — but also a splash pad for kids, parks, winding paths, a fitness club and, for the first time in the business park, housing.
The council is expected to review and comment on issues related to the proposed General Plan amendment, rezoning and the specific plan's economic analysis documents.
Some neighbors of the Safeway property have voiced concerns about economic blight, traffic and a loss of open space if a store in built there. Safeway hopes to begin construction by the end of 2014.
Contact Elisabeth Nardi at 925-952-2617. Follow her at Twitter.com/enardi10.


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Lock & Lock 4 Piece Container Set 4x 500ml
To add this product to your wishlist please login
The Lock & Lock Interlock Containers provide you with the perfect storage solutions to store and save food items. The containers have a unique interlocking system that allows you to stably stack the containers. The interlocking containers have a screw-type lid and the base of the containers have a unique design that allows you to interlock the base of the containers to the lids. Interlocking the containers provides you with a compact storage solution. The containers are BPA free and transparent making it easy to organise your pantry, fridge and home. The container has a space saving shape design. Not recommended for the dishwasher or microwave. The set includes 4 x 500ml containers.
• Interlocking containers
• Easy grip
• Containers can be stacked for compact storage
• Not recommended for the dishwasher or the microwave
• Keep away from fire, do not use abrasive cleaners or scrubbers
Additional Information
- Brand: Lock & Lock
- Size: 500ml


text:
Three Seconds
Anders Roslund, Borge HellstromPart of the DCI Ewert Grens Series
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Maxim Jakubowski's view...
One of our Great Reads you may have missed in 2011.
Winner of the CWA International Dagger 2011.
Further explorations of the dark side of Swedish society with a particularly brutal and vivid touch. An undercover cop is sent to a high security prison to investigate a drug-related murder. But all is not what it seems and a gallery of grotesques soon joins the dance in a plot that circles many times on itself and always comes up with the unexpected. The authors are a duo of investigative journalist and actual ex-criminal, so familiar with the dark side.
CWA Judges' comments: 'The Swedish duo's usual maverick cop takes a back seat to a riveting exploration of a deniable operation involving an undercover agent deep inside a criminal organisation. Their new character, doomed to betrayal by political manoeuvring, fights for his life with great intelligence and courage.'February 2011 Book of the Month.
A "Piece of Passion" from the Publisher...
"Roslund and Hellstrom's Three Seconds offers readers as tense and gripping, edge of seat experience as any novel I can remember reading. And a last twist that defies expectations and leaves the reader wrung out like an old dishcloth. It's what the very best thrillers can do.
Like Larsson's Girl with the Dragon Tattoo, Three Seconds starts in a pretty leisurely way and you have to be patient and find your way into this dangerous and terrifying world. Once there you find that the authors don't complicate matters - you are with the hero each step; you will find out why he has a way with tulips and what purpose he has with library books which no-one else will ever borrow, once he gets into the vicious Aspas high security prison.
I heard about these authors when their agent gave me a copy of their first novel to be translated into English and persuaded me to commission a translation of Three Seconds, which was just about to be published in Sweden. After some indecision we took the plunge and watched R&H become the true successors to Mankell and Larsson as their new novel dominated the bestseller lists and critical heights of the Scandinavian press.
And when the wonderful translation from Kari Dickson came in we felt intoxicated - with the book, with the potential of the book, with the authors whom we had met and knew would be great advocates of Three Seconds and the knowledge that for Quercus, Swedish crime would not end with the incomparable Larsson."
- Jon Riley, Editor at Quercus Books
If you like Anders Roslund, Borge Hellstrom you might also like to read books by Stieg Larsson, Jo Nesbo and Henning Mankell.Who is Maxim Jakubowski
Win a SIGNED FIRST EDITION of Damaged.
Get into the Spirit of Christmas with our special SIGNED FIRST EDITIONS competition! We are giving away ONE first edition hardback of Martina Cole's new crime thriller, Damaged, signed by the author!
To have a chance of winning this fantastic prize, click the button below. Please note that this draw is open only for UK residents and is free to enter, multiple entries from the same email address will only be counted once.
The draw closes on 4 December 2017. The winners will be notified by 11th December 2017.
Good luck!
Three Seconds by Anders Roslund, Borge Hellstrom
Piet Hoffmann is the best undercover operative in the Swedish police force, but only one other man is even aware of his existence. After a drug deal he is involved in goes badly wrong, he must face the hardest mission of his life - infiltrating Sweden's most infamous maximum-security prison. Detective Inspector Ewert Grens is charged with investigating the drug-related killing. Unaware of Hoffmann's real identity, he believes himself to be on the trail of a dangerous psychopath. But he cannot escape the feeling that vital information pertaining to the case has been withheld or manipulated. Hoffmann has his insurance: wiretap recordings that implicate some of Sweden's most prominent politicians in a corrupt conspiracy. But in Ewert Grens they might just have found the perfect weapon to eliminate him. Intelligent, gripping, brutal, Three Seconds is the new thriller from Roslund and Hellstrom, the heirs apparent to Stieg Larsson and Henning Mankell as the masters of Scandinavian crime.
'The action is non-stop and the tension unlimited'
The Times
'Gripping and intelligent ... Destined for the big screen'
Daily Mirror
'Three seconds is just about how long it feels it takes to read Three Seconds ... Terrific'
Time Out
'Extraordinarily compelling'
Daily Mail
About the Author
Book Info
Publication date3rd February 2011
AuthorAnders Roslund, Borge Hellstrom
More books by Anders Roslund, Borge Hellstrom
Author 'Like for Like'
Author's Websitewww.roslund-hellstrom.com/
Author's Facebook Latest
PublisherQuercus Publishing Plc
640 pages
Thriller / Suspense
All Shortlists and Winners
Crime / Mystery
eBook Favourites
eBook Favourites
Fiction in translation
Publisher ProfileQuercus Publishing Plc is an imprint of Quercus
Starting out in a small office in Dorset Street, London, the company quickly expanded, producing best sellers with each of its first two titles, Universe and Speeches that Changed the World.
As well as winning awards for their books Quercus also won Small Publisher of the Year at the British Book Industry Awards and the PLUS New Company fo the Year in the Fast Growth Business Awards.
Publisher's Websitewww.quercusbooks.co.uk
It's a lively, independent website with reviews, recommendations and more - with a huge range of books available to buy in all formats.Alison Layland
I love Lovereading for the wonderful like for like author recommendations and for highlighting new books, it's a great resource for readers!Lindsay
Insightful reader reviews and unbiased recommendations. I don't know how I chose books before Lovereading! An essential for all book lovers.Sarah Harper
Lovereading is like booking a holiday, you don't know what it will be like and it is a whole new experience.Sue Burton
Because of Lovereading I have broadened my reading horizons with some really great books that I probably would never have chosen myself.Susan Walsh
Lovereading helps me decide what real people read.Kerry Bridges
Lovereading has all the new books and also suggestions for 'similar' authors whilst waiting for your favourites new books.Carol Peace
Thanks to Lovereading I have discovered new writers and read books I would never had looked twice at - and enjoyed them.Angela Rhodes


text:
RISING COSTS: Fidelity Investments estimates a couple retiring this year will need $250,000, on average, to cover health care expenses in retirement. That's up 4.2 percent from last year.
BEHIND THE NUMBERS: The estimate is based on a 65-year-old couple with Medicare coverage, factoring in premiums, co-payments, deductibles, and out-of-pocket prescription costs.
WHAT'S LEFT OUT: Fidelity's estimate doesn't factor in long-term care or most dental services. It also doesn't account for prescription drug benefit savings that many retirees can expect from the new health care overhaul.


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Growing up, I never had a cat or dog. My first pet, after much pleading, was a hamster.
The hamster, who we named "Pooh" in reference to his golden-colored fur, didn't provide the companionship a dog or cat would, but he did teach us about caring for other living things.
He also brought his share of drama. A number of times we would come home to find his cage empty and find him stashed away under a bed in one of our rooms.
After Pooh died, there were a variety of other pets that passed through the Noel household such as the turtle named Yertle my sister got from her teacher and the orange-and-white tabby kitten we looked after for a couple of days.
But besides Pooh, I have never really had a pet that has been my own. I know this is probably drastically different from many of you, who probably grew up with a collection of pets who were considered members of your family.
For Karen Fling, owner and founder of the East Lake Veterinary Hospital, it was a cat she named "Q-tip" that helped inspire her interest in feline care.
At the recently opened East Lake Cat Care Center in Lake Highlands, the focus is all placed on cats from the design of the building to the medical practices and lodging.
I visited the cat care center to talk with Fling and get a closer look at the new facility, which resembles a Victorian-style house. Read more about the new facility in this week's cover story.
Fling has also started a weekly video blog which offers topics ranging from reviews on cat-related books to discussions on feline care.
Within the next month she also hopes to have a schedule for a lecture series to educate the public about cat care.
To view Fling's blog, visit eastlakecatcarecenter.com.
Also in this week's edition
In this week's edition you'll also find photos submitted by Lisa Rothberg of a recent Temple Shalom event to celebrate the installation of Rabbi Ariel Boxman. To view these photos, see Page 7 in this week's edition.
For this week's Sounding Off question, I asked contributors if they think Dallas and Collin counties do enough to prevent flu outbreaks and how they are planning on minimizing their chances of getting sick.
If you'd like to sign up to receive our weekly Sounding Off question, visit dallasnews.com/voices.


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The U.S. Army Corps of Engineers is racing against the clock to finish three stormwater outfall pipe extensions before tourists arrive in Rehoboth Beach.
Work on the Rehoboth Avenue stormwater outfall is underway; Laurel Street and Delaware Avenue pipes are still to come. The corps has no timetable for finishing the work, but officials are optimistic the job will be finished before Memorial Day.
Project engineer Ron Dooley said he hopes to finish within the next 30 days.
The $800,000 project to extend stormwater outfall pipes are designed to prevent sand from clogging the outfalls during future beach replenishments. As an add-on, Rehoboth officials authorized spending $200,000 on the Rehoboth Avenue pipe, where sand had been naturally eroding, to prevent future problems.
Subcontractor Channell Construction, under the supervision of the corps and primary contractor Reilly Construction, began work on the Rehoboth Avenue pipe by driving sheet pilings in the ocean to protect workers from the waves. Dooley said the workers will use sand to build a barrier to keep water out of the area where the extension will be placed.
The Rehoboth Avenue and Laurel Street pipes will be extended 18 feet further into the ocean, while Delaware Avenue will be extended 36 feet. Dooley said the Delaware Avenue pipe will be longer because that pipe lies deeper in the sand than the others.
"It just sits lower in the sand. It's going to get extended out further so it won't be a problem in the future no matter how wide we make the beach," he said.
Dooley said the old piles supporting the outfall pipes will be replaced. Rehoboth Avenue and Laurel Street will have a 30-inch pipe, while Delaware Avenue will have a 35-inch pipe, he said.
Early April rainstorms delayed the work, and the protective sheeting can only be installed during low tide, Dooley said. He said scheduling the work is up to the contractor; the corps contract gives a specified time for work to be completed, and it is up to the contractor to meet the deadline.
Dooley said as the spring weather has improved, construction crews have also had to deal with curious onlookers.
"It's never good to have construction equipment on the beach when people want to come down and relax, but these repairs have to be done. We'll do them as quickly and efficiently as possible and then clear out of the way," Dooley said.
Although Rehoboth Avenue was the first extension, Channell has not yet determined which outfall will be next. Dooley said Laurel Avenue is not as far out into the surf so it could be done next; the contractor is not required to work in any certain order.
Dooley said the crew is small, only five to 10 people, but all have experience in this kind of work.
When Rehoboth Beach was renourished in February 2012, five stormwater outfall pipes were buried in sand. Sand in pipes on Maryland and Virginia avenues eroded naturally, as engineers had anticipated. The Rehoboth Avenue pipe eroded somewhat, while Delaware Avenue and Laurel Street pipes did not erode as the corps expected, Throughout 2012, the corps had an excavator on standby to unclog the pipes as necessary at low tide. Those are the streets where stormwater pipes are now being extended.
Last year, clogging of the Delaware Avenue pipe caused flooding, first, during a late August storm that inundated the underground parking garage at Brighton Suites Hotel and then on Delaware Avenue during Hurricane Sandy.
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Wheatland, MO - On night number one of the Jesse Hockett/Daniel McMillin Memorial, Brady Bacon was unstoppable as the Broken Arrow native swept both the W.A.R Sprints and Lucas Oil ASCS presented by MAVTV A-Feature events this past Friday night.
"We'll see what happens tomorrow. That's the big night and it's the most important" commented Brady in Victory Lane. "I got that Jesse Hockett Helmet painted so maybe that has something to do with it. I'm glad to win in Jack's car and I'm glad to win in my car so hopefully we can do this again tomorrow."
Rolling off from the outside of the front row, the Fatheadz Eyewear No. 99 would ride second as Brian Brown assumed the point in the Casey's General Store / FVP / Searsboro No. 21. Riding the top shelf of the rough Lucas Oil Speedway, Bacon would see his opportunity on lap four as Brown slipped exiting turn four, allowing Bacon to diamond the corner for a bottom side pass entering turn one.
The races lone stop came on lap five for an incident that saw Marion, Arkansas's Derek Hagar flipping nearly half the length of the back straight-a-away after Justin Henderson was left with nowhere to go as Brad Loyet got crossed up exiting turn two. Loyet and Henderson would return with Hagar out with a 22nd place finish.
Underway with fifteen laps remaining, Bacon would quickly extend his lead as Brown worked to hold off the hard charging Tony Bruce Jr. Rollin the high side of the "Diamond of Dirt Tracks", the Oil Medics No. 18 would keep pace with Brown's Casey's General Store No. 21 who worked the middle to bottom of the racing surface.
Working through slower traffic, the battle for second would go from two to four as Wayne Johnson and eleventh starting Shane Stewart joined in the mix near the midpoint of the night's 25 lap feature event.
Finally clearing Brian Brown on lap sixteen, Bruce would run out of time to get a run for the lead as Brady Bacon stormed to his seventh A-Main win of the year. Tony Bruce Jr. crossed the line second with Brian Brown holding on to claim the final step on the podium. Using slower traffic, Shane Stewart grabbed fourth on lap twenty-two with Wayne Johnson landing just outside the lock-ins for Saturday's A-Feature in fifth.
Jonathan Cornell was sixth with Jason Johnson coming from fifteenth to seventh. Brandon Hanks also charged forward from eighteenth to eighth with Johnny Herrera and Jack Dover making up the top ten.
The 2nd annual Hockett/McMillin Memorial concludes Saturday September 29, 2012 with race time set for 6:30pm CDT.
hydrocodone apap 10mg 325mg tabs side effects Lucas Oil Speedway - Wheatland, Missouri
September 28, 2012
Car Count: 51
different types of hydrocodone pills Heat 1 (8 Laps): 1. 3G-Shane Stewart[1]; 2. 41-Jason Johnson[5]; 3. 5H-Logan Forler[2]; 4. 76-Jay Russell[6]; 5. 94-Jeff Swindell[8]; 6. 14-Andrew Turley[7]; DNF 21TX-Brandon Schure[3]; DNS 11C-Matt Covington
main page Heat 2 (8 Laps): 1. 45X-Johnny Herrera[1]; 2. 18-Tony Bruce Jr[4]; 3. 4-Jordan Boston[2]; 4. 38-Cody Baker[5]; 5. 04-Brad Graham[6]; 6. 9M-Mitchell Moore[3]; DNF 48X-Jon Corbin[8]; DNF 53-Jack Dover[7]
resource site Heat 3 (8 Laps): 1. 86-Kyle Bellm[2]; 2. 28-Jonathan Cornell[5]; 3. 2-Dustin Morgan[7]; 4. 33-Austin Alumbaugh[6]; 5. 3Z-Zach Davis[8]; DNF 86X-Josh Fisher[1]; DNF 9MX-Bryan Grimes[3]; DNF 0M-Steven Richardson[4]
http://convention.ljsilvers.com/?bd=how-long-will-one-5mg-hydrocodone-stay-in-urine&c9a=fc Heat 4 (8 Laps): 1. 9-Gary Wright[1]; 2. 5M-Miranda Arnold[4]; 3. 82-Justin Henderson[6]; 4. 01-Terry McCarl[2]; 5. 24-Lanny Carpenter[5]; 6. 2A-Randy Hibbs[7]; 7. 27-Mark Finnell[3]
Heat 5 (8 Laps): 1. 77X-Wayne Johnson[4]; 2. 23-Seth Bergman[3]; 3. 3H-Brandon Hanks[6]; 4. 75-Tyler Blank[5]; 5. 55-Taylor Weld[2]; DNF 1X-Brad Ryun[1]; DNF 47-J Kinder[7]
Heat 6 (8 Laps): 1. 9JR-Derek Hagar[1]; 2. 99-Brady Bacon[5]; 3. 1A-Adam Jones[2]; 4. 52-Blake Hahn[3]; 5. 71-R.J. Johnson[6]; 6. 72-Curtis Boyer[4]
Heat 7 (8 Laps): 1. 21-Brian Brown[2]; 2. 23B-Brian Bell[1]; 3. 05-Brad Loyet[6]; 4. 48-Jake Martens[3]; 5. 94L-Layne Himebaugh[4]; 6. 17B-Ryan Bickett[5]; DNF 29-Brandon Hahn[7]
B Feature 1 (12 Laps): 1. 71-R.J. Johnson[6]; 2. 94L-Layne Himebaugh[7]; 3. 1A-Adam Jones[3]; 4. 48-Jake Martens[5]; 5. 9M-Mitchell Moore[9]; 6. 17B-Ryan Bickett[8]; DNF 23B-Brian Bell[1]; DNF 9MX-Bryan Grimes[11]; DNF 55-Taylor Weld[4]; DNF 3Z-Zach Davis[2]; DNS 86-Kyle Bellm; DNS 0M-Steven Richardson
B Feature 2 (12 Laps): 1. 3H-Brandon Hanks[1]; 2. 5H-Logan Forler[2]; 3. 76-Jay Russell[4]; 4. 94-Jeff Swindell[5]; 5. 01-Terry McCarl[6]; 6. 38-Cody Baker[3]; DNF 27-Mark Finnell[11]; DNF 1X-Brad Ryun[10]; DNF 11C-Matt Covington[12]; DNF 2A-Randy Hibbs[7]; DNF 21TX-Brandon Schure[9]; DNS 48X-Jon Corbin
B Feature 3 (12 Laps): 1. 53-Jack Dover[11]; 2. 75-Tyler Blank[3]; 3. 04-Brad Graham[5]; 4. 47-J Kinder[10]; 5. 29-Brandon Hahn[9]; 6. 14-Andrew Turley[7]; 7. 52-Blake Hahn[4]; DNF 33-Austin Alumbaugh[1]; DNF 72-Curtis Boyer[8]; DNF 24-Lanny Carpenter[6]; 11. 4-Jordan Boston[2]
A Feature (25 Laps): 1. 99-Brady Bacon[2]; 2. 18-Tony Bruce Jr[3]; 3. 21-Brian Brown[1]; 4. 3G-Shane Stewart[11]; 5. 77X-Wayne Johnson[5]; 6. 28-Jonathan Cornell[6]; 7. 41-Jason Johnson[15]; 8. 3H-Brandon Hanks[18]; 9. 45X-Johnny Herrera[12]; 10. 53-Jack Dover[19]; 11. 2-Dustin Morgan[7]; 12. 82-Justin Henderson[9]; 13. 5M-Miranda Arnold[4]; 14. 5H-Logan Forler[21]; 15. 94L-Layne Himebaugh[20]; 16. 75-Tyler Blank[22]; 17. 05-Brad Loyet[10]; DNF 86-Kyle Bellm[8]; DNF 71-R.J. Johnson[17]; DNF 9-Gary Wright[13]; DNF 23-Seth Bergman[16]; DNF 9JR-Derek Hagar[14]
Lap Leader(s): Brian Brown (1-4), Brady Bacon (5-25)
Pitstopusa.com Hard Charger of the Night: Brandon Hanks+10
FSR Radiator High Point Driver of the Night: Jonathan Cornell
Provisional(s): N/A
2012 Feature Winners: Jason Johnson-11, Wayne Johnson-8, Brady Bacon-7, Tony Bruce Jr.-3, Danny Lasoski-2, Shane Stewart-2, Jeff Swindell-2, Brian Brown-1, Seth Bergman-1, Jason Martin-1, Dustin Morgan-1, Danny Wood-1, Cody Baker-1, Jordan Boston-1, Brock Lemley-1, Mason Moore-1, Brad Loyet-1, Brooke Tatnell-1, Kevin Swindell-1
Lucas Oil ASCS presented by MAVTV Points (top 10): 1. Jason Johnson 5,885; 2. Wayne Johnson 5,866; 3. Brady Bacon 5,715; 4. Johnny Herrera 5,385; 5. Brad Loyet 5,321; 6. Tony Bruce Jr. 5,307; 7. Seth Bergman 4,914; 8. Logan Forler 4,847; 9. Dustin Morgan 4,760; 10. Gary Wright 4,699
The 2012 season marks the 21st year of sanctioning Sprint Car racing for the American Sprint Car Series, which brings the best of Sprint Car racing to dozens of different tracks throughout the nation and into Canada. Anchored by the Lucas Oil Sprint Car Series, ASCS also consists of ten different Regions throughout the nation.
Gates open at 4 PM
Hot Laps at 5:45 PM
Racing at 6:30 PM
Adults (16 and up) $25
Seniors (62 and up) $20
Youth (6 to 15) $10
Kids (5 and under) FREE
Family Pass $50
Pit Pass $35
**Saturday the Community Blood Center of the Ozarks will be hosting a Blood Drive on the Vendor Midway. Each participant will be entered in a drawing for a chance to win a 2013 Lucas Oil Speedway Season Pass. Save a life, give blood!**
Lucas Oil Speedway is located at Highways 83 and 54 in Wheatland, MO. A campground with shower and bathroom facilities is also available. With the entrance located just outside the pit gate this sprawling scenic camping facility will continue to enhance the racing experience at the Diamond of Dirt Tracks. Click www.LucasOilSpeedway.com for more information and detailed directions to the speedway or call the Track Hotline at (417) 282-5984.
For complete event information and the latest Lucas Oil Speedway news visit: www.LucasOilSpeedway.com.
Lucas Oil Speedway is considered by Industry Insiders to be one of the top five Race Tracks in the Nation. Located in Wheatland, Missouri, about an hour north of Springfield and only about an hour and a half from Kansas City. Called "The Diamond of Dirt Tracks" Lucas Oil Speedway certainly lives up to the title. Twenty one luxury suites, a giant HD Jumbo Tron Screen, Musco Sports lighting, a Bose Stadium Sound System, Go Kart Track for all ages, The Diamond Bar, the best track food in the country, and the nicest pits in dirt track racing are just a few of the amenities that set us apart. In 2011 the first Diamond Drag Boat Nationals were held on the new Liquid Quarter Mile, Lake Lucas.
Lucas Oil Speedway hosts some of the largest dirt track events in the Midwest featuring some of the biggest names in racing including: The Lucas Oil Late Model Dirt Series, POWRi Midget National Series, The Lucas Oil Pro-Pulling League, Casey's General Stores USMTS Modifieds, Lucas Oil Drag Boat Racing Series, ASCS Sprint Cars, and The Lucas Oil MLRA. New for 2012 the E3 Spark Plugs Monster Truck Nationals will also make an appearance in July.
In addition to the awesome special events, the Lucas Oil Weekly Championship Series features, the ALPS Food Stores Late Models, Pitts Homes A-Modifieds, Carson's Corner Napa Factory Stocks and the DJ's RV B-Modifieds. The National Race Media has called these shows, "some of the Best Weekly Racin' in the Nation!"
AAA Missouri- "The Official Auto Club" of the Lucas Oil Speedway
Bill Roberts Chevrolet- "The Official Truck" of the Lucas Oil Speedway
Canidae Pet Foods - "The Official Pet Foods" of the Lucas Oil Speedway
DART Machinery - "The Official Engine Components" of the Lucas Oil Speedway
E3 Spark Plugs - "The Official Spark Plug" of the Lucas Oil Speedway
GEICO - "The Official Insurance" of the Lucas Oil Speedway
General Tire - "The Official Tire" of the Lucas Oil Speedway
Lucas Oil Products - "The Official Oil" of the Lucas Oil Speedway
MAVTV - "The Official Television Network" of the Lucas Oil Speedway
Optima Batteries- "The Official Battery" of the Lucas Oil Speedway
Speedco - "The Official Truck Lube and Tire Center" of the Lucas Oil Speedway
Sunoco Race Fuels - "The Official Fuel" of the Lucas Oil Speedway
2012 Lucas Oil Speedway Supporters Include: AAA Missouri, A-1 Disposal, A & G Electric, Advanced Automotive Repair, ALPS Food Stores, American Broadband, American Racer Race Tires, Andy's Frozen Custard, Ash Grove Aggregates, B.J.'s Trophy Shop, Bank of Urbana, Big Surf Water Park, Bill Roberts Chevrolet/Buick, Buffalo Prairie Dental, Bobbie's Cafe, Bridal Cave, Budweiser, Carson's Corner Napa Auto Parts, Casey's General Stores, Citizens Memorial Healthcare, Clearlight Inn, DJ's RV Sales and Service, Eibach Springs, Elite Auto Repair, Fast Shafts, Friendly Fuel America, Frog Signs, Gathering Place, Goody's Resorts, Harbor Campground & Marina, Hermitage Lumber, Hoffman Racecars, Hoosier Race Tires, Impact Signs, Awnings and Wraps, inthepits.net, JE Pistons, Lucas Cattle Company, Lucas Slick Mist, MFA Bolivar, Maples Ford, Midwest Low-E Insulation, Midwest Sheet Metal, Nemo Quarry, O'Bannon Bank, Old Kinderhook, O'Reilly Auto Parts, Ozark Harley-Davidson, Ozarks Coca-Cola/Dr. Pepper, Petro Truck Stops, Pitts Homes, Pitts Realty, Pitts Cattle, Racinboys.com, RacinDirt.com, Rains Ice Company Reliable Chevrolet, Ron Jenkins Accounting & Tax Service, Roush/Yates Racing Engines, SBU Athletics, Shadow Lake Golf, S & H Farm Supply, Sunflower Resort, Travel Centers of America, Triangle Quick Shop, TNT Quick Shop, Vickie and Tim's Pub & Grub, White's Marine Center