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The ISWI proteins form the catalytic core of a subset of ATP-dependent chromatin-remodeling activities. Here, we studied the interaction of the ISWI protein with nucleosomal substrates. We found that the ability of nucleic acids to bind and stimulate the ATPase activity of ISWI depends on length. We also found that ISWI is able to displace triplex-forming oligonucleotides efficiently when they are introduced at sites close to a nucleosome but successively less efficiently 30 to 60 bp from its edge. The ability of ISWI to direct triplex displacement was specifically impeded by the introduction of 5- or 10-bp gaps in the 3′-5′ strand between the triplex and the nucleosome. In combination, these observations suggest that ISWI is a 3′-5′-strand-specific, ATP-dependent DNA translocase that may be capable of forcing DNA over the surface of nucleosomes.
A general feature of eukaryotes is that their genomic DNA associates with proteins to form chromatin. In addition to providing a means of packaging DNA within nuclei, chromatin provides an additional level at which gene expression can be regulated. Active regions of the genome tend to be maintained in a more accessible state than inactive regions, and the manipulation of chromatin structure has been found to play an important role in the regulation of many genes. In order to be able to regulate gene expression at this level, eukaryotes have devised a number of strategies by which they can manipulate chromatin structure. These include the covalent modification of chromatin structure by posttranslational modification, the manipulation of the protein content of chromatin through the association of variant histone and nonhistone proteins, and the noncovalent alteration of chromatin structure by ATP-dependent chromatin-remodeling activities (2, 7, 38, 67).
It now appears likely that all eukaryotes encode multiple yet distinct ATPases with homology to the yeast SNF2 protein (Snf2p). These include the Sth1p, Chd1p, Isw1p, Isw2p, and Ino80p proteins in budding yeast and a spectrum of related proteins in higher eukaryotes (2, 66). Among these, the ISWI proteins represent a discrete class of ATPase involved in chromatin remodeling. The founding member of the ISWI group was identified in Drosophila melanogaster due to its similarity to Snf2p. As the homology is restricted to the helicase-like domain, it was named imitation SWI/SNF (ISWI) (21). Biochemical characterization of ISWI proteins began with the identification of ISWI as the catalytic subunit of three distinct complexes, nucleosome-remodeling factor (NuRF), chromatin accessibility complex (ChrAC), and the ATP-utilizing chromatin assembly and modifying factor (ACF) isolated from Drosophila embryo extracts (36, 60, 64). Subsequently, ISWI-related proteins have been found to be components of chromatin-remodeling complexes in organisms ranging from yeast to humans, suggesting that these proteins fulfill important functions conserved throughout evolution (5, 42, 43, 49, 61). Within each of these complexes, the ISWI protein is associated with a functionally conserved complement of additional proteins (2).
The biochemical fractionation of ISWI complexes has enabled their activity to be assessed by a variety of different assays. The ATPase activity of ISWI-containing complexes has been found to require nucleosomal histones for maximal stimulation (1, 12, 29). More recent studies have defined residues 16 to 19 of histone H4 as playing a critical role in the stimulation of ATPase activity (13, 33). Partial stimulation has been observed in the presence of complexes between the tail peptide and DNA (13). Thus, the ISWI ATPase is likely to recognize an epitope that includes H4 residues 16 to 19 within the context of a nucleosome. Recent studies have revealed that ISWI-driven ATP hydrolysis is capable of repositioning nucleosomes along DNA in cis (31, 34, 41). These observations, together with the finding that the dISWI protein alone is also able to carry out this reaction (41), suggest that nucleosome mobilization may be a common property of ISWI-containing complexes. This is an attractive proposition, as the ability to alter nucleosome positioning provides these complexes with a means to both organize nucleosomes into precisely spaced arrays (36, 64) and generate nuclease sensitivity within chromatin fibers (60, 64).
The ISWI proteins, like other ATP-dependent remodeling activities, contain seven motifs that are present in many helicase and helicase-like proteins (20). Recently, the high-resolution structures of a number of helicases have been determined. These include PcrA, which was shown to contain a tandem repeat of RecA-like domains (57). The structures of PcrA in two different DNA complexes show that the binding and hydrolysis of Mg2+ ATP results in a massive change in the relative orientation of the two RecA-like domains (65), which could drive the enzyme along the DNA backbone. This translocase activity can be physically uncoupled from the strand separation activity of the enzyme (54), raising the possibility that many of the helicase-like proteins that do not function in strand displacement assays could in fact be ATP-dependent duplex DNA translocases. Consistent with this, the structure of RecG, a member of the superfamily II helicase-like proteins (SF2) to which ATP-dependent remodeling enzymes are most closely related, also suggests that helicase motifs can function as part of a duplex DNA translocase domain (53).
Other members of the SF2 family which do not have a strand-separating activity also appear to be DNA translocases. An assortment of assays have been used to obtain evidence that the DNA translocating subunits of type I restriction enzymes can translocate along DNA (18, 22, 23, 27, 37, 48, 59, 63). Also of particular interest is recent evidence suggesting that the yeast Mot1 and Rad54 proteins, which are more closely related to the ATP-dependent chromatin-remodeling enzymes, are able to translocate along DNA (16, 50). This makes the concept that ATP-dependent chromatin-remodeling activities may also be able to translocate along DNA attractive. Supporting this, several ATP-dependent remodeling activities have been shown to generate unconstrained superhelical torsion within linear DNA or chromatin fragments, which was proposed to occur as a result of the translocation of remodeling enzymes along the helical DNA backbone (35). More recent studies of the yeast RSC and Drosophila ACF complexes support the idea that these complexes also translocate along DNA (see Discussion) (26, 51).
Here, we adapted a triplex oligonucleotide displacement assay (23) to study the action of ISWI on nucleosomal substrates. This provides new evidence to support the idea that ISWI is capable of distorting the DNA adjacent to nucleosomes. We also found that the ATPase activity of ISWI is stimulated to a low yet detectable level in the presence of single-stranded DNA. This suggests that ISWI is capable of functioning on one DNA strand. Consistent with this, the displacement of triplex DNAs was blocked with specific polarity by gaps in the 3′-5′ DNA strand. As these gaps were positioned between the triplex and the nucleosome, the results support the theory that ISWI functions as a nucleosome-stimulated DNA translocase oriented such that it forces DNA over the surface of nucleosomes.
Recombinant Drosophila ISWI and histones were purified as described previously by Corona et al. (14) and Luger et al. (44), respectively.
PCR-generated DNA was purified from oligonucleotide primers by fractionation over a 1-ml Source 15Q anion exchange column (Pharmacia). DNA fragments containing gaps and nicks were prepared by multiple-round primer extension as described previously (11), except that the resulting single-stranded DNA was purified by anion exchange chromatography. Templates were then prepared by annealing complementary single-stranded DNA in annealing buffer (10 mM Tris-Cl [pH 8.0], 50 mM NaCl). Further information about the DNA sequences used can be found at http://www.dundee .ac.uk/biocentre/owenhughes/MCB2003suppl.pdf.
A total of 10 pmol of 32P-radiolabeled TFO, TTCTTTTCTTTCTTCTTTCTTT, was incubated with 100 pmol of duplex DNA containing the triplex binding site in a 20-μl reaction volume containing 40 mM morpholineethanesulfonic acid (pH 5.5) and 10 mM MgCl2. The mixture was heated to 57°C for 10 min and allowed to cool slowly to room temperature. These sequences formed a parallel triplex, meaning there were no complementary sequences capable of annealing with the TFO to form a heteroduplex.
Standard reconstitution reactions were conducted with equimolar amounts (100 pmol) of duplex DNA and purified Xenopus histone octamer as described previously (44). Low-pH TFO reconstitutions were conducted by mixing 100 pmol of TFO-bound DNA with 100 pmol of recombinant Xenopus histone octamer in a 50-μl reaction containing 1.2 M NaCl, 40 mM Na-cacodylate (pH 6.0), and 5 mM MgCl2. This mixture was left to equilibrate at room temperature for 30 min and then placed in a microdialysis block containing Spectra/Por 1 dialysis tubing (cutoff, 11,000 to 15,000 Da; Spectrum). Stepwise dialysis was conducted at 4°C at 2-h intervals against buffer 0.85 CAM (850 mM NaCl, 20 mM Na-cacodylate [pH 6.0], 5 mM MgCl2) and then 0.65 CAM (650 mM NaCl, 20 mM Na-cacodylate [pH 6.0], 5 mM MgCl2), followed by 0.5 CAM (500 mM NaCl, 20 mM Na-cacodylate [pH 6.0], 5 mM MgCl2) and finally 0.1 CAM (100 mM NaCl, 20 mM Na-cacodylate [pH 6.0], 5 mM MgCl2). The level and homogeneity of reconstitutions were assessed by native gel electrophoresis. Reconstitutions were then stored at 4°C.
DNA and nucleosome binding were assayed by electrophoresis through 5% acrylamide-0.5× Tris-borate-EDTA (TBE) gels with 1× TBE as running buffer. Gels were run either in the cold room or at room temperature at 70 to 100 V as indicated in the figure legends. Binding reactions contained 3 mM MgCl2, 20 mM HEPES (pH 7.9), 400 μg of bovine serum albumin (BSA)/ml, 0.033% NP-40, 1 mM dithiothreitol, 5% glycerol or 2.5% Ficoll, and 60 or 120 mM NaCl as indicated in the figure legends.
ATPase reactions were carried out in the presence of 20 mM HEPES (pH 7.9)-3 mM MgCl2-0.033% NP-40-20% glycerol-1 mM dithiothreitol-400 μg of BSA/ml-5 or 10 μM ATP-2.2 μCi [α32P]ATP and differing amounts of nucleosomes, DNA, NaCl, and ISWI as indicated in the figure legends. Reactions were incubated at 30°C, and at the indicated time points, 1/10-volume aliquots were removed from the 10- or 20-μl reactions and stopped by addition to 10% sodium dodecyl sulfate. A total of 1 μl of each stopped reaction was analyzed by thin-layer chromatography (TLC) on polyethylenimine cellulose F TLC plastic sheets (Merck) with 0.75 M KH2PO4 (pH 3.5) as the running buffer to separate ATP from ADP. The extent of hydrolysis was determined by quantification of the spots corresponding to ATP and ADP with a PhosphorImager (Molecular Dynamics).
High-resolution nucleosome mapping was conducted as described previously (25). A total of 2 pmol of each 32P-end-labeled substrate was incubated for 30 min at 30°C in a 20-μl reaction containing 0.25 pmol of dISWI protein in the presence or absence of 1 mM Mg2+ ATP. The reaction was stopped by the addition of an excess of lambda DNA competitor and chilled on ice. Site-directed cleavage was initiated by the addition of 15 μM (NH4)2Fe(SO4)2, followed by 3 mM ascorbic acid and 0.05% H2O2 for 45 min. Reactions were stopped by the addition of 1 M thiourea and 10 mM EDTA. Samples were precipitated and analyzed by denaturing gel electrophoresis.
TFO displacement reactions were conducted in 20-μl reactions in 20 mM Na-HEPES (pH 7.9)-50 mM NaCl-3 mM MgCl2-10 μg of BSA (Promega)/ml-5% glycerol. All reactions contained 500 fmol of TFO-bound mononucleosomes. Mg2+ ATP or Mg2+ ATPγS was added to a final concentration of 1 mM. dISWI was added to a final concentration of 12.5 nM (250 fmol). All reactions were conducted for 30 min at 30°C. Displacement was assayed on a 1.5-mm-thick 5% (wt/wt) polyacrylamide gel containing 0.25× TAM (40 mM Tris-acetate [pH 7.0], 1 mM MgCl2). Electrophoresis was performed with 1× TAM buffer, after which the presence of the TFO was monitored with a PhosphorImager.
Previously, it was reported that the ISWI protein preferentially interacts with nucleosomes containing linker DNA (6). In order to investigate this further, the ability of the ISWI protein to bind to nucleosomal substrates containing defined lengths of linker DNA was studied. A prerequisite for this is the ability to position nucleosomes precisely. In order to achieve this, the well-characterized nucleosome A (NucA)-positioning sequence found in the mouse mammary tumor virus (MMTV) long terminal repeat was used (25). We adopted a nomenclature in which the DNA fragments are described by the term xAy, where x denotes the length of DNA attached to the upstream side of the nucleosome, A denotes the 147-bp positioning sequence, and y denotes the length of linker DNA attached on the downstream side of the nucleosome. For the nucleosome binding studies described here, we added linker DNA to one side of the nucleosome. The reason for this was that nucleosomes can be reconstituted to the end positions with a greater homogeneity than at central locations (data not shown). Figure Figure1A1A shows the binding of ISWI to 0A0 nucleosome core particles and 0A41-bp nucleosomal fragments. As reported previously, ISWI was found to form complexes stable enough to be resolved as discrete species during native gel electrophoresis with nucleosomes containing linker DNA but not with nucleosome core particles (6). However, we also noted that when the disappearance of free nucleosome was compared, there was little difference between the substrates. Thus, ISWI is capable of interacting with nucleosome core particles but discrete complexes are not formed.
We also noticed that the low levels of free DNA present in our nucleosome reconstitutions were shifted in the presence of ISWI. This suggests that ISWI has the ability to bind to free DNA. In order to investigate this further, we measured the binding of ISWI to DNA fragments of different lengths (Fig. (Fig.1B).1B). Although we could not detect ISWI binding to a 15-bp DNA fragment, it was able to bind to DNA fragments of 23, 32, and 41 bp with increasing efficiency. To characterize the length dependence of ISWI binding to DNA more carefully, ISWI binding was monitored over a range of DNA concentrations for fragments of 32, 41, and 77 bp. The results plotted in Fig. Fig.1C1C illustrate that, whereas the 41-bp fragment interacts with ISWI more efficiently than the 32-bp fragment, the 77-bp fragment does not further enhance ISWI binding. This suggests that the minimal DNA length for interaction with ISWI is between 15 and 23 bp and that a 41-bp fragment is capable of optimally interacting with ISWI.
The stable complexes formed during the binding of ISWI to the 41-bp fragment are similar to those observed during binding to nucleosomal DNA. This raises the possibility that the interaction of ISWI with DNA may contribute to the previously observed preference of ISWI for nucleosomes containing linker DNA (6, 40). Indeed, the nonspecific DNA binding properties of ISWI might be expected to bias the interaction towards linker DNA or relatively accessible DNA at the edge of the nucleosomes. This complicates the interpretation of the location of ISWI in bulk ISWI-nucleosome assemblies, as the most stable complexes may not be those in which the ISWI has engaged with the nucleosome in a functional orientation. For example, the functional interaction of ISWI might be transient and perhaps related to the smearing and aggregation we observed during the binding of ISWI to the 0A0 and 0A41 nucleosome core particles. In this case, the stable-shifted complexes observed with 0A41 nucleosomes could result from the nonfunctional binding of ISWI to the DNA adjacent to the nucleosome. Consistent with this, the presence of the H4 tail, which is critical for ISWI function, does not affect the ability of ISWI to gel shift nucleosomal DNA (12).
In order to gain further insight into ISWI function, we next determined the ability of nucleosomal substrates of different DNA lengths to function as substrates for ISWI-driven ATP hydrolysis. Figure Figure22 shows that, whereas nucleosome core particles produce low levels of ATPase activity, nucleosomes containing successively longer lengths of DNA stimulate progressively higher levels of activity. We also found that nucleosomes with linker DNA 61 bp in length did not further stimulate ISWI ATPase activity (data not shown). This implies that ATP hydrolysis involves the interaction of ISWI with DNA outside of the nucleosome core.
DNA fragments have also been reported to be capable of stimulating low levels of ISWI ATPase activity (14). Therefore, we next compared the ability of DNA fragments of increasing length to stimulate the ATPase activity of ISWI. Although the efficiencies of stimulation were lower than those observed with nucleosomal DNA, Fig. Fig.33 illustrates that DNA length does have an effect per se. While a 15-bp fragment was capable of only modest levels of stimulation above the background levels observed in the absence of DNA, 23-, 32-, and 41-bp fragments generated successively higher levels of stimulation. However, increasing the length of the DNA beyond 41 bp resulted in an only modest additional stimulation of ATPase activity.
Previously, it was reported that the ATPase activity of the SWI/SNF and RSC complexes can be stimulated by single-stranded nucleic acids (8, 9). For this reason, we next investigated whether single-stranded nucleic acids were also capable of stimulating the ATPase activity of the ISWI. We found that the stimulation of ISWI ATPase activity by single-stranded DNA was less efficient than that when using double-stranded DNA; however, by using higher concentrations of ISWI, this stimulation could be detected (Fig. (Fig.3A3A and C). We next investigated whether the stimulation of ISWI ATPase activity by single-stranded DNA was dependent on the fragment's length. Figure Figure3A3A and C illustrates that the ability of single-stranded DNA to stimulate the ATPase activity of ISWI displays a length dependence similar to that observed for double-stranded DNA fragments, with maximal stimulation occurring on fragments of approximately 40 bp.
The dependence of ISWI ATPase activity upon DNA length both within the context of a nucleosome and on free DNA suggests that DNA contacts over a region of ~40 bp are required for maximal stimulation. One possible explanation for this DNA length dependence is that ATP hydrolysis by the ISWI protein involves DNA translocation and that the shorter length of some of the templates we used reduces the length of DNA over which translocation is possible. An alternative explanation is that ISWI must contact DNA with a length of approximately 40 bp in order to fully activate its ATPase activity. Ideally, the next step would be to determine whether these contacts primarily affect the Km or Vmax of the reaction. Unfortunately, the activity of ISWI in ATPase assays is low, meaning that we were not able to perform ATPase assays over a broad enough range of concentrations and time points to accurately determine these kinetic parameters. Instead, we developed a new approach to monitor the action of the ISWI protein.
The DNA translocating subunits of type I restriction enzymes are, like the ATP-dependent remodeling activities, members of the SF2 family of helicase-like proteins. Recently, an assay was developed to measure the motion of the type I restriction enzyme EcoR124I along DNA (23). Briefly, this assay involves the annealing of a pyrimidine-rich triple-helix-forming oligonucleotide (TFO) to a purine-rich target sequence. The pyrimidine-rich TFO occupies the major groove parallel to the purine-rich strand and utilizes Hoogsteen hydrogen bonds to form T-AT and C+-GC triplets (30). The formation of the triple helix depends upon N3 cytosine protonation and is dependent on low pH; the triplex does not form at a neutral pH. However, this reliance on low pH can be circumvented by preforming the triplex at a low pH and then diluting it into reaction buffer and by the inclusion of millimolar concentrations of divalent cations, which stabilize the triplex indefinitely (39). Importantly, at a physiological pH, the TFO, once displaced, is unable to rebind. As a result, triplex displacement provides a means of monitoring the action of DNA translocases that are capable of displacing the TFO (32).
We have adapted this assay to study the action of ISWI. In order to do this, we first studied the displacement of TFOs from sites adjacent to centrally located nucleosomes. Although triplex displacement did occur, nucleosomes were also repositioned on these templates, making it difficult to distinguish whether ISWI or the movement of the nucleosome had caused the triplex to be displaced (data not shown). In order to study the action of ISWI in triplex displacement, we sought a means to distinguish between direct and indirect displacement. With this aim, we exploited the fact that ISWI has previously been reported to move mononucleosomes from central positions to the end position but not visa versa (41). The MMTV NucA-positioning sequence was used to generate nucleosomes that were positioned either centrally (61A61) or at the upstream end of the DNA fragments (0A75). To confirm whether nucleosomes were assembled at the expected locations, we used high-resolution nucleosome mapping (24). Briefly, this involves the tethering of cysteaminyl EDTA to a recombinant histone octamer via a thiol group introduced at H4 cysteine 47. The chelation of Fe2+ ions by the mapping reagent is capable of catalyzing the Fenton reaction, which results in the generation of hydroxyl radicals that are capable of cleaving DNA, but only within a range of approximately 1 nm. The site at which the reagent is attached on histone H4 comes into close contact with DNA approximately 4 bp on either side of the nucleosome dyad, meaning that the sites of cleavage can be used to deduce nucleosome positions. In the case of the 61A61 and 0A75 fragments, the major sites of cleavage prior to remodeling indicate that nucleosomes were predominantly located to the expected sites (Fig. (Fig.4A,4A, lanes 1 and 3). Mononucleosome templates were prepared that contained the derivatized histone octamer either in the middle or at the upstream end of the DNA fragment (Fig. (Fig.4A4A).
Following ISWI remodeling on the 61A61 fragment, much of the cleavage at the major initial location of the nucleosome was lost and new cleavage sites were observed that are consistent with the redistribution of this nucleosome to positions located close to either end of the DNA fragment (Fig. (Fig.4A,4A, lane 2). However, remodeling of the terminally located nucleosome (0A75) under the same conditions did not result in a similar reduction in cleavage at the initial location, suggesting that ISWI is not able to relocate a significant proportion of the nucleosomes assembled on this template (Fig. (Fig.4A,4A, lane 4).
Having established that the majority of the histone octamers residing at this terminal location are not relocated by dISWI, the ability of the enzyme to facilitate TFO displacement was investigated. Seven constructs were generated with a sequence capable of annealing to the TFO 0, 10, 20, 30, 40, 50, or 60 bp downstream from an end-positioned nucleosome. Radiolableled TFO was annealed to these DNAs, and they were then assembled into nucleosomes. During native gel electrophoresis in TAM buffer (see Materials and Methods), TFO remained associated with the DNA, enabling species with the mobility of the TFO-bound nucleosome and TFO-bound free DNA to be resolved (Fig. (Fig.4B).4B). Under these conditions, the free TFO migrated with the solvent front and was not shown. Restriction enzyme digestion was used to confirm that nucleosomes assembled onto the MMTV NucA-positioning sequence as expected when TFO was bound to the linker DNA (data not shown).
Incubation with dISWI and ATP resulted in efficient TFO displacement from constructs containing triplex DNA 0, 10, or 20 bp from the nucleosome. This was shown by a loss in intensity of the band corresponding to nucleosomal DNA (Fig. (Fig.4B,4B, lanes 2, 4, and 6). Note that TFO was not removed from free DNA as would be expected given that the ISWI ATPase activity was maximally activated by nucleosomal histones. However, when displacement was assessed on constructs 0A54(tfo30) and 0A54(tfo40), where the TFO begins either 30 or 40 bp away from the nucleosome, the displacement was reduced (Fig. (Fig.4B,4B, lanes 8 and 10). When triplex DNA was located 50 or 60 bp from the downstream edge of the nucleosome, no displacement was observed (lanes 12 and 14).
Figure Figure4C4C shows a plot illustrating the levels of ISWI-driven triplex displacement at each site. The fact that displacement becomes progressively less efficient further away from the edge of the nucleosome would be consistent with it occurring as a result of the DNA translocase activity originating from within or close to the edge of the nucleosome and oriented away from it. This would result in a collision between the ISWI and the triplex and, eventually, the displacement of the TFO. Limited processivity of this translocase could explain why displacement is more efficient close to the nucleosome than it is from further away. However, it also remains possible that some other form of DNA distortion could be responsible for triplex displacement.
If triplex displacement occurred as a result of ISWI translocase activity originating from within or close to the edge of the nucleosome, then insertion of lesions between the nucleosome and the TFO might be expected to interfere with triplex displacement. In order to test this, we generated a template in which a series of nicks, 5- or 10-bp gaps, were introduced into either DNA strand between the triplex and the nucleosome.
As expected, when supplemented with hydrolysable ATP, a control construct that contained no modification was able to facilitate dISWI-mediated TFO displacement (Fig. (Fig.5,5, lane 2). In a similar manner, both constructs containing nicks in either strand and 5- or 10-bp gaps in the top (5′-3′) strand showed normal displacement profiles (lanes 4 and 10). However, insertion of a 5- or 10-bp gap at the same position in the bottom (3′-5′) DNA strand was unable to support TFO displacement (lanes 12 and 14). This supports the involvement of one-dimensional DNA translocation in triplex displacement, as other forms of DNA distortion would not be expected to show this strand-specific polarity. The fact that gaps between the triplex and the nucleosome prevent displacement provides further evidence that translocation is oriented away from the nucleosome. Moreover, the observation that a gap in the 5′-3′ strand supports displacement suggests that translocation is occurring predominantly along the 3′-5′ DNA strand.
ISWI proteins form the catalytic core of a range of chromatin-remodeling activities that play important roles in gene regulation. In this study, we have used a range of different approaches to define the interaction between the ISWI ATPase and nucleosomal substrates. As reported previously, we found that ISWI forms more stable complexes with nucleosomes that contain additional linker DNA (6). However, we also found that ISWI binds to DNA fragments in a length-dependent fashion. Binding to a 23-bp fragment was weak and undetectable on a 15-bp fragment. ISWI bound to 32- and 41-bp fragments with greater affinity, and binding to these longer fragments was observed to result in the formation of more discrete complexes. We next investigated the dependence of ISWI ATPase activity on nucleosomal linker DNA length and found that this also displays length dependence. Although we found that ISWI ATPase activity is stimulated to a lesser extent in the presence of free DNA as reported previously, we also found that this stimulation showed a dependence on DNA length that peaked at approximately 40 bp. The ATPase activities of both SWI/SNF and RSC have previously been reported to be stimulated by both double- and single-stranded DNAs (9, 15, 51). Here, we report that the ATPase activity of ISWI is also stimulated by single-stranded DNA. In addition, we found that the stimulation of ISWI ATPase activity by single-stranded DNA shows a length dependence that correlates well with that observed using duplexes. The DNA length dependence that ISWI displayed in these assays raises the possibility that ISWI functions as an ATP-dependent DNA translocase.
In order to test this further, we adapted a triplex displacement assay to study the action of ISWI on nucleosomal templates. ISWI was able to displace TFOs most efficiently from sites close to the edge of the nucleosomes. However, this activity reduced to undetectable levels 50 bp from the edge of a nucleosome. This would be consistent with ISWI, exhibiting a DNA translocase activity oriented away from the edge of nucleosomes with limited processivity. Further evidence supporting the orientation of the translocase activity was obtained through the observation that the introduction of gaps between the nucleosome and the triplex could prevent triplex displacement in a strand-polarity-dependent manner.
While this work was in preparation, two reports were published presenting evidence that chromatin-remodeling activities translocate along DNA (26, 51). Fyodorov and Kadonaga used a template commitment assay to show that the ISWI-containing ACF complex remains associated with, and preferentially functions on, one template following exposure to an equivalent amount of a competing template. This commitment to the template to which ACF is exposed first depends upon ongoing chromatin assembly and may offer valuable new insight into the way in which ACF functions. Fyodorov and Kadonaga argue that the phenomenon of template commitment provides evidence that ACF functions as an ATP-dependent DNA-translocating motor. However, template commitment has been observed previously for many DNA and chromatin binding proteins that are not ATP-dependent DNA translocases (3, 46). In order to explain template commitment, it has been proposed that, during transient dissociation from a site on one template, a DNA binding protein will be more likely to reassociate with the same template, as the local concentration of the molecule to which it was originally bound will be far higher than that of other molecules in the solution (55). It is difficult to rule out the possibility that similar processes explain the observations made by Fyodorov and Kadonaga.
The study by Saha et al. investigated the DNA length dependence of ATP hydrolysis by the yeast RSC complex and its catalytic subunit Sth1p. Both of these activities are more potent in ATP hydrolysis than ISWI. This has enabled a more thorough investigation of the relationship between the DNA length and ATPase activity to be performed than has been possible with ISWI. Importantly, for both Sth1 and RSC, it has been possible to establish that DNA length affects ATP hydrolysis by affecting the maximal rate of hydrolysis, Vmax, rather than Km. This is consistent with the increased length of the template DNAs, allowing more rapid ATP hydrolysis before the translocase reaches the end of the DNA fragment. This form of length dependence is similar to that observed for bona fide helicases such as T4 gene 41 (68). In addition, Saha et al. performed an elegant experiment in which they showed that RSC and Sth1 have similar activities on short circular DNA molecules to longer linear ones. All of these approaches are consistent with these activities functioning as ATP-dependent DNA translocases. However, it should be noted that ATP hydrolysis need not necessarily provide a direct measure of DNA translocation.
In order to obtain additional evidence for DNA translocation, Saha et al. also used a triplex oligonucleotide displacement assay. They found that both Sth1p and RSC are capable of displacing TFOs in an ATP-dependent reaction. Although triplex displacement has been used previously to monitor the action of ATP-dependent DNA translocases and helicases (23, 39, 45), it is conceivable that TFOs could be displaced by other means. As a control for this, Saha et al. showed that triplex displacement is reduced from a triplex DNA that lacks double-stranded extensions and is still possible on one that contains a nick at the duplex-to-triplex interface.
Our studies of ISWI are entirely consistent with those described by Saha et al. and provide additional support for the model they present. We obtained two lines of evidence that support the involvement of a processive activity in triplex displacement. Firstly, triplex displacement decays with distance from the nucleosome. As an epitope within the H4 tail has been defined as playing a key role in the stimulation of ISWI ATPase activity, it is likely that at least a proportion of the ISWI protein functionally associates with this part of the histone octamer (13, 33). The fact that the TFO displacement decays with distance from this epitope supports a model in which translocation is orientated away from the nucleosome. Additional evidence for this orientation is provided by the observation that the insertion of 5- or 10-bp gaps in the 3′-5′ strand between the nucleosome and the triplex prevents triplex displacement.
In contrast to the effect of gaps in the 3′-5′ strand, the introduction of 5- or 10-bp gaps in the 5′-3′ strand has no effect on the ability of ISWI to function in triplex displacement assays. These observations are most readily explained if ISWI exhibits a strand-specific translocase activity. Strand specificity is a property of bona fide DNA helicases, which can be separated into two groups, 5′-3′ or 3′-5′, reflecting the polarity in which DNA transport occurs (10). Many members of the SF2 group, to which the ATP-dependent chromatin-remodeling activities are most closely related, display a 3′-5′-strand specificity. Our observation that 3′-5′, but not 5′-3′, gaps have an effect is consistent with this. Further evidence supporting the ability of remodeling activities to function on a single DNA strand stems from the observations that the ATPase activity of RSC (9), Sth1 (51), and ISWI (Fig. (Fig.3)3) is stimulated by single- as well as double-stranded DNA.
It is interesting to note that the ability of ISWI to function in the displacement of triplexes located up to 40 bp from the edge of a nucleosome bears similarities to studies of the yeast Mot1 protein. This protein has homology to the ATP-dependent remodeling activities and functions to displace the TATA binding protein from DNA in an ATP-dependent reaction. Mot1-driven TATA binding protein displacement requires contact with a DNA "handle" adjacent to the TATA box, like ISWI, and the introduction of single-stranded DNA gaps does not prevent Mot1 functioning (16).
Although the observations of Saha et al. together with the evidence presented here suggest to us that ISWI, RSC, and Sth1 are likely to function as ATP-dependent DNA translocases, it is important to note that it has not yet been possible to directly monitor the motion of any remodeling activity along DNA or determine the translocation rate as has been possible with EcoR124 (23). Until this has been achieved, it remains a formal possibility that ATP-dependent remodeling activities distort DNA in some way that does not involve translocation. This will be technically difficult, as it appears that RSC and Sth1 have a processivity limited to 80 bp (51), while in the case of ISWI, our own assays suggest that ISWI processivity is limited to 40 bp. This is a relatively short distance compared to previously characterized ATP-dependent nucleic acid translocases such as gp14 (68) and EcoR124I (58). In addition, this processivity limit is typically reached as a result of a stochastic dissociation event occurring at distances distributed around a mean length. This is expected to give rise to an exponential decay in processivity that fits to an equation hyperbolic for cofactor length. However, the work presented here and much of the data obtained by Saha et al. suggest that the length dependence displayed by these chromatin-remodeling enzymes is more abrupt than would be expected for an exponential decay process. This, combined with the fact that highly processive motion over long distances may not be required for the local distortion of chromatin structure, raises the possibility that a more precise mechanism regulates the processivity of remodeling enzymes. Other helicases, such as the RecBCD enzyme, have been observed to translocate in a stepwise fashion (4). Our observations of DNA binding, double- and single-stranded ATPase activity, and triplex displacement are all consistent with a processivity limit for ISWI of approximately 40 bp. However, unlike RecBCD, our observations would suggest that ISWI is able to dissociate from the template after each cycle of translocation.
Interestingly, Sth1 and ISWI appear to have different processivity limits. Whereas RSC and Sth1 appear to be capable of translocating for 80 bp, for ISWI, both the ATPase assays and triplex displacement assays suggest that the processivity of ISWI is limited to approximately 40 bp. This could explain differences in the activities of the two enzymes. While RSC is able to disrupt nucleosomes in addition to moving them along DNA, ISWI appears to be more specialized toward nucleosome mobilization. The distortion of less DNA by ISWI may suit it for a role in nucleosome mobilization without nucleosome disruption. It is also interesting that the 40-bp limit for ISWI is compatible with the average nucleosome spacing in many higher eukaryotes.
How might a DNA translocase activity stimulate nucleosome mobilization? Our data were generated by using chromatin templates, which provide a means of orientating the distortion of DNA driven by an ATP-dependent remodeling activity, in this case, ISWI, relative to that of a nucleosome. As ISWI requires residues 16 to 19 of the H4 tail for maximal ATPase activity, it is likely that the ISWI retains contact with at least this part of the nucleosome during ATP hydrolysis. Translocation of the ISWI protein along DNA, while it remains associated with the histone octamer, could cause DNA to be injected onto the surface of nucleosomes. This would result in the generation of a loop or bulge of DNA on the surface of the nucleosome, which might then be able to transit around the surface of the octamer in a fashion similar to that proposed for the translocation of SP6 RNA polymerase around nucleosomes (56).
Since our results suggest that ISWI has strand specificity, translocation would be expected to follow one DNA strand around the helical DNA backbone. A key consequence of this would be the generation of superhelical torsion between the translocase and the site at which DNA contacts the nucleosome surface. Consistent with this suggestion, the generation of superhelical torsion on chromatin templates by ISWI was previously detected (35). This provides a second means by which DNA might be moved over the surface of the nucleosome. Local defects to the superhelical twist of DNA on the nucleosome might be transmitted incrementally over the surface of the nucleosome, as proposed previously (62). This is also supported by the distortion of DNA in high-resolution structures of the nucleosome (17). Where rotation of the DNA on the surface of the nucleosome is not favored, for example, in the presence of strong rotational phasing, a buildup of torsion might be capable of impeding nucleosome mobilization. Consistent with this, it has been observed that the ySWI/SNF complex is inefficient in remodeling nucleosomes on small minicircles that cannot readily accommodate changes in twist (28). It has also recently been observed that the presence of nicks in a DNA template located at sites that could relieve torsion as it accumulates as a consequence of ISWI translocation stimulates the ability of ISWI to function in nucleosome mobilization (40). This is also consistent with a role for torsion in remodeling.
Neither of these mechanisms are necessarily mutually exclusive, and it is even possible to imagine a combination of the two. It is possible that the structural characteristics of the DNA, especially its flexibility and inherent curvature, play an important role in determining what combination of the two mechanisms is involved. Whatever mechanism is involved, it remains unclear why ISWI appears unable to move nucleosomes from the ends of some DNA fragments such as the one used in this study. It appears to be a general property of the movement of nucleosomes on short DNA fragments that they tend to accumulate toward DNA ends (25, 47). One explanation for this has been that there is an energetic penalty associated with the crossover of DNA duplexes entering and/or exiting the nucleosome in close proximity that is absent for nucleosomes at the ends of DNA fragments (52). However, this phenomenon has been characterized on only very few DNA fragments to date and we have observed cases where nucleosomes positioned at the ends of some DNA fragments can be relocated to other positions by ISWI or thermal incubation (A. Flaus, unpublished data). Thus, it is possible that the stability of end-positioned nucleosomes is a result of the combined energetics of DNA-sequence-dependent nucleosome positioning and the absence DNA duplex crossover at the entry and/or exit to the nucleosome such that some nucleosomes, like the one used here, strongly resist movement.
Although the ISWI protein provides an excellent model system to study mechanisms for ATP-dependent chromatin remodeling due to the fact it can be expressed as a recombinant protein and has ATPase activity that is targeted to a specific site on the histone octamer, it is likely that the means by which other ATP-dependent remodeling activities function may differ, at least to some extent. Indeed, the ISWI protein itself is normally found to be associated with other proteins that alter the way in which it moves mononucleosomes (19). Thus, it will also be important to determine how the assembly of ISWI into native complexes alters its action in nucleosome mobilization.
We thank Brad Cairns, Michael Bruno, Kristina Havas, Nicola Wiechens, Chantal Rencurel, and members of the Division of Gene Regulation and Expression for advice and support.
This work was funded by a Wellcome Trust Senior Research Fellowship (064414), Human Frontiers Programme grant, and an EMBO young investigator award to T.O.-H., a Wellcome Trust 4-year studentship to C.S., and a Wellcome Trust Career Development fellowship (053856) to M.D.S.
I.W. and C.S. contributed equally to this work.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
Fuel pump will not prime, replaced fuel pump and still wil not prime, all fuses and relay is good, relay clicks and is working, is there something that is telling the fuel pump to not kick on? Was driving fine and parked it, went to start it and wont start just turns over.
Monday, April 15th, 2013 AT 4:27 AM

subset: commoncrawl


text:
SanDisk's MP3 players have long been the popular alternative to Apple's all-conquering iPod players. Over the years they've managed to couple great value with decent usability and pretty slick design too. And Sansa players such as the venerable e-series and the more recent View have always crammed the features in.
Surprisingly, though, it has taken SanDisk nearly a year to respond to Apple's 'new' squat nano. We had the View months ago and the brilliant Clip, but neither took on the anodised iPod head on. The View was simply too big to rival its pocketability and the Clip too cheap and chirpy. Now that the Fuze is finally here, however, SanDisk at last has a direct competitor to Apple's most popular player.
If imitation really is the sincerest form of flattery, then the nano can feel itself well and truly idolised. The Fuze is almost identical in proportion to the nano, complete with short/fat profile. A landscape 1.9in screen sits above a circular control cluster, with even the sync and headphone connections in the same place, ranged along the bottom edge. The Fuze is slightly narrower, width-wise than the nano and a little taller too. It isn't as slim as the Nano, though, at 7.3mm compared to 6.5mm and it still can't quite match the Apple player's sense of style.
The Fuze is still a very nicely designed player, though, with a soft-touch rubberised rear and glossy front (available in more colours than just black, for the style conscious). Arguably the rubbery finish and slightly thicker profile makes it more comfortable to operate one handed, too. It isn't quite as soap-bar slippery as the nano.
Capacities go up to 8GB - the same as the nano - and even battery life is the same, quoted at 24 hours for music and five hours for video, but from here on the two players begin to diverge. The 1.9in screen, for instance, has a much lower resolution (220 x 176 pixels) than the QVGA (320 x 240 pixels) one found on the nano. And, apart from the fact that the quality isn't as good - colours aren't quite as natural and it's much more prone to reflections than the nano's screen - it doesn't support smooth 30fps video, forcing you to convert clips to a lowly, and much less watchable, 15fps.
Format support is limited to MPEG-4 files at native screen resolution too - a far cry from the excellent video support of the View, which supports WMV and MPEG-4 files at more than the native resolution of the screen (640 x 320). However, SanDisk does provide a free video converter tool for download from its website, which works very well and is easy to use.
SanDisk Sansa Fuze+ Black ( 8 GB ) Digital Media Player$40.00 View Item
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text:
This is probably the best hotel near the RAI centre but it is about 20 minutes by tram from the city centre.
If I was coming to Amsterdam as a tourist, I would probably look for something more central.
If your visit is for bussiness or for a conference at the RAI centre this hotel will be hard to beat. It is a fairly modern hotel and it shows in that everything has been purposefully designed.
- Official Description (provided by the hotel):
- Contemporary High-rise Hotel with a Dutch & Japanese flair located in the cozy neighborhood De Pijp and within walking distance of the RAI Convention Center. Hotel Okura Amsterdam facilitates four restaurants of which all have been awarded by Michelin. On the 23rd floor one can find two Michelin star Ciel Bleu Restaurant. On the ground floor, Teppanyaki Restaurant Sazanka is located, this restaurant has been awarded one Michelin star and here chefs prepare your dishes right in front of you on the hot griddle. Then, Yamazato Restaurant, awarded one Michelin star as well, serves the authentic Japanese kaiseki cuisine. And lastly, Serre Restaurant is inspired by Ciel Bleu and has received a Bib Gourmand by Michelin which awards the great price-quality relation. Furthermore, Hotel Okura Amsterdam facilitates Nagomi Spa & Health were guests can relax by means of treatments and workouts. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Hotels.com, Booking.com, Expedia, TripOnline SA, Orbitz, HotelsClick, Ctrip TA, Hotwire, Agoda, Travelocity, Priceline, getaroom.com, Orange Marketing Services, Japan Co., Ltd., Odigeo and Jetsetter so you can book your Hotel Okura Amsterdam reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Okura Hotel Amsterdam
- Amsterdam Okura Hotel


text:
Insightful look at a place that shows other systems are indeed possible,
Verified Purchase(What is this?)
This review is from: The Village Against the World (Hardcover)
I enjoyed reading Dan Hancox's account of Marinaleda. It is thorough and well-written. The author has a clear bias but he still raises important questions about the upcoming challenges for the village to maintain its structure. I found it interesting and inspiring to get to know a place that shows different models are possible.
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if these reviewers believe in supporting the local economy why are they buying the book from a multi-national like amazon
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text:
Who needs a man with friends like these? Kelly Brook parties with Katherine Jenkins and Myleene Klass after split from Danny Cipriani
She had been keeping a low profile after splitting from love rat
Danny Cipriani but it seems that Kelly Brook is back to being single and
ready to mingle.
The 33-year-old model threw on a polka dot dress, and a brave face, as she stepped out in London on Friday night.
Kelly showed that she doesn't need a man to make her happy as she was joined by close friends Myleene Klass and Katherine Jenkins for the night of revelry in Mayfair.
Butting on a brave face: Kelly Brook beamed as she headed out for drinks in Mayfair on Friday night, her first appearance since it emerged she had dumped love rat Danny Cipriani
What are friends for? Katherine Jenkins and Myleene Klass were on hand to give Kelly some support
The pretty brunette wore the demure spotty
dress, which boasted sheer paneling around the neckline and sleeves,
with a pair of T-bar sandals.
She kept her make-up simple and added a diamond bracelet around her wrist and wore glittering earrings.
Her lace dress, which was sheer at the back, meant that Kelly could not wear a bra to support her ample chest.
Not dwelling on it: The 33-year-old beamed as she headed out in Mayfair with friends on Friday
Putting her best fashion foot forward: Kelly teamed the dress with a pair of T-bar heels and diamond jewelery
Putting her best fashion foot forward: Kelly teamed the dress with a pair of T-bar heels and diamond jewelery
But Kelly had competition in the glamour stakes from her equally gorgeous girlfriends.
Katherine, 33, looked stunning in a nude bandage dress which clung to her enviable figure,teamed with strappy metallic sandals and a slick of bright red lipstick.
Myleene, meanwhile, opted for an eye-catching leopard-print trouser
suit, finished off with a black vest top and black T-bar heels.
Careful! Katherine watched her step as she angled herself into the cab after a night of partying
Dressed to impress: Katherine looked stunning in her nude bandage dress and metallic heels
Friends for life: Katherine posted a picture of herself and Kelly on her Instagram page
girls were no doubt doing their utmost to take Kelly's mind off of
ex-boyfriend Danny, who was recently the centre of claims that he
cheated with Kelly on numerous occasions during their relationship.
The smile on her face suggests that Kelly is wasting no time dwelling on the split from the rugby ace.
Kelly was reportedly furious when she found a string of message to six other women on rugby ace Danny's phone.
Worse for wear: Myleene looked slightly worse for wear as she got into her waiting car at the end of the night
Messing around: Myleene pulled some funny faces for the photographers
Posing up: Katherine later shared a picture of the trio inside Mahiki
seventh, Stacey Simcox, has since told The Sun that she received dozens
of text messages from the Sale Sharks star over an eight-month period,
despite never actually meeting him in person.
Danny – who has previously received treatment for depression – is now said to be undergoing therapy, and pals claim the transgressions that have damaged his relationship with Brook are connected to his past difficulties.
'There are deeper issues here that Danny has to sort out,' a source told the Daily Mail. 'He is going through the process of trying to get help. This is the catalyst he needs and he is prepared to do whatever it takes to resolve his behaviour.
We can see you! Preeya Kalidas and David Gandy were also seen during the night out with Kelly
'Danny is not a nasty person, he simply doesn't understand why he behaved like this, and it needs somebody with experience who can help treat this.'
Ironically the couple looked
blissfully happy during a recent break with pals in the Greek Islands,
from where Kelly posted a shirt Instavid clip for followers on her
Instagram account.
A caption accompanying the video,
which sees the pair relaxing on the deck of a boat and taking a dip in
the ocean, reads: 'last video #GreekIslands #Boats #Livin #Lovin
@kasiawolejnio @dannycipriani87.'
Who's your mate? Kelly left her home earlier in the evening with a male friend
Good spirits: Clearly the actress was hoping to forget her woes for a good night out in London
Staying in shape: Kelly looked fresh-faced on Saturday morning as she headed to the gym and to meet a friend
Trouble and strife: Kelly Brook's relationship with Danny Cipriani is on the rocks
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text: On Thu, Apr 14, 2011 at 12:58 PM, Louie Queral <louiethecuban at gmail.com> wrote: > This is fabulous, great job! > Curious though, what are your plans for the header? I feel like we've had > some Ubuntu Studio logo work done before, and we could possibly resurrect > that unless you have the logo under control. > Again, this is awesome. > -luis Yes. The new logo will be used. I'm sure the website will change a bit once we flesh the art direction. -- -Cory K.


text:
Should Covington be charged? This has been in the works since Ron left. Ron wanted to sell quicker than the board wanted. Covington was brought in to trim down the retail stores, to make the company look more profitable. This POS let the retail stores run down to barely functional operations. They fired every full time employee they could and ran the stores into the toilet. Shorty is rolling in his grave. NF was a formidable retailer until #$%$ like Ron and Alec were brought in downsize for sale.
Do not fool yourselves, this has been in the works for over ten years. The Board of directors announced "We are not for sale" while pounding a for sale sign into the ground at Edina. The BOD's should share a jail cell with Alec. I feel sorry for all NF stock holders, you have been used like a prison sock.
Merger huh? You cannot scrub a turd.
There will be tumbleweeds rolling through Edina, most of the building is already empty, Thanks Alec.
Remember him announcing his retirement??
The retail stores are now a crime riddled shell of a once great, thriving grocery store where the employees used to smile and would be friendly and helpful.
Now?? If you could find an employee for help, they are standing next to another employee talking about where they should apply for new jobs.
Damn shame. I would like to see Alec locked up for being an #$%$.
Sentiment: Strong Sell


text:
Julianna Goldman
Revolving Door
Who's coming and who's going. Tell us about your job move! Receive the Revolving Door Newsletter with expert analysis by email twice a week.
June 16, 2014: Julianna Goldman has been named Washington correspondent at CBS News. She had been White House correspondent at Bloomberg News. (TVNewser)


text:
South Korea and the United States on Monday began their annual two-week joint military exercise, according to the two countries' Combined Forces Command.
The exercise, Ulchi Freedom Guardian, is scheduled to last 12 days.
Yonhap News Agency said the exercise mobilizes about 50,000 South Korean forces and approximately 30,000 U.S. servicemen, including some 3,000 from the mainland and other bases in the Pacific region.
It is intended to examine the combined forces' ability to ensure security on the Korean Peninsula and maintain the joint defense capacities between the two allies.
The training is expected to include defense drills against North Korean provocations and cyber attacks that target the military network as well as contingencies involving weapons of mass destruction.
Meanwhile, President Park Geun Hye convened a National Security Council meeting Monday in conjunction with the start of the joint military exercise.
"Preparing for national contingencies is the most essential part for national security and the safety of the people," Park said during the meeting held at the presidential office, according to Yonhap.
"We cannot be negligent on that even for a moment," she told attendants at the meeting. Top-ranking officials responsible for national security attended Monday's meeting.


text:
1. price per ad, he said "200000 rmb" and it turns out to be $300000rmb.2. the number of ad on pages: He count the no. of co instead of link.If he ever bother to talk to the company, he would not have made a fool of himself.By the way, his skin is so thick that he would not get embarassed easily.
al the ex broker who was arrested before.. yes the same ignorant person i hope the occupy wall st peeps don't make their new home on als lawn.


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Dimple Dell Recreation Center
10670 South 1000 East, Sandy UT 84094 • 385-468-3355
Draper Outdoor Pool
SWIM LESSON REGISTRATION WILL BE ONLINE, JULY 22nd @ 6am
for August Swim Lessons
The Draper Outdoor Pool is a beautiful pool on South Mountain (657 Vestry Road) with a great view of the entire valley. We are open from Memorial Day through Labor Day every summer and offer a variety of programs and classes.
Questions? Call the Draper Pool at (385) 468-1909.
Or, if during off-season call Dimple Dell Fitness and Recreation Center at (385) 468-1902.
657 Vestry Road (14392 S.)
Draper, UT 84020
(385) 468-1909
|Learn more about our Adaptive Recreation and Inclusion Policy


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Crescent Villa
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I am a friend or relative of a resident
My mom stayed at Crescent Villa for about nine months, but it was not too desirable for a number of reasons. First of all, they had one general room which felt like a cafeteria and was not a warm environment. Secondly, we had a problem when another patient went into my mother's room and took some things. The food was OK. They did have some activities, like sing-alongs and card games. My mother was very unhappy there.
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The happiness of your life depends upon the quality of your thoughts . . .
The cure of anything is Salt water – Sweat, tears or the sea ♥
Art is the only way to run away without leaving home ♥
The sun at home warms better than the sun elsewhere . . .
Your lies do not comfort me… your truth will not demolish me . . .
What one loves in childhood stays in the heart forever♥
Travel is not a sprint. A journey is not a marathon. Chill out and enjoy the moment . . .
The Lake house ♥
There´s nothing more beautiful than the way the sea refuses to stop kissing the shoreline, no matter how many times it´s sent away…
When you arise in the morning, think of what a precious privilege it is to be alive – to breathe, to think, to enjoy, to love ♥
Autumn mornings: sunshine and crisp air, birds and calmness, year's end and day's beginnings…
The leaves fall, the wind blows, and the farm country slowly changes from the summer cottons into its winter wools…
Autumn, the year's last, loveliest smile…
If a cold heart feels no pain, then tonight i´ll sleep out in the rain…
Cooking its like love… it should be larger than what you think you need ♥
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‹‹ V.25 No.30 | July 28 - August 3, 2016
News Feature
Third Party Candidates Offer Variety
Presidential campaign filled with outliers
By Carolyn Carlson
Alibi political correspondent Carolyn Carlson takes a deeper look into third party presidential candidates and their various platforms.
Robert Maestas
Modern Day Slavery
Human trafficking still an issue in America
By Robin Babb
Come to New Mexico World Day Against Trafficking in Persons to learn how to stop slavery.
Rob M.
Baked Goods
Genius of Love
SWOP: the difference between sativa and indica
By Joshua Lee
Joshua Lee takes medical cannabis patients on a weekly tour of local dispensaries. This week he visits Southwest Organic Producers.
Odds & Ends
By Devin D. O'Leary
Funny because getting arrested at the gym happens to someone else.
Related Stories
EVENT HORIZON (Saturday, Jul 30)
Finish Him
Art Fight 21: Summer Block Party
By Maggie Grimason
Live music featuring Raashan Ahmad of Crown City Rockers, Burque SOL and Sol One as well as an art market with local vendors, art auctions, an art swap and more.
EVENT HORIZON (Saturday, Jul 30)
MuggleWatch 2016
Harry Potter Countdown to Midnight Magic Party
By Joshua Lee
Enjoy a night of activities and giveaways leading up to the midnight release of Harry Potter and the Cursed Child.
EVENT HORIZON (Saturday, Jul 30)
Viva Frida, Colorful Art Goddess
6th Annual Frida Fiesta
By Monica Schmitt
Celebrate the life of iconic Mexican artist Frida Kahlo with live music, a Frida Parade and a Frida birthday cake.
Today's Events
New Mexico World Day Against Trafficking in Persons at Civic Plaza
Learn about the dangers of human trafficking with Mayor Richard J. Berry, Attorney General Hector Balderas and others. Live music and dance, food trucks, and a performance by Graviel De La Plaga.
Zoob Club at Juan Tabo Public Library
LEAP Into Science: Drip, Drip, Drop at South Valley Public LibraryMore Recommended Events ››
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William "Bill" L. Sullivan, 69, of Linton, died at 6:48 p.m. Thursday, Feb. 5, at Greene County General Hospital.
Born April 24, 1934, in Linton, he was the son of Frank and Bertha (Fenson) Sullivan. He retired from Jack Wheeler Ford after working 32 years as a mechanic, and he owned Sullivan Auto for 20 years. He attended Saron United Church of Christ in Linton.
Survivors include his wife of 46 years, Hildred B. (Carpenter) Sullivan; daughter and son-in-law Peggy and Chris Sharp of Linton; son and daughter-in-law Rick and Rhonda Sullivan of Linton; a brother, Robert P. Sullivan of Linton; four sisters-in-law, Mary Joann Sullivan, Mary Walton and Dorothy Carpenter, all of Linton, and Violet Tincher of Middletown; a brother-in-law, Don Tincher of Middletown; three grandchildren, Chandra Sharp, Ryan and Josh Sullivan, all of Linton; and a great-grandchild.
He was preceded in death by five brothers, Clovis, Ora, Roy, Harold and John Sullivan; three sisters, Hazel Johnson, Betty Jo Sullivan and Bessie Johnson, and his parents.
Services will be 1 p.m. Monday, Feb. 9, at the Welch and Cornett Funeral Home Linton Chapel with Rev. Mike Roth officiating. Burial will be in Fairview Cemetery.
Visitation will be 4-8 p.m. Sunday and after 10 a.m. until the hour of the service on Monday.


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Come join us for some fun, friendly, and non-competitive games of euchre.
You must have a working knowledge of Euchre! Meetups for this group are every week including Sundays. People who like playing more are encouraged to get together and play.
Also, if there is an uneven number of people, some will have to sit out one game.
thinkin u might have to change my name to Flash seems I come and go so quickly lately...heheheh and I was playing soo good too had wonderful cards .. if only I could clone myself ...lol.. now what would the world do with 2 of me !!!!! c u all at the next one I will try and ground myself so I stay longer ....
1 · August 29, 2013
Hello Gay; I am so sorry I missed this meetup however I will attend the next one guaranteed.
0 · August 29, 2013
Gay; I lost my bus pass so I am GOING TO NEED A RIDE . . . Who lives near me?? I am off of N. Goodman by Bauch & Lomb . . . I live at #274 Randolph St.
0 · August 16, 2013
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On September 14, 2011, Harper's Magazine hosted The 9/11 Effect, a panel discussion at the City University of New York. Moderator Amy Goodman of Democracy Now! explored the lasting effects of the war on terrorism with lawyer and Harper's contributing editor Scott Horton, winner of a National Magazine Award for his exposé of inmate abuse at Guantánamo Bay; Fordham Law School's Karen Greenberg, former director of NYU's Center on Law and Security and author of "The Least Worst Place: Guantanamo's First 100 Days"; Petra Bartosiewicz, Harper's contributor, most recently of "To Catch a Terrorist," in the August 2011 issue; and Michael German, ACLU Policy Counsel and former FBI Special Agent.
The event was introduced by John R. MacArthur, publisher of Harper's Magazine.
More from Harper's Magazine:
Official Business — March 17, 2015, 4:01 am
Listen to the broadcast version of "American Hustle," Alexandra Starr's story, for the April 2015 issue of Harper's Magazine, about how elite youth basketball exploits African athletes.
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We defend Charlie Hebdo's right to publish its cartoons—and our right to critique them.
Memento Mori — September 2, 2014, 5:33 pm
In Havana, the past year has been marked by a parade of bold-faced names from the north — John Kerry reopening the United States Embassy; Andrew Cuomo bringing a delegation of American business leaders; celebrities ranging from Joe Torre, traveling on behalf of Major League Baseball to oversee an exhibition game between the Tampa Bay Rays and the Cuban national team, to Jimmy Buffett, said to be considering opening one of his Margaritaville restaurants there. All this culminated with a three-day trip in March by Barack Obama, the first American president to visit Cuba since Calvin Coolidge in 1928. But to those who know the city well, perhaps nothing said as much about the transformation of political relations between the United States and Cuba that began in December 2014 as a concert in the Tribuna Antiimperialista.
Estimated temperature of Hell, according to two Spanish physicists ' interpretation of the Bible:
Department of Applied Physics, University of Santiago (Santiago, Spain)
The ecosystems around Chernobyl, Ukraine, are now healthier than they were before the nuclear disaster, though radiation levels are still too high for human habitation.
A TSA agent in Seattle was arrested for taking up-skirt photos of women in the airport, a Maryland police officer was arrested for taking up-skirt photos of an off-duty colleague, and the Georgia Court of Appeals ruled that taking up-skirt photos is legal in the state.
Subscribe to the Weekly Review newsletter. Don't worry, we won't sell your email address!
"Matt was happy enough to sustain himself on the detritus of a world he saw as careening toward self-destruction, and equally happy to scam a government he despised. 'I'm glad everyone's so wasteful,' he told me. 'It supports my lifestyle.'"


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FreeBSD Is Getting Into The Magazine Business
The FreeBSD Foundation is sponsoring a new FreeBSD Journal publication that's about to begin publishing bi-monthly issues concerning BSD.
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Finding Solace in Solus LinuxThere are so many reasons why you might want to try a new distribution. For all intents and purposes, the desktop has very few jobs when it comes to the "average" user. You need a desktop that: Runs a web browser; and if you're a Google Docs user, you can skip the next item! Runs a productivity suite. (Optional) Can run a casual game or two. Can do these few jobs reliably and securely. That's it! And it doesn't hurt that it looks nice and runs fast. It's not that you're less demanding. You just want something that works. I know. We're all sick of hearing that expression. (Read the rest)


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MSNBC Live with Tamron Hall 08/01/14
MH17 investigators reach crash site despite heavy fighting
More than 60 international experts reached the MH17 site and are trying to recover the bodies of at least 80 people. NBC's Katy Tur joins NewsNation to discuss. ... more Duration: {{video.duration.momentjs}}
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A Window Into The Past; A Lesson For The FutureCheryl Kupfer
Friday, May 25th, 2012
Earlier this month, members of the Toronto Jewish community were given a rare opportunity to be visually transported back in time. The film, filmed in 1922, is called Hungry Hearts, and is based on the short stories of writer Anzia Yezierska, a Jewish woman born in Poland in the 1880s whose family immigrated to New York. Many of her writings are centered on her experiences and those of other immigrants living in the Lower East Side. Like all movies made at that time, it is silent, with dialogue conveyed by cue cards.


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Type of business: Salon
Myanmar, a Southeast Asian nation of more than 100 ethnic groups, consists of many disaster-torn communities and poverty-stricken residents. Without help from non-profit organizations like Miles 4 Myanmar, many would otherwise starve to death.
For over three decades the community of Hilmar has gathered at the end of June to pay homage to the community's agricultural lifeblood - dairy farming.
The California Highway patrol is investigating a collision where a Waterford teenager was killed when a Turlock man ran a red light.
On Tuesday the Turlock Irrigation District Board of Directors took a step forward in constructing a new electrical substation to be built off Washington Road.
Public transportation around Turlock is set to start making major changes starting in January with the City Council's approval of a new short range transit plan.
Downtown business owners and those looking to spend a night out on Turlock's Main Street may think that there is a lack of parking options in the downtown area, but according to TJKM Transportation Consultants, what the city really needs is improved parking management and not another parking lot.
The California Endowment announced they would grant the International Rescue Committee in Los Angeles (IRC) $306,344 to assist with the relocation of Syrian refugees across California.
With the Stanislaus County Fair just around the corner, residents of the 209 are growing more and more anxious for all the fun and entertainment lying ahead.
Although he may be Turlock's most famous quarterback, for the past four years Colin Kaepernick has taken time away from the gridiron one day each summer to swing iron at the Against All Odds Golf Tournament to benefit Camp Taylor.
A Stanislaus County resident who recently traveled outside of the country is the first local to test positive for the Zika virus.
The Merced County Library is hosting a fossil exhibit through the summer courtesy of the State of California and Caltrans.
Summer is the season of sunny days, ice cold refreshments and, unfortunately for many Californians, rising prices at the pump. With a wallet-binding combination of rising oil prices and high demand for gasoline, many Americans will notice the increasing cost when topping off their tanks.
Nicole Sanders of Turlock was crowned the District 6 Dairy Princess during ceremonies held Thursday at Our Lady of Assumption Hall in Turlock. Alexia Nunes of Turlock was named First Alternate and Marion Carpenter of Merced was named Second Alternate.
Fathers of Turlock are taking it upon themselves to be the next generation of doting dads. Although everyone has their own parenting style, most have just one goal: raise their children to be the best that they can be.
Page 4 of 315
Sixty-six years ago, American troops were fighting on Korean soil as they came to the aid of South Korea in the Korean War. Today, those ...
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In the wake of recent attacks on police officers across the nation, the Assyrian American Civic Club took it upon themselves to say "thank you ...
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Recent upgrades to Turlock's transit system were made in an effort to make it easier for passengers to ride the bus, but for one ...
For years, Turlockers have hoped for a bowling alley to roll back into the city. After endless rumors and failed attempts at building an entertainment ...
Page 2 of 2
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Air Conditioning problem
2002 Toyota Prius 4 cyl Two Wheel Drive Automatic 125000 miles
Air Conditioning cooling shuts off randomly. Can happen cat any time at any speed, while stopped or moving. Then it will work again. What might be causing this to happen and how can it be fixed? Thanks
Saturday, July 24th, 2010 AT 8:33 PM


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14 April 2013
10 December 2013 Last updated at 07:56 GMT
Canada has signalled its intention to claim the North Pole and some of the surrounding Arctic waters on its Atlantic coast.
The Canadian government has asked scientists to determine how far the country's continental shelf extends under the Pole.
The results of the survey will be submitted to the UN for consideration.
Emily Thomas reports.


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Chilean wines that are a delicious bargain
I met with Alex Rubio, one of the co-owners of Chile's PKNT, last week to try out their wine.
I have to admit I've never been a huge fan of Chilean vino – too many bad memories of bulk grapes being made into Acme brands by California wineries back in the '80s, I guess – but I was intrigued by the prices: $7 to $10 a bottle for the current vintages.
When I asked Rubio what PKNT stood for, he pointed at a label on one of his winery's bottles. It's crowned by a long chili pepper. If you look closely, you can see a map of Chile superimposed on it.
"The name isn't an acronym. It doesn't mean anything," Rubio said, although he hopes people will pronounce it "picante." "We were looking for something iconic, something that was easy to remember and would remind people of our country."
The gimmick must be working. PKNT has been marketed in the U.S. for only four years, but it's already the top-selling Chilean wine in several Midwestern states and is making inroads in California. It's even bigger in Canada. The company has grown from a small operation to a 200,000-case-per-year behemoth almost overnight.
Still, at these prices, how good could it be? I aimed to find out.
Over lunch at Antonello Ristorante in Santa Ana, Rubio opened several bottles from his silver and gold lines. (The former sells for $6.99 a bottle, the latter $9.99.) I was pleasantly surprised.
The 2009 Silver Chardonnay is blended with 15 percent Viognier, which adds a perfume-y nose and brings out some of the fruit of the Chard. I'd compare it to a cool-climate American Chardonnay – more lemony and orchard fruit than big and tropical, and not at all buttery. It's been aged in oak for ten months, but you barely notice. At this price, though, it was pretty impressive.
The 2008 Gold Cabernet Sauvignon was similarly well styled but a bit lacking in depth and tannins. Still, considering the cost, I was amazed by the quality PKNT can achieve.
The best was saved for last. PKNT makes a 100 percent Carmenère – the "lost" Bordeaux grape of France which survived in Chile because that country's vineyards never succumbed to the dreaded phylloxera that devastated European vineyards in the late 19th century.
That wine was a revelation: similar to Merlot in flavor, but silkier and with a wonderfully spicy note. And PKNT has managed a nice balance of fruit and earthiness. This is a wine you could drink all night, with or without food. For $9.99 a bottle, it's a steal, folks.
Also worth the prize is the 2008 Gold Shiraz, which compares well with good Australian examples of this varietal for a fraction of the price.
You can get PKNT wines locally. The 2006 Gold Carmenère goes for $10.99 a bottle at the San Clemente Wine Company, 212 1/2 Avenida Del Mar, San Clemente. (949) 429-7067 or www.scwinecompany.com. You can also find PKNT at Henry's Farmers Market outlets.
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China Begins G-20 Leadership With Ideas to Reduce Dollar's Role
- South Korea, France to lead working group exploring SDR use
- China exploring ways to make system more resilient to shocks
As China takes the reins of the Group of 20 for the coming year, the first indications are emerging of its agenda.
QuickTake The People's Currency
Among the priorities: making the global system more resilient to shocks and, perhaps, less reliant on the U.S. dollar. China is setting up a working group led by South Korea and France to develop proposals, including on ways to strengthen the role of the International Monetary Fund's reserve-currency unit, which is set to incorporate China's yuan as a component next year.
China also wants a discussion around whether some commodities should be priced in the IMF's reserve currency, known as Special Drawing Right or SDR, according to a European official involved in the G-20 talks.
Notably absent from a senior role so far is the U.S., owner of what's still the world's dominant currency. China's leadership has for years sought to strengthen the international use of the yuan, and encourage debate about lessening reliance on the dollar.
A surge in demand for dollars as a haven during the 2007-2009 global financial crisis first spurred China's calls. As chair of the G-20 in 2010, South Korea attempted to lead an effort to widen the international financial safety net, urging the adoption of permanent currency swap lines. The U.S. nixed the idea, with then-Federal Reserve Chairman Ben S. Bernanke saying officials shouldn't provide a "permanent service" to financial markets.
Chinese Imprint
Half a decade later, Chinese President Xi Jinping has the chance to put his imprint on a forum first set up during the Asian financial crisis of the late 1990s as a grouping of the largest emerging and developed markets to address systemic risks.
"We have seen China grasping every multilateral occasion to enhance its image and leadership role, be it regionally or globally," said Yun Sun, a senior associate with the East Asia Program at the Stimson Center in Washington. "There is little reason for China not to fully exploit the G-20 chairmanship."
China has an ever bigger stake in global financial stability as it endeavors to reduce its own limits on cross-border capital flows, part of a broader plan to give markets a more prominent role in the Communist-run country. Among the challenges for the coming year will be weathering the Fed's shift to monetary tightening, potentially sending emerging market currencies lower as money heads into higher-yielding dollars.
The G-20 under China's presidency will also need to consider whether to let expire $250 billion worth of its reserve unit issued in 2009 to boost liquidity during the global financial crisis.
Arms Length
Giving South Korea and France the job as chairs of the working group offers China some distance from the effort, after past failures to head off the repeated financial crises that afflicted the global economy since the G-20's 1999 foundation. South Korea said last month that the provisional agenda will include action plans to address capital-flow volatility, sovereign-debt restructuring and ways to enhance the IMF's unit of account, or SDR.
President Xi has called on the G-20 to do more to address global economic disruptions and secure balanced growth, according to a Xinhua Finance report.
China itself saw its regulators cast in poor light this year after doing little to counter a stock-market bubble, then mounting a rushed response including trading suspensions, as some $5 trillion worth of capitalization evaporated. A sudden move on Aug. 11 to change the yuan's exchange-rate mechanism also jolted global markets.
"Given the clumsy behavior of the government, what confidence can investors have that would lead them to buy securities denominated in the renminbi?" said Robert Aliber, professor of International Economics and Finance Emeritus at the University of Chicago, using another term for the yuan. "China's role as chair of the G-20 and the inclusion of the renminbi in the SDR are trivial sideshows compared with these market developments."
SDR Inclusion
China's G-20 chairmanship began at the start of the month, a day after the IMF said the yuan met the requirements for joining the dollar, euro, yen and pound as one of the currencies backing the SDR, a sort of overdraft account for IMF members. China central bank Governor Zhou Xiaochuan in 2009 advocated expanding the use of the SDR unit in calling for a "super-sovereign reserve currency."
Nothing came of Zhou's call six years ago, and changing the global financial architecture now remains difficult, analysts say.
"We will need another global crisis, and one whose roots can be clearly identified in the shortcomings of the current non-system, for this to happen," said William White, an adviser to the Organization for Economic Cooperation and Development. The G-20's agenda can also become dominated by pressing issues of the moment.
"I suspect that geopolitical issues will trump economic ones," White said.
Global Role
Whatever else, China's leadership of the group offers Xi a vehicle to promote his country's rising global role. The last time China hosted the G-20, in 2005, it was the world's fifth-largest economy. It's now No. 2, having surpassed the U.K., Germany and Japan. Its Asian rival will be chairing the Group of Seven developed nations next year.
G-20 members are Argentina, Australia, Brazil, Canada, China, France, Germany, India, Indonesia, Italy, Japan, South Korea, Mexico, Russia, Saudi Arabia, South Africa, Turkey, the U.S., the U.K. and the EU.
"China can and should look to be ambitious, and aim for actions that demonstrate global leadership," said Tristram Sainsbury, a research fellow at the G-20 Studies Center at the Lowy Institute for International Policy in Sydney. "But China needs to be realistic of the limitations of the G-20 forum -- it is consensus based, and has several design flaws, such as a rotating presidency and a lack of focus stemming from an inability to drop items off the agenda."