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The spatial arrangement of chromatin within the nucleus can affect reactions that occur on the DNA and is likely to be regulated. Here we show that activation of INO1 occurs at the nuclear membrane and requires the integral membrane protein Scs2. Scs2 antagonizes the action of the transcriptional repressor Opi1 under conditions that induce the unfolded protein response (UPR) and, in turn, activate INO1. Whereas repressed INO1 localizes throughout the nucleoplasm, the gene is recruited to the nuclear periphery upon transcriptional activation. Recruitment requires the transcriptional activator Hac1, which is produced upon induction of the UPR, and is constitutive in a strain lacking Opi1. Artificial recruitment of INO1 to the nuclear membrane permits activation in the absence of Scs2, indicating that the intranuclear localization of a gene can profoundly influence its mechanism of activation. Gene recruitment to the nuclear periphery, therefore, is a dynamic process and appears to play an important regulatory role.
For over a hundred years, it has been recognized that chromatin is distributed non-randomly within the interphase nucleus (Rabl 1885; Boveri 1909). More recently, three-dimensional fluorescence microscopy studies have established that chromosomes are organized into distinct, evolutionarily conserved subnuclear territories (reviewed by Cockell and Gasser [1999]; Isogai and Tjian [2003]). However, DNA is mobile and can move between these domains (reviewed in Gasser [2002]). Recent studies suggest that the subnuclear localization of genes can have dramatic effects on their chromatin state, rate of recombination, and transcription (Cockell and Gasser 1999; Isogai and Tjian 2003; Bressan et al. 2004). Heterochromatin, for example, is generally found concentrated in close proximity to the nuclear envelope. Several genes conditionally colocalize with heterochromatin under conditions in which they are repressed. The transcriptional regulator Ikaros, for example, interacts both with regulatory sequences upstream of target genes and with repeats enriched at centromeric heterochromatin. When repressed, these genes become colocalized with heterochromatin, suggesting that Ikaros promotes repression by directly recruiting target genes into close proximity with heterochromatin (Brown et al. 1997, 1999; Cobb et al. 2000). Consistent with this view, euchromatic sequences that become colocalized with heterochromatin are transcriptionally silenced (Csink and Henikoff 1996; Dernburg et al. 1996).
In Saccharomyces cerevisiae, genes localized in proximity to telomeres are similarly transcriptionally silenced (Gottschling et al. 1990). Silencing is due to Rap1-dependent recruitment of Sir proteins to telomeres (Gotta et al. 1996), which promotes local histone deacetylation and changes in chromatin structure (reviewed in Rusche et al. [2003]). Physical tethering of telomeres at the nuclear periphery through interactions with the nuclear pore is required for silencing (Gotta et al. 1996; Laroche et al. 1998; Galy et al. 2000; Feuerbach et al. 2002). When a reporter gene flanked by silencer motifs was relocated more than 200 kb away from a telomere, silencing was lost (Maillet et al. 1996). Silencing was restored to this gene by overexpression of SIR genes. Therefore it is thought that tethering serves to promote efficient recruitment of Sir proteins, which are enriched at the nuclear periphery and limiting elsewhere (Maillet et al. 1996). Another example of gene silencing at the nuclear periphery comes from experiments in which defects in the silencer of the HMR locus could be suppressed by artificially tethering this locus to the nuclear membrane (Andrulis et al. 1998). Thus, localization of chromatin to the nuclear periphery has been proposed to play a major role in transcriptional repression.
By contrast, we report here that dynamic recruitment of genes to the nuclear membrane can have profound effects on their activation. The gene under study here is INO1, a target gene of the unfolded protein response (UPR), which encodes inositol 1-phosphate synthase. The UPR is an intracellular signaling pathway that is activated by the accumulation of unfolded proteins in the endoplasmic reticulum (ER), which can be stimulated by treatment with drugs that block protein folding or modification or, in yeast, by starvation for inositol (Cox et al. 1997). These conditions activate Ire1, a transmembrane ER kinase/endoribonuclease (Cox et al. 1993; Mori et al. 1993), which, through its endonuclease activity, initiates nonconventional splicing of the mRNA encoding the transcription activator Hac1 (Cox and Walter 1996; Shamu and Walter 1996; Kawahara et al. 1997; Sidrauski and Walter 1997). Only spliced HAC1 mRNA is translated to produce the transcription factor; the Ire1-mediated splicing reaction, therefore, constitutes the key switch step in the UPR (Sidrauski et al. 1996; Ruegsegger et al. 2001).
Hac1 is a basic-leucine zipper transcription factor that binds directly to unfolded protein response elements (UPREs) in the promoters of most target genes to promote transcriptional activation (Cox and Walter 1996; Travers et al. 2000; Patil et al. 2004). However, a subset of UPR target genes uses a different mode of activation. Transcriptional activation of these genes, including INO1, depends on Hac1 and Ire1. These target genes contain an upstream activating sequence that is regulated by the availability of inositol, the UASINO element, in their promoters that is repressed by Opi1 under non-UPR conditions (Greenberg et al. 1982; Cox et al. 1997). Opi1 repression is relieved in a Hac1-dependent manner upon induction of the UPR (Cox et al. 1997). Positively acting transcription factors Ino2 and Ino4 then promote transcription from UASINO-containing promoters (Loewy and Henry 1984; Ambroziak and Henry 1994; Schwank et al. 1995). Our previous work established that the production of Hac1 by UPR induction functions upstream of Opi1, suggesting that the role of the UPR is to counteract Opi1-mediated repression (Cox et al. 1997).
To understand the regulation of UASINO-controlled genes by the UPR, we have examined the molecular events leading to the activation of INO1. We find that Scs2, an integral protein of the nuclear and ER membrane that was recently shown to play a role in telomeric silencing (Craven and Petes 2001; Cuperus and Shore 2002), is required to activate INO1. We observe dynamic INO1 recruitment to the nuclear membrane under activating conditions. Importantly, we find that recruitment requires Hac1 and is opposed by Opi1. Furthermore, we show that artificial recruitment of INO1 to the nuclear membrane can bypass the requirement for Scs2. Gene recruitment to the nuclear membrane therefore plays an instrumental role in INO1 activation.
To characterize the molecular basis of transcriptional activation of INO1, we first asked whether the steady-state levels of the known transcriptional regulators—the activators Ino2 and Ino4 and the repressor Opi1—were affected by induction of the UPR. To this end, we monitored the levels of myc-tagged proteins by Western blotting after UPR induction by inositol starvation (Figure 1A). Induction of the UPR did not result in a significant change of the abundance of any of the proteins. Thus, in contrast to what has been suggested in previous studies (Ashburner and Lopes 1995a, 1995b; Cox et al. 1997; Schwank et al. 1997; Wagner et al. 1999), INO1 transcription is not regulated through adjustment of the abundance of these regulators.
Next, we tested whether the subcellular localization of these regulators is modulated. We examined the localization of myc-tagged Opi1, Ino2, and Ino4 by indirect immunofluorescence (Figure 1A). Again, we observed no significant change upon UPR induction: Ino2 and Ino4 localized to the nucleus under both repressing and activating conditions. Localization of Opi1 also showed no change. Like Ino2 and Ino4, Opi1 localized to the nucleus under both conditions. However, in agreement with recent data by Loewen et al. (2003), we found that Opi1 was concentrated at the nuclear membrane and diffusely distributed throughout the nucleoplasm (Figure 1A). Furthermore, coimmunoprecipitation experiments showed that Ino2 and Ino4 heterodimerize under both conditions, suggesting that this interaction is not regulated (Figure 1B). Taken together, these observations therefore pose an interesting puzzle: How is regulation achieved when the localization and abundance of all three regulators is unchanged between activating and repressing conditions?
To begin to explore a possible functional significance of Opi1's unusual localization pattern at the nuclear membrane, we sought to identify binding partners that might tether Opi1 to the membrane. To this end, we immunoprecipitated myc-tagged Opi1 under nondenaturing conditions from mildly detergent-solubilized microsomal membranes. Bands that were enriched in the immunoprecipitated fraction from the myc-tagged strain were identified by matrix-assisted laser desorption ionization mass spectrometry (Figure 1C). This procedure identified Scs2, a bona fide integral membrane protein known to reside in nuclear membranes and ER (Nikawa et al. 1995; Kagiwada et al. 1998; Kagiwada and Zen 2003). To confirm that Scs2 and Opi1 interact, we performed coimmunoprecipitation analysis from extracts of strains expressing myc-tagged Opi1 and hemagglutinin (HA)-tagged Scs2. We observed specific recovery of Scs2-HA in Opi1-myc immunoprecipitates (Figure 1D). Recent results from a genome-wide immunoprecipitation study (Gavin et al. 2002) and in vitro peptide binding studies (Loewen et al. 2003) corroborate the interaction between Opi1 and Scs2.
In contrast to Opi1, the transcriptional repressor Scs2 has been implicated in the activation of INO1 transcription: Overexpression of SCS2 suppresses the Ino– growth phenotype in cells that cannot activate the UPR (Nikawa et al. 1995), and loss of Scs2 impairs activation of INO1 (Kagiwada et al. 1998; Kagiwada and Zen 2003). Therefore, either Scs2 is the downstream target of Opi1-mediated repression, or Scs2 functions upstream to relieve Opi1-mediated repression. To distinguish between these possibilities, we analyzed the growth of the double mutant in the absence of inositol. As shown in Figure 1E, opi1Δ cells grew in absence of inositol because INO1 is constitutively expressed. In contrast, scs2Δ cells did not grow under these conditions. Double mutant opi1Δ scs2Δ cells grew in the absence of inositol, indicating that Scs2 functions to regulate Opi1 and is dispensable in the absence of Opi1. Given that Scs2 is an integral membrane protein, these data suggest that regulation of Opi1 occurs at the nuclear membrane.
Ino2 and Ino4 have been shown by gel-shift analysis of yeast extracts to bind directly to the UASINO in the INO1 promoter (Lopes and Henry 1991; Ambroziak and Henry 1994; Bachhawat et al. 1995; Schwank et al. 1995). Binding was observed in extracts from cells grown under repressing or activating conditions, and was increased in the absence of Opi1 (Wagner et al. 1999). To monitor the interaction of Ino2 and Ino4 with the INO1 promoter in vivo, we used chromatin immunoprecipitation (ChIP) (Solomon et al. 1988; Dedon et al. 1991). Consistent with the gel-shift experiments, we found that Ino2-HA and Ino4-HA bound to the INO1 promoter under both repressing and activating conditions (Figure 2A). Real-time quantitative PCR analysis of immunoprecipitated DNA confirmed that both Ino2 and Ino4 associated with the INO1 promoter constitutively (Figure 2B). Although we observed an increase in the association of Ino2 with the INO1 promoter under inducing conditions compared with repressing conditions, these results argue that occupancy of the promoter by Ino2/Ino4 is not sufficient for activation but that it must be a subsequent step in the activation process that is regulated by the UPR.
The molecular mechanism by which Opi1 represses transcription is not understood. In particular, it is not clear whether Opi1 binds to the INO1 promoter directly. Early gel-shift experiments using yeast lysates suggested that Opi1 might interact with DNA (Lopes and Henry 1991). However, this association has not been confirmed, and its significance is unknown. We used ChIP analysis and real-time quantitative PCR to assess the interaction of Opi1 with the INO1 promoter in vivo. We observed specific enrichment of the INO1 promoter by immunoprecipitation of Opi1 from cells grown in the presence of inositol (repressing condition) but no significant enrichment of the INO1 promoter by immunoprecipitation of Opi1 from cells starved for inositol (activating condition; Figure 3). By contrast, when we performed the immunoprecipitations from either hac1Δ or scs2Δ strains, we observed greater enrichment of the INO1 promoter sequences from cells grown under both activating and repressing conditions. These results are consistent with the notion that Opi1 binds to chromatin at the INO1 promoter and that the function of Hac1 and Scs2 is to promote Opi1 dissociation.
In contrast to immunoprecipitation of Ino2 and Ino4, which specifically recovered the INO1 promoter and not the control URA3 sequences (see Figure 2), immunoprecipitates of Opi1 recovered significant amounts of URA3 sequences as well (Figure 3A, upper bands). It is clear from the quantitative PCR analysis that Opi1 binding to the INO1 promoter is specific (Figure 3B). The different conditions used in the qualitative gel analysis (measuring PCR products after many cycles) and the quantitative PCR (measuring PCR products in the linear range of amplification) are likely to account for this difference.
Since Opi1 dissociation from the INO1 promoter correlates with activation and requires Hac1 and Scs2, an integral nuclear membrane protein, we wondered whether activation might occur at the nuclear periphery and thus might be dependent on the subnuclear positioning of the gene. Consistent with this hypothesis, we found that a form of Scs2 (Scs2ΔTMD) lacking the transmembrane domain, which was localized throughout the cell and was not excluded from the nucleus (Figure 4A, compare cytosolic protein Rps2 to Scs2ΔTMD for colocalization with 4′,6′-diamidino-2-phenylindole), and was nonfunctional, rendering cells inositol auxotrophs, despite being expressed at levels comparable to full length Scs2 (Figure 4B and and44C).
If INO1 were regulated at the nuclear periphery, then the INO1 locus should colocalize with the nuclear membrane under activating conditions. To test this idea, we constructed a strain in which an array of Lac operator (Lac O in Figure 5) binding sites was integrated adjacent to the INO1 locus (Robinett et al. 1996). The strain also expressed a green fluorescent protein (GFP)-Lac repressor fusion protein (GFP-Lac I in Figures 5 and and6)6) that binds to the Lac operator array to allow localization of the INO1 gene. In a control strain, we integrated the same Lac operator array adjacent to the URA3 locus. Cells were fixed and GFP was visualized by indirect immunofluorescence. Most cells showed a single intranuclear spot localizing the tagged gene; the remaining cells showed two spots due to their post-replication state in the cell cycle. In both the tagged INO1 and the tagged URA3 strains, we simultaneously visualized the ER and nuclear membrane by indirect immunofluorescence against Sec63-myc using a different fluorophore (Figure 5A).
To ask whether INO1 associates with the nuclear membrane, we developed stringent criteria for scoring INO1 localization (Figure 5A). Using confocal microscopy, we collected a single z slice through each cell that captured the brightest, most focused point of the GFP-visualized Lac operator array. Images in which this slice traversed the nucleus (i.e., cells that showed a clear nuclear membrane ring staining with a "hole" of nucleoplasm), were binned into two groups: Cells in which the peak of the spot corresponding to the tagged gene coincided with nuclear membrane staining were scored as membrane-associated, and cells in which the peak of the spot corresponding to the tagged gene was offset from nuclear membrane staining were scored as nucleoplasmic. This procedure allowed us to determine the fraction of cells in a given population in which the tagged gene colocalized with the membrane, thus providing a quantitative measure for membrane association. Five examples of each group, with fluorescence intensity plotted along a line bisecting the nucleus and the spot, are shown in Figure 5A.
To confirm that our scoring criterion would identify nuclear membrane association in a meaningful way, we applied it to two controls. As a control for membrane association, we localized INO1 in a strain expressing GFP-Lac repressor fused to a peptide motif from Opi1 containing two phenylalanines in an acidic tract (FFAT motif), which serves as a nuclear membrane–targeting signal (Loewen et al. 2003). This motif was shown to bind to Scs2 and to be required for Opi1 targeting to the nuclear envelope (Loewen et al. 2003). Importantly, targeting of Opi1 to the nuclear membrane still occurred in the absence of Scs2 in an FFAT-dependent manner (Loewen et al. 2003), indicating that, in addition to Scs2, there must exist another, yet-unidentified receptor for FFAT in the nuclear membrane. As shown in Figure 5B, the localization of INO1 scored as 85% membrane-associated (Figure 5B, bar 1), confirming both our scoring criteria and the previous result that FFAT indeed promotes nuclear membrane targeting.
As a control for random distribution, we localized URA3 in a strain expressing GFP-Lac repressor without the FFAT targeting signal. URA3 scored as 23% membrane-associated (Figure 5B, bar 3). Induction of the UPR after depletion of inositol had no effect on the localization of either FFAT-tagged INO1 or URA3 in these strains (Figure 5B, bars 2 and 4). Given that 25% of the volume of the nucleus is contained in the outer shell represented by only 10% of the radius, this level of background is consistent with a random distribution of the URA3 gene throughout the nuclear volume. Based on the spatial resolution of our data (Figure 5A), a spot only 10% of the radius distant from the membrane signal would have been scored as membrane-associated. We therefore defined the mean frequency of membrane-association of the URA3 control between these two conditions (25% ± 3%) as the baseline for subsequent comparisons (Figure 5B, dashed line).
We next compared the membrane association of INO1 under repressing and activating conditions. Under repressing conditions, the membrane association of INO1 was only slightly greater than the baseline (32% ± 3%; Figure 5B, bar 5). In striking contrast, when INO1 was activated, the frequency of membrane association of INO1 increased significantly over baseline (52% ± 3%; Figure 5B, bar 6). Thus, we conclude that, in a significant portion of cells, the INO1 gene became associated with the nuclear membrane under UPR-inducing conditions.
To confirm that the observed recruitment was indeed due to UPR induction, we compared the membrane association of INO1 under repressing or activating conditions in the hac1Δ mutant. Because Hac1 is required for activation of INO1, we predicted that membrane association would be prevented in this mutant. Indeed, INO1 failed to become membrane associated in hac1Δ mutants starved for inositol (Figure 5B, bars 7 and 8). Our earlier experiments suggested that Hac1 functions to promote dissociation of Opi1 from the INO1 promoter. We therefore tested next whether the presence of Opi1 prevents membrane association. To this end, we determined INO1 localization in the opi1Δ strain, in which INO1 is constitutively transcribed (Cox et al. 1997). Indeed, we observed a high degree of membrane association, both in the presence and absence of inositol (68% ± 5%; Figure 5B, bars 9 and 10).
The experiments described above indicate that there is a correlation between membrane association of INO1 and its transcriptional activation. To establish causality, we examined the effect of artificially targeting INO1 to the nuclear membrane. In an otherwise wild-type background, artificial targeting of INO1 to the nuclear membrane via FFAT-Lac repressor binding (same strain as in Figure 5B, bars 1 and 2) had no effect on INO1 expression as assessed by Northern blot analysis (Figure 6A) or on the growth of the wild-type strain in the absence of inositol (Figure 6B; compare top two panels). This result suggests that membrane targeting per se is not sufficient to cause activation. In contrast, in the scs2Δ mutant we observed that the inositol-requiring growth phenotype of the strain was suppressed by expression of the membrane-targeted FFAT-Lac repressor (Figure 6B; compare bottom two panels). This effect was strictly dependent on having the Lac operator array integrated at the INO1 locus; expressing GFP-FFAT-Lac repressor in the absence of the array (Figure 6C)—or if the array was integrated at the URA3 locus (unpublished data)—did not improve the growth of the scs2Δ mutant in the absence of inositol. Consistent with the previous report that FFAT does not require Scs2 to promote nuclear membrane targeting, we observed approximately 50% membrane association of INO1 in the strain expressing the FFAT-Lac repressor (78 cells counted, unpublished data). Thus, the defect in transcription of INO1 in the scs2Δ mutant could be rescued, at least partially, through artificial targeting of INO1 to the nuclear membrane. This result demonstrates that nuclear membrane association is functionally important for achieving INO1 transcriptional activation.
It is becoming increasingly clear that the spatial arrangement of chromosomes within the nucleus is important for controlling the reactions that occur on DNA and might be regulated (reviewed in Cockell and Gasser [1999]; Isogai and Tjian [2003]). Here we have shown that activation of INO1 occurs at the nuclear membrane and requires the integral membrane protein Scs2. Moreover, artificial recruitment of INO1 to the nuclear membrane permits activation in the absence of Scs2, indicating that the precise intranuclear localization of a gene can profoundly influence its activation. Most importantly, we have shown that the localization of INO1 depends on its activation state; gene recruitment therefore is a dynamic process and appears to play an important regulatory role.
The nucleoplasm is bounded by the inner nuclear membrane, which provides a template that is likely to play a major role in organizing the genome. It is clear from numerous microscopic and biochemical studies that chromatin interacts with nuclear membrane proteins, associated proteins such as filamentous lamins, and nuclear pore complexes (DuPraw, 1965; Murray and Davies, 1979; Paddy, 1990; Worman et al., 1990; Belmont et al., 1993; Glass et al., 1993; Foisner and Gerace, 1993; Sukegawa and Blobel, 1993; Luderus et al., 1994; Marshall et al., 1996). Indeed, several transcriptionally regulated genes have been shown to colocalize with heterochromatin at the nuclear periphery when repressed (Csink and Henikoff 1996; Dernburg et al. 1996; Brown et al. 1997, 1999). Likewise, silencing of genes near telomeres requires physical tethering of telomeres to nuclear pore complexes at the nuclear periphery (Gotta et al. 1996; Maillet et al. 1996; Andrulis et al. 1998; Laroche et al. 1998; Galy et al. 2000; Andrulis et al. 2002; Feuerbach et al. 2002).
Thus, the nuclear periphery has been generally regarded as a transcriptionally repressive environment (Gotta et al. 1996; Maillet et al. 1996; Andrulis et al. 1998; Laroche et al. 1998; Galy et al. 2000; Andrulis et al. 2002; Feuerbach et al. 2002). In contrast, the work presented here shows that gene recruitment to the nuclear periphery can be important for transcriptional activation. This conclusion is supported by a recent study published while this manuscript was in preparation (Casolari et al. 2004). These authors found that a subset of actively transcribed genes associates with components of nuclear pore complexes and that activation of GAL genes correlates with their recruitment from the nucleoplasm to the nuclear periphery and pore-complex protein association (Casolari et al. 2004). The results presented here argue that recruitment of genes to the nuclear periphery is controlled by transcriptional regulators and is important for achieving transcriptional activation. Thus, together, the work by Casolari et al. (2004) and the work presented here demonstrate that gene recruitment to the nuclear periphery can have a general role in activating transcription.
This notion is consistent with the "gene gating hypothesis" put forward by Blobel (1985). As proposed in this hypothesis, transcription of certain genes may be obligatorily coupled to mRNA export through a particular nuclear pore complex. It remains to be shown for INO1, however, whether gene recruitment to the nuclear periphery involves interaction with nuclear pore complex components. Several other scenarios could explain why INO1 activation might require gene recruitment to the nuclear periphery. First, INO1 transcriptional activation requires the SAGA histone acetylase, and both the SWI/SNF and INO80 chromatin remodeling complexes (Kodaki et al. 1995; Pollard and Peterson 1997; Ebbert et al. 1999; Shen et al. 2000; Dietz et al. 2003). Conversely, repression requires the Sin3/Rpd3 histone deacetylase and the ISW chromatin remodeling complex (Hudak et al. 1994; Sugiyama and Nikawa 2001). Thus, if these factors have distinct subnuclear distributions, then the localization of genes regulated by them might influence their transcriptional state. Consistent with this notion, the SAGA complex interacts with nuclear pore complexes, and therefore might be concentrated at the nuclear periphery, where INO1 activation occurs (Rodriguez-Navarro et al. 2004). Second, because INO1 and many other UASINO-regulated genes are involved in the biosynthesis of phospholipids, it is possible that the state of the membrane itself plays a role, perhaps sensed by Scs2, in activating transcription. It has been shown that defects in phospholipids biosynthesis can disrupt regulation of INO1, although the mechanism of this regulation remains unknown (Greenberg et al. 1982; McGraw and Henry 1989; Griac et al. 1996; Griac 1997; Shirra et al. 2001). Third, inositol polyphosphates have been shown to regulate SWI/SNF-catalyzed chromatin remodeling, and it is possible that their production is spatially restricted (Shen et al. 2003; Steger et al. 2003).
Our current understanding of INO1 activation is summarized in a model in Figure 7. The positive transcription activators Ino2 and Ino4 constitutively associate with the INO1 promoter, which is kept transcriptionally repressed by Opi1. We do not currently understand the mechanism by which Opi1 prevents activation. Activation of the UPR leads to the production of Hac1, which, by an unknown mechanism, promotes Opi1 dissociation from chromatin. We propose that Scs2 at the nuclear membrane binds to Opi1 released from the DNA and thus keeps Opi1 sequestered and prevented from rebinding. Indeed, overproduction of Scs2 bypasses the requirement for Hac1 in activation of INO1 transcription and allows hac1Δ cells to grow in the absence of inositol (Nikawa et al. 1995), supporting the role of Scs2 as a sink for Opi1 and suggesting that Opi1 may cycle between chromatin-bound and free states.
Both Hac1 (Cox et al. 1997) and Scs2 (see Figure 1) are dispensable for INO1 activation in the absence of Opi1, suggesting that their role is to relieve Opi1 repression. However, our data suggest that Hac1 and Scs2 have distinct functions: While the absence of either protein prevents the dissociation of Opi1 from chromatin and the activation of INO1, we propose that the role of Hac1 is to promote dissociation and that of Scs2 is to prevent reassociation. This model explains why artificially tethering INO1 to the nuclear membrane suppresses the absence of Scs2 but not the absence of Hac1 (unpublished data): We propose that the environment of membrane-tethered INO1 promotes late steps in the transcription activation—such as chromatin remodeling, discussed above—permitting INO1 to be expressed upon transient Hac1-induced Opi1 dissociation. Therefore, we envision that dissociation of Opi1 from the INO1 promoter is coupled to the delivery of the gene to an environment near the nuclear membrane that is permissive for its activation.
The mechanistic role of Scs2 is currently not known. Its recently discovered function in promoting telomeric silencing (Craven and Petes 2001; Cuperus and Shore 2002) suggests that Scs2 may play a more global role in the regulation of transcription at the nuclear membrane. Scs2 contains a major sperm protein domain, named after a homologous protein in Ascaris suum sperm that forms a cytoskeletal structure and confers motility to sperm cells. It is thus tempting to speculate that Scs2 might similarly self-associate in the plane of the nuclear membrane, perhaps providing a two-dimensional matrix on which membrane-associated reactions could be organized. One suggestion from our data is that Scs2 may function as a local sink for Opi1. But it is also clear that other nuclear membrane components are likely to participate in the reaction. Opi1, for example, still localizes to nuclear membranes even in scs2Δ cells, indicating that another, yet-unidentified Opi1 binding partner must exist (Loewen et al. 2003; unpublished data). Similarly, artificial INO1 recruitment to the membrane via the FFAT motif suppresses the scs2Δ phenotype (see Figure 6)—i.e., it is sufficient to position INO1 in an environment permissive for its induction—yet the FFAT binding protein and the molecular nature of the permissive environment remain unknown.
Upon inducing the UPR, only 52% of the cells scored INO1 as membrane-associated (see Figure 5). Thus, under activating conditions, two types of cells are present in the population at any one time: those in which the INO1 gene is recruited to the membrane, and those in which the INO1 gene is dispersed throughout the nucleoplasm. This score correlated with the level of INO1 transcription; INO1 was membrane-associated in 68% of the cells in the opi1 mutant, which exhibits a correspondingly higher degree of activation than that observed in the wild-type strain. A quantitatively similar nuclear peripheral-nucleoplasmic distribution was observed upon activation of GAL genes (Casolari et al. 2004), suggesting that it may be a general feature of gene recruitment. There are at least two possible interpretations for the observed bimodal distributions. First, the distribution profiles might represent heterogeneity in the activation of INO1 among cells. In this case, activation of INO1 would be variable in individual cells exposed to identical conditions. Gene recruitment thus would stably trap INO1 in a permissive environment for activation, and the localization of INO1 would strictly correlate with its activation state. Alternatively, gene recruitment might alter the balance between two rapidly exchanging states; that is, stable membrane recruitment would not be required for activation. In this case, the observed distributions would represent snapshots of transient colocalization of INO1 with the nuclear membrane within a population of cells that are uniformly activating transcription. Dynamic measurements of gene recruitment and single cell activity assays will need to be developed to distinguish between these possibilities. But no matter which of these possibilities proves to be correct, gene recruitment emerges as a new mechanism regulating eukaryotic gene expression and may be crucial to the regulation of many genes.
Monoclonal anti- HA antibody HA11 was obtained from Babco (Berkeley, California, United States). Monoclonal anti-myc, anti-myc agarose and anti-HA agarose were from Santa Cruz Biotechnology (Santa Cruz, California, United States). Monoclonal anti-Pgk1, rabbit polyclonal anti-GFP, goat anti mouse IgG-Alexafluor 594, and goat anti-rabbit IgG Alexafluor 488 were from Molecular Probes (Eugene, Oregon, United States).
All restriction endonucleases and DNA modification enzymes were from New England Biolabs (Beverly, Massachusetts, United States). Unless indicated otherwise, all other chemicals and reagents were from Sigma (St. Louis, Missouri, United States).
All yeast strains used in this study were derived from wild-type strain CRY1 (ade2–1 can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–1 MATa). Tags and disruptions marked with either the kanr gene from E. coli or the His5 gene from S. pombe were introduced by recombination at the genomic loci as described (Longtine et al. 1998). Strains used in this study, with relevant differences indicated are JBY345 (OPI1–13myc::kanr), JBY350-r1 (scs2Δ:: kanr), JBY359 (SCS2-HA:: kanr), JBY356–1A (opi1Δ::LEU2), JBY356–1B (opi1Δ::LEU2 scs2Δ:: kanr ), JBY356–1C (scs2Δ:: kanr), JBY356–1D (wild-type control), JBY361 (scs2ΔTMD-HA:: kanr), JBY370(INO2-HA3::His5+), JBY371 (INO4-HA3::His5+), JBY393 (INO4-myc::His5+ MATa), JBY397 (SEC63–13myc:: kanr INO1:LacO128:URA3 HIS3:LacI-GFP), JBY399 (SEC63–13myc::Kar INO1:LacO128:URA3 HIS3:LacI-FFAT-GFP), JBY401 (ino4Δ::LEU2 SEC63–13myc::Kanr INO1:LacO128:URA3 HIS3:LacI-GFP MATα), JBY404 (opi1Δ::LEU2 SEC63–13myc::Kanr INO1:LacO128:URA3 HIS3:LacI-GFP), JBY406 (opi1Δ::LEU2 SEC63–13myc::Kanr INO1:LacO128:URA3 HIS3:LacI-FFAT-GFP), JBY409 (SEC63–13myc::Kanr URA3:LacO128:URA3 HIS3:LacI-GFP), JBY412 (INO2-myc::His5+), JBY 416 (hac1Δ::URA3 SEC63–13myc::Kanr LacO128:INO1 HIS3:LacI-GFP).
Plasmid pRS315-Opi1-myc was created by first amplifying the OPI1-myc coding sequence and 686 bp upstream from the translational start site from strain JBY345 using the following primers: OPI1 promoter Up (5′-GGGAGATACAAACCATGAAG-3′) and OPI1 down (5′-ACTATACCTGAGAAAGCAACCTGACCTACAGG-3′). The resulting fragment was cloned into pCR2.1 using the Invitrogen (Carlsbad, California, United States) TOPO TA cloning kit. The OPI1-myc locus was then cloned into pRS315 as a HindIII-NotI fragment. Plasmid pASF144 expressing GFP-lacI has been described (Straight et al. 1996). Plasmid pGFP-FFAT-LacI was constructed by digesting pASF144 with EcoRI and ligating the fragment to the following hybridized oligonucleotides, encoding the FFAT motif from OPI1: LacI_FFAT1 (5′-AATTGGACGATGAGGAGTTTTTTGATGCCTCAGAGG-3′) and LacI_FFAT2 (5′-AATTCCTCTGAGGCATCAAAAAACTCCTCATCGTCC-3′). The orientation of the insert was confirmed by DNA sequencing. Both pAFS144 and pGFP-FFAT-LacI were digested with NheI, which cuts within the HIS3 gene, and transformed into yeast.
Plasmid p6INO1LacO128 was constructed as follows. The INO1 coding sequence, with 437 bp upstream and 758 bp downstream, was amplified from yeast genomic DNA using the following primers: INO1_promoter_Up (5′-GATGAGGCCGGTGCC-3′) and INO1_3′down (5′-AAGATTTCCTTCTTGGGCGC-3′), and cloned into pCR2.1 using the Invitrogen TOPO TA cloning kit, to produce pCR2.1-INO1. INO1 was moved from pCR2.1 into pRS306 as a KpnI fragment, to produce pRS306-INO1. The Lac operator array was then cloned from pAFS52 into pRS306-INO1 as a HindIII-XhoI fragment, to produce plasmid 10.2. Because the Lac operator fragment was smaller than had been reported (2.5 kb instead of 10 kb), presumably reflecting loss of Lac operator repeats by recombination, the Lac operator array was duplicated by digesting plasmid 10.2 with HindIII and SalI and introducing a second copy of the 2.5-kb HindIII-XhoI fragment, as described (Robinett et al. 1996). The resulting plasmid, p6INO1LacO128, has a 5-kb Lac operator array, corresponding to approximately 128 repeats of the lac operator. To integrate this plasmid at INO1, p6INO1LacO128 was digested with BglII, which cuts within the INO1 gene, and transformed into yeast.
The INO1 gene was removed from this plasmid to generate p6LacO128. This plasmid was used to integrate the Lac operator array at URA3 by digestion with StuI and transformation into yeast.
Cells were lysed using glass beads in IP buffer (50 mM Hepes-KOH pH 6.8, 150 mM potassium acetate, 2 mM magnesium acetate, and Complete Protease Inhibitors [Roche, Indianapolis, Indiana, United States]). The whole cell extract was used for coimmunoprecipitation of Ino2-myc and Ino4-HA. For immunoprecipitation of Opi1-myc, microsomes were pelleted by centrifugation for 10 min at 21,000 × g and resuspended in IP buffer. Triton X-100 was then added to either whole cell extract (Ino2-myc; final concentration of 1%) or the microsomal fraction (Opi1-myc; final concentration of 3%) and incubated for 30 min at 4 °C; detergent-insoluble material was then removed by centrifugation at 21,000 × g, 10 min. Anti-myc agarose was added to the supernatant and incubated 4 h at 4 °C, while rotating. For the experiment in Figure 1D, a fraction of the total was collected after antibody incubation. After agarose beads were pelleted, an equal fraction of the supernatant was collected. Beads were washed either five (see Figure 1B and and1D)1D) or ten times (see Figure 1C) with IP buffer. A fraction of the final wash equal to the pellet fraction in Figure 1D was collected. After the final wash, proteins were eluted from the beads by heating in sample buffer and separating by SDS-PAGE (see Figure 1B and and1D).1D). Trypsin digestion, gel extraction, and mass spectrometry of proteins that coimmunoprecipitated with Opi1 were performed by the HHMI Mass Spectrometry facility (University of California, Berkeley, United States).
For immunoblot analysis, 25 μg of crude protein, prepared using urea denaturing lysis buffer (Ruegsegger et al. 2001), was separated on Invitrogen NuPage polyacrylamide gels, transferred to nitrocellulose, and immunoblotted. RNA preparation, electrophoresis, and labeling of probes for Northern blot analysis has been described (Ruegsegger et al. 2001).
Immunofluorescence was carried out as described (Redding et al. 1991), except that cells were harvested and fixed by incubation in 100% methanol at –20 °C for 20 min. Fixed, spheroplasted, detergent-extracted cells were probed with 1:200 monoclonal anti-myc (see Figures 1 and and5),5), 1:200 monoclonal anti-HA (see Figure 4), or 1:1000 rabbit polyclonal anti-GFP (see Figure 5). Secondary antibodies were diluted 1:200. Vectashield mounting medium (Vector Laboratories, Burlingame, California, United States) was applied to cells before sealing slides and visualizing using a Leica TCS NT confocal microscope (Leica, Wetzlar, Germany). For experiments localizing the GFP-Lac repressor, we first collected a single z slice through each cell that captured the brightest, most focused point of the GFP-visualized Lac operator array. This z slice was picked blind with respect to the nuclear membrane staining. Images in which this slice showed a clear nuclear membrane ring staining with a "hole" of nucleoplasm were then scored as follows: Cells in which the peak of the GFP-Lac repressor spot coincided with Sec63-myc nuclear membrane staining were scored as membrane-associated, and cells in which the peak of this spot was offset from nuclear membrane staining were scored as nucleoplasmic.
Chromatin immunoprecipitation was carried out on strains expressing endogenous levels of tagged Ino2, Ino4, and Opi1 as described (Strahl-Bolsinger et al. 1997), with the following modifications. The time of formaldehyde fixation was specific for each tagged protein. Strains expressing Ino2-HA were fixed for 15 min, strains expressing Ino4-HA were fixed for 60 min, and strains expressing Opi1-myc were fixed for 30 min. After lysis, cells were sonicated 15 times for 10 s at 30% power using a microtip on a Vibracell VCX 600 Watt sonicator (Sonics and Materials, Newtown, Connecticut, United States). After sonication, lysates were centrifuged 10 min at 21,000 × g to remove insoluble material and incubated for 4 h with anti-HA agarose or anti-myc agarose. After elution of immunoprecipitated DNA and reversal of crosslinks by heating to 65 °C for 8 h, DNA was recovered using Qiaquick columns from Qiagen (Alameda, California, United States). Eluted samples were analyzed by PCR using the following primers against the INO1 promoter or the URA3 gene: INO1_proUp2 (5′-GGAATCGAAAGTGTTGAATG-3′), INO1_proDown (5′-CCCGACAACAGAACAAGCC-3′), URAup (5′- GGGAGACGCATTGGGTCAAC-3′), and URADown (5′-GTTCTTTGGAGTTCAATGCGTCC-3′).
PCR reactions were carried out as described (Rogatsky et al. 2003) using a DNA Engine Opiticon 2 Real-Time PCR machine (MJ Research, Waltham, Massachusetts, United States), using 1/25 of the immunoprecipitation fraction and an equal volume of a 1:400 dilution of the input fraction as template. Primers used were: INO1up3 5′-ATTGCCTTTTTCTTCGTTCC-3′), INO1down2 (5′-CATTCAACACTTTCGATTCC-3′), URAup2 (5′-AGACGCATTGGGTCAAC-3′), and URAdown2 (5′-CTTCCCTTTGCAAATAGTCC-3′). Dilution of the input fraction from 1:25 to 1:12,800 in fourfold steps demonstrated that reactions were within the linear range of template. This dilution series was used as a standard curve of C(T) values versus relative template concentration for both primer sets. The concentration of the INO1 promoter and the URA3 gene were calculated using this standard curve. The ratio of INO1 promoter to URA3 was corrected for each sample to make the input ratio equal to 1.0.
The GenBank accession numbers of the genes and proteins discussed in this paper are Ire1 (NP_116622), INO1 (NP_012382), Trl1 (NP_012448), Opi1 (NP_011843), Hac1 (NP_011946), Ino2 (NP_010408), Ino4 (NP_014533), Scs2 (NP_009461), Rap1 (NP_014183), URA3 (NP_010893), SSS1 (NP_010371), Pgk1 (NP_009938) lacI (NP_414879), and Ikaros (Q03267).
The authors are very grateful to all the members of the Walter lab, in particular Gustavo Pesce, Tobias Walther, Jess Leber, and Tomas Aragon, for helpful discussions and inspiration; Isabella Halama for help with immunofluorescence; Hans Lueke for help with ChIP experiments and real time quantitative PCR; Doris Fortin for help with confocal microscopy; Joel Credle for technical assistance; John Sedat for providing the Lac operator and the GFP-Lac repressor plasmids; and Keith Yamamoto for helpful comments on the manuscript. This work was supported by a Helen Hay Whitney Postdoctoral Fellowship (JHB), by the Sandler Family Opportunity Fund, and by grants from the National Institutes of Health to PW. PW is an Investigator of the Howard Hughes Medical Institute.
Conflicts of interest. The authors have declared that no conflicts of interest exist.
Author contributions. JHB and PW conceived of the experiments. JHB performed the experiments. JHB and PW wrote the paper.
Academic Editor: Tom Misteli, National Cancer Institute
Citation: Brickner JH, Walter P (2004) Gene recruitment of the activated INO1 locus to the nuclear membrane. PLoS Biol 2(11): e342.
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Just so everyone knows. . .
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Bocanegra's understanding of kids and violence stems from his own history. He spent 14 years in prison for a gang-related murder, turned his life around and is now a graduate student at the University of Chicago. Through the Y, he mentors kids in communities where gangs are a constant presence.
He warns them of the consequences of their online activities. "I'll say, 'Don't you know you're creating a profile of yourself so police can see it?' ... How do you think this will impact you tomorrow, a month from now, five years from now?'... A lot of times, it's 'Who cares?'"
Some do.
Anderson Chaves, 17, changed his ways, removing a photo on his Facebook page of a man he once admired — Pablo Escobar, the Colombian drug lord killed in a gun battle with authorities. Chaves says he now avoids the back-and-forth online posturing.
"It's part of a macho display, 'Look at me. Look at who I am,'" he says. "They're not thinking that one day they might be standing in front of a judge and someone will pull all this stuff out. They don't think it will happen."
But it does.
Dawn Keating, a Cincinnati police officer who trains other law enforcement about social media, says by the time gang members appear in court, authorities have a dossier of their words and videos online that challenge how they want to portray themselves. "If a guy goes in and says, 'I'm a good person. I've never held a gun,' we can say, 'Look at what he puts out about himself on social media. Here he is with a gun.' It helps debunk a lot of things."
Despite those successes, police say monitoring social media is time-consuming and frustrating.
Eric Vento, a Houston police officer and gang specialist, says he sometimes creates aliases to befriend gang members online.
"You have to build your persona," he says. "That comes through countless, tedious hours of posting comments. You have to get to be friends with these people. You have to let them into your fake world. You have to build their trust. Only then, will they let you in. Until that time, you're there twiddling your thumbs."
Older gang members tend to restrict public access to their Facebook pages, but they can sometimes be found online through their wives and friends, he says. "They bite all the time," Vento says. "It's a question of keeping the bite. ... Depending on how high they are in the (gang) hierarchy, they're pretty suspicious. They know law enforcement is on Facebook. Maybe they're not thinking about it 24/7. They see there are enough news stories. They know there are fake profiles."
Younger gang members seem less cautious and more accustomed to sharing their lives online. And that can be a boon to police.
In Daytona Beach, Fla., in October, after a group of teens claiming to be in a gang pummeled and kicked a 14 year old, the victim's mother found the beating video posted on Facebook that night, says detective Scott Barnes.
After she contacted police, they quickly tracked down the suspects in their schools, Barnes says. The five, 13 to 18, were charged with aggravated criminal battery, he adds, and all pleaded guilty.
"Everybody wants to be cool and get the street cred they feel they deserve," he says. "It's not very smart, that's for sure. ... It's sad it's come to this. Some of these kids will be OK, but there's a group out there who are going to ruin their lives and end up in prison a long time or be dead. It's hard to get ahead of this, but we're trying."
Sharon Cohen is a Chicago-based national writer and can be reached at firstname.lastname@example.com.
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Supplier: CSA Group
Description: The requirements of this Standard apply to manually operated, general-use snap switches for connection to copper (Cu) or copper-clad conductors used in accordance with the National Electrical Code(NEC), ANSI/NFPA 70, or the Canadian Electrical Code (CEC), Part I, and intended for connection
Description: safety output Time delay output modules 1 to 30 seconds delay Fixed .5 second delay Two wire GO input interconnection Plug-in connection terminals CE UL applied BENEFITS Simple plug-in and use modular design Flexible from basic module with your design changes No programming Interconnection
- Maximum Current: 8.4 amps
- Maximum AC Switching Voltage: 250 volts
- Maximum AC Switching Power: 1500 VA
- Maximum DC Switching Power: 30 watts
Description: FM Approved for the storage of flammables; also meets NFPA specifications and OSHA regulations Fully-welded, double-wall, 18-gauge steel with 1.5" of insulating air space helps cabinet stay square Reduced air gaps provide excellent fire protection Welded-in dual vents with flame arrestors
- Height: 24 inch
- Width: 43 inch
- Depth: 18 inch
- Material: Steel
Supplier: Newson Gale Inc.
Description: Businesses with HAZLOC atmospheres are exposed to the risk of static electricity igniting flammable gas, vapor and dust atmospheres. This ignition risk can be eliminated by ensuring equipment is bonded and grounded in accordance with NFPA 77 "Recommended Practice on Static Electricity&rdquo
- Applications: Other
Description: due to an inadvertent machine shutdown. To meet current worldwide standards, safety interlock components cannot be easily bypassed. These switches have positive opening contacts (in accordance to NFPA 79) and redundancy to guarantee that they fail to a safe state. The TQ switch is Euchner's
- Normally Open Contacts: 1
- Maximum Current: 5 amps
- Maximum AC Voltage: 250 volts
- Maximum DC Voltage: 24 volts
Supplier: Dawg, Inc.
Description: . Haz-Drum models come with rollers for easy drum handling into cabinet. Optional Drum Cabinet Ramp makes loading and unloading safer and easier. Cabinets meet NFPA 30 regulations and OSHA 29 CFR 1910.106(d) 3 requirements.
- Type: Drum Dolly
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National Fire Protection Association (NFPA)
NFPA 30: Flammable and Combustible Liquids Code
For safety around flammable and combustible liquids, using the latest NFPA 30 is the first priority. Enforceable under OSHA and many state and local regulations, NFPA 30: Flammable and Combustible Liquids Code is the best practice widely used in industry and by insurers. The 2015 edition of the Code includes the latest and most complete criteria you need to protect your facility by ensuring safe storage, handling, and use of flammable and combustible liquids. Changes respond to recommendations... (read more)
Browse Codes, Standards, and Regulations Datasheets for National Fire Protection Association (NFPA)
Jamco Products, Inc.
Safety Flammable Cabinets - Manual Close Doors
and the manual close doors, which open to a full 180 degrees, also have a 3 point locking system and radius edges. This cabinet meets OSHA, and NFPA code 30 standards. Chemical storage maximum - Class I & II - 60 gallons, Class III - 120 gallons. Click here to learn more about Jamco Products ' entire line of safety flammable cabinets. Product Features: • All welded double wall 18 gauge construction with 1-1/2" insulating air space. • 2" leakproof sill to contain leaks... (read more)
Browse Safety Storage Cabinets Datasheets for Jamco Products, Inc.
Jamco Products, Inc.
Safety Flammable Cabinets with Manual Close Doors
and NFPA code 30 standards. Other special features include a 2 " leak proof sill to contain leaks, a lockable flush mounted handle with 2 keys and adjustable leveling feet and grounding connector. Click here to see Jamco's complete line of Safety Flammable Cabinets. Product Features: All sizes are FM Approved!. All welded double wall 18 gauge construction with 1-1/2" insulating air space. Doors are double walled, 14 gauge outside & 18 gauge inside. 2" leakproof sill to contain leaks... (read more)
Browse Safety Storage Cabinets Datasheets for Jamco Products, Inc.
U.S. Chemical Storage
FIREloc™ Fire-rated Chemical Storage
Fire-rated Chemical Storage Building. Our FIREloc ™ buildings are designed for storing, combustibles, and housing other flammable chemicals that require a 2-hour fire rating when located 10-30 feet from an occupied facility. Features. Multiple layers of fire resistant wallboard are encased between interior and exterior heavy gauge, hand welded steel. FIREloc ™ buildings are designed to meet EPA, OSHA and NFPA requirements. These buildings can be customized for climate control... (read more)
Browse Hazmat Storage Buildings Datasheets for U.S. Chemical Storage
Containment Solutions, Inc. (CSI)
Fire- Protected Vault Tanks
detection tube. Meets requirements of all National Fire Codes. UFC Articles 52 and 79, Appendix II and Standard A-ll-F-l. NFPA Standards 30 and 30A. BOCA. SBCCI. CARB Certified for Stage I and II Vapor Recovery. Underwriter 's Laboratories of Canada (ULC) Listed. 30-Year warranty. Factory installed equipment packages: Motor fuel dispensing, emergency generator/fuel oil,. used oil... (read more)
Browse Storage Tanks and Process Tanks Datasheets for Containment Solutions, Inc. (CSI)
Jamco Products, Inc.
Safety Flammable Cabinets for Pesticides
gauge outside & 18 gauge inside. The Model FL also features twin 2" flame arrester vents and adjustable leveling feet. This safety flammable cabinet also meets OSHA, and NFPA code 30 & UFC 79 standards. Click here to see Jamco's complete line of Safety Flammable Cabinets. Product Features: • Powder coated forest green finish. • Safety signage in large red letters. • Galvanized steel shelves adjust on 2-1/2" centers. • Polyethylene tray liner... (read more)
Browse Safety Storage Cabinets Datasheets for Jamco Products, Inc.
U.S. Chemical Storage
55 Gallon Drum Storage
for storage, mixing & dispensing or transferring chemicals. US Chemical Storage offers fire-rated construction: 2-hour fire-rated construction - allows you to store flammables and combustibles 10-30 feet from occupied facilities. 4-hour fire-rated construction - allows you to store flammables and combustibles 10 feet away or even inside your facility. Maintain hazardous chemical storage inside or nearby your operations to increase production capabilities with bulk storage on-hand. Fire-rated... (read more)
Browse Hazmat Storage Buildings Datasheets for U.S. Chemical Storage
Containment Solutions, Inc. (CSI)
Lube Cube Oil Tanks for new and used oils
volume in a given space than comparable cylindrical tank capacities. Double-Wall Containment for Added Security. Lube Cubes are available as either single or double-wall tanks. Double-wall tanks are UL listed as integral secondary containment and therefore do not require dikes for leak containment. Desert sand primer finish is standard. Special colors, coatings and interior linings, custom dimensions and equipment packages are available upon request. Standard Features: UL-142 listed. Meets NFPA 30... (read more)
Browse Storage Tanks and Process Tanks Datasheets for Containment Solutions, Inc. (CSI)
U.S. Chemical Storage
Outdoor Chemical Storage Locker
ventilation, lighting, flooring options, roll-up doors, and other explosion and non-explosion proof accessories. Meet The Regs. Outdoor chemical storage lockers by US Chemical Storage are designed to meet EPA, OSHA, NFPA, IBC, IFC regulations and FM or Warnock Hersey approvals. Non-Fire Rated Outdoor Storage Locker. Our non-fire rated line of outdoor chemical storage lockers allow storage of chemicals up to 30 feet away from existing structures and property lines. For safe chemical storage, outdoor... (read more)
Browse Hazmat Storage Buildings Datasheets for U.S. Chemical Storage
Parker Hannifin / Fluid Connectors / Parflex Division
CNG Packaged Hose Solutions Simplify Refueling
Ravenna, OH May 1, 2015 – Parker Hannifin Corporation, the global leader in motion and control technologies, is also a leader in the manufacture of hose solutions to dispense or transfer compressed natural gas (CNG). Specifically, Parker's Parflex Division offers a Parflex Division's 5CNG hose is offered in a CSA certified single-line assembly and as a packaged twin-line solution with hoses bonded into a compact, safe, easy-to-install assembly. These hoses can be up to 30% lighter than... (read more)
Browse Hydraulic Hose Datasheets for Parker Hannifin / Fluid Connectors / Parflex Division
Parts by Number Top
|Part #
|Product Category
|Allied Electronics, Inc.
|Not Provided
|Allen Bradley
|Not Provided
More Information Top
Vehicle Codes and Standards: Overview and Gap Analysis
Lack of data for existing codes and standards applicability to biodiesel storage systems National Fire Protection Association ( NFPA ) 30 and 30A and associated listing documents, IFC Provide data for the compatibility of Biodiesel with listed storage .
Bookshelf and Unified Agenda > Department of Energy > Chemical Safety Program > Chemical Handbooks > [FDBKF2005033008123185515] Chemical Management Handbook Vo...
NFPA 30 , 5.2; NFPA 45, 7.1; NFPA 45, 188.8.131.52, NFPA 430, 2.-1.1; NFPA 432, 4.7.1; 10CFR835.204(d)(2); 29CFR1910.106(e)(8); 29CFR1910.146(c)(1)- (d)(2); 29CFR1910.1450(e)(3...


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You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
40 year old man | Orlando, FL, USA | Seeking women 30-45 within 30 miles
Sarcastic smart ass looking for a long term commitment.
I`m a 38 year old father of two. My children are number one in my life. I enjoy movies, dinner with a nice conversation also.Love stupid funny movies (Super Troopers) is an all time favorite. Hoping to meet someone special and see what develops. With work being so hectic and having kids on the weekends it can be difficult to meet people. I figured this site would help, we'll see. I love kids, I coach my son's little league team. I'm not able to have anymore children so if that's an issue I apologize. I think being upfront and honest about it is best. Check out my profile if you like lets talk.
Coffee and conversation , Cooking , Fishing/Hunting , Movies/Videos , Museums and art , Exploring new areas , Video games , Watching sports
Baseball, Basketball, Football, Martial arts, Soccer, Weights / Machines
I exercise 1-2 times per week.
I have Dogs, Exotic Pets
Middle of the Road
Any place is a favorite if I'm with the right person.
Musical taste varies, blues, classical, break beats, country, acoustical to name a few. Spending time with my children.
When I have free time I like to hang out at home or go out.
Bourne Deception Robert Ludlum.
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|6'3" (190cms)
|5'5" (165cms) to 6'4" (193cms)
|Body type:
|About average
|Slender, About average, Curvy, Athletic and toned, A few extra pounds
|Black, Blue, Brown, Grey, Green, Hazel
|Auburn / Red, Black, Light brown, Dark brown, Blonde, Dark blonde, Platinum
|Occasionally, Daily, Yes, but trying to quit
|Social Drinker
|Never, Social Drinker, Regularly, Moderately
|Other profession
|Administrative / Secretarial, Artistic / Creative / Performance, Executive / Management, Financial / Accounting / Real Estate, Legal, Medical / Dental / Veterinary / Fitness, Retail / Food services, Sales / Marketing, Self-Employed / Entrepreneur, Education / Teacher / Professor, Technical / Science / Computers / Engineering, Travel / Hospitality / Transportation, Law enforcement / Security / Military, Fashion / Model / Beauty, Architecture / Interior design
|I'll tell you later
|No preference
|Never Married, Widow / Widower, Divorced
|Have kids:
|Yes, and they sometimes live at home (2)
|Yes, and they sometimes live at home, No, Yes, and they live away from home, Yes, and they live at home
|Wants kids:
|Not sure
|Not sure, Probably not, No, No, but it's OK if my partner has kids
|Latino / Hispanic
|Asian, Black / African descent, East Indian, Latino / Hispanic, Middle Eastern, Native American, Pacific Islander, White / Caucasian
|Christian / Other
|Agnostic, Atheist, Buddhist / Taoist, Christian / Catholic, Christian / LDS, Christian / Protestant, Hindu, Jewish, Muslim / Islam, Spiritual but not religious, Other, Christian / Other
|Arabic, Chinese, Dutch, English, French, German, Hebrew, Hindi, Italian, Japanese, Korean, Norwegian, Portuguese, Russian, Spanish, Swedish, Tagalog, Urdu, Other
|Some college
|Some college, Associates degree, Graduate degree, PhD / Post Doctoral


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Christmas Ornaments >>
David Strand
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Christmas Central brings a holiday experience to your home as unique as a snowflake with David Strand Designs Mouth Blown Christmas Ornaments. David Strand Designs are masterpieces of old world craftsmanship and each piece is hand painted by skilled artisans. David Strand takes classic Christmas themes and turns them upside down into a magical holiday experience. Santa Claus has never had so much fun as he is depicted riding in a Hot Air Balloon Ornament, Peacock Suit Ornament, Riding the North Pole Express Ornament and a series of other adventures. Snowmen get their opportunity to shine with Hot Air Balloon Ornaments, adorable Hand Cart Railroad Ornaments, and a Snowman wearing a Highland Plaid Sweater Ornament. David Strand has collections of bird ornaments that feature Cardinals, Blue Jays, Owls, and Peacocks. To really set your tree apart it is essential to check out the spectacular David Strand Tree Toppers for an incredible holiday experience. When you get a David Strand Designs Ornament from Christmas Central you have a family heirloom to treasure forever.


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Daemen College : Admissions : Admissions Staff : Meet our Tour Guide Staff : Admissions Assistants
Alexandrea Esquivel
Major: Nursing
Hometown: Los Angeles, California
Activities: Admissions Assistant
Click here to learn more about Alexandrea!
Marisa Mauro
Major: Physician Assistant
Hometown: New Hartford, NY
Activities: Lambda Chi Iota, Admissions Assistant
Click here to learn more about Marisa!
Justin Richmond
Major: Childhood Education English
Hometown: Bronx, NY
Activities: Admissions Assistant, Boys Club Volleyball
Click here to learn more about Justin!
Candice Roberts
Major: Psychology
Hometown: Cheektowaga, NY
Activities: Colleges Against Cancer, Psychology Club
Click here to learn more about Candice!
Caitlin Schuler
Major: Adolescent Education-History and Government
Hometown: Dryden, NY
Anthony Tojeck
Hometown: Buffalo, NY
Activities: PASS, Admissions Assistant
Cick here to learn more about Anthony!
Sarah Velarde
Major: Physical Therapy /Minor: Psychology
Click here to learn more about Sarah!
Daemen students can start their internship experiences at the beginning of their sophomore year.
Graphic Design
Nick held several student leadership positions at Daemen and also completed an internship for graphic design. He recently traveled abroad to Italy with students and the department chair of the program.
©2014 Daemen College : 4380 Main Street, Amherst, NY 14226 : 800.462.7652


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Credit : Getty ImagesEarn Points
True Blood
More True Blood Season 5 Premiere Party Pics (PHOTOS)
The stars were out last night at True Blood's season 5 premiere party. Along with the cast, celebrities from other TV shows supported the HBO drama. ;
Check out pics of True Blood's big night!


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Mario Chalmers
Ailing Wade, Aching Heat Find A Way To Beat Pistons, 109-102
Dwyane Wade scored 40 points one day after having fluid removed from his balky left knee, Udonis Haslem set season highs with 18 points and 13 rebounds, and the undermanned Miami Heat beat the Detroit Pistons 109-102 on Sunday night.
Injury-Riddled Heat Beat Celtics 93-86
Goran Dragic had 22 points and seven assists, and the banged-up Miami Heat beat the Boston Celtics 93-86 on Wednesday night.
Middleton Buzzer-Beater Sends Bucks To 89-88 Win Over Heat
Khris Middleton hit a 3-pointer at the buzzer as the Milwaukee Bucks snapped a six-game losing streak with an 89-88 comeback victory over Miami on Tuesday night in a key game for the sixth spot in the Eastern Conference playoff race.
Heat Stay Hot At Home, Pull Away From Nuggets 108-91
The Miami Heat stayed hot at home, beating the Denver Nuggets 108-91 on Friday night.
Wade Scores 32, Heat Rally To Beat Blazers 108-104
Dwyane Wade scored 15 of his 32 points in the fourth quarter, including a jumper with 13.6 seconds left to break a tie and help the Miami Heat beat the Portland Trail Blazers 108-104 on Wednesday night.
Top Reasons The Miami Heat Will Make The Playoffs
A lot has happened in the Miami Heat's world since losing in the NBA Finals last June.
Heat Start Fast And Finish Off Season Sweep Of Nets, 104-98
Follow CBSMIAMI.COM: Facebook | Twitter MIAMI (AP) — Dwyane Wade scored 28 points and added nine assists, Chris Andersen tied a career high with 18 points and set a season high with 14 rebounds, and the Miami Heat […]
Heat Look To Keep Pace In Playoff Race Against Visiting Nets
In the midst of an intense playoff hunt, the Miami Heat will be without one of their key players on Wednesday.
Heat Come Back And Get OT Win Over Kings
The Miami Heat's never say die attitude has become a major asset in recent games.
Banged Up Heat Try To Extend Home Winning Streak
While the Sacramento Kings have been the worst road team in the Western Conference since late November, their futility in Miami goes back much further than 3 1/2 months.


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Mount Carmel isn't the only football team that specializes in the split-back veer offense.
CHAMPAIGN The Illinois football faithful are not suffering alone.
Start-up schools usually struggle out of the gate. It takes at least a year or two or more to organize community support and establish relationships, a culture, an attitude, a tradition.
SOUTH BEND, Ind. -- As Notre Dame rolled past Michigan State and Michigan en route to a top-10 ranking, tight end Tyler Eifert's role within the Irish offense was been noticeably small.
SOUTH BEND, Ind. -- Brian Kelly said this week that he he intends to move ahead with Everett Golson as Notre Dame's starting quarterback, which should come as no surprise.


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A chapter by chapter reading experience for children and parents alike!
Purchase the paperback copy at SwordoftheLord.com
Danny Orlis and his friend are helping Danny's dad with a hunting expedition. One of the hunters doesn't follow instructions and appears to be lost. While searching for him, Danny finds evidence of illegal hunting. As the boys determine to find out who has been threatening the game population, they find themselves trapped by the criminals. Trusting God and doing the Christlike thing brings another opportunity for Danny to bear fruit for the Saviour.


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For additional details on the winning projects visit www.aginginplaceinitiative.org. A comprehensive report on the Miami Workshop: Transportation and Mobility Options: Developing Livable Miami-Dade and Monroe Counties for All Ages, will be available for download in the coming weeks.
Partners for Livable Communities (Partners) is a national, nonprofit organization working to renew communities for all ages. For more information on the other workshops planned in the national series, visit www.aginginplaceinitiative.org, or contact Kendall Alexander at email@example.com .
The National Association of Area Agencies on Aging (n4a) is a leading voice on aging issues for Area Agencies on Aging across the country. F
|SOURCE MetLife Foundation
Copyright©2009 PR Newswire.
All rights reserved


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A federal appellate court affirms dismissal of class-action lawsuit brought by home sellers who claimed that local brokerages had conspired to fix commission rates in violation of federal antitrust laws.
A group of home sellers (collectively, "Sellers") filed a lawsuit against a number of local brokerages and some of their corporate parent companies. The lawsuit alleged that the brokerage firms had entered into an illegal arrangement to fix commission rates, demonstrated by the fact that they all charged a similar rate and the commission rates hadn't decreased despite a rise in the average home sale price. Read More
A Texas appellate court has evaluated whether a buyer's representative was liable to his clients for inaccurate square footage information.
Bowen Zhu and Jian Yu (collectively, "Buyers") purchased a home. The Buyers had retained real estate licensee Kai Lam ("Buyer's Representative") of The Household Realty, Inc. ("Brokerage") to represent them in the transaction.
During the course of the transaction, the listing broker, the local government appraisal district, and the Buyer's Representative had all represented that the size of the property's living area as 2,722 square feet. While viewing the property, the Buyers had remarked that the property seemed smaller than their 2600 square feet residence, but the Buyer's Representative had responded that this was because the property had an open floor plan. Read More
859.263.7377 (local)
800.264.2185 (toll free)
859.263.7565 (fax)
161 Prosperous Place, Suite 100
Lexington, KY 40509
Sitemap | Related Links | Privacy Policy/Terms of Use | Advertising
Copyright© 2000-2014 Kentucky Association of REALTORS®


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Excellent service and very good food at a reasonable price. I tend to put salt and pepper on everything, but I didnt once think about reaching for the shakers.
Excellent service and very good food at a reasonable price. I tend to put salt and pepper on everything, but I didnt once think about reaching for the shakers.
Excellent service and very good food at a reasonable price. I tend to put salt and pepper on everything, but I didnt once think about reaching for the shakers.
I went with some friends on a Thursday Night for Dinner, We we seated at a booth in the bar area that was roomy and not cramped. We had the greatest server I have ever met, a young lady named Taylor who is a student at Kutztown State College. Everything we ordered was brought in a timely fashion and was... More
There were 6 of us that went there for my daughter's birthday. I was able to do call ahead seating which I love. Service was good and the food was delicious. We never feel rushed when we are there. All of our food was served hot.
The food was awsome.. I choose Ribeye medium well, super juicy and perfect term of the meat. The service was great also..
The atmosphere is your typical cheesy chain restaurant. The service is dead on average. Nothing good or nothing bad. I ordered a rib and pulled pork combo with a side salad and steak fries. The salad was nice and fresh. As far as the warm items; the steak fries were way oversalted, the ribs had little to no flavor as... More
What a crowded place on a Tuesday night! Great food brings out the people. The friendliness of the waitress to the quick service , explains why we enjoyed this place so much. The food was excellent from the great salad to the sweet baked potatoe and prime rib. Will come back soon again!
This was my first time here, although my spouse has been here several times and has enjoyed this restaurant immensely. When we were seated, I found it odd that the hostess asked us how our last visit was. When I told her that it was my first visit here, she didn't know what to say. The peanuts on the table... More
My family and I have always had a great meal here. We find ourselves going back all the time. The sirloins are always very tender and done to our liking. The wait staff always very friendly. The birthday song get annoying after awhile but can be overlooked. We end up going here at least once a month. You can do... More
Hello all you steak lovers out there Gregory Grookett here to review this locations Allentown location. Typical of this chain the establishment is great lives up to the name the company wants. Great steak choices among other things. Friendly wait staff and quick prompt service. Gregory Grookett went here with his girlfriend and we enjoyed our meal totally and will... More
We went to Texas Roadhouse on Veterans Day. All vets were eligible for a complimentary meal. At the entry way they had set up a "Missing Man" table to symbolize those who had not returned. I thought that was particularly touching. It was very crowded, but we we served in a timely fashion. Staff was very upbeat. Both the chicken... More
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Good luck to all the students taking this Saturday's March 1 SAT test. As we do for all the SAT test dates, here is our collection of last-minute advice and videos focused on SAT test day:
SAT Test Day: The Night Before and Morning Of
Real SAT Stories: Second Guessing on the SAT
SAT Directions: Optimize Your Time on Test Day
When to Guess on the SAT Writing Section
When to Guess on the SAT Critical Reading Section
When to Guess on the SAT Math Section
SAT Sentence Completion Questions: When to Guess?
Don't Blame the Chicken Pox for SAT Math Mistakes
Approach to SAT Math Problems
Habeus Answer, or Show Yourself the Answer!
Mastering Your SAT Test Weaknesses
SAT Questions…Easiest to Hardest
Autopilot - Don't Set a Course for a Lower SAT Score
A Lesson from a Course in Wills, Trusts and Estates
Bubbling: Avoid Mistakes in Your Answer Key
Please Put Down Your Pencil!
The Dreaded SAT Experimental Section
SAT Essay Rubric - Development of Position (Video 1 of 5)
SAT Essay Rubric - Organization (Video 2 of 5)
SAT Essay Rubric - Use of Language (Video 3 of 5)
SAT Essay Rubric - Sentence Structure (Video 4 of 5)
SAT Essay Rubric - Grammar and Word Usage (Video 5 of 5)
The SAT Essay - What Not To Do . . . Unless You "Want" a Zero
You can also see EVERY SAT post here. If you have an iPod, we encourage you to subscribe to our free podcasts and take ePrep with you on the go!


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There are 3 rooms, in the small one we have 2 beds but in the others 6 beds. We have a cozy yard and place to park... More Info »
Hostel has got perfect location 2 minutes walking distance from Metro Rustaveli. The sleeping rooms are with big... More Info »
Hostel Greenhouse, a two-storied house with a courtyard, huge balcony and a big common room with a piano and... More Info »
hostel More Info »
SkadaVeli Guest-house located in Kala - the oldest and most important residential district of Old Tbilisi - on the... More Info »
Probably the BEST PLACE for staying in Tbilisi. Tbilisi 'Old Town Hostel' is situated in the heart of Tbilisi. Next... More Info »
The best hostel in center of Tbilisi 1) Shower and clean sheets 2) Private rooms 3) Free Wi-Fi More Info »
Brand new hostel located in the heart of the city. Nice place to meat other travellers as well as experience georgian... More Info »
Easy to find, hard to leave! Fall in love with Tbilisi in this central, cozy and fun travelers haven. Enjoy a stay in... More Info »
If you are looking for the best ratio of price and quality our guesthouse is one of Tbilisi's most affordable and... More Info »
Please Note: Cancellation policy: 24h before arrival. In case of a late cancellation or No Show, you will be... More Info »
Home sweet home offers for visitors from all over the world comfortable and cheap accommodation right in Tbilisi... More Info »
Tbilisi Classic Hotel is located in Tbilisi, a 16-minute walk from old city centre and Medani Square. Free WiFi... More Info »
Vintage style Home #12 hostel is situated in excellent location - in city center of Tbilisi. We are offering you... More Info »
Hostel Konka Tbilisi offers rooms with air conditioning and central heating. There are two shower and toilets. There... More Info »
Do you want to explore the centre of Tbilisi, but stay somewhere unusual? Fox Hostel is there for you. A Cosy place... More Info »
Capital! City Center! Historic District ! 15 minutes walk to every central street of the city. Friendly... More Info »
2 bedroom fully furnished superior apartment in a luxury, newly built residential complex on prestigious Aleksandre... More Info »
A new hotel Check-In is located in the very center of Tbilisi. You will be met by cozy and pleasant atmosphere. The... More Info »
Lemon Hostel is located in Nadzaladevi. Free WiFi access is available. You can enjoy garden view from the room.... More Info »
Video guides, travel podcasts, city maps & free city guides. What to eat, where to stay and things to do and see - our travel guides have it all.Find a guide »
There's nothing like having your own transport to get around and when travelling the world it's no different.Read more »
We offer the most comprehensive selection of hostels on the internet with over 35,000 great places to stay in 180 countries. The ultimate online resource for hostels everywhere.Find great value hostels »