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The transcription factors TFIIB, Brf1, and Brf2 share related N-terminal zinc ribbon and core domains. TFIIB bridges RNA polymerase II (Pol II) with the promoter-bound preinitiation complex, whereas Brf1 and Brf2 are involved, as part of activities also containing TBP and Bdp1 and referred to here as Brf1-TFIIIB and Brf2-TFIIIB, in the recruitment of Pol III. Brf1-TFIIIB recruits Pol III to type 1 and 2 promoters and Brf2-TFIIIB to type 3 promoters such as the human U6 promoter. Brf1 and Brf2 both have a C-terminal extension absent in TFIIB, but their C-terminal extensions are unrelated. In yeast Brf1, the C-terminal extension interacts with the TBP/TATA box complex and contributes to the recruitment of Bdp1. Here we have tested truncated Brf2, as well as Brf2/TFIIB chimeric proteins for U6 transcription and for assembly of U6 preinitiation complexes. Our results characterize functions of various human Brf2 domains and reveal that the C-terminal domain is required for efficient association of the protein with U6 promoter-bound TBP and SNAPc, a type 3 promoter-specific transcription factor, and for efficient recruitment of Bdp1. This in turn suggests that the C-terminal extensions in Brf1 and Brf2 are crucial to specific recruitment of Pol III over Pol II.
In eukaryotes, the job of transcribing nuclear genes is divided among three different RNA polymerases: RNA polymerase I (Pol I), RNA Pol II, and RNA Pol III. A common step in transcription initiation by all three eukaryotic RNA polymerases is the recruitment of the polymerase to the proper promoters by a specific factor or complex. This transcription factor serves as a bridge between promoter-bound factors that nucleate preinitiation complex assembly and the RNA polymerase enzyme itself. For Pol II and Pol III transcription, the factors that accomplish this task are TFIIB and TFIIIB, respectively.
Although TFIIB is a single polypeptide, the TFIIIB activity is minimally composed of three polypeptides (see references 11 and 33 for reviews). In human cells, two forms of the TFIIIB complex have been characterized thus far: Brf1-TFIIIB, which functions on type 1 and 2 Pol III promoters, and Brf2-TFIIIB, which functions on type 3 Pol III promoters such as the human U6 small nuclear RNA (snRNA) promoter. Brf1-TFIIIB and Brf2-TFIIIB both contain the TATA-box binding protein TBP and the SANT domain protein Bdp1. They differ by the presence of different members of a family of TFIIB-related factors: Brf1 in Brf1-TFIIIB and Brf2 in Brf2-TFIIIB.
TFIIB, Brf1, and Brf2 each contain a Zn ribbon domain at their N terminus, followed by a core domain consisting of two imperfect repeats. In this region, human Brf2 is 20% identical with human TFIIB and 18% identical with human Brf1. In addition, Brf1 and Brf2 contain a C-terminal extension. In Brf1, this C-terminal segment contains two regions called homology domains II and III, which are conserved among yeast and human proteins. Moreover, a third region called homology domain I is conserved among several different yeast species (25, 28). Such domains have thus far not been characterized in Brf2.
Although all TFIIB family members share the function of bridging promoter-bound factors and the Pol, TFIIB and yeast Brf1 accomplish this function differently, even on TATA-box-containing Pol II and Pol III promoters. Indeed, each TFIIB family member can associate with a TBP/TATA box complex, but TFIIB and Brf1 do so primarily through different regions. TFIIB assembles with the TBP/TATA box complex through its core domain, which interacts directly with a region within the C-terminal stirrup of the TBP core domain (3, 12, 17, 30, 31, 42). The core domain of yeast Brf1 also interacts with the TBP/TATA box complex, but the highest-affinity anchorage point is between homology domain II within the C terminus of Brf1 and surfaces on the N-terminal lobe of the TBP core domain, on the face of the TBP/TATA box complex opposite the TFIIB binding site (9, 19, 20, 22, 36).
TFIIB and Brf1 also recruit the RNA Pol differently. With TFIIB, the Zn ribbon domain is crucial for the recruitment of RNA Pol II: it interacts with the dock domain of Pol II and allows the positioning of the TFIIB core domain on the polymerase (3, 5, 7, 8, 12, 17, 31, 42; see reference 13 for a review). In contrast, with Brf1 the zinc ribbon is not necessary for PolIII recruitment but instead plays an essential role at a later stage in transcription initiation, that of promoter opening (14, 20, 23). It is likely that interactions between the C34 subunit of Pol III and the Brf1 core and homology regions I and II (1, 25, 40, 41), as well as between the C17 subunit of Pol III and the Brf1 core (10), are strong enough for recruitment of the polymerase even in the absence of any protein-protein contact contributed by the zinc ribbon.
Like Brf1, Brf2 is a Pol III transcription factor, but it is specifically required for transcription from type 3 promoters such as the human U6 promoter. These promoters are located upstream of the transcribed region and their core region consists of a proximal sequence element (PSE) and a TATA box, which are recognized by a multisubunit complex we call SNAPc and by TBP, respectively. We have now examined the functions of various regions of Brf2 for U6 transcription and for assembly of a preinitiation complex containing SNAPc, TBP, Brf2, and Bdp1, and we have tested the ability of Brf2/TFIIB chimeric proteins to perform these functions. The results suggest that a common feature among TFIIB family members involved in Pol III transcription is the use of their C-terminal extension to associate efficiently with a TBP/TATA box complex and to recruit Bdp1.
Full-length Brf2 and TFIIB, as well as the Brf2 truncations and the four TFIIB/Brf2 chimeras, were all expressed in Escherichia coli with the T7 system from (37). The proteins contained an N-terminal FLAG tag and a C-terminal His tag, except for full-length Brf2, which had only the C-terminal His tag. The proteins were purified via their His tags by Ni-nitrilotriacetic acid (NTA) agarose (QIAGEN) chromatography and analyzed by sodium dodecyl sulfate (SDS)-12.5% polyacrylamide gel electrophoresis. The gels were stained with the GelCode Blue Stain reagent (Pierce) for 1 h according to the manufacturer's instructions and were scanned with an Odyssey Infrared Imager (LI-COR) to measure the intensities of the appropriate bands. The proteins were also analyzed by immunoblotting with the Odyssey Infrared Imaging System (LI-COR).
TBP was expressed in E. coli as a glutathione S-transferase (GST)-fusion protein and bound to glutathione agarose beads. The beads were washed, and TBP was released from the beads by thrombin cleavage at a site located just after the GST tag. Bdp1 was expressed in E. coli and purified first through a C-terminal His tag by Ni-NTA agarose chromatography and then through an N-terminal FLAG tag by anti-FLAG Antibody M2 agarose (Sigma) chromatography. Both TBP and Bdp1 were dialyzed against buffer D100 (50 mM HEPES [pH 7.9], 0.2 mM EDTA, 20% glycerol, 0.1% Tween 20, 100 mM KCl, 3 mM dithiothreitol [DTT], 0.5 mM phenylmethylsulfonyl fluoride).
SNAPc was expressed in Sf9 insect cells as described previously (15) and purified via a His tag fused to the C terminus of SNAP190 by Ni-NTA agarose (QIAGEN) chromatography. The RNA Pol III complex used in the reconstituted U6 transcription system was purified from a HeLa cell line expressing a FLAG- and His-tagged RPC4 (RPC53) subunit of Pol III, as described previously (18).
For the GST pull-down assay, GST, GST-tagged PCMT, and GST-tagged TBP were all expressed in E. coli with the T7 system from (37) and bound to glutathione-agarose beads. The beads were then washed four times with HEMGN150 buffer (25 mM HEPES [pH 7.9], 150 mM KCl, 12.5 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 0.1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 2 mM DTT, and protease inhibitors), resuspended in the same buffer, and stored at 4°C.
The reconstituted U6 transcription assay and RNA analysis were performed as described by (18).
The binding reactions contained the proteins indicated in the figure legends and 9 μg of fetal bovine serum, 25 ng of poly(dG-dC)-poly(dG-dC), and 75 ng of pUC118 in a buffer containing 20 mM HEPES (pH 7.9), 100 mM KCl, 10 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 1 mM DTT, 0.07% Tween 20, and 0.15 mM ATP. The total reaction volume was 20 μl. The mixtures were incubated at 4°C for 20 min before addition of the radiolabeled probe and further incubation at 30°C for 30 min. The samples were then fractionated on a 4% polyacrylamide gel (39:1 acrylamide-bisacrylamide) in TGE buffer (50 mM Tris base, 380 mM glycine, 2 mM EDTA).
Either full-length Brf2, full-length human TFIIB, the B/2/− chimera, or the B/2/2 chimera were mixed with 10 μl of glutathione-agarose beads bound by either GST, GST-PCMT, or GST-TBP in HEMGN150 buffer containing 400 μg of ethidium bromide/ml in a total reaction volume of 60 μl. The reactions were incubated for 2 h at 4°C, rotating end over end. The beads were then washed two times with HEMGN150, two times with HEMGN300 (containing 300 mM KCl instead of 150 mM KCl), and two times once again with HEMGN150, always in the presence of 400 μg of ethidium bromide/ml. The beads were then resuspended in 5× Laemmli buffer (325 mM Tris-HCl [pH 8], 10% SDS, 0.025% bromophenol blue, 50% glycerol, 500 mM β-mercaptoethanol) and boiled. The proteins eluted from the beads were then fractionated on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and visualized by immunoblot with an anti-His tag monoclonal antibody (QIAGEN) at a 1:3,000 dilution. The immunoblots were developed with the Odyssey Infrared Imaging System (LI-COR).
The top of Fig. Fig.1A1A shows a comparison of the structures of human TFIIB, Brf1, and Brf2. These three proteins each contain a zinc-binding domain (shown in blue) and a core domain (shown in purple). Brf1 and Brf2 additionally contain unique C-terminal domains. To determine the role of different parts of Brf2, we synthesized the truncated Brf2 proteins illustrated in the lower part of Fig. Fig.1A.1A. Brf2(66-419) lacks the region N-terminal to the core domain, which includes the Zn ribbon motif. The second truncation, Brf2(2-289), lacks the C-terminal domain of the protein. The full-length and truncated proteins all contain a histidine tag at their C terminus (indicated in red), which was used for purification after expression in E. coli. The resulting proteins are shown in Fig. Fig.1B1B and, as revealed by immunoblot with an anti-Brf2 antibody after normalization of the amounts, in Fig.Fig.1C.1C. These proteins were then tested for U6 transcription in a minimal in vitro transcription system, which consists of recombinant TBP, Bdp1, Brf2, and SNAPc combined with highly purified Pol III from a HeLa cell line expressing tagged RPC4 (RPC53) (18).
As seen in Fig. Fig.1D,1D, TBP, Bdp1, and SNAPc combined with purified Pol III in the absence of Brf2 did not give rise to any detectable U6 transcript in the reconstituted in vitro transcription assay (lane 1). As expected, addition of recombinant full-length Brf2 resulted in efficient U6 transcription (lanes 2 to 4). However, when either Brf2(66-419) or Brf2(2-289) were used in place of full-length Brf2, U6 transcripts were not detectable (lanes 5 to 7 and lanes 8 to 10, respectively), suggesting that both of the regions N and C terminal to the Brf2 core domain are important for transcriptional activity.
To enable us to study the functions of the N- and C-terminal regions of Brf2 in the formation of the transcription initiation complex, we set out to establish an electrophoretic mobility shift assay (EMSA) in which we could visualize the assembly of the various U6 transcription factors on the U6 promoter. We first focused on the Brf2-TFIIIB components. As shown in Fig. Fig.2A,2A, unlike Brf2 or Bdp1, TBP on its own was able to form a protein-DNA complex on a probe containing the wild-type U6 PSE and TATA box (lanes 2 to 4). As observed previously, the addition of both TBP and Brf2 resulted in the formation of a prominent complex (henceforth referred to as the "two-factor complex," labeled 2 in lane5; see also Fig. Fig.2E,2E, which summarizes the nomenclature used for some of the complexes), reflecting the cooperative binding of these two factors (6, 27, 43). With TBP and Bdp1, a weak complex migrating much more slowly than the TBP complex was obtained (lane 6, complex labeled TBP/Bdp1). Since Bdp1 on its own does not bind detectably to the probe (lane 4), this suggests protein-protein interactions between Bdp1 and TBP on the DNA. With TBP, Brf2, and Bdp1, a robust complex migrating even more slowly than the TBP/Bdp1 complex was obtained (henceforth referred to as the "three-factor complex," labeled 3 in lane 7; see Fig. Fig.2E).2E). This complex was supershifted by anti-Bdp1 and anti-TBP antibodies (which as expected did not bind to the probe on their own, see lanes 12 and 13), confirming that it contains Bdp1 and TBP (compare lanes 8 to 11 to lane 7). On the other hand, we did not observe a supershift with any of our anti-Brf2 antibodies (data not shown). Nevertheless, since the three-factor complex was not observed in the absence of Brf2 and migrated more slowly than the TBP/Bdp1 complex (compare lanes 6 and 7), it is highly likely that it indeed contains Brf2 but that the Brf2 epitopes recognized by the antibodies we tested are masked. None of the complexes were observed with a probe containing a mutated TATA box (lanes 15 to 17), indicating that they all depend on TBP-TATA box interactions.
We next combined the Brf2-TFIIIB components with SNAPc. As shown in Fig. Fig.2B,2B, as we titrated increasing amounts of SNAPc into a binding reaction containing the three Brf2-TFIIIB components, we observed a gradual increase in the formation of a large complex (henceforth referred to as the "four-factor complex," labeled 4 in lane 4; see Fig. Fig.2E)2E) migrating more slowly than the three-factor complex containing TBP, Brf2, and Bdp1 (compare complex 4 in lanes 4 to 6 with complex 3 in lane 1). This complex was not observed in the absence of the Brf2-TFIIIB components (lanes 7 to 11) and could be supershifted with anti-TBP, anti-Bdp1, and anti-SNAP190 antibodies (Fig. (Fig.2C,2C, compare lanes 3 to 8 with lane 2), confirming that it contains TBP, Bdp1, and SNAPc. Like complex 3, complex 4 was not supershifted by the anti-Brf2 antibodies we tested (data not shown), most likely because within the complex, the relevant Brf2 epitopes are not accessible to the antibodies.
In addition to complex 4, binding reactions containing SNAPc and Brf2-TFIIIB gave rise to four prominent smaller complexes (see, for example, Fig. Fig.2B,2B, lane 5). As shown in Fig.Fig.2D,2D, comparison with binding reactions containing various subsets of the four components on either a wild-type probe or a probe with a mutated TATA box indicated that, of these four smaller complexes, the one migrating the fastest is the two-factor complex containing TBP and Brf2 (compare lanes 3 and 6, complex 2, see also lanes 8 to 10), whereas the one above it corresponds to just SNAPc bound to the DNA (compare lanes 1 and 6; see also lanes 4, 7, 9, and 10). The third complex from the top in lane 6 comigrates with one formed by TBP, Brf2, and SNAPc (compare lanes 4 and 6, complex labeled with a white circle; see also lanes 9 and 10), whereas the complex just below the four-factor complex in lane 6 corresponds to the three-factor complex (compare lanes 5 and 6, see also lanes 8 to 10). Thus, by EMSA we can visualize a complex containing the basal transcription factors SNAPc and Brf2-TFIIIB, which are known to be sufficient to reconstitute U6 transcription when combined with a highly purified Pol III complex (18), as well as visualize various subcomplexes.
We used the EMSA system described above to determine whether the N- and C-terminal regions of Brf2 might be involved in initiation complex assembly. In the interest of space, all EMSA experiments presented hereafter show only the region corresponding to the upper half of the gel containing the various complexes. Lanes 1, 2, and 3 in Fig. Fig.3A3A show the two-, three-, and four-factor complexes (containing, respectively, TBP and Brf2; TBP, Brf2 and Bdp1; and TBP, Brf2, Bdp1 and SNAPc) obtained with full-length Brf2 (complexes labeled 2, 3, and 4). With the Brf2(66-419) truncated protein, the two-, three-, and four-factor complexes were all observed (lanes 10 to 12), indicating that the region N-terminal to the core domain is not required for binding to the TBP-TATA box complex (lane 10), recruitment of Bdp1 (lane 11), or assembly of the full complex (lane 12). In contrast, with a Brf2(66-289) truncated protein containing just the Brf2 core domain and with the Brf2(2-289) truncated protein, none of these complexes were obtained (lanes 4 to 9). In particular, we did not observe a two-component complex containing TBP and a truncated Brf2 (lanes 4 and 7). The only complexes observed were those containing either just SNAPc (compare lanes 6 and 9 with lane 16) or SNAPc and TBP (compare lanes 6 and 9 with lane 13), a finding consistent with previous observations that SNAPc and TBP bind cooperatively to the U6 promoter (27, 29). Thus, the Brf2 region C-terminal of the core domain is required for detectable binding of Brf2 together with TBP to the U6 TATA box in the EMSA. Moreover, it is required for the formation of all subsequent complexes containing both Brf2 and TBP.
Brf2, which does not bind to DNA on its own, can be recruited to the U6 promoter not only through protein-protein interactions with TBP binding to the TATA box but also through protein-protein interactions with SNAPc binding to the PSE (16). However, with the truncated Brf2 proteins missing the C-terminal region, we did not observe novel complexes that might contain Brf2 but lack TBP. We wondered, therefore, whether the C-terminal region of Brf2 might be required for cooperative binding with SNAPc. As shown in Fig. Fig.3B,3B, when only SNAPc and Brf2 were added to the U6 promoter probe, we observed a weak complex migrating more slowly than the SNAPc-only complex and slightly faster than the TBP/SNAPc complex (lane 4, complex labeled with a white arrowhead, compare with lanes 1 and 3). This complex most likely contains SNAPc and Brf2 and presumably forms due to interactions between Brf2 and the PSE-bound SNAPc. Notably, a similar, even darker complex was observed when full-length Brf2 was replaced by Brf2(66-419) but not when it was replaced by Brf2(2-289) (compare lane 4 with lanes 5 and 6). Thus, the C-terminal region of Brf2 is not only required for cooperative binding with TBP bound to the TATA box but also for cooperative binding with SNAPc bound to the PSE.
These results indicate that the C-terminal domain of Brf2 is necessary for the protein to form a complex with TBP, as well as with SNAPc, on a U6 promoter. Because of the failure to form these initial complexes, higher-order complexes in turn cannot form. Thus, the inability of Brf2(2-289) to direct U6 transcription (Fig. (Fig.1D1D above) can be ascribed to a failure of transcription initiation complex assembly. On the other hand, the inability of Brf2(66-419) to direct U6 transcription probably results from a defect at a step after transcription factor assembly, such as Pol III recruitment or promoter opening.
As discussed above, Brf2 and TFIIB have several features in common but they are used by different RNA polymerases—TFIIB by Pol II and Brf2 by Pol III. To learn more about the functions of the various Brf2 regions and the determinants of RNA polymerase specificity, we constructed a set of TFIIB/Brf2 chimeric proteins, which are shown in Fig. Fig.4A.4A. We divided Brf2 into core region and regions N and C terminal to the core and TFIIB into core region and region N terminal to the core and constructed four chimeras by swapping these regions between the two proteins. The chimeras were named based on the origin of the regions from which they were comprised. Thus, for example, 2/B/− consists of the N-terminal region of Brf2, the core domain of TFIIB, and no C-terminal domain, whereas B/2/2 consists of the N-terminal region of TFIIB and the core and C-terminal domains of Brf2. The four chimeras, as well as full-length Brf2 and TFIIB, were expressed in E. coli and partially purified through a C-terminal His tag. The resulting proteins are shown in Fig. Fig.4B4B as visualized by staining with the GelCode Blue Stain reagent (Pierce) and in Fig. Fig.4C4C as visualized by immunoblot with an anti-His tag antibody after normalization of the amounts.
We then tested the four chimeras for U6 transcription in the minimal in vitro transcription system, and the results are shown in Fig. Fig.4D.4D. Combining full-length Brf2, but not full-length TFIIB, with TBP, Bdp1, SNAPc, and purified Pol III reconstituted U6 transcription (compare lanes 2 to 4 to lanes 5 to 7 and to lane 1), consistent with previous observations showing that TFIIB is not active for Pol III-directed U6 transcription (26). Both the 2/B/− and B/2/− chimeras were inactive in this assay (data not shown) as was the B/2/2 chimera (lanes 8 to 10). The 2/B/2 chimera showed barely detectable activity (lanes 11 to 13). This prompted us to widen our titration of the 2/B/2 chimera, but in all cases the U6 signal was extremely weak (lanes 15 to 19), although clearly detectable in the original film. Thus, the chimeras are generally inactive for U6 transcription, except for the 2/B/2 protein, which displays extremely weak activity.
To understand the defects of TFIIB in terms of U6 transcription activity, as well as those of the chimeric proteins, we tested their capacity to assemble a U6 preinitiation complex by using EMSA. Figure Figure5A,5A, lanes 1 to 6, show the locations of the SNAPc complex alone (lane 1), the two-, three-, and four-factor complexes (lanes 3, 5, and 6) and the complexes containing TBP and SNAPc (lane 4, complex labeled TBP/SNAPc) and TBP, Brf2, and SNAPc (lane 4, complex labeled with a white dot on the left). We first focused on TFIIB. As expected, TFIIB alone was unable to bind to the probe but bound cooperatively with TBP to the U6 TATA box (lanes 7 and 8). However, upon addition of Bdp1, no additional complex was observed (lane 9), indicating that the TBP/TFIIB complex cannot recruit Bdp1. Interestingly, when TBP, TFIIB, Bdp1, and SNAPc were present in the binding reaction, we obtained a complex containing TBP, TFIIB, and SNAPc (lane 10, complex labeled with a white dot on the left). Thus, on the Pol III U6 promoter, TFIIB can assemble efficiently with TBP and SNAPc but is unable to recruit Bdp1.
We next examined the B/2/− and 2/B/− chimeras. In Fig. Fig.5B,5B, the locations of the two-, three- and four-factor complexes in the EMSA are shown (lanes 1 to 3), as are those of the complexes containing just SNAPc (lane 13); TBP and SNAPc (lane 10, complex labeled TBP/SNAPc on the left of the panel); and TBP, Brf2, and SNAPc (lane 11, complex labeled with a white dot on the left). The B/2/− chimeric protein contains the core of Brf2 but lacks its C-terminal region. Consistent with the deletion results obtained above, which indicate that the Brf2 C-terminal region is necessary for efficient cooperative binding with TBP, as well as with SNAPc (Fig. (Fig.3),3), the B/2/− chimera was unable to form any of the Brf2-dependent complexes (lanes 4 to 6). The only complexes visible were those containing either just SNAPc or both SNAPc and TBP (see lane 6). With the 2/B/− chimeric protein, which contains the core domain of TFIIB, we observed a two-factor complex containing TBP and 2/B/− (lane 7), a finding consistent with the known ability of the TFIIB core to bind to a TBP-TATA box complex (3, 12, 17, 42). This complex was, however, unable to recruit Bdp1 (lane 8), although it could assemble with SNAPc to form a complex containing TBP, 2/B/−, and SNAPc (lane 9, complex labeled with a white dot). This is consistent with the inability of full-length TFIIB to recruit Bdp1 (Fig. (Fig.5A).5A). Moreover, the results obtained above with Brf2(66-419) (Fig. (Fig.3A)3A) indicate that the N-terminal region of Brf2 is not required for Bdp1 recruitment but do not address the possibility that this region is capable of recruiting Bdp1 in a manner redundant with the core or C-terminal regions of Brf2. The results with the 2/B/− chimera show that the N terminus of Brf2 cannot recruit Bdp1 when targeted to a TBP-TATA box complex.
The activities of the B/2/− and 2/B/− chimeras in transcription complex assembly indicated an essential role for the Brf2 C-terminal domain in binding to the TBP-TATA box complex and perhaps in recruiting Bdp1. We therefore tested in the EMSA the B/2/2 and 2/B/2 chimeras, which differ from B/2/− and 2/B/− only by addition of the Brf2 C-terminal domain. As shown in Fig. Fig.5A,5A, both proteins were capable of forming a complex with TBP on the U6 TATA box (lanes 12 and 16, complex 2), although the complex formed with B/2/2 was weaker than that obtained with either Brf2 (lane 3) or 2/B/2. This is surprising since Brf2(66-419), which lacks the N-terminal region of Brf2 altogether, binds as efficiently as full-length Brf2 to a TBP/TATA box complex (Fig. (Fig.3).3). Moreover, as shown below, B/2/2 binds as efficiently as Brf2 to TBP in a GST pull-down assay. These differences may indicate that the presence of the N-terminal region of TFIIB in place of that of Brf2 somehow reduces the B/2/2 chimera's ability to assemble with TBP on the U6 TATA box but not off the DNA. We have not, however, pursued this observation further. For the 2/B/2 chimera, its binding to the TBP-TATA box complex may be due to the TFIIB core region, the Brf2 C-terminal region, or both since these two regions both contain TBP-binding activity. However, as with B/2/2, the two-factor complex obtained with 2/B/2 migrated as a tight band close to the position observed with full-length Brf2 (compare lanes 12 and 16 with lane 3) and very different from the diffuse and slower-migrating complex observed with TFIIB and TBP (lane 8). Although we do not know what causes the TFIIB-TBP-TATA box complex to be diffuse, this suggests that 2/B/2 binds to the TBP-TATA box complex in a manner more similar to Brf2 than TFIIB.
The promoter-bound B/2/2 and 2/B/2 proteins could recruit Bdp1 (Fig. (Fig.5A,5A, lanes 13 and 17, complex 3) and SNAPc (lanes 14 and 18, complex 4), although the recruitment of SNAPc by 2/B/2 is not efficient (lane 18). The B/2/2 protein's ability to promote assembly of the four-factor complex is consistent with the results above (Fig. (Fig.3)3) showing that the core and C-terminal regions of Brf2 together are sufficient for interaction with TBP, Bdp1, and SNAPc on the DNA. Its inactivity in U6 transcription indicates that the essential function(s) of the N-terminal region of Brf2 cannot be efficiently performed by the N-terminal region of TFIIB. In the case of the 2/B/2 protein, its ability to recruit Bdp1 contrasts with the inability of 2/B/− to perform this same function and indicates that the Brf2 C-terminal region is required for Bdp1 binding. Moreover, the formation of a four-factor complex with 2/B/2 indicates that when the C-terminal region of Brf2 is present the core region of Brf2 can be replaced with that of TFIIB. Indeed, the 2/B/2 chimera is capable of directing a very low level of U6 transcription (Fig.(Fig.4D).4D). However, the efficiency of both four-factor complex assembly and transcription is very low, indicating that either the TFIIB core region is not placed optimally for full function in the chimeric protein or that there is some function of the Brf2 core region that cannot be fully replaced by the TFIIB core region. One possibility is that the interactions between the 2/B/2 chimera and both Bdp1 and SNAPc together may not be stable enough to support efficient reconstituted U6 in vitro transcription. Another possibility is that the core domain of Brf2 is involved in Pol III recruitment, which was not assayed in our EMSA experiments.
To test which Brf2 region is required to bind to TBP off the promoter DNA, we performed the GST pull-down experiment shown in Fig. Fig.6.6. GST-TBP, as well as the negative controls GST and GST-protein carboxyl methyltransferase (GST-PCMT), were immobilized on glutathione-agarose beads, and the binding of various histidine-tagged proteins to these beads was visualized by immunoblotting with anti-HT antibodies. Lanes 1 to 4 show the proteins tested, namely, TFIIB, Brf2, B/2/−, and B/2/2. Both TFIIB and Brf2 bound specifically to GST-TBP in this assay, with Brf2 binding even more strongly than TFIIB (lanes 5 to 8). Strikingly, however, the B/2/− chimera, which contains the Brf2 core but not the Brf2 C-terminal region, bound very weakly to GST-TBP (lane 11), although the signal was clearly above the background seen with both GST alone and GST-PCMT (lanes 9 and 10). With the B/2/2 chimera, which contains the Brf2 C-terminal domain, very efficient and specific binding to GST-TBP was observed (lanes 12 to 14). These results provide further evidence for the crucial role of the C-terminal region of Brf2 in TBP binding and show that the region performs this function even off the promoter DNA.
TFIIB, Brf1, and Brf2 are all members of a family of transcription factors that share a number of common features, yet they are used by different classes of promoters: TFIIB by Pol II-recruiting promoters and Brf1 and Brf2 by Pol III-recruiting promoters. We have analyzed here the functions of various regions of Brf2 for U6 transcription complex assembly and in vitro transcription, and we have tested the ability of Brf2/TFIIB chimeric proteins to perform these functions. The results, which are summarized in Fig. Fig.7,7, reveal an essential role for the Brf2 C-terminal region and provide mechanistic insights on how RNA polymerase specificity is determined at the human snRNA promoters.
We find that the N-terminal region of Brf2 can be deleted or replaced by the TFIIB N-terminal region without major effects on the ability of Brf2 to bind to a TBP/TATA box complex, recruit Bdp1, or interact with SNAPc (Fig. (Fig.7).7). The N-terminal region is, however, required for U6 transcription, and for this function it cannot be replaced by the TFIIB N-terminal domain. These results are consistent with what is known about the roles of the N-terminal domains of human and yeast TFIIB and yeast Brf1. Indeed, in these proteins, the N-terminal domain is dispensable for association with a TBP-TATA box complex and, in the case of yeast Brf1, for recruitment of Bdp1, but plays essential roles at later stages (9, 20).
In TFIIB, the N-terminal domain associates tightly with PolII and is required for Pol II recruitment (3, 4, 12, 17, 31, 42). The crystal structure of a Pol II/TFIIB complex (5) and site-specific photo-cross-linking studies of both a Pol II/TFIIB complex and a complete preinitiation complex (7, 8; see reference 13 for a review) indicate that within the N-terminal region of TFIIB, the zinc ribbon is located within the center of the pocket formed by the clamp, wall, and dock domains of Pol II, most likely making contacts with one surface of the dock, whereas the B finger, which is located immediately downstream of the zinc ribbon on the linear TFIIB sequence, extends within the space from the RNA exit channel to the active site of Pol II. The zinc ribbon and the B finger appear to form the highest-affinity anchorage point between TFIIB and Pol II, which then allows, with the help of other components of the preinitiation complex, the proper positioning of the TFIIB core region on the polymerase. This in turn likely orients the promoter DNA over the central cleft of the polymerase (7, 8). Thus, in the Pol II preinitiation complex, the N-terminal region of TFIIB does not contribute essential contacts with TBP or the promoter DNA but is intimately associated with the catalytic core of the enzyme.
In yeast Brf1, the N-terminal domain is dispensable for recruitment of Pol III but is required at a later stage, during promoter opening (14, 20). In the case of human Brf2, the function performed by its N-terminal domain is not known, but it clearly cannot be performed by the TFIIB N-terminal region. It is possible that the N-terminal regions of both Brf1 and Brf2 contribute in fact to specific recruitment of Pol III over Pol II. The TFIIB, Brf1, and Brf2 zinc ribbon domains (but not the B finger regions) are highly conserved (14, 34, 38), and so are the Pol II and Pol III dock regions (see references 7), a finding consistent with the Brf1 and Brf2 zinc ribbons associating with Pol III in much the same way as the TFIIB zinc ribbon associates with Pol II. Nevertheless, the specific amino acids involved in the interactions may be different; indeed, it is striking that, for example, the yeast TFIIB N-terminal region fails to recruit human Pol II to the adenovirus 2 major late promoter, even though both the yeast and human TFIIB N-terminal regions can recruit yeast Pol II to this promoter (39). Similarly, subtle differences within the zinc ribbons of TFIIB, Brf1, and Brf2, as well as the lack of an obvious B finger in Brf1 and Brf2, may contribute to specific recruitment of the correct RNA polymerase. However, with Brf1, and perhaps Brf2, contacts between the core and C-terminal domain and Pol III (1, 10, 25, 41) are clearly strong enough to allow recruitment of the polymerase even in the absence of the N-terminal region.
The N-terminal region of Brf2 may also be required, like that of Brf1, for promoter opening, a function specific to PolIII transcription initiation complexes which, unlike Pol II transcription initiation complexes, do not require hydrolyzable ATP to form the transcription bubble (21, 23, 24).
In TFIIB, which does not contain a C-terminal extension, the core region is sufficient for efficient binding to the TBP/TATA box complex. This is different in yeast Brf1, where the association of a truncated protein missing the C-terminal domain is so weak that it is only detected after photochemical cross-linking (9, 20, 22, 35). Brf2 appears to be very similar to Brf1. Deletion of either the core (6) or the C-terminal domain (see Fig. Fig.33 and and7)7) prevents association with the TBP/TATA box complex as detected in an EMSA in the absence of cross-linking, indicating that the core is required but not sufficient for high-affinity association with the TBP/TATA box complex.
How does the Brf2 core associate with TBP? In the case of yeast Brf1, the weak association of a truncated protein corresponding to the N-terminal half of the protein (including the core domain) with TBP is prevented by radical mutations in the TBP surface that interacts with TFIIB, suggesting that the TFIIB and yeast Brf1 core domains contact human TBP in a very similar manner (35). However, such mutations affect the association of human full-length TFIIB family members differently. Radical mutations of TBP residues E284, E286, or L287 do not prevent association with full-length human Brf1 nor Brf1-dependent transcription, but they reduce association with TFIIB and change the mobility of the TBP/TFIIB/DNA complex in an EMSA, suggesting that they somehow affect the conformation of the complex (43). The different effects of TBP mutations observed with truncated yeast Brf1 and full-length human Brf1 may result from the ability of the Brf1 C-terminal region to compensate for loss of contacts with TBP within the Brf1 N-terminal region (35), or they may reflect different TBP contacts with yeast and human Brf1 in truncated and full-length proteins. Whichever the case, in the case of Brf2, radical mutations of TBP residues E284 and E286 have an effect very similar to that observed with TFIIB (43): reduction of the intensity of the complex and change in conformation, as suggested by a mobility change in the EMSA. Mutation of TBP residue L287, on the other hand, has little effect, as it does with Brf1 (43). Thus, the association of full-length Brf2 with TBP appears to share some features with TFIIB and others withBrf1.
When we replaced the core region of Brf2 with the core region of TFIIB, which has TBP/TATA box complex binding activity, the resulting chimera (2/B/2) was only able to sustain a barely detectable level of transcription, even though it was capable of recruiting Bdp1 to the promoter. The very low levels of transcription may be due to the very inefficient recruitment of SNAPc by this chimeric protein (Fig. (Fig.5A).5A). In addition, however, it may also result from inefficient recruitment of Pol III. Indeed, the yeast Brf1 core domain associates with both the C34 (1, 25) and the C17 (10) subunits of Pol III. The core domain of Brf2 may be similarly involved in interactions with Pol III-specific subunits.
In Brf1, the C-terminal region—more specifically, the conserved region II within the C-terminal region—provides the main anchor for binding to a TBP/TATA box complex (1, 9, 22, 25). The structure of a yeast Brf1 region II/TBP/TATA box complex has been solved and shows region II in Brf1 binding to the convex and lateral surfaces of TBP (19). We find that removing the C-terminal region of Brf2 inhibits U6 transcription (Fig. (Fig.1D)1D) and prevents Brf2 from associating efficiently with a TBP/TATA box complex (Fig. (Fig.3A)3A) and with TBP in solution (Fig. (Fig.6).6). An alignment of the C-terminal domains of human Brf2 (HsBrf2) and putative Brf2 homologues from mouse (Mm), rat (Rn), zebra fish (Dr), and fugu (Tr) is shown in Fig. Fig.8A8A and reveals a striking concentration of conserved residues close to the end of the protein. Such a high degree of sequence conservation between such varied species suggests an important function and prompted us to compare this segment to Brf1 conserved region II. One possible alignment is shown in Fig. Fig.8B.8B. The yeast and human Brf1 sequences are shown on top, together with the location of α-helices as seen in the yeast Brf1 region II/TBP/TATA box complex (19). Clearly, the Brf2 conserved sequence can be aligned with the Brf1 conserved region II, with identical and similar amino acids clustering in the region corresponding to Brf1 α-helices H22, H23, and the beginning of H24, which are all involved in interactions with the convex and lateral surfaces of TBP (19). This strongly suggests that the conserved Brf2 region near the end of the protein corresponds to the Brf1 conserved region II and provides the high-affinity contacts withTBP.
Apart from interactions with TBP, Brf2 is likely also involved in interactions with SNAPc because it can be recruited to the U6 promoter by a subcomplex of SNAPc referred to as mini-SNAPc and consisting of SNAP50, SNAP43, and the N-terminal third of SNAP190 (16). Although we have not previously observed recruitment of Brf2 by mini-SNAPc, perhaps because the mini-SNAPc/Brf2 complex was obscured by a comigrating complex containing what appeared to be an alternative conformation of mini-SNAPc (27), our present data indicate that the complete SNAPc can recruit Brf2 to the U6 promoter and that this is dependent on the C-terminal domain of Brf2 (Fig. (Fig.3B).3B). Thus, the C-terminal domain of Brf2 allows recruitment of the protein to the U6 promoter, probably through direct contacts with SNAPc, even in the absence of TBP.
In the absence of the entire TFIIB-related N-terminal half of the protein, the Brf1 C-terminal domain retains some ability to bind to a TATA box/TBP complex and to recruit Bdp1 (20). Our results suggest that the C-terminal domain of Brf2 is similarly involved in Bdp1 recruitment. Indeed, we find that the chimeric protein 2/B/− binds strongly to the TBP/TATA box complex but fails to incorporate Bdp1 (Fig. (Fig.5B).5B). However, when the C-terminal region of Brf2 is added to the 2/B/− chimera to form 2/B/2, Bdp1 can then be recruited (Fig. (Fig.5A).5A). Thus, the C-terminal domain of Brf2 appears to play a crucial role in the assembly of the preinitiation complex by providing stabilizing interactions with TBP, SNAPc, and Bdp1.
RNA polymerase specificity is determined at two levels. The first level is the arrangement of the promoter elements themselves. In the human snRNA promoters, it is the presence or absence of a TATA box downstream of the PSE that largely determines Pol III or Pol II transcription, respectively. This in turn somehow results in the specific recruitment of either TFIIB in the case of Pol II snRNA promoters or Brf2 in the case of Pol III snRNA promoters. Ironically, we do not yet understand how the presence of a TATA box specifies Brf2 recruitment over TFIIB recruitment. Indeed, we show here that it is possible to assemble efficiently a complex containing TBP, SNAPc, and TFIIB (or the 2/B/− chimeric protein) on the U6 promoter. This is somewhat reminiscent of the situation in the yeast U6 snRNA promoter, which consists of both a TATA box upstream of the transcription start site and a downstream binding site for the factor TFIIIC. The yeast U6 upstream promoter has no specificity for recruitment of Brf1 over TFIIB. Rather, it is the binding of TFIIIC to the downstream site that specifies Brf1-TFIIIB recruitment and Pol III transcription (32). It seems likely that, in human cells, as-yet-unidentified factors associated with either TFIIB, Brf1, TBP, or SNAPc prevent assembly of TFIIB and/or favor assembly of Brf2 into the U6 initiation complex.
The second level of RNA polymerase specificity determination is protein-protein interactions among factors recruited by the promoter elements. Thus, even though the U6 promoter is capable of assembling complexes containing either TBP, SNAPc, and TFIIB or TBP, SNAPc, and Brf2, only the latter complex (or complexes formed with TFIIB/Brf2 chimeras containing the Brf2 C-terminal region) is capable of recruiting Bdp1. Moreover, the C-terminal domain of Brf2 may also be involved in the recruitment of Pol III. With yeast Brf1, the C-terminal domain of the protein, and in particular homology domains II and III, have been shown to associate with the yeast Pol III subunit C34, which has no counterpart in Pol II (1, 25). Thus, the C-terminal extensions present in the TFIIB family members involved in RNA Pol III transcription, i.e., Brf1 and Brf2, are key to favoring recruitment of Pol III over that of PolII because they allow recruitment of Bdp1 and, probably, of Pol III itself through Pol III-specific subunits.
We thank Robert S. Haltiwanger, Patrick Hearing, and Winship Herr for helpful comments during the course of this project. We also thank Yuling Sun for generating the constructs expressing the TFIIB-Brf2 chimeras and J. Duffy for artwork and photography.
This study was funded in part by NIH grant GM38810. N.H. and B.M. are supported by the Howard Hughes Medical Institute.
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.


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University of North Texas Health Science Center At Fort Worth
Graduate School of Biomedical Sciences
Clinical Research Management
3500 Camp Bowie Blvd.
Fort Worth, TX 76107
United States
Program Information
Degrees Offered:
Clinical Research Management
Format: Campus
Program Description:
In depth knowledge of the CFR and GCP guidelines as well as International Guidelines specifically as they relate to protection of human rights, prevention and detection of fraud and the use of sound scientific principles, is a fundamental requirement for a clinical research professional. These individuals are key personnel involved in the conduct of clinical trials, which in turn are pivotal in getting new products approved and on the market.
The master's program in Clinical Research Management will provide a strong foundation upon which to build a career. The rigorous curriculum focuses on providing students a broad-based view of the biomedical sciences, as well as in depth knowledge of regulatory requirements (code of federal regulations, good clinical practices), ethical issues, and both the medical writing and administrative skills necessary to conduct clinical research. As part of the program, all students will complete an internship practicum in clinical studies and use this experience to write the thesis pursuant to receiving the Master of Science degree. The average time to complete the degree is eighteen months.
Accreditation:The UNT Health Science Center is regionally accredited by the Southern Association of Colleges and Schools.
Facts & Figures
Financial Aid: Yes
International Financial Aid: Yes
Classification: Specialized Institution—Medical school or medical center
Locale: Large City
Size & Settings: 1,000-2,999

subset: commoncrawl


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Race about on one of the largest fleets of Segways in Europe.
The UK's first Segway Polo team was started at this very company, so you and your group are playing with the best. The sport has been adapted from bicycle and horse polo – you still swing your mallet at the ball, just on a motorised vehicle… Segway Polo uses a smaller pitch, and you and your group will be split into two teams of five and taught all the basics, before one huge game. During the game, you'll be able to reach speeds of up to 14 miles an hour – it's seriously fast-paced.
You lot will also attempt a series of exciting Segway games. From Segway racing, to Frisbee, to even football – you and your group will be bombing about on the pitch in no time.
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VideoHive Crysis V.2 946341
After Effects Project Files \ Titles \ 3D Object
Crysis V.2 946341 by Sale @ VideoHive
After Effects Project Files \ Titles \ 3D Object
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Search the Community
Showing results for tags 'draft'.
Found 1 result
Will the browns finally turn it around
greenpassion posted a topic in Around the NFLwell, cleveland has been absolutely terrible for years upon years but they now have a very good opportunity to turn things around with a ton a early draft picks. i dont know exactly what they have but i think they have like two 1st, two 2nds just this year with 3 2nds next year in total i think its 22 picks over the next two drafts with most the extra picks coming in the early rounds. most fans seem to think they will just screw this up and i gotta say imo they are off to a terrible start if they pull the trigger on the deal with the pats for garropolo. i really dont understand why they want to do this with a guy that has a total of 2 starts and rumors are that NE is asking for a kings ransom


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One of our first products and most popular requests, our surfboard bar tops come with restaurant quality cast iron legs and sit at just over 40" tall. Each 8' long surfboard bar top is ideal for your tiki-bar, pool area, or beach themed room. Each redwood surfboard bartop includes extra layers of polyurethane finish for added protection and durability and features exotic hardwood details in the nose, tail, and fin. Customize your bar top with the choice of a vintage or a modern style fin. If you already have a base, we can provide just the surfboard counter and leave off the legs, call or email for details.
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Students in Amos Scully's Sophomore Studio toured Advantech, a low volume sheet metal and machining facility that does work in the defense, health care, and transportation industries. At their facility, students were impressed by the 240 ton metal brakes, robotic welding, CNC mills that automatically change out flutes, a laser cutter capable of cutting a 1.25″ armor plate with precision tolerances.
The students viewed New York subway car panels that were designed by Antenna Design, who also gave a guest lecture here at RIT last month.
Thank you, Marissa Allen and Advantech, for the fabulous tour.


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Carolyn Rose Shirk
- 58 years old
- Date of birth: Sep 17, 1956
- Date of passing: Aug 5, 2015
|Let the memory of Carolyn be with us forever
This memorial website was created in memory of our loved one, Carolyn Shirk, 58, born on September 17, 1956 and passed away on August 5, 2015. We will remember her forever.
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"I still find it difficult to comprehend that you're gone from this earth. We had so many happy times together and created a lifetime of memories. You were the sister I never had, in good times and the not-so-good times. I'm sure you are at peace now, and I hope that we will be reunited someday and be eternal friends. Miss & love you, Carol."
"I miss you today as much as I did the moment you passed.
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All my love."
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DEP Regulatory Requirements Chapter 78 Subchapter D. Dave English Division of Compliance and Data Management Bureau of Oil and Gas Management. Focus. Significant changes to Subchapter D. Relevant revisions to Subchapters A, C, and E.
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Dave English
Division of Compliance and Data Management
Bureau of Oil and Gas Management
Well Control
Well Construction (casing and cementing operations)
Mechanical Integrity of Existing Wells
Gas Migration Response
Well Reporting
Subchapter A: General Provisions
Subchapter A: General Provisions – new definitions added
Conductor pipe
Intermediate casing
L.E.L. (lower explosive limit)
Unconventional formations
Subchapter C: Environmental Protection Performance Standards
Plan must include operator's pressure barrier policy that identifies barriers to be used during specific operations
Plan must be available at the well site for review during drilling and completion activities
List of emergency contact phone numbers for the area in which the site is located must be prominently displayed at the well site during drilling, completion, and workover activities
BOP equipment to be used:
When drilling well intended to produce natural gas from an unconventional formation
When drilling out frac plugs
Where pressures are anticipated at the well site that may result in a loss of well control
Where operator is drilling in an area where there is no prior knowledge of pressure or natural open flow
When drilling conservation wells
When drilling within 200 feet of a building
Surface casing may not be set more than 200 feet below the deepest fresh groundwater except as necessary to set the casing in consolidated rock
Surface casing hole must be drilled using air, freshwater, or freshwater-based drilling fluid
Wellbore must be conditioned to ensure an adequate cement bond between the casing and formation prior to cementing
Centralizers: at least one within 50 ft. of the surface casing seat, then in intervals no greater than every 150 ft. above the first centralizer
Regulatory definition of "deepest fresh groundwater"
"The deepest fresh groundwater bearing formation penetrated by the wellbore as determined from drillers logs from the well or from other wells in the area surrounding the well or from historical records of the normal surface casing seat depths in the area surrounding the well, whichever is deeper. "
Buckwalter & Moore (2006)
Standard groundwater quality classification schemes
Fetter (1994)
Fresh 0 to 1,000 mg/l TDS
Brackish 1,000 to 10,000 mg/l TDS
Saline 10,000 to 100,000 mg/l TDS
Brine >100,000 mg/l TDS
Quiñones-Aponte & Wexler (1995)
Fresh <1,000 mg/l TDS
Slightly Saline (brackish) 1,000 to 3,000 mg/l TDS
Moderately Saline (brackish) 3,000 to 10,000 mg/l TDS
Very Saline (saltwater) 10,000 to 35,000 mg/l TDS
Brine >35,000 mg/l TDS
Olsthoorn (2008)
Some numerical considerations in Pennsylvania
Delaware River Basin Commission (DRBC)
Freshwater is "water containing less than 1,000 mg/l of dissolved solids, most often salt."
40 CFR 144.3 – United States EPA
"Underground source of drinking water (USDW) means an aquifer or its portion: (a)(1) Which supplies any public water system; or (2) Which contains a sufficient quantity of ground water to supply a public water system; and (i) Currently supplies drinking water for human consumption; or (ii) Contains fewer than 10,000 mg/l total dissolved solids; and (b) Which is not an exempted aquifer."
"10,000 mg/l is FAR TOO SALINE for drinking water supplies in this Commonwealth"
Numerical considerations elsewhere
Texas: 3000 mg/l TDS
Oklahoma: 10,000 mg/l TDS
Illinois: 10,000 mg/l TDS
New York: 1,000 mg/l TDS
Alberta: 4,000 mg/l TDS to a depth not to exceed 600 meters
Numerical considerations (31 states surveyed)
Numerical considerations (15 states with quantitative definition)
Techniques for defining base of deepest fresh groundwater aquifer
Estimating fracture zone yield and measuring specific conductance using a calibrated meter during drilling
Standard water well geophysical logging of tophole – specific conductance critical, but other logs may help corroborate water-bearing zones
More sophisticated geophysical logging of tophole per EPA UIC recommendations (SP log or resistivity/porosity log)
Installation of monitoring wells at well pad and groundwater testing
Information from offset wells including water well testing, geophysical log data, and surface casing set depths;considering water well offsets alone will typically not be enough
Williams (2010)
Policy: cement returns to surface followed by cement drop may be considered to be permanently cementedifthe DEP inspector determines an adequate amount of surface casing cement was placed above the seat.
Top of cement determination must be made and notification given to the DEP inspector for evaluation of casing cement adequacy and subsequent approval for remedial casing options. Must be done prior to continuation of drilling (e.g. no TOC determination after well drilled/completed to TD).
In addition to remedial casing options, the minimum amount of surface casing cement above seat and corresponding maximum amount of uncemented surface casing will be made on a case-by-case basis by DEP. In certain cases, the well may need to be plugged and abandoned if only a minimal amount of cement exists above the surface casing seat (a "catastrophic" loss of cement).
DEP may require remedial cementing from surface and/or pressure-testing of the casing string to determine integrity of the well and ensure protection of the surface casing seat.
Revised cement standards:
Cement must protect casing from corrosion and geochemical, lithologic and physical conditions of the surrounding wellbore
Gas-block additives and low fluid-loss slurries in areas of known shallow gas-producing zones are required
Zone of critical cement around surface casing seat
True eight-hour WOC (wait on cement) before casing may be disturbed
One-day notification to DEP prior to cementing of surface casing
Cement job log must be prepared and available at the well site during drilling operations and maintained for at least five years
Zone of Critical Cement:
Applies to bottom 300 ft. of surface casing cement, or entire cemented string if the surface casing string is less than 300 ft
Cement must achieve a 72-hour compressive strength of 1200 psi
Cement must achieve a free-water separation of no more than 6 milliliters of water per 250 milliliters of cement
Eight-hour WOC (wait on cement) – casing may be not be disturbedby:
Releasing pressure on the cement head; if check valves on float shoe are secure, the pressure may be released at a continuous, gradual rate after four hours
Nippling up on or in conjunction to the casing
Slacking off by the rig supporting the casing in the cement sheath
Running drill pipe or other mechanical devices into or out of the wellbore with the exception of a wireline used to determine the top of cement
Cement job log – required components:
Mix water temperature and pH
Type of cement with listing and quantity of additives
Volume, yield, and density in ppg of the cement
Amount of cement returned to the surface
Cementing procedural information including a description of the pumping rates in bbl/min, pressure in psi, time in min, and the sequence of events during the cementing operations
Logs must be available for all cement jobs done after 2/5/2011.
Quarterly monitoring program will begin first quarter after the Department develops a standard form for collecting mechanical integrity data
Key monitoring/testing provisions
Pressure monitoring associated with production casing
Pressure monitoring in annular space associated with production casing
Pressure monitoring at relevant casing seat
Checking well fluid level in production casing
Corrosion and equipment deterioration survey
Monitoring for leaking gas
Clear methodology for addressing over-pressured wells
Flexibility for Department to require additional testing
Report detailing results of quarterly inspections must be submitted to Department annually by January 31 of year following inspections
For wells not in compliance, the operator must immediately notify DEP and take corrective action to mitigate the excess pressure on the surface casing seat, coal protective casing seat, or intermediate casing seat when the intermediate casing seat is used in conjunction with the surface casing seat to isolate fresh groundwater
Corrective action occurs in the following hierarchy:
Operator must reduce the shut-in or producing back pressure to achieve compliance with 78.73(c)
Operator must retrofit the well by installing production casing to reduce pressure on the casing seat to achieve compliance with 78.73(c); the annular space surrounding the production casing must be open to the atmosphere; production casing must either be cemented in place or installed on a permanent packer
Operator must notify DEP 7 days prior to initiating corrective action
Potential well problems
Potential well problems: overpressuring
Harrison (1985)
Potential well problems: overpressuring (continued)
Harrison (1985)
Potential well problems: overpressuring (continued)
Harrison (1985)
Potential well problems: cement failures and inadequate casing/tubing
Some notable items
Operators will not be required to retrofit older wells for pressure monitoring
Overpressured conditions or problems noted during well corrosion/equipment deterioration survey must be reported immediately
7-day notification for wells that will be retrofitted with production casing
Some notable items (continued)
Water protection depth will apply in older wells where fluid levels can be determined
Pressure monitoring locations will vary as a function of well construction
Department projects underway or being considered to assist the industry
Development of comprehensive technical guidance/instructions to accompany form to ensure consistency and ease of implementation
Development of tracking system for problems noted to help identify what well maintenance procedures are critical during various points throughout operational history
Stray gas migration incidents continue to represent one of the most significant problems associated with oil and gas development in the Commonwealth
Previous discussion on well integrity highlighted some problems that result in stray gas migration incidents
Other contributing factor in Pennsylvania is the number of legacy/abandoned wells that were never properly plugged and whose locations remain unknown
Stray gas migration associated with Marcellus Shale development has been geographically isolated
The simplest of all paraffin hydrocarbon gas
Flammable, colorless, and odorless
Specific gravity: 0.555
Explosive range: 5-15%
Maximum solubility in water: 26-32 mg/l at standard temperature and pressure, but higher at depth due to pressure regime
Baldassare (2009)
Changes in barometric pressure
Soil and bedrock porosity/permeability
Pore water
Temperature contrasts
Other meteorological conditions including precipitation (rain vs. snow) and ground cover (layer of snow or frozen ground)
Figure courtesy of John Harper, PA Topographic and Geologic Survey
Subsurface microbial gas (deep- sea sediments and drift gas)
Near-surface microbial gas (marsh gas and landfill gas)
Thermogenic gas (natural gas and coalbed gas)
Baldassare (2009)
Locations: total number of stray gas cases since 1987 compared to all permitted drilling activity
Location: of Marcellus Shale stray gas cases since 2008 compared to Marcellus Shale drilling activity between 2008 and 2010
Recent trends in stray gas incidents: Marcellus Shale versus non-Marcellus Shale wells
Operators notified about a potential stray gas migration incident must immediately conduct an investigation to determine nature of incident, assess potential hazards, and mitigate hazards as needed
Response actions are tiered based on the severity of the incident
Investigation closure dependent upon Department approval
Category 1 (Immediate Threat): detectable concentrations equal to or greater than 10% of the lower explosive limit (LEL) or combustible gas in a building or structure(s), or otherwise deemed Category 1 by the Department.
Category 2 (Potential Threat): detectable concentrations less than 10% of the LEL of combustible gas in a building or structure(s), and/or combustible gas greater than 50% of the LEL in the headspace of a water well, and/or visual or audible evidence of stray gas bubbling through a water well column or surface body, and/or detectable concentrations of stray gas in the soils, and/or concentrations of dissolved methane in water at or above 25% of the lower solubility limit for methane (7 mg/l).
Category 3 (No Apparent Threat): none of the above conditions were met. If conditions indicate methane in groundwater at concentrations above 0.5 mg/l, but below 7 mg/l, continued monitoring is necessary to ensure that concentrations do not trend to a Category 2 potential threat.
Department projects underway or being considered to assist the industry
Development of stray gas migration technical guidance document to compliment new regulations
NCRO Stray Gas Prevention Program
Series of joint technical guidance and public outreach documents with Emergency Response staff
Subchapter E: Well Reporting
Subchapter E: Well Reporting – Revisions
78.121. Production reporting: Incorporates the requirements of Act 15 of 2010 which mandates semi-annual reporting of production of Marcellus Shale wells (8/15 & 2/15); Non-Marcellus wells report annually (2/15); Information is posted on DEP's website
78.122. Well record and completion report: Completion report to include: descriptive list of chemical additives used in the stimulation fluid; the percent by volume of those additives; a list of hazardous chemicals used in the stimulation fluid (MSDS/CAS #); the percent by volume of those hazardous chemicals; the total volume of water used; a list of water sources used pursuant to an approved water management plan; the total volume of recycled water used; and the pump rate and pressure used in completing the well
Operator must designate separate sheet as confidential or a trade secret; DEP will prevent disclosure of confidential information to the extent provided by the Right-To-Know Law
Well record adds certification by operator that well has been constructed in accordance with this Subchapter and any permit conditions imposed by DEP


text:
I've lived in Alaska since 1969. I love this wild land. The extremes of the far north shape and affect every living thing. Extremes of weather, elevation, daylight hours, isolation, flora and fauna, nothing in Nature is insignificant.I have a Davis Vantage Pro weather station, uploading to Wunderground, (Turks on the Tundra) the National Weather Service, the Citizen Weather Observation Program and MADIS (Meteorological Assimilation Data Ingest System). "May you have warmth in your igloo, oil in your lamp, and peace in your heart." ~ Indigenous Arctic Proverb
Uploaded by: katy99780
Tuesday September 10, 2013
Aberdeen, WA (Current Weather Conditions)
Caption: Love the massive giants of the Washington coast.
EXIF Data
Manufacturer: Apple
Model: iPad
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Compression: JPEG compression
Exposure Time: 1/267 sec.
FNumber: f/2.4
Exposure Program: Normal program
ISO Speed Ratings: 50
Exif Version: Exif Version 2.2
Components Configuration: Y Cb Cr -
Shutter speed: 8.06 EV (APEX: 16, 1/267 sec.)
Aperture: 2.53 EV (f/2.4)
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Metering Mode: Pattern
Flash: No flash function.
Focal Length: 4.3 mm
Subject Area: Within rectangle (width 699, height 696) around (x,y) = (1295,967)
FlashPixVersion: FlashPix Version 1.0
Color Space: sRGB
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Sensing Method: One-chip color area sensor
Exposure Mode: Auto exposure
White Balance: Auto white balance
Focal Length In 35mm Film: 35
Scene Capture Type: Standard


text:
Answers, much like victories, seem to be hard to come by these days for the Cincinnati Bengals.
A number of questions arose Sunday as the Bengals (3-7-1) were extending their winless streak to four games with a 19-14 loss at Baltimore, but head coach Marvin Lewis and offensive coordinator Ken Zampese ranged from vague to evasive when asked about various topics Monday afternoon.
Talking about the decision to replace starting center Russell Bodine, whose premature snap resulted in the first of two lost fumbles in the red zone, with T.J. Johnson, Zampese said, "(Johnson) did a nice job. It's good to have an experienced guy to go to when you need him."
Does the phrase "need him" mean Bodine was injured or benched?
"T.J played, and he played good," Zampese answered.
>>>>RELATED: Bengals Report Card from loss to the Ravens
Johnson's first action came on the 11-play touchdown drive in the third quarter. He also was in for the following series before Bodine went back in for the final two drives.
Lewis' explanation for the change was curious.
"It was to calm the troops a little bit," he said. "I think he did a good job of that. We felt good about what he did."
>>>>MORE: 7 things to know about Bengals loss to the Ravens
Also puzzling is the status of kicker Mike Nugent, who missed his third consecutive extra point and fourth in his last eight attempts, after Lewis attempted to address the situation.
"A missed PAT, those are things that affect the football team and affect us and our opportunity, but now we are where we are," Lewis said.
He was much more assertive in his support last week following the two misses against Buffalo, saying definitively that team was moving forward with Nugent as the kicker.
Lewis did say the Bengals will not be bringing in any kickers for tryouts Tuesday, but that doesn't necessarily mean the team is still intent on sticking with Nugent.
Follow Jay Morrison on Twitter
Four weeks ago after the 27-27 tie against Washington, in which Nugent missed an extra point and a field goal, the Bengals tried out Kai Forbath, Zach Hocker and Randy Bullock before electing to stick with Nugent.
The Vikings signed Forbath on Nov. 16, but Bullock and Hocker, who was with the Bengals during training camp but didn't get a chance to compete against Nugent due to a tendon injury in his plant foot, are still available and the Bengals don't need another tryout to make a decision.
The Bengals also are searching for the answer why the defense continues to struggle early, allowing opening drive touchdowns in six of the last eight games. And why offense falls apart late, with just one fourth-quarter touchdown in the last four games.
"They are areas that need to get done, Lewis said. "It's important to win that fourth quarter scoring, and we haven't been able to do that. We can move the ball, but haven't come away with points. We had two turnovers in the red zone yesterday. We had turnovers other times in the season too, and those are huge. Defensively, we just have to settle down and play. We're too excitable, or whatever it may be. We have some error, and we have to eliminate that."
One thing both Lewis and Zampese were eager to clear up was why the Bengals called a timeout that enabled Baltimore more time to look at replays and eventually challenge the spot of a third-down reception by James Wright that initially was ruled a first down.
After the review, the officials overturned the call, ruling Wright was a half yard shy of the marker, and the Bengals had to punt.
"They didn't re-start the (play) clock," Zampese said. "It was questionable whether we got the first down or not. They're deciding on it. Now all of a sudden the clock keeps going. And they didn't push the clock."
Like our Cincinnati Bengals News Now Facebook page
The 40-second clock started within seconds of Wright hitting the ground. The officials didn't signal first down until there were 31 seconds remaining. By the time the Bengals broke the huddle, there were only eight seconds left, forcing Dalton to call the timeout.
"I have to make sure we get a new clock in that situation, so I had to burn a timeout there," Lewis said. "They should see it. I shouldn't have to. We were down on time and so Andy didn't have an opportunity to get the play off. I was headed that way. We didn't get a new clock. Right now those things aren't working in our favor. We're not getting those breaks."
Roster moves: The Bengals re-signed defensive tackle DeShawn Williams on Monday and placed cornerback Chykie Brown on season-ending Injured Reserve.
Williams rejoins the team after he was waived Saturday when the Bengals brought long-snapper Tyler Ott off the practice squad to replace the injured Clark Harris.
An undrafted college free agent out of Clemson, Williams spent 2015 on the Bengals practice squad and was inactive for the first 10 games this season.
Brown suffered a season-ending knee injury in second quarter while covering a punt. He appeared in five games this season with one tackle on defense one on special teams.


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The 4WD Magna, which would sit above the new sporty VR-X model (pictured) to be launched next week, would take on the hot local offerings from Ford and Holden. A price tag between $45,000 and $50,000 is likely.
The car would capitalise on Mitsubishis success in world and local rally championships and the Ralliart organisation, which controls Mitsubishis motorsport operations, may be involved in developing the range-topping car.
The 4WD model would play a hero model role for the company. Mitsubishi Australias new boss, ex-Toyota executive Tom Phillips, sees changing the companys image as a top priority in lifting sales.
We should have a performance image, something that stands apart from the other local car makers. We have to make the It Is campaign mean something, he said.
The development process for the 180kW engine has already started, with a new camshaft delivering an additional 10kW over what is expected to be the 160-165kW delivered by the revamped 3.5-litre V6 engine that will power the VR-X.
But fitting a four-wheel-drive system to the Magna may prove tricky as the local car has a different front suspension and fuel tank to its Japanese sister model, the Diamante.
Local Mitsubishi engineers will discover how hard it is in a couple of weeks when a 4WD Diamante development car arrives from Japan.
Standard features will include five-speed automatic, climate-control air-conditioning, leather trim and an eight-speaker sound system.
No matter when the 4WD Magna arrives, the VR-X will be updated next year with 17-inch alloy wheels to replace the original 16-inch items. The 180kW engine is also expected to be fitted to the front-drive VR-X. To quell excess wheelspin, the updated VR-X is tipped to get a limited-slip differential.
Meanwhile, Mitsubishi Motors global president, Katsuhiko Kawasoe, will today in Queensland move to reassure the public that the company will continue to build cars in Australia now that its local offshoot is again making money.
The show of support from Kawasoe should allow the cash to develop an all-new Magna to go on sale in 2004 to flow.


text:
3D Digital Clock
Visit Publisher Site
3D Digital Clock Hosting Promo
Digital clock in pseudo-3D drawn from scratch using grass-roots AWT methods. compiled with jdk 1.0.Visit publisher site: 3D Digital Clock
Listing Details
- Version:
- jdk 1.0
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Scripts / Java / Applets / Clocks
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- Feb 17, 2003
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text:
Visit us at:
Customization Opportunity
The Modulating RetroReflector is highly customizable to suit the exacting requirements of your applications. The specifications listed (left) are for the default device that is intended for laboratory and research applications.
Please contact us to arrange a meeting with our engineering team to discuss how the MRR can be customized for you.
MRR Specifications
* Custom-sized apertures available upon request
Low Power MEMS Modulating RetroReflector for Free Space Optical Communication.
When mounted as one facet of a hollow corner cube retroreflector, the Boston Micromachines MEMS Optical Modulator is capable of passively returning light from an interrogating laser source while simultaneously modulating its intensity for asymmetric communication. In its unpowered state the device acts as a typical retroreflector, returning a high percentage of light to the source, while, in its powered state, most of this light is now scattered.
We are here to help with your questions on products, documentation downloads and getting you the latest software drivers.
Download our Whitepapers for more
information on products and applications.
Pair your Modulating RetroReflector with the
MB-200 Driver for a complete modulation solution.
Click here for specifications on this device.
The Modulating RetroReflector (MRR) system has been demonstrated to provide continuous asymmetric free space optical communication at a 180 kHz data rate. The MRR subcomponent is housed in a compact, easy-to-integrate package geared for laboratory technology demonstration, shown above. This technology can be used out of the box or integrated into a complete setup through the use of standard optical components. The MRR can be paired with the MB-200 Driver to enable immediate testing of an optical protocol using a standard TTL input. See below for complete device specifications.
E: email@example.com
Above: Front view of the Modulating RetroReflector
We support researchers in the grant application process.
Above: Modulating RetroReflector in a Gimbal mount
Take a look at our whitepapers for more information on our Modulating RetroReflector or Optical Modulation solutions.
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30 Spinelli Place | Cambridge, MA 02138
Phone: 617-868-4178 | Fax: 617-868-7996
Copyright 2016. Boston Micromachines Corporation. All rights reserved.
Above: Side view of the Modulating Retro Reflector


text:
I have witnessed the tremendous change Erika's Lighthouse has brought to a community over the last ten years. The educational programs offered have had a great impact on so many lives. Reducing the stigma surrounding depression so teens can get the help they need, and proceed with productive lives is the core of the organization's message. Programs offered for educators can be easily integrated into various curriculums and school cultures. There are videos featuring real teens speaking candidly about mental health and important skills needed to help themselves or a friend and let their parents know what they may be feeling. The Parent Handbook gives families much needed support and is full of valuable information. The website has lots of information about programs offered - www.erika's lighthouse.org
We started Erika's Lighthouse to change attitudes about mental health, and i think that slowly but surely, we make a difference in young peoples' lives. Join us, on our quest!
I have been involved with ELH for the past 5-6 years and it's been amazing to watch the impact the organization has had on the communities in the Chicago suburbs. In a relatively short time Erika's Lighthouse has provided area schools with the tools with which to educate students, faculty and parents about the signs and symptoms of adolescent depression. The best part is that the students in the ELH clubs (at quite a few schools now, and growing!) are the ones sharing this information with their peers and opening up dialogue that wouldn't have taken place just a few years ago.


text:
Lynx Racing, with drivers Buddy Rice and Mike Conte are prime contenders to hit the bulls-eye in the KOOL/Toyota Atlantic support race at the Target Grand Prix, August 21. Atlantic is the premier stepping-stone series to the CART...
Lynx Racing, with drivers Buddy Rice and Mike Conte are prime contenders to hit the bulls-eye in the KOOL/Toyota Atlantic support race at the Target Grand Prix, August 21. Atlantic is the premier stepping-stone series to the CART FedEx Championship, and Lynx Racing - the only championship-winning, woman-owned team in a major auto racing series - is one of the top teams. The past three Lynx drivers have graduated to CART: Patrick Carpentier to Player's/Forsythe, Alex Barron to Dan Gurney's All-American Racers and Marlboro Penske and Memo Gidley to Walker/Alpine Racing and Payton-Coyne/Herdez. Lynx driver Buddy Rice and Lynx graduate Memo Gidley teamed to win every oval track race on the Atlantic circuit in 1998, and Rice, with new teammate Mike Conte, has led the last four Atlantic races and scored three podium finishes in a row in 1999. "Lynx is used to winning, and we're planning to do just that at Chicago," says Rice, 23, of Phoenix, Arizona. "Since this is the first race ever held on this track, it's a level playing field for drivers and teams. We won every Atlantic oval track race last year, and we've been the most consistently competitive team this season. There are a lot of good, experienced drivers and teams in the Atlantic series this year, but not one of them wants a win in Chicago more than we do." Mike Conte, 30, of Seattle, Washington, is new to the Lynx team this year. He is a software designer who formerly worked for Microsoft and was a major force in creating several of their most popular programs, including Windows 95 and Internet Explorer. He now drives full-time with Lynx and also runs his own Porsche-based sports cars team - Contemporary Motorsports - that scored a podium finish in the 12 Hours of Sebring earlier this year. "Oval track racing is as much black art as science," says Conte. "So it's a big help that at Lynx we have both wizards and computer wonks to help us figure out the setup. And getting the car right is absolutely essential on an oval, particularly this oval, because with long straights and tight, constant radius turns, it looks we'll need to brake, and possibly downshift, going into the turns. You'll really have to drive it, not just circulate around with the gas pedal flat on the floor like some ovals. So this weekend will be a race of engineers and computers as much as drivers... until the green flag drops on Sunday, anyway." Lynx Racing is the only championship-winning racing team and driver development program owned by women that competes in a major auto racing series. Created by Peggy Haas and Jackie Doty, Lynx Racing is now in its ninth year of seeking out young drivers with championship potential and providing them with the funds, training and equipment to make it to the top levels of the sport. Lynx alumni include Patrick Carpentier, who won the 1996 Atlantic championship, was the 1997 CART Rookie of the Year, and now drives for Player's/Forsythe. Lynx's 1997 graduate, Alex Barron, won the Atlantic championship in his first and only year with the team, and was quickly signed by Dan Gurney's All-American Racers for the 1998 season. This year he also drove for Marlboro Penske in the U.S. 500 at Michigan, and will drive for them again in the season-ending race at Fontana. Lynx Racing's 1998, Memo Gidley, impressed CART insiders with four races as a substitute for the injured Naoki Hattori on the Alpine/Walker Racing team, and has now moved over to run in a second Herdez-backded car with Payton-Coyne. ______________________________
Target Grand Prix Schedule of Events:
Thursday, August 19 8:00 a.m. - 8:45 a.m. Atlantic practice 12:30 p.m. - 1:00 p.m. Atlantic practice 4:15 p.m. - 4:45 p.m. Atlantic practice
Friday, August 20 8:45 a.m. - 9:15 a.m. Atlantic practice 1:45 p.m. - 2:15 p.m. Atlantic practice 5:05 p.m. - 6:00 p.m. Atlantic single-car qualifying
Saturday, August 21 11:15 a.m. - 11:30 a.m. Atlantic warm-up 3:00 p.m. - 4:00 p.m. Atlantic race / 60 laps
Television Schedule:
ESPN2 Sunday, August 29 10:30 a.m. - 11:30 a.m. ESPN2 Tuesday, August 31 2:30 a.m. - 3:30 a.m. (repeat) ESPN2 Friday, Sept. 3 2:30 p.m. - 3:30 p.m. (repeat) CTV (Canada) Tuesday, August 31 8:00 p.m. - 9:00 p.m. RDS (Canada) Sunday, Sept. 9 Check local listings ESPN Intl. Sunday, August 29 10:30 a.m. - 11:30 a.m.
Next Event on Atlantic Schedule:
Molson Indy Vancouver / September 4


text:
https://web.liferay.com/web/robert.biter/profile/-/activities/rss 2017-11-18T04:53:02Z 2017-11-18T04:53:02Z Robert Biter commented on LPS-19139. 2011-07-26T07:31:11Z 2011-07-26T07:31:11Z You add this to portal-ext.properties default.user.public.layouts.lar=${liferay.home}/data/user_public.lar default.user.private.layouts.lar=${liferay.home}/data/user_private.lar You add the lars to the data directory (found next to tomcat and deploy) To see the contents of the lars, you can change their extension to .zip and browse through them like an archive. When you export them, have 3 public pages with some portlets and 2 private pages with other portlets. You will see it exports the same data. 2011-07-26T07:31:11Z Robert Biter commented on LPS-19139. 2011-07-25T11:34:40Z 2011-07-25T11:34:40Z Steps to reproduce (Revision 85838): 1 Login with firstname.lastname@example.com 2 Have a couple of public pages and private pages for him (I had the social networking portlets on them). 3 Go to my public pages 4 Manage Pages 5 Export (public.lar) 6 Go to my private pages 7 Manage Pages 8 Export (private.lar) The exported lars contain the same layouts and the same portlets (both private and public) even though they are different. Using this lars as default lars for a new user nets me a user that only has private pages and the pages are actually a copy of the pages I had as public pages when I exported the lar. If you can't reproduce this please tell me. 2011-07-25T11:34:40Z Robert Biter commented on LPS-19139. 2011-07-22T09:18:08Z 2011-07-22T09:18:08Z For me, it exported the private pages instead of the public pages. So, when I created the default lars for new users, they only got private pages even if I created the lars from public pages. There is definitely an issue there. If you get an exception in the console, you must check it out. 2011-07-22T09:18:08Z Robert Biter commented on LPS-19139. 2011-07-22T06:58:48Z 2011-07-22T06:58:48Z Indeed, I can not scroll down in the modal window that appears when clicking manage pages for example, but that is another bug (at least on Opera there is this behavior). 2011-07-22T06:58:48Z Robert Biter added LPS-19139. 2011-07-21T12:49:46Z 2011-07-21T12:49:46Z Exporting lars broken 2011-07-21T12:49:46Z Robert Biter added LPS-18819. 2011-07-12T07:43:41Z 2011-07-12T07:43:41Z Can't assign portlets to panel type pages 2011-07-12T07:43:41Z Robert Biter updated LPS-17267. 2011-05-16T07:44:38Z 2011-05-16T07:44:38Z Updated status to Contributed Solution.
 2011-05-16T07:44:38Z Lucian Ravac and Robert Biter are now friends. 2011-02-03T13:06:50Z 2011-02-03T13:06:50Z 2011-02-03T13:06:50Z Julio Camarero replied to Robert Biter's message board post, RE: Added Translation to Romanian, in Liferay.com. 2011-01-11T13:08:47Z 2011-01-11T13:08:47Z Go to Category 2011-01-11T13:08:47Z Robert Biter replied to Julio Camarero's message board post, RE: Added Translation to Romanian, in Liferay.com. 2011-01-11T09:48:50Z 2011-01-11T09:48:50Z Go to Category 2011-01-11T09:48:50Z


text:
Decades of disproportionate spending by states on road expansion at the expense of regular repair have left many state roads in poor condition. Federal Highway Administration data indicate that half of all major state roads were in "fair" or "poor" condition in 2008, and in 2009 the American Society of Civil Engineers gave the nation's roads a "D-," down from a "D" in 2005.
In spite of this enormous repair backlog, the vast majority of states continue to inadequately fund road repair projects. By underfunding repair and allowing roads to fall out of good condition, state leaders are choosing the most expensive type of repair possible, as rehabilitating a road that has completely deteriorated is substantially more expensive than keeping that road in good condition in the first place.
Adding further urgency to these budget concerns is that with every dollar spent on new construction, states add to a road system they are already failing to adequately maintain. As a result, states face a large and growing financial burden.
A smarter investment: road repair and preservation
Investing in repair and preservation does more than make headway on an inevitable problem; it actively reduces the scale of future costs. According to the American Association of State Highway and Transportation Officials, every $1 spent to keep a road in good condition avoids $6-14 needed later to rebuild the same road once it has deteriorated significantly. Prioritizing repair and preservation makes good fiscal sense and brings with it a host of additional benefits. This report examines road conditions and spending priorities in all 50 states and the District of Columbia and recommends changes at the state and federal level that would benefit taxpayers while creating a better transportation system.
Federal taxpayers have a significant interest in seeing that states keep the nation's roads in a state of good repair. Though much of the funding for repair and preservation comes from state and local budgets, billions of federal dollars are also spent each year on these roads. Federal funds built a large portion of these major state roads, and allowing states to under- invest in repair and preservation greatly reduces the value of these federal investments. In addition, roads in poor condition can negatively impact interstate trade and travel, the effects of which can be felt across large regions and across state lines.
State leaders make a number of decisions about transportation spending that shape their long-term financial trajectories. All 50 states and the District of Columbia can benefit from aligning transportation policies with capital spending decisions to make sure roads are kept in good repair. Doing so can prevent neglected preservation from undermining the value of their own infrastructure investments.
Download full version (PDF): Repair Priorities
View interactive map (SmartGrowthAmerica.org)
About Smart Growth America
"Smart Growth America is the only national organization dedicated to researching, advocating for and leading coalitions to bring smart growth practices to more communities worldwide. The coalition includes many of the best-known national organizations advocating on behalf of historic preservation, the environment, farmland and open space preservation, neighborhood revitalization and more."
About Taxpayers for Common Sense
"Taxpayers for Common Sense is a 501(c)(3) non-partisan budget watchdog serving as an independent voice for American taxpayers. Our mission is to achieve a government that spends taxpayer dollars responsibly and operates within its means. We work with individuals, policymakers, and the media to increase transparency, expose and eliminate wasteful and corrupt subsidies, earmarks, and corporate welfare, and hold decision makers accountable."
Tags: Reconnecting America, Smart Growth America, Taxpayers for Common Sense


text:
We designed and printed this A3 calendar for the Prestatyn Business Association to help them fundraise for the 1 Royal Welsh (Royal Welch Fusiliers) and the Welsh Guards.
David Bridle
Head honcho and teaboy at The Press Gang
Journalist, designer, print buyer and web designer – David started The Press Gang in 1991. Besides communing with Macs, he likes sailing, travelling, singing shanties, Spotify and keeping the customer satisfied.
Latest posts by David Bridle (see all)
- Big Banner for Winterfest - January 12, 2017
- Custom playing cards – they're a snap! - July 21, 2016
- Our posters promote Pasadena Roof Orchestra - May 3, 2016


text:
Draven Build Guide by ToxicRatt
This build has been archived and is for historical display only.
PLEASE NOTE: This build has been archived by the author. They are no longer supporting nor updating this build and it may have become outdated. As such, voting and commenting have been disabled and it no longer appears in regular search results.
Not Updated For Current Season
This guide has not yet been updated for the current season. Please keep this in mind while reading. You can see the most recently updated guides on the browse guides page.
Thank You!
Your votes and comments encourage our guide authors to continue
creating helpful guides for the League of Legends community.
Draven Build
Ability Sequence
Not Updated For Current Season
The masteries shown here are not yet updated for the current season, the guide author needs to set up the new masteries. As such, they will be different than the masteries you see in-game.
ToxicRatt with a complete Draven Guide. I have been playing him since he came out and got to tell you he is awesome!
I will make the guide all pretty later :)
Try this guide before commenting please!
Masteries are pretty standard for Draven going with 21 offense, 9 defense and 0 in utility.
Skill Sequence
Start off with Stand Aside for farming and slow. Get Blood Rush followed by Spinning Axe. Max spinning Axe as fast as possible.


text:
These cute little monsters like to keep you connected. Monster Gel Pens not only write well, they
snap together too; hand in monster hand.
These pens are your go-to tools for fun writing and creative drawing.
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Little Skye - Children's Boutique Clothing