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Most fundamental aspects of transcription are conserved among eukaryotes. One striking difference between yeast Saccharomyces cerevisiae and metazoans, however, is the distance over which transcriptional activation occurs. In S. cerevisiae, upstream activation sequences (UASs) are generally located within a few hundred base pairs of a target gene, while in Drosophila and mammals, enhancers are often several kilobases away. To study the potential for long-distance activation in S. cerevisiae, we constructed and analyzed reporters in which the UAS-TATA distance varied. Our results show that UASs lose the ability to activate normal transcription as the UAS-TATA distance increases. Surprisingly, transcription does initiate, but proximally to the UAS, regardless of its location. To identify factors affecting long-distance activation, we screened for mutants allowing activation of a reporter when the UAS-TATA distance is 799 bp. These screens identified four loci, SIN4, SPT2, SPT10, and HTA1-HTB1, with sin4 mutations being the strongest. Our results strongly suggest that long-distance activation in S. cerevisiae is normally limited by Sin4 and other factors and that this constraint plays a role in ensuring UAS-core promoter specificity in the compact S. cerevisiae genome.
Many aspects of transcription initiation are conserved between Saccharomyces cerevisiae and other eukaryotes. This includes a high degree of conservation for many fundamental classes of transcription factors, such as RNA polymerase II, general transcription factors, particular coactivators, chromatin remodeling complexes, and histone modification enzymes (6, 30). Other features are less well conserved, particularly the DNA regulatory sites that control transcription. While TATA elements are used at a significant number of promoters in both S. cerevisiae and metazoans, the TATA-start site distance is fixed in metazoans but not in S. cerevisiae, and the start site itself is more conserved in metazoans (68). In addition, downstream promoter elements, widely used in metazoans, are not found in S. cerevisiae (40).
Another significant difference between yeast and metazoans concerns yeast upstream activation sequences (UASs) and their metazoan counterparts, enhancers. Both serve as binding sites for gene-specific activators, yet while UASs are usually positioned within a few hundred base pairs 5′ of the TATA box or core promoter, enhancers are often located several kilobases away from or even 3′ of the promoter (5, 30). While there is some evidence that yeast UASs cannot function when moved too far from their promoters (31, 71), this area has not been extensively studied. As the S. cerevisiae genome is compact, and the majority of yeast promoters range from approximately 150 to 400 bp (28, 59), it seems logical that there would be restrictions on activation distance in order to maintain specificity between a UAS and its target gene. However, there is no clear understanding of the effect of distance on activation in S. cerevisiae in vivo and no knowledge concerning factors that might play a role in controlling this effect.
Different mechanisms have been proposed for the ability of enhancers to act over long distances (5, 11, 25, 62). One mechanism, looping, stipulates that proteins bound at an enhancer associate directly with proteins bound at a core promoter with a looping out of the intervening chromatin. For example, loops have been demonstrated between the locus control region of the murine β-globin locus and activated genes located 40 to 60 kb away (72). Another proposed mechanism, tracking, requires the movement or binding of molecules along the DNA between the enhancer and core promoter. The tracking model postulates that RNA polymerase is recruited at the enhancer and moves along the DNA until it reaches the target promoter. The strongest evidence for this mechanism comes from analysis of a bacterial enhancer-like element (32). In S. cerevisiae, evidence consistent with tracking came from experiments in which LexA binding or a transcription terminator blocked transcriptional activation (8). Current evidence from metazoans supports several of these mechanisms, including combinations of mechanisms at particular enhancers (for an example, see reference 76).
The looping mechanism has been demonstrated to occur in S. cerevisiae in some circumstances. Looping has been detected in yeast between the promoters and terminators of long genes and this event has been correlated with active transcription (1, 58). In addition, looping has also been demonstrated within yeast telomeres, bringing the UAS and TATA box of a reporter gene located within that region into close proximity (22). Finally, genetic evidence for looping facilitated by the Drosophila melanogaster GAGA protein has been described previously (60). No evidence for looping as part of transcriptional activation, however, has been found, most likely because most S. cerevisiae UAS elements are close enough to the core promoter not to require looping as part of gene activation.
To study activation distance in S. cerevisiae, we established a system using the well-characterized activator, Gal4, to examine the ability of the GAL1 UAS to function at different distances from a TATA box in S. cerevisiae. These experiments clearly show that activation diminishes with distance, confirming the suggestions from previous studies (31, 71) and indicating that long-distance activation is normally repressed. Unexpectedly, we have also found that when a UAS is far from a TATA, the UAS directs expression of nonfunctional transcription from nearby start sites regardless of the location of the UAS, suggesting that promiscuous activation may be a property of UAS elements. To gain insight into factors that control activation distance in S. cerevisiae, we screened for mutants that permit activation from the normally nonpermissive distance of 800 bp. These screens have identified a small set of factors known to play roles in chromatin structure: Sin4, Spt2, Spt10, and histone H2A/H2B. The mutations that allow the strongest level of long-distance activation, in SIN4, expand the range of transcription start sites, thereby allowing long-distance activation. Finally, experiments to test the mechanism of long-distance activation in sin4Δ mutants have provided strong evidence against a tracking model.
S. cerevisiae strains and plasmids.All S. cerevisiae strains used (Table 1) are isogenic with a GAL2+ derivative of S288C (79). Rich (yeast extract-peptone-dextrose [YPD]), minimal (SD), and synthetic complete drop-out media (for example, medium lacking histidine [SC-His]) were prepared as described previously (65). YPGal (1% yeast extract, 2% peptone, and 2% galactose) and SC-His Gal media contained 2% galactose as the carbon source. Where indicated, specified concentrations of 3-aminotriazole were added to SC-His Gal medium. Strains were constructed by standard methods, either by crosses or by transformation (4). The spt2Δ0::kanMX (57), (hta1-htb1)Δ::LEU2 (35), mot1-1 (21), spt6-140 (18), and gal11Δ::TRP1 (63) alleles have been described previously. The sin4Δ0::LEU2, sin4Δ0::URA3, spt10Δ0::LEU2, pgd1Δ0::LEU2, spt8Δ0::LEU2, med1Δ0::URA3, med2Δ0::URA3, nut1Δ0::URA3, srb2Δ0::URA3, srb5Δ0::URA3, srb9Δ0::URA3, srb10Δ0::URA3, soh1Δ0::URA3 and set2Δ0::URA3 deletion mutations were constructed by replacing their open reading frames (ORFs) with the auxotrophic marker LEU2 or URA3 (17). The spt2Δ0::natMX deletion mutation was constructed by replacing its ORF with the natMX marker, which confers resistance to nourseothricin (29). The spt21Δ0::kanMX deletion mutation was constructed by replacing its ORF with the kanMX marker which confers resistance to G418 (7, 75).
Long-distance activation reporters were made by a series of integrative transformations, resulting in either Gal4 or LexA binding sites integrated at different distances from a HIS3 gene with its adjacent TATA elements. Each reporter was made within a long coding region, chosen to avoid consensus TATA or other binding sites for transcription factors. The oligonucleotides used in the construction of all reporter strains are listed in Table S1 at http://genetics.med.harvard.edu/∼winston/Winston%20Lab%20Links.html . For the reporter in the BPH1 gene, construction began with the integration of URA3 at the 3′ end of the BPH1 ORF. Next, the HIS3 TATA and coding and terminator sequences were amplified by PCR from plasmid YIP55-ScHIS3A5 (36) with flanking homologies added so that the URA3 marker would be replaced entirely by the HIS3 sequences after transformation. Ura− transformants were selected by resistance to 5-fluoroorotic acid. To prevent transcription through BPH1, sequences from −500 to +49 (where +1 is the ATG) were deleted and replaced with copies of the sequence encoding the HA1 epitope, using a method described previously (66). This strain, FY2549, was used as the parent strain for all other BPH1 reporter strains. To insert Gal4 binding sites at specified distances 5′ of the HIS3 TATA in the BPH1 reporter, a cassette containing four Gal4 binding sites and the kanMX marker was amplified by PCR from the pFA6a-kanMX6-PGAL1 plasmid (47) using primers designed for recombination at specific distances 5′ of HIS3. To make strain FY2584 for use in synthetic genetic array analysis, integrative transformation was used to replace the kanMX marker with the natMX4 marker, which confers resistance to the drug nourseothricin (29). To insert the ADH1 terminator between the Gal4 binding sites and HIS3 TATA in FY2555, the ADH1 terminator was amplified from pFA6a-GFP(S56T)-kanMX6 (47) and this PCR product was used in an integrative transformation to replace a URA3 marker within BPH1 using the same method described above. To place LexA binding sites 5′ of the HIS3 TATA in FY2549, complementary oligonucleotides containing three overlapping LexA binding sites flanked by restriction sites were synthesized, annealed, and digested by BamHI and BglII. This LexA fragment was cloned into the BamHI and BglII sites of pFA6a-kanMX6-PGAL1, replacing the Gal4 binding sites and creating plasmid pFA6a-kanMX6-LexA. This plasmid was used as a template for PCR to generate DNA for integrative transformation into the BPH1 locus, and G418R transformants were selected. The LexA-GAL4 (8) and LexA-GCN4 plasmids were generously provided by Roger Brent and Mark Ptashne.
To construct the YBR281C reporter, we first marked HIS3 so that it could be integrated at the YBR281C locus in one step. To do this, the natMX4 cassette was amplified by PCR from pAG25 (29) and inserted 3′ of the HIS3 terminator at its genomic locus in a wild-type strain. Then the entire HIS3-natMX4 region was amplified by PCR and inserted by integrative transformation at the 3′ end of the YBR281C ORF, selecting for nourseothricin resistance. Subsequently, a cassette containing four Gal4 binding sites and the TRP1 auxotrophic marker was amplified from the pFA6a-TRP1-PGAL1 plasmid (47), with flanking homology to specific sites within YBR281C. These PCR products were used to insert Gal4 binding sites by integrative transformation at the specified distances 5′ of the HIS3 TATA within YBR281C. Transformants were selected by growth on SC-Trp. The design of the flanking homologous sequences was such that integration of the Gal4 binding sites within YBR281C simultaneously deleted all YBR281C sequences 5′ of that integration to −140 (where +1 is the ATG) in order to prevent transcription through YBR281C. Verification of all correct integration and recombination events was performed by PCR.
Isolation of long-distance activation mutants.Long-distance activation mutants were identified by four methods, a transposon insertion mutagenesis screen (12), spontaneous mutant selection, systematic genetic array analysis using the nonessential deletion set and the doxycycline-repressible essential allele set (53, 73), and testing of candidate genes. For insertional mutagenesis, a mutagenized yeast genomic library containing LEU2-marked transposon insertions (12) was digested with NotI and transformed into FY2555. Integrative transformants were selected by growth on medium lacking leucine (SC-Leu), and these colonies were replica plated onto SC-His-Leu Glu and onto SC-His-Leu Gal to screen for transformants that were His+ only when galactose was the carbon source. Of 11 His+ transformants, one was retested after purification. Vectorette PCR (2) was used to amplify the region where the transposon had integrated, and sequencing determined this location to be within the coding region of SIN4. Two methods of spontaneous selection were carried out using strains FY2554 and FY2555. For the first method, patches of FY2554 and FY2555 were grown on YPD and replica plated to SC-His Gal medium. Papillae that grew after 2 weeks at room temperature were purified and retested to check that the His+ phenotype was galactose dependent. Of the four mutants that were rechecked, two FY2555-derived mutants were complemented by a wild-type SIN4 plasmid and two FY2554-derived mutants remain unidentified. In the second method, four cultures of either FY2554 or FY2555 were grown overnight and 1 × 107 cells from each culture were plated on SC-His Gal and grown at 30°C. Three colonies were picked from each plate, making a total of 24 putative mutants, 19 of which were retested upon purification. Of the 10 FY2554 mutants, 6 failed to complement a deletion of SIN4, 1 failed to complement a deletion of SPT10, 1 failed to complement a deletion of HTA1-HTB1, and 2 others remain unidentified. Of the nine FY2555 mutants, five failed to complement a deletion of SIN4, two failed to complement a deletion of HTA1-HTB1, and two remain unidentified but are in the same complementation group. For the systematic genetic array analysis, we screened both the yeast nonessential deletion set (73) and a doxycycline-repressible set of essential genes (53) by crossing each set to strain FY2584 and screening for growth on SC-His Gal. Deletion mutants of SIN4, SPT2, and HTA1 were identified as having a His+ phenotype in these screens.
Northern analysis.RNA isolation and Northern hybridization experiments were performed as previously described (4). As indicated, strains were grown to mid-log phase in YPGal. Northern hybridization analysis was conducted with probes to the coding regions of HIS3 (−27 to +376, where +1 is the ATG) and ACT1 (+533 to +722) (see Table S1 at http://genetics.med.harvard.edu/∼winston/Winston%20Lab%20Links.html ).
5′ mapping of transcript start sites.5′ end mapping of RNAs was carried out using the rapid amplification of cDNA ends (RACE) method. Total RNA was prepared as described above (4) and treated with DNase using the RNeasy Mini kit (QIAGEN). cDNA synthesis and PCR were performed using the SMART-RACE kit (Clontech) as described previously (80). The primer used for mapping the 5′ ends of transcripts annealed to sequences from +263 to +282 of the HIS3 ORF (see Table S1 at http://genetics.med.harvard.edu/∼winston/Winston%20Lab%20Links.html ). The PCR products were cloned into the pCR2.1-TOPO vector (Invitrogen) and sequenced.
3C.To assay possible loop formation between the Gal4 binding sites and HIS3 TATA in the reporter, the chromosome conformation capture (3C) method was used (1, 23). We followed the method of Ansari and Hampsey (1) with some modifications. Fifty milliliters of cells was grown in YPD or YPGal to a density of 3 × 107 cells/ml and cross-linked with 1% formaldehyde for 30 min at room temperature. The reaction was quenched with the addition of glycine to 250 mM and 0.1% sodium dodecyl sulfate (SDS) and incubated for 5 min at room temperature. The cell pellet was washed with 10 ml of 1× Tris-buffered saline buffer plus 1% Triton X-100, resuspended in 1 ml 1× Tris-buffered saline, transferred to a 2-ml screw-cap tube, and pelleted. This pellet was resuspended in 750 μl of FA lysis buffer, approximately 500 μl of glass beads was added, and cells were lysed with vigorous shaking for 40 min at 4°C. Cell lysates were collected by puncturing the bottom of the tube with a 22-gauge needle and collecting the filtrate in a second 2-ml screw-cap tube set inside a 15-ml conical tube. The filtrate was then pelleted at top speed in a microfuge for 5 min at 4°C. The pellet was washed with 500 μl of FA lysis buffer and resuspended in 500 μl of 10 mM Tris, pH 7.5. Eighty microliters of this chromatin preparation was digested for 16 h, with shaking in a 125-μl digestion mixture with 10 μl each of enzymes BfaI (5,000 U/ml) and MseI (4,000 U/ml). Samples were pelleted at top speed in a microfuge for 5 min at room temperature and then resuspended in 90 μl of 10 mM Tris. Digestion was stopped by adding 10 μl of 10% SDS and incubating at 65°C for 20 min. To sequester the SDS, 75 μl of 10% Triton X-100 was added, and the reaction mixture was diluted to a volume of 750 μl. Ligations were carried out for 1 h at room temperature using 5 μl of Quick ligase (New England Biolabs). Two microliters of 10 mg/ml RNase was added, and the reaction mixture was incubated for 10 min at 37°C. Five microliters of 20 mg/ml proteinase K was then added, and the reaction mixture was incubated overnight at 65°C. The samples were extracted two times with phenol-chloroform and once with chloroform and ethanol precipitated in the presence of glycogen. The DNA concentration was determined, and 500 ng of DNA was used as the template for each PCR. PCRs (30 cycles) used same-strand oligonucleotides that anneal to the indicated regions of the reporter and require a cut between the two oligonucleotides to form a product (see Fig. 6A and Table S1 at http://genetics.med.harvard.edu/∼winston/Winston%20Lab%20Links.html ) (23). A PCR to control for the amount of template DNA was performed using convergent oligonucleotides that amplify a region of chromosome V lacking BfaI and MseI restriction sites (see Table S1 at the URL mentioned above). PCR products were visualized on a 1.6% agarose gel using ethidium bromide.
Construction and analysis of long-distance reporters.A small number of studies of S. cerevisiae have suggested that a UAS-TATA distance greater than 300 bp can impair transcriptional activation at some promoters (31, 71). To examine the effect of the UAS-TATA distance on transcriptional activation in a systematic fashion, we constructed reporter strains in which the UAS-TATA distance was increased in a stepwise fashion (Fig. 1A and Table 2). These reporters were constructed at two different locations within the genome, within the coding regions of the long, nonessential genes BPH1 and YBR281C. At each location, we integrated the HIS3 TATA, coding, and terminator sequences into regions of BPH1 and YBR281C that were devoid of consensus TATA or Gal4 binding sites. Then, the GAL1 UAS, with its four Gal4 binding sites, was integrated 5′ of HIS3 at distances that ranged from approximately 300 to 800 bp (Table 2).
The level of Gal4-activated expression of HIS3 in these reporter strains was initially assayed by testing growth on SC-His Gal. We also included tests on a range of concentrations of 3-aminotriazole, a competitive inhibitor of His3, as a further test of expression strength. Our results show that at BPH1, UAS-TATA distances less than or equal to 493 bp were strongly His+, a distance of 574 bp was weakly His+, and distances of 690 bp and greater were His− (Fig. 1B). The YBR281C location gave similar results, although this site was somewhat more permissive, with a UAS-TATA distance of 606 bp conferring a His+ phenotype and a distance of 806 bp being His− (Fig. 1C). The addition of different 3-aminotriazole concentrations revealed a progressive change of expression even at closer distances, with the greatest expression occurring at the smallest UAS-TATA distance. The expression of HIS3 was Gal4 dependent, as all reporters were His− when glucose was the carbon source instead of galactose.
To test whether other activators would function similarly to Gal4 as the UAS-TATA distance was increased, we constructed three additional reporter strains in which three overlapping LexA binding sites were integrated 123, 417, and 642 bp 5′ of the HIS3 TATA (Table 2). Using these reporters, we compared activation via two fusion proteins, LexA-Gal4 and LexA-Gcn4 (Fig. 1D). While the LexA-Gal4 fusion protein was able to activate transcription from distances of 123 and 417, similar to Gal4 alone, LexA-Gcn4 could activate transcription from only the shortest distance. None of the reporters were activated when transformed with a control plasmid that did not express an activator.
Isolation and characterization of mutants that allow long-distance activation.In order to discover factors that might determine the distance at which a UAS can activate in S. cerevisiae, BPH1 reporter strains with the UAS located either 690 or 799 bp 5′ of HIS3 were screened for factors which, when mutated, enable the expression of HIS3 in a galactose-dependent manner. Four methods were used: transposon insertion mutagenesis, spontaneous mutagenesis, a candidate gene approach, and synthetic genetic array analysis with both the yeast nonessential deletion set as well as the essential repressible allele set (described in Materials and Methods) (12, 53, 73).
We found a small number of factors affecting long-distance activation of gene expression, all of which have been previously implicated in chromatin structure (Table 3 and Fig. 2). Mutations in SIN4 were recovered in all our screens, and sin4 mutations conferred the strongest His+ phenotype (Fig. 2A). SIN4 encodes a subunit of the tail domain of the Srb/Mediator coactivator complex and plays both positive and negative roles in the regulation of transcription (15, 16, 19, 20, 37-39, 45, 48, 69, 77). SPT2 encodes an HMG-like protein, binds DNA in a sequence-independent manner, and has been implicated in both transcription elongation and the recruitment of mRNA cleavage/polyadenylation factors (33, 43, 44, 57). Both SIN4 and SPT2 were previously isolated in a screen for suppressors of a transcriptional defect at an HO-lacZ fusion in strains mutated for members of the Swi/Snf chromatin remodeling complex (69). SPT10 encodes a site-specific DNA binding protein that regulates histone gene transcription and is also a putative histone acetyltransferase (24, 26, 34, 56, 67). Finally, HTA1-HTB1 encodes the core histones H2A and H2B. Mutations in SIN4, SPT10, and SPT2 share other phenotypes with histone gene mutants, such as the suppression of Ty and δ insertions (Spt− phenotype), reinforcing their roles in controlling chromatin structure (18, 27, 38, 43, 55, 64, 69, 78). In all four cases, complete deletion of the gene allowed long-distance activation, showing that the phenotype is caused by a loss of function. In addition, for each mutant, long-distance activation occurred only in the presence of galactose, showing that the mutations did not merely bypass the requirement for a UAS. The rest of our analysis is focused primarily on long-distance activation in sin4Δ mutants, as they have the strongest phenotypes, with some analysis of spt2Δ for comparison.
To determine whether the long-distance activation observed in sin4Δ and spt2Δ mutants is specific for either location or activator, additional reporters were tested. First, we tested the effects of sin4Δ and spt2Δ mutations at YBR281C, testing the reporter in which the UAS-TATA distance is 806 bp. As can be seen by growth on SC-His Gal medium, sin4Δ and spt2Δ mutations allow long-distance activation at this site as well, showing that their phenotype is not location dependent (Fig. 2B). To discover whether HIS3 activation is activator specific, we tested our mutants with the reporter in which LexA binding sites are located 642 bases 5′ of the TATA. When testing sin4Δ and spt2Δ mutants, we found that both are His+ in strains expressing a LexA-Gal4 fusion protein; however, only spt2Δ mutants are His+ in strains expressing a LexA-Gcn4 fusion protein (Fig. 2C). This result suggests that long-distance activation in sin4Δ mutants may be more activator dependent than in spt2Δ mutants.
To determine whether activation at even longer distances could occur in sin4Δ or spt2Δ mutants, we constructed two additional reporters within BPH1 with UAS-TATA distances of 1,397 and 1,995 bp. While both reporter strains were His− in combination with spt2Δ, a low but visible level of growth was observed in sin4Δ mutants (Fig. 2D). This result confirms that sin4Δ mutants are quite permissive for long-distance activation. The ability to activate transcription from a distance of almost 2 kb makes the UASs in sin4Δ mutants behave similarly to Drosophila or mammalian enhancers.
Long-distance activation does not generally occur in Mediator mutants or other mutants that affect chromatin or transcription.Although we had screened both the yeast nonessential deletion set and the tet-repressible essential gene set for mutants that allow long-distance activation, our results prompted us to retest particular mutants. To do this, we directly examined additional genes for their role in long-distance activation by combining mutations in these genes with the BPH1 reporter having a UAS-TATA distance of 799 bp and examining growth on SC-His Gal medium. First, as Sin4 is part of the large Mediator coactivator complex, it was of interest to test whether mutations in other Mediator genes might also allow long-distance activation. Therefore, we tested mutations in almost all of the other genes encoding nonessential subunits of Mediator. With the exception of srb9Δ and srb10Δ mutants, which showed an extremely weak His+ phenotype, none of the other Mediator mutants tested were His+ (Fig. 2E and Table 4).
Some of the mutations that we isolated have also been shown to cause other transcription defects. For example, to various degrees, all four cause an Spt− phenotype (18, 27, 38, 78), some allow transcription from cryptic promoters within ORFs (41, 51, 57), and some suppress the requirement for a UAS (61). Therefore, to determine whether mutations that affect these phenotypes might also generally affect long-distance activation, we tested a set of mutations previously shown to cause these other phenotypes. Our results (Table 4) show that mutations in SPT8, SPT6, SPT21, SET2, and MOT1 do not allow long-distance activation. Furthermore, we note that there are qualitative differences that suggest that long-distance activation and these other phenotypes are caused by different mechanisms. For example, spt6 mutants are among the strongest cryptic initiation mutants (41); however, they fail to detectably allow long-distance activation. In contrast, sin4 mutants have the strongest known long-distance activation phenotype, yet have only a weak cryptic initiation phenotype (V. Cheung, personal communication). These data strongly suggest that we have identified a new class of transcriptional mutants with a distinct phenotype.
Northern and 5′ RACE analysis of long-distance activation.To assay long-distance activation at the transcriptional level and to correlate it with the His phenotype conferred by our reporters, we examined HIS3 mRNA levels by Northern analysis. We first examined HIS3 expression in the BPH1 reporter strains. As expected, in reporter strains that have a His+ phenotype, wild-type-length HIS3 mRNA is produced, with the level corresponding to the UAS-TATA distance (Fig. 3A, lanes 3, 4, and 5). At UAS-TATA distances of 690 and 799 bp, which are His−, extremely low levels of wild-type-length HIS3 mRNA are detected. Therefore, wild-type-length HIS3 mRNA levels correlate with the growth phenotypes conferred by the reporters, showing that greater distance decreases the level of activation. In sin4Δ and spt2Δ mutants, when we examined a UAS-TATA distance of 799 bp, we found that, as expected, wild-type-length HIS3 mRNA is produced at a level significantly greater than that in a wild-type background (Fig. 3A, compare lane 8 to lanes 9 and 10). Similar results were observed with the second reporter at YBR281C, where we observed even greater levels of HIS3 mRNA at the most proximal Gal4 binding site locations and in sin4Δ and spt2Δ mutants (Fig. 3B). Taken together, these results demonstrate that sin4Δ and spt2Δ mutations allow long-distance activation by Gal4.
In addition to the wild-type-length HIS3 mRNA, we were surprised to detect long, galactose-induced RNAs expressed in almost every case examined. This pattern was observed for the GAL1 UAS located at either BPH1 or YBR281C or for LexA or Ace1 binding sites inserted at BPH1 (Fig. 3; also data not shown). The sizes of these RNAs correspond to the location of the UAS and are consistent with transcription start sites just 3′ of the UAS, regardless of its position. As there are no consensus TATA sequences in any of these reporters except for the known HIS3 TATA, this transcription is TATA independent. Furthermore, Northern analysis showed that in sin4Δ mutants, while the level of the long transcripts was approximately the same as in the wild type, the band corresponding to the long transcripts was reproducibly more diffuse, suggesting that a greater range of start sites are used in this mutant (Fig. 3A, compare lanes 8 and 9). The identification of these long transcripts suggests that UAS elements, in the absence of a nearby promoter, have the potential to initiate promiscuous transcription nearby. This property suggests that it is beneficial for UAS elements to be positioned close to their target promoters.
To precisely determine the 5′ ends of all of the RNAs produced from a long-distance reporter, we used 5′ RACE, comparing wild-type and sinΔ strains (Fig. 4A). Our results lead to two main conclusions. First, in strains carrying sin4Δ, but not in wild-type strains, initiation occurs at the wild-type HIS3 start site (36, 70). Second, in both wild-type and sin4Δ strains, the long transcripts initiate over a range of positions, all 3′ of the GAL1 UAS; however, in the sin4Δ mutant, the degree of specificity for any site is reduced and the range is significantly greater. These results suggest that Sin4 normally constrains the distance over which transcription start sites can be used. Furthermore, in sin4Δ mutants, the distance barrier that exists in wild-type strains is surmounted, allowing accurate long-distance transcriptional activation to occur.
Construction and analysis of a reporter containing a terminator.The discovery of the long transcripts raised the possibility that this transcription might play a role either in the inhibition of long-distance activation in wild-type strains or in enabling long-distance activation in sin4Δ and spt2Δ mutants. For example, in wild-type strains, the long transcript might somehow prevent long-distance activation by transcriptional interference over the HIS3 TATA region. Alternatively, in sin4Δ and spt2Δ mutants, the long transcript might be required for long-distance activation by allowing RNA polymerase to track from the UAS to the HIS3 core promoter. To attempt to address these issues, we integrated the strong ADH1 terminator between the GAL1 UAS and the HIS3 TATA in the BPH1 reporter with a UAS-TATA distance of 839 bp (Fig. 5A). If transcriptional interference is the primary mechanism by which long-distance transcriptional activation is prevented, then integration of the terminator will result in the expression of the reporter and a His+ phenotype. Such a result was obtained in the case of transcriptional interference at the S. cerevisiae SER3 gene (50). We found, however, that a wild-type strain with the ADH1 terminator integrated between the GAL1 UAS and HIS3 TATA is still His−, supporting the hypothesis that distance, and not transcriptional interference, is the reason that activation does not occur at the HIS3 start site (Fig. 5B). We also tested the BPH1 reporter containing the ADH1 terminator in sin4Δ and spt2Δ mutants to determine whether transcription across the length of the BPH1 reporter was required for HIS3 expression in these mutants. Both sin4Δ and spt2Δ mutants are His+, even with the terminator between the UAS and TATA, suggesting that transcription through the BPH1 reporter is not required for long-distance activation (Fig. 5B). This result suggests that RNA polymerase II does not need to track along the template to activate at a distance.
Northern analysis of these strains confirms that the long transcript is efficiently terminated (Fig. 5C; also data not shown), and as expected, wild-type-length HIS3 mRNA is made in sin4Δ and spt2Δ mutants (Fig. 5C, lanes 4 to 7). An unexpected finding, however, was that in wild-type strains with this modified reporter, there is a transcript produced at a low level that appears to be the length of wild-type HIS3 mRNA (Fig. 5C, lane 3). This possibility seemed unlikely, since the strain has a tight His− phenotype. To analyze this class of RNAs more precisely, we performed 5′ RACE analysis to determine the 5′ ends, from both wild-type and sin4Δ backgrounds. Our results again showed there to be a range of transcription start sites, extending from just 3′ of the terminator to within the ORF of HIS3 (Fig. 4B). Unlike what was observed for the reporter strains lacking the terminator, in wild-type strains, transcription could now start over a wider range of positions, including a low level of initiation from the normal HIS3 start site. The insertion of the terminator between the UAS and TATA, then, does seem to help the UAS to communicate with the correct start site at a low level, suggesting that transcriptional interference may play some role in preventing HIS3 transcription. However, given the His− phenotype of this strain, there is not an adequate level of functional HIS3 mRNA to alter the phenotype. In sin4Δ mutants, transcripts started at the correct location significantly more often than in the wild-type strain (Fig. 4B). These results support the view that Sin4 normally functions to select the proper sites for transcription initiation.
3C analysis.Since the long transcript is efficiently terminated by the ADH1 sequences, we hypothesized that a loop might form between the UAS and the TATA for transcription to initiate 3′ of the terminator. To test for such a loop, we used 3C analysis (1, 23). In this technique, chromatin was cross-linked and digested with the restriction enzymes BfaI and MseI that cut on either side of, and at several sites between, the Gal4 binding sites and the HIS3 TATA (Fig. 6A). Digested chromatin was then ligated at low concentration to maximize intramolecular ligations over intermolecular ones, and PCR was performed to detect the ligation products by using primers that anneal to specific regions in the reporter gene (Fig. 6A). If the Gal4 binding sites and HIS3 TATA are held physically close to one another by a bridge of transcription factors, then these regions should remain physically associated after restriction enzyme digestion. Our results show that the growth of either wild-type or sin4Δ strains in galactose leads to an enrichment of a PCR product specific to a ligation between the restriction fragments containing the Gal4 binding sites and HIS3 TATA (Fig. 6B). Importantly, these PCR products require formaldehyde cross-linking and ligation (Fig. 6C). We do not detect a difference between wild-type and sin4Δ mutants, indicating that sin4Δ mutants affect start site selection and long-distance activation at a step subsequent to loop formation.
In this study, we designed a system using S. cerevisiae to examine long-distance transcriptional activation in vivo and have used it to demonstrate that activation by Gal4 diminishes with increasing UAS-TATA distance. Our results confirm those of previous studies that showed that increasing the distance between a UAS and TATA impedes transcriptional activation (31, 71). In addition, our studies yielded two new sets of information regarding S. cerevisiae UAS function and the potential for long-distance activation. First, we were surprised to discover that when the GAL1 UAS is located distant from a core promoter, it directs transcription initiation events just 3′ of its position, independently of both genomic location and activator and apparently in the absence of core promoter elements. This result suggests that UAS-proximal transcription is an inherent property of UAS elements. Such events would not normally be detected because of the compact nature of the S. cerevisiae genome. Second, our isolation of loss-of-function mutations that allow long-distance activation suggests that long-distance activation is normally repressed in S. cerevisiae. Our genetic evidence suggests that this repression depends upon a small number of factors, as our screens identified mutations in only four loci that cause this phenotype. Interestingly, other previously studied mutations known to suppress the requirement for a UAS or to allow initiation from cryptic promoters do not allow long-distance activation, indicating that the repression of long-distance activation that occurs in wild-type cells does not require several factors known to be required for a repressive chromatin state. Thus, long-distance activation may be normally repressed by an aspect of chromatin structure outside those that are currently known to regulate transcription initiation and elongation.
Our results have shown that the GAL1 UAS directs the expression of a long RNA whose transcription is independent of genomic location or activator. This finding suggests that promiscuous transcription initiation at proximal sites may be an inherent property of UAS elements. This conclusion is consistent with recent studies of transcription start sites in Drosophila, mouse, and human. Whole-genome analysis of Drosophila has discovered a large number of intergenic transcripts that correspond to newly identified 5′ exons of known genes (49). Additionally, an analysis of mammalian genomes has uncovered a number of "megatranscripts" that span megabases (13). Strikingly similar to what we have observed with our reporter genes, a transcript originates from the locus control region of the mammalian β-globin locus as those genes are actively being transcribed (3, 42). In these larger eukaryotes, it has been proposed that such intergenic transcription may keep chromatin structure in the region of the enhancer open and accessible for binding by transcription factors (11). Thus, the ability to direct nearby transcription initiation events may be a property conserved between UASs and enhancers.
The importance of strictly limiting long-distance activation in S. cerevisiae is evident from its genome organization. In contrast to what is seen for larger eukaryotes, the S. cerevisiae genome is very compact, with the distance between ORFs generally within a range of 150 to 400 bp, depending upon the gene configuration, with many coding regions under 1 kb (28, 59). The mutations we have identified enable communication between UAS and TATA regions that are at least 800 bp apart, and in the case of sin4Δ mutants, almost 2 kb apart. If the entire yeast genome were able to make efficient UAS-TATA interactions at this distance, there would be a significant disruption to normal regulation and transcription. Therefore, it is clearly critical for S. cerevisiae and other organisms with compact genomes to constrain activation distance.
Given the importance of constraining long-distance activation, one might expect that mutations that are permissive for this type of event would cause poor growth or inviability due to widespread aberrant gene expression. However, of the loci we identified, only HTA1-HTB1 is essential for growth (46). Furthermore, there is little correlation between the growth rate and the strength of long-distance activation in our mutants, as the strongest long-distance activation mutant, sin4Δ, has only a mild growth defect. Consistent with this observation, recent microarray analysis of sin4Δ suggests that it does not cause dramatic changes in transcript levels genome-wide (74). The fitness of these strains is most likely explained by the model that a UAS will preferentially act at a nearby core promoter even in a sin4Δ mutant, where it has the modest ability to activate at a distance. This preference for activation from proximal sites is supported by our observation that the GAL1 UAS in a wild-type strain will activate primarily from proximal sites, even in the absence of core promoter elements. Thus, in a sin4Δ mutant, nearby core promoters may serve as barriers or insulators for long-distance activation. It seems possible that mutations that allow a stronger level of long-distance activation might be able to overcome such barriers.
While our results have clearly shown that long-distance activation in sin4Δ and other mutants can occur, we do not yet understand the mutant alteration that allows this type of activation. Initial genetic tests suggest that a sin4Δ spt2Δ double mutant is similar to a sin4Δ single mutant with respect to long-distance activation strength, suggesting that they may work by similar means. In addition, the presence of the ADH1 transcription terminator does not inhibit long-distance activation in either sin4Δ or spt2Δ mutants, suggesting that tracking of RNA polymerase II along the template is an unlikely mechanism. A previous study of UAS-TATA interactions found that in a wild-type strain with a UAS-TATA distance of less than 300 bp, the same ADH1 terminator did block transcriptional activation (8). The difference between those results and ours seems likely to be caused by the sin4Δ and spt2Δ mutant backgrounds in which our experiment was performed. Given the nature of the genes identified, chromatin structure also seems likely to play a role in long-distance activation, at the level of either nucleosome positioning or histone modification. In addition, other mechanisms, such as localization of the gene within the nucleus (10, 14, 52), might play a role. As the level of transcription in our long-distance activation mutants is considerably lower than that in genes usually studied for chromatin effects, such as GAL1 or PMA1, it is conceivable that the events that allow long-distance activation may be below the level of detection. The isolation of stronger long-distance activation mutants, or enhancers of those already found, will facilitate molecular analysis.
Our studies also suggest that both local differences in chromatin structure as well as activator strength affect the potential for long-distance activation. Between the two reporters that we have studied, the one located at YBR281C on chromosome II is detectably more permissive for long-distance activation than the one at BPH1 on chromosome III, and LexA-Gal4 is a better activator than LexA-Gcn4, consistent with their relative strengths at short distances (9). Previous studies of PHO5 showed that activation of that gene occurs even after the insertion of several hundred base pairs between the UAS and TATA (54), indicating that the location of PHO5 or activation by Pho4 is better suited for long-distance activation than the cases we have examined. These results suggest that the threshold at which UAS-TATA distance is too great for activation is not static but varies depending upon local chromatin structure and activator strength.
Taken together, our data have contributed to a clearer understanding of the constraints on long-distance transcriptional activation in yeast. In addition, our results have provided evidence that proximal transcription is a common property of UAS elements, similar to metazoan enhancers. Our reporters, especially our LexA reporters, provide a system for the study of specific transcription factors in long-distance transcriptional activation. Further studies of our mutants should reveal the mechanisms by which wild-type cells are able to repress long-distance interactions between UAS and TATA elements.
We thank Vanessa Cheung and Iva Ivanovska for providing critical comments on the manuscript. We are grateful to Badrinath Singh, Michael Hampsey, Adriana Miele and Job Dekker for providing 3C protocols.
This study was supported by NIH grant GM45720 to F.W., and K.C.D. was a recipient of an NSF graduate research fellowship.
- Received 16 March 2007.
- Returned for modification 18 April 2007.
- Accepted 16 May 2007.
- Copyright © 2007 American Society for Microbiology
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text:
January 11, 2018
Paul Watkins, M.D., is the recipient of the 2018 American Society for Pharmacology and Experimental Therapeutics Division for Toxicology Career Award. Watkins is the Howard Q. Ferguson Distinguished Professor in the Division of Pharmacotherapy and Experimental Therapeutics and director of the Institute for Drug Safety Sciences at the UNC Eshelman School of Pharmacy at the University of North Carolina-Chapel Hill,
This award recognizes outstanding original research contributions to toxicology by an established investigator. Watkins was nominated by Alison Harrill, Ph.D., from the National Institute for Environmental Health Sciences, who said that his "ability to see value in developing a variety of experimental models is truly visionary and is reflective of Toxicity Testing in the 21st Century approaches."
Watkins received his M.D. at Cornell Medical College in 1979. After academic appointments at the Medical College of Virginia and the University of Michigan, he joined the faculty at UNC-Chapel Hill in 1999, where he has held many professorships and leadership positions.
Watkins has been a leading researcher in understanding the mechanisms underlying interactions between drugs and the liver and intestine. He is one of the most highly cited researchers in the field due to his discovery of mechanisms underlying many drug interactions and toxicities including inhibition of metabolism and transport and the effects associated with consumption of furanocoumarins within grapefruit juice. More recently, Watkins has continued to move the field of hepatotoxicity forward with the deployment of translational technologies that span preclinical models, quantitative systems toxicology and patients in clinical trials. His work continues to provide important information regarding detection and mechanistic understanding of drug-induced liver injury of a variety of etiologies.
An ASPET member for 12 years, he has served as on the editorial board of Drug Metabolism and Disposition.
The Division for Toxicology Career award will be presented during the ASPET Annual Meeting at Experimental Biology 2018 in San Diego, California, at the Division for Toxicology annual meeting on Tuesday, April 24, at 6 p.m.
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text:
After a frenetic month of tennis trying to align diaries with brief weather windows, the Club Championships culminated in a sunny but highly windy finals day on 31 August. Thank you to all 64 participants who took part over the course of the event and in particular to Stephen Davey and his spreadsheet skills for ensuring the draw ran smoothly. We all need to improve over the winter or we'll be seeing more pictures of Alfredo next year.
Congratulations to the following people who won:
Singles Shield: Dave Kerr beat Alison Fraser
Mixed Shield: Sally Cownie & Andrew Stirling beat Mandy Murray & David Gibson (for the second year in a row!)
Mixed Doubles: Barbara Moore & Alfredo Gonzales Sulser beat Alice Drysdale & Mark Atkinson
Ladies Doubles: Alice Drysdale & Julie Gillespie beat Laura Watson & Kirsten Stuart
Ladies Singles: Alice Drysdale beat Sophie Black
Men's Singles: Alfredo Gonzales-Sulser beat Ben Leslie
Vets (played over multiple days and surfaces!): Neil Ross beat Dave Jones


text:
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Measures: 46cm square. Actual Drawing measures 30cm square
Intricate picture drawn in pencil mounted and ready to frame. Limited edition 25/500
Nolon has been drawing all his life. Each drawing can take anywhere from 30 to 100 hours to complete and Nolon has spent the last 10 years developing the skills and techniques which has resulted in his unique and instantly recognisable style. His approach is to give life and character to each of his subjects while achieving a high level of realism.
Read Nolon Stacey's full biography HERE
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text:
Concept by: Iacopo Falciani
This course is designed for teachers who would like to further their knowledge on the use of ICT in class and to experiment with new enjoyable tools. The course will focus on web solutions such as Edmodo, Google educational tools, online Quizzes and others.
During the course, participants will
learn by doing: working in groups, they will have the opportunity to
test different tools directly and to share examples about how to use
them in class.
The techniques acquired in the course will enable teachers to make their lessons more engaging for their students thus making them more effective.
Upon completion of the course teachers will be aware of the strengths and weaknesses of many different ICT tools and will know how to exploit them in the classroom in a safe and useful way.
Learning Outcomes
The course will help the participants to:
- Use audiovisual platforms, making a search easier, creating playlists about different topics, sharing and embedding videos and playlists;
- Use online platforms for communication and sharing: managing a virtual classroom, sending assignments, searching and sharing useful materials, communicating with students and parents;
- Know Google Drive and Google Apps used as educational resources;
- Motivate students through serious digital games and online quizzes.
- Teachers (primary, secondary, vocational, adult, special needs);
- Teacher trainers;
- Headteachers;
- Principals;
- Managers of schools.
Head teacher of ICT Courses: Iacopo Falciani
Do you want to take more than one course? Build your own 2-week Course!
Is there any doubt you want to clarify? Contact us (firstname.lastname@example.com or by Facebook); we will try to answer all your questions as soon as possible.
If you like this course, you may also like:


text:
Tomorrow's post is going to be late too. The editor convinced me to delete 2 of the 3 final chapters. Which means the remaining chapter must be heavily rewritten. I worked on it today, and I nee... Read More
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How this book came about…. I wanted to thank you all for coming over to read Tera's story, and for leaving all those lovely comments here and on my FB page. For you to fully understand just how mu... Read More
(I'm posting the entire book here 1 chapter/day.)(Previously published as The Third Scroll.) CHAPTER ONE (Twelve Blue Crystals) "One eagle from the north. An omen for change," Koro sa... Read More
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text:
When designing a garden I have always given a lot of thought to how the garden looks in winter. When I designed gardens in the UK I often devoted the front garden to winter interest as I feel it gives a lot of pleasure to the owners and passers-by at a time when many front gardens are rather bare. The back garden was the space used most in summer and therefore I concentrated the other seasons there.
When I started gardening in Italy, between Rome and Siena I thought it would be possible to grow many plants to give pleasure during the winter months, I imagined that many bulbs would flower earlier here and so spring would be spread into the winter months. I already knew a lot of shrubs that flowered in winter imagined my garden as an oasis of flowers throughout the year. I WAS WRONG.
Many shrubs that flower in winter are triggered to flower by COLD; here the cold weather rarely begins before January so many plants that flower from November or December in the UK don't start into flower here until February.
A case in point is Lonicera fragratissima, a favourite of mine as, for me; it has the best perfume of any plant. In the UK it often begins to flower in November, maybe even October if there has been cold weather in September, here in Central Italy it doesn't really begin to put on a show until February, then flowering on through March and if it isn't too hot into April. The heat of spring soon brings flowering to an end so if it didn't have such a wonderful perfume I wouldn't grow it here.
Viburnum tinus is perhaps the most reliable winter plant in the UK; yes, it is used in every municipal planting but for a very good reason, it flowers for 9 months of the year. Not here! It is really only just beginning to open its tightly closed buds and by April it will be over – then the foliage looks dismal before the new leaves grow, so not such a useful plant, mine was burnt badly by last year's hot winds so is in need of a drastic prune, I had left the branches in the hope they might recover.
Some years daffodils don't flower at all, or if they do it is late, so late that summer plants detract from their beauty. Tulips, I won't give up, I adore their colour and form but they too flower only a little earlier than in the UK and are often accompanied by early roses throwing my ideas of the seasons into confusion.
So it is with very much pleasure that this winter two plants have flowered for months and don't seem to need cold temperatures to propel them into growth. What plants you may wonder; well I think this post is long enough so I'll describe these plants soon, can you guess what they are?


text:
'In short, this phone feels solid.'
'Nonetheless, if you require a smartphone for both professional and personal use (and don't mind a small screen), the Nokia E5 is an enticing proposition. '
'fine messaging E-series phone'
'This is probably the most comprehensive suite out there as the E5 could view, edit and create Excel, sheets, Word documents and PowerPoint presentations in the best possible way that you can do so on a mobile phone.'
'the phone felt great.'
'This is significantly more noticeable when opening the phone, and detracts from the phone's premium quality feel.'
'it has a front number pad and d-pad and can be used as a traditional phone, complete with T9-style text entry.'
'incredibly robust phone'
'phone flat and streamlined'
'The build quality of the phone is decent, yet not perfect.'
'decent multimedia moves'
'probably the worst wind noise test performance'
'The Nokia C6-01 is one of the most capable Nokia smartphones. '
'More worrying is that the top half of the phone sits a bit high on the bottom half, with the result that it feels a little wobbly when closed.'
'A good mid-range smartphone with promising hardware features and rich set of connectivity options. '
'Heavy and thick'
'fairly elegant phone'
'The 7230 is definitely one of the classier phones you'll find for under AU$200.'
'The phone's glossy finish also attracts finger-prints and provides a challenge in direct sunlight. '
'Nokia 7230 is an elegantly stylish and super slick slide phone. '
'rugged phone'
'An example of how rugged this phone is displayed in the fact that it has been designed for use by United States military personnel, so it can withstand just about any weather conditions.'
'the colors were dull and our images were fuzzy with too much noise.'
'Poor camera.'
'the phone is solid and slick, although a little on the heavy side at 110 grams.'
'the phone does not stay steady in the hand.'
'It's slow, it has just a VGA camera, MP3s sound like they're being played back from a sarcophagus under a pyramid, and, well, just about everyone in a second class train compartment can now be segregated into Nokia 6600 users and some Reliance users.'
'A speakerphone is useful for hands-free calling when you're driving or multitasking.'
'The handset is sleek and stylish which provides the user with an elegant and highly capable 3G mobile phone.'
'By adding support for CDMA/EV-DO Motorola should greatly increase the market for the MC70.'
'latest entry-level phone'
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'subtle, yet stylish design'
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text:
It was past 10p.m. and my hubby was craving for pizza. He looked for the number and ordered pizza. Actually, the pizza shop doesn't deliver until here in my place so hubby called to prepare the pizza and we picked it up. My hubby had 3 slices and I had one.
We ordered the cheese bite pizza with sweet potato mousse for only $10 at Nanta Pizza. Nanta 5000 Pizza is a korean pizza store that sells different kinds of pizza. You can have a pan of pizza for only 5,000 won around $5. I think it's where it got its name. But I don't recommend the 5,000won pizza. It's better to get the more expensive one which is more delicious of cours.


text: The original structure was located near the Cathedral Church, at the foot of the Motta (characteristic promontory located in the oldest part of the Guiscard town), a few years after the Council of Trent. Its current location dates back to the early 19th century, when it was moved to the former almshouse next to the Church of S. Francesco. It is a location of undisputed cultural significance (the poet and patriot Vincenzo Padula studied here, among others) and also contains a library collection of valuable books as well as important historic documents.


text: Bovis ISSN 2398-2993 Ketosis and fatty liver Synonym(s): acetonemia, slow fever, ketonemia, hyperketonemia, Contributor(s): Owen Atkinson, Mark Burnell , Georgios Oikonomou Introduction Pathogenesis Diagnosis Treatment Prevention Outcomes Introduction Cause: the blood accumulates high levels of ketones, usually when the cow is trying to release energy from her body fat and muscle reserves. Signs: weight loss Approach to the underweight cow, reduced appetite, ketonuria, nervous signs, vocalizing. Diagnosis: clinical signs in a recently calved cow, cow side blood test, ketonuria, response to treatment. Treatment: glucose and/or glucocorticoids. Prognosis: good. Clinical disease is often an indicator of more widespread subclinical ketosis in the herd. Pathogenesis EtiologyKetosis can be considered to be a result of modern farming practices. Modern dairy cows have been bred to produce extremely high milk yields. In early lactation, these huge milk yields can put an enormous strain on the cow and her body reserves, as her dry matter intakes of food are unable to match the energy output required for her production. The Krebs cycle of energy metabolism is illustrated in this simple diagram: A simple guide to energy metabolism. In periods of high energy demand, fat is mobilized and pathways resulting in the formation of 'ketone bodies' are upregulated. The diagrams, links below, illustrate the cycle whereby negative energy balance results in the formation of ketone bodies: Early lactation: using fat stores. What happens when a cow mobilizes fat?. The formation of ketone bodies can be illustrated in further detail: Ketone body formation. Glucose (from proprionate metabolism) is 'spared' as an energy source for the brain as this is the only form of energy that the brain can utilize. Predisposing factors Anything that limits dry matter/energy intake in freshly calved cows will act as a potential predisposing factor for ketosis: Management factors ie insufficient access to feed (lack of feed space, food running out/not 'pushed up', inappropriate ration composition/balance/palatability for stage of lactation. Cow factors ie reduced intake capability (excessive body condition/internal fat deposition, reduced rumen volume due to space occupied by calf and insufficient intakes when dry), heat stress. Disease/health factors eg, Hypocalcemia Hypocalcemia (milk fever), Dystocia Dystocia and sequelae eg, retained fetal membranes RFM, Metritis, mastitis, lameness etc (all affect dry matter intake). Genetic predisposition. Timecourse Clinical disease can appear to have a sudden onset, although this is often preceeded by a period of subclinical disease. Response to treatment is usually rapid, although relapse may occur without 'maintenance therapy'. Epidemiology Acetonemia is essentially a disease caused by an imbalance between energy intake and output, for maintenance and milk production. Acetonemia is likely to affect 20% or more of cows, calved up to 3 weeks, in many herds with yields over 8000 litres. These cows may have sub optimal production but are not necessarily affected by clinical ketosis. A proportion are likely to develop clinical disease. As production is reduced, energy balance is restored, and so in many cows this is a self curing condition and may go unnoticed. Diagnosis This article is available in full to registered subscribers Sign up now to purchase a 30 day trial, or Login Treatment This article is available in full to registered subscribers Sign up now to purchase a 30 day trial, or Login Prevention This article is available in full to registered subscribers Sign up now to purchase a 30 day trial, or Login Outcomes This article is available in full to registered subscribers Sign up now to purchase a 30 day trial, or Login Further ReadingPublicationsRefereed Papers Recent references from PubMed and VetMedResource. Berry et al (2011) Genetics of animal health and disease in cattle. Irish Veterinary Journal. 64 (5). Other sources of information Zoetis (2016) Building a healthier herd with Clarifide Plus [online]. Atkinson O (2015) Herd Health Management, a comprehensive guide [online]. Green M (2012) Ed Dairy herd health. 1st edn. CABI Publishing. ISBN: 1845939972. Organisation(s) Synergy Farm Health.


text:
June 2, 2015
Plenty of ways to celebrate the warm weather in this luxury rental community.
BORDENTOWN, NJ – Residents at Rivergate don't need to look far for fun – with abundant onsite amenities and a premier location on the Delaware River waterfront in Bordentown, leisure is just outside the front door.
The social hub of the Rivergate community, the clubhouse is home to a state-of-the-art fitness studio and a resort-style heated swimming pool, which offers a place to kick back and catch rays all summer. A spacious Residents Clubroom is perfect for enjoying time with friends and neighbors all year. Plus, professionally landscaped grounds and outdoor barbecues give residents the opportunity to entertain outside in the warm weather.
A short drive away from this waterfront community, you'll find vibrant downtown Bordentown, home to a variety of restaurants, boutiques, and services. Restaurant Row boasts cuisine from casual comfort food to fine dining. Visitors can kick back with a cocktail at Jester's Café, where window-lined walls with stained glass create a warm atmosphere and the outdoor patio allows you to take in the sights and sounds of the downtown while enjoying your favorite dishes before heading over to Smylie's for an ice cream cone.
Diverse boutiques and galleries create a shopping experience that's second to none. Locals love the Record Collector, voted the #1 favorite independent record store in the tri-state area by Details Magazine. Nearby Firehouse Gallery, a fine contemporary art dealer, offers art classes for kids and adults.
Just a stone's throw away from the luxury rentals at Rivergate, Crystal Lake Park offers residents a chance to relax in the great outdoors. Home to gorgeous Crystal Lake, this park encompasses over 370 acres of water, forests, and wetlands. Maintained to create a rugged wilderness feel, Crystal Lake Park allows residents to enjoy biking, hiking, fishing, and horseback riding just minutes from their apartments.
As summer heats up, the community at Rivergate Bordentown gears up for fun. Rivergate Bordentown features 159 rental residences with one-bedroom apartments from $1,400 per month and two-bedroom apartments from $1,600 per month.
To schedule a tour of Rivergate Bordentown, call 609.298.0303 or visit RivergateBordentown.com.
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For more than three decades, Sterling Properties has developed luxury apartments, single-family homes and townhomes in desirable locations throughout New York, New Jersey, Pennsylvania, Connecticut and Florida. Whether it's a rental or for sale community, Sterling Properties is committed to delivering upscale residences distinguished by award-winning designs, quality construction, sought-after features and first class amenities. For more information, visit SterlingPropertiesNJ.com.


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United States District Court, D. North Dakota
November 7, 2017
Damon John White Bird Solgado, Petitioner,v.Colby Braun, Warden, NDSP, Respondent.
Charles S. Miller, Jr., Magistrate Judge United States
District Court.
the court is a petition under 28 U.S.C. § 2254 for a
Writ of Habeas Corpus by a Person in State Custody filed by
petitioner, Damon John White Bird Solgado ("White
Bird"). (Doc. No. 2). Also before the court is a Motion
to Dismiss by respondent, Colby Braun ("the
State"). (Doc. No. 12). For the reasons set forth
below, the petitioner's writ of habeas corpus is
dismissed and the respondent's motion is granted.
April 2, 2013, White Bird was charged with attempted murder,
a class A felony, two counts of felonious restraint, class C
felonies, aggravated assault, a class C felony, and tampering
with physical evidence, a class A misdemeanor. (Doc. No.
11-1). On August 5, 2013, a jury found White Bird guilty of
all charges. (Doc. No. 11-1). White Bird was sentenced to ten
years in prison, with two five year terms to run
Direct Appeal
Bird appealed to the North Dakota Supreme Court on December
20, 2013. (Doc. No. 11-1). In that appeal, White Bird argued:
(1) he was not competent to waive his right to counsel; (2)
he did not receive a fair trial because the state district
court did not limit the evidence he introduced at trial; and
(3) there was insufficient evidence to sustain his
convictions. State v. White Bird, 2015 ND 41, 858
N.W.2d 642 ("White Bird I") (Doc. No.
8). The North Dakota Supreme Court affirmed White Bird's
conviction, concluding: (1) the state district court did not
err in concluding White Bird was competent to waive his right
to counsel; (2) the state district court did not err in not
limiting evidence White Bird presented; and (3) sufficient
evidence supported his convictions.
State Postconviction Proceedings
Bird next filed an application for postconviction relief on
December 6, 2015. (Doc. No. 11-9). In his forty-eight page
application, White Bird set forth seven grounds for relief.
(Doc. No. 11-10). Although not necessarily styled in this
fashion, White Bird essentially alleged: (1) an
unconstitutional search and seizure occurred at his
apartment; (2) White Bird was convicted with evidence
obtained pursuant to an unlawful arrest; (3) White Bird was
convicted in violation of his right to avoid
self-incrimination; (4) the prosecution committed a
Brady violation; (5) White Bird was denied effective
assistance of counsel during trial and appeal; (6) White Bird
was denied the right to appeal his conviction; and (7) White
Bird was convicted through use of a coerced confession.
state district court denied the application. (Doc. No.
11-10). After the State moved for reconsideration, the court
vacated its order as to the issue of whether White Bird
received ineffective assistance of appellate counsel. (Doc.
No. 11-10). After an evidentiary hearing limited to that
issue, (Doc No. 11-11), the court again denied White
Bird's petition. (Doc. No. 11-12).
Bird appealed that order on January 21, 2016. (Doc No. 11-9).
Of the fifteen issues raised by White Bird on appeal, the
majority focused on the conduct of his appellate counsel,
particularly with counsel not raising all the issues White
Bird wanted. (Doc. No. 11-13). On August 26, 2016, the North
Dakota Supreme Court affirmed the state district court's
order denying White Bird's application. White Bird v.
State, 2016 ND 47, 882 N.W.2d 727 ("White Bird
II") (Doc. No. 15). The court concluded White
Bird's appellate counsel's performance fell within
prevailing professional norms and White Bird did not suffer
any prejudice. The court also concluded the state district
court did not err in limiting White Bird's claim to the
effectiveness of his appellate counsel.
Federal Habeas Petition
Bird next filed his § 2254 petition with this court on
March 30, 2017. (Doc. No. 2). White Bird's forty-two page
petition contains six grounds for relief. In ground one,
White Bird alleges he was denied his right to effective
assistance of trial counsel when the state district court
refused to appoint him a new court-appointed attorney. In
ground two, White Bird alleges he was the victim of an
unconstitutional search and seizure that occurred when
authorities acted outside the scope of a search warrant. In
ground three, White Bird alleges the state district court
violated his right to compulsory process when the court
excused a witness subpoenaed by White Bird. In ground four,
White Bird alleges he did not receive a fair trial when the
alleged victims displayed scars to the jury and White
Bird's standby counsel did not object. In ground five,
White Bird alleges the state district court violated his
equal protection rights when it limited the postconviction
proceeding to the issue of whether White Bird received
ineffective assistance of appellate counsel. In ground six,
White Bird essentially alleges there was insufficient
evidence to support his convictions.
Evidence Supporting White Bird's Conviction
following factual summary, taken verbatim from the North
Dakota Supreme Court's decision on direct appeal in
White Bird I, at ¶¶ 2-6, provides context
in evaluating this petition:
[¶2] In April 2013, Fargo Police officers investigated
two separate violent assaults on two victims, which occurred
between twelve to eighteen hours apart at the same location,
White Bird's apartment. On the basis of the
investigation, White Bird was subsequently charged with
attempted murder, a class A felony; two counts of felonious
restraint, class C felonies; tampering with physical
evidence, a class A misdemeanor; and aggravated assault, a
class C felony. White Bird made his first appearance in the
district court on April 5, 2013, and he applied for and was
appointed a public defender.
[¶3] On April 30, 2013, on the basis of a defense
motion, the district court ordered White Bird committed to
the North Dakota State Hospital to evaluate his fitness to
proceed and criminal responsibility. A psychologist at the
State Hospital evaluated White Bird and prepared two reports,
which were both filed with the court on June 10, 2013. White
Bird was found both fit to proceed and criminally
responsible. In June 2013, White Bird and his attorney
appeared at the preliminary hearing. White Bird waived his
preliminary hearing and pled not guilty to all of the
[¶4] In July 2013, White Bird filed a document in which
he fired his attorney and moved to dismiss the case. On July
24, 2013, the district court held a hearing at which White
Bird's attorney said White Bird had fired him and wanted
to represent himself. His attorney raised the issue of White
Bird's ability to represent himself. The court then
discussed with White Bird his decision to represent himself.
White Bird said he did not believe his appointed counsel was
giving him an "adequate defense." The court
continued discussing the issue with White Bird, but White
Bird did not change his mind, insisting on representing
himself. The court informed him that trial was only six days
away, and he would have to subpoena his witnesses and be
ready to try his case.
[¶5] At another pretrial hearing on July 26, 2013, the
district court again addressed White Bird's desire to
represent himself. The hearing ended with White Bird's
consenting to permit his appointed counsel to represent him.
However, on July 29, 2013, the day before trial, at the final
pretrial hearing, White Bird again insisted that he wanted to
represent himself. The court again discussed with White Bird
the dangers of self-representation and his decision to fire
his appointed counsel. The court allowed White Bird to
represent himself and appointed his former attorney as
standby counsel. The court also denied White Bird's
motion to dismiss.
[¶6] At the five-day jury trial in late July and August
2013, White Bird represented himself with limited assistance
of standby counsel. The jury found White Bird guilty on all
five counts. In October 2013, White Bird moved for an
evidentiary hearing, alleging he was not competent to waive
his right to counsel. At a November 2013 hearing, the court
denied White Bird's motion and sentenced him.
Scope of Review
28 U.S.C. § 2254, a federal court may review state-court
criminal proceedings to determine whether a person is being
held in custody in violation of the United States
Constitution or other federal law. However, where the state
court has adjudicated the federal claim on the merits, this
court's review is limited by 28 U.S.C. § 2254(d) to
a determination of whether the state court's decision is
(1) directly contrary to, or an unreasonable application of,
clearly established federal law as determined by the United
States Supreme Court or (2) based on an unreasonable
determination of the facts based on the evidence presented in
the state-court proceeding. See 28 U.S.C. §
2254(d); see generally Harrington v. Richter, 562
U.S. 86, 97-100 (2011) ("Richter");
Williams v. Taylor, 529 U.S. 362, 399-413 (2000).
This highly deferential standard of review is often referred
to as "AEDPA deference" because it was enacted by
the Antiterrorism and Effective Death Penalty Act of 1996
(AEDPA). E.g., Pederson v. Fabian, 491 F.3d
816, 824-25 (8th Cir. 2007); see generally Renico v.
Lett, 559 U.S. 766, 773 n.1 (2010). The reasons for the
limited review are ones of federalism and comity that arise
as a consequence of the state courts having primary
responsibility for ensuring compliance with federal law in
state criminal proceedings. See, e.g.,
Richter, 562 U.S. at 103.
B.Exhaustion ...
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text: The year 2009 will live in infamy as the time when city officials at last began pushing back against one of S.F.'s most beloved genres of civic pastime — the flash mob event. These offbeat get-togethers, organized with the help of local bloggers and Web sites, include no-pants subway rides and gamboling crowds dressed in bridal attire. But the one that really gets the city's goat is the Valentine's Day Pillow Fight on Justin Herman Plaza. In fairness, you can't blame the naysayers for wagging their fingers at the spectacle of so many stray pillow feathers left for public employees to clean up; the city has even proposed shutting down the event. But that doesn't change the simple fact that wading into a throng of thousands, cheap pillow in hand and prepared for mortal combat, is one hell of a good time. Do posterity a favor and clean up after yourself in 2010.


text:
Marina Bruggeman and Sarah Baddeley have finished law school and are back today to commence their articles. We look forward to sharing their enthusiasm and intellect with our clients.
Brennan MacDonald and Mitchell McDade joined us on May 15th as Summer Law Students.
All four are exceptional students of law and have distinguished themselves both in school and the community.


text:
2019: the big 4-0
let's go year in review! it's always fun to go over what i wanted to do in the prior year, not only to see what i accomplished but also to see what i thought was important to accomplish (and what didn't happen). so, 2019, how did we fare? let's take a look at the good ol' to-do list:
- well, what would a new year's to-do list be without "take more photos" as number 1? i mean, it's almost expected at this point. so, take more photos.
- visit the state parks i want to visit. after last year's whirlwind visits, now i know which parks i want to go back to. i've got an itasca trip already planned. i'd like to head up to grand portage and actually spend time there. i also wouldn't mind camping at blue mounds.
- is this the summer i perhaps run TWO half marathons? maybe! we'll see what happens. i'm already signed up for the earth day half, and training starts in 3 weeks for that. seeing training on the horizon gives me hope that winter will come to an end.
- put in new floors in my house. i've got flooring for the entryway, which is my test subject. if that goes well, we'll give the kitchen a go and see what happens from there. this also means i've got to paint my cabinets.
- i signed up for a yoga program through fightmaster yoga and i hope it makes me more diligent in my yoga-ing. i do yoga pretty often, but it would be great to make it a daily thing.
- speaking of a daily thing, remember when i blogged EVERY DAY for a year? i don't know if i should try that again or if i should just think about it and dismiss it.
- do something interesting with nate – i like that on my list because it makes me think about something interesting to do with him. 2018 was a good year for doing stuff; let's make 2019 a good year too!
- I GUESS I'M TURNING 40. i hope something happens for it.
this wasn't a bad list! here's what happened:
- i don't think i took more pics. i DID take photos at two weddings and for a large family this year, though. i guess that's something! i take wedding pics about once every 4 years, and every time i remember why i don't do it that often! the wedding pics were two weekends in a row, too, which was pretty insane. well, it happened, i did it, and now it'll be another 4 years til another wedding.
- i did visit a few state parks this year! we went to itasca in early june and it was really fun to spend some time there. jane and i went up the north shore over labor day and camped at jay cooke and judge cr magney; we also checked out grand portage and drove up to canada while we were at it. on the way back or to somewhere, guess what i did? i picked up another state park passport. so i'm not a less-intense mission to visit them all once again.
- i did NOT run two half marathons. and that's ok!
- i also did NOT put new floors in the kitchen. and that's ok!
- i DID do yoga almost EVERY DAY this year. i bet there were maybe 20-30 days of non-yoga-ing due to vacations and other weird things, but overall i did a TON of yoga this year. you know what has benefited from this? MY ARMS. my arm muscles are glorious.
(please ignore the goo on the mirror)
- i did NOT blog every day. i had trouble doing kablpomo this year. i blogged twice – TWICE – in february. i don't know if there's just not enough interesting things I can think of that deserve a whole blog post or what, but i feel like it's hard to sit down and put 400 words to posterity. i don't know.
- did i do something interesting with nate? we went up to duluth over new year's 2019. he took a week off over thanksgiving and we drove to the cities and did some staycation stuff. we went to whole foods together (first and probably last time for both of us). we went to the state fair. is that interesting enough?
- on a side note, i did some traveling this year! while it wasn't with nate, i feel like it should still be noted because looking back, i should have put it on my list. i went to san antonio for a conference in march, and in october, i went to mexico!
- on a side-side note, i GOT MY PASSPORT. i haven't ever had a passport, and the last time i'd been out of the country had been in 1997. this past year i went to both canada and mexico, and now i can go to more countries! woo!
- on a side note, i did some traveling this year! while it wasn't with nate, i feel like it should still be noted because looking back, i should have put it on my list. i went to san antonio for a conference in march, and in october, i went to mexico!
- yep, turned 40. it happened!


text:
Last November, Netflix announced that Dave Chappelle would be releasing three stand-up specials in 2017. The first two, The Age of Spin and Deep in the Heart of Texas, came out March 21. The third one is managing to keep the promise Netflix made last year by coming at the last minute — Dave Chappelle: Equanimity will be released on the final day of 2017, the streaming service announced Friday.
No details besides date and title were released, but Chappelle did a 10-show residency at Radio City Music Hall this summer, so the special may be culled from those shows.
The date announcement video feels like it could be a very short and silly Chappelle's Show sketch in which Chappelle inserts himself into a scene from Stranger Things, begging Eleven (Millie Bobby Brown) to psychically slam him into a wall.
The special will cap a year or so in which Chappelle reentered the public eye after years out of the spotlight, starting with an Emmy-winning turn hosting Saturday Night Live last year, followed by the two specials and his Radio City run.
Dave Chappelle: Equanimity comes to Netflix Sunday, Dec. 31.


text:
2017 has been another fantastic year and, with Christmas approaching, we would like to take this opportunity to thank all our customers for your valued support throughout the year and wish you all a Merry Christmas and the very best of Health and Prosperity in 2018.
As you know for the past few years, rather than sending out Christmas cards, we have decided to put the money to a more charitable use. As such, we've made a donation to Blessings in Disguise. We certainly hope our support helps this admirable cause.
You can find their website here: http://www.blessingsindisguise.co.uk/
"Blessing in Disguise children's charity actively raises funds through events and through the kind generosity of its donors, support from the community and local and national businesses... helping to significantly improve the quality of life, encouraging social and recreational opportunities, creating many treasured memories to share with family and loved ones for terminally ill, disabled and disadvantaged children and young people across the UK."


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text: Lance Cpl. Thomas Waldburger, from Henderson, Nev., a drafter-surveyor with 8th Engineer Support Battalion, 2nd Marine Logistics Group (Forward), takes measurements prior to placing the bases of a non-standard bridge during a bridge operation in the Sistani Peninsula, Afghanistan, April 4 and 5. The Marines placed the bridge in support of 2nd Battalion, 3rd Marine Regiment, 2nd Marine Division (Fwd.) and International Security Assistance Force operations. The bridge will also benefit the Afghans by increasing their capability to transport more products and materials across the canal to local markets.


text:
Brian May is recovering from a heart attack and revealed that he "could've died" if doctors hadn't discovered he had several congested arteries.
On Sunday, the Queen guitarist posted a video on his Instagram filling fans in on exactly what happened to him over the past month.
May, 72, explained that it all began several weeks ago when he injured himself while gardening. They initially believed he had ripped a muscle in his butt, then discovered a week later that he actually had a "severely compressed" sciatic nerve.
The guitarist explained the pain was "excruciating" and like "putting a screwdriver in my back."
While still dealing with that pain, May said that he also suffered a "small heart attack," with around 40 minutes of pain and chest tightness.
In the video, May recounted how his own doctor drove him to the hospital, where they discovered he had three congested arteries that were in danger of blocking the blood supply to his heart.
While some suggested he have open heart surgery, the guitarist explained that he opted to have stents put in instead. May explained that he walked out with a heart that's "very strong now."
"I had no idea, I had great electro-cardiograms, nothing could tell me that I was about to be in real, real trouble cause I could've died from that, the blockages that were there," he described.
May told fans that he's in good shape now and there's no need for anyone to panic about him or his health. He added that one important lesson he's learned is that what seems to be a very healthy heart may not be.
"I'm incredibly grateful that I now have a life to lead again. I was actually very near death because of [my heart]...but the pain that I had was from something completely different. Funny how some things work," he said.


text:
Type: Multi Family
Taxes: 17000
Year Built:
Short Sale: No
Bank Owned: No
Financial Info:
Days on Market: 84
BEACHFRONT DUPLEX with 110 feet of ocean frontage download photos from iphone 6. Nestled in the dunes with spectacular views from Atlantic City to Avalon. Two four bedroom townhouse style units with spacious square footage for restructuring to your dream home on the ocean or to refurbish the existing units delicious games for free. m Currently deeded as condominiums units may be purchased individually. South Unit 1350000. North Unit 1250000.
Number of Units: 2
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Heating: Electric, Baseboard,
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Water: City
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Listing Office: FREDA REAL ESTATE


text:
Upward Bound (UB1 & UB2)
TRiO programs are designed to identify and provide services to individuals from disadvantaged backgrounds. The ECC TRiO programs serve and assist low-income individuals, first-generation college students, and individuals with disabilities to progress through the academic pipeline from middle school through postsecondary programs.
Counselors and teachers are encouraged to recommend students.
About the Upward Bound Programs
The Upward Bound Program at ECC is a college prep program for high school students who are potential first-generation college students or low-income. Funded by the U.S. Department of Education, the programs provide timely academic assistance and personalized guidance to qualified high school students who want to enhance their options for college by concentrating on their academic and personal development.
Upward Bound students meet weekly after high school for:
- Advising and tutoring with ECC students and advisors.
- Special activity sessions focused on group communication skills and personal development.
- Career and college workshops.
In addition, there are campus visits and sessions scheduled on Saturdays during the school year for all Upward Bound students and a summer non-residential component.
Program Eligibility
The Upward Bound Programs serve students in grades 9–12 at Elgin, Bartlett, Dundee-Crown, Larkin, South Elgin, and Streamwood high schools. These students must meet the following criteria:
- Are a U.S. citizen or permanent resident.
- Demonstrated financial need.
- Potential first-generation college student (neither parent(s)/legal guardian(s) graduated from a four-year college or university in the U.S.).
To apply for the program, contact the Upward Bound office for an application form. Forms must be printed and signed by the parent/guardian before submission to ECC.


text:
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text:
For the third year in a row, Embassy Luxembourg participated in the annual Heritage Days organized by the Luxembourg Administration des Service des Sites et Monuments in which historical monuments and buildings that are not otherwise opened may be viewed by the public.
The Embassy opened the Ambassador's residence – also called the Chief of Mission Residence – and almost 200 visitors participated in the guided tours given by the Public Affairs Section.
This year's theme of Heritage Days was "Personalities and Homes". Guests were able to enjoy a presentation of the historical aspects of the building dating back to its construction in the 1920s, throughout the occupation during the Second World War, as well as a special focus on five U.S. Government representatives and Ambassadors who had lived in the residence: George Platt Waller, Perle Mesta, Patricia Roberts Harris, John E. Dolibois and James C. Hormel.