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Yeast Spt16/Cdc68 and Pob3 form a heterodimer that acts in both DNA replication and transcription. This is supported by studies of new alleles of SPT16 described here. We show that Spt16–Pob3 enhances HO transcription through a mechanism that is affected by chromatin modification, since some of the defects caused by mutations can be suppressed by deleting the histone deacetylase Rpd3. While otherwise conserved among many eukaryotes, Pob3 lacks the HMG1 DNA‐binding motif found in similar proteins such as the SSRP1 subunit of human FACT. SPT16 and POB3 display strong genetic interactions with NHP6A/B, which encodes an HMG1 motif, suggesting that these gene products function coordinately in vivo. While Spt16–Pob3 and Nhp6 do not appear to form stable heterotrimers, Nhp6 binds to nucleosomes and these Nhp6–nucleosomes can recruit Spt16–Pob3 to form SPN–nucleosomes. These complexes have altered electrophoretic mobility and a distinct pattern of enhanced sensitivity to DNase I. These results suggest that Spt16–Pob3 and Nhp6 cooperate to function as a novel nucleosome reorganizing factor.
SPT16/CDC68 was identified in several genetic screens in Saccharomyces cerevisiae as a factor that affects transcription initiation globally (Prendergast et al., 1990; Malone et al., 1991; Rowley et al., 1991; Lycan et al., 1994), possibly by altering some property of chromatin (Schnell et al., 1989; Winston and Carlson, 1992; Xu et al., 1993; Wittmeyer et al., 1999). Pob3 forms a stable heterodimer with Spt16, and both proteins are localized to the nucleus (Xu et al., 1995; Wittmeyer and Formosa, 1997; Brewster et al., 1998) and partially associated with chromatin (Wittmeyer et al., 1999). pob3 mutations also cause defects in transcription, and display severe synthetic defects with spt16 mutations (Costa and Arndt, 2000; Schlesinger and Formosa, 2000). Along with a role in transcription, Spt16–Pob3 also appears to act in DNA replication since it binds to DNA polymerase α (Miles and Formosa, 1992; Wittmeyer and Formosa, 1997; Wittmeyer et al., 1999). Additionally, pob3 mutants are sensitive to the dNTP synthesis inhibitor HU, they are dependent on the Mec1 S phase checkpoint and spt16 or pob3 mutations interact genetically with DNA replication factors such as Pol1, Ctf4, Dna2 and Ctf18 (Wittmeyer and Formosa, 1997; Formosa and Nittis, 1999; Wittmeyer et al., 1999; Schlesinger and Formosa, 2000). Taken together, these results indicate that Spt16–Pob3 promotes both replication and transcription, perhaps by altering chromatin, the template for both processes.
Both SPT16 and POB3 are essential for viability in yeast, and are highly conserved among eukaryotes (Wittmeyer and Formosa, 1997; Evans et al., 1998). The formation of heterodimers is also conserved, since such complexes have been purified from both human (FACT; Orphanides et al., 1999) and frog (DUF1; Okuhara et al., 1999) cells. FACT allows RNA polymerase II to elongate past template sites incorporated into nucleosomes (Orphanides et al., 1998), suggesting a role in transcription elongation on chromatin. However, the Spt− phenotype, which arises from altered start site selection, suggests that Spt16 and Pob3 affect transcription initiation (Winston and Sudarsanam, 1998). Spt16–Pob3 associates with the histone acetyltransferase complex NuA3 (John et al., 2000) and human FACT interacts with the transcription initiation factor TFIIE (Kang et al., 2000). Spt16–Pob3/FACT therefore appears to have a complex role in transcription, which includes both initiation and elongation functions. In addition, depletion of DUF1 from frog oocyte extracts blocked DNA synthesis (Okuhara et al., 1999), indicating that the role in replication observed in yeast is also conserved. The processes affected by Spt16–Pob3/FACT/DUF1 all involve chromatin, and these diverse observations could all be explained by a single activity modulating the properties of nucleosomes.
Pob3 is a member of a conserved family, but it and the two other currently known proteins from this family that are encoded by yeasts lack a DNA‐binding motif found in other homologs. This motif was first noted in the high mobility group (HMG)1/2 chromatin proteins, and confers DNA binding, unwinding and bending properties (Bustin, 1999) on proteins such as SSRP1 and DUF87 (the Pob3 homologs in FACT and DUF1). Since this feature is broadly conserved in this family outside of yeasts and seems likely to provide an important activity for a chromatin‐associated factor, we considered the possibility that some other protein with an HMG1 motif acts with Spt16–Pob3. S.cerevisiae has several candidates, including Nhp6A, Nhp6B, Hmo1, Hmo2, Abf2 and Ixr1. Abf2 is localized to mitochondria, and null mutants cause phenotypes consistent with a role limited to these organelles (Diffley and Stillman, 1991). Ixr1 assists the repression of COX5b transcription under aerobic conditions and also enhances the damage caused by cis‐platin (Brown et al., 1993; Lambert et al., 1994), phenotypes that do not match the profile expected for an Spt16–Pob3 partner. Our initial characterization of genetic interactions between Spt16–Pob3 and Hmo1 or Hmo2 (Lu et al., 1996) indicated only minor effects (T.Formosa, unpublished data). However, we report here that Nhp6A/B (Kolodrubetz and Burgum, 1990) displays strong genetic interactions with Spt16–Pob3, suggesting that Nhp6 could provide HMG1‐motif function for Spt16–Pob3. Results consistent with this interpretation have also recently been reported by Brewster et al. (2001).
Nhp6A and Nhp6B are 92‐ and 98‐residue proteins representing single HMG1 motifs. They are 88% identical and are functionally redundant since only deletion of both genes causes significant phenotypes, which include aberrant transcription, slow growth and temperature sensitivity (Costigan et al., 1994; Paull et al., 1996; Sidorova and Breeden, 1999; Yu et al., 2000). We find that purified Nhp6 binds nucleosomes, and this structure can then bind Spt16–Pob3. The complex of Spt16–Pob3 and Nhp6 (SPN) with nucleosomes causes changes in the electrophoretic mobility and nuclease sensitivity of the nucleosomes. We therefore propose that SPN is a novel nucleosome modifying factor.
A screen for new spt16 alleles
spt16 mutations were isolated previously in three independent screens (Malone et al., 1991; Rowley et al., 1991; Lycan et al., 1994). Remarkably, all three obtained the same mutation, a G132D substitution (Evans et al., 1998). To examine a broader range of alleles, we isolated additional spt16 mutants. The G132D mutation was found in four of 18 temperature‐sensitive isolates (Figure 1A and Table I). While many of the remaining alleles were found to have multiple mutations, tight Ts− alleles with single substitutions were found (e.g. spt16‐6).
We tested the new alleles to see whether different mutations impact different processes. spt16‐G132D causes the Spt− phenotype, which is suppression of the lysine and histidine auxotrophies caused by insertion of Ty1 δ elements into LYS2 and HIS4 (Malone et al., 1991). This phenotype is associated with relaxation of the specificity of transcription start site selection (Winston and Sudarsanam, 1998). Most of the spt16 alleles cause a strong Spt− phenotype, as indicated by growth on media lacking lysine or histidine (Figure 1A and Table I). Since most mutants identified as Ts− are also Spt−, we conclude that full Spt16 activity is needed for normal transcription initiation, as was the case with Pob3 (Schlesinger and Formosa, 2000).
spt16‐G132D also causes sensitivity to 6‐azauracil (6‐AU; Figure 1B and Orphanides et al., 1999), which alters rNTP pools in yeast (Exinger and Lacroute, 1992), and is therefore considered to reveal defects in transcription elongation (Powell and Reines, 1996). The sensitivity of spt16 alleles to 6‐AU varied considerably, suggesting that different mutations affect elongation to different extents (Figure 1B and Table I; compare spt16‐7 with spt16‐8). Since none of the alleles of pob3 tested (pob3‐L78R, ‐2 and ‐7) displayed any 6‐AU sensitivity, this phenotype might reveal a unique function of Spt16.
Defects in POB3 lead to hydroxyurea (HU) sensitivity (Schlesinger and Formosa, 2000). HU inhibits ribonucleotide reductase, which is required for dNTP production (Kornberg and Baker, 1992), so this phenotype suggests a role in a process that requires dNTPs, presumably DNA synthesis. Some spt16 alleles also cause sensitivity to HU (Figure 1C and Table I; spt16‐9a shows that the HUs phenotype is separable from the Ts−), suggesting that SPT16 also functions in DNA synthesis. However, while pob3 mutations were found to cause dependence on the S‐phase checkpoint promoted by MEC1 (Schlesinger and Formosa, 2000), spt16 mutations did not have this effect. Strains with various spt16 alleles (including those causing HU sensitivity) died at 37°C at the same rates whether or not the MEC1 checkpoint was functional (our unpublished data). The nature or consequences of the defects caused by spt16 and pob3 mutations therefore appear to be different, at least with respect to this checkpoint.
We showed previously that spt16‐G132D displays a strong synthetic defect (a decrease in the maximal temperature permissive for growth) with all pob3 alleles tested, and that spt16‐4 is lethal when combined with most pob3 mutations (Schlesinger and Formosa, 2000). Table II extends these results to show that pob3‐L78R is lethal when combined with all spt16 alleles tested with the notable exception of the original spt16‐G132D, underscoring the importance of examining additional spt16 alleles. We also showed previously that spt16‐G132D displays synthetic defects with mutations in the nuclease–helicase encoded by DNA2, the catalytic subunit of DNA polymerase α encoded by POL1 and the Pol1‐binding protein encoded by CTF4 (Wittmeyer and Formosa, 1997; Formosa and Nittis, 1999; Wittmeyer et al., 1999). Table II extends these results to include other alleles of spt16. The previous results linking SPT16 to POL1, CTF4 and DNA2 were supported by this survey, with the interactions with dna2‐2 being particularly extensive, including complete lethality with spt16‐16. dna2‐2 alters a conserved helicase motif and causes both sensitivity to DNA damage and synthetic lethality with a CTF4 mutation (Formosa and Nittis, 1999). The data also demonstrate synthetic defects with a deletion of CTF18, whose similarity to RFC subunits (Cullmann et al., 1995; Kouprina et al., 1994) implicates some process involving the DNA polymerase processivity clamp protein PCNA. These interactions provide further evidence for a complex of Spt16 and Pob3 in vivo, and support the involvement of Spt16–Pob3 in a process that includes the activities of DNA replication factors.
The new alleles of spt16 therefore suggest that Spt16 acts in transcription initiation, transcription elongation and DNA replication. The varying phenotypes of the alleles show that these processes are affected to different extents by different mutations, suggesting that Spt16 and Pob3 have multiple distinct functional domains.
Spt16–Pob3 transcription activation is affected by histone acetylation
Mutating SPT16 or POB3 caused a varying decrease in expression from the HO locus (Figure 2A and Table I), ranging from no change to a 6‐fold drop with pob3‐1. In a W303 strain, spt16‐G132D and pob3‐L78R each caused reduced HO expression, but removal of the histone deacetylase encoded by RPD3 caused a return to at least normal levels (Figure 2B). This suppression extended to general growth as well, since the Ts− phenotype of a pob3 mutant was found to be partially suppressed by an rpd3 deletion (Figure 2C). Since deletion of a deacetylase restores activation lost when SPT16 or POB3 are mutated, at least part of the activation function of Spt16–Pob3 may involve changes in chromatin mediated by histone acetylation. Consistent with this, Spt16–Pob3 has been shown to be partially associated with the histone acetyltransferase complex, NuA3 (John et al., 2000). We conclude that Spt16–Pob3 is an activator of HO transcription that depends at least partly on chromatin modifications.
spt16, pob3 and nhp6 mutations share phenotypes and interact genetically
Since Pob3 lacks the HMG1 motif found in FACT and DUF1 subunits, we investigated the possibility that a protein with this feature might function with Spt16–Pob3. Nhp6A/B has this motif and, as expected for proteins that act together, Nhp6 has some functions that are similar to those of Spt16–Pob3. For example, Nhp6 is also an activator of HO transcription and this role is opposed by the deacetylase Rpd3 (Yu et al., 2000). Unlike SPT16 and POB3, NHP6A/B is non‐essential, so we examined cells lacking both NHP6 genes for phenotypes associated with spt16 or pob3 defects. An nhp6a/b his4‐912δ lys2‐128δ strain is phenotypically His+ Lys− (Figure 3A, and our unpublished data), which indicates a weak Spt− phenotype (Prelich and Winston, 1993; Sherwood et al., 1993; Jiang and Stillman, 1996). Like some spt16 mutants, nhp6a/b strains are also sensitive to the transcription elongation inhibitor 6‐AU (Figure 3A and Brewster et al., 2001), but not to HU (our unpublished data). The nhp6a/b deletion also displays a strong synthetic defect with a deletion of SPT4, such that either nhp6 or spt4 mutants are viable at 35°C, but a strain lacking all three genes is not (Figure 3B). spt4 mutants are 6‐AU sensitive, and Spt4 is a member of a complex required for transcription elongation (Hartzog et al., 1998). Spt5 is also found in this complex and an spt5 mutation also displayed a synthetic defect with nhp6a/b (Brewster et al., 2001). These results suggest that Spt4–Spt5 and Nhp6 each contribute to elongation, but in different pathways. Loss of Nhp6 therefore affects both transcription initiation and elongation, as also noted for Spt16 and Pob3.
We next tested whether removing Nhp6 exacerbates the phenotypes of spt16 or pob3 mutants, as expected if Nhp6 promotes the activity of Spt16–Pob3 in vivo. As shown in Table II and Figure 3C, deletion of NHP6A/B caused a dramatic defect when combined with some spt16 and pob3 mutations. An nhp6a/b strain grows slowly but remains viable at 33°C, as do strains with spt16‐8 and pob3‐11 mutations. However, combining these mutations caused as much as a 10 000‐fold decrease in viability (Figure 3C and Brewster et al., 2001). The effect was allele specific, ranging from no change in the maximal permissive temperature (MPT) to synthetic lethality with spt16‐16a (Table II and Figure 3D). In this case, a strain was constructed that had deletions of spt16 and nhp6a/b, and carried a plasmid with both URA3 and SPT16. Since strains with URA3 cannot grow on media containing 5‐FOA (Boeke et al., 1987), and the plasmid supplies the essential SPT16 function, this strain cannot grow on 5‐FOA. Introducing a LEU2 SPT16 plasmid allows loss of the URA3 plasmid and growth on 5‐FOA (Figure 3D, top line). However, a plasmid with spt16‐16a did not allow growth on 5‐FOA, indicating that spt16‐16a cannot support growth in a cell lacking Nhp6. The same plasmid supported growth in a strain with Nhp6 (Figure 3D, bottom line; the strain is Ts− as expected), demonstrating that spt16‐16a is synthetically lethal with the nhp6 deletion.
The strong, allele‐specific, synthetic defects caused by removal of Nhp6 from spt16 and pob3 mutants suggest that these proteins function together in vivo. In this case, some Spt16–Pob3 defects might be ameliorated by increasing the amount of Nhp6. While most alleles were not affected, the Ts− phenotypes of three spt16 alleles were partially suppressed by a high copy NHP6B plasmid (Figure 3E). Increased expression of Spt16–Pob3 did not affect the Ts− phenotype of an nhp6a/b deletion strain (our unpublished data). This pattern is consistent with formation of a complex; some spt16 or pob3 mutations cause diminished stability of this complex, but this can be suppressed by increasing the level of the binding partner Nhp6.
Weak physical interactions suggest that Nhp6 is not usually in stable complexes with Spt16–Pob3
Nhp6 does not copurify with Spt16–Pob3 (J.Wittmeyer, unpublished data), so these proteins do not appear to form a stable heterotrimer fully analogous to FACT or DUF1. To see whether Spt16–Pob3 and Nhp6 form less stable complexes, we fused the myc epitope to the C‐terminus of Spt16 or Pob3 (Longtine et al., 1998; expression is from the native promoters) and performed immunoprecipitations from lysates. Spt16–Pob3 did not coprecipitate with Nhp6 using standard conditions (P.Eriksson, unpublished data; Brewster et al., 2001 have also recently reported that these proteins coprecipitate only under conditions of relaxed stringency). To test for even weak interactions, we next added purified His10–Nhp6A to lysates and recovered the Nhp6 using antisera against the histidine tag. With this approach, Spt16 and Pob3 were enriched in the precipitated material only when His10–Nhp6 was added (P.Eriksson, unpublished data). No similar enrichment was observed when a protein that was not expected to interact with Spt16–Pob3 (His10–Swi5) was used, so the enhanced recovery of Spt16–Pob3 with His10–Nhp6 is not due to a spurious association with other components of the assay. We also tested various combinations of fusions in a two‐hybrid assay (Bartel and Fields, 1995), and obtained a weak but reproducible signal with Spt16‐DBD–Nhp6‐AD fusions relative to controls (P.Eriksson, unpublished data). We conclude that most of the Spt16–Pob3 and Nhp6 molecules in a cell are not stably associated with one another, but some complexes containing these three proteins do form. Such complexes could include additional proteins that mediate the interaction; for example, Spt16–Pob3 and Nhp6 could interact indirectly with one another by associating with chromatin. The genetic data therefore indicate that Spt16–Pob3 acts with Nhp6, but the physical data suggest that these proteins are not usually together in a free, stable heterotrimeric complex.
The migration of Nhp6–nucleosome complexes is altered by Spt16–Pob3
Our results lead to the hypothesis that the DNA‐binding protein Nhp6 (Yen et al., 1998) assists binding of Spt16–Pob3 to chromatin without first forming a free heterotrimeric complex. We tested this by examining the migration of DNA or nucleosomes on native polyacrylamide gels in the presence of purified His10–Nhp6 and Spt16–Pob3. Spt16–Pob3 alone had no effect on DNA or nucleosomes (Figure 4, lanes 2 and 6), whereas addition of Nhp6 significantly decreased the mobility of both (Figure 4, lanes 3 and 7). Addition of both Spt16–Pob3 and Nhp6 to DNA had about the same effect as Nhp6 alone (Figure 4, lane 4). In contrast, when both Spt16–Pob3 and Nhp6 were added to nucleosomes, a complex was formed with mobility different from that of the Nhp6–Nuc complex (Figure 4, compare lanes 7 and 8). Therefore, Spt16–Pob3 binds to Nhp6–Nuc complexes to form SPN–Nuc complexes with properties distinct from those of the Nhp6–Nuc form. Optimal complex formation required a ∼4‐fold molar excess of Spt16–Pob3 over nucleosomes but a ∼200‐fold excess of Nhp6 (Figure 4, lanes 9–16). Similar concentrations of Nhp6 were needed whether or not the protein was affinity tagged, purified from yeast or bacteria, or purified using trichloroacetic acid (TCA) precipitation or standard chromatographic methods (our unpublished data). Most of the Nhp6 is not associated with complexes in these experiments (see below), so we conclude that high concentrations of Nhp6 are needed because Nhp6 interacts weakly with nucleosomes (perhaps due to suboptimal reaction conditions, but we note that the concentrations of Spt16–Pob3 and Nhp6 in these assays are lower than in vivo).
Spt16 has an acidic C‐terminal domain and Nhp6 is basic. Increasing the NaCl concentration in the binding mixture from 30 to 380 mM (Figure 5A, lanes 7 and 8) or 600 mM (our unpublished data) caused minimal loss of the SPN–Nuc form. This stability at high ionic strengths suggests that SPN–Nuc complexes are not simply aggregates.
Nhp6 loads Spt16–Pob3 specifically to nucleosomes
Spt16–Pob3 migrates in a discrete band on a native gel in the absence of Nhp6 (Figure 5B, lane 2, 'SP'), and in two slower‐migrating bands when Nhp6 is added (Figure 5B, lane 3), with only the faster form being stable in high salt (Figure 5B, lane 4). This pattern was also observed when either DNA or nucleosomes were added (Figure 5B, lanes 5–8), and suggests that Spt16–Pob3 can interact with Nhp6 during native gel electrophoresis. Since the results above indicate that Spt16–Pob3 and Nhp6 do not form stable complexes in cell extracts, this interaction is likely to be non‐physiological. Very little Spt16–Pob3 comigrates with the Nhp6–DNA complexes, especially under high salt conditions (compare the regions near the asterisks in lanes 5 and 6 in Figure 5A and B), suggesting that this form contains only Nhp6 and DNA. In contrast, the SPN–Nuc form is associated with a salt‐stable band of Spt16–Pob3 protein (see lanes 7 and 8 in Figure 5A and B). Therefore, Spt16–Pob3 does not stably interact with Nhp6–DNA complexes, but it does bind to Nhp6–Nuc complexes. We conclude that Spt16–Pob3 does not simply bind to Nhp6 or even to Nhp6 bound to DNA, but instead specifically recognizes Nhp6–nucleosome complexes.
SPN–Nuc complexes contain about one molecule of Spt16–Pob3 per nucleosome
To test the stoichiometry of Spt16–Pob3 in SPN–Nuc complexes, we excised the regions of the native gels containing labeled DNA and subjected them to SDS–PAGE (Figure 5C). A small amount of Spt16–Pob3 was recovered from bands containing Nhp6–DNA complexes (Figure 5C, lane 4). We attribute this to contamination by protein that coincidentally migrates with the Nhp6–DNA complexes (see Figure 5B, lane 5; Nhp6–DNA partially comigrates with the trailing edge of the Spt16–Pob3 band). Excision of SPN–Nuc forms consistently yielded larger amounts of Spt16–Pob3 (Figure 5C, lane 8). Quantitation from several experiments revealed ∼0.93 molecules of Spt16 and 1.2 molecules of Pob3 per molecule of DNA at the SPN–Nuc position. While some of this material could also be due to spurious comigration, we conclude that each nucleosome is associated with no more than one heterodimer of Spt16–Pob3. This low stoichiometry is further evidence that Spt16–Pob3 does not simply aggregate with Nhp6 in this experiment. It was not possible to estimate the Nhp6 content of complexes using a similar approach since free Nhp6 was found to migrate throughout the region containing complexes both by examination of gel slices and by western blots (our unpublished data).
SPN causes changes in the organization of nucleosomes
The differing electrophoretic migration rates of Nhp6–Nuc and SPN–Nuc complexes suggest that these forms have altered properties compared with nucleosomes. We treated complexes with the nuclease DNase I to see if these alterations result in a different presentation of the DNA component. The 167 bp fragment of 5S DNA used for the nucleosome assembly is able to position histone octamers uniformly such that the unique label is near the entry point of the nucleosome, with a short duplex tail extending from the distal end. DNase I preferentially digested nucleosomes alone to produce a ∼143 nucleotide fragment, indicating removal of the unprotected tail (marked with a spot in Figure 6). Addition of Spt16–Pob3 alone did not affect the pattern significantly, but digestion at this exit point was diminished when Nhp6 was added. Since Nhp6 binds to DNA, it could simply inhibit digestion by limiting overall access. However, most sites were not affected by Nhp6 addition, so the entire nucleosome is not protected. Nhp6 alone also enhanced digestion somewhat at several sites (for example, at ∼110 nucleotides in Figure 6).
Addition of both Nhp6 and Spt16–Pob3 caused a more dramatic enhancement of DNase I digestion, particularly in the region proximal to the double‐stranded tail of these nucleosomes (marked with stars in Figure 6). Use of nucleosomes reconstituted with different DNA molecules (our unpublished data) revealed that the enhanced sites appear at a constant distance from the cut marking the exit of the DNA from the nucleosome (if the black spot is considered to be 0, the enhanced cuts are at about −13, −23, −33 and −42 nucleotides, with the central sites being the most pronounced). The ∼10 nucleotide periodicity of the enhanced sites indicates that the DNA is still associated with the nucleosome (this pattern is not observed with free DNA; see Figure 6), but in a more accessible form. Since many sites remain unaffected in the SPN–Nuc form, including sites an equivalent distance from the other end of the nucleosome, the alteration appears to be localized to the DNA near the extended tail. We conclude that Nhp6 binds to nucleosomes, probably through the duplex tail, causing a large change in electrophoretic mobility and a small change in accessibility of the nucleosomal DNA. This allows binding of Spt16–Pob3, which then causes a further reorganization of the nucleosome, changing the electrophoretic migration and altering the organization of the adjacent DNA within the nucleosome.
We present genetic and physical evidence that yeast Spt16–Pob3 functions together with the HMG1‐motif protein Nhp6 to bind nucleosomes and change both their electrophoretic mobility and the accessibility of their DNA. This suggests that these highly conserved factors participate in both replication and transcription by reorganizing nucleosomes in a way that changes the presentation of the DNA. Since accessibility of chromatin is also modulated by acetylation of histones, this also explains the functional overlap between Spt16–Pob3 and acetylation. Spt16–Pob3 could be associated with factors such as the acetyltransferase Nua3 (John et al., 2000) because both promote DNA accessibility in separate ways and would therefore be expected to act coordinately. Their combined function would be opposed by deacetylases like Rpd3, explaining the genetic suppression of spt16, pob3 and nhp6 mutants by the deletion of Rpd3 (Figure 2 and Yu et al., 2000).
New spt16 alleles support roles in replication and transcription
Spt16–Pob3 or its homologs have been associated with initiation of transcription (Malone et al., 1991; Rowley et al., 1991; Lycan et al., 1994; Costa and Arndt, 2000; John et al., 2000; Kang et al., 2000; Schlesinger and Formosa, 2000), elongation of transcription (Orphanides et al., 1998, 1999) and DNA replication (Wittmeyer and Formosa, 1997; Formosa and Nittis, 1999; Okuhara et al., 1999; Wittmeyer et al., 1999; Schlesinger and Formosa, 2000). This could indicate that Spt16–Pob3 has many functions, or that it has a single activity needed during many processes. We report here that multiple alleles of SPT16 have differential effects on the Spt− phenotype (transcription initiation), sensitivity to 6‐AU (transcription elongation), HU sensitivity (DNA replication) and synthetic defects with replication factors (DNA replication). If Spt16 had a single function and different mutations caused different degrees of impairment of that function, the same alleles should have the strongest effects in all tests. Since the allele‐specific effects among the phenotypes do not correlate in this way, we suggest that Spt16–Pob3 has a single central activity that is needed in multiple stages of transcription and replication, and that different mutations disturb the ability of Spt16–Pob3 to participate in discrete reactions. The simplest interpretation is that Spt16–Pob3 alters nucleosomes, and this is important first during the establishment of transcription and replication initiation complexes, and then again during elongation as both RNA and DNA polymerases encounter nucleosomes on their templates. Different mutations then disturb the ability of Spt16–Pob3 to function coordinately with factors that act in these different processes.
Spt16–Pob3 functions with Nhp6
Pob3 lacks the HMG1 DNA‐binding motif found in homologs from higher eukaryotes (Wittmeyer and Formosa, 1997), but we find in genetic tests that Spt16–Pob3 functions with a protein with this feature, Nhp6. Cells lacking both copies of NHP6 are viable (Costigan et al., 1994), so the essential Spt16 and Pob3 proteins cannot be entirely dependent on Nhp6 for their function. However, the robust genetic interactions we have observed demonstrate that Nhp6 supports Spt16–Pob3 function in some physiologically important way. Several approaches indicate that Nhp6 and Spt16–Pob3 are not typically associated with one another to form a free factor equivalent to FACT or DUF1. Instead, Nhp6 alone appears to interact with a nucleosome, and this forms a binding site for Spt16–Pob3. It has been suggested that HMG proteins deliver transcription factors to sites within chromatin (Bustin, 1999); our results indicate that they can also support the recruitment or action of other chromatin modulators.
Spt16–Pob3 binding might require contact with both Nhp6 and other features of the nucleosome, or it might require nucleosomes that are somehow repositioned by the binding of Nhp6. If other proteins are also capable of causing such a change in nucleosomes in vivo, subsets of these factors could be removed without blocking all Spt16–Pob3 activity. Candidates would include other known HMG1‐motif proteins, such as Hmo1 and Hmo2, which have been found to display minor genetic interactions with Spt16 (our unpublished data). However, since the quadruple deletion (nhp6a/b, hmo1, hmo2) is viable (Lu et al., 1996), if HMG1‐motif proteins play an essential role in yeast their redundancy must extend beyond this set of proteins. Spt16–Pob3 might also be able to function with other classes of DNA‐binding protein, or even alone, with reduced efficiency. The genetic results indicate that Nhp6 is more effective than other proteins at promoting Spt16–Pob3 function, but it remains to be determined whether the formation of SPN–nucleosome complexes involves specific protein–protein interactions between Spt16–Pob3 and Nhp6.
Nhp6 and SPN alter the properties of nucleosomes
Human FACT cosediments with nucleosomes or H2A–H2B dimers, and Orphanides et al. (1999) have suggested that FACT might act by releasing H2A–H2B from nucleosomes. We find that yeast SPN also binds to nucleosomes, and due to its modular nature we have been able to detect two distinct stages of binding. Nhp6 alone altered the electrophoretic mobility of nucleosomes, and addition of SPN caused both a further change in mobility and enhanced nuclease sensitivity. Notably, the strongest effects were in a region where H2A and H2B contact the DNA (Luger et al., 1997). This enhanced digestion suggests a reorganization of the nucleosome, specifically in the region of H2A–H2B, which is consistent with the proposed weakening of the protein–protein contacts between H2A–H2B and H3–H4. Alternatively, SPN could act by changing the contacts between histones and DNA. Nucleosome remodeling factors have been proposed to act in this way by displacing a loop of DNA (Kingston and Narlikar, 1999), which can then propagate around the nucleosome. FACT does not display standard remodeling activity (Orphanides et al., 1998), and Spt16–Pob3 does not have the ATPase activity normally associated with remodeling factors (Wittmeyer et al., 1999). However, FACT/SPN might still share some mechanistic features with these factors. Our data are consistent with a model in which the HMG1‐box factor associates with DNA near the entry/exit points of the nucleosome, then Spt16–Pob3 binds to this structure in such a way that the association of the DNA with the histone core is locally disturbed. Since the DNA passes the position of the H2A–H2B dimers twice, but we only see enhanced DNase I sensitivity in one of these regions, we prefer models in which the DNA–histone interactions are disrupted locally to those in which the protein core is disturbed, although these possibilities are not mutually exclusive. In either case, SPN is a novel remodeling factor that does not require NTP hydrolysis and does not reposition nucleosomes, but reorganizes them in a way that is important for replication and transcription machinery.
Altering the interactions between the components of nucleosomes could be an important step in preparing sites for initiation of DNA replication or transcription, or in allowing polymerases to progress on nucleosomal templates. This ability would therefore explain the broad range of effects caused by mutations in SPN components. The results reported here provide tools that will promote investigation of the role of Spt16–Pob3 and Nhp6 in chromatin‐mediated processes.
Materials and methods
Strains and plasmids
Strains are listed in Table III. spt16‐Δ(TRP1) lacks residues 8 through the stop codon. pCDC68 (Prendergast et al., 1990) and pTF125 are high copy URA3 SPT16 plasmids. pTF128 has 746 bp upstream of the SPT16 ORF and a modified but phenotypically normal SPT16 (which now terminates GSPR) in YCplac111 (Gietz and Sugino, 1988). NHP6A and NHP6B were deleted as described (Costigan et al., 1994). A construct that inserts KanMX into URA3 (Cross, 1997, D.J.Stillman, in preparation) was used to convert nhp6a‐Δ(URA3) to nhp6a‐Δ(KanMX). pTF146 is NHP6B with 1039 bp upstream and 786 bp downstream in YEplac195 (Gietz and Sugino, 1988).
SPT16 alleles were integrated using YIplac211 (Gietz and Sugino, 1988) derivatives digested with SnaBI. 5‐FOA‐resistant isolates (Boeke et al., 1987) were obtained from Ura+ transformants and then screened for the Ts− phenotype, tested by Southern hybridization and confirmed by sequencing relevant portions after PCR amplification. Other strains were as described previously (Malone et al., 1991; Formosa and Nittis, 1999; Schlesinger and Formosa, 2000; Yu et al., 2000) or derived from them using standard genetic methods.
spt16 mutant isolation and characterization
pTF128 was mutagenized with hydroxylamine as described (Schlesinger and Formosa, 2000) and used to transform strain 7737‐3‐2 (spt16‐Δ) carrying pCDC68 (Prendergast et al., 1990). Transformants were transferred to media containing 5‐FOA (Boeke et al., 1987), then replicates were incubated at 26 and 37°C. pTF128 derivatives were recovered from Ts− isolates and retested. The MPT was determined by streaking aliquots onto agar plates at 1°C increments covering a range from 26 to 37°C, with the MPT being the highest temperature producing at least 10% of the growth obtained at 26°C.
S1 assays
RNA levels were quantitated by S1 nuclease protection using HO and CMD1 probes followed by phosphorimager analysis as described (Bhoite and Stillman, 1998; Yu et al., 2000).
Nucleosome binding and nuclease sensitivity
DNA fragments containing the sea urchin 5S DNA gene were amplified by PCR and labeled with polynucleotide kinase and [γ‐32P]ATP (Sambrook et al., 1989). DNA and chicken histone octamers (Graziano et al., 1988; a generous gift from V.Graziano and V.Ramakrishnan) were mixed in 2 M NaCl and dialyzed as described (Luger et al., 1999). Nucleosomes were isolated by velocity sedimentation in fractions with 15% maltose, 0.1 mg/ml human serum albumin (Sigma), 25 mM HEPES pH 7.6, 1 mM Na2EDTA, 0.3 μg/ml leupeptin, 1.4 μg/ml pepstatin and 0.5 mM phenylmethylsulfonyl fluoride (gradient buffer).
Spt16–Pob3 was purified as described (Wittmeyer et al., 1999). NHP6A was amplified by PCR and inserted into pET16B, and the resulting His10–Nhp6 was expressed in Escherichia coli BL21(DE3)‐pLysS (Studier et al., 1990) and purified by chelated nickel chromatography as described (Brazas et al., 1995), with or without additional purification using DNA cellulose chromatography. Untagged Nhp6 was purified from E.coli carrying pRJ1228 (Paull and Johnson, 1995) or from yeast cells by differential precipitation with TCA and cation exchange chromatography essentially as described (Paull and Johnson, 1995). Binding assays were performed by mixing nucleosomes or DNA (in 5 μl of gradient buffer) with 2 μl of a solution containing 10% glycerol, 20 mM Tris–HCl pH 7.5, 100 mM NaCl, 1 mM 2‐mercaptoethanol, 1 mM Na2EDTA and Nhp6 or Spt16–Pob3 as indicated in each experiment. Reactions were incubated for 10 min at 30°C, then electrophoresed at 180 V for 4–5 h at 4°C through 4% polyacrylamide, 5% (w/v) glycerol, 2 mM MgCl2 and 0.5 × TBE (Sambrook et al., 1989). Regions containing labeled DNA were excised and separated by SDS–PAGE and stained with silver (Ausubel et al., 1994) or Coomassie Blue. The amount of DNA was determined by phosphorimaging, and the ratio of protein to DNA was quantitated using NIH Image software.
For immunodetection, proteins were transferred to nitrocellulose (Schleicher and Schuell BA83), probed with antisera directed against Spt16–Pob3 (Wittmeyer and Formosa, 1997) and detected with enhanced chemiluminescence as directed (Amersham‐Pharmacia Biotech).
For nuclease digestions, the 5S DNA was digested with EcoRI, labeled, then digested with ScaI, releasing a fragment with 167 and 163 nucleotide strands with the label at the 5′ overhang of the EcoRI cut. Nucleosomes and complexes were formed as above, then various amounts of DNase I (Boehringer‐Mannheim) were added, the concentration of MgCl2 was adjusted to 2 mM and the samples were incubated for 15 min at 30°C. EDTA and carrier DNA were added, the DNA was extracted with CHCl3, precipitated with ethanol and electrophoresed on 8% polyacrylamide gels containing 7 M urea along with size standards.
We thank V.Graziano and V.Ramakrishnan for histone octamers, R.Singer for pCDC68, the University of Utah Bioprocessing Center and D.Kolodrubetz for antisera, R.Johnson for pRJ1228 and B.Cairns for the 5S DNA plasmid, nucleosome expertise and helpful comments on this manuscript. This work was supported by grants from the NSF to T.F., the NIH to D.S., the Swedish Foundation for International Cooperation in Research and Higher Education to P.E. and the Huntsman Cancer Institute (through B.Cairns) for J.W.
- Copyright © 2001 European Molecular Biology Organization


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Condensé musical : Where Giants Once Stood vs The Body Politic
Let's see what we have here: two melodic metalcore bands releasing a cardboard EP, with a similar cover design, each of them being the second release of the band, the first one being released on September 2nd and the other on September 16th. Let's try something different and look at these two CDs at the same time.
In the red corner, we have Where Giants Once Stood, a name that resonates big and wide, and a reference to heaviness and gigantism. The young band comes from Toronto, thrash metal capital of Canada, and from the ashes of the scene's legacy they are ready to build something fresh and new.
In the blue corner, The Body Politic is a name that rightly sounds more intellectual: as a matter of fact, it's taken from a Clive Barker short story. Hailing from Vancouver Island, the members are proud to admit they shifted from jazz to progressive metal.
Live Above is a four-song EP. Where Giants Once Stood doesn't go into preliminaries: an EP has a short duration and they don't want to waste any time showing what they're capable of. "Living in Secrity" immediately catches the attention with its guitar solo à-la-Unearth and its clean vocals à-la-Avenged Sevenfold during the chorus. This great opener is followed by "Illuminate", a song that features Threat Signal's singer (and Live Above Producer) Jon Howard. To my humble opinion, this guest appearance weakens the song more than anything else, as his vocals doesn't match the power of the band's own set of vocals needed to carry on the attack. "The Damaged" brings in different influences and techniques–including a great guitar work throughout, and tams-tams and classical guitar at the end–resulting in a brilliant song structure. Last song "Myths, Lies, and Crimes" closes the EP the way it started: a powerful intro reminding me of Heaven Shall Burn, and then throwing in some grand features. Keyboards and bass guitar are particularly interesting on that last song, and they blend very well with the melodic chorus. While WGOS could have imitated their peers and abused breakdowns, they cleverly waited 'til the end of the album before throwing one in, and it's working pretty well.
With its six songs, Egressor is organized like a short album rather than a promo EP. It starts with "Vitam Agere", a keyboard-oriented instrumental intro, then switches gear on "Armature", where vocals come in. One thing that immediately struck me is how much The Body Politic's music breathes. It is apparent that the band knows how to gage the keyboard, bringing it in only when it adds something to the whole, while balancing technique with efficiency. The 7-strings attack stands out, as well as the mechanical math-grooves and the metronome-articulated songwriting. With its cold beauty, "All Hands" reminded me of Sybreed, aside from the clean vocals chorus. There are highlights for each instruments on this EP, for example drums have a particularly nice routine on "Swing for the Fences". While The Body Politic sounds and plays exactly like a metalcore band (think about Bring Me the Horizon or Parkway Drive), there is a clear venture on progressive territories and a mix of influences, including Opeth ("Colquoun") and Dream Theater ("Irradiate").
The Body Politic (photo credit Evan Nixon)
When we say melodic metalcore, we immediately think about short hair, shorts, wife beaters, dual vocals (harsh in verses, clean during chorus), a bit of a mathematical writing skill, and a sharp production. That's pretty much what we have on both releases, except that the guys put a matching t-shirt for the group photo. The production is huge, and the songwriting for both EPs is impressive. On WGOS's EP, I particularly enjoyed the quality of the drums recording, and the the lead guitars. On TBP's EP, I liked the compression on the bass drums, and the intelligent use of keyboards.
While WGOS has a more in-your-face approach, sending the infantry first in order to put the listener to fligth, The Body Politic uses its arsenal more strategically, bringing in its cavalry through clever technique. Both EPs are excellent, and despite the distance I would easily see them as a tag team on a tour.
Where Giants Once Stood (photo credit Logan Swinkels)
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photo Réjean Poudrette
Chevrolet's conventional twin-slot grille fronts a car filled with some very advanced technology. The grille area has shutters that close at speed to reduce wind resistance
photo Réjean Poudrette
First presented at the Detroit auto show in 2007, the Volt is GM's idea of an extended-range electric vehicle. The eye-catching shape of the concept car could not be produced in volume, but its battery-powered electric propulsion and range-extending gasoline engine acting as a generator made it to production -- a monumental feat. The production car survived GM's U.S. descent into bankruptcy.
The Volt went on sale in the U.S. in the fall of 2010, arriving in Canada a year later.
Key Competitors
With its range extending gasoline power, the Volt is unique. Only the new plug-in Toyota Prius also has the ability to operate under full battery power until depleted and then become a regular gasoline hybrid. However, the Prius' range on battery power alone is limited. Full electrics, like the Ford Focus BEV, Mitsubishi iMiEV and Nissan Leaf offer greater range under electric power but are stranded when the batteries are depleted.
One port for both 120 and 240 volt charging
Under battery power, the Volt's electric motor delivers brisk, instant, acceleration in near total silence. Interior noise is acceptable after the gasoline-powered range-extending generator starts up. Wind noise and road noise are noticeable, and perhaps more obvious because of the absence of noise from the engine when it is not running.
Under ideal conditions (about 20 degrees Centigrade), the Volt can travel about 65 kilometres under battery power before its gas generator kicks in. For the 2013 model year, small tweaks have extended the range in electric mode by a couple of kilometres. The Volt's electric reserve gauge consistently displayed an accurate estimate of the remaining range. A conventional gear selector reinforces the remarkably "normal" driving experience the car provides.
The Volt's electric power steering is nicely weighted and geared. The suspension combines a resiliently firm ride with crisp handling. However, the suspension is noisy; reverberating impact noises and clunking are constant when rolling over rough pavement, which hurts refinement and had the APA wondering about the durability of suspension components. The regenerative brakes require a long pedal stroke and heavy effort for quick stops.
Outward vision is less than ideal, hampered by a low seating position and big windshield pillars. The air conditioner is powerful.
Winter operation
The APA had an opportunity to experience the Volt in the winter, and the car performed a bit differently in cold weather. Even when the battery pack was charged, the Volt's gasoline-powered generator would come on for a few minutes after every start in cold weather. In a 25 kilometre one-way commute, the Volt consumed two to three litres of gasoline on each journey, with overall fuel consumption being roughly 6L/100 km. In summer, the gasoline-powered generator is virtually silent at the lower of its two fixed RPM cycles. In winter, the higher RPM cycle, which is noisier and transmits some vibration into the cabin, engages more frequently.
Braking is less satisfactory in winter, and the brakes tend to lock slightly at low speeds. The low-mounted front spoiler has limited ground clearance for snowy conditions. Windshield defrosting is efficient enough, but heater output is a weak in very cold weather and the fan needs to be run at its highest speed to warm up the occupants. The heater is helped somewhat by quick-acting and toasty-warm seat heaters.
The Volt features a remote starter that can warm the cabin and defrost the windows before you enter the car. If the car is plugged in, the cabin pre-heater runs off utility company power and doesn't drain the battery pack at all! The charger can be programmed to function when electricity rates are lowest, to reduce operating costs.
photo Réjean Poudrette
The Volt's 1.4L gasoline engine and electric motor are packaged together under the Volt's conventional hood
The Volt's cabin is fronted by a liquid crystal display (LCD) gauge package of remarkable visual brilliance. The gauge cluster has three main readouts, a speedometer in the middle, a battery life/kilometres-to-empty graphic on the left, and a display for real-time driving efficiency on the right.
The dash centre-stack is topped by a large LCD screen containing either the navigation display or various ancillary readouts, depending on driver preference. The centre-stack also houses the audio and climate controls. The controls for these two systems are co-mingled, leading to confusion, which is further undermined by touch-sensitive switches that don't always respond to driver inputs as they should.
While the controls aren't the easiest to operate, they, and the rest of the cabin, look great. Stung for years by comments about its shabby cabins, and knowing that the Volt was going to be expensive and THE technological standard-bearer for General Motors, GM crafted an elegant, futuristic interior for the Volt. Abundant soft-touch surfaces, matte finishes, chic two-toning and careful assembly are consistent with the Volt's high price and importance to GM.
The Volt's battery pack occupies a spine down the middle of the car, splitting both the front and rear into bucket seats, and making the car a four-seater. Seating is comfortable front and rear. Legroom is plentiful in front, but rear seat legroom is very tight. Rear seat headroom is also less than generous.
Though the Volt looks like a conventional sedan, it is actually a hatchback. Relatively deep below the window line, the Volt's adequate trunk space can be augmented by folding down the rear seatbacks. The audio system provides good sound, but excellence is expected from an upgraded unit like that installed in APA's test Volt.
photo Réjean Poudrette
Incredibly crisp liquid crystal display gauge package on the Volt. The green ball located between two parallel lines on the far right reflects real-time driving, migrating up or down depending on how gently or aggressively the car is being driven. The battery-like tube on the far left represents battery charge and the remaining range under battery power
photo Réjean Poudrette photo
Knowing that the Volt was going to be an expensive car that was also going to be the standard-bearer for GM's advanced technology, GM took considerable care when it created the Volt's cabin and used high quality materials. That said, the co-mingled climate and audio controls, and their touch-sensitive controls need improving. The conventional shifter works better than the mysterious joysticks used by some competitors
Living with the Volt
Other than a heavy, short cord that forced some APA staffers to use an extension cord (which got trapped in ice overnight), recharging the Volt is a snap. The Volt recharges in seven hours from a conventional socket, which is less than half the time needed to recharge a Nissan Leaf.
In Quebec, Hydro Quebec has introduced a network of recharging stations across the province it calls "The Electric Circuit". Electric car owners in major urban areas will be able to grab an hour or two of charge while shopping or dining at over 100 locations, concentrated in the Saint Hubert Rotisserie, RONA and Metro supermarket chains.
Not all points were operational in June 2012, and the APA discovered that the charging outlet at the suburban Montreal outlet where we planned to stop was not installed yet. Worse still, if you're only staying for an hour or two, the charging rate is so slow that the benefit is largely cancelled out by the drive to the charging station! The charging infrastructure will eventually have to be optimized for locations where an electric vehicle is likely to be parked for more than a couple of hours, and the downtime during charging will have to drop substantially to make recharging-on-the-go a reality.
photo Réjean Poudrette
A conventional silhouette hides a hatchback tail. The Volt is a four-seater, as its battery occupies a spine down the centre of the car. The trunk is reasonably deep below the window line. The two rear seatbacks fold to increase the length of the cargo bed.
Not rated. The Volt is a brand new design that uses a lot of unique technology. The Volt has a three year/60,000 km bumper to bumper warranty, five years/160,000 km on the powertrain, and eight years/160,000 on the electric powertrain. Routine servicing requirements are comparable to a conventional gas engine. The electric propulsion system is virtually maintenance-free. Regenerative braking means that conventional pads and rotors should last longer than on a conventional car. Repairs are likely to be dealer-only for some time. As with other EVs and hybrids, long-term reliability is unknown (except for the Prius, which has an enviable track record).
At a base price of $41,454, the Volt is much more expensive than a compact gasoline-powered hatchback of similar size. After-delivery rebates of $8,321 in Ontario and $8,000 in Quebec reduce the actual cost of the Volt considerably. Lease payments (48 months, $0 down) are virtually the same as financing (60 months $0 down) making leasing a non-starter. With the rebate, it's significantly cheaper than most entry-level luxury cars, but it doesn't appear to be attracting buyers in that segment.
The base Volt is priced nearly $6,000 higher than the base version of the Toyota Prius Plug-in, which is the car closest in concept to the Volt. The Volt costs nearly $6,000 more than a base Nissan Leaf, but benefits from a range-extending gasoline powered generator, and is a much more versatile proposition. The new all-electric Ford Focus BEV is priced only $356 less than the base Volt but lacks the Chevrolet's range-extending powertrain. Mitsubishi's iMiEV, a pure electric car, has an $8,547 lower MSRP than the base Volt, but is a less robust, less refined vehicle than the Chevrolet or all-electric Leaf.
The Volt is equipped with dual front, seat-mounted side, side-curtain airbags and knee area airbags for both front occupants. The Volt is an Insurance Institute for Highway Safety Top Safety Pick, and was rated Good in the IIHS's frontal, side, and rear crash tests, and in the roof strength test.
photo, Réjean Poudrette photo
The Volt's rear end contains a smoked-glass element which is the sole vestige of the glazed band that circumvented the passenger compartment of the original show car
Overall Assessment
After spending time behind the wheel in various battery powered and hybrid vehicles, the APA has come to realize that battery range is not the big limiter with electric vehicles. By way of example, motorcycles typically will not travel more than 150 km on a tank of fuel; that's similar to the better EVs, but riders think nothing of long distance travel. With EVs, the limiters are recharging times and the undeveloped charging infrastructure. A motorcycle rider can fill up in under five minutes every couple of hours at a gas station and get back on the road. With an EV, even in Quebec where a highway charging system is being installed, you'll be waiting for several hours before getting back on the road. To APA that's the real limitation, and one that only the gas-powered hybrid EVs can currently resolve.
After winter and summer tests, the APA came away very impressed with GM's latest electric car effort. The Volt is a more versatile package the could function as a family's primary or only vehicle, unlike the all-electric Leaf and more recent battery-only vehicles. It's well assembled, and the best handling of the "Green" cars. The Volt is an early ticket into the electric vehicle age, before the charging infrastructure and battery charging technology are fully mature. .
The Volt's principal limitations are its high price compared with a conventional car of similar size and utility, and a small back seat. Plenty of cars and SUVs offer less originality and features than a Volt, yet they command higher prices on account of the public's willingness to pay more for SUV and luxury features than "green" car benefits like very low fuel consumption. At this time, despite concern over fuel prices, sales of the Volt have been very weak in Canada; however, it is outpacing full-electric EVs like the Leaf and iMiEV.


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Hi Forums
I have a customer that uses the good old way of having a "WebPage" at
the users homedirectory like http://netwareserver/~username/something
Is that functionality still avalible with a OES2 - SP1 On Linux?
and if not would it be possible to reproduce this functionality
with apache and NSS volumes for users in a migration to Linux (OES2-SP1)?
- Michael


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Equine Science Update
Contact us.
Reports from the world of equine research.
Gait analysis in the field.
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A gait analysis system initially developed for the Great Britain dressage team is now available to all.
Haydn Price, farrier to the British Olympic team, started using video recordings in his work in 1999. Initially, he had only a hand-
held camera, but it allowed him to record the way the horse moved. He could then examine more closely any areas of concern.
But he wanted something more objective. "I could never prove that what we had done had helped the horse. I could see the effect, the owner could feel the effect, and the horse performed better. But I couldn't prove it."
To find a solution he turned to Professor John E Davies, consultant Physician in Sports Medicine at Guys Hospital, London. Together they developed a portable gait analysis system ("Equinalysis").
As the only equipment needed is a video camera and a laptop computer, the system is readily portable, making it possible to use it "in the field". A standard protocol has been devised.
Markers are placed at specific points of the horse's limbs -
mainly over joints. The horse is walked and trotted in hand past the camera. A video recording is made from in front and behind and from both sides. This can then be slowed down, examined and analysed.
What does Price look for in a recording? "The video clips should show clearly two full strides from a lateral view. The handler should not influence the horse, at both walk and trot, in any way. Of course the horse must move in a straight line."
The computer software records the movement of the markers, and produces data that can be analysed to give information on factors such as the range of movement, the angle, and degree of flexion of joints.
What are the most common reasons for examination? "The usual request is either poor performance or intermittent lameness" Price reports. " But also and interestingly, that the rider "feels something" but nothing clinical can be diagnosed at veterinary inspection "
"We have used the system to measure the effects immediately after the shoeing process has been executed, with some very interesting findings! This has highlighted the effects (or in some cases not!) of recognised treatment plans for specific conditions, such as rolled toe and raised heel."
"We can accurately measure differences in stride patterns and isolate it to particular segments of the limb"
Martyn Elliot, a farrier in Nottinghamshire, has been using the Equinalysis system since early 2006. "Most of the gait abnormalities can be identified by playing the video slowly" he explains. "But for complicated cases you can refer to the biomechanical measurements for interpretation."
"The big advantage to a farrier using this equipment is they can cut out the middle man. They can analyse, identify the problem, suggest a remedy, try it and reassess to see if it has had the desired effect."
He describes a Pony Club dressage horse that was referred to him by another farrier because of abnormal wear of the shoes that he could not explain. The video showed that the horse was markedly over-
tracking -
the hind foot would land three foot lengths in front of the fore foot. By studying the video, Elliot was able to see that the horse twisted the stifle in and the hock out just before breakover, at the end of the weight-
bearing phase of the stride. So he shod the horse with a squared toe with plenty of support at the quarters. Afterwards the horse moved much better. The rotation was much less marked and the overtracking was reduced to about one foot length.
Although the system was originally conceived as a means of assessing the effect of shoeing on the horse's gait, it has other applications. It can be used to assess other interventions such as manipulation, and can provide a record of the horse's action.
"We are about to train a chiropractor who wishes to measure the effects of treatment" Haydn Price explains. Indeed Martyn Elliot has already measured a horse following manipulation"
Haydn Price sees a place for the system as part of the prepurchase examination. "Some vets would prefer to incorporate it as part of the vetting procedure whilst others may prefer to use the facility afterwards. Just remember that the system does not diagnose and therefore can only be accurately used alongside present veterinary practices. Equally the information from the screening can assist in acting as benchmark in the same way as x-
rays or scans."
"We are collecting data from our business partners and from veterinary practices" he explains. "Whilst it is too soon to give statistical information, some very interesting findings are now beginning to appear. For example, with Equinalysis, I can prove that moving the front of the shoe back by 3mm results in a 6-
degree difference in knee flexion."
Price concludes " With Equinalysis we can optimise the trimming and shoeing so that the horse can move in the most efficient way. This should limit the strain on the joints, promote better performance and may delay the onset of degenerative joint disease."
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© Copyright Equine Science Update 2006


text:
I'm a hap hap happy hippo
Who plays out in the sun.
When all you children are at school,
I'm out there having fun.
I love to sit in water
Which keeps me cool, when hot,
And, wallowing in a muddy pond,
Is what I like a lot.
I'm glad I'm not a boy or girl
Who goes to school each day
And I think I'm very lucky
Because it's in my pool I play.
I'm a hap hap happy hippo
And a really friendly guy,
But now it's time to go to sleep
So I'll wish you all bye bye.
Copyright on all my poems


text:
More than 400 Canadian hospital patients die annually because they are admitted for urgent medical and surgical care on weekends, new research suggests.
The largest study of its kind to date in Canada — it looked at 4 million urgent hospital admissions over three years — found that patients admitted for unexpected problems on weekends have 4 per cent higher odds of dying compared to patients admitted on weekdays.
The "weekend effect," as it is known, is highest for those admitted for emergency surgery or heart attacks, according to an analysis by the Canadian Institute for Health Information, released Thursday.
Emergency surgery patients have 7 per cent higher odds of dying when admitted on weekends. And heart attack patients have 8 per cent higher odds of dying when admitted on Saturdays or Sundays.
There is no weekend effect for all other acute care patients, including children, mothers delivering babies and those receiving mental health services.
Overall, patients needing urgent surgery or medical care have 4 per cent higher odds of dying if admitted on a weekend instead of during the work week.
Canada's "weekend effect" isn't a bad as that of other countries. In the United States, it's 15 per cent and in the United Kingdom it's up to 16 per cent, according to other studies.
"If you have an urgent medical condition, you should feel confident coming to the hospital emergency department on a weekend and not waiting because time to treatment is so important for so many illnesses," said Kathleen Morris, CIHI's director of health system analysis and emerging issues.
"While the numbers that we found are statistically significant, they really are very small," she added, noting there are 75,000 in-hospital deaths in total annually in Canada.
Delays in accessing diagnostic tests may be a reason for the poorer outcomes for heart attack patients, researchers say. Both heart attack and stroke patients waited longer for diagnostics on weekends, yet only heart attack patients experienced the weekend effect.
Another possible cause of the weekend effect is the reduced staffing levels on Saturdays and Sundays, according to the online health policy magazine HealthyDebate.ca.
Also contributing to higher weekend death rates is the way people use hospitals. Those with less severe conditions may not come in on weekends as readily as they would on weekdays. This means the proportion of patients with more severe conditions becomes higher on weekend, contributing to an increase in mortality.


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2018 Audi A6 Release Date, Price, Redesign
2018 Audi A6 Release Date, Price, Redesign – The German automaker Audi is now preparing the next technology A6 mid-size sedan having an evolutionary design. The new model is stated to get the related interface with the new A8 luxury sedan. In the Audi vehicle lineup, the A6 is regarded as a significant item for the Audi brand mainly because it supplies an increased earnings for the company than the A3 and A4. Additionally, the Audi A6 is in foreign demand in Europe, United States Of America and China.
The 2018 Audi A6 is rumored to be biking on a new very versatile platform and presenting several of the newest technology features. This is an Audi MLB Evo modular-longitudinal principles that are likewise used by the approaching A8 and A7 sedans. The new platform enables Audi to offer AWD and FWD solutions on the up coming A6 sedan, as properly as making the weight about 100 kg lighter in weight. Furthermore, the fifth-technology A6 sedan will obtain a good deal of design vocabulary from the new Audi A4. So the after that A6 is expected to provide a thin design, refreshed design, two touch screens, as accurately as updated technological innovation.
2018 Audi A6 Engine
The new A6 should be the two more efficient and much more fun to operate than the old car thanks to a lighter weight MLB Evo chassis given to the impending A8 and A7. This advanced platform may make use of carbon dioxide-fibre and aluminum in place of traditional steel to help you the oncoming car consider considerably less than the old 1,535kg A6.
An economical 2.0-litre diesel engine must cater for most consumers but motorists right after a little more performance ought to start saving for further useful S6 models operated by either a 3.0-litre V6 or 4.0-litre V8 petroleum model. Audi ought to tease particulars of a seriously quick RS6 model after the A6's release which can be run by a dual turbo V8 shared with the most popular Porsche Panamera.
2018 Audi A6 Interior
The 2018 Audi A6 interior design is going to be significantly distinct. If you check out the modern interior cabin of the Audi A6, you will find a lot of switches to control the functions available in the car, for the 2018 version, most these switches will likely be removed. This car can have two various display screens with some other functionalities, and you start using these monitors to manipulate each feature seen on the car.
In addition to that; but the interior cabin of this new version is likewise provided with higher quality fabric for the seats and dashboard. The absent control buttons and much better good quality material implies much better comfortability; luckily there are nonetheless a lot more improvements created for its cabin. This new car is employing different platform to the final Audi A6 because it is said that the excess weight is about 50 weight lighter in weight and yes it offers more space available because of its greater dimension. There can also be a lot more contemporary safety functions accessible, most likely since some of the existing security capabilities are presently deemed as dated.
2018 Audi A6 Release Date and Price
Audi has yet to publicize precise specifics of the new A6's price or on purchase day. However, it's anticipated to be publicly unveiled on the marque's property turf, Germany's Frankfurt Motor Show, in September this year, with income starting in 2018. To stay very competitive in the industry, A6 costs are supposed to close to correspond with the provider model, which commences at around £35,000.


text:
Clue: Some platters
We have 1 answer for the clue Some platters. See the results below.
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- Contents of some sleeves
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- The Who's "Tommy" and others
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text:
Tesco has just announced a 10% fall in group profits to £1.6bn. The retailer's share price is already depressed and chief executive Philip Clarke has put the brakes on opening new stores while existing ones are spruced up.
Looks like he's applying the same logic to Clubcard, the loyalty scheme Tesco has doggedly maintained despite its huge cost.
Tesco is planning a brand extension called Clubcard Play, recruiting managers for the yet-to-be launched service aimed at giving Clubcard holders 'useful, fun access to their own data'.
Now, it's not often we get to see the words 'data' and 'fun' in the same sentence. As engagement propositions go, allowing consumers access to their transactional data isn't quite up there with free tickets to see Adele or even double Clubcard points.
But in launching Clubcard Play, Britain's biggest grocer can't be accused of missing an opportunity: Midata, the government-sponsored plan to encourage brands to give consumers access to their transaction records, will become mandatory next year.
Cynics will see Clubcard Play as a defensive move against an initiative the British Retail Consortium describes as an 'additional burden' on retailers and one that offers consumers 'little benefit'.
Yet if any brand was to make a virtue out of such a necessity, it would be Tesco. Since Clubcard's debut back in 1995, Tesco has mined the gold in the scheme by selling its data back to suppliers. Now it is seeing diamonds in that same data.
Rivals take note. Sainsbury's, we have no doubt, is working on its own Midata scheme with Nectar.
The challenge for Tesco will be to make the consumer – the right, elusive sort, not those who habitually respond to offers and games - bother to do something with their data.
But by investing in Clubcard Play now, Tesco is upping its personalisation game to ensure consumers won't see any point in passing their data to another brand's application.
At the very least, whatever the long term prognosis on Clubcard, the new scheme will provide reasons to engage once more with the Tesco brand, in a richer, less passive way than before.
Noelle McElhatton is editor of Marketing
firstname.lastname@example.com or tweet her at n_mcelhatton


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Impressive Attendance, Board Members Honored at uv.eb WEST 2015
UV+EB Technology
RadTechs Committees and Focus Groups are charged with expanding and sharing information about UV/EB technology. Many of these groups gathered for official face-to-face meetings during uv.eb WEST. If you are interested in joining a Committee or Focus Group, call 240-497-1242.
The uv.eb WEST 2015 tabletop exhibition was full of industry suppliers showcasing the latest equipment, technology and services.
Jo Ann Arceneaux of Allnex presented "Regulation Friendly UV-Curable Products" during the UV Inkjet for Food Packaging sessions, and "Mitigation of Oxygen Inhibition to Improve the UV LED Cure Process" during the What New Products and Processes Will UV LEDs Enable in 2015 sessions.
Attendees and exhibitors of uv.eb WEST 2015 were reflective of the diverse range of industries that UV/EB curing technology impacts.
RadTech International North America thanks its members and associates for their role in the impressive number of exhibitors and attendees at uv.eb WEST 2015, which took place in early March in Redondo Beach, California. This years conference featured a short course on The Chemistry of UV/EB and two days full of presentations, with sessions categorized under the following topics: 3D Printing; Printing & Packaging; UV Inkjet for Food Packaging; New Products & Processes in UV LEDs; UV/EB Curing Innovations.
Attendees reflected the diverse range of industries that UV/EB curing technology impacts – from brand owners and consumer product companies to packaging printers and converters, chemists and materials scientists. "We are particularly excited that a large percentage of our attendees were end users and/or first time RadTech participants," said Gary Cohen, executive director.
During a ceremony at the conference, Don Duncan of Wikoff Color, immediate past president of the Board of Directors, presented Presidential Awards to the co-chairs of the RadTech Editorial Board – Chris Miller of Estron Chemical and Jim Zawicki of Sartomer Americas. The two were honored for their efforts in launching RadTechs new magazine – UV+EB Technology – which made its official public debut during the show.
"Chris and Jim led a seamless transition from the RadTech Report to our new publication," said Peter Weissman of Quaker Chemical Corporation and current president of the Board. "Their hard work, guidance and leadership made possible our very successful launch with quality articles and a four-fold increase in distribution."
Also honored were several RadTech Board Members who have served their term of service, including Miller; Don Duncan of Wikoff Color, immediate past president of RadTech; Steve Lapin of PCT Engineered Systems division of Ebeam Technologies, Comet Group; Mike Sajdak of INX Intl; and Im Rangwalla of Energy Sciences Inc. For more information, visit www.radtech.org.


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This Exhibition encompasses different viewpoints of the expressive and visual perspectives for the sensual, affectional and emotional aspects of the meaning of love. It aims at exploring the components of individual and collective behavior in human relations, which is considered the main tool in tracing shapes, symbols and elements of artistic expression.
Love has various names, meanings and types. It is "the constant inclination of the roaring heart" in the Arabic and Islamic heritage, and its names include affection, fondness, passion, adoration, longing, friendliness, amorousness and infatuation. There are different types of love including divine, brotherly, platonic, romantic and physical. It is, in short, the essence of life.
When attempting to formulate and transform human emotions and verbal meanings of the concept of love to visual and expressive elements, impressions and accumulating images from the memory flow, as well as human and social relations. These form the framework and givens in setting up the various levels of expressions, in line with experimentation and searching for a context for a different visual, expressionist and cultural reading. Reduction becomes a main feature when dealing with shapes, elements and symbols within the expressionist context about people, wordings and shapes of human relations in an environment of cohesion and assembly, which in turn work on the dynamics of the scene, in addition to assure the linear overlap of shapes and elements.
The stress on the central positionality of the main elements in the scene was an attempt at the intensification of the collective cognitive narrative towards the part and the whole, where details and partial elements become themselves the main engine of the interaction between elements. The attempt is to tackle the dreamy human emotion through mutual inclusion - whether of humans, natural elements, or features from the geographical landscape - and within social and human relations, to highlight the symbolic and expressionist aspects of "the spirit of love".
Goerge Fikry Ibrahim


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Become part of the luxury found in the prestigious golf course community of Bay Oaks. Nestled on a cul-de-sac lot with a view of Horsepen Bayou, this magnificent home boasts a formal living room with breathtaking floor to ceiling windows, 2 gas log fireplaces, plantation shutters, granite counters, 3 staircases, and home theatre. The spacious island kitchen is an entertainer's dream with Sub Zero refrigerator, gas range, wine cooler, warming drawer, and chef's desk. Pamper yourself in the master suite featuring double tray ceiling, bay windows, walk-in closet, double sinks, whirlpool tub, and frameless shower. Home also includes game room with built-ins & mini kitchen, gated driveway entry, 3 car detached garage, and guest house with full kitchen, bath, laundry room, & elevator access. Step out into the privacy of the spacious backyard with stamped cement patio and lush landscaping. Spend afternoons at the country club relaxing by the pool or enjoying the premier tennis facility.
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Lance Loken
Listing Agent


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April 20th, 2016
by Danielle Miguel
Members of Generation Y, better known as the millennials, are currently on the rise in numerous fields across the globe. As heirs to this rapidly developing world, they must prepare themselves to usher in a new era of technology and innovation that vastly diverges from the experiences of their predecessors, Generation X and the Baby Boomers.
These technological advancements make their way into daily life and have allowed millennials to grow into the largest technically literate group. The influence of technology has changed how work is fundamentally conceived; not only does millennial work ethic differ from the high-collaboration style of Generation X and the cost-effective Baby Boomers, but their self-conception does as well. With 78% of millennials found to be tech savvy and 70% of them identifying as social media opportunists, it becomes evident that they have invested their efforts into making a name for themselves in a culture that stresses the hype of the start-up and marketing app. [1]
Accessibility to popular platforms and sites has positioned millennials in a phase of self-branding where names are promoted, relationships are made, and networks and communities are built. Looking into four different popular social media platforms, we can find a variety of ways that they impact the way millennial networks function.
As many are aware, Facebook is probably the biggest social media platform of the decade. With 76% of millennials scrolling through their newsfeeds to check up what their friends are up to, it becomes an inherent habit to make sure people see what one's life entails through constant status updates, video shares, and picture comments. [2] Simply pursuing relevance on the newsfeed sheds light on our initial self-branding — we create an image of how we live our lives, from showing off our latest foodie escapades to showcasing captured moments from our spring break trip to Cabo San Lucas, and we become consumers of each other's life stories.
Unlike Facebook, our self-branding on LinkedIn is tailored to be much more professionally appealing. José van Dijck describes the site as "the largest professional matchmaker site in the world." [3] Although LinkedIn provides features similar to Facebook's interface for its users, it has a far more professional twist. Using LinkedIn has become a career and work platform for millennials, and have given them the opportunity to brand themselves as reliable, hardworking, potential employees. Just as Facebook allows us to tap into the thoughts of others across the world, LinkedIn acts as an essential mode of expanding professional networks building one's connections to multiple sources of support across the globe.
The rise of Snapchat is different than Facebook and LinkedIn in that is by far the most informal of the three. In fact, it exceeds the standards set by Facebook in regards to spreading awareness between friends and keeping people updated with each other's lives. The app, in a sense, has broken the fourth wall of millennial self-branding by allowing users to send short, ten-second photos and videos out into the world as instantaneous and accessible commentary on their daily lives. Its uniqueness comes from its play on giving users up to 24-hour access and involvement to each other's lives regardless of proximity. It has also created a new kind of fame: pop culture successes like DJ Khaled's "Keys to Success" snaps, and the popular Damn Daniel clips have brought national attention to Snapchat users Daniel Lara and Joshua Holz.
Although Facebook and Snapchat undergird millennial social connections and LinkedIn facilitates professional bonds, millennials turn to Tinder to build a personal brand for their romantic and sexual encounters. The app involves creating a profile set to cater to certain ages and geographic locations and including a brief biographical statement and photos. [4] Tinder has revolutionized how millennials understand and brand themselves as "dateable" by linking dating to "likes" and "matches." Unlike previous generations, millennials have the capacity to set themselves up on dates, rather than relying on friends and family members to "match" them with others. While the app constantly updates with matches and conversations between potential dates, exposing the continually changing state of the millennial's social connections, it takes great strides to ensure the privacy of its individual members "likes," showing the additional care with which millennials regard their self-brand.
Danielle Miguel is currently a third year undergraduate studying Public Health, Public Policy, and Political Economy at the University of California, Berkeley. Authenticity, creativity, and opportunity are the core of her values, and she strives to apply them in all facets of life. You can reach Danielle at email@example.com
Image credit: Tanja Cappell https://flic.kr/p/dTZAW6
1. http://www.businessinsider.com/how-millennials-gen-x-and-boomers-shape-the-workplace-2013-9 ↩
2. https://www.americanpressinstitute.org/publications/reports/survey-research/millennials-social-media/ ↩
3.http://mcs.sagepub.com/content/35/2/199.short ↩
4.http://www.tandfonline.com/doi/full/10.1080/1369118X.2013.850527 ↩
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Credit card providers use 3-D Secure or 3Ds as an additional layer of security every time a Singapore cardholder uses a credit card or debit card for an online transaction. This extra authentication step protects merchants and cardholders against fraud. In other words, 3DS is a safe way to make sure that the person making the online transaction is the rightful owner of the card being used.
When a purchase is made at a merchant website that has 3DS technology, the cardholder receives a one-time password (OTP) on their mobile phone. They must then enter the OTP on the website to complete the transaction.
VISA first developed the 3DS system and offered it to consumers under the Verified by Visa service. Their goal was to improve the security of internet payments for both merchants and customers. MasterCard has since adopted the protocol under the MasterCard SecureCode, followed by JCB International as J/Secure. In 5 March 2010, the Association of Banks in Singapore adopted 3DS to further improve the security for card payments and better protect Singaporeans against fraud.
Air miles, also known as frequent flyer miles or travel points, are a type of credit card reward offered by airlines and card issuers. Cardmembers are rewarded every time they use their credit card in Singapore and overseas, by earning a certain number of miles for every dollar spent. When they accumulate a certain number of miles, they can redeem these for business class upgrades or even free flights with the card's partner airlines.
With an air miles credit card, cardmembers don't necessarily have to travel to earn miles. Because miles are awarded for every dollar spent on the card, cardmembers can start earning points towards their next trip by shopping, dining, or buying groceries. Since credit cards are accepted by virtually every merchant in Singapore, accruing miles becomes efficient when the card is used to pay for most expenses. Some credit cards also offer bonus points when online travel bookings are made through their partner merchants.
To redeem miles for flight tickets, cardmembers have to make the request through their card issuer's rewards portal. Depending on the card issuer, a redemption fee may be required to convert miles into air tickets. Co-branded airline cards usually credit the miles directly into the cardmember's frequent flyer account.
American Express is a top payment processing network and card issuer, with over 109.9 million cards running on their network worldwide. The number of cards they have in circulation makes them the 4th largest card network in the world and the largest card issuer based on purchase volume.
Most people know American Express by their iconic Green, Gold, and Platinum charge cards, as well as by the Centurion that graces the center. Cards of similar color levels are available in Singapore. They also have several co-branded cards which generally fall under the travel and retail categories of the local country they are present in. In Singapore, American Express has co-branded cards with Singapore Airlines and CapitaLand Malls.
Travel guides have noted that American Express credit cards are less accepted in Europe compared to Visa or MasterCard. Because American Express charges merchants 50% more than their rivals to carry their card, fewer retailers in Singapore take their cards as well.
Despite this, American Express was ranked the highest in Credit Card Customer Satisfaction by the 2015 and 2016 Singapore J.D. Power Credit Card Satisfaction Study. They were also the first card provider in Singapore to partner with Apple Pay, which allowed cardmembers to sync their personal or corporate credit cards to their Apple devices and used NFC technology to make payments as early as April 2016.
Also known as an Annual Membership Fee, a credit card annual fee is automatically billed to your statement for the convenience and privileges offered by the card issuer. They generally range from S$80 to S$500, depending on the type of credit card.
Not all credit cards in Singapore have an annual fee, but some types of cards are more likely to have it than others. Air miles cards, rewards cards, and premium cards are among those that have annual fees. Many of these cards waive the annual fee during the first year, then automatically charge it to your account in the subsequent year. Cards that have a high-value welcome offer, such as luggage or bonus miles, generally require cardmembers to pay the annual fee in order to claim the offer.
An annual fee waiver is when the card issuer cancels your annual fees on your credit card. Most credit cards that have an annual fee typically waive it during the first year or two of card ownership. However, you may need to pay the annual fee to claim a high-value welcome offer like luggage.
Annual fee waivers are up to the card issuer's discretion, and there are instances where they are more likely to grant it than others. Generally, if you charge a certain amount to your card every year, you have a better chance of getting the annual fee waived than if your card is barely used. Call your card issuer to find out what you need to do to get the fee waived.
A balance transfer lets you pay down the existing balance on a credit card by transferring it onto a new credit card account. The new credit card charges you 0% interest on the transferred balance for a certain period of time, usually 6 months, which must be fully paid off. If the transferred amount is not fully paid within this time period, the remaining balance is charged interest.
There are a few caveats to note before getting a balance transfer. Balance transfer credit cards are not entirely free. In some instances, you may be charged a fee of 3% - 5% on the transfer amount to complete the process.
The amount you can transfer must also be within the credit limit of the new card. For example, if the credit limit on the balance transfer card is S$8,000 and you want to transfer a balance of S$10,000, you will only be able to transfer up to S$8,000 (including fees).
Nevertheless, if used responsibly, a balance transfer gives cardmembers an opportunity to clear credit card debt without paying high interest rates.
A cash advance is a facility bundled with your credit card that allows you to withdraw cash from ATMs both locally and overseas.
There are fees and interest charges associated with a cash advance. Every cash advance you take will be subject to an upfront service fee (see Cash Advance Fee), which is typically 6% of the amount withdrawn, or S$15, whichever is higher. Also, the interest charged on your cash advance will be higher than the base interest rate of your credit card.
The amount of cash advance you can take depends on your available credit limit, and is capped at up to 90% of your total credit limit.
A cash advance is granted on your credit card's available credit limit; it is separate from the funds you have in your bank account.
Not all credit cards have the cash advance facility enabled by default. Depending on your provider, you may have to make a separate application to enable your cash advance facility.
This fee is charged by your card issuer each time you successfully carry out a Cash Advance (see above). The cash advance fee is charged as the higher of a flat fee, or a certain percentage of the cash advance amount. Typically, this is about S$15 dollars, or 6%.
Do note that like all finance charges, cash advance fees are subject to compounding interest if not paid on time.
A card payment network refers to a system that facilitates financial partnerships between merchant establishments and credit card issuers. The most common form of this relationship is the ability of a consumer to pay for a purchase using a credit or debit card.
There are many card payment networks in existence, with Visa, Mastercard, American Express and Union Pay as perhaps the most well-known in Singapore and the region.
Card payment networks usually charge a fee, known as an interchange fee, for their services. The bulk of this fee goes to the card issuing bank, with the remainder going to the card payment network and the retailer's merchant account provider.
Because it also issues its own cards, American Express does not charge an interchange fee. This is unlike other card payment networks, such as Visa and Mastercard, which actively offer their services to card issuing banks in exchange for an interchange fee.
Interchange fees are partly used to fund the various rewards programmes that cardholders are entitled to.
As interchange fees are typically hidden from the consumer, (being deducted from the merchants? accounts) there have been calls for greater transparency so consumers can make an informed choice in the use of credit cards.
A type of credit card reward in which a small percentage of the amount spent is credited back to the cardholder.
Cashback credit cards are usually designed to give cashback or cash rebates on specific categories of transactions, such as travel, dining, entertainment or groceries. There are also some credit cards that give cashback across all categories of spending.
Notwithstanding the above, there are certain transactions that do not earn rebates, such as recurring bills, insurance premiums or fines. Interest payments, instalment plans and fees are also usually not eligible for cashback.
The amount of cashback you can earn within a statement month is usually capped at a predetermined amount. However, there are some cashback cards that give unlimited cashback.
Often included as a credit card perk, credit card concierges can provide a variety of helpful services to eligible cardmembers.
When the need arises, a cardmember may contact their credit card's concierge service for help in a number of tasks. These may include (but are not limited to) making reservations at restaurants and other venues, re-booking a missed or cancelled flight, arranging travel insurance coverage, or finding a cheap hotel.
Concierge services are usually accessible via a dedicated phone number. Most concierge services are offered free of charge to the cardmember, but the cardmember is liable for any and all costs arising from bookings, purchases or transactions agreed upon.
A charge card is a type of payment card that differs from a credit card in significant ways. It does not have an interest rate nor a credit limit. However, you must clear the outstanding amount in full every statement month.
Failing to do so may incur high interest charges and late fee, and could even result in the charge card account being cancelled altogether.
Charge cards usually include similar perks and privileges as those offered by credit cards. However, charge cards usually have high income requirements and appeal to high earners and high spenders.
As you make use of unsecured credit facilities, such as credit cards and personal loans, your borrowing and spending activities will be collated, building up a record over time.
This record is known as your credit history, and is used by banks and other financial institutions to determine the likelihood of you paying back an unsecured loan or debt.
Consistently missing payments will result in a bad credit history, even if the amount of debt is low. Conversely, a habit of making payments on time creates a favourable credit history, even if you've incurred a significant amount of debt.
Your credit history is a main component used in the calculation of your credit score (see below).
A credit limit refers to the maximum amount you can charge to a credit card. Payments, fees, interest charges, cash advances and outstanding balances all count towards your credit limit.
Should you reach the credit limit on your card (aka 'maxing out' your credit card), you will no longer be able to use it to pay for your purchases. You may also be charged a penalty fee.
Generally, credit limits are determined at the time of application of your credit card. Credit limits are set at between 2 to 4 times your monthly income.
A card's credit limit is not set in stone. You may specify a credit limit during the application of your credit card, or you may request for it to be raised or lowered during use.
A credit score is a 4-digit numerical rating that indicates your likelihood of repaying your debts, or the chances of going into default.
Banks and other financial institutions look to your credit score when processing your application for a unsecured loan or credit facility. Your credit score Your credit score will help them guagto determine the risk involved in approving your application for a unsecured loan or credit facility. This helps them to gauge your likelihood of defaulting on your loan.
This is an indicator of risk that is based on statistical validation determined by credit card providers and credit bureaus. Your credit score represents your entire credit history in one numeric measure.
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INTERVIEW: Aggressive selling in UK car market – Toyota GB MD
While May's UK car market numbers looked impressive (market up 7.9%), Toyota's UK sales head Jon Williams has told just-auto that there is also widespread aggressive selling and 'big discounting' taking place.
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Nice to meet you! I'm Emily.
I first discovered photography in the black and white darkroom of my high school and knew then that I never wanted to leave it. Creating images with traditional film and historical processes is a love that I keep coming back to - there's just something about the craftsmanship that slows you down, makes you more intentional, and connects you to your work.
After studying everything from photogravure to Photoshop, I graduated with my BFA in Photography from Brigham Young University.
I would love to tell your story with this medium that has my heart and has taught me how to slow down, see, and create.
Photo by Jonathan Canlas


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Apr 15, 2017
For Immediate Release · By Monica Vega · email@example.com
Philadelphia Pa. – The St. Mary's College of Maryland women's rowing team travelled to Philadelphia on Saturday to compete on the Schuylkill River in 51st Annual Kerr Cup Regatta.
How It Happened
- The varsity eight A team would claim a spot in the grand final after coming in second in their heat with a time of 8:05.23. In the grand final, the Seahawks would finish in sixth place as they improved their time to 7:56.69.
- St. Mary's would also have a varsity eight team B that made it to the petit final following a third-place finish in their heat (8:23.67). In the petit final, the Seahawks would find themselves in second place with a time of 8:07.01, defeating Mid-Atlantic Rowing conference foes Washington College B, Franklin and Marshall College C, Johnson & Wales University B, and Cabrini University.
- The Seahawks' varsity four team would also see success in their heat, earning a second-place spot with a time of 9:08.71.
Seahawks Boat Line-Ups
- Varsity Eight A: Mira League (coxswain), Lynn Stevens (stroke), Kayla Waring (seven), Sarah Connell (six), Shannon Russ (five), Catherine Kardash (four), Mackenzie Begley (three), Katie Gill (two), Kendall Shriver (bow)
- Varsity Eight B: Anne Chernikoff (coxswain), Katherine Williams (stroke); Sarah Jeffrey (seven), Zoe Grabenstetter (six), Angelina Wilson (five), Thea Keppler (four), Clara Comstock (three), Emma Sigler (two), Alex Lundahl (bow)
- Varsity Four: Hana Yarbrough (coxswain), Kylee Naumann (stroke), Kat Bradshaw (three), Ellen Will (two), Hannah Roe (bow)
Up Next for the Seahawks
Apr. 29 – Mid-Atlantic Rowing Conference Championships (Lewisberry, Pa. – Gifford Pinchot State Park)
Facebook: StMarysAthletics
Twitter: @smcseahawks, @SMCMRowing, #GoHawks, #TalonsOut
Instagram: @smcseahawks, @smcmrowing