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Cell Reports, Volume 3, Issue 2, 485-496, 31 January 2013
Specificity and Function of Archaeal DNA Replication Initiator Proteins
Rachel Y. Samson1, 2, 6, Yanqun Xu3, 4, 6, Catarina Gadelha1, 7, Todd A. Stone2, Jamal N. Faqiri4, Dongfang Li5, Nan Qin5, 8, Fei Pu5, Yun Xiang Liang3, Qunxin She3, 4, , and Stephen D. Bell1, 2, ,
1 Sir William Dunn School of Pathology, Oxford University, South Parks Road, Oxford OX1 3RE, UK
2 Molecular and Cellular Biochemistry Department, Department of Biology, Indiana University, Simon Hall, 212 South Hawthorne Drive, Bloomington, IN 47405, USA
3 State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
4 Archaeal Centre, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark
5 BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen 518083, China
Corresponding author
6 These authors contributed equally to this work
7 Present address: School of Biology, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK
8 Present address: State Key Laboratory for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, Zhejiang University, Hangzhou 310003, PRC
Graphical Abstract
► The S. islandicus chromosome has three origins, each with its own initiator ► Two origins are Orc dependent, and one requires a Cdt1 homolog ► The ATP-bound form of Orc1 is proficient at MCM loading ► ATP binding remodels the protein structure, not that of the DNA template
- Chromosomes with multiple DNA replication origins are a hallmark of Eukaryotes and some Archaea. All eukaryal nuclear replication origins are defined by the origin recognition complex (ORC) that recruits the replicative helicase MCM(2-7) via Cdc6 and Cdt1. We find that the three origins in the single chromosome of the archaeon Sulfolobus islandicus are specified by distinct initiation factors. While two origins are dependent on archaeal homologs of eukaryal Orc1 and Cdc6, the third origin is instead reliant on an archaeal Cdt1 homolog. We exploit the nonessential nature of the orc1-1 gene to investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels the protein's structure rather than that of the DNA template.
Several archaeal species have multiple origins of replication per chromosome (Lundgren et al., 2004; Norais et al., 2007; Robinson and Bell, 2007; Robinson et al., 2004, Robinson et al., 2007). For example, chromosomes of species in the genus Sulfolobus are replicated from three origins (Lundgren et al., 2004; Robinson et al., 2004, Robinson et al., 2007). All three origins fire once per cell cycle in all cells (Duggin et al., 2008a). Two of the origins, oriC1 and oriC2, lie adjacent to the genes for the candidate initiator proteins Orc1-1 and Orc1-3, respectively (She et al., 2001). Orc1-1 and Orc1-3 share 35% amino acid sequence identity and are related to both eukaryotic Orc1 and Cdc6 (Bell and Dutta, 2002; Robinson and Bell, 2007). There is additionally a third Orc1/Cdc6 protein in Sulfolobus, Orc1-2, but it is not encoded adjacent to an origin. The third origin, oriC3, lies beside the gene for a crenarchaeal-specific protein, WhiP. WhiP is predicted to have a tandem winged-helix structure reminiscent of bacterial plasmid Rep proteins and has sequence similarity to the eukaryal replication factor Cdt1 (Robinson and Bell, 2007). Biochemical DNA footprinting studies have revealed that complex interactions among the Sulfolobus solfataricus Orc1/Cdc6 proteins, WhiP, and the replication origins are possible in vitro (Robinson and Bell, 2007; Robinson et al., 2004, Robinson et al., 2007). More specifically, Orc1-1, Orc1-2, and WhiP bind to all three origins, and Orc1-3 binds to oriC2 and oriC3. However, the precise requirements for origin function either in vivo or in vitro remain unknown. The Orc1 proteins are AAA+ proteins that undergo a single-turnover hydrolysis of ATP to ADP, but the functional consequences of ATP binding and hydrolysis are not yet known (Singleton et al., 2004). In the current work, we address these fundamental issues. First, we determine the initiator protein requirements for origin function in vivo in the genetically tractable strain Sulfolobus islandicus (Sis) (Guo et al., 2011; Zhang et al., 2010). We find that none of the Orc1/Cdc6 genes are essential for cell viability. Furthermore, we reveal that while oriC1 requires the orc1-1 gene product for function, oriC2 is dependent on Orc1-3. Finally, we find that oriC3 functions independently of all three Orc1/Cdc6 proteins but requires the Cdt1-related protein WhiP.
Next, we exploit the nonessential nature of Orc1-1 to express mutant forms of the protein and assay their phenotype with respect to oriC1 firing in vivo. Our data reveal that expression of a Walker B mutant form of the protein that can bind but not hydrolyze ATP leads to an overreplication phenotype, with elevated levels of initiation at oriC1. We further observe an enhanced ability of the Walker B mutant protein to recruit MCM to origin DNA in vitro. Finally, we demonstrate that the ATP-bound form of the protein, when bound to DNA, does not impinge on the geometry of the DNA but remodels its own conformation.
None of the orc1/cdc6 Genes Are Essential for Viability
To address the roles of the individual Orc1/Cdc6 paralogs, we generated mutant S. islandicus cell lines in which the relevant genes were deleted (Figure 1A). The resultant strains were viable and showed growth and flow-cytometry profiles that were essentially indistinguishable from the wild-type (WT) strain (Figures 1B and C). We assayed origin activity using two-dimensional (2D) neutral-neutral agarose gel electrophoresis. As can be seen in Figure 1D, deletion of orc1-1 abrogates firing of oriC1 but does not affect initiation at oriC2 or oriC3. Deletion of orc1-2 does not affect firing at any of the three origins, but deletion of orc1-3 abolishes oriC2 activity.
Next, we constructed double mutants lacking pairs of Orc1/Cdc6 proteins (Figure 2A). All three possible double mutants were generated, but attempts to generate a triple mutant were unsuccessful. The growth rates of all three double mutants were lower than WT or single mutants, and flow cytometry suggested an elevated S phase population of cells in the Δorc1-2/Δorc1-3 and Δorc1-1/Δorc1-3 strains (Figures 2B and 2C). The strain lacking both Orc1-2 and Orc1-3 phenocopies the single orc1-3 deletion, with no firing detectable at oriC2 but retaining oriC1 and oriC3 activity (Figure 2D). Similarly, as observed for deletion of orc1-1 alone, deletion of both orc1-1 and orc1-2 inactivated oriC1, but oriC2 and oriC3 still fired. (Figure 2D). Significantly, the Δorc1-1/Δorc1-3 double mutant only showed replication initiation at oriC3; no replication initiation activity was detected at either oriC1 or oriC2 in this strain (Figure 2D).
To exclude the possibility that hitherto-undetected cryptic origins might have been activated in the mutant backgrounds, we determined the genome-wide replication profile of the WT and Δorc1-1/Δorc1-3 mutant strains by marker frequency analysis (MFA) in conjunction with next-generation sequencing. In agreement with previous studies, three peaks can be observed in the WT strain, corresponding to origin activity at oriC1, oriC2, and oriC3 (Figure 2E). As previously seen, the peak at oriC2 is of lower amplitude due to the broader temporal window of firing of this origin compared to the other two. Significantly, only a single peak is observed in the Δorc1-1/Δorc1-3 mutant strain, revealing that the chromosome of this strain is replicated from a single origin, oriC3 (Figure 2F). There is a modest shoulder on the slope of the peak coincident with the position of oriC1, but potential firing here is clearly at a very low level, undetectable by 2D gel analyses and independent of Orc1-1 or Orc1-3. We further note that the trough corresponding to replication termination events is repositioned in this strain to 180° around the chromosome from oriC3.
The whiP Gene Is Required for oriC3 Function
Interestingly, none of the individual or double Orc1/Cdc6 gene deletions impinged upon firing of oriC3. We therefore speculated that this origin could be functioning independently of Orc1/Cdc6 paralogs. Notably, the Cdt1-related protein WhiP is encoded adjacent to oriC3 (Robinson and Bell, 2007). Further, we have previously shown that this protein binds to sequence motifs in the oriC3 region, immediately downstream of the whiP gene (Robinson and Bell, 2007). We therefore generated a strain lacking WhiP by introducing tandem stop codons following the start codon of the open reading frame (ORF). The absence of WhiP in the resultant line was confirmed by western blotting (Figure 3A). Cell growth and flow cytometry revealed that the mutant cells grew slower than either WT cells or any of the single orc1 deletion mutants and with a 2-fold increase in sub-1C signal in the flow cytometry (Figures 3B and 3C). Significantly, loss of the WhiP protein correlated with loss of firing at oriC3 but had no effect on initiation at oriC1 and oriC2, as adjudged both by 2D neutral-neutral agarose gel electrophoresis analysis and MFA (Figures 3D and 3E). Thus, while oriC3 is independent of individual and pairwise combinations of Orc1/Cdc6 proteins (Figures 1 and 2), WhiP is required for replication initiation at this origin (Figures 3D and 3E).
Binding of Initiators to Origins In Vivo
To address origin occupancy by the initiator proteins in vivo, we performed chromatin immunoprecipitation using antisera generated against Orc1-1, Orc1-2, and Orc1-3 and WhiP. In agreement with the lack of requirement for Orc1-2 for firing of any origin, we did not detect binding of Orc1-2 to any of the origins. Also in good agreement with the genetic analyses, we find that oriC1 is bound by Orc1-1, oriC2 shows strongest association with Orc1-3, and while no association of any Orc1/Cdc6 protein is observed at oriC3, the WhiP protein is specifically localized at this origin (Figures 4A–4D).
Archaea are believed to undergo coupled transcription and translation (French et al., 2007). We were, therefore, concerned that the ability to chromatin immunoprecipitate DNA corresponding to the origins located adjacent to the relevant initiator protein gene could reflect crosslinking of nascent polypeptides to the initiator ORF rather than to the origin itself. We therefore performed chromatin immunoprecipitation sequencing (ChIP-seq) analyses of the three Orc1/Cdc6 proteins and WhiP to give a higher-resolution analysis of the proteins' DNA occupancy. As can be seen in Figures 4E–4G and Figure S1, the relevant proteins showed strong association with the origin loci. Notably, the WhiP ChIP recovers most DNA at internal positions within the WhiP ORF. In addition, a broad but shallow peak was observed over the previously described in vitro binding sites (Robinson and Bell, 2007). These lie at the boundaries of the downstream intergenic region, to which initiation at oriC3 has been mapped (Robinson et al., 2007). The whiP ORF contains conserved AT-rich sequence repeats (Figure S1) and footprinting studies reveal that the protein binds to these with nanomolar affinity. We reiterate that the whiP mutation that we employed to inactivate the gene for the genetic studies was by introduction of stop codons at the start of the ORF, rather than by deletion of the ORF (and thus its internal repeats).
In agreement with the 2D gel analyses, we detect Orc1-3 binding at oriC2 but do not observe significant binding of Orc1-1 to this locus. This was surprising to us because previous ChIP analyses and biochemical and structural studies of the closely related organism, Sulfolobus solfataricus (Sso), had demonstrated that both Orc1-1 and Orc1-3 bind to oriC2 in that species (Dueber et al., 2007, Dueber et al., 2011; Robinson et al., 2004). However, examination of the sequence of S. islandicus oriC2 reveals that one of the characterized mini origin recognition box (mORB) Orc1-1 binding sites has several base substitutions in conserved positions in the binding site when compared with S. solfataricus oriC2 (Figure S2). Additionally, the second mORB element has substitutions immediately adjacent to the core consensus motif. We therefore tested the S. islandicus Orc1-1 and Orc1-3 proteins' DNA binding preferences in vitro. Remarkably, but in agreement with the ChIP analyses, Sis Orc1-1 binds with at least 10-fold lower affinity than Sso Orc1-1 to either of the mORB-like elements in Sis oriC2. Notably, Sis Orc1-1 has lower affinity for Sso oriC2 than has Sso Orc1-1. As expected from the ChIP studies, Orc1-3 binds to Sis oriC2-derived sequences (Figure S2). Thus, both origin sequence and initiator protein sequence define the differential binding of Orc1-1 to oriC2 between S. solfataricus and S. islandicus. Remarkably, even between two closely related species within the Sulfolobus genus, we can observe variation within initiator protein specificity and function.
Nucleotide Binding and Orc1-1 Function at oriC1 In Vivo
We wished to exploit the nonessential nature of Orc1-1 to perform structure-function analyses in living cells. We therefore sought to complement the chromosomal Orc1-1 deletion with a plasmid-encoded copy. Initially, we expressed the gene from its own promoter on a low-copy-number plasmid. We could detect Orc1-1 protein by western blotting (Figure 5A), and the protein localized to oriC1 as adjudged by ChIP experiments (Figure 6B). However, we were unable to detect any replication initiation at oriC1 by neutral-neutral 2D agarose gel electrophoresis (Figure 5B). We therefore expressed Orc1-1 from a strong arabinose-inducible promoter (Albers et al., 2006). Either basal or induced expression of the protein from the araS promoter led to initiation at oriC1 as detected by 2D gels (Figure 5B). Indeed, the intensity of the "bubble" arc initiation structure was reproducibly stronger than the rather weak arc observed in WT cells (Figure 5B). This enhanced intensity prompted us to evaluate the DNA content of these cells. Interestingly, cells that express the Orc1-1 protein from the arabinose promoter in either basal or induced conditions show an aberrant flow-cytometry profile with an elevated population of cells, with between 1C and 2C and greater than 2C genome contents (Figure 5C). Furthermore, fluorescence microcopy of these cells reveals altered cell morphologies, with 14% of cells extruding vesicle-like structures (Figure 5D).
Transcription of the orc1-1 gene in S. acidocaldarius and S. solfataricus is known to be regulated following perturbation of the cell cycle and after a number of cellular stresses (Fröls et al., 2007; Götz et al., 2007; Lundgren and Bernander, 2007; Ortmann et al., 2008). We therefore wished to determine the profile of expression of this and the other initiator protein genes during a minimally perturbed cell cycle. We employed our previously described "Baby machine" method for synchronizing cells (Duggin et al., 2008a). For these studies, we utilized S. acidocaldarius cells because they have given the best synchrony in these experiments. We observe a striking cyclic pattern of transcript levels for orc1-1, orc1-3, and whip (Figure 5E), although we have previously demonstrated that Orc1/Cdc6 protein levels remain constant across the cell cycle (Duggin et al., 2008a). Nevertheless, all three transcripts show lowest levels in S phase and peak in or just before G1 phase. Interestingly, orc1-3 mRNA levels peak slightly later than orc1-1, correlating with the broader temporal window of oriC2 firing (Duggin et al., 2008a).
The Orc1/Cdc6 proteins possess a AAA+ domain and previous work has shown that this catalyzes a single turnover ATP hydrolysis event, resulting in stable binding of ADP (Singleton et al., 2004). Indeed, Orc1/Cdc6s from a range of archaeal species, when purified as recombinant proteins from bacteria, are isolated exclusively in the ADP-bound form. By analogy with bacterial DnaA, we assumed that the ATP-bound form of Orc1/Cdc6 proteins is the active form (see also below) (Kaguni, 2011; Mott and Berger, 2007). We therefore speculated that the cell-cycle-dependent wave of orc1-1 transcription would result in transiently increased levels of Orc1-1⋅ATP and that this could create a permissive window for replication initiation. Expressing Orc1-1 constitutively during the cell cycle from the arabinose promoter would result in temporally ectopic Orc1-1⋅ATP and could thus account for the overreplication phenotype observed. To test this, we created mutant derivatives of Orc1-1 with alterations in the Walker A and Walker B motifs. The Walker A K69A is anticipated to be unable to bind ATP and the Walker B E147A mutant should bind but fail to hydrolyze ATP. These predicted properties were confirmed using purified recombinant proteins (Figure S3). To exclude the possibility of compounding effects due to overexpression, the proteins were then expressed from a plasmid using the native orc1-1 promoter in the Δorc1-1 strain (Figure 6A). Despite being expressed and binding to oriC1 as detected by ChIP, the Walker A mutant failed to support detectable initiation at oriC1 (Figures 6A–6C). In contrast, the Walker B mutant shows a bubble arc of greater intensity than seen in WT cells (Figure 6C). Furthermore, this strain shows an aberrant flow cytometry profile, with 22% of the population with greater than 2C genome content (Figure 6D). Additionally, microscopy reveals the presence of large cells with elevated DNA content; 13% of Orc1-1 E147A-expressing cells are greater than 1.5 μm in diameter, compared with less than 1% of WT (Figures 6E and 6F). Interestingly, microscopy of the population expressing the Orc1-1 Walker A mutant also shows aberrant large cells (3.8%); however, in agreement with the flow-cytometry profiles, there is not a discernable population of cells with elevated DNA content (as adjudged by DAPI staining). Taken together, our data therefore indicate that the ATP-bound form of Orc1-1 is proficient for origin firing and, furthermore, implicates the hydrolysis of ATP in regulating origin activity.
Biochemical Consequences of ATP Binding by Orc1-1
To investigate the molecular basis of the distinct properties of the proteins, we first performed gel-filtration analyses. These revealed an increase in retention time of the E147A protein compared to WT, suggestive of a conformational alteration (Figure 7A). Next, we performed analytical ultracentrifugation (AUC) with the WT and E147A proteins (Figure 7B; Figure S3). The AUC revealed that the WT protein migrated in two populations, both corresponding to monomer with distinct conformations. More specifically, 8.3% of the population had a sedimentation coefficient of 2.8 s and frictional ratio of 1.37, revealing a more extended configuration than the majority of the protein that resolved in a peak at 3.7 s with a frictional ratio of 1.07 and thus was more globular. Interestingly, very different behavior was observed with E147A. Although 65% of the E147A protein behaved as globular monomer (frictional ratio of 1.03), there was no peak corresponding to the extended monomer configuration. However, E147A generated a peak with a mass compatible with the formation of a dimer. Furthermore, the sedimentation coefficient and frictional ratio of the monomeric form of the E147A protein were lower than those of the majority WT species, indicating that it adopted a distinct conformation.
Structural studies have revealed that the ADP-bound forms of Orc1/Cdc6 proteins make bidentate interactions with DNA, binding via the C-terminal wH domain and the initiator-specific motif (ISM) in the AAA+ domain. To date, there is no structure available of the ATP-bound form of Orc1/Cdc6 on DNA. However, Wigley and colleagues were successful in denaturing Orc1-2 from Aeropyrum pernix (Ape), removing ADP and refolding in the presence of the nonhydrolyzable ATP analog, ADP-NP, prior to crystallization and structure determination (Singleton et al., 2004). The resultant structure revealed a significant alteration in the relative positioning of the wH and AAA+ domain compared to the various conformations adopted by the ADP-bound form (Singleton et al., 2004). Ape Orc1-2 has only 22% sequence identity to S. islandicus Orc1-1 and belongs to a distinct clade of Orc1/Cdc6s; nevertheless, our AUC and gel filtration data suggest that the identity of the nucleotide bound by Orc1-1 also modulates the conformation of this protein in solution.
Superimposition of the ADP-NP-bound Orc1-2 structure onto the Orc1-1⋅ADP–DNA structure, aligning via the principal DNA-binding wH domain, suggests three possible consequences of ATP binding (Figure S4). First, the ISM could disengage from the DNA, conceivably allowing the AAA+ domain to be accessible for interaction with another protomer or client protein; second, both wH and ISM could remain engaged with DNA with consequent remodeling of the intervening duplex DNA; third, both domains remain engaged with DNA and the protein undergoes an internal conformational alteration without modulating DNA structure. To test for the first two scenarios, we performed methidium propyl EDTA footprinting with the WT and E147A proteins on an isolated DNA binding site, the ORB2 element. This technique allows clear resolution of the DNA sequences contacted by wH and ISM. As can be seen from Figure 7C, no discernable difference is seen between the protection patterns mediated by the two proteins, indicating no significant disengagement from, or remodeling of, the DNA binding site. Similarly, there was no discernable difference in the protection pattern upon DNaseI digestion. The same result was observed with ORB1 and ORB3 DNA (data not shown). Furthermore, electrophoretic mobility shift assay (EMSA) analysis with full-length origin DNA fragments (Figure 7D) reveals no significant changes in the affinity for DNA or pattern of shifted complexes generated by either the Orc1-1⋅ADP or the Orc1-1 E147A⋅ATP forms of the protein. Additionally, we could not detect melting of origin DNA, by either potassium permanganate or nuclease P1 sensitivity, by either protein in the presence or absence of nucleotide (data not shown). In light of our AUC and gel filtration data revealing altered conformations of the monomeric protein, we therefore speculated that the principal effect of ATP binding was on the conformation of the protein itself rather than on that of the DNA. To investigate this further, we subjected WT and Walker B mutant Orc1-1 to limited proteolysis with trypsin in the presence and absence of saturating levels of ORB2 DNA. Under both conditions, we observe reduced levels of a ∼19 kDa species with the Walker B mutant form. In the presence of DNA, the digest patterns of both forms of the protein are altered, with a novel 23 kDa species appearing (Figure 7E). This species was also more abundant with the WT protein. We recovered the gel bands, digested them with chymotrypsin, and subjected the resultant peptides to mass spectrometry (Figure 7F). The upper band yielded two peptides that corresponded to the product of trypsin digestion at R193 and chymotrypsin digestion at Y208 or F185. This is in good agreement with the apparent size of the species of ∼23 kDa (trypsin cleavage at R193 would generate a C-terminal fragment of 22.8 kDa and a N-terminal fragment of 22.5 kDa). The ∼19 kDa band also yielded a peptide derived from trypsin cleavage at R193 and chymotrypsin at F185. Thus, the ATP-bound form of Orc1-1, both in DNA-bound and unbound form, displays altered sensitivity of R193 to trypsin digestion. Significantly, R193 corresponds to the arginine finger of the protein's AAA+ site. Thus, in the absence of any observable alterations to the affinity, geometry, or stoichiometry of the protein-DNA interaction between Orc1-1 and DNA, we propose that the differential sensitivity to trypsin reflects a conformational alteration of the ATP-bound form of the protein on DNA that renders it permissive to recruitment of the MCM helicase.
Finally, we established an in vitro assay for MCM recruitment to oriC1. In this assay, we immobilized origin or nonorigin DNA on paramagnetic beads, preincubated with WT (ADP-bound) or E147A Orc1-1 in the presence or absence of supplemental ATP and then added cell extract prepared from logarithmically growing S. islandicus cells. Following incubation at 50°C and washing, beads were recovered and bound proteins eluted by boiling in SDS-PAGE loading buffer prior to western blotting. As can be seen in Figure 7G, neither extract alone nor extract supplemented with WT (ADP-bound) Orc1-1 supports significant MCM recruitment in the absence of ATP. Extract with additional ATP supports recruitment, but interestingly this recruitment is lost by the addition of 1 μM WT Orc1-1⋅ADP, presumably as a consequence of competition with endogenous Orc1-1 for the 200 pM template. Importantly, however, the E147A mutant supported robust, ATP-stimulated recruitment of MCM in these assays.
Taken together, our data yield insight into the evolution, regulation, and mechanism of replication initiation in the chromosome of Sulfolobus species. We find that the chromosome is a mosaic of three distinct replicons, each with its own principal initiator protein. In particular, we note that initiation at oriC3 is not impacted upon by individual or combined Orc1/Cdc6 deletions but is abrogated by loss of expression of the Cdt1 homolog WhiP. Intriguingly, parallels can be drawn with metazoa: in Drosophila, the origin recognition complex (ORC) is dispensable for endoreduplication, whereas Cdt1 is essential for this process (Park and Asano, 2008). We note that it was not possible for us to generate a triple Orc-1 mutation in which all three paralogs were deleted. Given that Orc1-2 does not bind in vivo to any origin, we speculate that this synthetic lethality may reflect nonorigin roles for the Orc1/Cdc6 proteins. Indeed, our ChIP-seq analyses reveal multiple nonorigin genomic binding sites for the Orc1/Cdc6 proteins. Furthermore, the single mutants show significantly altered transcript profiles compared to WT cells (R.Y.S. and S.D.B., unpublished data). Thus, as with DnaA and eukaryal ORC proteins (Scholefield et al., 2011), it appears that archaeal Orc1/Cdc6 proteins play roles in transcriptional regulation.
We speculate that the mosaic nature of the Sulfolobus chromosome has arisen by integration of extrachromosomal elements into an ancestral chromosome that presumably contained a single origin of replication. Given the broad conservation of Orc1-1 orthologs and their DNA binding sites across the archaeal domain of life (Robinson et al., 2004), we suggest that the ancestral chromosome was dependent on Orc1-1 and oriC1. Because WhiP homologs are found in the Desulfurococcales and Sulfolobales and Orc1-3 is restricted to the Sulfolobales, we propose the sequential acquisition of first WhiP/oriC3 prior to the bifurcation of these two lineages, followed by integration of the Orc1-3/oriC2 replicon in the branch leading to the Sulfolobales. Interestingly, the genome replication mode of the bacterium Vibrio cholera also appears to have been sculpted by acquisition of extrachromosomal element replication origins. However, in that organism, the genome is split between two chromosomes, each with a single origin: one dependent on a classic bacterial oriC/DnaA system and the second having an origin with features reminiscent of plasmid origins. Indeed, this partitioning of bacterial genomes into multiple single-origin chromosomes is found in a range of bacterial species (Egan et al., 2005; Egan and Waldor, 2003). In contrast, multiple-origin single-chromosome genomes are found in a number of archaea. Why are archaeal chromosomes permissive in this regard? One explanation may lie in the contrasting mechanisms for replication termination in bacteria versus archaea (Duggin et al., 2008b; Toro and Shapiro, 2010). Whereas termination in bacteria involves polar fork traps, we have recently found that termination in Sulfolobus is by random fork collision (Duggin et al., 2011). In support of this, our MFA data on the mutant strains in which only a single origin fires reveal that termination is repositioned to occur 180° across the chromosome from the active origin (Figure 2F). An ectopic origin integrated into one replichore of a bacterial chromosome would result in premature arrival and prolonged stalling of a fork at a termination site. This may impact genome stability. In addition, because of the presence of the fork traps, the time taken to replicate a two-origin bacterial chromosome would still be dependent on the length of time it takes the fork arising from oriC to traverse the longest replichore. In contrast, the absence of active termination sites in Sulfolobus would mean that additional origins would simply result in repositioning of termination events to midway between active origins and a concomitant reduction in the total time spent in S phase, assuming similar timing of firing of the origins. The ability to accommodate and regulate multiple origins will have played a major role in permitting the increase in genome size seen in eukaryotic organisms (Diffley, 2011).
Our data also reveal mechanistic insight into the action of the Orc1-1 initiator. As with bacterial DnaA, we find that the ATP-bound form of the initiator is the active form (Kaguni, 2011; Mott and Berger, 2007). Our expression profiling reveals a wave of transcription of the genes for the Orc1-1 and Orc1-3 initiators immediately prior to the onset of replication. We propose that this imposes a temporal window for initiation in which the newly synthesized proteins bind to ATP and associates with origin DNA before hydrolysis of ATP to ADP inactivates the proteins. It may, therefore, be significant that Orc1-1 and Orc1-3 are encoded in the immediate vicinity of their cognate origins. Furthermore, expression of WT Orc1-1 driven by its native promoter from episomes, while resulting in origin occupancy (as adjudged by ChIP), fails to activate the origin, presumably due to hydrolysis of ATP in the cell prior to origin binding. However, expression of the Walker B ATP hydrolysis-defective mutant to the same level results in an overreplication phenotype.
In bacteria, the ATP-bound form of DnaA facilitates the formation of higher-order structures and is able to mediate localized DNA melting at the origin (Bramhill and Kornberg, 1988; Duderstadt et al., 2011; Erzberger et al., 2006; Katayama et al., 2010; Ozaki et al., 2008). Although we do observe a subpopulation of the Walker B mutant form of the protein existing as dimer in solution, we find that the ADP- and ATP-bound forms of Orc1-1 show equivalent DNA binding properties. Furthermore, we have been unable to observe any localized sensitivity to nuclease P1 or potassium permanganate, indicative of DNA melting, induced by either form of the protein (data not shown). Perhaps unsurprisingly, in this regard Orc1-1 is behaving in a manner more reminiscent of eukaryal ORC. There is no evidence for eukaryal ORC melting origins; rather, the available data support a model whereby MCM(2-7) is recruited to double-stranded DNA with duplex unwinding occurring at a later stage (Evrin et al., 2009; Heller et al., 2011; Remus et al., 2009). We propose that this is an evolutionarily conserved feature of the Orc1/Cdc6 proteins and that these proteins act principally to define loci for MCM recruitment. In light of our protease sensitivity assays, we suggest that ATP acts to modulate the conformation of Orc1/Cdc6 proteins on the DNA binding site such that the protein is able to effectively recruit MCM. Subsequent hydrolysis of ATP to ADP by Orc1/Cdc6 then renders the protein incapable of recruiting further MCM, thereby contributing to the fidelity of replication control. This simple binary switch model of archaeal Orc1/Cdc6 function is supported by our observation of the overreplication phenotype mediated by the Walker B mutant of Orc1-1. We note that in budding yeast, temporally distinct ATP binding and hydrolysis events by both the ORC complex and Cdc6 are required for iterative MCM(2-7) loading at replication origins (Randell et al., 2006). The increased organizational complexity of the eukaryotic origin definition and MCM recruitment system is likely a consequence of the increased regulatory demands imposed by the large number of replication origins found in eukaryotic chromosomes (Diffley, 2011). The simple binary-switch model that we propose for Orc1/Cdc6 function is clearly distinct from the mechanism of action of the bacterial initiator DnaA and provides a paradigm for the function of initiator proteins in archaea and eukaryotes.
Experimental Procedures
Strains, Media, and Growth Conditions
S. islandicus strain E233S (ΔpyrEF ΔlacS) constructed previously (Deng et al., 2009) was used as the host for genetic manipulations. All archaeal strains used in this work, including the genetic host and its transformants of diverse plasmids as well as the orc1/whip, are summarized in Document S1. Tables S1–S5. These archaeal cell lines were grown in SCVY (0.2% sucrose, 0.2% Casamino acids, 5 ml vitamin solution, and 0.005% yeast extracts) or TYS (0.1% tryptone, 0.05% yeast extracts, and 0.2% sucrose) medium at 78°C. If required, uracil was supplemented to 20 μg/ml while 5′-fluorooritic acid (5′-FOA) was added to 50 μg/ml, in SCVY medium. Microscopy was performed as described previously (Samson et al., 2011).
Protein Purification and Biochemical Analyses
Orc1/Cdc6 and WhiP proteins were expressed in E. coli with C-terminal Hexa-His tags and purified as described previously (Robinson and Bell, 2007; Dueber et al., 2007). EMSAs and footprinting were performed as described previously (Dueber et al., 2007; Robinson et al., 2004). The MCM recruitment assay was based on the approach initially established by Seki and Diffley, 2000. A total of 10 μg of a 5 kb plasmid (pCR Script containing a 2 kb sequence centered around oriC1 or a control locus, Sire_2328) was linearized by EcoRI and PstI digestion, the EcoRI-generated recessed end was filled in with Klenow fragment (New England Biolabs) and Biotin-14-dATP and dTTP. The DNA was recovered from the reaction mixture using QiaQuick column (QIAGEN) and immobilized on Dynabeads M-280 streptavidin beads following the manufacturer's recommendations (Life Technologies). Binding reactions were assembled in LoBind tubes (Eppendorf) and contained 200 pM DNA in 50 μl of binding buffer (20 mM Tris acetate, 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM DTT [pH 7.9] at 25°C) plus 20 ng/μl poly(dGdC). Reactions were heated to 50°C in a water bath and buffer or Orc1-1 protein (1 μM final) were added and supplemented with nucleotide to 2 mM as indicated. Following a 10 min incubation, 20 μl of a 5 mg/ml whole-cell extract of S. islandicus was added (see below). Then, 20 μl of water or supplemental ATP to 2mM as indicated was added and the reaction incubated for a further 30 min at 50°C before washing twice by magnet-mediated bead pelleting and resuspension in 100 μl of prewarmed binding buffer, followed by one wash in 100 μl binding buffer containing 500 mM potassium acetate. Following this final wash, beads were resuspended in 1xSDS PAGE loading buffer and subjected to SDS PAGE followed by western blotting. All wash steps employed preheated buffers and were performed in the 50°C water bath. We observed a very high background of recombinant Orc1-1 protein bound to both DNA substrates and even to beads alone (data not shown), but MCM was only efficiently recruited to the origin-containing DNA substrate.
The S. islandicus cell extract was prepared by growing cells to midexponential phase (OD 600 nm = 0.4) before harvesting and resuspension in 2x binding buffer (40 mM Tris acetate, 100 mM potassium acetate, 20 mM magnesium acetate, 2 mM DTT [pH 7.9] at 25°C). Cells were lysed using a FrenchPress (Thermo) and the extract was clarified by centrifugation at 23,700 g for 15 min at 4°C. Then, 100 μl aliquots were prepared in LoBind tubes and snap frozen on dry ice. Total protein concentration was determined by Bradford assay to be 5 mg/ml. Extracts were stored at −80°C.
Flow Cytometry
Flow cytometry of S. islandicus cells was performed essentially as previously described for S. acidocaldarius (Duggin et al., 2008a). Cells were fixed with 70% ethanol and their DNAs were stained with Mithramycin A and analyzed with A-40 Apogee flow cytometer.
Neutral-Neutral 2D Gel Electrophoresis
S. islandicus cells were collected by centrifugation and washed twice with TEN buffer (50 mM Tris-Cl [pH 8], 50 mM EDTA [pH 8], 100 mM NaCl). Cell suspensions were then mixed with low-melting-point agarose and dispensed into Bio-Rad plug molds. Genomic DNA preparation and 2D gel electrophoresis were performed as described previously (Robinson et al., 2004). For further details, please refer to Extended Experimental Procedures.
Chromatin Immunoprecipitation
ChIP was performed as described by Robinson et al., 2004. For further details, please refer to Extended Experimental Procedures.
Gene Knockout Plasmids
A novel gene deletion method recently developed for Sulfolobus islandicus REY15A (Deng et al., 2009) was employed to construct gene knockouts of this archaeon for the three orc1 genes and the other putative replication initiator gene whip. This method "Marker cassette Integration and target gene Deletion (MID)" employs three homologous sequence arms, which are target gene arm (G-arm), left sequence arm (L-arm) and right sequence arm (R-arm). The positions of these arms in pLRMG plasmids are illustrated in Figure S5. Primer pairs for each homologous sequence arm were designed to yield a PCR product of ca. 1 kb (Document S1. Tables S1–S5).
The marker cassette of pLRMG plasmids contained pyrE/F and lacS genes that code for orotate phosphoribosyltransferase, orotidine-5′-monophosphate decarboxylase and β-glycosidase, respectively. Using double markers enabled the transformants obtained on pyrEF selection to be verified by a simple X-gal (5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside) screening. These marker genes were obtained by PCR amplification from the Sulfolobus-E. coli shuttle plasmid pHZ2lacS with the primer pair of pyrEF+lacS Fwd and pyrEF+lacS Rev (Document S1. Tables S1–S5).
The E. coli vector pUC19 was the vector for all pLRMG plasmids construction. The first plasmid pLRMG-orc1-1 was constructed in three steps: First, L-arm and R-arm PCR products were cleaved with SalI + MluI and MluI +EcoRI, respectively and ligated to pUC19 at SalI and EcoRI sites in a triple ligation, yielding pUC-LR. Second, the marker cassette was inserted into the PstI and SalI sites of pUC-LR, giving pUC-LRM. Last, the G-arm was inserted into pUC-LRM at SphI and PstI sites, generating the knockout plasmid pLRMG-orc1-1.
Subsequent construction of other knockout plasmids to be used in this work was based on the pLRMG-orc1-1, following essentially the same strategy. Restriction sites used in pLRMG-orc1-2 and pLRMG-orc1-3 constructions included SphI and PstI (G-arm); SalI and NcoI (L-arm); and NcoI and BamHI (R-arm). During cloning, a DNA fragment containing the marker cassette and the orc1-1 G-arm was cleaved off from pLRMG-orc1-1 with SphI and SalI and the yielded fragment was inserted into the corresponding sites on pUC19, yielding the pUC-M-orc1-1. Then, the orc1-1 G-arm was replaced with orc1-2 or orc1-3, yielding the pUC-M-orc1-2 or the pUC-M-orc1-3. Finally, triple ligations of pUC-M-orc1-2/-orc1-3 with their own L-arm and R-arm yielded pLRMG -orc1-2 and pLRMG-orc1-3, respectively.
Construction of the Plasmid to Inactivate Genome Expression of WhIP Protein Expression
To study if the whip gene product could function in origin firing, it was essential to generate a mutant with complete whip gene sequence which lacks WhIP protein expression. For this purpose, we aimed to replace the WT whip gene with the mutant gene whip∗ which carried three tandem stop codons. The oligonucleotide 5′-ACGCGTTAATGATAA −3′ containing a Mlu I restriction site and three tandem stop codons was inserted between the first and the second codons of whip gene and expression from this whip mutant could only produce a peptide of three amino acids.
Mutant gene whip∗ was obtained by SOE-PCR with the four primers listed in Document S1. Tables S1–S5 (under whip∗). Then, the SOE-PCR product was inserted into pUC19 at SalI and BamHI sites, yielding pUC-whip∗. Marker cassette (cleaved off from pLRMG-orc1-1 with PstI and SalI) was inserted to the pUC-whip∗ to yield whip∗ replacement plasmid pUC-M-whip∗ (Figure S5).
Construction of orc1/whip Mutants of S. islandicus
Generation of pLRMG transformants
All pLRMG plasmids were linearized by restriction digestion and 0.9-1μg linearized plasmid DNA was used to transform the genetic host S. islandicus E233S by electroporation as described previously (Deng et al., 2009). After incubation at 78°C for 7-10 days, colonies were screened with X-gal. Then, a few blue ones were picked up and inoculated in 6 ml SCVY liquid medium in test tubes and incubated for 3–5 days. The cultures were harvested and genomic DNA extracted. The target gene alleles of these transformants were analyzed by PCR with verifying primer pairs (Document S1. Tables S1–S5).
Isolation of gene deletion mutants by counter-selecting transformants with 5′-FOA.
Once pLRMG-orc1/whip transformants were purified such that the WT allele of the target gene could not be amplified by PCR in the cultures, the transformant cells were diluted and spread onto plates containing uracil and 5′-FOA. Colonies appeared after 7-days incubation. These colonies were first screened with X-gal and a few white colonies were then grown in SCVY medium supplemented with uracil and 5-FOA.Target gene alleles were checked again by PCR and mutant cell lines were confirmed to possess only the deleted target gene allele.
Construction of orc1 double mutants
A second orc1 gene was to be deleted from a Δorc1 mutant strain to yield double orc1 knockouts. Δorc1-1 was the host for constructing Δorc1-1 Δorc1-2 and Δorc1-1 Δorc1-3 double mutants whereas Δorc1-2 was the host for constructing Δorc1-2 Δorc1-3 mutant and this was done by following single orc1 mutant construction as described above.
Construction of the Translation-Stop Mutant Gene whip∗
The construction of a whip∗ mutant with three tandem stop codons was a two-steps process (Figure S6). The first step was to select single crossover transformants of the constructed pUC-M-whip∗. 1 μg of the plasmid was used to transform the genetic host E233S. Upon entering the cell, homologous recombination between the host genome and the plasmid led to pUC-M-whip∗ integration either at Up-seq or Dn-seq. Recombination at Dn-seq was exemplified in Figure S6. After purification, the pUC-M-whip∗ transformants were subjected to counter selection. Cell lines carrying either the WT or mutant whip gene could both grow in the presence of 5-FOA and Uracil. Because a MluI site was introduced into whip∗, mutants carrying whip∗ were readily identified by digesting the PCR product of whip with MluI.
Analysis of Protein Levels by Western Blot Analysis
Sulfolobus cultures of exponential growth were adjusted to OD600 = 0.2 with SCVY. 10 ml of culture was taken and harvested by centrifugation. Cell pellets were resuspended in 0.2 ml H2O. After adding 0.2 ml 2 × SDS PAGE buffer, the samples were heated at 99°C for 10 min to lyse the cells and denature proteins. Total cellular proteins were then analyzed by SDS/PAGE and Western blot analysis was conducted using antisera against S. solfataricus Orc1-1, −2, −3, WhiP protein or PCNA2 or PCNA3 (the latter proteins serving as loading controls). The signals were detected with an enhanced chemiluminescence detection kit (Amersham Biosciences) and recorded with X-ray film.
Neutral-Neutral 2D Gel Electrophoresis
S. islandicus cells were collected by centrifugation and washed twice with TEN buffer (50 mM Tris-Cl, pH 8; 50 mM EDTA, pH 8; 100 mM NaCl). Cell suspensions were then mixed with low melting point agarose and dispensed into Bio-Rad plug molds. Genomic DNA preparation and 2D gel electrophoresis were performed as described previously (Robinson et al., 2004). Specifically, the agarose plugs were digested in NDS buffer (10 mM Tris, pH 9.5; 0.5 M EDTA, pH 8.0; 1% lauroyl sarcosine) containing 1 mg/ml Proteinase K for 48 hr at 37°C. The plugs were then equilibrated in an appropriate restriction buffer before digesting with 500-1000 units of restriction enzyme for approximately 16 hr. See Figure S3 for restriction maps of the oriC loci and probe position. A 0.4% agarose gel was poured around the plugs and run for approximately 20 hr at 1 V/cm at 4°C. Lanes containing DNA were excised from the gel, rotated 90°, and incorporated into a 1% agarose gel containing 0.3 μg/ml ethidium bromide. Electrophoresis in the second dimension was carried out at 5 V/cm for 6-7 hr at 4°C. The DNA was depurinated in 0.25 M HCl, then denatured and transferred in 0.5 M NaOH, 10X SSC to Hybond-XL membrane (GE Healthcare) by capillary transfer according to the manufacturer's instructions. 32P-labeled probes (Figure S7; Document S1. Tables S1–S5) were prepared according to the manufacturer's instructions using the NEBlot kit (New England Biolabs). Hybridizations were performed in Church-Gilbert buffer (0.5 M sodium phosphate, 1 mM EDTA, 1% BSA, 7% SDS) at 65°C and washes were performed as described in the Hybond-XL manual. Membranes were exposed to phosphoimager screens which were subsequently scanned on an FLA-5000 phosphoimager (Fujifilm).
Chromatin Immunoprecipitation
Chromatin immunoprecipitation (ChIP) was performed as described in (Robinson et al., 2004) Early logarithmic cultures of S. islandicus were crosslinked with 1% formaldehyde for 20 min. After quenching with 125 mM glycine, the cells were pelleted and washed with 1X PBS. The pellets were resuspended in TBS-TT (20 mM Tris, 150 mM NaCl, 0.1% Tween-20, 0.1% Triton X-100, pH 7.5), passed through a French press three times at 20,000 psi, and sonicated using a Diagenode Bioruptor to generate DNA fragments ranging from 200-1000 bp. The extract was then clarified by centrifugation. 10 μg of extract (based on protein concentration) were used in each 100 μl ChIP reaction. Samples were rotated for 2-3 hr with 3 μl of antiserum at 4°C. 25 μl of a 50% slurry of protein A sepharose were then added and the samples were rotated for another hour at 4°C. Each ChIP reaction was then washed five times at room temperature with TBS-TT, once with TBS-TT containing 500 mM NaCl, and once with TBS-TT containing 0.5% Tween-20 and 0.5% Triton X-100. Protein-DNA complexes were eluted from the protein A sepharose in 20 mM Tris, 10 mM EDTA, 0.5% SDS, pH 7.8 at 65°C for 30 min. Crosslinking was reversed and protein was digested by incubating the samples with 10 ng/μl Proteinase K for 6 hr at 65°C followed by 10 hr at 37°C. The samples were extracted with phenol/chloroform/isoamyl alcohol first, then chloroform alone and the DNA was precipitated in 100% ethanol containing 20 μg of glycogen. After washing with 70% ethanol and air-drying, the DNA was resuspended in 50 μl TE buffer. Recovered DNA were either subjected to quantitative PCR or prepared for next generation sequencing on an Illumina Genome Analyzer II (Source Bioscience, UK). Single end sequence reads were mapped to the S.islandicus genome using Genious (http://www.geneious.com). The resultant data were exported as .bam files and subsequently analyzed using SeqMonk (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/). Read counts were quantified in 20 bp windows and was normalized to input DNA.
For qPCR, SYBR detection assays were performed in triplicate using 1 μl of ChIP DNA as template, forward and reverse primers at 125 nM final concentration (Document S1. Tables S1–S5), and Stratagene 2X Brilliant II SYBR Green QPCR Master Mix according to the manufacturer's instructions. Standard curves were prepared in duplicate using ChIP input DNA as template. qPCR reactions and melting curves were performed and analyzed using a Mastercycler ep realplex2 machine and included analysis software (Eppendorf).
Analytical Ultracentrifugation Analysis of Orc1-1
Orc1-1 (WT and Walker B E147A) proteins were analyzed via sedimentation velocity using a Beckman-Coulter Optima XL-I ultracentrifuge. In each experiment, 450 μl of a 20 μM protein solution were loaded into a sector shaped graphite centrifugation cell. The protein was centrifuged at 50,000 rpm for eight hours while radial scans of absorbance captured the boundary distribution every two minutes until the sample had completely pelleted.
Boundary profiles from sedimentation velocity scans were collected and analyzed using the UltraScan II software suite (Demeler, 2005). Evidence of a highly pure system was observed via model independent enhanced van Holde-Weischet analysis of the data (Demeler et al., 1997) for WT as well as the Walker B mutant. This revealed a tight distribution of the sedimentation coefficient around a value of 3.7 s. Subsequent processing of the complete sedimentation velocity data sets employed 50 iterations of a Genetic Algorithm-Monte Carlo (GA-MC50) model (Brookes and Demeler, 2006). Figure S4 illustrates that the model is able to successfully reproduce the boundary distributions for both species.
Protease Sensitivity Assays
5 μg of purified, recombinant, WT and Walker B mutant Orc1-1 were digested with 0.5 ng/μl trypsin (Roche, sequencing grade) in 100 mM Tris (pH 8.5), 2 mM magnesium acetate, 2 mM ATP, and either 11 μM double-stranded ORB2 or an equivalent volume of water. Samples were removed at 0, 10, 30 min, 1, 3, 5, 7, and 22 hr for time course experiments and at 0 and 22 hr for endpoint digestions. Trypsin digestion was quenched by adding samples to 2.5X protease inhibitor (Roche Complete EDTA-Free Protease Inhibitor Cocktail). An equal volume of 2X SDS-PAGE loading dye was then added to the reactions and the samples were run on 4%–12% Bis-Tris gradient gels before being stained with Coomassie dye. All time course and endpoint experiments were performed a minimum of 5 times each. A representative gel is shown in Figure 7. Mass spectrometry was performed by the Oxford Proteomics Facility.
Work in Q.S.'s laboratory is supported by a special grant from Huazhong Agricultural University and the Danish Independent Research Councils (FTP/11-106683), and work in S.D.B.'s laboratory is funded by the Wellcome Trust (086045/Z/08/Z) and by the College of Arts and Sciences, Indiana University. We thank the staff at the Wellcome Trust Center for Human Genetics for performing the NGS described in Figure 3E.
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Publication Information
Received: May 2, 2012
Revised: October 9, 2012
Accepted: January 3, 2013
Published: January 31, 2013


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Metroid Prime Trilogy SEALED, 999 Promo Watch, Guides, Anime and more!
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Ratchet & Clank: A Crack in Time
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Cal Mil 1806-7-13 Tall Black Iron Iced Jar Display - 13 1/2" x 5 1/2" x 9"
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Keep butter portion packs, creamer packets, ice cream toppings, and more on ice in this Cal Mil 1806-7-13 tall black iron iced jar display! Its iron frame supports an elevated clear container that can be filled with a layer of crushed ice, then topped with the three included glass jars. Sleek and stylish, this versatile display is perfect for tabletops in buffets and restaurants. BPA free.
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I am a teacher, too, my daughter loved her ornament.
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Join Date: Jan 2013
Location: SE Texas
Posts: 650
Mini-project: Rebuilt my toilet. First time doing it.
My house was built in '83. I bought it in November '12. During the inspection, we noticed that the toilet screamed when you flushed it. Not a big deal to me, I figured it was something simple in the tank. It literally screamed like a pig in pain as the water filled the tank. It was loud enough that I could hear it in the garage, or on the opposite end of the house.
Since I had other projects ahead of it, I dealt with the noise until I could get to it. A couple days ago, I noticed that the toilet would keep refilling the tank every 10mins. I could hear what sounded like water slowly trickling passed the flapper valve. I now knew that it was time to do something about it. Having never done this before, I read around online of what the causes and problems could be. It seemed pretty straightforward, and was. First, I replaced the fill valve with the universal Fluidmaster fill valve. I adjusted it to the right height to get the water right back up to the water line in the tank. Replacing that fixed the loud screaming noise. Then, I took the flapper off and cleaned it up and the seat for the flapper valve. The toilet still ran every 10mins. I timed it, and it was 10mins every time. So, I went and got another flapper valve. Still had the same problem with the toilet running every 10mins. (Timed it again to see if the amount of water I was losing changed). Thinking that maybe I cheaped out on the flapper, I went with a better well known brand. Still, I was losing water, and the toilet would refill briefly every 10mins. While I was changing out this "better" flapper, I noticed something. There was a tiny bit of water still in the bottom of the tank. I happened to splash a little bit near the rubber seal of the Douglas Valve. I saw what looked like a seriously deteriorated rubber seal turn to dust and turn the water black. I then turned the water back on and refilled the tank. I pushed down evenly on top of the fill tube, and the sound of trickling water stopped. Off with the water, flushed the toilet to empty the tank once more, then used my wet/dry vac to get out any remaining water. Time to take off the tank. I actually looked forward to doing this. Considering the age of the house, I expected to find layers of paint behind the toilet tank since I doubt anybody would have removed the tank to paint behind it. I was right. 2 layers of white, 1 layer of tan, and some horrid looking wallpaper. IMGP4041 by r0ckstarr, on Flickr I scraped what I could of the wallpaper off for now, and slapped a quick coat of white over it for the time being. When I repaint the bathroom, I will do it all correctly. Judging by the nastiness under the tank, I would assume that the tank hadn't been taken off in years. Possibly, never. I checked Home Depot's website and it showed that they had a new Fluidmaster Douglas Valve in stock. I went down to pick it up, and found that they did not have it in stock. Instead, they had one made by a brand called Oatey. IMGP4049 by r0ckstarr, on Flickr I looked in the box and noticed that it came with a clear flapper, and thought that was cool, because then I would be able to see if water was leaking passed the flapper in the future. After cleaning up the nastiness of the toilet. I stripped everything from the tank and cleaned it inside and out. I then installed all of the new parts and put it back together. It's a very simple process, and I now have a much better understanding of how the toilet works. While I was at it, I replaced the flush handle as well. IMGP4044 by r0ckstarr, on Flickr I turned the water back on and gave it a test. I could clearly see water leaking passed the new flapper. I flushed 6 times, and every time this new flapper would sit down differently. I tried the chain in different positions, and still couldn't get it to seat down the same way every time. I currently have the chain directly above it, so that it lifts straight up on the flapper valve. I also timed it, and now the toilet refilled every 11 minutes! All that work, and all I gained was 1 minute? I was frustrated with the new parts, and made a video. http://www.youtube.com/watch?v=wvaTR5h6Sbg You can clearly see water dripping at the bottom of the flapper valve. I flushed during the video to show. Yes, the camera is underwater. The video does not change part of the way through it. That is the water level rising over the camera. ..And, on top of that, I now had another problem. A very very slow drip from one of the tank bolts. IMGP4051 by r0ckstarr, on Flickr I loosened up that side of the tank and was able to fit in a wrench in there. I tightened up that bolt half of a turn and the leak stopped. Then, I leveled the tank again and have not had a drip come from it since. My fault for not tightening it enough in fear of overtightening. I read some more online, and found some info where someone said to put a thin layer of Vaseline on the bottom of the flapper to help it seal until it wears in place. I did that, and am on my 12th hour of the toilet not running to refill again. I have flushed it at least 4 times since then and still not leaking passed the flapper. I still have the other 2 flappers I bought (cheapo and Korky). I will save those as backup parts for the future since this isn't the only toilet in my house. The old parts: IMGP4047 by r0ckstarr, on Flickr The rubber washer on the tank to bowl connection was so old and hardened, that I had to use channel locks to turn the entire thing to take it out of the tank. Last edited by r0ckstarr; 01-31-2013 at 10:16 AM. |
Solutions (handyman)
Join Date: Apr 2009
Location: Central Alberta Canada
Posts: 325
Mini-project: Rebuilt my toilet. First time doing it.
Without seeing you issue first hand, I can only venture a guess based on what you say. I don't think it's the parts, as you replaced most of them, but probably the tank. Some toilets have small flaws in the material that creates either a bump or a small dip. If the flaw is where the drain is, the flapper will never completely close. As to the leak by the bolts holding the tank and base together, it's either the washer inside, the samr flaw and previously suggested, or the bolt was screwed too tight and there is a small crack. Sinplest ssolution would be to replace the entire unit. It would be under warantee ... and would work. Frustrating, but you just may end up re-doing everything again and again unless you can find the source of the problem. Good luck
Join Date: Jan 2013
Location: SE Texas
Posts: 650
Mini-project: Rebuilt my toilet. First time doing it.Quote:
Join Date: Nov 2012
Location: Rochester NY
Posts: 51
Mini-project: Rebuilt my toilet. First time doing it.
Toilets are very frustrating things to replace, between floor/waste pipe to toilet differentials and wax rings, and porcelain tanks heating up an cooling down while you try to tighten them down.... it's just a drip, crack, puddle on your floor waiting to happen. Keep trying... Good luck!
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Join Date: Jan 2013
Location: SE Texas
Posts: 650
Mini-project: Rebuilt my toilet. First time doing it.Quote:
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text:
Population and Development Review Releases Supplement on Population and Public Policy
Influential journal celebrates career of founding editor and demographer
Paul Demeny
NEW YORK, NY (11 February 2013) — A new supplement to the influential journal Population and Development Review examines current themes in population and public policy—from low fertility and aging populations, to environment, technology, and wealth. Written by some of today's leading social scientists, the 21 essays present new insights and arguments on current population and development challenges. The supplement marks the retirement of noted demographer Paul Demeny, Distinguished Scholar at the Population Council. Demeny founded the journal in 1975 to address population issues with policy relevance and served as editor for the past 38 years.
"I have found great inspiration from Population and Development Review," says economist Nancy Folbre of the University of Massachusetts, Amherst. "It has created a platform for an open-minded and intellectually adventurous exploration of the nexus between fertility decisions and the broader forces shaping the relative well-being of women and men, young and old, parents and non-parents. We should be grateful for the many ways this collaborative project has moved the world of demography forward."
Essays in the supplement include:
- "The Intergenerational Welfare State," by Folbre and Douglas Wolf, explores the private and public routes through which families and societies provide for their young and old members. Folbre and Wolf discuss how public-sector transfers can better align the private costs and benefits of parenthood and contribute to greater equity between the generations.
- "Why Demographic Suicide? The Puzzles of European Fertility," by Lant Pritchett and Martina Viarengo, considers the enigma of ultra-low fertility and the future dwindling of population that it portends in many of today's rich countries. The usual economic explanations break down in the face of high rates of voluntary childlessness and fewer stable unions.
- In "Fertility Transition: Is sub-Saharan Africa Different?" John Bongaarts and John Casterline investigate the lagging birth-rate decline in sub-Saharan Africa, the world's last high-fertility region. Bongaarts and Casterline find that women in Africa have both a high ideal family size and a high unmet need for family planning. They conclude that the pace of fertility decline will not increase unless African policymakers champion the idea that smaller families are key to the health and prosperity of their countries.
- In "Working Misunderstandings: Donors, Brokers, and Villagers in Africa's AIDS Industry," Susan Cotts Watkins and Ann Swidler draw on their fieldwork in Malawi to argue that donor-sponsored HIV interventions have been largely ineffectual in terms of their immediate objectives but nonetheless have yielded a range of benefits for villagers and for the broader development landscape.
- "Peak Farmland and the Prospect for Land Sparing," by Jesse H. Ausubel, Iddo K. Wernick, and Paul E. Waggoner, examines human demands on the natural environment, and finds considerable reason for optimism: a near-term peaking of cropland area, followed by a downward trend. (This paper was published in advance of print as part of the 18 December 2012 symposium at The Rockefeller University in celebration of Paul Demeny's 80th birthday and to mark his retirement as editor of Population and Development Review.)
"The collection offers a rich sampling of contemporary thinking on policy-related population issues," says Geoffrey McNicoll, Population and Development Review co-editor.
Population and Development Review, a quarterly journal published by the Population Council, presents peer-reviewed scientific research on the relationships between population and social, economic, and environmental change and provides a forum for discussion of related issues of public policy.
Additional contents of the issue include:
- Reflections on Post-Transition Demography, by Geoffrey McNicoll
- Low Fertility in Historical Perspective, by Massimo Livi Bacci
- On Demographic and Democratic Transitions, by Tim Dyson
- A Demographic Perspective on Japan's "Lost Decades," by Reiko Aoki
- Population, Policy, and Politics: How Will History Judge China's One-Child Policy? by Wang Feng, Yong Cai, and Baochang Gu
- Patriarchal Demographics? China's Sex Ratio Reconsidered, by Susan Greenhalgh
- The Recent Fertility Transition in Rwanda, by Charles F. Westoff
- Fertility and Population Policy in Algeria: Discrepancies between Planning and Outcomes, by Zahia Ouadah-Bedidi and Jacques Vallin
- National Wealth, by Partha Dasgupta
- The Last Eighty Years: Continuities and Change, by Vaclav Smil
- Demographic Metabolism: A Predictive Theory of Socioeconomic Change, by Wolfgang Lutz
- Cross-Sections Are History, by Richard A. Easterlin
- Population Change among the Elderly: International Patterns, by Andrew Stokes and Samuel H. Preston
- The French School of Demography: Contextualizing Demographic Analysis, by Véronique Petit and Yves Charbit
- The Twilight of the Census, by David Coleman
Intergenerational Relations
Low Fertility, Population Aging, and the Body Politic
Policy and Program: China
Policy and Program: Africa
Environment, Technology, and Wealth
Population Theory and Measurement
Open-access contents available at: http://www.popcouncil.org/publications/books/2012_PDRSuppPopPublicPolicy.asp
About the Population Council
The Population Council confronts critical health and development issues—from stopping the spread of HIV to improving reproductive health and ensuring that young people lead full and productive lives. Through biomedical, social science, and public health research in 50 countries, we work with our partners to deliver solutions that lead to more effective policies, programs, and technologies that improve lives around the world. Established in 1952 and headquartered in New York, the Council is a nongovernmental, nonprofit organization governed by an international board of trustees.
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For 60 years, the Population Council has changed the way the world thinks about important health and development issues. Explore an interactive timeline of the Council's history, learn more about some of our key contributions, and watch a short video about why your support is so important to us.
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text: 000 FXUS63 KEAX 081148 AFDEAX AREA FORECAST DISCUSSION NATIONAL WEATHER SERVICE KANSAS CITY/PLEASANT HILL MO 548 AM CST Sat Mar 8 2014 .DISCUSSION... Issued at 352 AM CST SAT MAR 8 2014 Satellite imagery from this morning shows a moderately amplified pattern across the United States with a notable vorticity max sinking south through the Four Corners region towards Mexico. This will resulted in a a shear axis developing across the Central Plains as the circulation detaches from the northern stream. This shear axis in turn will interact with the elevated baroclinic zone being shoved south by the surface high oozing south through the Plains early this morning, thus resulting in a persistent chance at some light precipitation through the morning hours. Radar imagery from eastern Nebraska backs up this idea as band of light snow has developed within the frontogenic forcing expected to push south with the surface high. However, the elevated boundary is now expected to settle a little farther south today, thus limiting the northward extent of the precipitation. Otherwise, pinning down precipitation type will be a bit difficult today as thermal profiles continue to wander back and forth across the freezing line through the day. Carried a mix of precipitation types for the morning hours, though light accumulations ranging from a heavy dusting to a couple tenths of the inch of snow or sleet will be possible this morning. For the afternoon hours, dispute surface temperatures warming above freezing have kept a mix of wintry precipitation going as wet bulbing effects and strong vertical forcing might be able to force some frozen precipitation types to the ground. Potential accumulations are still expected to be rather light as the main focus for the afternoon hours will be across southeast Kansas and southern Missouri, though a few hundredths to a tenth of a inch of snow/sleet/rain could occur. After todays attempt at wintry weather Spring will makes its presents felt again quickly as temperatures rebound. The moderately amplified flow across the nation will flatten out late in the weekend, effectively keeping all that cold arctic air bottled up at higher latitudes through the coming work week. Only issue to watch for at this time appears to be another shearing trough, which will settle through the Plains States Tuesday night. Precipitation chances look iffy at this time as shearing, positively tilled, troughs are not the best precipitation generators for us. Besides today, temperatures this Wednesday look to be the coolest of the week as a cool surface high slides across the region behind the shearing trough, and even then we are expecting afternoon highs in the 40s. So, while overnight lows will retreat below freezing periodically at least afternoon highs will likely range above normal for much of the coming week. && .AVIATION...(For the 12Z TAFS through 12Z Sunday Morning) Issued at 547 AM CST SAT MAR 8 2014 A modest band of MVFR clouds will prevail at the terminals into the mid to late morning hours along with vicinity showers as an elevated frontal boundary presses south this morning. Skies are expected to begin clearing form north to south late this morning, but even if skies don`t clear out ceilings should lift well into the VFR range. Otherwise, our breezy north wind today will relax this evening before coming back around to the south Saturday morning. && .EAX WATCHES/WARNINGS/ADVISORIES... KS...NONE. MO...NONE. && $$ DISCUSSION...Cutter AVIATION...Cutter


text:
Evaluation Inhaled Corticosteroids on Exhaled Nitric Oxide Gas Exchange
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To evaluate the role of inhaled corticosteroids to suppress nitric oxide gas exchange in stable patients with moderate-to-severe COPD who are current non-smokers and not on oral corticosteroids.
Condition | Intervention |
Drug: fluticasone 100mcg/ salmeterol 50mcg Drug: fluticasone 250/salmeterol 50 Drug: salmeterol Drug: Salmeterol Drug: salmeterol and fluticasone propionate |
Study Type: | Interventional |
Study Design: | Allocation: Randomized Endpoint Classification: Bio-equivalence Study Intervention Model: Crossover Assignment Masking: Single Blind (Outcomes Assessor) Primary Purpose: Diagnostic |
Official Title: | Evaluation of the Effects of Varying Doses of Inhaled Corticosteroids on Suppression of Total Exhaled, Bronchial, and Alveolar Nitric Oxide as Markers of Endogenous Inflammation in Patients With Moderate-to-severe COPD |
- total exhaled, bronchial, and small airway/alveolar nitric oxide [ Time Frame: 3 months ] [ Designated as safety issue: No ]
- lung function [ Time Frame: 3 months ] [ Designated as safety issue: No ]
Enrollment: | 39 |
Study Start Date: | January 2006 |
Study Completion Date: | October 2013 |
Primary Completion Date: | October 2013 (Final data collection date for primary outcome measure) |
Arms | Assigned Interventions |
Active Comparator: long acting bronchodilator
salmeterol 50mcg bid by DPI, no fluticasone in patients with COPD/emphysema to evaluate effect on bronchodilation and exhaled nitric oxide
Drug: Salmeterol
salmeterol 50mcg bid X 3months
Other Name: serevent 50 mcg disk
Active Comparator: bronchodilator/ inhaled corticosteroid
fluticasone 250mcg/salmeterol 50 mcg bid X 3momths to evaluate effect on lung function and exhaled nitric oxide
Drug: fluticasone 250/salmeterol 50
inhaled fluticasone 250/salmeterol 50 X 3 months to evaluate effect on lung function and exhaled nitric oxide
Other Name: Advair 250/50
Drug: salmeterol and fluticasone propionate
salmeterol 50mcg/fluticasone 250mcg by DPI bid
Other Name: Advair 250/50
Active Comparator: C
Fluticasone 100mcg/salmeterol 50mcg
Drug: fluticasone 100mcg/ salmeterol 50mcg
inhaled fluticasone 100mcg/salmeterol 50mcg bid X 3 months
Other Name: Advair 100/50
Drug: salmeterol
salmeterol 50 mcg by dry powder inhaler disk bid to evaluated effect on lung function and exhaled nitric oxide
Other Name: Serevent 50mcg
Detailed Description:
Stable, non-smoking COPD patients not on oral corticosteroids will be randomized in single blinded study to evaluate nitric oxide gas exchange at baseline, and subsequent effects of fluticasone 100mcg/salmeterol 50mcg bid, and fluticasone 250mcg/salmeterol 50mcg bid. Primary end points will include measurements of nitric oxide at varying expiratory flow rates to calculate bronchial, and small airway/alveolar nitric oxide. Secondary end points will evaluate lung function. Exhaled nitric oxide production presumably reflects endogenous inflammation. Normal healthy, non-smoking controls will be used for comparison, with exhaled nitric oxide measured before and after 3 weeks of low dose inhlaed corticosteroids.
Ages Eligible for Study: | 40 Years to 85 Years |
Genders Eligible for Study: | Both |
Accepts Healthy Volunteers: | Yes |
Inclusion Criteria:
- Clinical diagnosis of copd
- Current non-smoker
- Not on oral corticosteroids
- Must be able to use Advair discus or salmeterol discus
Exclusion Criteria:
- Pregnancy
- Current smoker
- On corticosteroids
- Clinically unstable
United States, California | |
Arthur F Gelb MD | |
Lakewood, California, United States, 90712 |
Principal Investigator: | Arthur F Gelb, MD | Arthur F Gelb Medical Corporation |
Additional publications automatically indexed to this study by ClinicalTrials.gov Identifier (NCT Number):
Responsible Party: | Arthur F Gelb MD, Gelb, Arthur F., M.D. |
ClinicalTrials.gov Identifier: | NCT00568347 History of Changes |
Obsolete Identifiers: | NCT00569348 |
Other Study ID Numbers: | 20061697A |
Study First Received: | December 4, 2007 |
Last Updated: | March 1, 2014 |
Health Authority: | United States: Institutional Review Board |
Keywords provided by Gelb, Arthur F., M.D.:
chronic airflow obstruction |
Additional relevant MeSH terms:
Pulmonary Disease, Chronic Obstructive Lung Diseases, Obstructive Lung Diseases Respiratory Tract Diseases Nitric Oxide Salmeterol Fluticasone Bronchodilator Agents Autonomic Agents Peripheral Nervous System Agents Physiological Effects of Drugs Pharmacologic Actions Anti-Asthmatic Agents Respiratory System Agents Therapeutic Uses |
Free Radical Scavengers Antioxidants Molecular Mechanisms of Pharmacological Action Neurotransmitter Agents Endothelium-Dependent Relaxing Factors Vasodilator Agents Cardiovascular Agents Protective Agents Adrenergic beta-2 Receptor Agonists Adrenergic beta-Agonists Adrenergic Agonists Adrenergic Agents Dermatologic Agents Anti-Allergic Agents Anti-Inflammatory Agents |
ClinicalTrials.gov processed this record on March 09, 2014


text:
Playstation 3
This page contains a "KickBeat - Let's Play Trailer" for Playstation 3 and has been posted or updated on Aug 9, 2013 by EDF_Admin. Check this page periodically for the latest Video Game Trailers and Gameplay Videos.
- Developer: Zen Studios
- Publisher: Zen Studios
- Genre: Miscellaneous Rhythm Music
- Release Date: Sep 3, 2013
- ESRB: Teen
Video Game Trailers
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With this Cowgirl Tuff Ladies Western Black Short Sleeve Tee, believe anything is possible! This top is extremely soft and highly fashionable with burnout raglan dolman style sleeves. Dressed it up or go casual with this great piece for every woman.
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The Noble Equine Josie Top is perfect active wear for cool days! This shirt is made of performance knit, features thumbhole cuffs, and a 1/4 zip mock neck to ensure you stay at just the right temperature whether you're working hard or playing hard around the barn.
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text:
Chanticleer Sings Cole Porter, Joni Mitchell, Hildegard von Bingen and More
* Additional fees apply.
All offers for She Said/He Said have expired.
The last date listed for She Said/He Said was Friday September 27, 2013 / 8:00pm.
Most Popular Music Event Nearby:
Yoshi's Oakland: The Best Live Jazz in the East Bay
- Full Price:
- $15.00 - $49.00
- Our Price:
Yoshi's Oakland not only showcases the world's best jazz, gospel, world music and other entertainment, but its acclaimed chef Shotaro "Sho" Kamio is famed for creating some of the finest modern Japanese cuisine in the area. The live music club and upscale restaurant has been located in Jack London Square since 1997 and offers an unmatched entertainment experience. The venue has hosted such legendary jazz greats as Betty Carter, Max Roach, Dizzy Gillespie, Joe Williams, Diana Krall, Branford Marsalis, McCoy Tyner, Harry Connick Jr., Oscar Peterson and hundreds of others, including name comedians. Please see the full event description for the lineup of upcoming performers. Learn More


text:
Someone recently made a gif from a video where John hollers a question off-screen to Sarah about her dating preferences or some such thing and Sarah hollers back "obviously not" and John puts his head down and laughs. Anyone, anyone?
http://www.youtube.com/watch?v=PkoBoF9FDXg 1:37, I think this is the video you're talking about.
Darn, I was really chuffed I knew this. Beat me to the punch =P
You got a link to the GIF? I always thought that would make an awesome one!
That's the right video - thanks guys!! I've searched for the gif again as well and haven't found it - it was a good one.
Here are some links to help spread the awesome:
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text: Peter Friedman has been training as a baseball pitcher his entire life. He and his best friend A.J. have always planned on making and dominating their high school team. But you can't always count on your plans to work out. Curveball: The Year I Lost My Grip is one teen's journey to figure out what Plan B is.


text:
Class Notes
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Ashleigh Gage '10 and Michael Early '08 were married in a private ceremony in Los Gatos, Calif., on Feb. 19, 2011. They honeymooned where they fell in love—in London, England, where they studied abroad in 2006. They now live in Seattle, Wash., where Ashleigh works in sales for a small technology start-up.
Kaitlyn Devlin '10 serves with the Jesuit Volunteer Corps Northwest in Spokane, Wash., at St. Margaret's Shelter.
Ruben Dario Villa '10, is a graphic designer at Apple, Inc. As part of the iLife Team, Villa designs visual assets for software on the iPhone; he also corrects resolution issues on graphics and improves upon existing images for the purposes of retina display on the iPhone. Prior, he was a freelance graphic designer at Outloud Designs.
Heather Clayton '10 is going forward with her Master's of Public Health at Cal State Long Beach. She looks forward to visiting Santa Clara University soon.
Carolyn Chu '10 serving with the Jesuit Volunteer Corps Northwest in Gresham, Ore., for the second year at JOIN. She is working as a House Coordinator at a day center for homelessness. She provides basic services such as mail, laundry vouchers, and showers through a barter system: Clients contribute to the maintenance and community of JOIN to gain barter points, which they can use to trade for services. She also spends time directing people to other social services throughout the city of Portland.
Derek Chien JD/MBA '10 has joined Hoge Fenton Jones & Appel, a leading Northern California law firm. Chien is a new admittee to the California bar and is assigned to the firm's Intellectual Property and Corporate practice teams. His primary areas of focus are trademark and copyright prosecution and USPTO litigation, helping to manage significant IP portfolios for several of the firm's well known global clients. He also is involved in general corporate work, including mergers & acquisitions and the formation and dissolution of nonprofit organizations. Derek is fluent in Cantonese and speaks basic Mandarin and Spanish.
Sara Sorto '08, M.A. '10 married Dave Carvalho in Los Gatos, Ca on October 7, 2012. They live in Santa Clara where Sara teaches at Santa Clara High.
Gina Carnazzo MTS '10 is a volunteer English teacher at a mission school in Lome, Togo. She writes: "I have always wanted to come to Africa. I am drawn to the people and the land (and the animals—though there aren't any safari animals in Togo). But it always seemed like an impossible dream to travel here. Then, when I met so many people from African countries at JST, it began to feel more possible. I chose the Canossian Daughters of Charity because I liked what I read about their organization (VOICA), and I wanted to be involved with a Catholic organization. The core of my coming here is this: I have been showered with love and gifts all my life. I feel filled with love—overflowing, sometimes—and I just want to share that love. I did not come to Africa to convert anyone—just to share Christ's love with the people here. I am not bringing Christ's love, though, because His love is already here (something I learned at JST)."
Sarah R. Cafasso '10 completed a two-year Peace Corps service in October. She served as an Environment volunteer in rural Philippines where she promoted marine conservation and sustainability. She now works at the U.S. Environmental Protection Agency in San Francisco, where she is a Community Involvement Coordinator in the Region 9 Superfund Division.
Jeffrey Adams '10 is appearing in the new play Animals, presented by Wide Eyed Productions, which follows a group of "twenty-somethings" kicking around their suburban hometown. The play is running as part of the FringeNYC Festival 2012 at the Kraine Theatre in New York City in August. Adams is also working on his MFA at the New School for Drama.
Mikell Warms '11 is currently a graduate teaching assistant at Colorado State University.
Mark Vetto '11 is currently serving with the Jesuit Volunteer Corps in Syracuse, N.Y., with Catholic Charities of Onondaga County - Emergency Services.
Mark Vetto '11 is in his second year as a Jesuit Volunteer with Mental Health Advocacy Project, in San Jose, Calif. Based in four core values—social justice, simple living, community, and spirituality—Jesuit Volunteer Corps offers women and men an opportunity to work full-time for justice and peace. JVs serve the poor directly, working for structural change in the United States and in developing countries.


text:
For twelve years ELPH cameras have been coveted icons of design and innovation. Informed by that legacy yet stepping solidly into the future comes the PowerShot ELPH 530 HS. Beautifully sleek, this state-of-the-art camera is ready for the way you live now. Built-in Wi-Fi lets you share images and beautiful 1080p Full HD videos virtually anywhere, while the 3.2-inch wide touch panel PureColor System LCD makes operation easy and interactive. So slim, it's hard to believe it packs a 28mm Wide-Angle lens with 12x Optical Zoom to capture just the image you want. Image quality is superb, with a 10.1 Megapixel High-Sensitivity CMOS Sensor and DIGIC 5 Image Processor for the clear low-light imaging of Canon's HS SYSTEM. It also comes equipped with the latest technological advancements like Smart AUTO with Face ID to ensure that every shot you capture is amazing.
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Canon, Inc
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Product Name: |
PowerShot ELPH 530 HS Compact Camera
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Compact Camera
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0The items must arrive at our warehouse within 30 days of purchase. You must return items in their original condition to qualify for a full refund. We will issue a partial refund of up to 70 percent if returned items have been opened, used, or returned late. More details
Our standard shipping method is ground shipping. We also offer expedited shipping for most items. Your final shipping cost and estimated delivery timeframe will be available at checkout.
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* For your safety, some orders go through our loss prevention department. If we select your order for this process, we will immediately notify you that there may be up to a 3-day delay on your order.
** Most Oversize orders are delivered within 1-4 weeks. Some orders may take 6 weeks to be delivered.