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Tandem genetic duplications arise frequently between the seven directly repeated 5.5-kb rrn loci that encode ribosomal RNAs in Salmonella enterica. The closest rrn genes, rrnB and rrnE, flank a 40-kb region that includes the purHD operon. Duplications of purHD arise by exchanges between rrn loci and form at a high rate (10−3/cell/division) that remains high in strains blocked for early steps in recombination (recA, recB, and/or recF), but drops 30-fold in mutants blocked for later Holliday junction resolution (ruvC recG). The duplication defect of a ruvC recG mutant was fully corrected by an added mutation in any one of the recA, recB, or recF genes. To explain these results, we propose that early recombination defects activate an alternative single-strand annealing pathway for duplication formation. In wild-type cells, rrn duplications form primarily by the action of RecFORA on single-strand gaps. Double-strand breaks cannot initiate rrn duplications because rrn loci lack Chi sites, which are essential for recombination between two separated rrn sequences. A recA or recF mutation allows unrepaired gaps to accumulate such that different rrn loci can provide single-strand rrn sequences that lack the RecA coating that normally inhibits annealing. A recB mutation activates annealing by allowing double-strand ends within rrn to avoid digestion by RecBCD and provide a new source of rrn ends for use in annealing. The equivalent high rates of rrn duplication by recombination and annealing pathways may reflect a limiting economy of gaps and breaks arising in heavily transcribed, palindrome-rich rrn sequences.
THE most frequent tandem gene duplications in the Salmonella chromosome arise between directly repeated ribosomal RNA (rrn) loci (Anderson and Roth 1981; Reams et al. 2010). Duplications formed between such extensive repeats (5.5 kb) are generally thought to arise by unequal recombination between copies of the repeat in different sister chromosomes (Roth et al. 1996; Romero and Palacios 1997) (see Figure 1). Such exchanges are expected to depend heavily on the homologous recombination pathways that rely on the strand exchange enzyme RecA. In keeping with this expectation, duplications between chromosomal repeats of the lac operon form at high rates, comparable to those of rrn duplication, but depend heavily on RecA (A. Reams, M. Carter, and J. Roth unpublished results). In contrast, recent assays in two other situations suggest that RecA can be dispensable for formation of duplications between long repeats that are subject to frequent nicks or breaks (Reams et al. 2010, 2012). Duplication of regions not flanked by extensive repeats are also RecA independent, but arise at rates two to three orders of magnitude lower than those for rrn-mediated duplications (Reams and Neidle 2003, 2004; Kugelberg et al. 2006; Reams et al. 2010). Here we describe duplications arising by exchanges between directly repeated rrn loci that are separated by >40 kb and are normal residents of the Salmonella chromosome. The goal is to understand how these duplications arise at such high rates with or without RecA.
Seven rrn loci are scattered around the Salmonella enterica chromosome (Figure 2) and have nearly identical base sequences. Recombination between these loci generates a variety of chromosome rearrangements including duplications, inversions, and translocations (Anderson and Roth 1981; Liu and Sanderson 1998; Helm et al. 2003). Tandem duplications between rrn loci form at very high rates. For example, the argH gene, between rrnA and rrnB (see Figure 2), duplicates at 1.9 × 10−3/cell/division (Reams et al. 2010), and this rate is essentially unaltered in recA mutants (Reams et al. 2010). These duplications of argH are lost at a 10-fold higher rate (10−2/cell/division) by heavily RecA-dependent recombination between the extensive duplicated regions (>150 kb). Because of their high loss rate and fitness cost, argH duplications are carried as stable polymorphisms in unselected cultures and are maintained at a steady-state frequency of ∼1% (Reams et al. 2010). These high steady-state frequencies are likely to be a general property of all duplications in all organisms.
The surprising RecA independence of rrn-mediated duplication formation is examined here using the purHD locus. This region is flanked by the most closely spaced rrn cistrons, rrnB and rrnE (separated by 40 kb), and is held at the highest duplication steady-state frequency (∼3%) of any tested point in the chromosome (Anderson and Roth 1981). It is suggested that the high rate and apparent recombination independence of rrn duplications may reflect two features of rrn sequences. First, rrn cistrons are the most highly transcribed genes in the Salmonella and Escherichia coli chromosomes (Dennis 2004). Second, rrn sequences include many stem-loop structures that are responsible for folding of the ribosomal RNAs (16S, 23S, 5S) produced from each locus. The palindromic features of rrn DNA may allow untranscribed strands to form secondary structures that are subject to cutting or breakage. These unusual features may make rrn sequences prone to frequent gaps and breaks.
It is proposed here that blockage of early recombination steps (RecBCDA and RecFORA) can activate a single-strand annealing pathway of duplication formation that compensates for loss of recombination. When frequent DNA damage within rrn sequences remains unrepaired by recombination, these lesions can accumulate sufficiently that two different rrn loci can provide ends. Duplications can form by annealing when two single-strand ends are provided and neither strand is coated with inhibitory RecA protein. Activation of annealing pathways renders duplication formation independent of Holliday resolution activities (RuvC, RecG).
Materials and Methods
Strains and media
All strains were derivatives of S. enterica (Typhimurium) strain LT2 and are listed in Table 1. Rich medium was Luria broth (LB), used with antibiotics as described below.
Trapping duplications formed in overnight cultures
The drug-in-drug method for measuring duplication frequencies in unselected overnight cultures was described previously (Reams et al. 2010, 2012). For these assays, the strain to be tested carries the tetAR genes for tetracycline resistance (TcR) inserted as part of transposon Tn10 into the test locus (e.g., purH). These strains are then made sensitive to tetracycline by a kanamycin-resistance (KnR) determinant inserted into the tetA gene. The resulting strains are phenotypically Tcs KnR. Duplications are trapped by a Red-mediated recombineering cross in which a short single-strand fragment (80 bases) is introduced into the tested strain, which carries a pSIM5 plasmid and is grown at 30° (Court et al. 2002). This fragment, introduced at 42°, restores TcR by precisely excising the KnR determinant. Transformants are selected on LB–tetracycline agar plates. The majority of these transformants become TetR KanS. However, any recipient cells with a pre-existing duplication of the test locus become TetR, but retain their KanR phenotype and are detected by replica-printing to LB–tetracyline–kanamycin. Duplication frequencies are calculated by dividing the number of TcR KnR colonies by the total number of TcR colonies. Duplication of purHD by exchanges between rrn loci was verified by join-point PCR and whole-genome sequencing (see below).
The rate of duplication formation is defined here as "the duplication frequency attained by a culture during 33 generations of growth from a single cell without a duplication." This frequency is below the steady-state level, and the nearly linear accumulation of duplications in this period is taken as the initial rate of duplication formation. The course of duplication accumulation was described previously (Reams et al. 2010). The forces that drive duplication frequency toward a steady state minimize the effects of Luria–Delbruck fluctuation. Low viability of some rec mutant strains does not interfere with comparison of rates to those in wild type because each rate is based on the fraction of viable tester cells that carry a duplication. The 33-generation period is assured by a single colony isolating the strain to be assayed and using an entire small colony to inoculate a liquid culture (4 ml) that is grown to stationary phase. The final titer (2 × 109 cells) is achieved after 33 generations of growth of the cell that initiated the colony.
Duplication join-point analysis
Trapped purHD duplications were analyzed for the nature of their rrn-mediated join points using a divergent primer PCR method described previously (Helm and Maloy 2001). Two divergent primers direct synthesis in opposite directions away from purHD. The primers anneal to regions immediately adjacent to various rrn locus (Figure 2). Since these primers direct divergent replication, a PCR product will be amplified only in strains with a specific rrn-mediated duplication junction. The following primer pairs were used to test for the presence of the various hybrid rrn combinations possible for purHD duplications: rrnE/B (TP2306+TP2311), rrnH/B (TP2306+TP2315), rrnE/H (TP2311+TP2316), rrnE/A (TP2304+TP2311), rrnH/A (TP2304+TP2315), rrnE/C (TP2308+TP2311), rrnH/C (TP2308+TP2315). The sequences of these primers are TP2304 (5′ TGCCTTCATTTTGCGGTGGTTAGAG 3′), TP2306 (5′ CCAGGCGCTCAGTAGTTGTTGTTCG 3′), TP2308 (5′ GCTGTTAGGGCACTTCACTTTGGCG 3′), TP2311 (5′ CGATAGGGGCGATGTGGTGCTGTTC 3′), TP2315 (5′ CCATCCGCAGGGCAGCATAGAAGAG 3′), and TP2316 (5′ CGGCAATAGCCTTTTCCATCAACGG 3′).
Prior to PCR, both the parent and various trapped purHD duplication strains were grown overnight in 2 ml LB media and diluted 10-fold. For each divergent primer pair, a PCR reaction was run on each strain. All PCR products were separated on a 0.7% agarose gel using standard electrophoresis procedures and visualized after staining with ethidium bromide. Strains carrying a duplication specific to the applied primers generated an ∼7-kb PCR product, whereas the parent strains showed no PCR product. Using this method, the specific rrn locus involved in forming the duplication was determined for all 30 of the tested purHD duplications. Whole-genome sequencing of several strains verified their PCR-determined rrn join points and demonstrated the presence of a simple duplication.
The crossover points of the exchanges that generated the hybrid rrn duplication join points were determined by sequencing the gel-purified 7-kb PCR products derived from junctions of independently isolated purHD duplications. PCR products were sequenced using 12 different primers that covered the entire rrn region. The sequences of hybrid junction elements were compared to those of participating parent rrn loci. The crossover event was inferred to have occurred in the region where the hybrid rrn sequence of one rrn sequence (e.g., rrnE) transitioned to that of another (e.g., rrnE).
Normalization of read-depth plots
Plots of Illumina read-depth data are often noisy due to uneven amplification in the PCR step of library preparation and from the effects of G+C differences on detection chemistry and DNA fragmentation. Since the profile is remarkably uniform between isogenic strains, the noise can be filtered out by comparing data from an isogenic reference strain (preferably from the same run). A normalization factor is determined at each position and used to correct the depth of the test genome, yielding a graph that clarifies the features of interest. An example is shown in Figure 3. The duplication strain tested was a population in which half the cells contained a large duplication. The duplication was unrecognized initially, but became clear following normalization.
Duplications of the purHD operon arise between flanking rrn genes
Duplications were trapped as described previously (Reams et al. 2010), and junction points were classified by using PCR to determine which rrn loci recombined to form the duplication. For example, in identifying the rrnE/B junction (see Figure 1), one primer was directed clockwise toward rrnE and the other was directed counterclockwise toward rrnB as described previously (Helm and Maloy 2001). Cultures were diluted before DNA preparation to assure that the dominant structure was assayed and minimize contributions from any new rrn duplications appearing during growth. The presence of a rrnE/B duplication junction was demonstrated by production of a 7-kb PCR product using divergent primers (rrnE and rrnB). Other divergent primer pairs tested the presence of the various rrn join-point combinations studied here (e.g., rrnB/H, rrnA/E).
As seen in Figure 2, all 30 purHD duplications trapped in a recA+ strain showed hybrid rrn join points. Most (47%) involved the smallest interval (rrnE/B). Next most frequent (40%) were rrnH/B, and 13% involved rrnA/E (see Figure 2). None of the other rrn pairs flanking purHD were detected (rrnH/C, rrnH/A, rrnE/C). Several of the characterized rrn-mediated duplications were further analyzed by whole-genome sequencing. As seen in Figure 4, the Illumina read depth is elevated twofold for the genomic region between the corresponding rrn loci. This confirms that the trapped duplications carry two copies of the intervening region corresponding to the join point determined by PCR and no other obvious rearrangements.
Dependence of duplication rates on flanking rrn sequences
The duplication rate of the purHD operon is defined here as the duplication frequency attained during 33 generations of growth from a single cell lacking a duplication. During this period the frequency of purHD duplications reached 2.5%, a rate that corresponds to 3 × 10−3/cell/generation. This increase is nearly linear and is taken as an initial rate since it is far from the ultimate steady-state frequency.
The duplication rates of purHD and three nearby loci (zja, metA, and thrA) were assayed in strains lacking either or both of the rrnE and rrnH sequences. Positions of these loci are shown in Figure 2. Removal of both rrnE and rrnH caused a large decrease (170-fold) in the purHD duplication rate (Table 2). The duplication rates for the zja, metA, and thrA sites showed a similar drop after deletion of only rrnH. In all cases, the large drop in duplication rate resulted from removal of all the rrn loci from one side of the test site. The few residual duplications seen in these rrn-deficient backgrounds arose between diverse short sequence repeats and will be described elsewhere (A. Reams, E. Kofoid, and J. Roth, unpublished results).
As seen in Figure 2, roughly half of the purHD duplications arose by exchanges between rrnB and rrnE. Nevertheless, removal of rrnE alone had very little effect on the purHD duplication rate (Table 2), while removal of rrnH alone caused a fivefold reduction. The relatively large contribution of rrnH to the purHD duplication rate is not understood. We suggest that rrnH may be subject to more frequent chromosome gaps or breaks than rrnE, making rrnH/B duplications form more frequently than rrnE/B types. However, the larger (790 kb) rrnH/B duplication may have a higher fitness cost, leading to selective deletion of the region between rrnE and rrnH either before or after being trapped. Such remodeling would maintain the duplication state of purHD, but would leave a shorter (40 kb), lower-cost duplication with a rrnE/B join point.
Three sites in the chromosomal region between rrnE and rrnH (zja, metA, and thrA) were found to be duplicated at a slightly lower rate than purHD (see Table 2). Removal of rrnE had very little effect on the duplication rate of these loci, suggesting that most of these duplications form between rrnB and rrnH. The few duplications of this region that formed in strains without rrnH (for which rrn-mediated duplications are impossible) showed join points, suggesting that they arose by exchanges between diverse short sequences (A. Reams, E. Kofoid, and J. Roth, unpublished results).
Duplication rates are unaffected by defects in early steps of homologous recombination
The large size of the flanking rrn repeats and the high frequency of exchanges between them suggested that duplications of purHD are likely to form by homologous recombination. Strong RecA dependence has been seen in many situations in which duplications form between extensive perfect repeats (Roth et al. 1996; Romero and Palacios 1997). However, purHD duplication rates are essentially unaffected by mutations that eliminate enzymes important to standard pathways of recombination (see Figure 5). Furthermore, the distribution of junction types (rrnE/B, rrnH/B, and rrnE/A) for purHD duplications were the same in recA+ and recA mutant strains (see Figure 2, top). Taken at face value, these results suggest that RecA, RecB, or RecF enzymes are not involved in duplication formation. Despite these phenotypes, we suggest below that formation of these duplications in wild-type strains is heavily recombination dependent and occurs by single-strand annealing only in strains lacking these recombination functions.
The apparent RecA independence of rrn duplications suggested formation by single-strand annealing. Alternatively, RecA-independent events might be illegitimate exchanges mediated by topoisomerases that are likely to act within or near rrn loci in response to heavy transcription of these loci. The exchanges that produce duplications are not associated with formation of deletions or duplications within the rrn locus since the size of the rrn locus at the join point is as expected for a simple hybrid recombinant. To learn more about the nature of the exchanges, we analyzed hybrid rrn junction sequences to determine the approximate points within the rrn loci at which the genetic exchanges occurred. This is possible because the base sequences of different rrn loci are not identical. Each locus has sequence features that are unique, and every pair of rrn loci differs at several points. A tree of rrn sequence difference in the Appendix describes their differences. The aligned sequences of the three rrn loci involved in duplicating the purHD operon are diagrammed in Figure 6, where regions with many clustered sequence differences are boxed, and isolated single-base differences are indicated by colored triangles.
To determine the location of the crossover point that formed the duplication, the hybrid junction rrn loci were amplified from independently isolated duplications and their sequences were compared to those of the two parental rrn sequences. Figure 6 describes the exchange points of 26 independent rrnE/B duplications and 7 rrnH/B duplications isolated from recA+ and recA mutant strains. For each duplication, the exchange fell within one of three regions of extended sequence identity. Most crossover events occurred within region III, the largest window of perfect identity (2390 bp for rrnE/B or 2490 bp for rrnH/B), while fewer events occurred within the other two smaller windows, region I (756 bp for rrnE/B and rrnH/B) or region II (497 bp for rrnE/B and 413 bp for rrnH/B). Duplications trapped in rec+ and recA backgrounds showed a similar distribution of exchange points.
The results in Figure 6 suggest either that rrn-mediated duplications form by the same pathway in rec+ and recA strains or that homologous recombination and single-strand annealing pathways lead to the same distribution of exchange points. The results do not exclude the possibility that topoisomerase catalyzes exchanges within regions of shared rrn sequence identity. In discussing a model for rrn duplication, we favor the idea that DNA breaks often occur within the transcribed rrn region and produce gaps or breaks that can initiate duplications by two alternative pathways.
Duplication rates are strongly reduced in strains deficient for Holliday structure resolution
Standard pathways for homologous recombination (RecBC and RecFOR) involve early events that process breaks and gaps leading to RecA-catalyzed strand invasion. This produces Holliday junctions that are moved on recombining duplexes by RuvAB (Heller and Marians 2005; reviewed by West 2003). The later events that stabilize and ultimately resolve these Holliday structures are achieved by two alternative enzymes: (1) the RuvABC resolvasome and (2) the RecG branch-migration enzyme (Benson et al. 1991; Wardrope and Leach 2009). If one resolution pathway is inactivated by mutation, the other is still available. Cells lacking both RuvC and RecG are severely deficient in recombination, presumably due to their inability to stabilize and resolve Holliday junctions. As seen in Figure 7, a recG mutation alone had little effect on duplication rate. The RecG enzyme is associated with the RecBCD pathway, which below we argue does not contribute to duplication. Strains with a single ruvAB or ruvC mutation caused a significant drop in duplication rate. The largest decrease was seen in the ruvC, recG double mutant, which showed a 30-fold drop in duplication rate compared to that of wild type. This suggests that rrn-mediated duplications form by a recombination process that involves Holliday structures and requires their resolution by either RuvC or RecG. Of the residual duplications that formed at a reduced rate in the ruvC recG mutant, three junctions were sequenced and showed rrnE/B exchanges within the rrn sequence (see Figure 6), suggesting formation by single-strand annealing. The importance of Holliday structure resolution to duplication formation suggests that homologous recombination is normally involved in duplication formation despite the failure of recA, -B, or -F mutations to reduce the duplication formation rate. In addition to blocking recombination, the ruvC recG mutations may cause toxic accumulation of Holliday structures as seen previously in Neisseria (Sechman et al. 2006) and yeast (Fabre et al. 2002). This possibility will be discussed later.
Defects in early homologous recombination steps restore normal duplication rates to ruvC recG mutants
It was surprising that single recA, -B, and -F mutations and even a recB, recF double mutation, which block early steps in recombination, failed to affect a process that requires the late step of Holliday structure resolution. We expected a single recB or recF mutation to cause a less severe drop than a recA or a recB, recF double mutant. To pursue this, these early mutations were added to a ruvC, recG strain, defective for duplication. As seen in Figure 8, each of the single mutations restored a normal duplication rate. Thus, duplications of purHD formed at normal rates in strains lacking all of these major players in homologous recombination: RecA, RecB, RecF, RuvC, and RecG.
To explain the normal duplication rate in the suppressed ruvC recG mutants, we suggest that blockage of early recombination steps activates an alternative pathway that can form duplications by single-strand annealing and does not require RuvC or RecG. That is, duplications may form by homologous recombination in wild-type strains (despite the phenotypes seen in Figure 5). However, blockage of early recombination steps allows gaps and breaks to accumulate and provides ends that can form a duplication by single-strand annealing without need for strand invasion or Holliday structure resolution. When Holliday resolution is blocked, recombination cannot be completed, but the annealing pathway opened by early mutations supports duplication by an alternative annealing route for which Holliday resolution is irrelevant. A curious aspect of this suppression is that individual recB, recF, and recA mutations restored duplication. One might have expected that recA or the recB, recF mutation combination would be more effective than the recB or recF mutations individually. An explanation is suggested below.
Duplication by single-strand annealing raises a mechanistic problem. While it is easy to visualize use of an annealing pathway to form a deletion using resected ends at a chromosome break, formation of a duplication from one break would seem to require separation of strands for the entire duplicated region. Duplication by annealing is easier to imagine with two breaks, one near each participating rrn locus in different sister chromosomes. Because of frequent DNA breakage in rrn loci, delays in their repair caused by a recA (or recF or recB) mutation could allow damage to accumulate and assure coexistence of breaks in different rrn loci.
We suggest that, in the simple ruvC recG mutant, the early recombination functions (RecFORA and RecBCDA) continue to consume gaps and breaks that might otherwise provide ends for single-strand annealing. These early pathways try repeatedly but unsuccessfully to convert initial substrates into resolvable Holliday structures. Ultimately, these attempts may lead to loss of rrn sequences from the processed ends and thereby abort attempts at duplication. When RecA or the proteins that load RecA onto single strands (RecB or RecF) are eliminated, gaps and breaks remain unrepaired and can provide single-strand ends for use in annealing. These strands will not be coated with RecA protein and thus may be particularly active in annealing. In addition, these early blocks cause unrepaired gaps and breaks to accumulate, increasing the probability of non-allelic rrn loci in different sister chromosomes having simultaneous gaps or breaks. These simultaneous lesions may be essential to duplication formation by annealing as described below. The early blocks in recombination could also reduce any toxic effects of accumulated Holliday structures (see below).
The model described above is diagrammed in Figure 9 as it applies to duplication initiated by gaps. The gap may be generated by digesting one end of a nick or could be formed in one step following cutting of a hairpin structure in the nontranscribed strand of the rrn sequence (Figure 9, left). The RecFORA pathway starts repair of this gap using a sequence from a different rrn locus and generates Holliday structures that must a resolved by RuvC and RecG to complete recombinational duplication. When a recF mutation blocks early steps in gap repair, the gaps persist and accumulate such that two participating rrn loci can have single-strand ends with rrn sequences that can anneal (Figure 9, right). The annealing pathway activated by loss of gap repair (the recF mutation) replaces the lost recombinational duplication pathway and explains why recF mutants showed no duplication defect (Figure 5). This activated pathway also suppresses the defect seen in a ruvC recG double mutant, since the new pathway does not involve Holliday structures. This model does not explain why single recF or recB mutations did not show some partial loss of duplication ability or partial suppression of ruvC, recG. To approach this, we looked for reasons that one kind of damage (perhaps double-strand breaks) might be prevented from making duplications.
All seven rrn loci are devoid of Chi sites
One aspect of the results in Figure 8 does not fit with the model as described above. It seemed reasonable that blockage of both RecFORA and RecBCDA pathways might be required to fully suppress the RuvC RecG defect. In fact, duplication rates were fully restored to the ruvC recG mutant by either a recB or a recF mutation individually. This could indicate that RecB and RecF act together to generate the Holliday structure. However, an alternative explanation was suggested by examining the sequences of rrn loci.
Chi sites are required for RecBCD-mediated homologous recombination. Repair of a double-strand break by RecBCD involves degrading double-stranded DNA until a Chi sequence is reached (reviewed by Kreuzer 2005 and by Myers and Stahl 1994). At this point, only the 5′ end is further resected, leaving a 3′ overhang ending at the point Chi was encountered. A break within or immediately origin-distal to the rrn sequence could generate an exchange between non-allelic rrn sequences only if Chi sites were present within the rrn locus. Figure 10 shows the positions of all Chi sequences in the 75-kb regions surrounding each the seven rrn loci of S. enterica. It is apparent that Chi sites in the useful orientation are absent from all seven rrn loci and from all of their immediate origin-proximal 5-kb regions. Our calculations suggest that, given the frequency of Chi within the seven pictured regions (each 75 kb), the random chance of all rrn loci lacking Chi is ∼0.5% and the probability of having no Chi sites in either the rrn or the origin-proximal 5-kb regions is 3 × 10−5.
This Chi distribution suggests that, whenever RecBCD acts on a break within rrn sequences, it digests rrn material and produces a 3′ end that can initiate an exchange only outside of rrn. In the absence of Chi sequences, RecBCD can still repair DNA breaks occurring within the rrn region but does so by exchanges outside of the rrn sequence. With no Chi sites within rrn loci, double-strand breaks cannot initiate RecBCD-mediated duplications or deletions, which require a recombination exchange between the rrn sequences of two different rrn loci. Data in Figure 6 (above) show that all of the duplications analyzed did arise by exchanges within the rrn sequence. This suggests that RecBCD activity at double-strand breaks could not be responsible for duplications arising in wild-type strains. This explains why a single recB mutation did not reduce the normal duplication rate—RecBCD does not contribute to duplication. Any duplications made by recombination must reflect the activity of RecFORA at gaps. However, the ability of a recB mutation to fully suppress the ruvC recG mutations must be explained.
The processing of double-strand breaks within rrn is diagrammed in Figure 11. In a normal strain (top left) the break can be repaired by RecBCD, but the process removes the rrn sequence adjacent to the break and leads to an exchange point outside of rrn, allowing repair of the break, but no contribution to duplication. In the absence of RecBCD, breaks accumulate and different rrn loci can provide ends for single-strand annealing. In a simple recB mutant, this additional annealing makes only a small contribution to duplication rate above the background of recombinational duplication by gap repair. However, when late steps in recombination are blocked in a ruvC recG double mutant, the recB mutation makes a large contribution to duplication rate by allowing double-strand breaks to provide ends that initiate annealing. That is, the recB mutation opens a new annealing pathway that uses double-strand breaks within rrn as initiating structures. Such structures could not contribute in any RecBCD-proficient situation (wild type, recF, or recA) and make a small contribution in any background that already has an active pathway for duplication gap repair by either recombination or annealing.
Thus, there may be three ways of making an rrn duplication. 1) Gaps can initiate recombinational duplication (using RecFORA) in wild type. 2) Gaps can initiate duplication by annealing in recF or recA cells. 3) Double-strand breaks can initiate annealing only in recB mutants. By allowing use of a new initiating structure, a recB mutation might be expected to increase the duplication rate over that seen either in wild type or in recA or recF mutants. This makes a prediction that is tested below with mixed results.
Duplication rate is stimulated slightly by recB mutation
In the process of duplication suggested above, purHD duplications arise in rec+ strains predominantly by homologous recombination (RecFOR acting at gaps) and form exclusively by single-strand annealing in recA or recF mutants. Double-strand breaks within rrn cannot initiate duplications by recombination (using RecBCDA), but do initiate annealing in recB mutants because rrn sequences are left at the unresected break. The increased annealing allowed by a recB mutation under some circumstances should increase the rate of duplication formation. In a strain that has functional RecFORA, the effect of the recB mutation would likely be small, since it would enhance an already high rate of duplications due to RecFORA acting at gaps. Similarly, the enhancement might be small in a recF mutant because gaps provide a good source of duplication by annealing. The magnitude of duplication enhancement provided by a recB mutation would depend on the relative frequency of gaps and breaks. We have looked for evidence of this enhancement.
The recB mutation shows no significant enhancement in Figure 5, in wild type (above a background of gap-stimulated recombination), or in a recF strain (above a background of gap-initiated annealing). The increase in rate caused by recB in a recF, recA background does not appear significant (Figure 12). However, a slight effect was seen in a recA mutant background (Figure 12). This difference is on the border of significance, given the variability of these measurements, but the duplication rate seen in the recA, recB double mutant is the highest that we have measured in any strain. This rate may reflect annealing initiated by both gaps and breaks.
Effects of mismatch repair on duplication formation
The seven rrn loci of Salmonella differ at many points in their sequence (see Figure 6 and Appendix). Since duplications form by exchanges between different rrn loci, it seemed possible that recombination or even annealing might be subject to rejection of duplexes by the methyl-directed mismatch repair system (MMR) (Petit et al. 1991; Friedberg et al. 2005). This idea is supported by the observation (Figure 13) that a mutS mutation stimulated duplication in the recA+ background. No effect of mutS was seen in the recA mutant background, so we suggest that rrn heteroduplexes formed by single-strand annealing are not subject to MMR rejection. This result differs from earlier findings using shorter and more closely positioned repeats, for which MMR played a big role in annealing (Bzymek and Lovett 2001).
Testing the role of other recombination/repair functions in duplication
If duplications form efficiently by annealing in recA mutant strains, then mutations that inhibit annealing might decrease duplication rate in a recA mutant strain and might show no defect in recA+ strains, where duplications form primarily by homologous recombination. A series of mutations defective in various recombination and DNA repair functions were tested for their effect on duplication rates in recA+ and recA− strains (see Figure 13). While these effects are too small be interpreted, several seem interesting.
Most notable is the effect of recO mutations, which remove a central part of the gap repair mechanism (RecFORA). One might expect this mutation to behave as did the recF mutation described above. In a recA+ background, the recO mutation caused a significant drop in duplication rate, suggesting that it might eliminate the gap-initiated recombination that normally forms a duplication in a wild-type cell. The recF mutation (Figure 5) did not cause this drop, arguably because it activated the alternative annealing pathway. This might suggest that recO defects are less able to prevent loading of RecA on single strands and thus less able to activate annealing. In keeping with this idea, the recO mutation had no effect on duplication in a recA mutant, where no RecA protein is available to be loaded.
The sbcCD mutation caused a small reduction in duplication in an otherwise rec+ strain but not in a recA mutant. SbcCD degrades hairpin structures in single-strand DNA (Eykelenboom et al. 2008; Darmon et al. 2010) and might be expected to contribute to creation of gaps in ribosomal DNA (rDNA). Large gaps could be particularly important for recombination, which requires RecA loading, while smaller gaps are sufficient for annealing in a recA mutant where the only ends are needed and RecA loading is not an issue.
The sbcB and xonA mutations (dominant and null alleles of the gene for a 3′ exonuclease ExoI) may cause a small decrease in both pathways. ExoI and the RecJ single-strand 5′ exonuclease could be imagined to contribute by extending single-strand nicks to gaps, but recJ mutations showed little effect on duplication rate. The ligB gene encodes a nonessential homolog of the standard DNA ligase (ligA) (Sriskanda and Shuman 2001) and made no significant contribution to duplication formation.
There was also very little effect of lexANull and lexAInd mutations, which cause the LexA-repressed SOS DNA repair system to be either constitutively expressed or uninducible, respectively. The same result applied to both recA+ and recA strains. This suggests that none of the 40 enzymes controlled by the SOS response is limiting for rrn-mediated duplication by either recombination or annealing (Friedberg et al. 2005).
ruvC recG mutation combination does not affect duplication rate of the distant pyrD locus
The dependencies described above apply to duplications of the purHD locus, which is flanked by rrn loci. In contrast, the pyrD gene is located on the opposite side of the chromosome (see Figure 2) and is not closely flanked by rrn or any other major direct-order sequence repeats. The pyrD duplication rate is the lowest of all Salmonella loci tested at 4 × 10−6/cell/generation, 750-fold lower than the rate for rrn-mediated duplication of purHD (Reams et al. 2010). The pyrD duplication rate is unaffected by a recA mutation (like that of purHD locus). Unlike purHD, the pyrD locus duplicates at a normal rate in ruvC, recG mutants (see Figure 14).
In understanding the different effects of Holliday resolution in these two regions, it should be noted that these loci duplicate at very different rates (10−6 for pyrD and 10−3 for purHD). In addition, the pyrD locus is not flanked by extensive sequence repeats while purHD is closely flanked by the rrnB and rrnE loci. These differences suggest that pyrD normally duplicates by pathways that are independent of RecA and involve no Holliday structures (or RuvC, RecG activity). These duplications must use the very short available homologies, which has been verified by sequencing junctions of pyrD duplications (E. Kofoid, unpublished results). It seems likely that pyrD duplicates (at a low rate) by nonrecombination pathways that use single-strand annealing or the palindrome-processing pathway described previously for tandem inversion duplications (TIDs) (Kugelberg et al. 2006) and discussed below. While the duplication rate of pyrD is very low, this behavior seems likely to be representative of most areas of the chromosome, which have no flanking extensive sequence repeats and no mechanisms to stimulate formation of gaps or breaks. A purHD from which the repeated rrnB and rrnH loci were deleted behaved much like pyrD. Without flanking rrn repeats, the purHD duplication rate falls 100-fold (see Table 2) and is no longer affected by ruvC, recG mutations (A. Reams, unpublished results).
Three unexpected results are described here. First, formation rates of duplications arising between extensive repeated rrn sequences are unaffected by the absence of enzymes essential for homologous recombination (RecA, RecB, and RecF). Second, while strains lacking these early recombination functions form duplications at normal rates, mutants (ruvC recG) blocked for late recombination steps (Holliday structure resolution) show a 30-fold decrease in duplication rates. Third, the reduced duplication rates seen in the absence of RuvC and RecG are corrected by single blocks in earlier steps of homologous recombination (RecA, RecB, or RecF). These observations may reflect shifts in channeling a rich source of gaps and breaks between two alternative pathways of duplication formation–recombination and single-strand annealing.
In many assay systems, duplications mediated by large direct repeats depend heavily on RecA (Romero and Palacios 1997). However, these assays detect duplications by positive selections that demand multiple gene copies and may favor cells with higher amplifications. Since the amplification of a duplication to higher copy numbers is heavily RecA dependent (Kugelberg et al. 2006, 2010; Poteete 2009; Reams et al. 2010), we suspect that the frequently reported RecA dependency of duplication reported in other systems may reflect a need for higher amplification, which can depend on RecA even when the initial duplication event is RecA independent. The assay used here traps duplications without favoring higher copy numbers. In addition, this assay is not affected by the lethal effects of recombination mutations tested. That is, reduced viability of certain mutant strains (e.g., recA, ruvC) does not lower the measured duplication rate since the trapping assay detects the ratio of duplication to nonduplication cells in the viable population.
A different but not mutually exclusive explanation for the RecA independence of rrn-mediated duplications is that the repeated rrn sequences may be subject to frequent formation of gaps and breaks. A precedent for the of role of introduced breaks may be the low recombination dependence of duplications between IS3 elements, which depend on the availability of transposase to make nicks in the two recombining sequences (Reams et al. 2012). Nicks and or breaks in both recombining sequences can allow accumulation of the multiple simultaneous breaks needed for duplication by single-strand annealing and allow duplications to form without recombination. When these lesions are not processed by recombinational repair enzymes, they may be free to initiate an alternative annealing pathway for duplication formation.
The 5.5-kb rrn repeats are heavily transcribed and rich in palindromic sequences. These genes are GC-rich and GC-skewed such that the nontranscribed strand is G-rich and may be subject to formation of R-loops with hairpin structures. In Salmonella, several rrn sequences have extended palindromic intervening sequences (IVS in Figure 6) that extend natural palindromes that are inherent in the ribosomal RNA (rRNA) sequences. These extensions must be cut out of rRNA prior to ribosome assembly (Mattatall and Sanderson 1996, 1998). The distribution of these palindromic sequences among various rrn loci is described in the Appendix. Degradation of hairpin structures in the nontranscribed strand of rDNA would generate gaps in one step. The heavy transcription of rrn regions may temporarily expose single stands, making them available for pairing and susceptible to nucleases. The heavy transcription of rrn loci is likely to require activity of topoisomerases and gyrases, which, we speculate, may cause a significant rate of DNA breakage. These enzymes are able to catalyze illegitimate recombination (Ikeda et al. 1982) and make double-strand breaks in the presence of inhibitors or following collisions with replication forks (Gellert et al. 1977; Pohlhaus and Kreuzer 2005). While the frequent breakage of rrn sequences is speculative, it should be remembered that rrn duplications form at a very high frequency (10−3/cell/generation) and end in specific short regions (5.5 kb), suggesting a need for frequent internal initiating structures.
A model for formation of rrn duplications
We propose that the results described above are consistent with the idea that rrn loci are subject to frequent nicks and breaks that can be processed in alternative ways and form duplications by the several pathways described below.
In wild-type strains, duplications are made primarily by homologous recombination between sister strands using the RecFORA pathway (see Figure 9, left). Single-strand gaps within rrn sequences are loaded with RecA protein and invade a non-allelic rrn sequence of a sister chromosome, thereby generating Holliday structures that can be resolved to leave a duplication.
Elimination of the RecFORA pathway prevents recombinational formation of duplications, but allows unrepaired gaps to accumulate in both participating rrn loci. These gaps provide ends that can participate in an alternative single-strand annealing pathway (see Figure 9, right). The absence of RecA or failure to load RecA onto the gaps leaves single-strand ends free to engage in single-strand annealing. Evidence that RecA inhibits annealing was previously reported in E. coli (Bzymek and Lovett 2001), and the yeast RecA homolog Rad51 also inhibits single-strand annealing (McDonald and Rothstein 1994; Ivanov et al. 1996; Stark et al. 2004).
Double-strand breaks within rrn loci cannot initiate duplications because Chi sites are absent from all rrn loci (see Figure 10). Repair of rrn breaks by RecBCD is expected to occur by exchanges outside of rrn (Figure 11, left). Thus, a recB mutant shows no loss of duplication rate.
In a ruvC recG mutant, recombination is blocked by inability to resolve Holliday structures. (see Figure 9, left). In this mutant, however, the RecFORA enzymes continue to convert gaps into unresolvable Holliday structures. By processing gaps that might have led to annealing, the active RecFORA functions block the alternative annealing route to duplication. Repeated attempts to establish Holliday structures may ultimately lead to loss of rrn sequences from ends and thus prevent rrn rearrangements. The few rrn duplications formed in a ruvC recG mutant form by exchanges within rrn like those thought to form by annealing. The possibility of toxic Holliday structures is discussed below.
When recA or recF mutations are added to a ruvC recG mutant, gaps are allowed to accumulate and open the compensating annealing pathway, just as was seen in strains with only a recA or recF mutation. Thus, in the absence of these early recombination steps, single-strand annealing is stimulated and RuvC and RecG functions become irrelevant since there is no way to make a Holliday structure.
Addition of a recB mutation to a ruvC recG double mutant prevents digestion of rrn sequence from double-strand breaks within rrn and allows double-strand ends to initiate duplication by annealing. Thus, double-strand breaks within rrn, which do not normally contribute to recombinational duplication (due to lack of Chi), can contribute to duplication by annealing in the absence of RecBCD degradation.
A remarkable aspect of the reported results is that a similar rate of duplication was seen regardless of the pathway used. If the activated annealing pathway makes up for a loss in recombination events and leaves the duplication rate largely unchanged, the two pathways must function at the same rate. Similarly, when a recB mutation is added to a ruvC, recG mutant, the final rate is very similar to that seen in wild type, in recA, or in recF. We suggest that the similarity in rates reflects dependence on a limiting resource, the gaps and breaks that initiate the exchange, regardless of the pathway used. That is, gaps form at a limiting rate and are essentially all ultimately repaired by either one pathway or the other. The similarity of rates when double-strand annealing is allowed by a recB mutation may suggest that the rate of forming breaks within rrn is equivalent or a bit lower to than that of forming gaps. It should be kept in mind that the exchange events measured here arise at a very high rate—10−3/cell/division.
Requirements for activation of annealing pathways
This model suggests that normal recombination functions not only contribute to duplication by homologous recombination, but also minimize the likelihood of single-strand annealing by processing structures such as nicks or gaps and coating single strands with RecA protein. This inhibition may minimize rearrangements between long direct repeats that are prone to frequent damage, such as rrn sequences. The model also suggests that annealing can be initiated either by single-strand nicks and gaps or by double-strand breaks. In the case of rrn loci, formation of these lesions may be stimulated by the high transcription rate and abundance of included palindromic sequences. A previous report on E. coli has shown that deletions arising by single-strand annealing are strongly enhanced by intervening palindromes (Bzymek and Lovett 2001).
The ability of recombination pathways to block rearrangement by annealing may also involve coating single strands with RecA protein. Coated single strands (i.e., RecA filaments) are impaired for annealing. Evidence for the stimulation of single-strand annealing by the absence of a functional RecA has been previously reported in E. coli (Bzymek and Lovett 2001). The activation of the annealing pathway may require elimination of RecA or the proteins that load RecA onto a single strand (i.e., RecFOR or RecBCD). This fits with a recF mutant, which can accumulate unrepaired nicks and gaps as outlined above, and also fails to load RecA on single-strand regions. Similarly, a recB mutant fails to remove rrn sequence from ends at a break and also fails to load RecA onto single-strand ends.
While the genes involved in homologous recombination have been studied extensively in E. coli and Salmonella, relatively very little is known about the annealing process. Several proteins have been associated with single-strand annealing in bacteria, including SbcCD (Bzymek and Lovett 2001; Goldfless et al. 2006). Figure 11 shows the effects of removing these functions from a recA mutant strain, in which duplications are believed to arise exclusively by annealing (Figure 13). Of the enzymes tested, only recQ and recJ mutations appeared to reduce duplication by annealing, but their effect was small. No effect was seen for SbcCD, although this nuclease might have been expected to cut palindromic structures in rrn genes and contribute to initiation by both pathways (Connelly et al. 1998). Duplication formation in a wild-type strain, presumed to involve RecA, was stimulated by a mismatch-repair defect (mutS), suggesting rejection of mismatched heteroduplexes, whereas the RecA-independent annealing pathway was unaffected. These results are consistent with the idea that rrn-mediated duplications arise by two alternative pathways, each dependent on different sets of genetic components.
Lethal effects of accumulated Holliday structures
The ruvC recG mutation combination is known to severely reduce recombination rates and lowers cell viability to 20% in an overnight culture of E. coli (Lloyd 1991). Recently, lethality has been attributed to the toxic effects of a failure to resolve Holliday structures in both yeast (Fabre et al. 2002) and Neisseria (Sechman et al. 2006). The general loss of viability in E. coli ruvC recG mutants is thus likely to reflect toxic Holliday structures formed by sister-strand exchanges following occasional spontaneous damage. This general loss of viability is not likely be responsible for the reduced duplication rate described here (Figure 7 and Figure 8) because the measured duplication rate is based on the relative frequency of cells with and without a duplication, which are subject to the same loss of viability. However, it is possible that the 30-fold drop in duplication rate is associated with lethal effects of Holliday structures formed in the course of recombination between rrn loci. That is, ruvC and recG mutations block recombination between rrn sequences and cause accumulation of a lethal intermediate. Such lethality is specific to the production of rrn duplications, but is peripheral to the general model presented here as long as it requires rrn-rrn recombination and duplication would be prevented with or without the lethal effect. The recA, recB, and recF mutations that suppress ruvC, recG not only allow initiating structures to accumulate and activate an annealing pathway, but also prevent formation of toxic Holliday structures. In Neisseria, pilin variation is achieved by recombination between an expression site and silent copies of the pilin gene (Sechman et al. 2006). These exchanges are lethal in strains unable to resolve Holliday structures, and lethality requires both RecA and a mechanism to stimulate the exchange (Cahoon and Seifert 2011). Thus lethality seen in pilin variation and possibly in rrn recombination may reflect high exchange rates in specific restricted sequences.
Similarity to previously reported results for bacterial conjugation
A pattern of genetic dependencies similar to those reported here was seen previously for plasmid conjugation in E. coli (Benson et al. 1991). The conjugative F′lac+ plasmid is transferred between cells as a single strand, whose recombination with the chromosome is low. Acquisition of the plasmid is reduced in ruv mutants and this defect is corrected by an added recA mutation. These results suggest that plasmid circularization normally occurs by recombination requiring Holliday structure resolution, but removal of recA activates an alternative RecA-independent pathway that may involve annealing.
Comparing these results to previous studies of rDNA recombination in yeast
In budding yeast, rrn loci are present as a tandem array of >100 identical copies of a 9-kb repeat, in which copy number is subject to frequent change (Linskens and Huberman 1988). Duplication of an rDNA sequence has been attributed to gene conversion rather than unequal recombination because addition of a copy is seldom accompanied by a corresponding deletion. That is, the exchange that generates a duplication is usually nonreciprocal (Gangloff et al. 1996). While standard conversion events would be expected to require canonical recombination functions, the rate of rDNA copy-number increase is not reduced by lack of Rad51, Rad52, or Rad59 (Houseley and Tollervey 2011). Although the process of yeast rDNA copy-number control is complex (Fierro 1999; Pâques and Haber 1999), it may resemble the process described here in that copy-number changes are caused by homologous recombination, but can also arise by alternative annealing pathways that are activated when recombination is prevented. In addition, the copy-number changes in yeast rDNA copies may be more similar to the second step of amplification (e.g., increasing from a tandem duplication to three tandem copies). In bacteria, multiple, closely positioned substantial repeats interact to cause copy-number changes by a process that is highly RecA dependent. In contrast, the de novo bacterial rrn duplications described here involve interactions between two separate imperfect repeats that are highly transcribed and have the potential to form secondary structures.
Alternatives to duplication by the single-strand-annealing pathway
Single-strand annealing is proposed as the RecA-independent alternative pathway for duplication formation. Annealing avoids the need for strand invasion, but requires simultaneous breaks or nicks to generate single strands from different rrn sequences. A completely different RecA-independent duplication pathway has been proposed to explain the origin of short duplications that amplify during growth under selection in the Cairns system (Cairns and Foster 1991; Roth et al. 2006). In this pathway, a quasi-palindromic sequence initiates repair replication from a snap-back structure that eventually switches template strands, perhaps stimulated by a second palindrome, to make three copies of the intervening region (Kugelberg et al. 2010). The resulting triplication has direct-order copies flanking a central inverse order copy—a symmetrical tandem inversion duplication (sTID). The two internal quasi-palindromic sTID junctions are prone to deletions because of their propensity to form secondary hairpin structures. Such structures, singly or together, can be deleted to leave a TID with asymmetric junctions or a simple direct repeat duplication. The first step of this pathway has been clearly demonstrated by Leach and coworkers (Darmon et al. 2010), and palindromes have been shown to stimulate deletion formation in several systems (Sinden et al. 1991; Bzymek and Lovett 2001).
Palindrome processing by the TID pathway is attractive for rrn duplications because palindromes within the locus could be used for both initiation and strand switching. However, the large duplicated regions, such as rrnB-rrnH (790 kb), would necessitate repair synthesis of extensive chromosomal regions. Observed TID amplifications are generally shorter (10–30 kb) (Araya et al. 2010; Kugelberg et al. 2010). Palindrome processing also does not easily explain the reduced duplication rate seen in a ruvC recG double mutant. While the TID mechanism remains a formally possible way to form rrn duplications, we suggest that this pathway is more likely to apply to the RecA-independent duplications that arise at low rates in regions without long direct-order flanking repeats.
We thank Sophie Maisnier-Patin, John Paul Aboubechara, Semarhy Quinones-Soto, Douglas Huseby, Wolf Heyer and Steven Kowalczykowski for helpful suggestions. This work was supported by National Institutes of Health grant GM27068.
Appendix: Sequence Differences Between rrn Loci in Salmonella enterica
The figure below describes the sequence variation between the seven rrn loci of S. enterica. The central cluster of most similar rrn genes (rrnA,- B,-C, -D, -E) contains those located nearest the replication origin of the Salmonella chromosome (see Figure 2). All of the loci in this cluster have an intervening sequence in helix 25 of their 23S RNA gene that is removed from the messenger RNA prior to ribosome assembly (Mattatall and Sanderson 1996, 1998). The most dissimilar loci (rrnH and rrnG) show ∼1.5% sequence difference. The rrnG locus has an intervening sequence only in helix 45 of its 23S RNA gene. The rrnH gene has an intervening sequence in both helix 25 and helix 45.
Communicating editor: S. Sandler
- Received August 23, 2013.
- Accepted October 17, 2013.
- Copyright © 2014 by the Genetics Society of America


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Long Story Short
The latest Apple rumours confirm some long-held suspicions and add a new wrinkle: an updated iPhone X, a lower-priced model with similar features and a redesign of the "Plus" line that could be the largest phone Apple's ever produced.
Long Story
The introduction of the iPhone X last fall should have been a watershed moment for Apple (it was the iPhone's 10th anniversary, after all), but was more like a wet fart: while no one would accuse them of doing poorly, new phone sales still failed to reach projections in Q4 2017.
While the company is far from struggling, the lagging sales seemed to confirm what anecdotes suggested: the iPhone X was nice, but not nice enough to justify the the price tag. The iPhone 8, on the other hand, didn't offer enough upgrades to justify buying it at all.
But if there's one thing that's true of being the richest company on Earth, it's that you can't rest on your laurels when your bread isn't getting buttered. To that end, a recent Bloomberg report suggests that Apple will come out with guns blazing this fall, offering three completely new phone models.
We've heard these rumours before. Repeatedly, in fact -- 2018 will bring an upgraded iPhone X, a lower-priced model and... something else. The only difference now is that, if the Bloomberg report is to be believed, we have an idea of what that "something else" is: a gargantuan friggin' iPhone with a 6.5" screen that even Apple will be forced to refer to as a "phablet."
While a 6.5" screen would make it one of the biggest smartphones on the market (let alone ever produced by Apple) the overall handset size will be kept in check with the same tech that gave the iPhone X a large screen in a small package -- an edge-to-edge screen with no home button. The final product is said to be about the same size as the current iPhone 8 Plus, and it's also rumored to have two SIM cards. That's a big plus for people who travel a lot.
That's good news for people who need that kind of functionality, but there's still hope for people who don't feel the need to walk around with a small TV in their pocket. The iPhone X will get some internal upgrades, though not much else. There's no word on pricing for any of the 2018 models, at least not yet.
More promising is the rumoured introduction of a cheaper phone. The problem with the fall 2017 launch was that people were given two options: a sleek, all-new phone that many couldn't afford, or a very un-sleek phone that was materially the same as the one they already owned, but still cost more money. It was not an ideal choice, hence the flagging sales.
But according to this report, the third iPhone will retain the features people like about the iPhone X (edge-to-edge screen, FaceID) while dispensing with some of the more expensive details (OLED screen, stainless steel frame). This model is instead rumored to opt for a traditional LCD screen and the aluminum frame found on other iPhones.
Again, no pricing information is available yet, but if this newer, cheaper iPhone is actually affordable, I smell a hit.

subset: commoncrawl


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Workflow Automation
We apply today's technologies to re-engineer clients' business processes, especially those linking together separate existing systems.
Project Management
We transfer our project management experiences to you via workshops and hand-on execution.
Employee Administration
In today's sophisticated and demanding world of employee administration, planned employee management strategy has evolved into a complex life form all its own. Ageis Technologies designs, implements, and integrates employee rolex replica management solutions for your organization using an approach that emphasizes delivering the accurate content, to the right people, at the right time, with the correct methodology.
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Enable B2B and B2C by using appropriate web technologies.


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Maltese Natural windows
Malta's coast line is quite the exiting one, first of all it is surrounded by watch towers dating back to the war where they used to signal incoming enemy ships. Then there are the white sandy beaches attracting thousands of tourists every year.
But what catches my eye is the rugged coast line separating the sandy beaches filled with salt pans, and natural rock formations such as arches. One can find many naturally occurring arches and they come in all shapes and sizes and can be found in all 3 major Maltese islands. They range from the famous ones such as blue grotto to ones which are less known such as the small (but still impressive) one found at tal- hamrija at Qrendi or the one found at lahrax tal Mellieha known as Marks and dukes window which is pretty large. These structures are so abundant thanks to the everlasting natural forces such as sea movements and wind continuously eroding the soft rock the Maltese islands are mostly made off but sadly the same forces that creates them will also one day break them.
Below are some of the arches.Read More


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The Variable Body in History published
The essays in this book explore the different ways the body has been experienced and interpreted in history, from the medieval to the modern period. Challenging the negative perceptions that the term 'disability' suggests, the essays together present a mosaic of literary representations of bodies and accounts of real lives lived in their particularity and peculiarity. The book does not attempt to be exhaustive, but rather it celebrates the fact that it is not. By presenting a group of individual cases from different periods in history, the collection demonstrates that any overarching way of describing bodies, or unifying description of the experience of the myriad ways of being in a body, is reductive and unhelpful. The variability of each body in its context is our subject.
Available for purchase here.
Chris Mounsey worked for several years in theatre before an accident and four months' immobility, in which reading was the only possible occupation, led to an academic career. Degrees in philosophy, comparative literature and English from the University of Warwick followed, and a doctorate on Blake founded an interest in the literature of the eighteenth century. Professor Mounsey, who now teaches at the University of Winchester, is author of Christopher Smart: Clown of God and Being the Body of Christ and editor of Presenting Gender, Queer People, Developments in the Histories of Sexualities and The Idea of Disability in the Eighteenth Century.
Stan Booth is a PhD student at the University of Winchester. He worked for many years as a local government manager advising members of the community of a disadvantaged area of London. Seeing the credit crunch coming and the obvious impact it would have, he returned to learning, undertaking an MSc in Health and Disease and from there re-entered academia. His doctoral project explores the discourse of physical paralysis in the eighteenth century with a cultural studies emphasis that allows scope to explore individuals and to go beyond the notion of the typical and the predicted models.


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10 things to do in Singapore for tourists
Whether you're staying for a day or a week, no trip to Singapore is complete without doing these ten things
Get sky high
See the whole of Singapore from the Marina Bay Sands SkyPark. At 200 metres above ground, the boat-shaped SkyPark took three months to lift up to the 57th storey where it now sits prettily atop three hotel towers and is home to the famous infinity rooftop pool and chi-chi club Ce La Vi.
Walk the talk
Put on your walking shoes and join an Original Singapore Walk by Journeys, as guides talk you through Singapore's lesser-known history. From atmospheric night walks through the old red-light district of Chinatown to fascinating war trails, expert storytellers shine a light into the nooks and crannies of the city even locals don't know about, bringing its fascinating history and culture to life (6325 1631, www.journeys.com.sg/singaporewalks).
Find a unique treasure in Haji Lane
Budget bohemia thrives in Haji Lane, a sliver of an alley sandwiched between Arab Street and Bali Lane. Mini-boutiques mix with Middle Eastern cafes and design houses. Pick up some monochrome accessories at Salad (27 Haji Ln, 6299 5808) and for kitsch appeal, don't miss Pluck. For an even more exciting fashion experience you'll need to leave ground level and make your way to the second floor of the shop houses. Soon Lee is one such must-visit shop offering an eclectic range of books, one-of-a-kind clothes, jewellery, hats and accessories (73 Haji Ln, 6297 0198).
Feel the beat of Clarke and Boat Quay
Down by the riverside of the Colonial District is where much of Singapore have their fun, with a number of the most popular bars, pubs and nightclubs perched on the edge of the river along Clarke and Boat Quay. Discover a little Latin flavour at Cuba Libre or rub shoulders with the beautiful people at Attica. If your dance moves leave a little to be desired, you can always get your thrills beyond the bars. Try the G-MAX Reverse Bungee or GX5 Extreme Swing instead – no rhythm required (3E River Valley Road, 6338 1146).
Give your feet a fish pedicure
Don't inflict your stinky feet on some poor pedicurist. Instead, let the fish take care of them. A shoal of industrious little Turkish fish wait in a pool to nibble away dead skin and leave your toes fresh and exfoliated. Best avoided by those with ticklish feet! Fish reflexology is $35 for 20 minutes in the pool and 20 minutes foot reflexology. Kenko Reflexology and Fish Spa (313 Orchard Rd, 6887 0303; www.kenko.com.sg).
Go wild at the Night Safari
Through the use of special lighting, the night safari allows you to observe what 900 nocturnal animals get up to after dusk. The 45-minute narrated tram ride is the best way to see the animals from the safety of your seat. After the trip, brave the dark walking trails to get a closer look at smaller animals such as flying squirrels and dangling bats.
Join Singapore's culture club
Catch a performance by international touring companies like the Royal Shakespeare Company or local talents like the Singapore Symphony Orchestra at the iconic landmark and world-class artistic venue, The Esplanade.
See how the locals live
Venture beyond the hub of the city to visit the heartlands and suburban neighbourhoods where locals reside, shop and socialise. Toa Payoh features a bustling local mall as well as the grand Buddhist temple, Lian Shan Shuang Lin monastery. Katong and Joo Chiat are also worth a visit for their melting pot of cultures – from Malay to Indian to Chinese to Peranakan – making them brilliant areas to visit for sampling the local food stalls alone.
Buy lunch with your spare change…
Start with a dirt-cheap yet delicious lunch from one of Singapore's many hawker centres. Think delicious roast pork or fluffy pancakes at Tiong Bahru Market for around $3 (no kidding) or fabulous seafood at Lau Pa Sat. Or if you are in a shopping mall, check out the food courts such as Vivocity's Food Republic.
…then blow the budget for dinner
Splurge on a lavish and memorable dinner at Tetsuya's Waku Ghin, which offers multi-faceted dining at its quirkiest. Take in the opulent surroundings as you work your way through the incredible 10-course degustation menu, which starts at $325 (The Shoppes at Marina Bay 2/L; 6688 8507).


text: For years I have been staring at my vintage wooden drying racks trying to figure the best least invasive method to repair loose dowels. When the wood ages the pins shrink in the sockets and won't stay put. However because it's a hinge, they can't be repaired with glues or nails etc… I have previously more »


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Plus Size Brides at Ivory & Beau
Here at Ivory & Beau, we believe that no matter what body type a bride is, she deserves to find her perfect gown. That's precisely why we've updated our inventory to include a selection of plus size wedding dresses by Maggie Sottero and Rebecca Ingram.
Lots of plus sized brides believe that designers won't appeal to the plus size bride looking for a little personality in her dress, but these gowns are anything but basic, with styles ranging from classic and romantic to sleek and sexy. Take a peek below to see what we're offering in the boutique!
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These gowns serve as the perfect base for the bride looking to add a pinch of personality to her gown. Sweet and simple Rosamund (left) is a great choice for the modern bride, featuring a bold lace print that would pair perfectly with a blingy belt of your choice. Speaking of bling, don't forget about Hannah (center)! This ball gown is best for brides who would like to sparkle from head to toe. Rylie (right) also features a classic A-line silhouette, offset by a lace hemline, tulle skirt, and sweetheart neckline.
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Written by Paige Grieninger


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14 Sunday Apr 2013
Posted Baby, Motherhood, Photographin
14 Sunday Apr 2013
Posted Baby, Motherhood, Photographin
20 Wednesday Mar 2013
Posted Baby, Family, Motherhood, parent, Son, Uncategorizedin
I realized while taking William's eight month photographs that we really hadn't spent any time on the front porch or in the yard since he was a tiny baby. Those times he was usually asleep. That seems a distant memory now.
We started on the porch swing which led to fast disaster when he spit up everywhere. Brandon graciously offered to photoshop out the evidence on William's outfit…I thought for the sake of these pictures we would "keep it real."
Post disaster, we moved to the top of the stairs which quickly led to William wanting to eat the plants, lean over the stairs and otherwise make me a nervous wreck. So, we landed in the front yard. All I had to do was keep him from eating the mulch. It was quite a testament to how different things are once the baby becomes mobile. We shall never rest again.
19 Tuesday Feb 2013
Posted Uncategorizedin
14 Thursday Feb 2013
Posted Uncategorizedin
Baby, Chihuly, photography, son, VMFA
Last week we had the pleasure of attending the Chihuly exhibit at the VMFA. On Friday, William and I went with our friend Susan. She graciously spent the afternoon with us – offering me much needed support in my current path toward figuring out what is next. On Sunday, we went as a family with my parents and younger sister. The first time William and I went he was more interested in the people than the actual exhibit. The colors didn't attract his attention like I had anticipated. On Sunday, however, he was captivated by one part of the exhibit. You walked through a hallway and over 1,000 pieces of glass were displayed through a glass wall above you. It was breathtakingly beautiful. Looking at the display above, instead of in front, made the colors and designs look even more amazing.
There was something really emotional for me in sharing that moment with William. I was very attracted to the exhibit, but seeing William truly experience all of those colors and shapes made it that much more intense for me.
13 Wednesday Feb 2013
Posted Uncategorizedin
Baby, family, photography, son
It's been really hard to keep up with the blog lately. One might think with the abundance of time I now have (jobless) it would be easy. Instead, I find myself wrapped up in spending every minute I get with William, or perusing job sites.
I've never been one to participate in the act of giving something up for Lent. It just doesn't work for me because I know I would not be doing it with the right intention. Instead, I will participate in the act of taking a few things on. Living intentionally. One of those items is to write here daily. Our family will not always be doing something interesting, and William will not make new strides every day (though sometimes it really seems like babies do). So, these entries may not be all that interesting but they will be my full effort of tracking our family's life during this precious time. Because each day really is precious. That's one thing I do start to understand as I get older.
In the words of one of my most favorite poets, Mary Oliver, "Tell me, what is it you plan to do with your one wild and precious life?"
That's the question on my heart these days. I consider myself lucky that while I ponder that question, I also get to experience the joys of being William's mother.
21 Wednesday Nov 2012
Posted Baby, Photographin
William had his four-month check-up this week. He weighed in at 13 lbs 8 ounces and 25 inches long. He is in the 15% for weight and 40% for height. So many people, including his daycare teachers, comment on what a big baby he is for his age. I can only assume it's because he is so alert. That and those big blue eyes.
We've now officially reached the point in time where we can take his monthly pictures without a binky and without any fuss. This makes his camera toting mother and father most happy.
19 Friday Oct 2012
Posted Baby, Photograph, Sonin
This series of three month pictures cracks me up. We waited until later in the evening to take the pictures. This meant that William was torn between falling asleep and fussing. Brandon was balancing himself between the couch and coffee table. I was standing behind him talking in goofy high-pitched voices and shaking a rattle. The things you do to get a good picture of your baby…..


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During a weekend trip to Chiang Mai we visited the Rajanagarindra Institute of Child Development.
This center provides care for children from all parts of Thailand
with developmental disabilities and behavioral problems. Some treatments at
RICD include physical, occupational, and music therapy. However, the "music therapist"
at this institute is not trained in music therapy. She went to school for music
education and implements music activities at RICD with the children. In
America, we stress advocacy of music therapy and require board certification. We
often look down upon those who practice music therapy without the proper qualifications.
In Thailand, my views about music therapy had to change to meet the culture's
views about music therapy.
|Rajanagarindra Institute of Child Development
While RICD may not implement what those trained in the United States call music therapy, their openness to the use of music as therapy left me astonished. The institute is in the process of building a new hospital in the shape of a grand piano with a giant violin-playing panda featured in the lobby.
|Musical panda located in the lobby of the new hospital.
I am an active supporter of research-based music therapy. I believe that music can be used in many different ways to reach non-musical therapeutic goals that have been tested in various circumstances. In the American culture this outlook of music therapy is extremely important. Music therapists are constantly advocating for their career as a legitimate healthcare profession. However, roadblocks are created as a result of the society we are a part of. Some people view music therapy as an illegitimate medical practice and collecting research that shows its benefits is important to furthering the progress of music therapy. In Thailand, the culture as a whole is more open to alternative medical practices. Acupuncture and types of ambient music listening are often implemented in medical institutions or hospitals. As a result, the Thai people in the medical field have a desire to execute music therapy but many do not have access to the resources needed to create a comprehensive music therapy program. The differences in the appreciation of music as therapy in correlation to access to the product between the United States and Thailand strikes me as unfortunate.
|New group music therapy room.
Although America and Thailand view music therapy in different ways, I believe that both countries can learn from the philosophies of the other. Where the United States is abundant in standards for music therapy practice, Thailand is abundant in openness to the practice of music therapy. Instead of focusing on the differences I hope that music therapists and non-music therapists alike can broaden their worldview and seek improvements in working with others who may not have the same outlook, in any situation. Viewing a comfortable situation in another light provides insight to untapped wisdom, in music therapy or any other profession.
|Learning how to play a Thai traditional instrument.
What do you think music therapists in America can learn from music therapists in Thailand and vice versa? What types of cultural differences have you experienced within your own profession? Please answer in the comment section below.


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VANCOUVER - The Department of National Defence and Canadian Armed Forces say they take child luring charges against a former B.C. naval cadet program volunteer "very seriously."
Twenty-seven-year-old Victor Overfield of North Vancouver was arrested last December and has been charged with one count of child luring and one count of sexual touching.
Overfield volunteered with the Royal Canadian Sea Cadets in North Vancouver from 2007 to 2012 and with the Navy League Cadets in Vancouver from 2012 until his arrest.
The Royal Canadian Sea Cadets is a national youth program administered by the armed forces and funded by the Department of National Defence.
A spokesman for the national defence department says cadets' safety is the organization's "first priority," and all volunteers are screened every five years, including through a police records check.
Maj. Doug Keirstead says sexual harassment and abuse are not tolerated and any allegations are immediately investigated by authorities and followed up with appropriate action.
Meantime, a spokesman for the Navy League Cadets says Overfield was suspended pending the outcome of the investigation and directed to refrain from contacting cadets.
Andrew Warden says volunteers are screened carefully and all cadets receive training to recognize and report harassment and abuse. He adds a policy is in place to prevent one-on-one contact between volunteers and cadets.
"The Navy League continues to be vigilant and to protect the youth in our program," he says in an emailed statement.
"Whenever any suspect behaviour is noticed, it is immediately reported to the appropriate authorities and every effort is made to safeguard those participating in our program."
Court documents indicate the charges relate to an incident alleged to have occurred on Nov. 1 of last year in Surrey, located southeast of Vancouver.
Overfield has been released from custody on conditions including no contact with his victim and any other young people, RCMP have said.
They said several other unspecified conditions intended to ensure the safety of the public were also in place.
The allegations against Overfield have not been tested in court.


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The developers behind Dead Island originally pushed back Dying Light's release date, saying, "The new date ensures that we can fully realize our vision of an innovative open-world game. We won't need to make compromises or trade-offs on any of the five platforms we're working on. For you, it means an outstanding, original game that makes the wait more than worthwhile."
The first-person zombie survival horror finds the player scavenging for food, supplies, and weapons during the daylight to use after nightfall, when the infected population becomes more aggressive. The game will be available for Xbox 360, Xbox One, PlayStation 3, PlayStation 4, and PC on January 27.
For more on Dying Light, check out impressions from the E3 2014 demonstration.
Rachel is a freelance writer for IGN. You can follow her on Twitter at @haasrachel.


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Union Minister Ram Vilas Paswan has said that the upper castes are the backbone of the ruling BJP and that they should be given 15% reservation.
Union Minister Ram Vilas Paswan has said that the upper castes are the backbone of the ruling BJP and that they should be given 15% reservation. In an interview to Hindustan Times, Paswan who is seen as the senior-most Dalit leader in the Modi government said that the recent protests against the government's decision to reverse the Supreme Court's order on SC/ST Act will have no impact on the vote bank of the BJP. He said that the upper castes are the hardcore supporters of the BJP and that they will never vote for RJD of Lalu Prasad Yadav, Akhilesh Yadav (Samajwadi Party) or Mayawati (BSP).
Paswan said that it is wrong to call the Modi government 'anti-Dalit' and 'anti-backwards'. He said that there was a perception problem but it has changed now. Paswan who is the president of Lok Janshakti Party (LJP), said that the government has done a lot of work to uplift the marginalised sections of the society, adding that PM Narendra Modi gave Dr BR Ambedkar his due.
"Six months ago, we faced so much protest. It had become difficult for ministers to move. The Modi government was called 'anti-Dalit' and 'anti-backward'," he said. "A lot of work was done but there was a perception problem. Today, the perception has changed. I often say that only two leaders – VP Singh and Narendra Modi – gave BR Ambedkar his due."
When asked about the protests by the upper castes, he said that the unrest will die down in a few months from now. He said that "there is no protest and whatever is there, it is temporary".
"The upper castes are hardcore BJP voters, and they will remain loyal to the BJP," he said.
"Do you think they will go to Lalu Prasad, who abuses them on a daily basis? Or will they go with Mayawati or Akhilesh Yadav?" Paswan asked.
To a question whether he feels something needs to be done for upper castes too, he replied: "Why 'something'? They should be given 15% reservation."
On Supreme Court's decision to dilute stringent provisions in the SC/ST Act, Paswan said that had the government brought an ordinance on the first day, "Things would not have turned this bad". The Minister said that the committee headed by Home Minister Rajnath Singh of which he is also a part, decided to bring an ordinance, but bureaucracy has its own speed.
"Whatever happens, happens for the good. Had there been no protests, the people would not have known what Modi has done," he said.
Paswan also attacked the Congress party for attempting to paint a wrong image of the Modi government. He said that today, the perception has not only changed but the same people have started dubbing us as anti-upper-caste.
"The BJP can never have an anti-upper-caste perception. Upper castes are the backbone of the BJP. They are the party's natural allies," Paswan said.
The Central government had last month brought a bill in the Parliament to reverse the Supreme Court's earlier decision that had diluted stringent provisions in the SC/ST Act. The court's February order had triggered a nationwide violent protest by Dalits, leaving at least 10 dead.
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She said she loved getting home before I was done with my shower because that was the only time she got to hear me sing. Said my voice filled the bathroom, overflowed into our bedroom, where she could feel the air vibrating with my sorrow or happiness…
Good Girl, the first in the Quick & Steamy Collection of Lesbian Erotica author Olivia M. Hamilton, offers quick, hot shorts that put readers right in the middle of the action! CAUTION: May be too hot to handle!
Available From:
Amazon US • Amazon CA • Amazon UK • Amazon AU


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Edmonton Golf Course Real Estate
Do you enjoy 18 holes of golf without having to drive long distances? Looking for a home or property near a golf course in or around Edmonton?
Imagine living near a resort-style residential community where you spend your day's off golfing, the gym, out for dinner, and many more activities with your entire family.
There are many choices of golf courses you and your family can enjoy in Edmonton, such as the Victoria Golf course Clubhouse, Riverside Golf Course, Rundle Park Golf Course, Twin Willows Golf, Lewis Estates Golf Course, The Derrick Golf and Winter Club, and many more!
Some of the more popular communities in Edmonton near golf courses are Trumpeter, Windermere, Windsor Park, Riverview, Westbrook Estate, Glenora, Hays Ridge, Magrath Heights, Keswick.
With many beautiful homes in our Edmonton real estate market near golf courses, choosing one can be really exciting! With one of Edmonton's top agents, we can assist you in finding the right home for you and your family!
Displayed below are homes and lots for sale near golf courses.
For more information about any the golf course Real Estate listings, click the "Request More Info" button located on the detail page of each property.


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Back in November 2008, we reviewed the Chevrolet Epica, an enigmatic saloon that was part of the American company's push into Europe.
Back in November 2008, we reviewed the Chevrolet Epica , an enigmatic saloon that was part of the American company's push into Europe. And the large saloon did indeed up the ante for not budget, but cost-effective motoring.
The all-new Chevrolet Cruze is in much the same vein and although it is effectively a replacement for the Lacetti, it has more in common with the Chevrolet Epica. Especially when it comes to looks. This is a generalisation, of course because in detail, the two are quite different.
The front end of the Chevrolet Cruze displays a prominent, body-coloured, bar across the upper part of the front grille, onto which, the large, 'bowtie' emblem barely fits. The bar follows the bonnet line, which is not unusual but, the upper part of the grille eats into the bonnet and that is a little different. Depending on the trim level, the edges of the grille are finished in matt chrome giving a distinctive 'face', which is part of Chevrolet's new 'global design'.
Either side of the bonnet, the top, outer corners of the otherwise rectangular headlight clusters, stretch to a point along the side of the lid, above the wheelarches. When viewed from the side, they seem to extend to the base of the window-line, as they follow the contours of the wing. The integrated front bumper has a flexible lower lip which can cope with minor bumps and manages to miss speed bumps. This area houses the optional fog lights in black recesses that compliment the lower grille.
The Chevrolet Cruze is longer and wider than the Chevrolet Lacetti and it shows in the elegant, planted lines that make this four-door saloon look more like a four-door coupe. The car sits low on wheels that fill the arches perfectly and this element, together with the tight shut-lines, adds to the 'up-market' impression.
The idea that the Chevrolet Cruze has ideas above its station is no bad thing and one that Chevrolet was aiming for, but for me, the rear end goes a long way to spoiling the effect. All is well as we follow along the broad, concave shoulder-line that blends into the rear light clusters. However, these large units are in two parts which extend into the boot lid and each section contains a circular moulding. Chevrolet says that this is a traditional element, but it is somewhat garish. The sculpted rear bumper emphasises the size of the taillights and generally this whole aspect is just too fussy.
Chevrolet Cruze Road Test Data
|Model Reviewed
|Chevrolet Cruze 2.0 VCDi LT
|Body Type
|Super Red
|Performance (manufacturers data)
|0 - 62 mph
|8.7 seconds
|Top Speed
|130 mph
|5-Speed Manual
|Fuel Type
|CO2 Emissions (NEDC Figures)
|Economy (NEDC Figures)
|40.4 mpg
|Extra Urban
|58.9 mpg
|50.4 mpg
|Insurance Group
|Euro NCAP Rating
|3 years / 60000 miles
|Price (when tested on the 09/03/10)


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No matter where you come from, what language you speak, how you dress, how old or young you are or feel, whether you are religious or simply just curious, you are welcome at Holy Trinity. As Jesus called his friends 2,000 years ago, he calls us today, "Come and see."
send us an email
If you have a question or are just curious and want to know about who we are, please click the button below to send us an email. We look forward to meeting you soon.
visit us in person
With four services on Sunday and one on Wednesday, we would invite you to come anytime and meet us. We would be happy to talk with you, pray with you, and we do pray that God blesses you today.


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Justin Bieber fans queued early to get a good spot for their hero performing on the main stage.
Bieber performs a headline slot later today at V Festival at Weston Park after headlining Hylands Park yesterday.
The gates to the arena opening sparked a mad dash towards the main stage to get the best spot on the barrier.
A commendable effort considering Justin Bieber isn't due on stage until 9pm and they will have been there since 11am. Hopefully they've taken plenty of water.
Meanwhile, Rihanna headlined the festival in style on day one of the festival in Weston Park.
There were also performances from the likes of David Guetta and Years & Years in the build-up.
The weather was inconsistent throughout the day with punters likely at risk on sunburn as well as a good soaked.
Watch the Justin Bieber fans rush for the main stage in the video above.


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Coventry is a city David Fox knows very well. It is full of outstanding venues which are favourite haunts of live performers. David recently performed at Butts Park Arena near Coventry city centre and left an indelible impression on all the people who witnessed his extraordinary feats of mind-reading, sleight-of-hand and illusion.
Fantastic wedding venues such as the Ricoh Arena, St. Mary's Guildhall and the Village Hotel in Tile Hill are all ideal for David Fox's unique magical act. He will always endeavour to meet your requirements for your very special day.
The centre of the city of Coventry is full of interesting places to visit and historical landmarks like Coventry Cathedral. Some wedding parties may even decide to get their photographs taken in the dramatic shell of the cathedral whilst a magician like David Fox entertains the remaining guests at the venue of the reception. The cathedral was never restored after the intense bombing campaign conducted by the Luftwaffe during World War Two. It serves as a constant reminder of the incredible struggle the residents of Coventry conducted against Nazi tyranny.
Coventry is also a vibrant node of learning and the University of Warwick and the University of Coventry attract many students to the city. Magic is excellent for student events throughout the year. Whether it is for a sporting event, freshers function or graduation ball, the magic of David Fox will always fit the bill. David regularly seizes the opportunity to entertain young audiences and delights in their reactions to his performance of mystery, magic and wonder.
If you are organising an event in Coventry, David Fox's magic will guarantee success and amazement. Phone this master magician now for free planning and advice.


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WE CAN Leadership Team
- David Wade lives in Friendly Neighborhood and he loves walking to the Friendly Street Market and throwing frisbee at the Friendly City Park. For him the more the neighborhood has coffee shops, tea and cider bars, as well as delicious food options the better. He's convinced Tam's Place has the best chicken wings on earth. When not hanging with his neighbors you can find promoting tiny homes and bicycle trips to wineries.
- Dennis Casady is a life-long Eugene resident. He is an alumi of the University of Oregon, and is a property manager and landlord in the Eugene area. He also serves as Vice-President for the Rental Owners Association of Lane County.
- Dylan Lamar is an architect-developer and founder of Cultivate, a benefit corporation promoting small footprint livability and access to homeownership for all. He has witnessed his generation being priced-out of location-efficient neighborhoods as a result of zoning regulations that compel socio-economic segregation.
- Eliza Kashinsky is a resident of the Jefferson Westside Neighborhood, and a former vice-chair of the Eugene Active Transportation Committee. When not working on WE CAN, she is the HR Manager for a non-profit mental health clinic in Cottage Grove.
- Josh Newman is a resident of the Friendly Neighborhood and a former co-chair of the Friendly Area Neighbors. Josh has taken part in various campaigns advocating for active transportation and transportation choice. He is also an all season bicycle commuter. Professionally, Josh is an Environmental Engineer in local government whose work focuses on improving water quality in our local urban waterways in the Eugene/Springfield metro area and the Upper Willamette River Basin.
- Laurie Hauber is new to Oregon and lives in the Friendly Neighborhood. She enjoys the bike and walk-centric life style, pace of life and proximity to the stunning beauty Oregon has to offer. She has practiced community and economic development law for much of her legal career and recently joined Lane County Legal Aid/Oregon Law Center to focus on affordable housing issues.
- Seth Sadofsky has served on the City of Eugene Active Transportation Committee (formerly BPAC) since 2014 and was briefly on the Board of Southeast Neighbors in 2016. When not advocating for transportation or development issues Seth works in Environmental protection for the State of Oregon. He has live in Southeast Eugene for 10 years.
- Sue Wolling lives in a condominium complex on the edge of downtown, where she enjoys the benefits of "Missing Middle" housing in a location that puts groceries, theaters, restaurants, shopping, entertainment and a huge park within a 10-minute walk or 5 minute bike ride. Sue serves on the Eugene Sustainability Commission, is on the board of the Downtown Neighborhood Association, and advocates for bicycling and walking with numerous groups. She works as an RN in the Intensive Care Unit at Riverbend Hospital, and loves bicycling, hiking, cross-country skiing and backpacking when she's not at work.