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The Kluyveromyces lactis ter1-16T strain contains mutant telomeres that are poorly bound by Rap1, resulting in a telomere-uncapping phenotype and significant elongation of the telomeric DNA. The elongated telomeres of ter1-16T allowed the isolation and examination of native yeast telomeric DNA by electron microscopy. In the telomeric DNA isolated from ter1-16T, looped molecules were observed with the physical characteristics of telomere loops (t-loops) previously described in mammalian and plant cells. ter1-16T cells were also found to contain free circular telomeric DNA molecules (t-circles) ranging up to the size of an entire telomere. When the ter1-16T uncapping phenotype was repressed by overexpression of RAP1 or recombination was inhibited by deletion of rad52, the isolated telomeric DNA contained significantly fewer t-loops and t-circles. These results suggest that disruption of Rap1 results in elevated recombination at telomeres, leading to increased strand invasion of the 3′ overhang within t-loop junctions and resolution of the t-loop junctions into free t-circles.
Telomeres are the nucleoprotein structures at eukaryotic chromosome termini that cap the chromosome ends. They consist of tandem G-rich repeats that terminate in a single-stranded 3′ overhang and proteins that interact with both the single-stranded and double-stranded regions of telomeric DNA. Disruption of telomere capping can lead to one or more outcomes, including telomere fusions, increased telomeric recombination, and replicative senescence (12). In higher eukaryotes, telomeres are commonly arranged into a structure termed a telomere loop (t-loop) that involves strand invasion of the 3′ overhang into internal telomeric repeats of the same chromosome end (14). Such loops have been seen in human, murine, chicken, plant, and ciliate telomeres (5, 14, 24, 27). T-loop formation in vitro is facilitated by human TRF2 protein (1, 14, 29), and inhibition of TRF2 in vivo results in telomere dysfunction and chromosome fusions (3, 35), suggesting that the ability to stabilize t-loops may be an important part of telomeric capping.
Telomeres in the budding yeast Saccharomyces cerevisiae have been proposed to assume a fold-back structure mediated by protein-protein interactions, although the variable telomeric-repeat sequence may or may not allow strand invasion of the 3′ overhang to generate a t-loop (10, 11). The Taz1 protein, from the fission yeast Schizosaccharomyces pombe, is an ortholog TRF2, and in vitro studies have shown that, like TRF2, it has the ability to arrange model telomere substrates into looped structures (32). However, direct examination of native yeast telomere architecture by electron microscopy (EM) to confirm the presence or absence of t-loops in yeast has proven problematic, due to the difficulty of isolating the short telomeres in these species. This is because long telomere restriction fragments (TRFs), which can be separated from the short genomic restriction fragments by gel filtration chromatography, are a technical requirement for observation of native telomeric DNA by EM.
While telomere attrition is most often counterbalanced by telomerase activity, homologous recombination-dependent telomere maintenance mechanisms also exist. A subset of human tumor and immortalized cells use a telomerase-independent mechanism termed alternative lengthening of telomeres (ALT) that is believed to be dependent on recombination (16), and recombinational telomere elongation (RTE) is well characterized in yeast mutants with telomerase deleted (23). Such mutants display gradual telomere shortening, leading to senescence. However, survivors can emerge from senescence if chromosome ends have become elongated by recombination (20, 22). Two types of survivors are characterized in S. cerevisiae: type I has short telomeres and exhibits amplification of the subtelomeric Y′ elements, while type II is similar to human ALT cells, exhibiting elongated, heterogeneous telomeric DNA (7, 20, 30). Survivors in the related budding yeast, Kluyveromyces lactis, are exclusively type II (22).
Current data support a model of type II RTE termed "roll-and-spread" in which extrachromosomal telomeric repeat (ECTR) DNA circles (t-circles), created by telomeric recombination, act as a template for rolling-circle replication, elongating one (or more) telomere (26). The elongated telomere(s) then spreads to other chromosome ends via nonreciprocal recombination events. Support for this model comes mainly from K. lactis, in which exogenous t-circles were shown to promote RTE and a DNA sequence from one elongated telomere was shown to spread to other telomeres following RTE onset (25, 26, 33). Evidence of a roll-and-spread mechanism was also observed in the mitochondria of the yeast species Candida parapsilosis, which contains linear mitochondrial DNA capped by telomeres maintained in a telomerase-independent fashion (28). Consistent with a role in RTE, t-circles are a conserved feature in human ALT cells (4, 36), S. cerevisiae survivors (18, 19), and C. parapsilosis mitochondria (31), and as described below, they likely reflect a t-loop origin. Recent data also indicate that Ku70 deletion in Arabidopsis thaliana results in t-circle formation and the growth of callus cultures with ALT-like telomeric phenotypes (39).
K. lactis provides an excellent opportunity for investigating telomere structure in a yeast species. Unlike S. cerevisiae, the 25-bp K. lactis telomeric repeat contains TA dinucleotides that allow interstrand cross-linking by psoralen (Fig. 1D), a necessary step for maintaining t-loop structure during isolation (14). K. lactis mutants with telomeres long enough to permit isolation from the bulk genomic DNA following restriction enzyme fragmentation are available, either with or without obvious telomere-capping defects (34). The ter1-16T mutant, in particular, is well suited for such studies. Mutation of the telomerase RNA gene in ter1-16T generates telomeric repeats with a base change that alters the binding site for Rap1, a protein crucial to telomere length regulation and capping (Fig. 1D) (34). The ter1-16T mutation leads to long, uncapped telomeres with abnormally long 3′ overhangs, both of which can be suppressed by RAP1 overexpression (34). ter1-16T cells have also been shown to produce small t-circles using homologous recombination, and this can be inhibited by RAD52 deletion (15).
In this study, we have isolated telomeric DNA from the K. lactis ter1-16T strain for direct examination by EM and 2D gel analysis. We show that telomeric DNA from ter1-16T cells contains looped molecules of telomeric DNA with the physical characteristics of t-loops previously isolated from mammalian and plant cells, as well as abundant t-circles ranging up to the size of an entire telomere. In contrast, telomeric DNA from ter1-16T RAP1OE (overexpressing RAP1) and ter1-16T rad52Δ cells contained significantly fewer t-loops and t-circles. Cumulatively, these data suggest that increased recombination at K. lactis telomeres in the absence of Rap1 results in increased strand invasion at t-loop junctions and resolution of t-loops by homologous recombination into free t-circles.
Yeast strains.The telomerase mutant ter1-16T and the double mutant ter1-16T rad52Δ were constructed as described previously in a His-positive revertant of the 7B520 strain (34). The ter1-16T RAP1OE strain was generated by transforming ter1-16T with pCXJ3+RAP1 (a gift from Anat Krauskopf). ter1-16T and ter1-16T rad52Δ strains were transformed with pCXJ3 lacking a transgene. Transformants were selected on media lacking uracil and then grown in liquid yeast extract-peptone-dextrose medium containing 125 μg/ml G418, previously shown to maintain >100 copies of plasmid per cell (37).
Psoralen cross-linking and genomic-DNA isolation and digestion.K. lactis cells were grown to an optical density at 600 nm of 15 (postexponential phase). One liter of cells was spheroplasted, and nuclear isolation was performed as described previously (17) using 100 μg/ml Zymolyase 100T (Seikagaku, Tokyo, Japan). Nuclei were treated with psoralen and UV light, and the genomic DNA was isolated as described previously (5, 14). Prior to restriction digestion, genomic DNA was incubated with Escherichia coli single-stranded DNA binding protein (SSB) to protect single-stranded DNA segments (5, 6) and then was digested for 2 h at 37°C with AluI, HpaII, and NlaIII (New England Biolabs [NEB], Beverly, MA) at 1 unit/μg of DNA each. These enzymes were chosen for their lack of single-stranded nuclease activity (NEB technical support). The mixture was supplemented with an equal amount of enzyme plus 20 μg/ml RNase A for an additional 2 h of incubation. Digestions were checked for completeness on a 1% agarose gel, and if required, more enzyme was added. The digested DNA was purified and prepared for gel filtration chromatography as previously described (5, 14).
Gel filtration chromatography.TRFs were fractionated as described previously (5). The telomeric contents of the eluted fractions were determined by slot blot analysis (5) using a 32P-labeled oligonucleotide K. lactis C-strand telomere probe and quantified using a Storm 840 PhosphorImager and associated software (Molecular Dynamics, Piscataway, NJ).
EM.DNA samples were prepared for EM by surface spreading on a denatured protein film (14). When DNA from the gel filtration column was subsequently digested with RsaI or MseI (NEB), the sample was brought to 0.1 μg/ml SSB in 1× NEB buffer 1, incubated on ice for 30 min, and then treated with 0.5 unit of restriction enzyme for 15 min at 37°C. EDTA was added to 5 mM, and then the mixture was passed through a G50 Sephadex (Pharmacia, New York, NY) spin column and prepared for EM by surface spreading. Staining of single-stranded DNA with SSB was done on ice for 30 min in 20 mM HEPES, pH 7.6, 50 mM NaCl, with 0.2 μg/ml SSB. The mixture was fixed with 0.6% glutaraldehyde for 5 min at room temperature and then directly mounted onto carbon foils for EM (13). All samples were examined in a Philips/FEI Tecnai 12 transmission electron microscope (Eindhoven, The Netherlands), and molecule measurements were done using Digital Micrograph software (Gatan, Pleasanton, CA). Statistical analyses were done using GraphPad software (GraphPad, San Diego, CA), and images were prepared for publication using Adobe Photoshop (Adobe, San Jose, CA).
Agarose gel electrophoresis.DNA isolation and detection of single-stranded G-rich telomeric DNA by in-gel hybridization without denaturation were done as previously described (34). Measurement of TRF length by pulsed-field gel electrophoresis (PFGE) was done using aliquots from non-cross-linked preparations for future EM analysis, and the telomeric material was detected by denaturation and in-gel hybridization as described previously (5). For standard two-dimensional (2D) gel electrophoresis, restriction fragments were separated in the first dimension in 0.6% gold agarose (ISC Bio Express, Kaysville, UT) in 0.5× TBE (44.5 mM Tris base, 44.5 mM boric acid, 1 mM EDTA) at 1 V/cm for 13.5 h. The gel was stained with ethidium bromide and visualized by UV light, and the appropriate lanes were excised. A 1.1% agarose (Invitrogen, Carlsbad, CA) gel in 0.5× TBE containing 300 ng/ml ethidium bromide was poured around the excised slab. Second-dimension electrophoresis was carried out in 0.5× TBE containing 300 ng/ml ethidium bromide at 6 V/cm for 3 h. The telomeric content was determined by in-gel hybridization or Southern blotting, following a denaturation step with a 32P-labeled oligonucleotide K. lactis C- or G-strand telomere probe. Circular-DNA markers were made by diluting HindIII-digested λ DNA to 10 ng/μl and ligating it overnight at 16°C with T4 DNA ligase. High-percentage agarose-chloroquine 2D gel electrophoresis and Southern blotting were done as previously described (15). All hybridizations were visualized using a PhosphorImager.
ter1-16T has a RAD52- independent telomere-capping defect that is suppressed by overexpression of RAP1.In this study, we employed K. lactis strains that possess a mutation in the 16th position within the telomerase RNA subunit (ter1-16T). This mutation confers telomerase-mediated addition of mutant telomeric repeats that are poorly bound by Rap1 onto the chromosome ends. Two of the strains used in this study, ter1-16T and ter1-16T rad52Δ, possess long, uncapped telomeres, with the latter strain not exhibiting telomere recombination and ECTR DNA due to the absence of RAD52 (34). Additionally, we used the RAP1-overexpressing strain ter1-16T RAP1OE, in which the telomere-uncapping phenotype is suppressed due to an abundance of Rap1p (34).
Undigested genomic DNA from ter1-16T, ter1-16T rad52Δ, and ter1-16T RAP1OE cells was isolated, separated by standard gel electrophoresis, and subjected to in-gel hybridization under conditions that detected native single-stranded G-rich telomeric DNA (Fig. 1A). In ter1-16T and ter1-16T rad52Δ, abundant single-stranded G-rich telomeric DNA was present, consistent with previous results (34) and indicative of an uncapped telomere state. We noted that ter1-16T rad52Δ showed a diminished amount of the small single-stranded G-rich material found in ter1-16T (Fig. 1A) (34). In the ter1-16T RAP1OE strain, the single-stranded G-rich telomeric signal was absent, indicating effective suppression of the uncapping phenotype (Fig. 1A).
Telomere length was assayed in all three strains by separation of uncut or AluI/HpaII/NlaIII (A/H/N)-digested genomic DNA using PFGE. A/H/N digestion of K. lactis genomic DNA leaves an average of 370 bp (estimated range, 145 to 450 bp) of subtelomeric DNA on the resulting telomere fragments. Consistent with previous results, ter1-16T contained elevated levels of ECTR DNA, exhibited by a smear of low-molecular-weight telomeric DNA between 5 kb and 0.5 kb in the uncut lanes (Fig. 1B), while the ter1-16T rad52Δ and ter1-16T RAP1OE strains lacked similar signals in the uncut material, indicating a reduction in ECTR DNA material. The ter1-16T and ter1-16T rad52Δ strains exhibited heterogeneous A/H/N-derived TRFs up to greater than 20 kb in length. A/H/N-derived TRFs from ter1-16T RAP1OE were shorter, with the bulk of the signal between 1.75 and 5 kb, which is still significantly greater than the ∼300-bp telomeres in wild-type K. lactis.
Enrichment of TRFs from ter1-16T, ter1-16T rad52Δ, and ter1-16T RAP1OE by gel filtration chromatography.Observation of t-loop structures by EM is dependent upon first psoralen photo-cross-linking the nuclei, which is proposed to establish covalent bonds between the invading 3′ overhang and the duplex telomeric DNA (8, 14). Crude nuclei from ter1-16T, ter1-16T rad52Δ, and ter1-16T RAP1OE were psoralen photo-cross-linked, and the DNA was purified and reduced to TRFs by treatment with A/H/N. At least 0.5 mg of A/H/N-digested DNA from psoralen cross-linked or non-cross-linked samples from each of the strains was chromatographed over Bio-Gel A-15m. Elution profiles comparing the DNA concentrations and telomere contents indicated that for all preparations, the telomeric DNA preceded the bulk genomic DNA, providing the required enrichment for EM analysis (Fig. 1C).
Visualization of TRFs in ter1-16T and related strains.DNA from the high-molecular-weight, telomere-enriched fractions was prepared for EM. We quantified fractions starting at the peak of the telomere signal as determined by blotting and then examined earlier-eluting (higher-molecular-weight) fractions until the DNA concentration within the fractions became too low for EM analysis (Fig. 1C). Higher-numbered (lower-molecular-weight) fractions beyond the telomere peak contained increasing amounts of small DNA molecules, presumably representing the bulk genomic DNA. Tables 1 and 2 summarize the results for each preparation and statistical comparisons between conditions.
The sizes of DNA molecules in the earliest-eluting telomere-enriched fractions in ter1-16T and ter1-16T rad52Δ ranged up to 30 kb, with an average size of approximately 7 kb, while fractions containing the peak telomeric signal had an average DNA molecule size of approximately 4 kb. In the telomere-enriched fractions from ter1-16T RAP1OE, the average DNA length was smaller, 2 to 4 kb, consistent with a decreased telomere length in this strain. As observed in previous studies of t-loops, linear DNA molecules were the most common structural form in the telomere-enriched fractions and are consistent with telomeres that were not arranged into a t-loop, telomeres in which strand invasions were not maintained during the enrichment procedure, or nontelomeric contaminants (4, 5, 14). Given that A/H/N digestion of whole K. lactis DNA is expected to produce only three nontelomeric restriction fragments of greater than 1,500 bp in size (genomic fragments of 1,787 and 1,731 bp and a 1,546-bp mitochondrial fragment), our observation of DNA fragments consistently larger than 2 kb suggests that the vast majority of the DNA molecules we observed were telomeric in nature.
Looped molecules are present in the psoralen cross-linked, telomere-enriched fractions from ter1-16T.Lariat molecules consistent with the t-loop structure, described as a double-stranded DNA circle with a double-stranded DNA tail, were found in the telomere-enriched cross-linked fractions from ter1-16T (Fig. 2 and Table 1). A 3.6-fold-greater abundance in the percentage of looped molecules was observed in the cross-linked ter1-16T samples than in non-cross-linked samples, indicating a significant psoralen contribution to the stabilization of the loop structure (Fig. 3A and Tables 1 and 2). Additionally, when the cross-linked samples from ter1-16T RAP1OE and ter1-16T rad52Δ were compared to the cross-linked fractions from ter1-16T, they had significantly fewer looped molecules, 3.2- and 2.5-fold, respectively (Fig. 3A and Tables 1 and 2).
The sizes of the looped molecules (loop plus tail) in ter1-16T ranged from 0.7 to 27.2 kb (n = 94; mean = 6.1 kb; median = 3.6 kb) (Fig. 3B). Forty-eight percent of looped molecules were less than 3 kb, with the remaining 52% in a distribution ranging from 3 to 30 kb. The latter fraction correlates well with the range of telomere sizes as measured by PFGE, consistent with their being telomeric DNA (compare Fig. 1B and 3B). The smaller loops are consistent with the low-molecular-weight ECTR DNA or possibly the shortest chromosomal telomeres in ter1-16T. A lower abundance of looped molecules was observed in ter1-16T RAP1OE; however, analysis of a large number of total molecules provided enough looped molecules to measure and analyze their size distribution (Fig. 3B). Consistent with the shortened telomeres, the majority of looped molecules in ter1-16T RAP1OE were small, with 96% less than 3 kb (n = 91; range, 0.5 to 20.1 kb; mean = 1.51 kb; median = 1.08 kb). Not enough looped molecules were observed in ter1-16T rad52Δ for accurate size quantification.
Looped molecules from ter1-16T are composed of telomeric DNA and are structurally similar to t-loops from higher eukaryotes.The 25-bp telomeric repeat in K. lactis contains a single RsaI site and no sites for MseI (Fig. 1D, MseI digests 5′-TTAA-3′). Incubation of multiple fractions from ter1-16T cross-linked preparation 2, with MseI, resulted in an increase in the abundance of looped molecules (Fig. 3C). Because these fractions correspond to the peak of the telomere signal in their respective elution profiles, we believe that this reflects the digestion by MseI of contaminating nontelomeric DNA in these late-eluting fractions, effecting a further purification step. In contrast, digestion of the same fractions with RsaI resulted in the complete disappearance of looped molecules and the almost complete reduction of the DNA within these fractions to sizes smaller than the limit of EM detection (Fig. 3C). The resistance of looped molecules from ter1-16T to MseI and their sensitivity to RsaI strongly suggests they are composed of telomeric DNA.
In the t-loop structure, a segment of single-stranded DNA is predicted to be present at the t-loop junction, due to strand invasion of the 3′ overhang to generate the displacement loop (14). Consistent with this, E. coli SSB localized to the loop junction in 35% of t-loops isolated from human and mouse cells (14). We incubated DNA from the ter1-16T telomere-enriched, cross-linked fractions with E. coli SSB and directly mounted the samples onto carbon-coated EM grids. In these experiments, the SSB protein localized to the loop junctions of the majority of the looped molecules, suggestive of strand invasion-mediated telomere looping (Fig, 4A to C). The amounts of the SSB at the loop junction varied between a single SSB particle and large amounts of protein, suggesting considerable variability in the amount of single-stranded DNA present. This variability is likely due to heterogeneity in the elongated 3′ overhangs in ter1-16T, which strand invade to form the displacement loop. Consistent with this, we also observed linear molecules from ter1-16T with variable amounts of SSB on one end (Fig. 4D and E).
Heterogeneous telomere circles in ter1-16T.Genetic data from K. lactis suggests that RTE in ter1Δ survivors is dependent on rolling-circle replication initiated on circular telomeric DNA as small as 100 bp (26). Previous examination of gel-isolated low-molecular-weight ECTR DNA from ter1-16T indicated that in this strain, small single-stranded and double-stranded t-circles were present, and that they ranged upward in size from ∼100 bp/nucleotide (15). In the telomere-enriched, cross-linked fractions from ter1-16T, we observed an abundance of circular DNA molecules (Fig. 5), ranging from 0.5 to 19.3 kb, with 84%, of the circles measuring <3 kb in size (n = 102; mean = 2.1 kb; median = 1.2 kb). DNA circles in psoralen cross-linked preparations from ter1-16T cells were significantly more abundant, 4.7- and 7.3-fold more prevalent on average, than in the psoralen cross-linked preparations from ter1-16T RAP1OE and ter1-16T rad52Δ, respectively (Fig. 3D and Tables 1 and 2). Circular molecules in ter1-16T RAP1OE were also generally small, with 96% measuring <3 kb (n = 83; mean = 1.2 kb; median = 0.8 kb).
To determine if the circular material observed by EM contained telomeric sequences, we separated A/H/N-digested genomic DNA from all three strains by standard 2D gel electrophoresis and probed in gel with telomere-specific probes. TRFs from ter1-16T contained a strong band of telomeric material migrating in an arc consistent with open circular-form DNA, in both the psoralen cross-linked and non-cross-linked samples, suggesting that the DNA circles observed by EM were t-circles (Fig. 6A and data not shown). Circle abundance in ter1-16T as assayed by both EM and 2D gels was enriched by psoralen cross-linking (Fig. 3D, Table 1, and data not shown). This might indicate that some of the circles are not covalently closed but instead associated by homologous base pairing that was stabilized by psoralen cross-linking. Equivalent t-circle arcs were not observed in any of the psoralen cross-linked or non-cross-linked samples from ter1-16T RAP1OE or ter1-16T rad52Δ (Fig. 6B and data not shown). In addition, we observed an arc of telomeric signal derived solely from the G-rich strand, with the bulk of the material migrating between 2 and 0.5 kb, which ran below the arc of linear double-stranded DNA in ter1-16T and ter1-16T rad52Δ, but not ter1-16T RAP1OE (Fig. 6B). This is consistent with material in this arc being single-stranded G-rich ECTR DNA, suggesting that the increased single-stranded G-rich signal associated with telomere uncapping in budding yeast may be derived from both increased overhang length and ECTR DNA.
Because the resolution of open circular forms in standard 2D gels is limited to circles of >2 kb in size, we separated uncut DNA from ter1-16T and ter1-16T RAP1OE on 4% agarose-chloroquine 2D gels to visualize the smaller t-circles. In these experiments, a small amount of ECTR DNA consistent with t-circles was observed in DNA isolated from ter1-16T RAP1OE cells, though an exposure three times longer was required to see a signal compared to DNA isolated from ter1-16T (Fig. 6C). The significant decrease in circles in ter1-16T RAP1OE as assayed by 2D gels is in agreement with the EM results.
Circular molecules were also quantified in the MseI and RsaI digestion experiments detailed above. In these experiments, RsaI digestion of telomere-enriched, cross-linked fractions from ter1-16T resulted in the complete disappearance of circles in one fraction and an 88% reduction in another, consistent with the circles containing telomeric DNA (Fig. 3E). MseI digestion also resulted in some reduction in circles (28% in one fraction and 21% in the other) (Fig. 3E), suggesting that treatment with MseI disrupted some t-circles or that some circles from ter1-16T observed by EM contained nontelomeric sequences.
The observation of RAD52-dependent t-circles in ter1-16T is consistent with a mechanism of circle formation due to homologous recombination resolution of the t-loop junction, resulting in a truncated telomere and a free t-circle (36). We therefore predicted a correlation in size between the loop portions of t-loops and t-circles, as observed previously in the human GM847 ALT cell line (4). In ter1-16T, there is good correlation between the sizes of t-circles and the loop portions of t-loops, with the greatest amount of discrepancy at the smaller size ranges (Fig. 3F). This likely reflects the loss of the smallest circular molecules during the gel filtration step.
In this study, we used EM and gel electrophoresis to probe the structure of yeast telomeric DNA. We observed looped structures in K. lactis ter1-16T, a mutant that develops abnormally elongated telomeres as a result of a defect in the Rap1 binding site within telomeric repeats. Four lines of evidence suggest the looped molecules observed here are composed of telomeric DNA and primarily represent t-loops, structures in which a 3′ telomeric overhang has strand invaded a more internal region of the same telomere. First, the total length of the looped molecules observed by EM in the high-molecular-weight fractions tightly correlates with the telomere lengths in the strain of DNA origin. Second, the looped molecules in ter1-16T are sensitive to RsaI and resistant to MseI, consistent with the K. lactis telomeric DNA sequence. Third, the looped molecules in ter1-16T are significantly more abundant in the psoralen cross-linked fractions, in accordance with previous observations indicating a requirement for psoralen cross-linking to maintain the t-loop structure (14). Fourth, SSB localizes to the loop junctions of looped molecules in ter1-16T, as seen in previous observations in mammalian cells, suggesting the presence of single-stranded DNA at the loop junction due to the presence of a D-loop (14). Two lines of evidence also suggest the circular molecules observed in these studies by EM contain telomeric DNA. First, like the looped molecules, circles in ter1-16T are sensitive to RsaI and resistant to MseI, and second, 2D gel electrophoresis of TRFs derived from ter1-16T revealed an arc of telomeric signal migrating in a pattern consistent with double-stranded DNA circles. We therefore conclude the looped and circular molecules observed here are bona fide t-loops and t-circles.
Several lines of evidence have led to the hypothesis that t-circles form from a recombination reaction involving t-loops. RAD52-dependent telomere rapid deletion (TRD) of overly elongated S. cerevisiae telomeres occurs via intramolecular recombination that is proposed to involve a t-loop intermediate (2, 21). Sudden telomere shortening in mammalian cells following TRF2ΔB expression was dependent upon the recombination proteins XRCC3 and NBS1 and was accompanied by the formation of t-circles (36). t-circle formation in mice following POT1A deletion is also NBS1 dependent (38). In human ALT cells, t-circles have a size distribution similar to that of the loop portions of t-loops, suggesting they are derived from the same source (4), and t-circles were repressed in ALT cells following NBS1 and XRCC3 inhibition via RNA interference (9).
The data presented in this report strongly support the idea that t-circle formation in K. lactis is dependent on both recombination and t-loops. Not only are the frequencies of t-loops and t-circles in ter1-16T cells greatly diminished in the absence of RAD52, but the sizes of t-circles also correlate with the sizes of the loop portions of t-loops. Additionally, the small number of RAD52-independent t-loops and t-circles we observed could also be related to TRD, as a subset of TRD events were previously shown not to require RAD52 (2). One implication of our link between t-loops and t-circles is that both the formation and the resolution of t-loops are likely to be critical steps in any RTE that is driven by t-circles. A second implication is that t-loops occur in any of the budding yeasts or mutants in which t-circles have been observed (18, 19). As the resolution of t-loop junctions by homologous recombination is predicted to generate t-circles that have a nick or gap on both strands, this may explain why psoralen appears to stabilize t-circles in our preparations, as well as the exclusively open circular form of t-circles observed in this study.
When RAP1 is overexpressed in ter1-16T cells, there is a sharp decrease in the incidence of both t-loops and t-circles. This, however, does not indicate that t-loops are solely associated with an uncapped-telomere phenotype in K. lactis, as the very long overhangs present at ter1-16T telomeres are expected to greatly improve the efficiency of psoralen cross-linking within the t-loop junction. Therefore, even if the short overhangs of wild-type and ter1-16T RAP1OE K. lactis cells are sequestered within t-loops, our isolation protocol might be poorly able to cross-link these structures and recover them intact. Thus, while we cannot assign an architecture to wild-type K. lactis telomeres, we can conclude that in ter1-16T RAP1OE the telomeres are structurally arranged in a way that prohibits excessive strand invasion at a t-loop junction, leading to a diminished recovery of t-loops or t-circles. We propose that poor occupancy of Rap1 protein at the mutant telomeric repeats present in ter1-16T cells leads to an altered telomere structure that renders the telomeres more recombinogenic, including a greater amount of strand invasion at the t-loop junction (Fig. 7). Strand invasion of a telomeric end under the recombinogenic state of telomeres lacking Rap1 would be prone to some or all of the processes that might act on a strand-invaded broken DNA end that has formed a D-loop, including branch migration at the t-loop junction, nucleolytic cleavage resulting in TRD and a free t-circle, or extension by a DNA polymerase.
Roger R. Reddel (CMRI, Westmead, Australia) is thanked for the use of his laboratory's apparatus and reagents to complete some of the 2D gels shown here.
This work was supported by grants from the National Institutes of Health to J.D.G. (GM31819 and ES 013773) and to M.J.M. (GM61645). C.G.-V. was supported through a training grant from the National Institutes of Health (5T32GMOO&10330) and S.A.C. though a National Institutes of Heath postdoctoral training grant (GM077900-01).
- Received 24 June 2007.
- Returned for modification 20 July 2007.
- Accepted 10 October 2007.
↵▿ Published ahead of print on 29 October 2007.
- American Society for Microbiology


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Hassan Mneimneh
Hassan Mneimneh is a contributing editor with Fikra Forum and a principal at Middle East Alternatives in Washington. Previously, he participated in several research institutions as a senior fellow and assumed leading functions at the Iraq Memory Foundation, the Iraq Foundation, and the Iraq Research and Documentation Project.
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November 6, 2018

subset: commoncrawl


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These friendships will be there for you and last through times of difficulty. You shall maintain chastity, not committing adultery, nor taking secret lovers. Do not consummate the marriage until their interim is fulfilled. And they maintain their chastity.
Let me break the suspense by saying that I have yet to find that one verse. The real challenge to your faith starts when precious attention shifts away from the needs of your soul to the needs of your body and drains your efforts to increase your remembrance of God. That's a pretty good argument that I've tried to use myself more than once. Let's look at if from another angle.
Indeed, with pain there is gain. All you need to do is place your trust in God, have patience, and stay firm in your commitment to keep your purity of body and mind. Best places to visit in Bellary. Increasing your physical and emotional contact happens naturally since your convinced mind can't hear the weak calls from your soul.
Search and find Abohar girls phone number for friendship, love and chat by. You can start having many meaningful friendships instead of spending most of your time with only one person. Tour Search Online Join us. The ultimate triumph belongs to the righteous. Believe me, how to write a I have spent many a long night trying to figure this one out for myself.
He knows the best time for you to get married and He has already picked out the perfect mate for you. Aaj koi fre hai abohar hanumangarh sai hu jis k pass place ho top or more top mujhe sms kare aj mai. Anyone who rejects faith, all his work will be in vain, and in the Hereafter he will be with the losers. You will experience the beauty of this diversity, bisexual dating online the fun of having such different personalities in your life and the blessing of being able to turn to a strong social network in the time of need.
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For some, there may be a sincere intention to have only a social interaction with a person. This feeling is extremely hard to tolerate and the challenge is difficult, but the outcome is worth every effort. Yes, I can certainly relate that it's innocent and very common, but I can assure you that it's more involved than you imagine.
In Seeking abohar beautiful woman a. If you leave any wiggle room or gray area in your thinking, you will soon find yourself trying to convince your mind that certain aspects of dating are just fine. Avoid one-to-one encounters until you know deep inside that the relationship is clearly friendship.
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Mahindra and Mahindra is headquartered in Mumbai and has two manufacturing facilities, one in Mumbai and the other in Igatpuri, Maharashtra. Your path is rare and requires you to work hard and strive. Read more Mahindra and Mahindra is one of the oldest Indian car manufacturers. The process spins out of control and becomes very hard to control. Speaking of consequences, what does the Quran say about this issue?
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Abohar, Ferozepur, Punjab Abohar now anyone wanna meet? Abohar, Ferozepur, Punjab Hunk. Some we teach at Abohar city photos. Passing your test can as simple as avoiding dating and controlling your physical urges.
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Dating bloomington indiana you have to find someone with dating site for. Do not meet them secretly, unless you have something righteous to discuss. Drop the idea from your mind and analyze the situation as if you are asking God for advice.
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- Even then, it's always better to involve your other friends when you want to do something.
- It seems to me that any way I look at it, I see dating as being a harmful process for the soul no matter how you try to justify your action.
- Mahindra also singles to the lifestyle clench with the Mahindra Stir, the off-road reserve, along with its clean in bond citg racing through Mahindra Seclusion.
You begin to ask yourself why you should have to endure such temptation. These romantic affairs and tearjerkers we see in the movies are made to sell tickets and popcorn, hamilton dating services not our souls. Abohar now anyone wanna meet? God has given you the chance to strive for an everlasting life that radiates with happiness and peace.
Your boat is in perfect condition and is not going anywhere without you. For this reason, following the example of a submitter is a rare and beautiful honor that we should be proud uphold. The Mahindra Bolero utility vehicle is the highest selling Mahindra yet and is followed by its other stalwart, the Mahindra Scorpio.
Typically, the word dating is used when a guy and a girl develop both an intimate physical and social relationship together. The beauty of God's system is that He not only rewards you for your efforts in the Hereafter but also showers you with all sorts of rewards in this life for your commitment. The least important is the fact that you are probably years away from being ready to tie the knot. Then, you're in the right et Someone.
Such thoughts can cloud your judgment and can make the smallest encounter appear as the start of a great love story. Dating agency cyrano ost for friendship - Is the number one destination for. As you're probably excited to find a sex contact and other adult friends, get started today on our get it on site. Meet a woman in Abohar from thousands of singles girls and dating women seeking men Abohar looking for love, friendship, chat and serious relationship. But why should you bring yourself to the point of having to repent when you have the power to remain steadfast from the very beginning?
Previous Article dating site. Abohar is full of single men dual voice coil hook up and women like you looking for dates, lovers, friendship, and fun. God promises a deep and meaningful happiness that is far greater than any temporary romance can ever bring you. These descriptions definitely caught my attention too!
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If you are giving in to your urges and satisfying your physical desires, I suggest you fasten your seat belt before continuing to read some of the verses I come cross in the Quran. Is dating against the Quran? Join online chat rooms without registration and chat with strangers in Abohar chat.
After putting these pieces of the puzzle together, the dangers of dating start becoming much more clear. What is the alternative to dating? She is looking for Friendship, Chat and Workout Partner.
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The physical part is as simple as holding hands and evolves into hugging, kissing and other forms of sexual relations. Based on your reasoning, sexual relations with someone who is rightfully yours is not as bad and makes you feel less guilty about your actions. Keep in mind also that the example you set when you date as a submitter is very damaging since you are supporting something that is against the faith you are practicing everyday. Chastity is defined Quranically in as avoiding sexual relations.


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Can you generate income from the internet to aid you make it through, even flourish, doing so legally and also without tearing people off or by enjoying illegal activities? The response is yes, naturally, or else there would not be much point in creating this short article, would certainly there?.
You can make money legally by supplying info of high value to web searchers inquiring on that particular certain subject.
Know that I am not a computer developer. I do not have comprehensive understanding of html, though given that I have actually been "playing" online I could not help yet find out some. Bad Credit Car Loans Miami I am not a graphic artist but I such as to experiment with paint programs on my computer system. I like electronic photography, and also it is pleasing to use my very own pictures on my very own website must I want to. My software application library does not contain a lot in the means of programs for websites advancement. I could not develop a web site from scratch of my life depended on it. My "know-how" is exclusively pertaining to the areas in which I have gained my living in the years prior to becoming a website author.
I believe that the web will certainly remain to grow which every person in the world that visits for the very first time enhances the capacity for anyone else that wishes to generate an earnings from the internet to be able to do so.
I think that the major players of the web (Google, MSN, Yahoo and so on) will remain to create their web programs to continually remove those that try to make the most of people by means of the internet.
So how? How does one make genuine loan? The typical method; hard work, honesty, effort, simpleness, hard work, and a systematic approach. Oh, and also did I state hard work?.
All kidding apart, the 'job' involved with generating income from the internet involves listing info concerning a subject with which you are already quite familiar. You will be telling people concerning an interest you have. So, it's not hard, yet it does take time. There is no get rich system that works– that, death as well as tax obligations are the only certainties in life that I understand of.
One method to be in company online is by supplying an online "store". Several firms world wide currently provide their products on-line, and this is a genuine method of generating earnings. Providing you have something that desire, and that you are positively located (price/ service/ product mix and so on) you will earn money as folks will certainly get from you. I have no passion in producing an online store, as I'm not interested in having stock, a storage facility, delivery and also getting centers and so on
. There is constantly internet site consulting as an additional internet site revenue resource. From my web wanderings it would seem that there's virtually one specialist for every website online! Much excessive competition for me, though the good consultants can profit, the negative ones will certainly not. Like those that market hard goods, a working as a consultant requires to be well positioned to survive. I do not have the skills to be a reliable expert to people looking for internet expertise, so I do not even try.
You may take into consideration a web site with the sole purpose of offering marketing on it as many of the "fringe" internet site around do now. How many times have you gone to a site to obtain details on a topic only to locate ads, and also web links to extra advertisements? Very aggravating, isn't it? Those that undertake this type of endeavor will certainly find that the major online search engine are doing whatever in their power to ensure that "edge" websites like their own aren't noted with them. If the site isn't provided, no one will discover it, and if nobody can find the website, income will go away. I select not to be entailed with this kind of chicanery.
So what is it that I do to legally generate income by means of the net?
Making use of software application I discovered on the internet I "fill in the spaces" with expertise. I use my expertise of a specific subject and the system transforms that knowledge right into a web site. The software application enables me to create the site in plain English using basic word processing program software. It helps me figure out if there is a market for the info I intend to supply about my topic, and after that it transforms what I write right into a quite neat internet web site, if I do say so myself!
Coming to my site will achieve 2 things for the visitor. As they roam through it they will certainly see what an ill-skilled internet site contractor has actually accomplished using some conveniently available and affordable software application. They will not recognize that I'm ill-skilled nonetheless, since the website looks truly great. Further, as they review the material in my site they will see ads that show up every now and then on those pages. Not many, not invasive, yet advertisements none the less.
The advertisements are placed for me courtesy of Google. Google gave me software that can "review" my website, and position "material" ads on the pages, ads that associate with the basic web content of that particular web page and also to my website in general.
Google pays me when individuals legally click the ads on my pages. And Google does pay me. Consistently.
The more individuals that see, the extra possible for site visitors to click the advertisements on my website, as well as the better capacity for earnings for me, and a lot more significantly, revenues for the advertiser's that trust their ad spending plan to Google.
Individuals visit my website because I write high content worth pages regarding my subject. They find my site by utilizing search words relating to that subject because they have an interest in getting details about those words. I compose pages about the search words people utilize, since my software program informs me which words that they are using to locate information on my subject. No methods, it's an easy as that. Car Loans for Bad Credit Miami If you create web pages regarding points that individuals are looking for, they will locate your pages.
When individuals reach my website, I attempt to ensure that they will certainly discover enormous, useful web content regarding the subject they have an interest in. The web content they locate will entices them to linger and visit various other web pages on my website. As they do this, the advertisements that appear will certainly encourage them to check out those on-line advertisers, as the advertisements describe the goods as well as solutions that they were currently trying to find, or they wouldn't have also discovered my site in the first place.
See exactly how all of it ties together?
Ask on your own, what is your location of expertise? What are you passionate about? What is your work history? What pastime do you have that you already recognize scads of people want? What can you write pages of details on that particular truly and also absolutely offers useful info to the people that read it?
Oh, do not fret about creating your web pages in a purposeful way. The software application reveals you how to do that, too.
If you compose words that provide useful material concerning any kind of subject, and that subject is one that individuals have an interest in (with a world populace in the billions, search for a topic that some people aren't interested in!) and you make use of an easy program to turn your composed words right into a rather neat internet site (helping you stay clear of taking the time and expense to come to be a website programmer yourself) then you can earn money on the internet.
As I have actually said, placing words to paper requires time. Putting those web pages into your site takes time. Research to ensure that you are targetting your page content to words that people are searching for about your topic takes some time. And if you are encumbered a "dial-up" web connection as I am, waiting for data transmission sure takes some time!.
Trying to find a rapid dollar, an easy ride to get great deals of cash from the net? After that please look in other places.
Are you taking into consideration making some loan for straightforward job, possibly thinking about working from house, perhaps dealing with retired life and also searching for something to fill your days, perhaps a stay-at-home Mommy or Dad, or you simply intend to aid others discover your special interest? You can generate income doing so.
Whether those profits are a whole lot or a little relies on the amount of individuals are seeking info on your subject and also the number of web pages of valuable details you provide when they locate your site.
Whatever the amount is, understand that you can make money on the net. That's specific.
Currently, get composing!


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When I search for a better driver, none is ever found. I was worried like you. Doubled by the dual-channel architecture, the widest memory bus bandwidth 8. Brand and product names mentioned are trademarks of their respective companies. On the Asus website it states that this on board sound card is not compatible with Windows 7 and there are no drivers available for Windows 7 as well. Do you already have an account?
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Yes, my password is: How satisfied are you with this reply? All of these are done on one computer. I would recommend you to install the drivers in windows XP SP2 compatibility mode.
You must log in or sign up to reply here. Letting Windows p5ad2-ee to solve the problem then p5da2-e in an attempt to reinstall the device driver, which fails with the message "High Definition Audio Device has a driver problem". P5ad2–e on the box, and downloaded the zip file. Thanks for your feedback, it helps us improve the site. It's worth a thousand words. Specifications and features vary by model, and all images are illustrative.
Search for the a sound driver that will be compatible with windows 7, that will be found in the website of the motherboard brand. Posted on Jan 02, I went to this sight www. Posted by kondesz on Feb 03, Lately during the filling cycle water hammer is occurring.
Asus P5AD2-E audio driver(CMI9880)Windows7 – ASUS P5AD2-E Deluxe Motherboard
Please enter a valid email address. Brand and product names mentioned are trademarks of their respective companies. ArendRensink Created on May 22, Browse to the page below and type the model number of this laptop in the search box on the left pane.
Join the community hereit only takes a minute. I have freestanding Series 8 dishwasher. Applications such as 3D games and video editing demand a huge chunk of system resource. Add your comment to this article You need to be a member to leave a comment. This motherboard provides excellent compatibility and flexibility by supporting either bit or bit architecture.
Hi there, Save hours of searching online or wasting money on unnecessary repairs by talking to a 6YA Expert who can help you resolve this issue over the phone in a minute or two. So now it plays audio, but not as audo as it did in Win XP.
The drivers are actually meant for windows xp but they are working fine for me and im very happy now. Tell us about your experience with our site. The email address entered is already associated to an account.
P5AD2-E | Motherboards | ASUS Global
When I search for a better driver, none is ever found. Then change the compatibility mode of the Setup. Thank You in advance.
You can follow the question or vote as helpful, but you cannot reply to this thread. Join the community here. Shekhar S Replied on May 23, Please refer specification pages for full p5ad2.
Right click on the setup file.


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« Back to What's in Bloomsbury Fashion Business Cases
Cases in Other Areas:
Consumer Markets & Behavior
Design / Product Development
Law / Ethics
Merchandising and Management
Supply Chain
Introductory: Foundation and first year undergraduates
Attracting Millennials to the Luxury Market, Serra John and Lorynn Divita (Public case)
Brand Identity as a Strategy for The Financial Sustainability of New Emerging Fashion Enterprises, Rosita binti Mohd. Tajuddin, Siti Fatimah binti Hashim (Scenario case)
Brand Repositioning Within the Fashion Industry, Jongeun Rhee and Kim K. P. Johnson (Scenario case)
Dealing With Toxic Consumers: New Balance's Dilemma, Myles Ethan Lascity (Public case)
Descente's Multi-brand and Localization Management Strategies, Koji Yoshimoto (Public case)
Diego's Faces Fashion Slump, Nancy J Rabolt and Judy Miler (Scenario case)
International Strategies for Cosmetics Companies: KOLMAR Korea, Janghyun Kim and Ilhyun Bae (Public case)
Middleton's Department Store Loyalty Programme, Nancy J Rabolt and Judy Miler (Scenario case)
Only Bright Lights for Uniqlo?, Myles Ethan Lascity (Public case)
Rsport: Quality Over Quantity for the Plus Sized Market, Addie Martindale (Field case)
Intermediate: Mid to upper level undergraduates
'D&G Go Home Now!' A Case Analysis in Cultural Sensitivities, Local Awareness and Customer Relations for Luxury Brands Operating in Overseas Markets, Anne Peirson-Smith (Public case)
Abercrombie & Fitch's Brand & Store Positioning, Nancy J Rabolt and Judy Miler (Public case)
Adoption of the "See Now Buy Now" Business Model in the Fashion Industry: A Fashion Calendar Revolution, Jennifer Kyungeun Lee (Scenario case)
Anna Laurel Designs, Inc.: Marketing Strategy for the Future, Nancy A. Oliver (Scenario case)
Applying Circular Economy Principles in Luxury Fashion: Petit h, Patsy Perry and Hakan Karaosman (Public case)
Bigger than the Brand: When a Social Media Messenger Overshadows Its Fashion Brand, Camille Kraeplin, Rosanne Hart (Public case)
Brand Identity as a Strategy for The Financial Sustainability of New Emerging Fashion Enterprises, Rosita binti Mohd. Tajuddin, Siti Fatimah binti Hashim (Scenario case)
Branding Strategies for Handsome: Increasing Brand Value, Ho Jung Choo and Hyunok An (Public case)
Building a Lifestyle Brand Through Extensions: Bhumi Organic Cotton, Anjum Khan and Michael B. Beverland (Public case)
Building Online Engagement at Sephora, Jingyi Wang and Michael Beverland (Public case)
Conscious Step: Marketing Socks to Fight Poverty, Leslie Davis Burns (Public case)
Death by Nylon: Managing Communications in a Facebook Brand Community, Tony Cooper (Public case)
Digital Heritage Storytelling at Mulberry, Amanda Grace Sikarskie (Public case)
Di$count Universe: Luxury Brand in a Post-Modern World, Michael Beverland (Public case)
Giorgio Armani and Utilizing the Red Carpet, Thea Macdonald (Public case)
Indochino, The Customer Experience from Online to Brick and Mortar: Shopping of the Future: Customization and Personalization in Menswear, Flora Brunetti (Public case)
Issues in Implementing 3D Technology: Good People Co. Ltd., Kyunghi HONG (Field case)
JCPenney's 'Fair & Square' Pricing Strategy a Bust: Back to the Basics for the Win!, Amy J. Shane-Nichols & Jessica L. Hurst (Public case)
Marketing Ethical, Sustainable, and Fair Trade Brands, Andrea Reyes (Public case)
Max Wild's Sporting Goods, David Loranger (Scenario case)
Patagonia: Creative Sustainability Strategy for a Reluctant Fashion Brand, Rosemary Varley, Natascha Radclyffe-Thomas and William Webb (Public case)
Project September: The New Diversity in the Social Marketing of Cosmetics, Amanda Grace Sikarskie (Public case)
Refinery29 Recharged: Brand Boosting the Digital Mediasphere, Patti Jordan (Public case)
Stella McCartney's Sustainable Luxury: The End of The Kering / Stella McCartney Partnership and New Beginnings on Old Bond Street, Natascha Radclyffe-Thomas and Rosemary Varley (Public case)
The Case of Ancient Greek Sandals: Ethnic Heritage and Celebrity Endorsement, Elena Chatzopoulou (Field case)
The Denim Dilemma at Velvet Sun Boutique, Nancy J Rabolt and Judy Miler (Scenario case)
The Fashion Retail Market in Singapore: Discovering the Challenges of Local Brands Through a Collaborative Work Structure, Michael Charles Daniel Rezandi and Cheong Wei Quan (Field case)
The Impact of the Influencer, Anna Fenlon (Scenario case)
The Lime-Green Colored Glove: Product Design and Development, and Buyer Liaison, Valerie Wilson Trower (Scenario case)
The Role of Heritage in Contemporary Global Luxury Branding, Tim Jackson (Public case)
To Commit or Not, the Retail Market in India, Nancy J Rabolt and Judy Miler (Public case)
Advanced: Postgraduate
Fabindia: Fashion with Social Objectives: Strategy for an Iconic Indian Fashion Brand, Dinesh Kumar and Punam Gupta (Public case)


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Not all the excitement was on stage at this year's MTV Video Music Awards, after Pamela Anderson's exes Kid Rock and Tommy Lee got into a fight.
MTV VJ Sway said that he saw Pammy's most recent ex-husband walk up to Motley Crue drummer Tommy, who was sitting next to P Diddy, and "deck him".
The incident was confirmed by Palms Hotel and Casino public relations representative Larry Fink.
Another eyewitness, rap producer Rich Nice, said although Kid Rock threw the only blows that landed – a backhand slap and a punch – Tommy Lee was the instigator.
"It looked like Tommy Lee initiated it because Kid Rock was ignoring him," said Nice, who was two tables away.
"And Tommy Lee kind of antagonised him. And then when Tommy Lee stood up, it looked a little weird, like, 'Yo, what?'
"When Tommy stands up is when Kid hit him the first time with a backhand. And then Tommy Lee looked like he was trying to get at him to aggressively retaliate and then Kid Rock hits Tommy Lee again – bong.
"And then security grabs Tommy Lee in a headlock and pulls him off."
Security guards let Kid Rock stay for the rest of the show while his adversary was dragged out.
The incident inspired plenty of mock rebukes from the hip-hoppers in attendance.
"They say it's only rappers," said Sway. "I told you rockers fight too."
And Jamie Foxx tutted: "Stop all this white-on-white violence."


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It leads us to another Freud's concept — dichotomy virgin Madonna — whore, when all women divided into two categories which never overlap. Pabst's The Joyless Streetargues: My goal is to analyse specific ways of representation of prostitutes in Weimar cinema. At Frau Greifer's place she gets noticed by Herr Rosenow's friend Don Alfonso Canez, who is also responsible for the intrigue which drawn Greta's father bankrupt.
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New schools were frequently established in Weimar Germany to engage students in experimental methods of learning. I will clarify my viewpoint on this problem and I will support my claims with references to the research already made.
An English version was planned but never materialized.
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An "urban petty bourgeoisie " along with a growing middle class grew and flourished in wholesale commerce, retail trade, factories and crafts.
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- It was easy to find a tenant but after a small embarrassing incident the contract was terminated and Greta and her family find themselves with no income again. Weimar
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- Psychology Press. Weimar


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North Queensland Cowboys vs Cronulla Sharks Live Stream & TV Channel NRL 2019 Full HD
North Queensland Cowboys vs Cronulla Sharks Live Stream Free Online
The North Queensland Cowboys are with full confidence and will be looking to put up an impressive performance when they take on Sharks in a stunning Matchup.
The Cronulla Sharks on the other Side will be eyeing the very best Points when they lock horns the Cowboys side and also put smile on their fans face
Clearly, North Queensland were left counting their woes last weekend after a crushing 29-10 defeat against the Brisbane Broncos.
Surely, The Cowboys came into last weekend's game confident of a good performance after a Round 1 victory over the St George Illawarra Dragons.
And last week was far from the cakewalk they had in the opening game and the tough contest against the Broncos took its toll on them
The Townsville club have also dropped Tom Opacic after an indifferent performance against his old club last week.
Te-Maire Martin moves from five-eighth to full back, and they've added youngster Jake Clifford to the halves.
Cronulla put a disappointing Round 1 performance behind them last Saturday when they sent their fans home happy with a 20-6 victory against the Gold Coast at Shark Park.
They looked average in their opening round match against the Knights when they fell to a 14-8 defeat
And last weekend the Sharks were again not at their peak, but were made to look good at times by the hapless Titans.
The men from the Shire will need to put in a full 80-minute performance up against the Cowboys
Given the fact that they have failed to score points in two of the four halves of football they have played this season.
After announcing his retirement mid-week, Paul Gallen will be looking to inspire his team to victory as he has done for so long.
In two matches this season, 'Gal' has averaged 90 running metres per game and his efforts will be key yet again if the Sharks want to take victory in Townsville.
Streaming: WatchESPN, Strikeout.me, FOX
Also, Taumalolo missing from the Cowboys line up and Cronulla's solid record up north see the visitors assume the favourites mantle.
For the Cowboys, they are Unable to gain any momentum and at 24-0 down in the second half
Definitely, the game was already gone for the Cowboys despite a late mini-resurgence.
Obviously, injuries troubled the Cowboys and have had a big impact on the lead-up to this game
And of course, With Paul Green having to cope with the loss of superstar Jason Taumalolo for up to 10 weeks with an MCL injury.


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HSJ100: New health aristocracy takes shape
Find your next role
A Frost and Sullivan Technical Brief
Enterprise communications platforms have undergone a fundamental shift, migrating from traditional, onpremises, TDM-based PBX to highly agile, IP-based unified communications and collaboration platforms. This evolution has not only enhanced the means and efficiency of communications among employees, partners and, most importantly, customers, but has also enabled highly flexible and innovative deployment opportunities. Today, businesses can choose from a number of delivery models, from on-premises solutions deployed within a data center environment to fully hosted unified communications as a service (UCaaS) solutions implemented within public or private clouds.
Converging voice and data communications and collaboration applications on a shared IP-based network affords businesses a wealth of integration and cost-saving opportunities, but this level of convergence requires unique considerations. Specific attention must be paid to the quality of wide area network (WAN) links that connect remote locations to the business and all locations to the outside world. This technical brief outlines the benefits UCaaS can offer businesses and identifies key architecture considerations for deploying cloud-based enterprise communications that ensure the highest levels of service quality and reliability.
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Health Blogs » AllAfrica Health » Nigeria: Over 850,000 Nigerians in 12 States At Risk of Poisoning From Mercury Use
Posted date: 2019-05-21 11:59:58
Tags: None
Abuja — In Nigeria, not less than 850,690 people are at risk of mercury poisoning. In at least 12 Nigerian States, particularly Niger, Osun and Zamfara, artisanal and small-scale gold mining (ASGM) is one of their key source of income generation; however, ASGM is also the main source of the largest release of mercury emissions around the world.
The others are Kebbi, Katsina, Kaduna, Kwara, Borno, Kaduna, Jigawa, and Yobe States and Federal Capital Territory.
Dr Edwin Edeh, National Consultant Public Health and Environment (PHE) of World Health Organization (WHO) pointed out that "mercury, which is widely used to extract gold in Artisanal and Small-Scale Gold Mining (ASGM); is
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