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DNA polymerase δ plays an essential role in chromosomal DNA replication in eukaryotic cells, being responsible for synthesising the bulk of the lagging strand. In fission yeast, Pol δ is a heterotetrameric enzyme comprising four evolutionarily well-conserved proteins: the catalytic subunit Pol3 and three smaller subunits Cdc1, Cdc27 and Cdm1. Pol3 binds directly to the B-subunit, Cdc1, which in turn binds the C-subunit, Cdc27. Human Pol δ comprises the same four subunits, and the crystal structure was recently reported of a complex of human p50 and the N-terminal domain of p66, the human orthologues of Cdc1 and Cdc27, respectively.
To gain insights into the structure and function of Cdc1, random and directed mutagenesis techniques were used to create a collection of thirty alleles encoding mutant Cdc1 proteins. Each allele was tested for function in fission yeast and for binding of the altered protein to Pol3 and Cdc27 using the two-hybrid system. Additionally, the locations of the amino acid changes in each protein were mapped onto the three-dimensional structure of human p50. The results obtained from these studies identify amino acid residues and regions within the Cdc1 protein that are essential for interaction with Pol3 and Cdc27 and for in vivo function. Mutations specifically defective in Pol3-Cdc1 interactions allow the identification of a possible Pol3 binding surface on Cdc1.
In the absence of a three-dimensional structure of the entire Pol δ complex, the results of this study highlight regions in Cdc1 that are vital for protein function in vivo and provide valuable clues to possible protein-protein interaction surfaces on the Cdc1 protein that will be important targets for further study.
Three evolutionarily-conserved DNA polymerase enzymes play essential roles at the eukaryotic replication fork [1]. DNA polymerase α-primase is essential for the initiation of leading strand synthesis at individual replication origins and for the initiation of each Okazaki fragment on the discontinuously-synthesised lagging strand. Extending from the RNA-DNA primers laid down by DNA polymerase α (Pol α)-primase, recent results indicate that DNA polymerase ε (Pol ε) is likely to replicate the bulk of the leading strand [2] while DNA polymerase δ (Pol δ) synthesises the bulk of the lagging strand [3], reviewed by [4,5]. In the absence of catalytically-active Pol ε, Pol δ is apparently able to synthesise both leading and lagging strands [6]. Completion of lagging strand synthesis requires subsequent processing of the nascent Okazaki fragments to remove the RNA primer and ligation to form a continuous DNA strand [1].
Each of the three replicative polymerases α, δ and ε is a member of the B family of DNA polymerases [1] and each is a multi-subunit entity, comprising an essential catalytic subunit and a number of smaller subunits, some of which are also essential for cell viability based on the results of genetic analysis in the yeasts [1]. Until recently, very little high-resolution three-dimensional structural information on the three polymerases was available, as the only solved structures were those of a 38 amino acid zinc finger derived from the carboxy-terminal end of the catalytic subunit of human Pol α and 75 amino acids of the amino-terminal domain of the B-subunit of human Pol ε, both determined by NMR [7,8]. A low-resolution structure of the budding yeast Pol ε complex was also obtained by cryo-EM reconstruction [9]. However, high-resolution structures are now available for the complex of p50 (the second subunit) and the N-terminal 144 amino acids of p66 (the third subunit) of human Pol δ (designated p50•p66N) [10,11] and for the complex of the C-terminal domain of budding yeast Pol1 protein, the catalytic subunit of Pol α, and the C-terminal PDE and OB fold domains of the Pol α B-subunit Pol12 [12].
The fission yeast Schizosacchromyces pombe is an excellent model for studying Pol δ structure and function [1]. In this organism, DNA polymerase δ is a heterotetrameric complex, comprising the catalytic subunit Pol3 and three smaller subunits Cdc1, Cdc27 and Cdm1 [13,14]. Pol3, Cdc1 and Cdc27 are essential proteins [15-18] whereas Cdm1 is not [19]. Orthologues of all four subunits make up human Pol δ. In contrast, Pol δ in the budding yeast Saccharomyces cerevisiae is a three-subunit enzyme; no Cdm1 orthologue is present in this organism. Pol3 binds directly to the B-subunit Cdc1 [20] via one of two conserved zinc finger modules located at the C-terminus of Pol3 [21]. Cdc1 binds the C-subunit Cdc27 [20] which interacts directly with two other key components of the replisome – the catalytic subunit of Pol α-primase [22] and the sliding clamp processivity factor PCNA [23]. Cdc27 has an unusual structure: a globular N-terminal domain of ~160 amino acids is followed by a highly extended C-terminal region of ~210 amino acids [14]. The interactions with Pol α-primase and PCNA both involve short conserved protein-protein interaction motifs located within this C-terminal region, but neither interaction is essential for replisome function [22,24]
In contrast to PCNA and Pol α-primase, Cdc1 binds the N-terminal domain of Cdc27 [23]. The structure of p50•p66N complex [10,11] (where p50 and p66 are human orthologues of Cdc1 and Cdc27) now enables the mapping of the interactions within the Cdc1Cdc27 assembly. Figure 1 provides a summary view of the human p50•p66N complex (Figure 1A) together with a schematic representation of the domain structure of the two proteins in humans and yeast (Figure 1B). Three domains are apparent: p50 comprises a phosphodiesterase-like domain (PDE domain) and an oligonucleotide/oligosaccharide binding domain (OB domain) while p66N comprises a winged helix-turn-helix (wHTH) domain. The PDE domain is located in the centre of the complex with the OB domain on one side and the wHTH on the other (Figure 1A). The PDE and OB domains are present in the B-subunits of all three eukaryotic replicative polymerases as well as in the essential second subunit of the archaeal DNA polymerase PolD [25,26] found exclusively in the Euryarchaeota and Thaumarchaeota phyla [27]. In the case of the euryarchaeal proteins the PDE domain is active as a 3'-5' exonuclease, affording the PolD enzyme proofreading activity. Key residues required for nuclease activity are absent from the eukaryotic proteins; in the case of Pol δ and Pol ε, proofreading activity resides in the catalytic subunit of the complex [1].
Figure 1. Structure of the human p50•p66N complex. A. Structure of the human p50•p66N complex showing the p50 PDE and OB domains (cyan and magenta respectively) and the p66N wHTH domain (gold). The figure was prepared with PyMol software (Delano Scientific) using the coordinates of p50•p66N complex (Protein Data Bank with accession code 3E0J) [10]. B. Schematic showing the predicted location of the PDE and OB domains in fission yeast Cdc1 in relation to their sequence numbering.
This study describes the results of mutational analysis of fission yeast Cdc1. Despite its essential cellular role and widespread evolutionary conservation, the precise biochemical function of the Cdc1/p50 protein is not known nor is it clear how the structure of the protein is related to this. In fission yeast, a small number of temperature-sensitive mutant alleles of cdc1 have been isolated and characterised, and several truncated Cdc1 proteins tested for function in vivo [20]. The temperature-sensitive mutations were shown to result from single nucleotide changes in the gene sequence, resulting in a single amino acid changes in the encoded proteins. These changes mapped towards the C-terminus of the Cdc1 protein, within the PDE domain [20]. Similar results were seen with budding yeast pol32/hys2 mutations [28,29]. Analysis of truncated mutant Cdc1 proteins showed that removal of 20 amino acids from the C-terminus of Cdc1 abolished the in vivo function of the protein, while removal of the first 25 amino acids from the N-terminus led to dominant negative cell cycle arrest that could be reversed by simultaneous overproduction of Cdc27, suggesting that the mutant Cdc1-NΔ2–25 protein exerted its function by titrating Cdc27 from the cell [20].
To investigate further the structure and function of Cdc1, a collection of thirty mutant cdc1 alleles were generated by directed and random mutagenesis methods. Each of the mutations was mapped onto the three-dimensional structure of human p50 [10] and each mutant protein was then tested for function in fission yeast cells, and for binding to Pol3 and Cdc27 using the two-hybrid system.
Pentapeptide insertion mutagenesis
Random mutagenesis of the cdc1+ gene was carried out using the pentapeptide scanning mutagenesis (PSM) method [30,31]. This method makes use of a modified form of the Tn3-related transposon Tn4430 of Bacillus thuringiensis. Tn4430 transposes efficiently in E.coli and in doing so duplicates 5 bp of host sequence at the insertion point. The transposon also contains KpnI restriction sites 5 bp from the outer end of its terminal repeats. If, following insertion of Tn4430 into the target gene, the bulk of the transposon is deleted by in vitro KpnI restriction and re-ligation, a 15 bp sequence insertion remains. Since Tn4430 inserts with low sequence specificity, the pentapeptide insertions will vary in composition. The usefulness of this system has been demonstrated through its application for random mutagenesis of, amongst others, TEM-1 β-lactamase [30,32], XerD recombinase [33] and phospholipase D1 [34].
The strategy for mutagenesis of cdc1+ is outlined in Figure 2. Plasmid pBR322-Cdc1 (see Methods) was transformed into E.coli FH1046 containing plasmid pHT385 which carries the Tn4430 transposon [30]. Individual transformant colonies were then mated with E.coli DS941 and resistant colonies identified and plasmids recovered. Restriction mapping was then used in order to identify clones in Tn4430 had inserted into cdc1+ rather than the plasmid backbone, and the bulk of the transposon sequences then removed by cleavage with KpnI followed by ligation under conditions that promoted self-ligation (see Methods).
Figure 2. Pentapeptide scanning mutagenesis (PSM) strategy. A schematic representation of the mutagenesis and cloning strategies used to create the cdc1 PSM alleles. Plasmid pBR322-Cdc1 (top left) carries a cdc1+ cDNA on a BamHI-BamHI fragment. This plasmid was transformed into E.coli FH1046 containing pHT385. Following mating with E.coli DS941(see Methods), plasmids in which the Tn4430 transposon had inserted into the cdc1+ ORF (indicated as pBR322-Cdc1-Tn, top right) were identified by restriction mapping and the bulk of the Tn4430 sequences excised by KpnI digestion and self-ligation of the digested plasmids. The mutant alleles were then transferred into pREP3XH6BN, a fission yeast expression vector, and either pBTM116 or pBTM116BN, budding yeast two-hybrid bait vectors.
Number and distribution of insertion mutations
In total, 31 mutant alleles were generated by the PSM method (Table 1, Figure 3). Despite their independent origins, DNA sequence analysis revealed that these corresponded to only 20 different mutant alleles (Table 1), indicating that insertion of Tn4430 is not entirely random, as noted previously [30]. The insertions also clustered towards the 5' end of the cdc1+ gene, with 26 of the 31 insertions mapping within the first 420 bp of the 1386 bp ORF. Clustering towards the 5' end of the ORF was observed in a similar study of the S.pombe rfc2+ gene cloned into pBR322 [35]. Thirty of the mutant alleles contained a 15 nt insertion when compared with the wild-type cdc1+ sequence. The remaining allele (Cdc1-E2) contained a 16 nt insertion, presumably the result of an inexact transposition event. This insertion leads to the Cdc1 protein being truncated after amino acid 26. Another insertion also led to production of a truncated protein: Cdc1-J17 was truncated after amino acid 27. Two of the insertions resulted in different insertions at the same amino acid position in the protein (Cdc1-J6 and Cdc1-E9, between amino acids 124 and 125).
Table 1. Pentapeptide insertion and site-directed mutants: a summary
Figure 3. Location of mutants in Cdc1 protein. Sequence alignment of the fission yeast Cdc1 protein and its human orthologue p50 in single-letter code. Residues identical between the two proteins are highlighted in yellow. The sites of insertions and point mutations in Cdc1 are indicated by colour-coded triangles (pentapeptide insertions, E and J mutants) and circles (point mutations, A mutants). Grey circles/triangles indicate fully-functional mutants; green circles/triangles indicate partially-functional mutants; magenta circles/triangles indicate non-functional mutants. Note that the A9 and A10 mutants see two adjacent amino acids mutated and are therefore indicated by side-by-side (magenta) circles. Where the same insertion was found more than once, only one allele appears on the figure (see Table 1 for further information). The ten conserved regions defined in the previous study [36] are underlined and indicated by Roman numerals (I – X). The red, cyan and grey bars correspond to positions of α-helices, β-strands and disordered regions in human p50 [10]. The amino acid residues in the p50 protein that are involved in interactions with p66N are shown in green type. See text for details.
Site-directed mutagenesis
In addition to the random pentapeptide insertion mutations, ten additional cdc1 mutations were constructed by oligonucleotide-directed in vitro mutagenesis (see Methods). Eight of these alleles resulting in the encoding proteins (Cdc1-A1 to -A8) differing from the wild-type Cdc1 by a single amino acid substitution. The remaining two alleles encoded proteins (Cdc1-A9 and Cdc1-A10) that featured substitutions of two adjacent amino acids. All the mutated amino acids were amongst those that are found conserved between the eukaryotic DNA polymerase B-subunits and the archaeal PolD small subunit [26,36]. Mutations A1 – A4 and A9, which combines mutations A1 and A2, cluster in conserved region VI [36], whereas mutations A5, A6 and A10, which combines A5 and A6, map to conserved region VII. A7 and A8 map to conserved regions VII and VIII, respectively (see Figure 3).
Analysis of mutant function in fission yeast
Expression in cdc1Δ cells
In order to test for function in S.pombe, each allele was subcloned to plasmid pREP3XH6BN to permit expressing of the mutant proteins under the control of the thiamine-repressible nmt1 promoter (Figure 2, see Methods for details). The resulting plasmids were then transformed into a cdc1+/cdc1::ura4+ h-/h+ diploid strain and diploid transformants isolated at 32°C on supplemented EMM plates with and without 5 μg/ml thiamine. Transformant colonies were then picked and subjected to treatment with helicase to kill vegetative cells and degrade the walls of the asci present in the nutritionally-depleted colony centres. Following washing, the spores were plated on supplemented EMM media with and without 5 μg/ml thiamine and incubated at 32°C for 4–6 days (see Methods for further details).
Figure 3 summarises the properties of the Cdc1 mutants. When overexpressed (in cells grown in the absence of thiamine), twenty-three of the thirty mutant proteins were able to substitute for complete loss of wild-type Cdc1 function (grey/green circles/triangles in Figure 3). Given that all the pentapeptide insertion sequences include a proline residue that might be expected to have a significant effect on the secondary structure in the vicinity of the insertion, it is perhaps surprising that only seven of the insertions abolish Cdc1 function outright (purple circles/triangles in Figure 3). However, related studies of two other essential fission yeast DNA replication factors Rfc1 [37] and Rfc2 [35] have revealed similar ratios of functional to non-functional mutations, as have published studies of unrelated proteins [30,32-34]. Of the twenty-three functional alleles, nine were able to support growth only in cells grown in the absence of thiamine, indicating that high level expression of the mutant proteins is required for rescue (Figure 3, green circles/triangles).
Expression in cdc1-P13 cells
In addition to testing the function of the mutant proteins in a cdc1Δ background, each was expressed in wild-type cells and in cells carrying the temperature-sensitive cdc1-P13 allele [15]. Expression of the mutant proteins had no detectable effect on wild-type cells. In cdc1-P13 cells, the proteins were tested for their ability to rescue at the restrictive temperature of 36.5°C in cells grown in the presence and absence of thiamine. The results obtained broadly mirrored those seen with the cdc1Δ strain but with the following exceptions: at low expression levels, several of the mutant proteins that could not rescue cdc1Δ could rescue cdc1-P13 at 36.5°C, namely Cdc1-A6, -J3, -J4, -J5, -J10, -J11, -J13 and -J21, and two of the mutants, Cdc1-A10 and -J7, displayed dominant negative properties at 28°C, described further below.
Analysis of protein-protein interactions using the two-hybrid system
As described in the Introduction, the Cdc1 protein interacts directly with both the catalytic subunit of the Pol δ complex Pol3 and also with the C-subunit Cdc27. These interactions can be conveniently monitored using the yeast two-hybrid system [20,23]. Each cdc1 mutant allele was therefore cloned into the two-hybrid bait pBTM116 (or the related pBTM116BN plasmid) to allow expression of Cdc1 fused to the DNA binding domain of bacterial LexA protein (see Methods). The resulting plasmids were initially co-transformed into S.cerevisiae CTY10-5d along with the prey plasmid pGAD2F-Cdc27, which expresses Cdc27 fused to the yeast Gal4 transcriptional activation domain. Co-transformants were monitored for β-galactosidase activity by X-gal overlay assay (data not shown) and ONPG liquid assay (Figure 4A).
Figure 4. Quantitative two-hybrid analysis. A. The Cdc1 mutants, arranged depending on the position of the mutation within the Cdc1 protein, from N- to C-terminus, were tested for their ability to interact with Cdc27 using the two-hybrid system in S.cerevisiae CTY10-5d. Quantitative values were obtained using an ONPG-based β-galactosidase assay from liquid cultures (see Methods). Values are expressed in arbitrary units with the wild-type Cdc1-Cdc27 interaction being set at 100 units (~65 Miller units of β-galactosidase activity, see [20]). B. Similar analysis of Cdc1-Pol3 interactions, performed in the presence of Cdc27 (see text).
To examine whether the mutant Cdc1 proteins interacted with Pol3, each pBTM116-Cdc1 plasmid was transformed into CTY10-5d along with the prey plasmid pACT-Pol3 and pAA-Cdc27. The latter directs expression of Cdc27 as a c-myc epitope-tagged fusion protein. Previously it was shown that interaction between LexA-Cdc1 and Gal4-Pol3 could only be detected when Cdc27 was simultaneously co-expressed in the cells [23]. Presumably Cdc27 binds to Cdc1 to stabilise the protein or alter its conformation, allowing interaction with Pol3 to take place at a level that can be detected under two-hybrid assay conditions. As above, β-galactosidase activity was monitored by X-gal overlay assay (data not shown) and ONPG liquid assay (Figure 4B). Those mutant Cdc1 proteins that were unable to bind to Cdc27 in the two-hybrid system would be predicted to be also incapable of binding Pol3.
Analysis of dominant negative Cdc1 mutants
Two of the Cdc1 mutant proteins exerted a strong dominant negative phenotype when expressed at high level in cdc1-P13 cells. Transformed cdc1-P13 cells carrying either pREP3XBN-Cdc1-J7 or -A10 were viable at 28°C only when grown on medium containing thiamine to repress the nmt1 promoter (Figure 5). When transferred to medium lacking thiamine, high-level expression of the mutant proteins led to cell cycle arrest (elongated cell phenotype) and a failure to form colonies. Similar behaviour was observed when these mutant proteins were expressed in cdc1–223 cells at 28°C, although the phenotype was somewhat weaker (data not shown).
Figure 5. Dominant negative Cdc1 mutants. cdc1-P13 cells carrying plasmids pREP3X, pREP3XH6BN-Cdc1, pREP3XH6BN-Cdc1-J7 or pREP3XH6BN-Cdc1-A10 were grown up in EMM medium at 28°C before being plated onto EMM plates with (+T, right hand panels) or without 5 μg/ml thiamine (-T, left hand panels) and incubated for 4 days at 28°C.
The two-hybrid analysis described above (Figure 4) suggests that both Cdc1-J7 and Cdc1-A10 retain the ability to bind to Cdc27 but cannot bind Pol3, suggesting that the dominant negative phenotype may be the result of the mutant proteins interacting with Cdc27 to form non-functional Cdc1-Cdc27 dimers, thus titrating Cdc27 from the cell, impairing Pol3-Cdc1-Cdc27 (Pol δ) complex formation and inhibiting chromosome replication. If this were the case, simultaneous overproduction of Cdc27 would be expected to reverse the toxic effects of Cdc1-J7 and Cdc1-A10, by providing excess Cdc27 to interact with non-mutant Cdc1.
To test this, cdc1-P13 cells expressing Cdc1-J7 and Cdc1-A10 were co-transformed with plasmid pREP4X-Cdc27 which expresses the full-length Cdc27 protein, also from the nmt1 promoter [23]. However, no rescue of the dominant negative phenotype was observed: cdc1-P13 cells expressing Cdc27 and either Cdc1-J7 and Cdc1-A10 were still unable to grow on plates lacking thiamine at 28°C. We also tested whether overproduction of Pol3 or a nuclear-targeted form of the C-terminal ZnF domain of Pol3 would rescue the toxic effects of Cdc1-J7 and Cdc1-A10, despite their apparent inability to bind Pol3 as gauged by two-hybrid assay (Figure 4): again, no rescue was seen. Previously, overproduction of an N-terminally truncated form of Cdc1, Cdc1-NΔ25, has been shown to exert a similar effect on cdc1-P13 cells [20]. In the case of Cdc1-NΔ2–25, however, simultaneous overproduction of Cdc27 was sufficient to rescue the phenotype. This suggests that the Cdc1-J7 and -A10 proteins exert their effects through a mechanism distinct from that of Cdc1-NΔ2–25, perhaps by binding to, and titrating out, an as yet unidentified Pol δ interacting factor. Screening for high copy suppressors of the Cdc1-J7 and Cdc1-A10 dominant negative phenotypes might allow this factor to be identified, although negative regulators of the nmt1 promoter are also likely to be identified in such a screen.
In this report, we describe the results of mutational analysis of the fission yeast Cdc1 protein using both random and site-directed mutagenesis techniques. In total, thirty mutant proteins were expressed and assayed for function in vivo and for binding to Pol3 and Cdc27 by two-hybrid. The results of these assays are summarised in Table 1.
For the purposes of the following discussion, we divide the mutations in four separate groups based on the two-hybrid interaction data presented in Figure 4 and use the structure of the human p50 protein [10] as the basis for discussion of possible structural effects of the mutations. In the absence of a crystal structure for the fission yeast Cdc1 protein, the human p50 structure represents the only available option for this type of analysis but it is important to note that the two proteins are only ~35% identical at the primary sequence level and that this could affect some of the conclusions drawn. An additional point to note, particularly when considering mutations that appear to disrupt Cdc1 function altogether, is that possible effects of individual mutations on protein stability have not been addressed in this study. For technical reasons we have been unable determine the expression levels of the mutants, even in cells cultured in the absence of thiamine. Figure 6 shows the locations of the mutations in each group mapped onto the structure of human p50 according to the protein sequence alignment shown in Figure 3.
Figure 6. Mapping of Cdc1 mutants to the structure of human p50•p66N complex. The secondary structure elements are colour-coded as follows: α-helices, β-strands and coils are red, yellow and green in p50, and cyan, magenta and light pink in p66N. The sites of pentapeptide insertions and amino acid substitutions are highlighted by double and single grey spheres, respectively. In panel B the blue sphere indicates the position of Gln347. The labels in magenta, green and grey correspond to non-functional, partially functional and fully functional pentapeptide insertions or amino acid substitutions. The disordered region d1 was modelled for panels A, C, and D. The positions of disordered regions d1-d4 are drawn by dotted lines in panel B. The figure was prepared with PyMol software (Delano Scientific) using the coordinates of p50•p66N complex (Protein Data Bank with accession code 3E0J) [10].
Mutations that disrupt both Pol3-Cdc1 and Cdc1-Cdc27 interactions
The E2, J1, J16, J21, J17 and J6 mutations disrupt Cdc1 interactions with both Pol3 and Cdc27 in two hybrid-assays (Figure 4); the locations of the mutated residues on the three-dimensional structure of human p50 are shown in Figure 6A.
Five of the six mutations in this group are pentapeptide insertions (J1, J16, J21, J17 and J6, see Table 1). The J21 insertion is in the middle of α5 (Figure 6A) and likely interferes with the folding of this helix, which is an important feature of PDE domain. Several residues within α5 are also involved in Cdc27 binding, raising the possibility that loss of Pol3-Cdc1 interaction in two-hybrid assays is the caused by disruption of the Cdc1-Cdc27 interaction since Pol3-Cdc1 interactions were only detectable by two-hybrid in yeast cells co-expressing Cdc27. In contrast, the J1, J6 and J16 insertions are located within, and appear likely to disrupt the folding of, the OB domain (Figure 6A). The fact that none of the three latter insertions are close to the Cdc1-Cdc27 interface (based on the p50•p66N structure) appears to rule out the possibility that Pol3-Cdc1 two-hybrid interaction is impaired by direct modification of Cdc1-Cdc27 interaction interface. Instead, the disruption of OB domain fold by J1, J6 and J16 insertions is likely to affect the Cdc1-Cdc27 interaction in part by destabilizing the PDE domain and in part by preventing the correct positioning of N-terminal residues which are important for the function of Cdc1 [20]. The consequences of the J1 and J16 mutations for Cdc1 activity in vivo were more severe than for J6 and J21, since neither J1 nor J16 was able to substitute for loss of Cdc1 in cdc1Δ cells under any conditions, while J6 and J21 were able to rescue when overexpressed (Figure 3).
For the remaining two mutations in this group (J17 and E2), it is not surprising these the mutant protein fail to interact with both Pol3 and Cdc27, as both mutations result in incorporation of a stop codon into the cdc1 sequence and premature termination of translation in the flexible β2α2 linker, after amino acids 26 and 28 respectively. When expressed at near wild-type levels in S.pombe, from the repressed nmt1 promoter, neither protein is able to support growth of cdc1Δ cells. However, when overexpressed (nmt1 promoter depressed in the absence of thiamine), cdc1Δ cells expressing Cdc1-J17 were able to grow (Figure 3). This is most likely a consequence of low-level readthrough of the amber (UAG) stop codon in the cdc1-J17 allele. Although E2 is also an amber mutation, the sequence insertion in this allele comprised 16 nucleotides rather than the usual 15, so that expression of a full-length Cdc1 protein would require the unlikely combination of readthrough of the AUG codon and translational frameshifting.
Mutations that disrupt Pol3-Cdc1 interactions only
This group contains a total of thirteen mutations that can be further divided into two sub-groups on the basis of the ability to substitute for wild-type Cdc1 in cdc1Δ cells. The first sub-group contains six mutations that display either impaired function in vivo (rescuing cdc1Δ cells only when overexpressed from the derepressed nmt1 promoter) or which cannot rescue cdc1Δ cells under either of the conditions tested. This sub-group comprises the pentapeptide insertions J7 and J10, the single point mutations A3 and A6, and the double point mutations A9 and A10 (Figure 6B). A9 is a combination of A1 and A2; A10 of A5 and A6 (Figure 3). Five of the six mutations are located in the PDE domain and one (J10) in the OB domain. Each of the six mutant proteins is able to bind Cdc27 in two-hybrid (although in the case of A6 binding is reduced to <10% of that seen with the wild-type Cdc1 protein) but is greatly impaired in Pol3 binding ability (< 0.5% of binding seen with wild-type protein, see Figure 4). Strikingly, all six mutations map to the same side of Cdc1, identifying a possible Pol3-interacting surface. In support of this, glutamine 345 in human p50 (glutamine 347 in Cdc1) also maps to this region (indicated in Figure 6B). A mutation in the corresponding residue in budding yeast Pol31 (lysine 358 mutated to glutamate) was identified in a screen for dominant extragenic suppressors of the temperature-sensitive pol3–13 mutation, in which one of four cysteines of the second C-terminal zinc finger of Pol3 (designated ZnF2) is replaced by serine [38,39]. Subsequent work showed that the ZnF2 domain was both necessary and sufficient for Pol31 binding by Pol3 and that in two-hybrid assays, the K358E mutant of Pol31 interacted more strongly with both wild-type and mutant forms of ZnF2 [21]. Taken together, these results suggest that the region defined by these mutations constitutes a Pol3 binding site on Cdc1. This suggestion is consistent with the recent crystal structure of interacting domains of the budding yeast Pol1 and Pol12 proteins, the catalytic and B-subunits of Pol α respectively [12]. The C-terminal domain of Pol1, comprising the two zinc fingers ZnF1 and ZnF2 and a three α-helix bundle, makes extensive contacts with the PDE and OB fold domains of Pol12 over a strikingly large area that is equivalent to that defined by our study of Cdc1-Pol3 interactions (Figure 6B). Intriguingly, the disordered regions d1-d4 in the human p50 structure (indicated by the grey bars in Figure 3) are localised to the same region [10]. It is tempting to speculate that these disordered regions also have a part to play in binding Pol3 and become structured only once Pol3 is bound.
The second sub-group of mutations unable to bind to Pol3 comprises mutations J8, J12, J15, A2, A4 and A5. These display >20% binding to Cdc27 in the Cdc1-Cdc27 two hybrid assay but with the exception of A5, display < 0.5% binding to Pol3 in the Pol3-Cdc1-Cdc27 two hybrid assay (A5 displayed ~5% binding compared to wild-type Cdc1, Figure 4). Despite their extremely limited ability to bind to Pol3, however, these mutant proteins were able to function in vivo, even when expressed at near wild-type levels from the repressed nmt1 promoter (Figure 3). The reason for this discordance is unclear but one possibility is that the mutants benefit from the presence of the fourth subunit of the Pol δ complex in fission yeast, Cdm1 [19]. In both fission yeast and in mammalian cells, the Cdm1 protein (designated p12 in mammals) appears to have a stabilising effect on the Pol δ complex [13,40-42] possibly through direct binding to both Pol3 and Cdc1 [43]. There is no Cdm1 orthologue in the budding yeast S.cerevisiae, the host for the two-hybrid analysis presented here [44]. Mapping of the J8, J12, J15, A2, A4 and A5 mutations onto the structure of human p50 reveals that J8, A2, A4, and A5 may directly affect the disordered loops (Figure 6B), which we propose to form an area of protein-protein interactions. The remaining two mutations, J12 and J15, are apart from the suspected interaction area and are likely to exert their influence to Pol3-Cdc1 interactions via an allosteric effect: J12 via an N-terminal residues to a disordered region d1, and J15 via a loop β8-α3 to the disordered region d2.
Mutations that disrupt Cdc27 interactions only
As noted above, in the two-hybrid assay system used here and described previously [21,23], Pol3-Cdc1 interactions were only detectable when Cdc27 was co-expressed in the reporter cells. Under these conditions, Cdc27 presumably serves to stabilise or alter the conformation of Cdc1 to facilitate its binding to Pol3. It is therefore surprising that five of the insertion mutant Cdc1 proteins generated in this study (E1, J2, J3, E9, J4) were able to interact with Pol3 in the Pol3-Cdc1-Cdc27 two-hybrid assay (displaying >20% of wild-type binding activity) despite being unable to interact with Cdc27 in the Cdc1-Cdc27 assay. Interestingly, four of these map to the OB domain and the fifth (J2) to the β2α2 disordered linker. All five are therefore located some distance away from the Cdc1-Cdc27 interface (Figure 6C) making it unlikely that they have a direct effect on Cdc27 binding. It may be the case that disruption of the OB domain and β2α2 linker results in a widespread perturbation of the structure of Cdc1 and disruption of the Cdc1-Cdc27 interface. When Pol3 is present (as in the Pol3-Cdc1-Cdc27 two-hybrid assay), the structure of the Cdc1 protein, and more specifically the putative Pol3 binding region described in the preceding section, may be stabilised, effectively insulating the Cdc1-Cdc27 interface from the effects of disruption of the OB domain and β2α2 linker. Cdc1-Cdc27 interactions are also impaired by the J22 insertion (<10% binding in two-hybrid assay) which maps close to the Cdc1-Cdc27 interface (Figure 6C).
Mutants unimpaired in Pol3 or Cdc27 binding
Five mutations fall into this group: the insertions E4, E5 and J9, and the single point mutations A1 and A8. Four of these (the exception is E4) map to loop regions in the human p50 protein (Figures 3 and 6D) and probably do not disrupt the secondary structure of Cdc1. The E4 insertion is on the N-terminus of β2. However, the two amino acids preceding the insertion site (proline 9 and cysteine 10) are identical with the residues at the positions 4 and 5 of the pentapeptide insertion (see Table 1). This has the effect of shifting the insertion from the N-terminal end of β2 to the β1β2 linker, which likely explains the low impact of this insertion.
Using both site-directed and random mutagenesis strategies, thirty new mutant Cdc1 proteins have been test for in vivo function and for Pol3 and Cdc27 binding using the two-hybrid system. The results obtained can be rationalised in the context of the recently published crystal structure of human p50 bound to the N-terminal domain of human p66 [10] and, in addition, offer new insights into the potential location of interaction surfaces on Cdc1 for Pol3.
Bacterial strains and methods
E.coli FH1046 and DS941, used as the donor and recipient strains for pentapeptide mutagenesis, have been described elsewhere [30] and were the generous gift of Dr F. Hayes (now at UMIST, UK). Otherwise, E.coli JM109 and DH5α were used for routine cloning work. Standard molecular cloning methods [45] were used throughout, unless otherwise stated.
Yeast strains and methods
The fission yeast strains used in this study were as follows: cdc1–223 (mis1–223) his2 leu1–32 h+, cdc1–64 (mis1–64) ade6–704 leu1–32 h-, cdc1-A24 leu1–32 ura4-D18 h-, cdc1-P13 leu1–32 h+, cdc27-P11 leu1–32 h+, leu1–32 ura4-D18 h- and cdc1+/cdc1::ura4+ leu1–32/leu1–32 ura4-D18/ura4-D18 ade6-M210/ade6-M216 h-/h+ [20]. The cdc1/mis1 strains [46] were a generous gift of M.Yanagida (Kyoto University, Kyoto, Japan); cdc1-A24 was a generous gift of K.L. Gould (Vanderbilt Uinversity, Nashville, USA). Standard growth conditions [47] were used throughout. For two-hybrid analysis, S.cerevisiae CTY10-5d and standard methods were used [20].
Pentapeptide mutagenesis method
For pentapeptide mutagenesis, a 1.5 kb BamHI fragment containing a cdc1+ cDNA was sub-cloned from pBTM116-Cdc1 [20] into pBR322, at the unique BamHI site in the latter, to generate plasmid pBR322-Cdc1. pBR322 was chosen as it is a low copy number plasmid that is devoid of restriction sites for KpnI, two features that are important for the mutagenesis strategy [30]. The orientation of the cdc1+ insert in pBR322-Cdc1 is such that the 3' end of the insert is proximal to the unique HindIII site in pBR322. Mutagenesis was carried out as described previously [30]. Briefly, pBR322-Cdc1 was transformed into the donor E.coli strain FH1046 containing the kanamycin-resistant Tn4430-carrying plasmid pHT385. Transformant (donor cell) colonies obtained after overnight growth at 37°C on LB agar plates containing kanamycin (50 μg/ml) and ampicillin (50 μg/ml) were picked and resuspended in 100 μl of LB broth (no antibiotics) and 2.5 μl spotted onto LB agar plates. Next, 2.5 μl aliquots of recipient cells (from an overnight culture of E.coli DS941 in LB supplemented with 50 μg/ml streptomycin) were applied to the donor spots, after which the plates were air-dried and incubated for 3 hours at 37°C to allow mating. The mating mixes were then resuspended in 300 μl of LB broth, and 100 μl plated onto LB agar plates containing ampicillin, kanamycin and streptomycin. Colonies were then screened either by preparing plasmid DNA and digesting this with BamHI to determine if the Tn4430 had inserted within the vector or the BamHI insert, or by PCR analysis of individual colonies using oligonucleotides flanking the BamHI site in pBR322, in which case those isolates in which a 1.6 kb band corresponding to a Tn4430-less cdc1+ insert were discarded. Each pBR322-Cdc1-Tn plasmid was then digested with KpnI, re-ligated and transformed into E.coli JM109. Plasmids were prepared from ampicillin-resistant, kanamycin-sensitive colonies and checked for absence of the transposon by restriction mapping. The 1.5 kb BamHI-NotI fragments from the transposon-free plasmids were then cloned into plasmid pREP3XH6N (below) to facilitate expression in S.pombe, sequence analysis and subsequent sub-cloning to pBTM116.
Oligonucleotide-directed mutagenesis
Oligonucleotide-directed in vitro mutagenesis was carried out using the Mutagene II in vitro mutagenesis system (BioRad). The template used was pTZ19R-Cdc1-cDNA [20]. Oligonucleotides used were as follows: mutant A1, oligonucleotide AANO1 (5'-TAGATGTCACTTTAATGGCTGGTCCTTATG-3', with mutant alanine codon underlined); A2, AANO2 (5'-GTCACTTTAATGCCTGCTCCTTATGATTAC-3'); A3, AANO3 (5'-CCTGGTCCTTATGCTTACAGTTCAACTATC-3'); A4, AANO4 (5'-GTTCAACTATCCTTGCTCAACAGCCTTTGC-3'); A5, AANO5 (5'-ACAAACAGTTACGGCTCCCACTTGGCTTTC-3'); A6, AANO6 (5'-AAACAGTTACGAATGCCACTTGGCTTTCTC-3'); A7, AANO7 (5'-TGGCTACTAGCGCCCAAAACATTAATGATC-3'); A8, AANO8 (5'-AATCATATCACAGCTACCAGCCCTGATACC-3'); A9, AANO1/2 (5'-GTCACTTTAATGGCTGCTCCTTATGATTAC-3'); A10, AANO5/6 (5'-ACAAACAGTTACGGCTGCCACTTGGCTTTC-3'). The resulting mutants were then subcloned to pREP3XH6BN and either pBTM116 or pBTM116BN, as below.
Expression in yeast
Each pentapeptide insertion allele was subcloned as a BamHI-NotI fragment from pBR322-Cdc1-Tn into pREP3XH6BN, a derivative of pREP3XH6 [48] in which a NotI site was introduced just 3' to the existing BamHI site by cutting the vector with BamHI and SmaI and ligating in a short linker made up from the following oligonucleotides: 3XH6Not-1 (5'-gatcCATCATCGCGGCCGCATCG-3', with NotI site underlined and BamHI 5' overhang in lower case) and 3XH6Not-2 (5'-CGATCGCCGGCGGATGATG-3'). When cloned into pREP3XH6BN, the Cdc1 protein is expressed as a hexahistidine fusion with the 13 amino acid sequence MRGSHHHHHHGIL N-terminal to the native initiating methionine [48]. The resulting plasmids were then transformed into S.pombe cdc1+/cdc1::ura4+ leu1–32/leu1–32 ura4-D18/ura4-D18 ade6-M210/ade6-M216 h-/h+ [20] by electroporation [49] and transformants obtained on EMM medium [47]. Individual colonies were then patched overnight at 32°C on ME medium to induce sporulation, before being treated overnight with SRP-Helicase enzyme (BioSepra, France) to break down the ascus walls and eliminate vegetative cells. Spores were then washed with water before being plated on EMM plates supplemented with adenine (EMM+A), uracil and adenine (EMM+AU), adenine and 5 μM thiamine (EMM+AT) and adenine, uracil and 5 μM thiamine (EMM+AUT) at 32°C. Leucine was omitted from all plates to facilitate selection of pREP3X plasmids which carry the LEU2 selectable marker. Adenine is required to permit the growth of haploid cells either the ade6-M210 or ade6-M216 alleles. The addition of uracil permits growth of cdc1+ haploids; in the absence of uracil, only cdc1::ura4+ haploids expressing functional Cdc1 proteins can grow. The presence of 5 μM thiamine represses the nmt1 promoter in pREP3X, reducing cdc1 expression by a factor of 80–100 compared to cells grown on EMM without thiamine. Following 4–6 days growth at 32°C, colonies were analysed by microscopy and by replica plating to confirm predicted genotypes.
Two-hybrid analysis
For two-hybrid analysis, mutant cdc1 alleles were sub-cloned as a BamHI fragment into pBTM116 [20] or as a BamHI-NotI fragment to pBTM116BN, a modified form of pBTM116 in which a NotI site was added to the multiple cloning site by digesting pBTM116 with BamHI and PstI and ligating in a short duplex linker comprised of the following oligonucleotides: PBTMMOD1 (5'-gatcCGATCCGGCGGCCGCTtgca-3', with NotI site underlined and BamHI and PstI overhangs, 5' and 3' respectively, in lower case) and PBTMMOD2 (5'-AGCGGCCGCCGGATCG-3'). The plasmids were then co-transformed into S.cerevisiae CTY10-5d along with either pGAD2F-Cdc27 (for analysis of Cdc1-Cdc27 interactions) or pGAD2F-Pol3 and pAA-Cdc27 (for analysis of Cdc1-Pol3 interactions). β-galactosidase activity was determined by an X-gal agar plate overlay assay and later by ONPG liquid culture assay, as previously described [20,21]. Liquid culture assays were done in triplicate with the values for individual cultures being within 10% of one another. The results shown in Figure 4 are mean values from the triplicate assays.
Authors' contributions
The A and J mutant alleles were constructed and analysed in yeast by JSG who also performed all the two-hybrid analysis. The E mutant alleles were constructed and analysed in part by EVK. The remaining analysis was performed by FCG. AGB and THT contributed to the mapping of mutants to the three-dimensional structure of p50•p66N and analysis of the possible effects of Cdc1 mutations to Cdc1-Pol3 and Cdc1-Cdc27 interactions. The project was initiated and overseen by SAM. All authors read and approved the final manuscript.
We would like to thank Dr Finbarr Hayes (UMIST, Manchester, UK) for supplying the components required for pentapeptide mutagenesis and advice on their use. JSG was supported by a BBSRC Research Committee studentship, EVK by the Darwin Trust of Edinburgh, and FCG and SAM by a Wellcome Trust Senior Research Fellowship in Basic Biomedical Sciences. AGB and THT research was supported by UNMC Eppley Cancer Center Pilot Project LB595 to THT.
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The number of plants in Mr McGregor's magic potting shed increases
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Mr McGregor has a magic potting shed. Overnight, the number of
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Special clue numbers related to the difference between numbers in
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This Sudoku puzzle can be solved with the help of small clue-numbers on the border lines between pairs of neighbouring squares of the grid.
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First Connect Three game for an adult and child. Use the dice numbers and either addition or subtraction to get three numbers in a straight line.
Find out about Magic Squares in this article written for students. Why are they magic?!
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A student in a maths class was trying to get some information from
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designed to meet. . . .
There is a long tradition of creating mazes throughout history and across the world. This article gives details of mazes you can visit and those that you can tackle on paper.
Four friends must cross a bridge. How can they all cross it in just 17 minutes?
The puzzle can be solved by finding the values of the unknown digits (all indicated by asterisks) in the squares of the $9\times9$ grid.
An extra constraint means this Sudoku requires you to think in
diagonals as well as horizontal and vertical lines and boxes of
Four numbers on an intersection that need to be placed in the
surrounding cells. That is all you need to know to solve this
This sudoku requires you to have "double vision" - two Sudoku's for
the price of one
How have "Warmsnug" arrived at the prices shown on their windows? Which window has been given an incorrect price?
The puzzle can be solved with the help of small clue-numbers which
are either placed on the border lines between selected pairs of
neighbouring squares of the grid or placed after slash marks on. . . .
Use the differences to find the solution to this Sudoku.
60 pieces and a challenge. What can you make and how many of the
pieces can you use creating skeleton polyhedra?
Solve this Sudoku puzzle whose clues are in the form of sums of the
numbers which should appear in diagonal opposite cells.
This pair of linked Sudokus matches letters with numbers and hides a seasonal greeting. Can you find it?
Four small numbers give the clue to the contents of the four
surrounding cells.
A Sudoku with a twist.
A man has 5 coins in his pocket. Given the clues, can you work out
what the coins are?
Ben passed a third of his counters to Jack, Jack passed a quarter
of his counters to Emma and Emma passed a fifth of her counters to
Ben. After this they all had the same number of counters.
Whenever a monkey has peaches, he always keeps a fraction of them each day, gives the rest away, and then eats one. How long could he make his peaches last for?
A Sudoku with clues as ratios.


text:
2013 World Series Media Day
Similar imagesView all
Koji Uehara of the Boston Red Sox warms up with his son Kazuma during... News PhotoAdult,Baseball - Sport,Baseball Player,Baseball Team,Baseball World Series,Bestof,Boston - Massachusetts,Boston Red Sox,Exercising,Fenway Park,Horizontal,Koji Uehara,Males,Massachusetts,Media Day,Men,Only Japanese,Son,Sport,Sports Team,Sportsman,Topics,Topix,USA,Warming UpPhotographer Rob CarrCollection: Getty Images Sport 2013 Getty ImagesBOSTON, MA - OCTOBER 22: Koji Uehara (R) #19 of the Boston Red Sox warms up with his son Kazuma during team workout in the 2013 World Series Media Day at Fenway Park on October 22, 2013 in Boston, Massachusetts. The Red Sox host the Cardinals in Game 1 on October 23, 2013. (Photo by Rob Carr/Getty Images)


text:
Optimism and tension mark the mood of integration of combatants
At the furthest end of the Maoist People's Liberation Army third division cantonment in this central Nepal town, make shift tents are put up. More than two dozen young men and women, in blue jackets which announce their status as members of the monitoring team, have lined up for lunch and are chatting loudly. In one corner, their chief, and coordinator of the Secretariat for Special Committee for Supervision, Integration and Rehabilitation, Lt Gen (retired) Balananda Sharma, is sitting with the divisional commander, Dhan Bahadur Maskey 'Rajesh'.
Mr. Sharma is the man in charge of operationalising the seven-point agreement signed by major parties on November 1, which outlines that a maximum of 6500 Maoist combatants would be integrated while the rest can take rehabilitation packages or choose to voluntarily retire with cash packages. The first step of the process, which will commence on Saturday across seven cantonments across Nepal, is to regroup combatants into the three categories, depending on their preferred option.
The retired Army general expressed his optimism, and hope, that this will mark the conclusion of the peace process — five years after the Comprehensive Peace Agreement was signed on November 21, 2006 by Maoists and other political parties. Mr. Rajesh echoed him, tellingThe Hindu, "From tomorrow, combatants will make their respective choices and fill out related forms. We consider respectful integration an essential part of the process, and eventually PLA will not exist but become a part of history."
Integration forms ask for physical standards of combatants, and educational levels. Rehabilitation forms offer them training options in spheres ranging from formal and technical education, foreign employment, vocational training, to specializing in agriculture and livestock.
But there is recognition on all sides that the process will not be easy.
Dipendra Basnet 'Sangharshshil' is a company vice commander. Basnet said he is unhappy with the compromises made by his party leadership, and has decided to opt for voluntary retirement. "Instead of the Security Sector Reform (SSR) Model, the leaders have accepted a Demobilisation, Disarmament and Rehabilitation (DDR) Model. We will not get a constitution that delivers rights to the people."
PLA commanders accept that the top level divisions in the party, where chairman Prachanda and Prime M Baburam Bhattarai have backed the agreement while vice chairman Kiran has opposed it, is reflected in their ranks.
Mr Rajesh says, "We will abide by the seven-point agreement, but the deal is incomplete. If it is not implemented in a constructive, creative and respectful manner, the process will not move forward." He hinted about the need for another deal which will address issues like rank and education. Other parties have said educational levels of combatants at the time of their verification by U.N. in 2007 should be the criteria; Maoists have asked for current educational levels to be considered.
A senior source in the monitoring team said he could sense anxiety, irritation, and general tension among the combatants.
"This emotional state is a natural human condition. After all, they are making long-term decisions about their life." He said the resentment was directed against the party leadership, which had not been able to communicate the decision well enough. "This raises questions about their chain of command." But both members of the monitoring team, and Maoist commanders, said that despite challenges, the process of regrouping will take place.


text:
The mutual fund industry has lost around 4 % or 16 lakh retail folios during the second half of the past financial year, making it the sixth consecutive half-yearly decline, a Crisil release said.
According to the data released by the Association of Mutual Funds in India (AMFI), the industry had 3.95 crore folios, including institutional and high networth individuals, in H2 of FY14 compared to 4.13 crore folios in H1.
During the second half, equity category saw maximum decline in the number of retail folios as investors booked profit due to steep rise in the segment coupled with expectations of volatility ahead.
Interestingly, HNIs too turned away from equity during this period.
"After rising almost three times in H1 of FY14, the number of folios held by HNIs (individuals investing Rs 5 lakh or more) declined by 5 % or 1.5 lakh folios in the second half of FY14 as the segment chose to book profit," the rating agency said.
It, however, added the overall decline in the segment was offset by an increase in the number of folios in the debt and money market oriented funds.
In the gold exchange-traded funds (ETFs), the retail folio count fell by nearly 7 % to 4.89 lakh during the second half of the last fiscal compared to 5 % decline in H1 of FY14.
However, debt funds category continue to see rise in the number of retail folios.
"Debt funds added 2.91 lakh retail folios over the past six months, highest since September 2012," Crisil said.
During this period, retail equity AUM that stayed invested for more than two years, dipped to 62 %, lower than 68 % seen in the preceding six months.
"Out of the Rs 1.22 lakh crore of retail investment in equity-oriented funds, Rs 75,403 crore of investment were held for over 24 months," the report said.
Giving a segment wise data, the rating agency said while corporates continued to dominate the industry AUM with 49 % share in March 2014, HNIs were the second biggest contributors with 29 % share followed by retail with 19 % share.


text:
Rebecca Skloot has an essay in today's New York Times discussing the recent publication of the genome sequence of a widely used human cell line. Skloot, as most of you already know, wrote a book about the history this cell line – known as HeLa for Henrietta Lacks, the woman from whom they were obtained. In […]
Michael Eisen
I'm a biologist at UC Berkeley and an Investigator of the Howard Hughes Medical Institute. I work primarily on flies, and my research encompases evolution, development, genetics, genomics, chemical ecology and behavior. I am a strong proponent of open science, and a co-founder of the Public Library of Science. And most importantly, I am a Red Sox fan. (More about me here).
I can be reached at:
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Recent Posts
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text:
HBO disrespected Floyd...
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C Money
Paul McCloskey
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Re: HBO disrespected Floyd...
Originally Posted by
Did HBO disrespect Floyd? I think so. Hes gotta be the most hated boxer in the game especially on this forum. Did he get called out by Hatton and those ****ed up comments by Max Kellerman (way outta line). It just seems just like when they started to hate Bernard when Jermain started to rise. It just aint right for these gifted fighters that have done so much for the game. That was supposed to be Hattons night, Floyds says he's retired. What yall yelling his name for?
Why's Floyd call himself the best but doesnt fight the best comp or DEFEND like he is a true CHAMPION??? Floyd says a lot of **** and only some of it's true, Floyd LOVES MONEY and he's the "man"now
So I guess no one KNOWS until its PROVEN but there's plenty of money to be had!!
Kellerman sux Floyd's nuts, if anything his commentary was biased against Hatton.
Dont compare Hops to Floyd. Hops defended and fought the best and is a respected and UNIFIED CHAMP. One's got nothing to do with the other. HBO?? With so much $$$$$ around Floyd?? What do you expect??
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Cheap TFN to Liege flights
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Browse the most popular airlines servicing the Santa Cruz de Tenerife (TFN) airport. Visit their flights pages to learn more about the most popular routes these carriers fly.


text:
Investment club
|This article needs additional citations for verification. (March 2014)
An investment club is a group of less than 100 individuals who meet for the purpose of pooling money and investing; members typically meet on a periodic basis to make investment decisions as a group through a voting process and recording of minutes, or gather information and perform investment transactions outside the group.[1] The upper limit for an investment club is 25M on the sum of funds invested as individuals or as a group.[citation needed] There is no lower limit. Investment clubs provide members a means to learn about markets, while meeting and working with people who have similar interests.[2]
- 1 History
- 2 Starting a Club
- 3 Club Types
- 4 Conduct of business
- 5 Legal structure
- 6 Tax implications
- 7 See also
- 8 External links
- 9 Further reading
- 10 Reference Section
Although people have been investing in groups for thousands of years, the world's first investment club was allegedly established in Texas in 1898 back in the days of the Wild West when few investments could be considered safe. Investment clubs were seen as an ideal way of spreading the risk – away from just cattle.[3]
While the first investment club on record dates back to the 1800s in Western America, Various online communities devoted to this type of investing have recently emerged and have contributed to the personal investing boom in the United States. One of the reasons that people come together in investment clubs is to learn how to invest.
Starting a Club[edit]
Industry observers recommend prospective members adhere to these guidelines:[4]
- Consult a tax adviser on the taxation implications of the club's selected legal structure
- Establish an investment mission, policy and goals that allow active participation before investing
- Draw up a written operating or membership agreement covering asset management rules and membership changes
Club Types[edit]
|This section needs additional citations for verification. (March 2014)
High School Extracurricular Investment Clubs[edit]
This form of investment club usually meets to develop interest in investing and the intricacies of investing for minors, usually high school students. They typically are led by an adult who has vast experience in professional investing. An archetypal example is the Hopkins School Investment Club, which features an exclusive membership of high standing scholars at the Hopkins School in New Haven. Other elite prep schools, such as Choate or Kent, likely have Investment Clubs, as many graduates from the prep schools of New England go on to further their interest in economics as a major or minor in college.
Stock, Mutual Fund or Bond Investment Clubs[edit]
These clubs normally buy stocks, mutual funds or bonds. Prior to 2008 many of these type of clubs existed in major cities and were disbanded as they incurred significant losses from changes in the economy. Members and club owners were typically unsophisticated investors making investment decisions based on biased advertising paid for by the same companies who were seeking additional capital. Typical members have very little knowledge of: fundamental analysis, technical analysis, call and put options, financial analysis, and value investing to make wise investment decisions consistently. Club owners and members are not normally required to hold a securities license as long as they refrain from soliciting compensation in exchange for financial advice or soliciting the sale of stock, mutual funds or bonds in third party companies. One allowed exception is that a CEO / President or CFO of a C Corporation is allowed to solicit stock or bonds in their corporation as long as they provide a private placement memorandum that complies with the law to their new shareholders. One additional requirement is that non-accredited investors must be Directors of the C Corporation in addition to being shareholders.
Real Estate Investment Clubs[edit]
Clubs of this type are most commonly publicized as a real estate investment group or company, rather than a club. The legal distinction according to the SEC is having 100 members or less. Whereas, a real estate investment group would normally have more than 100 investors. These clubs normally buy real estate or notes (loans originated by a licensed third party) to benefit from: cash flow, appreciation of assets, instant equity, tax benefits from deductions or qualified dividends, group buying power, monthly or daily compounding, higher liquidity and diversification of risk.[5] Real estate transactions are limited to 35 participants to meet the legal requirements of many states for being closely held, and can be legally organized as a sole proprietorship transferring assets to a group living trust, limited liability company, limited liability partnership, general partnership or C corporation.
Business Investment Clubs[edit]
These clubs are often called incubators and are formed to purchase businesses that generate cash flow and equity. Investment types range from a group of people buying lower risk franchises with at least two years of significant revenues and positive cash flow like major fast food franchises, gas stations and hotels to higher risk businesses without an income history like start-ups, inventions, or product patenting and prototype development.
Hybrid Investment Clubs[edit]
Hybrid clubs are a combination of two or more of the above types of investment clubs. Typically, real estate investment clubs are lower in risk and provide higher returns of 21% to 70% than stock, mutual fund or bond investment clubs due to the ongoing need for housing and population growth.[6] Business investment clubs have about equal risk when compared to stock, mutual fund or bond investment clubs. However, business investment clubs have several additional benefits which include significantly higher returns and lower operating expenses measured by the net profit per year divided by the amount of cash necessary to purchase the business. Additionally, you can also hire yourself or family members to work in the business as employees.
Conduct of business[edit]
In order to operate an investment club, business must be conducted in a somewhat orderly fashion. The level of formality will vary based on the club type. A typical club will have informal channels of communication via mailing lists, Twitter accounts or message boards. In additional to the informal channels of communication, an investment club must set up formal channels of communication to conduct business. Typically, this is done with monthly meetings. A typical meeting agenda will include all the normal activity you would expect in an organization with elected officials. A typical meeting agenda:[citation needed]
- Roll call
- Old business – list of any unfinished business from the previous meeting.
- Summary of votes – Summary of all voted decisions from the previous meeting
- NAV presentation – NAV or the Net Asset Value of the Club will indicate what each club member is worth on paper.
- Available funds – a presentation of Available funds to invest.
- Presentation of current research – each member assigned to present an investment option will present his/her research.
- Vote to hold or invest – A vote to hold cash, sell current assets or invest in one of the investment options
- Investment vote – A final vote on an investment strategy specifying an exact amount to invest and strategy to execute that investment.
- Other business – Typically, other business will include proxy voting needs or other day-to-day business operational needs. This could also include options to change a brokerage house, or discussions of counter party risk.
- Formal close of meeting
Legal structure[edit]
Investment clubs are generally formed as general partnerships, but could also be formed as limited liability companies, limited liability partnerships, corporations, or sole proprietorship that transfer real estate assets to a group living trust (similar to a family trust). While an investment club could incorporate, the double tax treatment on corporate distributions makes the corporate structure less desirable than a partnership except in the case when a C Corporation pays out qualified dividends after deducting allowable expenses. Typically, a general partnership does not generate any tax liability on its own; instead, any tax liability is passed through to members each year. However, income taxes are generally much higher than taxes on qualified dividends.
In order to understand the legal structure that an investment club should choose, the club should first understand its club type. Each of the different club types will have different legal requirements as well as different reporting requirements. Typically, the SEC only requires reporting for investment groups with over 100 members, which is reclassified as an investment group, not an investment club. Publicly held offerings like a Real Estate Investment Trust as known as a REIT also have additional reporting requirements.
Tax implications[edit]
In the United States Investment club partnerships must file Form 1065 and Schedule K-1s with the IRS each year, and with states that require partnership filings.[citation needed] In the United Kingdom investment clubs and their members are required to submit form 185(new) to HMRC each year.[citation needed] Investment club accounting software can facilitate the management of a club's books and the preparation of tax filings.
See also[edit]
- List of finance topics
- National Association of Investors Corporation (also known as NAIC and BetterInvesting)
External links[edit]
- Investment clubs and the SEC - A FAQ on investment clubs provided by the SEC
- Stories from over 100 investment clubs about their experiences
Further reading[edit]
- Investment Clubs for Dummies by Douglas Gerlach and Angele McQuade (ISBN 0-7645-5409-3), published in 2001
Reference Section[edit]
- ^ sec.gov
- ^ Dunnan, Nancy (May 1, 2007). How to invest $50-$5,000: the small investor's step-by-step plan for low. Harper Paperbacks; 9th edition. p. 58. ISBN 978-0-06-112982-7.
- ^ "Investors are slowly joining the club". The Independent (London). June 3, 1995.
- ^ Barns, Roger (September 1990). Black Enterprise, Personal Finance. pp. 34–35.
- ^ Nase, Daniel (2013-08-28). "Real Estate Investment Club". Bakersfield Investment Club.
- ^ Nase, Daniel (2013-08-28). "Business Investment Club, Hybrid Investment Club". Bakersfield Investment Club.


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Globe 10.0: What do the Bruins need?
(Boston Globe) In this episode of Globe 10.0, Kevin Dupont and Scott Thurston debate whether the Bruins can win the Stanley Cup without making a trade. (By Alan Miller, Globe Staff)
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Stoner/Doom Bassists Thread Pt. 45: Rule 34: Handwired Gear Pronz
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Return to Mar Tesaro: Court Intrigue
The players ride hard through the day, arriving in Woodhurst after nightfall. Not wanting to draw attention to themselves, they sneak into town and drop their prisoner off in a safe house.
There was a brewing discussion about where to hide the prisoner. Although the town is loyal to the Brotherhood, parading one of the Queen's lieutenants through the streets would draw too much attention, as would grabbing rooms at the local inn. I didn't want the game to grind to a halt over the issue, so I told them the Brotherhood had provided a small home for just such an occasion. Never be afraid toThey see to Fariba's comfort before going to confront the Brotherhood's council. The council meets in a private hall; the players being the group's top lieutenants, their entry is welcomed warmly, but the Hawk has no interest in mincing words.
|"All right you bastards, time to politick."


text:
|Nov 10, 2011, 11:14 AM
Help on how to: Apple TV and Surround Sound
Greetings all.
First, let me preface my question with a bit of background info.
Last night I discovered the "Remote" app in the AppStore. I learned that I can control my iTunes music library with my new iPhone 4s (first time iOS user, loving it so far, btw).
I hooked up my laptop via AUX, to my Bose SoundDock. I was able to play my music from my iTunes library onto my SoundDock this way, which was great.
Then I thought, that perhaps there was a way I can do the same thing, without having the need for the laptop. Here is where Apple TV comes in. I just called Apple's tech support, but I am not sure the rep knew what he was saying.
So, onto my question:
Is there a way, that I can hook up Apple TV to surround sound, WITHOUT having my laptop powered on ? I don't want to hook up Apple TV to my TV or anything like that. I just want the Apple TV to go DIRECTLY into my surround sound, so that I can control my iTunes library with my iPhone.
Is this possible ?
Thanks all !
|Nov 10, 2011, 11:20 AM
1st problem is that the current gen apple tv doesn't have an analog output, only optical and hdmi. So the question is what you want to hook it up to? The sounddock? I believe that only has an analog input. I'm not sure what you mean by "Surround Sound".
You might be a better candidate for an Airport Express. It has an analog out and doesn't involve a TV at all.
|Nov 10, 2011, 11:24 AM
But, there is a cable on Amazon that is Optical Audio to 3.5mm AUX, so I was going to try that cable. (I think its called a TOSLINK cable)
I don't know what Airport Express is, but I am about to check it out on Apple's site.
Thanks !
Last edited by PassMeAGuinness; Nov 10, 2011 at 11:40 AM.
|Nov 10, 2011, 11:33 AM
They're usually $99, but HERE is a refurbed model for $69.
iPhone 6; iPad Air 2
|Nov 10, 2011, 11:43 AM
I think the airport express is what you need. Hook that up to the sounddock and you can stream music from iTunes or from you iphone using Airplay.
If there is no TV, avoid the apple tv, because at the very least you will need a TV to set it up.
|Nov 10, 2011, 11:59 AM
Wirelessly posted (Mozilla/5.0 (iPhone; CPU iPhone OS 5_0 like Mac OS X) AppleWebKit/534.46 (KHTML, like Gecko) Version/5.1 Mobile/9A334 Safari/7534.48.3)
Is your iTunes library on your laptop?
iphone 5 32gb - iPhone 6+ 64gb - MacBook Air i7 - MacBook Pro 2012 2.5ghz - mac mini 2011 dgpu i5 2.5Ghz
|Nov 10, 2011, 12:06 PM
I just spoke to another Apple Tech Support rep, and he said that I can completely bypass using my iTunes Library, IF I want to use the library stored on my iPhone.
Which is fine, because it is the exact same library (they are synced)
So, apparently, all I have to do is initially set up Apple TV (through a television, which is no problem) to sync with the iPhone. But you are telling me that Airport Express does this ? Without having the need to have my laptop on ?
Thanks again.
EDIT: Also, on the TOSLINK cable, according to the reviews on Amazon, it seems like people are using the cable to move sound from their TV to their surround sound.
Wouldn't this be the same case, in moving sound from Apple TV to my SoundDock ?
Again, thanks !
here are the reviews:
|Nov 10, 2011, 05:29 PM
If you just want audio from your device why don't you just get one of these http://www.belkin.com/IWCatProductPa...duct_Id=508754
|Nov 11, 2011, 07:31 AM
The fact that Apple TV has all the extras (Netflix, YouTube, photo slideshow, etc) is just a huge bonus, and would be nice to have. But then again, if it doesn't stream my music from my phone to my surround, (without the need of a TV or my laptop) then its useless to me.
Nonetheless, I am going to look into that link you gave me. Thanks
|Nov 11, 2011, 09:34 AM
You (OP) have a lot of varying wants here.
Consider getting a new receiver with HDMI and optical inputs. The Sound dock is OK but it's a little locked down (as you are experiencing right now) as your audio/video flexibility wants expand. A robust receiver should let you do just about anything you want to do for years to come. But paired with a current generation TV, a good receiver won't resolve the need to have that computer running. The current TV demands the link to a computer. If you need to be able to turn that computer off and/or take it out of the house and still access your content, the current TV is not your answer.
Consider TV version 1. It includes analog audio out and is almost as robust hardware-wise (720p 25fps vs. 720p 30fps). Version 1 won't have Netflix and a few other (newer) niceties and Apple has apparently abandoned it for any additional software updates. But it still does the job and just about everything version 2 can play can be played on version 1. Also, the remote app will run version 1 too, but no airplay. And it has more storage than the iPhone so if you can sync all of your media on an iPhone you could sync it all onto the TV hard drive and turn the computer off.
Consider a non-Apple set-top box like Roku and others. Some of them come with analog audio out. None of them link as tightly with iTunes but that may not matter that much (to everyone). Generally, you can put all your media on an attached USB drive and it is always ready to play without any computers running. If you can fit all of your media on an iPhone, even a smallish (cheap) attached hard drive should do the trick. If you happen to have some old storage sitting around doing nothing, pull it out of the retired computer and put it in an cheap case. Even older IDE drives can be resurrected for this kind of use (and work fine).
Lastly, (even though you say you don't want to do this) if you have a TV with output options (audio out), consider linking an TV directly to the TV and then using those output jacks to send the audio back to the Sound Dock. Generally, this means you'll need to turn your TV on for the pass through, but it might be a way to go from digital audio out (of TV version 2) to digital audio in (on the TV) to analog out (of the TV) to analog in on the Sound Dock. You should test this to see if it works as it may not (but I bet it will). The big downside here is having to leave your TV on to do this audio pass through but it would be one of the cheapest ways to do what you want (though still not solving the "leave the computer off" desire if you go with TV version 2).
Based on your stated wants, I think you should look to Roku or similar or maybe the first generation TV. That "turn the computer off" want just about begs you to choose something other than the current TV. But again, with those options, there's no Airplay and while Roku can handle Netflix and many others, the first gen TV doesn't have Netflix.
Last edited by HobeSoundDarryl; Nov 11, 2011 at 09:51 AM.
|Nov 11, 2011, 09:54 AM
|Nov 11, 2011, 10:01 AM
|Nov 11, 2011, 12:09 PM
Thanks again everyone, for your input.
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San Bernardino County Sheriff's Department
Press Release
LOCATION: Cities
of Loma Linda and Redlands
Huy Age: 23
San Bernardino, CA
VICTIM(S): Bank
of America
On July 29, 2014 at approximately 2:15 p.m., deputies assigned to the Loma Linda Police Department responded to Loma Linda University Medical Center reference a forgery and stolen check report. The recipient of the check, who resides in the city of Redlands, discovered the check was stolen from her mailbox. During the investigation, the suspect was identified as Huy Pham. Pham is on work release through the Riverside County Sheriff's Office for a previous criminal charge and conviction not related to this investigation. He was arrested and booked into the Central Detention Center in San Bernardino.
The San Bernardino County Sheriff's Department would like to remind citizens that mail theft and crime related to identity theft is a common occurrence.Citizens should check their mail box daily for important documents. Arrange to have billing invoices, bank account statements and mail with personal information emailed to them, when possible. This is one way to deter the theft of your personal information through the mail.
John McMahon, Sheriff-Coroner
San Bernardino County Sheriff-Coroner Department
Sheriff's Public Affairs Division
655 E. Third Street
San Bernardino, California 92415-0061
Telephone: (909) 387-3700


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RICHARD BRIGHT (1789-1858) was the son of a wealthy banker of Bristol, England and received a good education with many opportunities to travel. Bright studied at Guy's Hospital in 1810, and in 1813 received his medical degree from the University of Edinburgh. After a period of travel, he began to practice in London, and was appointed assistant physician to Guy's Hospital, where he worked and taught for forty years.
In Reports of Medical Cases (1827) Bright pointed out the association between diseased kidneys, dropsy and albuminous urine and clearly defined the disease since known by his name. The beautiful illustrations added to the value of the work.


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13.02.05 - By Jim Amato: As the month of February closes, HBO will showcase Miguel Cotto defending his W.B.O. 140 pound title against "Chop Chop" Corley. The junior welterweight division is red hot right now and the hard hitting Cotto is right in the thick of it. With the upcoming Kostya Tsyzu-Ricky Hatton fight being a sell out.. Also the tremendous interest in a possible Arturo Gatti-Floyd Mayweather Jr. battle, there is some SERIOUS money to be made.
Cotto has been a star on the rise for a while now. One would have to be blind not to see the tremendous talent in this young man. Now though the time is getting close to see if Miguel can lay claim to being the BEST in his weight class.
After the Corley bout, assuming he wins he will probably be matched with the winner of Tsyzu-Hatton or the Gatti-Mayweather Jr. winner. That's if that fight does come off. Floyd has some legal issues to tend to first. Corley is no cream puff but at this stage of their respective careers Cotto should emerge victorious. After that we'll just have to wait and see what the future holds for Miguel.
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