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Bacteria occur in facultative association and intracellular symbiosis with a diversity of eukaryotic hosts. Recently, we have helped to characterise an intracellular nitrogen fixing bacterium, the so-called spheroid body, located within the diatom Rhopalodia gibba. Spheroid bodies are of cyanobacterial origin and exhibit features that suggest physiological adaptation to their intracellular life style. To investigate the genome modifications that have accompanied the process of endosymbiosis, here we compare gene structure, content and organisation in spheroid body and cyanobacterial genomes.
Comparison of the spheroid body's genome sequence with corresponding regions of near free-living relatives indicates that multiple modifications have occurred in the endosymbiont's genome. These include localised changes that have led to elimination of some genes. This gene loss has been accompanied either by deletion of the respective DNA region or replacement with non-coding DNA that is AT rich in composition. In addition, genome modifications have led to the fusion and truncation of genes. We also report that in the spheroid body's genome there is an accumulation of deleterious mutations in genes for cell wall biosynthesis and processes controlled by transposases. Interestingly, the formation of pseudogenes in the spheroid body has occurred in the presence of intact, and presumably functional, recA and recF genes. This is in contrast to the situation in most investigated obligate intracellular bacterium-eukaryote symbioses, where at least either recA or recF has been eliminated.
Our analyses suggest highly specific targeting/loss of individual genes during the process of genome reduction and establishment of a cyanobacterial endosymbiont inside a eukaryotic cell. Our findings confirm, at the genome level, earlier speculation on the obligate intracellular status of the spheroid body in Rhopalodia gibba. This association is the first example of an obligate cyanobacterial symbiosis involving nitrogen fixation for which genomic data are available. It represents a new model system to study molecular adaptations of genome evolution that accompany a switch from free-living to intracellular existence.
A diversity of extracellular and intracellular symbiotic interactions occurs between bacteria and eukaryote hosts. The degree of interconnection between partners ranges from the weak dependence of some extracellular associations to permanent or obligate intracellular symbiosis. In the latter case, the endosymbiont is transmitted vertically to the next generation without any need for re-infection. The dependence on the host can be stabilised by loss or inactivation of genes, whose products are no longer required in the partnership [1,2]. Consequently, intracellular obligate symbionts loose their autonomy and therefore the capacity for a host-independent life style. Isolated from free-living populations, vertically transmitted endosymbionts have limited possibilities for genetic exchange through processes such as conjugation or transformation. Typically, endosymbiont genes diverge rapidly in comparison to their homologues in free-living relatives, a phenomenon that perhaps reflects genetic drift operating on small population size [3,4] and/or relaxation of structural/functional constraints on endosymbiont protein evolution [5]. The genomes of obligate intracellular bacteria often show an accumulation of deleterious mutations and a higher AT-ratio, accompanied with reduction in genome size when compared to their free-living relatives [6]. The dimension of these processes can be as extreme as seen in the reduced genome of Buchnera sp., an endosymbiont of aphids with a genome size of 641 kbp [7,8] and Carsonella, a γ-proteobacterial symbiont of phloem sap-feeding insects with a genome size of only 160 kbp [9]. Others, like the endosymbionts of the rice weevils Sitophilus zeamais (SZPE) and Sitophilus oryzae (SOPE) as well as Sodalis glossinidius, a symbiont of tsetse flies [10-12] represent the other extreme, and exhibit only slight reduction in genome size in comparison to free-living close relatives. Unlike the genomes of Buchnera and Carsonella, the genomes of these endosymbionts do not exhibit unusually high AT content.
Intracellular symbionts including SZPE and SOPE, have lost at least one of the recombinational repair enzyme genes encoded by recA and recF, a characteristic found in all other bacterial intracellular symbionts. The only known exception to this finding is the observation of intact genes for recA and recF in S. glossinidius. The occurrence of genes for flagella apparatus still encoded in the genome of S. glossinidus might indicate that this symbiosis has only recently been established [12]. Cyanobacterial interactions with plants and protists are also well known [13-17]. These associations are in most cases facultative, and do not involve vertical transmission. In these cases, the cyanobacterial symbiont re-infects the host every generation. As with other facultative symbioses, genetic modification of the endosymbiont genome is yet undetected [17].
The pennate diatom Rhopalodia gibba harbours endosymbionts closely related to extant cyanobacteria. Some of the closest free-living relatives of these so-called spheroid bodies are diazotrophic cyanobacteria of the Cyanothece sp. group [18]. The spheroid bodies encode genes for nitrogen fixation and have the capacity to fix molecular nitrogen [18,19]. Although the spheroid bodies are of cyanobacterial origin, they lack the typical photosynthetic pigmentation; and thus have been assumed to be photosynthetically inactive. Unlike all other unicellular nitrogen fixing cyanobacteria, they fix nitrogen under light conditions only [18-20]. We observe one to four spheroid bodies per host cell depending on culture conditions which are transmitted vertically to the daughter cells during host cell division [15]. Altogether, these findings have led to the supposition that the spheroid bodies of R. gibba are obligate endosymbionts. Physiological adaptation to an intracellular endosymbiotic association is expected to result in genome modification and this expectation has motivated our investigation of gene structure, content and organisation in the spheroid body's genome of R. gibba. In order to investigate this, we have constructed fosmid libraries of the spheroid body and Cyanothece sp. ATCC 51142 and analysed genomic regions of special interest. Here we describe observations and analyses of the nif-gene region and also loci relevant to the question of the obligate nature of the spheroid body endosymbiosis. Our investigations show massive genomic changes introduced into the spheroid body's genome. These include inactivation and losses of genes and the creation of large non-coding AT-rich areas. Our observations confirm the obligate nature of the spheroid body endosymbiont and provide insight into the nature of genome changes that accompany endosymbiosis and organelle formation.
The nif-gene region of spheroid bodies and Cyanothece sp. ATCC 51142
For this study we cloned and sequenced the nif-operon and flanking regions from the genomes of the intracellular symbiont of the diatom R. gibba, the spheroid bodies, and a close free-living relative, the diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 (Figure 1). Altogether we sequenced and analysed a contiguous 63,362 bp Cyanothece fragment comprising the nif gene region and a contiguous 51,475 bp fragment including the corresponding region of the spheroid body's genome. Additionally, we sequenced and analysed 140,000 bp of non-contiguous genomic DNA of the endosymbiont. Using these additional datasets we characterised sequences in the spheroid body's genome for recA, recF, psbC and psbD. For further analyses we also used information from the genome of the recently sequenced Cyanothece strain CCY0110 and other closely related cyanobacteria whose genomes have been sequenced and are available in the NCBI nr database. To obtain a phylogenetic framework for making inference of genome modification in the spheroid body's genome we reconstructed maximum likelihood gene trees for all homologues greater than 200 amino acids in the 63,362 bp region. Where possible, trees were outgroup rooted using homologues from Synechocystis sp. PCC 6803. Figure 2 shows supernetworks [21] built for these taxa. These networks summarise the relationships in individual gene trees which do not necessarily need to have the same taxon sampling. In this analysis, with some proteins (NifB, NifN, NifS), the spheroid body's genome was found to be most closely related to Cyanothece sp. ATCC 8801 (Figure 2a) but with other proteins (NifD, NifH, NifK, NifE), spheroid body sequences have a closer phylogenetic relationship with Cyanothece sp ATCC 51142, Cyanothece strain CCY0110, Crocosphaera watsonii WH 8501, and Gloeothece sp. KO68DGA (Figure 2b).
Figure 1. Gene content in, and downstream of, the nif gene region of Cyanothece sp. ATCC 51142 and spheroid body of R. gibba. Blue and red bars represent orfs coded on the leading or lagging strand of DNA, respectively. The locations of pseudogenes in the spheroid body fragment have been indicated with green bars. Genes have been named either according to homology matches in BLAST analyses or numbered consecutively for each organism (see also additional files 1 and 2). A GATA [29] plot is shown and indicates regions of high synteny between both organisms. GATaligner settings were: Window size: 100; Match: 5; MisMatch:-4; Gap Creation:-10; Gap Extension:-4; Raw Score Cut Off: 80. GATAPlotter score settings: Max: 141 bits, expect 1E-33; Min: 46.8 bits, expect 5E-5. GATAPlotter scores have been represented using a greyscale bar. Regions of the spheroid body genome showing modifications of special interest have been indicated. A) Gene inactivation by pseudogenisation (e.g. fdxN*); B) Gene deletion with DNA loss (e.g. cyl0012); C) Gene deletion without DNA loss resulting in large non-coding regions (e.g. cyl0016); D) Gene deletion with DNA loss resulting in gene fusion (e.g. cyl0019). See text for further description of individual modifications.
Figure 2. Supernetworks displaying relationships inferred from phylogenetic analysis of different gene regions. A. Supernetwork reconstructed using strict consensus maximum likelihood trees for NifB, NifN, NifS and ABC, HesA, NdhK, ModC, PerM, UvrD, SAM, Sbr0014 of different cyanobacetria (see Materials and Methods). B. Supernetwork reconstructed using strict consensus maximum likelihood trees for NifD, NifH, NifK, NifE, and ABC, HesA, NdhK, ModC, PerM, UvrD, SAM, Sbr0014. The networks are outgroup rooted using Synechocystis PCC 6803. A reticulation occurs in the centre of both graphs because of the local instability in the placement of Synechocystis PCC 6803.
Figure 1 summarises the nif-operon-related regions of Cyanothece sp ATCC 51142 and the spheroid body's genome. In both, components of the nitrogenase dependent cyanobacterial nitrogen fixation machinery are encoded, including structural genes of the nitrogenase nifH, nifD and nifK, cofactors (nifB, nifN, nifE, nifV, nifW) and processing proteins for metal centre biosynthesis (nifU, nifS). As shown in figure 1, synteny of the nif-genes including the size of intergenic regions is very high. There is an overall G/C-content of 40.8% for this region of Cyanothece genome and a G/C-content of 37.2% for the spheroid body sequence. Codon usage is nearly equivalent in both genome regions with a slight AT-bias at the third codon position in the spheroid body genes (see Table 1).
Table 1. Genome features of obligate intracellular symbiotic and parasitic bacteria
Although the spheroid body's genome contains the same set of genes at the nif locus as in Cyanothece ATCC 51142, remarkable differences are apparent. Notably, between nifB and nifS, the functional fdxN gene is replaced by a pseudogene (fdxN*) in the spheroid body's genome. The coding sequence is interrupted by several stop-codons. In contrast, an intact reading frame for fdxN is conserved in all other close free-living cyanobacterial relatives, indicating formation of the pseudogene is a derived feature of the spheroid body lineage (Figure 3). The presence of a truncated nifU gene is also derived on the endosymbiont lineage, corresponding to approximately 170 amino acids of the N-terminus (Figure 4). This nifU homologue still encodes an intact open reading frame for the [2Fe-2S] binding and C-terminal NifU-domain. Interesting, in more distantly related cyanobacteria (Synechocystis sp. PCC 6803 and Gloeobacter violaceus PCC 7421) a truncated homologue is also present. Based on the phylogenetic analyses reported in Figure 2 and also comparative analyses of the NifU protein (not shown) it appears that truncation of nifU in spheroid bodies is a derived feature of the endosymbiont lineage.
Figure 3. Analysis of Ferredoxin N. A. Multiple alignment of cyanobacterial FdxN proteins. Accession numbers: CyaATCC51142: AAW56985.1 (Cyanothece sp. ATVV51142); CyaPCC8801: AAC33373.1 (Cyanothece sp. PCC8801); CyaCCY0110: ZP_01727762.1 (Cyanothece sp. CCY0110); GloeK068DGA: BAF47148.1 (Gloeothece sp. KO68DGA); NostPCC7120: AAA22005.1 (Nostoc punctiforme PCC 7120); AnabATCC29413: YP_324413.1 (Anabaena variabilis ATCC 29413). B. Alignment of Cyanothece sp. ATCC51142 FdxN protein with the spheroid body fdxN* pseudogene translated in 3 forward reading frames. Evidence of homology, at the level of amino acid similarity, is distributed across all 3 reading frames of the pseudogene, indicating multiple substitutions and single nucleotide deletion events.
Figure 4. Truncation of nifU in spheroid bodies. A. GATA plot for Cyanothece and spheroid body nifU indicating conserved regions. GATaligner settings: Window size: 100; Match: 5; MisMatch:-4; Gap Creation:-10; Gap Extension:-4; Raw Score Cut Off: 92.0. GATAPlotter score settings: Max: 141 bits, expect 9E-38; Min: 28 bits, expect 9E-4. GATAPlotter scores are indicated. B. Multiple alignment of predicted amino acid sequences for NifU indicating an N-terminal truncation in the homologue from the endosymbiont. NifU accession numbers: Cya51142: AAW56987.1 (Cyanothece sp. ATCC 51142); Cya0110: ZP_01727764.1 (Cyanothece sp. CCY0110); GloKO68DGA: BAF47150.1 (Gloeothece sp. KO68DGA); Cya8801: AAC33371.1 (Cyanothece sp. PCC 8801); Lyn8106: ZP_01620769.1 (Lyngbya sp. PCC 8106); TriIMS101: AAF82636.1 (Trichodesmium sp. IMS101); NdCCY9414: ZP_01628437.1 (Nodularia spumigena CCY9414); Nos73102: ZP_00112317.1 (Nostoc punctiforme PCC 73102); SynJA2: YP_476679.1 (Synechococcus sp. JA-2-3B'a(2–13)); sb8: AAW57048.1 (spheroid body); Syn6803: NP_442853.1 (Synechocystis sp. PCC6803); Glo7421: NP_925823.1 (Gloeobacter violaceus PCC 7421); Cro8501: ZP_00516385.1 (Crocosphaera watsonii WH 8501).
Gene deletions and modifications downstream the nif-region
Conserved downstream of the nif-operon genes in Cyanothece ATCC 51142 and the spheroid body are genes encoding subunits of the NADH-dehydrogenase and ferredoxin as well as transporters for Fe and Mo (Figure 1). However, genes encoding photosynthetic proteins, which are precursors for cytochrome c6 (petJ) and plastocyanin (petE), are absent in the spheroid body in this genome region. Interestingly, elsewhere in the genome, the photosystem II protein genes psbC (CP43) and psbD (D2) exist in an operon-like structure, similar to that of Cyanothece sp. CCY0110 and other cyanobacteria. However, in the spheroid body these genes are highly truncated or disrupted by several stop codons and thus exist as pseudogenes. This finding is consistent with the lack of photosynthetic activity previously reported for the spheroid body [18,19].
Downstream of the highly conserved nif-gene region, other genetic modifications can be inferred. In Cyanothece ATCC 51142, and other closely related cyanobacteria the open reading frame (orf)cyl0012 is flanked by the genes for an iron-transporter (feoA) and a Mo-ABC-transporter (modC). This orf has been deleted in the spheroid body's genome (Figure 1). In this case the whole gene has been removed without trace of pseudogenisation or local sequence conservation. The orf identified as cyl0019 in Cyanothece ATCC 51142 has also been lost from the nif-region. In this species and other close relatives, this orf is flanked by two conserved orfs cyl0018 and cyl0020. In the spheroid body's genome, cyl0019 is deleted and the flanking orfs cyl0018 and cyl0020 are fused to give to the hypothetical protein sbl0010 (Figure 5). Not all deletions of genes in the spheroid body's genome have resulted in genome compaction as some genome regions appear replaced by non-coding regions as described in the following section.
Figure 5. Spheroid body orf sbl0010 encodes a fusion protein derived from homologues of the Cyanothece sp. ATCC51142 Cyl0018 and Cyl0020 proteins. A. Alignment of predicted amino acid sequences for spheroid body protein Sbl0010 and Cyanothece proteins Cyl0018 and Cyl0020. Deletion in the endosymbiont genome of cyl0019 in the creation of sbl0010 can be inferred during reductive genome evolution. In Sb10010, homologues of Cyl0018 and Cyl0020 have been conserved in full length and are separated by a 17 amino acid residues. Cyl0018: green, Cyl0020: orange, Sbl0010: black. B. Cyl0018 and Cyl0020 are highly conserved in cyanobacteria closely related to the spheroid body. They are aseparated by 1–5 genes when they co-occur at the same locus, but in some cases they are encoded at different loci of the genome (indicated by x).
Extensive modifications lead to large non-coding regions in the spheroid body's genome
A significant difference between the genome of Cyanothece sp. ATCC 51142 and that of the spheroid body is the extent of non-coding DNA stretches greater than 500 bp (Figure 6). In the former there are three non-coding stretches at the nif locus (including downstream region). There are seven such regions in the spheroid body's genome fragment. One of these additional non-coding regions is located adjacent to the nif gene region, in a region of high synteny between both genomes. The non-coding regions of the spheroid body's genome are characterised by elevated levels of A and T nucleotides. Several pseudogenes are also located within these genome regions (Figure 1 and 6).
Figure 6. A/T-G/C frequencies in Cyanothece sp. ATCC 51142 and spheroid body genome fragments. A/T-G/C-plot for the genome region shown in Fig. 1 (red: AT; blue:G/C), indicating a high AT composition in the large non-coding regions (black arrows) of the spheroid body fragment.
In Cyanothece ATCC 51142, the gene fdxIII and an orf coding for a conserved hypothetical protein (cyl0018) flank four open reading frames (cyl0014/vagC, cyl0015/pilT, cyl0016, cyl0017/vapC). Three of these have highest similarity to virulence associated proteins (vap-proteins) and the fourth has greatest similarity with proteins containing a PIN-domain (Figure 1). In spheroid bodies, fdxIII and the homologue to cyl0018 (as part of sbl0010, see above) frame a non-coding region of about 2000 bp which contains two pseudogenes: for a flavodoxin, long-chain hypothetical protein and an orf conserved in Crocosphaera sp.(CwatDRAFT_1967). This non coding region shows an increased AT-ratio of 73.5% (Figure 6). Its presence is the result of extensive genome modification which has led to the deletion of multiple genes. Unlike other regions where there has been gene deletion (e.g. as with cyl0012), this modification is not accompanied by the deletion of the genomic regions. It is possible that these regions might be the outcome of multiple mutations leading to the loss of genes, no longer recognizable as pseudogenes, with preservation of the genomic locus, which might be subsequently eliminated [22]. Because the whole genome size of spheroid bodies is not experimentally determined so far, it is not known if these modifications have lead to an overall decrease of the spheroid body genome size. As described in more detail in the discussion part, we used a bioinformatic model to predict the size of the whole spheroid body genome. Using this prediction, the genome size is estimated to be approximately 2,6 Mb.
To investigate whether intact copies of missing or pseudogenised genes (e.g. after genome rearrangement or gene duplication events) are present elsewhere in the spheroid body's genome, we performed PCR analysis with specific or degenerate primers for the cyanobacterial genes fdxN, petJ, psbC, cyl0012 and cyl0017. As shown in Figure 7, no products were amplified using either spheroid body or R. gibba DNA as template, indicating that the identified (pseudo)genes do not have functional counter parts encoded elsewhere in the endosymbiont's genome.
Figure 7. PCR analysis of missing or pseudogenisised genes. PCR analysis of Cyanothece sp. ATCC 51142 (CY), R. gibba (RG) and spheroid body (SB) DNA. Amplification with primers specific for the cyanobacterial fdxN, petJ, psbC, cyl0012 and cyl0017 genes show that the analysed genes are not encoded in R. gibba and the endosymbiont's genome. CYrecF and SBrecF were used as positive controls. GeneRuler™ Express DNA Ladder (Fermentas) was used as molecular weight standard.
RecA and RecF are encoded by the spheroid body's genome
The proteins RecA and RecF play an important role in recombinational repair of DNA as well as roles in other repair pathways like nucleotide excision (reviewed in [23]). From the study of insect-bacterium symbioses it is known that gene loss and pseudogene creation is often associated with defects in recA, and/or recF [10]. In order to investigate whether the observed inactivation and deletion of genes in the spheroid body's genome might be explained by defects in the bacterial repair systems, we searched the genomes of the spheroid body and of Cyanothece ATCC 51142 for recA and recF. As shown in Figure 8, intact and highly conserved orfs can be identified in both genomes, indicating that repair pathways are active in the R. gibba endosymbiont.
Figure 8. Pairwise alignments for RecA and for RecF. Alignment of RecA (left) and RecF (right) homologues from the spheroid body (SB) and Cyanothece sp. ATCC51142 (CY) genomes. The spheroid body encodes complete full length recA and recF orfs.
Accumulation of pseudogenes in the spheroid body's genome
As already mentioned, by analysing the contiguous spheroid body's genome fragment (Figure 1) several pseudogenes could be readily identified, among them a mutated ferredoxin gene (fdxN*) within the nif-operon as well as other genes downstream of the nif-gene region. Additional pseudogenes for conserved cyanobacterial proteins were also identified when screening 140,000 bp of non-contiguous genome sequences from the spheroid bodies. These were found either via BLAST homology search (minimum e-value: 5e-10) or by analysis of spheroid body's genome regions corresponding to operons conserved in other cyanobacteria. Genetic regions with homologues in other species, divided into two fragments by one stopcodon or frameshift or just truncated were considered pseudogenes if the similar gene region was less than half the length of its homologue [12]. Deleterious mutations leading to pseudogenes were found in genes encoding proteins that affect cellular processes such as cell wall biosynthesis and transposon controlled genome rearrangement (summarized in Table 2). In Cyanothece sp. ATCC 51142, only one pseudogene was identified within the genome fragment that contained the nif-operon (Table 2). In contrast, six pseudogenes were found in the spheroid body's genome fragment. No pseudogenes were identified in the genome of Cyanothece sp. strain CCY0110 which is a very close relative of Cyanothece sp. ATCC 51142.
Table 2. Pseudogenes in all sequenced spheroid body genome fragments (totalling 192335 bp) and pseudogenes identified in the Cyanothece sp. ATCC 51142 genome region shown in Figure 1 (63344 bp)
An intriguing example of an obligate intracellular symbiotic interaction is the cyanobacterium-diatom symbiosis found in Rhopalodia gibba [18]. Here the symbiont (spheroid body) can fix nitrogen for its eukaryotic host, and we have hypothesised that this capacity has been a driving force for establishing the intracellular endosymbiotic relationship [17]. The spheroid body of Rhopalodia gibba provides an opportunity to investigate changes in endosymbiont physiology and genome evolution during adaptation of a symbiont to an intracellular environment.
Previous studies have reported changes in the genomes of bacteria following development of symbiotic relationships. In bacteria that are thought to have recently or transiently become symbiotic, changes include occurrence of multiple transposable elements and deletions of important components of recombinational DNA repair mechanisms [1]. In longer established symbiotic and parasitic eukaryote-bacterium interactions, significant gene losses have been observed, and these have been accompanied by reduction of genome size and generation of AT rich genomes [3,6,24]. Changes that have occurred in the spheroid body's genome can not be categorised as an obvious example of the former or latter relationship. For example, the spheroid body's genome encodes several transposase genes, all with disrupted reading frames, indicating that these are pseudogenes. This finding is consistent with stability of the diatom-spheroid body endosymbiosis and a long term host-endosymbiont interaction, which can be traced back to the Miocene [25]. Contrasting with the occurrence of transposase pseudogenes is evidence suggesting a functional DNA repair system in the spheroid body's genome. This is a finding more consistent with a relatively young endosymbiotic relationship. In nearly all intracellular bacteria studied to date, at least one of the genes encoding the DNA repair proteins RecA and RecF has been eliminated. It is thought this might be necessary to facilitate restructuring of the symbiont genome (for the exception see [12]). In the spheroid body's genome both recA and recF are present and have intact open reading frames. Thus the genome modifications that we report for the spheroid body's genome have all occurred against a background of a presumably intact DNA repair system. These modifications suggest that selective pressure for certain genes has changed upon establishment of the interaction, and the challenge is to attempt to understand the potential relevance of these for necessary and redundant functions in an obligate endosymbiotic relationship. For example, gene truncations as detected e.g. in nifU, would remove genes redundant for diazotrophic growth [26], and such deletion might be an early event in genome reduction of the symbiont. A subsequent or perhaps parallel step would include the inactivation of genes whose gene products are no longer needed for the initial symbiotic association. For this to occur, various different possible scenarios could be hypothesized: inactivation of genes by deleterious mutations resulting in the accumulation of pseudogenes or loss of genome fragments by deletion of larger DNA portions via rearrangements [27]. Another hypothesis posits a "domino-effect" of initial pseudogenisation triggering subsequent large-scale gene loss [28]. In this scenario, random pseudogenisation might lead to the inactivation of a pathway due to mutation of a single essential factor, followed by large-scale deletion of other genes involved in this pathway. In each case, the selective pressure would be different for genes coding for different functions, and loss would depend on whether function could be compensated by other genes in the endosymbiont or host cell genome. In the latter case, as in highly adapted interactions, signal-dependent transport of the protein from the host cytoplasm to the endosymbiont would be necessary.
We detected several examples for the disruption of coding regions by mutations (Table 1), in which the original gene is still detectable by analysis of all three possible frames. This includes psuedogenisation of fdxN (fdxN*), a gene which has been found to be non essential for nitrogen fixation in Anabaena variablis [20] and several other genes on spheroid body's genome fragments that we have sequenced (Table 2, Figure 1). Such observations provide further evidence that pseudogenisation of genes, which are non-essential for endosymbiotic life-style, is an important feature in the early reductive genome evolution of obligate intracellular cyanobacteria. Gene loss through independent DNA deletion events could also be inferred in comparative analysis of the spheroid body's genome fragment; among these the deletion of factors conserved in diverse cyanobacterial lineages (cyl0012, cyl0019). Due to elimination of the immediate DNA region, these modifications have led to a localised increase in gene density. In one extreme, deletion has produced a fusion of non-adjacent genes on the endosymbionts genome (sbl0010). In other cases of gene deletion, genes have been removed and replaced with non-coding sequence that is much higher in AT-content than occurs in the coding regions (Figure 6). It is unclear whether this difference in composition reflects a shift in substitutional bias favouring A and T residues, and/or whether an existing bias becomes more apparent in de-novo regions that are under reduced structural/function constraint. In either event, the existence of these AT rich non-coding regions suggests that pseudogenisation and DNA deletion are not inevitably linked events in a sequential process of degenerative genome evolution in spheroid bodies. However, non-coding regions are rare in genomes of free-living bacteria. Since DNA can be introduced in several ways into prokaryotic genomes, their compactness is maintained by the deletion of harmful DNA. Given the intracellular existence of spheroid bodies, it is possible that their genome is less exposed and less susceptible to introductions of foreign DNA through mechanisms of horizontal gene transfer and lysogenic bacteriphages in comparison to those of free-living bacteria. If so, processes excluding non-coding DNA and pseudogenes from the spheroid body's genome may well be less efficient than those operating in free living bacteria. Such a hypothesis might help explain the greater extent of non-coding DNA and pseudogenised genes in the spheroid body's genome. Increased mutation rates, thought to be associated with reductive genome evolution would contribute to accumulation of these genome features [29]. The genome modifications observed in the spheroid body are in some respects comparable to those of Sodalis glossinidius. A large fraction (49%) of the Sodalis genome is composed of non-coding DNA that has accompanied reductive genome evolution. Moreover, the Sodalis chromosome contains many unusual pseudogenes [12]. The spheroid body's genome differs from Sodalis with respect to their generally higher AT-content.
The diverse features of reductive genome evolution in obligate intracellular symbionts (and pathogens) include a significant reduction of overall genome size in these organisms. However, the experimental determination of the spheroid body's genome size using standard molecular techniques is difficult due to the extreme stability of the host-spheroid body interaction and the limited amount of intact and purified endosymbionts that can be obtained from R.gibba. Recently in a study on the dynamics of reductive evolution, exponential relationships were inferred between genome size and SSU rDNA GC-content in mitochondria, free-living and obligate intracellular bacteria [30]. Based on the model these authors propose, and using 16S sequence data previously published [18], we have estimated that the genome size of spheroid bodies is approximately 2.6 Mb. The genome size of free-living Cyanothece sp. CCY0110 is 5.8 Mb. Hence if our estimate of the spheroid body's genome size is accurate, this estimate suggests that reduction has produced a genome currently similar in size to that of Synechococcus (2.2–2.6 Mb), and may indicate that the endosymbiosis is still at an early state of development.
Our comparative analyses of spheroid body's genome fosmid sequences indicate that the photosynthetic genes psbC and psbD have been inactivated by mutation in the endosymbiont genome. These gene products are essential factors in the photosynthetic light reaction of photosystem II [31]. According to the "domino-effect" hypothesis [28] initial deletion of components such as PsbC and PsbD is expected to lead to mass deletion of other genes involved in photosynthetic light reactions. Consistent with this prediction, additional photosynthetic factors that occur in closely related cyanobacteria are either absent (e.g. the cytochrome PetJ and the plastocyanine precursor PetE) or appear as a non functional pseudogene (e.g. the flavodoxin fldA*) in the spheroid body's genome.
Aside from gene loss resulting from reductive genome evolution, the absence of certain genes within the analysed genome region could also be explained by gene duplications or rearrangements. Without the complete sequence of the spheroid body's genome we can not exclude the possibility that following duplication, pseudogenisation has affected copies of some genes within the analysed genome region, while functional copies are retained elsewhere. However, the phenotypic loss of photosynthetic pigmentation indicates a complete loss of at least one essential factor of photosynthesis in the endosymbiont's genome. In addition, PCR analysis did not identify intact psbC and psbD genes present anywhere else in the spheroid body's genome (Figure 7).
The diverse modifications in the analysed spheroid body's genome fragment are not equally distributed over the whole sequence but accumulate downstream of the conserved nif gene region (Figure 1 and 5). This skewed distribution of degenerative modifications possibly reflects purifying selection acting across this genome region during the molecular adaptation process [32]. Aside from the mutation of fdxN* – a protein unimportant for nitrogen fixation -and the truncation of nifU, all proteins for nitrogen fixation are conserved in the region without signs of degenerative genome evolution. This conservation of nif genes is consistent with the hypothesis that molecular nitrogen fixation has been an important driving force for the endosymbiotic interaction.
It can be expected that endosymbiont and host biochemistry will change with the development of the symbiotic interaction. Genes whose products become superfluous for symbiont-host coexistence are expected targets for mutation. At earlier stages of accumulation of deleterious mutation, holomologues will still be identifiable by BLAST homology searches. Table 2 lists many pseudogenes that may fit this category.
A diverse range of genetic modifications have occurred in the genome of R. gibba spheroid bodies and these would compromise the ability of the endosymbiont to exist as a free-living cyanobacterium, thereby confirming their suspected obligate status. Our findings provide insight into the genome evolution of a nitrogen-fixing endosymbiontic cyanobacterium living within a unicellular eukaryotic host. These are of special importance, as past inferences about processes of reductive genome evolution have mainly been based on the study of insect-bacteria interactions. In these, the symbionts reside within special cells or organs and thus their genomes may haven been subject to selection pressures different from those acting on the genomes of intracellular endosymbionts found in unicellular host organisms. Further analysis of the whole spheroid body's genome and comparison with free-living cyanobacteria will provide additional important information on the age of the interaction and the importance of different molecular processes and genetic modifications. Since the spheroid body is derived from cyanobacterial-like ancestors, the interaction could also serve as useful model system for understanding early events in the evolution of chloroplast genomes.
Symbiont Isolation and Purification
Spheroid bodies of R. gibba were isolated and DNA was purified as described by Prechtl [18]. Cyanothece sp. ATCC 51142 genomic DNA was isolated using standard procedures [33].
Construction of gDNA libraries
Fosmid libraries of spheroid bodies and Cyanothece sp. ATCC 51142 were prepared using the fosmid library construction kit (Epicentre). After physical shearing, the DNA was blunt-end repaired and gel-fractionated to a fragment size of approximately 40 kbp as described by the manufacturer. Insert-DNA was ligated in the pCC1-Fos™ vector, constructs were in vitro packaged into phage particles and transfected into Escherichia coli EPI 300™-T1R.
Screening for nif-gene region, recA and recF
Screening for clones containing the nitrogen fixing operon and flanking sequences and clones containing the recA and recF genes was performed using colony-PCR screening with oligonucleotides specific for spheroid body and the Cyanothece nifD-, recA- and recF-gene, respectively. Primer sequences were SBnifD_uni: 5'-CGG ACA AAG AAA ACG CAG AAT TTG-3', SBnifD_rev: 5'-CAG AAC GTC ATC ACA CTG TTT TTG-3', CynifD_uni: 5'-CCG TCA CGT TGT TCC TGC TTT C-3' CynifD_rev: 5'-CCA AGG GGT GCC AAT TAA TCC C-3', SBrecA_uni: 5'-CTA CTC TCG CTC TCC ATG CGA TTG-3', SBrecA_rev: 5'-CGG CGA ATA TCT AAA CGG ACT GAG-3', CyrecA_uni: 5'-GAT CGC AGA GGT GCA AAA GGC TG-3', CyrecA_rev: 5'-CAG TTC CTC CGG TGG TGA CTT C-3', SBrecF_uni: 5'-TCG GAC CTC AGC ATT ATC-3', SBrecF_rev: 5'-TCG ATG AGGTCC TAC TAA GC-3', CyrecF_uni: 5'-GCC GTC GAA TTA TTA GCA ACC C-3' and CyrecF_rev: 5'-GAA TTC GAC ATC ATC TCG ATG GG-3'. Clones for the upstream and downstream regions of positive nif-fosmids were obtained using the primers F13A12/3_uni: 5'-GAA CTC TAC AAT ACA GAT TAA CCG C-3', F13A12/3_rev: 5'-CAC TAA TCC ATC TAG ATT AGC CAC T-3', F13A12/5_uni: 5'-GGG CAT TCC AGA ATT AGA AGT AGG-3' and F13A12/5_rev: 5'-CTG TAG CCA AGC CAA AGT CGT TAT G-3' for the spheroid bodies and F4D10/5_uni: 5'-CAA GCT GTC TTT GGA CAA AAG-3', F4D10/5_rev: 5'-CGT TGA AGG TTT CCT CAA AAC-3', F4D10/3_uni: 5'-GAT ATC GTT GAA ACC TAT CGA G-3' and F4D10/3_rev: 5'-GAA TGT TAG GAC GAG CAA AAG G-3'. PCR reactions were performed using standard procedures.
Cloning of positive fosmids
Preparation of fosmids and other DNA was performed according to standard protocols [33]. For subcloning, fosmid DNA was physically sheared by sonification. After blunt-end repair using the DNA Terminator Kit (Lucigen) and size fractionation by gel electrophoresis, fragments between 1000 and 1500 bp were isolated. The fragments were ligated in the pEZSeq™-vector (Lucigen) as described by the manufacturer and used to transform E. coli XL1blue MRF' cells. Sequencing of shotgun plasmids was carried out using cycle sequencing with 700 and 800 nm fluorescent labeled oligonucleotides and the LICOR™ sequencing system. 5'- and 3'-end sequencing of positive fosmid clones was performed using the primers M13 (700): 5'-GTA AAA CGA CGG CCA GT-3' and a modified pCC1™/pEpiFOS™ RP-2 (800): 5'-GCC AAG CTA TTT AGG TGA G-3'. Shotgun inserts in the pEZSeq™-vector were sequenced with M13_for: 5'-AGC GGA TAA CAA TTT CAC ACA GGA-3' and M13_rev: 5'-CGC CAG GGT TTT CCC AGT CAC GAC-3'.
PCR analysis of missing or pseudogenised genes in the spheroid body's genome
PCR analysis of missing or pseudogenised genes in the spheroid body's genome was performed with primers specific for cyanobacterial fdxN, petJ, psbC, cyl0012 and cyl0017 genes. Primer sequences were fdxNuni: 5'-AGT TAC ACT ATC ACC AAT G-3', fdxNrev: 5'-ATT TCT TGG GAG TAA GCA TC-3', CYpetJuni: 5'-ATG AAA AGA TTA TTG TCC CT-3', CYpetJrev: 5'-TGC TTG ACT TAA RAC ATA AG-3', psbCuni: 5'-ACG TAG TTA AAG GAG TTA ACG-3', psbCrev: 5'-TTC GGC TAT CTG CTG AAA GC-3', CY0012uni: 5'-CCT CTC AAC TTA GCC ATT AG-3', CY0012rev: 5'-AAG CTT TGC TGT GTA GAA AC-3', CY0017uni: 5'-ATN RTN GGN TGY MGN AAY AA-3' and CY0017rev: 5'-GCD ATN ARN SHR TCN GGD AT-3'. CYrecF and SBrecF were used as positive controls, with the same primers used for the fosmid screening experiments. PCR reactions were performed using standard procedures.
Sequence homology determinations and annotation
We assembled, finished and annotated sequences using the Sequencher [34] and Sequin software to allocate data and facilitate annotation. Identification of orfs was accomplished using BLAST analyses. Comparison of genome fragments was performed using BLAST analysis and the GATA tool for comparative sequence analysis [35]. Pseudogenes with one or more mutations were identified by BLAST searches or direct analysis of all open reading frames. Genome fragments of both Cyanothece sp. ATCC 51142 and R. gibba spheroid bodies are annotated in GenBANK under the accession numbers AY728386 and AY728387, respectively. Genes and orfs identified in both organisms were named according to BLASTp protein homologue names. Those orfs with homology to uncharacterised conserved hypothetical proteins and hypothetical orfs without any BLAST hit are numbered and referred as conserved hypothetical proteins and hypothetical proteins, respectively. Orfs oriented in the forward or reverse direction of the analysed fragment are named cyl or cyr for Cyanothece, sbl or sbr for spheroid bodies and follow consecutive numbering (see additional files 1 and 2).
Additional File 1. BlastP analysis of identified and annotated orfs of Cyanothece sp. ATCC 51142 (accession number AY728386). The table provides information on all annotated orfs of the analysed Cyanothece sp. ATCC 51142 genome fragment.
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Additional File 2. BlastP analysis of identified and annotated orfs of Rhopalodia gibba spheroid bodies (accession number AY728387). The table provides information on all annotated orfs of the analysed spheroid body fragment genome fragment.
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Phylogenetic tree building
Orthologues for spheroid body proteins greater than 200 amino acids from the genome region shown in Figure 1 were identified in closely related cyanobacteria using BLAST. These were aligned using CLUSTALX [34] and edited so that only conserved blocks of residues were used for evolutionary tree building. PHYML [36] was used to build maximum likelihood trees, assuming a JTT model of substitution and non-parametric bootstrapping (100 replicates). Strict consensus trees were built for each gene using the 100 gene trees produced from bootstrap replicates. SplitsTree 4.0 [37] was then used to build the supernetworks shown in Figure 2. Some proteins greater than 200 amino acids in length did not produce resolved strict consensus trees or were problematic for other reasons and these were omitted from the analysis (these included DapF, PyrF, Sbr0016, Sbl0019, FeoB1, NifP and Sbl0010). Protein sequences, additional to those reported in the present study and used for phylogenetic anlayses were those inferred from nucleotide sequences in both complete and unfinished genome projects. Genbank accession numbers for these are: Cyanothece sp. CCY0110 (AAXW00000000), Crocosphaera watsonii WH 8501 (AADV00000000), Nodularia spumigena CCY9414 (NZ_AAVW00000000), Nostoc punctiforme PCC 73102 (NZ_AAAY00000000), Nostoc sp. PCC 7120 (NC_003272), Anabaena variabilis ATCC 29413 (NC_007413), Lyngbya sp. PCC 8106 (NZ_AAVU00000000), Trichodesmium erythraeum IMS101 (NC_008312), Synechocystis sp. PCC 6803 (NC_000911).
Authors' contributions
CK and CV performed the molecular studies, sequences alignments and drafted the manuscript. PL participated in drafting the manuscript and performed the phylogenetic analyses. UGM conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Our work is supported by the Deutsche Forschungsgemeinschaft (SFB 395, TP B9), the Alexander von Humboldt Foundation, and New Zealand Marsden Fund.
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Posts Tagged 'AQS Publishing'
Congratulations to The Quilt Patch
Congratulations to Leslie Pfeifer, owner of The Quilt Patch in Fairfax, VA, who won this beautiful raffle quilt displayed at the AQS Quilt Market booth. Quilt: Seaworld MagicThe quilt blocks are from two AQS books: Beyond the Block by Linda K. Johnson & Jane K. Wells [Read More]
Winners are Announced at AQS Quilt Expo – Des Moines
The winners of the second annual AQS Quilt Expo in Des Moines, Iowa were announced at 8:30 a.m. on Wednesday, October 28, just prior to the opening of the show. The following winners were present (left to right): Karen Kay Buckley – Best of Show; Marilyn Badger [Read More]
International Quilt Market Update
Oh what fun we had at Quilt Market! This is the fall wholesale trade show where we get to show our new AQS publications to the retail shops…and see what is new for the coming season from fabric companies to notions. The colors are yummy and can't wait to make [Read More]
AQ Classifieds = Quick Results
Did you know that AQS members are entitled to one free classified ad (up to 30 words) per year, a $24.00 value? Starting with the November 2009 issue of American Quilter, the classified ads will be accessible on our Web site, www.AmericanQuilter.com. Read this [Read More]
Your opinion counts
If you are a regular reader of American Quilter magazine, please take a few minutes to complete our online survey by June 30, 2009. Your feedback will help us choose the articles and projects that YOU want in future issues of AQ, plus you could win a very nice prize just [Read More]
It's Baa-aack!
Anita Shackelford's very popular book on feather quilting patterns, Infinite Feathers, is back. AQS has reprinted her book due to popular demand. That, and the fact that it's still one of the best how-to-feather-quilt books around. Whether you quilt by hand or [Read More]
Quilt Market Update
Just back from the Spring 2009 Quilt Market trade show only two days and my feet have finally recovered! Wearing sensible shoes is a must to stand on cement with a layer of carpet and walk several miles. So much to see and so little time. Everyday we met many charming [Read More]
What a Show!
There aren't enough "wows!" to describe the 25th Anniversary Celebration in Paducah last week. From the incredible Best of Show quilt by Mariya Waters of Melbourne, Australia to the excellent entertainment by Ricky Tims, Eleanor Burns, Nancy Zieman and [Read More]
Executive Inn Telephone Number
Several quilters who have rooms at the Executive Inn during the quilt show have been trying to call the hotel. The hotel has a new telephone number: 270-415-9960. This week they do not have staff there all of the time; you may need to leave your number so they can call you [Read More]
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Author Alice Wilhoit has brought crazy quilting into the 21st century with our latest book, Crazy Patchwork. She uses batiks to create the foundation blocks, and this makes all the difference! Forget the dark, heavy look of Victorian crazy quilts; Alice shows you how to [Read More]

subset: commoncrawl


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Detroit ranks near last as energy-efficient city, report finds
Detroit came in 48th of 51 cities in a report that assessed energy-efficiency programs, according to the second annual City Energy Efficiency Scorecard.
Crain's Detroit Business is a metered site. Print and digital subscribers have unlimited access to stories, but registered users are limited to eight stories every 30 days. After viewing three metered stories, you'll be asked to register or log in. After eight more stories in 30 days, you'll be asked to subscribe.
State leaders need to protect the Great Lakes, our communities and our economy with a plan to retire or decommission the pipelines in the straits, writes Chris Shepler, owner and operator of Shepler's Mackinac Island Ferry.
Detroit came in 48th of 51 cities in a report that assessed energy-efficiency programs, according to the second annual City Energy Efficiency Scorecard.
An energy company says an oil well in Shelby Township has failed to produce as hoped and it's ending the project.
Brader, a former senior policy adviser to Gov. Rick Snyder, becomes the executive director of the new Michigan Agency for Energy
The state of Michigan gave Francine Pegues a three-year contract in February to operate the golf facility on Belle Isle, with the intent of using it as a tool to bring golf to underserved populations, especially children, but unanticipated expenses and troubles have made that a difficult goal.
Community solar is an alternative to popular rooftop solar panel systems and theoretically allows customers, investors and utilities – or a combination – to build large solar panel arrays on neutral sites to share power generation and cost savings.
With the 1.1 megawatt solar plant added to its inventory, DTE says it is now the largest investor in solar power in Michigan. The equivalent of 185 homes can be powered by the solar array.
Novi-based ITC Transmission has finished the third and final phase of a $510 million transmission line project to bring up to 5,000 megawatts of lower-cost wind energy to 10 million Michigan customers.
Ecotelligent Homes ran into trouble when it expanded into commercial work. Here's why.
Trends toward greater reliance on renewable energy technologies and the increasing importance of natural gas seem to have solidified. The question is, how will we respond?
The Nuclear Regulatory Commission approved the project Thursday, although the utility said it has no immediate plan to build an additional unit at the Fermi plant in Monroe County.
The U.S. Supreme Court said natural-gas suppliers, including Duke Energy Corp. and Jackson-based CMS Energy Corp., must defend against lawsuits claiming they manipulated prices during the 2000-02 California energy crisis.
A few spaces are open for exhibitors at Michigan's largest conference devoted to energy conservation and advanced energy technology at the Suburban Collection Showplace in Novi.


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JPMorgan Jap Smllr (JPS) - Half Year Results RNS Number : 4382R JPMorgan Japan Smaller Co Tst PLC 19 November 2012 LONDON STOCK EXCHANGE ANNOUNCEMENT JPMORGAN JAPAN SMALLER COMPANIES TRUST PLC UNAUDITED HALF YEAR RESULTS FOR THE SIX MONTHS ENDED 30TH SEPTEMBER 2012 Chairman's Statement Performance The Japanese smaller companies market continued to be very challenging during the first half of our current financial year. Concern over the prospects for the global economy, the ongoing uncertainty surrounding the Eurozone, the recent US Presidential elections and the strength of the yen were all factors which weighed on sentiment. Positively, the Company outperformed its benchmark index, the S&P/Citigroup Japan Extended Market Index (total return net), with its undiluted net asset value falling by 0.8% compared to the benchmark's decline of 6.1%. The diluted total return on net assets, which assumes that all of the Subscription shares were exercised at the current rate of 174 pence per share, on the other hand, rose by 1.1%. Over the same period, the Company's Ordinary share price fell by 7.5%, reflecting the widening of the discount from 10.4% to 18.0%. Subsequent to the period end, the discount has tightened somewhat and, as at the time of writing, stood at 15.8%. In my year end statement, I highlighted the measures your Board had taken to address the poor performance delivered in recent years, not least the appointment of a new management team based in Tokyo. It is pleasing to see an immediate marked upturn in relative performance following these changes, albeit in a dull market, and the Board will continue to monitor progress carefully. A more detailed review of the Company's performance is given in the Investment Manager's report below. Gearing The Company has a ¥2.0 billion secured credit facility with Scotiabank Europe plc, which gives the Investment Managers the ability to gear tactically. During the six months to 30th September 2012, the Company's gearing ranged from net cash of 2% to 5% geared. At the time of writing, the company was 99% invested. Subscription Shares During the six months to 30th September 2012, the Company issued 33,417 new Ordinary shares following valid applications to exercise Subscription shares. At the time of writing, the Company's shares were trading at 136 pence per share, somewhat below the current exercise price of 174 pence per share. Further details of the Subscription shares can be found on the Company's website at www.jpmjapansmallercompanies.co.uk Outlook Our Managers remain cautious regarding the near term outlook for the Japanese economy, but are confident of continuing to identify companies with attractive positioning. The Portfolio is focused on high quality Japanese stocks with good earnings progression, strong balance sheets and, in many cases, exposure to the faster growing economies of Asia. We believe a Portfolio of such stocks offers considerable promise. Alan Clifton Chairman 19th November 2012 Investment Managers' Report Market review The Japanese market fell during the review period, and the return on S&P/Citigroup Japan Extended Market Index (total return net) was -6.1% in sterling terms. Investors became increasingly risk averse as the outlook for both global and domestic economies deteriorated, the yen continued to strengthen against most other currencies and tensions rose between Japan and China over the disputed Senkaku Islands. During the three months to end June, the market focused on news emanating from Europe with elections in Greece and France, as well as soaring borrowing costs for Spain and Italy at centre stage. In addition, the economic outlook deteriorated for the US and China, the two largest economies in the world. Employment data in the US, bellweather for the health of the economy, showed that the pace of the recovery has been painfully slow. In China, the local Shanghai Composite index fell to a new post February 2009 low due to poor economic data and lack of decisive policy response. Although investor sentiment improved markedly in the July to September period, thanks to aggressive monetary easing in Europe, the US and Japan among other countries, the Japanese market failed to participate in the global rally. A stronger yen, rising tensions with China, the failure of the domestic economy to rebound strongly from the adverse shocks of the earthquake and tsunami, as well as the floods in Thailand that disrupted the supply chain for many Japanese exporters in 2011, are all to blame for the underperformance of Japan relative to other markets. Performance review The key focus of the Company's holdings was little changed - Japanese companies that have an opportunity to grow in Asia and other emerging economies, those that are restructuring and those that are in structural growth niches within the domestic economy. We focus on companies and industries that are able to show sustained business expansion and that trade at attractive valuations. Typically the balance sheet is strong, operating margins are high, as are returns on assets and equity. Overall there is a very strong focus on business quality. Over the six months to September, the Company produced a return of -0.8% in sterling terms, while the return of the benchmark S&P/Citigroup Japan Extended Market Index (total return net) was -6.1%. The outperformance over the benchmark is attributable largely to stock selection, although sector allocation also contributed positively. The positive contribution was achieved in a range of sectors, in each of which our focus on quality of earnings and quality of balance sheet helped produce superior returns than the broader market. The top contributors to the outperformance were Ship Healthcare Holdings which operates dispensing pharmacies and consulting services to hospitals, an online medical portal operator M3, Seven Bank which is an ATM network operator mainly through Seven Eleven convenience stores, Sho-Bond Holdings which specialises in the maintenance and repair of bridges and other social infrastructure, and Sysmex, a medical equipment company. All of them are core holdings whose medium-term growth potential is, we believe, not fully appreciated by the market. In addition, all of them have robust balance sheets and generate strong cash flow. On the other hand, our holdings in cyclical sectors, such as Daido Metal, Skymark Airlines and Eagle Industries, detracted from performance. With respect to sector allocation, our focus on quality of balance sheet and long-term, as opposed to cyclical, growth potential has resulted in an overweight stance in sectors such as healthcare and internet and a cautious stance towards materials, capital goods and technology. The Company was fully invested at the end of September and the turnover was less than 20% during the six months, down significantly from the same period a year ago. Outlook and portfolio strategy We believe that equity market volatility will remain high, due to uncertainties related to the outlook for the economy as well as policy risks around the world. It is our view that global economic growth will be subdued for a protracted period, and the risks are skewed to the downside due to deflationary forces arising from de-leveraging. The Bank of Japan does not appear to be prepared to engage itself in aggressive easing, nor is the government any closer than before to addressing the structural issues of budget deficit, declining working population and deflation. It is likely that there will be another general election before spring next year, and the consensus view is that the ruling Democratic Party of Japan will lose its majority and a new coalition government centred around the Liberal Democratic Party will succeed. Under such circumstances, we do not intend to change our strategy. In the last report, we wrote the following: 'There are many long-term trends in Japan, both positive and negative. We believe that the differences between those companies that will succeed and those that will not are set to become ever greater. Our local presence on the ground in Tokyo should be a strong competitive advantage in identifying long-term country, sector and stock specific themes. Although there are many macro-economic headwinds we are positive on the outlook for the Company's holdings in the long-term view.' Indeed, the Japanese equity market has played out almost exactly as we anticipated above. Those companies that are expected to deliver long-term growth have generally performed strongly. On the other hand, companies that lack clear competitive advantage and companies whose balance sheets are highly geared, have seen their share prices fall significantly. Some of the themes that we have focused on include the internet, an ageing population in Japan and the rise of the middle class in emerging economies. Although we are cautious on the long-term trend for overall domestic demand, we believe we can identify clear winners and losers. The Portfolio comprises stocks which are well positioned to prosper, notwithstanding the challenges facing the Japanese economy. Shoichi Mizusawa- Nicholas Weindling Naohiro Ozawa Investment Managers 19th November 2012 Interim Management Report The Company is required to make the following disclosures in its half year report. Principal Risks and Uncertainties The principal risks and uncertainties faced by the Company fall into the following broad categories: investment and strategy; market; accounting, legal and regulatory; corporate governance and shareholder relations; operational; foreign currency; and financial (including credit risk). Information on each of these areas is given in the Business Review within the Annual Report and Accounts for the year ended 31st March 2012. Related Parties Transactions During the first six months of the current financial year, no transactions with related parties have taken place which have materially affected the financial position or the performance of the Company during the period. Directors' Responsibilities The Board of Directors confirms that, to the best of its knowledge: (i) the condensed set of financial statements contained within the half yearly financial report has been prepared in accordance with the Accounting Standards Board's Statement 'Half-Yearly Financial Reports'; and (ii) the interim management report includes a fair review of the information required by DTR 4.2.7R and 4.2.8R of the UK Listing Authority Disclosure and Transparency Rules. For and on behalf of the Board Alan Clifton Chairman 19th November 2012 For further information, please contact: Andrew Norman For and on behalf of JPMorgan Asset Management (UK) Limited, Secretary 020 7742 6000 Please note that up to date information on the Company, including daily NAV and share prices, factsheets and portfolio information can be found at www.jpmjapansmallercompanies.co.uk Income Statement for the six months ended 30th September 2012 (Unaudited) (Unaudited) (Audited) Six months ended Six months ended Year ended 30th September 2012 30th September 2011 31st March 2012 Revenue Capital Total Revenue Capital Total Revenue Capital Total £'000 £'000 £'000 £'000 £'000 £'000 £'000 £'000 £'000 (Losses)/gains on investments held at fair value through profit or - (478) (478) - 4,495 4,495 - 936 936 loss Net foreign - (91) (91) - (441) (441) - (411) (411) currency losses Income from 636 - 636 555 - 555 1,295 - 1,295 investments Other interest receivable and similar - - - - - - 1 - 1 income Gross 636 (569) 67 555 4,054 4,609 1,296 525 1,821 return/(loss) Management fee (349) - (349) (494) - (494) (873) - (873) Other (188) - (188) (188) - (188) (372) - (372) administrative expenses Net return/(loss) on ordinary activities before finance 99 (569) (470) (127) 4,054 3,927 51 525 576 costs and taxation Finance costs (49) - (49) (97) - (97) (124) - (124) Net return/(loss) on ordinary activities 50 (569) (519) (224) 4,054 3,830 (73) 525 452 before taxation Taxation (45) - (45) (39) - (39) (91) - (91) Net return/(loss) on ordinary activities 5 (569) (564) (263) 4,054 3,791 (164) 525 361 after taxation Return/(loss) per Ordinary share (note 3) - undiluted 0.01p (1.44)p (1.43)p (0.67)p 10.27p 9.60p (0.42)p 1.33p 0.91p - diluted 0.01p (1.51)p (1.50)p (0.67)p 10.32p 9.65p (0.42)p 1.34p 0.92p All revenue and capital items in the above statement derive from continuing operations. No operations were acquired or discontinued in the period. The 'Total' column of this statement is the profit and loss account of the Company and the 'Revenue' and 'Capital' columns represent supplementary information prepared under guidance issued by The Association of Investment Companies. The Total column represents all the information that is required to be disclosed in a Statement of Total Recognised Gains and Losses ('STRGL'). For this reason a STRGL has not been presented. Reconciliation of Movements in Shareholders' Funds Called Capital up Six months ended share Share Other redemption Capital Revenue 30th September capital premium reserve reserve reserves reserve Total 2012 (Unaudited) £'000 £'000 £'000 £'000 £'000 £'000 £'000 At 31st March 4,017 716 314,775 1,836 (243,203) (12,263) 65,878 2012 Repurchase of shares held in Treasury - - (310) - - - (310) Issue of Ordinary shares on exercise of Subscription 3 46 - - - - 49 shares Net (loss)/return - - - - (569) 5 (564) on ordinary activities At 30th September 4,020 762 314,465 1,836 (243,772) (12,258) 65,053 2012 Called Capital up Six months ended share Share Other redemption Capital Revenue 30th September capital premium reserve reserve reserves reserve Total 2011 (Unaudited) £'000 £'000 £'000 £'000 £'000 £'000 £'000 At 31st March 4,058 710 314,823 1,794 (243,728) (12,099) 65,558 2011 Cancellation of shares held in Treasury (42) - - 42 - - - Issue of Ordinary shares on exercise of Subscription - 6 - - - - 6 shares Net return/(loss) - - - - 4,054 (263) 3,791 on ordinary activities At 30th September 4,016 716 314,823 1,836 (239,674) (12,362) 69,355 2011 Called Capital up Year ended share Share Other redemption Capital Revenue 31st March 2012 capital premium reserve reserve reserves reserve Total (Audited) £'000 £'000 £'000 £'000 £'000 £'000 £'000 At 31st March 4,058 710 314,823 1,794 (243,728) (12,099) 65,558 2011 Repurchase of - - (48) - - - (48) shares into Treasury Cancellation of (42) - - 42 - - - shares in Treasury Issue of Ordinary shares on exercise of Subscription 1 6 - - - - 7 shares Net return/(loss) - - - - 525 (164) 361 on ordinary activities At 31st March 4,017 716 314,775 1,836 (243,203) (12,263) 65,878 2012 Balance Sheet at 30th September 2012 (Unaudited) (Unaudited) (Audited) 30th September 30th September 31st March 2012 2011 2012 £'000 £'000 £'000 Fixed assets Investments held at fair value 64,330 66,263 69,273 through profit or loss Current assets Debtors 493 1,432 1,490 Cash and short term deposits 5,073 10,305 554 5,566 11,737 2,044 Creditors: amounts falling due within (67) (8,645) (5,439) one year Net current assets/(liabilities) 5,499 3,092 (3,395) Total assets less current liabilities 69,829 69,355 65,878 Creditors: amounts falling due after more than one year (4,776) - - Net assets 65,053 69,355 65,878 Capital and reserves Called up share capital 4,020 4,016 4,017 Share premium 762 716 716 Other reserve 314,465 314,823 314,775 Capital redemption reserve 1,836 1,836 1,836 Capital reserves (243,772) (239,674) (243,203) Revenue reserve (12,258) (12,362) (12,263) Total equity shareholders' funds 65,053 69,355 65,878 Net asset value per Ordinary share (note 4) - undiluted 165.8p 175.8p 167.1p - diluted 165.8p 171.3p 164.0p The Company's registration number is 3916716 Cash Flow Statement for the six months ended 30th September 2012 (Unaudited) (Unaudited) (Audited) Six months ended Six months ended Year ended 30th September 30th September 31st March 2012 2011 2012 £'000 £'000 £'000 Net cash inflow/(outflow) from operating activities (note 5) 190 (1) (59) Net cash outflow from return on investments and servicing of finance (53) (101) (125) Net cash inflow from capital expenditure and financial investment 4,521 10,294 2,325 Net cash outflow from financing (261) (6,233) (7,928) Increase/(decrease) in cash in 4,397 3,959 (5,787) the period Reconciliation of net cash flow to movement in net funds/debt Net cash movement 4,397 3,959 (5,787) Net loans repaid - 6,239 7,887 Exchange movements (91) (441) (411) Movement in net funds in the 4,306 9,757 1,689 period Net debt at the beginning of the (4,009) (5,698) (5,698) period Net funds/(debt) at the end of 297 4,059 (4,009) the period Represented by: Cash and short term deposits 5,073 10,305 554 Debt falling due after more than (4,776) (6,246) (4,563) one year Net funds/(debt) at the end of 297 4,059 (4,009) the period Notes to the Accounts for the six months ended 30th September 2012 1. Financial statements The information contained within the Financial Statements in this half year report has not been audited or reviewed by the Company's auditors. The figures and financial information for the year ended 31st March 2012 are extracted from the latest published accounts of the Company and do not constitute statutory accounts for that year. Those accounts have been delivered to the Registrar of Companies and included the report of the auditors which was unqualified and did not contain a statement under either section 498(2) or 498(3) of the Companies Act 2006. 2. Accounting policies The accounts have been prepared in accordance with United Kingdom Generally Accepted Accounting Practice ('UK GAAP') and with the Statement of Recommended Practice 'Financial Statements of Investment Trust Companies and Venture Capital Trusts' issued in January 2009. All of the Company's operations are of a continuing nature. The accounting policies applied in these half year accounts are consistent with those applied in the accounts for the year ended 31st March 2012. 3. Return/(loss) per Ordinary share (Unaudited) (Unaudited) (Audited) Six months ended Six months ended Year ended 30th September 30th September 31st March 2012 2012 2011 £'000 £'000 £'000 Return/(loss) per Ordinary share is based on the following: Revenue return/(loss) 5 (263) (164) Capital (loss)/return (569) 4,054 525 Total (loss)/return (564) 3,791 361 Weighted average number of Ordinary shares in issue during the period used for the purpose of the basic (undiluted) calculation 39,379,850 39,455,653 39,446,389 Weighted average number of Ordinary shares in issue during the period used for the purpose of the diluted calculation 37,514,172 39,267,442 39,134,213 Basic (undiluted) Revenue return/(loss) per 0.01p (0.67)p (0.42)p Ordinary share Capital (loss)/return per (1.44)p 10.27p 1.33p Ordinary share Total (loss)/return per (1.43)p 9.60p 0.91p Ordinary share Diluted Revenue return/(loss) per 0.01p (0.67)p (0.42)p Ordinary share Capital (loss)/return per (1.51)p 10.32p 1.34p Ordinary share Total (loss)/return per (1.50)p 9.65p 0.92p Ordinary share The diluted return/(loss) per Ordinary share represents the return/(loss) on ordinary activities after taxation for the period divided by the weighted average number of Ordinary shares in issue during the period, as adjusted in accordance with the requirements of Financial Reporting Standard 22 'Earnings per share'. 4. Net asset value per Ordinary share (Unaudited) (Unaudited) (Audited) Six months ended Six months ended Year ended 30th September 30th September 31st March 2012 2012 2011 Undiluted Ordinary shareholders' funds 65,053 69,355 65,878 (£'000) Number of Ordinary shares in 39,229,318 39,456,287 39,421,401 issue Net asset value per Ordinary 165.8p 175.8p 167.1p share Diluted Ordinary shareholders' funds assuming exercise of dilutive Subscription shares and reissuance of any Treasury shares 65,053 79,987 76,510 (£'000) Number of potential Ordinary 39,229,318 46,688,796 46,653,796 shares in issue Net asset value per Ordinary 165.8p 171.3p 164.0p share The diluted net asset value per Ordinary share assumes that all outstanding dilutive Subscription shares were converted into Ordinary shares at the period end and any shares held in Treasury at the period end were reissued in accordance with the Board's policy on the reissuance of Treasury shares. At 30th September 2012, the Subscription and Treasury shares have no dilutive effect, as the Ordinary share price was below the Subscriptions share conversion price and the Treasury shares will only be reissued at a premium to net asset value per share. 5. Reconciliation of net (loss)/return on ordinary activities before finance costs and taxation to net cash inflow/(outflow) from operating activities (Unaudited) (Unaudited) (Audited) Six months ended Six months ended Year ended 30th September 30th September 31st March 2012 2011 2012 £'000 £'000 £'000 Total (loss)/return on ordinary activities before finance costs and taxation (470) 3,927 576 Less capital loss/(return) before 569 (4,054) (525) finance costs and taxation Decrease/(increase) in accrued 164 182 (66) income Decrease in other debtors 2 20 9 (Decrease)/increase in other (30) (37) 38 creditors Overseas withholding tax (45) (39) (91) Net cash inflow/outflow from 190 (1) (59) operating activities JPMORGAN ASSET MANAGEMENT (UK) LIMITED ENDS A copy of the half year has been submitted to the National Storage Mechanism and will shortly be available for inspection at www.morningstar.co.uk/uk/NSM The half year will also shortly be available on the Company's website at www.jpmjapansmallercompanies.co.ukwhere up to date information on the Company, including daily NAV and share prices, factsheets and portfolio information can also be found. - This information is provided by RNS The company news service from the London Stock Exchange END IR BKLFFLFFEFBQ -0- Nov/19/2012 10:59 GMT
JPMorgan Jap Smllr JPS Half Year Results
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Make a Mini Plant Press at the Tulsa Botanic Garden
May 22, 2015
Kids can be a junior botanist as they learn how to press plants and make a mini-plant press to take home ($3 for materials). The Botanic Garden is open Fridays and Saturdays from 9 a.m. to 4 p.m. Suggested admission is $2 for adults;; 50¢ for kids 3-12 yrs. For more information, call 918-289-0330.
Phillips 66 Big 12 Baseball Championship
May 22, 2015 - May 24, 2015
The Tulsa Sports Commission and ONEOK Field are hosting the 2015 Phillips 66 Big 12 Baseball Championship. The top eight men's baseball teams from the Big 12 Conference will compete in tournament.
Tulsa Historical Society and Museum: 1930's Exhibit
May 22, 2015 - Jun 18, 2015
This event occurs weekly on Tuesday, Wednesday, Thursday, Friday and Saturday.
The Tulsa Historical Society & Museum has opened its featured 1930's exhibit. The exhibit explores Tulsa as it was in the 1930's and allows those of all ages to come and enjoy artifacts and photos from this time period. Admission is required and the museum is open 10 a.m. to 4 p.m., Tuesday through Saturday (Closed Sundays, Mondays, and Holidays).
ONEOK Fan Fest
May 22, 2015 - May 24, 2015
The ONEOK Fan Fest will take place at the Guthrie Green in downtown Tulsa. Guthrie Green is an urban park and entertainment space in the heart of Tulsa's Brady District, located two blocks from ONEOK Field. The ONEOK Fan Fest will be open from Wednesday, May 20 through Sunday, May 24. Activities will begin daily at 11am and close at 11:30pm. The ONEOK Fan Fest is open to the public and is free...
Tulsa Comic Con Expo
May 22, 2015
The Tulsa Comic Con Expo is the regions go to Comic Convention for all things Geek, Movie, Comics related. With over 100,000 square ft of space open to the fans of all genres we welcome you to this event. At The Tulsa Comic Con Expo you can meet TV and film celebrities along with experience celebrity Q & A's, panels, celebrity photo ops, and parties.. You can also take...
Tulsa Shock Home Game
May 22, 2015
The Shock are in the WNBA Western Conference along with the Seattle Storm, Los Angeles Sparks, Phoenix Mercury, San Antonio Stars, and Minnesota Lynx. The Eastern conference consists of the Atlanta Dream, Indiana Fever, Washington Mystics, New York Liberty, Chicago Sky, and Connecticut Sun.
Book-A-Librarian (Broken Arrow Library)
May 22, 2015 - May 30, 2015
This event occurs daily, every 1 day(s).
Need individualized instruction on a library service or product? Whether you want to learn how to download digital content or improve your job skills, we are here to help. Book-A-Librarian appointments are available on a first-come, first-served basis and generally last 30-60 minutes. Call 918-549-7500 to reserve your time. For adults.


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Night Optics Gen 3 4x100mm Night Vision Rifle Scope
night vision rifle scopes are built using state of the art materials and technology, reducing weight ... The Night Optics 4x100mm Night Vision Rifle Scope utilizes Generation 3 night vision for a relatively ... rifle scopes is ideal for medium range tactical situations, and they feature Mil-Dot reticles ... riflescope, the NightOptics Dual Color 4x Gen 3 NV Riflescope is the way to go for professional night ... vision. Night-Optics D740 Generation 3 NV 4x Rifle Scope w/ Advanced Tube, Manual Gain, Gated ... Power Supply Specifications for Night-Optics Gen 3 Mil-Dot 4x100 Infrared Rifle Scope: Night ... Features of Night Ops Generation 3 4x Multi-Coated Night Vision Riflescopes: Professional-grade...
Night Optics Gen 3 6x165mm Night Vision Rifle Scope
Night Optics D760 Gen 3 6x Night Vision Rifle Scope is a high magnification rifle scope designed ... have gone into the manufacture of these night vision rifle scopes, which boast multi-coated optics ... and a waterproof, purge-capable housing with a lighter weight than many other rifle scopes. Generation 3 ... The Night Optics 6x165mm Night Vision Rifle Scope features a dual color Mil-Dot reticle that you can ... and elevation knobs in standard 1/4 MOA increments, just like a standard rifle scope. You'll rarely have ... the 6x magnification on these Night Optics riflescopes is better suited to the heavier load-outs ... Riflescopes: Night Vision: Generation 3 Resolution: 57-72 lp/mm Power Supply: Non-Gated (760-3ST,...
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lightweight Night Vision Rifle Scope is designed to save space on your rifle and reduce the weight ... load. Waterproof and rugged, the Night Optics D730 Superlight Rifle Scope features superior ... caliber weapons. The Night Optics Superlite NV Rifle Scope comes with an illuminated Mil-Dot ... Features of Night Optics D730 Night Vision Rifle Scope - 3.7x NV Scope: 3.7x Magnification...
Starts at $3,089.00
Night Optics Gen 2+ High Performance 4x100mm Night Vision Rifle Scope
dependable rifle scope for use by law enforcement and military professionals, preferred for its rugged ... night vision tubes on these rifle scopes give you an accurate night vision image with little ... Performance NV Mil-Dot Rifle Scope utilizes modern materials for a highly recoil-resistant body ... that will stay sighted while firing almost any caliber. These Night Optics rifle scopes have a waterproof ... Optics has equipped the NightOps 4x100mm Night Vision Waterproof Riflescope with a throw-lever quick ... precise control over you rifle scope. Manual gain control allows you to reduce the blooming effect ... rifle scopes are dependable enough for long-range tactical and surveillance observations,...
Night Optics Gen 2+ High Performance 6x165mm Night Vision Rifle Sight
objective lens of the rifle scope combine to give it a clarity you'd be hard pressed to find ... on a standard riflescope, let alone a night vision device. The Generation 2+ night vision tubes in the scopes ... High Performance NV Mil-Dot Rifle Scope can be taken into any combat zone, thanks to its waterproof ... it simple to attach and release the NightOps Gen 2+ HP 6x165mm Night Vision Rifle Scope to your rifle ... have been installed in these Night Optics rifle scopes, both illuminated to stand out against your ... vision scope technology when you mount the Night Optics 6x165mm Gen 2+ Night Vision Mil-Dot Riflescope to ... for Night Optics 6x100mm Gen 2+ High Performance Rifle Scope: Night Vision: Generation 2+ Resolution:...
Night Optics NS-520 2.8x Generation 2+ Night Vision Riflescope
Gen2+ NS-520-2S delivers amazing performance. This NV Riflescope from Night Optics uses pristine ... Plus NS 520 NV Riflescope. Night Optics NS-520-2S: NS-520 2.8x Generation 2+ Night Vision Scope, ... Package Contents: Night Optics NS520 Night Vision Riflescope - 2.8x, Gen2+ NS-520-2S Storage Pouch Operator Manual ... With it's small and lightweight package, the Night Optics NS520 Night Vision Riflescope - 2.8x, ... Riflescope is packed with features like Mil Dot Reticle with adjustable brightness control; ... Black Specifications for Night Optics NS-520 2.8x Generation 2+ Night Vision Scope, Black NS-520-2S ... : Night Optics Part #: NS-520-2S Resolution: 28-32 lp/mm Magnification: 2.8x Lens System: 80...
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The Night Optics D-750 4x Gen 3 Night Vision Rifle Scope has it all: powerful magnification ... D750 NV Rifle Scope easy and quick to use. The Night Optics 4x NV Rifle Scope comes...
Night Optics D-740 4x Generation 2+ Black and White Night Vision Scope
The Night Optics D-740 4x Generation 2+ Black and White Night Vision Rifle Scope 740-2BW ... Optics D-740 Rifle Scope: Night Optics Part #: NS-740-2H Resolution: 51+ lp/mm Magnification: 4x...
Night Optics Gladius 760 6x Gen. 2 Night Vision Sight
professionals. Night Optics NS-760-2S: Gladius 760 6x Generation 2 Standard Night Vision Weapon...
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'Desperate' Rout mailer tries linking Stanton to Ford, Jackson
Democrat Ed Stanton Jr. says a recent mailer trying to link him to convicted former state senator John Ford and indicted General Sessions Court Clerk Otis Jackson is a "desperate," divisive tactic.
However, Republican Rick Rout's campaign says it mailed the campaign piece to persuade voters that Democrats have a record of poor performance in the Shelby County General Sessions Court clerk's office.
In the mailer, mock newspaper headlines announce "Former Court Clerk John Ford Convicted of Bribery" and "General Sessions Clerk Otis Jackson Indicted."
Underneath is the line: "They've been caught with their hands in the cookie jar over and over again" while to the left, a hand reaches in a jar for chocolate chip cookies.
Candidate Rout was unavailable for comment, but his brother, Jimmy Rout III says although Stanton was not mentioned in the mailer, party affiliation ties Stanton, Ford and Jackson together.
"The track record for Democrats has not been so good, whereas Chris Turner was clerk for 12 years and left with a surplus," Jimmy Rout III said.
Stanton, who worked for Turner, says he doesn't believe voters will buy the argument -- they are "smarter than that," he said.
"It has nothing to do with me and we will not comment on that because we're running on our own record, a record of service and experience and accountability and hopefully people will say, our integrity," Stanton said.
Stanton has spent 29 years on court clerks offices, working for 20 years in the criminal court clerk's office, leaving as director of operations. He spent eight years in general sessions as administrator and was asked last year by Chancery Court judges to come out of retirement to run the office.
But Stanton has not served as an elected official, Jimmy Rout III said.
"He's only served as pro temp and has never had to answer to the electorate," he said.
Rick Rout, the son of former Shelby County Mayor Jim Rout, has also never held public office.
"His record -- you've been shown the concerns and problems he's had in his own record," Stanton said.
Rout was suspended twice from his job as a process server for the Juvenile Court Clerk's office, including a 10-day suspension earlier this year on charges he cursed his boss, did not follow instructions, falsified a court document and lied.
Rout has said he was framed by his supervisor and has been targeted for harassment
The headlines about Ford and Jackson are true, though neither was ever charged with taking taxpayer money.
Ford was convicted of bribery while a state senator, years after serving as general sessions court clerk. Jackson was elected in 2008 and indicted in 2011 for allegedly attempting to force employees to contribute to his re-election fund.
Their actions have nothing to do with Stanton, said Van Turner, chairman of the Shelby County Democratic Party.
"I don't think it has any place in today's conversation where we're trying to heal divides and not create them," Turner said.
Justin Joy, chairman of the Shelby County Republican Party, contends that the mailer has relevance.
"I think it goes back to the fact that Rick is running to restore trust in the clerk's office," Joy said. "And the mail piece highlights instances in the past when the public trust has been violated by those who have been elected general sessions court clerk."
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A huge night of boxing in Belfast is on the way as Paul McCloskey and Prizefighter Middleweights II take over the Kings Hall on May 5, live on Sky Sports.
McCloskey is looking to keep in World title contention following his brave victory over Breidis Prescott in September and will be gunning for a second World title shot later this year but first he will face a stern test against two-time World Champion Diaz.
The Mexican has held the International Boxing Federation (IBF) lightweight crown twice and challenged for the WBC and WBO versions, and the 32 year-old known as The Kidd carry plenty of top level experience into his 46th clash on May 5.
The Kings Hall will host the 24th edition of Prizefighter on the same night as McCloskeys showdown with Diaz as the Irish Middleweights get it on, with unbeaten Commonwealth gold winner King Kane Eamonn OKane, EBA champ Anthony Fitzer Fitzgerald, Mark Kid Dynamite Heffron, Joe Sugar Rea, and Darren Cruise the first named confirmed by show promoter Eddie Hearn.
If you imagine the best atmosphere of any Prizefighter to date, then crank it off the dial, I still don't think you are close to what we are about to witness on May 5, said Hearn. Couple that with Paul McCloskey's faithful army of incredible fans and I believe this has the potential to go down in history as one of the great Irish fight nights.
Hearn also revealed that ticket details will be release at a press conference in Belfast next week.
February 23, 2012 Mexico City.
From the office of WBC President Dr. José Sulaimán:
The World Boxing Council ratified unanimously the implementation of the partial open scoring rule to be used in WBC-sanctioned bouts. The rule allows the announcement of the official scoring of the judges after the 4th and 8th rounds.
The WBC has used open scoring for six years all around the world. The results have been extremely positive, without a single incident that would have compromised the integrity of the sport or the bout. To the contrary, the use of the rule has provided the fans at the arena and TV spectators great drama in the last four rounds. More importantly, the use of open scoring has given both fighters the fair opportunity to adjust their strategy during the fight.
The boxing community in general, and especially the fighters and trainers who have experienced the open scoring rule, are overwhelmingly in favor of its use.
The WBC Board of Governors also approved the option to use a limited form of open scoring. Under the limited rule, only the corners would receive the official scores after the 4th and 8th rounds. The limited rule intends to cover instances when the jurisdiction does not permit the announcement of the scores to the public.
The WBC would like to work with the Association of Boxing Commissions in the U.S. and with the commissions of every U.S. jurisdiction toward the implementation of some form of open scoring during WBC-sanctioned fights.
The State of Arkansas used open scoring during the Jermain Taylor versus Kassim Ouma fight on December 9, 2006. More recently, the State of Texas used the limited open scoring rule during the Julio Cesar Chavez Jr. versus Marco Antonio Rubio WBC middleweight championship fight.
I am very happy with this practice. I thought I was winning with a clear advantage but in reality the judges had a much closer fight, so I gave it all in the last four rounds and I was victorious. - Julio Cesar Chavez Jr.
It is a wonderful rule. I had used it in Mexico before and it helped my strategy tremendously in this fight against Chavez. - Hector Sanchez, trainer of Marco Antonio Rubio.
The links immediately below include very positive statements from Sugar Ray Leonard and Lennox Lewis about the open scoring rule upon being asked about it.
http://vimeo.com/37327389 (Sugar Ray Leonard)
http://www.youtube.com/watch?v=XoZVcv46uII (Lennox Lewis)
The WBC respects the authority and jurisdiction of each country, state, and local commission, and will work together with those agencies to try to establish the open scoring rule in the U.S. The WBC believes that the rule is a great tool for fighters to strive toward a just result. It also allows the fighters to act and react based on accurate and non-speculative information about the judges scores while the fight is still ongoing. In short, the open scoring rule prevents the surprise and shock that can come with learning that the actual scores are totally different from what each corner thought they were.
The 2012 National Novice Championships begin at the National Stadium in Dublin this Friday evening.
The weigh-in for the Championships will be held between 8.30am and 10am and 12 noon and 1pm at the National Stadium Friday followed by the draw at 1.30pm.
Boxing - contingent on entries, details will be announced at draw.
Men Kg: 48,51,54,57,60,64,67,71,75,81,85,91,91+
Women Kg: 46,48,50,52,54,57,60,63,66,70,75,80,86
Visit www.iaba.ie for entries updates, draws and results.
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text:
Pork With Watercress
12 Oct 2009
|400 Gram (4 Large Bunches)
|Salted water
|1 Cup (16 tbs) (As Required)
|Peanut oil
|1⁄4 Cup (4 tbs)
|Garlic clove
|1 Small, chopped
|Pork shoulder
|1 Pound, cut in thin slices
|Soy sauce
|3 Tablespoon
1. Wash the watercress well and drain. Cut off the bottoms of the stems. Soak in cold salted water to cover thirty minutes. Drain, rinse in fresh water and dry gently.
2. In a skillet heat the oil, add the garlic and pork and brown the meat quickly on all sides.
3. Add the soy sauce and watercress. Cook, stirring, until the mixture reaches a boil. Cover and cook two minutes longer.
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Pork With Watercress Recipe


text:
Pronunciation: Ohope
Added on: 2012-05-20 Listened: 45 timesin: Towns - New Zealand
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