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The genomes of salmonids are considered pseudo-tetraploid undergoing reversion to a stable diploid state. Given the genome duplication and extensive biological data available for salmonids, they are excellent model organisms for studying comparative genomics, evolutionary processes, fates of duplicated genes and the genetic and physiological processes associated with complex behavioral phenotypes. The evolution of the tetrapod hemoglobin genes is well studied; however, little is known about the genomic organization and evolution of teleost hemoglobin genes, particularly those of salmonids. The Atlantic salmon serves as a representative salmonid species for genomics studies. Given the well documented role of hemoglobin in adaptation to varied environmental conditions as well as its use as a model protein for evolutionary analyses, an understanding of the genomic structure and organization of the Atlantic salmon α and β hemoglobin genes is of great interest.
We identified four bacterial artificial chromosomes (BACs) comprising two hemoglobin gene clusters spanning the entire α and β hemoglobin gene repertoire of the Atlantic salmon genome. Their chromosomal locations were established using fluorescence in situ hybridization (FISH) analysis and linkage mapping, demonstrating that the two clusters are located on separate chromosomes. The BACs were sequenced and assembled into scaffolds, which were annotated for putatively functional and pseudogenized hemoglobin-like genes. This revealed that the tail-to-tail organization and alternating pattern of the α and β hemoglobin genes are well conserved in both clusters, as well as that the Atlantic salmon genome houses substantially more hemoglobin genes, including non-Bohr β globin genes, than the genomes of other teleosts that have been sequenced.
We suggest that the most parsimonious evolutionary path leading to the present organization of the Atlantic salmon hemoglobin genes involves the loss of a single hemoglobin gene cluster after the whole genome duplication (WGD) at the base of the teleost radiation but prior to the salmonid-specific WGD, which then produced the duplicated copies seen today. We also propose that the relatively high number of hemoglobin genes as well as the presence of non-Bohr β hemoglobin genes may be due to the dynamic life history of salmon and the diverse environmental conditions that the species encounters.
Hemoglobin, one of the most well-studied proteins to date, is responsible for oxygen transport from the lungs or gills to the tissues of vertebrates. The hemoglobin molecule is comprised of two α and two β subunits that non-covalently bond to form a tetramer [1, 2]. The genes encoding the hemoglobin subunits are abundantly present and show relatively high similarity in structure (i.e., consisting of three exons and two introns) throughout the vertebrate lineage . These characteristics, combined with the relative ease of isolating and studying hemoglobin proteins and their suspected role in adaptation to variable environmental conditions, have made the hemoglobin genes major targets for evolutionary studies [1–4].
Examinations of the genomic organization of chromosomal hemoglobin gene regions suggest that all hemoglobin genes evolved from a single monomeric form when gnathostome fish evolved from the more primitive agnathan fish approximately 500-700 million years ago [5, 6]. The entire hemoglobin gene region then appears to have undergone a series of tandem duplications and divergence, giving rise to the modern α and β hemoglobin genes. Initially the α and β genes were adjacent on the same chromosome, and expansion of this region, including lineage-specific gene gain and loss, produced the multiple copies of α and β genes seen in Gnathostomata today [4, 5, 7, 8].
The current genomic organization seen in mammals and birds is such that α and β hemoglobin gene clusters are located on different chromosomes and transcribed from the same strand in order of temporal expression [8, 9]. The most parsimonious explanation of this arrangement involves a disruption in the α-β linkage by translocation of part of the hemoglobin gene cluster and subsequent gene silencing of α and β hemoglobins on respective chromosomes prior to the lineage leading to birds and mammals approximately 300-350 million years ago [9, 10]. Studies of the genomic organization of hemoglobin genes in the mammalian and avian lines examined this hypothesis by looking for evolutionary "footprints" of silenced hemoglobin genes as well as conservation and divergence patterns of genes surrounding the α and β gene clusters along the mammalian line [9, 11].
The disruption in the α and β hemoglobin gene linkage in mammals and birds appears to have occurred after their divergence from the poikilothermic jawed vertebrate taxa. Rather, with the exception of some extreme cold-adapted Antarctic icefish that retain only remnants of α hemoglobin genes and have completely lost the β hemoglobin genes, and thus do not express hemoglobin [12, 13], the fish and amphibians studied to date exhibit intermixed α and β hemoglobin genes on the same chromosome. For example, within the amphibian line, the genomes of both Xenopus laevis and X. tropicalis exhibit linked α and β hemoglobin genes .
The teleosts, or the ray-finned fish, are a diverse group that comprises most living species of fish, including more than 20,000 extant species covering more than 40 orders . Despite significant differences in the number of hemoglobin genes and within-chromosome arrangements, model teleosts whose genomes have been studied to date, including the Japanese pufferfish (Fugu rubripes) , the zebrafish (Danio rerio)  and medaka (Orzias latipes)  are reported to exhibit two hemoglobin gene clusters located on distinct chromosomes. These observations support the hypothesis that the teleost lineage experienced a whole genome duplication (WGD) event subsequent to the divergence from tetrapods .
The Salmonidae, a family of teleosts that includes the salmon, trout, charr, grayling and whitefish, are of considerable environmental, economic and social importance. Indeed, more is known about the biology of salmonids than any other fish group . The common ancestor of salmonids underwent a WGD event between 20 and 120 million years ago [20, 21]. Thus, the extant salmonid species are considered pseudo-tetraploids whose genomes are in the process of reverting to a stable diploid state. The Atlantic salmon (Salmo salar) has been chosen as a representative salmonid for genomics studies, and an international collaboration to sequence the Atlantic salmon genome has been established .
Wolff and Gannon  provided the first sequence of an Atlantic salmon α hemoglobin from a kidney cDNA library. Subsequently, reports of the organization of the Atlantic salmon hemoglobin gene cluster described six lambda phage genomic clones comprising two sets of α and β hemoglobin genes oriented 3' to 3' on opposite strands [24–26]. This was the first evidence of this type of hemoglobin gene arrangement for any vertebrate species. The six clones comprised four unique α hemoglobin gene sequences and six unique β hemoglobin genes, including a β hemoglobin containing the characteristic amino acid changes that eliminate the Bohr effect, as well as one partial β hemoglobin gene (GenBank accession numbers X97284-X97289) . It remained unknown, however, whether these represented all hemoglobin-like genes within the Atlantic salmon genome. In addition, it was not known whether the clusters were on separate chromosomes, or whether, as would be predicted by the salmonid-specific 4R WGD hypothesis , there were actually four hemoglobin gene clusters in salmon. Furthermore, the relative locations and orders of the clones to one another were not established, and the sequences of intergenic regions as well as the genes surrounding the hemoglobins were not determined. Finally, these investigations were not able to identify putative pseudogenes, incomplete hemoglobin genes or footprints of historical hemoglobin genes within the hemoglobin gene clusters or elsewhere in the genome. Thus, a full characterization of the Atlantic salmon hemoglobin gene repertoire is needed to provide insight to the evolution of the organization and function of the Atlantic salmon hemoglobins, particularly in light of the teleost and salmonid-specific WGD events.
We used oligonucleotide probes specific for Atlantic salmon α, β and non-Bohr β hemoglobin genes as well as probes designed from rainbow trout (Oncorhynchus mykiss) embryonic hemoglobin cDNAs  to locate these genes within the Atlantic salmon bacterial artificial chromosome (BAC) library, CHORI-214 . Four BACs, representing two genomic locations on different chromosomes and comprising the entire Altantic salmon hemoglobin gene repertoire were sequenced and annotated. Fluorescence in situ hybridization (FISH) and linkage analyses were performed to assign these BACs to chromosomal locations within the Atlantic salmon genome. Here we present the first description of an entire salmonid α and β hemoglobin gene repertoire. We also discuss our results in terms of the fate of the hemoglobin genes during and after the salmonid WGD event, and how this fits into the evolution of the hemoglobin gene family in teleosts.
All 32P-labelled 40-mer probes for α, β, non-Bohr β and embryonic hemoglobins (probe and primer sequences are provided in Additional file 1, Table S1) hybridized to Atlantic salmon BACs belonging to two fingerprint scaffolds (fps), within the Atlantic salmon physical map [30, 31]. Fps1046 contains 21 BACs and spans an estimated 458.8 kb; fps135 is comprised of 391 BACs spanning approximately 3.473 Mb. PCR was used to confirm the hybridization results and narrow down the regions within the fps that contained hemoglobin genes by screening all BACs surrounding the hybridization-positive BACs for the presence of hemoglobin genes. PCR primers were designed for sequence tag sites (STS) within the BAC-end sequences (SP6 and T7 ends) of suspected overlapping BACs spanning the hemoglobin gene region, and overlaps were checked by PCR amplification of the STS within the putative overlapping BACs. The overlapping BACs S0014B03 and S0055H05 were determined to span the hemoglobin gene region of fps1046, while S0155C07 and S0079J05 spanned that of fps135, thus creating BAC tiling paths for the hemoglobin regions of these fps. Individual shotgun libraries were generated for all four BACs and sequenced.
The CHORI-214 BAC library was made from a diploid male Atlantic salmon individual, meaning that BAC inserts originated from either maternal or paternal chromosomes and therefore, overlapping BACs could exhibit allelic differences. This appeared to be the case for BACs S0014B03 and S0055H05, for which the overlapping region covered the hemoglobin genes within fps1046. Thus, although this overlapping section assembled into one contiguous section, reads containing allelic differences assembled into independent contigs that aligned to homologous regions along the solid contig. Nevertheless, the full BACs assembled very well, and only three contigs > 1000 bp and two gaps remained after hand finishing. These gaps presumably span repetitive regions in the Atlantic salmon genome . Furthermore, given that the entire hemoglobin gene region was assembled with no gaps, it can be assumed that the six putatively functional α and six putatively functional β hemoglobin genes, two of which were defined as non-Bohr β hemoglobins, along with the two putative α hemoglobin pseudogenes and three putative β hemoglobin pseudogenes that were annotated within fps1046 represent all hemoglobin genes within that cluster (note that the solid contig was used for sequence annotation; Figure 1A). The total size of the assembly for the two BACs, not including allelic contigs (i.e., the non-redundant sequence), was 242,883 bp, with approximately 49,000 bp of overlap between them and the hemoglobin genes spanning approximately 87,000 bp.
Genomic organization of the Atlantic salmon hemoglobin gene clusters. A) Schematic representation of the region of Atlantic salmon chromosome 6 containing the hemoglobin genes. Sequence reads for this region assembled into one solid sequence contig (ctg 41). B) Schematic representation of the region of Atlantic salmon chromosome 3 containing the hemoglobin genes. Sequence contigs are indicated by horizontal green lines. β hemoglobin genes are indicated in blue; α hemoglobin genes are indicated in red. Arrows indicate strand of transcription. All hemoglobin gene names begin with SsaChr6 or SsaChr3 for chromosome 6 and chromosome 3, respectively, followed by α or β and a number indicating the order of the genes. SP6 and T7 ends of overlapping BACs are indicated by grey arrows. Thus, the regions between the arrows indicate BAC overlapping regions. bN: Non-Bohr β hemoglobins.
Sequence reads from the overlapping region between the BACs S0155C07 and S0079J05 of fps135 assembled into one sequence contig with no apparent allelic differences. However, the remainder of the fps135 BACs proved much more difficult to assemble, and unfortunately, the repetitive nature of the sequences made it impossible to further improve the assembly by sequencing PCR products to fill gaps because we were unable to design specific PCR primers that would amplify a single product (i.e., numerous bands, or smears on agarose gels were obtained). Thus, a total of 23 sequence contigs > 1000 bp remained after hand-finishing of the assembly. The relative orders of some of the sequence contigs of fps135 were determined by matching paired-ends of sequence shotgun clones. Six sequence contigs contained hemoglobin genes, and the relative order of these fps135 contigs with respect to one another was estimated by aligning the contigs against the completed assembly of the two BACs from fps1046. This was based on the assumption, given the highly similar nature of the non-coding regions, as well as that of the genes flanking the globins, that there has not been a major disruption in the form of an inversion to either of the hemoglobin regions (i.e., that of fps135 or fps1046). Our sequence annotation identified seven putatively functional α hemoglobin genes and one putative α hemoglobin pseudogene, as well as eight putatively functional β hemoglobin genes, four of which were defined as non-Bohr β hemoglobins, and three putative β hemoglobin pseudogenes within the fps135 hemoglobin BACs (Figure 1B). This, however, must be considered a minimum estimate of hemoglobin genes within this region given the possibility that gaps between sequence contigs could contain additional hemoglobin genes. The total size of the assembled sequence contigs for the two BACs was 421,907 bp, with approximately 33,000 bp of overlap. The hemoglobin genes spanned approximately 130,000 bp not including gaps between contigs.
All sequences were deposited in the NCBI GenBank database with the assembled sequence contigs for BACs S0155C07 and S0079J05 (fps135) under the accession number GQ898924 and those for BACs S0055H05 and S0014B03 (fps1046) under the accession number GQ898925.
All previously published Atlantic salmon hemoglobin sequences  were identified within the annotated hemoglobin clusters; however, there were two examples of possible allelic differences. Specifically, Clone 3 α hemoglobin (GenBank accession number X97286.1) exhibited 99% similarity at the nucleotide level to SsaChr6α6, which resulted in one amino acid change from a methionine to a leucine at amino acid 32, and the Clone 5 and Clone 6 β hemoglobins (Bohr; X97288 and X97289) showed 99% similarity at the nucleotide level with SsaChr6β3, which resulted in one amino acid change from valine to leucine at amino acid 143. However, as both of these changes were caused by single nucleotide substitutions, each resulting in a single amino acid change, and given the depth of sequencing coverage of the Atlantic salmon BACs (i.e., > 18× coverage in both cases), it is more probable that they reflect sequencing errors in the published clones rather than allelic differences.
To provide further evidence that the two Atlantic salmon hemoglobin gene clusters encompassed all previously identified Atlantic salmon α and β hemoglobin genes, we compared all identified putatively functional Atlantic salmon hemoglobin genes against all full-length Atlantic salmon cDNA clones . Indeed, all unique full-length cDNA clones annotated as β hemoglobins were accounted for within the identified putatively functional β hemoglobin genes. This was also true for the α hemoglobins, with the exception of the cDNA clones with accession numbers BT046755.1 and BT046550.1, which are highly similar to one another but not to the identified hemoglobins. An alignment of these clones using BLASTn  against the nr/nt database revealed similarity to hemoglobin subunit α-D, a distinct type of hemoglobin present in birds, mammals and reptiles that is predicted to have arisen via duplication from a gene that had larval/embryonic function . This gene is apparently found in Atlantic salmon given the presence of the ESTs, but is not in the regions of the α and β hemoglobin genes.
Additional file 2, Table S2 lists all annotated α hemoglobin genes (Table S2A) and β hemoglobin genes (Table S2B) with the source chromosome, strand of transcription, start location, whether the entire hemoglobin gene matches one of the Atlantic salmon hemoglobin clones published by McMorrow et al.  at the amino acid level. It also identifies which genes are non-Bohr β hemoglobins, and lists the top hemoglobin EST cluster hit, if any, with the percent identity from the salmonid EST database [33, 36] and whether the hemoglobin matches one of the full-length cDNA clones with the corresponding NCBI accession number. Table S2C (Additional file 2) lists all putative pseudogenes with the chromosome name, strand of transcription, start location and a description of each exon, with explanations of why the gene was classified as a pseudogene.
The identified putatively functional α hemoglobin genes SsaChr6α2 SsaChr3α2 as well as the β genes SsaChr6β1, SsaChr6β2, SsaChr6β3, SsaChr6β6, SsaChr3β1, SsaChr3β2 and SsaChr3β8 did not have matching EST clones. This could mean that these represent newly identified hemoglobin genes, or that these genes are rarely or never transcribed and thus are not represented in the Atlantic salmon cDNA libraries. Interestingly, several of these genes lie in regions where the tail-to-tail alternating order of the hemoglobin genes is disrupted and they would be transcribed on opposite strands than expected (see below for more details). Future studies using expression profiling of the Atlantic salmon transcriptome at various time points throughout the species' life cycle will provide further insight to this.
Wagner et al.  first reported the tail-to-tail orientation and alternating order of the Atlantic salmon α and β hemoglobin genes. We found that this orientation was fairly well conserved, with the α hemoglobins transcribed on the negative strand and β hemoglobins transcribed on the positive strand, and the alternating α-β order was mostly maintained in both chromosomes with some notable exceptions. On chromosome 6 (fps1046), the alternating α-β pattern is conserved throughout (including putative pseudogenes), but there are some apparent disruptions to the strand of transcription at the 5' end of the cluster. Specifically, SsaChr6β2 as well as all putative β hemoglobin pseudogenes, including SsaChr6βψ1, SsaChr6βψ2, SsaChr6βψ3, were predicted as being transcribed from the negative strand, whereas all putative α hemoglobin pseudogenes (SsaChr6αψ1, SsaChr6αψ2) as well as SsaChr6α2 would be transcribed on the positive strand (Figure 1A). On chromosome 3 (fps135), the β hemoglobin pseudogene SsaChr3βψ1 as well as the putatively functional genes SsaChr3β1 and SsaChr3β2 were predicted to be transcribed from the negative strand, and SsaChr3βψ2 and SsaChr3βψ3 disrupt the otherwise conserved alternating α-β order and orientation of the genes, SsaChr3β2 and SsaChr3β3 being adjacent (Figure 1B). In terms of α hemoglobin genes on chromosome 3, SsaChr3αψ1 and SsaChr3α2 were predicted as transcribed in the positive direction, whereas all others are transcribed on the negative strand. Note again that, for chromosome 3, the order and orientation of the sequence contigs was predicted based on homology with that of chromosome 6 (see dot plot in Additional file 3, Figure S1). Thus, it is possible that inversions or rearrangements may have taken place, and that the resulting predicted order and orientation of the hemoglobin genes is incorrect.
As indicated above, it is interesting to note that all of the putatively functional α hemoglobin genes predicted to be transcribed from the positive strand and all β hemoglobin genes predicted to be transcribed from the negative strand are lacking a corresponding EST at this time. It is possible that the apparent rearrangements have contributed to a global shutdown of transcription in these regions of the genome, allowing several of the hemoglobin genes to degenerate into obvious pseudogenes and silencing the remainder. This should be further explored using expression profiling by qPCR across all life stages of Atlantic salmon.
Microsatellite marker Ssa10067BSFU, representing fps1046 was informative in both the Atlantic salmon SALMAP families (Br5 and Br6) [37, 38] and was mapped to linkage group 4. Microsatellite Ssa0516BSFU was informative in the Br6 family and mapped to linkage group 11 (Figure 2). FISH analysis revealed that fps1046 is found within Atlantic chromosome 6 and fps135 is within chromosome 3 (see  for chromosome nomenclature). The FISH and linkage mapping of chromosomes 6 and 3 to linkage groups 4 and 11, respectively, contributed to the integration of the Atlantic salmon karyotype and linkage map . Primer sequences for the microsatellite markers used for linkage analysis are provided in Additional File 1, Table S1 and within ASalbase, the Atlantic salmon genomic database .
Merged female linkage maps for Atlantic salmon SALMAP families Br5 and Br6 showing linkage groups 4 and 11. Microsatellite marker Ssa10067BSFU (underlined), representing fps1046 was informative in both the Altantic salmon SALMAP families (Br5 and Br6) and mapped to linkage group 4. Microsatellite Ssa0516BSFU (underlined) was informative in the Br6 family and mapped to linkage group 11.
We examined the regions surrounding the hemoglobin gene clusters in the available teleost genomes and compared them against one another as well as to those predicted to surround the Atlantic salmon hemoglobin gene clusters to gain insight to the nature of the teleostean hemoglobin gene containing chromosomes. We found that the genes surrounding the hemoglobin gene clusters are well conserved. Figure 3 shows a schematic diagram of the hemoglobin gene regions and the predicted surrounding named genes in medaka, zebrafish, stickleback and tetraodon compared to those of Atlantic salmon. Note that, for the hemoglobin gene containing BACs within Atlantic salmon chromosome 3, only the order and orientation of the sequence contigs that aligned with those from the BACs within chromosome 6 could be predicted. That is, given the extensive overlap between the two BACs that cover fps1046 (chromosome 6), the total sequenced region is much shorter than that of fps135 (chromosome 3); therefore, any sequence contigs from fps135 that did not fall within the coverage of fps1046 could not be ordered or oriented. Thus, for any sequence contigs that fell outside of this region, we were only able to establish their relative location compared to those that aligned with chromosome 3 based on their source BAC. Within Figure 3, solid lines between predicted genes indicate that the order and orientation of the predicted gene relative to those neighboring it is known, whereas a single black dot between predicted genes indicates that the relative location of the predicted genes compared to those joined by solid lines is known, but their order and orientation (i.e., that of the sequence contigs on which they reside) relative to one another is not. Arrows in Figure 3 indicate the direction of transcription of the gene relative to the location of the hemoglobin gene cluster; lack of an arrow indicates that the relative direction of transcription cannot be determined.
Comparative synteny of hemoglobin gene clusters among sequenced teleost species. Schematic representation of annotated genes within the regions surrounding the hemoglobin gene clusters for Atlantic salmon and four annotated teleost genomes (O. latipes, D. rerio, G. aculeatus, T. nigroviridis). Colored blocks indicate shared or common genes as specified in the Figure legend. Black blocks indicate genes that are not shared within the indicated regions of any other species. Distances between genes vary (i.e., figure is not to scale); the start and end of the chromsome/group region is shown in base pairs (bp) for each of the annotated teleost genomes. Solid lines between predicted genes indicate that the order and orientation of the predicted gene relative to those neighboring it is known, whereas for Atlantic salmon fps 135 (chromosome 3), a single black dot between predicted genes indicates that the relative location of the predicted genes compared to those joined by solid lines is known, but their order and orientation (i.e., that of the sequence contigs on which they reside) relative to one another is not. Arrows indicate the direction of transcription of the gene relative to the location of the hemoglobin gene cluster; lack of an arrow indicates that the relative direction of transcription cannot be determined. For D. rerio chromsome 3, the gene for ELAV-like protein was found distantly downstream of the nearest common gene (Arylakylamine N-acetyltransferase 2), as indicated by the distance shown, with numerous predicted genes in between.
Briefly, medaka, zebrafish and tetraodon and Atlantic salmon exhibit two distinct hemoglobin gene clusters on separate chromosomes or linkage groups, whereas stickleback has only one. Although there are some rearrangements in terms of the positioning of genes relative to the hemoglobin genes and direction of transcription as well as some apparent gains, losses and duplications of genes, all of the organisms possess one similar cluster (hereafter Cluster 1) that contains, among others, the shared genes UPF0171 protein C16orf35, Rhomboid family member 1, Dedicator of cytokinesis protein 6, ELAV-like protein 3 and DNA-3-methyladenine glycosylase (MPG; see Figure 3). Note these results are consistent with those of Patel et al. , who report that MPG and C16orf35 surround the α hemoglobin gene cluster in frog, chicken and human, and one of the α and β hemoglobin clusters in platypus and opposum. However, whereas this cluster appears twice in Atlantic salmon, the second cluster in zebrafish, tetraodon and medaka (hereafter Cluster 2) is characterized by a different set of shared genes; specifically, the presence of Aquaporin-8 and Rho-GTPase-activating protein, although tetraodon is lacking the former and zebrafish is lacking the latter. In addition, tetraodon exhibits a copy of Rhomboid family member 1 on Cluster 2 as well as Cluster 1. Stickleback and Atlantic salmon, however, appear to have lost Cluster 2 entirely. Instead, the stickleback genome only has one hemoglobin cluster (Cluster 1), whereas that of Altantic salmon shows two copies of Cluster 1.
A dot plot generated using the JDotter software  comparing the sequenced BACs from Atlantic salmon chromosomes 3 and 6 showed that the regions surrounding the hemoglobin genes are > 95% similar between the two chromosomes, with variations only within the hemoglobin gene regions (Additional file 3, Figure S1). This further suggests that the two Atlantic salmon hemoglobin gene containing chromosomes or regions are homeologous (i.e., represent duplicated copies of the same cluster as the result of a WGD event). Thus, we hypothesize that the WGD at the base of the teleost lineage produced Cluster 1 and Cluster 2, which remain in the zebrafish, medaka and tetraodon lineages, that Cluster 2 was lost in the stickleback lineage, and that Cluster 2 was lost within the salmonid lineage prior to the WGD, which yielded two copies of Cluster 1. This hypothesis is also supported by the fact that the Atlantic salmon chromosome arms 3q and 6q (where the hemoglobin gene clusters are located) share nine duplicated genetic markers .
The results of the phylogentic analysis (Figures 4 and 5 for α and β genes, respectively) suggest that the hemoglobin genes cluster according to functional similarity, which corresponds to sequence similarity. This is expected given the high sequence similarity and short nature of the hemoglobin genes. Specifically, in Figure 5, all of the non-Bohr α hemoglobin genes (SsaChr3β3, SsaChr3β5, SsaChr3β6, SsaChr3β8, SsaChr6β4 and SsaChr6β6) form a distinct clade with no other hemoglobin genes, further supporting that there are no β globin genes lacking the Bohr effect in the other fish species examined (see Discussion). Additionally, many genes that were annotated as embryonic within Ensembl (identified with "emb" following the species name) clustered closely, which provides some suggestion as to candidate Atlantic salmon embryonic hemoglobin genes (see Discussion).
Phylogenetic tree of teleost and Xenopus tropicalis α hemoglobins. The α hemoglobin cDNAs (exclusive of untranslated regions) annotated within the Ensembl 54 database for medaka, zebrafish, tetraodon, stickleback and X. tropicalis, as well as those identified in Atlantic salmon here and the hemoglobin genes identified as embryonic within rainbow trout  were independently aligned using EBioX . Phylogenetic trees were constructed using the a Bayesian approach with (5 runs, 100,000 generations, 40% burn-in period) within the TOPALi V.2 software package  running the MrBayes program  under the best selected model (SYM). For simplicity, as well as to clearly indicate the source chromosome of the gene, the teleostean hemoglobin genes were named using the same system used to name those of Atlantic salmon. That is, an abbreviated three letter (g enus sp ecies) name followed by chromosome/linkage group name followed by α or β followed by a number indicating the sequential order of the genes from 5' to 3' as defined by Ensembl (Additional file 4, Table S3). Hemoglobin genes that were previously identified via expression analysis as being expressed exclusively during embryogenesis, and that are identified as embryonic within the Ensembl 54 database are denoted with "emb" following the assigned gene name. Branch numbers indicate posterior probabilities.
Phylogenetic tree of teleost and Xenopus tropicalis β hemoglobins. The β hemoglobin cDNAs (exclusive of untranslated regions) annotated within the Ensembl 54 database for medaka, zebrafish, tetraodon, stickleback and X. tropicalis, as well as those identified in Atlantic salmon here and the hemoglobin genes identified as embryonic within rainbow trout  were independently aligned using EBioX . Phylogenetic trees were constructed using the a Bayesian approach with (5 runs, 100,000 generations, 40% burn-in period) within the TOPALi V.2 software package  running the MrBayes program  under the best selected model (SYM). For simplicity, as well as to clearly indicate the source chromosome of the gene, the teleostean hemoglobin genes were named using the same system used to name those of Atlantic salmon. That is, an abbreviated three letter (g enus sp ecies) name followed by chromosome/linkage group name followed by α or β followed by a number indicating the sequential order of the genes from 5' to 3' as defined by Ensembl (Additional file 4, Table S3). Hemoglobin genes that were previously identified via expression analysis as being expressed exclusively during embryogenesis, and that are identified as embryonic within the Ensembl 54 database are denoted with "emb" following the assigned gene name. Branch numbers indicate posterior probabilities.
In both trees, the X. tropicalis hemoglobin genes formed their own clade, although they did not form distinct outgroups, which again, may be a function of the high similarity between the hemoglobin genes across species. All annotated medaka, zebrafish, stickleback and tetraodon α and β hemoglobin genes that were used to generate the phylogenetic trees (i.e., all that were identified within the Ensembl 54 database) are provided in Additional file 4, Table S3 by species with the corresponding name assigned by us for comparison purposes (see Methods), as well as the Ensembl gene ID, chromosome/linkage group, start and stop location and strand of transcription.
Notably, there are not four clusters of hemoglobin genes in Atlantic salmon even though the hemoglobin clusters were already duplicated within the teleost lineage prior to the salmonid-specific WGD event that took place between 20 and 120 million years ago [20, 21]. Furthermore, our comparative genomic analysis, as well as the high similarity in the non-coding regions of the two fps, suggests that Atlantic salmon exhibit a duplicated copy of one cluster (Cluster 1), and are missing the second cluster (Cluster 2), which is still seen in medaka, zebrafish and tetraodon (see Figures 3 and 6). Figure 3 shows a schematic diagram of the predicted genes surrounding the hemoglobin genes in Atlantic salmon as well as four annotated teleost genomes, while Figure 6 depicts the phylogenetic relationships of the studied teleost fishes (adapted from ), and illustrates our hypothesis of the evolutionary events that took place to produce the observed chromosomal arrangements of the teleost hemoglobin genes. With respect to the other teleosts studied, subsequent to the teleost WGD, which produced Clusters 1 and 2, zebrafish and medaka and tetraodon appear to have maintained both hemoglobin gene clusters, whereas stickleback and Atlantic salmon have lost Cluster 2. In addition, zebrafish exhibits an apparent inversion in Cluster 1 such that ELAV-like protein 3 is located on the opposite side of the hemoglobin genes, far downstream from Rhomboid family member 1 with several unshared genes between them. Within the tetraodon genome, Cluster 2 also exhibits some shuffling compared to those of the other genomes, and, interestingly, contains Rhomboid family member 1, which is also found on Cluster 1. These relationships will be clarified by further analysis and in-depth annotation of the full-length hemoglobin gene repertoires of other teleost species as more of them undergo full genome sequencing.
Schematic representation of the evolution of teleostean hemoglobin gene clusters. Whole genome duplication (WGD) events are indicated by grey diamonds. The two hemoglobin gene clusters resulting from the teleost WGD are represented as (1) and (2) for Cluster 1 and Cluster 2, respectively (see text). Loss of a hemoglobin gene cluster by excision is indicated by a diagonal slash across that cluster. Although the genome sequence is available for the pufferfish, T. rubripes, the fugu genome was not included in this analysis because the published hemoglobin arrangement of two hemoglobin gene clusters, one containing only α hemoglobin genes and one containing both α and β hemoglobin genes  did not agree with the annotation results of the latest fugu genome assembly reported within the Ensembl database.
With respect to the salmonid lineage, we propose that Atlantic salmon lost Cluster 2 prior to the salmonid-specific WGD, which duplicated Cluster 1, thus producing the two copies of Cluster 1 and lack of Cluster 2 seen within the Atlantic salmon genome today. We also recognize the possibility of an alternative pathway, which involves tetraploidization of hemoglobin gene Clusters 1 and 2 as predicted by the salmonid-specific WGD (i.e., producing two copies of each cluster), followed by subsequent loss of both copies of Cluster 2 within the salmonid lineage. However, this involves two separate excision events subsequent to the salmonid WGD, whereas the former hypothesis only involves one such event prior to the salmonid WGD, and is thus the more parsimonious route. Note that the loss of the two clusters must have taken place by excision of the entire regions or chromosome loss as opposed to gradual degradation of the hemoglobin genes or gene silencing, or we would have expected our hemoglobin probes to hybridize to footprints of old hemoglobin clusters in other regions of the genome.
Our results revealed that the Atlantic salmon genome contains substantially more paralogous α and β hemoglobin gene copies than previously published [24–26]. Furthermore, there are more copies of the α and β hemoglobin genes within the Atlantic salmon genome than in other teleosts whose genomes have been sequenced. Specifically, of the teleost genomes examined, that of zebrafish contains the most hemoglobin genes, with six β hemoglobin genes and seven α hemoglobin genes, compared to 13 and 14 putatively functional α and β hemoglobin genes, respectively, in Atlantic salmon. Note, however, that some of the putatively functional hemoglobin genes do not have an EST associated with them, perhaps suggesting that the actual number of functional genes should be reduced accordingly. However, this would still leave the Atlantic salmon with more α and β hemoglobin genes than any of the other teleosts examined to date.
Numerous reports suggest that mammalian hemoglobin levels are implicated in the increased oxygen affinity of blood in situations of adaptation to altitude-induced hypoxia (reviewed by ), thus suggesting that hemoglobin levels contribute to survival in low oxygen environments. Further, Hoffman et al.  suggest that variation in hemoglobin gene copy number may be a source of regulatory variation affecting physiological differences in blood oxygen transport and aerobic energy metabolism in mammals. Indeed, it has been proposed that the capacity of fish to colonize a wide range of habitats is directly related to their hemoglobin systems . In addition, a study using real-time quantitative PCR demonstrated that hypoxic conditions induce complex responses in hemoglobin gene expression in zebrafish . Thus, the extensive array of hemoglobins in Atlantic salmon may reflect the diverse range of environmental conditions that an individual salmon must endure throughout its lifecycle as it migrates from freshwater streams to open ocean and back.
The Bohr effect is the phenomenon that the affinity of hemoglobin for O2 is affected by pH. Specifically, an increase in the blood pCO2 shifts the oxygen dissociation curve to the right, resulting in the release of O2 and thereby enabling more efficient gas exchange between blood and tissues. This is the result of the oxy-deoxy conformational change and allosteric interactions between O2 and H+/CO2 binding sites of the hemoglobin molecule. Hemoglobin molecules lacking the Bohr effect are able to retain O2 under conditions of elevated acidity caused by increased oxygen consumption . Therefore, the non-Bohr hemoglobin may function as an emergency oxygen supplier when an organism is exercising vigorously, such as when a fish is escaping a predator, catching prey, or swimming against a current.
The Bohr effect depends on the intricate arrangement and interactions of all cation and anion binding sites in the hemoglobin molecule and involves a number of contributing amino acid groups that have not yet been fully elucidated . Indeed, a comparison of mammalian, avian and teleost fish hemoglobins suggested that several different histidine and non-histidine sites contribute to the Bohr effect in different species to varying degrees . It is widely accepted, however, that a greater overall histidine content in the hemoglobin molecule correlates with an increased Bohr effect , with the C-terminal histidine residue accounting for up to 50% of the effect [45, 47, 48]. In Atlantic salmon, the non-Bohr β hemoglobin exhibits phenylalanine at this position . In addition, in Atlantic salmon, the non-Bohr β hemoglobin has 147 amino acids (vs. 148 in the Bohr molecule), including the initiator methoinine, and the amino acid at position 93 is alanine [26, 49]. We used these three characteristics to identify a total of six putatively functional non-Bohr β hemoglobin genes within the Atlantic salmon genome (Figure 1; Additional file 2, Table S2B). Conversely, no β hemoglobin genes identified within the medaka, zebrafish, stickleback or tetraodon genomes exhibited all three of these hallmarks of the non-Bohr β hemoglobin. In addition, a recent PCR-based exploration of the Atlantic cod genome found no β hemoglobin genes exhibiting these characteristics , implying an absence of the non-Bohr hemoglobins in this species. All of the Atlantic salmon non-Bohr β hemoglobins formed a distinct clade in the phylogenetic analysis, which reflects their common structural elements and therefore highly similar sequences, and lends further support to the finding that no other fish species examined possesses non-Bohr hemoglobin genes [Figure 5].
This apparently high number of non-Bohr β hemoglobin gene copies within the Atlantic salmon genome may be attributable to the Atlantic salmon life history, with its extensive migratory range and the need to swim upstream into fresh water habitats to spawn. In contrast, all of the model teleosts studied so far inhabit relatively consistent environments with little variation in depth, temperature or salinity. Additionally, although Atlantic cod, a non-model teleost that does not appear to possess non-Bohr β hemoglobin genes, inhabit depths from the surface up to 600 m and undertake seasonal migration, at no point in their lifecycle do they inhabit freshwater . Thus, future studies of the hemoglobin gene repertoires of other migratory salmonids, such as the Pacific salmon species, as well as land-locked freshwater salmonids, in addition to expression profiling of hemoglobin genes at different life stages will provide further insight to this phenomenon.
To date, there has been no published study examining temporal expression of hemoglobin genes in Atlantic salmon. Indeed, the most closely related species for which this has been done is rainbow trout , for which there is no published genome sequence as well as no comprehensive examination of the rainbow trout hemoglobin repertoire such as this one. Given the complex life history of salmon and the lack of available expression data, we could not confidently assign the title of embryonic to any Atlantic salmon hemoglobins at this time. However, it is noteworthy that SsaChr3α1 and SsaChr6α1 form a clade with Omyα2emb, as well as Omyα1emb and an embryonic Danio rerio α globin, DreChr3α5emb (Figure 4), and that SsaChr3α2 and SsaChr6α2 cluster closely with this clade. Figure 5 shows that SsaChr3β1, SsaChr6β1, SsaChr3β2 and SsaChr6β2 form a clade with Omyβ1emb. These phylogenetic relationships suggest that these Atlantic salmon hemoglobin genes may be embryonic. Also worth noting is that all of these are the first genes in the 5'-3' direction on their respective chromosomes. In mammals, temporal expression of hemoglobin genes correlates with spatial location on the chromosome, with the first upstream hemoglobin gene being the first expressed . This further suggests that these genes (i.e., SsaChr3α1 and SsaChr3α2, SsaChr6α1, SsaChr6α2, SsaChr3β1 SsaChr3β2, SsaChr6β1 and SsaChr6β2) encode candidate embryonic hemoglobins. However, further analysis, in particular, detailed expression profiling of hemoglobin genes during all life stages, is required to examine these hypotheses.
We found that, despite the Atlantic salmon genome having gone through at least two WGD events relative to tetrapods, which would result in four predicted hemoglobin gene clusters, only two such clusters were present. Furthermore, the Atlantic salmon genome appears to exhibit two copies of one of the duplicated ancestral teleost hemoglobin gene clusters, and has presumably lost the other cluster. We also found that the Atlantic salmon genome harbors substantially more hemoglobin genes than the other teleosts for which the hemoglobin gene repertoires have been identified, and that they possess several hemoglobin genes that appear to encode non- Bohr β hemoglobins. We suggest that these characteristics of the Atlantic salmon hemoglobin genes reflect the dynamic life history of Atlantic salmon.
As part of the Genomic Research on All Salmonids Project (GRASP), an Atlantic salmon BAC library was produced from a partial EcoRI restriction enzyme digest of DNA from a Norwegian aquaculture strain male fish (CHORI-214 segments 1-3). There are 312,000 BAC clones in the library with an average insert size of 190,000 bp, which have been arrayed onto nylon membranes, thus representing an 18.8-fold coverage of the Atlantic salmon genome . BACs were fingerprinted using HindIII and arranged into contigs to create the first physical map of a salmonid genome . Approximately 210,000 BAC end-sequences have been determined, corresponding to approximately 3.5% of the Atlantic salmon genome. Information on the Atlantic salmon BACs and physical map can be found at .
To identify the Atlantic salmon BACs containing the hemoglobin genes, oligonucleotide probes (~40-mers) were designed from the published Atlantic salmon Clone 6 (GenBank accession number X97289) for α, β and non-Bohr β hemoglobins. PCR primers sets were also designed to span intron 1 of all three hemoglobin types. In addition, primer sets were designed to span intron 1 of the embryonic α and β hemoglobins of rainbow trout, with a ~40-mer forward primer that was used for hybridization probing (GenBank accession numbers: α: AB015448; β: AB015450). All primers and probes were designed using Primer3 ver. 0.4.0  and are provided in Additional file 1, Table S1. The oligonucleotide probes were end-labeled with 32PγATP using T4 polynucleotide kinase and hybridized to six BAC filters at a time as described by Johnstone et al. . Briefly, prehybridization was carried out in 5× saline-sodium citrate buffer (SSC), 0.5% sodium dodecyl sulfate (SDS), and 5 × Denhardt's solution at 65°C. The filters were washed three times for 1 hr at 50°C, in 1 × SSC and 0.1% SDS. Filters were exposed to phosphor screens that were scanned using the Typhoon Imaging System and visualized using ImageQuant software, giving an image of the 32P-labeled hybridization-positive BACs containing the hemoglobin markers. The hybridization-positive BAC clones were picked from the library, cultured in 5 mL LB media containing chloramphenicol (50 μg/mL) overnight at 37°C shaking at 250 rpm and made into glycerol stocks for subsequent PCR verification that they indeed contained hemoglobin genes. Hybridization and PCR-positive BACs for the hemoglobin genes were matched to two fingerprint scaffolds (fps) within the Altantic salmon physical map (fps135 and fps1046; [30, 31]).
Using a combination of hybridization probing and PCR (see above) to screen all BACs within the suspected hemoglobin gene containing regions, we identified two overlapping BACs from each of fps1046 (BACs S0055H05 and S0014B03) and fps135 (BACs S0155C07 and S0079J05) spanning the entire Atlantic salmon hemoglobin gene repertoire. That is, all primers amplified hemoglobin gene products within these BACs, and no additional BACs that were not contained within the four BACs as determined by the Atlantic salmon physical map yielded PCR products using the hemoglobin gene primers. The four BACs were sequenced using standard Sanger sequencing of a shotgun library as previously described . Briefly, BAC DNA was isolated from each of the hemoglobin-containing BACs using Qiagen's Large Construct kit as per the manufacturer's directions (Qiagen, Mississauga, Ont. Canada). The kit includes an exonuclease digestion step to eliminate E. coli genomic DNA. The purified BAC DNA was sheared by sonication and blunt-end repaired. The sonicated DNA was size fractioned by agarose gel electrophoresis and 2-5 kb fragments were purified using the QIAquick Gel Extraction Kit (Qiagen, Mississauga, Ont. Canada). DNA fragments were ligated into pUC19 plasmid that had been digested with SmaI and treated with shrimp-alkaline phosphatase to produce de-phosphorylated blunt ends. The ligation mixture was used to transform supercompetent E. coli cells (XL1-Blue; Stratagene, La Jolla, CA. USA). Transformed cells were cultured overnight at 37°C on LB/agar plates supplemented with ampicillin (200 μg/mL) and 1,920 (5 × 384 well plates) clones were sent to the Michael Smith Genome Sciences Centre, Vancouver, BC Canada, for sequencing. The sequences were analyzed for quality using PHRED , assembled using PHRAP , and viewed using Consed version 15.0 . BAC assemblies were complicated by the repetitive nature of the Atlantic salmon genome . Assemblies were hand-finished to fill gaps (i.e., join sequence contigs) as best as possible using primer walking; however, primers could not be designed to join some sequence contigs that ended in repetitive sequence, or often primers amplified multiple products (i.e., showed a multiple bands or a smear on an agarose gel).
The sequences of BACs S0055H05 and S0155C07 (representing fps 1046 and 135, respectively) were screened for microsatellite markers that were variable (i.e., informative) within the two Atlantic salmon SALMAP mapping families, Br5 and Br6, each of which contains two parents and 46 offspring . Markers Ssa10067BSFU and Ssa10051BSFU were identified within S0055H05 and S0155C07, respectively. PCR primers were designed to amplify the region containing the microsatellite. The forward primer for each pair contained an M13 sequence tag that was used for genotyping analysis. Genotyping results were analyzed with LINKMFEX ver. 2.3 .
A single end-sequenced BAC containing the α, β and non-Bohr β hemoglobin genes was chosen from each of fps135 and fps1046 (S0155C07 and S0055H05, respectively) to be used for chromosome assignment. Approximately 1 μg of BAC DNA was purified (Qiagen mini-prep kit; Qiagen, Mississauga, Ont., Canada) and used for FISH analysis to identify the Atlantic salmon chromosomes containing the hemoglobins. Comparison of the results of the linkage and FISH analysis of the Atlantic salmon hemoglobin BACs contributed to the recent integration of the Atlantic salmon linkage map and karyotype .
The genes were predicted to contain three exons and two introns.
The predicted exons were of the appropriate sizes, meaning that predicted splice junction sites aligned with those of known functional hemoglobins and start and stop codons were present in the appropriate places.
The final predicted protein included 147 or 148 amino acids for β hemoglobins and 143 amino acids for α hemoglobins.
The sequences of any predicted ORFs that aligned to hemoglobins but failed to meet any one of the above criteria were examined by eye for potential miss-calling by our annotation procedures. Specifically, we looked for historical footprints of missing exons that were not recognized by the pipeline, interruptions to splice sites as well as insertions and deletions of stop and start codons, potential sequencing errors and frame-shift mutations caused by insertions or deletions. We also examined by eye any putative three-exon ORFs identified by our pipeline that were not recognized by a BLAST search as encoding hemoglobins to determine whether they may be remnant hemoglobin genes or previously undefined hemoglobin-like genes. If, after this hands-on annotation, predicted proteins still did not meet the above criteria, the sequences were defined as putative pseudogenes. Furthermore, any regions for which the predicted orientation (i.e., α hemoglobin genes transcribed on the negative strand and β hemoglobin genes on the positive) and alternating order of the α and β hemoglobin genes was disrupted were examined by eye for putative remnant hemoglobin exons and introns. All such regions were aligned against intact hemoglobin genes using ClustalW2 , and predictions were made as to whether these regions represented footprints of historical hemoglobin genes.
All annotated hemoglobin genes were assigned an Ssa ( S almo sa lar) name followed by Chr3 for fps135 and Chr6 for fps1046 to denote its chromosomal location, then α or β to identify the gene encoded, and finally a number corresponding to its order relative to the other α or β genes on that chromosome from 5' to 3'.
We defined β hemoglobin genes exhibiting three hallmarks of a lack of the Bohr effect were defined as putative non-Bohr β hemoglobin genes. These hallmarks include: 1) the non-Bohr β hemoglobin has 147 amino acids, including the initiator methionine; 2) the C-terminal amino acid is phenylalanine; 3) the amino acid at position 93 is alanine [48, 27].
Atlantic salmon BAC sequences surrounding the hemoglobin gene clusters were annotated using our in-house annotation pipeline described above. This provided a preliminary prediction of the genes lying within the sequenced regions. However, note that different components of the pipeline can differ in their gene predictions, and that a full, comprehensive annotation of these regions as well as the rest of the Atlantic salmon genome will be completed with sequencing of the whole genome.
The genes surrounding the hemoglobin clusters in four annotated teleost genomes, medaka, zebrafish, tetraodon (Tetraodon nigroviridis) and stickleback (Gasterosteus aculeatus), were identified using the Pfam ID for the hemoglobin protein family (PF00042)  available within Biomart . Specifically, the Ensembl 54 Genes database was searched using the appropriate genome-specific dataset for hemoglobins. Once the genomic locations of the hemoglobin genes were determined, the region surrounding the hemoglobin gene clusters was expanded until at least five predicted genes were identified on either side of the hemoglobin gene cluster, or until no additional common or shared genes could be identified. This allowed us to examine the synteny of the regions surrounding the hemoglobin genes, and thereby generate hypotheses of hemoglobin gene evolution in teleost fishes. Note that the fugu genome was not included in this analysis because the published hemoglobin arrangement of two hemoglobin gene clusters, one containing only α hemoglobin genes and one containing both α and β hemoglobins  did not agree with the annotation results of the latest fugu genome assembly reported within the Ensembl database. Instead, only one apparent hemoglobin cluster containing both α and β hemoglobin genes could be identified on fugu scaffold 3, and when the genes surrounding this cluster were compared to those of the other genomes examined, no shared genes (i.e., no conserved synteny) could be found.
The α and β hemoglobin cDNAs (exclusive of untranslated regions) annotated within the Ensembl 54 database for medaka, zebrafish, tetraodon and stickleback, as well as those identified in Atlantic salmon here and the hemoglobin genes identified as embryonic within rainbow trout  were independently aligned using EBioX . We examined the relationships among the gene products by constructing phylogenetic trees using the a Bayesian approach with (5 runs, 100,000 generations, 40% burn-in period) within the TOPALi V.2 software package  running the MrBayes program  under the best selected model (SYM). For simplicity, as well as to clearly indicate the source chromosome of the gene, the teleostean hemoglobin genes were named using the same system used to name those of Atlantic salmon. That is, an abbreviated three letter (g enus sp ecies) name followed by chromosome/linkage group name followed by α or β followed by a number indicating the sequential order of the genes from 5' to 3' as defined by Ensembl (Additional file 4, Table S3). Note that hemoglobin genes of medaka , zebrafish  and rainbow trout  that were previously identified via expression analysis as being expressed exclusively during embryogenesis, and that are identified as embryonic within the Ensembl 54 database are identified within the phylogenetic trees (denoted with "emb" following the assigned gene name) as well as within Additional file 4, Table S3.
We are grateful to the Michael Smith Genome Sciences Centre for sequencing the BAC shotgun libraries. This work was supported by funding from Genome Canada, Genome BC, and the Province of British Columbia (WSD and BFK) and the United States Department of Agriculture Grant # 2006-04814 (to RBP) as well as graduate scholarships from Weyerhaeuser Corporation (Weyerhaeuser Molecular Biology Scholarship) and Simon Fraser University (Molecular Biology Graduate Fellowship and President's Research Stipend; NLQ).
NLQ contributed to the study design, identification and isolation of BACs, sequence assembly and hand-finishing, sequence annotation, data analysis and manuscript preparation. KAB and WC conducted the bioinformatics and contributed to sequence annotation and data analysis. KPL assisted with sequence assembly and data analysis. EAD performed the linkage analysis and RBP conducted the FISH analysis. BFK and WSD contributed to the study design, data analysis and manuscript preparation. All authors read and approved the final manuscript.

industry_corpus2: Biopharmaceuticals


text:
The unorganised manager series comes in three parts. Parts 1 and 2 work together in showing that, no matter how efficient managers may think they are, they cannot be fully effective until they learn how to manage their time.
In Part 1: Damnation, a hard-working manager is so disorganised that he unwittingly makes the lives of his family and colleagues a misery. His lack of organisation leads to an early heart attack and a confrontation with St Peter. By being given a second chance on Earth, he is able to learn the principles of time management in a way that viewers, many of whom will have been unaware of their own shortcomings, can easily relate to.
Ideal for incorporating into time management and delegation courses at junior management level, this engaging and humorous plot uses memorable, wrong-way, right-way scenarios to help managers learn the practical skills of effective time management.

industry_corpus2: Subject Education


text:
Sophie Parkes | Writer: 500 words on folk?
The ever-lovely Dan and Hannah at PYT set me the ultimate challenge - to write something (anything!) about the folk scene... in 500 words. So it ended up 886. Quickly and smartly reduced to 700 by Hannah. Thanks, guys - if only everyone was as free and easy as you! I'll let you know when the next issue of Pull Yourself Together is out and where it can be found.
I've also had the pleasure of interviewing the delectable Hannah Peel for the High Voltage fanzine. I'd seen Hannah play as part of the expanded lineup with The Unthanks, but this was the first time I'd heard her solo stuff and my, what a voice.

industry_corpus2: Film & Entertainment


text:
NCA a charitable organisation has been a vital part of the Northmoor community for over 18 years. Our Northmoor Community Centre hub provides a wide portfolio of quality social, welfare, self-help, educational, leisure and creative activities for everyone 0-99+ based on bringing different diverse people together and learning in a fun environment – which sees us support 1300+ people per year.
We pride ourselves on the warm, open door, welcoming approach with caring staff and volunteers who understand the needs of those who use the services available. .
Since 2000 NCA has provided facilities and services managing Northmoor Community Centre offering play, learning, and social group activities for everyone in our community – from Under 5’s to older people. In 2005 our offer of services and facilities expanded to include the development and management of a Social Enterprise, Northmoor Community Laundrette. Throughout our history, we’ve worked to support and promote grassroots development and provide accessible, affordable opportunities that reflect the needs of our community who live in poverty.
For more information please see our history page.
To achieve these goals we recognise we need to work in different ways to meet the needs of the many groups, cultures and individuals in our community. That’s reflected in the broad portfolio of quality assured services we deliver from our Community Centre supporting people from birth through to old age.

industry_corpus2: Others


text:
Even the name sounds healthy. I came across this line of baby and kid furniture and found it intriquing.
Celery creates simple, modern furniture for babies and kids. Their unique designs require no hardware.
They also give consideration to the environment by using, "bamboo with non-toxic adhesives, and 100% recycled formaldehyde-free medium density fiberboard combined with zero to low-VOC finishes affirm our commitment to lasting and eco-friendly materials for all our furniture."
Their website mentions that a line of grown-up furniture is planned as well.
I am so digging the chalkboard bed...!

industry_corpus2: Manufacturing


text:
We will be the global No.1 through unchanging sincerity and effort with challenge and creation.
We introduce detail contents about outstanding technical materials that only JINYANG has below.
We are trying to find a outstanding people who will make a new step to the world with JINYANG. We wish you have interest in it.
We introduce the JINYANG which has grown to the best of sealing products since 1991.


text:
DIn accordance with the provisions of the current legislation on Personal Data Protection, we inform you that your data will be included in the processing system owned by MARINA ASJAL SL with tax identification number B07693948 and business address at GERMANS MONTCADA 11 07181, PALMANOVA (ILLES BALEARS), in order to facilitate, speed up and meet the commitments established between both parties. In compliance with current legislations, MARINA ASJAL SL informs that the data will be kept for the period deemed strictly necessary to comply with the aforementioned precepts.
Unless stated otherwise, we will assume that your information remains unchanged, that you agree to notify us of any changes and that we have your consent to use them for the aforementioned purposes.
MARINA ASJAL SL informs that it will proceed to use this information lawfully, fairly, transparently, adequately, relevantly, limitedly, accurately and that it will be kept up to date. Therefore, MARINA ASJAL SL agrees to adopt all reasonable measures to ensure that they are promptly removed or rectified where inaccurate.
In accordance with the rights conferred by current data protection regulations, you may exercise your rights of access, rectification, limitation of processing, deletion, portability and opposition to the processing of your personal data and the consent given for their processing, by sending your request to the postal address indicated above, or via email to marinapalmanova@gmail.com.
You may also contact the competent authority to make any complaint you deem appropriate. Sending these details confirms you accept this clause.


text:
I teach piano lessons and music theory from my home in Faversham. Pupils have regular opportunities to perform, compose, play in ensembles, participate in competitions and festivals, and work towards graded exams.
Please email me for current availability.
Well done to all the performers at this year’s Summer Recital at the Assembly Rooms! Here are a few highlights, more pictures can be found on the gallery page.
Well done to everyone who performed at the Assembly Rooms last Saturday, 7th May. Big thank you to all the friends and relatives who came along to support you. Everyone did a wonderful job and I hope you all enjoyed yourselves! Some pictures and videos can be found on the gallery page. Copies of all will be emailed to parents this week.
This year’s Summer Recital was held at The Assembly Rooms on Preston Street. It’s always a highlight of the year- this year we had 26 performers playing solos, duets and singing songs!
It’s never too late to learn… here are two very brave adult performers with their certificates!
Two spaces still available on Wednesday and Friday- early evening!
Scientists have discovered what childhood piano lessons can do for your brain!


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Our systems utilize the latest Redhat server technology to allow our servers to be continually up to date with Redhat system & security patches. Read more on Red Hat Linux here.
We use the latest Windows server operating systems for our Windows based web hosting packages.
Updates and patches are applied as and when they are released making sure that your website and server is always secure and protected.
Umbrella Host ‘s network operates on industry-leading network equipment from Cisco Systems. All our switches and routers are developed by Cisco.
Our network is based on gigabit technology, which means that it runs at a speed of 1000 Mb per second – most web hosting networks are limited to 100 Mb per second.
We use only Dell servers in our web hosting platform. Utilizing state-of-the-art technology we have built a web hosting platform that is second to none.

industry_corpus2: Computing & Telecommunications


text:
For the first Bank Holiday in May, Dave and I headed to North Cornwall and Devon for a weekend by the coast with friends.
Have you ever heard of Weston-Super-Mare’s Carnival? I hadn’t. But after some of my family went last year and came back raving about it, I know I’d have to go to the next one. Fast forward a year and I’m very glad that I tagged along!

industry_corpus2: Travel & Geography


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If you need Air Cargo in Queen Alia International Airport or move Air Cargo from or to Queen Alia International Airport Freight Tender is the ideal place to meet your requirements. Freight Tender is a free freight exchange with Air Cargo for Queen Alia International Airport and many other locations throughout Europe and the rest of the world.
We currently have 11 companies registered who are located in Jordan that have Air Cargo to move from and to Queen Alia International Airport.
We currently have 6 shipping companies registered who are located in Jordan that move Air Cargo from and to Queen Alia International Airport.
There has been 1 adverts for Air Cargo to and from Queen Alia International Airport placed in the past on Freight Tender excluding any current live adverts.
We currently have 0 live adverts for Air Cargo from or to Queen Alia International Airport. See below for details of up to 5 adverts, click on the link below the adverts to view more.
Although we currently have no live adverts for Air Cargo from or to Queen Alia International Airport, we have new adverts being placed daily so if you are a transport company and have not registered please register so that you are emailed when any new adverts are placed that meet your requirements. Remember its free to use Freight Tender!

industry_corpus2: Transportation


text:
Come and cheer for your favorite racers at Southern California’s best MotoGP watch party! Round 2 takes the series to our home nation of Argentina at Termas de Rio Hondo Circuit. Last year Cal Crutchlow nabbed the win ahead of Johann Zarco and Alex Rins in what was one of the most surprising podium lineups of the season. Who will it be this year?! Free to attend. Coffee, pastries, food, and wine will be available for purchase at the event.
Sorry, you missed Argentina MotoGP Watch Party at Doffo Winery.
Demand that Doffo Winery gets added to the next tour!
You missed Argentina MotoGP Watch Party at Doffo Winery.
We're generating custom event recommendations for you based on Argentina MotoGP Watch Party right now!

industry_corpus2: Sports


text:
The institute offers specialized programmes in the following areas - Finance, Human Resource Management, Information Technology; Money, International Banking and Finance; Planning, Control and Marketing; Rural Finance and Development Research. The institute offers Post-Graduate Diploma in Management (Banking & Financial Services) - PGDM (B&FS) and Doctoral Programme.
National Institute of Bank Management recruitment offers great career opportunities for several posts such as Hotel Supervisor. Interested candidates can pursue courses in Graduate + General written and oral communication skills + Well versed with MS Office - Word & Excel + Knowledge of English, Hindi & Marathi, for a great career at National Institute of Bank Management.
Checkout the recent employment oppurtunities for National Institute Bank Management in Government Sector. We have the latest jobs for National Institute Bank Management in India. We are the first to announce any recruitment for National Institute Bank Management as soon as it is announced.
Start your job search for National Institute Bank Management today - for FREE!
Register now, and keep informed about all the government jobs for National Institute Bank Management for FREE. You can also get National Institute Bank Management Government Job alerts through Mobile.
Never miss a job at National Institute of Bank Management.

industry_corpus2: Finance & Economics


text: We love Captain Jack Sparrow. He’s a devilishly handsome adventurer who has an unparalleled way with words, especially when he’s searching for eternal youth. He’s such a wonder with words that when we started putting together our list of Captain Jack zingers we felt like there was too much epicness for one article. As such, we’ve narrowed our focus and compiled our favorites from Pirates of the Caribbean: On Stranger Tides for your entertainment. (Never fear, swashbucklers… we’ll bring you more Oh Snap moments from Jack’s other feature film fantasticalness at some time in the future. So stand by.) For now, enjoy the Tides: Oh, Captain. Please don’t ever change.


text: This beautiful baby girl has her first play date today and she is very excited to make a new friend. You have to help her get ready for the big day. First she has to take a bath, so put her in the tub and fill it up with water. Also put some rubber toys in so she does not get bored. Wash her hair with a shampoo to get it fresh and clean and her body with a delicate soap. Finally wipe her with a soft towel and blow dry her hair, which you should brush afterwards. Now you can choose for her a cute haircut and dress her up in a colorful ruffled dress with polka dots or flower prints. Accessorize it with a wonderful pair of shoes with little bows or roses, a headband in her hair and a pastel necklace. It is time to hit the playground! Have an incredible time!


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Assess value of land, building, structures, machinery, equipment and property improvements for purposes of taxation, grants and regional planning.
Classify the property by type, and determine the taxable status of the property.
Review and analyze data such as past sales, title searches, engineering and alignment maps, soil maps, subdivision plans, water and sewer plans, leasing cost data and easements.
Advise and consul in a specific field of assessment such as residential, commercial, industrial, institutional, or farm lands.
Collect and analyze financial records of a business, such as financial statements, budgets, projections and sales data, to assess its competitiveness or to estimate its market value.
Appear as an expert witness before courts, security regulators and other regulatory boards and commissions.
Explain the assessment process to ratepayers.
Who Employs Real Property Assessors?
Assessors may be employed by government agencies, real estate firms and other private companies; or they may be self-employed.
Property assessment and appraisal are multidisciplinary vocations centered on the study and valuation of land, businesses, estates and other real property for the purpose of ad valorem taxation.
Both mass appraisal and real property assessment require specialized skills in valuation, in addition to focused study in finance, urban land economics, real property law, statistics, and building construction.
The educational requirements for AAOM accreditation reflect current industry standards for real property appraisal.
What distinguishes Real Property Assessment as a profession?
To become an accredited assessor in Manitoba, a combination of experience, education, and adherence to a professional code of conduct is required.

industry_corpus2: Real Estate & Construction


text:
Win vinyl artwork of your favorite quote or rodeo saying.
Submit your favorite rodeo saying or motivational quote! We are looking for original, inspiring, and or poetic sayings or quotes. Be creative but no obscenities please. Submit your entry by leaving a comment on our website rodeomomsclub.com or facebook page or e-mailing Lisa at lisa@lisasvinylcreations.com. Be sure to leave us your contact information. Entries must be submitted by April 15th.
3 winners will be chosen at random. We would like to publish them for other moms to share. So if it is not your original please list the author.
"Attitude IS Everything" my kids lived by this in all the sports the did.
"Can't never done anything!" This is what Josh is always telling the kids when they are whinning and saying I Cant!!
Ok ONe more.....Don’t go where the path may lead. Go instead where there is no path. . . and leave a trail.


text:
Shirt bodysuit with paisley Timberland print. Navy blue shirt with white paisley print. Short sleeves. Navy blue bow tie, removable through buttons. Pocket on left side. Metal buttons with Timberland print. White bodysuit with light grey trim. Popper buttons underneath and in the front. Short sleeves. Apparel composition: 100% cotton. Size: 6 M - 67 cm.
Timberland 1973 cap. Classic baseball cap, colour light grey with blue shaded visor. Timberland 1973 logo printed in navy blue in the front. Double lining inside. Elasticated adjustment. Composition: 100% cotton. Size: 44 - 6 M.
Presented in round wool knit box with plaited cable pattern and matching lid.


text:
BB1, is likely to descend to seal level as a low pressure by tonite. Initially, at around 998 mb, it will form off the Orissa coast, with clouds in the southern and SW quadrant of the system.
Since a seasonal trough today runs from west to east, with its western end over Upper Sindh and running thru Rajasthan, U.P. and the eastern end into the system.
Normally, BB1 will track along this trough. Presuming the trough to remain in the same position, which it should, the low, on deepening into a well marked low by Thursday, will track into North M.P. and thence into U.P.
But, bearing in mind the clouding is in the south and SW of the centre of BB1, rainfall will be more in Eastern Vidharbha initially, and moving into east M.P. from Thursday/Friday. Similarly, the eastern end pf the trough will get active to precipitate heavy rains in the NE states, and presumably move SWM into more areas of the east and Nepal.
In line with BB1, west winds will be attracted towards the low on the west coast. Heavy rain along North Konkan accordingly from Thursday. Mumbai forecast put up yesterday remains intact !
As-1 is now out and over, done with. Precipitation is dying out off the Saurashtra coast. I understand there were traces of rain in Karachi yesterday. Dont think more can be expected from "ex-AS1"
Next update with forecast map tonite at 10.30 pm.
All India Toppers SWM Rainfall from June 1 to 12 - 2011. Compiled and sent by Pradeep. Thanks.
heavy rain in borivali since 4.30pm. but the santacruz airport "metar" does not indicate any such activity. just "haze". what about south mumbai?
any chance of bb1 coming upto delhi?
@sset. Thu/fri rains should be around 40-50 mms per day. But much less than As1, when it poured 180 mms in Scruz and 103 mms at Colaba. I'll put up previous june figs.
@RK..i have my doubts abt BB1 reaching Delhi..


text: These days one of the most popular automatic strains. Enormous buds which start off with an open structure which then fill out completely from bottom to top. A fruity, skunky aroma with a relaxant effect and full of flavour.

industry_corpus2: Agriculture & Fisheries


text:
THE NEWEST LUXURIOUS 4 STAR BUSINESS & LIFESTYLE HOTEL IN BRAȘOV. KRONWELL.Special Rates New Year. LIFE IN STYLE. PLEASURE FOR BUSINESS.
• Kids are warmly welcomed!

industry_corpus2: Hospitality & Catering


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Deadweight loss is the lost welfare because of a market failure or intervention. In this case, it is caused because the monopolist will set a price higher than the marginal cost. This means there will be people willing to pay more than the cost of production which will not be able to purchase the good because the monopolist is maximizing profit.
Qm = Quantity produced by a monopoly.
Therefore, to find the value of the deadweight loss (DWL) we will need to find the values for MC, P, Qc, Qm which we will do in the following example.
Example - Calculate deadweight loss with numbers!
Suppose that the demand curve is represented by P = 10 - 2Q and MC = 2.
To find Qc we need to find the point where MC = the demand curve.
Therefore, we let 2 = 10 - 2Q. We solve for Q and find that Q = 4.
Firstly, we need to know what the marginal revenue equation is. Well, if the demand curve is linear (a straight line) then it will always have a slope twice the size of the demand curve and the same intercept term. Since demand is: P = 10 - 2Q this means that MR = 10 - 4q.
Now we equate MR = MC such that 2 = 10 - 4Q and re-arranging we will find Q = 2.
To find the price, we get our function P = 10 - 2Q and we substitute in our value for Qm.
therefore, our dead weight loss will be 4. And that's how we calculate the size of the deadweight loss!
Marginal Cost should not be horizontal!!!
Firstly, this is just an abstraction to make the problem a little bit easier. In reality the marginal cost curve might be slightly increasing, but for simplicity it makes sense to just assume that it is flat. We do the same thing when we assume that the demand curve is a straight line... Remember, these are just models and models often abstract away unnecessary detail.
There are also a lot of circumstances where it might make sense to assume that the marginal cost curve is horizontal, too!
Consider a firm producing pharmaceutical goods.
The most significant component of their costs are fixed costs. Even if the marginal cost curve is increasing, it is so insignificant compared to their total costs that it does not really matter if we assume that the curve is flat (i.e, if the price of manufacturing a drug is 10 cents for the first customer and it is 20 cents for the thousandth customer, but the price they charge is $80 per unit, does it really matter if we just assume that the marginal cost curve is flat?).


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Europe is often seen as an over-populated, over-travelled continent. But it can still serve up huge helpings of mind-blowing natural beauty – and the peace and quiet with which to contemplate it at leisure. To prove it, we’ve listed eight of Europe’s wildest, most exhilaratingly and away-from-it-all spots – and the best ways to explore them.
When it comes to jaw-dropping natural beauty, few places can compare with the Lofoten archipelago, whose clustered mountains tower above deeply indented bays. It’s not exactly empty of people, with quaint fishing villages now playing host to a burgeoning tourist industry. But untrammelled nature is never far away.
A plethora of hiking trails, cycling routes and fjord cruises provide access to some truly heart-stopping scenery. The islands are well within the Arctic Circle too, so there’s every chance that the midnight sun will add to the drama.
When it comes to European wetlands, few can compete in size and diversity with the Danube Delta. Here, the continent’s greatest river splits into myriad channels before entering the Black Sea. It’s a unique landscape of sandbar islands, semi-sunken forest and dirt-road villages, the majority of which can only be reached by boat.
Disembark at the fishing village of Crişan in the heart of the delta and you’ll be able to follow trails into reed-beds frequented by all manner of birds. Sfântu Gheorghe, the end-of-the-river settlement on the delta’s southern branch, offers more reeds, more birds and several kilometres of stark white beach.
The last significant swathe of primeval woodland left in Europe, Białowieża Forest straddles the border between Poland and Belarus. This emerald world of trees, grasses, mosses and lichens is also home to a 900-strong herd of European bison, re-introduced in the 1920s after the last indigenous specimens had been killed in World War I.
Certain parts of the forest are off limits to casual visitors and can only be explored with a guide. But there’s still a wealth of free-to-wander trails radiating out from the main access point, the pretty village of Białowieża itself.
Running for some 100km along Croatia’s Adriatic coast, the Velebit massif is one of the most brutally rugged mountain chains in southern Europe. While the canyon-riven Southern Velebit (site of the Paklenica National Park) is packed with summer trippers, it’s the less-trodden Northern Velebit that offers the most exhilarating hiking.
Towering Mount Zavižan marks the start of the Premužić trail, the 57km-long holy grail of Croatian hikers. However much of the route you manage to tackle, you’ll be rewarded with stunning views of both the coast and the inland karst.
Nothing screams “wilderness” more than a Baltic peat bog, its squelchy surface covered with mosses, lichens, cranberry bushes and dwarf confers. One of the best places to explore them is Estonia’s Soomaa National Park, where a patchwork of grassland, bog and riverine forest hosts a lively community of elk, beavers, flying squirrels and lynx.
Boardwalk paths such as the Riisa Trail lead out into this swamp-like realm. The spring thaw brings flooding and with it the possibility of canoe trips organised by local outfits such as Sooma.
Mountain ranges are routinely described as wild and unspoilt – but few are genuinely as wild and unspoilt as Durmitor. This limestone massif takes up a large chunk of northern Montenegro. It offers a huge variety of stunning scenery, from moon-grey peaks to grassy plateaux and lakes of eerie beauty.
Hiking possibilities are endless, with a network of trails beneath the 2523m-high summit of Bobotov Kuk. But it’s the rafting trips along the Tara Gorge – Europe’s deepest canyon – that really earn the superlatives. Local agencies such as Summit can book you a place in a dinghy.
In many ways the whole of Finland is a bit of a wilderness, with pristine lakes and huge silent forests lying within easy reach of even the biggest cities. To experience the country at its most awesomely empty, head north to Lapland’s Urho Kekkonen National Park. The park is a 2250-square-kilometre expanse of bare fells, birch forests and tundra-like heath.
Settlements such as Saariselkä, on the western rim, offer access to marked trails suitable for walks of half a day or more. However it’s the longer, 2–3 day trails in the uninhabited heart of the park that will truly put your frontier spirit to the test.
Never heard of High Cup Nick? Or the Nine Standards? That’s probably because the Lake District gets all the tourists, leaving the majestically bleak and boggy hills of the neighbouring Upper Eden Valley comparatively off radar.
Green-brown fells, stone barns, hardy sheep and horizontal rain are the area’s main visual signatures. Two of England’s best-known long-distance trails – the Coast to Coast Walk and The Pennine Way – penetrate parts of the Upper Eden, ensuring that its wind-blasted trails are well documented and easily accessible. Head for the market town of Kirkby Stephen or the heritage village of Dufton to get started.


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To celebrate the milestone, Rhino will release two versions on September 21, a day before the anniversary of the album’s original release on September 22, 1978. The 3CD/1LP boxed set ROAD TO RUIN: 40th ANNIVERSARY DELUXE EDITION will be available, as well as a newly remastered version of the original album will also be released as a single CD and LP. All titles will also be available digitally and via streaming services. Listen to the previously unreleased “Take 2 (Basic Rough Mix)” of “I Wanna Be Sedated” HERE.
ROAD TO RUIN: 40th ANNIVERSARY DELUXE EDITION contains two different mixes of the album, unissued rough mixes for every album track, and an unreleased 1979 concert recording of the Ramones in New York. It will be produced in a limited and numbered edition of 7,500 copies worldwide and comes packaged in a 12 x 12 hardcover book. Along with the music, the set also features rare photos and artwork, including the unused, alternate cover image, plus essays by former New York Rocker writer Roy Trakin, album cover artist John Holmstrom, and Road To Ruin producer Ed Stasium, who details the making of this classic record.


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Colonic irrigation is a specialist treatment for the colon. All the water in our colonics is ionised to increase oxygen content, thus enabling the speedy eradication of bacteria and parasites.
A specialised pregnancy massage is designed to ease the tension and aches that occur during pregnancy.
Microscopy offers a brief window into our inner world. Many strange phenomena occur within this internal ocean. It is an excellent Primary Screening / Bio- Feedback / Visual Education Tool.
Using the “Perrin Technique” we are able to effectively and successfully treat these debilitating illnesses. The Willows Clinic are a licensed therapist in Ireland and we have helped many clients achieve amazing results.
We offer a wide range of procedures to treat whatever life throws at you. From general health & wellbeing to speclialised treatments for infertility, debilitating illnesses, with our highly qualified therapists you are in safe hands.
We boast a range of advanced qualifications and accreditations which enables us to offer some of the most effective and successful treatments in Ireland.

industry_corpus2: Health & Medicine


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Crawded NTSE challenge will prepare 10th standard school students for the NTSE (National Talent Search Exam) examination. NTSE is conducted every year by NCERT in two stages first being at state level and second at National level.
Students who pass this examination will get scholarship every month by the government. Crawded NTSE Preparation Challenge is organized in a manner to test and improve your Mental and Scholastic Ability. Top ten winners of this challenge will get scholarship from Crawded.


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CHESTNUT HILL, Mass. - At 6:42 p.m. Saturday night, an hour and a half before kickoff of the Notre Dame-Boston College game, an announcement came over the Alumni Stadium press box.
Eagles coach Frank Spaziani would start freshman Chase Rettig at quarterback, a move that had been speculated since early in the week.
"Finally," someone in the press box proclaimed.
Spaziani had played coy all week, declining to say whether it would be Rettig or sophomore Mike Marscovetra replacing the benched Dave Shinskie.
"I know this," Spaziani said Wednesday during the ACC media conference call, "on Saturday night at 8 we'll have one of those guys out there. That's the timetable."
Rettig made his debut in a less-than-ideal circumstance, taking his first snap with the Eagles in a 7-0 hole.
It didn't get much better. Before Retting had played a full quarter as a starter, he faced a 21-0 deficit. And a left ankle injury sent him to the locker room before he could complete a half.
His first pass, which would have picked up a first down, was thrown behind receiver Clyde Lee. It wasn't the only errant throw. Some were dropped. Another was nearly intercepted. And his first completion, on a third-and-23, lost a yard.
Still, Rettig threw his first touchdown late in the quarter when wide receiver Bobby Swigert slipped behind ND cornerback Gary Gray and caught a 58-yard touchdown pass.
Rettig, however, suffered an ankle injury and was replaced in the second quarter by Marscovetra. With Marscovetra under center, the Eagles kicked field goals on consecutive drives to cut the deficit to 21-13 before a David Ruffer field goal made it a 24-13 ND halftime lead.
Rettig complete 5 of 10 passes and the touchdown in the first half while Marscovetra connected on 4 of 7.
The Notre Dame-Boston College series has included some testy moments through the years.
Eagles players have taken home chunks of the ND Stadium grass as souvenirs after victories, and the BC media release this week pointed out that in the 1992 game, with ND up multiple touchdowns, Lou Holtz called a fake punt leading to another score in a 54-7 Irish victory.
Yes, it's still a heated rivalry, but the two teams have been able to put the game in perspective. Last year, then-Irish coach Charlie Weis and receiver Golden Tate reached out to BC linebacker Mark Herzlich, who at the time was battling a rare form of cancer.
This year, Boston College defensive end Alex Albright switched his number from 98 to 78 for the ND game to honor Matt James, the Irish recruit who died during a spring break accident last spring.
Albright played at the same high school - St. Xavier in Cincinnati - as James.
"We always try to stick together as a community, so it's something I want to do," Albright told The Heights, BC's student newspaper.
Boston College safety Wes Davis has been a mainstay in the Eagles secondary the last few years. He's probably a mainstay on the list of players requested by the media. His thoughts on talking to the press?
"One day I'll have a book of quotes and then all my thoughts will be for sale, but until then that's the only way I can get anybody to listen to me - people who are paid to listen," he explained.
"Otherwise people get tired of my long-windedness. The media love to listen to me so I love to talk to them. It's a good relationship. I think it's a great opportunity. If it was up to me I'd have them around all the time to boost my confidence."
Davis delivered a big hit on a BC kickoff. He hit ND return man Bennett Jackson that stood Jackson up and essentially ended Jackson's return on the spot.
How big is the ND-BC rivalry? Big enough that two trophies go to the winner. One is the Frank Leahy Memorial Bowl, in recognition of the Boston College and Notre Dame coaching legend.
The second is the Ireland Trophy, which is presented by the opposing school's student government.
Entering Saturday night's game, Boston College was 47-7 in non-conference games since 2000, 23-3 since 2005.
BC sophomore linebacker Luke Kuechly answers to a number of nicknames - Boy Wonder, Superman and Clark Kent - because of his glasses and studious look.
Boston College starting defensive tackle Kaleb Ramsey was suspended for Saturday's game for an undisclosed violation of team rules. He was replaced in the starting lineup by sophomore Conor O'Neal.


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From gryphons to gargoyles and wyverns to wolves, come along and explore the hidden beasts lurking in the walls of Temple Newsam.
Inspired by the rich ornamentation of realistic and imaginary animal motifs on the furniture, interiors and decorative arts at Temple Newsam, the Fantastical Beasts exhibition combines a focused trail around the house, a display of art and artefacts in the exhibition room, including items from the social and natural history collections, and an interactive themed space for family visitors.
Take a trail around the house and delve into the weird and wonderful collection of ceramics, furniture and art work inspired by fantastical beasts of myth, legend and reality, and see our exciting new acqusition by artist Kate MccGwire.


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The game is starting to be playable, the other night I started a new group, fought in the bar, sleep in the inn, visited the sage, attacked the king, fought the king’s guards.
combat not giving any exp, I know kind of a big one!
I must admit, I have loved my Raro all my life, but I’m a little concerned that my work mates will claim that this is the final evidence needed, that I only eat sugar.
Yesterday after spending time trying to win a $25 dollar cheese voucher and failing, I decided to investigate how the auctions work a little.
The first thing I noticed was that I was getting snipped by people in the last few seconds, yet it was hard to know when the last few seconds were.
this can be hit every second, giving you quite good time count down.
I’ve shown a picture here due to the actual result is 9kB (1.7kB html 7.3kB inline styles).
Also of interest is that the winners time is shown 30 minutes before the previous bid. WTF!
I had planned to pull the past cheese auctions and show the history of the winning prices. But my own Internet went funny last night, and Mintshot is down this morning, sighting heavy traffic.
When watching an auction, only the time left refreshes. This means you have to reload the whole page to see if the current leader or auction history has changed.
There is a large amount of debugging code left in the live site, but commented out. You can almost see the developers figuring out how to get it working by what is left in there.
Explicit styles set on every element. Not only does it bloat the page, and makes more work for the servers, but horror if they decide to re-theme the site.
After a bit of reading I found mod_rewrite (Linux hosting) was the tool to fix the problem.
I downloaded the /blog/.htaccess file and used the above mentioned manual to decode the meaning and start hacking me a new file.
Much wasted time latter, I worked out that WordPad was messing with the Unix new-line markers, and changing to Visual Studio was met with success (or lack of server failure).
And magically the link now redirects to the correct page.
After some vanity Googling today, I noticed MDCrack, a project that I helped with back in ~2001 has been resurrected by the lead developer Gregory Duchemin. Well it was resurrected in 2006, but I’ve been busy….
Makes me want to restart tinkering with the MD2/MD4/MD5 hashes, and build my dream cracker.

industry_corpus2: Gaming


text:
Struggling thru .NET: App Store Race - MS the winner??
App Store Race - MS the winner??
I was looking at an article on The Register and they were comparing the Windows 8 App store and Apples iOS App store. Microsoft has over 3,000 apps (which 1 is mine) and iOS store has over 650,000 (11 are mine). This got me to thinking that this is actually bad journalism and that things are not being reported correctly. The true comparison to new Windows Store is actually the Mac App Store, not the iOS one. So here is a comparison of the 2 desktop app stores.
As a developer I look at both stores, Microsoft is launching with over 3x the number of apps that Apple did. Even after over a year, there are only around 10,000 Mac apps in the store and Microsoft is launching with over 1/3 of that. I can see that the number after a year will be a lot higher than 10,000.
Also as someone that is developing apps for mobile and tablets, I personally like the idea that I can have a trial app and then people can just purchase it and then they have the full one. Right now I have some iOS apps where I have to maintain a free version and a paid version and rev both of them when a new SDK is out or new things that I want to put in. Having a single app is very appealing to me. I know that some of the iOS developers actually use this to their advantage to put out multiple versions of a single app - this one does animal sounds, this one does English voices, etc and then flood the category with multiple free and paid versions of each. In the Microsoft store, this could be a single app with in-app purchases for each different thing. Easier to maintain and enhance. A friend of mine has close to 80 iOS apps and many of them are very similar like this. But to me, submitting updates for all 80 would be very tedious.
As far as reporting that Apple has over 650,000 apps compared to Microsoft's 3,000 apps, be careful on how you read stories like that. Are they comparing apples to apples (no pun intended) or are they comparing apples to pumpkins?
Microsoft will have more apps in the store at launch time than Apple did. Microsoft is making it easier for the app developers to get people to try their apps with the trial/buy method built into the SDK. Microsoft is taking less money after you start making a good amount. Microsoft is supporting the open source movement better than Apple. Microsoft is giving store developer accounts to people with MSDN subscriptions for free, where Apple will make sure that you pay for it.
Now go make some more apps for the store.
Not really surprised that MS is winning the desktop app race, but I'm fairly certain Apple will continue to win the mobile one. And let's be real, no one really cares about desktop apps anyway. Besides, if it wasn't so tricky to force quit Mac apps, they'd probably be winning the desktop race too.


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Update: The Funday PawPet Show, Megaplex, and for the Furry Costume Information Exchange have been restored.
A RAID storage facility holding data and code for several furry sites has failed, leading to downtime and uncertainty over their recovery.
Sites known to be affected center around the Florida Furs, and include the Funday PawPet Show, Megaplex, the Furry Costume Information Exchange (hosting the FURSUIT mailing list and an archive of fursuit-related stories), JR's Poinkcasts (also available elsewhere), and resources for furry ham radio enthusiasts.
IRC server lion.anthrochat.net is also affected. As it was the provider of IRC services for the Anthrochat network, channel and nickname registrations may be lost. The network itself remains online, and a temporary replacement is in place on another server.
According to server administrator Yappy Slyfox, no separate backup had been taken, as the design was meant to ensure security of the data. Recovery operations are in progress at the hosting data center.
Data recovery on the affected array has recovered major portions of the web sites for the Funday PawPet Show, Megaplex, and for the Furry Costume Information Exchange. Image and video archives for these sites are still not yet fully available, however the textual data content for the sites is mostly intact and has been restored to each domain. The respective web sites: pawpet.tv, megaplexcon.org, and fursuit.com are back online.
The Funday Pawpet Show art jam archives, chat logs and all other remaining damaged areas have been restored. The web site is now fully recovered/restored and online.