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The limited (2X) coverage of the tammar wallaby (Macropus eugenii) genome sequence dataset currently presents a challenge for assembly and anchoring onto chromosomes. To provide a framework for this assembly, it would be a great advantage to have a dense map of the tammar wallaby genome. However, only limited mapping data are available for this non-model species, comprising a physical map and a linkage map.
We combined all available tammar wallaby mapping data to create a tammar wallaby integrated map, using the Location DataBase (LDB) strategy. This first-generation integrated map combines all available information from the second-generation tammar wallaby linkage map with 148 loci, and extensive FISH mapping data for 492 loci, especially for genes likely to be located at the ends of wallaby chromosomes or at evolutionary breakpoints inferred from comparative information. For loci whose positions are only approximately known, their location in the integrated map was refined on the basis of comparative information from opossum (Monodelphis domestica) and human. Interpolation of segments from the opossum and human assemblies into the integrated map enabled the subsequent construction of a tammar wallaby first-generation virtual genome map, which comprises 14336 markers, including 13783 genes recruited from opossum and human assemblies. Both maps are freely available at http://compldb.angis.org.au webcite.
The first-generation integrated map and the first-generation virtual genome map provide a backbone for the chromosome assembly of the tammar wallaby genome sequence. For example, 78% of the 10257 gene-scaffolds in the Ensembl annotation of the tammar wallaby genome sequence (including 10522 protein-coding genes) can now be given a chromosome location in the tammar wallaby virtual genome map.
Stimulated by the human genome project and the need to further understand species genome architecture, more divergent mammals are being sequenced [1-7], providing more complete and detailed comparative genomic information, and contributing to our understanding of mammalian genome evolution.
Marsupials are a particularly interesting lineage of mammals, being only distantly related to eutherian (placental) mammals such as human and mouse. They have many major features common to all mammals, such as bearing fur and suckling young, but they show many unique characteristics in reproduction, lactation, sex determination and immunology [8]. Marsupials last shared a common ancestor with eutherians about 150 million years ago (mya) [9], an evolutionary distance sufficient to exclude the conservation of most non-functional sequence, but not too wide to lose the conservation of most functional sequence [10].
Two marsupial genomes have been sequenced to date, representing lineages of marsupials living in South America and Australia that diverged about 70 mya [11]. The Brazilian opossum (Monodelphis domestica) was sequenced with more than 7× coverage in 2007 [5]. The Australian model kangaroo, the tammar wallaby (Macropus eugenii), has recently been sequenced at about 2× coverage [12]. The first version (Meug_1.0) of the tammar wallaby assembly, released in December 2008, comprised relatively small contigs (N50 = 2.5 kb) and scaffolds (14.5 kb), consistent with the relatively low coverage. In July 2009, Ensembl released its annotation of these segments [13], but there remains insufficient genome structure information to enable any of these sequence segments to be assembled onto M. eugenii chromosomes.
The situation is comparable to that of genomes of other species sequenced at low coverage. Sequence (1.9×) of the domestic cat (Felis catus) [14] was assembled first by reconstituting cat contigs on the basis of their alignment with the dog assembly, then aligning these revised contigs with a cat radiation hybrid (RH) map. Although this approach was productive, it largely neglected other cat mapping resources such as the cat linkage map. The sheep genome, too, has been sequenced to approximately 2× coverage (primarily for SNP discovery) [15]. In this species, great use was made of sheep BAC-end sequences (BES), which were assembled in a consensus manner based on their alignments to cattle, human and dog sequence assemblies. A virtual sheep genome was then created by transferring relevant human assembly segments onto the consensus alignment of sheep BES [16].
In a non-model, non-domestic species such as the tammar wallaby, there are insufficient BES available for the creation of a consensus alignment, and no closely related species with which to align. However, there are considerable gene mapping data available from long-running efforts to produce a linkage map and a FISH-based physical map of this species [17,18], although there is no RH map. We therefore used all the available tammar wallaby mapping data to create an integrated map, using the Location Database (LDB) tool.
LDB is a bioinformatics tool first created to integrate all available human maps into a single map before the human sequence assembly was available [19]. Inputs can include linkage maps (in cM), RH maps (in cR) and fluorescent in situ hybridisation (FISH) data (cytogenetic locations). The output is a single map comprising the loci from all of the input data. Each locus is allocated a cM, cR and cytological band location, even if it has actually been mapped using only one of these methods. Most importantly, the position of each locus on a chromosome in the genome of that species is estimated in kb, just as in an actual sequence assembly. This map-integration strategy has been shown to be very powerful and accurate, and has been applied to livestock species not then sequenced [20].
For the tammar wallaby, there are cytogenetic and linkage-mapping resources that can be integrated to provide a framework for the genome assembly. A linkage map has been under construction for many years: the first-generation map was published in 2002, incorporating 64 loci on all autosomes and the X chromosome [17]. Since then, many loci have been added, creating a second-generation linkage map comprising 148 loci, with a total size of 1402.4 cM, and an average inter-locus distance of 10.9 cM (Wang et al., unpublished data). The chromosomes of the tammar wallaby have been thoroughly characterized [18], and have been mapped by FISH using BACs containing functional loci with human orthologues. The strategy of screening for tammar orthologues of genes that flank regions with a conserved gene content in both humans and opossum (regions of conserved synteny) [21] has delivered the locations of 492 genes in the tammar wallaby, including nearly all the new loci in the second-generation linkage map [22] (Deakin et al., unpublished data). The initial focus in the present paper is therefore to integrate all available tammar wallaby mapping information to provide the "backbone" for a tammar map that is based only on tammar wallaby information.
The integration of these hard-won tammar wallaby resources produces a map that is valuable, but not sufficiently complete or high-resolution to be adequate for genome assembly, e.g. some of the FISH locations on small chromosome arms cannot be specified to a greater precision than a whole arm. Comparative mapping information can be utilised to refine the relatively crude FISH locations of some tammar wallaby genes. Thus, without compromising the "backbone" of the integrated map that is based on tammar wallaby linkage and cytogenetic maps, comparative information from the most closely related sequenced genome is used to fine-tune the location of genes in the integrated map for which only crude tammar locations are available. Finally, a tammar wallaby virtual genome map is created by assuming conservation of synteny in the intervals between genes in the integrated map, again using primarily information from the tammar wallaby's nearest sequenced evolutionary neighbour, opossum, and (where this information is insufficient) from human. Although this latter step provides only a null hypothesis of the actual order and relative location of genes not yet mapped in the tammar wallaby, in the absence of any more direct information, this strategy has produced a virtual map that can be used as the framework for the initial assembly of the tammar wallaby genome sequence.
The aims of this paper, therefore, are (a) to identify blocks of conserved synteny and hence evolutionary breakpoints in the tammar wallaby genome; (b) to create a first-generation integrated tammar wallaby map by combining all available tammar mapping information for genes that have been mapped in tammar wallaby, making use of orthologous marker locations in opossum and human to fine tune loci FISH-mapped in tammar wallaby with low resolution; and (c) to create a first-generation tammar wallaby virtual genome map by utilising comparative opossum and human mapping information, for genes that have not been mapped in tammar wallaby.
The integrated and virtual maps of the tammar wallaby genome were created from the second-generation linkage map, comprising 148 loci [22], together with FISH-mapping data for 492 loci (Deakin et al., unpublished data), using the strategy described by Liao et al [20]. Firstly, we estimated the physical size and linkage size of each tammar wallaby chromosome, and the total genome size. We then identified blocks of conserved synteny between tammar wallaby and opossum (or human when no clear conserved syntenic blocks between tammar wallaby and opossum could be observed), and identified breakpoints between them. Next we estimated the size of each chromosome arm, and estimated band locations in kb by linear interpolation. We then created files of FISH locations, linkage-map locations and breakpoints for each chromosome, and also an orthologue-location file for FISH-mapped markers, all of which were used to create the integrated map. Finally, orthologue-location files were created for all the conserved syntenic blocks, and these were used to create the virtual genome map. Chromosome nomenclature follows the usual practice: HSA (human), MEU (tammar wallaby) and MDO (opossum).
Step 1: Estimation of Mb and cM size of each chromosome
The tammar wallaby genome consists of seven pairs of autosomes and one pair of sex chromosomes, X and Y. The physical size (in Mb) of each tammar wallaby chromosome was determined by direct comparison with the well established sizes of human chromosomes. The direct comparison was performed by bivariate flow karyotyping and the results are presented in Table 1. Tammar wallaby chromosomes 1-6 are all larger than human chromosomes. For instance, MEU1 is about twice the size of HSA1. Notable is the size of MEUX of 150 Mb, which is a value between HSA8 and HSAX. Despite MEUX having a much smaller gene content than HSAX and being regarded as the ancestral therian X [21], its DNA content is comparable with HSAX, due to the addition of repetitive sequences of this NOR-bearing chromosome. The estimated total linkage length for each chromosome, also shown in Table 1, was estimated as its cM length in the second-generation linkage map, divided by the estimated proportion of chromosome covered by that linkage map (Wang et al. unpublished).
Table 1. Chromosomes sizes and genome size (Mb and cM).
Step 2: Estimation of genome size
The tammar wallaby genome size is estimated to be 2.457 Gb and 1698.10 cM determined by adding up the chromosome sizes given in Table 1. This physical size is smaller than the human genome size of 3.08 Gb [23].
Step 3: Identification of blocks of conserved synteny and evolutionary breakpoints between tammar wallaby, opossum and human
For every gene FISH-mapped in the tammar wallaby, the location of its orthologue in the opossum and human genomes was determined. Blocks with conserved gene content were identified, as were genes that marked the end of each block and hence evolutionary breakpoints. Estimates were obtained of the size (in opossum or human in some cases) of each block of conserved synteny, and sizes of the blocks were summed over chromosomes and scaled to the estimated total size of the relevant tammar wallaby chromosome, creating a location (in kb) in tammar wallaby for each breakpoint. These blocks were defined to reflect the FISH-mapping results for all markers. Orientation of each block on the tammar wallaby chromosome was deduced from their FISH-mapping data wherever possible. Amongst the total of 84 conserved syntenic blocks, there were 23 with only one orthologous gene, 19 of which are on MEU3. When FISH data did not suggest a clear orientation of the block and for the one-probe blocks, the gene orders were retained the same as in the reference genome (opossum or human). With the one-probe blocks comprising only 8 Mb, compared with 58 Mb coverage of multi-gene blocks, this assumption concerning gene order was not critically important. A summary of the estimated breakpoints for all autosomes is presented in Table 2. The X chromosome was excluded from this table because, even though all the loci FISH-mapped to MEUX are located on MDOX, there were no obvious regions of conserved synteny, so it was not possible to estimate the breakpoints. Figure 1 illustrates the blocks of conserved synteny, and the breakpoints, for a typical tammar wallaby chromosome, MEU5. Similar illustrations for each of the other tammar wallaby chromosomes are presented in Additional File 1, Figure S1, Additional File 2, Figure S2, Additional File 3, Figure S3, Additional File 4, Figure S4, Additional File 5, Figure S5, and Additional File 6, Figure S6.
Table 2. Breakpoints between blocks of conserved synteny (with respect to opossum or human [in italics]) in tammar wallaby autosomes.
Figure 1. Comparative map between tammar chromosome MEU5, and corresponding opossum (MDO) and human (HSA) chromosomes. The different coloured regions in MDO4 and MDO7 indicate blocks of opossum-human conserved synteny (with the largest human chromosome blocks identified in the figure, and all relevant human chromosomes indicated in the colour-scheme box). Letters A to I indicate blocks of tammar-opossum/human conserved synteny, with each of the eight breakpoints (and the orientation of each block) indicated by flanking markers. The two markers to the left of MEU5 flank the centromere identified by FISH mapping. The sections of MDO7 shadowed as MEU6 indicate that all those sections of MDO7 except the block conserved with MEU5, are conserved with MEU6.
Additional file 1. Figure S1. Comparative map between tammar chromosome 1 (MEU1) and opossum chromosomes 1 and 6 (MDO1, MDO6). Note: For easier comparison with MEU1, MDO6 is shown with its q telomere at the top and its p telomere at the bottom.
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Additional file 2. Figure S2. Comparative map between tammar wallaby chromosome 2 (MEU2) and opossum chromosome 2 (MDO2). Note: For easier comparison with MEU2, MDO2 is shown with its q telomere at the top and its p telomere at the bottom.
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Additional file 3. Figure S3. Comparative map between tammar wallaby chromosome 3 (MEU3) and opossum/human chromosomes (MDO6, MDO8, HSA12, HSA22). Note: For easier comparison with MEU3, MDO6 is shown with its q telomere at the top and its p telomere at the bottom.
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Additional file 4. Figure S4. Comparative map between tammar wallaby chromosome 4 (MEU4) and opossum chromosome 3 (MDO3).
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Additional file 5. Figure S5. Comparative map between tammar wallaby chromosome 6 (MEU6) and opossum chromosomes 5 and 7 (MDO5, MDO7).
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Additional file 6. Figure S6. Comparative map between tammar wallaby chromosome 7 (MEU7) and opossum chromosomes 1 and 5 (MDO1, MDO5).
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As an example of the approach, MEU5 comprises nine blocks of conserved synteny, indicated as blocks A-I in Figure 1, with their boundaries defined by eight breakpoints. Loci on the right-hand side of MEU5 are end markers for each conserved syntenic block, which were FISH-mapped in tammar wallaby. The last three blocks (G, H and I) have no clear opossum counterparts, but show marked conserved synteny with regions of HSA 11, 1 and 19, respectively. However, their FISH-mapping locations in tammar wallaby are too close to resolve, so we could not determine the orientation of these blocks in tammar wallaby. Different tammar wallaby chromosomes showed different degrees of evolutionary rearrangement, the number of blocks of conserved synteny ranging from 2 (MEU7) to 28 (MEU3), with an average of 12.
The only difficulty encountered in identifying blocks of conserved synteny in autosomes concerned the p arm of MEU2 (Additional File S2), for which only approximately 6 Mb (out of a total estimated length for 2p of 44 Mb) could be identified as being orthologous to human or opossum chromosomal segments (HSA11 or MDO5 and MDO8). Therefore, breakpoint analyses were not carried out on MEU2p.
Step 4: Estimation of centromere position and arm lengths
In all tammar wallaby chromosomes except MEU5, the FISH-mapping data from tammar wallaby indicated that the centromere was located between two blocks of conserved synteny. Thus it was placed at the breakpoint between those two blocks. For MEU5, the FISH-mapping data suggested the centromere is located within a conserved block, between markers EGFL6 and CTPS2. The location of this centromere was first estimated as the mid-point (in the opossum assembly) between these two markers. The distances between each of these markers and the two breakpoints defining this conserved block in opossum were then rescaled onto the tammar wallaby version of this conserved block, to provide an estimate of the location of this centromere in the tammar wallaby.
The size of each arm of each autosome was then estimated in Mb from the chromosome sizes from Step 1 and the relative conserved syntenic block sizes on each side of a centromere. The arm sizes in cM were firstly calculated using the same p and q arm ratio as in Mb. Where necessary, they were then minimally adjusted to ensure that the centromere was located between two flanking markers, one of which had been FISH-mapped to the p arm and the other to the q arm. Table 3 shows the estimated size of each autosome arm. No orthologous regions were identified in the short arm of MEU2, so arm sizes in Mb and cM for MEU2 p and q were estimated from the arm ratio of 12:88 in the published tammar wallaby karyotype [17]. An arm file with the p and q arm Mb and cM lengths was then created for each chromosome.
Table 3. Arm size estimated for each autosome.
Step 5: Estimation of the kb location of the beginning and end of each band in the idiogram
The proportional location of each border of each band was estimated from the standard idiogram [18] and translated into kb locations by linear interpolation within the total kb size of the relevant chromosome arm, as determined in step 4. A band file was created for each chromosome and, after an iterative curation process as described in the Methods section, the final band definitions was determined, as presented in Additional File 7, table S7.
Additional file 7. Table S7. Band location estimated for each autosome.
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Step 6: Creation of an input file from each source of mapping data
Linkage map and FISH-map input files were created for each chromosome, as described in the Methods section. A FISH-orthologue file was created for each block of conserved synteny with either opossum (preferably) or human, to enable the fine-tuning of the location of FISH-mapped markers that would otherwise be located via the LDB process in the centre of their band(s). A breakpoints file was also created for each chromosome.
Step 7: Creation of an integrated map
For each chromosome, the integration process was run using the LDB software [20], with the integration order being FISH-map file, linkage-map file, breakpoints file and FISH-orthologue file(s), followed by manual curation of the band definitions by checking the discrepancies between the LDB predicted and the FISH-mapped band locations, as described in the Methods section, and running the integration process again until no further discrepancies existed. This produced an integrated map for each chromosome, combining all available FISH-mapping and linkage-mapping data. For markers whose only tammar wallaby data comprised FISH locations, their predicted locations in the integrated map were fine-tuned by the comparative information contained in the FISH-orthologue files.
As an example, portions of the integrated map of MEU5 are shown in Table 4. The map extends from the 5p telomere (5ptr) at 0 kb to the 5q telomere (5qtr) at 340000 kb. The 5qtr location corresponds to the size of chromosome 5 (Table 1). Similarly, the predicted locations in the linkage map of MEU5 range from 0 cM to 232.68 cM, the latter location corresponding to the estimated cM length of MEU5 (Table 1). Included in Table 4 are each of the eight breakpoints in this chromosome, together with loci flanking these breakpoints and the loci adjacent to the telomeres. Complete integrated maps for all MEU autosomes are presented in Additional File 8, table S8, and are also available through the Comparative Location Database [22]. In total, there are 553 markers in the first-generation integrated map across all autosomes.
Table 4. Portions of the integrated map for tammar wallaby chromosome 5 (MEU5).
Additional file 8. Table S8. The tammar wallaby integrated map.
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Step 8: Creation of a virtual genome map
Opossum and human orthologue files, containing opossum (wherever possible) or human orthologous locations for all mapped and unmapped genes in each conserved syntenic block, were constructed for each block of conserved synteny, as described in the Methods section. A virtual genome map for each chromosome was then constructed by incorporating the relevant orthologue files into the integrated map for that chromosome, using the same LDB software.
The virtual genome map for a tammar wallaby chromosome therefore comprises all genes whose location on that chromosome is supported by available tammar wallaby and orthologous information, including the many genes that have not yet been mapped in this species. In this virtual genome map, three confidence levels are defined. The highest (confidence level 2) is assigned to loci that have been mapped (linkage and/or FISH) in the tammar wallaby. The middle level (confidence level 1) is applied to those loci not mapped in the tammar wallaby but which occur in a block of conserved synteny whose orientation in tammar wallaby can be deduced from FISH-mapping data. The lowest level (confidence level 0) is given to those loci, which are neither mapped in tammar wallaby, nor have any evidence from tammar wallaby supporting their order within their conserved syntenic blocks.
To illustrate this outcome, portions of the virtual genome map for MEU5 are presented in Table 5. The total length of the chromosome is the same as in the integrated map, namely 340000 kb and 232.68 cM. It can be seen in Table 5 that two opossum markers (indicated by the prefix ENSMODG) have been interpolated between CXADR and SAMSN1. Similarly, 13 opossum markers have been interpolated between the centromere and CTPS2. These two sets of interpolated opossum markers have a confidence level of 1, indicating that although they have not been mapped in tammar wallaby, in each case their orientation could be deduced from the known orientation of the two flanking markers that are also in that group in the opossum assembly (CXADR and SAMSN1 for the first group, and cen and CTPS2 for the second group). Next, seventeen human markers (indicated by the prefix ENSG) have been interpolated between Breakpoint 7 and, DFFB and four human markers between LENG8 and qtr. These two sets of markers have a confidence level of 0, indicating that although they are predicted (from comparative mapping data) to be located between the relevant markers shown in bold, their orientation and order are not known with any certainty.
Table 5. Portions of the virtual genome map for tammar wallaby chromosome 5 (MEU5).
Across all autosomes, the virtual genome map comprises 14336 loci and has a size of 2457 Mb. Because of its size, the entire tammar wallaby virtual genome map could not be included in the Additional Files. It is available through the Comparative Location Database [22].
Given the large quantity of data incorporated, the first-generation integrated and virtual genome maps reported here will enhance significantly genome research in the tammar wallaby (a valuable model kangaroo species), and facilitate the assembly of the genome sequence of this species.
Whenever comparative data were required, we have been conservative in using wherever possible the genome of the most closely related sequenced marsupial. The tammar wallaby and the opossum diverged around 70 mya [11], comparable with the divergence within eutheria, amongst which much use has been made of comparative information, e.g. dog and human [24]; sheep and human [16]. The next-best choices are eutherians, which are more than twice as distant (diverging 150 mya) [9,10]. These realities provide a strong justification for the present strategy of drawing comparative information from opossum in preference to eutherians.
In addition, the remarkable conservation of chromosome arrangement in marsupials makes this approach particularly appropriate for the tammar wallaby. Whereas the eutherian genome has been grossly rearranged in many lineages, there is very strong conservation of synteny between tammar wallaby and opossum [25], even to the extent of whole chromosome arms being conserved [26]. When the integrated map was used to create the virtual genome map, the preferred strategy was, once again, to rely as much as possible on the tammar wallaby's nearest sequenced evolutionary neighbour, and then to turn to one of the most mature genome assemblies, namely human, only in the minority of cases where the opossum information was not sufficient.
Of course there will be errors in the order and relative location of loci, especially in the virtual genome map: the integration of data from conserved blocks of synteny means that the location of most tammar wallaby genes in the virtual genome map is predicted on the basis of their order in other species. However, as argued above, in using whenever possible comparative mapping data from opossum, we are, in effect, relying on better comparative information then has been the basis of the utilisation of comparative mapping information within eutherians.
Estimation of conserved-synteny blocks is not a simple process, and errors are certain to have been made, given the relative paucity of information available in the tammar wallaby. For the markers with the lowest confidence level, it is important to note that close-range locus order presented is just one of several equally likely possibilities.
As more sequence-level comparative data become available, these blocks will be better defined.
In principle, the overall aim of creating an integrated map is to combine together in a rational manner all available mapping information in the species of interest, without recourse to any information from other species. In the creation of the integrated map of sheep, for example [20], comparative data were used only in the local repositioning of loci that had all been FISH-mapped to the same chromosomal band. As discussed by Liao et al. [20], this did not compromise the essential integrity of the integrated map in reflecting all available sheep data: it simply provided a first estimate of the order of a set of loci that are known to be located within a particular band. In the case of the tammar wallaby, there was a lack of orthologues mapped with sufficient resolution in this species, which precluded the local ordering of loci that had been FISH-mapped to a particular band. The best solution, given the lack of resources to create a denser physical map, was to estimate evolutionary breakpoints in the tammar wallaby with respect to the opossum (wherever possible) and human assemblies. Whilst this provides an additional compromise to the integrity of the integrated map, this does not alter any mapping data gleaned from the wallaby alone. Therefore, in practice, it does not provide any additional compromise to the integrity of the wallaby-mapping data.
How does this first-generation virtual genome map compare with the resources used in genome assemblies in other species? The bovine genome sequence [27] was assembled onto a single RH map [28]. The opossum genome assembly [5] was assigned to chromosomes based primarily on FISH-mapping of BACs from scaffolds [29], with support from the second of two linkage maps whose terminal markers had also been FISH-mapped [30]. Our tammar wallaby first-generation virtual genome map is more comprehensive than either of these strategies, since it is based on all available mapping information from the species itself, combined in a rational manner, supplemented by comparative mapping data. This integrated map is better and more useful than either of its components considered alone. Obviously it would be desirable to obtain more mapping information (both linkage and physical) for the tammar wallaby. As such data become available in the future, they will be used in the construction of second-generation integrated and virtual genome maps. In the meantime, the maps described in this paper are the best available at this time; they utilize all available information to create the most complete maps of the tammar wallaby chromosomes that can be produced at this time.
As pointed out by Lewin et al. [31], "Every genome sequence needs a good map". Genome sequence itself is not sufficient to enable a chromosome assembly or construction of good comparative maps to reveal hidden evolutionary stories. Good genome maps (e.g. physical maps, RH maps, linkage maps) are a necessary complement to genome sequence. However, they are of limited use in isolation. What is needed is a means of integrating all available mapping data for a species into a single map. The first-generation integrated map reported in this paper achieves this aim for the tammar wallaby, and has enabled the creation of a first-generation virtual genome map for this species, combining the integrated map with comparative mapping data from species with more mature chromosome assemblies.
By combining the first-generation virtual genome map presented in this paper with the Ensembl annotation [13] of the initial (Meug_1.0) tammar wallaby assembly, it is now possible to construct the first draft chromosome assembly for the tammar wallaby. In their annotation process, Ensembl were able to create 10257 "gene-scaffolds" comprising two or more Meug_1.0 scaffolds. Of these, 7027 have one gene in common with the virtual genome map, and an additional 953 have more than one gene in common with the virtual map, giving a total of 7980 gene-scaffolds that can be incorporated into a chromosome assembly, based on the virtual genome map. Thus 78% of the Ensembl gene-scaffolds can be incorporated into a tammar wallaby chromosome assembly, and 9% of the gene-scaffolds can be orientated in this chromosome assembly.
In addition to the Ensembl gene-scaffolds, there are another 1175 Meug_1.0 scaffolds that have at least one gene in common with the virtual genome map, and 54 Meug_1.0 scaffolds that have multiple genes in common with the virtual genome map. The total size of gene-scaffolds and scaffolds that can be incorporated into a chromosome assembly is 533,684,520 bp, which is 22% of the estimated tammar wallaby genome size (2457 Mb). This chromosome assembly includes 10522 of the 15290 protein-coding genes identified in the Ensembl annotation. In other words, the virtual genome map enables the creation of a chromosome-based tammar wallaby genome assembly that includes a high proportion (69%) of protein-coding genes identified in the sequence data. This compares with the few gene-scaffolds whose location can be determined solely from the integrated map built almost exclusively from tammar wallaby mapping information: only 265 gene-scaffolds have one gene in common with the integrated map and three gene-scaffolds have more than one gene in common with the integrated map. Also the virtual genome map has been tested in the recent tammar wallaby genome sequence assembly attempt and has significantly enhanced the N50 of the assembly [32].
Construction of a tammar wallaby first-generation integrated map has allowed prediction of the genomic content and organization of the wallaby genome via a first-generation virtual genome map, which will be useful as a resource for assembly of the wallaby genome sequence. Since marsupial chromosomes are highly conserved [25,26,33], the results from this study, when combined with the opossum assembly, will inform studies on the genome of other marsupial species. In particular, the detailed breakpoints between opossum/human and tammar wallaby revealed in this study will be useful for studies of genome evolution in marsupials, construction of an ancestral marsupial karyotype, and comparisons with the genomes of eutherians, and with non-mammal vertebrates.
Estimating chromosome and genome size by flow cytometry
Tammar wallaby chromosome sizes were determined by bivariate flow karyotyping according to Trask et al. [34], Boschman et al. [35], Langford et al. [36]. Chromosome preparations of tammar wallaby and human were measured together and separately but sequentially with the same flow cytometry settings, allowing a direct comparison between the tammar wallaby chromosomes and the human chromosomes. Following the protocols described in the above reports, chromosomes were isolated from mitotic cells obtained by blocking with colcemid. The chromosomes were stained with Hoechst 33258 and chromomycin A3, which have a preferential binding to AT- and GC-rich DNA, respectively. The "DNA-line" in the flow karyotypes was drawn from the origin through human chromosome 4. Each human and tammar wallaby chromosome peak was projected onto this line, and the distance from the origin to this projection was an estimate of the DNA content of that particular chromosome. The established human chromosome sizes (Ensembl) were used as references for the tammar wallaby chromosomes taking into account both conversion and offset. Tammar wallaby genome size was determined by adding up the chromosome sizes.
Breakpoint analysis: estimation of blocks of conserved synteny and evolutionary breakpoints between tammar wallaby, opossum and human
For each of the 492 genes FISH-mapped in the tammar wallaby, the location of its orthologue in the opossum and human genomes was determined. Blocks of conserved synteny were identified. Genes that mark the end of each block were noted. These genes indicate evolutionary breakpoints. The size of each block of conserved synteny was estimated in opossum or, in the few cases where this was not possible, in human. For each tammar wallaby chromosome, the total size of all blocks of conserved synteny that comprise that chromosome was calculated. This total was then scaled to the estimated total size of the relevant tammar wallaby chromosome, creating a location (in kb) in tammar wallaby for each breakpoint.
Creation of input files and their use in creating the integrated map
An arm file and a band file were created for each chromosome. Each arm file comprises four columns: the first column identifies the chromosome arm (i.e. p or q), the second column is the arm size in Mb, then cM length in male and cM length in female. To combine the available linkage data with the maximum number of markers in the present context, the sex-pooled cM size of the arm was used in the last two columns. Each band file also has four columns, namely the band name (e.g. p1, q2), band size in Mb, band start location in Mb, and band end location in Mb.
A FISH-map file for each chromosome comprised three columns for each FISH-mapped locus, namely locus name, and then two fields indicating the ID of the band or bands to which that gene has been FISH-mapped. If the gene had been FISH-mapped to just a single band, then the second and third columns both have the ID of that band. If the gene had been mapped to a region encompassing two or more bands, the second column contained the ID of the band at the left (p telomere side) end of that region, and the third column contained the ID of the band at the right (q telomere side) end of that region.
For each chromosome, a linkage-map file comprised three columns, namely locus name, male cM location and female cM location. Similar to the arm file, the sex-pooled cM location of each locus was inserted in each of the second and third columns of the linkage-map file. The cM locations entered in the file are slightly different from those in the second-generation linkage map [22], having been scaled to correspond to the full-coverage map lengths shown in Table 1 by adding an offset value to each marker location corresponding to the cM equivalent of the p-telomeric end of the chromosome not covered by the second-generation linkage map. To calculate this for each chromosome, we used the estimate of the size of the uncovered p-telomere end of the chromosome as a percentage of the size of the chromosome covered by the second-generation linkage map, as calculated for the second-generation linkage map (Wang et al, unpublished data), where it is called m%. The offset value for a chromosome was then calculated by multiplying the m% by the length of the second-generation linkage map. The offset values for MEU1 to MEU7 were 12.21, 29.10, 7.33, 16.34, 3.60, 3.53 and 1.49 respectively. In addition to actual loci, the linkage-map file also contained rows for the p telomere, the q telomere and the centromere.
A breakpoints file was also created for each chromosome, comprising two columns, the first being breakpoint ID and the second being breakpoint location in kb (identified in the breakpoint analysis). These files also contained rows for both telomeres and the centromere.
A FISH-orthologue file was also created for each block of conserved synteny with either opossum (preferably) or human, as gleaned from Ensembl. These FISH-orthologue files enable the fine-tuning of the location of FISH-mapped markers that would otherwise remain located, via the LDB process, in the centre of their band(s). The three columns in each FISH-orthologue file were name of locus FISH-mapped in tammar wallaby, kb location of the orthologue of that locus in opossum/human, and opossum/human chromosome name. To enable correct integration, each FISH-orthologue file also contained relevant breakpoints, the p telomere, the q telomere and the centromere.
The integrated map was built with LDB software and the above input files. This map creation process involved initial running of the LDB software, manual curation of the band definitions by minimally extending some band boundaries to include all FISH-mapped markers where these fell outside the LDB predicted band positions, and repeating this process until no further discrepancies existed between the predicted and FISH mapped band locations.
Creating the virtual genome map
The construction of the virtual genome map required the creation of one more type of input file, namely orthologue files. Orthologue files contain opossum or human orthologous locations for all mapped and unmapped genes in each conserved syntenic block. Prior to the construction of orthologue files, the list of loci in each conserved block was compared between opossum and human. For those blocks that were clearly visible in both species, a very small number of loci within those blocks had locations that were not consistent with the block. These so-called "orphans" were excluded from the blocks prior to creation of the orthologue files. An opossum or a human orthologue file was thus constructed for each block of conserved synteny. Each file comprised three columns, namely locus ID (in this case the Ensembl ID of the opossum or human locus), the bp location of that locus in opossum or human, and the opossum or human chromosome on which that locus resides. In addition, rows were inserted for the two telomeres, the centromere, and the breakpoints for that conserved block.
BAC: bacterial artificial chromosome; BES: BAC-end sequences; cM: centimorgan; cR: centiRay; FISH: fluorescence in-situ hybridization; HAS: human (Homo sapiens); kb: kilobase; LDB: Location DataBase; Mb: megabase; MDO: opossum (Monodelphis domestica); MEU: tammar wallaby (Macropus eugenii); pg: picogram; RH: radiation hybrid; SNP: single nucleotide polymorphism.
Authors' contributions
CW wrote the first draft of the manuscript. CW and FWN drafted subsequent versions, with substantial input from JAMG and JED. CW constructed the integrated and virtual maps under FWN's close supervision. JED gave extensive feedback and suggestions on the maps, which greatly helped the development of additional strategies required for the creation of the maps. WR calculated genome and chromosome sizes using flowcytometry data. KB provided formal supervision in the latter stages of CW's postgraduate candidature, and provided very useful feedback on the manuscript. JAMG and KRZ provided the initial impetus for this project. JAMG provided financial and laboratory support. KRZ provided close supervision in the early stages of the project. All authors read and approved the final manuscript
We thank ARC Centre of Excellence for Kangaroo Genomics and the Cooperative Research Centre for Innovative Dairy Products for funding support. We gratefully acknowledge the following colleagues who have contributed to the linkage mapping data and FISH data used in this paper: Lee Webley, Ke-jun Wei, Matthew J. Wakefield, Hardip Patel; Margaret Delbridge and Amber E. Alsop. We thank Patricia O'Brien for flow cytometry analysis.
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Reviews - The Grand Atlantic Hotel3
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Graham McPhee spells out some of the cold, hard facts about the dismal state of ME research and politics, and has some suggestions as to what we can do about it ...
Discuss the article on the Forums.
b.p. - there is a tie to hypothyroid
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by Lauren Busser Direct from the Designers™ Contributing Writer
Choosing a floor plan for your new home can be an exciting, and maybe just a bit intimidating, process. As you start combing through your different options it is important to know how your family will use the space and how it fits with your lifestyle. The right floor plan will greatly improve how you experience your new home, and everything from the shape to the style of your floor plan should be accounted for. You might be overwhelmed by your options but we're here to help. Here are some tips to help you better decide which house plan is perfect for you.
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Constructing Masculinity: De Utero Patris (from the Womb of the Father)
Collins, Paul M., Journal of Men, Masculinities and Spirituality
Feminist critique of the male language and patriarchal implications of the classic formulation of the Christian doctrine of the Trinity in terms of a father--son relationship has elicited a variety of responses. This paper is framed within an analysis of the workings of the metaphorical and analogical ascription of language to the divine. What does the metaphorical and analogical description of the divine in terms of Fatherhood-Sonship mean? And how do metaphor and analogy work in this context?
I have chosen the phrase de utero Patris (from the womb of the father) as a point of departure to investigate possible constructions of masculinity in relation to a present-day reception of the Father--Son relationship found in the classic formulation of the Christian doctrine of the Trinity. In undertaking this investigation I use a cross-disciplinary method in which the disciplines of theology, philosophy, psychology and social, cultural and media studies will be brought together in an analytical critique to address instances of the construction of masculinity in four contemporary representations. Such a method is particularly required for this enquiry, but I also want to defend this kind of method for all forms of contextual theological discourse. I will test an hypothesis which emerges from within the discourse of feminist critique of the doctrine of the Trinity through an analysis of the reception of four contemporary examples of the construction of masculinity. The hypothesis emerges from Luce Irigaray's (1985, 1984) understanding that symbolic changes follow on from psychological changes, and is rooted in Lacan's schema of: the real, the imaginary and the symbolic (Miller, 1988, pp. 73-159), which is often used in the theorization of the media. I will assess the impact and reception of four examples of the construction of masculinity in an attempt to discern if the changes they may instantiate indicate the kind of psychological change which assist a new understanding of the Trinitarian Father--Son relationship. From the outset I want to suggest that reception of understandings of masculinity and the theorization of the intra-divine relations is a "two-way street." That is to say there is a reciprocity in the reception of the theorization of the intra-divine Father--Son relationship and the impact of the construction of representations of masculinity.
The four examples are contemporary representations in the media and arts, and the evidence for the reception of them is taken from within the media (with particular reference to the UK newspaper The Guardian), and as such the evidence base for the analysis may be said to be "populist." In undertaking this analysis of the reception of these representations, there needs to be an explicit recognition on the part of those receiving and interpreting the representations--as well as of myself--that in conducting the analysis there are overlapping gender constructs and stereotypes in use. There needs also to be a recognition that the categories used in the analysis of the representations are themselves constructs which are in constant need of re-evaluation and/or deconstruction: masculine, feminine; divine, material; gay, straight; heteronormative, homoerotic; being, having.
In addition to the above constructs and categories, I will appeal to the concepts of vulnerability and pathos. In making this appeal I am not wishing to suggest that vulnerability is a characteristic to be applied to one gender more than another. Rather I am suggesting that while each of the four constructions present masculinity in terms of physical strength and beauty they also suggest a susceptibility to the physical and emotional power of others. It is this combination of strength and susceptibility which intentionally evokes a complex emotional response from the audience. This I have understood in terms of pathos.
Finally there needs to be recognition of the particular limitations of this study. …


text: Does anyone have any experience with the black powder revolver conversion cylinders? Are they worth it? I have a 1858 Remington (Cabelas I think) and was wondering if they work good, are junk or are the best thing since sliced bread. Any feedback would be good. Thanks. I was thinking of the Kirst kind.


text:
HANNA Instruments Primo TDS Tester provides a fast and dependable way to measure the Total Dissolved Solids in water samples. Features a large, easy-to-read LCD and automatic temperature compensation to ensure your readings are consistent to the standard or reference temperature.
Easy to operate, just press the button on top of the meter once and it's powered and ready for use. Press and hold the button for a few seconds and Primo automatically enters calibration mode. Simply open a sachet of the calibration solution, dip the probe and within seconds the meter calibrates itself!
Includes batteries and complete instructions. One year warranty.
Size: 7.1″ long
Instruction Manual – PDF format


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tattooed woman
My Favorite Ink: Michelle Mae TinOctober 25 2013
NAME: Michelle Mae Tin OCCUPATION: Bartender and SuicideGirl (Ayumi Suicide) HOMETOWN: Chicago, IL "I wanted a cherry blossom tree before I could even get tattoos. A lot of people get them, but I've never seen it done on a leg… [ read more ]
Carley AquilinoSeptember 3 2013
Carly Aquilino If you like beautiful girls with tattoos and a sense of humor, this comic will be your new crush. I fell asleep with the TV on, and when I stirred hours later, there was a pixie with vibrant… [ read more ]
Training Day (Massy Arias)August 29 2013
NIKE BRA; BLACK MILK LEGGINGS; SKECHERS SNEAKERS; VALEO LIFTING BELT. Training Day (Massy Arias) "I think society has set a standard of how women should look, but I feel like we are all different and unique," says Massy Arias, a.k.a… [ read more ]
Vanessa LakeAugust 14 2013
American Apparel shorts, Office London heels. Vanessa Lake VANESSA LAKE IS A MAKEUP ARTIST, hairdresser, and model, when asked if it is better to dazzle someone than to be dazzling, she answered, "They are equal. I like being the one… [ read more ]
Mustang GirlsJuly 18 2013
Mustang Girls The women racing with the INKED logo in this year's Bullrun Rally from Montreal to New Orleans moved rapide. In a very specific type of Road Trip Bingo along Eastern byways this June, it took a light blue,… [ read more ]
Freyja VedaJuly 12 2013
Ampere bra and underwear; Uranium body chain; Bones and Feathers bracelet; stylist's own bracelets and ring; model's own heels, rings and headbands. Freyja Veda Cindy Crawford is recognized for her mole, Angelina Jolie has those lips, and Pam Anderson has,… [ read more ]
NOCTURNIA: DOMINATRIX I worked on the business side of the music industry for some years and engaged in BDSM in my personal life with my partner. Being a professional dominatrix wasn't in my crosshairs. It happened for a short time… [ read more ]


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Weird sound coming from under the hood
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MvZiC MaN
Dodge Ram Forum Senior Member
Join Date: Aug 2011
Location: Port Charlotte Fl
Age: 21
Posts: 140
Gender: Male
Vehicle: 2001 Dodge Ram
Trim Level: Base
Color: Matte Black
Engine: 1994-2001 239ci (3.9L) Magnum V6 175hp
Rep Power:
Weird sound coming from under the hood
I noticed a weird sound comin from under the hood of my truck today
, Im not sure how to explain it but my buddys 1990 chevy blew a rod and about 6 months before, it was making a simalar sound and it got louder as it went un-fixed since this is my First and only vehicle im Really worried about this because i have no money for another Engine/Truck now im very unsure if its a rod or what... But upon further inspection, I did notice that the Pully that the fan is attatched to is moving back and forth about
of an inch....
is this supposed to happen?
if not could this be the source of the noise?
Please help im concerned about my truck.....
i will try to get a video of the pully so you can see it
If guns kill people than i should start blaming my Keyboard for Spelling Errors
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Munson: Razing the ruins in small-town Iowa
Van Meter is one of several communities using state funds to wipe away eyesores
Apr. 27, 2013
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Munson: Razing the ruins in small-town Iowa
V One way to survey Iowa's small-town landscape is to browse through all the pretty economic development brochures and scan the quaint scenes of water tower-topped skylines on city websites.
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An air strike in Yemen by the Saudi-led coalition has killed at least 40 people and wounded about 200 at a camp for internal refugees, a human rights group has said. Of the injured, 25 were severely injured in the raids, a spokesman for the International Organisation for Migration (IOM) …Read More »
Tag Archives: yemen
US Drone strike in Yemen kills 12-year-old student
Details on the Monday US drone strike against Yemen have begun to emerge, and one of the three "al-Qaeda militants" slain turned out to have been a 12-year-old student named Mohammed Saleh Qayed Taeiman. Taeiman's father and older brother had been killed in US drone strikes back in 2012, and another …Read More »
Memo authorising drone attacks on US citizens released
The memo used to justify the legal killing of American citizens on foreign soil by drone strikes has finally been released Monday by the Department of Justice after months of legal wrangling and pressure from the ACLU and New York Times. The legal request for the memo came about after …Read More »
Drone operator speaks out: "This is what you are not told"
Whenever I read comments by politicians defending the Unmanned Aerial Vehicle Predator and Reaper program – aka drones – I wish I could ask them a few questions. I'd start with: "How many women and children have you seen incinerated by a Hellfire missile?" And: "How many men have you seen crawl across a …Read More »
Drone strike kills 15 wedding party-goers in Yemen
Fifteen people who had been heading to a wedding in Yemen have been killed in an air strike. Local media reported that a drone attack had been responsible, and the party-goers had been hit instead of an Al-Qaeda convoy. "An air strike missed its target and hit a wedding car …Read More »
Obama waives ban on sending military aid to countries with child soldiers
The White House on Monday afternoon announced that it had issued blanket waivers to three countries, allowing them to receive military aid despite their ongoing use of child soldiers despite a 2008 law to the contrary. The Child Soldiers Prevention Act of 2008 (CPSA) is meant to bar the United States from …Read More »
The drone that killed my grandson
I learned that my 16-year-old grandson, Abdulrahman — a United States citizen — had been killed by an American drone strike from news reports the morning after he died. The missile killed him, his teenage cousin and at least five other civilians on Oct. 14, 2011, while the boys were …Read More »
'Underwear bomber' was working for the CIA
A would-be "underwear bomber" involved in a plot to attack a US-based jet was in fact working as an undercover informer with Saudi intelligence and the CIA, it has emerged. The revelation is the latest twist in an increasingly bizarre story about the disruption of an apparent attempt by al-Qaida to …Read More »
US sets records with number of drone strikes in Afghanistan this year
The United States has carried out more drone strikes this year in Afghanistan than it has during its eight-year-long air war in Pakistan, launching 447 strikes and killing thousands while also reducing its air surveillance. At a time when the US is transitioning military power to the Afghan government and …Read More »
Yemenis rally against US assassination drone attacks
People have taken to the streets in Yemen's northern Sa'ada Province to denounce the US assassination drone attacks in their country. The Yemeni demonstrators on Sunday called on their government to sever its ties with Washington. The protesters chanted anti-US slogans, condemning the most recent drone attacks which killed three …Read More »


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We usually associate Boston University with brain studies and bat-chasing UAVs; we'll have to add technology patent lawsuits to the list. Following similar claims versus the likes of Amazon and Samsung, the university has sued Apple for allegedly infringing on a 1997 patent for making gallium nitride thin films used in semiconductors. The university wants both financial compensation (likely the real objective) and a ban on US sales of the purportedly offending iPad, iPhone 5 and MacBook Air. Neither side is commenting on the case, although we suspect that it will end with a whimper, not a bang. Like many big tech firms, Apple tends to fight patent lawsuits when it expects to win, and settle out of court when it doesn't. We'd add that the patent expires in 2015 -- a Boston University victory would have relatively little effect on Apple's future.
- Key specs
- Prices
- Discussions
- Form factor Tablet
- Operating system iOS (8)
- Screen size 9.7 inches
- Storage type Internal storage (16 GB, Flash)
- Maximum battery life Up to 10 hours
- Dimensions 9.4 x 6.6 x 0.24 in
- Weight 0.96 lb
- Announced 2014-10-16
Apple iPhone 6s
Apple iPhone 5
Apple MacBook Air 13-inch (early 2015)


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The email address is already associated with a Freelancer account. Enter your password below to link accounts:
Link your account to a new Freelancer account
Email address:
Valid username
Link to your existing Freelancer account
Need help with friends germany? Hire a freelancer today! Do you specialise in friends germany? Use your friends germany skills and start making money online today! Freelancer is the largest marketplace for jobs in the world. There are currently 17,764 jobs waiting for you to start work on! (suggest changes)
|Price (USD)
|Price (USD)
|Facebook Accounts with friends from Germany
|...to buy 25 facebook accouts each with 50 friends from Germany You need to provide full login details...from other countries which e.g. study or work in Germany All accounts must have logical background stories...picture, and logical personal information - All friends must be real people with active facebook pages
|Internet Marketing, Facebook Marketing
|Jun 11, 2011
|Jun 11, 2011Ended
|3000 facebook friends USA, Germany, UK only
|Hello! - Need 3000 REAL facebook friends for our fanpage! - You will not have access to my account...account infos. - I want ONLY REAL FACEBOOK Friends (USA, Germany, UK only) - Timeline 2 Weeks - 2 Milestones
|Internet Marketing, Social Networking, Facebook Marketing, Marketing
|Feb 17, 2012
|Feb 17, 2012Ended
|Ge facebook friends
|get facebook friends from uk, netherlands, belgium, germany, frence
|Social Networking, Facebook Marketing
|Mar 27, 2015
|Mar 27, 2015Ended
|Social Networking, Facebook Marketing
|May 17, 2011
|May 17, 2011Ended
|USA & Germany facebook accounts with 1000+ real friends available
|I have USA & Germany facebook accounts. There has 800 - 1000+ real friends in each id. All account with
|Sales, Google Adwords, Marketing
|Apr 13, 2010
|Apr 13, 2010Ended
|Facebook friends
|Hey to all, I need 1000 Germany facebook fans for my wesbite Project requirements: *Fans must...account. * All fans must be from Germany/and should have at least 50+ friends * All fans must be real people
|Internet Marketing, Social Networking, Facebook Marketing, Marketing
|May 16, 2011
|May 16, 2011Ended
|Facebook likes from Germany
|...Likes for our site from the folowing countries: Germany. "All likes must come from real people with real...real active accounts who have at least 20 real friends on their facebook page. ************ NO PAYMENT...likes must be from people who have at least 20+ friends in their facebook page (real profiles) - Obtained
|Internet Marketing, Link Building, Marketing, Bulk Marketing, Advertising
|Jan 19, 2013
|Jan 19, 2013Ended
|2K Germany Fans
|We need 2,000 Germany Facebook Fans for our page - All fans must be real people with active facebook...FAKES, NO BOTS, NO SPAM! - All fans must be 50+ friends - All fans must have status updates that go back
|Social Networking, Facebook Marketing
|Dec 8, 2011
|Dec 8, 2011Ended
|Need 5000 Facebook fans Germany
|Requirements: - all friends/fans from Germany - all friends/fans at least 18 years old -
|Facebook Marketing
|Apr 2, 2010
|Apr 2, 2010Ended
|Facebook Fans (Germany)
|...Requirement for Fans: - 100% of Fans must live in Germany - Fans must be 18+ - All fans must push our Like...of 15 days. - All fans must have at least 50 friends or more, profiles must be at least 20 days old...MUST be REAL Facebook accounts With at least 50 friends. - All accounts must have at lease two photos
|Internet Marketing
|Mar 27, 2011
|Mar 27, 2011Ended
|Need Germany Facebook Fanpage
|I need 5000-10000 Facebook Fans from Germany country If you can deliver fans from german speaking countries...hat/grey Hat techniques. • All fans must have 25+ friends and a minimum of 3 photos • All fans must have
|Internet Marketing, Social Networking, Facebook Marketing, Advertising
|Aug 1, 2011
|Aug 1, 2011Ended
|need 5k facebook like in germany
|hello, friends i need 5k facebook like for germany in 5 days i will pay thish project 200$
|Accounting, Finance, Quickbooks & Quicken, Payroll
|Dec 26, 2011
|Dec 26, 2011Ended
|Need Germany Mobile Subscriptions
|...my time if you cannot follow these ) : *Only Germany quality traffic and sign-ups *Only unique and...people to fill them for you *No Do not get your friends to sign up *No Rotating IP addresses *No fake
|Internet Marketing, Marketing, Bulk Marketing, Advertising, Leads
|Dec 12, 2012
|Dec 12, 2012Ended
|1000 Facebook and twitter friends
|I need 1000 marketing professionals facebook friends added to a Facebook page and twitter page IMPORTANT: ...fans must be from the either Australia , USA, germany or Japan - no other fans mixed in 2) All fans
|Internet Marketing, SEO, Link Building
|Jan 27, 2011
|Jan 27, 2011Ended
|1K Germany Fans
|We need 1,000 Germany Facebook Fans for our page - All fans must be real people with active facebook...FAKES, NO BOTS, NO SPAM! - All fans must be 50+ friends - All fans must have status updates that go back
|Social Networking, Facebook Marketing
|Oct 5, 2011
|Oct 5, 2011Ended
|Facebook friends for company
|...work at we would like to have a lot of Facebook friends. To make the brand more popular. The country's...Most important: - Netherlands - USA — - Germany - England - Spain Hope to hear form you
|Social Networking, Facebook Marketing
|May 13, 2011
|May 13, 2011Ended
|500 Likes on Facebook from Germany
|...these requirements: 1. 100% of Fans must be in Germany or Austria. 2. All fans must be active and have...least 50 friends or more 4. All Accounts MUST be REAL Facebook accounts With at least 50 friends. 5. All
|Facebook Marketing, Marketing
|Feb 17, 2011
|Feb 17, 2011Ended
|Freelancer Agent in Germany
|...100 !! PLEAS TO NOT BIT IF YOU ARE NOT LIVING IN Germany !!!!!! This part of cooperation is based on searching...8 weeks) courses 2. German Language courses in Germany or Austria We have short (2 weeks) medium ( 6 weeks)...one is not compulsory.. If you have any other friends interested in cooperation we will be glad if you
|German, English (US)
|Jan 11, 2014
|Jan 11, 2014Ended
|2000 Facebook Fans (Germany and Austria)
|...Requirement for Fans: - 100% of Fans must live in Germany or Austria. - Fans must be 18+ - All fans must...of 15 days. - All fans must have at least 50 friends or more, profiles must be at least 20 days old...MUST be REAL Facebook accounts With at least 50 friends. - All accounts must have at lease two photos
|Internet Marketing, Social Networking, Facebook Marketing
|Mar 23, 2011
|Mar 23, 2011Ended
|IT Germany
|Dear Sir/Madame, Please see below the specific requirements of the German IT Company which is looking to recruit Freelancers with the following skills. If you meet all or most of these criteria, kindly , please send us again your CV or Company portfolio if you are a Company (but again with the CVs of your team ) The candidates should include in their resumes their birth dates as well...
|Web Security
|Jul 10, 2015
|Jul 10, 2015Ended


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Planning Permission :
It is our understanding that the following are issues concerning planning permission:
- The Cabin should be more than 5 metres from the main dwelling and up to 50% of the remaining garden can be utilised with this type of building.
- The roof line to be lower than 4metres, if a pitched roof
- Overall height from ground level to ridge must not exceed more than 2.5 metres within a 2 metre range of any boundary. A building with overall height exceeding 2.5 mtrs in height and placed within 2 mtrs of any boundary will require planning permission.
- Height of the eaves must not exceed 2.5 mtrs
- No verandas higher than 30cms from ground level.
Other Restrictions :
- Covenants may exist to restrict siting of a Cabin.
- If you are in a conservation area or a listed property it is best to check with your Local Authority
Use the Planning, A Guide for Householders and the Planning Permission for Outbuildings to assist your enquiries.
If there's any doubt, you should contact your local planning authority before you begin work.
Disclaimer: this is an introductory guide and is not a definitive source of legal information.
NOTE: This guidance relates to the planning regime for England. Policy in Wales may differ. If in doubt contact your Local Planning Authority.
Felt Shingles
Our professional quality felt shingles can be supplied as a stand alone product. You don't need to buy a building from us to benefit from their superior quality.Take a look .....
Custom Builds
Made to order bespoke or customised log cabins to compliment your garden making you the envy of your friends and family! Find out more.......Take a look .....
Professional Installation
We offer a full installation service by trained and experienced fitters. Have your cabin installed by a professional team for less than you think.Take a look .....


text:
Is it harmful to breathe 100-percent oxygen?
We need oxygen to live, so it seems to me that if we got more oxygen, we would be better off. Is it harmful to breathe 100-percent oxygen? What will happen?
We need oxygen to live, so it seems to me that if we got more oxygen, we would be better off. Is it harmful to breathe 100-percent oxygen? What will happen?
Indium, a rare chemical element. Indium is a soft, silvery-white metal, easy to melt and mold. See more »
Iridium, a hard, white, metallic element of the platinum family. It is found in mineral deposits with platinum and osmium. See more »
Krypton, a colorless, odorless, tasteless element. It is a noble, or inert, gas (one that ordinarily will not combine with other elements). See more »
Lawrencium, a radioactive, metallic chemical element. It does not occur in nature. See more »
Lithium, a silvery-white metallic chemical element. Lithium is the lightest metal and the third lightest of all elements. See more »
Magnesium, a silvery metallic element. It is very light, weighing only two-thirds as much as aluminum. See more »
Manganese, a hard, brittle, gray-white metallic element. It is chemically active; it corrodes readily in air and is soluble in water and dilute acids. See more »
Mendelevium, a transuranium chemical element. All isotopes of mendelevium are radioactive. See more »
Mercury and its compounds are highly poisonous. They are released into the environment by such activities as the burning of fossil fuels and the use of certain pesticides, and by such natural processes as volcanic eruptions. See more »
Metal, a member of the largest class of chemical elements in the Periodic Table. See more »


text:
Women Find Humble Guys To Be the Sexiest
Macho can only get a guy so far it seems.
Men's Health Magazine says the way to a woman's heart is through humility, and that's more attractive than a cocky attitude. Here's how to make it happen guys, so you can get that hot date and keep her around.


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The Way I See It!
I am an Ultra-Conservative, Alpha-Male, True Authentic Leader, Type "C" Personality, who is very active in my community; whether it is donating time, clothes or money for Project Concern or going to Common Council meetings and voicing my opinions. As a blogger, I intend to provide a different viewpoint "The way I see it!" on various world, national and local issues with a few helpful tips & tidbits sprinkled in.
Unions Better For Workers?
Workers don't do better in unions
History Repeating?
Will the UAW Ruin the Domestic Car Industry Again?
More Chicago Teachers Union
Chicago Teachers Union joins growing national opposition to deeply flawed Common Core Standards
Chicago Teachers Union
Chicago Teachers Union to Mayor Rahm: Improving failed schools full of black kids is RACIST
UAW Rejected at Tennessee Volkswagen AG's Assembly Plant
UAW loss in Tennessee will prompt strategy rethink
Volkswagen AG Not Keen on UAW?
U.S. senator drops bombshell during VW plant union vote
Where Did the Disabled Children Governmental Money Go?
DNC and Granholm agree: Disabled kids' Medicaid money is for Democratic candidates
Happy Labor Day!!!
As you celebrate Labor Day remember history and remember today!
So Much for Being Outspent!
Big labor's contribution to Barrett during the recall? $23 million. Add that to his declared $6 million. That's $29 Million
Important Info to Read & Understand
WMC Talking Points: State Budget Helps Families, Schools
What Percent Are Paying Public Sector Union Dues Now?
From the Wisconsin State Journal
I Guess They Didn't Think We Knew This!
Union officials: Reforms will affect political activities
The Good Public Union?
AFSCME at Entitletown (Madison).
Guest Blog By Paul B. - Want To See Fake Democracy In Action?
You will all find this article very informative! Once again, those who think what is happening in
Hmm Those Privileges
One of the flee 14 wants an amendment on collective bargaining. So, he admits that collective bargaining is NOT a right then.
NJ Teachers' Union vs. Gov. Chris Christie
New Jersey Gov. Calls Teachers' Union 'Bullies and Thugs'
Today's Hmm Moment!
If someone says something to the sort of:
Think Of This Statement
"I don't defend the actions of my union or its members; I am just defending my union!"
Another One
Ronald Reagan on Public Unions
Earlier Parts:
One of the Unionista Protests You Might Have Missed
Gov. Walker Protests May Move Into Grocery Stores
The Trickle Down Effect!
The left has been up in arms since the 1980's over the trickle down. Their claim is it doesn't work and we don't need to even talk about it.
Referendum on Governor Walker and His Policies?
I am getting so tired of hearing that every election is a "referendum" on Walker and his policies. I can see the left and public sector union party "PUP" claiming that the Wisconsin State Supreme Court election with David Prosser was.
Translating Public Union Liberal Speak
It is being said:
What Was That, No The Fleeing 14 Never Got Info From Outside Wisconsin?
National union spokesman sent talking points to Senate Democrats
More on the Event You Might Have Missed
Please remind yourself that many on the left said, "She was a plant, it will end up being a repub, this is no story, who cares!"
Did you see this?
Wanggaard wants probe of union letters campaign
Who is George Meany?
This is important to have some history so when I give you the Ronald Reagan videos and transcripts of him talking about unions you have a reference and perspective.
An Event You Might Have Missed
DOJ raises concerns with Madison DA over e-mail threat
Such Nice People!!
'Koch brothers' confusion results in death threat for
Public Sector Workers
By various people I have been accused of relentless attacks on the public sector workers, told I am just piling on, told that I am unwilling to be fair to the public sector workers, and told that my blog posts are one sided in showing only the Walker view (which I do hold and support).
Opting Out Of the Union
**** The things you "say out of anger" do not come with a return policy. Watch what you say and how you say it. ****


text:
Chris Busby on GEnomic Instability from Radiation, Japan Genomic Rice Fukushima Fallout
- Uploaded by Fal2grace on Aug 1, 2013
- Views: 56
Chris Busby explains the dangers of radiation on plants, animals, humans, etc. I've posted this interview a couple of times before, but I thought it was time to give it a little more air time to express the severity of radioactive fallout and gene mutations for Generations - Genetic Instability."Japanese rice is famous for its quality. For many consumers around the world, however, it costs too much. Now Japanese researchers are cross-breeding varieties to create new types that are less expensive, but just as tasty." Genomic Rice Study...http://www3.nhk.or.jp/nhkworld/newsline/201307260817.htmlhttp://tinyurl.com/kmdg7pyGenomic instability and radiation.http://www.ncbi.nlm.nih.gov/pubmed/12875549http://tinyurl.com/mpc7cj7Genomic instability is a hallmark of cancer cells, and is thought to be involved in the process of carcinogenesis. Indeed, a number of rare genetic disorders associated with a predisposition to cancer are characterised by genomic instability occurring in somatic cells. Of particular interest is the observation that transmissible instability can be induced in somatic cells from normal individuals by exposure to ionising radiation, leading to a persistent enhancement in the rate at which mutations and chromosomal aberrations arise in the progeny of the irradiated cells after many generations of replication. If such induced instability is involved in radiation carcinogenesis, it would imply that the initial carcinogenic event may not be a rare mutation occurring in a specific gene or set of genes. Rather, radiation may induce a process of instability in many cells in a population, enhancing the rate at which the multiple gene mutations necessary for the development of cancer may arise in a given cell lineage. Furthermore, radiation could act at any stage in the development of cancer by facilitating the accumulation of the remaining genetic events required to produce a fully malignant tumour. The experimental evidence for such induced instability is reviewed.Genome instability (also "genetic instability" or "genomic instability") refers to a high frequency of mutations within the genome of a cellular lineage. These mutations can include changes in nucleic acid sequences, chromosomal rearrangements or aneuploidy. Genome instability is central to carcinogenesis[1] but also is a factor in some neurodegenerative diseases such as amyotrophic lateral sclerosis or the neuromuscular disease myotonic dystrophy.The sources of genome instability have only recently begun to be elucidated. A high frequency of externally caused DNA damage[2] can be one source of genome instability since DNA damages can cause inaccurate translesion synthesis past the damages or errors in repair, leading to mutation. Another source of genome instability may be epigenetic reductions in expression of DNA repair genes. Because endogenous (metabolically-caused) DNA damage is very frequent, occurring on average more than 10,000 times a day in the genomes of human cells, epigentically reduced DNA repair is likely an important source of genome instability.http://en.wikipedia.org/wiki/Genome_instability