Sulfolobus solfataricus was purified to homogeneity for biophysical characterization.The cluster of orthologous group COG2042 has members in all sequenced Eukaryota as well as in many Archaea. The cellular function of these proteins of ancient origin remains unknown. PSI-BLAST analysis does not indicate a possible link with even remotely-related proteins that have been functionally or structurally characterized. As a prototype among COG2042 orthologs, SSO0551 protein from the hyperthermophilic archaeon 73-Arg78 is hyper-sensitive, and thus accessible and flexible. Lysine labeling with NHS-biotin and cross-linking with DTSSP revealed that the 35 amino acid RLI motif at the N terminus is solvent exposed. Cross-links between Lys10-Lys14 and Lys23-Lys25 indicate that these residues are spatially close and in adequate conformation to be cross-linked. These experimental data have been used to rank multiple three-dimensional models generated by a de novo procedure.The untagged protein is thermostable and behaves as a monomeric protein in gel filtration experiment. Several mass spectrometry-based strategies were combined to obtain a set of low resolution structural information. Kinetic data from limited proteolysis with various endoproteases are concordant in pointing out that region GluOur data indicate that COG2042 proteins may share a novel fold. Combining biophysical, mass-spectrometry data and molecular model is a useful strategy to obtain structural information and to help in prioritizing targets in structural genomics programs. Saccharomyces cerevisiae COG2042 homolog. Deletion of the Yor006c gene was shown to result in a viable phenotype but some apparent moderate growth defects were noticed on a fermentable carbon source , [35–166], [56–166], [57–166], [76–166], [79–166], [101–166] . MALDI-TOF mass spectrometry was used to determine cleavage sites by following the time course generation of peptides. Several protease/substrate ratios were assessed to confirm which preferential sites on entire protein were first attacked (earliest cleavage), thus corresponding to a native state of the protein. The two fragments generated by such cleavage may be more vulnerable to subsequent attacks than native protein and therefore late proteolytic sites are considered less informative. Both small and large peptides generated during proteolysis were evaluated. Partial proteolyzed products obtained with trypsin were first resolved by reverse-phase chromatography and analyzed by MALDI-TOF mass spectrometry. Results recorded from direct analysis of the digestions without prior separation were almost similar to those obtained with separation. Therefore, the latter cost-effective strategy was used for analyzing the numerous conditions tested. Figure m/z: 8614.6 amu, 10603.4 amu, 6489.8 amu, and 12724.1 amu, were attributed to fragments [1–75] (Δmass: -178 ppm), [76–166] (+89 ppm), [1–56] (+145 ppm), [57–166] (+200 ppm), respectively (data not shown). These data clearly indicate that Lys75 and Arg56 are two sites of early cleavage by trypsin. Identification of peptides Val32-Lys166  and Gly35-Lys166  also indicates that Arg31 and Lys34 could be two other initial nick-sites.During the earliest events of the trypsin proteolysis analyzed in various conditions for detection of large products but also smaller peptides, monocharged cations with following m/z of 1920.9 amu ([1-15] +70 ppm) and 17296.8 amu  on one hand, 8998.1 amu  and 10217.5 amu  on the other hand. These data indicated that Arg78 and Arg15 are the main proteolyzed sites when ArgC enzyme was used. Chymotrypsin attacks SSO0551 native protein mainly at Phe74 because two complementary peptides with m/z of 8487.0 amu  and 10734.2 amu  were clearly evidenced. Glu73 is the main proteolyzed site when GluC protease was used, as peptides with m/z of 8338.7 amu  and 10880.0 amu  were detected. For all these analysis, smaller peptidic fragments that accumulated over time could be attributed from further proteolysis of the products arising from initial attacks (data not shown). All these results are concordant in pointing out that Glu73-Arg78 and Glu28-Arg31 are two accessible solvent-exposed regions of the protein as they can be proteolyzed by several endopeptidases, the first cited being definitively hyper-sensitive. Local unfolding not just surface exposure is necessary for efficient in vitro proteolysis because the polypeptide segment being cleaved must form a specific structure with the associated protease  (Δmass tolerance: +16 ppm), [123–138] (-7 ppm), [57–78] (+61 ppm) and [56–78] (+82 ppm). Since trypsin does not cleave after a modified lysine, we conclude that Lys25, Lys75 and Lys128 were modified. After DTT treatment, peaks corresponding to the expected products  were detected at m/z 1065.46 (-109 ppm), 1973.03 (0 ppm), 2611.21 (-28 ppm) and 2767.26 (-45 ppm), respectively. Fig. + ions at m/z 1491.76 and 1835.84 corresponding to intrapeptide cross-linked peptides: [21-31] (+31 ppm) and [3-15] (+3 ppm), respectively. These peptides contain two proximal lysine residues (Lys23-Lys25 and Lys10-Lys14). As shown in Fig. m/z 1493.69 and 1837.81 appears at the expected increment . An additional peak at m/z 2502.22 could be attributed to peptide [35–55] (+37 ppm) with an intrapeptide cross-link between Lys49 and Lys51. However, the corresponding reduced peak was not detected. Strikingly, every lysines that were reactive with DTSSP were also detected by NHS-biotin labeling. Most modifications are located at the N terminus of the protein where five modified residues belong to the RLI motif.Using a lysine cross-linking reagent, DTSSP, it is possible to assess intra- or inter-molecular protein contacts ,32. DTSS73-Arg78 region being revealed hyper-sensitive to various proteases . The RLI domain is also present at the N terminus of another group of orthologous proteins, namely COG1245. Remarkably, COG1245 proteins only occur in two domains of life (Archaea and Eukarya) similarly to COG2042 proteins. Although co-occurrence of protein members is not strictly identical , such occurrence pattern may reflect a functional link between the two protein families.Although COG2042 proteins are distributed among a large number of organisms, no experimental evidences have yet been reported concerning their biochemical characterization and function. As they are not related, even remotely, to any other family of proteins, COG2042 members can be phylogenetically considered as orphans. Figure ses Fig. . It probses Fig. . The lenab initio models of SSO0551 using the fully-automated ROBETTA server based on ROSETTA procedures  (DTSSP) were obtained from Pierce. Matrices for Matrix-assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry and calibration standards were purchased from Bruker Daltonics. Sequencing grade proteolytic enzymes were from Roche Applied Science.S. solfataricus P2 genome (NC_002754)) and an N-terminal extended version of SSO0551 (starting with an ATG codon at nucleotide 484916). For both proteins, an N-terminal 6His tag was added to render the purification of the recombinant products easier. For this purpose, synthetic oligonucleotide primers were oAB22 (5'-gctagcATGAAGCCCAAACCC-3') and oAB49 (5'-gctagcATGAAGGTATATATTATAGAC-3') that both contain an engineered NheI site, oAC34 (5'-cggatcctacTCATTTTTCAAGTATTTTC-3') and oAE62 (5'-ggatcctcaTCATTTTTCA AGTATTTTCTC-3') that both contain an engineered BamHI site . Oligonucleotide pairs oAB22/oAC34 and oAB49/oAC34 were used for two distinct PCR amplifications of SSO0551 with S. sulfolobus total DNA as template. A 643-bp fragment (N-ter 6His-tag extended version of SSO0551) and a 517-bp fragment (N-ter 6His-tag SSO0551) were obtained, respectively. They were cloned into pCRScript-cam (Stratagene), resulting in plasmids pSBTN-AB36 and pSBTN-AB37, respectively. The two inserts were removed by digestion with NheI and BamHI and ligated with T4 DNA ligase into plasmid pSBTN-AB23 , a derivative of pCR T7/NT-topo (Invitrogen) containing a T7 promoter and 6 His-tag, previously digested with the same endonucleases. The resulting plasmids pSBTN-AB30 and pSBTN-AB31, respectively, were verified by DNA sequencing in order to ascertain the integrity of the nucleotide sequence. Hyperexpression of the recombinant SSO0551 constructs was achieved with E. coli Rosetta(DE3)pLysS strain (Novagen), freshly transformed with the plasmids described above. Cultures were carried out at 30°C as described earlier  (DTSSP) was used to cross-link two ε-amino groups of SSO0551 lysines, essentially as described in . The masAb initio modeling was performed using the ROBETTA server ; IPTG, isopropyl-γ-D-thiogalactopyranoside; HPLC, high performance liquid chromatography; EDTA, Ethylenediaminetetraacetic acid; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; HEPPS, N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid);IMAC, immobilized metal ion adsorption chromatography; MALDI-TOF, Matrix-assisted Laser Desorption/Ionization Time-of-Flight; NHS-biotin, N-hydroxysuccinimide-biotin; PSI-BLAST, Position-Specific Iterated Blast; Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.ab initio modeling of SSO0551 and drafted the original manuscript. AD actively participated in conception of the mass spectrometry strategies, advised OS on execution and interpretation of mass spectrometry experiments and assisted in figure design. OS performed and interpreted all mass spectrometry experiments. JLP contributed its experience for the modeling aspects of the project. EQ contributed its experience in mass spectrometry-based topology. All authors participated in manuscript preparation, read and approved the final manuscript.JA conceived, coordinated the study and participated in all its experimental aspects. He analyzed the genomic distribution of this family of proteins, designed and engineered the recombinant SSO0551 molecule, conceived the mass spectrometry strategies and interpreted the data. He proposed


Emerging Themes in Epidemiology (ETE) is a new, online, Open Access peer-reviewed journal. The Journal is unique in that it was conceived and is managed by research degree students in epidemiology and related public health fields. The Journal's management is overseen by its Editor-in-Chief and Associate Faculty Editors, all of whom are senior members of faculty. ETE aims to encourage debate and discussion on the theoretical, methodological and practical aspects of epidemiologic research and practice. In addition, ETE is dedicated to the promotion of Open Access publication and the training of research students in the scientific publishing process. This editorial, to coincide with the launch of ETE, sets out the Journal's philosophy and aims. Epidemiology is a rich and innovative science that has much to gain from broader discussion of the causal frameworks that underpin it. ETE aims to be a major forum for such discussion. Causality. There is no escape from it, we are forever slaves to it. Our only hope, our only peace is to understand it, to understand the 'why'["the 'why'."- The MerovingianAs far as we know, the Wachowski brothers' Merovingian was not an epidemiologist, but his sentiments should resonate well with those in the field. Probably more than those in any other profession, epidemiologists are slaves to causality. Our professional lives are dedicated to the pursuit of pumphandles, spiderwebs and causal pies ,3. AddinDictionary of Epidemiology does not include the term 'cause', opting instead to give a definition of 'causality' that involves a brief discussion of necessity and sufficiency [The epidemiologic literature on causality certainly makes for stimulating reading, but it would be interesting to know how many of us have causal pies and fractals on our minds as we reach for that red folder labelled "Logistic regression 101". Discursive articles on the usefulness of such metaphors are widely regarded as philosophical flights of fancy that we might eventually get around to reading after clearing that backlog of papers waiting to be written in the next two months. Yet what are our alternatives? The newly-qualified epidemiologist leaves their degree with a solid grasp of error, bias, confounding and statistical methodology, but with perhaps a single lecture on Koch-Henlé postulates and Bradford-Hill 'criteria' as the extent of their training into causal thinking. It is interesting to note that Last's ficiency . Any reaficiency , or admiFinal Destination [Rocky Mountain High ironically playing in the background, she tries to calm herself by making some tea. Moving to the kitchen sink, she fills the kettle with water, spilling some down the side. She wipes the kettle, turns towards the gas stove and tosses the towel carelessly behind her, which catches onto a knife block. With the kettle whistling, she picks up a school coffee mug, drops two tea bags inside and fills it with boiling water. Picking up the mug, she suddenly recognizes the school logo and, in shock, reflexively throws the mug's contents into the sink. Opting now for something stronger to calm her nerves, she takes some ice cubes from the freezer, drops them into the still-warm mug and re-fills it, this time with vodka. Oblivious to the crack that has appeared in the mug, she walks towards the living room, leaving a trail of vodka behind. As she stands by her computer monitor, vodka drips into the circuitry. An electrical surge creates a spark that ignites the alcohol, causing the monitor to explode and sending out flying shards of glass that slash her throat. Shocked and bleeding, she stumbles towards the kitchen sink, chased by a trail of burning alcohol. Reaching the stove, the trail of flames ignites the gas burners, lighting up her clothes and hair. Falling to the ground and still bleeding, she rolls around violently trying to put out the flames. In an act of desperation, she reaches up and grabs the dangling towel, tilting the knife block and sending a half dozen blades cascading into her stomach while flames catch the curtains and set the house on fire.James Wong's 2000 teen horror movie tination  is unlikSuppose you were the investigator arriving on the scene. There is a half burnt-down house, a blown-up computer, a broken mug and a corpse with third-degree burns, stab wounds and a cut throat, but no signs of struggle. What would you determine was the cause of this tragedy? The severe burns, the protruding knives and the neck wound would be pretty obvious choices. But perhaps it is more complicated than that. Perhaps there were extenuating circumstances without which this tragic outcome might not have occurred. The exploding computer maybe, or what about the vodka that burned leaving no trace, or the cracked mug? No, maybe it was the towel, complicitly catching onto the knife block. Or maybe we should blame the teacher's drinking habit.The point of this rather unsavoury story is that without the benefit of such extrinsic observation, detailed reconstructions of causal processes are unattainable. A reasoned observer might conclude that all of the above factors were in some way responsible. They all contributed to the process in their own small way, and had any one of them not been involved things may not have turned out the way they did. They were all what one might call 'component causes'. But is this enough to convince us of what the real cause was? Clearly not.Suppose now that you were an audience member and were somehow able to communicate directly with the characters in the movie. You might wish to warn Valerie of her impending ill fate. At which point in the whole sequence would you alert her so that her death could be prevented? Clearly you would not deny her a mug of tea and you would most likely have no way of knowing that the towel would land on the kitchen block with dire consequences just a few moments later. You might, of course, realize this at the last moment and warn her against reaching up and grabbing the towel, but she might still have died from her neck wound. You might have shouted for her to grab a fire extinguisher and put out the trail of flames, but it is unlikely she would have listened to you as she tried hopelessly to stop the bleeding from her neck. You might, with better foresight, have pointed out to her that vodka was dripping from her mug. Or perhaps with hindsight, you might have recognized that the best thing would have been to provide some moral support and consolation in her time of grief, with which the whole sorry incident might have been avoided altogether.One thing is now clear. There are steps within causal processes on which we can act to try and alter their course- the trail of flames might have been extinguished, and the consequences of the dripping vodka might have been avoided. There are other steps on which we can have no influence, eg. the tossed towel landing on the knife block. Another important thing is also apparent: causal processes have hierarchies. Depending on what happens at one stage, a number of alternative events may result at the next. Had Valerie been able to stop the trail of flames, she might have reached the kitchen sink, realized she could not stop the bleeding and called an ambulance. Or she might have run out of the house shouting for help and her neighbour, trained in first aid, might have saved her life. Saving victims of horror movies, however, is not an easy job. Knowing when best to step in is not necessarily that simple, as we have seen with Valerie. In some cases we may be given a number of opportunities and intervening at any of them might lead to a positive outcome. But in other cases, once certain factors are in place there will be an air of inevitability in everything that follows, and all our attempts to intervene thereafter may prove to be little more than an exercise in futility.Perhaps it is now time to admit that I have extended this fanciful analogy far beyond what is appropriate. I make no apology if in so doing I have in some way managed to convey the idea that epidemiology thrives on causal processes, on elucidating their complexities and identifying the most effective points for intervention no matter at what level of their elaborate hierarchies. If this is a worthwhile venture, then the discussion of how we conceptualize and study these causal processes surely is so too.Emerging Themes in Epidemiology (ETE) was born out of this ideal- that contrary to common belief, epidemiology is not merely a collection of standardized tools to be applied at will to any health-related situation, but that it is a rich and innovative science that aims to describe reality in all its complexity, spanning the molecular to the global, with the ultimate goal of improving the health of individuals and populations. And that in order for this to be achieved, we need to improve our understanding of how factors, at any level of biological or social organization, eventually lead to ill health.ETE is founded on three core principles:• That epidemiology and epidemiologists have much to gain from a broader and more fundamental discussion of the concepts and theoretical frameworks that underpin the practice of epidemiologic research• That Open Access publication has a crucial role to play in reducing the current inequities in access to scientific information, which should be a universal and freely-available resource for the benefit of the whole of society• That students of epidemiology and related fields can make substantial contributions to the introduction of new concepts and ideas into mainstream epidemiologic research, not only through writing, but also through having a direct influence over what is publishedETE will not generally publish research reports, although exceptions may be made in cases where the results can be placed within a broader public health context to present a new concept or theoretical framework.In keeping with this philosophy, we recognize that epidemiology has much to gain from developments in other fields and we welcome contributions from all public health professionals. We will consider articles that comment critically on current epidemiologic theory and practice, either generally or within a specific specialty, including articles from other fields that have implications for the conduct of epidemiologic research. Emerging Themes in Epidemiology a global forum for the discussion of new developments in epidemiologic thinking and practice that will benefit the global public health community. In doing so, we recognize that there is much that the scientific community can do to support Open Access publication. The health consequences of inequitable access to scientific information remain largely ignored, yet for years we have adhered to a system of publication that is restrictive and largely subsidized by institutions and libraries at great expense. Open Access is an important step towards making the publication process, and its associated costs, more transparent. We thus call on individuals, institutions, funding bodies and governments to engage in the Open Access movement by promoting and supporting Open Access publication. This will involve a major shift not just in publishing costs, but also in thinking. Our current measures for assessing the impact of academic research are inherently intertwined with our inequitable publication tradition. Developing new ways of assessing the quality of scientific research that are independent of journals' perceived 'impact' are imperative for the wider recognition of Open Access.By making all of this material freely available online under the auspices of the Open Access publisher, BioMed Central, we aim to make ETE to be a training ground for postgraduate students, providing them with an opportunity to be involved at every stage of the publication process, including commissioning, reviewing and writing articles. Our editorial board is formed principally of research students, who operate the Journal with support from an international group of associate faculty. Our collaborations extend to a growing number of students from diverse institutions serving as article referees. We welcome suggestions for extending these collaborations in the future.In promoting the role of students in the publication process, we intend for Emerging Themes in Epidemiology will be a tool for epidemiologists confronting that challenge.The third millennium has brought with it exciting challenges for epidemiologists. An explosion in the availability of genetic and molecular information, the development of bioinformatics, the increasing application of sophisticated statistical analyses to model complex systems and the gradual incorporation of sociological approaches to understanding health inequalities have arrived together with sobering statistics on the state of global public health. Knowing when and how to apply these and other developments at a time of rapid political, social and biological change while maintaining clear sight of our public health goals will be the major challenge for current and future generations. It is our hope that


The 156 breeds of dog recognized by the American Kennel Club offer a unique opportunity to map genes important in genetic variation. Each breed features a defining constellation of morphological and behavioral traits, often generated by deliberate crossing of closely related individuals, leading to a high rate of genetic disease in many breeds. Understanding the genetic basis of both phenotypic variation and disease susceptibility in the dog provides new ways in which to dissect the genetics of human health and biology.in situ hybridization (FISH). The two data sets show excellent concordance. Excluding the Y chromosome, the map features an RH/FISH mapped BAC every 3.5 Mb and an RH mapped BAC-end, on average, every 2 Mb. For 2233 markers, the orthologous human genes have been established, allowing the identification of 79 conserved segments (CS) between the dog and human genomes, dramatically extending the length of most previously described CS.To facilitate both genetic mapping and cloning efforts, we have constructed an integrated canine genome map that is both dense and accurate. The resulting resource encompasses 4249 markers, and was constructed using the RHDF5000-2 whole genome radiation hybrid panel. The radiation hybrid (RH) map features a density of one marker every 900 Kb and contains 1760 bacterial artificial chromosome clones (BACs) localized to 1423 unique positions, 851 of which have also been mapped by fluorescence These results provide a necessary resource for the canine genome mapping community to undertake positional cloning experiments and provide new insights into the comparative canine-human genome maps. Three major advances in the development of resources for mapping canine disease genes have been: 1) the development of a radiation hybrid (RH) map composed of large numbers of microsatellite markers and genes that link the canine and human genomes -5; and 3The above mapping efforts are complemented by the recent release of a 1.5x sequence of a Standard Poodle genome . The seqUsing the above resources, canine researchers have undertaken genome-wide screens for linkage to a variety of disease loci as well as morphological traits defining differences between breeds. Many of these studies have met with success. Disease loci have been genetically mapped or otherwise localized in the dog for several disorders including: vision-associated disease such as progressive rod cone degeneration, early retinal degeneration, cone degeneration, and collie eye anomaly -11, kidnTo facilitate positional cloning efforts in the canine community, we have localized a large set of randomly selected canine-specific BACs onto the canine 5000-rad RH map, a subset of which we have also localized cytogenetically. The resulting map of 4249 markers includes 1760 mapped BAC-ends, 851 of which have been FISH mapped, to generate a dense, accurate and highly integrated map of the canine genome.This novel RH map of the dog genome contains 4249 markers of three different types: 900 genes, 1589 microsatellites and 1760 BACs. The map was generated by genotyping 1092 new BAC-end markers on the RHDF5000-2 panel, and recomputing the new vectors with those from the previous map  and TSP/Analysis of the entire dataset of 4249 RH vectors by MULTIMAP  at a Lod. Again, the chromosome with the fewest is CFA 38, with 16 BACs localized to 14 unique positions. This resource provides a dense scaffold on which to build regional physical maps and search for new genes. Of particular interest to disease gene mappers will be the 26 BACs that are RH mapped to the X chromosome, 13 of which are also localized by FISH. Only two BACs were localized to the Y chromosome, one of which is also FISH mapped. See Table The resulting 4106 linked markers defined 3090 unique positions regularly spaced across all autosomes. The largest chromosome, canine chromosome 1 (CFA 1), contains 220 markers, while CFA 38, the smallest chromosome at 38 Mb, has only 32 markers  between the human and the dog genomes is 79. CS were identified by both FISH localization and RH mapping with four chromosomes showing four human/dog CS, and the remainder showing one to three. The number of markers and the resolving power of the 5000 rad panel allowed us to detect several instances in which CS appear to be split in sub-segments. In several such cases, the order of markers in the two adjacent sub-fragments is conserved, although the two sub-fragments are oriented in opposing directions. Following the previously agreed definitions, these sub-fragments are termed CSO for "conserved segment order" . Such a As shown in Table Canidae. Finally, with the imminent release of the 7x canine genome assembly, the resources described in this paper will provide a long-awaited means to translate canine cytogenetics data into canine DNA sequence data, thus advancing our knowledge of canine and comparative genomics.Of interest to many researchers will be the multiple applications that a cytogenetically defined and RH verified set of canine BAC clones can provide. For example, the ordered set of 981 FISH mapped canine BAC clones with a unique cytogenetic location will be welcomed by cancer geneticists as a means to characterize chromosome aberrations in canine tumors. The dense cytogenetic coverage available for most chromosomes allows the use of serial differential labeling of some or all clones in chromosome-specific panels designed to 'tile' structurally aberrant chromosomes. This approach will allow a much more accurate assessment of chromosome rearrangements than is possible with single color whole chromosome paint probes. In addition, the ordered clones will allow expansion of the current canine BAC microarrays, which in turn may be used for a variety of applications including array-based Comparative Genome Hybridization (CGH) analysis of canine tumors , array-pThe presentation of a map containing 900 canine specific genes, 1589 microsatellites and 1760 BACs provides the canine genetics community with nearly all the resources it needs to undertake experiments aimed at both mapping and cloning traits of interest. A dense set of microsatellite markers (MSS-2) for undertaking genome wide scans was provided in the previous iteration of the map [Escherichia coli (E. coli) and repetitive DNA sequences. BAC sequences were also compared using Cross_Match with the complete E. coli genome to remove contaminating sequences of bacterial host origin. Finally, sequences were examined for interspersed repeats and regions of low sequence complexity using RepeatMasker . Primers defining each BAC-end were designed to regions of high quality sequence data using Primer3 software . Primers of 25 bp lengths were preferentially designed in order to minimize problems associated with non-specific amplification, to generate amplicons of 200 to 500 bp, and to work under a single optimal set of PCR conditions.Canine BACs were randomly selected from the RPCI-81 canine BAC library  and wereGenotyping was performed using the RHDF5000-2 panel, which is comprised of 118 cell lines. The panel was constructed by fusing dog fibroblasts irradiated at 5000 rads with TK-HTK3 hamster cells and has an experimental retention frequency of 22% with a theoretical resolution limit of 600 Kb .PCR reactions were carried out at the University of Rennes and the Fred Hutchinson Cancer Research Center (FHCRC) as described previously in 15 μl volumes -35 usingDuplicate genotypes were obtained for the 1092 new BACs added to the map. Data were considered consistent when the number of discrepancies between data sets was ≤ 16%, a value determined to correspond to a distance lower than the resolution limit of the RHDF5000-2 panel. In the rare cases where two independent typings yielded >16% discrepancies, a third typing was done and the resulting vector was either integrated into the map construction, or the marker was discarded if no agreement was observed between two of three genotypes.RH vectors corresponding to the BAC-end markers and marker vectors of the previous map  were comIndividual linkage groups were analyzed using the multipoint approach of the rh_tsp_map version 2.0 of TSP/CONCORDE, as described previously ,29. Inte.A total of 851 BAC clones from the RH map were also localized by multi-color FISH analysis. DNA from each clone was prepared from 2.5 ml cultures using a BAC RealPrep  protocol. Two hundred nanograms from each sample were labeled using nick translation to incorporate one of five fluorochromes, Spectrum Red/Orange/Green dUTP , diethylaminomethylcoumarin (DEAC)-5-dUTP , or Cy5-dUTP . Typically, 25 ng of each of five differentially labeled probes were pooled and precipitated in the presence of 15 μg of sonicated genomic dog DNA as competitor. Chromosome preparation, probe hybridization and post hybridization washes were performed as described previously ,36. Chro). For 648 of the BACs (34%), each of the paired end-sequences gave a best hit to human genomic locations that are separated by 50–500 Kb, and aligned head-to-head (mean span of human genomic DNA = 191 Kb). The remaining 1262 BAC-end sequences were searched against scaffolds of the 1.5x assembly [Nineteen 384-well plates of BAC clones from the RPCI-81 library  were sel and .Accession numbers, PCR conditions, primers and BAC-end sequences are available for all markers at: BAC-Bacterial Artificial Chromosomein situ HybridizationFISH-Fluorescence PCR-Polymerase Chain ReactionRH-Radiation HybridCGH-Comparative Genomic HybridizationcR-centiRaysMb-megabasesKb-kilobasesBp-base pairMin.-minutesE. Coli-Escherichia coliSOM-Supporting Online MaterialDAPI-Diamidino-2-phenylindoleCS-Conserved SegmentsCSO-Conserved and Ordered SegmentsTIGR-The Institute for Genomics ResearchFHCRC-Fred Hutchinson Cancer Research Centerin situ hybridization, and microscopy. MB performed all the cytogenetic analysis to generate detailed probe ordering. MB and RT completed the merging of RH and FISH data, and prepared the drawing of Figure MB, RT, AS, and RH all contributed to the FISH mapping aspects of the manuscript, including isolation of BAC DNA samples, generation of canine chromosome preparations, probe labeling and purification, fluorescence FISH Information: RH Map Information:  and .


S. cerevisiae the β-1,4-linked N-acetylglucosamine polymer, chitin, is synthesized by a family of 3 specialized but interacting chitin synthases encoded by CHS1, CHS2 and CHS3. Chs2p makes chitin in the primary septum, while Chs3p makes chitin in the lateral cell wall and in the bud neck, and can partially compensate for the lack of Chs2p. Chs3p requires a pathway of Bni4p, Chs4p, Chs5p, Chs6p and Chs7p for its localization and activity. Chs1p is thought to have a septum repair function after cell separation. To further explore interactions in the chitin synthase family and to find processes buffering chitin synthesis, we compiled a genetic interaction network of genes showing synthetic interactions with CHS1, CHS3 and genes involved in Chs3p localization and function and made a phenotypic analysis of their mutants.In CHS1, CHS3, CHS4, CHS5, CHS6, CHS7 and BNI4 in a synthetic genetic array analysis we assembled a network of 316 interactions among 163 genes. The interaction network with CHS3, CHS4, CHS5, CHS6, CHS7 or BNI4 forms a dense neighborhood, with many genes functioning in cell wall assembly or polarized secretion. Chitin levels were altered in 54 of the mutants in individually deleted genes, indicating a functional relationship between them and chitin synthesis. 32 of these mutants triggered the chitin stress response, with elevated chitin levels and a dependence on CHS3. A large fraction of the CHS1-interaction set was distinct from that of the CHS3 network, indicating broad roles for Chs1p in buffering both Chs2p function and more global cell wall robustness.Using deletion mutants in CHS3 are involved in the amelioration of cell wall defects and in septum or bud neck chitin synthesis, and we newly assign a number of genes to these functions. Our genetic analysis of genes not interacting with CHS3 indicate expanded roles for Chs4p, Chs5p and Chs6p in secretory protein trafficking and of Bni4p in bud neck organization.Based on their interaction patterns and chitin levels we group interacting mutants into functional categories. Genes interacting with  Saccharomyces cerevisiae, chitin, a linear polymer of β-1,4-linked N-acetylglucosamine (GlcNAc) residues, is selectively concentrated at the bud neck and is also found as a minor component of the mature lateral cell wall. Chitin is also the main constituent of the primary septum, a structure that separates mother and daughter cells .CHS3, is responsible for synthesis of the chitin ring at the bud neck and for chitin in the lateral wall. CSII synthesizes the chitin of the primary septum, and is encoded by CHS2, a gene that is essential in many strain backgrounds , the interacting gene mutation [SD-His/Arg + canavanine/G-418], and both the query-gene and interacting gene mutations [SD-His/Arg + canavanine/Nourseothricin/G-418] then incubated at 30°C for ~2 days. Cell growth under the three conditions was compared and double mutants were scored as synthetic sick (SS), synthetic lethal (SL) or no interaction (No) [CHS3-7 or SHC1 [CDC3,CDC11, CDC12 and CHS2) or genes whose deletion leads to systematic growth defects in our conditions  and these genes were used in our network analysis. No synthetic interaction between GAS1 and CHS4 or CHS7 was found by the SGA. These discrepancies with other's data [CHS1 or a set of 16 non-reciprocal interactions (that is gene A found in screen for genes interacting with gene B and gene B not found in the set of genes interacting with gene A).Synthetic genetic array analysis (SGA) was used to identify genes required for the optimal growth of strains deleted for escribed ,19. From or SHC1 ,25,56,572HPO4/0.1 M citric acid, pH 6.0) was added to pellets. Extracts were then stored at -20°C until processed for chitin measurements. Samples were thawed and subjected to two digestions with 4 μl of purified Streptomyces plicatus chitinase-63 (4 μg/ul in PBS) at 37°C for 36–40 h and then for 20–24 h. The amounts of chitin were then determined by using the modified Morgan-Elson procedure as described previously [p < 0.01 in a Student's t-test, see Stationary phase cultures were diluted 1:100 into 3 ml of YEPD and grown again for 22-24 h at 30°C. Cells from 1.5 ml culture were colleted by centrifugation . Pellets were then frozen at -20°C until used for alkali-extraction. Dry weights were determined after a 2 day incubation at 37°C. Cell pellets were resuspended in 1 ml 6% KOH and heated at 80°C for 90 min with occasional mixing. Alkaline insoluble material was pelletted , neutralized with phosphate-buffered saline for 10–20 min with occasional mixing. After centrifugation , 200 μl of McIlvaine's Buffer  containing 10 μg/ml or 50 μg/ml Calcofluor white , and control plates. Plates were incubated at 30°C for 48 hours, photographed, and then rechecked after 72 hours. A literature search indicated that the phenotype we found agreed with that previously reported for 29 mutants (22 interacting mutants + 7 query mutants). In 4 cases however , we found a wild type sensitivity for mutants that had previously shown altered Calcofluor white sensitivity [Sensitivity to Calcofluor white was assessed using a modified version of the method described by Ram et al. . Cells wsitivity ,58,59. TGL participated in the design of the study and its coordination, collected and analyzed data, and drafted the manuscript. JS carried out confirmations of SGA screens and Calcofluor white sensitivity assays. CAS carried out chitin determinations and data analysis. AMS, PM, SH and AHYT carried out SGA screens. HB and CB conceived the study, and oversaw its design and coordination. HB participated in data analysis and manuscript writing. All authors read and approved the final manuscript.Title: Interactions reported previously and not included in our global analysis.Click here for fileTitle: Sensitivity to Calcofluor white and chitin levels of mutants.Click here for file


The biological potential of prostate cancer is extremely variable. Particular interest is focused on markers not expressed in normal prostatic tissues. pS2 protein expression has been demonstrated in a range of malignant tissues in an oestrogen-independent pathway. Recently, it has been demonstrated that pS2, in prostate cancer, is closely associated with neuro-endocrine differentiation. In the present study, we have analyzed, the potential of Neuro-endocrine and pS2 (TFF1) expression in human prostate cancer determined by immunohistochemistry, in primary adenocarcinoma of the prostate and attempted to correlate this with the clinico-pathologic features of the patient and neuroendocrine expression.Ninety-five malignant prostatic specimens from primary adenocarcinoma, obtained from either transurethral resection of prostate or radical retropubic prostatectomy, from 84 patients between January 1991 and December 1998 were evaluated by immuno-histochemical staining using selected neuroendocrine tumor markers i.e. chromogranin A (CgA) and estrogen inducible pS2 protein. The relationship between the expressions of pS2 was studied with CgA expression, clinical stage (TNM) and tumour grade (Gleason system). Fischer exact test was used for statistical analysis.The mean age of the patients was 70 + /- 9.2 years. The pS2 expression was seen in 10% of primary prostate cancers. Worsening histological grade was associated with greater expression of pS2 (p < 0.001). The expression of CgA was noted in 31% of malignant prostatic tissue. In pS2, positive cases 2/3rd of patients were also CgA +ve. However, there was no significant correlation between pS2 expression and the stage of disease.pS2 expression in prostate cancer significantly correlates with histological grade and the neuroendocrine differentiation, as demonstrated by Chromogranin A expression but not with the clinical stage of the disease. However, the overall expression was low consequently; no definitive conclusions can be drawn. We feel further work is required in a larger series, both in primary and metastatic cancer. The biological potential of prostate cancer is extremely variable . It is pMaisakowski et al. first described the pS2 gene in the MCF-7 human breast cancer cell line . The pS2In the present study, we have investigated the expression of pS2 in malignant primary prostatic tissue in specimens obtained from transurethral, open prostatectomy, and correlated this with neuro-endocrine differentiation and clinical stage and grade. This is a preliminary report on pS2 expression in prostate cancer, a larger study will better define the correlation between stage, grade of cancer with pS2 and CgA expression.Ninety-five malignant consecutive primary prostatic specimens were obtained from 84 patients by either trans-urethral resection of prostate (n = 69 patients) for urinary obstruction or from radical retro-pubic prostatectomy (n = 15 patients) between January 1991 and December 1998. These tissue specimens were taken from the archived records of the department of pathology. The age ranged from 52–93 years (mean 70 + 9.2 years).Immuno-histochemical staining for pS2 and Chromogranin A Sections were stained for H & E as well as for pS2  and Chromogranin A  by immunohistochemistry using indirect immunoperoxidase technique.Briefly, 3 μm thick tissue sections were cut and mounted on poly-L-lysine (sigma) coated slides. Sections were deparaffinized in xylene and re-hydrated through graded alcohol series followed by water. Antigen retrieval was done in case of pS2 with 10 mM citrate buffer, 6.0 in a microwave oven 3 × 5 seconds at 450 W, then gradually cooled down to room temperature.Sections were washed with water followed by Phosphate buffer saline (PBS) rinse.Endogenous peroxidase in the sections was blocked for 30 minutes with 0.3% H2 02 in methanol. Sections were washed with PBS. All sections were treated with Normal Swine serum (NSS) prediluted 1:10 in PBS for 5 minutes. The sections were then incubated with the primary antibody to pS2 diluted with NSS (1:100) and Chromogranin A (1:20) for 90 minutes at room temperature. Slides were washed with PBS and incubated with peroxidase-conjugated swine anti rabbit secondary antibody (DAKO) at a dilution of 1:150 for 45 minutes at room temperature. 3, 3'-diaminobenzidine (DAB) was used as a final Chromogen. Harris Haemtoxylin was used as a counter nuclear stain. Positive and negative controls were used with all batches of IHC staining. A prostatic adenocarcinoma specimen section expressing pS2 was used as a positive control. Same case exhibiting the primary antibody was used a negative control with each staining procedure. The extent of pS2 reactivity was semi quantitatively assessed by estimating the percentage of positive acini present in the whole mounted sessions. Expression was graded ++ if more than 50% of the tissue showed expression, + if between 5 and 49% showed expression and focal if <5% showed expression.Histological grading The Gleason system was used for grading of the cancer specimens; a senior histopathologist (SP) blinded of previous Gleason grading and clinical course did this. Based upon the Gleason score patients were divided into three groups i.e. well differentiated (Gleason sum 2–4), moderately differentiated (Gleason sum 5–7) and poorly differentiated (Gleason sum 8–10).To study correlation and determine the p value Student t test was applied.The cancerous lesion composed of 35% (n = 29) stage T1, 32% (n = 27) stage T2, 25% (n = 21) stage T3 and 6% (n = 5) stage T4 disease according to the TNM classification. Based upon the stage of the disease patients were divided into three groups i.e. organ confined (T1-2), locally invasive (T3-4 and N1) and metastatic (M1) cancer.In 95-cancer specimen from transurethral resection (n = 69) for urinary obstruction and radical retropubic prostatectomy for organ-confined cancers n = 15), pS2 reactivity was detected in the adjoining normal or hyperplastic acini in only 4.2%. The pS2 expression in cancer was found in 10% . The expression of pS2 figure  in varioIn the present study, we investigated the expression of pS2 protein in the adenocarcinoma of prostate and in the surrounding normal prostate tissue. We used a standard immunohistochemical method to assess pS2 expression in tissue sections of adenocarcinoma prostate instead of instead of biochemical or immuno-radiometric assay. The immuno-histochemical method for detection of pS2 expression has drawbacks in comparison to biochemical and immunoradiometric assay on tissue extracts. Both of the later methods allow precise quantification of levels of expression for a better correlation with other parameters studied.However, as we are interested in the clinical utility of pS2 expression in our prostate cancer population, we used immunohistochemistry, which allows appreciation of intra-tumoral heterogeneity of expression and of both cancerous and non-cancerous cells. pS2 protein expression has been demonstrated in a range of malignant and benign pathologies. It is highly expressed in receptor positive human breast cancer  but exprA significant implication of pS2 in prostate cancer is the close association of this marker with Neuroendocrine (NE) differentiation. There is increasing evidence that focal NE differentiation frequently occurs in prostatic adenocarcinoma and it may have significant prognostic implications -14. NE dOur results demonstrate that although the expression of pS2 protein was noted in only 1/10th of prostate cancers, it significantly correlates with the histological grade and NE differentiation; both have independent and interdependent prognostic value. There is dearth of data exploring the correlation of pS2 expression and aggressiveness of prostate cancer cell behavior. Limited literature available at present show significant association of pS2 expression with prognosis in prostate cancer, however more work is required to explore the utility of this marker in defining the biological potential of prostate cancer.The author(s) declare that they have no competing interests.MHA, conceived of the idea, wrote the manuscript and conducted clinical part of the study. FA, helped in designing the study and reviewed the draft of the manuscriptNF, helped in conducting study, helped in data collection and analysis. MI, conducted the pathological part of the study. SP, conducted and supervised the pathological aspects of the study and reviewed the manuscript and wrote methods and results related to the pathology. All authors' have read and approve of the final manuscript.The pre-publication history for this paper can be accessed here:


Due to differences in current socio-economical situation and historically shaped values, different societies have their own concepts of high-quality life. This diversity of concepts interferes with quality of life (Qol) research in health sciences. Before deciding to apply a Qol assessment tool designed in and for another society, a researcher should answer the question: how will this instrument work under the specific circumstances of my research. Our study represents an example of the utilization of qualitative research methods to investigate the appropriateness of the Rheumatoid Arthritis Quality of Life Scale (RAQol) for the assessment of Qol in Estonian patients.Semi-structured interviews were conducted with the rheumatoid arthritis (RA) patients of Tartu University Hospital and these were analyzed using the principles of the grounded theory.We described the significance of the questionnaire's items for our patients and also identified topics that were important for the Qol of Estonian RA patients, but that were not assessed by the RAQol. We concluded that the RAQol can be successfully adapted for Estonia; the aspects of Qol not captured by the questionnaire but revealed during our study should be taken into account in future research.Our results show that qualitative research can successfully be used for pre-adaptation assessment of a Qol instrument's appropriateness. With this article we are introducing our experience of how qualitative research can be utilized in the process of adapting of a quality of life (Qol) assessment instrument. We will argue the unique benefits of the qualitative method for assuring the validity of an adapted measure.Qol cannot be treated as something uniform and stable. One reason for its variability in time and space is the social nature of the "quality". Different societies with their current socio-economic situation, values and traditions, carry their own, to certain degree dissimilar understandings of high-quality life. Therefore Qol can be seen as a social construct. For further discussion on Qol domains see Schalock, 2004 .Inclusion of Qol assessment into a set of health outcome measures is, regarding the achievements in improving the survival of chronically ill patients, well justified. But the social complexity of the construct means that its evaluation is also complex compared to traditional outcome measures e.g. symptoms or results of lab tests. Restricting Qol assessment to condition-bound groups (disease-specific Qol) permits potential influences on everyday life to be homogenized; problems with the various individual significances of these condition-characteristic impacts remain. Different Qol concepts in health sciences try to improve assessment's generalizability. Functionalistically oriented health-related Qol concentrates on restrictions in performance of everyday activities . AlthougBefore deciding to use an existing instrument for Qol assessment, a researcher should answer the question: how will this questionnaire work in the particular circumstances of the research? Even an instrument that has performed excellently in the country of origin can loose some validity when applied in a different social context. There are three categories of topics that should be recognized for every Qol instrument: first, those important for the patients being investigated, and already assessed by the instrument ; second, those unimportant (or not so important) for the patients being investigated, but still assessed by the instrument; and third, those important the for the patients being investigated, but not assessed by the instrument. It is evident that an appropriate questionnaire consists mostly of the first category items, the content of the second category items is minimized and all topics important for patients are included.The items belonging to the two first categories can be determined during a standard adaptation process. The drawback of this approach is the amount of adaptation work that usually has to be done before receiving evidence about the merits of the questionnaire. Delimiting of the third category requires a deeper insight into the society-specific determinants of Qol.Because of the smallness of Estonia (population about 1.4 million) studies involving only our patients may demonstrate a lack of statistical power; also some restrictions concerning scientific potential and funding should be admitted. Therefore a promising choice for Estonian clinical and health sciences can be seen in cooperative research projects. Reasoned selection, judicious adaptation and application of internationally approved assessment instruments could be crucial for success. As a part of the Soviet Union, Estonia stayed isolated from Europe for years and quite strong ideological pressure was brought to bear on the citizens. After establishing independence in the 1990s, Estonian society has undergone abrupt ideological and economic changes. A researcher working in the Qol assessment field should take into account the suspected impact of these factors on understanding life quality among the local population.Our work offers an example of the application of qualitative interviews for exploring the essence of quality of everyday life for Estonian rheumatoid arthritis (RA) patients. We will illustrate the evaluation of the three topic categories using an example of RA specific Qol assessment scale.Rheumatoid Arthritis Quality of Life Scale (RAQol) was developed in the 1990s as an outcome measure to assess the impact of RA and its treatment on QoL. The content was derived from interviews with 50 RA patients conducted simultaneously in Great Britain and the Netherlands ,5. The t• food, drink, sleep, activity, sex, pain avoidance• warmth, shelter, security, safety, freedom from fear, stability• affection, love, physical contact, intimacy, attachment, communication, sharing experiences, sharing goals, affiliation• curiosity, exploration, play, stimulation, enjoyment, creativity, meaningfulness• identity, status, recognition, approval, appreciation, usefulness to others, respect, competence, self esteem, mastery, achievement, power, independence, freedom• time structure• self actualizationAs a RA specific instrument, the RAQol was designed to assess the fulfillment of only those needs whose importance for RA patients emerged during the interviews. The original wording from the interviews was retained as much as possible. The final RAQoL is a 30-item measure where each item is in the form of a simple statement to which patients indicate whether or not it is true for them at that moment. The following example of the RAQol items demonstrates the wide range of everyday areas covered by the instrument: self-care, different indoor and outdoor activities, emotions and conditions, and interpersonal relations.• Item 18. I have problems taking a bath/shower• Item 7. Jobs about the house take me a long time• Item 6. I find it difficult to walk to the shops• Item 16. I often get depressed• Item 12. I find it hard to concentrate• Item 13. Sometimes I just want to be left alone• Item 29. I avoid physical contactet al. 1997  but it might come in the future; I don't know what will happen next year /.../ you're afraid that if you really get so poorly that you can't take care of yourself who will take care of you (4)• I still try to be like a human being but don't know how long I can manage (5)• there's really nothing for granted in this world of course, but I am preparing a back-up solution in case things get worse (8)No patients reported that they were disturbed by continuously thinking about the disease. Three interviewees with a disease duration of over 10 years talked about not thinking of their condition as a positive phenomenon connected with adaptation to the disease. We concluded that item number 23: My condition is always on my mind, can be treated as significant, although our interviews revealed no evidence that the problem is recognized when present.• I don't think that I'm an ill person at the moment, just when these hands are painful or I just can't cope with everything; I don't think that I'm ill, that I'm so miserable, I don't think about it (4)• I have to change and re-adjust my basic values, but I don't think about it all of the time, maybe I have adjusted them subconsciously /.../ I then, subconsciously, not thinking about it, eat something softer or don't go biting on a big apple if my jaw joint is painful (7)• I don't' even think about my disease at this point, it's like a husband now, day and night, its there in everything I do and I know that as long as I live I will have it and I cant get rid of it and I don't make it a problem anymore (9)To this group we included four items.In one interview, the impact of pain on attention was described. We did not find this evidence sufficient for designating item number 12: I find it hard to concentrate, as significant. In our opinion, these expressions describe switching of attention to another stimulus and do not refer to concentration difficulties. One reason why the interview data did not support the importance of this item can be the lack of currently active intellectual workers in our sample.• You can't think about anything but pain; crossing the road you might get hit because you're only thinking about the pain and forget that you have to keep an eye on the road (6)No data corresponding to item number 1: I have to go to bed earlier than I would like, emerged from the interviews. Because tiredness is a characteristic feature of RA, one explanation for the inability of our data to show this item's importance is associated with the paucity of absorbing nighttime activities, especially in Estonian rural areas.Item number 24: I often get angry with myself, had no matches in our interviews. It is difficult to find any obvious explanations for this particularity but we can suggest that in some cases anger was rechanneled against the medical system – a phenomenon that we will discuss later.Our data failed to demonstrate the importance of item number 28: I avoid physical contact. We believe that intimacy connected with this topic could be the reason our respondents avoided openly discussing it. If so, due to the greater impersonality of a questionnaire format, the item can retain its significance as a part of the instrument.As a result of the analysis, three groups of topics connected with satisfaction of the needs included in the list of those crucial for Qol and important for our patients, but not assessed by the questionnaire, were described.Next we will name these needs and present the evidence of the limitations in their fulfillment. Our deeper inquiry into the reasons for these peculiarities will be presented in the discussion section.Adaptation to a disease is a difficult process comparable to passing through phases of grief. An inevitable and hurtful part of it is abandoning of old roles and the recognition of new ones.Changing role functioning was a common motif in the interviews. Regret and anxiety due to inability to perform in the roles of a healthy person were expressed by the majority of respondents.• What man isn't disturbed by the inability to take care of himself, then you're like a kid not a man (8)• My appearance isn't as attractive [as a woman] anymore as it could have been without the disease (4)• I can't even go help my sister in the country with potato planting, a bit sad (2)• just sad because of him [the son], he asks me why I can't come outside to play soccer with him, well, I really can't (7)• totally like a small kid, someone else has to help you all the time, whatever it is, meals or something else (1)• I walked with a stick, it was a catastrophe, such an old granny (3)• I want to do something, just to make something and do a job, my hands want to work; people drop by and try to rope you in – I can't, there's no doer, all the time "I can't", other days I can't at all (2)• I am afraid of going to work soon; this hand is so ugly; what a hairdresser with such a horrible hand! (4)The new role of a diseased person was generally interpreted as something deprecatory; being ill was considered as opposite to being normal.• I can't move; my movements aren't like they should be, not like healthy and normal people have (1)• before I used to wear high heel shoes like normal people (3)• you're like some prehistoric creature, everybody goes by modern means but you like going back to the stone age (6)Feeling ashamed of the disparity led to preoccupation with concealing the condition. Surrounding people were often seen as appraisers; their opinions were valued more highly than success in coping was. As a result, even the simplest aids that could be noticed by others were rejected. In some cases the fear of being labeled as different elicited the preference of social isolation.• I don't want to see things that point to my condition, don't want these to be seen, and of course, don't want to have to always hide everything (6)• I control myself so that others won't notice the way I am – I have so many acquaintances that for a long time didn't know I was ill (9)• I'm even afraid to tell anyone that this hand is so ill, I'm so quiet (4)• [a special cup with two ears] I have it at home, but I don't use it; at this point I still try to be humanlike (5)• you don't go shopping with a crutch, no way (8)• I feel very uneasy eating in company; I still want to eat like people do, with regular tableware (9)• if I still find it impossible to eat or drink in company, then I don't; I won't go into company [to eat] like this (6)A well functioning medical system should strengthen the feelings of security and stability of its clients. In the words of our respondents the medical system constituted an enemy. The system was something to blame, to vent anger on; at the same time it was described as an inevitability that had to be obeyed and against which protection was required.• the system is wrong, the sick funds and all, why can't I get procedures done that are necessary for me (3)• you sign up there [for a rheumatologist], you wait and wait, a lot changes in that time; you wait a month and a half; you get there; by that time the drugs have run out, later you're at fault for not having taken them (2)• I went to a private clinic, almost like crying at the door, there are no vacancies, there's no one I could talk to, and I didn't. I went away to the country/.../now I'm here and now I'm told that this Achilles' has been broken for at least a month (10)• [left alone with the disease] just can't be such a thing, not alone, but life is like this, don't know what to wish (2)• helplessness, no person to protect me [against the system] (3)non grata turns in the course of the disease. One of the respondents regarded physicians as co-victims of the system, incapable of defending their patients.Physicians, some of them named as saviors and supporters, were still more often seen as being an impersonal part of the system. Professional incompetence and superficiality were connected with • if I had been sent to the rheumatologist right away then maybe things wouldn't have gotten this bad; thus, when my neck was stiff at first, it was thought I had caught a cold and it would pass (1)• the troubles started when my leg trauma was labelled as radiculitis (2)• initially no action was taken and consequently the general attitude [of the doctors] became such that nobody had the guts to do anything with me, and because it was such a awkward situation because they didn't have the right doctor who would have done something, I was left a bit high and dry (10)• the kidney doctor said: not to come to me, I am out of money, go to the GP /.../you go to the GP, she can't help you, prescribes what she can, she can't prescribe rheuma-drugs (2)The ability to do necessary shopping is directly assessed by item number 6: I find it difficult to walk to the shops. Difficulties with walking to the shop were described by our respondents and the item was considered to be important. But other problems connected with shopping emerged even more often in the interviews – managing in the shop, checking out, and carrying the purchases.• I don't want to stand in a line, my feet start to ache (7)• [shopping trolley] is very big, you have to push it hard and it's narrow there [in the shop] (9)• checking out at the counter you're in a hurry and then comes this psychological moment that you get nervous and can't handle it (7)• I open my wallet, they take what they need (5)• when the bag is too heavy then my feet can't stand it; you have calculate how much you buy (6)• sometimes you would like to buy more but you can't carry it; I just put stuff that I really need in the cart (9)For our interviewees, a topic closely related to shopping was the capability to use public transport. The obstacles encountered when entering, leaving and riding a bus were described.• I can't get onto the bus, even worse with the tram; the steps are very steep, I can't manage however I try (3)• I can't get off, I ride to the next stop; the bus doesn't pull over to the sidewalk but stops further away (6)• very bad to ride, I just stagger, lose my balance, sometimes you can fall quite badly; the worst is when you have to stand (9)• sitting down and getting up [on bus] is difficult for me, it's better I hold on to something for some time (5)The lack of money was mentioned by all of our respondents. Its interference with the satisfaction of the majority of needs was described.• I wouldn't say my spending has increased a lot, but my income is, yes, notably lower; before I even paid more income tax than I get paid now; the habits I had before have become impossible for me, financially (1)• a handicapped person could also make his/her life comfortable and tolerate everything if it was possible financially (6)• the pension is so tiny, I can't do anything with this; I can't manage a family with this (7)Our results showed the high significance of the majority of the RAQol items for the interviewees. This allows us to state that the RAQol can successfully be adapted into Estonian for usage in international research projects. Our results also highlight the difficulties in translating some specific items (number 9 and 28), which should be reckoned with during the adaptation process.Three Qol aspects that were important for Estonian RA patients but were not evaluated by the RAQol – the issues concerning changes in role performance, safety and stability of communication with the medical system, as well as some issues of procuring the necessities of life-were revealed by the analysis of interviews.Next we would like to discuss the reasons for these peculiarities and suggest some additional resources for in-depth reading.homo sovieticus mentality was the placing of collective interests above personal ones; individuals were appreciated by their contribution to the common good. Successful performance in social roles approved by the regime was honored; inability to meet validated ideals was considered shameful. Although we can talk of a dramatic change of approbated values and ideals after the end of the Soviet era – an independent, successful, competitive young individual is idealized now –, but the tendency to disapprove of the inability to fit expectations has remained.Only 13 years have elapsed since the end of Soviet rule in Estonia. Our respondents spent a considerable part of their lifetimes in the Soviet ideological environment and this has undoubtedly influenced their values and beliefs. An obligatory component of the We believe that this potpourri of old and new values and attitudes could explain the high significance of themes connected with roles and role functioning in our respondents' conversations. A person disabled because of disease could not perform successfully in acknowledged Soviet-time roles ; the same is true for present-day Estonia. Being categorized as "socially uncompetitive" would alter the identity and self-esteem of the disabled individuals and concealment of the condition could be seen as defense reaction. In the light of this, topics connected with role functioning and social acceptability should be included among those observed during a Qol investigation. See also -21.The transition from one economic and ideological system to another in Estonia has caused social instability, which is reflected in an increase in people's subjective sense of insecurity and fear. The most insecure time for the Estonian population was in the early 90ties; since that time, due to stabilization of the economy and economic development, a sense of security is returning. Still, there is remarkable disaffection with different spheres of public administration; future-oriented reorganizations that do not provide immediate benefits are treated with caution. The health care system, which has undergone fundamental reforms in the post-soviet period, is the favored object of criticism for the Estonian media. In the Soviet era health care was funded from the state budget and all citizens had free access to health services. Today's health care delivery system in Estonia is financed through health insurance; the private sector is growing. The introduction of family practitioners, with novel financing principles and responsibilities, in the mid 1990ties has changed previously existing relations between patients and medical specialists; access to consultants has lost its immediacy. Bureaucracy, long waiting lists, visit charges (something unthinkable for Soviet medicine) and open discussion of the financial difficulties of the health service in the Estonian media have all made their contributions to lowering confidence in the health care system in the eyes of our patients. Communication with health care deliverers constitutes a significant part of everyday life for RA patients. Therefore the impact of this communication on the everyday life of patients cannot not be ignored. In the case of a transitional society like Estonia's, the effect of health care delivery on patients' needs for stability and safety should be considered. See also -26.In health care planning, the strategic military interests of the Soviet Union were given priority; this resulted in the development of an excessive large hospital network. Habilitation and rehabilitation were upstaged and drastically under-funded. In the Soviet era the people with special needs were partly institutionalized as disabled; their problems and even their existence (with the exception of war veterans) were simply hushed up by the authorities and media. There were no adaptations for people with physical special needs in public places and in the physical environment of towns in Soviet Estonia; elementary facilities were not accessible. People in wheelchairs began to be seen on our street with arrival of western tourists in early 90ties; they were joined shortly afterwards by our own special needs persons. During the following years adaptations considering the requirements of people with special needs have little by little been introduced into the physical environment. However, due to limited resources the improvements are coming slowly and conditions have not reached the level where the comfort of special needs persons can be guaranteed. Coping with errands and chores should be taken into account as a distinct Qol topic in Estonia today. See also . the average monthly pension for the disabled in Estonia in 2003 was 1110 Estonian kroons (approximately 71 EUR). This constituted one sixth of average monthly gross wages and salaries, and was 301 kroons (19 EUR) lower than estimated minimum means of subsistence for 30 days in the same period. From these figures we can see that financial troubles can overshadow every aspect of the everyday life of our patients.According to the Statistical Office of Estonia In our opinion adding the assessment of these three aspects – changes in role performance, safety and stability of communication with the medical system, and some aspects of procuring the necessities of to life – should be considered by Estonian researchers, especially when carrying out disease-specific Qol studies at the national level.In our research we used qualitative interviews for assessing the appropriateness of a RA-specific Qol instrument, the RAQol, for adaptation for use in Estonia. We described the importance of the items to our patients and identified Qol topics that were significant for our respondents but that were not assessed by the questionnaire. We also discussed the nature of the discrepancies in significance of Qol topics for Estonian patients.Our results show that the utilization of a qualitative study as an introductory part of Qol assessment instrument adaptation provides possibilities for more thoroughly considered Qol research. By evaluating the significance of items in the particular context, it allows the mechanical acceptance of instruments just because they have performed well in other societies and cultures to be avoided. Moreover, it offers an opportunity to identify topics that are not included in the instrument but are important for local interpretation of Qol, which are otherwise often overlooked. For researchers, qualitative studies offer a deeper understanding of the instrument in question and of the research topic – Qol of the patients.The data collected during this qualitative research process also has the potential to be used for a wider analysis of the Qol of Estonian RA patients. Still, our current interests were centered on the appropriateness of the adaptation of a particular Qol instrument, and therefore the use of the gathered data was quite limited. But we believe that the knowledge gained will be beneficial for our forthcoming research projects.MT: the idea and conceptual construction of the research, recruitment of the participants and conduction of the interviews, coordination of the coding and analysis processesJS: methodological construction and supervision of the research, active participation in the coding and analysis processesKM and EH: participation in discussions and decision-making throughout the whole course of the researchAll authors have read and approved the final manuscript.


Immune deficiency, Centromeric region instability, and Facial anomalies syndrome). In normal cells the inactive X has characteristic silencing type histone modification patterns and the CpG islands of genes subject to X inactivation are hypermethylated. In ICF cells, however, genes subject to X inactivation are hypomethylated on the inactive X due to mutations in the DNA methyltransferase (DNMT3B) genes. Therefore, if DNA methylation is upstream of histone modification, the histones on the inactive X in ICF cells should not be modified to a silent form. In addition, we determined whether a specific methyl-CpG binding protein, MeCP2, is necessary for the inactive X histone modification pattern by studying Rett syndrome cells which are deficient in MeCP2 function.In mammals, there is evidence suggesting that methyl-CpG binding proteins may play a significant role in histone modification through their association with modification complexes that can deacetylate and/or methylate nucleosomes in the proximity of methylated DNA. We examined this idea for the X chromosome by studying histone modifications on the X chromosome in normal cells and in cells from patients with ICF syndrome (We show here that the inactive X in ICF cells, which appears to be hypomethylated at all CpG islands, exhibits normal histone modification patterns. In addition, in Rett cells with no functional MeCP2 methyl-CpG binding protein, the inactive X also exhibits normal histone modification patterns.These data suggest that DNA methylation and the associated methyl-DNA binding proteins may not play a critical role in determining histone modification patterns on the mammalian inactive X chromosome at the sites analyzed. Although it has been known for some time that histone modifications play a role in gene expression , it is oNeurospora, histone methylation appears to determine DNA methylation patterns [Arabidopsis, non-CpG DNA methylation also appears to be determined by histone methyltransferases, whereas CpG methylation does not [MECP2 mutations exhibit histone hyperacetylation [Mecp2 mutations [In patterns ,4. In Ardoes not ,6. In madoes not -11. Mutadoes not , supporttylation . Histoneutations . Thus, Dutations .We are especially interested in the X chromosome with respect to the question of the relationship between DNA methylation and histone modification. The mammalian X chromosome is unusual in that about a thousand gene-associated CpG islands are hypermethylated on the inactive X and hypomethylated on the active X. Except for imprinted loci, methylation patterns at most other regions of the genome are similar between homologs. Histone modification differences known to be associated with either silent or expressed chromatin also distinguish the active and inactive X chromosomes -19. ThusImmune deficiency, Centromeric region instability, and Facial anomalies". In most cases, the molecular defects result from mutations in the DNMT3B methyltransferase gene [MECP2. This suggests that this specific methyl-DNA binding protein does not have a major role in silencing the inactive X through histone modification.To examine more fully the possible relationships between DNA methylation and histone modification, we have utilized cell cultures from individuals with a human hypomethylation disease called the ICF syndrome. This disease is clinically characterized by "ase gene -22. Certase gene  and LINEase gene . If DNA We examined histone modifications known to be associated with the inactive X chromosome in two ICF cell lines and normal control cells. Specific histone modifications including histone H3 and H4 acetylation, and histone H3 methylation at K4 and K9, were examined using antibodies to stain nuclei and metaphases -17. We aThe expected patterns of macroH2A1 histone concentration and histone modification on the inactive X were found in cells derived from ICF individuals and in control normal cells  on the inactive X, and in the second case, we had one clone with the wild type MECP2 on the inactive X (1789V) [We examined histone modifications and macrohistone association in clones from two individuals heterozygous for a mutation in tivation . MECP2 mtylation ,13, as w (1789V) . For allG6PD, NEMO, MECP2, SYBL1, AR, FMR1, and PGK1) in ICF cells so that we could employ allele-specific chromatin immunoprecipitation (ChIP) analysis. We restricted our search for polymorphisms to the promoter region, as several reports have indicated that marked differences in histone modifications between active and inactive alleles are seldom detected at other regions [SYBL1  and AR (androgen receptor in Xq12).To verify our cytological histone modification results at the gene level, we searched for promoter polymorphisms at seven X-linked loci  was informative in one ICF sample (PT 4) and in several controls, thus permitting separation of the active and inactive alleles in cloned cells, and in cultures with highly skewed X inactivation. The antibodies were highly specific under the amplification conditions chosen, so that a strong signal was seen for the pull-down experiment with antibody and little or no signal for the "no antibody" control  has reported on a ChIP study at the ive gene . Here wethylated . In the rol Fig. .AR gene were then separated on an automated sequencer according to CAG repeat number. Two major peaks are detected in the input control DNA, corresponding to the active (A) and inactive (I) X alleles, differing in CAG repeat number  also showed normal histone H3 hypoacetylation and K4 H3 hypomethylation in spite of the very low levels of DNA methylation in this CpG island region  is the sensitivity of the cytological histone modification assay too low to detect active-type histone modifications on the ICF inactive X? and (2) is the extent of methylation on the ICF inactive X greater than is suggested by CpG island and LINE-1 methylation patterns?AR, in ICF cells  has previously reported this to be the case for the SYBL1 gene  and harvested in trypsin:EDTA (Gibco Invitrogen Corp.) under standard conditions ,48.For analysis of interphase stages, cells were plated on alcohol-washed 22 mm square cover slips in 35 × 10 mm Petri dishes. On the following day the medium was removed and the cells were washed once with PBS followed by fixation in 95% ethanol:5% acetic acid for 1 min at room temperature. The rest of the procedure followed the "Immunocytochemistry Protocol" of Upstate . DAPI-stained slides mounted in antifade were examined with a Nikon Microphot FXA microscope and images were captured with a Nikon Coolpix 995 digital camera. The inactive X was recognized under DAPI staining as sex chromatin. Absence of a particular histone modification on the inactive X was seen as a hole or gap at the sex chromatin location.For analysis of metaphase cells, we plated cells in 150 × 25 mm tissue culture dishes and added colcemid (Gibco Invitrogen Corp.) 48 h later (0.1 μg/ml for 2 h). The medium was removed and the cells were washed once with Hanks' balanced salt solution, followed by trypsinization (Gibco Invitrogen Corp.) with slight agitation to collect metaphase cells. Serum was added to stop tryptic action and the cells were recovered by centrifugation, then placed in hypotonic solution (3 mg/ml KCl and 1 mg/ml sodium citrate) at 37°C for 15 min. Cells were collected on to slides using a Cytospin centrifuge and then fixed in 95% ethanol:5% acetic acid for 1 min. The rest of the procedure followed the Upstate protocol mentioned above, followed by DAPI staining, mounting in antifade, and examination with a Nikon Microphot FXA microscope.Antibodies used to detect histone modifications were obtained from Upstate and included: "Anti-acetyl-Histone H4," recognizing acetylated lysines 5, 8, 12, and 16, "Anti-acetyl-Histone H3 (Lys 9)," "Anti-dimethyl-Histone H3 (Lys 9)," "Anti-dimethyl-Histone H3 (Lys 4)," and "Anti-Histone macroH2A1."2 tissue culture flask (about 3 × 106 cells) was washed with PBS and treated with 4% formaldehyde (pH > 7) for 10 min at 37°C. Protease inhibitor cocktail (Complete) from Roche Diagnostics  was used in place of individual inhibitors, and Protein A-Separose 4B  was used to collect immune complexes. After elution of immune complexes, they were heated at 65°C for 4 h to reverse crosslinks, and the DNA was recovered with a "QIAquick" PCR purification kit from Qiagen Inc. .Chromatin immunoprecipitation was performed using the protocol of Upstate with slight modifications. For each experiment, a near-confluent 75 cmAR DNA was across the polymorphic 5' CAG repeat region as previously described [Primary amplification of escribed , using 2escribed , using 6escribed .SYBL1 gene was performed similarly to that of AR, except the allele-specific reaction entailed XhoI digestion of the primary amplification product followed by these products being separated by agarose gel electrophoresis. Conditions for PCR amplification and XhoI digestion were as previously described [Analysis of the 5' region of the escribed .ac = acetylatedChIP = chromatin immune precipitationDAPI = 4,6-diamidino-2-phenylidoleFB = fibroblastFAM = 5-carboxyfluoresceinFITC = fluorescein-isothiocyanateICF = immune deficiency, centromeric region instability, facial anomaliesH3 = histone 3H4 = histone 4K4 = lysine 4K9 = lysine 9LINE-1 = long interspersed nuclear element 1MeCP2 = methyl-CpG binding proteinWT = wild typeXa = active X chromosomeXi = inactive X chromosomeSMG and RSH conceived the study design, supervised and coordinated its progress, and drafted and prepared the final manuscript. KRV and PL carried out the cell culture and cytological studies. TKC carried out the ChIP analyses. JT and UF developed the cloned Rett cell cultures. All authors read and approved the manuscript.


It is a major challenge of computational biology to provide a comprehensive functional classification of all known proteins. Most existing methods seek recurrent patterns in known proteins based on manually-validated alignments of known protein families. Such methods can achieve high sensitivity, but are limited by the necessary manual labor. This makes our current view of the protein world incomplete and biased. This paper concerns ProtoNet, a automatic unsupervised global clustering system that generates a hierarchical tree of over 1,000,000 proteins, based solely on sequence similarity.In this paper we show that ProtoNet correctly captures functional and structural aspects of the protein world. Furthermore, a novel feature is an automatic procedure that reduces the tree to 12% its original size. This procedure utilizes only parameters intrinsic to the clustering process. Despite the substantial reduction in size, the system's predictive power concerning biological functions is hardly affected. We then carry out an automatic comparison with existing functional protein annotations. Consequently, 78% of the clusters in the compressed tree  get assigned a biological function with a high confidence. The clustering and compression processes are unsupervised, and robust.We present an automatically generated unbiased method that provides a hierarchical classification of all currently known proteins. The explosive growth in the number of sequenced proteins has created a glut of proteins that are sequenced but whose structure and function are as yet unknown. A common way to tackle this problem is to use database searches to find proteins similar to a newly discovered protein, thus inferring protein function. This method is generalized by protein clustering or classification where databases of proteins are organized into groups or families in a manner that attempts to capture protein similarity. Such classification into families is a critical component in structural and functional genomics -4. The nClassifying the entire protein space into families serves not only as a method for large-scale protein annotations but also to support functional and structural genomic initiatives . Some pr. For the SwissProt-based tree, there are 227,436 clusters (including the proteins as singletons). The classification provided by ProtoNet provides the full range of cluster granularity: from single proteins to huge protein clusters that carry no biological relevance (the root clusters). We test the biological validity of ProtoNet, by its examination from different perspectives, using external-defined protein keyword annotations. Four different annotation sources are used  in ordeAs mentioned, ProtoNet contains 227,436 clusters, which is obviously much more than the upper estimate of 30,000 protein families ,26. TherIn this paper we describe methods for the unsupervised compression of the ProtoNet tree, by using intrinsic tree-based parameters of the clusters that correlate well with biological validity. By preserving the unsupervised nature of the ProtoNet data, we prevent biasing towards previously discovered findings and better allow for future generalizations, in addition to maintaining the automation of the process.Finally, automatic functional annotation to proteins is of great importance. In ProtoNet, an automatic method for assignment of biological annotation to the protein clusters is used, yielding high-confidence functional assignments for a large majority of the proteins' clusters.In order to measure the correspondence between a given cluster and a specific annotation, and allow for supervised validation of the ProtoNet clusters, we define the notion of a correspondence score (CS). The CS for a certain cluster and a given keyword measures the correlation between the cluster and the keyword, using the well-known intersect-union ratio.c be the set of annotated proteins in cluster C; Let k be the set of proteins in the system annotated by keyword K; We define:Let C be a cluster in the ProtoNet tree, and K be a keyword (from a specific source) that annotates (some of) the proteins in the system; Let best cluster). The score for a given cluster on keyword K ranges from 0 (no correspondence) to 1 .The cluster receiving the maximal score for keyword K is considered the cluster that best represents K within the ProtoNet tree , GO (Molecular Function) and ENZYME . The results Table  show a h-100). Note that even for the SCOP fold level, which is associated with proteins that may be extremely remote in sequence, ProtoNet's relative success is extremely high .To avoid trivial correspondences between a keyword and a cluster, such as the assignment of a keyword that annotates only one protein to its singleton cluster, we tested our success only with keywords that annotated at least two proteins (for SCOP and ENZYME keywords). For InterPro and GO, we selected a threshold of 20 proteins per keyword, as the majority  of the annotations is included above this threshold, thus allowing the test to focus on the more significant keywords.In order to validate the hierarchical structure of ProtoNet, we compare it with the hierarchical structure of GO as described in Figure A total of 1577 GO terms were selected as described, with 1798 edges between them according to the GO hierarchy. 771 out of 1291 (60%) edges that were produced by the ProtoNet hierarchy appear in the GO hierarchy. This number is highly significant considering the fact that there exist over 1,200,000 possible edges between the 1577 vertices in the graph (considering it as a DAG). It should be noted that there are some terms in GO that are connected to many other vertices. These vertices may bias the results of this test. In order to confirm that ProtoNet performs well without these vertices as well, the vertices were removed manually and the test was repeated, with similarly significant results (33% of the edges were correct).In order to allow unsupervised automatic compression of the ProtoNet tree, we searched for an intrinsic parameter of the clustering process that would specify clusters of biological validity. By applying such a parameter one could dispose of clusters that do not pass a certain threshold value, remaining with clusters of high biological validity. Once we remain with a subset of biologically valid clusters, the new hierarchy amongst them can be reconstructed according to the original tree hierarchy.The agglomerative hierarchical clustering scheme defines a set of terms that are intrinsically associated with the process. In such a scheme, each cluster is created from smaller clusters, which are captured as its descendants in the clustering tree. The ProtoLevel (PL) ranges from 0-100 and is used as a standard quantitative measure of the relative height of a cluster in the merging tree. The PL of a cluster is defined as the arithmetic average of the BLAST E-score of the pairs of its proteins. The PL of the leaves of the tree is defined as 0, whereas the PL of a root equals 100. The larger the PL, the later the merging that created the cluster took place. Therefore, the PL scale is considered as an internal monotonic timer of merging, during the clustering process. As mentioned above, a cluster is said to be created when the merging of its two children clusters forms it. The PL at this point is said to be the birthtime of this cluster. The deathtime of a cluster is defined as the PL at its termination, i.e. the point at which it merges into its parent cluster (or 100 if it has no parent). The lifetime (LT) of a cluster is defined as:LT = deathtime - birthtimeTherefore, the LT of a cluster reflects its remoteness from the clusters in its "vicinity" in protein sequence space.We examined the LT distribution of the set of InterPro best clusters in comparison with the LT distribution of all clusters in ProtoNet Figure . The resIn order to search for a reasonable LT threshold value , several threshold values were examined Figure . The resThe mean best CS was examined for all four external sources Table . The resThe following scheme was used to annotate the protein clusters: For each cluster C and keyword K we define the following score:TP is the amount of true positives (proteins in C that have the keyword K), FN is the amount of false negatives (proteins not in C that have the keyword K) and FP is the amount of false positives (proteins in C that do not have the keyword K).Where For each cluster, we search against all keywords of GO and InterPro for the keyword with the highest AS. If the AS of the cluster is greater than 0.25, the cluster is assigned that keyword as its annotation. The logic behind the score and the threshold is as follows: the score is determined by two parameters, the specificity and the sensitivity; let us consider the two worst-case limit cases. In the first case, specificity>0.5 and sensitivity = 1: a majority of the proteins of the cluster share the keyword, and there exist no other known proteins that have the keyword. In the second case, specificity = 1 and sensitivity>0.25: all proteins of the cluster share the keyword and they constitute more than 1/4 of the total proteins known to have this keyword. In both cases, the keyword can be assigned to the protein cluster with a high degree of confidence. All other clusters fall in between these cases.Using this method, all 6,879 clusters that contain 20 or more proteins and that remain after the compression were tested. 5,355 (77.8%) clusters passed the high confidence threshold and were therefore given an annotation. Figure Figure The challenge of protein classification by using sequence similarity has been addressed extensively by several different methods. In order to assign function to proteins, advanced methods (such as Hidden Markov Models implemented in Pfam) have been used to learn sequence-based patterns on "seeds", manually validated alignments of known protein families. The widely-used BLAST algorithm is considered to be a reliable tool for sequence alignment, but has been shown to lack sensitivity for weak similarities that may be detected by signature detection methods. We show here that by using an unsupervised bottom-up clustering method based on BLAST E-values, we have been able to construct a global hierarchy of the SwissProt proteins that can be validated by external biological sources, merely by undertaking a global view of the protein space.The four different external sources that were used for validation reflect different aspects of the protein space: InterPro annotation is predictive and is based on various signature detection methods; GO annotation assignments are both based on prediction and from known research, while the GO hierarchy was constructed completely manually; SCOP is a semi-manual classification of structures that is not necessarily reflected in sequence; the ENZYME database supplies Enzyme Commissions, which constitute a hierarchy that is based on the enzymes' chemical reactions. ProtoNet successfully constructs clusters that correspond highly to all four of the sources. Even high levels of SCOP (such as the Fold classification), which are considered to have no detectable sequence similarity, are partially matched . NotablAfter validating the biological relevance of the ProtoNet clusters by using external sources, we examined the hierarchy of ProtoNet. The test showed that the hierarchy presented by ProtoNet significantly corresponds to the manually-constructed biological hierarchy of GO. It is important to note that the method used by ProtoNet is not expected to fully recapture the GO hierarchy due to the fact that ProtoNet is structured as a collection of trees while GO is structured as a DAG. In this sense, the approach of ProtoNet may be limited in the portrayal of evolutionary complexity (as in cases of multiple domains). However by using a domain-based clustering approach, allowing multiple entities of each protein in the hierarchy, a substantial improvement in the CS quality measure may be achieved (unpublished results).An intrinsic parameter that reflects the stability of clusters during the clustering process was used in order to compress the cluster sets, leaving 16.4% of the clusters; removing the singletons clusters as well leaves 12.2% of the clusters. As mentioned above (see Methods), the entire ProtoNet scaffold contains 227,436 clusters that are represented by 630 roots; following this condensation, there are only 27,823 clusters that are represented by 2,236 roots. We show that although a massive portion of the clusters is discarded, very little performance is lost by this condensation process. It is obvious that prior to the condensation process, ProtoNet holds within it both clusters that correctly represent biological groups and clusters that are irrelevant artifacts of the clustering process (e.g. the large root clusters that are constituted of tens of thousands of proteins). Therefore, by allowing a major reduction without significant loss of biological coherence ProtoNet seems to present a more correct view of the protein world.An automatic unsupervised method for the classification of proteins has some important advantages over supervised methods : First, an unsupervised method is unbiased in automatic assignment of function to proteins, a major goal in bioinformatics. Also, it allows high-throughput analysis of whole genomes and enhances understanding of global biological systems without the need for the manual annotation of every protein. Using an automatic annotation method, we are able to successfully annotate 77.8% of the major protein clusters (of size 20 or more) that remain after the compression of the ProtoNet tree. The annotation uses a relatively conservative threshold and therefore yields high-confidence annotations. This further suggests that the clusters remaining after the condensation process are relevant biological clusters and not mere artifacts.One aspect that we have rigorously examined is the robustness of the ProtoNet tree: given a different set of proteins to cluster or a different clustering method, would the resulting tree be significantly different, or are its properties maintained? Interestingly, changing the underlying protein databases , the substitution matrices used for the preliminary BLAST, or the merging strategy  producedProtoNet version 2.4 which was used for the analyses described in this paper is based on classification of the SwissProt database (version 40.28) that contains 114,033 proteins. The entire ProtoNet scaffold contains 227,436 clusters that are contained in 630 trees. Most trees (611) are singletons and only one contains most (>99%) of the proteins. For more details on the construction of the ProtoNet hierarchy see . ProtoNeSeveral external sources were used as a biological reference for validation of the ProtoNet tree: InterPro  is an exWe have used EBI mappings of InterPro and GO to SwissProt proteins.Annotation Score (AS), Correspondence Score (CS), Directed Acyclic Graph (DAG), Enzyme Commission (EC), Gene Ontology (GO), Lifetime (LT).ML conceived of the compression of the ProtoNet tree. NK, MF and MF participated in implementation of the condensation and in the design and implementation of the various tests. All authors participated in the analysis of the results. All authors read and approved the final manuscript.