There has been tremendous interest in the study of biological network structure. An array of measurements has been conceived to assess the topological properties of these networks. In this study, we compared the metabolic network structures of eleven single cell organisms representing the three domains of life using these measurements, hoping to find out whether the intrinsic network design principle(s), reflected by these measurements, are different among species in the three domains of life.S. cerevisiae and the six Bacterial species in almost all measured higher-level topological properties. Our findings also indicate that the metabolic network in Archaeal species is similar to the exponential random network.Three groups of topological properties were used in this study: network indices, degree distribution measures and motif profile measure. All of which are higher-level topological properties except for the marginal degree distribution. Metabolic networks in Archaeal species are found to be different from those in If these metabolic network properties of the organisms studied can be extended to other species in their respective domains (which is likely), then the design principle(s) of Archaea are fundamentally different from those of Bacteria and Eukaryote. Furthermore, the functional mechanisms of Archaeal metabolic networks revealed in this study differentiate significantly from those of Bacterial and Eukaryotic organisms, which warrant further investigation. Classification of biological organisms is of fundamental importance to evolutionary studies. It is commonly believed that there are three domains of life: Archaea, Bacteria and Eukaryote. Currently, the most popular classification method is the so called "molecular approach", in which polymorphism information in DNA or protein sequence is exploited to assess the phylogenetic relationships among species ,2. To a γ function, which measures the similarity between rankings of nodes in two networks. They studied metabolic and information network structures of 43 organisms using these two measures under the hypothesis that network structure and the network design principle(s) behind them contain phylogenetic information. Ma and Zeng [Podani and co-workers  proposedand Zeng  conducteand Zeng . These dRestricted by the theoretical network structural studies, there are not many deterministic and informative topological measurements available -11. The E. coli and S. cerevisiae have been studied intensively in biochemistry, cell biology and genetics; hence the rebuilt networks in those organisms present the best chance to approximate the true underlying network. Moreover, single cell organisms are less likely to have experienced the Whole Genome Duplication (WGD), which might drastically change the network structure [S. cerevisiae; six Bacteria: E. coli, V. cholerae, R. solanacearum, B. subtilis, L. lactis, S. coelicolor; and four Archaea: S. solfataricus, S. tokodaii, M. acetivorans, T. acidophilum.Single cell model organisms such as tructure ,18. As ain vivo pull down assay or DNA microarray, which are subject to high uncertainties, and the resulting networks may not be good approximation to biological complexity [There are three main types of intracellular networks: the protein-protein interaction network, the transcriptional regulation network and the metabolic network. The first two are rebuilt by using high throughput techniques such as yeast two-hybrid system, mplexity -22. On tmplexity . It is wmplexity ,25 and amplexity . The formplexity . Therefo2O, should be removed from metabolic networks. We followed their suggestions by removing such "current metabolites" before conducting the following analysis.In constructing metabolic networks, Ma and Zeng  argued tS. cerevisiae and the six Bacterial species appear much more heterogeneous than Archaeal metabolic networks. It is well known that the so-called exponential random network  appears homogeneous while scale-free network  appears more heterogeneous and modular [Before checking different types of network topological measurements, we visually compared different metabolic networks Fig. . Metabol modular .S. cerevisiae andthe six Bacterial species are more clustered and modular than those in the four Archaeal species  also indicate that the metabolic networks in es Table , Fig. 2.S. cerevisiae and the six Bacterial metabolic networks are much higher than those in Archaeal metabolic networks , see Methods for details) of three-node subgraphs and four-node subgraphs. The concentration of subgraphs is an objective measure of the extent of clustering and modularity of the network ,9. It isrks Fig. .R2 and 95% individual confidence intervals are summarized in Table γ is around -0.3 in all cases and the estimated log-transformed scaling parameter α ranges within 2.0 to 2.5. These indicate that marginal degree distribution, which is a low-level  topological property measure, although showed some distinction, is not enough to effectively differentiate networks from different domains. Overall, metabolic networks in most of the species we studied seem to follow the power-law distributions and thus are "scale-free". The fact that the incoming degree distributions of most Archaeal species we studied do not follow power-law well  topological properties such as average clustering coefficient . In metaN is defined as the number of edges connecting nodes of connectivity K0 to those of connectivity K1. For metabolic networks, which are directed, N is used to measure the number of arches where substrate (node) with out-connectivity Kout transforms to product with in-connectivity Kin. This quantity reflects intrinsic properties of the network and can be used to distinguish different types of networks. For instance, we can test whether N of a particular network differs significantly from that of the random network. To be specific, we calculate , where  represents the mean of random variable N in a large number  of random networks simulated by an edge-rewiring algorithm proposed by Maslov and Sneppen [ denotes the estimated standard deviation of N. The p-value can then be obtained by compare Z to a standard normal distribution. Comparing with "properly" randomized network ensembles allows us to concentrate on those statistically significant patterns of the complex network that are likely to reflect the design principle(s) [Joint degree distribution measures and describes correlation between connectivities of neighboring nodes.  Sneppen ,  .S. cerevisiae and the six Bacterial species. Although the dark red regions of the Z-score profiles in Archaeal species are quite different in scale, they all seem to differ significantly from the random network preserving the corresponding marginal degree distribution in a similar way . Looking into the correlation profiles more carefully, we found that the number of statistically significant positive  increases in the order of S. cerevisiae, the six Bacterial species and the four Archaeal species. The significant Z-score of certain observation N implies that the chemical reaction between substrates with out-degree Kout and products with in-degree Kin are statistically significant. We define substrates whose Kout >= 2 or products whose Kin >= 2 as versatile metabolites. Thus, the above trend implies that the preference to employ reactions involving versatile metabolites increases in the order of S. cerevisiae, the six Bacterial species and the four Archaeal species. Correspondingly, the variety of metabolites decreases in the above order and so does the number of distinct enzymes or variety of enzymes because of the high specific binding of metabolites and enzyme. This is consistent with the biological facts that S. cerevisiae (Eukaryote) encodes a greater variety of enzymes than Bacterial and Archaeal species.We calculated statistically significant correlation profiles  for the metabolic network in each organism Fig. . The Z-sThe network motif is defined to be recurring and non-random building blocks of the network ,15. JustE. coli transcription regulation network are covered by network motifs [E. coli transcriptional regulatory network, "individual motifs aggregate into homologous motif clusters and a supercluster forming the backbone of the network and play a central role in defining its global topological organization." More importantly, network motifs capture the information that is likely to be missed by the correlation profiles because motif actually describes the number of appearances of certain configurations of multiple nodes, and therefore nicely complement with the correlation profiles [Network motif has been shown to be informative of network design principle(s) and network structure. It was found that over 80% of the nodes in the k motifs . Dobrin k motifs  recentlyprofiles . One migS. cerevisiae and the six Bacterial species are identical while there is no three-node motif found in any of the four Archaeal networks  are shared by the latter  to neurology and engineering fields was not found in any of the four Archaeal metabolic networks  motif found in networks from biology  in Archaeal species might also enabled them to survive in those extreme physiological conditions. Archaeal species might employ some biologically significant subgraphs  which can not be detected by current motif searching algorithm [The metabolic networks in Archaea are both "random-like" and "scale-free", which might exert profound influences on their adaptability to the hostile environment. Archaeal species are typically restricted to marginal habitats such as hot springs or areas of low oxygen concentration and can assimilate different kinds of inorganic carbon and nitrogen sources. Indeed, the chemical structure and component of the macromolecules such as protein and lipid make significant contributions to the organism's adaptability to the environment. The seemingly lgorithm . This maOur comparison results showed that many network structural properties measured in Archaeal species are different from those of non-Archaeal species. However, the hidden anthropomorphic factors might account for some of the differences observed. Specifically, the drastic differences of topological profiles between the metabolic networks of Archaeal species and non-Archaeal species may be partially explained by the fact that significantly less extensive metabolic pathway studies have been conducted in Archaeal species . RobustnS. cerevisiae and the six Bacterial species. This provides further evidence that the metabolic network structures and consequently the design principle(s) in the four Archaeal species are very different from those in S. cerevisiae (Eukaryote) and the six Bacterial species. Our finding that the metabolic networks in Archaeal species possess many properties of the exponential random network begs for better understanding of the design principle(s) in biological networks, which may be revealed by further systematic analyses. For example, locate and align conservative pathways such as glycosis between E. coli or S. cerevisiae and Archaeal species to understand the functional mechanisms of Archaeal metabolic networks.Our network analysis results showed that in most of higher-level  topological properties measured, metabolic networks in the four Archaeal species are similar to each other but significantly different from those in reaction, enzyme, react, connect and organism. We compiled a new table from this database excluding any inconsistent or redundant connections between metabolites (details below). SQL was used to query the database.Chemical reaction data was obtained from metabolic database in Ma and Zeng , which c+ and Nicotinamide is a pair of metabolites in two reactions: 1) NAD+ + L-Arginine = Nicotinamide + N2 (ADP-D-ribosyl)-L-arginine 2) NAD+ + H2O ->Nicotinamide + ADPribose. . Reaction 1 is a reversible reaction while reaction 2 is not. We annotated an edge between the two metabolites as long as there was at least one reversible reaction that both of them were involved. For example, the type of connection between NAD+ and Nicotinamide is edge (undirected connection). This step could be summarized as "edge ← edge + arc".Inconsistent connections refer to pairs of metabolites that have conflicting reversibility annotation. It is caused by the fact that a pair of metabolites can be in more than one reaction and the reversibility of these reactions can be different. For example, NADN-Acylsphingosine = UDP + Glucosylceramide 2) Glucosylceramide + H2O = D-Glucose + N-Acylsphingosine. (N-Acylsphingosine and Glucosylceramide is a pair of metabolites that switch their roles in two irreversible reactions). In case of redundancy, we annotated an edge between the pair of metabolites rather than the two arcs because they could be converted to each other through two reactions. This step could be summarized as "edge ← arc + arc".There are also numerous redundant connections where the same pair of metabolites switch their roles between substrate and product in two or more different irreversible reactions. For example: 1) UDPglucose + Cin and Cout. Cin measures the average clustering coefficient of the node representing the product that can be generated from its first-order "nearest neighbors" through chemical reactions. Cout measures the average clustering coefficient of the node that generate its first-order "nearest neighbors" through chemical reactions. The larger the coefficients, the more clustered and modular the network appears to be.We define two kinds of clustering coefficients for each node in the directed metabolic networks, i.e. ni in the network is defined as , where gjk is the number of shortest paths between node j and node k. gjk(ni) is the number of shortest path between node j and node k containing node ni, g is the total number of nodes with edges/arcs. CB(ni) needs to be multiplied by two in the case of directed network [. Higher value of betweenness indicates the network is more clustered and modular.The betweenness for any node  network . The ave, where d is the shortest path length between node j and node k (distance), V represents the set of all nodes with edges/arcs of the graph, and g is the number of nodes with edges/arcs.Watts and Strogatz  defined G is the longest geodesic between any pairs of nodes. The geodesic is the shortest path between a pair of nodes. Pajek [The diameter of the directed graph s. Pajek  was usedGs is a subgraph of G if the set of nodes of Gs is a subset of the set of nodes of G, and the set of lines in Gs is a subset of the lines in the graph G. Let M be the number of subgraphs, and N be the number of nodes with edges or arcs. Then the "concentration of subgraph" is defined as C = M/N. A high value of C indicates the network is more clustered and modular. Mfinder1.1 [M and N.Wasserman and Katherine  defined inder1.1  was usedA, a matrix of 0 or 1. Zero means there is no connection between nodes, and 1 the opposite. The outgoing degree of the node i, kout(i) is defined as , where . The incoming degree of the node i, kin(i) is defined as .The marginal degree distribution of each network is calculated from the Boolean adjacency matrix P(Ki) = γ log(Ki) + log(α) + εi , γ and α are parameters, εi is the residual. Ki is the degree and P(Ki) is the corresponding probability. Based on the fitted linear model, we made statistical inference including parameter estimation and individual confidence intervals on the estimates using the Least Square method.The power-law degree model was first log transformed into linear model, i.e. log Statistically significant correlation profiles were calculated using Matlab code downloaded from Dr. Maslov's website . The adjp-value is smaller than a pre-specified threshold, e.g. 0.01. 2) The number of appearances in real networks with distinct sets of nodes is larger than another pre-specified cut-off value, e.g. 4. 3) The number of appearances in real networks is significantly larger than that in randomized networks, i.e. . Nreal and Nrand represent the number of certain subgraphs detected in real-life network and randomized networks, respectively. This is to avoid the situation where some common subgraphs are detected as motifs that have only slight differences in Nreal and Nrand but have a narrow spread of distribution in randomized networks [According to Milo et al. , a subgnetworks ,15. Motinetworks .DZ and ZSQ conceived and designed the study; DZ wrote the computer code, analyzed the data and draft the manuscript. Both authors read and approved the final manuscript.Four-node motifs found in the metabolic networks in different species. The number of connecting nodes for each network is shown. For each motif, the numbers of appearances in real networks  and in randomized networks  are shown. The p-values of all motifs are less than 0.01, as determined by comparing to 1000 randomized networks. Each motif occurs at least four times in one network. Other restrictions apply. Motifs were detected and generated using program found in Milo et al. [o et al.  and the Click here for file


Practice management education continues to evolve, and little information exists regarding its curriculum design and effectiveness for resident education. We report the results of an exploratory study of a practice management curriculum for primary care residents.After performing a needs assessment with a group of primary care residents at Wright State University, we designed a monthly seminar series covering twelve practice management topics. The curriculum consisted of interactive lectures and practice-based application, whenever possible. We descriptively evaluated two cognitive components (practice management knowledge and skills) and the residents' evaluation of the curriculum.The mean correct on the knowledge test for this group of residents was 74% (n = 12) and 91% (n = 12) before and after the curriculum, respectively. The mean scores for the practice management skill assessments were 2.62 before (n = 12), and 3.65 after (n = 12) the curriculum . The residents rated the curriculum consistently high.This exploratory study suggests that this curriculum may be useful in developing knowledge and skills in practice management for primary care residents. This study suggests further research into evaluation of this curriculum may be informative for practice-based education. Practice management education for residents has traditionally included training physicians in management issues related to the practice environment, including fiscal management, leadership skills, business and management skills, and managed care concepts . ManagedA few studies on practice management curricula exist for resident education, but much more information is needed on successful curriculum design and evaluation -13. We dWe developed the curriculum for the University Medicine/Pediatrics Practice (UMP). This practice is a primary care-oriented, faculty-resident practice on the campus of Wright State University. Thirteen internal medicine/pediatrics residents, five general internist faculty, two internal medicine/pediatrics faculty, and one pediatrics faculty practice here. The practice is managed by Premier Healthnet, a 100 physician multi-site primary care group. Although UMP's mission includes addressing the needs of indigent patients in the Dayton area, it is modeled after a community-based, teaching practice model. Therefore, faculty and residents are expected to use effective practice management skills in their individual practices. A typical resident from our program enters a small (1–5 physician) community practice upon graduation and practices both internal medicine and pediatrics.In 2001, two faculty members (GEC and RJS) at the Wright State University Departments of Medicine and Community Health were identified as lead faculty for this curricular project. Primary care faculty in the Departments of Pediatrics and Medicine tasked these two lead faculty members to development a practice management curriculum to reflect the evolving practice theme. By "evolving practice theme," we mean teaching practice management topics similar to the Tuft's curricular theme.One lead faculty member (GEC) performed a needs assessment on the Internal Medicine/Pediatrics residents at UMP in the spring of 2001. The assessment used qualitative analysis via informal interviews of two senior residents, one of whom was chief resident. The interviews included open-ended questions on the need for practice management knowledge (example question: "What do you need to learn this year to help prepare you for running a community practice?"). The needs assessment also included an open feedback session with the residents after discussion of potential topics at the monthly resident education meeting (majority of residents present).The results of the need assessment were uniform; the residents felt inadequately trained in practice management. The lead faculty concluded that these residents had some training in a few specific content areas , but lacked an overall basic practice management knowledge or skill.The lead faculty met again in mid-2001 to design the curriculum. The goals of the curriculum were to give the residents a basic understanding of practice management concepts and skills in the evolving practice environment. The lead faculty were free to select the most effective methods to meet their goals. They did face some challenges. They were given only 30-minute time slots each month and had 12 months to accomplish these goals. They also had to show some objective evidence of its effectiveness and have support of the residents at the end of the 12 months to continue the curricular project.The design process resulted in a series of seminars covering 12 topics, listed in Table The respondents were a convenience sample of Internal Medicine/Pediatrics residents from all four years of training. We used a pre-experimental (one-group pretest/posttest) design for this exploratory study. We descriptively evaluated the curriculum on two cognitive components: practice management knowledge and skills. We also assessed the residents' evaluation of the curriculum.To evaluate practice management knowledge, we used a knowledge test consisting of identical 12 item , and each question covered one objective from each topical area from Table To evaluate practice management skills, we devised a survey of self-assessed practice management skills. The survey consisted of 12 statements, and each statement queried the residents to respond on their assessment of their own practice management skills. Each statement consisted of one specific skill from an objective from each topical area listed in Table To explore the residents' evaluation of the curriculum, we devised another survey. This survey consisted of four statements querying the residents on their overall assessment of this curriculum and practice management education in general. The statements from the survey are given in Table The process of test instrument development was the same for both the knowledge test and the self-assessed skills survey. One lead faculty member (GEC) would generate a list of candidate items based on each objective in Table th-year; 3, 3rd-year; 4, 2nd year; and 4, 1st-year residents). A third year resident failed to complete the 0-month tests and surveys, and a first year resident failed to complete the 12-month tests and surveys. This left 12 responses for both sets (0- and 12-month) tests and surveys. The average attendance for the sessions was 12, with a range of 10–13 attendees.The participants were the 13 Internal Medicine/Pediatrics residents, and represented all four years of training .Second, we were interested if the missing data on the 0-month and 12-month data could have impacted the results. Since one third-year resident completed the 12-month but not the 0-month test, we were interested in exploring if his responses on the 12-month test could have caused a larger difference between these two tests. After censoring his data, there appeared to be little impact on the 12-month results . Additionally, the first year resident who failed to complete the 12-month test may have also impacted the results. Due to loss of identity links, we could identify her data to censor from the 0-month test. However, we censored the lowest score on the 0-month test as representing hers . After censoring this data, we found no significant change in the 0-month results .The results for the self-assessed skill survey are given in Table The results of the curriculum evaluation survey are given in Table The practice environment continues to evolve . AlthougWe were interested in whether a curriculum design with this evolving theme may be useful in primary care education. We describe, in detail, a curriculum design similar to the evolving theme designed for a small group of primary care residents. The advantage of such a program as ours is its detailed design based on general and specific needs assessments and a description of evaluation methodologies. Our data suggests that this intervention may have had an impact on resident knowledge scores and self-assessed skills. Additionally, the residents appeared remarkably positive towards this practice management curriculum and practice management education in general.A few studies have been published on practice management curricular design and evaluation for primary care residents. In a response to the growing need physician-managers, both Zoorob and Taylor and Johnson described curricular designs they proposed would fill this need ,14. LyncOur study does have some limitations. Because our small sample size, formal hypothesis testing was not possible and our data remains descriptive only. Stronger conclusions of these results would need to be re-evaluated with more subjects using formal hypothesis testing methods. Our experience suggests that internal motivation was probably one key factor to acceptance and apparent acceptance of this curriculum; the request for developing the curriculum came from our residents themselves. Also, the UMP faculty is uniformly positive towards developing these skills in themselves and in the residents, and this probably influenced residents' motivation to learn the subject matter. Since our faculty-resident practice is based on a primary care, community model, it may be difficult to generalize it to hospital-based practices or specialty residency training.We conclude that an extended curriculum in practice management with an evolving practice theme may be useful in primary care education. We also believe that attention to instructional design, including performing a needs assessments, using many teaching methods, and applying the concepts learned in learners' practices, may contribute to its acceptance and success. Future educational designs for this curriculum include its continued expansion, exploring more educational opportunities for implementation, and addressing specific characteristics of success and failure. Future educational research in this area would require a more formal research design to derive stronger conclusions regarding its effectiveness.None declared.GEC participated in the curricular needs assessments, curricular design, curriculum implementation, and drafting of the manuscript.RJS participated in the curricular design, curriculum implementation, and drafting of the manuscript.The pre-publication history for this paper can be accessed here:


To study the cellular distribution of CCN3(NOV) and to determine if the carboxyterminus of CCN3 is hidden or masked due to high affinity interactions with other partners. CCN3 was detected using affinity purified antibodies (anti-K19M-AF) as well as a Protein A purified anti-K19M antibodies (anti-K19M IgG) against a C-terminal 19-aminoacid peptide (K19M) of human CCN3 protein. The antibodies were applied in indirect immunofluorescence tests and immunoenzyme assays on glial tumor cell line, G59, and its CCN3-transfected variant G59/540 and the adrenocortical cell line, NCI-H295R.Anti-K19M-AF antibodies reacted against K19M peptide in ELISA and recognized two bands of 51 kDa and 30 kDa in H295R  cell culture supernatants by immunoblotting. H295R culture supernatants which contained CCN3 as shown by immunoblotting did not react with anti-CCN3 antibodies in liquid phase. Anti-CCN3 antibodies stained the surface membranes of non-permeabilized H295R and cytoplasm in permeabilized H295R cells. Similarly, anti-CCN3 stained surface membranes of G59/540, but did not react with G59 cells. Prominent cytoplasmic staining was observed in G59/540, as well as the cell footprints of G59/540 and H295R were strongly labeled.The K19M-AF antibody directed against the C-terminal 19-aminoacid peptide of CCN3 recognized the secreted protein under denaturing conditions. However, the C-terminal motif of secreted CCN3 was not accessible to K19M-AF in liquid phase. These anti-CCN3 antibodies stained CCN3 protein which was localized to cytoplasmic stores, cell membranes and extracellular matrix. This would suggest that cytoplasmic and cell membrane bound CCN3 has an exposed C-terminus while secreted CCN3 has a sequestered C-terminus which could be due to interaction with other proteins or itself (dimerization). Thus the K19M-AF antibodies revealed at least two conformational states of the native CCN3 protein. The CCN3 protein belongs to an emerging family of growth regulators referred under the CCN acronym  .The CCN3 (NOV) gene had been initially characterized as an integration site for the myeloblastosis associated virus MAV  which inThe CCN proteins share a strikingly conserved multimodular organization with distinctive functional features . From thThe multimodular structure of CCN3 and other CNN proteins raises interesting questions as to participation of each individual module in conferring the biological properties to the full length proteins. Either the biochemical functions of the individual IGFBP, VWC, TSP and CT modules are indeed conserved and in sum determine the ultimate function of the full length protein, or each module confers on the whole protein specific biological functions which may vary from the conserved function, and either substitute or add to those of individual modules.Application of the yeast two-hybrid system and co-precipitation strategies to identify proteins interacting with CCN3 has revealed that full length CCN3 interacts with several receptors, signaling molecules, and proteins of the extracellular matrix (16–19), suggesting functional involvement of CCN3 in cell signaling and adhesion regulation.Our results also established that truncated isoforms of CCN3 could bind specific targets and pointed the CT domain of CCN3 as a critical determinant for protein interaction. This led us to hypothesize that truncated isoforms of CCN3 could also modulate its biological activity (3). The question therefore arises whether different conformational states exist due to multiple protein interactions and thereby the presentation of known antigenic epitopes.In the present study we have used an immunological approach to establish the cellular distribution of CCN3 in cell lines representing adrenocortical and glioblastoma tumors and to ask whether the CT module of CCN3 exists in different conformational states depending on its involvement in protein interactions and cellular location. We now provide evidence that the CT end of CCN3 exists in more than one conformational state.Cell culture supernatants and cellular lysates from the H295R, G59/540, and parental G59 cell lines were electrophoresed under denaturing conditions and immunoblotted with anti-K19M IgG antibody. Immunoblot analysis revealed secreted forms of CCN3 for the H295R and G59/540 cell lines, consisting of two distinct bands at 51 kDa and 30 kDa Figure . The latWhen tested in ELISA, pre-incubation of the anti-K19M-AF antibodies with CCN3-containing H259R supernatant did not affect the binding of anti-K19M-AF antibodies to the K19M peptide coated on microtitre plates .Paraformaldehyde fixed, non-permeabilized H259R cells treated with the anti-K19M antibody (Protein A purified) exhibited immunofluorescent membrane specific staining distributed over the cell surface Figure.  while siIn summary, the results produced using an immunological approach would suggest multiple conformations of the C-terminal end which harbors the immunogenic epitopes. Furthermore, these variations are associated with cell bound and secreted forms of CCN3. Cytoplasmic localization indicated abundant CCN3 protein localized with ER and Golgi networks.In this study we exploited the range of binding affinities present in polyclonal antibodies raised against the C-terminal peptide of CCN3 and analyzed with different immunoaffinity methods to ask whether CCN3 exists in alternate conformational forms in cell cultures and supernatants. Whereas immunocytochemistry of fixed, permeabilized and non-permeabilized cells yielded evidence of both cell surface membrane and cytoplasmic expression and topographical distribution of native CCN3, ELISA method and western immunoblot revealed different possible conformational forms of CCN3. Taken together the results of the assays suggest that native CCN3 assumes different configurations that either expose or sequester the C-terminal peptide depending on whether CCN3 is cell associated or free within the culture supernatant. When considered in the light of recent evidence indicating that CCN3 can associate with specific integrins at the cell membrane ,24 the qin vitro that CCN3 is sequestered in cell footprints representing a secreted extracellular matrix. Cell footprints can be deposited by various cell types in different arrangements and consists of extracellular matrix, including a variety of basement membrane proteins  maintaining cell side up. Cells were further permeabilized in 0.5% Triton X-100 in PBS for 15 min and then blocked in 5%FBS/PBS for 30 min.Anti-K19M IgGs antibodies were applied at 1:1000 dilution in 5%FBS/PBS for 1 hour with intermittent rotation, followed by 5 washes in PBS containing 0.1% Tween 20. Subsequently, the cells were incubated in anti-rabbit IgG serum conjugated with either Alexa 488 (green fluorescence) or Alexa 594 (red fluorescence), in 5%PBS/BSA for 1 hour. After final washes in PBS/Tween 20 followed by one wash in PBS cells were mounted with antifade mounting medium , excess liquid adsorbed with filter paper, and coverslips sealed with clear nail polish. Immunofluorescence images were captured on 400 ASA film and processed further with Adobe Photoshop (version 7.0).None.None declared.SK carried the affinity chromatography and immunoenzyme experiments;HY carried out immunofluorescence labeling experiments;AMB carried out IgG purification and Western blots;BP conceived the study design and coordinated and edited the manuscript


Erythroleukemia K562 cells show narrow signals at 1.3 and 0.9 ppm, corresponding to mobile lipids , which are reduced when K562 cells are multidrug resistant (MDR). While the significance of the mobile lipids is unknown, their subcellular localization is still a matter of debate and may lie in the membrane or the cytoplasm. In this study, we investigate the role of cholesterol in the generation of mobile lipid signals.Most cancer cells are characterized by mobile lipids visible on proton NMR  cell line and its MDR counterpart (K562adr), using methyl-β-cyclodextrin, was accompanied by a rise of mobile lipids in K562wt cells only. The absence of caveolae was checked by searching for the caveolin-1 protein in K562wt and K562adr cells. However, cholesterol was enriched in another membrane microdomain designated as "detergent-insoluble glycosphingomyelin complexes" or rafts. These microdomains were studied after extraction with triton X-100, a mild non-ionic detergent, revealing mobile lipid signals preserved only in the K562wt spectra. Moreover, following perturbation/disruption of these microdomains using sphingomyelinase, mobile lipids increased only in K562wt cells.These results suggest that cholesterol and sphingomyelin are involved in mobile lipid generation via microdomains of detergent-insoluble glycosphingomyelin complexes such as rafts. Increasing our knowledge of membrane microdomains in sensitive and resistant cell lines may open up new possibilities in resistance reversion. When studied by NMR proton spectroscopy, most cancer cells are characterized by increased narrow signals at 0.9 and 1.3 ppm corresponding, respectively, to methyl and methylene resonances that belong to lipid acyl chains moving isotropically. This so-called "mobile lipid signal"  has been studied for several decades [(K562adr), whereas(K562adr). Thus, a(K562adr), we showIn vivo, cellular-free cholesterol is located in the plasma membrane [In model membranes, free cholesterol interacts with phospholipids and sphingolipids to influence membrane fluidity . In vivomembrane , which emembrane , LM and membrane ,14. Moremembrane .The aim of this study was to investigate a possible contribution from the cholesterol-containing membrane domain to the ML detected in the NMR proton spectra of wild-type K562 and K562 adriamycin-resistant cells.In a first set of experiments, cell membrane cholesterol was extracted by methyl-β-cyclodextrin (MCD). MCD is an oligosaccharid able to pump cholesterol out of the cell . MoreoveK562wt and K562adr cells do not differ in their total cholesterol contents. The proportion of cholesterol-ester were found to be lower in the resistant cells than in the wild type cells Table . This imThe cell proliferation results for K562wt and K562adr are presented in Figs. When K562adr cells were resuspended in R10 containing daunorubicin (0.25 nM), they were able to proliferate at this daunorubicin concentration. However, after treatment with MCD, the K562adr proliferation rate was decreased as shown in figure Caveolin-1 expression was studied in K562 cells by SDS-page and western-blotting. Caveolin-1 is a protein of 21 kD . As a po3/Ct and CH2/Ct ratios in MCD-treated K562wt cells compared with non-treated cells.Figure 2/Ct ratio was increased for K562wt cells and, to a lesser extent, for K562adr cells. N-trimethyl to creatine peak ratios were decreased in both cell lines. After treatment of these microdomains with sphingomyelinase, the spectra showed an increased CH2/Ct ratio compared with the previous spectrum obtained on "DIGCs" in K562wt cells. However, the spectra of K562adr cells treated with Smase remained unchanged compared with spectra obtained on DIGCs from K562adr cells.spectra obtained after treatments with PFA, triton and sphingomyelinase are presented in Figures We studied the modification of cell spectra using cholesterol and sphingomyelin because: i) cholesterol levels affect membrane fluidity; ii) membrane microdomains, rafts and caveolae are all enriched in these lipids. Western-blot did not detect the presence of caveolin-1 in either K562wt or K562adr. This confirms previous results showing the absence of caveolin-1 in K562wt . Even ifRafts have been described as corresponding to membrane microdomains enriched in cholesterol and sphingomyelin lipids . In our Nevertheless, we observe that MCD treatment affected the K562adr cell line. This is demonstrated by the reversion of daunorubicin resistance. In the plasma membrane, K562adr cells express the P-gp glycoprotein responsible for the extrusion of drugs. When K562adr cells were treated with MCD, the resistance to daunorubicin was decreased, showing that the P-gp is unable to extrude daunorubicin from the cells. P-gp has been widely reported to be sensitive to the lipid content of plasma membrane . The preThis first set of experiments suggests that ML signals are generated during the disruption of membrane microdomains in K562wt cells, because MCD has been shown to extract cholesterol preferentially from membrane microdomains . OverallIn a second set of experiments, we isolated rafts using triton X-100 at 4°C, a mild detergent in which they are insoluble . We applIn the final set of experiments, we incubated detergent insoluble fractions with sphingomyelinase, which was supposed to metabolise the sphingomyelin included in the rafts. Only the K562wt spectra exhibited an increase in ML signal compared with the raft spectra. Again, this suggests that microdomains are differently organized between K562wt and K562adr. Several hypotheses may be proposed to explain the difference between K562wt and K562adr: i) sphingomyelin concentrations are higher in K562adr; ii) lipid microdomains in K562adr may be organized in such a way that sphingomyelin is inaccessible to sphingomyelinase and iii) there are more microdomains in K562adr, as suggested previously by some authors .These results suggest that, while isolated rafts generate ML signals within the membrane, the presence of rafts seems to maintain a relatively tight organization. When this organized structure is disrupted by MCD or sphingomyelinase, the ML signal may be detected in K562wt cells and the P-gp function can then be modulated in K562adr cells. Taken together, the results suggest that ML signals may be sensitive to modifications in the lipidic organization of the cells and membrane, in which microdomains behave as rafts enriched in cholesterol and sphingomyelin. A better knowledge of these microdomains, as well as the differences in behaviour between sensitive and resistant cells, could open up new therapeutic perspectives for reversing the drug-resistance phenotype.2O) and all chemicals for western-blot analysis were provided by Sigma-Aldrich . Triton X-100 was supplied by Merck , and methanol by Acros Organics .Methyl-β-cyclodextrin, sphingomyelinase, cholesterol, chloroform, isopropanol and paraformaldehyde, as well as deuterium oxide (D2. The K562 resistant variant cells (K562adr) were a gift from F. Calvo, Université Paris 7, and were grown in the same medium with 0.5 μg/ml adriamycin.Human erythroleukemia K562 cells were grown in the culture medium RPMI 1640  supplemented with fœtal calf serum (FCS) (10%) and glutamine (2 mM) (R10 medium) at 37°C and 5 % COLipids were extracted as previously described , and the2. Controls were incubated in RPMI, glutamin 2 mM, with or without FCS 10% for 2 hours.Cells were incubated in RPMI, glutamin 2 mM, containing methyl-β-cyclodextrin 5 mM for 2 hours at 37°C, 5% CO2. Proliferation was assessed by counting cells with a cell coulter  at 24, 48 and 72 hours after seeding.After incubation with methyl-β-cyclodextrin, with RPMI+ FCS or with RPMI , cells were collected, washed and resuspended at 150,000 cells/ml in RPMI, glutamin 2 mM, FCS 10%. Cells were seeded in triplicate on 96-well microplates and incubated at 37°C and 5% CO3 0.1 M; MgCl2, 6H2O 1 mM) with tetrazolium nitro-blue 3% in N,N-dimethylformamide 70% and bromo-4-chloro-3-indolylphosphate p-toluidine 1% in N,N-dimethylformamide 70% for 30 minutes at room temperature in the dark.Cells were collected in late log-phase and washed twice in PBS 150 mM and were resuspended in loading buffer  for 5 mn at 95°C. They were centrifuged at 12500 g, 4°C for 10 minutes, and 50 μg of extracted protein were deposited per well on an 8% acrylamide gel. Migration was performed in an electrophoresis buffer  for 1 hour at 125 V with a size marker . Protein was transferred on a nitrocellulose membrane for 2 hours at 80 V and 4°C in a transfer buffer . Non-specific sites were blocked with 1% milk  in TBS 1X . The nitrocellulose membrane was incubated for 1 hour at room temperature with 1 μg/ml anti-caveolin 1 rabbit antibody  and washed three times for 10 minutes with TBS before incubation for 1 hour at room temperature with goat anti-rabbit and conjugate secondary antibody (1:3000). After three washes in TBS for 10 minutes, staining was developed in a phosphatase alkaline buffer  3% in PBS 150 mM for 30 minutes at 4°C and washed twice with 1 ml PBS 150 mM at 4°C to be resuspended in triton X-100 1% in PBS 150 mM at 4°C for 30 minutes. Cells or triton-insoluble fractions were collected and washed for NMR experiments at 25°C.After triton treatment, cells were collected, washed with PBS 150 mM and treated for 35 mn at 37°C with 0.5 units of sphingomyelinase. Cells were washed as described for NMR experiments at 25°C.2O, centrifuged at 250 g and counted. Then, 107 cells were resuspended in 400 μl PBS/D2O before transfer to a 5-mm Shigemi NMR tube. Experiments were performed without rotation, and the ?analysed cell pellet was maintained in the coil volume in the Shigemi NMR tube.Cells were washed twice in 1 ml PBS 150 mM, twice in PBS/D3 at 0.9 ppm), methylene group (CH2 at 1.3 ppm), choline N-trimethyl group (N+(CH3)3 at 3.2 ppm) and creatine (CH3 at 3.05 ppm). The values obtained for the different treatments of the two cells lines were compared by ANOVA analysis followed by a Student-Neumann-Keuls test for group-to-group comparison. P < 0.05 was considered as a significant value.The NMR proton spectra of whole cells were obtained at 25°C on a Unity Inova spectrometer  working at 500 MHz. One-dimensional runs were performed by accumulating 128 transients of 90° pulse with 2 s relaxation time. The signal from the residual water was suppressed by the presaturation technique, by using 0.03 mW irradiation for 2 s. Acquisition time was 1.34 s on 16K data points, corresponding to a spectral width of 6 kHz. The Fourier transform was applied without zero-filling using an exponential window multiplication function corresponding to 1 Hz line broadening. The resonances were integrated after automatic baseline correction. Two-dimensional COSY runs were performed with 2K data points in the F2 direction and 256 data points in the F1 direction. The two-dimensional Fourier transformation was applied after zero filling to 512 data points in the F1 direction with a sine-bell function in both directions. Each run consisted of a one-dimensional acquisition and a two-dimensional COSY spectrum. Peak assignments were based on data from the literature. The peak areas were measured by manual integration with the Bruker WINNMR software using a manual tangential baseline correction for each peak, and the assigned peak areas were normalized to the creatine peak area. The following resonances were integrated: methyl group . The runs consisted of a 1D acquisition with similar parameters as the previous 1D spectra, and the spectrum analysis was performed as previously with Bruker WINNMR software.2 peak area. As both groups belong to fatty acyl chains, this ratio remained constant since lactate did not significantly contribute to the increase of the CH2 signal [The possible contribution of lactate to the 1.3 ppm signal was eliminated by analysing 2D Cosy spectra in whole cells, which resolved the resonances of lactate from fatty acid chains. In fixed cells, we calculated the ratio of the double-bond signal (CH = CH at 5.4 ppm) to CH3: methyl; CH2: methylene; N(CH3)3: N-trimethyl; Ct: creatineNMR: Nuclear Magnetic Resonance; MDR: multi-drug resistance; ML: mobile lipid; K562wt: K562 wild type; K562adr: K562 adriamycin-resistant; MCD: methyl-β-cyclodextrin; DIGC: detergent-insoluble glycosphingolipid complex; FCS: fœtal calf serum; R10: RPMI culture medium with 10% FC; PBS: Phosphate buffer saline; TRIS: Tris(hydroxymethyl)-1,3-propanediol; TBS: TRIS buffer Saline; PFA: paraformaldehyde; COSY: correlated-spectroscopy; CHall authors contributed equally to this work


Saccharomyces cerevisiae. This complex was designated SWR1-Com in reference to the Swr1p subunit, a Swi2/Snf2-paralog. Swr1p and six other subunits were found only in SWR1-Com, whereas six other subunits were also found in the NuA4 histone acetyltransferase and/or the Ino80 chromatin remodeling complex. H2A.Z and SWR1 were essential for viability of cells lacking the EAF1 component of NuA4, pointing to a close functional connection between these two complexes. Strikingly, chromatin immunoprecipitation analysis of cells lacking Swr1p, the presumed ATPase of the complex, revealed a profound defect in the deposition of H2A.Z at euchromatic regions that flank the silent mating type cassette HMR and at 12 other chromosomal sites tested. Consistent with a specialized role for Swr1p in H2A.Z deposition, the majority of the genome-wide transcriptional defects seen in swr1Δ cells were also found in htz1Δ cells. These studies revealed a novel role for a member of the ATP-dependent chromatin remodeling enzyme family in determining the region-specific histone subunit composition of chromatin in vivo and controlling the epigenetic state of chromatin. Metazoan orthologs of Swr1p  may have analogous functions.The conserved histone variant H2A.Z functions in euchromatin to antagonize the spread of heterochromatin. The mechanism by which histone H2A is replaced by H2A.Z in the nucleosome is unknown. We identified a complex containing 13 different polypeptides associated with a soluble pool of H2A.Z in  The histone variant H2A.Z localizes specifically to euchromatin and prevents spreading of silent chromatin into adjacent euchromatin. This study shows that the chromatin-remodeling complex SWR1-Com helps deposit H2A.Z onto euchromatin in yeast Saccharomyces cerevisiae Swi2/Snf2 protein, originally identified for its role in promoting transcription.Histones are the major constituent of chromatin and exert a profound influence on most if not all aspects of chromosome behavior. The functional state of chromatin is regulated, in part, by histone modifying enzymes and ATP-dependent chromatin remodeling enzymes. Members of the latter enzyme class alter the structure of nucleosomes or slide them along DNA in vitro  is essential for the assembly of the kinetochore  and the Ino80-C chromatin remodeling complex.Even less is known about the mechanisms of deposition of variant histones. Understanding the mechanism by which euchromatin that contains H2A.Z is formed requires the identification of the machinery that catalyzes its deposition. The results of this study identify a multisubunit protein complex, SWR1-Complex (SWR1-Com), which contains a Swi2/Snf2 paralog and is required for H2A.Z deposition and function in S. cerevisiae  tag  are also subunits of NuA4 . ConsistThe initial purifications suggested that Swc4p and Bdf1p, both of which have domains that are involved in recognition of histone tails, copurified with H2A.Z-TAP and might be part of SWR1-Com. Independent assessment of the composition of the complexes deduced by mass spectrometry was obtained by analytical-scale affinity purifications of Yaf9-TAP, Esa1-TAP, Rvb2-TAP, Swr1-TAP, and Ino80-TAP from cells containing a version of Swc4p that was fused at its carboxyl-terminus to a triple hemagglutinin (HA) tag. These analytical-scale affinity purifications were more stringent than the initial TAP purifications and therefore served to eliminate false-positive results and to provide independent tests of interactions. Anti-HA epitope antibodies and antibodies against Tra1p, the largest subunit of NuA4, were used to analyze the copurified material. Both Yaf9-TAP and Esa1-TAP associated with comparable amounts of Tra1p and Swc4p, supporting the assignment of Yaf9p and Swc4p as new subunits of NuA4. Likewise, Rvb2-TAP and Swr1-TAP copurified with a substantial amount of Swc4-HA, but Ino80-TAP did not. Rvb2-TAP, Swr1-TAP, and Ino80-TAP were not associated with Tra1p A. These The number of peptides corresponding to Bdf1p in the TAP purifications was low, and Bdf1p was found only in the H2A.Z-TAP and the Yaf9-TAP . Bdf1p'sTo determine whether subunits of SWR1-com associated specifically with H2A.Z or both H2A.Z and H2A, TAP-tagged versions of H2A.Z and H2A were purified from cells containing HA-tagged versions of the five different SWR1-Com components Swr1p, Swc2p, Swc3p, Swc4p, and Swc7p, and the nuclear import factor Kap114p. The composition of the copurifying material was then evaluated by immunoblotting with antibodies against the HA tag, Bdf1p, and Tra1p. Yaf9-TAP served as a positive control for recovery of SWR1-Com and NuA4.H2A.Z associated with a substantial fraction of the SWR1-Com as judged by the comparable intensity of the signal for SWR1-Com subunits in the material copurified with H2A.Z-TAP and Yaf9-TAP, whereas no NuA4 copurified with H2A.Z-TAP based upon the absence of Tra1p in the H2A.Z-TAP sample A. In conCanonical H2A that is not bound to chromatin is usually found in a H2A/H2B dimer . The preswr1Δ cells were compared to the profiles of htz1Δ cells  Swr1p-dependent genes were within 20 kb of a chromosome end, which is less than 3% of the genome  and even more impressive for telomere-proximal genes. These data suggested that Swr1p and H2A.Z shared a common function in regulating gene expression.Comparison of the transcript profile across the genome of  overlap A. Ninetyin htz1Δ A. This rswr1Δ and htz1Δ datasets grouping the genes described in swr1Δ cells but were not reduced enough to meet the stringent significance cutoff. However, there also were clear examples of genes that required H2A.Z for expression but not Swr1p. Likewise, there were clear examples among the 94 genes that required Swr1p for expression but did not require H2A.Z  did not appear to require Swr1p for expression. A color representation of the re H2A.Z .The evidence linking H2A.Z and Swr1p function and the association of both H2A.Z and H2B with SWR1-Com suggested that SWR1-Com was responsible for depositing H2A.Z onto chromatin in vivo, perhaps in the form of an H2A.Z/H2B dimer. . Consistent with H2A.Z being in a stable complex with Swr1p that protected it from protein degradation, the level of HA3-H2A.Z in swr1Δ strains was reduced approximately 2- to 3-fold (PRP8 open reading frame [ORF]) in samples derived from each whole cell extract and precipitate  experiments comparing wild type to o 3-fold . To normtate see . We firssome XIV A. In wilsome XIV B; HA3-H2 regions B. In addswr1Δ cells, the enrichment (relative to the PRP8 locus) of HA3-H2A.Z at every locus tested approached a ratio of one . Thus, Swr1p was broadly required for the deposition of HA3-H2A.Z, even in regions distant from silenced domains. It is worth noting that for several of the loci examined in the swr1Δ mutant, the ChIP enrichment was significantly less than one, suggesting that there may exist some residual deposition of HA3-H2A.Z at the PRP8 locus in these cells.H2A.Z is also deposited at several loci that are not near silenced regions . The funYDR359C was a subunit of NuA4, consistent with some earlier results from high-throughput studies  and the microtubule poison benomyl and to caffeine and formamide, reagents that elicit a number of cellular responses . Whereas Swc4p was shared with SWR1-Com, Eaf1p was not. However, cells lacking EAF1 were sensitive to DNA-damaging drugs and genotoxic stress conditions, as were cells lacking subunits of SWR1-Com and H2A.Z. While NuA4's involvement in DNA damage survival was known . Crude extracts were prepared by centrifugation in a SS34 rotor for 20 min at 14,000 rpm. These were then further clarified by ultracentrifugation . NP-40 was added to a final concentration of 0.15%, and the extract was incubated with 200 μl of IgG Sepharose beads  for 90 min at 4 °C. The beads were then washed with 800 μl of TAP-B2 . After washing, the TAP tag was cleaved by adding 10 μl of TEV protease  in 200 μl of TAP-B2 to the beads and incubating at 16 °C for 90 min. Cleaved protein complexes were eluted with an additional 200 μl of TAP-B3 Purifications of native protein complexes were performed using extracts from strains with a segment encoding the TAP tag fused in-frame to the 3′ end of the chromosomal gene of interest . In gene2, 0.15% NP-40) followed by 200 μl of TAP-B5 . Finally, the proteins were eluted by adding 600 μl of TAP-EB  to the beads and incubating for 30 min at room temperature, and were then precipitated with trichloroacetic acid.The material eluted by the TEV protease cleavage from the first affinity matrix was incubated with 200 μl of Calmodulin beads  for 60 min at 4 °C. Beads were washed with 400 μl of TAP-B4 . Acid-washed glass beads were added, and the cells were disrupted mechanically using a bead beater  for 5 min. Insoluble material after cell disruption was removed by centrifugation in a microfuge at 14,000 rpm for 20 min. The supernatant was incubated with 25 μl of IgG sepharose beads (Amersham Biosciences) for 90 min at 4 °C. Beads were then pelleted and washed three times with 0.6 ml of TAP-IPB. After washing, the beads were resuspended in SDS sample buffer and subjected to SDS PAGE and immunoblotting with anti-HA-Peroxidase antibody  and antibodies against Tra1p (a generous gift from J. Workman), Bdf1p (a generous gift from A. Ladurner), and Act1p (a generous gift from D. Drubin).For coprecipitation assays, we prepared extracts from 150 ml yeast cultures harvested at an ODMATα swr1Δ::kanMX4 his3Δ1 leu2Δ0 ura3Δ0 lys2Δ0  and YM1769 MATα his3Δ1 leu2Δ0 ura3Δ0 lys2Δ0. Exponentially growing cultures were diluted to OD600 0.1 in yeast extract-peptone-dextrose medium (YPD)  supplemented with tryptophan and adenine. Each mutant culture was paired with a wild-type (wt) culture placed in an adjacent slot in a shaker. Four such pairs of cultures were grown at 30 °C to OD600 0.8. Cultures were harvested at identical optical densities by vacuum filtration onto nitrocellulose filters , and snap-frozen in 15 ml conical tubes in liquid nitrogen. Total RNA was extracted as described (http://www.microarrays.org), and mRNA was prepared using oligo-dT coupled to latex beads, using the manufacturer's protocol . mRNA was then reverse-transcribed into cDNA.The strains used for expression analysis were derived from S288c: YM1823 http://www.microarrays.org.Microarrays were fabricated as described by After chemical coupling to Cy5 and Cy3 fluorescent dyes, mutant and wt cDNA samples were mixed and hybridized to microarrays at 63 °C for 12–16 h. Two of the four hybridizations were performed with fluor-reversed samples to avoid artifacts arising from differences in coupling efficiency of the two dyes. After washing and drying, the arrays were scanned on a Genepix 4000B scanner  and the images analyzed using Genepix 3.0 software to determine the ratio of median fluorescence intensity (above background) for each spot. After flagging poor quality spots, the ratios were normalized for total signal in the two samples. After filtering the data for dim and uneven spots, genes with at least three good measurements were retained for statistical analysis.swr1Δ/wt mRNA ratios were analyzed using the SAM  statistical package , acetylated K5 of H4 , acetylated K8 of H4 (Serotec # AHP415), acetylated K12 of H4 (Serotec #AHP416), acetylated K16 of H4 (Serotec #AHP417), and acetylated K9 of H2A (Upstate Biotechnology #07-289).Figure S1Fractions collected from glycerol gradient centrifugations of whole cell extracts containing HA-tagged SWR1-Com subunits (shown on the right) were analyzed by immunoblot with an anti-HA antibody. The gradients were from 10% to 40 % glycerol and 22 0.1-ml fractions were collected in each case, starting at the top (Fraction 1). A percentage of the total cellular pool of all six SWR1-Com subunits that were tested was present in the same fractions, consistent with their association in one complex.(263 KB PDF).Click here for additional data file.Figure S2swr1Δ strains were tested for levels of 3HA-H2A.Z using an anti-HA antibody. Equal amounts of total protein extract were present at each dilution, as seen by the immunoblot with the antibody against Vma1p. The level of H2A.Z in the swr1Δ mutant was reduced approximately 2- to 3-fold. This suggested that the SWR1-Com contributed to the stability of H2A.Z, likely by protecting it from protein degradation.Three different dilutions of whole cell extracts from wt or (54 KB PDF).Click here for additional data file.Table S1(54 KB PDF).Click here for additional data file.Table S2(95 KB PDF).Click here for additional data file.Table S3(60 KB PDF).Click here for additional data file.Saccharomyces genome database (http://www.yeastgenome.org) accession numbers of the proteins discussed in this paper are actin (SGDID S0001855), Arp4 (SGDID S0003617), Asf1p (SGDID S0003651), Bdf1p (SGDID S0004391), CAF-I (SGDID S0006222), Cse4p (SGDID S0001532), Eaf3p (SGDID S0006227), Epl1p (SGDID S0001870), Esa1p (SGDID S0005770), H2A (SGDID S0002633), H2B (SGDID S0002632), Kap114p (SGDID S0003210), Nap1p (SGDID S0001756), Rvb1p (SGDID S0002598), Rvb2p (SGDID S0003118), Rvb2p (SGDID S0006156), SAS-I HAT (SGDID S0005739), Sir2p (SGDID S0002200), Sir3p (SGDID S0004434), Sir4p (SGDID S0002635), Swc2p (SGDID S0002893), Swc3p (SGDID S0000009), Swc4p (SGDID S0003234), Swc6p (SGDID S0004505), Swc7p (SGDID S0004377), Swi2/Snf2 (SGDID S0005816), Swr1p (SGDID S0002742), Tra1p (SGDID S0001141), Yaf9p (SGDID S0005051), and Yng2p (SGDID S0001132). The Saccharomyces genome database accession numbers of the genes discussed in this paper are AAD3 (SGDID S0000704), HML (SGDID S0029214), HMR (SGDID S0029655), HTZ1 (SGDID S0005372), PRP8 ORF (SGDID S0001208), YDR359C (SGDID S0002767), and YNR074C (SGDID S0005357). The http://www.ncbi.nih.gov/Genbank/index.html) accession number of EAF1 is AY464183.The GenBank (


Coeliac disease in adolescents has been associated with an increased prevalence of depressive and disruptive behavioural disorders, particularly in the phase before diet treatment. We studied the possible effects of a gluten-free diet on psychiatric symptoms, on hormonal status  and on large neutral amino acid serum concentrations in adolescents with coeliac disease commencing a gluten-free diet.Nine adolescents with celiac disease, aged 12 to 16 years, were assessed using the semi-structured K-SADS-Present and Lifetime Diagnostic interview and several symptom scales. Seven of them were followed at 1 to 2, 3, and 6 months on a gluten-free diet.Adolescent coeliac disease patients with depression had significantly lower pre-diet tryptophan/ competing amino-acid (CAA) ratios and free tryptophan concentrations, and significantly higher biopsy morning prolactin levels compared to those without depression. A significant decrease in psychiatric symptoms was found at 3 months on a gluten-free diet compared to patients' baseline condition, coinciding with significantly decreased coeliac disease activity and prolactin levels and with a significant increase in serum concentrations of CAAs.Although our results of the amino acid analysis and prolactin levels in adolescents are only preliminary, they give support to previous findings on patients with coeliac disease, suggesting that serotonergic dysfunction due to impaired availability of tryptophan may play a role in vulnerability to depressive and behavioural disorders also among adolescents with untreated coeliac disease. Coeliac disease is an under-diagnosed autoimmune type of gastrointestinal disorder resulting from gluten ingestion in genetically susceptible individuals. Non-specific symptoms such as fatigue and dyspepsia are common, but the disease may also be clinically silent. Diagnosis is based on small-bowel biopsy, and a permanent gluten-free diet is the essential treatment. Undetected or neglected, coeliac disease is associated with serious complications. -3 DepresImprovement in state anxiety , in behaThe present work is a preliminary prospective psychiatric follow-up study of adolescents with newly diagnosed coeliac disease measuring psychiatric symptoms, hormonal status , and large neutral amino acid (LNAA) serum concentrations repeatedly after their commencement of a gluten-free diet, testing the hypothesis that the treatment of coeliac disease may increase the availability of tryptophan and alleviate psychiatric symptoms.The study sample comprised all nine adolescents  consecutively diagnosed with coeliac disease between January 1999 and December 2000 in the Department of the Gastrointestinal Services of the Hospital for Children and Adolescents, Helsinki University Central Hospital, in Finland. None of the patients had a history of, or current psychiatric treatment. Duration of coeliac disease symptoms and signs  leading to a biopsy was 2.3 (± 1.5) years. The study was approved by the institutional Ethics Committee. Written informed consent was obtained from each patient and a parent.Baseline psychiatric evaluation was conducted 1 to 4 weeks after the diagnostic biopsy, during the wait for the diagnosis of coeliac disease to be established by the pathologist. The adolescent and a parent were interviewed separately by an adolescent psychiatrist (PP) using a semi-structured diagnostic interview, the Schedule for Affective Disorders and Schizophrenia for School-Age Children – Present and Lifetime version (K-SADS-PL) . Seven pCoeliac disease activity was followed by determining serum tissue transglutaminase (S-tTGAbA) and endomysium (S-EndoAbA) autoantibodies . Pre-dieStatistical analysis was carried out with parametric and non-parametric tests as appropriate; tests for two-independent groups , for repeated measures of two-related groups (Wilcoxon signed-ranks test), for three-related groups (Friedman test) and Spearman's rank correlation testing were used. P-values (2-tailed) < 0.05 were regarded as significant.At baseline, three adolescents  had a depressive disorder: two girls had major depressive disorder (MDD), one with a learning disorder not otherwise specified (NOS), and another with comorbid conduct disorder; one girl had the depressive disorder NOS. Further, one boy had a phobic disorder plus attention-deficit hyperactivity disorder, and another conduct disorder NOS. Four adolescents (44%) had no diagnosis.Pre-diet free L-tryptophan was positively correlated with pre-diet levels of tTGAbA , and negatively with vitamin B-6  and S-T4 . Prolactin levels Table  from theDepressive patients (n = 3/9) had significantly higher pre-diet prolactin levels  and pre-diet S-T4 levels , and significantly lower L-tryptophan/CAA ratios  and free L-tryptophan concentrations . Pre-diet free L-tryptophan correlated negatively with biopsy morning S-T4 level . No significant differences appeared in L-tryptophan (36.3 ± 5.1 vs. 43.3 ± 6.1) or in L-tyrosine concentrations, nor in BCAA and CAA levels.Two adolescents with conduct disorders, one a girl with concomitant MDD, did not adhere to the gluten-free diet and dropped out of the psychiatric follow-up. Among others (n = 7), a significant decrease in most of the problem and symptom scores of YSR and CBCL, and in BDI, BAI, and Hamilton scales was evident after 3 months on the gluten-free diet, compared to baseline Table .Celiac disease-associated antibody titres had decreased in all by the first month on a gluten-free diet, and had already normalised (= S-EndoAbA titre < 5 and S-tTGAbA titre < 8) in 4 of 7 patients by 6 months. Boys had lower biopsy morning prolactin levels , but higher levels after one month on the diet. In the first month, the S-T4/TSH ratio (nmol/l:mU/l) reflecting thyroid function increased significantly .  had significantly lower pre-diet tryptophan/CAA ratios and free tryptophan concentrations and significantly higher biopsy morning prolactin levels. Adolescents with coeliac disease showed improvement in psychiatric symptoms after starting a gluten-free diet, and this improvement coincided with a significant decrease in coeliac disease activity and in prolactin levels, and with a significant increase in serum concentrations of L-tyrosine and other CAAs. The increase in free L-tryptophan levels was approaching significance. The findings of this study – improvement in depressive and behavioural symptoms after the start of a gluten-free diet – are supported by the findings of our larger previous retrospective case-control study . AlthougThe decrease observed in psychiatric symptoms took place regardless of the stress accompanying being diagnosed with a chronic and restrictive illness, and improvement was not explainable in terms of physical symptoms, since both in the present and in our previous study , the preSince the free tryptophan and the tryptophan/CAA ratios in plasma determine the availability of tryptophan to the brain , our finIn the present study, stress-induced biopsy-morning prolactin levels were significantly higher among depressive patients  and correlated negatively with L-tryptophan/CAA levels. This finding is only preliminary, but it is, however, interesting: Although disturbances in the central serotonergic system have been associated with depressive and impulse-control disorders among adults [see ], and chOn the other hand, in the present study also non-depressed adolescents with coeliac disease had higher than normal biopsy morning prolactin levels. Significantly higher prolactin levels among untreated coeliac children (5–18 years) compared with treated patients has been reported by Reifen et al. . They suOur preliminary findings on amino acid levels in adolescents with coeliac disease with or without depression are unlikely to be explained by malabsorption, since pre-diet free L-tryptophan and tryptophan ratios were not correlated with the BCAA levels that reflect the level of protein nutrition. It is of theoretical interest that increased production of interferon-γ (IFN-γ), known to be the predominant cytokine produced by gluten-specific T-cells in active coeliac disease , can supThe alleviation of psychiatric symptoms found among adolescents with coeliac disease after commencement of a gluten-free diet coincides with a rapid decrease in antibody titres indicating coeliac disease activity and in their prolactin levels, and with a significant increase in L-tyrosine and other CAA serum concentrations, and with a nearly significant increase in the free fraction of L-tryptophan. Although these findings are only preliminary, and more research is needed, they give support to previous findings on patients with coeliac disease, suggesting that serotonergic dysfunction due to impaired availability of tryptophan may play a role in vulnerability to depressive and behavioural disorders, also among adolescents with untreated celiac disease. And since diet treatment may alleviate psychiatric symptoms, and earlier diagnosis may have beneficial effects on psychological and even on neurobiological vulnerability to depression, the possibility of psychiatric complications of coeliac disease needs to be taken into account in differential diagnosis of depressive and behavioural disorders.The author(s) declare that they have no competing interests.PAP, ETI, MAV, ES, VAA contributed to the conception and design of the study, and PAP, MAV, ES to acquisition of the data. All authors  contributed to the analysis and interpretation of the data, were involved in drafting and revising the article, and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Bovine chromosome (BTA) 15 contains a quantitative trait loci (QTL) for meat tenderness, as well as several breaks in synteny with human chromosome (HSA) 11. Both linkage and radiation hybrid (RH) maps of BTA 15 are available, but the linkage map lacks gene-specific markers needed to identify genes underlying the QTL, and the gene-rich RH map lacks associations with marker genotypes needed to define the QTL. Integrating the maps will provide information to further explore the QTL as well as refine the comparative map between BTA 15 and HSA 11. A recently developed approach to integrating linkage and RH maps uses both linkage and RH data to resolve a consensus marker order, rather than aligning independently constructed maps. Automated map construction procedures employing this maximum-likelihood approach were developed to integrate BTA RH and linkage data, and establish comparative positions of BTA 15 markers with HSA 11 homologs.The integrated BTA 15 map represents 145 markers; 42 shared by both data sets, 36 unique to the linkage data and 67 unique to RH data. Sequence alignment yielded comparative positions for 77 bovine markers with homologs on HSA 11. The map covers approximately 32% of HSA 11 sequence in five segments of conserved synteny, another 15% of HSA 11 is shared with BTA 29. Bovine and human order are consistent in portions of the syntenic segments, but some rearrangement is apparent. Comparative positions of gene markers near the meat tenderness QTL indicate the region includes separate segments of HSA 11. The two microsatellite markers flanking the QTL peak are between defined syntenic segments.Combining data to construct an integrated map not only consolidates information from different sources onto a single map, but information contributed from each data set increases the accuracy of the map. Comparison of bovine maps with well annotated human sequence can provide useful information about genes near mapped bovine markers, but bovine gene order may be different than human. Procedures to connect genetic and physical mapping data, build integrated maps for livestock species, and connect those maps to more fully annotated sequence can be automated, facilitating the maintenance of up-to-date maps, and providing a valuable tool to further explore genetic variation in livestock. Genome maps for livestock species are necessary to identify genes affecting economically important production traits. Linkage maps, based primarily on highly polymorphic, anonymous microsatellite markers, have been important for identifying chromosomal regions influencing economically important traits in cattle -3. BecauIntegrated analysis of both linkage data and RH data allows each source of information to complement the other, providing coarse to intermediate scale maps of the bovine genome, populated with gene markers to facilitate discovery of positional candidate genes for QTL. These integrated maps will lack the fine scale of complete genome sequence, but represent a resource useful for gene identification through comparative mapping approaches, using more complete genome sequence and annotation from other organisms. Similarity between segments of bovine DNA and genomic sequence from other species may supplement integrated data to predict the location of unmapped genes in the bovine genome . A comprAn integrated map can also provide a framework for assembling bovine genomic sequence as data becomes available. A well-ordered map of sequence-tagged-sites (STS) was essential for assembling the human sequence . The NatIntegration of linkage and RH maps has been reported for a number of species -14 and iA large volume of data are being generated in cattle and other livestock species that is not rapidly reflected in current map representations. The result is a lack of truly up-to-date maps of any livestock species, as the maps may lag by months or years in their representation of existing data. It is not feasible to devote significant human resources to constantly maintain and update these maps, so it is critical that automated procedures be developed to free human map curators from many of the time-consuming, error-prone tasks experienced in the mapping process. Existing map construction software is automated to the extent that the likelihoods of many alternative marker orders can be evaluated with a single command, but the entire process of gathering and formatting raw data, constructing maps, examining results and publishing on the internet, or elsewhere, requires human intervention at several stages. Automated procedures will streamline the process in order to focus human effort on the critical stages of verifying raw data and examining the resulting maps.Bovine chromosome (BTA) 15 provides an interesting example to study the integration of linkage and RH data, and comparison of the bovine to the human genome. A QTL for meat tenderness has been reported on bovine chromosome 15 ,23. CompThe low resolution of the bovine linkage map is indicated by multiple markers sharing the same map position, even when they may be separated by a substantial physical distance. Inclusion of RH data provides additional evidence by which markers that are inseparable only with linkage data can be ordered. The BTA 15 linkage map Figure  shows 78Markers common to both the linkage and RH data sets provide a basis for integrating the data and constructing maps representing both types of data. Primer sequences associated with the RH and linkage markers indicated 42 common markers in the two data sets, with 36 markers unique to the linkage data and 67 unique to RH, for a total of 145 markers represented on the integrated linkage-RH map.Four sets of markers with different primer sequences matching the same bovine sequence were identified. In two instances , markers in the set were placed adjacent to each other by the map building routine. In the two other cases , markers in the set were separated by several markers after initial map construction. In both cases, the map could be reordered so markers in each set were placed next to each other without decreasing likelihood of the map. The final integrated order includes these manual adjustments, so that in all cases of different markers matching the same sequence, the markers are adjacent on the map.Comparison of the integrated map to independently solved linkage Figure  and RH  maps indComparison of the integrated map to the RH map shows the markers remained in the five blocks identified by Gautier et al. , and theprimersearch [Comparative map positions for 77 markers mapped to BTA15 were established using ersearch  to identersearch  were retersearch ,23. Our Markers more recent ,24 than Order is well conserved within syntenic group segments S1, S3, S4' and portions of S2 and S4. The most notable rearrangements within segments are an inversion of several markers in the center of S2, and inconsistent ordering within a subset of S4. The internal rearrangements within syntenic groups found here, pig-human rearrangements , and mouChallenges for building high-resolution integrated maps and leveraging data from various sources, both within and across species, will be to determine regions where additional data may be informative and placing appropriate emphasis on the different sources of information at different levels of resolution. Linkage maps can provide the scaffold for ordering an entire chromosome, so linkage data may receive the greatest emphasis for initially determining a coarse order. Increased emphasis should be given to higher resolution RH and other physical mapping data to resolve order where placement of linkage markers is uncertain, and markers are too close to provide definitive order. Comparative sequence and mapping information from other species should be most useful to position markers within regions where physical data have insufficient resolution and within-species sequence data are not available. Using appropriate weights to combine genetic and physical mapping data, within-species sequence and comparative sequence data should allow the different data sources to complement each other, resulting in consensus maps supported by the combined sources of information.Genome maps of livestock species need to represent current information in order to maximize utility of the maps. Positions of putative QTL may become misleading if QTL positions are not updated to reflect subtle rearrangements resulting from new mapping data. Genes associated with phenotypic variation will be more readily identified if available information to link mapping data to genes and their function is maintained. Continually updating the maps to depict relevant existing information will be facilitated by automation, but a number of issues must be addressed for implementation of automated procedures to be fruitful.Access to dynamic sources of mapping data must be maintained, so that new information can be incorporated into the maps soon after it is generated. Information to connect data from various sources must be available to expedite integration. Map computation strategies deserve some attention, to minimize the delay between acquiring new data and appearance of those data in subsequent maps.Procedures developed to integrate BTA15 linkage and RH data can be applied to available data for the entire bovine genome. The integration effort will be more valuable, however, if sources of data for the integrated map are periodically updated. Success of a comprehensive integration effort will also depend on information available to establish connections between the data sets. One alternative is to resolve marker nomenclature, perhaps by developing and maintaining a database of marker names and synonyms. A more straightforward, and easily automated, approach is to use primer sequences as universal identifiers to establish connections between mapping data sets. Database curation efforts to associate mapping records  with primer pairs may be more worthwhile than attempts to resolve all possible names for a given marker.Primer sequence can also be used to establish connections to sequence databases. Sequence similarity searches should reveal connections to STS sequences associated with markers; the process will also identify connections to other sequences, including more completely annotated and assembled sequence. Sequences identified in this process can be used to establish connections with human and other well annotated, assembled genomic sequence for comparative mapping. Similar associations between mapping and sequence data may be established using marker and locus names, provided that marker nomenclature can be resolved Sequence-based connections between mapping data sets, integrated maps, and genomic sequence may be more reliable and are more amenable to automation than attempts to connect sources using names and other information.Connections between maps and annotated sequence can accelerate positional candidate gene discovery if the sequence annotation includes functional information. Harhay and Keele  used GO Placement of new markers on integrated maps must keep pace with new marker development, if integrated maps are to remain current with available mapping and sequence data, The basic concept of map construction, finding the most likely marker order out of all possible orders, is conceptually simple but computationally demanding, because the number of possible orders increases factorially with the number of markers. Evaluating all possible marker orders is not feasible when mapping data represents more than twenty or thirty markers on a chromosome. Cost and time constraints limit map construction to strategies that evaluate a sufficient number of possible orders to ensure that a reasonably good order is identified.As bovine sequence data becomes available, methods to exploit that resource to refine both the integrated maps and sequence assemblies must be implemented. Advent of whole-genome sequence assemblies has not diminished the value of maps in sequenced species. Discrepancies between human maps and sequence assemblies have been noted ,33, althThe strategies employed must be sufficiently flexible to allow manual manipulation of the resulting maps. Some evidence, such as STS markers sharing the same sequence, and ordering information from other species, is not easily represented in linkage and RH mapping data. In some cases of markers sharing the same sequence, markers can be forced to share the same position, or data from multiple markers can be combined to create a single haplotype representing multiple markers. Marker orders suggested by maps of other species may be compared with likely orders identified from within-species data. Incorporating information not directly represented in mapping data can require manually evaluating additional orders, and making some judgement about which results are most acceptable.In exploratory analyses merging BTA15 linkage and RH data, simulated annealing and taboo search algorithms in CarthaGene were explored as methods of initially ordering the integrated map, before refinement with the polish and flips routines. Resulting maps were similar to the map presented, but required more than 24 hours to compute. The map presented was initially ordered by placing each marker against a pair of markers common to both data sets, and was constructed in less than four hours. Another approach involved initially placing markers against the set of all markers common to both the linkage and RH data sets, in the linkage map order. While map construction was somewhat faster using this approach, the resulting map was less likely than the map initiated from a pair of markers and showed greater disagreement with the linkage map.Parallelization of the mapping algorithms can substantially increase the speed of map construction. Likelihoods of a number of alternative orders must be computed at several steps during the map building process. If these calculations are distributed across multiple processors, time required to compute all likelihoods and arrive at a final order will be reduced because computations are performed simultaneously. Increased parallelization should also increase the feasibility of implementing more thorough algorithms that examine a larger number of possible orders, therefore increasing the probability of identifying more likely maps.Linkage and radiation hybrid maps are powerful tools to facilitate discovery of genomic regions and ultimately genes influencing livestock production traits. Combining linkage and RH data can provide more accurate, consolidated maps representing more information, especially if the maps are connected to well annotated genomic sequence. Automating map construction and comparative mapping procedures will expedite construction of whole-genome integrated maps and maintaining a comprehensive resource as new data becomes available. Success of automated procedures to connect data from various sources and construct integrated maps depends on information available to establish connections between data; sequence-based approaches to connect data are preferrable.Linkage data for 78 markers in the BTA15 linkage group were obtained from the U.S. Meat Animal Research Center (MARC) reference population  assemblx . Other x , and hux .primersearch tool. The longest matching sequence having one or fewer mismatches and an amplimer less than 600 bp was selected for homology search against HSA11 contigs. The selected sequences were examined for gaps, and where gaps occurred, only the ungapped pieces matching a primer pair was used in the homology search. Connections between the individual sequences matching bovine markers and human sequence were then determined via BLASTN [-20, and default values for other parameters.Connections between data sets are necessary for integrated analyses of those data to be meaningful. Because some marker names were ambiguous, connections between markers in the linkage and RH data were established using primer sequence. Markers with identical primers were considered to be the same, regardless of marker name. Primer sequence was also used to establish connections with human sequence. Primer pairs were matched against bovine sequence from GenBank, NT and BTGI databases using the EMBOSS primerseaa BLASTN  with an Connections between human position and functional GO annotation were extracted from the downloadable LocusLink database . Proceduchrompic option of CRIMAP [Observations for RH data are binary (0/1), indicating absence or presence of a particular marker in a cell line, where each cell line represents a relatively short segment of DNA on a chromosome. Physically close markers are more likely to be observed on the same cell line than distant markers. Linkage data includes pedigree information and marker genotypes, where individual genotypes represent alleles inherited from each parent. Alleles for physically close markers on a single chromosome are more likely to be inherited from the same grandparent; the likelihood of marker alleles with different grandparental origin appearing on the same chromosome increases with distance between markers. These chromosomes can be represented in a binary, RH-like format that can be merged with RH data using CarthaGene. Analagous to RH data representing presence or absence of a marker in a cell line, binary representation of linkage data indicates presence or absence of a maternal allele on an individual chromosome. The f CRIMAP  was useddsmergor). Two markers shared by the linkage and RH data sets are identified, and all other markers are inserted, one at a time, into the most likely position using the CarthaGene buildfw procedure. Once all markers from both data sets are placed, the marker order is refined iteratively, cycling through polish and flips routines until likelihood does not improve. The polish procedure individually tests each marker in all alternative positions, and flips evaluates permutations of all sets of six adjacent markers.An interface to the CarthaGene shared library was developed using perl and the perl Inline modules  to automAfter convergence using the map construction script, further evaluation of alternative orders was carried out with the backcross linkage data merged with a diploid model of the RH data, again assuming common marker order. Marker orders consistent with available sequence information were evaluated. Where primer paris for different markers matched the same bovine sequence, but the markers were separated by one or more other markers by the map construction routine, likelihoods of orders with the matching markers placed adjacent to each other were determined. Likelihoods of marker orders consistent with human sequence within each syntenic segment were also computed. The sequence-based orders were used in the final integrated map if they did not decrease likelihood of the map. Log-likelihoods of the final integrated map order were computed with the RH and linkage data sets for comparison to the independent maps, using CarthaGene for the RH map and CRIMAP for the linkage map.dsmergen. Marker order was set to the final integrated order, map distances computed, then scaled to range from zero to 100.The final integrated marker order was projected onto a common relative scale representing all markers. This was accomplished by merging the linkage data with RH data, modeled as backcross, using All computation was performed on a 10-node Linux cluster, each node configured with 2 AMD 1900+ CPUs and 3 Gb RAM. When practical, computation was parallelized using perl scripts and open source Grid Engine software  to distrWMS developed perl scripts for automated analyses, conducted analyses to match markers from linkage and RH data sets, constructed the linkage and integrated maps, and drafted the manuscript. MG and AE developed RH markers and constructed the RH map. AE and JWK conceived the research, and contributed to planning analyses and evaluating results. RTS and TPLS assisted with evaluation of the integrated map. GPH conducted BLAST analyses and associated bovine sequence with human GO annotation. NI, AT, HT developed linkage markers. YS and GLB coordinated linkage map data collection. GLB curates the MARC linkage data and linkage maps.linkage, RH and integrated maps of BTA 15Click here for fileperl interface to CarthaGene, requires perl Inline::Tcl moduleClick here for fileperl script to construct integrated maps using CarthaGene, requires carthaPerl.pl interface to CarthaGeneClick here for file


Sexual differentiation appears to be an ancient, and potentially homologous, feature of animal biology, and yet the pathways that underlie the process exhibit bewildering variety Hox genes in Drosophila melanogaster and the subsequent discovery of their conservation and similar expression in different Metazoans led to the revolutionary realization that many of the mechanisms critical to basic animal development have been conserved across more than 500 million years of evolution. Many other developmental pathways, such as those specifying the heart and the central nervous system, have since been elucidated and promptly subjected to successful comparative analysis.Evolutionary developmental biology is motivated by the premise that the differences we see between species are caused by changes that have occurred in the genes that regulate their developmental programs. Beginning in the 1980s, general principles began to emerge about the evolution of development in animals. The identification of the These celebrated discoveries illustrate ways that very different organisms are, at a fundamental level, similar to one another. But not all developmental processes are so conservative; an outstanding example is sex determination. The majority of animal species produce two sexes, and current phylogenies  suggestThe variety of primary sex determination cues was appreciated long before the advent of molecular genetics . The twoDrosophila, Caenorhabditis, and mice have revealed a clear lack of conservation, underscoring the diversity. For example, although the primary sex determination signal in both D. melanogaster and C. elegans is the X:A ratio, the fruit fly pathway consists of a cell-autonomous cascade of regulated mRNA splicing, while that of the nematode follows a Hedgehog-like intercellular signaling pathway [Sry) encoding a transcription factor. In the face of such impressive differences, perhaps we should question our assumption of homology: could it be that sex determination in different taxa has arisen independently over and over again in evolution? Until 1998, this seemed like a good bet.Molecular genetic investigations of GSD in model systems such as  pathway . GSD in  pathway ) upon a doublesex in Drosophila and mab-3 in C. elegans provided the first evidence for a common evolutionary basis of sex determination in animals [doublesex-mab-3 (DM)-family genes with roles in male sexual development were discovered in vertebrates and even cnidarians [The discovery of the homology of the key sex-determining genes  animals . Soon, ridarians ,8. Here To understand how such diversity came to be, we need to look at the differences between closely related species. This approach allows the discovery and interpretation of small-scale sex determination changes before they are obscured by subsequent changes. The processes discovered in this way might then be reasonably extrapolated to explain the seemingly unrelated systems of more deeply diverged taxa. Work in dipterans  and nemaCaenorhabditis, xol-1. The xol-1 orthologues of the closely related nematodes C. elegans and C. briggsae are a mere 22% identical [xol-1 are much better conserved . Similar results have also been found for the housefly, Musca domestica [Sxl in sex determination may be restricted to Drosophila and its closest relatives. In contrast, tra and dsx are key sex regulators in all dipterans examined thus far.In a series of groundbreaking papers, Saccone and colleagues investigated the pathway in the more distantly related heterogametic Mediterranean fruit fly omestica , indicatCeratitis tra homologue was characterized [Drosophila, only females productively splice tra mRNA. However, this splicing difference can be explained nicely by a positive feedback, similar to that seen in Drosophila Sxl, in which Tra protein regulates its own splicing. In 2002, Pane et al. proposed that the dominant, male-specifying M factor on the Y chromosome inhibits this autoregulation [tra mRNA). A detailed scenario for how this might occur has recently been proposed [A further surprise came when the cterized . In the gulation . As a reproposed .Caenorhabditis nematodes have shed light on the genetic basis of the convergent evolution of sex determination related to mating system adaptations. An important factor in this area are new phylogenies of the genus [C. elegans and C. briggsae acquired self-fertilization independently, from distinct gonochoristic  ancestors (Finally, recent studies of he genus ,18, whicncestors . AlthougC. elegans, Clifford et al. [fog-2, a gene required for spermatogenesis in hermaphrodites but not in males. Upon doing so, it became clear that fog-2 is part of a large family of F-box genes and was produced by several recent rounds of gene duplication. The C. briggsae genome sequence suggested that while C. briggsae possesses a similarly large family of F-box proteins, the duplication event giving rise to fog-2 was specific to the C. elegans lineage. In this issue of PLoS Biology, Nayak et al. [fog-2 is indeed absent in C. briggsae. The authors also identify a short, C-terminal domain that makes FOG-2 uniquely able to perform its germline sex-determining function. This domain is probably derived from a frame-shifting mutation in an ancestral gene. Working with C. briggsae, Stothard et al. [C. elegans requires the male-promoting genes fem-2 and fem-3 to produce sperm in hermaphrodites, C. briggsae requires neither. Given that both genes have conserved roles in male somatic sex determination, this suggests that C. briggsae evolved hermaphroditism in a way that bypasses these genes.Working with d et al.  cloned fk et al.  extend td et al. , Haag etd et al. , and HilThe long-standing mystery of sex determination and its diversity began by comparisons between distantly related species. Recent work on closer relatives has uncovered processes that through a reasonable extrapolation enable the connection of these disparate dots into a fascinating picture of developmental evolution. Though the divergence is extreme, it is likely that a better understanding of the evolution of sex determination genes and pathways holds lessons about the evolution of development in general. The next major challenge will be to integrate the comparative developmental data with the ecological and population processes that are driving the evolution of sex determination. Only then will we be able to say that the picture is complete.