We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.The recessive lethal mutation  flotte lotte (flo) mutants. Intestinal apoptosis occurs in the absence of the DNA damage marker γH2X, and levels of chromatin bound Mcm2, a component of the DNA replication helicase, were also reduced in flo mutants. These findings suggested that flo intestinal cells cannot repair endogenous replication errors. Consistent with this idea, flo mutants were highly sensitized to treatment with DNA replication inhibitors such as hydroxyurea, UV irradiation, or cisplatin, but not agents that cause DNA double strand breaks, such as γ-irradiation or camptothecin. These data point to a conserved role for nucleoporins in the cellular response to replication stress in eukaryote cells.DNA replication is a complex process that requires activation of cell cycle checkpoints and DNA repair pathways. Genetic analyses in fungi have suggested that nucleoporins, the proteins that make up the nuclear pore complex (NPC), play a role in the cellular response to agents that disrupt cell proliferation or damage DNA. Here we show that mutation of the Elys nucleoporin causes widespread apoptosis in the intestine and other tissues of zebrafish  Programmed cell death is believed to function in two contexts during early mammalian development. Prior to implantation and near gastrulation, apoptosis eliminates embryonic cells rendered unfit by growth factor deficiency Although apoptosis is normally activated in only a small number of cells of early mammalian embryos, gene targeting experiments have demonstrated the susceptibility of surviving cells. Genomic instability is speculated to be one possible underlying cause of this predisposition, since mutation of DNA repair Elys is a conserved mammalian gene that is required for embryonic survival during early development Elys are resorbed at peri-implantation stages (e5.5–e7.5) and inner cell mass cells from cultured Elys−/−blastocysts undergo apoptosis soon after hatching from the zona pellucida. Elys is expressed throughout the developing mouse embryo and in a wide range of adult tissues http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Hs.300887#Legend). Recently human and frog Elys proteins were shown to be orthologs of the gene encoding Mel-28, a protein required for nuclear integrity in C. elegans flotte lotte (flo), a previously described recessive zebrafish mutant with retinal, neural and digestive organ defects elys. Strong maternal elys expression enables cells of early flo embryos to survive to larval stages. However, at later stages proliferative cells in tissues in which NPC assembly is disrupted, such as the retina, optic tectum and intestine, undergo cell cycle arrest and ultimately succumb to apoptotic cell death via p53-dependent and p53-independent mechanisms flo intestine is associated with activation of the Chk2 protein kinase flo larvae, but not their heterozygous or homozygous wild type siblings. Homozygous flo larvae, but not their siblings, were also sensitized to DNA replication inhibitors, but not agents that induce DNA double strand breaks. Finally, we also found that loss of Elys reduces levels of chromatin bound Mcm2, but not Mcm3, Mcm4 or phospho-Mcm4 in the wild type or irradiated flo intestine. Together, these and other data support a role for Elys in DNA replication and the cellular response to replication stress, independent of its role in NPC assembly.Here, we show that flotte lotte  is a recessive lethal mutation that was identified as part of a large scale ENU mutagenesis screen flo mutant phenotype that are first evident in live fish on the fourth day post-fertilization . To further assess the effect of the flo mutation on organ progenitors, we also measured cell proliferation in the intestinal and retinal epithelium. Both sets of data were consistent with G1 arrest of rapidly proliferating cells in these tissues , a gene recently shown to be required for NPC assembly and nuclear integrity in worms Elys is expressed in a wide range of tissues and is essential for early mammalian development elys encodes a predicted protein that consists of 2527 amino acid residues. Computational analysis of the Elys protein revealed two WD-40 repeats and three nuclear export signals in the N-terminus plus a coiled-coil region (involved in protein-protein interactions) and several nuclear localization signals in the C-terminus , was not evident in the intestine of the elys-morpholino injected larvae despite clear evidence of NPC disruption. Although partial intestinal phenocopy by the Elys knockdown was not unexpected because morpholino knockdowns are often transient, these data raise the possibility that apoptosis caused by loss of Elys function may occur independently of altered NPC assembly seen in flo mutants.Antisense knockdown using morpholinos targeting either the acceptor  phenocop retinal  and tect retinal . Phenocoved mRNA . Combines tissue  as do flelys expression in zebrafish embryos and larvae is consistent with nearly all aspects of the flo phenotype  show strong elys expression in the brain (Elys expression in the zebrafish digestive organs persists through 5 days post-fertilization (dpf), when cell proliferation in these organs is low, but at a greatly reduced level compared to 48 hpf, when the rate of cell proliferation is high . These data show that disruption of NPCs activates the DNA damage response in a tissue-specific manner.To gain a better understanding of how NPC disruption caused apoptosis and cell cycle arrest in tivation . To furtflo intestine to determine whether they could account for tp53-independent apoptosis. Mammalian Chk2 is activated by the ATM kinase in response to double strand DNA breaks and other types of DNA damage flo intestine, and morpholino knockdown confirmed the specificity of the anti phospho-Chk2 antibody . By contrast, concomitant treatment was lethal by 24 hours post-treatment for all sibling homozygous flo larvae analyzed (n = 86 larvae), most likely as a result of widespread intestinal injury. Unlike flo, Chk2 inhibition was well tolerated by another zebrafish mutant, slim jim  in which intestinal epithelial cell proliferation is reduced to a level comparable to flo as a result of altered RNA Polymerase III activity slim jim mutants is expected because in contrast to flo, phospho-Chk2 levels are not elevated by this mutation . Although a modestly more pronounced effect was evident in the flo larvae, the difference in sensitivity to ATM inhibition was minor in comparison to the differential effects of Chk2 inhibition. Treatments with caffeine, an inhibitor of the mammalian ATR-Chk1 signaling pathway was tolerated by flo and sibling larvae, but caused significant developmental delays in both groups (data not shown).To further interrogate the ATM-Chk2 pathway, flo larvae that have only low γH2AX levels suggested that Elys could be required to maintain replication forks in highly proliferative tissue progenitor cells. To test this hypothesis, we treated wild type and flo larvae with hydroxyurea, a DNA replication inhibitor that can activate Chk2 (as well as Chk1) and H2AX in mammalian cells flo larvae were sensitized to hydroxyurea treatment. Overnight or 6 hour exposure to hydroxyurea doses (250 mM) was lethal for all homozygous flo larvae analyzed (n = 86 larvae) but was well tolerated by all sibling wild type and heterozygous flo larvae analyzed (n = 302 larvae), as well as slim jim larvae (n = 60 mutant larvae). Survival of flo mutants, but not their wild type siblings or slim jim mutants, was also reduced following treatment with the DNA cross-linker cisplatin  as well as UV-irradiation, another cause of replication stress, but not the DNA topoisomerase 1 inhibitor camptothecin, even at doses that caused widespread intestinal apoptosis and wild type larval death , or γ-irradiation (30 Gy). Together, these data suggest that the Elys protein may be required to prevent replication fork collapse in response to specific types of replication inhibitors. The effects of Chk2 inhibition and replication inhibitors on flo mutants are listed in Chk2 dependence of flo retinal and intestinal epithelial cells, we found no evidence of promiscuous firing of licensed replication origins in these highly replicative cells, as arrest in G1 rather than S phase was evident in both tissues , plays an essential role in mediating and regulating this process elys, a gene recently shown to be required for NPC assembly in human cells that was previously known to be essential to early mouse development flo embryos survive early development because of the high level of maternal elys expression. As a result, the effects of the loss of Elys protein function during the later developmental stages, such as organogenesis, can be assayed. The principal findings of our study regarding the role of Elys protein in NPC assembly are in agreement with the reported role of Elys in human cells and in vitro assays of NPC assembly using siRNA knockdown and competitive binding assays Here, we report the phenotype arising from mutation of zebrafish floelys point mutation introduces a premature termination codon into the Elys cDNA, upstream of the coiled-coil domain, the AT hook region and 3′ nuclear localization signals. Absent an antibody that recognizes the zebrafish Elys protein, we cannot determine if such a protein is actually translated from reduced levels of mRNA we can detect in flo mutants. Furthermore, whether such a truncated protein might retain activity was not addressed in this study. Based on comparison with the NPC phenotype arising form Elys knockdown in cultured cell lines, the floelys allele appears to be either a severely hypomorphic or null allele. In the future, further analysis of this or other elys alleles may be used to dissect functional domains in the Elys protein that might mediate pore and non-pore related functions (discussed below).The flo mutant phenotype. We focused on understanding the cause of cell death in the flo intestinal epithelium because of our prior work using the zebrafish to study digestive organ development. Our experiments showed that apoptosis in the flo intestine was not dependent on p53, whereas it was in the retina and tectum. Instead Elys deficiency activated Chk2, a checkpoint protein that can induce cell cycle arrest and apoptosis independently of p53. Checkpoint activation is an important component of the cellular response to replication stress, because it allows stabilization of stalled replication forks, thereby averting replication fork collapse and activation of apoptotic programs flo mutants was unexpected. γ-irradiation of zebrafish larvae induced Chk2 phosphorylation, thus showing an evolutionarily conserved role for this arm of the DNA damage response pathway in zebrafish larvae. Chk2 was also activated by replication stress induced by hydroxyurea, whereas Chk1 phosphorylation was unchanged from its baseline level of activation with either treatment. From this, we conclude that hydroxyurea induced replication stress activates Chk2 in the zebrafish intestine because Chk1 is already maximally activated by endogenous replication errors that occur in these highly proliferative progenitor cells Apoptosis is a prominent feature of the flo larvae even though we found evidence of only low levels of DNA damage in the mutant larvae at this stage . This argued that checkpoints were activated in response to physiological stimuli; i.e. - replication stress. Second, survival of flo mutants was reduced following treatment with doses of replication inhibitors, such as hydroxyurea, UV-irradiation and cisplatin, that had only minor effects on wild type siblings, or another mutant, slim jim, in which intestinal progenitor cells proliferation is also reduced. These data show that Elys deficient cells cannot tolerate additional genotoxic insults induced by stalled DNA replication forks, as reported for cells carrying mutations of DNA repair genes flo mutants with γ-irradiation or the topoisomerase inhibitor camptothecin did not compromise survival compared with wild type siblings. Non-sensitivity to these agents, which induce DNA double strand breaks flo larvae retain the ability to activate repair pathways involving homologous recombination or non-homologous endjoining, whereas mechanisms to maintain replication forks in the setting of nucleotide deprivation (hydroxyurea) or intrastrand DNA crosslinks  are compromised by Elys deficiency. Collectively, these data argue that sensitivity of Elys deficient cells to hydroxyurea and other agents is not a non-specific effect incurred by cells that lack nuclear pores.Although we initially postulated a number of mechanisms to explain how mutation of Elys activated the DNA damage response, several lines of evidence support a model in which Elys protein is required for the repair of replication errors that occur in rapidly dividing cells. First, Chk2 inhibition was lethal to flo intestinal cells compared with both wild type siblings and slim jim mutants that had a comparable reduction of intestinal cell proliferation. These data argue that reduced Mcm2 levels are caused by Elys deficiency, rather than reduced proliferation, as previously reported Levels of Mcm2-7 complex proteins in eukaryotic cells far exceed the number of active replication origins flo. However, G1 arrest induced by Elys deficiency would be expected to increase rather than decrease levels of chromatin bound Mcm2-7 complex flo mutants. Also arguing against this mechanism, we found that Mcm levels were not reduced in slim jim mutants that had a comparable reduction in the percentage of cycling cells that had arrested in G1 Mcm protein levels vary with the cell cycle, and we initially considered this as a possible explanation for reduced Mcm2 levels in Mcm2 allele have reduced numbers of proliferating progenitor cells in the subventricular zone of the brain flo mutants were only affected by hydroxyurea.We speculate that reduced levels of chromatin bound Mcm2 prevents firing of dormant replication origins that sustain DNA replication stalled by endogenous replication errors, or stress arising from exogenous agents, such as hydroxyurea. When this compensatory mechanism is impaired, stalled replication forks are more likely to collapse, leading to cell death. Consistent with this idea, mice homozygous for a non-lethal hypomorphic in vivo, but only the Mcm4,6,7 subcomplex, and not the Mcm2,3,5 subcomplex has helicase activity in vitro. Recent work suggests that the Mcm2,3,5 subcomplex may regulate the association of hexameric Mcm2-7 complex with chromatin, and its helicase activity in vitro, through the phosphorylation of Mcm2 and or Mcm5 by the CDC7/DBF4 kinase Mcm2-7 proteins form a hexameric complex flo tissue progenitor cells to both endogenous and exogenous replication stress. Reduced nuclear export stabilizes p53 in response to genotoxic stress flo intestinal and retinal cells showed no evidence of gross chromatin defects, however, this does not exclude the possibility that zebrafish NPC disruption alters chromatin organization, such as the location of heterochromatin – euchromatin boundaries, which can epigenetically alter gene transcription.We recognize that the lack of nuclear pores could play a significant role in the response of flo retinal epithelium even though the NPC defect persisted in all of the double mutant cells we analyzed. Second, morpholino knockdown of elys fully phenocopied the NPC and differentiation defects present in flo mutants, but did not cause apoptosis. Thus, in both the eye and intestine, the nuclear pore defect associated with Elys deficiency could be dissociated from apoptosis. These findings are interesting in light of genetic studies in yeast and other fungi that first indicated a role for nucleoporins in DNA repair: non-lethal nucleoporin mutations can cause either sensitization to DNA damaging agents Despite these considerations, two experiments from this study argue that a lack of nuclear pores may not be sufficient to affect a cell's ability to respond to replication stress. First, p53-mutation rescued apoptosis in the flo phenotype, one way to further address the role of Elys in DNA replication would be to generate elys alleles that preferentially sensitize progenitor cells to replication stress, rather than NPC assembly. This may identify regions of the Elys protein that are required for Mcm-chromatin interactions, but not NPC assembly. We plan to pursue this type of genetic screen and other experiments that can help characterize other interesting features of the flo mutant phenotype, such as the tissue specificity of p53-dependent apoptosis and Chk2 activation, in future studies.Although it may be difficult to fully exclude a role for the NPC in the flo  and p53 (zdf1tp53) adult fish were maintained as described flo larvae were treated with hydroxyurea (Sigma Aldrich) and the Chk2 inhibitor (Sigma Aldrich) at the indicated concentrations for 4 hours and overnight respectively. To identify apoptotic cells, larvae were soaked in acridine orange (5 ug/ml) for 30 minutes, rinsed in embryo media and examined under a fluorescent dissecting microscope (Olympus MVX 10).Wild type, heterozygous flo locus was performed using bulk segregant analysis as previously described flo locus are presented as Chromosomal localization of the flo larvae were identified via PCR amplification of genomic DNA using primers designed to create Afl III and Hph I recognition sites at the site of the flo mutation using dCAPS Finder 2.0 (http://helix.wustl.edu/dcaps/dcaps.html). Primer sequences are supplied in Homozygous flo larvae were identified by molecular genotyping or retinal acridine orange staining. Primers used for RT-PCR reactions are listed in RNA recovery and RT-PCR was performed as described elys and tp53 morpholinos are supplied in Morpholino antisense oligonucleotides  were injected into one-cell stage wild type embryos as described Histological and immunohistochemical analyses were performed as described Nuclear protein was recovered from the intestine or eye of fifty embryos or larvae and pooled. Standard methods for protein recovery and Western analyses were utilized. Detailed descriptions of these methods and the specificity of all antibodies used for Western analyses are presented in flo larvae (∼80 hpf) were treated with Chk2 inhibitor, ATM inhibitor, caffeine, hydroxyurea, camptothecin and cisplatin at the doses and times indicated in flo larvae in embryo media were placed 12 cm from a Sankyo Denki UV germicidal bulb (15W) for 10 minutes. γ-irradiation of wild type and flo larvae were irradiated as previously described Wild type and sibling Figure S1flo locus. A physical contig comprising five BAC clones spanned markers that defined the critical region surrounding the flo locus (markers (TAAA)7 and (GAT)6). The number of mutant larvae  that were recombinant for each marker is listed. Zero mutants were recombinant for the (GAT)6 marker located within the coding region of the elys gene. This marker is located 3079 bp from the flo mutation.Combined physical and genetic map of the (80 KB TIF)Click here for additional data file.Figure S2ti262cflo mutation is highlighted in light blue.Mouse-zebrafish Elys protein alignment. Amino acid alignment of the mouse and zebrafish Elys protein showing the following protein domains: NES, nuclear export signal; WD-40 repeats; coiled-coiled domain; AT hook domain; NLS-nuclear localization signal. The arginine residue (R) targeted by the (5.3 MB PDF)Click here for additional data file.Figure S3elys expression in flo larvae. Results from real time quantitative PCR amplification of elys cDNA fragments. These data show reduced elys expression in 5 dpf flo vs. sibling wild type larvae. The Elys-1 primers are located in exons 5/6. The Elys-2 primers are located in exons 22/23. The flo mutation is located in elys exon 30. Reduced elys expression in flo larvae is consistent with non-sense codon initiated mRNA decay induced by the ti262cflo mutation (23).Reduced (166 KB TIF)Click here for additional data file.Figure S4elys cDNA truncation. (A) Ethidium bromide stained agarose gel showing 1 kb truncation of an elys cDNA fragment amplified from 1 dpf and 2 dpf wild type embryos that had been injected with the elys exon 30 splice junction morpholino. This morpholino targets genomic sequence at the intron 29/exon 30 splice acceptor. Successful targeting induces deletion of exon 30, as revealed by sequence analysis (B) of the 0.6 kb fragment.Morpholino induced (434 KB TIF)Click here for additional data file.Figure S5flo mutants. (A-D) Histological sections of flo mutants and wild type siblings immunostained with anti-FG Nup antibody (mAb414) showing nuclear pore defects in the 36 hpf flo retina  and 48 hpf intestine . Blue, Dapi stained nuclei.Nuclear pore defects in early (1.5 MB TIF)Click here for additional data file.Figure S6flo mutants.  Quantitative PCR reveals increased p53, p21, and mdm2 expression in flo mutants. Note that tp53 knockdown in flo (B) abrogates increased p21 and mdm2 expression. (C) Western blot showing comparable levels of phospho-Chk2 in the flo and wild type eye (48 hpf). (D) Western blot showing native and phospho-Mcm4 (*) in the zebrafish wild type (wt), flo intestines before and after γ-irradiation (30 Gy). Note that there is very little native Mcm4 in wt and flo following γ-irradiation (lanes 2 and 3). Phosphatase treatment (λ) of the wt sample from lane 2 dephosphorylates nearly all of the phospho-Mcm4 protein such that only native Mcm4 is present in the sample. (E) Confirmatory Western blot showing reduced chromatin bound Mcm2 in the intestine of 84 hpf flo larvae compared with sibling wt larvae. Far right lane labeled “A/G” shows undetectable levels of Mcm2 and Histone 3 recovered from Ig fraction of the wild type intestinal protein prep prior to anti-histone immunoprecipitation. Presumptive phospho-Mcm2 bands on this gel are denoted by the asterisk (*). (F) Western blot showing reduced Mcm2 in 96 hpf flo larvae, but normal levels in 96 hpf slj larvae compared with control wild type larvae. (G) Western blot showing inhibition of Chk2 phosphorylation in flo larvae treated with the Chk2 inhibitor. (H) Western blot showing specificity of the anti-phospho Chk2 antibody. Phospho-Chk2 levels are elevated in 84 hpf flo larvae but are reduced when injected with splice morpholinos  or morpholino designed against the Chk2 translation initiation site (atg) but not larvae injected with vehicle control (cntrl). (I) Western blot showing specificity of the anti-phospho Chk1 antibody: abundant phospho-Chk1 is present in irradiated Hela cells and the non-irradiated 96 hpf flo intestine, but reduced levels are present in the intestine of 96 hpf flo treated with the ATR inhibitor caffeine ; the intestine of 96 hpf flo larvae treated with caffeine (10 uM) and a commercially available ATM inhibitor ; but not in the intestine of 96 hpf flo larvae treated with ATM inhibitor alone (12 uM beginning at 84 hpf). His3, anti-Histone 3; A/G, IG fraction recovered following protein A/G precipitation.DNA damage response activation in (1.1 MB TIF)Click here for additional data file.Table S1flo intestine. Percentage of S-phase cells is reduced in the 75 hpf flo intestine vs. wild type siblings as determined by BrdU immunohistochemistry. The percentage of phospho-Histone H3 cells is comparable in flo and wild type larvae. These data are consistent with G1 arrest.Reduced cell proliferation in the (33 KB PDF)Click here for additional data file.Table S2flo retina. Data derived from FACS of freshly dissociated retinal epithelial cells from flo and sibling wild type larvae. Sorting performed as described in Reduced cell proliferation in the (26 KB PDF)Click here for additional data file.Text S1Supplementary data.(47 KB DOC)Click here for additional data file.


Arabidopsis includes more than 130 genes with an embryo-defective (emb) mutant phenotype. Many of these essential genes remain to be cloned. Hundreds of additional EMB genes have been cloned and catalogued (www.seedgenes.org) but not mapped. To facilitate EMB gene identification and assess the current level of saturation, we updated the classical map, compared the physical and genetic locations of mapped loci, and performed allelism tests between mapped (but not cloned) and cloned (but not mapped) emb mutants with similar chromosome locations. Two hundred pairwise combinations of genes located on chromosomes 1 and 5 were tested and more than 1100 total crosses were screened. Sixteen of 51 mapped emb mutants examined were found to be disrupted in a known EMB gene. Alleles of a wide range of published EMB genes  were discovered. Two EMS mutants isolated 30 years ago, T-DNA mutants with complex insertion sites, and a mutant with an atypical, embryo-specific phenotype were resolved. The frequency of allelism encountered was consistent with past estimates of 500 to 1000 EMB loci. New EMB genes identified among mapped T-DNA insertion mutants included CHC1, which is required for chromatin remodeling, and SHS1/AtBT1, which encodes a plastidial nucleotide transporter similar to the maize Brittle1 protein required for normal endosperm development. Two classical genetic markers  were identified based on similar map locations of known genes required for thiamine (THIC) and chlorophyll (PDE166) biosynthesis. The alignment of genetic and physical maps presented here should facilitate the continued analysis of essential genes in Arabidopsis and further characterization of a broad spectrum of mutant phenotypes in a model plant.The classical genetic map of  Hundreds of mutants with a wide range of defects in seed development have been described www.seedgenes.org. This database includes more than 350 genes and 600 mutant alleles emb mutants disrupted in genes that remain to be identified is not available. An initial dataset of 250 emb mutants analyzed in our laboratory, including many that have not been cloned, was published 15 years ago Arabidopsis Biological Resource Center (ABRC) and can be found at TAIR, the central Arabidopsis database (www.arabidopsis.org). Another 1400 mutants derived from T-DNA insertion lines Embryo-defective  mutants analyzed in our laboratory EMB genes defined by mutation is around 30% to 50% saturated as potentially indicated by recent estimates, then a similar percentage of mapped (but not cloned) mutants should be allelic to cloned (but not mapped) mutants. We describe here the results of a study focused on the two largest chromosomes (1 and 5) that pursued this approach in considerable detail. This work provided us with an opportunity to update the classical genetic map, integrate this map with the known physical map, uncover the identities of 16 mapped EMB genes not previously cloned, identify four new EMB genes from mapped mutants tagged with T-DNA, and provide further information on the total number of genes with a loss-of-function phenotype affecting seed development in Arabidopsis.One valuable resource of Arabidopsis was published 25 years ago by Koornneef et al.2 plants produced from self-pollination of heterozygotes. Other genes with mutant phenotypes were added to the map over the years. Precise gene orders were often left unresolved because map positions were typically determined by comparing two-point recombination frequencies rather than by analyzing definitive three-point backcrosses. The most recent version of the map published 10 years ago The first comprehensive genetic map of EMB genes have been cloned, even when results of the present study are included. Over 90 mapped EMB genes remain to be cloned, including 34 on the two chromosomes examined here in detail. These represent promising candidates for EMB genes not represented in the SeedGenes database. et al. Three hundred and thirty-five genes are included on the updated genetic map summarized in Arabidopsis genes with a mutant phenotype of any kind A complete list of 335 genes included on the updated map, along with known locus numbers, is presented in EMB genes that remained to be cloned. This information was required before we could compile lists of cloned emb mutants that were logical candidates for allelism tests with mapped emb mutants localized to similar regions of the genome. First we needed to plot the estimated genetic position versus known physical location for all mapped and cloned genes regardless of mutant phenotype. We decided against using sequence coordinates to track physical distances because this value was cumbersome and not readily derived from the locus number. We also chose not to use locus numbers to measure physical distance, in part because this made it difficult to visualize the number of genes within a defined physical region. We devised instead a simple measure of physical distance, the estimated gene number (EGN), which was derived by subtracting 1000 from the locus number, because for chromosomes 1, 2, 3, and 5, locus numbers begin with 01010 and not 00010, and then dividing by ten because locus numbers for adjacent genes are typically separated by ten. The EGN for At1g02580 is therefore 158 (02580 – 01000 divided by 10). For chromosome 4, we simply dropped the final digit because locus numbers on that chromosome begin with 00020. Although the EGN is an imperfect measure of physical distance, it can be easily derived, readily visualized, and has minor inconsistencies when compared with genetic map distances. The utility of this measure of physical distance was also confirmed by the results described here.We decided to update the genetic map and identify additional examples of mapped genes already cloned in order to estimate more precisely the physical locations of mapped 2 = 0.930 vs 0.928 and 0.959 vs 0.952, chromosome 1a, 1b; and 0.945 vs 0.934 and 0.921 vs 0.924, chromosome 5a, 5b) when physical distance was determined using the sequence coordinate midpoint (TAIR 7.0) of each mapped gene instead of EGN. These results provide further confirmation of the validity of the approach used here to estimate physical distance. Information about mapped genes flanking each centromere is presented in PDS1 (At1g06570), VAR2 (At2g30950), FPA (At2g43410), TT6 (At3g51240), and FAS2 (At5g64630). Placement of these loci on the genetic map appears to be inaccurate but the gene identities are correct. Another mapped locus (CHL2) with contradictory mapping information and a questionable gene assignment was also removed.Scatter plots showing the relationship between genetic and physical distances are shown in emb mutants was to limit complementation tests to mutants disrupted in genes that mapped within 5 cM of each other EMB genes that were 200 EGN above or below the projected physical location of a mapped (but not cloned) EMB gene based on data presented in The approach we took in the past when looking for duplicate alleles among mapped EMB genes that appeared to be located in the vicinity of each mapped EMB, we next asked whether the two mutant phenotypes appeared to be consistent with potential allelism. This was often difficult to resolve with confidence because the intragenic locations of mutations in mapped (but not cloned) emb mutants remained unknown. However, we usually excluded combinations that seemed unlikely, such as a cloned, putative null allele that exhibited a much later embryo phenotype than a mapped, untagged mutant. We also included some questionable combinations, especially when there were few other candidates available. Nevertheless, some of the mapped mutants that were tested but remain unresolved may be disrupted in a known EMB gene that was simply not chosen for crosses.After identifying the cloned 1 siliques with the expected frequency of defective seeds are listed in EMB genes) suggested a level of saturation (30%) consistent with past estimates of 1000 total EMB genes Approximately 200 sets of complementation tests and more than 1100 individual crosses involving pairwise combinations of candidate alleles were performed. Fifty-one mapped loci  and 115 cloned loci  were included. Crosses that failed to demonstrate allelism are summarized in EMB genes as revealed through genetic complementation tests are presented in emb9 and emb16) were part of the initial collection of embryo lethals described in the 1970s by Meinke and Sussex EMB genes emb64) was identified from a collection of X-irradiated seeds generated in the 1980s by Joseph Ecker emb24, emb36, emb71) identified about the same time were part of a widespread effort to characterize the response of mutant embryos in culture EMB156, EMB86, EMB161) were shown before The identities of 16 mapped EMB2284, EMB2775) and translation (EMB1030) to signal transduction (YDA), folate biosynthesis (GLA1), and chloroplast protein import . Ankyrin repeat proteins , PPR proteins , cysteine and aspartic proteases , and proteins with uncertain cellular functions  are also included. Duplicate mutant alleles for 11 of these cloned genes have been isolated and characterized in considerable detail EMB loci.Most of the cloned gene identities presented in emb24) was particularly intriguing because mutant embryos cultured on a basal medium developed into viable plants that produced 100% defective seeds following self pollination EMB24 likely performed an embryo-specific function. This hypothesis has now been confirmed by demonstrating that emb24 is disrupted in AtASP38, which encodes an embryo-specific aspartic protease that has been postulated to limit programmed cell death during reproductive development emb24 allele exhibits a consistent seed phenotype (linear cotyledon stage embryo) and a simple inheritance pattern . In contrast, the AtAsp38 allele exhibits a combination of embryonic and gametophytic lethality, a wide range of seed phenotypes, and a complex inheritance pattern. The new allele described here appears to have more residual function than the original AtAsp38 knockout, which may be useful in future efforts to understand the precise role of this gene in plant growth and development.One mutant studied 20 years ago (emb88) that was problematic in the past EMB gene positioned on chromosome 1. This gene has now been identified through allelism tests as EMB2279, which encodes a pentatricopeptide repeat (PPR) protein required for normal embryo development. The abundance of these putative RNA binding proteins among collections of cloned EMB genes has been noted before emb88 chromosomal rearrangement ArabidopsisAnother example of an informative allele identified here is a tagged T-DNA mutant (emb71), described before as external YODA, a gene that has received considerable attention in recent years. This locus encodes a component of a MAP kinase cascade that promotes proliferation of the basal cell lineage (suspensor) during embryo development YODA in the zygote is activated by the product of another gene (SSP) with an unusual parent-of-origin effect in which a transcript produced transiently in mature pollen is not translated until after fertilization emb71 allele identified here increases the diversity of yoda loss-of-function mutants available. The dramatic seed phenotype is shown in emb71), where wild-type seeds are consistent in size and shape, than in the Landsberg (erecta) accession . The yda-1 allele obtained from ABRC (CS6392) also segregated for a second (unlinked) emb mutation with a white seed phenotype. Two classes of mutant seeds were identified in siliques of emb71 heterozygotes. One was dark green, often escaped detection unless seeds were dissected, and contained a distorted linear embryo with rounded cotyledons and a reduced hypocotyl. The second class was composed of pale yellow seeds of varying sizes and a disorganized embryo positioned at the base of the seed. This class gave rise to the external embryo phenotype later in development. Both classes were observed in F1 siliques produced from genetic complementation tests. We could not determine whether the external embryo phenotype resulted from unusual expansion of a developing embryo stuck in the micropylar region of the seed without a normal suspensor or from inappropriate activation of a degradation pathway within the seed coat.The mapped mutant with the most unusual phenotype  PCR was used to recover plant sequences flanking T-DNA inserts in several mapped emb86 were particularly interesting because two different TAIL products were confirmed from chromosome 5. One identified the EMB1030 locus (At5g22800) responsible for the mutant phenotype; the other revealed a linked locus (At5g20935) at the expected distance inferred from genetic analysis EMB/EMB) transplants observed following selection on kanamycin EMB locus (At5g22800) and a linked insert in another gene (At5g20935) not required for seed development.Results obtained with EMB genes identified among mapped insertion lines were of particular interest because weak alleles with post-embryonic phenotypes had been described before EMB104; SHS1; AtBT1) involved two emb mutants with similar map locations that were known to be allelic emb104-1); the other  was generated with EMS. We used TAIL-PCR to recover flanking sequence from emb104-1 and direct PCR to confirm that the insert was located in the third (last) exon of At4g32420, which encodes a nucleotide transport protein. Another T-DNA mutant (shs1) with an upstream insertion site exhibits a salt-hypersensitive, ABA-insensitive phenotype that was at first attributed to a complete loss of function of a protein thought to be found in the endoplasmic reticulum emb104. The essential nature of this gene has therefore been confirmed. Terminal phenotypes of the SALK and GABI mutants were similar; arrested embryos were pale and ranged from the globular to elongate (triangular) stages of development. Giant suspensors and twin embryos were occasionally found. The phenotypes of both mapped alleles included distorted embryos at more advanced (linear cotyledon) stages of development, consistent with a somewhat higher level of normal gene function. Some of these embryos featured single or multiple cotyledon initials.Two emb262) contained a T-DNA insert localized within the 5′UTR of a gene  known to play an important role in chromatin remodeling Agrobacterium-mediated root transformation emb262 seed phenotype because additional insertion lines were not available and plants homozygous for the RNAi construct did not exhibit a consistent defect in seed development. We suspect that the 35S promoter controlling RNAi expression was not active at a level sufficient to inhibit embryo development. Perfect linkage was nevertheless found between a kanamycin resistance marker and the mutant phenotype (0 EMB/EMB plants among 204 resistant transplants screened), consistent with T-DNA tagging emb262 phenotype is somewhat unusual in that mutant embryos, which reach the cotyledon stage of development, are paler than expected based on the appearance of mutant seeds. Whether these embryos can be rescued in culture and produce plants that are resistant to Agrobacterium-mediated root transformation remains to be determined.Another tagged mutant  after completing the map alignments shown in emb) or pigment-defective embryo (pde) mutant in the SeedGenes collection. By crossing alb1 heterozygotes to four potential alleles  based on similar map locations, we showed that ALB1 is identical to PDE166, which encodes the CHLD subunit of the Mg-chelatase enzyme involved in chlorophyll biosynthesis ALB1 gene has therefore been identified.One future application of this work may be found in associating classical genetic loci that remain to be cloned with genes examined through reverse genetics that exhibit similar loss-of-function phenotypes. We encountered one such example while updating the genetic map. The alb1 mutant allele (V157) was obtained from a collection of chlorina (yellow-green rosette) mutants generated by Robbelen starting in the 1950s angustifolia genetic marker alb1 allele we examined (ABRC stock CS26), which appears to define the null phenotype, was first used by Van der Veen and colleagues uvh1) plants pde166  insertion alleles from the Syngenta collection ch-42; cs; chli2) with milder defects The original Arabidopsis were recently tracked by constructing a high-resolution genetic map of single feature polymorphisms based on whole-genome array hybridization of recombinant inbred lines We did not attempt here to document localized differences in recombination rates across the entire genome. This has been addressed before in a number of comprehensive studies involving humans Arabidopsis, which is limited to genes with mutant phenotypes mapped relative to each other, has outlived its usefulness as a reference for genetic analysis. The specific application described here represents a notable exception. Unlike other model organisms where the genetic map was constructed decades before genome sequencing was initiated and efficient methods of gene isolation were developed, the Arabidopsis classic map enjoyed a rather brief period of influence. Few genes have been added to the map in recent years and most new mutants are localized relative to molecular markers on the physical and recombinant inbred maps or recovered from T-DNA insertion lines amenable to rapid gene identification without mapping. Furthermore, the orders of many closely-linked genes on the classical map are inconsistent with their known physical locations, and there is no simple solution to this problem.In some respects, the classical genetic map of Arabidopsis, which we concluded would eventually replace the classical genetic map ArabidopsisSeveral years ago, we published the first sequence-based map of genes with mutant phenotypes in EMB loci examined in our laboratory were obtained from internal stocks. Duplicates of these stocks are available through the Arabidopsis seed stock centers at Columbus, OH (ABRC) and Nottingham, UK (NASC). Several additional SALK tps1 ; dom1, zeu1, cyl1, cyl2 ; oep80 ; win1 ; and pas2 . Seeds for EMB3000 series mutants were obtained from Fumiyoshi Myouga and Kazuo Shinozaki .Mutant alleles used in genetic complementation tests are listed in In order to track germination rates, observe segregating seedling phenotypes, and produce uniform populations of plants, we first plated mature seeds on a germination medium composed of Murashige and Skoog salts , 3% glucose, and 0.8% agar. Seeds were surface-sterilized by a 30 sec exposure to 95% ethanol followed by a 6 min treatment with 50% Clorox (containing 1 drop of Tween 20 detergent per 10 ml), and then several rounds of washing with sterile water. Plates were stored in the refrigerator (4°C) for 2 days before being placed under fluorescent lights (16 hr light/8 hr dark cycles) at room temperature (23°C). After 14 to 17 days, seedlings were transplanted to pots containing a mixture of soil, sand, and vermiculite, watered daily with a nutrient solution, and maintained under long days in a plant growth room as described by Berg et al. www.seedgenes.org/tutorial.html). Late floral buds with non-dehiscent anthers  were carefully emasculated with fine-tipped (Inox No. 4) forceps under a Wild (M7) dissecting microscope. A bent paper clip was often used to position the inflorescence on the microscope stage. Mature anthers from other heterozygous plants  were then applied to the stigma surface of the emasculated bud, covering it with pollen. Two or three open flowers immediately below the cross were removed to minimize pollen contamination. The number of flowers removed was recorded and each stem used for a cross was marked with colored thread for tracking purposes. Excess lateral branches were removed from the female parent to help direct nutrients to the inflorescence with the cross. The plant was then placed under fluorescent lights in a Percival  plant growth chamber (AR-36L) and watered with the same nutrient solution used in the main growth room. We found that using a growth chamber improved the overall success rate of crosses performed, most likely due to increased humidity and light. The optimal time for screening each cross was dependent to some extent on the seed phenotype involved. However, most crosses were screened 10 days after the cross was performed. Complementation resulted in siliques with all normal seeds. Failure to complement resulted in siliques that continued to segregate for normal and mutant seeds. Three siliques were examined per inflorescence: the expected cross and one silique above and below. Typically, three sets of reciprocal crosses were performed for each cross combination to increase the reliability of results obtained.Desired heterozygotes were first identified by screening siliques from selfed plants for the presence of mutant seeds with the expected phenotype as explained in the tutorial section of the SeedGenes website (Five cauline leaves from heterozygous plants germinated on a selection medium containing 50 µg/ml kanamycin were collected and genomic DNA was extracted using a modified cetyl trimethyl ammonium bromide (CTAB) protocol Table S1EMB genes) not cloned.Classical genetic map loci (excluding (0.02 MB XLS)Click here for additional data file.Table S2Arabidopsis arranged by cM  or locus number (cloned genes).Classical genetic map of (0.11 MB XLS)Click here for additional data file.Table S3Estimated map locations of centromeric regions.(0.01 MB XLS)Click here for additional data file.Table S4Details on cross combinations that complemented .(0.05 MB XLS)Click here for additional data file.Table S5Details on cross combinations that failed to complement .(0.03 MB XLS)Click here for additional data file.Table S6PCR primers used in the analysis of T-DNA insertion mutants.(0.03 MB XLS)Click here for additional data file.Table S7emb mutants.Locations of T-DNA inserts in mapped (0.02 MB XLS)Click here for additional data file.


Peripheral blood lymphocytes from female patients with early breast cancer were examined before surgery for their ability to develop a primary antibody response in vitro against sheep red blood cells in soft agar cultures containing autologous plasma. After 6 days incubation, foci of proliferating hemolysin-forming cells surrounded by a lytic area were detected on the surface of the plates and counted with a dissection microscope; this response was antigen-dependent and antigen-specific. We applied this assay to a group of women suffering from early breast cancer and devoid of distant metastases. From our data, it appears that if all the patients are grouped together, cancer-bearing women produce somewhat fewer (P less than 0.05) haemolytic foci than healthy controls. However, division of the cancer patients into two subgroups, according to the TNM pretreatment clinical classification of regional lymph nodes, generated an interesting finding: N1 patients (N1b or N1a) produced definitely fewer foci than N0 patients, and the difference was highly statistically significant (P less than 0.001). The depression of anti sheep red blood cell antibody production observed in N1 patients was unrelated to the presence or absence of metastatic growth in their regional lymph nodes.


Trichomonas vaginalis has an unusually large genome (∼160 Mb) encoding ∼60,000 proteins. With the goal of beginning to understand why some Trichomonas genes are present in so many copies, we characterized here a family of ∼123 Trichomonas genes that encode transmembrane adenylyl cyclases (TMACs).Trichomonas strains (S1 and B7RC2). Each TMAC is composed of a series of N-terminal transmembrane helices and a single C-terminal cyclase domain that has adenylyl cyclase activity. Multiple TMAC genes are transcribed by Trichomonas cloned by limiting dilution.The large family of TMACs genes is the result of recent duplications of a small set of ancestral genes that appear to be unique to trichomonads. Duplicated TMAC genes are not closely associated with repetitive elements, and duplications of flanking sequences are rare. However, there is evidence for TMAC gene replacements by homologous recombination. A high percentage of TMAC genes (∼46%) are pseudogenes, as they contain stop codons and/or frame shifts, or the genes are truncated. Numerous stop codons present in the genome project G3 strain are not present in orthologous genes of two other Trichomonas is the presence of unstable families of genes such as those encoding TMACs that are undergoing massive gene duplication and concomitant development of pseudogenes.We conclude that one reason for the unusually large genome of  Trichomonas vaginalis is the only medically important protist (single-cell eukaryote) that is sexually transmitted. The ∼160-Mb Trichomonas genome contains more predicted protein-encoding genes  than the human genome. To begin to understand why there are so many copies of some genes, we chose here to study a large family of genes encoding unique transmembrane cyclases. Our most important results include the following. More than 100 transmembrane cyclase genes do not result from chromosomal duplications, because for the most part only the coding regions of the genes, rather than flanking sequences, are duplicated. Almost half of the transmembrane cyclase genes are pseudogenes, and these pseudogenes are polymorphic among laboratory strains of Trichomonas. Messenger RNAs for numerous transmembrane cyclases are expressed simultaneously, and representative cyclase domains have adenylyl cyclase activity. In summary, the large family of Trichomonas genes encoding transmembrane adenylyl cyclases results from massive gene duplication and concomitant development of pseudogenes. Trichomonas vaginalis, the most important sexually transmitted protist, causes vaginitis in women and urethritis in men Trichomonas increases the risk of HIV transmission, pelvic inflammatory disease, and spontaneous abortion Trichomonas lives under microaerophilic conditions in the lumen of the vagina by means of fermentation enzymes that are present in a modified mitochondrion called the hydrogenosome Trichomonas causes vaginitis when the protist adheres to the host epithelium and changes from a flagellated to an ameboid form Trichomonas genome encoding ∼60,000 proteins Trichomonas genome is the presence of hundreds of DNA transposons that include mariner elements and Mavericks Trichomonas. Introns are rare and short, so the presence of large non-coding regions in Trichomonas genes cannot be an explanation for the large genome size Recent whole genome sequencing showed an ∼160-Mb Trichomonas genes are present in so many copies and focused on one a large family of predicted transmembrane adenylyl cyclases (TMACs). These TMACs are of particular note because (1) they have a predicted topology different from those of other metazoan and protist transmembrane cyclases, and they appear to have originated via gene duplication in Trichomonas and closely related species  We were interested in why some Trichomonas vaginalis strain G3 has been sequenced to ∼6× redundancy, so that it is likely that the majority of genes have been predicted Trichomonas present at the NCBI or at TrichDB Dictyostelium discoideum, Homo sapiens, and Trypanosoma brucei, as well as those of the TMGCs of Homo sapiensTrichomonas TMAC protein sequence (TVAG_350120) and BLASTP to search the predicted proteins of Trichomonas or used this TMAC and TBLASTN to search Trichomonas scaffolds in the database at J. Craig Venter Institute (JCVI) or the WGS database at the NCBI. Intact TMAC genes, apparent TMAC pseudogenes (see below), and partially sequenced TMAC genes due to assembly problems are listed in Tritrichomonas foetus and Pentatrichomonas hominis.The genome of Transmembrane helices (TMHs) of TMACs were predicted using the Phobius combined transmembrane topology and signal peptide predictor Trichomonas scaffolds As described above, phylogenetic trees were drawn using cyclase domains to determine the number of ancestors for the present set of TMAC genes. To determine whether duplication of segments of chromosomes contributed to the large number of copies of TMAC genes, we aligned whole scaffolds  containing TMAC genes with each other Trichomonas at the JCVI or NCBI. Pseudogenes contained in-frame stop codons (nonsense mutation) and/or frame shifts that we could confirm by examining multiple independent primary sequence reads. In addition, we amplified the DNA around numerous of these stop codons by PCR to confirm their presence in the genome project G3 strain and to assess their occurrence in the B7RC2 and S1 strains. We also mapped the location of the various stop codons and frame shifts to determine whether any of them were present in more than one TMAC gene. This result would suggest that a pseudogene was duplicated. TMAC genes that were incomplete because they were at the edge of a contig were not considered pseudogenes.To identify TMAC pseudogenes, we took advantage of the absence of introns in any of the TMAC genes and the strict conservation of N-terminal TMHs and C-terminal cyclase domain in the predicted transmembrane cyclases Trichomonas, many of which were given different names , were identified as paralogs or orthologs of the complete TMAC (TVAG_350120). TMAC pseudogenes were strongly suggested when these paralogs were present in an array of short proteins that spanned the length of a complete TMAC gene. In this case, the in-frame stop codons and/or frame shifts could be inferred by the prediction of multiple short proteins rather than a single full-length protein. Because stop codons and frame shifts in these pseudogenes identified using the paralog data base were not checked versus single reads, these pseudogenes are listed as putative in File S1.Additional pseudogenes were identified using the paralog and ortholog function at TrichDB While TMAC pseudogenes were identified by inspection, pseudogenes in cyclic nucleotide phosphodiesterases and other proteins in Trichomonas vaginalis, was received from Dr. B. N. Singh , while the genome project G3 strain and B7RC2 strain were from Patricia Johnson (UCLA). Trichomonas was grown at 37°C and sub-cultured every 24 hr in TYI-S-33 medium containing 10% adult bovine serum Trichomonas was diluted in medium to 102–3 cells/ml and cloned on plates containing 0.6% agarose Trichomonas was grown for seven days under anaerobic conditions. Individual clones were picked and sub-cultured in liquid medium in 48-well tissue culture plates, and RNA was isolated as described in the next section.The S1 strain of Trichomonas RNA isolated using the RNAqueous-4PCR kit (Ambion) was treated with DNAse1 for 1 hr at 37°C. First strand cDNA synthesis was performed with RETROscript (Ambion), using oligo dT primers for 1 hr at 42°C on ∼1 g RNA. PCR of Trichomonas cDNAs was performed using SYBR Green Master Mix with Rox from Roche Applied Science. Reverse transcriptase and template were separately omitted from negative controls, while primers to an actin gene (TVAG_094140) were positive controls for RT-PCR. For primer sequences used in the RT-PCRs, please see Total 6) of Trichomonas, using the Wizard Genomic DNA purification kit (Promega). PCR primers were designed to isolate representative DNAs encoding cyclase domains of two Trichomonas TMACs (TVAG_013980 and TVAG_456550). These PCR products were cloned into the pGEX-6p vector (Amersham Biosciences) Escherichia coli BL21 cells transformed with pGEX-6p were grown in LB medium and induced with 1 mM IPTG for 3 hrs at 30°C. Recombinant glutathione-S-transferase (GST)-cyclase fusion-proteins were purified with glutathione-agarose beads and released with 10 mM glutathione.Genomic DNA was isolated from one confluent flask  Using cyclase domains from TMACs of chomonas  [8], , [3Trichomonas cAMP phosphodiesterase genes also appears to be independent of flanking sequences or repetitive elements (data not shown).In about a dozen occasions, two TMAC genes each have the same flanking sequences that contain multiple open reading frames and short segments of repetitive DNA . In the Trichomonas TMAC genes (∼46%) are pseudogenes, as they contain stop codons and/or frame shifts (the vast majority) or are truncated (the minority)  . With onStop codons in both groups A and B are less frequent in regions of the TMAC genes that encode the conserved domain of unknown function and cyclase domain . A possiTrichomonasWhile the transmembrane cyclases have the highest percentage of pseudogenes (46%), 32% of ABC family transporters appear to be pseudogenes . Other gTrichomonas strains (S1 and B7RC2)  are present in orthologous genes of two other d B7RC2) . This rerecently . Finallyrecently . Strict Giardia, Plasmodium, and TrypanosomaGiardia and Plasmodium variant expression occurs in part because there are different adherence functions to the surface proteins. Similarly, Trichomonas TMACs may have different functions in signal transduction. To begin to answer this question, we prepared mRNAs from two clones of Trichomonas that were isolated on soft agar Trichomonas clone . Variant expression has been described for surface antigens of as clone . We useds widely . We founTrichomonas transmembrane cyclases, one arbitrarily chosen from group A (TVAG_456550) and one from group B (TVAG_013980), were expressed as glutathione-S-transferase (GST)-fusion enzymes in bacteria and incubated with ATP or GTP Trichomonas cyclase showed adenylyl cyclase activity but no measurable guanylyl cyclase activity. For the group A cyclase, the Km for ATP is 520±10 µM, and the specific activity is 6.1×10−12 mol/min/µg. For the group B cyclase, the Km for ATP is 710±10 µM, and the specific activity is 8.5×10−12 mol/min/µg. We conclude that the Trichomonas transmembrane cyclases are adenylyl cyclases and have similar kinetics.Two cyclase domains from TrichomonasThe very large genome of Trichomonas proteins, we could not determine an overall rate of pseudogenes in Trichomonas. Based on the data in Trichomonas pseudogenes is at least 5%. In GenBank there are 1354 Trichomonas genes annotated as pseudogenes  Trichomonas pseudogenes include 97 BspA genes, 42 kinases, 227 ankyrin repeat proteins, and 696 hypotheticals. However, only 5 of the 56 TMAC pseudogenes identified here are annotated as such in GenBank, suggesting the number of Trichomonas pseudogenes has been grossly underestimated. Regardless, the percentage of pseudogenes in Trichomonas is much greater than the percentages of pseudogenes (<0.1% in each) of protists with a similar microaerophilic life-style (Giardia and Entamoeba) Trypanosoma cruzi and Trypanosoma brucei that show variant expression Because we were unable to make good models for many of the unique Trichomonas. The TMAC pseudogene sequences provide more precise information than methods that use restriction fragment length polymorphisms or pulse-field gel electrophoresis Trichomonas strains, as has been suggested Trichomonas, the protist appears to have some of the conserved machinery for meiosis Giardia, another microaerophilic protist, suggest there is reassortment of markers consistent with sex Stop-codons of TMAC pseudogenes are surprisingly polymorphic  and 4 miTrichomonas cAMP-mediated signal transduction system predicted here differs in two fundamental ways from those of metazoans and DictyosteliumTrichomonas TMACs and cyclic nucleotide phosphodiesterases are unique. Second, Trichomonas TMACs and cyclic nucleotide phosphodiesterases are present in more copies than in metazoans, while predicted Trichomonas G protein-coupled receptors (GPCRs) are fewer than in metazoans (data not shown) Trichomonas may be explained by their rapid duplication and concomitant conversion to pseudogenes, we cannot easily explain the relative paucity of GPCRs in Trichomonas. One possible explanation for the low rate of GPCRs is that the heterotrimeric G-proteins are activated independent of GPCRs, as has been noted in Caenorhabditis elegansThe Trichomonas genes lack introns Trichomonas BspA genes are arranged in clusters with as many as 17 genes, consistent with several tandem duplication events The absence of synteny around most TMAC genes  suggestsTrichomonas) (The present studies cannot determine whether the TMAC pseudogenes are “junk” or have some function homonas)  and 3, Thomonas)  may be iTrichomonas gene families do not have nearly the percentage of pseudogenes (46%) observed in TMAC genes Because there are so many TMAC genes, we assume that they play a role in pathogenesis Trichomonas, we are a long way from relating these findings to pathogenesis.In summary, while the bioinformatic and experimental methods here have generated numerous novel findings concerning gene duplication and pseudogene development in Data S1Best estimate of the number of TMAC genes and pseudogenes.(0.04 MB DOC)Click here for additional data file.Data S2Trichomonas TMAC genes.Primers used for RT-PCR of (0.03 MB XLS)Click here for additional data file.Figure S1Trichomonas TMACs shows conserved domains. Seventy TMAC sequences were aligned, and the amino acid conservation (shown by the height of each position) was determined using WebLogo Sequence logo of aligned (1.20 MB TIF)Click here for additional data file.Figure S2Trichomonas. Pseudogenes are marked in red, while incomplete genes due to assembly issues are marked in grey. Branch lengths are proportional to differences between sequences, while numbers at nodes indicate boot strap support for 100 iterations. Nodes with less than 50% support are collapsed.This figure, which complements (0.37 MB TIF)Click here for additional data file.Figure S3Trichomonas transmembrane cyclase genes.This figure, which complements (0.23 MB TIF)Click here for additional data file.


The human endometrium undergoes cyclical regeneration throughout a woman's reproductive life. Ectopic implantation of endometrial cells through retrograde menstruation gives rise to endometriotic lesions which affect approximately 10% of reproductive-aged women. The high regenerative capacity of the human endometrium at eutopic and ectopic sites suggests the existence of stem/progenitor cells and a unique angiogenic system. The objective of this study was to isolate and characterize putative endometrial stem/progenitor cells and to address how they might be involved in the physiology of endometrium.in vitro, whereas in the same medium, EMP cells differentiated only into stromal cells. Furthermore, ESP cells, but not EMP cells, reconstituted organized endometrial tissue with well-delineated glandular structures when transplanted under the kidney capsule of severely immunodeficient mice. Notably, ESP cells generated endothelial cells that migrated into the mouse kidney parenchyma and formed mature blood vessels. This potential for in vivo angiogenesis and endometrial cell regeneration was more prominent in the ESP fraction than in the EMP fraction, as the latter mainly gave rise to stromal cells in vivo.We found that approximately 2% of the total cells obtained from human endometrium displayed a side population (SP) phenotype, as determined by flow cytometric analysis of Hoechst-stained cells. The endometrial SP (ESP) cells exhibited preferential expression of several endothelial cell markers compared to endometrial main population (EMP) cells. A medium specific for endothelial cell culture enabled ESP cells to proliferate and differentiate into various types of endometrial cells, including glandular epithelial, stromal and endothelial cells These results indicate that putative endometrial stem cells are highly enriched in the ESP cells. These unique characteristics suggest that ESP cells might drive physiological endometrial regeneration and be involved in the pathogenesis of endometriosis. Retrograde shedding and ectopic implantation of menstrual endometrial cells and tissue fragments give rise to endometriotic lesions outside of the uterus. Endometrial cells prepared from the human endometrium are also capable of reconstituting functional endometrium in xenograft models of endometriosis +PDGFRβ+ stromal cells +CD73+CD90+ stromal cells in vitro. No studies have yet explored the in vivo regenerative capacity of these putative endometrial stem/progenitor cells. Candidate tissue-specific stem cells have been identified in several tissues based on the SP phenotype. This characteristic is due to the unique ability of the primitive cells to pump out the DNA binding dye Hoechst 33342 via the ATP-binding cassette transporter G2 (ABCG2) in vivo when xenotransplanted into the uteri of NOG mice Several groups have identified a number of endometrial cell subpopulations as candidate endometrial stem/progenitor cells. These include clonogenic endometrial cells in vivo regeneration assay and SP isolation procedure to characterize the properties of human endometrial SP (ESP) cells. These cells were able to differentiate into endometrium-like tissue and a variety of endometrial cell components when xenotransplanted into NOG mice. This is the first in vivo evidence in support of the existence of stem/progenitor cells in the ESP.In the present study, we adapted our We first dissociated human endometria mechanically and enzymatically and purified epithelial-enriched and stromal-enriched fractions as previously described We found that each preparation contained a small subset of cells in the SP fraction . SP cellWe next examined whether the proportion of ESP cells varied across the menstrual cycle. As shown in in vivo, we transplanted ESP cells under the kidney capsule of ovariectomized NOG mice, after which they were treated with a 17β-estradiol (E2) pellet for eight to ten weeks. As was seen in the endometrial regeneration model To investigate the stem cell-like regenerative capabilities of ESP cells + cells were dominant, together with some hVm+ cells migrating into the mouse kidney parenchyma (+ cells were positive for CD31 (+ endothelium was surrounded by a layer of ESP-derived smooth muscle cells positive for α-smooth muscle actin (αSMA)  antibody (clone V9) . As V9 crenchyma . We conffor CD31 . Furthern (αSMA) . In the n (αSMA) . This tyer, CD13 . Notablyer, CD13 , as the 2 stimulation. In agreement, PR was expressed prominently in the E2-exposed endometrial tissue (The glandular structure of the well-organized tissue was positive for cytokeratin (CK), an epithelial marker . The regl tissue . These rl tissue . Indeed,l tissue .in vitro, we attempted to cultivate and expand the cells using conventional media supplemented with various hormones and/or growth factors; however, ESP cells alone never proliferated efficiently. Furthermore, while SP cells derived from human myometrium are able to proliferate preferentially under hypoxic condition in vitro in conventional media  was significantly less than that of EMP cells . It has Immunofluorescence studies revealed three distinct types of colonies in the ESP cultures. The most frequent type was comprised of fibroblastoid stromal cells positive for CD13, an endometrial stromal cell marker . The secTo further characterize ESP cells, gene expression analysis of ESP and EMP cells was performed using RT-PCR. ESP cells preferentially expressed mRNA of universally SP-associated markers, ABCG2 and multidrug resistance 1 in vivo capacity to preferentially give rise to ECs  containing ESP cells can generate functional endometrial tissue comprising glands, stroma, immune cells and vascular components when they are transplanted under the kidney capsule of severely immunodeficient mice functionalis and basalis layers of the human endometrium et al. demonstrated that endometrial surface epithelial regeneration takes place as a consequence of cellular differentiation from stromal cells and not by direct extension from the residual basal epithelial glands basalis but also in the functionalis endometrium.Human and primate endometrium regenerates from the lower functionalis endometrium may increase, presumably resulting in the decline in the proportion of ESP cells from menstruation towards the late secretory phase.The percentage of SP cells derived from cultured epithelial-enriched fraction was significantly greater during menstruation than at any other cycle stage in vivo as well as in vitro culture. Successful proliferation of ESP cells in the presence of EMP cells in conventional media  onto the deconstructed eutopic endometrium. In both eutopic and ectopic implantation, endothelial ESPs might give rise to various endometrial cell components in the process of ESP-driven angiogenesis. Our eutopic reimplantation hypothesis does not contradict the current paradigm but rather provides an additional mechanism for endometrial regeneration. We describe previously that a certain type of cells in endometrium could migrate, invade, form chimeric vasculature in the host kidney of NOG mouse and establish the functional circulatory systemIn view of these findings, we here propose a single model for ESP-driven endometrial regeneration and establishment of endometriosis . In thisWe have shown that ESP cells, but not EMP cells, express ERβ but do not express ERα or PR. A similar expression pattern is observed in endometrial vascular ECs which express ERβ, but neither ERα nor PR in vitro and in vivo. In our hands, ESP cells were not able to proliferate from a single cell in vitro. Without single cell analyses, it remains uncertain whether individual ESP cells posses multi-lineage differentiation potential at the clonal level or, instead, the cells consists of a mixed population of progenitors or stem cells. The study of the ESP, however, will improve the understanding of endometrial physiology and provide insight into the pathogenesis and treatment of endometrium-derived diseases such as endometriosis.In summary, we have demonstrated that undifferentiated ESP cells are present in human cycling endometrium. Purified ESP cells, but not EMP cells, contain putative endometrial stem/progenitor cells with potentials for differentiation into multiple types of endometrial cells Detailed protocols can be found in in situ. Written informed consent was obtained from each patient and the use of these human specimens was approved by the Keio University Ethics Committee.Endometrial specimens (n = 78) were collected from women with normal menstrual cycles undergoing total abdominal hysterectomy for benign gynecological diseases, or cervical carcinoma +) and suspended at 2×106 cells/mL in HBSS+ and stained with 5.0 µg/mL Hoechst33342  for 90 min at 37°C, as described previously +. Cells were incubated on ice for 30 minutes, pelleted, and washed with HBSS+. The antibodies we used are listed in SI Methods. After collecting 1×105 events, the SP population was defined as previously reported Endometrial specimens were separated and dissociated into endometrial stromal and glandular epithelial single cell fractions as described previously To track the fate of SP cells in co-culture with MP cells, the SP and MP cells were labeled with two different fluorescent dyes-Vybrant DiO (green fluorochrome) and Vybrant DiI (red fluorochrome) . These reagents allow two-color labeling of cell populations for identification after mixing and co-culture. DiI-labeled SP cells and DiO-labeled MP cells were mixed at a ratio of 1∶1 and co-cultured in DMEM containing 1% antibiotic-antimycotic, and 10% FBS.2 in 35 mm dishes and cultured in EGM-2MV medium . After 14 days of culture, clusters of cells were considered colonies when they were visible macroscopically and had more than 50 cells. Colonies were counted and the cloning efficiency (CE) was determined from the formula: CE (%)  =  (number of colonies/number of cells seeded) ×100.SP and MP cells were separately seeded at a clonal density of 400 cells/cmTotal RNA was extracted from cell cultures and subjected to RT-PCR. First-strand cDNA was synthesized and amplified using specific PCR primers . The PCR4 to 105 cells), freshly isolated from human endometria, were immediately transplanted under the kidney capsules of NOG mice as described previously The same numbers of SP and MP cells  or antibodies against Vm and CD13 (B). Bars, 100 µm.(4.16 MB TIF)Click here for additional data file.Figure S2Expression of hematopoietic stem cell marker and mesenchymal stem cell marker on ESP cells. Flow cytometric analysis of ESP cells stained with antibodies against hematopoietic stem cell markers (CD34 and CD133) and mesenchymal stem cell markers .(3.15 MB TIF)Click here for additional data file.Figure S3Remaining basalis layer of endometrium after endometrial tissue has been scraped off. Histological and immunofluorescence images of the uterine interface between the endometrium and myometrium stained with H&E or antibodies against CK and αSMA. Bars, 500 µm. Black and yellow arrowheads indicate endometrial glands present adjacent to or inside the myometrium.(0.96 MB TIF)Click here for additional data file.Table S1List of antibodies used for flow cytometric analysis.(0.03 MB DOC)Click here for additional data file.Table S2Sequences of the primers used for detection of various genes.(0.04 MB DOC)Click here for additional data file.Table S3List of antibodies used for immunofluorescence staining.(0.04 MB DOC)Click here for additional data file.Methods S1(0.07 MB DOC)Click here for additional data file.


Ewing's sarcoma's relapse rarely occurs more than two years after the initial diagnosis. We report the case of a 26-year-old man with a history of Ewing's sarcoma of the left maxillary sinus at the age of 10 who presented with a very late local relapse, 16 years after the first occurrence of disease. Ultimate control was achieved after multimodal therapy including surgery, high-dose chemotherapy, and radiotherapy. This report indicates that local relapses of Ewing's sarcoma can be treated with curative intent in selected cases. Ewing'ssarcoma usually occurs before 30 with a peak incidence at 15 years of age. Itrepresents 5% of all primary bone tumors and is the second most commonmalignant bone tumor in children. It is extremely rare in the head and neck asit represents 3% of osseous Ewing's sarcoma (OES) . ChildreA 26-year-old man self-referred for isolated moderately paced painless swellingof the left nasal ala with no physical status deterioration. His past medicalhistory was remarkable for Ewing's sarcoma of the left maxillary sinus at theage of 10  and Musculoskeletal Tumor Society (MSTS)staging systems, resp.). The patient had been treated according to theSociété Française d’Oncologie Pédiatrique (SFOP's) protocol with chemotherapy,hemimaxillectomy and palatine prosthetic reconstruction, and postoperativechemotherapy without radiation therapy because of the absence of residualviable tumor cells in the tumor resection specimen. The protocol wasadapted from the SFOP guidelines and consisted of (1) initial chemotherapy withcyclophosphamide-doxorubicin, (2)radical surgery if possible  were considered goodresponders and were omitted local radiotherapy), and (3) maintenance chemotherapy(for up to 9 months) with actinomycin-vincristine.Swelling had worsened over the past three months anddischarge appeared over the past month. Examination was consistent with a large mucocele of the left nose area. On computerized tomography (CT) scan, a 19-mL contrast-enhancedlesion with central sunburst pattern and calcifications  was noteMultimodality therapy for Ewing'ssarcoma is associated with markedly improved survival rates. Surgery followedby adjuvant radiotherapy and multiagent chemotherapy has dramatically improved fiveyears survival rates to 70%, compared with single—or even dual—modalitytherapy . In our Early metastatic relapse has the poorest prognosis . CollinsFive-year survival after relapse is less than 10% butselected patients benefit from aggressive salvage treatment . In partFinally, the patient is well and free of disease 15months after relapse. While followup following treatment of relapse is stillshort, intensive salvage multimodality treatment including radiotherapy forlocal relapse was feasible and ultimate tumor control was achieved.Such pastmedical history as Ewing'ssarcoma must not beoverlooked when facing apparently benign tumor since very late local relapse ispossible. Ultimate control can be achieved for local relapses of Ewing'ssarcoma in the adulthood with aggressive salvagetreatment protocols. Further data are needed to assess the role of aggressivelocal treatment.


Runt genes are central regulators of skeletogenesis. Runx2 was shown to be essential for osteoblast differentiation, tooth development, and bone formation. Both Runx2 and Runx3 are essential for chondrocyte maturation. Furthermore, Runx2 directly regulates Indian hedgehog expression, a master coordinator of skeletal development. To clarify the correlation of Runt gene evolution and the emergence of cartilage and bone in vertebrates, we cloned the Runt genes from hagfish as representative of jawless fish  and from dogfish as representative of jawed cartilaginous fish (ScRunx1–3). According to our phylogenetic reconstruction the stem species of chordates harboured a single Runt gene and thereafter Runt locus duplications occurred during early vertebrate evolution. All newly isolated Runt genes were expressed in cartilage according to quantitative PCR. In situ hybridisation confirmed high MgRunxA expression in hard cartilage of hagfish. In dogfish ScRunx2 and ScRunx3 were expressed in embryonal cartilage whereas all three Runt genes were detected in teeth and placoid scales. In cephalochordates (lancelets) Runt, Hedgehog and SoxE were strongly expressed in the gill bars and expression of Runt and Hedgehog was found in endo- as well as ectodermal cells. Furthermore we demonstrate that the lancelet Runt protein binds to Runt binding sites in the lancelet Hedgehog promoter and regulates its activity. Together, these results suggest that Runt and Hedgehog were part of a core gene network for cartilage formation, which was already active in the gill bars of the common ancestor of cephalochordates and vertebrates and diversified after Runt duplications had occurred during vertebrate evolution. The similarities in expression patterns of Runt genes support the view that teeth and placoid scales evolved from a homologous developmental module.The skeleton is one of the most important features for the reconstruction of vertebrate phylogeny but few data are available to understand its molecular origin. In mammals the  Runt gene family (Runx1–3) is of extraordinary importance for skeletogenesis. Runx2 deficient mice completely lack bone. Runx2 and Runx3 are essential for cartilage development and Runx2 regulates the key factor Indian hedgehog, which coordinates skeletogenesis. Here, we reconstructed Runt gene evolution in correlation to skeletal evolution. By analyzing lancelets, one of the closest living relatives of vertebrates, we revealed that the single Runt and Hedgehog family founder genes were co-expressed in primitive skeletal elements of the chordate stem species. Interestingly, at this stage the Runt and Hedgehog pathways were already directly linked to one another. Furthermore we isolated two Runt genes from a representative of jawless cartilaginous fish (hagfish) and three Runt genes from jawed cartilaginous fish (dogfish) which were all expressed in cartilage. The dogfish Runt genes were also found in teeth and placoid scales. This study suggests that Runt genes were involved in all ancient processes of chordate skeletogenesis. Furthermore the analysis supports the theory that most likely the gut was the tissue that originally secreted an acellular gill gut skeleton in the chordate ancestor.Important molecular mechanisms underlying mammalian skeletogenesis have been described but knowledge about the evolutionary origin of these gene networks is limited. The  The skeleton is a hallmark of vertebrates and has been widely used over the past decades for phylogenetic analyses Col2α1), which is also the characteristic matrix protein for jawed vertebrates (gnathostomes) Sox9, which directly regulates Col2α1 in mammals, was shown to be expressed in cartilage of the lamprey SoxE  was found to be co-expressed with fibrillar collagen in the hemichordate Saccoglossus bromophenolosus Descriptive data are available for the matrix proteins produced by the cells that constitute the skeleton in jawless vertebrates . Beside species specific proteins Runt gene expression has been described in skeletal elements of lancelets, agnathans and jawed cartilaginous fish in spite of the fact that Runt transcription factors (Runx1–3 synonyms: Aml1–3/Cbfa1–3/Pebp2αa–c) are central regulators of skeletal development in higher vertebrates Runt gene has two isoforms with different N-termini starting with a MASNS-like motif under the distal P1 promoter and a MRIPV sequence under the proximal Promoter P2. Furthermore the 3′ end has a conserved VWRPY-motif Runx2 is indispensable for osteogenesis as mice bearing a homozygous mutation in Runx2 completely lack bone Runx2 is together with Runx3 essential for cartilage differentiation Indian hedehog (Ihh), which coordinates cartilage differentiation, endochondral ossification and limb outgrowth Hedgehog (Hh) family  also Sonic hedgehog (Shh) signaling is influenced by Runx2 during tooth morphogenesis Runx2 haploinsufficiency causes the human bone disease cleidocranial dysplasia, further substantiating its importance for skeletal development Runt genes are expressed in cartilage and have been shown to play a role in the formation and differentiation of skeletal elements Runt genes in the mouse are involved in tooth formation Up to now no Hox genes, which are important for patterning, Runt genes are essential for features that represent evolutionary innovations of vertebrates such as bone Runt genes for skeletogenesis in higher vertebrates we hypothesized that these genes played a role in the evolution of cartilage, bone and teeth and thus might be instrumental to understand skeletal evolution in chordates. We therefore analyzed number and expression of Runt genes in hagfish (Myxine glutinosa) as a representative of jawless vertebrates, in dogfish (Scyliorhinus canicula) as a representative of cartilaginous fish and lancelets (Branchiostoma lanceolatum and B. floridae) as representatives of celphalochordates to reconstruct if Runt genes were already expressed in the developing skeleton of the chordate, vertebrate and jawed vertebrate stem species. In addition, we tested if Runt and Hh are co-expressed in lancelets and if a functional interaction between the Runt and Hh pathways might have evolved before the cellular cartilage of vertebrates evolved.In contrast to the extensively studied Runt gene, whereas three Runt genes were present before the emergence of gnathostomes. Runt genes are expressed in developing cartilage, teeth and placoid scales of cartilaginous fish and cartilage of jawless vertebrates. In adult lancelets the Runt gene is expressed together with Hh, in the endo- and ectoderm of the gill bars. Furthermore, we demonstrate that the lancelet Runt protein can directly bind to and activate the lancelet Hh promoter. This suggests that beside SoxE and fibrillar collagen two other key factors for vertebrate skeletogenesis (Runt and Hh) were part of an ancient gene network for skeletogenesis in the gill gut stabilizing the gill bars of the common ancestor of vertebrates and lancelets approximately 700 million years ago. Our finding that the gut is an ancient Runt expression domain of deuterostomes is in accordance with the hypothesis that endodermal secretion was the ancestral mode of making pharyngeal cartilage In this study we show that the stem species of chordates harboured a single Runt genes in lower vertebrates. This led to the detection of two Runt genes in hagfish  and three Runt genes in dogfish (ScRunx1–3). All of these newly detected Runt genes had a 3′ end with the characteristic VWRPY-motif. The two different 5′ ends of the Runt genes amplified from embryonal dogfish cDNA were homologous to the 5′ mammalian promoter variant-1 (MASNS-like) and variant-2 (MRIPV-like) motifs, respectively. In the two hagfish Runt genes amplified from adult hagfish cDNA only a single 5′ gene end was detected. According to our Blast searches against the Ensembl pre-genome sequences of lamprey (Pteromyzon marinus) the two hagfish 5′ ends represent most likely the promoter variant 2. Because of the unavailability of hagfish embryos it could not be clarified if two Runt gene promoter 1 variants are expressed during early hagfish development.We used a PCR-based approach using cDNA as well as genomic DNA to identify Runt gene in cnidarians (Nematostella vectensis), nematodes (Caenorhabditis elegans), cephalochordates (B. floridae), and tunicates  Runt genes in sea urchin (Strongylocentrotus purpuratus) Runt genes in skate (Raja eglanteria) Runt genes in mammals Takifugu rubripes) Danio rerio) including a duplicated Runx2 gene Gallus gallus) three Runt genes were found. An alignment of all newly detected Runt genes together with other deuterostome Runt genes is provided as supporting information  revealed that there are most likely two Runt genes in the lamprey genome, and one ormation  and the Runt loci Runt genes are followed by a Clic gene on the complementary strand. The chicken Runx1 on chromosome 1 is followed by a Clic6 homologous gene, the chicken Runx2 on chromosome 3 by a Clic5 homologous gene and the chicken Runx3 on chromosome 23 is followed by a Clic4 homologous gene. In lancelet the Runt and Clic genes are located on different scaffolds (JGI assembly vers 1.0). However, in the genome of the tunicate C. intestinalis a Clic homologous gene was found in proximity to Runt on chr_12q . This strongly suggests that the entire Runt locus was triplicated during the evolution of chordates.Comparable to the human Runt gene, whereas the last common ancestor of jawed vertebrates harboured three Runt genes. In addition our results indicate that the dogfish ScRunx1–3 genes are orthologous to the Amniota Runx1–3 genes. In contrast to this, the two hagfish Runt genes did not cluster with any of the three paralogous Runt genes from higher vertebrates. As outlined in Runt genes are a result of a Runt gene duplication in the stem species of vertebrates or evolved by a separate gene duplication event in the hagfish lineage.Our phylogenetic analysis  suggestsRunt genes in the skeleton we asked the question if Runt genes are expressed in skeletal elements of hagfish. Using quantitative Reverse Transcriptase PCR (qRT-PCR) from dissected tissues we found that the MgRunxA gene had its highest expression in hard cartilage, followed by the gill region and soft cartilage  a diffuse Runt staining, indicating a maternal Runt expression, was detected, comparable to the description of maternal Runx1, –2b, and –3 expression in zebrafish. Runt gene variant starting with exon 1  and the Runt gene variant starting with exon 2 . These two probes showed overlapping staining patterns (To be able to reconstruct the patterns .Runt gene variant P1 was expressed at the 8 somite stage (16 h) in the posterior part of the gut, the notochord and the developing neural tube .Our analysis had shown that  33 h PF . Howevert region . To detesections . We deteill bars  but not Runt and Hh showed co-expression in the gill gut region, we analyzed whether a functional relationship between both genes, as it is known for the mouse B. floridae Hh promoter revealed several putative Runt binding sites , and bony fish (duplication of Runx2). A chordate stem species with only a single Runt gene is the most parsimonious assumption since the genomes of cnidarians, nematodes, cephalochordates and tunicates harbour also only a single Runt gene. The presence of two Runt genes in sea urchin is most likely a result of a tandem duplication, as we found both genes on a single genomic contig and they cluster together in our phylogenetic analysis  were identified in hagfish (as a representative of jawless vertebrates). The phylogenetic tree  genome, an enigmatic fourth Runt domain gene (FrRunt) was detected in addition to the orthologs of the Runx1, –2 and –3 genes, which appeared to represent either a pufferfish-specific fast evolving derivative of Runx2 or a direct descendant of the ancestral chordate Runt gene FrRunt gene represents a direct descendent of the ancestral chordate Runt gene which evolved in parallel with the vertebrate Runt genes FrRunt orthologous gene in tunicates, lancelets, hagfish and dogfish. Instead our phylogenetic analysis  Runt genes are followed by Clic genes together with the evidence that the FrRunt gene represents a fast evolving Runx2 gene suggests that during chordate evolution the entire Runt locus was triplicated.Our findings that beside the human α1 as the predominant matrix protein. Differentiation of this cartilage is regulated by a molecular network including Sox9, a transcription factor that directly regulates Col2α1 expression Sox9 is a target of PTH related protein (PTHrP) that controls chondrocyte differentiation through a negative feedback loop with Indian hedgehog (Ihh). Runx2 in turn directly regulates IhhRunx2 also Runx1 and Runx3 genes are expressed during murine and zebrafish cartilage formation. However Runx2 and Runx3 appear to be the most important Runt genes for skeletogenesis Cartilage has evolved multiple times in metazoa MgRunxB gene is only weakly expressed in both types of cartilage. However, the MgRunxA gene had its strongest expression in hard cartilage and its third highest expression in soft cartilage  the Runt genes are expressed in the developing placoid scales , as well as the cells that deposit dentin (odontoblasts) are derived from the neural crest Whereas the homology of the gill gut in lancelets and vertebrates is well established Runt and SoxE gene expression in adult lancelets. Our analyses revealed that both genes were highly expressed in the gill bar region  were collected by shovel and sieve in water of 1 m in depth in Tampa Bay, Florida and in vitro fertilization, embryo culture and fixation were performed as previously described B. lanceolatum were obtained from the Biologische Anstalt Helgoland. Hagfish (M. glutinosa) were collected by S.E. Material from adult dogfish (S. canicula) was obtained from the Biologische Anstalt Helgoland and dogfish embryos from the Aquazoo (Düsseldorf).Lancelets (All primers and oligonucleotides employed in our study are given as supporting information. Primers for dogfish sequences can be found in B. floridae (larvae), B.lanceolatum (adult), M. glutinosa (adult), S. canicula . Runt genes were amplified by a strategy reported previously, using degenerated primers to amplify the conserved Runt domain followed by RACE PCRs to amplify the full length Runt genes MgRunxB 5′ end which was obtained by inverse PCR with gene specific primers Total RNA was isolated as described previously Runt sequences from O. dioica (AAS21356.1), C. intestinalis , B. lanceolatum and B. floridae , M. glutinosa , S. canicula , D. rerio . T. rubripes , G. aculeatus , M. musculus  and H. sapiens , while using the sea urchin Runt genes from S. purpuratus  as an outgroup.Alignments were obtained with ClustalW from 28 full length Runt amino acid sequences B. lanceolatum tissue sections. Non-radioactive in situ hybridization on cryo-sections of B. lanceolatum was carried out using the GenePaint System MmIhh and ScRunx3 were used as hybridization controls for B. lanceolatum.Whole mount in situ hybridizations with lancelet larvae were performed as previously described SoxE expression by qRT-PCR first a SoxE cDNA fragment was amplified by employing primers which were designed according to a SoxE sequence of B. floridae. Expression of target genes was normalized using 18S rRNA as reference.QRT-PCR was performed on an ABIPrism 7900HT Cycler  using SYBR Green PCR Master Mix (Applied Biosystems). TaqMan Reverse Transcription Reagents (Applied Biosystems) were used to synthesize the cDNA and primers were generated using the Primer Express software (Applied Biosystems). Quantification was performed using the standard curve method with dilutions of plasmids containing the sequence to be amplified in a known copy number as a standard. For the analysis of For immunohistochemistry on paraffin sections citrate antigen retrieval was performed. Anti-human Ihh antibody (Santa Cruz) was applied 1∶50 over night. Secondary antibody  was applied 1∶500 for one hour. Subsequent staining was performed with the Vectastain ABC kit from Vector laboratories according to the manufacturers′ instructions.Runt cDNA from B. lanceolatum. Specific binding was confirmed with a labeled oligo containing the putative binding site and using either wild type oligos or oligos with mutated binding sites as competitors.Electrophoretic mobility shift assays for putative binding sites were performed as described in B. floridae Hh promoter (AC150424) were cloned into the pGL3-basic reporter vector. NIH3T3 cells were transfected in 24-well plates with the reporter constructs (250 ng per well) together with an expression vector containing either the cDNA for BlRunt or MmRunx2 or an empty vector as control (100 ng per well). 5 ng per well of pRL-CMV were co-transfected for normalization. Cells were lysed with 100 µl passive lysis buffer . 5 µl of the lysate were measured using the Dual-Glo Luciferase Assay Kit (Promega) with 25 µl of the assay reagents each. Measurements were performed on a 1450 MicroBeta Scintillation and Luminescence Counter . The result of a representative experiment is shown which was confirmed five times independently.PCR amplified fragments of the ScRunx1 Acc-Nr DQ990011, ScRunx2 DQ990013, ScRunx3 DQ990015 and MRIPV-like-motifs promoter variant 2, ScRunx1 DQ990010, ScRunx2 DQ990012, ScRunx3 DQ990014. Hagfish: MgRunxA DQ990008, MgRunxB DQ990009. Lancelet: SoxE EF051347.The sequences reported in this paper have been deposited in the GenBank databases. Dogfish: MASNS-like-promoter variant 1, Figure S1http://www.mbio.ncsu.edu/BioEdit/bioedit.html) of newly detected Runt genes in hagfish  and dogfish  with other deuterostome Runt genes. The conserved sequence blocks used for the phylogenetic analysis are underlined with #. Parameters used with Gblocks 0.91b were: Minimum number of sequences for a conserved / flanking position: 15/15; Maximum number of contiguous nonconserved positions: 8; minimum length of a block: 5; allowed gap positions: all. 338 (52%) of the original 645 alignment positions were used in the phylogenetic analysis.Alignment used for Phylogenetic Analysis. Alignment Click here for additional data file.Figure S2Synteny Analysis.FrRunt locus (Ensemble: Scaffold 39) than previously analyzed (Ensemble: Scaffold 835FrRunt gene and chromosome 1 of zebrafish comprising Fstl1 and Gja5 (A). Furthermore we detected in the stickleback (G. aculeatus) genome a FrRunt orthologous gene with a genomic environment almost identical to the FrRunt gene locus (B). The gene orthologous to Clic 5 located 3′ of Runx2a in the zebrafish genome was found by Blast searches on group 1 in the stickleback genome. Together these results suggest that a translocation between a region of the 3′ end of the FrRunt locus and chromosome 1 had occurred in the common stem species of pufferfish and stickleback.A search for cross-species conserved gene orders was performed as previously described (0.05 MB DOC)Click here for additional data file.Table S1Dogfish Primers.Runt genes in dogfish. PA: Primary amplification, RA: Reamplification.Primers employed to amplify and analyze the expression of (0.08 MB DOC)Click here for additional data file.Table S2Hagfish Primers.Runt genes and analyze Runt gene expression in hagfish. PA: Primary amplification, RA: Reamplification.Primers employed to detect (0.07 MB DOC)Click here for additional data file.Table S3Amphioxus Primers.Sox9, Hedgehog and Runt genes in lancelets.Primers employed to analyze (0.08 MB DOC)Click here for additional data file.Table S4EMSA Oligos.Oligos employed for the electrophoretic mobility shift assays.(0.07 MB DOC)Click here for additional data file.