A challenging goal in biology is to understand how the principal cellular functions are integrated so that cells achieve viability and optimal fitness in a wide range of nutritional conditions.Bacillus subtilis, is very strong as some metabolic alterations fully restore viability to replication mutants in which a lethal arrest of DNA synthesis otherwise occurs at a high, restrictive, temperature. Full restoration of viability by such alterations was limited to cells with mutations in three elongation factors  out of a large set of thermosensitive mutants affected in most of the replication proteins. Restoration of viability resulted, at least in part, from maintenance of replication protein activity at high temperature. Physiological studies suggested that this restoration depended on the activity of the three-carbon part of the glycolysis/gluconeogenesis pathway and occurred in both glycolytic and gluconeogenic regimens. Restoration took place abruptly over a narrow range of expression of genes in the three-carbon part of glycolysis. However, the absolute value of this range varied greatly with the allele in question. Finally, restoration of cell viability did not appear to be the result of a decrease in growth rate or an induction of major stress responses.We report here a tight link between glycolysis and DNA synthesis. The link, discovered during an analysis of suppressors of thermosensitive replication mutants in bacterium Our findings provide the first evidence for a genetic system that connects DNA chain elongation to glycolysis. Its role may be to modulate some aspect of DNA synthesis in response to the energy provided by the environment and the underlying mechanism is discussed. It is proposed that related systems are ubiquitous. Bacillus subtilis and related micro-organisms, this machine contains two different polymerases which are thought to be specialized in leading (PolC) and lagging (DnaE) strand polymerization The replisome is a multiprotein machine that replicates DNA in living organisms Glycolysis, gluconeogenesis, the pentose phosphate pathway and the tricarboxylic acid pathway are major elements of the central carbon metabolism in which nutrients provided by the environment are converted into building blocks and used for generating energy and reducing power for biomass synthesis. Glycolysis is a nine reactions pathway that is conventionally split in two parts. In the first part, glucose 6-phosphate is converted into glyceraldehyde 3-phosphate. This set of reactions, directly fuelled by glycolytic nutrients, is efficiently shunted by the pentose phosphate pathway. In the second part, glyceraldehyde 3-phosphate is transformed into pyruvate. These reactions, termed thereafter the three-carbon part of glycolysis, are required for degradation of virtually all carbon sources and cannot be efficiently shunted in most organisms. Thus, they play a key role in cell metabolism. The gluconeogenesis pathway operates when carbon sources feed the bottom part of the central carbon metabolism. It uses most of the glycolytic reactions in the opposite direction to produce glucose 6-phosphate.Escherichia coli, this coupling is achieved by regulating the frequency of initiation of replication and, in slow-growing cells (generation time ≥70 mn), by modulating the rate of DNA chain elongation E. coli and arrests DNA elongation at specific sites in the chromosome of B. subtilis (Saccharomyces cerevisiaeS. cerevisiae MCM1 protein B. subtilis. Key elements of the link have been identified. They are the DnaE (the lagging strand polymerase), DnaG (primase) and DnaC (helicase) replisomal enzymes and the activity of the three-carbon part of the glycolysis/gluconeogenesis pathway. The mechanism and function of the link are discussed.Several studies indicate that DNA replication might be linked to cell metabolism. First, the rate of replication is coupled to nutrient richness in bacteria. In subtilis . This resubtilis . Third, dnaE thermosensitive (Ts) mutations  ensuring the terminal reactions of glycolysis  into parental dnaE(Ts) strains led to Tr transformants. Second, four dnaE(Ts) mutants carrying a suppressive mutation in pgk, pgm, eno or pykA became Ts when expressing a WT copy of the corresponding glycolytic gene from a xylose inducible promoter (as exemplified in pgm8 suppressor). Third, a deliberately generated deletion of pykA caused dnaE(Ts) suppression (see below).To identify proteins functionally connected to the lagging strand DnaE polymerase, a search was conducted for extragenic suppressors of four ycolysis . Three sdnaE(Ts) suppressors. First, the seven suppressed strains carrying a mapped suppressive mutation grew vigorously at restrictive temperature on plates for at least 20 generations, as they formed thick, wild-type-like colonies and had a plating efficiency of 100% in these growth conditions (dnaE(Ts) strains did  grew as fast as the corresponding metabolic mutants at restrictive temperature  alleles and to search for other suppressive mutations, three of the above suppressors  and 16 other metabolic mutations (mainly deletions) in genes of the central carbon metabolism (see dnaE(Ts) strains. Control experiments showed that none of the metabolic mutations affect viability of DnaE WT cells at high temperature in LB. Most metabolic mutations (15/19) were not suppressive as they did not restore cell viability on plates at high temperature  was also investigated. Only the mutations in the three-carbon part of glycolysis were suppressive. Full suppression (i.e. on plates and in liquid) was detected for three new dna(Ts) mutations mapping in the elongation factors DnaG  and DnaC  (dnaC8133), two PolC (dnaF33 and dnaF69) and two DnaX (dnaX8132 and dnaH51) mutants was also observed in some strains (the reason for different suppression efficiency on plates and liquid is not fully understood) (dna(Ts) mutations suggest that the link is particularly strong with the DnaE, DnaG and DnaC enzymes.To further circumscribe the glycolysis/replication relationship, the effect of 9 metabolic mutations on 14 Ts mutations mapping in initiation and elongation replication genes was assessed (93 strains were tested). The effect of  dnaC30) . Partialerstood) . The remerstood) . Taken tpgm8, pgmIP, ΔgapA, pgkEP, pykAJP and ΔpykA) had a moderate growth rate (1.5–2 doublings per hour). Interestingly, some non suppressive mutations grew at a similar  or even lower  rate. Moreover, suppression was observed in fast growing cells  or carried the ΔpykA mutation and were grown in a gluconeogenic medium (see below). Altogether, these data suggest that a mere decrease in growth rate is not a prerequisite for suppression.Mutations in genes of the central carbon metabolism often reduce cell growth. To test whether this reduction correlated with suppression, the doubling time of 20 metabolic mutants, WT for replication functions, was measured at high temperature in LB. A large range of growth rates, extending from the optimal value (∼3.8 doublings per hour) to a significantly lower rate (∼1 doubling per hour) was observed . SuppresdnaE2.2, dnaE2.4, dnaE2.6, dnaG20 and dnaC30 strains, WT for metabolic functions, was assessed on LB plates supplemented (separately or in combination) with chemicals causing osmotic (2% NaCl), energetic (125–500 µg/ml sodium azide) or nutritional  stresses. In no case was suppression observed. Control experiments showed that, at the concentration used, the chemicals alter growth of WT cells but not their viability and plating efficiency. In case of arginine hydroxamate, WT cells did not survive to a 2 times higher concentration of the drug. This suggests that stress-like responses are not key determinants in suppression.Mutations in genes of the central carbon metabolism cause unscheduled alterations in concentration of metabolites and/or energetic compounds. Because (i) unbalanced metabolism might be sensed by cells as a stress, (ii) some nutritional stresses interfere with DNA replication pgk or pykA) or a group of genes  of the bottom part of glycolysis under the control of an inducible promoter were constructed. As glycolytic enzymes are abundant in living cells, we first tested whether the selected promoters (Pspac-I - IPTG dependent - or Pxyl - xylose dependent) were strong enough to modulate gene expression and hence glycolysis throughout a substantial range at high temperature in LB. Results with the Pspac-I::pykA strain (WT for replication functions) showed a significant modulation of PykA activity (from about 0.04 to 0.45 unit per mg of protein) (ΔpykA (0.006 unit/mg) and wild-type PykA+ (2.03 units/mg) strains, presumably because of some leakage and insufficient strength of Pspac-I (the weak increase in PykA activity and possibly growth rate at 0–15 µM IPTG could result from a non linear response of the Pspac-I promoter at low IPTG concentrations). Despite this limitation, Pspac-I allowed a growth rate modulation extending from that of the PykA+ and ΔpykA strains (compare gapA operon expressed from Pspac-I and with pgk or pykA expressed from Pxyl (not shown). This shows that the inducible promoters are strong enough to substantially modulate cell metabolism.To further identify metabolic alterations required for suppression, strains expressing a gene (protein) . However compare  and 5A. pykA expression on the Ts character of the dnaE2.6 mutation, fresh dnaE2.6 Pspac-I::pykA cells grown at 30°C were spread on plates containing various IPTG concentrations and grown at restrictive temperature. Colony counting showed an abrupt shift from the Ts to Tr phenotype between 60 and 30 µM IPTG (dnaE2.6 mutation was observed at a high growth rate when the whole gapA operon was expressed from Pspac-I or when pykA or pgk were expressed from Pxyl (not shown). Taken together, these data suggest that the Ts/Tr shift of the dnaE2.6 strain occurs abruptly upon a moderate reduction (∼2 times) in expression of the genes encoding enzymes of the bottom part of glycolysis.To evaluate the impact of  µM IPTG . This sh µM IPTG . They we µM IPTG  and 5A. Pspac-I::pykA fusion was then introduced in two other DnaETs mutants (dnaE2.2 and dnaE2.4), one DnaC mutant (dnaC30) and in the dnaG20 strain and suppression was assessed on plates as above. All mutants shifted from the Ts to Tr phenotype upon modulation of PykA expression (dnaE2.2 mutant shifted at low PykA concentration and growth rate while dnaE2.6 and dnaC30 shifted at high PykA concentrations and growth rates (The pression . In the th rates . It is ipykA or gapA genes grew at a slower (∼2.5 times) rate than WT cells or strains deleted for the gluconeogenic pckA or gapB genes  in glycolytic carbon sources and strongly induced  on gluconeogenic nutrients ΔpykA and pgkEP) and non suppressive  contexts  prevented (or strongly reduced) growth in CM, while inactivation of the glycolytic PykA enzyme had no effect on growth rate (not shown). The plating assay showed that dnaE2.2, dnaE2.4, dnaE2.6, dnaG20 and dnaC30 strains, WT for metabolic functions, were Ts in CM. The isogenic ΔpykA strains were all but one (the dnaC30 ΔpykA strain) Tr on plates. Taken together, these data showed that suppression of some Ts mutations can occur in the neoglucogenic regimen.In order to test whether suppression can also take place in gluconeogenesis, suppression was assessed in CM, a minimal medium supplemented with gluconeogenic carbon sources  that allows a high growth rate (∼2.9 doublings per hour at 46°C) to WT cells. This medium is gluconeogenic as mutations in the three-carbon part of the gluconeogenesis pathway . Hence, survival at high temperature of suppressed strains requires at least partially active Ts proteins.Suppression could have resulted from changes in the replisome that make Ts proteins dispensable for DNA synthesis. Two sets of data showed that this is not the case. First, most of the suppressed strains exhibited a residual thermosensitivity at extreme tures cf  and 3B. dnaE, dnaC and polC promoters with the lacZ reporter gene indicated that suppression does not result from dna(Ts) genes over-expression -SPA PykA+ cells were Ts and that the isogenic PykA− strains were Tr at high IPTG concentrations . This suggested that DnaETs-SPA proteins respond to glycolytic mutations like the original DnaETs proteins. Despite the contrasted phenotype of the PykA+ and PykA− strains, both cell types contained similar amount of DnaETs-SPA fusion proteins at saturating amount of IPTG (1000 µM) as shown by Western blot analysis with anti-SPA antibodies (dnaE2.2-SPAΔpykA strain exhibited a similar IPTG dependence and intracellular DnaETs-SPA concentration at permissive and restrictive temperature (dnaE2.4-SPAΔpykA strain (not shown). Taken together, these results suggest that (i) suppression does not depend on Ts protein accumulation and that (ii) the DnaTs proteins are Tr in the PykA− background.Transcriptional fusions of the pression . In ordetibodies . Moreoveperature . Similardna(Ts) suppressors, is very strong as some metabolic alterations fully restore growth of dna(Ts) mutants at a temperature at which a lethal arrest of DNA synthesis otherwise occurs. Two major lines of evidence show that this link is highly specific in terms of both replication and metabolism. In the case of replication, full viability is restored to mutants affected in 3 DNA elongation factors out of 9 replication functions tested (3 specific for DNA initiation and 6 mainly involved in DNA elongation). These factors are the lagging strand polymerase DnaE, the DnaC helicase and the DnaG primase. These enzymes probably act on the same DNA strand (the lagging strand template) in the replication fork and two of them (DnaG and DnaC) interact DNA replication and carbon metabolism are fundamental to life. Here, we report a robust genetic link between them. This link, discovered during an extensive analysis of dnaE, dnaG and dnaC mutants has not been fully analyzed. However, two mutants (dnaE2.10 and dnaE2.6) are defective in the elongation phase of DNA replication as they cease incorporation of labeled precursors shortly after a shift to restrictive temperature (47°C) dnaE2.2 and danE2.4 mutations are also considered to affect elongation, as cells carrying these mutations exhibit a strong decrease  in the rate of precursor incorporation (unpublished data) and form long filaments at restrictive temperature  mutants at high temperature. In one hypothesis, growth restoration is due to DnaTs proteins being protected from heat-inactivation by a stress-like response induced by metabolic alterations. However, the following observations make this possibility unlikely. First, separate or combined induction of the general stress regulon and the stringent response by chemicals inducing nutritional, energetic or physical stresses was not suppressive. Second, efficient suppression is observed with Ts mutations occurring in only three replication proteins while a stress-like-induced mechanism would be expected to cause a more random profile of suppression because of the relaxed effect of thermoprotector agents on protein conformation Several hypotheses can account for the viability of dna(Ts) cells results from dna(Ts) genes over-expression and DnaTs protein accumulation. This hypothesis is based on recent evidence that several metabolic enzymes  play a role in the organization and packaging of bacterial and mitonchondrial genomes, and thereby, can modulate DNA supercoiling and expression of genes with promoters sensitive to DNA topology dna gene over-expression  nor DnaTs protein accumulation (3 dnaE(Ts) alleles were tested) are detected in suppressed cells. Topological changes might also rescue cell growth by stimulating expression of genes involved in the stabilisation, repair or restart of altered replication fork A second hypothesis is that the restoration of viability of E. coli, transcription of the RNR encoding gene is stimulated during initiation of DNA synthesis and this stimulation is required for DNA elongation A third hypothesis relies on the role played by the ribonucleotide reductase (RNR) and the dNTP pool on DNA replication. The synthesis of dNTP depends on RNR that converts NDPs into dNDPs. In E. coli, mutations that restore viability to a thermosensitive DNA elongation mutant (dnaX2016) have previously been isolated dnaA, a gene that encodes the major initiator of DNA replication. During initiation, DnaA binds to short repeated sequences located in the chromosomal oriC region, partially unwinds oriC, and recruits the helicase. The helicase then interacts with the primase that synthesizes primers at which the replisome is assembled for initiating DNA synthesis [reviewed in dnaX(Ts) dnaA double mutants at high temperature does not result from a strong stimulation of DNA elongation. Indeed, in 3/3 suppressed strains analyzed at high temperature, DNA synthesis remained strongly disturbed, leading to a high proportion of filamentous cells containing an incorrectly compacted and distributed nucleoid dnaX dnaA double mutants was shown to result from a decrease in the efficiency of initiation. This decrease is a direct consequence of dnaA suppressive mutations that significantly weaken the initiation activity of DnaA dnaA mutations restore viability at high temperature by reducing the number of chromosome per cell to a level that allows the partially active DnaX protein to support ongoing chromosomal replication and cell growth E. coli cells to mild inhibition of chain elongation by drugs also relies on mutations that reduce the efficiency of initiation B. subtilis dna(Ts) cells results mainly from stimulation of DNA elongation rather than from modifications in initiation efficiency. First, suppression can occur at temperatures at which DNA synthesis is arrested. This is at least the case for the dnaE2.10 allele which can be suppressed at temperatures ranging from 47–50°C while DNA synthesis is already arrested at 47°C in Ts cells WT for glycolytic genes dna(Ts) mutants, these cells do not form filament at high temperature and their nucleoid is properly compacted and distributed. It should also be noted that despite the relatively large study conducted here, no suppressive mutations mapping in any of the four initiation genes  were isolated despite the total size of the DNA in these potential target genes being similar to that of the suppressive glycolytic genes . These observations therefore suggest that the restoration of cell viability in E. coli and B. subtilis elongation mutants depends on different mechanisms and that in B. subtilis, this mechanism mainly operates via stimulation of DNA chain elongation.A fourth hypothesis is that the restoration of viability is the result of a change in the characteristics of initiation or of chain elongation or of both. In B. subtilis suppressed cells might result from changes in replisome requirements. One possibility is that there is a change in the composition of the replisome that allows replication to occur despite the absence of the DnaTs proteins. There is no precedent for this, most likely because the function of DnaC, DnaG and DnaE  are essential for DNA synthesis and cannot be carried out by other replisomal proteins. Nevertheless, they might theoretically be ensured by alternative replication factors as those encoded, for example, by prophages. While attractive, this possibility can be discounted because (i) studies on dnaE(Ts) mutants have all been carried out in cells devoid of the three main B. subtilis prophages  dnaC(Ts) and dnaG(Ts) mutants, initially observed in prophage-containing cells, occurs with the same efficiency in prophage-free strains (not shown). It is also discarded that the putative alternative replication factors are of chromosomal origin since B. subtilis does not encode structural and/or functional homologues of the DnaC helicase and DnaG primase that could replace efficiently these proteins at the fork dna(Ts) cells requires DnaTs proteins. First, most of the suppressed strains are thermosensitive at extreme temperatures. Second, the dnaE gene cannot be inactivated in cells carrying a suppressive mutation. Hence, restoration of viability cannot be explained satisfactorily by changes in the composition of the replisome.Stimulation of DNA elongation in dna(Ts) cells is a functional change in the replisome, a hypothesis consistent with all our results. Such a functional change might be due to suppressive metabolic mutations either making some activities of DnaTs proteins dispensable for DNA synthesis or protecting these activities from heat-inactivation. It may therefore be significant that (i) suppressed and non-suppressed strains contain the same amount of DnaETs protein at restrictive temperature and (ii) the minimal DnaETs concentration required for survival of (PykA−) suppressive strains is similar at permissive and restrictive temperature. As mentioned above, DnaC, DnaG and DnaE have catalytic activities that are essential for DNA synthesis and cannot be efficiently ensured by alternative proteins. Their heat inactivation can thus only be countered by a process allowing their thermo-protection. However, replication enzymes might also be required at the fork for structural and/or regulatory purposes. If the loss of these additional functions could be tolerated in certain conditions, this loss might be suppressed not only by thermo-protection but also by processes making these functions dispensable at the fork. The function(s) defective in DnaTs proteins at high temperature need to be identified to further dissect the molecular basis of stimulation of DNA elongation in suppressed cells. However, irrespective of the detailed mechanism, an attractive hypothesis is that suppression involves conformational changes in replisomal enzymes.An alternative possibility for stimulating DNA elongation in Our physiological studies identify the activity of the metabolic linker region as important in restoration of viability. This restoration occurs abruptly over a narrow range of metabolic linker activity and the absolute value of this range varies greatly with the Ts allele in question. However, viability can be restored without major change of the growth rate or the direction of carbon flow through the metabolic linker. The absence of effect of reversing the carbon flow rules out catabolite repression, a genetic system that regulates expression of numerous genes in highly energetic glycolytic carbon sources B. subtilis using the yeast two hybrid technology A neuronal receptor Our suppression assay provides the first evidence for a genetic system that connects DNA chain elongation to glycolysis. Its role in WT cells may be to modulate some aspects of DNA synthesis in response to the provided energy. The molecular mechanism of the linker effect on the postulated conformational changes in replisomal enzymes remains to be elucidated. We viewed the system as comprising the metabolic linker, a signal-generating activity of this linker, and signal-mediated conformational changes in replisomal proteins, possibly the lagging-strand DnaE polymerase, DnaC helicase and DnaG primase . These cSeveral reports in the literature suggest that the link between DNA replication and cell metabolism is ubiquitous (see above citations). Understanding the underlying mechanisms is thus of general interest. Such studies might also be of medical importance as early events in carcinogenesis, which generally include up-regulation of glycolysis (the Warburg effect) and a decrease in DNA stability and replication fidelity B. subtilis strains are listed in Supplementary E. coli strain used for plasmid constructions was DH5α (supE44 supF58 hsdS3(rB−mB−) dapD8 lacY1 glnV44 Δ47 tyrT58 gyrA29 tonA53 Δ(thyA57)). B. subtilis strains carrying the dna(Ts) mutations in a prophage-free background (DGRM1-4) were constructed by transforming TF8A competent cells with total DNA of EDJ strains. Transformants were selected at 30°C on phleomycin (Pm) containing plates (the phleomycin gene - pmr - is located just upstream of dnaE) and the presence of the dna(Ts) mutation was checked by analyzing cell growth and filamentation at restrictive temperature by plating and optical microscopy analysis. To construct dnaH51, dnaC8133 and ts-6 prophage-free strains , a similar strategy was used except that a Trp+ DNA was mixed to the transforming dna(Ts) DNA. Transformants were first selected for Trp+ at 30°C and then toothpicked to select Ts colonies (note that the procedure cured the spectinomycin (Sp) marker of BD54 ts-6). To construct strains (WT for DNA replication functions) lacking Zwf, Pps or PycA metabolic enzymes , the corresponding genes, cloned into an E. coli vector, were interrupted by an erythromycin (Em) cassette. The mutations were then introduced in the B. subtilis genome by transforming TF8A cells with the plasmid cut in the vector backbone. Strains resulting from a double crossover event were selected by PCR. Metabolic mutations carried by strains provided by different laboratories were transferred in the TF8A context using transformation and appropriate marker selection . To label spontaneously isolated suppressors mapping in genes of the gapA operon, the spectinomycin marker (spc) was inserted downstream of the operon (in araE) by transforming spontaneously suppressed strains  with a PCR product composed of spc flanked by the 5′ and 3′ region of araE. The yielded strains were named DGRM61, 62, 64, 83, 102 and 105. To label the pykAJP suppressive mutation, the corresponding suppressed strain (DGRM7) was transformed to erythromycin resistance with DNA of the BFS67 strains (from the European collection of B. subtilis mutants ery) in the vicinity of pykA. The presence of the PykA mutation in a representative transformant (DGRM65) was confirmed by showing that the constructed strain grew as slowly as the parental strain in LB and in minimal medium supplemented with glucose. To evaluate the impact of spontaneously isolated suppressive mutations on metabolic enzyme activity, suppressive mutations labeled by the spc or ery markers were transferred into the WT TF8A strain by transformation yielding strains DGRM18–24. Cell slow growth in LB at 45°C was used as a criteria for the presence of the metabolic mutation in the produced strains. A large (∼200) collection of strains carrying a metabolic mutation and a replication or division Ts mutation (apart the spontaneously isolated strains) was generated by transforming Ts cells with total DNA extracted from strains containing a labeled metabolic mutation. The genotype of the constructed strains was checked by analyzing their Ts at 51°C , growth rate at 30°C , back-cross studies, PCR analysis and/or Southern blotting. To construct strains (WT or Ts for replication functions) expressing PykA from the IPTG inducible promoter spac-I, a segment corresponding to the 5′ region of pykA (extending from position −42 to +412 according to the first base of the ORF) was generated by PCR and cloned between the HindIII-BamHI sites of the E. coli (EmR) plasmid pMUTIN2 pykA gene of TF8A. This puts pykA under the control of Pspac-I. The resulted strain (DGRM26) was verified by PCR and by analyzing growth rate and PykA activity as a function of IPTG concentration. The fusion was then transferred to various dna(Ts) strains yielding DGRM260–265. For complementation studies, WT copies of the pgk, pgm, eno or pykA genes were PCR amplified and cloned downstream of the xylose inducible promoter pxyl of plasmid pAX01, at the BamHI site spc marker and delimited by the front and back regions of the B. subtilislacA locus. The labeled fusion was then introduced at the lacA locus of the WT (TF8A) strain by double crossover. The structure of the yielded strains (DGRM242–246) was controlled by PCR. The construction what then introduced by transformation into strains carrying a labeled glycolytic mutation or into dnaE(Ts) strains mutated in a glycolytic gene (DGRM247–259). Conditional expression of the metabolic genes was checked by following cell growth in LB complemented or not with 0.5% xylose. To measure the effect of metabolic mutations on carbon flux polarity, competent cells of the GM1514 strain that contains lacZ downstream from the gapB promoter at the amyE locus were transformed with the total DNA extracted from strains carrying metabolic mutations . Transformants were selected on plates supplements with appropriate antibiotics. The presence of the metabolic mutations and of the fusion were verified by analyzing cell growth in LB at 37°C and by PCR, respectively. For strains carrying the pgkEP mutation, the presence of a functional PgapB::lacZ fusion was verified by measuring β-galactosidase activity upon transfer of the structure in the 168 strain. The constructed strains were termed DGRM239–241 and 285. To measure replication promoter activity in suppressed and non suppressed strains, we introduced by transformation the ΔpykA::spc mutation (from strain DGRM25) into strains carrying the PdnaE::-, PpolC::- or PdnaC::lacZ fusions and β-galactosidase activity was measured in the parental PykA+  and yielded PykA− (DGRM322–324) strains. To investigate whether suppression depends on accumulation of DnaETs proteins, we first constructed PykA+ or PykA− strains carrying the dnaE(Ts) genes under the control of Pspac-I. For this, we transformed the dnaE(Ts) pmr strains (DGRM1-3) with the DNA extracted from cells carrying the Pspac-I-dnaEery construction (HVS614). Transformants were selected at 30°C on plates containing EmR and 500 µM IPTG. A toothpicking analysis on 100–200 transformants allowed us to identify Ts EmR PmS cells. Growth analysis at 30°C  in liquid and solid rich medium supplemented or not with 500 µM IPTG showed that the selected strains containas expected dnaE under the control of Pspac-I. The strains were termed DGRM230–232. To construct the isogenic PykA- strains, competent spac-I-dnaE(Ts) cells were transformed to SpR by the DNA extracted from the pykA::spc strain (DGRM25). Representative IPTG-dependent, slow growing EmR transformants were selected (DGRM233–235). That these cells carry the dnaE(Ts) mutation was controlled by transferring the EmR region into the 168 strain and showing that most of the transformants were Ts. We then fused the DnaETs proteins to the SPA tag dnaE-SPA fusion labeled with a neo marker. A Western blot analysis of KmR transformants was carried out using the anti-SPA monoclonal anti-FLAG M2 antibody . Clones encoding an IPTG inducible fusion protein of the expected size were thus identified. Among these, a genetic screen allowedthe selection of representative strains containing the dnaE(Ts) mutation. The genetic analysis indicated that the SPA fusion decreases the activity of the DnaETs proteins as the corresponding cells were more IPTG dependent than the isogenic counterpart lacking the fusion. The structure of the dnaE region in the constructed strains (DGRM304–306 and 308–310) was checked by PCR. Plasmids used in the above mentioned constructions are available upon request.pta mutation, acetate (1%) was added to LB in order to improve cell growth and prevent appearance of fast growing variants (control experiments showed that this complementation does not interfere with suppression). The ΔgapA strain was grown in the presence of 1 mM IPTG in order to allow Pspac-I-driven expression of the metabolic genes located downstream of gapAB. subtilis and E. coli competent cells were prepared as previously described and antibiotics were added to the media at regular concentrations Cells were grown in LB or minimal medium supplemented with glucose 0.2% or malate 0.5% dnaE(Ts) strains (DGRM1–4) cured of the three major prophages  were used. Tr derivatives were selected at the lowest temperature reducing 104–105 fold the platting efficiency of the Ts mutants (see below the temperature used). For each mutant, 20–40 cultures of 5 mL of LB were inoculated with 104 cells and grown at 30°C under shaking. At saturation, 106 to 107 cells were spread on LB plates and incubated at restrictive temperature. After 48–72 hours of growth, a large and a small colony from each plated culture were selected and streaked for further growth at restrictive temperature. An isolated colony was then cultivated at 30°C in liquid LB and stocked at −80°C. To identify strains carrying an extragenic suppressor, chromosomal DNA of Tr variants was extracted and used to transform the WT 168 strain to PmR. Twenty to fifty transformants selected at 30°C were then toothpicked on Pm plates and grown at high or low temperature. Suppressive mutations were assumed to be extragenic when the linkage between the Tr marker and pmr was ≤85%. This procedure allowed us to identify 29 independent extragenic suppressors.In order to search for suppressive mutations, four JP) carried by strain DGRM7 and located in the vicinity to dnaE2.4 (genetic linkage of ∼80%), a collection of cells carrying an EmR marker at various positions in the dnaE region was used  and BFA91 (45%) suggesting that the JP mutation maps ∼5 kb downstream of dnaE. This was confirmed by transforming the original dnaE2.4 strain to thermoresistance with PCR fragments generated from the suppressed strain and containing overlapping sequences downstream of dnaE. DNA sequencing of the region revealed a 81 bp deletion in the central part of the glycolytic pykA gene. This mutation was termed pykAJP.To precisely map the suppressor , was carried out by random transposition as described previously Tn) in the vicinity to sup8, DNA of a large pool of cells that had suffered transposition was extracted, used to transform the parental dnaE2.6 strain to CmR (the Tn marker) at 30°C and CmR Tr transformants were selected by replica-plating. This second generation CmR Tr cells was then pooled and processed as above for further enrichment of Tn-sup8 linked genomes. Genetic linkage analysis showed that a representative CmR Tr strain carried Tn in the vicinity of sup8 (linkage of 24%). To further map Tn, the DNA of this strain was restricted with several enzymes, run on agarose gel along with DNA ladders, and analyzed by Southern blot using as a probe a 32P labeled DNA homologous to Tn. The size of the Tn-containing segments was then compared to the predicted restriction map of the B. subtilis genome using a software developed in the laboratory (available upon request). This allowed us to map Tn in a 585 bp segment located between coordinates 3,444 862 to 3,445 447 of the B. subtilis genome. This location was confirmed by PCR. To map sup8, mutants of the BFA collection carrying the EmR determinant in the Tn region were used (see R-sup8 linkage (90%) was observed with strain BFA1079 which carries the EmR determinant in yvbK. DNA sequencing of the region revealed a single mutation (termed pgm8) in the glycolytic pgm gene, ∼8 kb downstream of yvbK.Mapping of used see . The higR marker located in the vicinity of metabolic genes in BFA mutants . As the suppressed strains grew poorly on minimal medium supplemented with glucose, it was speculated that they carry a mutation in the glycolytic genes located downstream from yvbK. Consistently, single mutations were found by sequencing in pgk (pgkEP), pgm (pgmIP and pgm25) and eno (enoLP). All the suppressive mutations are described in In order to determine whether additional suppressive mutations map in genes of the central carbon metabolism, the genetic linkage between suppressors and an Emants see  was inve4–105 fold the plating efficiency of Ts mutants on LB (for the plating assay) or reducing 101–103 cell viability after 2.5 hours of incubation in LB broth (for the filamentation assay), were as follows : dnaE2.2 and dnaE2.4 ; dnaE2.6 ; dnaE2.10 ; dnaG20 ; dnaC14 (P: 49°C: F: 45°C); dnaC30 ; dnaC8133 ; dnaX8132 ; dnaF33 (P: 37°C); dnaF69 (P: 42°C); dnaH51, ts-6, dnaB37 and dnaD23 (P: 45°C); dnaG5 and dnaI2 (P: 47°C). For studies carried out in MM-CM, selective temperatures were as above except for dnaE2.2  and dnaE2.4 .Restrictive temperatures, defined as the lowest temperatures reducing 10600 = 0.3 were centrifuged, resuspended in 75 µl of lysis buffer , incubated 20 mn on ice, 5 mn at 37°C, 15 mn at 20°C, centrifuged 10 mn at 12000 g and the supernantant was stored at −20°C. The standard assay system contained in 1 ml: 50 mM Imidazole HCl pH 7.1, 10 mM MgCl2, 50 mM KCl, 5 mM ADP, 5 mM phosphoenolpyruvate, 0.2 mM NADH, 10 units lactate dehydrogenase. Reactions were initiated by the addition of 0.5 to 10 µl of crude extract in a 1-cm quartz cuvette, and kinetics of NADH oxidation was followed at 340 nm in a Lambda 20 Perkin Elmer spectrophotometer at 25°C. One unit of enzyme is defined as the amount required to convert 1 µmole of phosphoenolpyruvate to pyruvate per min in the standard assay.DNA extraction and manipulations were carried out according to standard procedures To check for cell filamentation, parental Ts strains incubated 2–3 hours at restrictive temperature and suppressed strains grown for at least 5 generations were stained 5 mn in the presence of 4′,6′-diamidino-2-phenylindole (DAPI) and FM5-95 (Molecular Probes) for visualizing the nucleoid and membrane, respectively. They were then deposited on glass slides covered with 1.2% agarose in minimal medium, and examined with a Leica  microscope with a 100× magnification oil-immersion objective and a CDD camera . Cell pictures were captured with METAMORPH V5.0 .Table S1(0.05 MB XLS)Click here for additional data file.Table S2(0.03 MB XLS)Click here for additional data file.Table S3(0.05 MB XLS)Click here for additional data file.


The glucocorticoid receptor (GR) associates with glucocorticoid response elements (GREs) and regulates selective gene transcription in a cell-specific manner. Native GREs are typically thought to be composite elements that recruit GR as well as other regulatory factors into functional complexes. We assessed whether GR occupancy is commonly a limiting determinant of GRE function as well as the extent to which core GR binding sequences and GRE architecture are conserved at functional loci. We surveyed 100-kb regions surrounding each of 548 known or potentially glucocorticoid-responsive genes in A549 human lung cells for GR-occupied GREs. We found that GR was bound in A549 cells predominately near genes responsive to glucocorticoids in those cells and not at genes regulated by GR in other cells. The GREs were positionally conserved at each responsive gene but across the set of responsive genes were distributed equally upstream and downstream of the transcription start sites, with 63% of them >10 kb from those sites. Strikingly, although the core GR binding sequences across the set of GREs varied extensively around a consensus, the precise sequence at an individual GRE was conserved across four mammalian species. Similarly, sequences flanking the core GR binding sites also varied among GREs but were conserved at individual GREs. We conclude that GR occupancy is a primary determinant of glucocorticoid responsiveness in A549 cells and that core GR binding sequences as well as GRE architecture likely harbor gene-specific regulatory information. The glucocorticoid receptor (GR) regulates a myriad of physiological functions, such as cell differentiation and metabolism, achieved through modulating transcription in a cell- and gene-specific manner. However, the determinants that specify cell- and gene-specific GR transcriptional regulation are not well established. We describe three properties that contribute to this specificity: (1) GR occupancy at genomic glucocorticoid response elements (GREs) appears to be a primary determinant of glucocorticoid responsiveness; (2) the DNA sequences bound by GR vary widely around a consensus, but the precise sequences of individual GREs are highly conserved, suggesting a role for these sequences in gene-specific GR transcriptional regulation; and (3) native chromosomal GREs were generally found to be composite elements, comprised of multiple factor binding sites that were highly variable in composition, but as with the GR binding sequences, highly conserved at individual GREs. In addition, we discovered that most GREs were positioned far from their GR target genes and that they were equally distributed upstream and downstream of the target genes. These findings, which may be applicable to other regulatory factors, provide fundamental insights for understanding cell- and gene-specific transcriptional regulation. The great challenge of metazoan transcriptional regulation is to create specialized expression pathways that accommodate and define myriad contexts, i.e., different developmental, physiological, and environmental states in distinct organs, tissues, and cell types. This is achieved by a network of transcriptional regulatory factors, which receive and integrate signaling information and transduce that information by binding close to specific target genes to modulate their expression. For example, the glucocorticoid receptor (GR) associates selectively with corticosteroid ligands produced in the adrenal gland in response to neuroendocrine cues; the GR-hormone interaction promotes GR binding to genomic glucocorticoid response elements (GREs), in turn modulating the transcription of genes that affect cell differentiation, inflammatory responses, and metabolism ,2. Expre(IL8) is just upstream of the promoter [NRIP gene [Fkbp5 promoter that appears to serve as an androgen response element [An intriguing feature of GREs and other metazoan response elements is that their positions relative to their target genes are not tightly constrained ,6. Althopromoter , whereasIL is just RIP gene , and an  element . It has  element . HoweverEvidence from numerous anecdotal, gene-specific studies indicates that native response elements are typically composite elements that encompass distinct sequence motifs recognized by two or more regulatory factors. In turn, the bound factors recruit non-DNA binding coregulatory factors, forming functional regulatory complexes that remodel chromatin and modify the activity of the transcription machinery. In this scheme, the structure and activity of the regulatory complex at a given response element would be specified by at least three determinants: the sequence motifs comprising the response element; the availability of those sequences for factor binding; and the availability and activity levels of regulatory factors present in the cell. For example, primary GREs, defined as those at which GR occupancy is required for glucocorticoid-responsive regulation, are a diverse family of elements that bind GR together with an array of additional factors defined by the above three determinants. Such composite response elements provide a powerful driving force for combinatorial regulation ,12, vastGR binds to a family of related sequences that defines a consensus motif: an imperfect palindrome of hexameric half sites separated by a three-bp spacer –15. WithIn the present work, we sought to define and characterize a set of GREs in A549 human alveolar epithelial cells. Thus, we determined in A549 cells the presence of GR at specific GREs close to genes that are steroid regulated across a range of cell types. We assessed whether the GR-occupied GREs were limited mainly to genes that are GR regulated in A549 and measured within and between species the conservation of GRE sequences, architecture, and genomic positions.To assess the correlation of GR occupancy with glucocorticoid responsiveness, we examined GR binding at three classes of genes in A549 human lung carcinoma cells: first, genes regulated by GR in A549 cells; second, genes regulated by GR in U2OS human osteosarcoma cells but not in A549; third, genes regulated by GR or the androgen receptor (AR) in cells other than A549 or U2OS. The AR-responsive genes were of interest because AR is closely related to GR and shares similar DNA-binding specificity in vitro ,16,22. TTo identify GR binding regions (GBRs), we used chromatin immunoprecipitation-microarray (ChIP-chip) to interrogate 100-kb genomic segments centered on the transcription start sites (TSSs) of our set of 548 genes. This ~55-Mb sample of the genome also included or impinged upon an additional 587 genes not previously reported to be regulated by GR; thus, we assessed GR occupancy in the vicinity of more than 1,000 genes. Immunoprecipitated chromatin samples from A549 cultures treated for one hour with the synthetic glucocorticoid dexamethasone (dex) (100 nM) or ethanol were hybridized onto the ChIP-chip tiling arrays. Independent biological replicates were hybridized onto two separate arrays, and GBRs were identified using the SignalMap detection program; we detected a 3.4% false positive rate for the GBRs found in both arrays as assessed by conventional ChIP and quantitative PCR (qPCR) analysis. Importantly, we did not detect GR occupancy at 22 regions that showed no GR binding in the arrays (unpublished data). The ChIP-chip experiments revealed a total of 73 GBRs adjacent to 61 genes , which wOf the 73 A549 GBRs identified in the present study, 64 (88%) were associated with genes regulated by GR in those cells . AlthougTo test whether the A549 GBRs can confer glucocorticoid-directed transcriptional responses, we cloned 500-bp DNA fragments encompassing the GBRs into luciferase reporter plasmids. Of the 20 GBRs randomly selected from the 73 GBRs identified in this study, 19 were dex responsive in A549 cells as assessed by reporter analysis A. We defWe determined the positions of the A549 GREs relative to TSSs of their respective target genes A. For thOur finding concerning GRE distribution is supported by two indirect analyses using nuclease sensitivity and sequence conservation. Sabo et al. found that DNAse I hypersensitive sites, indicative of chromatin-bound factors, are broadly distributed with a majority located >10 kb from the nearest TSS . FurtherNative GREs, defined as naturally evolved genomic elements that confer glucocorticoid regulation on genes in their chromosomal contexts, are likely to be “composite elements,” made up of binding sites for GR together with multiple nonreceptor regulatory factors . To asseIL8 gene. Importantly, the AP-1 binding site is known to be crucial for regulation of IL8 by the AP-1 factor [IL8 GRE region [In addition, we found that motifs similar to AP-1, ETS, SP1, C/EBP, and HNF4 binding sequences were enriched in the 500-bp GRE fragments B. For ex1 factor ; similarE region . Thus, aE region .To estimate the extent of GRE conservation, we measured sequence identity in human and mouse across 4-kb regions centered on the core GR binding sites see  averaged were indeed dex inducible in the C3H10T1/2 cells  GR occupancy at a GRE is generally a limiting determinant of glucocorticoid response in A549 cells; (2) the core GR binding sequences conform to a consensus that displays substantial GRE-to-GRE variation as anticipated, but the precise binding sequences at individual GREs are highly conserved through evolution; (3) GREs appear to be evenly distributed upstream and downstream of their target genes; (4) most GREs are positioned at locations remote from the TSSs of their target TSSs; and (5) native GREs are commonly composite elements, comprised of multiple factor binding sites, and they are individually conserved in position and architecture yet very different from each other. We shall consider the implications of these conclusions in turn.We began by surveying more than 1,000 genes, with half of them candidates for steroid regulation, and a specific subset known to be GR-regulated in A549 cells. We found that GR occupancy of A549 GREs correlated strongly (nearly 90%) with genes that are glucocorticoid responsive in A549, suggesting that GR binding is generally a limiting determinant for response in these cells. In a small number of cases, we observed GR occupancy close to genes that were GR-unresponsive in A549 cells, but were steroid regulated in other cells [We examined sequence conservation of a set of GREs that are occupied by GR both in human lung epithelial cells and in mouse mesenchymal stem cells. We found that the 15-bp core GR binding sequences varied greatly among the different GREs B, whereaTo characterize the architecture of GREs, we took several approaches. In unbiased computational analyses, we identified enriched sequence motifs within 500-bp segments encompassing core GR binding sites. Sequence motifs resembling binding sites for GR, AP-1, ETS, SP1, C/EBP, and HNF4 were overrepresented relative to a background of unbound GR regions, consistent with the notion that native GREs are composite elements. For most of these GREs, the role of these factors in GR transcriptional regulation remains to be tested, but it is notable that ETS-1, SP1, and HNF4 have been shown at other genes to augment glucocorticoid responses –39. More. For example, Carroll et al. detected an interaction between the NRIP-1 promoter and its distal estrogen response element [To investigate the distribution of regulatory elements relative to their target genes, we monitored GR occupancy across 100 kb regions centered on the TSSs of glucocorticoid responsive genes. We found that GREs were evenly distributed upstream and downstream of their target genes with the majority located >10 kb from their target promoters; other metazoan regulatory factors, such as estrogen receptor (ER) and STAT1, have similarly been reported to act from sites remote from their target genes ,42–45. I element . It willRemote response element locations can complicate assignment of cognate target genes. An extreme example is olfactory receptor gene expression, which is governed by a regulatory element that can operate on target genes located on different chromosomes . In thisGenomic response elements orchestrate transcriptional networks to mediate cellular processes for single- and multicellular organisms. The present study advanced our understanding of the organization, evolution, and function of GREs and at the same time raised a series of interesting questions. Among the more intriguing: How is GR occupancy restricted to a small subset of potential GREs in a given cell context? What is driving the strong conservation of virtually every base pair within the core GR binding sequence at individual GREs? Addressing these and other questions raised in our study will contribute additional new insights about gene regulation by GR and by other regulatory factors.http://www.promega.com). The 20 reporters tested (http://www.stratagene.com) was used for reporter mutagenesis. The 13 core GR binding sites that were mutated in the reporters (http://www.invitrogen.com). After overnight transfection, cells were treated with hormone, harvested, and luciferase activity was measured as described for the dual luciferase reporter system (Promega) using a Tecan Ultra Evolution plate reader .A549 and C3H10T1/2 cells were grown in DMEM supplemented with 5% or 10% FBS, respectively, in a 5% carbon dioxide atmosphere. Before hormone treatment, media was replenished with DMEM containing charcoal stripped FBS, which depletes endogenous steroids. Plasmid PGL4.10 E4TATA (generously provided by Yuriy Shostak) was created by insertion of the E4TATA minimal promoter into pGL4.10 vector . The ligation-mediated PCR (LMPCR) process was adapted from Oberley et al. [Hsp70 (see http://genome.ucsc.edu) NCBI Build 35, and qPCR primers were designed using Primer3 [http://www.nimblegen.com). SignalMap was utilized to find peak enrichments with both window threshold detection  and second derivative peak detection  (NimbleGen).ChIP assays were performed as described  with they et al. . We used Primer3 . For theThe RNA isolation, reverse transcription, and qPCR steps were performed as previously described . PrimersThe experiments were adapted from previous described protocol  with thep-values for enrichment were Bonferroni corrected to identify putative factor binding sites [For computational analysis of enriched motifs, all repeat-masked DNA sequences were downloaded from the UCSC genome browser (NCBI Human Build 35). BioProspector analysis was initially performed using nucleotide widths (w) 14 and 16 on GREs to identify GR binding sites and the top motifs were masked to identify other motifs . For Mobng sites . The topp-values less than 0.05, which were used to measure similarity to known binding sites in TRANSFAC [We built position weight matrices (PWMs) of those motifs with TRANSFAC . We meashttp://pipeline.lbl.gov/cgi-bin/gateway2).The human–mouse conservation score was calculated as described  using a Figure S1ChIP-chip analysis revealed GR occupancy at genes not previously recognized as GR targets in A549 cells. Analysis using qPCR demonstrated that 28 of these genes were dex responsive after four or eight hours of treatment. Induction or repression is represented as fold changes in positive or negative values, respectively, averaged over at least three independent experiments. The data shown represent largest fold change obtained from four or eight hours of hormone treatment.(109 KB PDF)Click here for additional data file.Figure S2All 73 GRE sequences were scored for putative GR binding sites using a predicted position weight matrix representative of the GR binding site. Percent of sequences predicted to contain a GR binding site with varying score cutoffs is plotted as red squares. The false positive rate (blue triangles) was calculated by randomly sampling unbound sequences at varying score cutoffs.(126 KB PDF)Click here for additional data file.Figure S3Identity scores were determined for human–mouse aligned sequences and are plotted as in (498 KB PDF)Click here for additional data file.Figure S4(A) A sequence comparison of human GRE 10.5 with human GRE 6.1 is shown.(B) A sequence comparison of human GRE 6.4 and X.2 is shown. The sequences were pair-wise aligned using ClustalW  and simi(142 KB PDF)Click here for additional data file.Table S1Quantification of relative mRNA levels by qPCR of a subset of the 587 genes (denoted at ChIP-chip Spanned) included in the ChIP-chip arrays  showed t(23 KB XLS)Click here for additional data file.Table S2The distances were calculated based on coordinates of the mouse aligned GREs (mGREs) and TSSs of the GR-regulated mouse homolog genes obtained from UCSC Genome Browser. The TSS of the longest transcript was used for this calculation when a gene has multiple variants. Bold letters represent the distance of the GREs relative to the adjacent gene in mouse.(21 KB XLS)Click here for additional data file.Table S3Capitalized letters represent the restriction digestion sites used for cloning the constructs into pGL4.10 E4TATA.(25 KB XLS)Click here for additional data file.Table S4FO primer and RE primer represent the forward and reverse primer, respectively, for the corresponding amplified genomic regions or cDNA sequences of the indicated genes.(39 KB XLS)Click here for additional data file.


Rousettus aegyptiacus) in Gabon as shown by finding virus-specific RNA and IgG antibody in individual bats. These Marburg virus positive bats represent the first naturally infected non-primate animals identified. Furthermore, this is the first report of Marburg virus being present in this area of Africa, thus extending the known range of the virus. These data imply that more areas are at risk for MHF outbreaks than previously realized and correspond well with a recently published report in which three species of fruit bats were demonstrated to be likely reservoirs for Ebola virus.Marburg and Ebola viruses can cause large hemorrhagic fever (HF) outbreaks with high case fatality (80–90%) in human and great apes. Identification of the natural reservoir of these viruses is one of the most important topics in this field and a fundamental key to understanding their natural history. Despite the discovery of this virus family almost 40 years ago, the search for the natural reservoir of these lethal pathogens remains an enigma despite numerous ecological studies. Here, we report the discovery of Marburg virus in a common species of fruit bat ( Forty years after the discovery of Marburg virus as the cause of a hemorrhagic fever outbreak among laboratory workers in Germany, the natural reservoir for this highly pathogenic filovirus remains unknown Rousettus aegyptiacus) as evidenced by the presence of virus specific RNA and antibody.Here, we report testing of bats collected from Gabon and Republic of Congo and demonstrate Marburg virus infection in a common species of fruit bat  to inactivate virus. Each tissue was then diluted to 1X and homogenized for 2 minutes, at 1500 strokes/min using a ball-mill tissue grinder . Total RNA was extracted from ∼150 ul of the homogenate Epomops franqueti (N = 47) were used as the negative control group.IgG was detected from bat sera diluted 1∶100 using a previously described protocol modified for Marburg virus Bat derived sequence fragments (Genbank accession numbers EU068108-13) were concatenated then aligned with 18 MBG virus genomes  Maximum likelihood analyses (bootstrap 500 replicates) were completed . Genetic distances were calculated using the Wisconsin package of GCG version 10.3. .Bat species were morphometrically determined in the field using the species identification key developed by Bergman Rousettus aegyptiacus. Homogenized liver and spleen samples of 1138 bats were first analyzed using a Marburg virus-specific real-time RT-PCR assay to the VP40 gene recently used for human diagnostic testing during the 2005 Angola MHF outbreak Rousettus aegyptiacus, were positive at low levels  in the real-time assay. All four of these bats were trapped near caves in 2005 and early 2006 at two geographic locations in Gabon 250 km apart and approximately 700 km north of Uige, Angola. These four samples were then subjected to further analysis by conventional nested RT-PCR targeting the virus VP35 and NP genes  (Rousettus aegyptiacus while none of the other species tested (N = 196) had corrected OD values greater than this threshold value. Sera from three of the 29 bats had OD values greater than 0.13 when diluted 1∶400 while another five bats, including 1448 and 2296, met the same criteria at dilutions of 1∶1600  using a goat anti-bat conjugate made by immunization with IgG from multiple diverse bat species (both micro and mega-chiropterans). The results of this secondary testing were identical to the initial serological findings using protein A/G (data not shown). These data indicate a substantial fraction, almost 9%, of Rousettus aegyptiacus trapped at these locations may have low-level antibody to Marburg virus, while another 3% have more significant Marburg antibody titers. Among the R. aegyptiacus population tested for which age determinations could be made, evidence of Marburg infection in bats favored adults (24/138) over juveniles (4/86), 17.4% to 4.6% respectively . However, firm conclusions about the proportion of infected adult versus juvenile populations are difficult because the majority of ‘positive’ bats show only low titers of Marburg antibody while those bats with more conclusive evidence of Marburg infection, by being either PCR positive and/or having IgG titers greater than or equal to 1∶400 (N = 8), are more equivalently distributed (five adults and three juveniles). In addition, there could be residual maternal Marburg-specific antibody in the juvenile bat population. Fifteen of the bats, with OD values greater than 0.13, were males while among the three PCR-positive animals, two were adults  and one was a juvenile .After screening the collection of bats by real-time RT-PCR, we tested for Marburg virus specific antibody in sera (if available) from all bats trapped at the two locations (438 bats) from which PCR positive bats were found . Interesanqueti) . Moreovef 1∶1600 . UnfortuR. aegyptiacus, it is worth noting that all of the animals caught appeared clinically healthy and were strong enough to leave their roost to forage for food. Virus isolation attempts and antigen detection tests on the same liver/spleen organ extracts were negative, which along with the quantitative PCR data, indicate low levels of Marburg virus in these organs.The serological data, combined with the PCR data, are suggestive that these bats may represent a bon-a-fide reservoir species. However, we cannot rule out periodic contact by the bats with an as yet unnamed reservoir. The presence of both virus RNA and IgG antibodies in three (or possibly four) of the animals is consistent with extended viremias, but may well represent late acute phase infections. While it is difficult to determine from these data if Marburg virus causes significant morbidity in R. aegyptiacus representing a natural host for Marburg virus. Cave roosting is not generally observed with most fruit bats R. aegyptiacus is known to roost in caves, a behavior that correlates well with the epidemiologic linkage of greater than 80% of human cases in the Durba MHF outbreak to mining activity in a gold mine R. aegyptiacus encompasses the geographic origin of all known sources of Marburg virus outbreaks (Pteropodinae may serve as filovirus hosts and 3) the species of fruit bats that harbor Ebola and Marburg viruses are likely distinct, yet their home ranges may have large areas of overlap. Identification of the reservoir host should allow development of risk reduction measures to help mitigate the potential of future disease outbreaks.Although inconclusive, several other lines of evidence are consistent with utbreaks  [13] as


GSAO (4-[N-[S-glutathionylacetyl]amino] phenylarsenoxide) is a hydrophilic derivative of the protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO). It inhibits angiogenesis and tumour growth in mouse models and may be evaluated in a phase I clinical trial in the near future. Initial experiments have implicated GSAO in perturbing mitochondrial function. Other molecular effects of GSAO in human cells, for example on the phosphorylation of proteins, are still largely unknown.Peripheral white blood cells (PWBC) from healthy volunteers were isolated and used to profile effects of GSAO vs. a control compound, GSCA. Changes in site-specific phosphorylations, other protein modifications and expression levels of many signalling proteins were analysed using more than 400 different antibodies in Western blots.in vivo conditions. This eliminated detectable GSAO effects on most, but not all signalling proteins analysed. Activation of the MAP kinase Erk2 was still observed and the paxillin homologue Hic-5 still displayed a major shift in protein mobility upon GSAO-treatment. A GSAO induced change in Hic-5 mobility was also found in endothelial cells, which are thought to be the primary target of GSAO in vivo.PWBC were initially cultured in low serum conditions, with the aim to reduce basal protein phosphorylation and to increase detection sensitivity. Under these conditions pleiotropic intracellular signalling protein changes were induced by GSAO. Subsequently, PWBC were cultured in 100% donor serum to reflect more closely in vitro. Low serum culture, which is typically used in experiments analysing protein phosphorylation, is not suitable to study GSAO activity in cells. The signalling proteins affected by GSAO under high serum conditions are candidate surrogate markers for GSAO bioactivity in vivo and can be analysed in future clinical trials. GSAO effects on Hic-5 in endothelial cells may point to a new intracellular GSAO target.Serum conditions greatly influence the molecular activity profile of GSAO  The term 'cancer' encompasses a wide variety of distinct, multigenic diseases. Even within a specific tumour type, a remarkable degree of heterogeneity on the level of DNA lesions and affected signalling pathways is apparent. Many cancer relevant signalling molecules, but also many molecular targets of anti-cancer drugs, therefore remain unknown. Prominent examples of signalling protein classes long known to be involved in generating cancer pathologies include GTPases, protein kinases and transcription factors. By contrast, protein phosphatases have only recently entered the stage as known players in cancer development. At least 30 protein phosphatases are now implicated in cancer development and other human diseases -3. In soin vivo markers of drug activity are available before or very early during the clinical trial.The modulation of specific cellular signalling pathways to treat human cancers has only recently developed into an area of intense clinical research activity. A large number of clinical trials for novel signal transduction modulator (STM) drugs are currently planned or under way. STM drugs often have relatively low toxicity, so determination of the maximum tolerated dose (MTD) may not be a prime goal for phase I clinical trials. Instead, identification of an optimal biologically active dose (OBD) is essential . Rapid dThis study identifies several proteins in PWBC which are affected by the novel anti-cancer compound GSAO (4-[N-[S-glutathionylacetyl]amino] phenylarsenoxide)  Figure . They main vitro and in vivo [per se explain the low toxicity and anti-angiogenic activity of GSAO.GSAO has anti-angiogenic activity  in vivo . Mitochoin vivo activity of GSAO in endothelial cells. Nevertheless, it is well known that practically all drugs in clinical use have multiple molecular in vivo targets. They depend on the affected cell type and applied doses. Some of these targets are intimately linked to the adverse effects of the drug. In cancer cells, with their heterogeneous genetic lesions, drug targets can even be present in different combinations or at different expression and activity levels when comparing individual patients. Identification and monitoring of novel molecular targets could thus help to understand why a drug is more effective or toxic in some patients compared to others. PWBC from donated blood were chosen for the analyses, since they are easily obtainable from healthy volunteers prior to clinical trial and later from the patients enrolled in the trials. A main focus of the experiments was to detect changes in protein phosphorylation induced by GSAO treatment, because GSAO is a hydrophilic derivative of phenylarsine oxide (PAO), a well known protein phosphatase inhibitor [in vivo applications and toxic for cells at nanomolar concentrations [Recent studies have revealed that differences in multidrug resistance proteins (MRP1 and 2) and cellular glutathione levels  appear tnhibitor ,13. In ctrations . It pene2, before treatment with the compound of interest. Cells were treated with either GSAO, the active drug, or 4-N-(S-glutathionyl acetyl) amino) benzoic acid (GSCA) in which the trivalent arsenic of GSAO has been replaced by a carboxylic acid, as a control, for the concentrations and durations indicated. Stock solutions of both GSCA and GSAO were made at a concentration of 3 mM in RPMI 1640 and, since GSAO is prone to oxidation, stored in aliquots at -80°C unless used immediately. PAO was freshly dissolved in cell culture grade DMSO at a concentration of 10 mM and diluted to 1 μM with medium.PWBC were obtained from 14 healthy volunteers not taking any medication  with informed consent. Ethical approval was granted by the Oxfordshire Ethics Committee (study number 05/Q1605/59). Cell preparations from more than 40 different blood donations were used. All blood donations took place between 9 and 11 a.m. to minimise potential circadian changes of signalling proteins. Whole blood (54 ml) was collected from a peripheral arm vein into 6 ml of 46.7% (v/v) trisodium citrate  and syringes immediately placed on a nutator at room temperature for at least 5 min to ensure complete mixing. Batches of 20 ml citrated blood were then carefully layered onto 20 ml Polymorphprep™ density gradient media  and centrifuged at 500 × g for 30 min at 20°C to remove erythrocytes. PWBC were collected from the medium/serum interface, transferred into a 50 ml centrifuge tube and gently resuspended in sterile PBS. PWBC were then centrifuged for 20 min at 600 × g at 20°C, PBS was aspirated and the PWBC pellet stored in ice. PWBC were subsequently cultured in either 100% donor serum or RPMI1640 supplemented with 0.5% foetal bovine serum (FBS), 100 μg/ml penicillin-streptomycin and 2 mM L-glutamate. Donor serum was simultaneously prepared freshly from peripheral blood. For this, blood was collected without anticoagulant and centrifuged for 30 min at 3700 × g at 20°C. The supernatant was transferred into a fresh centrifuge tube and incubated for 2 h at room temperature with nutation to allow coagulation, before centrifugation at 3700 × g for 30 min at 20°C to obtain serum. Pelleted PWBC were resuspended in either growth medium or donor serum and equilibrated for 2 h at 37°C in a humidified cell culture incubator with 5% COTreated or mock-incubated PWBC were scraped from tissue culture flasks, transferred to a 50 ml centrifuge tube filled with PBS and centrifuged at 3700 × g for 20 min at 20°C to pellet the cells. The PBS was completely aspirated from the PWBC pellet and the pellet lysed directly in boiling SDS-PAGE sample buffer  β-mercaptoethanol, 40% (v/v) glycerol, 3%(w/v) SDS, 0.05% (w/v) bromophenol blue). The samples were then boiled for a further 5 min, cooled to room temperature and sonicated for 30 seconds to fragment DNA contained in the cell lysate. Proteins in the lysate were resolved by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to PVDF membranes . The membranes were blocked and probed overnight at 4°C with different phospho-epitope specific or other antibodies according to the antibody manufacturers instructions. Bound primary antibodies were detected using horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulins (Jackson ImmunoResearch Inc.) and ECL (Amersham Biosciences). Equal loading of different protein extract samples was routinely confirmed by Coomassie-blue staining of an SDS-PAGE-separated aliquot prior to blot analyses. Antibodies used in this study are compiled in the additional files 2. Cells were lysed with boiling sample buffer. Protein analysis, SDS PAGE and Western blots were carried out as described for the PWBC.Human umbilical vein endothelial cells (HUVEC) were purchased from TCS Cell Works,  and cultured according to the manufacturers conditions with recommended growth media, cell attachment factor and passage conditions. Cells were cultured in 10 cm plates until 80% confluent before treatment with GSAO or GSCA in growth media with high serum (15% FBS) for 24 hours at 37°C with 5% COIn initial experiments it was determined how rapidly protein phosphorylation changes were detectable upon GSAO treatment of freshly isolated PWBC from healthy donors under low serum culture conditions. For this, PWBC were incubated with GSAO or its more hydrophobic relative PAO. As previously reported in the literature for various cell types, increased tyrosine phosphorylation was rapidly observed with PAO. Incubation of PWBC with 1 μM PAO for 1 h resulted in a hyper-phosphorylation of protein-tyrosyl residues that was comparable to an incubation of PWBC with 50 μM GSAO for 8 h . This waIn a subsequent set of experiments, the effects of GSAO vs. GSCA on specific signalling proteins in PWBC were investigated. The results are described and shown in additional text file and additional files in vivo [From this large data set we conclude that GSAO has rather pleiotropic effects on the cell signalling protein network of PWBC cultured in low serum conditions. Since it is very unlikely that a drug which affects numerous key signalling proteins would show the low toxicity profile seen in vivo , we modiFigure Subsequent analyses focussed on GSAO effects on high serum-cultured PWBC with those phospho-epitope-specific antibodies, which had shown phosphorylation changes under low serum culture conditions. From these experiments, it is obvious that GSAO is affecting only a much smaller number of proteins in high serum culture. For example, p90Rsk phosphorylation on Ser380 and phosphorylation of JNKs is at best barely observable in high serum culture figure . Howeverhydrogen oxide inducible clone-5; [The paxillin homologue Hic-5  array-format assays or FACS-based HTP analyses like multiplex-bead assays difficult to properly evaluate.Our experiments clearly show that classical low serum culture, often used to reduce background signals prior to addition of a drug or factor, is not ideal to find potential markers for GSAO bioactivity Monitoring of drug effects on total protein expression levels, is simpler, but appears to be overall less informative as many phospho-changes are not mirrored by changes in overall protein expression. Nevertheless, it can give some initial leads and many good quality monoclonal antibodies are readily available. For optimal retrieval of information from western blot type assays it is necessary to perform non-automated visual re-inspections to detect all changes. The GSAO-induced Hic-5 mobility shift was found through this approach and would have been missed using a spot array or another technique not separating proteins by apparent molecular weight.Hic-5 is an interesting candidate for further analyses of GSAO actions in cells. It is a member of the paxillin family ,20 and hThe consistent activation of the MAP kinase Erk2 by GSAO under high and low serum conditions is also intriguing, since no activation of its regulator kinases MEK1/2 could be observed (data not shown). Apart from a direct effect on Erk-inactivating phosphatases, like MKPs, other more indirect changes in PWBC signalling may lead to Erk2 activation. Since the intracellular GSAO uptake is very slow and effects are usually seen after many hours, a plethora of possible indirect mechanisms, including changes in the transcription of certain genes may underlie the observed Erk2 activity change. Erk2 activation is thought to trigger its dimerisation and nuclear translocation , possiblin vivo activity of GSAO in this cell type.Changes in one of the three candidate surrogate markers for GSAO activity found in PWBC is detectable in endothelial cells, but additional changes in tyrosine phosphorylation were observed in a multitude of proteins at 15 μM GSAO treatment in HUVEC. This suggests that overlapping, but distinct groups of signalling pathways are affected by GSAO in the different cell types. Further studies into the biological mechanisms of GSAO interference with endothelial cell proliferation are needed to elucidate the This study has shown that GSAO effects on the PWBC phosphoprotein signalling network are quite selective, provided the appropriate experimental conditions are used. The results also give suggestions for the development of new reagents, which may be useful to support future clinical studies and to possibly gain further insight into the cellular targets of GSAO. Analyses with substrate epitope-specific antibodies show that further GSAO-affected proteins remain to be identified. Variation in phosphorylation patterns between PWBC and HUVEC suggest distinct pathway patterns become activated by GSAO in different cell types. As PWBC are easily obtainable they are more probably suitable for measuring surrogate markers in future clinical studies, but endothelial cells are clearly also an important cell type that needs to be analysed further with molecular and functional assays.All authors declare not to have any competing interests. Cancer Research UK, which funds the work of VAC, SMF and ALH, is a sponsor and organiser of the planned GSAO clinical trial.VAC performed most of the experiments and participated in the drafting of the manuscript. PJH provided the GSCA and GSAO and critically commented on the drafted manuscript. ALH participated in the design of some experiments and critically commented on the drafted manuscript. SMF designed the study, drafted the manuscript and performed some initial experiments.The pre-publication history for this paper can be accessed here:GSAO and phenylarsine oxide (PAO) induce similar patterns of tyrosine phosphorylation in peripheral white blood cells (PWBC). Freshly isolated PWBC were equilibrated in medium with 0.5% FBS for 2 h and then treated with the indicated concentrations of PAO or GSAO. PWBC were subsequently lysed and proteins analysed for protein tyrosine phosphorylation by western blot. Uptake of the hydrophilic GSAO into cells is, as expected, very slow. Treatment of PWBC with 50 μM GSAO for 8 h results in protein tyrosyl-hyperphosphorylation similar to that seen upon incubation with 1 μM PAO for 1 h. Hyperphosphorylated protein bands are indicated by arrows.Click here for fileDose-dependent increase of PWBC protein threonine phosphorylation induced by GSAO. PWBC were treated with GSAO or GSCA where indicated for 24 h and threonine phosphorylation detected by western blot. A 50 μM GSCA does not alter the protein pThr levels of PBWC detectably. B Changes in protein pThr patterns are apparent with as little as 1.5 μM GSAO. GSAO-affected proteins are indicated with arrows.Click here for fileGSAO induces phosphorylation of c-Jun N-terminal kinases (JNKs) at the kinase activity-regulating epitope (Thr 183/Tyr 185) in PWBC. PWBC were treated with GSAO or GSCA, then lysed and analysed by western blot for JNK expression or phosphorylation. The phospho-specific antibody detects JNK protein only effectively when doubly phosphorylated on Thr183 and Tyr185. A Cells were treated with 50 μM GSAO or GSCA for 24 h where indicated and analysed for phospho-JNK (upper panel) or total JNK (lower panel) B Cells were treated with different concentrations of GSAO for 24 h as indicated and analysed for phospho-JNK.Click here for fileEffects of GSAO on the MAP kinase cascade. Cells were treated with GSAO or GSCA as indicated for 24 h and lysates analysed for the phosphorylation of Erk1/Erk2 (pThr202/pTyr204) or c-Raf-1 . Phosphorylation of Erk1/Erk2 at the epitope Thr202/Tyr204 correlates closely with Erk kinase activation. The phospho-epitopes analysed for c-Raf-1 also contribute to the regulation of c-Raf-1 kinase activity, but numerous other regulatory sites have been described in c-Raf-1. A Effect of GSAO treatment on the phosphorylation of Erk1/Erk2 (pThr202/pTyr204) detected by a polyclonal antibody . Apart from the major Erk1/2 phospho-bands, several additional phospho-bands of different sizes, some possibly representing other Erk family members, are visible. B Effect of GSAO treatment on the phosphorylation of Erk1/Erk2 (pThr 202/pTyr204) detected by a monoclonal antibody . C Effect of GSAO treatment on the phosphorylation of c-Raf-1 at Tyr340/Tyr341. Phosphorylation of this epitope can contribute to the activation of Raf kinase. D Effect of GSAO treatment on the phosphorylation of c-Raf-1 at Ser259. Dephosphorylation of this epitope is thought to be important for activation of the kinase. The c-Raf-1 band shift observed with 15 and 50 μM GSAO, probably resulting from phosphorylation events at other c-Raf-1 epitopes, makes it difficult to determine if a partial dephosphorylation of pSer259 occurs at these GSAO concentrations.Click here for fileEffects of GSAO on the phosphorylation of p90Rsk at Ser380 in PWBC obtained from different volunteers. PWBC were treated with GSAO or GSCA as indicated for 24 h and lysates analysed for the phosphorylation of Ser380 in p90Rsk. Cells from three different donors were used. The male donor was analysed on two different occasions. Increased Ser380 phosphorylation is clearly visible at 5 μM GSAO in all cases. With 50 μM GSAO a major mobility shift of p90Rsk (pSer380) is visible. This could indicate the occurrence of additional phosphorylation events.Click here for fileDetection of proteins hyperphosphorylated after treatment of PWBC with GSAO using a PKA substrate-directed antibody. Cells were incubated for 24 h with GSAO or GSCA as indicated and analysed with an antibody made to recognise multiple PKA substrate proteins in their PKA-phosphorylated forms . A Strong effect of GSAO on phosphoproteins detected with the PKC substrate phospho-antibody using protein extracts from low serum cultured PWBC. The control compound GSCA does not detectably change the basal phospho-protein pattern. B Effects of 15 μM GSAO on high serum cultured PWBC. Arrows indicate bands that are slightly hyperphosphorylated in GSAO-treated cells.Click here for fileDetection of proteins hyper-phosphorylated after treatment of PWBC with GSAO using a PKD substrate-directed antibody. Cells were incubated for 24 h with GSAO or GSCA as indicated and analysed with an antibody made to recognise multiple PKD substrate proteins in their PKD-phosphorylated forms  A Effect of GSAO on phosphoproteins detected with the PKC substrate phospho-antibody using protein extracts from low serum cultured PWBC. The control compound GSCA does not detectably change the basal phospho-protein pattern. B Effects of 15 μM GSAO on high serum cultured PWBC. Arrows indicate three bands that are clearly hyperphosphorylated in GSAO-treated cells.Click here for fileCompiled results of the Powerblot™ comparison of PWBC cultured in low serum and treated with 50 μM GSAO or GSCA.Click here for fileAntibodies used for analysis of GSAO effects on PWBC.Click here for fileSummary of GSAO-induced protein expression changes in PWBC cultured with low serum medium. Of 240 individually selected signalling proteins, which were analysed by the commercially available Powerblot™, the 24 proteins listed here showed an apparent change in protein expression considered to be highly significant according to the company. Proteins in blue color are upregulated, proteins in black downregulated with GSAO vs. GSCA. From this list it is obvious, that kinases and PTPases and their targets make up the majority of proteins affected in their expression level by GSAO. At least two of the signals  are, however, clearly questionable, as the detected band size in the sample varies greatly from the expected protein size. In other cases, for example Hic-5, an expression change is detected by the automated system. Upon visual inspection this supposed expression change results from a mobility shift of Hic 5. A full list of all proteins analysed and changes detected are compiled in Click here for filePleiotropic effects on PWBC in low serum conditions.Click here for file


Nodal activity in the left lateral plate mesoderm (LPM) is required to activate left-sided Nodal signaling in the epithalamic region of the zebrafish forebrain. Epithalamic Nodal signaling subsequently determines the laterality of neuroanatomical asymmetries. We show that overactivation of Wnt/Axin1/β-catenin signaling during late gastrulation leads to bilateral epithalamic expression of Nodal pathway genes independently of LPM Nodal signaling. This is consistent with a model whereby epithalamic Nodal signaling is normally bilaterally repressed, with Nodal signaling from the LPM unilaterally alleviating repression. We suggest that Wnt signaling regulates the establishment of the bilateral repression. We identify a second role for the Wnt pathway in the left/right regulation of LPM Nodal pathway gene expression, and finally, we show that at later stages Axin1 is required for the elaboration of concordant neuroanatomical asymmetries. Structural and functional asymmetries are common features of the nervous systems of both invertebrates and vertebrates . The besDuring early development in zebrafish, bilaterally located parapineal cells migrate leftward from the pineal complex to form a left-sided nucleus that sends ipsilateral axonal projections to the left habenula . The paiThe parapineal influences the elaboration of habenular asymmetries. For instance, the parapineal modulates gene expression in the left habenula, and ablation of parapineal cells results in the left habenula adopting some right-sided character . On the The earliest known indication of brain asymmetry in zebrafish is the expression of Nodal pathway genes within the left epithalamus from about 18 hpf . EpithalXenopus can lead to cardiac left-right reversals  embryos carry a mutation in Axin1 that disrupts the binding of GSK3β, reducing the ability of GSK3β to degrade β-catenin and consequently leading to overactivation of Wnt/β-catenin signaling in the anterior neural plate  and lft1 (n = 25 of 39) expression . This phenocopies mbl mutants, lending support to the conclusion that overactivation of Wnt signaling in the anterior neural plate during late gastrulation removes repression of epithalamic Nodal pathway gene expression. At no other stage from late gastrula until 22 somite stage did LiCl treatment affect epithalamic Nodal asymmetries without concomitantly affecting gene expression in the LPM  or lft1 (n = 47 of 47). Thus, by mid-somite stages, activation of epithalamic Nodal signaling is dependent upon LPM Nodal activity.The laterality of expression of Nodal pathway genes in the brain was concordant with expression in the LPM in these experiments, suggesting that by mid-somite stage, the activation of Nodal signaling in the brain is dependent upon Nodal signaling emanating from the LPM. To test this, we treated To further validate our conclusions suggesting a temporally restricted role for Wnt signaling and to assess whether such a role is conserved across species, we performed comparable experiments in the distantly related teleost, medaka . Asymmetwnt1 in a Tg line at 2–4 somite stage results in bilateral Nodal pathway gene expression in the epithalamus and LPM . These data indicate that Axin1 is required for the correct onset of parapineal cell migration.Analysis of igration . In contal cells . Expresslls . Even accounting for the variable Nodal expression phenotype of mbl embryos, which predicts 64% left-sided parapineal cells, the greater leftward bias in parapineal cell migration in mbl embryos is statistically significant . To verify this result, we generated a double transgenic mbl mutant line, in which GFP is expressed under the control of both the lft1 promoter  showed 35% with parapineal migration at 2 days, two-thirds of which were positioned to the left and one-third to the right. Together with our analysis of gfi and otx5 expression  and therapineal ). Analyspression , these dmbl embryos develop symmetrically. Expression of the superficially symmetrical habenular markers cxcr4b (n = 45/53) and zic2a (n = 15/15) is not obviously affected in mbl mutants  or nonmigrated parapineal cells (n = 6) were selected for further analysis. At 4 days of development, all 12 of these embryos exhibited a migrated parapineal. Irrespective of whether parapineal cells showed normal or delayed migration, parapineal neurons projected axons to one or the other habenula, or in one case to both habenulae . However, to look for subtle differences in lov expression, we incubated the brains in the color reaction substrate for a further 24 hr. Following this procedure, we observed slightly higher levels of lov expression in the habenula innervated by parapineal projections. This suggests that, in mbl embryos, there is still residual communication between the parapineal and habenular cells.To determine the relationship between the various epithalamic asymmetries in individual mbl embryos may compromise the ability of parapineal axons to influence habenular neuron connectivity.The mechanism by which the parapineal influences lateralized gene expression in the habenulae is not understood. One possibility is that the parapineal axons that innervate habenular neurons influence gene expression and efferent connectivity of these neurons. We speculated that if this is the case, then delayed innervation of the habenulae in mbl embryos have projections by 46 hpf comparable to wild-types  as in wild-type embryos  and found that migrated and nonmigrated parapineal neurons in ld-types M and 5N. embryos O and 5P.In this study, we show that Wnt/Axin1/β-catenin signaling is involved reiteratively during the establishment and concordant elaboration of brain and body asymmetries. Activation of Wnt signaling during late gastrula stages abrogates asymmetry of expression of Nodal pathway genes in the brain but not the LPM, and we suggest that this occurs through the loss of bilateral repression of Nodal pathway activation in the prospective epithalamus. Widespread activation of the pathway during mid-somite stages results in concordant disruption to asymmetric Nodal pathway gene expression in both the LPM and brain, suggesting that by this stage Nodal activation in the brain is dependent upon Nodal signaling in the LPM. Downstream of Nodal pathway activity in the epithalamus, Axin1 is required both for development and concordance of parapineal and habenular asymmetries.mbl embryos frequently exhibit bilateral activation of Nodal pathway genes in the brain despite establishment of normal visceral situs. Axin1 is a critical component of the protein complex that degrades β-catenin  and other events that lead to the establishment of LPM asymmetries  showing that Six3b/Six7 activity in the neural plate during late gastrulation is required to repress epithalamic Nodal pathway gene expression. Enhanced Wnt signaling, as seen in mbl embryos, suppresses Six gene expression .Finally, the model provides a likely explanation for an unusual aspect of the ndomized . Howeverwnt1 overexpression in medaka during a narrow time window at mid-somitogenesis stages frequently leads to bilateral activation of Nodal pathway gene expression in both LPM and brain. At these stages, key events such as midline development and formation of KV have already occurred. Indeed, KV is beginning to regress at the time at which we manipulate Wnt signaling . On the other hand, habenular neurogenesis does appear to be slightly delayed by approximately 2 hr between 32 and 34 hpf as judged by the appearance of HuC+ neurons (data not shown). As described previously  mutant lines and tm213mblxTg(foxD3:GFP) and tm213mblxTg(foxD3:GFP); Tg(lft1:GFP) transgenic fish were used. All mutant analyses in this study were performed using transgenic mbl mutants derived from an initial cross of tm213mbl to Tg(foxD3:GFP) transgenic fish, which were kept in an AB background This background resulted in mild mbl mutant phenotypes in subsequent generations  transgenic fish [formerly named (gfp:HSE:Wnt1)] were used . Stages wnt7b cDNAs, the following degenerated primers were designed: Wnt7b-up (5′-GTGGTVGCYYTGGGHGCVARCATCAT-3′) and Wnt7b-low (5′-CCAKTGGAAYTTRCAGTTRCAYTGCC-3′). RT-PCR reactions were performed using somitogenesis stage zebrafish RNA. The resulting cDNA fragment was cloned into TOPO-TA vector and subcloned into pBluescript, and several clones were sequenced. The obtained sequences showed highest homology with wnt7b. As we found slight differences in the sequence and also embryonic expression of two cDNAs (data not shown), we named them wnt7b and wnt7bl after approval by ZFIN (http://zfin.org/zf_info/nomen.html).To clone All of the cDNAs used in this work for RNA synthesis were cloned in pCS2+. Capped mRNA was generated using a Message Machine RNA synthesis kit (Ambion) according to the manufacturer's instructions.Antisense RNA probes were generated using digoxigenin/fluorescein RNA labeling kits (Boehringer-Mannheim). GFP protein was detected by anti-GFP antibody  and acetylated tubulin using antiacetylated tubulin antibody (Sigma T6793). Whole-mount in situ hybridization  and antitcf3a Mo  encoding GFP:HS:lef-VP16 (The following MOs (Genetools) were used in this study: lef-VP16  was injelef-VP16 .LiCl treatments were performed as described . EmbryosIt is important to note that compounds dissolved in DMSO cannot be used for the study of asymmetry, as DMSO alone disrupts Nodal gene asymmetry even at very low concentrations .Heat shock treatments in zebrafish and medaka were performed at 39°C for 1 and 2 hr, respectively.


RPB1 from representatives of Glaucocystophyta, the third eukaryotic group with primary plastids.Evolutionary analyses of the largest subunit of RNA polymerase II (RPB1) have yielded important and at times provocative results. One particularly troublesome outcome is the consistent inference of independent origins of red algae and green plants, at odds with the more widely accepted view of a monophyletic Plantae comprising all eukaryotes with primary plastids. If the hypothesis of a broader kingdom Plantae is correct, then RPB1 trees likely reflect a persistent phylogenetic artifact. To gain a better understanding of RNAP II evolution, and the presumed artifact relating to green plants and red algae, we isolated and analyzed RPB1 trees can be explained as phylogenetic artifacts; however, these apparent artifacts also appear in regions of the tree that support widely held views of eukaryotic evolution. In fact, most of the RPB1 tree is consistent with artifacts of rate variation among sequences and co-variation due to functional constraints related to C-terminal domain based RNAP II transcription.Phylogenetic analyses incorporating glaucocystophytes do not recover a monophyletic Plantae; rather they result in additional conflicts with the most widely held views on eukaryotic relationships. In particular, glaucocystophytes are recovered as sister to several amoebozoans with strong support. A detailed investigation shows that this clade can be explained by what we call "short-branch exclusion," a phylogenetic artifact integrally associated with "long-branch attraction." Other systematic discrepancies observed in Our results reveal how subtle and easily overlooked biases can dominate the overall results of molecular phylogenetic analyses of ancient eukaryotic relationships. Sources of potential phylogenetic artifact should be investigated routinely, not just when obvious "long-branch attraction" is encountered. RPB1 sequences consistently recover a polyphyletic kingdom Plantae, with independent origins of red algae and green plants )C. paradoxa (CCAC 0074) was obtained from the Culture Collection of Algae (CCAC) at the University of Cologne, Germany. Cells were grown in bubbling cultures of soil water medium with barley seeds  under constant fluorescent light at 25°C. Glaucocystis nostochinearum (UTEX-B 1929)was obtained from UTEX culture collection  and grown under the same conditions, but in AlgaGro freshwater medium . Cells were pelleted in a table-top centrifuge and stored at -80°C for nucleic acid extraction.An axenic culture of Glaucocystis samples were placed in a chilled mortar, flash frozen with liquid nitrogen, pulverized with a pestle to a fine powder and suspended in an equal volume of nucleic acid extraction buffer. Because Cyanophora lacks a cell wall, no grinding was required. DNA extractions were performed using a CTAB extraction method  and Cyanophora [DQ223186] were aligned with a data set of RPB1 sequences from organisms present in GenBank and genome-sequencing databases (see Cyanophora (regions A-G). A second smaller alignment, representing 30 taxa, was constructed by removing sequences with large amounts of missing data, as well as sequences demonstrated to produce phylogenetic artifacts in previous analyses.Inferred RPB1 amino acid sequences from ases see . SequencMaximum-likelihood parameters  were estimated in TREEPUZZLE 5.0  under a Analyses were performed using MRBAYES 3.1 , with tha priori phylogenetic hypotheses were examined with RPB1 data by implementing the Kishino-Hasegawa (KH), as well as the more conservative Shimodaira-Hasegawa (SH) tests [One thousand distance bootstrap replicates also were run using in PROTDIST and NEIGHBOR (Phylip 3.6), with a JTT substitution matrix. Parsimony bootstrap was carried out in PAUP  with 100H) tests ,83 in PRRPB1 tree, and specific differences between that topology and trees recovered from other data sets, we analyzed "long-branch" tendencies of sequences in the 30 taxon data set. We used three different methods, each independent of a priori assumptions about relationships among distinct eukaryotic lineages. 1) A χ2 test was performed in TREEPUZZLE to ascertain which sequences deviated significantly from average amino acid composition. 2) Unique autapomorphies at otherwise highly conserved sites were scored for all individual sequences, using MACCLADE 3.06 [RPB1 alignment and used in parsimony analyses with 20 random sequence additions to determine the empirical tendency of each RPB1 sequences to attract "long-branches." Sequences were deemed to be prone to long-branch artifacts if they attracted a random sequence in 5% or more of parsimony replicates. These sequences were removed from the alignment, and the analyses repeated with three progressively smaller subsets of RPB1 genes with decreasing apparent long-branch tendencies. With the smallest of these sub-alignments (five taxa), 1000 bootstrap replicates were performed with each of 10 random sequences (20 random additions each), to determine the distribution of their points of attachment when sequences with stronger "long-branch" tendencies were removed.To assess the bases for the overall topology of the ADE 3.06 . Unique LH sequenced glaucocystophyte RPB1 genes and cDNA, performed bioinformatics searches for other eukaryotic sequences, annotated intron positions, and was primarily responsible for multiple sequence alignments. JWS performed analyses of long-branch indicators. Both authors contributed ideas contained in the paper, worked on phylogenetic analyses and contributed to authorship of the manuscript. Both authors read and approved the final manuscript.Supplementary Table. Database sources for sequence used in this investigationClick here for fileBayesian inference tree. Consensus Bayesian tree inferred from the alignment of 30 RPB1 sequences. Branch lengths and posterior probabilities were recovered using the sumt command in MrBayes. See methods section and legend to figure Click here for file