These phylogenetic studies did not, however, sample two additional groups traditionally classified within Rafflesiales (Apodantheaceae and Cytinaceae). Here we provide molecular phylogenetic evidence using DNA sequence data from mitochondrial and nuclear genes for representatives of all genera in Rafflesiales.The phylogenetic relationships among the holoparasites of Rafflesiales have remained enigmatic for over a century. Recent molecular phylogenetic studies using the mitochondrial Mitrastema, ascertained using mitochondrial matR, are congruent with results from nuclear SSU rDNA when these data are analyzed using maximum likelihood and Bayesian methods. The relationship of Cytinaceae to Malvales was recovered in all analyses. Relationships between Apodanthaceae and photosynthetic angiosperms varied depending upon the data partition: Malvales (3-gene), Cucurbitales (matR) or Fabales (atp1). The latter incongruencies suggest that horizontal gene transfer (HGT) may be affecting the mitochondrial gene topologies. The lack of association between Mitrastema and Ericales using atp1 is suggestive of HGT, but greater sampling within eudicots is needed to test this hypothesis further.Our analyses indicate that the phylogenetic affinities of the large-flowered clade and matR gene tree, thus providing compelling evidence for organismal relationships. Horizontal gene transfer appears to be influencing only some taxa and some mitochondrial genes, thus indicating that the process is acting at the single gene (not whole genome) level.Rafflesiales are not monophyletic but composed of three or four independent lineages (families): Rafflesiaceae, Mitrastemonaceae, Apodanthaceae and Cytinaceae. Long-branch attraction appears to be misleading parsimony analyses of nuclear small-subunit rDNA data, but model-based methods  recover a topology that is congruent with the mitochondrial  Combining gene sequences from multiple subcellular compartments continues to provide increasingly well-resolved flowering plant phylogenies  and thesRafflesia, whose meter-wide flowers are the largest among all angiosperms, and Pilostyles, whose flowers are less than a centimeter in diameter. Such wide morphological variation has resulted in classifications that comprise four families: 1) the "small-flowered clade" (Apodanthaceae) with Apodanthes, Berlinianche, and Pilostyles, 2) the "large-flowered clade" (Rafflesiaceae s. str.) with Rafflesia, Rhizanthes, and Sapria, 3) the "inflorescence clade" (Cytinaceae) with Bdallophyton and Cytinus, and 4) the "hypogynous clade" (Mitrastemonaceae) with Mitrastema [Rafflesiales are a fascinating and enigmatic group of holoparasitic plants that includes trastema ,8.matR to identify the closest photosynthetic relatives of two clades within Rafflesiales. Three genera, representing two of the four families in the order, were used in that study: Rafflesia and Rhizanthes (Rafflesiaceae s. str.) and Mitrastema (Mitrastemonaceae). Analyses of the matR data placed Rafflesiaceae s. str. within Malpighiales, an order that includes passionflowers (Passiflora), willow , and violet (Viola). Mitrastemonaceae was placed within Ericales, an order containing blueberries (Vaccinium), primroses (Primula), and tea (Camellia). The authors argued that these results were robust because they were congruent using different analytical methods (parsimony, neighbor-joining, Bayesian) and were not affected by long-branch attraction artifacts [Recently, Barkman et al.  used DNArtifacts . MoreovematR, 2) mitochondrial atp1 and 3) a "3-gene" data set consisting of nuclear SSU rDNA plus two chloroplast genes: rbcL and atpB (the latter two only from nonparasites).In this study we expand upon the previous analysis  by inclumatR resulted in trees congruent with each other and with those previously generated [Rafflesia, Rhizanthes, and Sapria were placed with strong support in Malpighiales. Mitrastema was placed in Ericales sister to Vaccinium. The Cytinus and Bdallophyton clade (Cytinaceae) was strongly supported and this clade was sister to one composed of four genera of Malvales, an order that contains cotton (Gossypium), rockrose (Cistus) and chocolate (Theobroma). For Apodanthaceae, Apodanthes and Pilostyles were sister taxa and derived from within Cucurbitales, an order that contains squash/pumpkin (Cucurbita) and Begonia. For Berlinianche, sequences homologous to matR could not be obtained using several primer combinations.Maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) analyses of mitochondrial enerated  , although this relationship does not receive strong support. This is remarkable given that 15 taxa from Ericales were included, yet a relationship with this order (as seen with matR) was not obtained with atp1. The large-flowered clade was strongly supported as monophyletic in all analyses, however, its position within the eudicots did not receive strong support. Parsimony analysis placed Pilostyles as sister to Pisum  and this clade was sister to Berlinianche, but both with low bootstrap support. Apodanthes was strongly suported (90% bootstrap) as sister to Polemonium  with MP but with ML this long-branch clade received lower support  to strong (BI) support.All three analytical methods of the e Figure . Clades t Figure . The twoPilostyles were sister to a clade composed of Pavonia and Gossypium, also with low bootstrap support. In constrast, BI analysis of the 3-gene matrix placed Mitrastema with Ericales and the large-flowered clade was a component of Malpighiales, the latter with strong support. The inflorescence clade  and the small-flowered clade (Pilostyles) were allied with Malvales  and reduced (77 taxon) 3-gene matrices were generally congruent and both resulted in all taxa of Rafflesiales being associated with Malvales  placed this clade in Malpighiales. Thus, the positions of the parasite clades inferred in four separate MP analyses matched the positions found for these clades in the single ML tree.MP analyses of SSU data sets from which all but one parasite group had been removed resulted in phylogenetic placements that matched those found in the ML tree. MP analysis of a data set from which all Rafflesiales except rbcL that are typically used to infer phylogenetic relationships among angiosperms but have not yet been amplified from any Rafflesiales and are presumed lost [Rafflesia and Cytinus [atp1 and matR were used, in combination with nuclear rDNA and chloroplast genes, to reliably place Hydnoraceae with Aristolochiaceae [Determining the photosynthetic relatives of Rafflesiales has long presented a challenge owing to the extreme reduction and/or modification of morphological structures that have accompanied the evolution of this lineage ,11. Molemed lost . Increasmed lost ,12. Simi Cytinus . DespiteRafflesia was several times longer than any other branch, and that this branch was attracted to the second-longest branch in the tree – the one between gymnosperms and angiosperms. For these reasons, they argued that nuclear SSU rDNA sequences are of limited utility for assessing the phylogenetic position of Rafflesia.Long-branch attraction, a bias in certain phylogenetic inference methods in which similarity due to convergent or parallel changes produces an erroneous phylogenetic grouping of taxa , is oftematR topology presented by Barkman et al. [Rafflesia is closely related to Malpighiales and Mitrastema is a member of Ericales , recovers a nearly monophyletic Rafflesiales containing all of the longest terminal branches in the tree  but he also considered other groups such as Aristolochiaceae , Sterculiaceae  and Cucurbitaceae . In general, different characters supported relationships with one or another group and therefore he left the subject as unresolved. Three proposed synapomorphies between Passifloraceae and Rafflesia were cited by Barkman et al. [Rafflesia), an androgynophore (gynostemium or column in Rafflesia), and an annular corona (diaphragm in Rafflesia). Whether these structures are homologous is not clear and will likely require further morphological studies, possibly examining the floral development genes themselves. These hypotheses require scrutiny because the apparent similarities in structure are not clear when examined in detail. For example, the androgynophore of Passiflora is composed of a stalk that bears the androecium and gynoecium. In Rafflesia, the ovary is inferior , hence the central column must involve other gynoecial parts. The corona of Passiflora is very different in structure and function from the diaphragm of Rafflesia [Passiflora caerulea and Euonymus [Rafflesia. Whether this association represents parasitism or not is a matter of semantics [Cissus and Opuntia growing on Yucca and Opuntia on Cercidium and Idria. In all of these cases, a true haustorium does not form and more likely these represent forms of grafting. It is difficult to state whether such rare occurrences have any bearing on the origin of parasitism in Rafflesiales or other parasitic flowering plants.Barkman et al.  suggestert Brown  represenn et al. : a hypanafflesia . The obsEuonymus  was discEuonymus  as a posemantics , for othMitrastema in Ericales, a result congruent with that obtained using mitochondrial matR. As noted by Barkman et al. [Mitrastema has bisexual, protandrous flowers with a collar-shaped, four-merous perianth tube. The stamens are connate into a tube (androphore) crowned by a fertile zone of pollen-bearing locules. The staminal tube, open at the top by a small hole, circumscissally separates from the flower as it is pushed up by the growing gynoecium. The apical portion of the staminal tube is sterile, but below this is a series of vertical rings of ca. ten minute, pollen sacs each. The gynoecium is hypogynous, one-locular, with a thick, conical stigma. Placentation is parietal with 8–15 (-20) unequal placental lobes filling the locule. The numerous ovules are small (190 by 120 μm), anatropous, unitegmic (but with two cell layers), and tenuinucellar. Although some floral morphological features of Mitrastema are not in conflict with those seen in Ericales, such as extrorse anthers and cellular endosperm, features such as decussate leaves, circumscissile fruit dehiscence, and parietal placentation are too general to draw specific associations. Given that Mitrastema is an achlorophyllous holoparasite and that one clade of Ericaceae (Monotropoideae) contains achlorophyllous mycotrophs, it is intriguing to ask whether these groups share a common ancestor or evolved independently. The most specialized morphological feature found in Mitrastemonaceae, the athecal androecium, is not found in Ericales but in Malvaceae, the only angiosperm family that shows the entire gamut from taxa with normal stamens, to taxa with stamens deviating only slightly from the common pattern [Maximum likelihood and Bayesian analyses of the 3-gene and nuclear SSU rDNA data partitions placed n et al. , this re pattern ,28, to a pattern .matR results. These results and those obtained from the simulation study indicate that the large-flowered clade and Mitrastema are artifactually attracted to Cytinaceae when parsimony is utilized.The most consistent phylogenetic signal that is seen across all data sets and types of analyses is a relationship between Cytinaceae and Malvales Table . BecauseRafflesia and Sapria (but not Rhizanthes), is lacking in Cytinaceae. Bdallophyton is dioecious and Cytinus is either dioecious (C. capensis, C. sanguineus) or monoecious (C. hypocistis). The perianth is tubular, composed of four to nine imbricate organs. The androecium is connate, forming a compact synandrium with extrorse anthers and the pollen is 2-, 3-, or 4-porate. The female flower is epigynous with a columnar style terminated by a globose or capitate, viscous stigma with commissural lobes [Unlike other Rafflesiales, members of Cytinaceae have multiple flowers arranged in an inflorescence. The floral structure called the diaphragm, seen in al lobes . The ovaPilostyles (the only Apodanthaceae for which SSU rDNA sequences are available) within Malvales, however, a sister relationship with Cytinaceae is not consistently obtained. A 3-gene alignment that included additional representatives of Malvales (16 taxa) gave similar results as shown in Figure Pilostyles on a clade separate from other Rafflesiales). These data, in conjunction with the results from the mitochondrial genes, support an evolution of Apodanthaceae independent from Rafflesiaceae s. str. The well-supported relationship between Pilostyles and Apodanthes using matR is expected given their very similar floral morphology [Begonia and Cucurbita). Contamination with host tissue is excluded because neither parasite is known to currently occur on a member of Cucurbitales. Apodanthaceae are grouped with Pisum  and Polemonium  on the atp1 tree, but no atp1 sequences from representatives of Cucurbitales were available from GenBank to test the matR result. The sister relationship between Apodanthes and Polemonium is strongly supported on the MP tree . Additional sampling within the eudicots will be required to better understand the atp1 gene tree topology.Maximum parsimony and likelihood analyses of the 3-gene data set and nuclear SSU rDNA sequences alone also place rphology , yet thiPilostyles and Apodanthes male flowers, a tubular synandrium surrounds and fuses with a central structure that could be interpreted as a sterile gynoecium. Support for the concept that such a central structure is a pistillode comes from Berlinianche where the upper portion of the synandrium is free from the central part. In female flowers of Apodanthaceae, there is no rudiment of an androecium, hence the central tissue is apparently entirely gynoecial.Morphological features shared between Apodanthaceae and Cytinaceae are: unisexual flowers, a connate androecium, an inferior ovary, and a unilocular ovary with four parietal placentae bearing numerous, anatropous, tenuinucellate ovules ,31. FlormatR data indicate Apodanthaceae are related to Cucurbitales, an order with seven families, 129 genera and 2300 species. Hosts for Apodanthaceae are generally legumes, although Apodanthes occurs most frequently on Casearia . Thus, neither recent HGT nor contamination explains this result. Apodanthaceae shares some morphological features with members of Cucurbitaceae, subfamily Cucurbitoideae: unisexual, five-merous flowers (Berlinianche); carpellate flower with a unilocular, inferior ovary with parietal placentation; anatropous, bitegmic ovules; staminate flower with connate filaments (monadelphous) and a rudimentary gynoecium (pistillode) [In contrast to the above discussion, the tillode) . ConflicA requirement of the molecular phylogenetic approach to inferring evolutionary histories of organisms is vertical transmission of genetic material from parent to offspring. In contrast, horizontal gene transfer (HGT) describes the movement of genetic material between organisms of no direct ancestor-descendant relationship. Although the frequency of HGT is currently not well understood among prokaryotic and eukaryotic organisms, it is clear that HGT can compromise accurate inference of genealogical history. In plants, lateral movement of genetic material has been documented for mobile genetic elements such as introns -37 but oIncongruence among gene trees derived from different data sets can derive from a number of factors such as technical causes (insufficient data, gene choice, sequencing error, taxon sampling and identification), gene/genome-level processes, and organism-level processes (e.g., hybridization/introgression, lineage sorting, and HGT) . HGT hasApodanthes and Pilostyles on the atp1 tree as sister to Pisum (a legume, the family of hosts for Pilostyles) represents a likely case of HGT. The atp1 data conflict with those from matR that associates Apodanthaceae with Cucurbitales. Moreover, we infer that the SSU rDNA tree better represents the organismal phylogeny because it seems less likely that nuclear genes would be influenced by HGT [We believe that incongruence between the the mitochondrial and the nuclear gene trees Table  stem notd by HGT ,45. The matR tree closely matched results from other molecular phylogenetic investigations of angiosperms. The present study confirms that HGT is not implicated for the two lineages studied by Barkman as well as Cytinaceae, but this process could be invoked for Apodanthaceae. More recent work by these authors [atp1. These included Dalea to Pilostyles, Tetrastigma to Rafflesia, and Lithocarpus to Mitrastema. In addition, HGT of another mitochondrial gene, nad1, has been reported for Rafflesia and Sapria, both of which occur on the same clade as their hosts (Tetrastigma) on a gene tree [matR gene in Pilostyles and Apodanthes. Moreover, present-day hosts of parasitic angiosperms do not represent the only conduit for HGT if host choice has shifted through time as the parasite lineage evolves. For example, Barkman et al. [Mitrastema only parasitizes Fagales (e.g., Lithocarpus and Castanopsis, both Fagaceae) but this parasite has also been recorded from Aquifoliaceae, Asteraceae, Elaeocarpaceae, Juglandaceae, and Myrtaceae [In their study of Rafflesiaceae s. str. and Mitrastemonaceae, Barkman et al.  discount authors  identifiene tree . These en et al.  state thyrtaceae . Host lamatR tree  were found to be identical to these "parasite" sequences, strongly suggesting contamination. In the case of Rafflesia, the DNA was obtained from a bud still attached to the host vine, both of which had been sectioned longitudinally. Disruption of these tissues likely resulted in transfer of host sap to the bud region where the tissue was sampled. Other samples of R. tuan-mudae from the same population, obtained as floral bracts with no host tissue, resulted in matR sequences that were similar to the other two Rafflesia species.Contamination of parasite DNA with DNA from the host plant is an issue that must be given serious attention. Indeed, two sequences shown on the e Figure , TetrastBerlinianche, whose flowers are much smaller than those of Rafflesia (5 mm in diameter), extreme care (using a stereo microscope) was exercised to remove floral parts devoid of any host tissue. Despite this, the matR sequence obtained from the first sample was that of the host, Julbernardia. Later, silica gel dried samples of other populations of the parasite were extracted, again using extreme care in avoiding host contamination. PCR products were obtained using several mitochondrial matR primers, but none were found to be homologous to this gene following BLAST searches. This result shows that host DNA was not present in this sample in sufficient amounts to amplify and that the parasite matR gene, if present, is highly divergent at the priming sites used.For Pilostyles was extremely difficult. Despite PCR products of the correct sizes using a variety of primer combinations, the sequences obtained from genomic DNA derived from flowers were deemed contaminants following BLAST searches that showed them to be most similar to monocots, fungi, etc. Only when sequences from two accessions of Pilostyles  both were most similar to Malvales was this considered good evidence for their true phylogenetic affiliation. Retrospectively, it is likely that the sticky flowers had accumulated wind-dispersed pollen (e.g., grasses) and that this DNA, despite being in low concentration, had less divergent priming sites than the parasite target DNA, allowing PCR to preferentially amplify the contaminant DNA.For all three Apodanthaceae genera, the conical style in female flowers is papillate and heavily secretory . This stPilostyles: P. hamiltonii [P. thurberi [Cuscuta [Pilostyles suggest that intact, mature mitochondria are too large to pass through heteroplastic plasmodesmata, however, mitochondrial genomes or portions of the genome are certainly small enough for transmission. Once inside the parasite cell, there are various fates for the host gene. It could become incorporated into the parasite mitochondrial genome, and then either replace the parasite copy or exist as a duplicate, or the host gene could reside in the parasite nuclear genome. For the latter case, the gene would likely become a pseudogene given the requirement of mitochondrial-specific patterns of RNA editing. Two forms of atp1 are present in the primitive angiosperm Amborella trichopoda [matR mRNA would be useful to determine whether the gene is expressed and whether mitochondrial-specific RNA editing patterns are present.Given the accumulating molecular evidence for HGT from host to parasitic plant, it is worthwhile to consider potential mechanisms, along with their constraints, that may suggest further research. Relatively little information exists on the structure of the endophyte of Rafflesiales. Ultrastructural studies have been conducted on two species of miltonii  and P. tthurberi . These a[Cuscuta  and inde[Cuscuta . Given tichopoda , one of Rafflesia, Rhizanthes, and Sapria) representing the large-flowered clade are monophyletic and are related to Malpighiales. The monogeneric family Mitrastemonaceae, the only member of the order with a superior ovary, is related to Ericales. The first of the remaining two families that have previously not been sampled is Cytinaceae  which is strongly supported as a member of Malvales. The last remaining unsampled family, Apodanthaceae (Apodanthes, Berlinianche, and Pilostyles) is either related to Malvales or Cucurbitales. Our simulation studies indicate that Mitrastema, Bdallophyton/Cytinus, and Rafflesia/Rhizanthes/Sapria have branches that are long enough to mislead parsimony. All of these relatively long branches appear to be attracted toward the Cytinaceae clade within Malvales. When nuclear SSU rDNA sequences are analyzed with ML, results fully congruent with those previously reported for two Rafflesiales clades using mitochondrial matR are obtained. If the phylogenetic affinityof Apodanthaceae are with Malvales, the results from the mitochondrial matR gene must represent a case of horizontal gene transfer (HGT) from Cucurbitales. If this proves to be the case, this provides an example of HGT from a nonhost plant to a parasitic angiosperm.In this study we have used data derived from nuclear, mitochondrial and chloroplast DNA and a variety of analytical approaches to address long-standing questions about the holoparasitic flowering plant order Rafflesiales. We show that Rafflesiales are not monophyletic but composed of at least three and possibly four independent lineages. Rafflesiaceae . Comparisons among gene trees allows for the identification of HGT, a phenomenon that requires further investigation to determine its modes of action and frequency among taxa and through evolutionary time.® 377 automated DNA sequencer, Applied Biosystems) according to manufacturer's protocols.DNA was extracted, amplified, cloned, and sequenced by using methods formerly reported -54. The matR alignment incorporated all of the Rafflesiales parasites and the nonparasite sequences previously published [Ginkgo, Pinus, and Zamia), 28 monosulcates, 63 nonparasitic eudicots, and 15 Rafflesiales. For two taxa (Mitrastema and Rhizanthes), our sequences, as well as those previously published, were from the same species but different accessions to test for consistency. Taking into account codon information, an alignment of 2177 sites was constructed manually using SeAl version 2.0 [atp1 alignment was similarly constructed and included the same gymnosperm outgroup genera as above, 24 monosulcates, 32 nonparasitic eudicots and 12 Rafflesiales. All of the monosulcate genera in the atp1 alignment were also represented in the matR data set, whereas eudicot sampling for atp1 was constrained by sequences available on GenBank (12 of the same genera as with matR or placeholders from same family).The initial ublished  as well sion 2.0 . The fulrbcL and atpB was constructed that included 103 taxa . Sampling across angiosperm orders was very similar to the matR matrix, differing only by the presence of 11 placeholders and a second accession of Pilostyles. For the holoparasites, only nuclear SSU rDNA sequences were included; the chloroplast gene data for these taxa were coded as missing. The two chloroplast genes were included to add stability to the tree topology given that nuclear SSU has been shown to contain lower phylogenetic signal when used alone [matR, the 103-taxon matrix was truncated to 77 taxa by removing all but five monosulcate taxa to facilitate likelihood analyses. All alignments reported in this paper have been deposited with TreeBASE [study accession number S1177, matrix accession numbers = M2034–M2037.To test the position obtained for Rafflesiales taxa using mitochondrial genes with an independent data set derived from different compartments, a 4646-site "3-gene" matrix combining sequences from nuclear SSU rDNA and chloroplast ed alone . As withTreeBASE : study aAll three data sets were analyzed using maximum parsimony (MP) and maximum likelihood (ML) methods in PAUP* 4.0b10  and BayeBdallophyton + Cytinus, Mitrastema, Pilostyles or the large-flowered clade comprising Rafflesia, Rhizanthes and Sapria) was removed to determine the position of each parasite group in the absence of other long-branch parasite taxa in the analysis. This is a form of the test proposed by Siddall and Whiting [All MP searches were performed using 100 random addition sequence replicates with tree-bisection-reconnection (TBR) branch-swapping, holding ten trees at each addition step, with all sites equally weighted. For the 77-taxon SSU data set, a series of four MP analyses were performed in which all but one parasite group . This submodel employed four (rather than six) relative rate parameters: one for A-C transversions and A-G transitions, one for A-T and C-G transversions, one for C-T transitions, and one for G-T transversions; the PAUP* LSET option used for analysis was "RCLASS = (a a b b c d)". Likewise, the models chosen by ModelTest for the matR data set were TVM+Γ (hLRT) and TIM+Γ (AIC), but a simpler statistically equal model  was used for analysis. This model employed three relative rate parameters: one for A-C, A-G, and G-T substitutions; one for A-T and C-G substitutions; and one for C-T substitutions; "RCLASS = (a a b b c a)", with among-site rate heterogeneity modeled with a gamma distribution. These simplified models were chosen to reduce computational time and to avoid estimation of unnecessary parameters, which can lead to greater variance in parameter estimates and higher topological uncertainty.For ML analyses, a MP tree was used in PAUP* to evaluate 56 nucleotide substitution models. ModelTest 3.06  was usedA successive approximations approach was used for all ML analyses ,64. SubsmatR and atp1 data sets; 10 million generations, with the first 5 million discarded as burn-in and trees sampled every 500 generations for the 3-gene data set) [matR and atp1 data sets instead. A partitioned model was used for the 3-gene data set (see below). Two Bayesian runs were performed for all analyses in an attempt to determine if stationarity was reached, and plots of log likelihood and parameter convergence were also evaluated; log-likelihood plots alone are insufficient for monitoring chain mixing and convergence [Nodal support for all data sets was estimated using one or more of the following methods: equal-weights MP bootstrap analysis (100 pseudoreplicates, each consisting of a heuristic search using 100 random sequence addition replicates), ML bootstrap analysis  ,66, and ata set) . The GTRvergence ,68.atpB first, second and third codon positions; rbcL first, second and third codon positions. Appropriate substitution models for each data partition were chosen by computing likelihood scores for each partition on a MP tree for the 3-gene data set under 56 substitution models in PAUP* and comparing the scores in ModelTest. The GTR+Γ + I model was the best-fitting model for all partitions. The Bayesian analysis was performed with all model parameters (except branch lengths) unlinked across partitions.The 3-gene data set was also analyzed in MrBayes 3.0. A "fully partitioned" analysis was used in which the 3-gene data set was divided into seven partitions: nuclear SSU; For the nuclear SSU rDNA data, constrained analyses were also performed. A constraint tree for 63 nonparasitic taxa was constructed using the MP topology of the "B series" tree from Soltis et al.  with relTo investigate possible long-branch attraction in parsimony analyses of the SSU rDNA data set, two sets of simulations were performed. For the first set of simulations, a reduced data set of SSU rDNA sequences for 20 taxa  was constructed and analyzed under ML (GTR+Γ + I model) in PAUP*. The tree resulting from this analysis, with its associated ML branch lengths and model parameters, was used as the model tree on which 100 data sets of length 1766  were simulated in Seq-Gen 1.2.7 . For theΓ + I – gamma + invariant sites distributionatp1 – ATP synthase alpha subunitatpB– ATP synthase beta subunitBI – Bayesian inferenceGTR – general time reversible modelHGT – horizontal gene transfermatK – maturase KmatR – maturase RML – maximum likelihoodMP – maximum parsimonyrbcL – ribulose bisphosphate carboxylase/oxygenase, large subunitSSU – small subunitTBR – tree bisection-reconnection branch swappingatp1 and matR sequencing and revised the text regarding morphological character comparisons. YQ provided primers, introduced AB to the field of molecular systematics, and supervised his Ph.D thesis. RVR conducted the PCR experiments showing host contamination of Rafflesia DNA and generated the matR sequences for several taxa. FEA performed the phylogenetic analyses. All authors read and approved the final manuscript.DLN coordinated all aspects of the study, obtained many of the genomic DNAs, generated all the nuclear SSU rDNA, conducted the sequence alignments, and drafted the manuscript. AB conducted the majority of the mitochondrial matR MP strict consensus tree from mitochondrial Strict consensus of 200,000+ trees obtained from maximum parsimony (unconstrained MP) analysis of the 77-taxon mitochondrial matR matrix. Bootstrap percentages are shown above the lines. Rafflesiales taxa are shown in bold italics.Click here for fileatp1 Strict consensus MP tree from mitochondrial Strict consensus of 328 trees resulting from a MP analysis of the 71-taxon mitochondrial atp1 matrix. Rafflesiales taxa are shown in bold italics. Bootstrap percentages are given above the branches.Click here for fileMajority rule consensus BI tree from 3-gene data set Majority rule consensus of 20,000 trees (10 million generations, 5 million burn-in) resulting from Bayesian analysis of the 77-taxon nuclear 3-gene matrix. Clades with Bayesian posterior probabilities are indicated above the clades. Rafflesiales taxa are shown in bold italics.Click here for fileStrict consensus constrained MP tree from nuclear SSU rDNA Strict consensus of 6 trees resulting from the constrained MP analysis of the 77-taxon nuclear SSU rDNA matrix. Rafflesiales taxa are shown in bold italics. Bootstrap percentages are given above selected nodes .Click here for fileTaxa used in this study MS Excel file giving taxon names and GenBank numbers for all genes used.Click here for file


Whether a person inherits a defective gene or acquires genetic damage by chance, two types of genes typically play a role in transforming a healthy cell into a cancer cell. Oncogenes and tumor suppressor genes are normally involved in cell growth, development, and cell differentiation. Both functions can be appropriated to ill effect by mutations. Single mutations in these genes rarely cause cancer on their own, but they predispose cells to additional insults that precipitate malignant transformation.Susceptibility to cancer depends, among other things, on age. Though cancer in children is rare, the most common childhood cancers strike the hematopoietic system (leukemia), nervous system, and skeletal muscle system, while solid tumors of the lung, breast, prostate, and colon are more common in adults. This age differential suggests that an oncogene's ability to cause cancer in a particular cell type might depend on that cell's developmental stage.  In a new study, Dean Felsher and colleagues show that age matters: activating oncogenes at different developmental time points in mouse liver cells produces different results.MYC oncogene to initiate liver cancer (hepatocellular carcinoma) in a transgenic mouse model varies with the age of the mouse.Typically, once a cell is transformed, it stays in its “differentiative” state, that is, it stays in whatever developmental stage it was in when it became a tumor cell. But in a previous study, Felsher and colleagues found that turning off oncogenes in tumor cells allowed them to differentiate; these mature cells did not resume tumorigenesis after the oncogenes were reactivated. In this study, Felsher and colleagues show that the ability of the MYC overexpression in the liver cells of embryonic, neonatal, and adult mice, the authors used a biotech trick  that controls gene expression dose and timing with a drug. The system relies on the interplay of two elements: a gene (in this case, MYC) fused to a regulatory enhancer, and a transcription factor that binds to the enhancer and activates the gene. Administering a tetracycline-like drug (in this case, doxycycline) prevents the transcriptional activation of the gene.To study the consequences of MYC oncogene in mice during embryonic development or at birth occasioned their demise fairly quickly (ten days and eight weeks after birth, respectively). In contrast, overexpression of MYC in adult mice resulted in tumorigenesis only after a long latency period. When the authors evaluated the cellular effects of MYC overexpression, they found that hepatocytes from neonatal transgenic mice showed evidence of increased proliferation (replicated DNA content) compared to normal hepatocytes, while transgenic adult hepatocytes showed increased cell and nuclear growth (some nuclei had as many as twelve genome copies instead of two) without dividing. Since these adult cells eventually developed into tumors, some clearly acquired the ability to divide, which the authors show is facilitated, among other events, by the loss of the p53 tumor suppressor.Overexpressing the MYC activation, Felsher and colleagues conclude, depend on the cell's developmental program, which determines whether a cell can grow and divide, or simply grow. In adult hepatocytes—which are normally quiescent—MYC requires additional genetic events to induce cell division and tumorigenesis; in immature hepatocytes—which are already committed to a program of cellular proliferation—MYC activation alone is sufficient. The next step will be to identify the epigenetic developmental factors, both internal and external, that lead to tumor formation, and how to prevent it.Altogether these results suggest that whether oncogene activation can support tumor growth depends on the age of the host, which in turn suggests the role of genetically distinct pathways in young and adult mice. The consequences of


There is accumulated evidence that plasma concentration of the sulfur-containing amino-acid homocysteine (Hcy) is a prognostic marker for cardiovascular morbidity and mortality. Both fasting levels of Hcy and post methionine loading levels are used as prognostic markers. The aim of the present study was to investigate the existence of a daily rhythm in plasma Hcy under strictly controlled nutritional and sleep-wake conditions. We also investigated if the time during which methionine loading is performed, i.e., morning or evening, had a different effect on the resultant plasma Hcy concentration.® formula).Six healthy men aged 23–26 years participated in 4 experiments. In the first and second experiments, the daily rhythm in Hcy as well as in other amino acids was investigated under a normal or an inverse sleep-wake cycle. In the third and fourth, Hcy concentrations were investigated after a morning and evening methionine loading. To standardize food consumption in the first two experiments, subjects received every 3 hours 150 ml of specially designed low-protein liquid food (EnsureIn both the first and second experiments there was a significant daily rhythm in Hcy concentrations with a mid-day nadir and a nocturnal peak. Strikingly different 24-h patterns were observed in methionine, leucine, isoleucine and tyrosine. In all, the 24-h curves revealed a strong influence of both the sleep-wake cycle and the feeding schedule. Methionine loading resulted in increased plasma Hcy levels during both morning and evening experiments, which were not significantly different from each other.There is a daily rhythm in plasma concentration of the amino acid Hcy, and this rhythm is independent of sleep-wake and food consumption. In view of the fact that increased Hcy concentrations may be associated with increased cardiovascular risks, these findings may have clinical implications for the health of rotating shift workers. Experimental results accumulated in recent years have revealed that plasma concentration of the sulfur-containing amino-acid homocysteine (Hcy) is a prognostic marker for cardiovascular morbidity and mortality -5. PlasmStudies conducted during the 1960s have demonstrated that plasma levels of several amino acids vary in a daily manner. Feigin, Klainer and Beisel  were theMore recently, plasma Hcy levels were also shown to vary in a daily manner in humans with an evening peak and a morning nadir . SignifiIn the present study, we further investigated the possible existence of a daily rhythm in plasma Hcy under strictly controlled nutritional and sleep-wake conditions. We also investigated if the time during which methionine loading is performed, i.e., morning or evening, had a different effect on the resultant plasma Hcy concentration.2). They were instructed to avoid alcohol and coffee beverages during the 24 hours that preceded each of the experimental periods. The study was approved by the local Human Ethics Committee, and subjects gave written informed conset before being enrolled in the first experiment. Subjects were paid for their participation.Six healthy men aged 23–26 years participated in 4 experiments. All were students who maintained a normal and regular sleep-wake cycle for at least three months prior to the studies. They were screened to ensure an adequate state of health by physical examination, detailed medical history and blood testing. All had a normal body weight (mean body mass index (BMI) = 23.5 ± 1.6 Kg/mIn the first and second experiments, daily rhythms in Hcy as well as in other amino acids were investigated under a normal or an inverse sleep-wake cycle. In the third and fourth experiments, Hcy concentrations were investigated after morning and evening methionine loading.® formula) with the following composition: proteins , fat , carbohydrates , vitamins and minerals, in 77 ml water. No other food except for water was allowed.Subjcts were admitted to the laboratory at 1800 for a period of 24 hours, after having a normal day. A catheter was inserted into an antecubital vein and was kept patent by a drip of saline. Electrodes were attached for polysomnographic monitoring to determine sleep stages. These included EEG, EMG, EOG, respiration by respiratpry belt and nasal thermistor, and oximetry. Starting at 1900, 5-ml blood samples were drawn every hour until 1900 on the next day. Thoughout this period subjects were either in a supine or a sitting position in individual rooms where they could read, use their personal computers or watch television. From 2300 to 0800 the room lights were turned off during the sleep period. Blood samples were collected into EDTA treated tubes, immediately centrifuged at 4°C, and plasma was stored at -70°C until assay. Hourly blood sampling during sleep continued with minimal disturbance to subjects' sleep. To standardize food consumption and to provide adequate energy intake, subjects received every 3 hours 150 ml of specially designed liquid food . At the start of each methionine loading test, subjects were administered 100 mg/kg body weight methionine, mixed in fruit juice. Blood samples (5 ml) were taken into EDTA treated tubes before methionine loading, designated as time 0, and then at +2, +4, +6 and +8 hours after methionine administration. Light carbohydrate rich meals were provided at +1 and +6 hours after the methionine loading in each of the test periods.12 were also measured in all samples of all subjects using commercially available kits from Abbott. The assays were performed on an Abbott IMX analyzer that utilizes ion capture technology for folate determination and microparticle enzyme immunoassay (MEIA) technology for B12. The assays were performed according to the manufacturers' instructions and used quality control sera supplied by Abbott.Plasma amino acids levels (Hcy, methionine, leucine, isoleucine and tyrosine) were measured in duplicates using a Biochrom 20 Amino-Acid analyzer (Pharmacia Biothech, Cambridge, UK) as described before . The meaRepeated measurements ANOVA was used to compare the means of the amino acids between the first two experiments. To obtain the average 24-h Hcy curves, each individual data point was replaced by a z-transformation based on the individual 24-h mean and standard deviation, before averaging across subjects. Then, each of the individual time series was subjected to Cosinor analysis to determine its amplitude and acrophase. Since Experiment 1 was perfomed during the summer (August) and Experiment 2 was performed during early winter (late November), approximately 2 months after the change from Summer daylight-saving time to Winter time, during which the clock in Israel was advanced by one hour, the 24-h curves of the first experiment were advanced by 1 hour before the analysis. Then repeated measurements ANOVA was used to determine differences in acrophase between the experiments. In the third and fourth experimens, the concentrations of Hcy at times 0, 2, 4, 6, and 8 hours after methionine loading were analysed by repeated measures ANOVA to determine if there were any significant morning-evening differences in Hcy levels.All subjects successfully completed the four experiments. In experiment 1 when they slept from 2300 to 0700, average sleep latency was 22.2 ± 7.3 min, total sleep time was 407.3 ± 51.8 min, and sleep efficiency was 77.7 ± 9.2%. In experiment 2 when they slept from 0720 to 1500, average sleep latency was 4 ± 3.1 min, total sleep time was 371.5 ± 59.4 min, and sleep efficiency was 83.6 ± 12.2%. In spite of the reversal of the sleep-wake cycle, the 24 h means and coefficients of variation of Hcy in the two experiments were very similar to each other, 8.82 μmol/L and 29.7% and 8.51 μmol/L and 27.7%, in experiments 1 and 2, respectively. None of the subjects had abnormal Hcy levels (>15 μmol/L) at any point across the 24 hours.Figure Strikingly different 24-h patterns were observed for the other amino acids: methionine, leucine, isoleucine and tyrosine. In all, the average z-transformed 24-h curves revealed a strong influence of both the sleep-wake cycle and the feeding schedule. Their level was notably lower during the sleep period, regardless of its timing, and increased every two hours in synchrony with the times of feeding. This pattern is exemplified in Figure 12 and folic acid. While folic acid showed a linear increase throughout the study period, the 24-h pattern of B12 was rather constant with slight elevation during the night time (data not shown).We did not find any evidence for rhythmicity in the concentrations of BAs expected, methionine loading resulted in increased plasma Hcy levels during both morning and evening experiments Figure . AnalysiThe present study demonstrated that under strictly controlled dietary conditions plasma levels of Hcy shows significant daily rhythmicity, which is independent of the 24-h cycle of sleep and wake, with a peak at around 2200 to 2400. Previously, similar rhythmicity in Hcy with an evening peak was reported in obese diabetic patients by Bremner et al  and with12 dependent while the reaction with betaine is not. In the transsulforation pathway, Hcy condenses with serine to form cystationine in an irreversible reaction catalyzed by the pyridoxal-5'-phosphate (PLP)-containing enzyme, cystationine beta synthase. Although we do not have any information as yet on the underlying mechanism responsible for the daily rhythm in plasma Hcy, it is most probably related to the balance between its rates of production and disposal. A high Hcy concentration could be due to an elevated production rate, a decreased rate of transsulforation, a decreased rate of remethylation to methionine, or any combination of these processes.Homocysteine is a non-protein sulfur containing amino acid, and an intermediate in the metabolism of the essential amino acid methionine. The metabolism of Hcy is accomplished by two major pathways, remethylation into methionine and transsulfuration to cystationine . In reme12 deficiencies, or by methylenetetrahydrofolate reductase thermolability. The variations in plasma vitamin concentrations, however, could not provide an explanation for the daily rhythms in Hcy. The 24-pattern of folate levels showed a linear increase from the beginning to the end of the study. Although the plasma concentrations of vitamin B12 varied across the 24 hours – in contrast however to what was expected if B12 were involved in the daily rhythm in Hcy, ie, increasing levels of B12 associated with decreasing levels of Hcy – the 24-h pattern in B12 was parallel to that of Hcy with a daytime nadir and a night time peak. Thus, it is unlikely that a daily rhythm in plasma vitamin concentrations can  explain the daily rhythm in Hcy.The fact that the range of the daily variations in the plasma levels of Hcy is on the same order of magnitude as those seen in mild hyperhomocysteinemia, may suggest that the two phenomena share a common underlying mechanism. Mild hyperhomocystenemia seen under fasting conditions is due to mild impairement in the methylation pathway. This may be caused by folate or BThe methionine loading test has been used to test the individual's ability to dispose of methionine through the transsulforation pathway . The facA different possibility that cannot be ruled out at this point is the involvement of the Hcy cellular export mechanism. The small amount of plasma Hcy is the result of a cellular export mechanism that is essential for keeping intracellular concentrations low to avoid potentially Hcy cytotoxic effects. Thus the daily rhythm in plasma Hcy may reflect variations in the activity of the cellular export mechanism, which result in varying levels of Hcy disposed to the plasma at different phases of the 24 hours rather than in its rate of metabolism. Further studies are needed to test this possibility.Finally, what may be the clinical implications of the present findings? We would like to suggest that the existence of a daily rhythm in Hcy concentration may have possible health-related consequences to shift workers, who were shown to be at an increased cardiovascular risk . FirstlySecondly, we do not know how the desynchronization between the circadian system and the enviornment which occurs in rotating shift workers may affect the rhythm in Hcy concentrations and its overall plasma concentration. Recently, Martins et al  reportedIn view of the fact that Hcy is a risk factor for cardiovascular morbidity, more research is needed on the possible role of hyperhomocysteinemia as a cardiovascular risk factor in shift workers.12 and folic acid. Methionine loading at 9 AM and 9 PM produced a comparable time-dependent increase in Hcy concentrations with a tendency toward a higher increase in the evening during the first 2 hours after loading. In view of the possible involvement of Hcy in cardiovascular morbidity, and of the increased cardiovascular morbidity in shift wokers, these findings may have implications to shift workers health.Our results demonstrated a daily rhythm in plasma concentrations of Hcy with a nocturnal peak that was independent of sleep-wake cycle and food consumption. There were no comparable rhythms in the concentrations of methionine, leucine, isoleucine and tyrosine, nor in the concentrations of BHcy – homocysteineEEG – ElectroencephalographyEMG – electromyographyEOG – electrooculographyEDTA – ethylanediaminetetraacetic acidCV – coefficient of variationANOVA – analysis of varianceNone declared.PL and LL co-designed the study, supervised the data collection and data analysis and wrote the paper.


The past few years have seen an explosion of data on the mouse genome, widely hailed as a guidebook to the genetic origins of human disease. Scientists are particularly interested in charting the location of SNPs—single nucleotide polymorphisms—throughout the genome. SNPs are DNA sequence variations, or mutations, that change a single nucleotide in the genome, replacing a cytosine (C) base, say, with thymine (T). Many SNPs, even those found within genes, have no functional effect. Others, however, can increase risk of specific diseases or alter a person's response to pathogens and drugs. Whether or not they are involved directly in a disease, SNPs are attractive markers for population studies aimed at identifying the multiple genes underlying complex diseases like diabetes and cancer.Extensive data exist on multiple inbred strains of mice linking their genetic makeup (genotype) to physical traits (phenotype), and scientists have used these data to guide investigations of gene function and disease. Many data have been gathered by crossing mouse strains and painstakingly analyzing their progeny, to tease out the relative contributions different genes make to pathogenesis. But these efforts take time. Investigators would greatly benefit from high-throughput methods to scan the mouse genome and flag markers for candidate disease genes. In 2001, Andrew Grupe et al. introduced an “in silico”  approach to do that very thing. The method scanned mouse SNP data to home in on chromosomal areas regulating complex traits and reduce the time needed to analyze mouse disease models from “many months” to “milliseconds.”For a number of reasons, however, it wasn't clear whether in silico mapping could deliver on its promise. For one thing, the density of SNP maps was sufficient to provide meaningful markers for only a few mouse strains, and phenotype information was lacking for many strains. In a new study, Tim Wiltshire and colleagues have addressed these limitations by mapping nearly 11,000 SNP probes to 48 mouse strains. They have also been able to use this dataset for in silico mapping to predict genomic regions with functionally important phenotypes.Wiltshire and colleagues first show that their method can predict the genomic location of a Mendelian trait (controlled by a single gene), in this case coat color, which the authors acknowledge is a “minimum requirement for a viable in silico mapping method.” They go on to map complex “quantitative” traits —gallstone development and plasma levels of high-density lipoprotein cholesterol—and find that their predictions fall in line with loci identified by traditional mouse disease studies. Noting a high correlation between QTLs predicted in silico and those identified experimentally, the authors argue that loci predicted using this method are “very likely to be biologically relevant.”Wiltshire and colleagues are careful to point out that in silico mapping is meant to complement, not replace, traditional gene mapping models. After all, computers are no match for living organisms in modeling the subtleties inherent in biological reactions. But this approach is a good starting point for identifying significant genomic areas in a new strain. And as new strains are genotyped and phenotyped, and refinements are made to the SNP database, the robustness of this method should only get better.


Fugu rubrices in order to investigate 1) the relationships between three major lineages of mammals: rodents, artiodactyls and primates, and 2) the rate of evolution and the occurrence of positive Darwinian selection using codon based models of sequence evolution.The availability of abundant sequence data from key model organisms has made large scale studies of molecular evolution an exciting possibility. Here we use full length cDNA alignments comprising more than 700,000 nucleotides from human, mouse, pig and the Japanese pufferfish We provide evidence that the evolutionary splits among primates, rodents and artiodactyls happened shortly after each other, with most gene trees favouring a topology with rodents as outgroup to primates and artiodactyls. Using an unrooted topology of the three mammalian species we show that since their diversification, the pig and mouse lineages have on average experienced 1.44 and 2.86 times as many synonymous substitutions as humans, respectively, whereas the rates of non-synonymous substitutions are more similar. The analysis shows the highest average dN/dS ratio in the human lineage, followed by the pig and then the mouse lineages. Using codon based models we detect signals of positive Darwinian selection in approximately 5.3%, 4.9% and 6.0% of the genes on the human, pig and mouse lineages respectively. Approximately 16.8% of all the genes studied here are not currently annotated as functional genes in humans. Our analyses indicate that a large fraction of these genes may have lost their function quite recently or may still be functional genes in some or all of the three mammalian species.We present a comparative analysis of protein coding genes from three major mammalian lineages. Our study demonstrates the usefulness of codon-based likelihood models in detecting selection and it illustrates the value of sequencing organisms at different phylogenetic distances for comparative studies. Fugu rubrices is used as an outgroup to estimate the branching order of the three species relative to each other.Large scale sequencing projects of many different species allow us to investigate phylogenetic issues in much more detail and to identify whether certain genes have had an extraordinary evolution in one or more species and thus gain insight into the actions of natural selection. Despite the sequencing of an increasing number of mammalian genomes and the implementation of more sophisticated evolutionary models using maximum likelihood and Bayesian methodology, the branching order within the mammalian phylum is still not completely resolved. The main reason for this uncertainty is that the diversification of these orders occurred over a short period of time, making the inference of branching order a difficult problem. One of the highly debated issues concerns the relative order of branching among primates, artiodactyls and rodents -9. Here,Codon based models ,11 allowa in Figure In a recent study of cDNA trios of human, mouse and chimpanzee a codon based branch-site specific model was used to search for human genes that have undergone positive selection since our divergence from other primates . Here, aThe distribution of sequence lengths of the 1120 three-species alignments is shown in Figure The relative branching order of the three mammalian species was investigated with the individual genes as well as with a concatenated super gene. Using the empirical amino acid substitution model of Whelan and Goldman  we maximWe used the baseml program of PAML to compare the three topologies in a nucleotide based framework. Different nucleotide based substitution models were used to maximize the likelihood on the three topologies for each of the three codon positions separately. The results of using different models of nucleotide evolution were highly similar so here we only discuss the results obtained with the HKY85 model . The resThe internal branch is rather short in all cases. Therefore in the remaining analyses we treat the mouse, human, pig split as a trifurcation. Depending on which topology is actually the right one, the only bias introduced by treating the topology as a star tree, as shown in Figure The three-species alignments were used to estimate the synonymous and nonsynonymous substitution rates of the three branches under the free ratio model, see Table The dN/dS ratios on the three different lineages were estimated under the free ratio model Figure . Most geThe one ratio model averages over sites and lineages, which makes this an extremely conservative method of detecting positive selection. Only four of the 1120 three-species alignments have an average dN/dS ratio larger than one, see Table Several studies have shown that averaging over sites is more conservative when searching for positive selection than is averaging over lineages. The branch-site specific model A and model B  were oriThe molecular function of the genes predicted to be under positive selection was determined using the Panther server  and the Several different models have been developed that allow for heterogeneity of ω over sites in an alignment. We used the M4 model  which alThe concatenated super gene was also used to investigate the patterns of codon usage in the three species; the results of this investigation are summarized in Table The phylogeny of the early mammalian radiation has been extensively debated over the last two decades. The classical view based on fossil evidence states that all major orders of placental mammals first appear right after the Cretaceous-Tertiary (KT) boundary approximately 65 million years ago . This suGallus gallus) shows that the average value of dS between human and chicken genes is approximately 1.66 [The incongruence of single gene phylogenies was investigated in a recent study of eight yeast species . The phyely 1.66 , which iely 1.66 ,32. (2) ely 1.66 ,39. (4) ely 1.66 ,36.The rate of synonymous substitution was estimated to be almost three times higher in rodents than in other mammals, in agreement with previous investigations that also showed an elevated rate in rodents -42. ThisPrevious studies of the occurrence of positive selection based on pair wise comparisons have revealed a very low occurrence of positive selection. In a study of 3595 alignments only 17 genes showed evidence of positive selection . The braCodon based branch-site models similar to the ones used here were used in a paper based on a three way comparison among chimpanzees, humans and mice . They reThe multiple testing and the small number of taxa used in a study like this imply that the results presented should not be taken as conclusive evidence for positive selection, but more as an approach to searching among the thousands of genes to look for genes that may have evolved in a biologically interesting manner. Comparative approaches such as the one we use here can only be a first step towards showing that positive Darwinian selection may be a key part in the evolution of many different gene families. Further experimental and computational analyses must then be used to investigate the suggested candidates more thoroughly.During the course of our investigation a large fraction of the genes were re-annotated as putative pseudogenes: 188/1120 ~16.8%. However, all these genes have uninterrupted reading frames in all three species; only a tiny fraction of all codons seems to have evolved in a neutral-like fashion (ω~1), and the distributions of the synonymous as well as the nonsynonymous rates of these putative pseudogenes are almost identical to the distributions of the remaining genes (results not shown). The only difference is a slight increase in the dN/dS ratio in the human lineage, which is actually due to a few genes that experience an unusually high dN/dS ratio. Omitting these genes from the analysis removes the observed differences completely. Thus, if all these genes are indeed pseudogenes in human, the loss of function must have occurred quite recently and they may not be pseudogenes in pig and mouse.The collection of a large set of pig cDNA sequences has enabled us to study long term evolutionary trends in mammalian genes. Our results indicate that the codon models are able to detect evolutionary signals indicating adaptive evolution in several genes. Our phylogenetic investigation of the primate, rodent, artiodactyl split disagree with most recent findings in favouring a primate, artiodactyl clade with rodents as an outgroup. Our study indicates that several genes that are not classified as genes in the most recent human annotation might after all be real genes; or at least they have become pseudogenes very recently, and the orthologous genes in mouse and pig might still be functional. This shows the potential of comparative methods in identifying functional regions of the genome.Sus scrofa was assembled at the Danish Institute of Agricultural Sciences (DIAS) from cDNA libraries from 100 different tissues constructed at DIAS and the Royal Veterinary and Agricultural University in the following way. Total RNA was purified from selected tissues using Rneasy (Qiagen) or Tri ReagentR and poly(A+) mRNA was selected using Oligotex (Qiagene) or PolyATract (Promega). Directional cloneable cDNA was synthezised from Poly(A+) mRNA using the cDNA Synthesis Kit (Stratagene) and was ligated into Eco RI/Xho I digested pTrueBlue (GenomicsOne) or pBluescript (Stratagene) followed by electrotransformation into E. coli XL1-Blue MRF' (Stratagene). 5'-EST sequencing was performed using standard protocols (Applied Biosystem). The sequences were trimmed to the longest open reading frame and the termination codons were removed.Complete cDNA from the domesticated pig Fugu rubrices were obtained with the blastall program with default parameters; the E-score was set to 10-8. We constructed two different datasets, one with and one without Fugu rubrices. Individual alignments were made using ClustalW version 1.83 with default parameters [Sus scrofa have been deposited in Genbank with the following accession numbers: AY609387-AY610506.Homologues sequences from human, mouse and the Japanese pufferfish rameters . We keptNine hundred and eighty-eight four-species alignments were concatenated into a super gene. The three topologies were compared using the super gene as well as each individual gene. Both nonsynonymous nucleotide substitutions and amino acid substitutions were investigated with PAML v. 3.14 . The nonUsing the 1120 three species alignments, the synonymous and nonsynonymous rates of evolution were estimated with the codeml program (seqtype = 1) using the free ratio model (model = 2) with the transition/transversion ratio estimated from the data (fix_kappa = 0).i substitutes into another codon j during the time interval t is determined by the rate matrix Q = (qij) with entriesThe different tests for positive Darwinian selection are all based on extensions of the basic codon based likelihood model . Likelihi ≠ j, with corresponding substitution probability matrix given by exp(Qt). Here πj is the equilibrium codon frequency of codon j, κ is the transition/transversion ratio and ω is the dN/dS ratio. All parameters are estimated independently for each gene. The star topology of the three species is used to estimate the branch lengths (τhuman, τpig, τmouse) for synonymous and non-synonymous substitutions.for 2 distribution with 2 degrees of freedom. The genes found in one or more lineages evolving with a dN/dS ratio > 1 are compared to a nested model where the dN/dS ratio is fixed at 1 in the lineages shown to have a dN/dS ratio larger than one to see whether the result can be attributed to natural selection or just relaxation of selective pressures.Positive selection was tested in two different ways. Test 1 averages over sites but differentiates among lineages. The LRT compares the free ratio model where all three lineages have a different value of ω estimated from the data with the one ratio model where all three lineages share a common value of ω . We notep1 of sites where ω1 are estimated from the data and is forced to lie between 0 and 1, and a proportion p2 of neutrally evolving sites where ω1 = 1 (p1 + p2 = 1). Model A furthermore allows a pre-specified branch to have a proportion of sites that evolve with a different value of ω estimated from the data. This value cannot be smaller than 1. The LRT follows a χ2 distribution with 2 degrees of freedom. If the value of ω in the foreground lineage is estimated to be equal to one the model collapses to the neutral model.Test 2 is based on a new and improved version of the branch-site method presented in . We willPAML v. 3.14  was usedA simple extension of the F3 × 4 codon equilibrium frequency model can incorporate CpG avoidance by adding an extra parameter that penalizes a C followed by a G in the second and third codon position. The new model is parameterised as followsi11 represents the frequency of nucleotide i1, at codon position 1, and ψ(0 < ψ < 1) is a CpG penalizing parameter. The scaling factor cψ ensures that the codon frequencies sum to one.Here πFGJ carried out the analyses and was the primary writer of the text. AH and FGJ together implemented some of the analysis tools. FGJ, AH and MHS together developed the ideas and discussed the interpretation of the results. MF and CB gathered the EST data used. HHJ assembled the cDNAs and carried out Blast searches. All authors read and approved the final manuscript.


Clinical descriptions of Parkinson's disease remain remarkably similar to those first described by James Parkinson nearly 200 years ago. Patients with “shaking palsy” experience a progressive loss of muscle control, increased muscle rigidity, inhibited movement, and tremors. These symptoms, it was later discovered, result from the loss of dopamine-producing neurons specifically in an area of the ventral midbrain called the substantia nigra. Midbrain dopamine neurons relay chemical signals that regulate motor control and less quantifiable attributes like mood and motivation, and therefore the loss of these cells is predicted to lead to the symptoms of Parkinson's.Despite the well-characterized cellular basis of Parkinson's disease, the molecular mechanisms responsible for dopamine neurodegeneration remain unknown. There is evidence that both genetic and environmental components are involved. That a person with Parkinson's disease is three to four times more likely than an unaffected individual to have a close family member with “parkinsonian” symptoms suggests a genetic factor; furthermore, several genes have been associated with relatively rare, familial forms of the disease. For example, mutations of the protein alpha-synuclein (α-synuclein), which is found to aggregate in the brains of patients with Parkinson's, lead to a familiar parkinsonism syndrome. Mutations in a second gene called DJ-1 were recently found in two families with an inherited form of Parkinson's. Importantly, mutations in DJ-1 have previously been linked to the pesticide paraquat in unrelated research on cell stress and reactive oxygen species, and have been linked to dopamine neuron toxicity. Reactive oxygen species are molecular byproducts of oxygen metabolism that react with and damage cellular components like proteins and DNA, and there is evidence from postmortem studies that reactive oxygen species may play a role in Parkinson's disease.PLoS Biology, Asa Abeliovich and colleagues make the case that a model based on mouse embryonic stem cells offers a promising platform for dissecting the disease mechanism of Parkinson's. Working with these cells, the researchers report that DJ-1-deficient cells—and especially DJ-1-deficient dopamine neurons—display heightened sensitivity to oxidative stress. In a second paper, they link DJ-1 dysfunction to alpha-synuclein aggregation.Part of the challenge of untangling the relative contributions of all these components stems from the difficulty in finding a model that can adequately mimic the loss of dopamine cells. In two papers published in Oxidative stress has long been associated with neuronal cell death and neurodegenerative diseases like Parkinson's. Proving a causal relationship between oxidative stress and neurodegeneration, however, requires establishing a molecular mechanism.In the first paper, to explore the hypothesis that DJ-1 contributes to the cellular response to oxidative stress, Abeliovich and colleagues created mouse embryonic stem cells lacking functional copies of DJ-1 and exposed them to hydrogen peroxide, a powerful oxidizer. Compared to normal cells, DJ-1 mutants showed signs of greater toxicity and higher levels of cell death. These defects were corrected when the researchers reintroduced the protein in the mutants, confirming DJ-1's responsibility for the defects. DJ-1 protects against oxidative damage, the results show, not by inhibiting the accumulation of the reactive oxygen species associated with hydrogen peroxide, but by mitigating the damage created by them.Abeliovich and colleagues then explored DJ-1's function in dopamine neurons by inducing mutant and control embryonic stem cells to differentiate in cell cultures. Production of dopamine neurons was significantly reduced in the DJ-1-deficient cultures relative to the control cultures. And like DJ-1-deficient embryonic stem cells, DJ-1 dopamine mutants were vulnerable to oxidative stress. “DJ-1 deficiency,” the authors conclude, “leads to reduced dopamine neuron survival and predisposes these cells to endogenous and exogenous insults.” Inhibiting DJ-1 activity in neurons from the embryonic mouse midbrain produced the same results.In the second paper, Abeliovich and colleagues go on to probe the molecular basis of DJ-1's activity. There have been several leads regarding how DJ-1 functions, based on homology to related genes, including a potential role as a molecular protein chaperone; protein chaperones assist in the folding and refolding of damaged proteins, and thus play a central role in the cellular response to oxidative stress. Abeliovich and colleagues found that DJ-1 acts as an unusual molecular chaperone that is specifically induced under oxidative conditions, and acts to prevent the aggregation of cellular proteins. Interestingly, the researchers go on to show that one substrate of DJ-1 activity is alpha-synuclein, thus providing a possible mechanism linking these two molecules implicated in Parkinson's disease. Altogether, these results support a link between toxin-induced oxidative damage and disease, and provide a tractable model for studying the molecular mechanisms of neurodegenerative disease.


H. pylori.Horizontal gene transfer is central to evolution in most bacterial species. The detection of exchanged regions is often based upon analysis of compositional characteristics and their comparison to the organism as a whole. In this study we describe a new methodology combining aspects of established signature analysis with textual analysis approaches. This approach has been used to analyze the two available genome sequences of ftsK, xerD and polA. In some cases it was possible to determine which strain represented the 'parent' and 'altered' states for insertion-deletion events. Different signature component lengths showed different sensitivities for the detection of some horizontally transferred genes, which may reflect different amelioration rates of sequence components.This gene-by-gene analysis reveals a wide range of genes related to both virulence behaviour and the strain differences that have been relatively recently acquired from other sequence backgrounds. These frequently involve single genes or small numbers of genes that are not associated with transposases or bacteriophage genes, nor with inverted repeats typically used as markers for horizontal transfer. In addition, clear examples of horizontal exchange in genes associated with 'core' metabolic functions were identified, supported by differences between the sequenced strains, including: New implementations of signature analysis that can be applied on a gene-by-gene basis for the identification of horizontally acquired sequences are described. These findings highlight the central role of the availability of homologous substrates in evolution mediated by horizontal exchange, and suggest that some components of the supposedly stable 'core genome' may actually be favoured targets for integration of foreign sequences because of their degree of conservation. Helicobacter pylori is a bacterial pathogen associated with gastritis, peptic ulcers, gastric adenocarcinoma, and rare lymphomas [E. coli from Salmonella can be attributed to point mutation genes but are broadly attributable to horizontal exchange [H. pylori is relatively unusual in that it is a naturally transformable Gram-negative species that does not appear to have a species-specific DNA uptake sequence and appears to rely upon its niche separation as a transformation barrier [H. pylori strains have been divided into two types, type I being those that carry the cag pathogenicity island [cag PAI), which has a foreign species origin, and are associated with more severe disease.ymphomas . It has ymphomas . The acqymphomas -5. In thexchange . H. pylo barrier . Diseasey island   and longer signatures. In addition, use of a new approach based upon classical text analysis that allows analysis of genomes gene-by-gene is described. Analysis of cag pathogenicity island (6 and 5), vac and related toxins (3 and 4), and restriction-modification genes (2 and 4). On the basis of the similarities determined in the H. pylori strain J99 sequence annotation, 7 of the most divergent genes as determined by DNS are not present in strain 26695. Likewise, 2 of the 50 most divergent genes in strain 26695 are not present in strain J99. This is consistent with the identification of genes acquired from other species that have not extended to both sequenced strains. It also suggests that a significant proportion of the 6 to 7% of genes unique to one or other strain [Helicobacter gene pool, but are variably present in different strains rather than reflecting recent foreign origins. Comparisons of a selection of identified orthologous genes in the two strains are shown in Figure Genes were ranked on the basis of their divergence from the mean genome composition. The degree of divergence that is indicative of acquisition from other species is not an absolute. The frequency with which genes are acquired, the untypicality of the donated material, and the rate at which they are ameliorated to the host sequence composition influence it. Strains J99 and 26695 had 53 Table  and 60  genes rer strain  are inhecag PAI gene (the 1st and 2nd most divergent gene in the whole genomes of strain 26695 and J99 respectively, JHP0476/HP0527) has a highly complex repetitive structure and the size of the large divergent peak associated with this island using previous methods is largely due to the presence of this gene.It cannot be assumed that all genes identified in this manner have been recently acquired. It is necessary to assess the nature of the sequence to determine if its divergence might be accounted for on the basis of features of the encoded protein. For example, JHP0476/HP0527, JHP1300/HP1408 and JHP0074/HP0080 include repetitive sequences likely to account for their DNS divergence. This type of analysis cannot be used to determine the possible foreign origin of such genes. Notably, the most divergent While a significant proportion of the genes identified in this analysis are associated with regions including several such genes and which share characteristics of islands of horizontal transfer or pathogenicity islands, this is far from universally true. There are many instances of single genes or small numbers of genes that are present that are not associated with any features that might otherwise have been used as indicators of horizontal acquisition such as transposases and flanking repeats.mutS, ftsK, xerD, and polA. The comparisons of the latter three between the sequence strains are shown in Figure Our initial goal was to identify recently acquired and exchanged genes as candidates likely to be important in niche-adaptation, host interactions, and alterations in bacterial fitness. It has been argued that essential genes are unlikely to be transferred successfully since recipient taxa would already bear functional orthologues, which would have experienced long-term co-evolution with the rest of the cellular machinery. In contrast, it is proposed that those under weak or transient selection – like those associated with nonessential catabolic processes, new operons, and those providing new niche-adaptive changes are likely to be successfully transferred and retained . This leTetranucleotide composition has been used for the consideration of the presence of palindromic sequences that might be substrates for restriction systems and Chi sites and the presence of unstable repeats mediating phase variation , but therd, 13th and 41st most divergent genes by HNS, were 319th, 857th and 750th most divergent by DNS, respectively. In some instances the TNS gave intermediate results and in others identified other genes as more divergent than the other methods. The TNS was most sensitive for the detection of rpoB (HP1198 / JHP1121) which is associated with a significantly different gene length in the two strains  that were not in the 53 (>2 SD) most divergent by DNS, these included 11 restriction-modification system genes and 6 others that were not annotated within the strain 26695 genome sequence. The identification of genes of a type known to be horizontally exchanged, and different between the gene complements of the strains, is strong corroboration for the foreign origin of the additional genes identified by HNS. In several instances  and the 5' 26695 gene (HP0079, 751st in divergence), and the high divergence of the 3' 26695 gene (HP0078, 68th in divergence), indicate that the most likely event is an insertion into strain 26695 . When the homologous genes were compared between the strains, extensive divergences were evident between more than one region of each protein. That these genes have divergent signatures in both strains suggests that neither has a wholly native composition. This observation is consistent with the models of rapid evolution which suggest that transient competitive advantages are enjoyed by organisms that are hypermutators under conditions of environmental stress and transitions, and that these states which can be produced by mutations in DNA repair genes [mutS and recN suggest that H. pylori is another species that can make use of this strategy for diversification under stressful conditions.The inclusion of two DNA metabolism genes associated with recombination and repair is notable. Both ir genes -26. Howeir genes -29. The The identification of RNA polymerase genes, with associated differences between the strains, is striking. The divergence of phylogenetic trees based upon different sequences has been highlighted, and particularly the differences between the trees associated with RNA polymerase genes and rRNA ,31. It hThe spectrum of recently horizontally acquired sequences identified emphasizes the two driving forces of horizontal exchange: the transfer of a phenotype which alters or enhances bacterial fitness resulting in increased competitive fitness or altered niche adaptation, and the presence of a substrate for homologous recombination. Because of the focus upon, and relative ease of identifying, large islands associated with readily identifiable features and phenotypes, the importance of the latter component has perhaps been underestimated. The genes that have been considered to code for 'core metabolic' 'house-keeping' functions are amongst those most likely to be changed by horizontal transfer events because of the presence of homologous substrates, and changes are likely to persist even when the change is phenotypically neutral. Equally, changes in the genes involved in core functions such as gene expression and DNA metabolism may have pleotropic effects and there may be significant differences in strain behaviour, that are not simply the consequence of differences in their respective gene complements. The selection of genes for phylogenetic analysis on the basis of their coding for conserved core functions is also problematic because these are also frequently the genes most likely to share the high homology that facilitates recombination and horizontal exchange.k equal-sized subsequences (or 'windows'). The mathematical basis for the signature is an odds ratio – pi – calculated by dividing the frequency of a length-L oligonucleotide by its expected frequency. The odds ratios for each of the 4L oligonucleotides in each window (w) are compared with the odds ratios for the overall sequence (s) [δ is plotted and thus a nucleotide signature consists of a k-length sequence of δ values: δ(w,s) = (1/4L)Σ(4L,i:x)|pi(w) - pi(s)|, where x is the set of all permutations of length L and i is one such permutation.A traditional nucleotide signature is generated by segmenting a sequence of DNA into ence (s) ,10,33. Tweighted cusum (cumulative sum) [, is modified so that n denotes the number of ORFs in the genome, Xi the number of oligonucleotides in ORF i, and wi the number of nucleotides in ORF i. The results are scaled according to ORF size using the standard error σ = √(*#ORF). In this way false positives are abrogated by normalizing for over-representation of lower order peptides.There are interesting parallels between signature-style genome analysis and stylometric techniques previously used to determine the authorship of controversial literary texts. This is analogous with the biological problem and it is from this that our method is derived. Rather than using a fixed-window signature, signature scores are calculated for each coding open reading frame (ORF) and weighted with variance estimates so that the scores for shorter ORFs confer with their longer counterparts. Bissell's ive sum) , , is moO(N) (N = sequence length) refinement: probabilities for the complete sequence are calculated in O(N) steps for any length-L oligonucleotide, and maintain O(N) when 4L>N through a hashing function; the second part of the program calculates σ for each ORF using a loop flattening technique, thereby avoiding the program having to recalculate overlapping sub-expressions. The program is available from  and .The method is implemented in Java and efficiency is maintained through an Sequence alignments, as shown in Figure ORF, Open Reading Frame; DNS, Dinucleotide Signature; TNS, tetranucleotide signature; HNS, hexanucleotide signature.NJS initiated the project, performed the genome sequence analyses, compared the two strains, interpreted the results, and prepared the biological aspects of the manuscript. PB was a DPhil student who worked on the coding aspects of the new methodology. JFP contributed to the bioinformatics discussions and planning stage of this project. SAJ directed and primarily developed the analysis strategy and the implementation of the new computational basis of the methodology, and prepared the computational aspects of the manuscript.


One of the major interests in our laboratory has been to develop a scientific framework for dietary advice for patients with diabetes. Knowledge regarding the metabolic consequences and potential effects on health of protein in people with type 2 diabetes has been a particular interest.We recently have completed a study in which dietary protein was increased from 15% to 30% of total food energy. The carbohydrate content was decreased from 55% to 40%, i.e. dietary protein replaced part of the carbohydrate. This resulted in a significant decrease in total glycohemoglobin, a decrease in postprandial glucose concentrations and a modest increase in insulin concentration. Renal function was unchanged.Currently we also are determining the metabolic response to a diet in which the carbohydrate content is further decreased to 20% of total food energy. The %tGHb decrease was even more dramatic than with the 40% carbohydrate diet.From these data we conclude that increasing the protein content of the diet at the expense of carbohydrate can reduce the 24-hour integrated plasma glucose concentration, at least over a 5-week period of time. The reduction was similar to that of oral agents. Renal function was not affected significantly. Thus, increasing the protein content of the diet with a corresponding decrease in the carbohydrate content potentially is a patient empowering way of reducing the hyperglycemia present with type 2 diabetes mellitus, independent of the use of pharmaceutical agents. Our research group has been and continues to be interested in the metabolic response of people with type 2 diabetes to macronutrients in the diet in general. More recently, we have been particularly focused on the metabolic response to a high protein diet. The reason for this is three fold: First, for several years, one of our major goals has been to develop a scientific framework for dietary advice based on sound metabolic principles. Second, we have data that suggest that an increase in dietary protein may be salutary for people with diabetes. And lastly, knowledge regarding the metabolic consequences and potential effects on health of dietary protein has lagged far behind that of dietary fats and carbohydrates.In this paper we will focus on the concept that an increase in dietary protein may be salutary for people with diabetes, and particularly for the control of blood glucose.The concept that an increase in dietary protein may be useful in controlling the blood glucose would appear to be counterintuitive, since amino acids derived from ingested or endogenous proteins are major net gluconeogenic substrates.2 to form urea. The remaining deaminated product is largely converted into glucose through gluconeogenesis, although a small amount is converted into other products. .The reason that 5 weeks was chosen for the study is because this is the time required for the % total glycohemoglobin to decrease 50% of its ultimate value after a rapid stable decrease in blood glucose concentration  .Urine urea, normalized to the urine creatinine, was measured as an index of compliance. Since the protein content of the diet was doubled, one would expect that the urine urea:creatinine ratio also would approximately double if the subjects were compliant. The ratio on the standard diet was ~7 and was stable throughout the 5 weeks. When the same subjects were given the 30% protein diet, the urine urea:creatinine ratio was ~13–14, i.e. a value that one would expect with good compliance with the diet.The fasting glucose concentration did not change when the subjects received the 30% protein diet. However, the postprandial glucose concentrations were lower throughout the day .Although the differences in postprandial glucose values were not very large, when integrated over the 24-hour period, there was a 38% decrease in postprandial glucose area response. If the 24-hour integrated area is considered to be 100% when the subjects ingested the 15% protein diet, when they ingested the 30% protein diet it was 62%  the %tGHb would have decreased by only 50% of the expected final decrease  .None declared.Both authors were equally responsible for designing the experiments, evaluating the statistics, interpreting the data, writing the manuscript, and organizing the figures and tables.


Likewise, we determined that cushion formation is blocked in −/−)cardiofunk . We show that in yeast this mutation negatively affects actin polymerization. Because the lack of cushion formation in sih- and cfk-mutant embryos could be due to reduced myocardial function and/or lack of blood flow, we approached this question pharmacologically and provide evidence that reduction in myocardial function is primarily responsible for the defect in cushion development. Our data demonstrate that early myocardial function is required for later steps of organogenesis and suggest that myocardial function, not endothelial shear stress, is the major epigenetic factor controlling late heart development. Based on these observations, we postulate that defects in cardiac morphogenesis may be secondary to mutations affecting early myocardial function, and that, in humans, mutations affecting embryonic myocardial function may be responsible for structural congenital heart disease.Function of the heart begins long before its formation is complete. Analyses in mouse and zebrafish have shown that myocardial function is not required for early steps of organogenesis, such as formation of the heart tube or chamber specification. However, whether myocardial function is required for later steps of cardiac development, such as endocardial cushion (EC) formation, has not been established. Recent technical advances and approaches have provided novel inroads toward the study of organogenesis, allowing us to examine the effects of both genetic and pharmacological perturbations of myocardial function on EC formation in zebrafish. To address whether myocardial function is required for EC formation, we examined s cardiofunkCardiac anomolies can result from very early defects in heart development. In zebrafish, such defects have been shown to be caused by a new gene called  The genetic programs and developmental processes that lead to organ formation are still poorly understood. We are currently witnessing an expansion in research that aims to identify the genes responsible for the structural development of organs and their later function. Among the organs of the body, the heart is unique because it begins to function mechanically before structural development is complete, begging the important question of whether myocardial function is required for the morphogenetic events that occur after the heart begins beating. One of the late steps of heart development is the formation of the endocardial cushions (ECs), which are tissue swellings that develop in characteristic locations along the anterior–posterior (AP) extent of the heart tube and contribute to valves and, in four-chambered hearts, to septae. Because of the clinical significance and prevalence of EC defects in humans , an undeDuring the process of EC and valve development, specific endocardial cells undergo multiple poorly understood specification, differentiation, and migration events en route to becoming functional heart valves. The genes involved in one substep of this process, epithelial–mesenchymal transformation (EMT), are gradually being identified. Analysis of EMT during cardiac-cushion development has implicated molecules such as Fibronectin, Transferrin, ES-130, hLAMP-1, TGF-β2, TGF-β3, BMP-2 (reviewed in To examine the role of epigenetic factors involved in EC formation, tie2 promoter (−/−)silent heart (sih embryos, which establish neither a heartbeat nor blood flow due to mutation of cardiac troponin T (tnnt2), but do undergo looping morphogenesis  boundary. This process is easily visualized at 48 h postfer-tilization (hpf) by examining the endocardial cells expressing green fluorescent protein (GFP) under the control of the mouse promoter  , that fails to accumulate tie2::GFP-positive cells at the AV boundary by 48 hpf  extent of the heart tube before becoming localized to the AV boundary at 42 hpf  zC202M22 , and fv49b10 (−/−cfk mutants and reverse genetic techniques using morpholino antisense oligonucleotides. Injections of morpholinos against each of the four coding sequences showed no obvious cardiac phenotype, indicating that we might not be working with a loss-of-function mutation or that cfk has overlapping function with another gene. Subsequently, sequencing of the actin gene (zeh0631) showed a change of arginine 177 to histidine. The three embryos recombinant at either end of the 52 kb region were homozygous at the site of the R177H lesion, suggesting that we had isolated cfk.To understand better the molecular basis for the d Z10582 . AnalysizC202M22 A. Follow GenScan  and BLAS fv49b10 B. Compar fv49b10 C and hum fv49b10 D and concfk mutation supported our hypothesis that cfk corresponded to an actin gene. Analyses of Cfk indicate that it is a sarcomeric actin by virtue of its homology to zebrafish α-cardiac and α-skeletal actins and lack of homology to zebrafish cytoplasmic actin, as well as its conservation of synteny with the human skeletal actin. Further evidence that cfk encodes a sarcomeric actin and not a cytoplasmic actin includes the presence of an extra residue at the N-terminus of Cfk, which is seen in all sarcomeric actins but no cytoplasmic actins, as well as the presence of residues that are stereotypic for sarcomeric actins at all 20 locations where sarcomeric and cytoplasmic actins have unique amino acids , which is distinct from the 3′ UTR sequences of zebrafish α-cardiac and α-skeletal actins. These data showed that cfk is expressed in the myocardial but not endocardial cells of zebrafish embryos from 24 to 48 hpf  leads to unstable filaments, occasionally giving rise to a phenotype in heterozygous embryos.During our work with e cfk+/− A, suggese cfk+/−  demonstr−/−cfk embryos have a dilated heart and lack blood flow and because subsequently approximately 50% of these embryos fail to form ECs, we wondered whether these two phenotypes were causally related or whether lack of EC formation could occur independently of poor early myocardial function. To address this question, 379 embryos from multiple cfk clutches were assayed for cardiac dilation and lack of blood flow at 36 hpf and for lack of endocardial ring formation at 48 hpf. Embryos that were phenotypically wild-type at 36 hpf invariably developed endocardial rings over the next 12 h, whereas all embryos that failed to form endocardial rings had previously shown a functional phenotype at 36 hpf  and fail to generate blood flow, it is impossible to conclusively state whether EC formation is affected in these embryos directly as a result of poor myocardial function or indirectly as a result of the perturbation in blood flow and shear stress caused by poor myocardial function. n = 74) formed an endocardial ring, indicating that blood flow is not required for the initial steps of cushion formation. When myofibril function was further decreased by treatment with 10 mM 2,3-BDM, the percentage of embryos with an endocardial ring decreased to 13% (n = 68). Studies with the anesthetic tricaine confirmed the observation that ECs may form in the absence of blood flow and that the likelihood of forming cushions is inversely proportional to the concentration of tricaine (data not shown). In summary, the results from the 2,3-BDM and tricaine treatments suggest that it is poor myocardial function, and not lack of blood flow, which is primarily responsible for the loss of EC formation in −/−cfk and −/−sih embryos.Because −/−sih embryos fail to beat yet undergo looping morphogenesis and AV boundary specification. The specific absence of EC formation in these embryos clearly demonstrates that myocardial function is required for EC formation. Precisely how myocardial function is required for EC formation is unclear. Prior work has demonstrated a requirement for AV boundary myocardium in the formation of ECs, and several signaling molecules emanating from the myocardium at the AV boundary have been identified , abc-b10 (5′-TCATTCGCAACATTGTCCCATACAT-3′), fv49b10.y1 (5′-CCGACCTAATTCGCTTGGT-CACCAT-3′), and cfk (5′-CATCTTGATGTATTCTTTCTCTGCT-3′). The morpholinos were injected at 4 ng and 8 ng into tie2::GFP embryos at the one-cell stage and examined at 24 to 72 hpf for myocardial function and EC phenotypes. Morpholinos against abc-b10, rab4a, and fv49b10.y1 did not affect myocardial function or EC formation. The cfk morpholino caused no phenotype, an expected result given the ability of actin genes to compensate for each other when down-regulated (cfk with α-cardiac actin.We designed morpholino oligonucleotides (Gene Tools, Philomath, Oregon, United States) to bind to the initiation codon and flanking sequences of the following zebrafish genes: sih , rab4a (egulated  and the s11cfk-AB/SJD hybrid strain using SSLP with various CA repeat markers and SSCP with various ESTs. SSCP was performed by denaturing PCR products at 95°C for 10 min in 0.7 mM EDTA and 36 mM NaOH and placing on ice before loading on nondenaturing acrylamide gels. BAC library filters for the CHORI-211 (zC) library were obtained from P. de Jong at BACPAC . Linkage of the R177H mutation to the s11cfk allele (through genotyping of mutant embryos) was accomplished by performing PCR across the mutation (forward primer = 5′-ATCGTGCTGGACTCTGGTG-3′ and reverse primer = 5′-GAAAGAATAACCGCGCTCAG-3′) and digesting with NsiI, which cuts only the H177 allele. Synteny analysis was performed through http://genome.jgi-psf.org/fugu3/fugu3.home.html (fugu) and http://genome.ucsc.edu (human).We genotyped diploid mutant embryos from a s11cfk mutant embryos to extract mRNA (Trizol, GIBCO–BRL, Gaithersburg, Maryland, United States) and synthesize cDNA . PCR was then performed from the 5′ UTR to 3′ UTR and multiple clones sequenced. To confirm that the amino acid change we saw was a mutation and not a polymorphism in the strain used for mutagenesis, we examined the DNA of four F1 females from the screen .We used a pool of 25 e screen  in whichClutches of dechorionated embryos were placed in 2,3-BDM (Sigma B-0753, Sigma–Aldrich, St. Louis, Missouri, United States) at 24 hpf at concentrations between 2 and 20 mM. This concentration range has been shown to affect myofibrillar ATPase in a dose-dependent manner, with 25% of activity remaining at 10 mM . 2,3-BDM2, and 0.1 mM DTT) and used within 2 d. Actin polymerization was induced by the addition of MgCl2 and KCl to final concentrations of 2 mM and 50 mM, respectively, and polymerization was assessed by the increase in light scattering as a function of time at 25°C in a 120 μl volume in a thermostatted cuvette using a SPEX Fluorolog 3 fluorimeter with excitation and emission wavelengths set at 360 nm.Site-directed mutagenesis of the yeast-actin coding sequence in a centromeric plasmid and construction of haploid cells producing only the mutant actin were carried out as described previously . Wild-tyVideo S1−/−cfk hearts have complete regurgitation of blood from the ventricle to the atrium and therefore no forward blood flow.Wild-type hearts propel blood through the vasculature, whereas (2.91 MB MOV).Click here for additional data file.Video S2−/−cfk embryos (two different embryos shown) have dilated hearts with weak contractions and are not capable of generating circulation.Wild-type embryos have a narrow, strongly contracting heart and generate effective blood flow. (2.9 MB MOV).Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/index.html) accession numbers discussed in this paper are for cfk (AY222742), zebrafish α-cardiac actin (AF116824), zebrafish α-skeletal actin (AF180887), and zebrafish cytoplasmic actin (AF057040).The GenBank (