Our results are not limited to Ti-Pd-Cr SMAs but potentially provide a strategy for searching for SMAs with small thermal hysteresis.The large thermal hysteresis (ΔT) during the temperature induced martensitic transformation is a major obstacle to the functional stability of shape memory alloys (SMAs), especially for high temperature applications. We propose a design strategy for finding SMAs with small thermal hysteresis. That is, a small ΔT can be achieved in the compositional crossover region between two different martensitic transformations with opposite positive and negative changes in electrical resistance at the transformation temperature. We demonstrate this for a high temperature ternary Ti-Pd-Cr SMA by achieving both a small ΔT and high transformation temperature. We propose two possible underlying physics governing the reduction in ΔT. One is that the interfacial strain is accommodated at the austenite/martensite interface via coexistence of B19 and 9R martensites. The other is that one of transformation eigenvalues equal to 1, i.e.,  Shape memory alloys (SMAs) undergo a reversible martensitic phase transformation from the high symmetry austenite (A) to low symmetry martensite (M) phase upon the influence of temperature or stress field, giving rise to the shape memory effect (SME) and superelasticity (SE), respectively13357834101112U, which maps the martensite lattice to the austenite lattice16U, λ1 ≤ λ2 ≤ λ3, represent the presence of an invariant habit plane between austenite and martensite16λ2 = 1, as means to reduce ΔT, so that there is a perfect coherent interface (unstressed and untwinned) between austenite and martensite10λ2 = 1 and ΔT has been confirmed for Ti-Ni-Au, Ti-Ni-Pt and Ti-Ni-Pd ternary systems by various authors51819In searching for thermoelastic SMAs with small thermal hysteresis (ΔT), several different methodologies have been utilized. Experimentally, combinatorial synthesis of SMA thin films has been employed to screen the various compositions and select the best candidates1011135a priori knowledge of crystal symmetry and lattice parameters, or the relationship between lattice parameters and alloying elements so that λ2 can be evaluated in advance. The access of those information beforehand requires a lot of experimental efforts, especially for multicomponent systems. Thus, a simple strategy that can use the knowledge available to guide the design of SMAs with small ΔT is highly desireable.However, the use of GNLTM theory to design new SMAs requires R), whereas in other SMAs the martensitic transformation is accompanied by a decrease in resistance at the martensitic transformation, as denoted in R. Different martensites can be characterized by the distinct behavior they show with respect to ΔR. A +ΔR is due to the higher electrical resistance of M over A; and −ΔR indicates that M has a lower resistance than A. Examples of martensites with +ΔR include Ti50Ni50 (aged), Ti50Nix50−Cux, Ti50Nix50−Fex, Ti50Pdx50−Crx (x > 5%at.), whereas those with −ΔR include Ti50Ni50, Ti50Nix50−Pdx, Ti50Pdx50−Crx (x < 4%at.)R can gradually change to those with −ΔR by either defect doping or thermal treatment21R = 0, as shown in R continuously or monotonically varying with either defect doping or thermal treatment, there must exist a point or regime where ΔR = 0. In such a case, a martensitic transformation occurs without any change in resistance both on cooling and on heating, thus the thermal hysteresis would be expected to be zero  is much more severe in high temperature SMAs and as the transformation temperature increases, the thermal hysteresis should also increase dramatically. For example, Ti50Pdx50−Crx system between 4 at.% and 5 at.% Cr. All the samples were fabricated in the present study under the exact same conditions to avoid the possible effects from microstructure and defects. We first checked the crystal structure of our samples. Typical X-ray diffraction profiles of all the samples are shown in at.% Cr, may possess 9R martensite structure as wellat.% Cr, indicating something special happens and will be discussed in the discussion part.To verify our strategy, we fabricated the Ti50Pdx50−Crx system between 4 at.% and 5 at.% Cr. R(T) curves with −ΔR can be found in samples with Cr concentration near 4 at.%, and R(T) curves with +ΔR are observed in samples with Cr concentration near 5 at.%. The former −ΔR situation corresponds to the B2 to B19 martensitic transformation that is typically found in Ti50Pd50 alloy, whereas the latter +ΔR is due to the B2 to 9R martensitic transformation which appears after heavily doping the Ti50Pd50 alloy with Cr, V, or Mn24at.% and 4.7 at.%, the R(T) curves do not possess the usual “S” shape at the transformation temperature, instead, show a tendency of flattening and merging together. Such evolution of ΔR from a positive value through almost zero to a negative value is exactly the situation expected in T.Ti50Pdx50−Crx. The ΔT is calculated using Ms and Mf are the martensitic transformation start and finish temperatures, and As and Af are the reverse transformation start and finish temperatures. These start and finish temperatures are determined using the tangent method as shown in the inset of at.% with −ΔR, and then decreases with increasing Cr concentration. It reaches a minimum at Cr concentration of 4.7 at.%, which is just within the composition crossover region mentioned above. ΔT then increases again for Cr concentration around 5.0 at.% with +ΔR. We also determined ΔT from DSC measurements via endothermic and exothermic peaks, as shown in As, Af, Ms and Mf; and ΔT was calculated using the same equation as for the R(T) curves. The ΔT from DSC measurements behaves identically to that obtained from R(T) curves, emphasizing the consistency of our approach. In addition to the tangent method, we also calculated ΔT from the exothermal/endothermic peak temperatures, ΔT = Texo − Tend, as shown in the left inset to at.%, which is still in the crossover composition region. The “V” behavior in R and −ΔR correspond to the B2-B19 and B2-9R martensitic transformations, respectively. The optimized compositions just sit in the crossover region between B2-B19 and B2-9R transformations. Thus the optimal, smallest ΔT is likely to be due to instabilities associated with the three phases which include B2, B19 and 9R. At the phase transformation temperature in the crossover region, the three phases can be degenerate in energy, allowing the possibility of multiple transformation pathways. Most likely, the preferred pathway in the energy landscape is one that include a saddle point between the maxima of B19 and 9R. Microstructurally, the saddle point and pathway would correspond to coexisting B19 and 9R martensite at the A/M interface so that the strain at the A/M interface is easily accommodated via both B19 twins and 9R twins. However, for direct B2-B19 and B2-9R transformations, energy barriers need to be overcome for the phase transformations to take place. This is accompanied by larger strains at the habit planes for B2-B19 and B2-9R, resulting in larger ΔT. This explanation is consistent with the initial decrease in ΔT from a larger value to a fairly small value followed by an increase again to a relatively large value. Another possible physical origin relies on the eigenvalues of transformation strain matrix. Due to the strong correlation between λ2 = 1 and small ΔT, it is also natural to expect that the samples with smallest ΔT, i.e., 4.6 at.% Cr and 4.7 at.% Cr, may possess λ2 = 1. The abnormal unit cell volume change within 4 at.% and 5 at.% Cr shown in λ2 is closely related with the lattice parameters of martensite. Consequently, a perfect coherent interface (unstressed and untwinned) between austenite and martensite should be present in those TiPd samples with smallest ΔT. Although we provided two possible explanations for the low thermal hysteresis of our newly found alloy, the exact underlying mechanism still needs to be clarified in the future work.It is known that +Δ50Ni50 and our best alloy, Ti50Pd45.3Cr4.7, by thermal cycling using DSC. Ti50Pd45.3Cr4.7 alloy  alloys were made by arc melting a mixture of 99.9% pure Ti, 99.9% pure Pd and 99.9% pure Cr in an argon atmosphere. Specimens for measurement were spark cut from the ingots that were hot rolled to 1 mm thick. They were then solution treated at 1273 K for 1 hour in evacuated quartz tubes and quenched into ice water. In order to remove the affected surface layer, the specimens were mechanically polished, and followed by chemical etching.Base ingots of Kmin−1 to detect the resistance change (+ΔR or −ΔR) at the martensitic transformation. Differential scanning calorimetry (DSC) measurements were also employed with a cooling/heating rate of 10 Kmin−1 to measure the martensitic transformation by exothermal/endothermic peaks.A temperature dependent resistance (R(T)) measurement was made with a cooling/heating rate of 2 How to cite this article: Xue, D. et al. Design of High Temperature Ti-Pd-Cr Shape Memory Alloys with Small Thermal Hysteresis. Sci. Rep.6, 28244; doi: 10.1038/srep28244 (2016).


There were few citations of my proposal—and virtually none of viroids—beyond plant virology unil 1994, when Cheles-Flores critically examined the hypothesis and pointed out a serious difficulty, as well as a process by which this difficulty could be overcome. In 2013, when investigations by Koonin and Dolja revealed that of extant RNAs, viroids “strikingly” display some of the molecular properties posited for the earliest evolving, selfish RNAs , but, because extant organisms, aside from higher plants, appear not to harbor viroids, they cannot be regarded as primordial fossils, but appear to have evolved post LUCA . Here, I review whether some evidence nevertheless is compatible with the original postulate of the 1989 hypothesis. My analysis reveals no unequivocal evidence for an ancient origin of viroids, but suggests, alternatively, that viroids may have evolved de novo more recently, probably by novel processes similar to those suggested by each reviewer.These results are important, because they help illuminate a little understood period of abiogenesis—after the abiotic synthesis of life’s chemical building blocks, which is, in principle, understood, and before the evolution of DNA and proteins in the late RNA World. Beyol agents –7 becausl agents .In 1989, I hypothesized , 11.Here, I report how the biological community reacted to my hypothesis and by what means scientists suggested to test its plausibility.While my hypothesis was frequently cited in the biological literature with little or no comment, it was first critically examined in 1994 by Cheles-Flores , who poiIn a second 1994 paper , Chela-FIn 1998, Jeffares. et al.  reportedGiven the strong trend RNA --- > RNP --- > protein, Jeffares et al. examined the phylogenetic distribution of RNA in modern organisms for relics and thus developed a model for complexity in the RNA world. Candidates were RNAs which fit at least one of their criteria: “catalytic, ubiquitous (or at least conserved within the eukaryotic lineage…), or central to some aspect of metabolism.”Most importantly, in the context of the present assessment, is Jeffares et al.’s conclusion that viroids, plant satellite RNAs, and “hammer heads” are, indeed, ancient relics of the RNA world. It is not clear, however, on which of the parameters this conclusion is based. Viroids are listed confusingly (together with “hammer heads”) at the bottom of table 2—titled “RNA functions in modern cells”— under the rubric “Function,“ as “Various,” under the rubric “Distribution” as “Plant satellite RNA,“ and under the rubric “In the RNA world?” as “In RNA world (see text)” but this referral in a footnote is not illuminating.By the authors’ parameters, many, if not most, extant RNAs (or their precursors) were also already present in the RNA world, including precursors of the three major cellular RNAs: rRNA, mRNA, and tRNA.final complexity of the RNA World,”— just prior to the evolution of translation and proteins—it was actual properties of viroids, listed in 1989, which I considered to suit them for survival in a prebiotic “soup” far less hospitable than that envisioned by Jeffares et al., i.e., for an earlier stage in the RNA World.A distinction must be made, however, between Jeffares et al.’s chosen criteria for relics and those chosen in my 1989 publication. Whereas Jeffares et al. developed their “model for the Therefore, if correct, Jeffares et al.‘s results would not only be in accord with the relic hypothesis, but more accurately define the stage of the RNA World, in which viroids (or their precursors) could presumably have prospered. However, Jeffares et al.’s choice of parameters is, by necessity, subjective and any substitutions would likely alter the conclusions. Even given the existence of an ancient RNA World, there are problems with understanding how, without DNA or proteins, cellular life could have evolved. One of the major problems is the question as to how one and the same kind of RNA molecule could serve simultaneously as both information carrier and biocatalyst, which would require a combination of features: good “templating” ability (for replication) and stable folding (for ribozymes). This poses a paradox, because well folded sequences are poor templates for copying, but poorly folded sequences are unlikely to be good ribozymes .In 2013, Ivica et al.  describeThe investigators demonstrated this strategy by use of computational simulations of RNA sequences (including 40 viroid sequences) and their folding as experimental models of early replication, “involving non-enzymatic, template-directed, RNA primer extension.” The investigators recognized the fact that “interestingly, viroid RNA sequences…show significant asymmetry in folding energy between the infectious (+) and template (-) strands due to G: U pairing, suggesting that this strategy may even be used by replicators in the present day world,” as well, as postulated, in the RNA world. If so, this viroid-suggested process should be amenable—beyond computer simulation—by experimentation with actual RNA molecules.Also in 2013, Ma et al.  cited myForterre’s revolutionary proposal  to dividTheoretical studies  indicatecis signals for replication.” Koonin and Dolja [Koonin and Dolja  studied nd Dolja  also desKoonin and Dolja  concludeProperties of Hepatitis delta virus may supply a clue: While the origin of HDV is unknown and while several different theories of its origin have been proposed , HDV RNAIf so, why have they disappeared? Conceivably, the metabolism of animals, but not that of plants, changed with time, such as to make animals less hospitable for the survival of viroids, and they eventually became extinct—the only exception being HDV RNA, which, as an undoubtedly rare event, evolved to “find refuge” in the capsids of a virus (HBV) and thus became protected from the presumed damaging animal metabolism.While apparently no evidence exists of viroid-inhibiting metabolites present only in particular hosts, not in others, such compounds have not been actively searched for and could easily have been overlooked—in which case, absence of a particular viroid in some, but not other hosts or host families, would not necessarily permit one to draw conclusions regarding the evolutionary age of that viroid.Both reviewers’ reports penetratingly and convincingly state reasons why my hypothesis faces severe difficulties and may be wrong. Why, if viroids are of primordial provenance, have none been isolated from cyanobacteria or from any other organisms in the evolutionary line older than angiosperms? Have they been sufficiently searched for in such “out-of-the-way” plants? Maybe not, because most viroid-investigating plant pathologists are, and have been, concerned with viroids’ agricultural, not evolutionary significance.With these considerations in mind, and encouraged by both reviewers’ statements that my 1989 hypothesis is still alive, I propose that it should not be abandoned, but held in abeyance, awaiting future pertinent experimental evidence for or against it.Sophisticated, newly conceived alternative hypotheses (see reviewers’ reports) convincingly claim that viroids could have evolved de novo at more recent than primordial times.However, such hypotheses leave unexplained why extant viroids so “strikingly” display several crucial properties of posited primordial RNAs. Being common features of viroids, presence of these properties in extant viroids is unlikely to be a multiple coincidental occurrence.Clearly, a decision between these opposite theories is not possible at this time; only considerable, dedicated new research could eventually solve the dilemma.Hopefully, this paper will stimulate novel research, in which case both reviewers’ reports, with their original ideas, would be an essential help, by suggesting exciting new investigations, be there in line with Dr. Koonin’s novel protocol for studying a possible de novo origin of viroids, which, based on the early, seminal work of Dr. Sol Spiegelman, offers a viable alternative explanation for the origin of viroids, or Dr. Dolja’s similarly promising suggestions to the same effect. Such investigations may also help illuminate an early period in the evolution of life, a state of abiogenesis that, located between the first stage---the abiotic, chemical synthesis of amino acids and nucleotides, which is relatively well known ---and thNot applicable.Not applicable.Not applicable.Reviewer Recommendation Term: Endorse publicationThis manuscript is useful in attracting attention to poorly investigated problem of the viroids’ origin, but provides insufficient support to the concept of direct descent of viroids from primordial RNA world. I fully support Dr. Diener’s effort to revitalize discussion on the origins of viroids, the smallest known replicons that encode no proteins and possess ribozyme activity, thus recapitulating two features of the postulated primordial replicons of the RNA world. It should be emphasized, however, that extant viroids, as well as Hepatitis delta agent, lack an even more critical feature, i.e. ability to self-replicate, instead relying on polymerase proteins provided by the host. My other major reservations with the hypothesis of viroid’s direct descent from RNA world are as follows. First, to the best of our knowledge, viroid-like parasitic agents are found only in two types of organisms, higher plants (angiosperms) and vertebrates, but in none of their presumed ancestors, algae and more primitive animals, respectively. Furthermore, there is no data on any such agents in a vast variety of unicellular eukaryotes whose evolutionary history predates that of plants or animals by hundreds of millions of years. Worst yet, no viroid-like agents were reported in any of the prokaryotes, either archaea or bacteria that existed long before emergence of eukaryotes. Thus, according to author’s hypothesis, primordial viroid ancestors should have treaded a very narrow evolutionary path leading straight to land plants, but being eliminated from other organisms, even those that believed to be direct plant ancestors, such as Characean algae. To my opinion, a hypothesis allowing de novo origin of viroids in the higher plants, perhaps, via recombination between a pre-existing, cellular, ribozyme-possessing RNA with one that endowed the product with DNA-dependent RNA polymerase recognition signal, appears to be a simpler and thus more plausible scenario. This not to say that the original idea of Dr. Diener that viroids, as the smallest known autonomous replicons are most reminiscent of the RNA world replicons is not valid; it is, and very much so. I am not so fond of the hypothesis 2 for one simple reason: the data, supporting hypothesis 2 are not yet peer-reviewed, published, or independently confirmed. It feels premature to use these data until that happens. In conclusion, I am in favor of publishing this manuscript because the problem of viroids’ origin is extremely important and grossly under-investigated. The author’s hypotheses broaden the discussion and, hopefully, could stimulate the relevant experimental research.Response to Dr. Dolja’s review of my manuscriptI wish to thank Dr. Dolja for his thoughtful and constructively critical review, as well as for his excellent suggestions for improving the manuscript. His idea of creating an alternative hypothesis to the primordial RNA origin of viroid-hypothesis, by allowing de novo origin of viroids in the higher plants via recombination of a preexisting, cellular, ribozyme-possessing RNA, with one that endowed the product with DNA-dependent RNA polymerase recognition signals—promises to be a made-to-order solution to the viroids’ serious descent dilemma. There would be left, however. a plausible explanation, why the de novo-created viroids “strikingly” resemble their posited primordial RNA-like features, which would now be bereft of functional significance.Dr. Dolja is quite correct: the preliminary data, as well as hypothesis 2 in toto are not suitable for publication. I have deleted both, including mention of the unpublished data.Theodor O. Diener.Reviewer Recommendation Term: Endorse publicationThis Hypothesis article by Theodor Diener, the discoverer of viroids, revives his 25 year old hypothesis that viroids are living fossils of the primordial RNA World. The hypothesis is worth attention for two important reasons: first, it comes from the discoverer of these minimal replicons and will be of interest to many for that fact alone, and second, the idea of the primordial origin of viroids is a “natural” and as such, should be explicated, discussed and revisited. Indeed, among all known replicons, viroids come the closest to what one would envisage as a vestige of the RNA world: they are small RNA molecules that encode no proteins yet replicate autonomously and vigorously. Moreover, just as expected of putative survivors of the RNA World, viroids possess ribozyme activity that is required for their replication. These are striking arguments pro the hypothesis of the primordial origin of viroids. In fact, so obvious and (seemingly) persuasive are these arguments that, contrary to the rather surprising statement of Diener that his hypothesis had been ignored for 20 years, it had been lauded as a fundamentally important and best available scenario for the origin of viroids . There are two serious contra arguments: first, viroids employ their ribozyme activity only for the processing of the replicative intermediates whereas replication itself is catalyzed by an elaborate host enzyme, the DNA-dependent RNA polymerase (nuclear or chloroplast), and second, the host range of viroids, to the best of our current knowledge, is limited to plants. The first counter-argument is not as damning as it might seem. Indeed, it would be unreasonable to expect that any RNA World survivor could retain the ancestral, RNA-only mode of replication which undoubtedly was much less efficient than any of the varieties of the modern, protein-catalyzed replication. The second counter-argument is harder to refute with as the limited host range of viroids implies numerous losses of viroids not only in animals (the focus of Diener’s discussion) but also in numerous lineages of bacteria, archaea and unicellular eukaryotes. This is a decidedly non-parsimonious evolutionary scenario  which is the main reason I hold the opinion that Diener's hypothesis is false and an alternative scenario for the origin of viroids should be sought (see below). First, however, I have to address Diener’s evidence that is deemed to be supportive of the primordial hypothesis. The first one comes from the mathematical model of Ma and colleagues (Diener’s Ref. 17) that is purported to demonstrate the plausibility of the hypothesis on the primordial origin of viroids. With all my respect for and interest and involvement in mathematical modeling of evolution, this does not come even close to testing the hypothesis. The model includes a variety of far-fetched assumptions  and at best is of interest as a conceptual exploration of replication processes in and RNA World that contained viroid-like molecules. It says nothing on the origin of the actual modern plant pathogens. Actually, I strongly doubt that any mathematical model, even a more realistic and carefully constructed one, could do anything substantial in that direction. The second line of evidence presented by Diener has to do with the purported replication of HDV in plant cells and of viroids in animal cells, in the presence of delta-antigen. Regrettably, this argument is based solely on unpublished experiments of Pelchat that are quoted in Ref. 22. This is troubling for purely formal reasons but more important, because in the absence of essential technical data, in particular, on the specificity of the stimulation of viroid replication in animal cells by delta-antigen, the reader cannot assess the validity of the claims. Assuming that these experiments are fully controlled and completely reproducible (a big assumption), such findings will provide us with a valuable insight into viroid-host interaction. However, even under this best case scenario, this will not buttress the primordial hypothesis at all. Indeed, it is completely reasonable to propose, as Diener does, that for some reasons, animal cells are inhospitable to viroids  and become permissive only in the presence of proteins with certain properties such as those of delta-antigen. Should this be the case, further molecular exploration of this system will be of considerable interest. However, this has no bearing on the origin of viroids. Furthermore, Diener assumes that, if there is one viroid-like agent (HDV) in animals, there should be more, and furthermore, that such agents should be ancestral to animals. This is, however, a non sequitur: why not horizontal transfer of a viroid from plants to mammals, e.g. via an insect vector? Actually, given the rarity of viroid-like agents in animals, I find this to be the most likely scenario. So what is the origin of viroids? We have no way of knowing but I think alternative scenarios that are more credible than the primordial one are not too difficult to conjure. In my brief discussion of such possibilities, I would like to first quote Diener: “Nevertheless, viroids seem to recapitulate the principle features of the selfish elements from the ancient RNA world”— thus leading to an internal contradiction, in that by one criterion (molecular properties), viroids “strikingly” appear to be descendants of primordial RNAs, whereas by another criterion (apparent evolutionary age), they clearly are not. To decide between them, I have conceived a new hypothesis.’ . In this argument, there is a strong hidden assumption: ‘resembling’ = ‘descendants of’. Is this assumption valid? Definitely, viroids resemble primordial RNA genomes in some of the basic properties. Does this imply that the former actually descend from the latter? This depends on the complexity of the entity involved. In the case of viroids, the answer, I believe, is: not at all. As Diener correctly points out, citing the seminal work of Szathmary and Maynard Smith (Ref. 19), parasites inevitably evolve in any replicator system. Moreover, a universal trend in the evolution of parasites, with examples abounding, from viruses to parasitic worms, is genome reduction. There is one that might be especially relevant to the origin of viroids and hence worth citing, namely the classic early experiments of Spiegelman and colleagues on the in vitro evolution of RNA bacteriophages. In these serial transfer experiments, where phage encoded proteins did not contribute to genome replication given that the replicase was provided externally, the size of the genome rapidly dropped about 10-fold. What remained, was a minimal replicon, a RNA molecule of approximately 400 nucleotides, resembling viroids in size and the extent of secondary structure :213-253; Mills DR, Kramer FR, Spiegelman S. Complete nucleotide sequence of a replicating RNA molecule. Science. 1973 Jun 1;180(4089):916-927). In other words, under the appropriate selective pressure, the RNA virus genome readily evolved into a small non-coding RNA that effectively retained only the signals for the replicase recognition. These experiments might serve as a model of the evolutionary processes that led to the emergence of viroids. The key intermediate could be satellite RNAs of plant viruses of so-called group 3 which are viroid-like circular RNA molecules that replicate via a rolling circle mechanism similar to that employed by viroids but utilizing the RNA-dependent RNA polymerase of the host virus . Such satellite RNAs could have evolved from the viral RNA by genome reduction, losing the protein-coding genes, in a path mimicking Spiegelman’s experiments. Clearly, under this scenario, the signals for replicase recognition were already present in the ancestral RNA, so the evolution of the viroid-like satellite would only require the acquisition of a hammerhead ribozyme required for the circular RNA replication. Alternatively, both viroids and satellite RNAs could have evolved from small host RNA, for example, intronic ones, which would not involve the loss of protein-coding genes but would require acquisition of both the replicase recognition signals and the ribozyme. Importantly, it has been shown that the simple hammerhead ribozyme can easily evolve on multiple occasions :82-84). Thus, the path to the de novo evolution of viroids seems to be rather straightforward. Does the above argument falsify the primordial hypothesis? Certainly, not. To do so, it would be necessary to discover evolutionary intermediates of the de novo route of evolution. This seems to be a possibility but so far, there is no evidence of such intermediate forms. Moreover, it is easy to imagine new findings that would favor the primordial hypothesis, in particular, discovery of viroid-like agents in Cyanobacteria. However, in the absence of such evidence, I find the hypothesis of in planta origin of viroids to be more plausible that the primordial scenario. My position with regard to the hypotheses presented in the paper is discussed above. The author might want to cite some references where the hypothesis on the primordial origin of viroids is discussed and actually positively assessed. The claim in the current version of the manuscript, that this hypothesis had been ignored for decades, is inaccurate. This is particularly strange because the author fails to acknowledge the credit given by others to his own ideas. The use of unpublished data of other researchers is problematic (even though a permission is mentioned). I wonder if there is by now any publication, preprint or at least abstract that could be cited. In the very least, a formal acknowledgement is necessary. The reference list was apparently prepared manually and contains some errors and typos: Ref. 4 - page range cannot be correct Ref. 6 - year missing Ref. 8 - typo Ref. 10 - Mimivirus not Minivirus On several occasions, ‘hospital’ is used where it should be ‘hospitable’.Response to Dr. Koonin’s review of my manuscriptI wish to express my deep gratitude to Dr. Koonin for honoring me with this very detailed, scientifically most informative and, last but not least, beautifully organized and written review, which stands by itself as a well documented essay in its own right. Needless to state, I shall not try to describe Dr. Koonin’s brilliant hypothesis of de novo origin of viroids in my revised manuscript, but only to refer the reader to its description in his review of my original manuscript—hopefully expecting, its timely publication.But now to Dr. Koonin’s suggestions for improving my manuscript. Most importantly, I have deleted hypothesis 2 and all references to Pelchat’s preliminary results. Also, I assume a neutral attitude between the old primordial RNA and the new de novo origin theory, now that Dr.Koonin has convinced me that the former is probably false.I am sorry, if I have failed to cite important, relevant publications. which may have been due to my relying on PubMed to keep me up-to-date, but have now learned that this source is not infallible. Thus, for example, PubMed failed to cite the one early effort to obtain more convincing arguments for the primordial RNA hypothesis, written by Cheles-Flores in1994 (Refs. 12 and 13). I have checked all 132 papers in Google Scholar, which cited my 1989 hypothesis paper. I determined that 34 were from the Ricardo Flores’ group, today arguably the foremost viroid-investigating group in existence , 16 concerned Hepatitis Delta Virus, 76 were assorted reviews and papers not otherwise involved with viroid origin, and only 6 touched directly on viroid origin—5 of which I had already cited, leaving only the Cheles-Flores papers.Theodor O. Diener


RNA molandscape . Studyinandscape . A challandscape  but the Projecting information from a 3D volume into a single image plane, for example, using astigmatism, double helix spiral phase microscopy or similar Fourier-space engineered approaches .rd dimension  [In order to reliably identify and track individual RNA molecules, the image reconstruction from the acquired 3D snapshots is critical and non-trivial . We solvial - z) . The new-actin mRNA [-actin mRNAs are able to access every part of the nucleus freely and that the transcripts were not excluded from heterochromatin-rich regions. Furthermore, most mRNAs were located within 0.5 μm of a nuclear pore, caused by the nucleus' disc-like shape which can only be observed in 3D images.The 3D-SMRT is a powerful tool to answer fundamental questions that cannot be addressed by conventional microscopy, such as visualizing various RNA regulatory processes in living cells. In our work, we imaged the nuclei of live mouse fibroblasts expressing fluorescently labeled nuclear pores and βtin mRNA . We foun+D microscopy will enable us to define the rules that govern the kinetics, locations, and interactions of proteins and nuclear acids that might be exploited for the treatment of human disease.Going forward, we use 3D-SMRT to study how dynamic movements of nuclear products like RNA relate to the nuclear structure and function. One prominent reporter system is the HIV-1 viral genome. HIV-1 RNAs have a minimal set of proteins that are crucial to its function , but it


The genomes of large multicellular eukaryotes are mostly comprised of non-protein coding DNA. Although there has been much agreement that a small fraction of these genomes has important biological functions, there has been much debate as to whether the rest contributes to development and/or homeostasis. Much of the speculation has centered on the genomic regions that are transcribed into RNA at some low level. Unfortunately these RNAs have been arbitrarily assigned various names, such as “intergenic RNA,” “long non-coding RNAs” etc., which have led to some confusion in the field. Many researchers believe that these transcripts represent a vast, unchartered world of functional non-coding RNAs (ncRNAs), simply because they exist. However, there are reasons to question this Panglossian view because it ignores our current understanding of how evolution shapes eukaryotic genomes and how the gene expression machinery works in eukaryotic cells. Although there are undoubtedly many more functional ncRNAs yet to be discovered and characterized, it is also likely that many of these transcripts are simply junk. Here, we discuss how to determine whether any given ncRNA has a function. Importantly, we advocate that in the absence of any such data, the appropriate null hypothesis is that the RNA in question is junk. Starting with the discovery of transfer RNA and ribosomal RNA in the 1950s, non-coding RNAs (ncRNAs) with biological roles have been known for close to 60 years. Even in the late 1970s and early 1980s the existence of other functional ncRNAs was known, including RNAse P , snRNAs At present, the distinction between functional ncRNAs and junk RNA appears to be quite vague. There has been, however, some effort to differentiate between these two groups, based on various criteria ranging from their expression levels and splicing to conservation. Ultimately these efforts have failed to bring consensus to the field.A similar problem has plagued the investigation of whether transposable elements (TEs), which make up a significant proportion of most vertebrate genomes, have been exapted for the benefit of the host organism. Although some have claimed that many TEs are functional, a few groups have offered a much more balanced view that is in line with our current understanding of molecular evolution .In this article we explain several concepts that researchers must keep in mind when evaluating whether a given ncRNA has a function at the organismal level. Importantly, the presence of low abundant non-functional transcripts is entirely consistent with our current understanding of how eukaryotic gene expression works and how the eukaryotic genome is shaped by evolution. With this in mind, researchers should take the approach that an uncharacterized non-coding RNA likely has no function, unless proven otherwise. This is the null hypothesis. If a given ncRNA has supplementary attributes that would not be expected to be found in junk RNA, then this would provide some evidence that this transcript may be functional.types of transcripts with their abundance in a typical cell. Many of the putative functional ncRNAs are present at very low levels and thus unlikely to be of any importance with respect to cell or organismal physiology. Importantly, the abundance of an ncRNA species roughly correlates with its level of conservation . Although there is less tRNA by mass, their small size results in their molar level being higher than rRNA . Other abundant RNAs, such as mRNA, snRNA, and snoRNAs are present in aggregate at levels that are about 1–2 orders of magnitude lower than rRNA and tRNA . Certain small RNAs, such as miRNA and piRNAs can be present at very high levels; however, this appears to be cell type dependent.At steady state, the vast majority of human cellular RNA consists of rRNA . Although the estimated number of different types of human lncRNAs has ranged from 5,400 to 53,000 (Table 2), only a small fraction have been found to be present at levels high enough to suggest that they have a function. According to ENCODE’s own estimates, fewer than 1,000 lncRNAs are present at greater than one copy per cell in the typical human tissue culture cell line . As a whole, these are present at levels that are two orders of magnitude less than total mRNA . These are thought to act as scaffolds that regulate the 3D architecture of chromosomes in the vicinity of their transcription site . eRNAs aTable 1).There are still other more exotic species of RNAs , includiIn addition to all of the mentioned species, ENCODE and other groups have found transcripts that map to the rest of the genome termed “intergenic RNA” . Most ofRegardless of these concerns, it is clear that most of the ncRNAs in question  are typically present at very low levels when compared to known functional RNAs. These observations are consistent with the idea that the eukaryotic genome produces a vast amount of spurious transcripts.1 (Table 2). These would originate from <1% of the human genome. Needless to say, this is a very small fraction of the total. Even if we compiled all of the putative lncRNAs using the most optimistic analysis (Table 2). Thus far, only a small minority of lncRNAs have been shown to be important for organismal development, cell physiology, and/or homeostasis. As of December 2014, the LncRNA Database2, a repository of lncRNAs “curated from evidence supported by the literature,” lists only 166 biologically validated lncRNAs in humans  arise from only a minute fraction of the genome. Again it appears that the vast majority of the genome that falls outside of these loci is transcribed into junk RNA that is present at very low levels at steady state.in vivo . Therefore if a hypothetical ncRNA were present at 10 copies per cell at steady state, they would increase the pool of intergenic/lncRNAs by 0.1%, and would increase the total pool of RNA by a negligible amount .First the mere presence of the ncRNA may act as a burden. The typical mammalian tissue culture cell has on the order of 500,000 mRNA molecules. Other RNAs with unknown function  are at levels between 1 and 4% those of mRNA  and thusSecond, there is a cost to synthesizing the RNA. One study that investigated the energetics of synthesizing long introns has estimated that for an mRNA that is expressed at a level of 30 copies per cell and a half-life of 1 h (resulting in the generation of 360 new RNA molecules/cell per day), an intron would have to be roughly 83,000 nucleotides long for it to be a significant burden, given the effective population size of humans . Using tThird, the ncRNA may have some associated activity that may be deleterious. Most often the major concern is whether it will be translated into short random peptides . Although ncRNAs are poorly translated, most studies have found that they can be engaged by the ribosome at low levels . This caWe might imagine that our putative non-functional RNA was transcribed due to the fortuitous action of one or more TF binding events. As described above, it is likely that many such sites exist in the mammalian genome as the number of active transcriptional start sites exceed the number of protein-coding genes by an order of magnitude . Since tIn determining how our putative non-functional RNA would be distributed intracellularly, there are several facts to take into account. First, if this RNA were to be exported to the cytoplasm, it is reasonable to believe that it would be a substrate for the translational machinery, as long as the RNA is free of extensive secondary structures. Second, this RNA would be translated into a random polypeptide. Unlike nucleic acids, unstructured polypeptides have a high tendency to aggregate and activate cellular stress . Lastly,From this discussion it makes sense that our non-functional ncRNA would be nuclear, but what about its localization to a specific sub-nuclear compartment? Again, some have used localization to sub-nuclear loci as proof of functionality . In expeWe would expect that the non-functional ncRNA would lack strong processing signals, as these regions would be expected to be under strong purifying selection only in functional spliced transcripts. For example in most mRNAs, introns are not only flanked by splicing donor and acceptor sites but are also defined by location of intronic and exonic splicing elements . HoweverPolyadenylation signals are also likely to be present in our putative junk RNA. These sites are quite abundant – to the extent that many of these sites are present in introns but are normally suppressed by the action of the spliceosome. These cryptic 3′cleavage sites become quite heavily used in cells with reduced U1 snRNA levels . As withCertain groups, such as the HUGO Gene Nomenclature Committee , have deThe important distinction between functional and non-functional RNAs is that processing signals are under a high level of selection pressure in the former but not in the latter. Thus, although non-functional ncRNAs may be processed, they will likely have weak signals. Interestingly, introns in lncRNAs tend to be spliced post-transcriptionally, while those in mRNAs tend to be removed co-transcriptionally . This maIt has been demonstrated for the last 50 years that sequence conservation is a reliable indicator of function. In line with this thinking, many commentators have declared that conservation should be the only criterion for identifying functional genomic loci . In agreThere are, however, various circumstances that may give the appearance of conservation. For example, the transcribed loci may also contain some conserved functional element, such as a critical TF binding site. If the region of conservation is confined to a pseudogene or TE sequences, one may simply be detecting these entities, which are typically non-functional.Saccharomyces cerevisiae genome , plays some critical role.As stated above, certain commentators have championed selection as the primary arbiter of whether a genomic locus is functional. These same individuals have dismissed any evidence that is based on causal roles, which is defined as “the way(s) in which a component contributes to a stated capacity of some predefined system of which it is a part: what it in fact does” . The proIn light of these problems, the question clearly becomes, can a non-functional ncRNA be distinguished from one that is functional, simply on the basis of an experiment that demonstrates a “causal role”? In our opinion the answer is yes, as long as the appropriate causal role is chosen. By definition, elimination of functional ncRNAs should affect homeostasis, development or other important biological processes that would impact the fitness of the organism. In contrast, other causal role events that could potentially be associated with non-functional ncRNAs would be insufficient to qualify as evidence of functionality.There are some problems with relying on causal roles to determine function, in that it is not always clear whether an activity could occur by chance with an RNA with a random sequence. For example, if the overexpression of an ncRNA promotes oncogenesis, would this provide evidence of functionality? This hypothetical ncRNA could simply be sequestering an RNA binding protein that has a pro-apoptotic function, and in this instance this type of evidence would be weak. If, on the other hand, the ncRNA in question acted as a ribozyme that generated free radicals which caused DNA damage, this would then be much stronger evidence, as this activity would not be expected from a random RNA. Other evidence, such as the association of lncRNAs with certain protein complexes , seems Ultimately, the ideal experiment is to determine whether the elimination of an ncRNA affects a biological process that is required for the proper development or homeostasis of the organism. This has become more feasible with the advent of CRISPR/Cas9 technology . One serprovisionally labeled as non-functional. Subsequently, if the ncRNA displays features/activities beyond what one would expect for the null hypothesis, then we can reclassify the ncRNA in question as being functional.To date, projects such as ENCODE, LNCipedia and the HUGO Gene Nomenclature Committee have distinguished lncRNAs from junk RNA primarily based on expression levels and RNA-processing. In contrast, we believe that researchers need to evaluate whether any putative functional ncRNAs have properties that are beyond what one would expect from a non-functional ncRNA, given our knowledge of biochemistry, genomic evolution and current empirical data. Evidence for function can consist of expression levels that are very high , a high degree of conservation, and/or experimental evidence that the ncRNA is required for some important biological process. Importantly, ncRNAs should be evaluated on a case-by-case basis. In the absence of sufficient evidence, a given ncRNA should be It is clear that the human genome contains a large number of functional ncRNAs. Indeed it is likely that the list of biologically validated ncRNAs, as listed in the LncRNA Database , will coThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Results displayed a mix of deleted and full-length coxsackievirus B3, characterized by a low viral RNA load (8.10 Enteroviruses (EVs) are common human pathogens that are small, naked, single-stranded, positive-sense RNA viruses of ≈7,400 nt. Their genome is flanked on the 5′ end by a noncoding region (NCR), which is crucial for the initiation of the replication and translation of the virus genome  that began 1 month before. Transthoracic echocardiogram revealed a decreased left ventricle ejection fraction (LVEF) (45%) and a dilated left ventricle with a telediastolic diameter of 36 mm/m2. Coronary artery disease was ruled out by coronarography; no other etiologic causes of DCM were indicated by heart magnetic resonance imaging  to RNA negative-sense (RNA–) ratio value of 4.8  according to the manufacturer’s instructions. Real-time quantitative reverse transcription PCR was performed for quantitative detection of total and strand-specific EV-genus genomic RNA according to previously published protocols ; nucleotide and amino acid sequence homologies were 79% and 92%, respectively, with the CV-B3 Nancy strain (GenBank accession no. JX312064.1)  PCR followed by an in-house NGS strategy. RNA-backboned oligonucleotides (trP1 and trP1c) were linked on 5′ extremity of EV RNA+ and 3′ extremity of RNA–, respectively . A retroOur results estimated a major proportion (84.8%) of reads presenting with a terminal 48-nt deletion associated with minor proportions of reads deleted of 15 nt (14.3%) and nondeleted (0.9%) . BecauseOur study demonstrated the existence of major 5′ terminally deleted CV-B3 populations in endomyocardial tissues taken from a patient with IDCM at the time of her clinical diagnosis. We observed low viral RNA load values associated with a low viral RNA+ to RNA– ratio (<5), indicating that these viral forms were compatible with persistent endomyocardial EV-B populations (One previous study reported a detection of 5′ terminally deleted CV-B2 populations ranging in size from 22 to 36 nt in cardiac tissues taken from an immune competent patient in Japan who died of fulminant myocarditis . These dThe clinical data associated with our molecular results argued for a CV-B3–induced DCM stage that developed several years after an undiagnosed clinical CV-B3–related myocarditis event. Taken together with the unique published CV-B2–induced myocarditis case, our findings support the hypothesis that the emergence and the selection of terminally deleted EV-B forms could occur during the early acute viral replication phase and might explain the pathophysiological progression from acute viral myocarditis to the DCM phase, in which cardiac persistent terminally deleted virus populations would be advantaged (Schematic representation of the next-generation sequencing library generation steps performed on total nucleic acid extracted from a flash-frozen cardiac tissue of a patient with idiopathic dilated cardiomyopathy, Reims, France, September 2011.


ETV6 (12p13) are among the most common structural abnormalities in pediatric B-cell acute lymphoblastic leukemia (B-ALL) and involve numerous partner genes. Additionally, the t, which can result in the placement of CEBPD (8q11.2) near the regulatory regions of IGH@ (14q32) and consequent overexpression of CEPBD, occurs at a higher frequency in individuals with Down syndrome-associated ALL (DS-ALL) compared to both the general and pediatric population. The coexistence of cytogenetically detectable ETV6 abnormalities and t is a rare occurrence in B-ALL and has only been reported in a single case in the literature.Rearrangements involving IGH@ and ETV6 in these translocations, respectively. Additionally, interphase FISH revealed a clonal subpopulation bearing biallelic IGH@ rearrangements not observed by conventional cytogenetic analysis.Herein, we present a case of B-ALL in a 9-year old male with Down syndrome in which conventional cytogenetic analysis revealed two reciprocal translocations: a t and a t. Interphase and metaphase fluorescence in situ hybridization (FISH) analysis using break apart probes confirmed the involvement of ETV6 translocation with a partner gene on the short arm of chromosome 2 confirmed by FISH. Additionally, it is the second reported case of t-ALL bearing a concomitant, cytogenetically detectable abnormality involving ETV6. This case provides insight into a novel translocation involving ETV6 as well as potentially unique and understudied mechanisms of clonal evolution in pediatric B-ALL.To the best of our knowledge, this is the first reported case of B-ALL bearing an  ETV6/RUNX1], hyperdiploidy, t [PBX1/TCF3], t [AFF1/MLL], hypodiploidy, and t [BCR/ABL1]  is the most common of these rearrangements, other translocations involving ETV6 with greater than 20 partner genes have been observed, including protein tyrosine kinases and transcription factors, many of which can act by distinct mechanisms to promote leukemogenesis .ETV6 in 50.7 % (152/300) of the nuclei examined, suggesting a potential ETV6 (12p13) gene rearrangement , consistent with the constitutional genetic makeup of this patient (Down syndrome). FISH on previously G-banded metaphase spreads using the Vysis LSI ETV6 Dual Color, Break Apart Probe Kit revealed a fusion signal (F) corresponding to an intact ETV6 gene on the normal chromosome 12 and a split signal (1R1G) corresponding to a rearranged ETV6 gene, with a red signal (5′/telomeric region of ETV6) on the derivative chromosome 2 and a green signal (3′/centromeric region of ETV6) on the derivative chromosome 12, ultimately confirming the t observed by conventional cytogenetic analysis , and the latter suggests rearrangements of both IGH@ genes . FISH on previously G-banded metaphase spreads also revealed the former signal pattern (1F1R1G) consisting of a fusion signal and a split signal (1F1R1G), with the fusion signal corresponding to an intact IGH@ gene on the normal chromosome 14, the red signal (3′/telomeric region of the IGH@ gene) on the derivative chromosome 8, and the green signal (5′/centromeric region of the IGH@ gene) on the derivative chromosome 14, ultimately confirming the t observed by conventional cytogenetic analysis x3[152/300]/[148/300].x2)(3′IGH@ sep 5′IGH@x1)[269/300]/(IGH@x2)(3′IGH@ sep 5’IGH@x2)[20/300]nuc ishnuc ish(MLLx2)[300]nuc ish(PDGFRBx2)[300]nuc ish 4cen(CEP4x2)[300]nuc ish10cen(CEP10x2)[300]IGH@ gene and the CEBPD gene, placing CEBPD in close proximity to the regulatory regions of IGH@ and resulting in its overexpression [The cytogenetic findings in this case highlight a unique combination of rearrangements that has not been previously described in B-ALL. The t8;14)q11.2;q32) is a recurrent translocation in B-ALL that generally causes exchange of the regulatory regions of the pression . Althoug;q32 is a;14q11.2;pression . It has IGK (2p12) and CCND2 (12p13) genes, and results in overexpression of CCND2 due to the placement of the CCND2 gene near the regulatory regions of the IGK gene [ETV6 gene, which is also located at the 12p13 band, confirmed that the t we observed involves the ETV6 gene, and not CCND2. Additionally, since both ETV6 and CCND2 localize to 12p13, we confirmed that the ETV6 break apart probe  we utilized in our analysis bears no overlap with the CCND2 gene, minimizing the possibility of a false-positive result. The break apart probe we utilized spans chr12: 11321260–12578058, whereas the CCND2 gene spans chr12: 4382902–4414522. We queried the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer for cases of B-ALL bearing a t and were only able to identify seven cases in addition to our case, indicating that it is an extremely rare cytogenetic abnormality in B-ALL  with ETV6 involvement confirmed by molecular analysis. Of note, each of the previously reported cases and our case occurred in pediatric patients, and five out of eight of the cases carried chromosome 21 abnormalities, including constitutional and/or acquired +21 and i(21q).The t2;12) is a recurrent, albeit rare, translocation in cyclin D1-negative mantle cell lymphoma (MCL). In these cases, it involves the IGK gene . However2 on the short arm of chromosome 12 [ETV6 gene material, confirming the amplification, and immunohistochemical analysis confirmed overexpression of ETV6 compared to case-matched controls [ETV6 that are known or predicted to result in its loss of function were observed in these cases. These two cases provide evidence against the hypothesis that ETV6 functions exclusively as a tumor suppressor gene and suggests that in certain contexts, it can function as an oncogene via its overexpression, a rare phenomenon that has been observed in other genes such as the Wilms tumor 1 (WT1) gene [CCND2 due to placement of the gene near the regulatory regions of IGK, it is possible that in our case, a similar mechanism could result in overexpression of ETV6 in the context of this translocation due to the placement of ETV6 near the regulatory regions of IGK. Additionally, Lu et al. reported the first case of B-ALL bearing a t involving ETV6 and IGH@ confirmed by metaphase FISH using probes specific to both genes, showing that translocations involving ETV6 and immunoglobulin genes can occur in B-cell neoplasias [ETV6 in the evolution of this malignancy.However, two reported cases – one case of myelodysplastic syndrome and one case of B-ALL – showed amplification of the osome 12 , 5. In bcontrols , 5. FurtT1) gene . Becausetp;q32 invoIGH@ and ETV6 genes and the variation in the frequencies of these abnormalities as detected by interphase FISH provides evidence for an underlying cytogenetic evolution in the development of this malignancy. First of all, clones bearing a monoallelic IGH@ rearrangement, evidenced by a 1F1R1G signal pattern by interphase FISH using an IGH@ break apart probe, were observed in 89.7 % of interphase nuclei analyzed. Conversely, only 6.7 % of cells showed evidence of biallelic IGH@ rearrangements (2R2G) using the same probe and 50.7 % of interphase nuclei showed evidence of an ETV6 rearrangement when analyzed with a dual color, dual fusion translocation probe, suggesting that a monoallelic IGH@ rearrangement, caused by t, was the primary cytogenetic abnormality in this malignancy. Because we were not able to detect biallelic 14q32 rearrangements by conventional cytogenetic analysis but were able to observe both a monoallelic t and t in the same metaphase spread, the evolution of this malignancy likely happened via one of two mechanisms: (1) acquisition of a primary t, followed by t in the same clone, followed by an additional IGH@ rearrangement in the same clone or (2) acquisition of a primary t, followed by t in one clone and an additional IGH@ rearrangement in a different clone  and an additional IGH@ rearrangement, but not a t. It is important to note that because we did not observe biallelic 14q32 rearrangements in any metaphase spreads by conventional cytogenetic analysis or metaphase FISH, the additional IGH@ rearrangement could be due to an additional t or an IGH@ rearrangement involving a different gene or locus. Due to the relatively low frequency of the clone bearing biallelic IGH@ rearrangements, it is more likely that this malignancy evolved via the former, linear mechanism, although both possibilities must be considered.The variation of abnormalities of the et al., was found to bear a concomitant cytogenetic abnormality involving the 12p13 locus, described as follows: 47, XX, t, del(12)(p12p13),+21c. Further FISH studies confirmed both a monoallelic IGH@ rearrangement and a monoallelic ETV6 deletion [ETV6 in the evolution of t-ALL is an extremely rare occurrence that presents a number of questions regarding molecular mechanisms and clinical implications that remain understudied. We emphasize the importance of utilizing both conventional cytogenetic and molecular genetic tools to elucidate relevant abnormalities and molecular mechanisms in such cases, ultimately to determine the clinical implications of rare cytogenetic abnormalities in pediatric ALL such as those presented in this case. In summary, this case provides insight into a novel translocation involving ETV6 as well as potentially unique and understudied mechanisms of clonal evolution in pediatric B-ALL that warrant further investigation.Among all reported cases of t8;14)-ALL, only one case, reported by Harrison (q11.2; q


Caenorhabditis elegans hermaphrodite, the stereotyped migration pattern of two somatic distal tip cells (DTCs) is responsible for shaping the gonad. Guidance receptor UNC-5 is necessary for the dorsalward migration of DTCs. We found that BLMP-1, similar to the mammalian zinc finger transcription repressor Blimp-1/PRDI-BF1, prevents precocious dorsalward turning by inhibiting precocious unc-5 transcription and is only expressed in DTCs before they make the dorsalward turn. Constitutive expression of blmp-1 when BLMP-1 would normally disappear delays unc-5 transcription and causes turn retardation, demonstrating the functional significance of blmp-1 down-regulation. Correct timing of BLMP-1 down-regulation is redundantly regulated by heterochronic genes daf-12, lin-29, and dre-1, which regulate the temporal fates of various tissues. DAF-12, a steroid hormone receptor, and LIN-29, a zinc finger transcription factor, repress blmp-1 transcription, while DRE-1, the F-Box protein of an SCF ubiquitin ligase complex, binds to BLMP-1 and promotes its degradation. We have therefore identified a gene circuit that integrates the temporal and spatial signals and coordinates with overall development of the organism to direct cell migration during organogenesis. The tumor suppressor gene product FBXO11 (human DRE-1 ortholog) also binds to PRDI-BF1 in human cell cultures. Our data suggest evolutionary conservation of these interactions and underscore the importance of DRE-1/FBXO11-mediated BLMP-1/PRDI-BF1 degradation in cellular state transitions during metazoan development.Spatiotemporal regulation of cell migration is crucial for animal development and organogenesis. Compared to spatial signals, little is known about temporal signals and the mechanisms integrating the two. In the  C. elegans gonad. How the spatiotemporal migration pattern is regulated is not clear. We identified a conserved transcription factor BLMP-1 as a central component of a gene regulatory circuit required for the spatiotemporal control of DTC migration. BLMP-1 levels regulate the timing of the DTC dorsal turn, as high levels delay the turn and low levels result in an early turn. We identify and characterize upstream regulators that control BLMP-1 levels. These regulators function in two ways, i.e. by destabilization of BLMP-1 through ubiquitin-mediated proteolysis and by transcriptional repression of the blmp-1 gene to down-regulate BLMP-1. Interestingly, blmp-1 also negatively controls these regulators. Our data suggest that a dietary signal input acts together with a double-negative feedback loop to switch DTCs from the “blmp-1-on” to the “blmp-1-off” state, promoting their dorsal turn. Furthermore, we show that some protein interactions in the circuit are conserved in C. elegans and humans. Our work defines a novel function of the conserved blmp-1 gene in the temporal control of cell migration, and establishes a gene regulatory circuit that integrates the temporal and spatial inputs to direct cell migration during organogenesis.The migratory path of DTCs determines the shape of the  Cell migration is important for organogenesis and development of animals. Numerous extracellular guidance cues and receptors for the spatial control of cell migration have been identified and characterized C. elegans hermaphrodites develops from a four-cell primordium positioned in the ventral midbody in vivodaf-12, dre-1, and lin-29lin-29, daf-12, and dre-1 encode, respectively, a zinc-finger transcription factor, a steroid hormone receptor similar to the vertebrate vitamin D and liver-X receptor, and an F-Box protein of an SCF ubiquitin ligase complex The bilobed gonad of unc-40 appears to be transcribed in the DTCs throughout their migration unc-5 is transcriptionally up-regulated at the time when the dorsal turn is initiated unc-5 cDNA at the time when DTCs have not yet turned dorsalward induces unc-6-dependent precocious dorsal migration unc-5 is both necessary and sufficient for DTC dorsal migration and that the increase in unc-5 transcription is responsible, at least in part, for the initiation of DTC dorsal migration unc-5 is temporally regulated to direct dorsal migration precisely at the late L3 stage is unclear.The dorsal migration of DTCs is regulated by the guidance receptors UNC-5 and UNC-40  blmp-1 mutants. Our results showed that blmp-1 is a heterochronic gene that acts with daf-12, dre-1, and lin-29 in a regulatory circuit to control the correct timing of DTC migration. We also showed that this regulatory circuit is, at least in part, conserved in C. elegans and humans.In this study, we performed genetic screening for mutants defective in DTC migration and isolated tp5 and tk41. A genetic complementation test showed that tp5 and tk41 were allelic, and that either allele failed to complement the previously identified mutation dpy-24(s71)  To identify genes that are important for the spatiotemporal regulation of DTC migration, we performed genetic screening for mutants defective in DTC migration, as described in the dpy-24(s71) to chromosome I, near stp124, by sequence tag site (STS) mapping unc-40 and unc-75 and subsequent SNP mapping positioned dpy-24(s71) within the region between cosmids F45H11 and F37D6. Cosmids covering this region were microinjected into the dpy-24(s71) mutant, and cosmid F25D7 rescued the DTC migration defect (dpy-24(s71) mutant and only F25D7.3, which contained a single blmp-1 gene, had a rescue effect (dpy-24(s71) mutant  We mapped n defect . The gene effect . In addi) mutant . These rblmp-1 gene encodes a protein with 27% identity to mouse B lymphocyte-induced maturation protein 1 (Blimp-1) and 26% identity to human positive regulatory domain I-binding factor (PRDI-BF1) (The RDI-BF1) . Both Bls71, tk41, and tp5 have, respectively, a non-sense mutation in codon 281, 381, or 434, and are predicted to encode truncated proteins without zinc fingers , has an 810 bp deletion, removing part of exon 3 and part of intron 3  in blmp-1(s71) in blmp-1 genomic DNA rescued the dumpy phenotype and embryonic lethality of the blmp-1(s71) mutant (blmp-1.The four ) mutant , showingblmp-1 mutant DTCs did not migrate significantly slower than the wild-type DTCs  mutant, using the division stages of the vulval precursor cell P6.p as temporal developmental markers, as described previously blmp-1(s71)  posterior gonadal arms in early L3 (top panels) and late L3 (bottom panels). s71 mutant, the anterior DTCs in 36% of worms and the posterior DTCs in 66% of worms had turned dorsalward before P6.p divided. These results demonstrate that loss of blmp-1 causes a precocious initiation of DTC dorsal migration  and that blmp-1 functions to prevent a precocious DTC dorsal turn.To examine whether the abnormal DTC migration pattern of the blmp-1 mutations , which are loss-of-function recessive alleles by genetic tests  blmp-1, unc-5 and unc-40 function cell-autonomously in the control of DTC migration blmp-1 may prevent DTC precocious dorsalward turning by regulating unc-5 and/or unc-40. unc-40 appears to be transcribed in the DTCs throughout their migration unc-5 is transcriptionally up-regulated at the time when the dorsal turn is initiated unc-5(1 kb) gfpP, in which an approximately 1 kb sequence upstream of the unc-5 coding sequence was used to drive the GFP reporter, we confirmed this unc-5 expression pattern, i.e. unc-5 was expressed during and after, but not before, the DTC dorsal turn ::gfpP transgene (blmp-1(s71) mutants, DTCs at the same stage had performed the dorsal turn and displayed precocious unc-5 transcription  mutant (blmp-1 (s71); unc-5 double mutant, showing that the unc-5(e53) mutation blocked the precocious dorsal turn phenotype of the blmp-1 (s71) mutant and that the blmp-1 precocious dorsal turn phenotype required unc-5.Next, we tested whether the ) mutant . No precblmp-1 down-regulation and its effect on the initiation of DTC dorsalward turning. To this end, we overexpressed blmp-1 using the transgene blmp-1::blmp-1::gfpP, in which the fusion protein BLMP-1::GFP was expressed under the control of the promoter blmp-1P. In the resulting transgenic line, 10% of transgenic worms had DTCs that displayed normal BLMP-1::GFP down-regulation, underwent a normal dorsal turn ::mCherryP reporter. In the resulting transgenic line, 10% of transgenic worms had DTCs that displayed normal BLMP-1::GFP down-regulation, underwent a normal dorsal turn, and showed unc-5(1 kbP)::mCherry expression ; blmp-1(s71) double mutant was very sick and could not be maintained as homozygote, we used viable alleles and/or RNAi for dre-1, blmp-1 and lin-29 to analyze the genetic interactions of these genes. The precocious phenotype of the blmp-1(s71) mutant was partially suppressed by a single mutation of lin-29, dre-1, or daf-12 or combined mutations of any 2 or all 3 (blmp-1(s71) mutants had a precocious DTC migration defect, whereas 54% of the blmp-1(s71); lin-29(RNAi) double mutants, 73% of the blmp-1(s71); dre-1(dh99) double mutants, and 31% of the blmp-1(s71);daf-12(rh61rh41) double mutants displayed a precocious turn defect. This suggests that the blmp-1 precocious phenotype may require the activity of lin-29, dre-1, or daf-12, and that blmp-1 may function upstream of, or in parallel with, lin-29, dre-1, and daf-12 to prevent a precocious DTC dorsal turn during early larval development. Conversely, the blmp-1 mutation also partially suppressed the retarded dorsal migration of the double or triple mutants of lin-29, dre-1, and daf-12 (dre-1(dh99); daf-12(rh61rh411) double mutants showed retarded DTC migration, whereas 12%, 73%, and 15% of the blmp-1(71); dre-1(dh99); daf-12(rh61rh411) triple mutants showed, respectively, wild-type, precocious, or retarded DTC migration. This suggests that the retarded phenotype of the lin-29, dre-1 and daf-12 double and triple mutants may require blmp-1 activity and that lin-29, dre-1, and daf-12 may function upstream of, or in parallel with, blmp-1 to prevent a delay in the DTC dorsal turn during the late larval stage. Thus, two distinct regulatory hierarchies in these heterochronic genes are likely employed to specify the temporal identities of DTCs at the early and late larval stages, and the switch from the “blmp-1-on” state to the “blmp-1-off” state during developmental progression may determine the timing of the DTC dorsal turn.The heterochronic genes or all 3 . For exad daf-12 . For exadaf-12, dre-1, or lin-29 partially suppressed the blmp-1-related precocious DTC dorsal migration defect , the modENCODE Consortium has shown that BLMP-1 binds to the upstream sequence of lin-29, but not that of daf-12 or dre-1, at the early larval stage lin-29 expression. To test this possibility, we analyzed and compared lin-29 transcription in the wild-type and blmp-1 mutants using the transcriptional reporter lin-29::gfpP, in which gfp was expressed under the control of the lin-29 promoter lin-29P ; lin-29(n546) (100% of 30 worms scored), dre-1(dh99); daf-12(rh61rh411) (20% of 60 scored), and lin-29(RNAi); daf-12(rh61rh411) (∼5% of 106 scored) (examples shown in lin-29(RNAi); daf-12(rh61rh411) double mutant than in the other double mutants and bleached too quickly to be photographed by our imaging system. The daf-12(rh61rh411) allele is molecular null, while lin-29(n546) and dre-1(dh99) are partial loss-of-function As mentioned above, in the wild-type control, BLMP-1 was not detected in DTCs at the L4 stage  and a silin-29, daf-29, and dre-1, we used two gfp reporters in an in vivo expression assay to examine whether blmp-1 expression was repressed at the transcriptional level by transcription factors LIN-29 and DAF-12 and at the post-transcriptional level by the F-Box protein DRE-1. We first generated a transcriptional reporter blmp-1::dgfpP (destabilized GFP) was controlled by the blmp-1P promoter. dGFP contains a PEST sequence and has a shorter half-life than normal GFP blmp-1::dgfpP transgene, the DTCs of 75.6% of worms expressed GFP in the early and mid L3 stages, while only 4.3% expressed GFP in the late L3 and L4 stages  worms had dGFP-expressing DTCs. This result shows a differential requirement for lin-29 (stronger) and daf-12 (weaker) for blmp-1 transcriptional repression during, and after, the late L3 stage (daf-12(rh61rh411); lin-29(RNAi) double mutants had DTCs expressing dGFP  mutation was introduced into the wild-type or the daf-12mutant  worms had DTCs expressing dGFP.We also tested the involvement of 12mutant . Thus, ddre-1 RNAi, RNAi for skp-1, rbx-1, or cul-1 caused a retarded DTC migration phenotype in the daf-12(rh61rh411) single mutant Previous genetic and biochemical data have shown that the F-Box protein DRE-1 functions in an SCF ubiquitin ligase complex, which contains CUL-1 (a cullin scaffold protein), SKP-1 (a SKP1-like adaptor that binds to the F-Box protein), and RBX-1 (an RBX ring finger that bridges to the E2 ubiquitin conjugating enzyme), and is important for the temporal control of somatic and gonad development lag-2::gfp::blmp-1P, in which BLMP-1 was tagged with GFP and expressed under the control of the lag-2P promoter , although this difference could be due to different sensitivities of the gfp reporters. The notion that lin-29 is more significant than daf-12 in the repression of blmp-1 was further supported by the observation that, using the blmp-1::dgfpP reporter, loss of lin-29 increased blmp-1 transcription in the late L3 to L4 stages, but loss of daf-12 had no detectable effect on blmp-1 expression . Interestingly, wild-type worms carrying lag-2::blmp-1P had normal DTC migration ::gfpP transgene, we found that inactivation of either daf-12 or lin-29 slightly reduced unc-5 transcription, while loss of both genes completely abolished it , raising the possibility that DAF-12 may directly bind to unc-5 for its transcriptional activation.Using ChIP-seq, the modENCODE Consortium showed that BLMP-1 binds to the upstream sequence of ished it . These rblmp-1-on” state (high BLMP-1 and low UNC-5 levels) and phase II dorsal migration occurs in the“blmp-1-off” state (low BLMP-1 and high UNC-5 levels). A double-negative feedback loop, in which two genes mutually repress each other directly or indirectly, is commonly utilized to generate switch-like bistable responses during the progression of cellular and developmental processes lin-29 and blmp-1 act in a double negative feedback loop that helps maintain DTCs in one of the bistable “blmp-1-on” and “blmp-1-off” states. On the basis of our results, we propose a molecular model for the switch-like process of the DTC dorsal turn. In L2 and early L3, BLMP-1 is expressed and represses lin-29 transcription  are generated from dietary cholesterol and bind to the ligand binding domain of DAF-12 blmp-1 transcription. During early to mid L3, dre-1 expression is initiated blmp-1 off). In late L3, accumulation of LIN-29 locks the DTCs in the “blmp-1-off” state through the negative feedback loop, thus allowing DTCs to switch to dorsal migration. This gene regulatory circuit integrates the temporal and spatial signals and coordinates with overall development of the organism to direct DTC dorsal migration during organogenesis.The DTC dorsal turn can be characterized by a specific transition in which the DTC moves irreversibly from phase I centrifugal migration into phase II dorsal migration. Phase I centrifugal migration occurs in the “cription . The doulin-29 is negatively regulated by lin-42lin-42 regulates the expression of lin-29 is not clear. lin-42 also genetically interacts with daf-12. During development, when conditions are unfavorable, due to starvation, crowding or high temperature, daf-12 promotes diapauses and formation of duaer larvae daf-12 represses expression of lin-42 during dauer formation lin-42 antagonizes the dauer-inducing signal from ligand-free DAF-12 daf-12 and lin-42 also regulate each other in the timing of DTC dorsalward turn during gonadaogenesis needs to be examined in the future.Expression of blmp-1 affects DTC migration along the AP axis. As shown in blmp-1 mutants, suggesting simultaneous execution of centrifugal phase I and dorsal phase II movement. This result implies that the mutual exclusion of dorsalward and centrifugal migration in wild type animals was impaired in the blmp-1 mutants. Moreover, as shown in blmp-1 mutants. Expression of unc-5 by the lag-2 promoter from transgene lag-2::unc-5P resulted in DTC migration defects, including the reversal of the migration direction during the centripetal phase III migration , similar to those seen in blmp-1 mutants  mutants had incomplete alae and the rest had no alae in the adult stage (blmp-1(s71) mutant was found to have a normal number of seam cells at the late L4 and early adult stage  mutants had adult alae at the early L4 stage (dre-1 in preventing the adult fate of the seam cell dre-1(dh99) mutants had incomplete alae  mutants have a weak uncoordinated movement phenotype blmp-1 expression has been previously observed in neurons using reporter transgene assays blmp-1 revealed by the transgene may be ectopic and not reflect the endogenous expression pattern.blmp-1 mutations, lin-42 RNAi causes both a precocious DTC dorsal turn lin-29 expression in L2, one stage earlier than in the wild-type lin-42(RNAi) and blmp-1 mutations induce precocious dorsal migration at different larval stages, i.e. in L2 for lin-42(RNAi)blmp-1 blmp-1 . Similarhttp://www.ebi.ac.uk/Tools/msa/clustalo/, we found that Bcl6 shares sequence similarity with BLMP-1 (20%) and PRDI-BF1 (24%). These observations and our result showing that DRE-1/FBXO11 and BLMP-1/PRDI-BF1 are associated in human cell cultures indicate that DRE-1, FBXO10 and FBXO11 target different proteins for degradation in different cellular or developmental contexts, i.e. DRE-1 targets BLMP-1 and CED-9, FBXO10 targets Bcl2 and FBXO11 targets PRDF-BF1 and Bcl6. It will be interesting to determine how target specification is regulated.DRE-1 has two human orthologs FBXO10 and FBXO11, which are localized in the cytoplasm and nucleus, respectively C. elegans strains were cultured at 20°C on NGM agar inoculated with E. coli OP50, as described previously blmp-1, lin-29(n546); LGIII, unc-119(ed3); LGV, dre-1(dh99); LGX daf-12(rh61rh411). The blmp-1(tm548) mutant was generated and provided by the National Bioresource Project in Japan. Strains CB4856 and RW7000 were used for single-nucleotide polymorphism (SNP) mapping lin-29::GFP was kindly provided by S. Shaham tk41, tm548, s71 or tp5 mutation is recessive or dominant, homozygous mutant hermaphrodites were crossed to wild-type males carrying the integrated sur-5::gfp transgene. The cross-progeny hermaphrodites carrying the sur-5::gfp transgene were scored for the dumpy (Dpy) and DTC migration phenotypes using Nomarski microscopy. In these crosses, all heterozygous cross-progeny hermaphrodites were wild-type, showing that these mutations are recessive.To test whether the tk41, tm548 or tp5 homozygous hermaphrodites were crossed to s71 males carrying the sur-5::gfp transgene, the cross-progeny hermaphrodites with the transgene were scored for the Dpy and DTC migration phenotypes using Nomarski microscopy. In these crosses, all of the mutations tested failed to complement s71.In complementation tests, the blmp-1(s71) on the basis of the Dpy phenotype within the region between snpF45H11 and snpY106g6h, which correspond to cosmids F45H11 and F37D6, respectively, using three-factor mapping with unc-40 and unc-75 and subsequent SNP mapping as previously described blmp-1 is located between unc-40 and snpY106g6h: from a blmp-1(s71)unc-75/++ (CB4856) hermaphrodite 37/37 Dpy non-Unc recombinant progeny segregated neither snpY106g6h nor snpF59C6, and 6/6 nonDpy Unc recombinant progeny segregated snpY106g6h and snpF59C6. From an unc-40 blmp-1 (s71)/++(CB4856) hermaphrodite 85/91 nonDpy Unc recombinant progeny segregated snpY106g6h and snpF59C6, 1/91 recombinant progeny segregated snpY106g6h and 5/91 recombinant progeny segregated neither snpY106g6h nor snpF59C6. The following cross demonstrates that blmp-1 is located right of snpF45H11: from an unc-40 blmp-1(s71)/CB4856 hermaphrodite 3/171 Dpy nonUnc recombinant progeny segregated snpF45H11.We positioned tk41. In an independent screen for mutants with a DTC migration defect using Nomarski microscopy, we isolated the mutation tp5.We performed genetic screening for mutants defective in DTC migration under a dissecting microscope, as described in Nishiwaki, 1999, and isolated the mutation 3 and observed on a microscope with Nomarski optics. For the time course analysis of DTC migration, wild-type and blmp-1(s71) mutants at the indicated time points were collected and scored under the Nomarski microscope.The DTC migratory patterns of the wild-type and mutants were determined by observing the shape of the gonadal arms in the adult stage. Worms were mounted on a 4% agar pad containing 20 mM NaNblmp-1 cDNA, the first three exons were amplified by RT-PCR using a forward primer corresponding to the SL1 sequence and the reverse primer blmp-1_exon_3/r (see blmp-1 cDNA fragment from the yk487b7 clone by fusion PCR blmp-1 cDNA.To obtain the 5′ end of lag-2::blmp-1P (pYW798), blmp-1 cDNA was PCR-amplified from pYW687 using primers DPY-24-KpnI/f and DPY-24-KpnI/r and the product inserted into pPD49_26/Plag-2 via the KpnI site.To construct unc-5 transcriptional gfp constructs were generated. The unc-5(4.6 kb)P DNA fragment was PCR-amplified from C. elegans genomic DNA using primers unc-5P_4.6 kb/f and unc-5P_4.6 kb/r and inserted into the vector pGEM-T Easy. The unc-5(4.6 kb)P fragment of the resulting construct was then inserted into the vector pPD95.77 (A. Fire) via SphI/SalI sites to generate unc-5(4.6 kb)::gfpP. A similar approach was used to generate unc-5(1 kb)::gfpP. The unc-5(1 kb)P fragment was PCR-amplified from genomic DNA using primers unc-5P_1 kb/f and unc-5P_1 kb/r and cloned into the vector pGEM-T Easy, then the unc-5(1 kb)P fragment was excised from the resulting plasmid and inserted into the vector pPD95.75 (A. Fire) via the XmaI site. To generate unc-5(1 kb)::mCherryP, the gfp fragment of the plasmid unc-5(1 kb)::gfpP was replaced by mCherry cDNA.Two unc-5(1 kb)P fragment was expected to contain the regulatory region for proper unc-5 function in guiding DTC migration, as the unc-5(1 kb)::unc-5::gfpP plasmid is sufficient to rescue the DTC migration defect of the unc-5 mutant (our unpublished data). The unc-5(1 kb)::unc-5::gfpP plasmid was generated by fusion PCR by fusing two PCR-amplified products corresponding to unc-5(1 kb)::unc-5(cDNA)P and gfp::unc-54 3′ UTR from plasmid pU5HA (J. Culotti) or pPD95.75, respectively.The blmp-1::dgfpP fragment was generated by fusion PCR by fusing two PCR products corresponding to blmp-1P and the region containing dgfp and the unc-54 3′ UTR. blmp-1P was PCR-amplified from genomic DNA using primers D24-5end5kb and d24Pgfp/r, and the region containing dgfp and unc-54 3′UTR was PCR-amplified from pPD95.75PEST (pYW807), which contains dgfp, using primers GFP/f and D. The two PCR products were then mixed and fused by fusion PCR using primers d24-5kbf-nest and D′. The blmp-1::blmp-1::gfpP fragment was generated by fusion PCR by fusing two PCR products corresponding to blmp-1 genomic DNA and the region containing gfp and unc-54 3′ UTR from the pPD95.75 plasmid blmp-1 genomic DNA was PCR-amplified from wild-type genomic DNA using primers D24-5end5kb and d24gfp/r, and the region containing gfp and the unc-54 3′ UTR was PCR-amplified from the pPD95.75 plasmid using primers GFP/f and D, then the two PCR products were fused by fusion PCR using primers d24-5kbf-nest and D′.The The primers used in this work are listed in blmp-1::blmp-1::gfp (20 ng/µl) was co-injected with the unc-119 rescuing plasmid (100 ng/µl) unc-119(ed3) mutant to generate tpEx49 transgenic worms. For the unc-5 transcription assay, unc-5(4.6 kb)::gfpP, unc-5(1 kb)::gfpP, or unc-5(1 kb)::mCherryP (20 ng/µl) was co-injected with the marker myo-2::gfpP (2 ng/µl), which expresses gfp in the pharynx blmp-1 was down-regulated during larval development, the indicated plasmids (20 ng/µl) were co-injected with myo-2::gfpP (2 ng/µl) into wild-type worms to generate transgenic worms, and the resulting transgenes were crossed to the indicated heterochronic mutants. For the blmp-1 cell autonomy assay, lag-2::blmp-1P or blmp-1::blmp-1P (20 ng/µl) was co-injected with myo-2::gfpP (2 ng/µl) into wild-type worms to generate worms carrying the respective transgenes, then the blmp-1(s71) mutation was introduced into these transgenic worms by crossing to generate the blmp-1(s71) mutant carrying either the lag-2::blmp-1P or blmp-1::blmp-1P transgene.Transgenic worms were generated by microinjection of the indicated plasmid, as described previously The transgenes used in this work were listed in blmp-1 cDNA fragment from the plasmid pYW687 was digested with EcoRI and cloned into the vectors pGEX5X-1  and pRSET B (Invitrogen) at their EcoR I sites to generate the respective constructs pYW802 and pYW691. The GST-BLMP-1 and HIS-BLMP-1 fusion proteins were present in inclusion bodies and were purified using standard methods lin-29(RNAi);daf-12(rh61rh411) mutants, the worms were mounted on a gelatin-chromic potassium sulfate-subbed slide and immunostained with anti-BLMP-1 antibodies, as described previously The blmp-1, skr-1, cul-1, rbx-1, dre-1, or lin-29, RNA interference (RNAi) was performed either by feeding worms with bacteria expressing the double stranded RNA for the indicated genes or by injecting the double stranded RNA for the indicated genes as described previously lin-29 RNAi construct, the region corresponding to exons 3–9 of lin-29 was PCR-amplified from the yk1430g04 plasmid, which contains lin-29 cDNA , and cloned into the L4440 vector. The blmp-1, srk-1, cul-1, and rbx-1 RNAi constructs were obtained from the Ahringer RNAi library.To knock down HEK293T cells were grown in Dulbecco's modified Eagle's medium (Thermo Inc) containing 10% fetal bovine serum, 100 U/ml of penicillin, and 100 µg/ml of streptomycin  and were transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen). MG132 (20 µM) was added to the medium 2 h before the cells were harvested to block protein degradation by the proteasome.Cells were harvested using pre-chilled PBS and lysed using 1% NP-40 lysis buffer with protease inhibitor (Roche). HA-tagged or Myc-tagged fusion proteins were immunoprecipitated by incubation overnight at 4°C with anti-HA or anti-Myc agarose beads (Sigma) and bound protein identified by Western blotting using antibodies against HA (Covance), Myc (BD Biosciences), or CUL1 (Zymed). Antibodies against tubulin (Sigma) or GAPDH (Abnova) were used on the loading controls.Figure S1blmp-1. (A) The genetic map near the dpy-24/blmp-1 locus on chromosome I is shown above. The cosmid clones shown in the middle and the open reading frames of the cosmid F25D7 shown below were tested for their abilities to rescue the blmp-1(s71) mutant phenotypes. The gene structure and transcription direction (indicated by an arrow) for each open reading frame is shown. (B) Rescue of the phenotype responsible for the DTC migration defect, dumpy phenotype or embryonic lethality of blmp-1(s71) mutants by germline transformation using genomic DNA clones. Plus sign, rescue; minus sign, no rescue. n.d., not determined.Molecular Cloning of (TIFF)Click here for additional data file.Figure S2blmp-1 mutant alleles are indicated by arrowheads.Sequence alignment of BLMP-1 and related proteins. Sequence alignment of BLMP-1, mouse Blimp-1, and human PRDI-BF1. The light gray and black shading indicates, respectively, residues that are identical in two or three proteins, while the dark gray shading with white letters indicate similar residues in two proteins. The PR domain is indicated by double underlining and the zinc fingers by circles, the predicted NLS is boxed by a rectangle, and the positions of the (TIFF)Click here for additional data file.Figure S3blmp-1 results in partial penetrant embryonic lethality, a dumpy phenotype, and a defect in alae formation. (A) blmp-1 mutants are dumpy. DIC images of wild-type and blmp-1(s71) worms 24 h after reaching adulthood. Scale bar, 100 µm. (B) blmp-1 embryos have a partial penetrant embryonic lethality. The table shows the percentages of embryos that did not hatch after 24 h. The progeny derived from three worms was counted for each genotype. (C) DIC images of wild-type (a), blmp-1(s71) (b), dre-1(dh99) (c) and blmp-1(s71); dre-1(dh99) (d) adults. Alae are indicated by arrows. Scale bar, 10 µm. (D) The seam cell phenotype of the indicated mutants. The seam cell numbers and alae were scored at the indicated developmental stages. The seam cell-specific marker SCM::GFP allows visualization of seam cell nuclei and was used to assay seam cell number. The apical junction marker AJM-1::GFP was also present in the transgene.Loss of (TIFF)Click here for additional data file.Figure S4unc-5(1 kb)::gfpP is expressed in DTCs during, and after, the dorsal turn. DIC and GFP images at different migration phases of DTCs in worms carrying the transgene unc-5(1 kb)::gfpP. DTCs are indicated by arrows.(TIFF)Click here for additional data file.Figure S5unc-5 transcription in a redundant fashion. (A) DIC and GFP images in the late L3 stage of a wild-type worm (a), lin-29(RNAi) (b) worm, daf-12(rh61rh411) worm (c), or daf-12(rh61rh411); lin-29(RNAi) worm (d) carrying the unc-5(4.6 kb)::gfpP transgene. Scale bar 20 µm. The intensity of the GFP signal is also weaker in the lin-29 or daf-12 single mutants than in the wild-type worms. (B) Percentages of worms of the indicated genotype carrying the unc-5(4.6 kb)::gfpP transgene with posterior DTCs expressing GFP after late L3. At least 50 worms were scored.DAF-12 and LIN-29 activate (TIFF)Click here for additional data file.Table S1blmp-1 mutants.DTC migration patterns of (TIFF)Click here for additional data file.Table S2blmp-1 mutants.The speed of DTC migration is similar in wild-type and (TIFF)Click here for additional data file.Table S3blmp-1 and unc-5.Genetic interactions of (TIFF)Click here for additional data file.Table S4DRE-1 functions in an SCF complex to promote the DTC dorsal turn.(TIFF)Click here for additional data file.Table S5List of transgenic strains and transgenes used in this work.(TIFF)Click here for additional data file.Table S6Sequences of the primers used in the PCR reactions for cloning.(TIFF)Click here for additional data file.