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Oligogalacturonides (OGs) released from the plant cell wall are active both as damage-associated molecular patterns (DAMPs) for the activation of the plant immune response and regulators of plant growth and development. Members of the Wall-Associated Kinase (WAK) family are candidate receptors of OGs, due to their ability to bind in vitro these oligosaccharides. Because lethality and redundancy have hampered the study of WAKs by reverse genetics, we have adopted a chimeric receptor approach to elucidate the role of Arabidopsis WAK1. In a test-of-concept study, we first defined the appropriate chimera design and demonstrated that the Arabidopsis pattern recognition receptor (PRR) EFR is amenable to the construction of functional and resistance-conferring chimeric receptors carrying the ectodomain of another Arabidopsis PRR, FLS2. After, we analyzed chimeras derived from EFR and WAK1. Our results show that, upon stimulation with OGs, the WAK1 ectodomain is capable of activating the EFR kinase domain. On the other hand, upon stimulation with the cognate ligand elf18, the EFR ectodomain activates the WAK1 kinase, triggering defense responses that mirror those normally activated by OGs and are effective against fungal and bacterial pathogens. Finally, we show that transgenic plants overexpressing WAK1 are more resistant to Botrytis cinerea. damage-associated molecular patterns | elongation factor tu receptor | pectin-mediated signaling | plant immunity | chimeric receptors doi: 10.1073/pnas.1000675107

Recent years have seen an increasing appreciation of the pervasive (genome-wide) transcription that occurs in most genomes and the multiple functional roles of RNAs within cells (1,2). Many individual laboratories [...], Deep sequencing of 'transcriptomes'--the collection of all RNA transcripts produced at a given time--from worms to humans reveals that some transcripts are composed of sequence segments that are not co-linear, with pieces of sequence coming from distant regions of DNA, even different chromosomes. Some of these 'chimaeric' transcripts are formed by genetic rearrangements, but others arise during post-transcriptional events. The 'trans-splicing' process in lower eukaryotes is well understood, but events in higher eukaryotes are not. The existence of such chimaeric RNAs has far-reaching implications for the potential information content of genomes and the way it is arranged.

Since publication of the first Thermotogales genome, Thermotoga maritima strain MSB8, single-and multi-gene analyses have disagreed on the phylogenetic position of this order of Bacteria. Here we present the genome sequences of 4 additional members of the Thermotogales (Tt. petrophila, Tt. lettingae, Thermosipho melanesiensis, and Fervidobacterium nodosum) and a comprehensive comparative analysis including the original T. maritima genome. While ribosomal protein genes strongly place Thermotogales as a sister group to Aquificales, the majority of genes with sufficient phylogenetic signal show affinities to Archaea and Firmicutes, especially Clostridia. Indeed, on the basis of the majority of genes in their genomes (including genes that are also found in Aquificales). Thermotogales should be considered members of the Firmicutes. This result highlights the conflict between the taxonomic goal of assigning every species to a unique position in an inclusive Linnaean hierarchy and the evolutionary goal of understanding phylogenesis in the presence of pervasive horizontal gene transfer (HGT) within prokaryotes. Amino acid compositions of reconstructed ancestral sequences from 423 gene families suggest an origin of this gene pool even more thermophilic than extant members of this order, followed by adaptation to lower growth temperatures within the Thermotogales. classification | horizontal (lateral) gene transfer | thermoadaptation | taxonomy | phylogenomic

During mouse development, primordial germ cells (PGCs) that give rise to the entire germ line are first identified within the proximal epiblast. However, long-term tracing of the fate of the cells has not been done wherein all cells in and around the germ-cell lineage are identified. Also, quantitative estimates of the number of founder PGCs using different models have come up with various numbers. Here, we use tetrachimeric mice to show that the progenitor numbers for the entire germ line in adult testis, and for the initiating embryonic PGCs, are both 4 cells. Although they proliferate to form polyclonal germ-cell populations in fetal and neonatal testes, germ cells that actually contribute to adult spermatogenesis originate from a small number of secondary founder cells that originate in the fetal period. The rest of the 'deciduous' germ cells are lost, most likely by apoptosis, before the reproductive period. The second 'actual' founder germ cells generally form small numbers of large monoclonal areas in testes by the reproductive period. Our results also demonstrate that there is no contribution of somatic cells to the male germ cell pool during development or in adulthood. These results suggest a model of 2-step oligoclonal development of male germ cells in mice, the second step distinguishing the heritable germ line from cells selected not to participate in forming the next generation. stem cells | testis | premordial germ cells | apoptosis chimeras

Bacteriophages [phi]29 and Nf from Bacillus subtilis start replication of their linear genome at both DNA ends by a protein-primed mechanism, by which the DNA polymerase, in a template-instructed reaction, adds 5'-dAMP to a molecule of terminal protein (TP) to form the initiation product TP-dAMP. Mutational analysis of the 3 terminal thymines of the Nf DNA end indicated that initiation of Nf DNA replication is directed by the third thymine on the template, the recovery of the 2 terminal nucleotides mainly occurring by a stepwise sliding-back mechanism. By using chimerical TPs, constructed by swapping the priming domain of the related [phi]29 and Nf proteins, we show that this domain is the main structural determinant that dictates the internal 3' nucleotide used as template during initiation. chimerical terminal protein | polymerase | linear genomes | protein-primed replication | sliding-back

From bacterial viruses to humans, site-specific recombination and transposition are the major pathways for rearranging genomes on both long- and short-time scales. The site-specific pathways can be divided into 2 groups based on whether they are stochastic or regulated. Recombinases Cre and [lambda] Int are well-studied examples of each group, respectively. Both have been widely exploited as powerful and flexible tools for genetic engineering: Cre primarily in vivo and [lambda] Int primarily in vitro. Although Cre and Int use the same mechanism of DNA strand exchange, their respective reaction pathways are very different. Cre-mediated recombination is bidirectional, unregulated, does not require accessory proteins, and has a minimal symmetric DNA target. We show that when Cre is fused to the small N-terminal domain of Int, the resulting chimeric Cre recombines complex higher-order DNA targets comprising > 200 bp encoding 16 protein-binding sites. This recombination requires the IHF protein, is unidirectional, and is regulated by the relative levels of the 3 accessory proteins, IHF, Xis, and Fis. In one direction, recombination depends on the Xis protein, and in the other direction it is inhibited by Xis. It is striking that regulated directionality and complexity can be conferred in a simple chimeric construction. We suggest that the relative ease of constructing a chimeric Cre with these properties may simulate the evolutionary interconversions responsible for the large variety of site-specific recombinases observed in Archaea, Eubacteria, and Eukarya. bacteriophage | [lambda] recombinase | site-specific recombination | evolution

[K.sup.+] channels operate in the plasma membrane and in membranes of organelles including mitochondria. The mechanisms and topogenic information for their differential synthesis and targeting is unknown. This article describes 2 similar viral [K.sup.+] channels that are differentially sorted; one protein (Kesv) is imported by the Tom complex into the mitochondria, the other (Kcv) to the plasma membrane. By creating chimeras we discovered that mitochondrial sorting of Kesv depends on a hierarchical combination of N- and C-terminal signals. Crucial is the length of the second transmembrane domain; extending its C terminus by [greater than or equal to] 2 hydrophobic amino acids redirects Kesv from the mitochondrial to the plasma membrane. Activity of Kesv in the plasma membrane is detected electrically or by yeast rescue assays only after this shift in sorting. Hence only minor structural alterations in a transmembrane domain are sufficient to switch sorting of a [K.sup.+] channel between the plasma membrane and mitochondria. algal viruses | dual targeting | [K.sup.+] channel sorting | PBCV-1 | Esv-1

New genes can originate by the combination of sequences from unrelated genes or their duplicates to form a chimeric structure. These chimeric genes often evolve rapidly, suggesting that they undergo adaptive evolution and may therefore be involved in novel phenotypes. Their functions, however, are rarely known. Here, we describe the phenotypic effects of a chimeric gene, sphinx, that has recently evolved in Drosophila melanogaster. We show that a knockout of this gene leads to increased male-male courtship in D. melanogaster, although it leaves other aspects of mating behavior unchanged. Comparative studies of courtship behavior in other closely related Drosophila species suggest that this mutant phenotype of male-male courtship is the ancestral condition because these related species show much higher levels of male-male courtship than D. melanogaster. D. melanogaster therefore seems to have evolved in its courtship behaviors by the recruitment of a new chimeric gene. chimeric gene | gene duplication |I new functions | phenotype evolution

RecAX53 is a chimeric variant of the Escherichia coli RecA protein (RecAEc) that contains a part of the central domain of Pseudomonas aeruginosa RecA (RecAPa), encompassing a region that differs from RecAEc at 12 amino acid positions. Like RecAPa, this chimera exhibits hyperrecombination activity in E. coli cells, increasing the frequency of recombination exchanges per DNA unit length (FRE). RecAX53 confers the largest increase in FRE observed to date. The contrasting properties of RecAX53 and RecAPa are manifested by in vivo differences in the dependence of the FRE value on the integrity of the mutS gene and thus in the ratio of conversion and crossover events observed among their hyperrecombination products. In strains expressing the RecAPa or RecAEc protein, crossovers are the main mode of hyperrecombination. In contrast, conversions are the primary result of reactions promoted by RecAX53. The biochemical activities of RecAX53 and its ancestors, RecAEc and RecAPa, have been compared. Whereas RecAPa generates a RecA presynaptic complex (PC) that is more stable than that of RecAEc, RecAX53 produces a more dynamic PC (relative to both RecAEc and RecAPa). The properties of RecAX53 result in a more rapid initiation of the three-strand exchange reaction but an inability to complete the four-strand transfer. This indicates that RecAX53 can form heteroduplexes rapidly but is unable to convert them into crossover configurations. A more dynamic RecA activity thus translates into an increase in conversion events relative to crossovers.

In allogeneic hematopoietic cell transplantation (HCT), donor T cell-mediated graft versus host leukemia (GVL) and graft versus autoimmune (GVA) activity play critical roles in treatment of hematological malignancies and refractory autoimmune diseases. However, graft versus host disease (GVHD), which sometimes can be fatal, remains a major obstacle in classical HCT, where recipients are conditioned with total body irradiation or high-dose chemotherapy. We previously reported that anti-CD3 conditioning allows donor CD[8.sup.+] T cells to facilitate engraftment and mediate GVL without causing GVHD. However, the clinical application of this radiation-free and GVHD preventative conditioning regimen is hindered by the cytokine storm syndrome triggered by anti-CD3 and the high-dose donor bone marrow (BM) cells required for induction of chimerism. Histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA) are known to induce apoptosis of cancer cells and reduce production of pro-inflammatory cytokines by nonmalignant cells. Here, we report that SAHA inhibits the proliferative and cytotoxic activity of anti-CD3-activated T cells. Administration of low-dose SAHA reduces cytokine production and ameliorates the cytokine storm syndrome triggered by anti-CD3. Conditioning with anti-CD3 and SAHA allows induction of chimerism with lower doses of donor BM cells in old nonautoimmune and autoimmune lupus mice. In addition, conditioning with anti-CD3 and SAHA allows donor CD[8.sup.+] T cell-mediated GVA activity to reverse lupus glomerulonephritis without causing GVHD. These results indicate that conditioning with anti-CD3 and HDAC inhibitors represent a radiation-free and GVHDpreventative regimen with clinical application potential. anti-CD3 mAb | graft versus host disease | hematopoietic cell transplantation | systemic lupus erythematosus | suberoylanilide hydroxamic acid

Mutations in the gene encoding the transcription factor Foxp3 lead to fatal autoimmune pathology in mice and humans, which is associated with a deficiency in [Foxp3.sup.+] regulatory T cells ([T.sub.reg]). It has also been proposed that Foxp3 inactivation in nonhematopoietic tissues, particularly in thymic epithelium, is required for the pathogenesis, because Foxp3 mutant scurfy bone marrow cells fail to transmit the disease to lethally irradiated WT hosts. We demonstrate here that the lack of pathology in these radiation chimeras is due to the presence of radioresistant endogenous [Foxp3.sup.+] [T.sub.reg] of the host. In addition, chimeras carrying the scurfy mutation only in nonhematopoietic cells exhibit no evidence of autoimmune pathology. Thus, Foxp3 deficiency in nonhematopoietic cells does not contribute to the scurfy disease. Furthermore, our analyses of radiation chimeras revealed that the peripheral [T.sub.reg] pool is fully and specifically restored and maintained by radioresistant endogenous [T.sub.reg] or adoptively transferred exogenous [T.sub.reg] through 'homeostatic' proliferation in the absence of [T.sub.reg] production from scurfy donor bone marrow cells. These results thus provide evidence that the autoimmune pathology in scurfy mice results indeed from a [T.sub.reg] deficiency and illustrate a robust homeostatic mechanism that strictly controls the size of peripheral [T.sub.reg] pool by fine-tuning of homeostatic proliferation. autoimmunity | immune homeostasis | immunological tolerance

The formation of viable genetic chimeras in mammals through the transfer of cells between siblings in utero is rare. Using microsatellite DNA markers, we show here that chimerism in marmoset (Callithrix kuhlii) twins is not limited to blood-derived hematopoietic tissues as was previously described. All somatic tissue types sampled were found to be chimeric. Notably, chimerism was demonstrated to be present in germ-line tissues, an event never before documented as naturally occurring in a primate. In fact, we found that chimeric marmosets often transmit sibling alleles acquired in utero to their own offspring. Thus, an individual that contributes gametes to an offspring is not necessarily the genetic parent of that offspring. The presence of somatic and germ-line chimerism may have influenced the evolution of the extensive paternal and alloparental care system of this taxon. Although the exact mechanisms of sociobiological change associated with chimerism have not been fully explored, we show here that chimerism alters relatedness between twins and may alter the perceived relatedness between family members, thus influencing the allocation of parental care. Consistent with this prediction, we found a significant correlation between paternal care effort and the presence of epithelial chimerism, with males carrying chimeric infants more often than nonchimeric infants. Therefore, we propose that the presence of placental chorionic fusion and the exchange of cell lines between embryos may represent a unique adaptation affecting the evolution of cooperative care in this group of primates. callitrichid | genetic chimerism | genomic conflict | social behavior

In colonial marine invertebrates, allorecognition restricts somatic fusion and thus, chimerism, to histocompatible individuals. Little is understood, however, about how invertebrates respond to chimerism formed across histocompatibility barriers or whether embryonic exposure to histoincompatible cells induces allotolerance. We here evaded natural allorecognition barriers by generating well mixed embryonic chimeras of Hydractinia symbiolongicarpus (Cnidaria:Hydrozoa) and developed molecular markers to detect chimerism in both histocompatible and histoincompatible settings. Histocompatible chimeras exhibited markedly higher growth rates and survivorship than histoincompatible pairings. Histoincompatible chimeras were unstable, with chimerism being undetectable by 4 wk of age. In contrast, colonies generated from histocompatible pairings remained chimeric at markedly higher frequencies and longer durations. Histoincompatible chimeras that lost detectable chimerism retained the fusibility/rejection characteristics of the remaining component of the chimera but not that of the lost component. Chimerism across histocompatibility barriers in an invertebrate model organism was unstable and did not induce tolerance. evolution | transplantation | hydractinia | allorecognition

Type I diabetes in both humans and nonobese diabetic (NOD) mice results from autoreactive T cell destruction of insulin-producing cells. Cure of type 1 diabetes may require both reversal of autoimmunity and regeneration of [beta] cells. Induction of chimerism via allogeneic hematopoietic cell transplantation has been shown to reestablish tolerance in both prediabetic and diabetic NOD mice. However, it is unclear whether this therapy augments [beta] cell regeneration. Furthermore, this procedure usually requires total body irradiation conditioning of recipients. The toxicity of total body irradiation conditioning and potential for graft-versus-host disease (GVHD) limit the application of allogeneic hematopoietic cell transplantation for treating type 1 diabetes. Here we report that injection of donor bone marrow and [CD4.sup.+] T cell-depleted spleen cells induced chimerism without causing GVHD in overtly diabetic NOD mice conditioned with anti-CD3/CD8 and that induction of chimerism in new-onset diabetic NOD mice led to elimination of insulitis, regeneration of host [beta] cells, and reversal of hyperglycemia. Therefore, this radiation-free GVHD preventive approach for induction of chimerism may represent a viable means for reversing type 1 diabetes. hematopoietic cell transplantation | type 1 diabetes | anti-CD3-conditioning | reversal of diabetes | reversal of autoimmunity

Fetal progenitor cells enter the maternal circulation during pregnancy and can persist for decades. We aimed to determine the role of these cells in tissue inflammation during pregnancy. WT female mice were mated to males transgenic for the EGFP (ubiquitous) or the luciferase gene controlled by the VEGF receptor 2 (VEGFR2; V-Luc) promoter. A contact hypersensitivity reaction was triggered during such pregnancies. Fetal cells were tracked by using real-time quantitative amplification of the transgene (real-time PCR), Y chromosome in situ hybridization (FISH), immunofluorescence or in vivo bioluminescence imaging. Real-time PCR disclosed fetal cells in the inflamed areas in all tested mice (17/17) with higher frequency and numbers in the inflamed compared with the control areas (P = 0.01). Double labeling demonstrated CD31 + EGFP+ fetal cells organized as blood vessels. In WT pregnant mice bearing V-Luc fetuses, a specific luciferase activity signal could be detected at the hypersensitivity site only, demonstrating the elective presence of VEGFR2-expressing fetal cells. In conclusion, using various techniques, we found the presence of fetal endothelial cells lining blood vessels in maternal sites of inflammation. These results imply that fetal endothelial progenitor cells are acquired by the mother and participate in maternal angiogenesis during pregnancy. angiogenesis | chimerism | gestation | fetal stem cells

The role of nonthymic epithelial (non-TE) MHC in T cell repertoire selection remains controversial. To analyze the relative roles of thymic epithelial (TE) and non-TE MHC in T cell repertoire selection, we have generated tetraparental aggregation chimeras (B6-nude [left and right arrow]BALB/c and B6 [left and right arrow] BALB/c-nude) harboring T and B cells from both parents, whereas TE cells originated exclusively from the non-nude donor. These chimeras mounted protective virus-specific TE and non-TE MHC-restricted T cell responses. To further evaluate whether non-TE MHC alone was sufficient to generate a functional T cell repertoire, we generated tetraparental aggregation chimeras lacking MHC class II (B6-nude [left and right arrow] [MHCII.sup.-/-]) or both MHC molecules (B6-nude [left and right arrow] [MHCII.sup.-/-][II.sup.-/-]) on TE cells, but not on cells of B6-nude origin. Chimeras with MHC-deficient TE cells mounted functional virus-specific CD[8.sup.+] but not CD[4.sup.+] T cell responses. Thus, maturation of functional CD[4.sup.+] T cell responses required MHC class II on thymic epithelium, whereas CD[8.sup.+] T cells matured in the absence of TE MHC. T cell selection | tetraparental aggregation chimeras

Encephalisation is the most important characteristic in the evolutionary transition leading from protochordates to vertebrates. This event has coincided with the emergence of a transient and pluripotent structure, the neural crest (NC), which is absent in protochordates. In vertebrates, NC provides the rostral cephalic vesicles with skeletal protection and functional vascularization. The surgical extirpation of the cephalic NC, which is responsible for building up the craniofacial skeleton, results in the absence of facial skeleton together with severe defects of preotic brain development, leading to exencephaly. Here, we have analyzed the role of the NC in forebrain and midbrain development. We show that (i) NC cells (NCC) control Fgf8 expression in the anterior neural ridge, which is considered the prosencephalic organizer; (ii) the cephalic NCC are necessary for the closure of the neural tube; and (iii) NCC contribute to the proper patterning of genes that are expressed in the prosencephalic and mesencephalic alar plate. Along with the development of the roof plate, NCC also concur to the patterning of the pallial and subpallial structures. We show that the NC-dependent production of FGF8 in anterior neural ridge is able to restrict Shh expression to the ventral prosencephalon. All together, these findings support the notion that the cephalic NC controls the formation of craniofacial structures and the development of preotic brain. Fgf8 | exencephaly | preotic vesicles | prosencephalic organizer | quail-chick chimeras

The computer program Mallard can screen whole 16S rRNA gene libraries for chimeras and other artifacts and it enables a graphical approach for visualizing phylogenetic relationships among these gene sequences. An analysis of large libraries of cloned bacterial 16S rRNA gene sequences submitted to the public repository during 2005 was undertaken to illustrate the use of Mallard and the study underlined the importance of researchers adequately screening their libraries before submission.

Chlorocatechol 1,2-dioxygenase (CCD) is the first-step enzyme of the chlorocatechol ortho-cleavage pathway, which plays a central role in the degradation of various chloroaromatic compounds. Two CCDs, CbnA from the 3-chlorobenzoate-degrader Ralstonia eutropha NH9 and TcbC from the 1,2,4-trichlorobenzene-degrader Pseudomonas sp. strain P51, are highly homologous, having only 12 different amino acid residues out of identical lengths of 251 amino acids. But CbnA and TcbC are different in substrate specificities against dichlorocatechols, favoring 3,5-dichlorocatechol (3,5-DC) and 3,4-dichlorocatechol (3,4-DC), respectively. A study of chimeric mutants constructed from the two CCDs indicated that the N-terminal parts of the enzymes were responsible for the difference in the substrate specificities. Site-directed mutagenesis studies further identified the amino acid in position 48 (Leu in CbnA and Val in TcbC) as critical in differentiating the substrate specificities of the enzymes, which agreed well with molecular modeling of the two enzymes. Mutagenesis studies also demonstrated that Ile-73 of CbnA and Ala-52 of TcbC were important for their high levels of activity towards 3,5-DC and 3,4-DC, respectively. The importance of Ile-73 for 3,5-DC specificity determination was also shown with other CCDs such as TfdC from Burkholderia sp. NK8 and TfdC from Alcaligenes sp. CSV90 (identical to TfdC from R. eutropha JMP134), which convert 3,5-DC preferentially. Together with amino acid sequence comparisons indicating high conservation of Leu-48 and Ile-73 among CCDs, these results suggested that TcbC of strain P51 had diverged from other CCDs to be adapted to conversion of 3,4-DC.

Author(s): Insoo Hyun (corresponding author) [1] After more than a decade of controversy, the United States is nudging towards approving research on human-animal embryos. Last week, the National Institutes of [...]

Novel cellulosome chimeras displaying atypical geometries and binding modes are designed, wherein the targeting and proximity functions are directly incorporated as integral parts of the enzyme components. The analysis has shown that cellulosome components are used for generating higher-order functional composites and has suggested that enzyme mobility is a key parameter for cellulosome efficiency.

The utility of the Greengenes data assembly process and examination of the validity of prokaryotic candidate phyla was illustrated by aligning and chimera checking more than 90,000 public 16S small-subunit rRNA gene sequences. Putative chimeras were identified in 3 percent of environmental sequences, which were classified into 100 phylum-level lineages in Archea and Bacteria.

Killer peptide was expressed in plants by using Potato virus X (PVX)-derived vector as a fusion to the viral coat protein, yielding chimeric virus particles (CVPs) displaying the heterologous peptide. The results indicated that the PVX-based display system is a high-yield, rapid and efficient method to produce and evaluate antimicrobial peptides in plants, representing a milestone for the large-scale production of high-scale production of high-added value peptides through molecular farming.

A novel in vivo method of generating a random chimera library based on the combination of highly frequent deletion formation in the Escherichia coli ssb-3 strain with an rpsL-based chimera selection system is described. This system enables the selection of chimeras without the target gene expression and can therefore be applied to the chimeragenesis of cytotoxic targets even in their intact form.

A study using the cassette PCR technique to isolate a C23O exhibiting improved activity against 4-mthylcatechol is presented. Cassette PCR is a PCR method that allows a variety of chimeric genes to be isolated without using cloned genes.

Since September 2015, researchers have been banned from receiving funding from the US National Institutes of Health (NIH) for adding human stem cells to animal embryos, creating blends called chimaeras. [...]

More than 40 years after scientists bred mice by combining cells originating from different embryos, researchers have finally succeeded at creating the world's first chimeric monkeys. By fusing cells from [...]