The coronavirus E protein is a small membrane protein with a single predicted hydrophobic domain (HD), and has a poorly defined role in infection. The E protein is thought to promote virion assembly, which occurs in the Golgi region of infected cells. It has also been implicated in the release of infectious particles after budding. The E protein has ion channel activity in vitro, although a role for channel activity in infection has not been established. Furthermore, the membrane topology of the E protein is of considerable debate, and the protein may adopt more than one topology during infection. We previously showed that the HD of the infectious bronchitis virus (IBV) E protein is required for the efficient release of infectious virus, an activity that correlated with disruption of the secretory pathway. Here we report that a single residue within the hydrophobic domain, Thr16, is required for secretory pathway disruption. Substitutions of other residues for Thr16 were not tolerated. Mutations of Thr16 did not impact virus assembly as judged by virus-like particle production, suggesting that alteration of secretory pathway and assembly are independent activities. We also examined how the membrane topology of IBV E affected its function by generating mutant versions that adopted either a transmembrane or membrane hairpin topology. We found that a transmembrane topology was required for disrupting the secretory pathway, but was less efficient for virus-like particle production. The hairpin version of E was unable to disrupt the secretory pathway or produce particles. The findings reported here identify properties of the E protein that are important for its function, and provide insight into how the E protein may perform multiple roles during infection. Coronaviruses are enveloped viruses that bud and assemble intracellularly, and therefore must use the host secretory pathway for release. Coronavirus E is a small protein that contains a single predicted hydrophobic domain and is targeted to the Golgi region. The E protein has been implicated in the assembly of coronavirus particles, as well as in virus release after assembly. The mechanism of action is not understood, but may involve ion channel activity. The membrane topology of the E protein is also unclear, and the protein may adopt distinct topologies that have different functions. We previously showed that the E protein from the infectious bronchitis virus could disrupt the secretory pathway to the apparent advantage of the virus. Here we have mapped this activity to a single, essential residue within the hydrophobic domain. Additionally, we developed mutant versions of IBV E that adopt a single membrane topology, and showed that a transmembrane topology is required for disruption of the secretory pathway. Our results broaden the understanding of E protein function and will impact the development of antiviral strategies. Coronaviruses (CoVs) are enveloped, positive strand RNA viruses that infect a variety of mammalian and avian species. In humans, CoVs are responsible for nearly 20% of common cold cases. CoVs can also lead to more serious disease as seen during the outbreak of the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003. To better prepare for the emergence of another highly pathogenic CoV it is important to increase our understanding of CoV biology.The CoV virion consists of a helical nucleocapsid, made up of the CoV N protein and the genome, surrounded by a lipid envelope. Three structural proteins are embedded in the virion envelope. The CoV S protein is a type I transmembrane protein and is responsible for the attachment and fusion of the virion during entry. The CoV M protein has three transmembrane domains and drives the organization of the virion through its interactions with the other structural proteins In addition to its role in assembly, CoV E may have other functions during infection. Studies in planar lipid bilayers have shown that CoV E has ion channel activity exo, Ccyto) cyto, Cexo) The membrane topology of CoV E is of considerable debate. CoV E has a short (∼10aa) hydrophilic N-terminus followed by a long hydrophobic domain (∼25aa) and a hydrophilic C-terminus. The N-terminus does not contain a canonical ER signal sequence Since CoVs assemble intracellularly, their virions must pass through the host secretory pathway for egress. How or if the secretory pathway is modified in infected cells is not well understood, but may involve the E protein. A version of TGEV lacking the E protein was unable to produce infectious particles, but electron microscopy revealed that immature virions were present in secretory organelles of infected cells In the present study we set out to determine what properties of the HD of IBV E were important for disrupting the secretory pathway. We performed alanine scanning mutagenesis on the HD and identified a key residue required for disrupting the secretory pathway. We also addressed the role of topology in disrupting the secretory pathway by designing mutant versions of IBV E that adopted either a transmembrane or a membrane hairpin topology. This allowed, for the first time, functional analysis of the two specific forms.When the HD of IBV E (GenBank ID: CAC39117) is modeled as an alpha helix and viewed in a helical wheel projection, polar uncharged amino acids cluster on one side . If the In addition to disrupting protein trafficking, IBV E expression disrupts Golgi morphology We next determined if there was any flexibility in the amino acid required at position 16 in IBV E. A multiple sequence alignment of several different CoV E proteins showed that a polar uncharged residue is conserved at position 16 . We intrPreviously we reported that replacing the sequence of the HD of IBV E does not affect virus-like particle (VLP) production CoVs fall into three distinct groups based on genome similarities, alpha, beta and gamma. IBV is a gamma-CoV, and we wanted to determine if the effect on the secretory pathway was a property of other CoV E proteins. We transiently expressed the E proteins from the beta-CoVs SARS-CoV (GenBank ID: NP_828854.1) and MHV (GenBank ID: ACO72886) as well as the alpha-CoV TGEV (GenBank ID: ABG89321) in HeLa cells. Using antibodies directed against the various CoV E proteins or GM130, the morphology of the Golgi complex was examined. Somewhat surprisingly, none of the other CoV E proteins caused the Golgi complex to disassemble . Other mMultiple groups have proposed different membrane topologies for the CoV E protein, either as a transmembrane protein or as a membrane hairpin . For IBVHaving developed versions of IBV E that adopt a unique orientation in the membrane, we determined if topology was important for disrupting the Golgi complex. IBV E, ssIBV E, and FLAG-IBV E were transiently expressed in HeLa cells and subjected to indirect immunofluorescence microscopy. Staining for IBV E and GM130 revealed that ssIBV E disrupted Golgi complex morphology to a similar degree as IBV E . HoweverNext we tested whether expression of the topology constructs affected protein trafficking. We found that ssIBV E disrupted protein trafficking similar to IBV E . FLAG-IBThe addition of a FLAG tag to the N-terminus of IBV E could result in a number of effects beyond changing the topology of IBV E. Thus, we generated a version of IBV E that had a canonical signal sequence, followed by a FLAG tag on the N-terminus (ssFLAG-IBV E). When transiently expressed in HeLa cells, ssFLAG-IBV E was not targeted as well to the Golgi complex as the other constructs . Also, sTo test how the membrane topology of IBV E affects particle assembly, we assayed IBV E, ssIBV E and FLAG-IBV E in a VLP assay. We co-expressed the E constructs along with plasmids encoding IBV M and IBV N in HeLa cells and determined the amount of VLPs released into the supernatant by immunoblotting . Cells eWe reported previously that replacing the entire HD of IBV E with a heterologous sequence eliminated disruption of the secretory pathway in transfected cells, and dramatically reduced the release of infectious virus from infected cells trans-Golgi to prevent the premature activation of the fusion protein Many viruses encode small membrane proteins that have ion channel activity While T16 in IBV E is required for disrupting the secretory pathway, it is not important for virus assembly as judged by VLP production. Our VLP results also suggest that disruption of the secretory pathway is not required for virus egress, since the T16A mutant produced the same level of VLPs as the wild-type E protein. However, the VLP assay does not allow measurement of infectivity, which was greatly reduced for particles released from cells infected with IBV carrying an E protein with a heterologous HD If CoV E is important for the release of infectious particles, why do some CoVs show only a modest reduction in infectivity when E is deleted exo, CcytoPrevious reports on CoV E protein topology have suggested that it may exist either as a transmembrane protein or as a membrane hairpin with both the N- and C-termini in the cytoplasm. The ability to adopt multiple membrane topologies could be a mechanism to increase the number of protein functions within the constrictions of genome size. Here, we generated mutant versions of IBV E that adopted either a membrane hairpin or transmembrane topology. We found that the transmembrane version of the protein behaved largely like IBV E, with the exception that it was unable to drive VLP production to the same degree. The membrane hairpin version of IBV E was unable to disrupt the secretory pathway or drive VLP production. These data suggest that IBV E largely functions as a transmembrane protein, with no apparent role for the membrane hairpin. However, such conclusions should be drawn with caution. While we determined that ssFLAG-IBV E behaved largely like ssIBV E, addition of the FLAG tag onto the N-terminus of IBV E could have any number of off-target effects, especially when considering the interaction of the E protein with M. We attempted to generate a membrane hairpin using several different strategies, including altering the charge distribution on either end of the HD, extending the N terminus with different tags, and shortening the C-terminus. Our only successful strategy was adding the FLAG tag onto the N-terminus. It should be noted that all reports of CoV E demonstrating that it adopts a membrane hairpin upon expression have been carried out using N-terminally tagged proteins The IBV E protein is a multifunctional viral protein that plays a role in both the assembly and release of infectious virus. The exact mechanism by which the protein alters the secretory pathway to facilitate infectious particle release is still unknown, but may depend on a single amino acid in the HD. Identification of the mechanism will be a big step in understanding the interplay between the secretory pathway and CoV trafficking. Also of interest is how E protein function varies among CoVs and what underlies any difference(s). Understanding these questions will provide insight into both therapeutic approaches to CoV infection and increase our understanding of how CoVs use the host secretory pathway to their advantage.2. Transient transfection of HeLa cells was performed using Fugene6 or XtremeGene 9 (Roche) according to the manufacturer's protocol. Experiments were performed 18–22 hours post transfection unless noted otherwise.HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) with 10% Fetal Bovine Serum (FBS) , and 0.1 mg/ml Normocin (InvivoGen) at 37°C under 5% COThe expression plasmids for IBV E, VSV G and IBV M have previously been described The following antibodies have been previously described: Rabbit and rat antibodies recognizing the C termini of IBV E, rabbit antibody recognizing the N terminal portion of IBV E Multiple sequence alignment of CoV E proteins was carried out using ClustalW2 at the European Bioinformatics Institutes server 35S35S [35S]-methionine-cysteine (Perkin Elmer) in cysteine-methionine free DMEM for 20 min, and chased in normal growth medium. Prior to collection, labeled cells were washed with PBS. Samples were lysed in detergent solution with protease inhibitor cocktail and clarified at 20,000×g. SDS was added to 0.2% and the samples were pre-cleared with Staphylococcus aureus Pansorbin cells. Rabbit anti-VSV antibody was added to each sample and incubated for 20′. Immune complexes were collected with 20 µl of washed Staphylococcus aureus Pansorbin cells and washed two times in RIPA buffer . Immune complexes were eluted in 1% SDS [pH 6.8] at 100°C and digested in 75 mM Na-citrate [pH 5.5] with 0.2 µl endo H (100 units) (New England Biolabs) at 37°C overnight. Concentrated sample buffer  was added to each sample prior to separation on 10% SDS-PAGE. Labeled proteins were visualized by using a Molecular Imager FX phosphorimager (Bio-Rad) and quantified using Quantity One software (BioRad).HeLa cells were transfected with pCAGGS VSV G (1 µg) along with either a control plasmid (0.5 µg pCAGGS IBV M) or a pCAGGS E construct (0.5 µg). Cells were incubated in cysteine-methionine free DMEM for 15 min, labeled with 50 µCi of ExpreHeLa cells plated on glass coverslips were processed for immunofluorescence 18–22 h after transfection. For assaying Golgi disruption in cells expressing IBV E or the HD mutants cells were fixed in 3% paraformaldehyde for 10 min. The fixative was quenched with PBS containing 10 mM glycine (PBS/Gly). The cells were permeabilized in 0.5% TX-100 for 3 min and washed in PBS/Gly. Cells were stained with rabbit anti-IBV E (1∶1000) and mouse anti-GM130 (1∶1000). Secondary antibodies were Alexa Fluor 488 conjugated anti-rabbit IgG (1∶1000), Alexa Fluor 568 conjugated anti-mouse IgG (1∶1000). DNA was stained prior to imaging with Hoechst 33285 (0.1 µg/ml). All images were collected using an Axioscop microscope (Zeiss) equipped for epifluorescence using an ORCA-03G charge-coupled-device camera . Data analysis was done using iVision software (BioVision Technologies) and Microsoft Excel. To determine if the Golgi complex was disrupted in cells expressing IBV E or its mutants, the staining for the Golgi complex (as judged by the GM130 staining) was outlined. The area encompassing the Golgi complex was measured for cells expressing IBV E, the HD mutants, or non-transfected cells. A normal Golgi was determined to be the average area of non-transfected cells +/−1.5 standard deviations. Cells with a staining area larger than this were scored as disrupted. The percent disrupted was calculated by dividing the number of cells scored as disrupted by the total number of cells measured.3, 2 mM Mg(OOCH3)2) and kept on ice. The cells were permeabilized with 75 µg/ml digitonin (EM Sciences) for 10 min. The digitonin solution was removed and the cells were rinsed twice with cold KHM. The cells were moved to room temperature and fixed with 3% paraformaldehyde for 10 min. The control cells were incubated with mouse anti-Myc (1∶2) and rabbit anti-GFP (1∶500). The C-terminus of IBV E, ssIBV E, and FLAG-IBV E were detected using a C-terminal rat anti-IBV E antibody (1∶500). The N-terminus of IBV E and ssIBV E was detected using a rabbit anti N-terminal IBV E antibody (1∶100). The N-terminus of FLAG-IBV E was detected using a mouse anti-FLAG antibody (1∶500). Secondary antibodies were The Alexa Fluor 488 conjugated donkey anti-rabbit IgG (1∶1000), Alexa Fluor 568 conjugated anti-mouse IgG (1∶1000), and Texas Red conjugated donkey anti-rat (1∶500). DNA was stained prior to imaging with Hoechst 33285 (0.1 µg/ml).HeLa cells were transfected with CFP-KDEL (0.2 µg) and golgin160-Myc (1 µg) for control samples or with IBV E (0.5 µg), pCAGGS-ssIBVE (1.5 µg), pCAGGS-FLAG-IBV E (0.5 µg), pCAGGS ssFLAG-IBV E (0.5 µg), pCAGGS IBV E T16A (0.5 µg). For the Triton samples the protocol listed above was followed. For selective permeabilization of the plasma membrane, cells were washed with a cold KHM  around an untransfected cell and measuring the fluorescence intensity. The exact same ROI and measurement was then made on the corresponding N-terminal image. Next, an ROI was drawn around a cell showing C-terminal staining and the mean fluorescence intensity was measured. Again, the exact same ROI was overlaid onto the corresponding N-terminal image and the mean fluorescence intensity was measured. The ratio of N- to C-terminal staining was calculated by first subtracting the background from each measurement, and then dividing the N-terminal value by the C-terminal value. For the data shown, the final ratios were normalized so that the signal ratio in the Triton X-100 samples was equal to 1.HeLa cells were plated in 6 cm dishes and transfected with a combination of plasmids encoding IBV M (2 µg), IBV N (1.5 µg), IBV E (0.1 µg), ssIBV E (0.4 µg), FLAG-IBV E (0.2 µg), ssFLAG IBV E (0.2 µg), T16A (0.1 µg), T16S (0.1 µg), T16N (0.1 µg) and T16N (0.1 µg). Samples were prepared 42–48 hours post transfection. The medium was clarified via centrifugation at 4500×g for 20 min. The supernatant was loaded onto a 20% sucrose cushion and centrifuged at 234,000×g in a TLA-110 rotor for 60 min. The supernatant was discarded and the pellet containing the VLPs was resuspended in 1× glycoprotein denaturation buffer (New England Biolabs) containing 100-fold concentrated protease inhibitor cocktail (Sigma). To collect the cell fraction, dishes were washed with cold PBS. The cells were scraped off the dish in 1 ml PBS and pelleted at 4000×g for 2 min. The pellet was resuspended in detergent solution and insoluble material was pelleted at 20,000×g for 1 min. 10× glycoprotein denaturation buffer was added to 1×. Both the VLP and cell fractions were heated at 100°C for 1 min. Both samples were digested with PNGase F (New England Biolabs) according to the manufacturer's protocol. After digestion concentrated sample buffer was added to a final concentration of 50 mM Tris [pH 6.8], 2% SDS, 0.05% bromophenol blue, 15% glycerol. Samples were separated on 15% PAGE gels  and transferred to polyvinylidene fluoride Immobilon membranes (Millipore). Proteins were detected using rabbit anti-IBV N , rabbit anti-IBV M (1∶5000) and rabbit anti-IBV E  primary antibodies and horseradish peroxidase conjugated donkey anti-rabbit IgG  secondary antibody. After incubation in secondary antibody, the membrane was incubated with HyGlo Quick Spray chemiluminescent detection reagent (Denville Scientific Inc.). Images were collected using a Versa Doc model 5000 (Bio-Rad) and Quantity One software.HeLa cells were treated with 100 µg/ml cycloheximide (Sigma) diluted into culture media at 18 hours-post transfection. Cells were fixed and prepared for immunofluorescence as described above at 0, 3, and 6 hrs after cycloheximide treatment. Images were collected from each time point at the same exposure time, and the mean fluorescence intensity was determined for cells expressing the E protein. The half-lives of each E protein were calculated by plotting the signal intensity versus time on a semi-log graph.Figure S1Expression of other CoV E proteins does not disrupt Golgi complex morphology. Indirect immunofluorescence microscopy on cells expressing TGEV E, MHV E, or SARS-CoV E. The E protein is shown in green, GM130 is shown in red, and nuclei are shown in blue. Scale bars, 10 µm.(TIF)Click here for additional data file.Figure S2t-test (p≤2.5×10−7).IBV E T16A has the same topology as IBV E. Selective permeabilization was carried out on cells expressing IBV E T16A. The N- and C-termini were detected using antibodies specific to each terminus. The histogram shows quantification of topology as a ratio of the N-terminus to C-terminus fluorescence signal see . The dat(TIF)Click here for additional data file.Figure S3t-test (p≤1×10−8). (C) Quantification of Golgi complex disruption in HeLa cells expressing ssFLAG-IBV E (see t-test (p<1.4×10−4).ssFLAG-IBV E behaves similarly to ssIBV E. (A) Indirect immunofluorescence microscopy on cells expressing ssFLAG-IBV E. The E protein is shown in green, GM130 is shown in red, and nuclei are shown in blue. Scale bar, 10 µm. (B) Selective permeabilization was carried out on cells expressing ssFLAG-IBV E. The N- terminus was detected using an anti-FLAG antibody and the C-terminus was detected using a Rat anti-IBV E antibody. The histogram shows quantification of topology as a ratio of the N-terminus to C-terminus fluorescence signal see . The dat(TIF)Click here for additional data file.


Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis.The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against  Acetylcholinesterase AChE, EC 3.1.1.7) terminates cholinergic neurotransmission by rapidly catalyzing hydrolysis of the neurotransmitter, acetylcholine, at neuronal and neuromuscular synapses .1.1.7 te. At the Torpedo californica AChE (TcAChE) numbering), which make thin walls between the active site pocket and the outside solvent in the putative backdoor region (BDR), have been visualized by molecular dynamics simulations . Th. Th32]. 2 and 510 Å2, respectively, to the buried interfacial surface area. The combining site of Fab403 traps the long Ω loop in EeAChE encompassing Ser75, with residues Tyr101-Trp103 in the very hydrophobic CDR-H3 being anchored central to the binding interface  form . Two and three ensembles of models were respectively obtained, from which the first and second best solutions generated by HADDOCK matched the reference Fas2-mAChE complex structure, with rmsd values in the 0.6-1.1 Å range (data not shown). Relevance of the modeled Fab403-EeAChE complex was further assessed by comparison with the crystal structures of mAChE or TcAChE complexes with the organic PAS ligands propidium  and acet2 combining site of Fab410 is markedly electropositive, and its dipole moment, of 892 Debyes , is associated with a vector roughly aligned along H-chain strand β4 and oriented toward the H-chain CDRs, instead of the L-chain CDRs as found in Fab403 . As observed for Fab403 (cf. above), analysis of the Fab410 CDRs reveals a distinctive repartition of theoretical pI values: for CDR-H1, CDR- H3 and CDR-L1 these values are in the 4.0-5.2 range, while for CDR-H2 and CDR-L2 they are in the 8.7-11.0 range; only CDR-L3 has a theoretical pI value close to neutrality . As a ren Fab403 . This obn Fab403  and its Experimental information that can help delineate the Fab410 binding site at the EeAChE surface points to i) absence of binding competition between Fab410 and the smaller, organic PAS ligands, ii) a residual activity of the Fab410-EeAChE complex slightly higher than that of the Fab403-EeAChE complex, iii) critical contribution of Ser75, but not Gln279 and Leu282, to Fab410 binding , Figure S2 and Figure S3.(PDF)Click here for additional data file.


IGH) and kappa-chain (hIGK) germline loci (named as κHAC) are capable of producing functional hpAbs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, are homozygously inactivated (double knockouts or DKO). However, B lymphocyte development in these Tc cattle is compromised, and the overall production of hpAbs is low. Here, we report the construction of an improved HAC, designated as cKSL-HACΔ, by incorporating all of the human immunoglobulin germline loci into the HAC. Furthermore, for avoiding the possible human-bovine interspecies incompatibility between the human immunoglobulin mu chain protein (hIgM) and bovine transmembrane α and β immunoglobulins (bIgα and bIgβ) in the pre-B cell receptor (pre-BCR) complex, we partially replaced (bovinized) the hIgM constant domain with the counterpart of bovine IgM (bIgM) that is involved in the interaction between bIgM and bIgα/Igβ; human IgM bovinization would also improve the functionality of hIgM in supporting B cell activation and proliferation. We also report the successful production of DKO Tc cattle carrying the cKSL-HACΔ (cKSL-HACΔ/DKO), the dramatic improvement of B cell development in these cattle and the high level production of hpAbs (as measured for the human IgG isotype) in the plasma. We further demonstrate that, upon immunization by tumor immunogens, high titer tumor immunogen-specific human IgG (hIgG) can be produced from such Tc cattle. Therapeutic human polyclonal antibodies (hpAbs) derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc) cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin heavy-chain (h Immunotherapy with human polyclonal antibodies (hpAbs) or immunoglobulins (hIgs) prepared from the plasma of normal and convalescing human donors have been shown to be an effective treatment for several human diseases . For exain vitro antibody production system based on recombinant DNA and mammalian cell culture technologies has been in the development by Symphogen A/S [To address the medical demand for hpAbs, different alternatives have been explored to substitute or even replace human plasma-derived hpAbs. For example, IgG or Fabs (fragments of antigen binding), prepared from the plasma of immunized horses or sheep has been widely used to treat severe envenomation resulting from snake and spider bites ,8. Howevogen A/S . This apogen A/S . IGH) and kappa light-chain (hIGK) with homozygous double knockouts (DKO) of the two bovine Ig mu heavy-chain loci (bIGHM-/- and bIGHML1-/-) [To harness the power of natural humoral immune response not only for its unparalleled diversity but also for its capability to respond rapidly after antigen exposure, we have been developing a transchromosomic (Tc) bovine system that quickly produces diverse hpAbs in large quantities . PreviouHML1-/-) . In thesHML1-/-) . FurtherHML1-/-) .IGH, hIGK and hIGL chromosome loci that carry the entire human immunoglobulin gene repertoire, the human VpreB (hVPREB1) and λ5 (hIGLL1) genomic loci from human chromosome 22 (hChr22) was incorporated, and part of CH and TM domains, CH2-TM, of hIGHM gene, was replaced by the corresponding bovine gene sequence (bovinization of the CH2-TM domains of hIGHM). As IgM interacts with surrogate light-chains and Igα/Igβ via its variable (V) and CH-TM domain, respectively, such modifications would restore the natural intra-species protein-protein interactions in the pre-BCR (The pre-B cell receptor (pre-BCR), comprising of IgM and invariant surrogate light-chains VpreB and lambda5 (λ5), plays critical roles for pre-B cell proliferation and maturation during B lymphogenesis . It tran pre-BCR . We alsoAL512355 locus (about 300 kb centromeric to the hIGH locus) and the other at AL391156 locus (about the 85 Mb centromeric to the AL512355 locus), through homologous recombination for deleting the 85 Mb sequences on hChr14 between these two loci (AL512355 and the promoter-less puromycin (puro) gene along with the second lox511 sequence and a hygromycin selection cassette at locus AL391156, respectively (hisD)  for chromosomal modification as described previously . We empltwo loci . In ordey (hisD) . Post elZeo) expression cassette flanked by loxP sequences  was inserted into the intron between CH 4 and TM1 for positive selection and a dT-A selection cassette was added outside of the 3’-homologous arm for negative selection , we set out to modify hChr22 as outlined in AP000350 locus with the targeting vector pTELCAGzeoSLFR and then was further modified with the targeting vector p553CAGlox2272bsrDT to integrate the lox2272 and the CAG promoter at the locus AP000553. Through extensive genomic PCR screening, a DT40 clone, STL54, was identified to carry the correctly modified hChr22  for the correctly modified hChr2, and clone K53 was identified and selected for the final HAC construction  to generate DT40 hybrid cells. Colonies derived from WCF were screened for the presence of both hChr2 and hChr22 with genomic PCR and FISH by using human COT-1 DNA as the probe as described in Materials and Methods. Clone SLK2 was identified as a positive clone  were screened by genomic PCR with CAGpuro-F3R3 primers, followed by direct sequencing of the PCR product. Clone SLKH6 was identified to contain the correctly generated SLK HAC  domain. Recombinants were enriched by sorting of GFP positive DT40 cells, as GFP expression was conferred by reconstitution of the PGK promoter-loxP-GFP cassette at the translocation site (data not shown). cKSLDH22 was finally identified as a DT40 hybrid cell line retaining the cKSL-HACΔ and subjected to extensive genomic PCR (data not shown) and three-color FISH by DNA probes recognizing the hIGH, hIGK and hIGL loci, respectively, for structural confirmation. The cKSL-HACΔ was constructed in chicken DT40 cells as outlined in The constructed cKSL-HACΔ was transferred to Chinese hamster ovary (CHO) cells by MMCT to establish CHO-based master cell banks. The structural integrity of the constructed cKSL-HACΔ was further examined by extensive genomic PCR, array comparative genomic hybridization and three-color FISH (data not shown). To our knowledge, this is the first report of construction of an artificial chromosome composed of structurally defined human chromosome fragments from three different chromosomes. -/- and bIGHML1-/-) bovine fibroblast cells via MMCT. Cells from the fibroblast colonies derived from MMCT were used as donors for somatic cell chromatin transfer (CT) as previously described [To clone cKSL-HACΔ/DKO cattle, we established cKSL-HACΔ/DKO bovine fibroblast donor cells by transferring the newly constructed cKSL-HACΔ from CHO cells into the DKO  and IgM/CD21-double positive (IgM+/CD21+) B cells  in newborn animals with flow cytometry. We used anti-hIgM antibody to detect IgM animals , cKSL-HA B cells .We measured the serum concentrations of total hIgG, i.e. all the hIgG regardless of whether it pairs with human lambda or kappa (hIgG/hIgλ/κ) light-chains or bovine lambda or kappa (hIgG/bIgλ/κ) light-chains, as well as fully hIgG (hIgG/hIgλ/κ only), when the Tc calves reached to 5-6 months of age. Even though the total hIgG levels varied among these Tc calves, the overall total hIgG production was found to be dramatically improved compared to the κHAC/DKO Tc animals, at levels comparable to the physiological hIgG levels found in healthy humans . NeverthAs our ultimate goal is to produce high titer antigen-specific hpAbs from the Tc cattle, we conducted an immunization study by immunizing two Tc cattle with a human oral squamous cell carcinoma cell line as an antigen. To evaluate antibody titers specific to the tumor cells, we collected sera from each of the immunized Tc cattle at 14 days post second vaccination (V2) and performed flow cytometry-based immunofluorescence assay. As shown in There are numerous technical challenges in producing fully hpAbs totally independent of the human immune system. First, as the human immunoglobulin genes and gene segments exist as gene clusters spanning large regions on three chromosomes, the only feasible way currently of delivering such a large size of human genomic DNA into a biological system for hpAbs production is through the engineering of HACs that comprise all or most of the human Ig genes. We report here the successful engineering of a HAC comprising the entire hIg gene repertoire that reside on three different human chromosomes, i.e. the IgH locus from hChr14, the Igk locus from hChr2, and the Igλ and surrogate light-chain loci from hChr22. The second challenge for fully hpAbs production by a recombinant system is to have a biological mechanism to efficiently tap into the genetic information provided by the immunoglobulin gene repertoires for generating the seemingly unlimited diversity of hpAbs. The mammalian humoral immune system is currently the only conceivable biological system that has such functionality. Therefore, we set out to develop a Tc animal system for fully hpAbs production. We chose bovine for the large body size, which makes it feasible to collect large volumes of plasma, a prerequisite for producing large amounts of therapeutic hpAbs. +/CD21+ B cells in newborn Tc calves  and are likely much less immunogenic than fully animal derived antibodies for therapeutic applications, it is obvious that fully human antibodies are more desirable as a biotherapeutic. This is especially true for long-term, repetitive treatments. Therefore, we have recently knocked out the bovine lambda light genes by deleting the entire bovine lambda light-chain gene cluster (manuscript in preparation) and are in the process of knocking out the bovine kappa light-chain genes. Accompanying our efforts to genetically improve our Tc bovine system, we also have developed robust immunization protocols in Tc bovine for producing high titer immunogen-specific fully hpAbs. The fact that high titer antigen-specific hpAbs can be produced from Tc animals through hyper-immunization will offer great advantages over the human plasma derived hpAbs in that the titers to certain pathogens from human plasma derived hpAbs tends to be low and sources of convalescent plasma are often difficult to obtain. In summary, our improved Tc bovine system for the production of fully hpAbs has further advanced achieving the goal of producing large quantities of therapeutic hpAbs as an alternative for plasma derived hpAbs.GuidefortheCareandUseofLaboratoryAnimals and the GuidefortheCareandUseofAgriculturalAnimalsUsedinAgriculturalandResearchTeaching for animal care standards. Animal care and use and facilities are inspected by the IACUC on a semiannual basis. Animals may have to be euthanized due to unfortunate events like terminal illness and trauma. Methods of euthanasia must follow the guidelines given by the American Veterinary Medical Association (AVMA) 2013. These are acceptable methods by the AVMA and they have been approved by the IACUC. All protocols and procedures used in the Animal Care and Use Program that may cause discomfort, distress and pain, and injury are reviewed by the IACUC for appropriate management practices. Analgesic, anesthetics and tranquillizing drugs are used when appropriate under supervision of Veterinary Services. Veterinarians and herdsmen use cattle chute for restraining when needed. Cattle are not restrained for more than four hours per day. The animal protocols contained in the study were approved by the Hematech (previous name of Sanford Applied Biosciences) Institutional Animal Care and Use Committee (IACUC) . Care of all vertebrate animals is subject to regular review by the IACUC and complies with Animal Welfare laws and regulations of the United States. Periodic health evaluations  are made by Veterinary Service to ensure that Tc cattle are healthy. All Tc bovine receive adequate housing, feed, access to water and bedding. Daily observations are made by herdsmen to ensure that appropriate standards of animal care are being met. Sanford Applied Biosciences uses the Genomic DNA isolated from primary bovine fibroblast cell lines established from Holstein dairy cattle was used to construct the bovine genomic library. Each λ phage-based genomic library was constructed using λFIX II vector through a custom library construction service . Library screening and λ phage DNA extraction/purification was done as described previously . puro gene with the hisD gene, Eco RI site with Srf I, and Spe I site with Pme I site to generate plasmid pTEL'hisDPm. A pPURlox2272 plasmid was constructed by cloning the double strand lox2272-containing oligo DNA (Oligo DNA pair 1) into HindIII site of plasmid pPUR . In parallel, a 7.3kb homologous arm  was subcloned into Bam HI site of plasmid pTEL’hisDpurolox2272. Then the Bam HI fragment from the pPURlox2272 was blunted and cloned to Pme I site of the pTEL'hisDPm to generate the pTEL’hisDpurolox2272F9R9 vector. pTEL’hisDpurolox2272F9R9: A previously described plasmid pTELpuro  was modiEco RI site to Pme I and by replacing the puro gene with CAGzeo gene to generate vector pTELCAGzeo(Sr)Pm. Then the second homologous arm (7.4kb)  was cloned into Bam HI site of the plasmid pTELCAGzeo(Sr)Pm to generate pTELCAGzeoSLF2R2. pTELCAGzeoSLF2R2: plasmid pTELpuro was modified by converting the HCF2 gene with that of the AP000553  to generate p553loxPHyg(F). This plasmid was Not I-digested and self-ligated to remove loxPHyg cassette, followed by cloning of dT-A fragment into Srf I site. In parallel, pDRIVE-CAG  was modified by replacing the lacZ fragment (Bsr GI-Eco RI) with the loxP-containing oligo DNAs (Oligo DNA pair 2). Then Sda I-Swa I fragment was liberated from this plasmid and cloned into Pst I/Sma I-digested pBluescript SK−  to generate pCAGloxP. The loxP sequence was then replaced by the lox2272-containing sequence that was generated after annealing Oligo DNA pair 3. Then the bsr gene was added to Spe I site to generate pCAGloxP2272bsr. Finally, the Not I-Kpn I fragment (CAG-lox2272-polyA-bsr) was cloned into Not I site to complete p553CAGlox2272BsrDT. p553CAGlox2272BsrDT: A previously described targeting vector pHCF2loxPHyg  was modiSpe I site of a plasmid pBluescript where the Kpn I site was converted to Srf I site to generate pSC355F3R3. The pCAGloxP plasmid was similarly modified by replacing the loxP sequence with the lox511-containing sequence (Oligo DNA pair 4) and by adding the hisD gene to Spe I site to generate pCAGlox511hisD. The Not I-Kpn I fragment (CAG-lox511-polyA-hisD) from pCAGlox511hisD was cloned into the Eco RV site of pSC355F3R3. Finally, the dT-A cassette was cloned into Not I site to complete pSC355CAGlox511hisDDT. pSC355CAGlox511hisDDT: a genomic DNA fragment (10.2 kb) as one of the homologous arms was amplified by using a PCR primer pair, SC355-F3 and SC355-R3, in 40 cycles of 98 °C for 10 s and 68 °C for 15 min. This PCR product was cloned into Bam HI site of a plasmid pBluescript where the Kpn I site was converted to Pme I site to generate p14CEN(FR). The modified lox511-containing oligoDNA (Oligo DNA pair 5) was cloned into Hin dIII site of a plasmid pPUR  to generate plasmid pPURlox511. The Bam HI fragment from the pPURlox511 was cloned to Bam HI site of pBluescript SK− , followed by cloning of the hyg gene to Eco RV, to generate pHygPurolox511. The Not I-Kpn I fragment (puro-lox511-hyg) was cloned into the Hpa I site of p14CEN(FR). Finally, the dT-A cassette was subcloned into Pme I to complete p14CEN(FR)hygpurolox511DT. p14CEN(FR)hygpurolox511DT: a genomic DNA fragment (10.2 kb) for another homologous arm was amplified by using a PCR primer pair, 14CEN-F and 14CEN-R, in 40 cycles of 98 °C for 10 s and 68 °C for 15 min. This PCR product was cloned into dT-A cassette. pRNR2loxPbsrDT: the previous vector pRNR2loxPbsr  was modiIGHM constant region by using a probe that was a PCR product . A 1.7 kb of Pml I fragment from one of the positive phage clones was cloned into Sma I site of pBluescript to generate pCH1S (F). Then a 1 kb of Sac I-Pml I bovine IGHM genomic fragment from plasmid pBCμAY37-95 (a clone containing IGHM bovine genomic fragment derived from lambda phage based genomic library [Pst I site of the pCH1S (F) to generate pCH1SSP (F). A 7.4 kb of the Sma I-Eco RI fragment from another positive phage clone was cloned into Eco RV/Eco RI-digested pCH1SSP (F) to generate pCH1SL. In parallel, a 3.5 kb of Sac I fragment from pBCμAY37-95was cloned into pBluescript and then the Xho I fragment of floxed CAGzeo was cloned into Van91 I site to generate pmAYSazeo (F). The Sac I fragment from the pmAYSazeo (F) was then cloned into blunted Eco RI site of pCH1SL resulting in pCH1zeo (F). As a final step, the dT-A cassette was cloned into Not I site of the pCH1zeo (F) to complete pCH1CAGzeo(R)DT(F). pCH1CAGzeo(R)DT(F): a genomic λ phage library was constructed from CHO cells containing the κHAC using λFIX II vector through a custom library construction service . The genomic library was screened for h library  was subcPst I site of pBluescript. From the pBCμAY37-95, a 2 kb of Sph I-Bam HI fragment was cloned into Sph I-Bam HI site of the plasmid generated by cloning SeSp into the Pst I site, generating pmAYSpB. Similarly, a 2 kb of Bam HI-Pml I fragment from the pBCμAY37-95 was cloned into Bam HI-Pme I site  resulting in pmAYSpBPml. A 0.6 kb of Eco RI-Sex AI fragment from the above phage clone was cloned into Eco RI-Sex AI site of the pmAYSpBPml to generate pRISe. Then the floxed CAGzeo was cloned into Van91 I site of the pRISe to generate pRISeCAGzeo (R), of which Not I site was converted to Eco RI site, generating pRISeCAGzeoE. Meanwhile, 1.7 kb of Pml I fragment from the above clone 4 was subcloned into Sma I site of pBluescript of which Eco RV site was converted to Mlu I site, generating pCH2S (F). 6.6 kb of Mlu I-Eco RI fragment from the above clone 1 was cloned into Mlu I-Eco RI of the pCH2S (F), generating pCH2LS. Then, the Eco RI fragment from the pRISeCAGzeoE was subcloned into Eco RI site of the pCH2LS, generating pCH2CAGzeo (F). As a final step, the dT-A cassette was subcloned into Not I site of the pCH2CAGzeo (F) to complete the pCH2CAGzeoDT. pCH2CAGzeoDT: an annealed oligo DNA pair, SeSp, which contains human and bovine junction region at CH2 flanked by SexAI and Sph I site, was cloned into blunted HAC vector construction was carried out as previously described ,17,20. BMMCT was done with each HAC vector as described previously ,17,20.These analyses were implemented as previously described ,17,20,21Array probe design, chip hybridization, and data analysis were performed by Roche NimbleGen (www.nimblegen.com). IGH, hIGK and hIGL loci, probes were synthesized from DNA derived from BAC clones RP11-417P24, RP11-316G9 and RP11-22M5 (MBL-EBI and Sanger Institute: http:// www.ensembl.org/index.html), respectively. Human COT-1 FISH and hChr-specific multi-color FISH were performed as previously described . To specCloned fetuses and calves were produced using chromatin transfer procedure as described previously .6 cells were incubated with primary antibody on ice for 20 min. After washing twice, the cells were incubated with appropriate secondary antibody for 20 min on ice if the primary antibody is not directly labeled with a fluorochrome. Finally, the cells were washed followed by re-suspend in FACS stain media for reading.Flow cytometry analysis on B cell development in newborn Tc calves was performed as previously described , except Total hIgG ELISA assay was performed as previously described . For ful8 cells/dose formulated with Montanide ISA 25 adjuvant  as water-in-oil emulsion plus Quil A  as immune stimulant. The Tc calves were immunized two times at 3-week intervals (primary immunization followed by the booster after 3 weeks). Vaccine was administered by intramuscular injection in the neck region. Serum samples were collected as previously described [cKSL-HACΔ/DKO calves were immunized with X-ray-irradiated human oral squamous cell carcinoma (DSMZ) antigen at 2 x10Sera collected from Tc calves immunized with human carcinoma cells were used as the primary antibody to stain the human carcinoma cells. Pre-immune Tc calf serum  was used as the negative controls. AF488-conjugated goat anti-hIgG Fc  at 1:80 dilution and PE-conjugated mouse anti-hIgκ  at 1:8 dilution were used to detect bound hIgG/hIgκ antibody. The assay was performed in PBS supplemented with 4 % horse serum, 0.1% sodium azide and 2 mM EDTA. The results were expressed as mean fluorescence intensity (MFI) as measured by FACSAria flow cytometer .Table S1Oligo DNA information.(XLSX)Click here for additional data file.


L-plastin is a leukocyte-specific protein that cross-links actin filaments into tight bundles, increasing the stability of actin-based structures such as podosomes and lamellipodia. While first identified as an abundant cytoplasmic protein in hematopoietically derived cells over 25 years ago, the requirement for L-plastin in multiple functions critical for immunity, such as antigen receptor signaling, adhesion, and motility, has only recently become clear. L-plastin has been identified as an important component in cellular processes critical for neutrophil, macrophage, osteoclast, eosinophil, and T- and B-lymphocyte biology. Following a brief description of the structure and function of L-plastin, the regulation of immune cell functions by L-plastin will be reviewed in detail. The actin cytoskeleton enables numerous cellular processes required for the mammalian immune response. Actin is rapidly polymerized in response to T-cell receptor signaling, and F-actin provides stability for the contact site between antigen-presenting cells and T cells, termed the immunological synapse –4. ActinPlastins, or fimbrins, are actin-bundling proteins critical to actin regulation in eukaryotes. Human fimbrin can complement yeast SAC6 in endocytosis, suggesting a high degree of conservation . Three iα-actinin family. Other α-actinin family members include α-actinin, filamin, spectrin, dystrophin, and actin-binding protein 120 [Plastins bind F-actin through ABDs that each consists of two calponin-homology domains, placing plastins in the tein 120 , 21. Platein 120 . An atomtein 120 , 24. Bunα-actinin [While LPL is thought to bundle actin in a manner similar to the homologous T-plastin , direct -actinin , 26. It -actinin , 26. Whi-actinin .No molecular function beyond actin bundling has been described for LPL. However, as outlined below, peptides derived from the N-terminus of LPL lacking ABD domains can promote changes in cell functions, such as integrin avidity and cell adhesion. These results suggest that LPL may play a role as an adaptor or a scaffolding protein in signaling in addition to its actin-bundling activity, roles that require further experimental elucidation. Also, as will be discussed below, the details of mechanisms by which actin bundling may promote or restrain immune cell functions have not yet been fully described.γR ligation, and PMA [Regulation of the bundling of activity of LPL has been demonstrated to occur through both calcium binding and serine phosphorylation. LPL is phosphorylated in multiple cell types primarily at serine residue 5 following a variety of stimuli, including IL-1, IL-2, lipopolysaccharide, fMet-Leu-Phe, Fc and PMA –31. To s and PMA . Wild-ty and PMA . Analysi and PMA . Phospho and PMA . Thus, win vitro assay, it was determined that calcium concentrations of greater than 10−6 M reduced the binding of LPL to F-actin, while LPL binding to F-actin was intact at concentrations of less than 10−7 M calcium [LPL may differ from I- and T-plastin in that calcium binding negatively regulates LPL-bundling activity . In an i calcium . Calcium calcium . Phospho calcium .LPL may also be regulated through direct binding by other proteins. For instance, binding of the protein-ionized calcium binding adaptor molecule 1 (Iba1) to LPL in macrophages enhanced the bundling capability of LPL, independently of the intrinsic bundling capability of Iba1 . Iba1 isDespite much work on the various mechanisms by which LPL may be regulated, an integrated description of how these mechanisms interact during a specific cell process in a specific cell type, such as macrophage adhesion, has not emerged. Such an integrated description is challenging, because the regulation of LPL likely varies from cell type to cell type and from stimulus to stimulus. In fact, different receptors for the same stimulus can regulate LPL via different signaling pathways . HoweverγR in some systems.The most extensive characterization of a role for LPL in hematopoietic cells has been performed in neutrophils. Critical to the immediate, innate host defense, neutrophils rely on complex interactions between Fc receptors, chemoattractant receptors, and integrins to guide neutrophil maturation, trafficking, and degranulation . RegulatγR signaling when LPL was identified as a target of bromophenacyl bromide (BPB) [γR-mediated calcium flux and diminished neutrophil degranulation, adherence, and phagocytosis. BPB-mediated inhibition of FcγR signaling correlated with the binding of BPB to LPL. Intriguingly, BPB did not prevent the FcγR-stimulation binding of LPL to F-actin, suggesting that LPL may have a function beyond that of actin bundling in FcγR signaling in neutrophils [γRII activation through bead- or plate-bound immune complexes induced phosphorylation of the N-terminal headpiece of LPL, while stimulation with soluble immune complexes did not [αMβ2  did not affect LPL phosphorylation, but did reduce the translocation of LPL to the podosomes on immune complex coated surfaces [γR, and the precise regulation and function of LPL in neutrophils adhering to surface-bound ligands remain to be fully described.LPL was initially recognized in neutrophils as a target of phosphorylation following stimulation with IL-8 or the chemoattractant peptide fMet-Leu-Phe , 46. LPLde (BPB) . BPB inhtrophils . Further did not . Immune  did not . Furthersurfaces . Thus, iαMβ2 in neutrophils was revealed in human neutrophils treated with a cell-permeable synthetic peptide derived from the N-terminal headpiece of LPL [γR signaling and by PI3K-independent f-Met-Leu-Phe and PMA stimulation. The serine 5 residue in the N-terminus of LPL was identified as the primary target of phosphorylation in these signaling pathways. A synthetic peptide derived from residues 2–19 of the N-terminus of LPL was fused at the carboxy terminus to the HIV tat protein, which enabled spontaneous translocation of the LPL peptide across the cell membrane. Treatment of neutrophils with the wild-type sequence of LPL was sufficient to induce adhesion of neutrophils to surfaces coated with fetal calf serum, a process mediated through the activation of the integrin αMβ2. Treatment with a constitutively phosphorylated synthetic LPL peptide also induced adhesion, while mutation of the serine residue at position 5 to alanine to prevent phosphorylation abrogated the induction of neutrophil adhesion. Induction of adhesion by wild-type LPLtat but not by constitutively phosphorylated LPLtat could be inhibited by blocking PI3K and PKC, indicating that phosphorylation of LPL through these signaling pathways was likely necessary for the ability of LPL to induce activation of αMβ2 and thus neutrophil adhesion [αMβ2 [A mechanistic link between phosphorylation of LPL and activation of the integrin e of LPL . Signalsadhesion . Additioon [αMβ2 .αvβ3 in the human erythroleukemic cell line K562, which enabled analysis of the function of LPL in a well-established system of activable integrin adhesion [αMβ2 in neutrophils [αvβ3 with the LPLtat peptide promoted integrin activation and cellular adhesion to surfaces coated with the integrin ligand vitronectin [tat activation of αvβ3 was independent of the tyrosine phosphorylation of the cytoplasmic tail of the integrin β chain, indicating that LPL activates integrins in a mechanism different from that of PMA stimulation. LPLtat treatment of cells triggered a conformation change in the integrin, revealing ligand-induced binding sites on the integrin that increased its avidity for its ligand. Changes in integrin avidity required actin depolymerization [αMβ2 and αvβ3 was not examined in these studies. However, LPL has recently been reported to exist in complexes with either β1 or β2 integrins in breast and prostate cancer cells, consistent with the proposal that LPL can directly regulate integrin activity [Induction of integrin activation was confirmed and extended through the examination of activation of adhesion . Similartrophils , treatmeronectin . LPLtat rization . These ractivity .Phosphorylation of LPL has also been linked to generation of NADPH-oxidative burst in neutrophils following stimulation with PMA. Both nonphosphorylated and hyperphosphorylation of LPL correlated with inhibition of PMA-generated NADPH oxidative burst. Optimally phosphorylated LPL correlated with induction of NADPH oxidative burst . These rThe kinases that phosphorylate LPL in neutrophils vary with cell stimulus . Immune tat peptide, it was somewhat of a surprise to find that neutrophils from the LPL−/− mouse were not defective in integrin-mediated adhesion, nor was chemotaxis of LPL−/− neutrophils diminished [−/− neutrophils were defective in integrin signaling to the kinase Syk. Deficient activation of Syk following integrin ligation led to a defective respiratory burst, resulting in an inability to kill Staphylococcus aureus, and LPL−/− mice were more susceptible to staphylococcal skin abscesses. Signaling through both FcγR and PMA to the respiratory burst in LPL−/− neutrophils was intact, indicating that integrin signaling was specifically dependent upon LPL [Given the increase in integrin activation and adhesion triggered by the LPLminished . Howeverupon LPL . These rIn addition to its role in neutrophil biology, LPL has also been recognized as a substrate of phosphorylation during T-cell receptor signaling to T-cell activation as early as 1994 . PhosphoT-cell activation is dependent upon the creation of a specialized contact site between the antigen-presenting cell (APC) and the responding T-cell , 58. Des−/− murine T cells were defective in proliferation and cytokine production when stimulated with plate-bound anti-CD3 or with peptide-pulsed APCs, but not with soluble anti-CD3. Interestingly, proximal TCR signaling events, such as calcium flux, tyrosine phosphorylation of the adaptor protein LAT, and ERK activation and were intact in stimulated LPL−/− T-cells. However, spreading of LPL−/− T cells on anti-CD3 coated surfaces, formation of LPL−/− T-cell: APC conjugates, and generation of the IS in LPL−/− T cells were impaired. These data are all consistent with a model in which LPL is required for later stages of IS formation, stabilization, or maintenance, but is dispensable for early TCR signaling events. A requirement for LPL in later stages of signaling would also be consistent with its identified functions in stabilizing F-actin structures. Defective formation or maintenance of the IS likely contributed to the downstream defects in proliferation and cytokine production of LPL−/− T cells [A requirement for LPL in the formation of the immune synapse was independently confirmed through the analysis of murine T cells genetically deficient for LPL . LPL−/−  T cells . It is n T cells , have no T cells .−/− mice were also found to have diminished chemoattractant-mediated motility, as assessed by two-photon microscopy in explanted lymph nodes as well as in transwell assays [−/− T cells despite normal activation of the small GTPase Rac, induction of a burst of actin polymerization, and upregulation of cellular adhesiveness thought to be due to integrin activation. Motility reduction correlated with diminished polarization of LPL−/− T cells, which failed to generate clearly delineated uropods and lamellipods seen in chemokine-stimulated WT T cells. Phenotypically, the reduced motility of LPL−/− T cells resulted in diminished thymic egress in LPL−/− mice [Lymphocyte trafficking is dependent upon an array of integrins and chemoattractant receptors that promote changes in cell polarity, adhesion, and motility , 63–66. l assays . The red−/− mice . While i−/− mice , a requi−/− mice were found to be deficient for a specialized population of B cells, called marginal zone B cells. Development of MZ B cells is exquisitely sensitive to chemotactic and adhesive signaling, and LPL−/− B cells were found to exhibit reduced motility towards the chemoattractants CXCL12, CXCL13, and sphingosine-1-phosphate. Interestingly, the integrin-mediated increase in cellular motility required LPL, while the chemokine-mediated increase in integrin adhesiveness did not. As seen before, some downstream elements of chemoattractant receptor signaling did not require LPL, as activation of ERK and p38 were intact in CXCL12-stimulated LPL−/− B cells. However, total protein levels and phosphorylation of the integrin-associated kinase Pyk2 were diminished in LPL−/− B cells, providing a possible molecular explanation for the requirement for LPL in lymphocyte motility [B cells also require LPL for chemotaxis-mediated motility . LPL−/− motility .Osteoclasts are highly specialized, hematopoietically derived cells that maintain bone mass and remodel bone structure through the tightly regulated process of bone resorption . LPL hasα-actinin [μM calcium reduced the affinity of LPL for actin by threefold. LPL was also identified as a heavily phosphorylated protein in macrophages stimulated with LPS [LPL was identified as one of the major constituents of cytoplasmic gels isolated from alveolar macrophages, along with actin, filamin, and -actinin . LPL waswith LPS . As seenwith LPS , 74. A swith LPS .The abundance of LPL in macrophage cytoplasm has enabled the imaging of macrophage movement through fluorescently tagged LPL . Fluores−/− neutrophils, grancalcin-deficient neutrophils were able to kill Staphylococcus aureus in vitro, and grancalcin was not required for other immune functions, such as macrophage recruitment to sites of inflammation or resistance to fungal infections [LPL has also been reported to bind to macrophage-specific proteins that may be critical to macrophage function. For instance, LPL was found to bind Iba1 in a two-hybrid screen . The expfections . The funβII and two subunits of the GM-CSF receptor. Treatment with GM-CSF resulted in increased expression of the integrin αMβ2, increased sensitivity to eotaxin in a chemotaxis assay, prolonged survival of eosinophils, and primed eosinophils for degranulation. Inhibition of PKCβII resulted in the reduction of LPL phosphorylation and the loss of these GM-CSF-stimulated effects. Most importantly, internalization of a constitutively phosphorylated LPL peptide (residues 2–19) by eosinophils resulted in increased αMβ2 integrin expression and sensitization to chemotaxis, suggesting a mechanistic link between phosphorylation of LPL and GM-CSF-induced sensitization to chemotaxis [αMβ2 integrin expression by the N-terminal portion of LPL, which does not contain ABDs, suggests that LPL may have intrinsic signaling capabilities that do not require actin-bundling functions.A function of LPL in receptor signaling events beyond its capacity as an actin-bundling protein has been suggested by a recent report that LPL may regulate GM-CSF-mediated sensitization of eosinophils . GM-CSF emotaxis . The indα [LPL is ectopically expressed in many tumor cells, and its function in the potential promotion of invasive potential of transformed cells has been extensively reviewed . In brieα . The invα . Blockadα . Finallyα . Combineα .Despite 25 years of research, an integrated understanding of the function of LPL in hematopoietic cells remains elusive. A clear description of the role of LPL has been frustrated by the inherent technical difficulties of analyzing the rapid, transient, and subcellular events of receptor signaling, adhesion, and motility that are dependent upon the highly dynamic actin cytoskeleton. Advances in imaging technology will address these difficulties. Additionally, the requirement for LPL may vary with cell type and cell stimulus, as LPL was required for chemoattractant-mediated motility of lymphocytes , 70, butβ1 and β2 though the ABDs [β1 and β2 may be regulated by μ-calpain cleavage [−/− T cells was performed on a surface coated with integrin ligand [A function for LPL beyond actin-bundling has been recently suggested by the observation that LPL binds directly to the cytoplasmic portion of integrins the ABDs . This incleavage . LPL wascleavage . A criticleavage , 56, 77.cleavage , 56. Then ligand . Whethern ligand , but uprn ligand , 70. Intn ligand , 68, 70.Mutations in cytoskeletal proteins, such as Wiskott-Aldrich Syndrome protein, coronin, DOCK8 and Rac2, result in human immunodeficiency –92. Whet


From September 14 to 16, 2011 the 12th symposium on Plant Protein Phosphorylation was held in Tübingen, Germany. The topic is as broad as the name suggests and covers all aspects of this important means of protein modification in plants. I have had the pleasure of attending the 2007 and the 2011 symposia. The interesting concept behind these meetings is to hear about the same biochemical mechanism operative in a multitude of experimental systems. The meetings are quite informal and present an excellent mix ranging from technology to biochemical experience and novel findings and tools.++ pathways and phosphoproteomics. It was hosted by the Zentrum für Molekularbiologie der Pflanzen (ZMBP) and the organizing committee chaired by Klaus Harter.The two-and-a-half-day program was divided into five double sessions: biotic interactions, hormone signaling, abiotic interactions, Mitogen Activated Protein Kinase (MAPK), and CaIn the biotic interactions session the role of phosphorylation in Pathogen-Triggered Immunity (PTI) was the central topic. Several speakers discussed differential phosphorylation events after turning on the PTI pathways. The first presentation was by Yuelin Zhang (Beijing) who discussed recent work on identifying targets of the MAPK pathway using a suppressor screen. To what extent cross talk between growth and PTI pathways exists at the level of the receptors was discussed by Cyril Zipfel (Norwich). Important points raised were whether the dynamics of receptor activation as observed in brassinosteroid receptors are directly applicable to the non-Arginine Aspartic Acid (RD) type of kinases that seem to feature in most PTI pathways. A very intriguing report was made by Iris Jarsch (Munich), suggesting a role for remorins to sequester receptors in small membrane micro domains. Looking in the very first events of membrane receptor activation, it appears that changes in heteromeric state of Flagellin Sensing 2 (FLS2) and BRI1 Associated Kinase (BAK1) can be seen a mere few seconds after ligand application . Speed was also the topic of a presentation from the host , showing evidence for direct activation of plasma membrane adenosine triphosphatases (PM ATPases) by the BRI1 receptor. Down-regulation by endocytosis of the FLS2 receptor, negative control elements such as phosphatases appear to play more and more important roles in controlling receptor activity and sensitivity  while genes have been identified that modulate the calcium signature of the PTI response .A much-explored theme in the signaling session was to isolate receptor-interacting proteins by direct pull downs or yeast interaction screens. This was used in approaches by Sorina Popescu (Ithaca) investigating reticulon-like proteins that regulate trafficking and activity of FLS2 and Sacco de Vries (Wageningen) looking at the interaction properties of members of the Somatic Embryogenesis Receptor-like Kinase (SERK) family of non-ligand binding coreceptors that includes BAK1(SERK3). Other work dealt with control of cellulose synthase by phosphorylation , subcellular relocalization of Pin-formed (PIN) proteins through phosphorylation by PINOID kinases  and several talks on the direct control of abscisic acid (ABA) signaling by Protein Phosphatase 2C (PP2C) enzymes  including interesting kinases that lack the conserved lysine in the catalytic domain called With No K (WNK)s; Esther Görlich, Heidelberg). Using an alternative approach by comparing phosphorylation patterns in the presence or absence of a specific inhibitor of the salicylic acid (SA) response-mediating Casein Kinase 2 (CK2), an SA-induced phosphorylated p23 protein was identified that may act in a Heat Shock Protein (HSP) 90 complex . Clearly, molecular approaches using phospho-site mutants are generally applied to unravel functional relevance of the proteins under investigation.Arabidopsis S-phase Kinase Associated Protein 1 (SKP1)-like 5 (ASK5) kinase in linking adaptive regulation of the redox balance via control of the phosphorylation status of the pentose phosphate entry enzyme glucose 6-phosphate dehydrogenase (G6PD). To enlarge the scope of the downstream changes in phosphorylation patterns upon stress induction several talks  used ABA signaling as a starting point to describe genome-wide changes in the phosphoproteome. The challenges in these large-scale projects are obviously the reliability of the predicting software, the coverage obtained, and the required biological verification of the changes encountered. The last two talks in this session were given by Markus Teige (Vienna) and Thomas Guérinier (Orsay). Teige described an elegant approach to purify kinases that give specific changes to target transcription factors, in this case a member of the basic region Leucine Zipper (bZIP) family involved in metabolic reprogramming. This approach also highlighted the temporal and site specific dynamic changes in the phosphorylation pattern. A different set-up was used by Guérinier to identify peptide-based kinase assays to identify novel modulating kinase effectors such as nucleotides using Sucrose Non-fermenting 1 Related Protein Kinase 1 (SnRK1) as a model.The next session was devoted to abiotic signaling and started with Jay Thelen (Columbia) introducing a number of nice generally applicable tools to discover phosphorylation sites MUSite and Kinain vivo phosphoproteomics was used to identify direct targets of the CDPKs. How calcium is sensed intracellularly by Calcineurin B-like Proteins (CBLs) and associated CBL-Interacting Protein Kinases (CIPKs) was discussed by Jörg Kudla (Münster) and showed a highly complex signaling network present at the PM and capable of decoding local changes in calcium and effecting directly sodium and potassium channels. From the same lab and in association with the group of Harter (Tübingen), Kenji Hashimoto presented results showing that the CBL1/CIPK1 pair acts as an important switch in translating light signaling into transcriptional changes. Then the MAPKs took center stage in talks by Souha Berriri (Evry) who used a gain-of-function screen for MAPKs first in yeast, as a tool to identify targets of the MAPK pathway. MAPK4 GOF mutants were then used to help delineate PTI and Effector-triggered Immunity (ETI) targets that run through the same MAPK cascade. The differences and similarities between human and plant innate immunity systems was discussed by Heribert Hirt (Evry) showing that Arabidopsis can be infected by Salmonella typhimurium, the major cause of food poisoning. Two kinases were identified that appear to act in a PTI pathway different from the MAPKs directly under control of PTI-related kinases. Genetic approaches were also employed by Roman Ulm (Geneva) having identified a phosphatase that mediates the UV-B stress response and by Marie Boudsocq (Harvard). In that work, CDPKs were identified that could mimick 22 amino acid flagellin fragment (FLG22) signaling and suggesting that alternative routes exist besides the classical MAPK pathway in PTI signaling. A constitutive CDPK under control of an inducible promoter was used to identify changes in the phosphoproteome .The following session dealt with a series of experimental systems used to gain more insight into the subcellular specificity in signaling systems. The calcium-dependent protein kinases (CDPKs) were presented by Tina Romeis (Berlin) as mediators and perhaps integrators of ABA and Pathogen Associated Molecular Pattern (PAMP) responses. Here The last sessions were devoted to technological advances in phosphoproteomics as being applied to a number of different systems. Themes that emerged were the necessity to use well-defined induction systems, the use of biological and technical replicates and the transient qualitative changes that could be captured.Arabidopsis that clearly showed the improvement of these methods coupled to improved search algorithms. In particular tyrosine phosphorylation appears to be as widespread in Arabidopsis as in our own cells! The effects on the phosphoproteome that can be distinguished after nutrient uptake can be separated into early and late events, involving both different proteins and different phospho-sites. The tools are available through improved web-based applications  were employed to provide reliable data. Further improvements in these procedures were presented by Wolfram Weckwerth (Vienna) using better software prediction coupled to metal oxide purifications and by Pururawa Mayank (Zürich) using similar approaches but now to describe the pollen phosphoproteome. The last talk was given by Thomas Stratil (Münich) returning to the exciting remorin proteins  of which one was also found to be important for Medicago root nodule development. It was directly associated with one of the symbiotic RLKs.Naoyuki Sugiyama (Tsuruoka) introduced Hydroxy Acid Modified Metal Oxide chromatography (HAMMOC) reported to increase the yield and specificity of phosphopeptides from complex digests. A number of examples were shown in both rice and Fifty-six posters of excellent quality were on display in front of the auditorium during the entire meeting and formed the central point of discussion during the coffee and tea breaks. In the short space allotted to this report it is unfortunately not possible to describe them all .in vivo determination of particular phospho-sites is feasible. An example of that can be found in a recent publication of the Jenny Russinova Lab  and better software have all resulted in a situation that Realization that most phosphorylation changes upon or during activation of a pathway are transient and also multiple will require time-lapse recording experiments at the second and minute time scale. So far this is only possible in single or few protein analyses; for an example of such an analysis using the Epidermal Growth Factor Receptor (EGFR; see Dengjel et al., Efforts to visualize first general and next specific phosphorylation events as is now relatively common in mammalian signaling systems (EGFR; Verveer et al.,


E. coli (Escherichia coli), yeast (Saccharomyces cerevisiae) and Arabidopsis  and that it is essential in Arabidopsis as in both micro-organisms. Using a RNA interference strategy, plant lines were generated with a diminished dUTPase activity as compared to the wild-type. These plants are sensitive to 5-fluoro-uracil. As an indication of DNA damage, inactivation of dUTPase results in the induction of AtRAD51 and AtPARP2, which are involved in DNA repair. Nevertheless, RNAi/DUT1 constructs are compatible with a rad51 mutation. Using a TUNEL assay, DNA damage was observed in the RNAi/DUT1 plants. Finally, plants carrying a homologous recombination (HR) exclusive substrate transformed with the RNAi/DUT1 construct exhibit a seven times increase in homologous recombination events. Increased HR was only detected in the plants that were the most sensitive to 5-fluoro-uracils, thus establishing a link between uracil incorporation in the genomic DNA and HR. Our results show for the first time that genetic instability provoked by the presence of uracils in the DNA is poorly tolerated and that this base misincorporation globally stimulates HR in plants.Deoxyuridine triphosphatase (dUTPase) enzyme is an essential enzyme that protects DNA against uracil incorporation. No organism can tolerate the absence of this activity. In this article, we show that dUTPase function is conserved between  Deamination of cytosine (C) results in the formation of uracil (U) in DNA which will code for adenine in the following replication cycle, thus giving rise to a C to T (thymine) transition Uracils in DNA are repaired by the base excision repair (BER) pathway. They are first specifically recognized and excised by the uracil DNA glycosylase (UDG), leaving abasic sites E. coli (Escherichia coli) and yeast (Saccharomyces cerevisiae) and thus only leaky point mutations have been isolated for this gene E. coli, the dut-1 mutant exhibits an increased homologous recombination (hyper-Rec) phenotype and is synthetically lethal with mutations in RecA or RecBC, two proteins that are central to the homologous recombination (HR) pathway DUT1 gene (dut1-1) triggers a spontaneous mutator phenotype The gene coding for the dUTPase function is essential in E. coli RecA, is essential for these steps. In C. elegans (Caenorhabditis elegans), partial loss of dUTPase activity results in the accumulation of RAD51 foci in the chromatin suggesting that DSBs have been created and are being repaired by HR Trypanosoma (Trypanosoma brucei), also results in the introduction of DSBs, as chromosome fragmentation is observed Arabidopsis .HR is one of the mechanisms by which DNA DSBs can be repaired, even though it is not preponderant in plants Arabidopsis DUT1 gene can functionally complement the E. coli dut-1 mutant and yeast dut1-1 mutant and that this gene is essential in Arabidopsis. In order to knock-down dUTPase expression, as no dut1 mutant was available from the Arabidopsis collections of insertion lines, we produced RNAi/DUT1 lines DUT1 plants were effectively depleted in dUTPase activity, and showed sensitivity to 5FU. These plants exhibited RAD51 and PARP2 induction, and an increased frequency of HR events in their somatic cells, suggesting the presence of DNA damage and HR stimulation.In this article, we describe the consequences of a reduction in dUTPase activity in plants, in the continuity with our very preliminary published data dutΔ (deletion) mutant is lethal in E. coli, but the dut-1 partial inactivation (point mutation) mutant is viable and sensitive to the presence of uracil (10 mM) or an analog of uracil, 5FU (50 µM) Arabidopsis DUT1 gene, we transformed the E. coli dut-1 mutant with a plasmid expressing the AtDUT1 cDNA. The transformants were tested for their capacity to grow on uracil or 5FU containing media. Wild-type (WT) E. coli transformed with an empty vector remained sensitive to the presence of uracil or 5FU, whereas the growth of this same dut-1 mutant on uracil or 5FU containing medium was restored to a similar level as the WT E. coli upon transformation with the AtDUT1 cDNA (recA(Ts) dut-1 or recBC(Ts) dut-1 double mutants are dead when grown at restrictive temperature (37°C). This lethality is rescued by AtDUT1 expression  is viable DUT1 cDNA were examined for their growth on 5FU-containing medium. The yeast dut1-1 mutant transformed by an empty vector remained sensitive to 5FU, while yeast dut1-1 transformed with the AtDUT1 cDNA grew as well as the WT on 5FU-containing media . Furthermore, it was partially alleviated upon the use of the pDMC1 promoter DUT1, DMC1 or EMB506) set under the pAlcA promoter and thus aimed at expressing the RNAi constructs under ethanol exposure. Only the lines containing the pAlcA:RNAi/DUT1 construct died when grown on ethanol containing media (0.05%)  . These dDUT1 construct on DUT1 mRNA levels in the two independent RNAi/DUT1-4 and RNAi/DUT1-5 transgenic lines, chosen as 5FU sensitive, quantitative RT-PCR analyses were carried out. RNA extractions were performed on young leaves. The amount of DUT1 mRNA detected in the RNAi/DUT1-4 and RNAi/DUT1-5 lines was decreased to 12% and 34%, respectively compared to WT plants  after the addition of dUTP. The dUTPase activity in both RNAi/DUT1 plants was found to be reduced by 40% of that measured in control plant (RNAi/0) extracts  and AP site (AP∶G) repair activities in protein extracts comparing WT to RNAi/DUT1 plants, as described in DUT1 seedlings. It should be noted that to measure UNG activity, a substrate that contains a uracil (U∶G) is added to the extract, and this substrate must first undergo uracil excision, and then apurinic site endonucleotytic cleavage for the UNG activity to be detected. Therefore, UNG activity is the sum of both uracil excision and apurinic site incision. Unexpectedly, we observed that the level of UNG activity was diminished by 30 to 40% in the RNAi/DUT1 plant protein extracts compared to the WT  of WT plants. Compared to non-irradiated plants, RAD51 was induced 230 times in irradiated plants and 13- to 11- fold, respectively in the RNAi/DUT1-4 and RNAi/DUT1-5 plants, compared to WT  DUT1 construct was introduced into plant line 58F by transformation. The sensitivity to 5FU was assessed in selected plants in order to establish the RNAi silencing efficiency of the DUT1 gene in these new transformants , whereas one (58F/RNAi/DUT1-luc9) was as 5FU resistant as plants transformed with the empty vector, and was thus considered as not depleted in dUTPase activity. As a control, we selected three independent lines called 58F/RNAi/0-luc1, -luc2 and -luc3, which were transformed with an empty RNAi vector. The level of HR was monitored as the frequency of luciferase foci per seedling . The effectiveness of our dUTPase complementation also suggests that this protein has no critical partners, the independent evolution of which would have precluded it to be functional in different backgrounds.Our data clearly show that dUTPase activity is essential in Arabidopsis, we obtained, via a RNAi strategy, plants in which dUTPase activity was partially decreased. This decrease was attested at the transcription and the enzyme activity levels. It led to reproducible phenotypes that corroborate the fact that a lack of dUTPase is deleterious to cells in plants as previously shown in E. coli and yeast. It should be noted that such a conservation of phenotype between E. coli and Arabidopsis is not always observed. These plants were extremely sensitive to 5FU, grew poorly, died when the RNAi construct was induced, showed DNA breaks as indicated by the “TUNEL assay”, and induced genes specific to the DNA-damage response  in response to these DNA breaks.Since no insertional mutants were available in DUT1 lines with an Arabidopsis rad51 mutant showed that they were compatible. This is not always the case in micro-organisms such as E. coli where the dut1-1 mutation becomes lethal when accompanied by either recA(Ts) or recB(Ts) mutations when grown at non-permissive temperature Arabidopsis are not repaired via the HR pathway, as previously discussed E. coli. This appears to be an important difference between prokaryotes and eukaryotes since the absence of a functional homologous recombination repair pathway is most detrimental to E. coli while it is well-tolerated in higher eukaryotes such as Arabidopsis.However, crossing our RNAi/DUT1 plants provided, and only when, the RNAi is efficient at decreasing the dUTPase activity (which is ascertained by the 5FU sensitivity of the transgenic RNAi/DUT1 plants). This occurs consequently to a modification of the DNA itself without the introduction of any recombinase. A comparable result was observed when similar reporter lines were exposed to genotoxic stresses such as methyl-methone-sulfonate or bleomycin that are known to induce double strand breaks DUT1 plant cells, DSBs were formed as an effect of the reduced dUTPase activity. HR was increased seven-fold in the RNAi/DUT1 plants even though it was quantified with a construct that only presents 1146 bp of homology. The chances of having a DSB occurring in this region are lower than when an I-SceI site is introduced and used as an initiator for HR once cut, which increased HR by a factor of up to two orders of magnitude in tobacco cells via a reduction in dUTPase activity might become very efficient. Therefore, knocking down DUT1 may be a good tool to obtain gene targeting in plants.Finally, our data show that HR is probably increased in the RNAi/DUT1 cDNA was cloned into pSE380 (Promega) for expression in E. coli (Escherichia coli) and into p415GAL1 for expression into yeast (Saccharomyces cerevisiae) DMC1 (meiotic) expression in plants are described in Siaud et al.DUT1 is named K68 in Siaud et al. (2010) et al.DMC1 or pAlcA promoters in the different experiments.The E. coli dut-1 mutants were provided by A. Kuzminov − l− rac−thi-1 hisG4 Δ(gpt-proA)62 argE3 thr-1 leuB6 kdgK51 rfbD1 araC14 lacY1 galK2 xylA5 mtl-1 tsx-33 supE44(glnV44) rpsL31(strR); dut-1 (AK105 dut-1 zic-4901::Tn10: AB1157×P1 PK4001); the WT, recA(Ts) and recBC (Ts) ± the dut1-1 mutation were as in dut1-1 mutant was provided by S. Boiteux MATa leu2-3,112 trp1-289 his7-2 ura3-52 lys1-1) and dut1-1 to BG217 .Yeast E. coli were selected as ampicillin resistant. Depending on the assay, plates were examined in the presence or absence of uracil (10 mM) or 5FU (50 µM) at 37°C. When examining thermosensitive mutations, plates were incubated for 24–36 h at the required temperature (20°C or 37°C). Transformed yeasts were selected on SD-Leucine, their promoter was induced with galactose and they were examined in the presence or absence of 5FU (50 µM). The plates were incubated for 48 h at 30°C.Transformed Arabidopsis thaliana plants  were grown in the greenhouse with a photoperiod of 16 h light and 8 h dark at 23°C or in vitro on MS 0.5  with or without 5FU (50 µM). The pAlcA promoter constructs were examined in vitro with or without ethanol at 0.05%. Plants carrying p35S::AlcR construction with a Nos terminator (pSRN) are described in Arabidopsis rad51 mutant is described in DUT1 ; ACTIN ; RAD51 ; PARP2  and 1 µL of cDNA in a total volume of 25 µl. Each experiment, from RNA extraction to cDNA synthesis, was performed twice independently, and quantitative RT-PCR was performed using triplicate samples. The relative level of mRNA reported in the figures was normalized using the ACTIN mRNA level.Total RNA from young leaves were isolated with the Nucleospin kit (Macherey-Nagel). Reverse transcription was performed using the 〈〈SuperScript™ First-Strand Synthesis System for RT-PCR〉〉 kit (Invitrogen) according to the manufacturer instructions. Real-time amplifications were performed in a LightCycler® 480 Real-Time PCR System (Roche) with LightCycler® 480 SYBR Green I Master gene using specific primers: 2, 100 µM dUTP (Sigma-Aldrich) at 30°C. The reaction was initiated by adding protein extract and aliquots were taken at different times. The reaction was stopped by heating to 100°C. After centrifugation, the supernatant was subjected to reverse-phase ultra performance chromatography  using a UPLC HSS T3 1.8 µm (100 mm•2.1 mm i.d.) column (Waters). dUMP formed in the course of the reaction was eluted using a linear gradient of 5% methanol in 15 mM Triethylamine pH 7.9 (0.6 mL.min-1) and detected at 260 nm using a diode array detector (Waters 2696 PDA detector). Peak areas were calculated using the Empower software (Waters), using commercial dUMP as a standard (Sigma). To ensure that steady-state conditions were applied, dUMP was measured at three time points. Product formation was linear for at least 45 min.Total soluble proteins were extracted from 0.3 g of leaves from two month-old plants. Leaves were collected and crushed in the presence of liquid nitrogen and then 1 ml of extraction buffer was added: 100 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 5 mM DTT and 1 µl of protease inhibitor . The suspension was centrifuged at 14 000 g for 10 min at 4°C and supernatants were desalted on a NAP-5 column  and eluted with extraction buffer. Protein concentrations were measured according to Bradford et al (1976) 2, 250 mM Saccharose, 10% Glycerol, 1 mM DTT) with 16 µL of protease inhibitor cocktail (Sigma-Aldrich). After one hour in ice and a centrifugation, supernatant was filtered and dialyzed in dialysis buffer . Protein concentrations in the extracts were measured according to Bradford and collaborators  0.5 g of leaves were crushed in the presence of liquid nitrogen and 1.5 mL of extraction buffer was added  were incubated at 30°C for 3 hours in a reaction mixture containing, 2.5 mM Hepes KOH at pH 7.8, 10 mM KCl, 2.5% Glycerol, 1 mM DTT, 0.2 mM EDTA and 10 µg of protein extract in a total volume of 50 µl. Reactions were stopped by adding EDTA to 20 mM, sodium dodecyl sulfate to 0.6%, and proteinase K to 200 µg/ml, and the mixtures were incubated for 30 min at 37°C. DNA was extracted with phenol∶chloroform∶isoamyl alcohol (25∶24∶1) and ethanol precipitated at −20°C in the presence of 0.3 mM NaCl and 16 µg/ml glycogen. Samples were resuspended in 10 µl of 90% formamide, and heated at 95°C for 5 min. Reaction products were separated in a 12% denaturing polyacrylamide gel containing 7 M urea. Fluorescein-labelled DNA was visualized using the blue fluorescence mode of the FLA-5100 imager and analysed using Multigauge software (Fujifilm).The TUNEL assay was conducted as in The detection of bioluminescence was performed on two-week-old T2 generation seedlings of independent lines. Seedlings were sprayed with a solution of Luciferin (1 mM Luciferin (Biosynth AG), 0.05% Triton X-100) and visualized with a LCD camera used with the Photolite software. The number of spots per plant was then measured. Spots indicate the presence of a reconstituted active luciferase gene and hence occurrence of recombination events.