Previously, antigens expressed from DNA vaccines have been fused to the VP22 protein from Herpes Simplex Virus type I in order to improve efficacy. However, the immune enhancing mechanism of VP22 is poorly understood and initial suggestions that VP22 can mediate intercellular spread have been questioned. Despite this, fusion of VP22 to antigens expressed from DNA vaccines has improved immune responses, particularly to non-secreted antigens.Bacillus anthracis, the causative agent of anthrax. Protective immunity against infection with B. anthracis is almost entirely based on a response to PA and we have generated two constructs, where VP22 is fused to either the N- or the C-terminus of the 63 kDa protease-cleaved fragment of PA (PA63).In this study, we fused the gene for the VP22 protein to the gene for Protective Antigen (PA) from B. anthracis strain STI spores, no difference in protection was evident in groups immunised with the DNA vaccine expressing PA63 and the DNA vaccines expressing fusion proteins of PA63 with VP22.Following gene gun immunisation of A/J mice with these constructs, we observed no improvement in the anti-PA antibody response generated. Following an intraperitoneal challenge with 70 50% lethal doses of 63.VP22 fusion does not improve the protection of A/J mice against live spore challenge following immunisation of DNA vaccines expressing PA The VP22 protein is a major component of the amorphous tegument region of the Herpes Simplex Virus type I HSV-1). Composed of 301 amino acids, it has become known as a protein transduction domain able to mediate intercellular spread. Like other translocatory proteins such as antennapedia and the HIV Tat protein, it is highly basic, it is able to bind heparin or sialic acid and all three proteins have an almost identical predicted pI [. ComposeThe ability to 'piggyback' proteins or peptides into cells may be particularly useful for gene therapy. Thymidine kinase and p53 have benefited from fusion with VP22 . VP22 hain vitro studies that use methanol fixation. Because methanol dissolves cellular membranes, it may produce an artefact interpreted as cell to cell spread [However, the ability of VP22 to mediate intercellular spread has been questioned, based on l spread . In furtl spread  and a ful spread . A critil spread .in vivo responses to a number of antigens not only in the context of gene therapy, but also when fused to antigens within a DNA vaccine. This could be particularly useful because although DNA vaccines can offer protection against a wide variety of pathogens in small animal models, their efficacy in larger animal models and primates is insufficient. In this study, we evaluate the potential of VP22 to enhance DNA vaccines against anthrax.Irrespective of whether VP22 can mediate intercellular spread however, VP22 can enhance Bacillus anthracis causes the disease anthrax. The current UK-licensed vaccine is an alum-precipitated filtrate of a B. anthracis Sterne strain culture, administered by the intramuscular route, which occasionally causes some transient reactogenicity in vacinees [The spore-forming bacterium vacinees . The US-vacinees .The key component in both these vaccines is the protective antigen (PA), which along with lethal factor (LF) and edema factor (EF) forms a tripartite toxin and is one of the virulence factors of the bacteria . Host ceB. anthracis expressing either the 63 kDa fragment of PA [DNA vaccines against nt of PA ,20 or thnt of PA . Protectnt of PA -22. Atte63) attached to a secretion signal. The VP22 protein, which has previously been shown to improve the performance of DNA vaccines [63. We show that following gene gun administration of these vaccines, fusion with VP22 does not improve anti-PA antibody responses to the PA63 DNA vaccine, nor does it increase protection against anthrax lethal spore challenge.The aim of this study was to assess the potential of VP22 to enhance the immunogenicity of a DNA vaccine expressing the 63 kDa fragment of PA  using primers VP22 F9 (5' ACTCTAGCTAGCACGGCGCCAACCCGATCCAAGACA 3') and VP22 R8 (5' ATTGTCACGGTCTGGAACCGTAGGAGCAGCTGGACCTGGACCCTCGACGGGCCGTCTGGGGCGAGA 3'). Additionally, the gene for PA63 was PCR amplified from pGPA using primers PA F8 (5' CCTACGGTTCCAGACCGTGACAAT 3') and PA R9 (5' CGCGGATCCTTATCCTATCTCATAGCC 3'). The two sequences were then fused together by PCR [The DNA vaccine pGPA contains the signal sequence for human plasminogen activator fused to the N-terminus of the gene for the 63 kDa fragment of PA  and was r by PCR  using pr63 sequence was PCR amplified from pGPA using primers PA F11 (5' CTAGCTAGCCCTACGGTTCCAGACCGTGACAAT 3') and PA R10 (5' TGTCTTGGATCGGGTTGGCGCCGTAGCAGCTGGACCTGGACCTCCTATCTCATAGCC 3'). The gene for VP22 was PCR amplified from pCR®T7/VP22-1 (Invitrogen) using primers VP22 F10 (5' CGGCGCCAACCCGATCCAAGACA 3') and VP22 R11 (5' CGCGGATCCTTACTCGACGGGCCGTCTGGGGCGAGA 3'). The two sequences were then fused together using VP22 F10 and VP22 R11 by PCR fusion [63 proteins, to allow folding of the fusion protein. Using restriction sites NheI and BamHI, the PA63 gene was exised from pGPA and the gene fusions were ligated into the vector to form pSTU-22-PA  and pSTU-PA-22 . These constructs were verified by sequencing and are schematically represented in figure To create the C-terminal fusion, the gene for the PAR fusion . The PCRThe control DNA vaccine expressing VP22 only (pSTU22) has been previously described . The pla5 cells well-1 into 6 well plates (Corning). Cells were transfected with 1 μg of plasmid DNA using the transfection reagent Polyfect (Qiagen) according to the manufacturer's guidelines. Transfected cell lysates were separated by 4–20% polyacrylamide gel electrophoresis , using XCell SureLock™ Mini-Cell apparatus (Invitrogen) according to the manufacturer's protocol. Protein from the gel was then transferred to nitrocellulose by electroblotting (Invitrogen). An ECL Western blotting kit (Amersham Biosciences) was used with antibody to PA  or VP22  to detect expression from DNA vaccines.African Green Monkey Kidney COS-7 cells  were plated at 1–5 × 10Groups of 10 female A/J mice (Harlan OLAC) were immunised with 1 μg of DNA coated onto gold particles and delivered using a Helios™ gene gun (BioRad) as described previously . Mice we-1 recombinant PA (Aldevron) in phosphate-buffered saline using 50 μl well-1 and incubated overnight at 4°C. Three columns on each plate were coated with anti-IgG (Fab) (Sigma) in order to produce a standard curve for quantification of IgG concentration. After washing three times with PBS containing 0.2% Tween-20, non-specific binding was blocked with 5% (w/v) powdered skimmed milk in PBS and the plates were incubated for 2 hours at 37°C. The plates were washed three times and serum was added at a starting dilution of 1:50 in blocking buffer, and double-diluted down the plate. IgG or isotype standards (Sigma), diluted in blocking buffer, were added to wells which had been coated with anti-IgG (Fab) (Sigma), and double diluted as before. Plates were incubated for 1.5 hours at 37°C before washing and the addition of goat anti-mouse IgG (or anti-mouse IgG isotype) conjugated to horseradish peroxidase (Sigma), diluted in blocking buffer. Plates were incubated for 1 hour at 37°C, then washed 3 times before addition of the substrate ABTS (Sigma). Absorbance at 410 nm was measured after 20 minutes incubation at room temperature and analysed using Ascent software.Microtitre plates were coated with 5 μg mlB. anthracis STI (Tox+ Cap-) spores. Sufficient spores for the challenge were removed from stock cultures, washed in sterile distilled water, and resuspended in PBS to a concentration of 7 × 105 spores ml -1. Mice were challenged with 100 μl volumes containing 7 × 104 spores per mouse  and wer.One-way ANOVA with Tukey's multiple comparison post analysis test and statistical analysis of survival using the Mantel-Haenszel Logrank test were performed using GraphPad Prism version 3.02 for Windows, GraphPad Software, San Diego, California, USA 63, VP22-PA63, PA63-VP22 or VP22 .Groups of 10 female A/J mice were immunised three times by gene gun administration of 1 μg plasmid DNA at two weeks intervals. Serum samples were collected 17 days after the third immunisation . Sera from individual mice were assayed for PA-specific total IgG Figure . Mice imB. anthracis strain STI by the intraperitoneal route. The DNA vaccine expressing PA63 conferred 70% survival to the immunised mice. In comparison, 80% and 50% of the mice survived following immunisation with the DNA vaccines expressing VP22-PA63 and PA63-VP22, respectively .Mice were challenged three weeks after the final dose with 70 50% lethal doses of y Figure . Thus inin vitro studies of this protein remain inconclusive, with reports that the apparent effects of VP22 can be attributed to an artefact produced by methodology [in vitro studies, most in vivo work shows that VP22 has a beneficial effect, particularly in the gene therapy field. Fusion of VP22 to either the pro drug-activating enzyme thymidine kinase [The Herpes Simplex virus type I VP22 protein has been suggested to mediate intercellular spread by exit from cells in a golgi-independent manner and entry to adjacent cells by a non-endocytic mechanism [hodology ,10,31,32e kinase  results e kinase , Enhancee kinase  and the e kinase ,25,33,34B. anthracis. This antigen expressed from a DNA vaccine is protective against challenge with either lethal toxin (PA plus LF) in Balb/c mice [63 at either terminus failed to significantly enhance anti-PA antibody responses compared to the PA63 DNA vaccine. Following challenge, all three DNA vaccines expressing either PA63, VP22-PA63 or PA63-VP22 offered significant protection against 70 LD50's of B. anthracis compared to unimmunised control mice. However, the inclusion of VP22 did not significantly increase or decrease the protection afforded when compared to PA63-expressing DNA vaccine alone. This suggests that the fusion of VP22 to either the N- or C-terminus of PA63 within a DNA vaccine, does not alter either the antibody response elicited in vivo or the protection afforded to A/J mice following spore challenge. The longevity of the immune response or the ability of these DNA vaccines to initiate long-term protection was not evaluated in this study.In this study, the VP22 protein has been fused to either the N- or C-termini of the Protective Antigen (PA) of b/c mice ,20 or spb/c mice . We used63 contrasts with other YFP, EGFP or HPV E7 antigens expressed from DNA vaccines where improvement is evident [B. anthracis challenge contrasts with studies involving DNA vaccines expressing HPV E7 protein where protective anti-tumour immunity was increased with VP22 fusion [63-expressing DNA vaccine used here does contain a secretion signal.The failure of VP22 fusion to increase antibody responses to PA evident -26. Furt2 fusion ,34. Howe2 fusion . The PA6The inclusion of a secretion signal is a commonly used strategy for DNA vaccination as liberation of the protein from the cell can increase immune responses -37. The 63 of B. anthracis. The VP22 protein has been shown previously to enhance the performance of DNA vaccines expressing non-secreted proteins. In this case, the PA63-expressing DNA vaccine contains the human plasminogen activator signal sequence [63-expressing DNA vaccine encoding a secretion signal.This study investigates the inclusion of the VP22 protein in a DNA vaccine expressing PAsequence . InclusiThe author(s) declare that they have no competing interests.SDP, HCF-S, HSG, AEE-L carried out the studies. FKS, RJP participated in the design of the study. All authors read and approved the final manuscript.


Retroviruses by definition insert their viral genome into the host cell chromosome. Although the key player of retroviral integration is viral integrase, a role for cellular cofactors has been proposed. Lentiviral integrases use the cellular protein LEDGF/p75 to tether the preintegration complex to the chromosome, although the existence of alternative host proteins substituting for the function of LEDGF/p75 in integration has been proposed. Truncation mutants of LEDGF/p75 lacking the chromosome attachment site strongly inhibit HIV replication by competition for the interaction with integrase. In an attempt to select HIV strains that can overcome the inhibition, we now have used T-cell lines that stably express a C-terminal fragment of LEDGF/p75. Despite resistance development, the affinity of integrase for LEDGF/p75 is reduced and replication kinetics in human primary T cells is impaired. Detection of the integrase mutations A128T and E170G at key positions in the LEDGF/p75–integrase interface provides in vivo evidence for previously reported crystallographic data. Moreover, the complementary inhibition by LEDGF/p75 knockdown and mutagenesis at the integrase–LEDGF/p75 interface points to the incapability of HIV to circumvent LEDGF/p75 function during proviral integration. Altogether, the data provide a striking example of the power of viral molecular evolution. The results underline the importance of the LEDGF/p75 HIV-1 interplay as target for innovative antiviral therapy. Moreover, the role of LEDGF/p75 in targeting integration will stimulate research on strategies to direct gene therapy vectors into safe landing sites. Viruses have a limited genome and therefore exploit the cellular machinery of infected host cells to complete the replication cycle. Today there is a growing interest to unravel these virus–host interactions. Lentiviral integrases use the cellular protein LEDGF/p75 to tether the preintegration complex to the chromosome, although the existence of alternative host proteins substituting for the function of LEDGF/p75 has been proposed. Here, we used virus evolution to investigate the role and importance of LEDGF/p75 in HIV integration and replication. We selected a virus that is resistant against overexpression of the integrase binding domain of LEDGF/p75. This virus displays a reduced affinity for its own cofactor but still depends on the cofactor for replication. We demonstrate the unique and essential role of LEDGF/p75 in HIV replication and support the LEDGF/p75 HIV-1 interplay as target for innovative antiviral therapy. Moreover, elucidation of the role of LEDGF/p75 in targeting integration will stimulate research on strategies to direct gene therapy vectors into safe landing sites. Although a dynamic interplay between the virus and host factors occurs at all steps of the HIV replication cycle, the identity and role of these cellular cofactors are poorly understood. LEDGF/p75 is a cellular cofactor of HIV-1 integrase (IN), which catalyses integration of the HIV-1 DNA into the host cell genome . The tigSeveral studies have addressed the question whether LEDGF/p75 is an important cofactor for viral replication in vivo. The Q168 residue in HIV-1 IN was found to be critical for the interaction with LEDGF/p75 . A virusIn this study, we show that HIV can develop resistance to overexpressed truncation variants of its own cellular cofactor, although at the expense of a reduced affinity to LEDGF/p75 and highly reduced replication kinetics. To our knowledge, such resistance phenotype to a cellular cofactor has not been described before. Detection of mutations at key positions in the LEDGF/p75–IN interface provides critical evidence of the importance of LEDGF/p75 for viral replication.IN gene of this virus (unpublished data). Therefore, we started to select resistant strains by repeated passaging of HIV-1 (NL4.3) in MT-4 eGFP-Δ325 cells. As a control, virus was passaged in parallel in MT-4 eGFP-Δ325 D366A cells. By reducing the volume of the inoculum , selective pressure was progressively increased during the experiment.Overexpression of LEDGF/p75 truncation mutants containing the IBD severely inhibits viral replication . Figure IN gene. Since MT-4 cells better resemble natural host cells of HIV-1 infection, subsequent experiments focused only on the specific IN mutations found by selection in MT-4 eGFP-Δ325 cells. After 21 passages (#21), two mutations, V38A and S113N, were also found in the gp120 encoding regions of the selected virus population; however, they were present in only 25% and 50% of the virus population, respectively. Moreover, these mutations were also detected in the gp120 gene of the virus population passaged on MT-4 eGFP-Δ325 D366A cells, although in a lower percentage of the virus population. These mutations likely represent adaptation of the virus to cell culture and infection conditions.To verify whether the selected HIV-1 strains were indeed less susceptible to the inhibitory effect of the overexpressed LEDGF/p75 C-terminal domain, MT-4 eGFP-Δ325 and MT-4 eGFP-Δ325 D366A cells were infected with the parental HIV-1 strain (NL4.3), the HIV-1 strain selected for 21 passages on MT-4 eGFP-Δ325 cells (NL4.3/MT-4 eGFP-Δ325 [#21]), and the HIV-1 strain selected for 21 passages on MT-4 eGFP-Δ325 D366A cells (NL4.3/MT-4 eGFP-Δ325 D366A [#21])  Figure A and 2B.IN mutations are necessary and together sufficient for the strong rescue phenotype of the selected HIV-1 strain in MT-4 eGFP-Δ325 cells.To evaluate the importance of the described mutations for the observed resistant phenotype, these mutations were cloned in the HIV-1 NL4.3 molecular clone (pNL4.3) as single or double mutants . Susceptibility of the molecular clones to eGFP-Δ325 overexpression was determined by studying multiple round HIV-1 replications in MT-4 eGFP-Δ325 A and in IN gene have not been described before. Therefore, we studied the impact of the IN mutations on the enzymatic level in an oligonucleotide-based overall integration assay  B. All muTo determine the step at which the replication cycle of eGFP-Δ325 resistant virus is affected, real-time quantitative PCR analysis was performed to quantify the formation of the different DNA species in the cell. To ensure single-round infection, 293T cell lines were infected with vesicular stomatitis virus G protein (VSV-G) pseudotyped virus. DNA was extracted at several time points postinfection. As shown in To analyze the rescue of viral replication by real-time quantitative PCR, 293T eGFP-Δ325 cells and control 293T eGFP-Δ325 D366A cells were infected with WT VSV NL4.3 or VSV NL4.3 A128T/E170G. As shown in s (mRFP-INs) fusion protein localized to the nucleus of WT HeLaP4 cells  LEDGF/p75. First, confocal imaging was used to study the cellular localization of the different mutant INs A. As expP4 cells A 21–26]–26s  and IN containing the A128T mutation (IN-A128T) were able to pull down LEDGF/p75, MBP-IBD, and MBP-Δ325. On the contrary, almost no interaction was detected with the E170G or A128T/E170G double mutation. The same results were obtained when N-terminal WT and mutant His-tagged INs for pull-down were used (unpublished data). The highly reduced interaction of MBP-Δ325 with IN-E170G and IN-A128T/E170G was analyzed in more detail with different IN and MBP-Δ325 concentrations using AlphaScreen technology  with WT mRFP-INs was readily detected (p < 0.01), indicating that both proteins interact with WT IN in the cytoplasm of the living cell (p < 0.001). The cross-correlation signal for the interaction of A128T/E170G mRFP-INs with eGFP-Δ325 was more affected than that for interaction with full-length LEDGF/p75, suggesting that the mutations in viral IN differentiate between Δ325 and full-length LEDGF/p75.To further characterize these interactions in vivo, we applied fluorescence cross-correlation spectroscopy (FCCS) in HeLa-P4 derived cell lines. This method allows the quantification of the interaction of two fluorescently labeled proteins in cellulo by measuring simultaneous diffusion. Because the diffusion of the complex mRFP-INlization , and meaing cell . The binA crystal structure of the dimeric catalytic core domain (CCD) of HIV-1 IN complexed to the IBD of LEDGF/p75 was previously reported . A128 isHow does HIV overcome the replication block induced by the C-terminal fragment of its cofactor? FCCS measurements suggest that by altering the affinity of IN for the cofactor, HIV-1 may be capable to discriminate between endogenous LEDGF/p75 and the inhibiting C-terminal fragment (eGFP-Δ325). Alternatively, HIV-1 that has become deficient for interaction with LEDGF/p75 may replicate independently from LEDGF/p75, by employing another cofactor. The latter explanation has been put forward recently after analysis of integration sites in LEDGF/p75 knockdown cells . LEDGF/p.In line with these results and to further strengthen our model that the A128T/E170 virus is still capable of interacting with LEDGF/p75 in vivo, we speculated that poor replication of the double mutant virus in WT cells could be rescued by overexpression of LEDGF/p75. For this purpose, MT-4 cells stably overexpressing LEDGF/p75 and MT-4 cells stably overexpressing eGFP were generated. Overexpression was verified by Western blot analysis A. These We have previously characterized LEDGF/p75 mutants containing the IBD but lacking the chromosome attachment site (eGFP-Δ325 and eGFP-IBD) that strongly impair HIV-1 replication by competing for the interaction with IN . We haveIN gene compared with other viral genes ATP (Amersham Biosciences). The DNA substrate and target for IN reactions were made by annealing INT1 and INT2. An equimolar mixture of the two oligonucleotides in the presence of 100 mM NaCl was heated shortly at 95 °C and allowed to cool slowly to room temperature.The HPLC-purified deoxyoligonucleotides, INT1 (5′-TGT GGA AAA TCT CTA GCA GT) and INT2 (5′-ACT GCT AGA GAT TTT CCA CA), corresponding to the U5 end of the HIV-1 LTR, were purchased from Amersham Biosciences . Then, 6 μg of recombinant C-terminal His6-tagged IN was incubated with 40 μl of Ni-NTA-agarose for 10 min at 4 °C. Next, 6 μg of LEDGF/p75 in 200 μl of binding buffer supplemented with 4 μg of BSA was added to the mixture. Following 4-h incubation at 4 °C, the agarose beads were recovered by centrifugation for 2 min at 13,000 rpm at 4 °C and washed several times with 300 μl of binding buffer. Bound proteins were eluted in 20 μl of binding buffer supplemented with 200 mM imidazole and 1% SDS and analyzed by 11% SDS-PAGE followed by staining with Coomassie Blue R-250 [Hiss et al. , with miue R-250 .2, 0.01% Tween-20, 0.1% BSA). The mixture was incubated for 90 min at room temperature. For subsequent binding of the proteins to the beads, Ni-chelate–coated acceptor beads and streptavidin-coated donor beads premixed with biotinylated anti-MBP antibody  were added to a final concentration of 20 μg/ml. All dilutions were done in assay buffer and, if necessary, the final volume of the reaction was adjusted to 25 μl. In order to bind the anti-MBP antibody to the streptavidin-coated donor beads, the antibody was dialyzed overnight against assay buffer without BSA, added to the beads in a final concentration of 50 nM, and incubated for 1 h at room temperature. After addition of the beads to the binding assay, light exposure was omitted and the incubation was carried out for an additional 60 min before analyzing the interaction in the EnVision plate reader (Perkin Elmer).To confirm the loss of interaction of the mutant INs to the Δ325 fragment, AlphaScreen technology (Perkin Elmer) was performed. In a 384-well plate, 0, 30, 100, or 300 nM of IN was added to 0, 10, 30, or 100 nM MBP-Δ325 at room temperature in assay buffer  Schematic representation of HIV-1 integration in MT-4 and MT-4 eGFP-Δ325 cells. In MT-4 cells, association with LEDGF/p75 tethers IN to the chromatin, facilitating the integration of the viral cDNA in the host genome. When eGFP-Δ325 is overexpressed, eGFP-Δ325 competes with endogenous LEDGF/p75 for binding to IN and thereby inhibits viral integration. Note that the concentrations of LEDGF/p75 and eGFP-Δ325 proteins shown are arbitrary.(B) HIV-1 (NL4.3) replication in MT-4 eGFP-Δ325 D366A cells (filled diamonds) and MT-4 eGFP-Δ325 cells (open diamonds). MT-4 cells were infected at an MOI of 0.1, and viral replication was followed by measurement of p24 antigen in the supernatant.(4.1 MB TIF)Click here for additional data file.Figure S2(A–C) 293T cells were infected with VSV-G pseudotyped viral clones: WT NL4.3 (diamonds), NL4.3 A128T (boxes), NL4.3 E170G (triangles), or NL4.3 A128T/E170G (crosses). DNA was extracted at different time points postinfection, and the amounts of (A) total viral DNA, (B) 2-LTR circles, and (C) proviruses were determined by quantitative PCR.(D–F) 293T eGFP-Δ325 and 293T eGFP-Δ325 D366A cells were infected with VSV-G pseudotyped viral clones: WT NL4.3 (diamonds) or NL4.3 A128T/E170G (crosses). DNA was extracted at different time points postinfection, and the amounts of (D) total viral DNA and (E) 2-LTR circles were determined by quantitative PCR. To quantify integrated DNA (F), the total viral DNA was measured 144 h postinfection . Quantifications were performed in duplicate. Averages ± SD are shown.(4.1 MB TIF)Click here for additional data file.Figure S3s, 24 h before laser scanning microscopy.HeLaP4, HeLaP4 eGFP-Δ325, and HeLaP4 eGFP-Δ325 D366A were transfected with different mRFP-INss expression in HeLaP4 cells.(A) mRFP-INs expression in HeLaP4 eGFP-Δ325 and HeLa P4 eGFP-Δ325 D366A cells.(B) mRFP-INs expression upon overexpression of eGFP-LEDGF/p75 in HeLaP4 cells.(C) mRFP-IN(6.4 MB TIF)Click here for additional data file.Figure S4http://www.pymol.org). IN chains A and B are colored blue and green, respectively, while the IBD subunits are violet. Selected residues are shown as sticks, and hydrogen bonds are indicated by dotted lines.Models were drawn with PYMOL  Binding of the two WT catalytic core domains with the IBD (PDB code 2B4J). A128 is part of the α3 helix in the IN-CCD, which forms a hydrophobic patch that accommodates the side chains of the LEDGF residues I365, F406, and V408 [(B) Representation of the model showing the effect of mutating A128 to T128 and E170 to G170. For A128T, the bulky side chain of threonine causes sterical hindrance, thereby forcing the I368 residue of the IBD to adapt a different conformation. This occurs without a drastic influence on the conformation of residues in the LEDGF/p75 IN binding interface. The E170G mutation has a more drastic effect. This mutation results in a different conformation of the loop disrupting two interactions. First, the double hydrogen bond between the hydrogens of the loop backbone at residues E170 and H171 and D366, a critical interacting residue on LEDGF/p75, is broken. Second, the electrostatic interaction of E170 (IN) with K364 (LEDGF/p75) is abolished.(6.5 MB TIF)Click here for additional data file.Figure S5(A) Western blot analysis of LEDGF/p75 expression levels in MT-4 cells (lane 1) and MT-4 cells stably overexpressing LEDGF/p75 (lane 2). As a control, MT-4 cells expressing eGFP were made (MT-4 eGFP) (lane 3). Equal loading was controlled by α-tubulin detection.(B) MT-4 LEDGF/p75 cells and MT-4 eGFP cells were infected with 10 pg/ml p24 antigen: WT NL4.3 (diamonds) or NL4.3 A128T/E170G (crosses). The replication kinetics for the viruses was determined by measuring viral p24 antigen levels in the supernatant. Experiments were performed in duplicate. Averages ± SD are shown.(1.3 MB TIF)Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the proteins discussed in this paper are LEDGF/p75 (NM_033222) and HIV-1 NL4–3 IN .The Genbank (


The effects on experimental melanoma of a combination of recombinant human tumour necrosis factor alpha  and carmustine (BCNU) were studied in vitro and in vivo. In vitro, BCNU alone was cytotoxic to murine B16 melanoma cells, and at all concentrations of BCNU this toxicity was increased by the addition of TNF. In vivo, BCNU and TNF, when given separately, caused tumour growth delay of B16 melanoma and of human melanoma xenografts in immune-deprived mice. The combination of TNF at low dose 2.5 x 10(5) U kg-1 = 122 ng kg-1) with BCNU (35 mg kg-1) resulted in significant growth delay  in B16 melanoma (P = 0.005). There was no significant increase in toxicity as assessed by weight loss and peripheral blood counts. Experiments with human melanoma xenografts yielded similar results (P = 0.001) but only at higher doses of TNF (1 x 10(6) U kg-1 = 489 ng kg-1). The enhancement of BCNU cytotoxicity by TNF may be important if it can be translated into patients with melanoma. A randomised study is now underway to investigate the clinical potential of this observation.


Mei11,Dmc1, Dmc1 encoding a missense mutation in the L2 DNA binding domain that abolishes strand invasion activity. Meiosis in male heterozygotes arrests in pachynema, characterized by incomplete chromosome synapsis and no crossing-over. Young heterozygous females have normal litter sizes despite having a decreased oocyte pool, a high incidence of meiosis I abnormalities, and susceptibility to premature ovarian failure. Mei11Dmc1 exposes a sex difference in recombination in that a significant portion of female oocytes can compensate for DMC1 deficiency to undergo crossing-over and complete gametogenesis. Importantly, these data demonstrate that dominant alleles of meiosis genes can arise and propagate in populations, causing infertility and other reproductive consequences due to meiotic prophase I defects.DMC1 is a meiosis-specific homolog of bacterial RecA and eukaryotic RAD51 that can catalyze homologous DNA strand invasion and D-loop formation in vitro. DMC1-deficient mice and yeast are sterile due to defective meiotic recombination and chromosome synapsis. The authors identified a male dominant sterile allele of  Dmc1 that causes male-specific infertility due to defects in meiosis. Dmc1 encodes a key protein required for meiotic recombination; the mutation causes a single amino acid change that prevents genetic exchange, or crossing-over, in males, abolishes its recombination activity, and abrogates the production of sperm. Though heterozygous females are fertile, they have fewer oocytes due to a high incidence of meiosis I abnormalities, and show susceptibility to premature ovarian failure. Importantly, these data demonstrate that dominant alleles of meiosis genes can arise and propagate in populations, and produce meiotic prophase I defects that cause infertility and other reproductive abnormalities.About 10%–15% of couples are infertile due to defects in meiosis (the process by which egg or sperm cells containing a single copy of each chromosome are produced). Because studying the genetics of meiosis in humans is difficult, we performed genetic screens in mice and identified a novel mutation in  Meiosis occurs in both the male and female germ lines; here the authors describe a mutation that affects only male meiosis, causing sterility. Genetic recombination occurs in all organisms and is critical for repair of DNA damage, proper chromosome segregation during meiosis, and genetic diversification. Recombination in yeast and mice is initiated by the formation and processing of double-strand breaks (DSBs). Meiotic DSBs are repaired by proteins that mediate homologous strand exchange, mismatch repair, and resolution of recombination intermediates. As these activities are occurring, homologous chromosomes undergo pairing and synapsis, which are completed by the pachytene stage of meiosis. The ability of germ cells to complete meiosis, and to undergo proper segregation of chromosomes in the subsequent meiotic divisions, hinges on the fidelity of these events. Defects in recombination and meiosis have been shown to underlie aneuploidy syndromes such as Downs  and azooOur understanding of the genetic control of meiotic recombination in mammals has depended largely on studies of model organisms such as yeast. Mice with null mutations in orthologs of recombination genes often have meiotic defects that are very similar to yeast. However, it is clear that mammals have many genes required for meiosis that do not have orthologs in yeast, and that there are substantial differences between the sexes in the response to mutations in meiotic genes –5. AdditMei1, a novel vertebrate-specific meiosis gene subsequently implicated in human male infertility [Mei11)Dmc1  formation [Mei11Dmc1 has the unusual property of causing autosomal dominant male sterility. Our studies of this allele provide insight into the biochemical properties of DMC1, underscore stark sexual dimorphism in meiotic recombination and response to errors thereof, and provide experimental proof that autosomal dominant meiotic mutations can arise, propagate, and cause mammalian infertility.t manner , and humt manner . In Saccormation –13. In c1) daughter of a chimeric male, a fully penetrant, dominant male-specific sterility trait was discovered and mapped to Chr 15 [(Mei11). Since Mei11 can be transmitted only through females, yet the pedigree was initiated by a male chimera, it is possible that the mutation arose not in embryonic stem cells but in the G1 female or one of her descendants.In previous work, phenotype-driven screens were conducted on pedigrees of mice derived from chemically mutagenized embryonic stem cells . During o Chr 15  (see MatMei11 in the C3HeB/FeJ (C3H) strain background (≥N3) was arrested in meiotic prophase, with an absence of postmeiotic spermatids  in all affected Mei11/+ males and carrier females . To verify this mutation causes the mutant phenotype, we generated mice containing Mei11 in trans to a Dmc1 null allele. This resulted in female sterility concomitant with ovarian defects (see below). Furthermore, we found that B6-Mei11/−Dmc1 testes underwent complete prophase I meiotic arrest equivalent to that in −Dmc1−/ males (not shown), without signs of postmeiotic differentiation as in B6-Mei11/+ males  or aberrant pachytene-like morphology (62%), containing a mixture of synapsed, partially synapsed, and asynapsed chromosomes , Dmc1Meomosomes B. Consisomosomes E. Similastaining C on someWe also monitored synapsis using an antibody against phosphorylated histone H2AX (γH2AX). While serving as a marker of leptotene DSBs , H2AX phMei11/+Dmc1, Mei11Dmc1/−Dmc1, and wt zygotene nuclei  compared with wt  spermatocytes, suggesting that DMC1Mei11 is deficient for RN incorporation . We observed no obvious difference in numbers of RAD51/DMC1-positive foci between e nuclei . Normalle nuclei G. The ra pattern H, but RA pattern I, arrows pattern J. When wporation .Mei11/+Dmc1 spermatocytes, we probed meiotic chromosomes with an antibody against the mismatch repair protein MLH3, a marker of chiasmata [Mei11Dmc1/+ chromosomes . We observed approximately wt levels of more developed follicles, reflecting the ability of ovaries to recruit and mature a “normal” number of oocytes from a smaller resting pool.To examine effects of ckground , and theckground A and 3B.ckground C and 3D.Mei11Dmc1/Dmc1− females were sterile, regardless of genetic background. Interestingly, ovaries of such females fell into two phenotypic classes. Approximately 60% (8 of 13) exhibited a relatively normal ovarian morphology with developing and antral-stage follicles  had fully synapsed meiotic cores .To determine whether the reduced oocyte number in mutant adult females was related to recombination and synapsis defects, we examined prophase I chromosomes from embryonic ovaries. Whereas the majority of embryonic day 17.5 oocytes from wt embryos were at mid-pachynema  with fully synapsed autosomes as indicated by colabeling with anti-SYCP1 and anti-SYCP3 A, only 1ic cores B, whereaic cores C. Dmc1−/ene-like D. Intereic cores . These aMei11/Dmc1+ mid-pachytene stage oocyte nuclei were devoid of RAD51/DMC1 foci , although the distribution of crossovers on different chromosomes varied , lying within the counterpart of the structurally disordered “Loop 2” (L2) domain of RecA [Mei11, we constructed an equivalent A272P mutation within the human protein (HuDMC1Mei11) and purified it in bacteria , even after addition of the HOP2-MND1 complex  or presence  that occurs in response to asynapsis, and/or failure of sex chromosome inactivation in mid-pachynema , the latMei11Dmc1/+ females were fertile despite exhibiting a high percentage of abnormal meioses, and that most mutant oocytes were capable of normal crossing-over compared with the complete lack thereof in spermatocytes, underscores the stark sexual dimorphism with respect to checkpoint control and recombination mechanisms. A key underlying difference may have to do with the timing of checkpoint activation in meiotic prophase I. For example, an earlier checkpoint activation may occur in spermatocytes before expression of one or more essential components of late RNs, preventing them from being made or modified in a way that is required for late RN formation. Notably, MLH3 foci are apparent prior to pachynema in human oocytes [RAD51 or RAD54 can partially rescue meiosis and sporulation of dmc1 yeast [The fact that  oocytes , and com oocytes . Later c oocytes . Anotherc1 yeast ,39.−/−Dmc1 spermatocytes are essentially devoid of homologous synapsis, Mei11Dmc1/−Dmc1 spermatocytes have a mixture of pachytene-like synapsed homologs and asynapsed chromosomes. Therefore, DCM1Mei11 clearly retains some activity that is sufficient to drive some degree of synapsis. Though it is completely unable to catalyze D-loop formation in vitro, the mutant protein might facilitate recombinant activities of other molecules so as to initiate interhomolog pairing. In mammals, DMC1 has been shown to interact with many proteins, including RAD51, RAD54B, BRCA2, MSH4, MND1, and HOP2 [Mei11 protein may retain recombinogenic activity in vivo by virtue of protein–protein interactions that are not recapitulated in the in vitro system. Alternatively, other molecules such as RAD51 may be able to drive some amount of interhomolog pairing independently of DMC1, but DMC1Mei11 retains an additional, independent role in synapsis per se. Notably, S. cerevesiae is capable of low levels of crossover recombination in the absence of DMC1 [Whereas and HOP2 ,41. Ther of DMC1 .Mei11 did not exert a dominant negative effect on D-loop formation in vitro. One possibility for this discrepancy is that DMC1Mei11 forms dominant negative interactions with another key protein(s) in vivo, resulting in defective DSB processing. The mutant protein did not impair the ability of wt DMC1 to catalyze strand invasion in the presence of HOP2-MND1, ruling them out as the target of dominant-negative interactions. However, as stated above, mammalian DMC1 is known to interact with several other meiotic proteins involved in recombination and DNA repair. Since the Mei11Dmc1 mutation decreased DNA binding efficiency in vitro to approximately 30% of wt activity, consistent with a decreased number of DMC1 foci in mutants, it is possible that the dominant-negative interaction may result from binding to a key protein(s) that depends upon DMC1 for localizing to sites of DSB repair.Despite acting in a genetically dominant fashion in vivo, recombinant HuDMC1−/−Dmc1 mutant females, oocytes are depleted around birth prior to primordial follicle formation, resulting in residual ovaries that are depleted of ovarian follicles and oocytes [Mei1 or Spo11 in DMC1-deficient mice permits further meiotic progression through metaphase I in females [Mei11Dmc1/+ females were normally fertile, but had a deficit of primordial and primary stage follicles. Depletion of early-stage follicles in response to unrepaired chromosome breaks and/or asynapsis has also been reported for mice with mutations in Spo11 and Sycp3 as well as mouse models of Turner's syndrome [Mei11Dmc1/+ females are able to compensate by recruiting a near-wt number of oocytes for ovulation. This type of compensation has also been reported for mouse models of Turner's syndrome, busulfan-treated rats, and ovariectomized mice [Dmc1 [In  oocytes ,12. The  females ,44. Younsyndrome ,43,45. Ozed mice –48. The zed mice . Notablyce [Dmc1 .Mei11Dmc1/+ oocytes revealed that a significant proportion had unrepaired breaks and asynapsed chromosomes at the time when most oocytes normally reach pachynema and diplonema. It is likely that these meiotic defects (particularly unrepaired DSBs) are sufficient to trigger checkpoint-mediated apoptosis in a subset of oocytes, leading to the observed shortfall of follicles. Some or most of the oocytes that proceeded past this checkpoint were capable of fertilization and development. We do not know if this entire cadre of “successful” oocytes was completely normal, or whether some escaped checkpoint elimination with a subcritical level of aberrations. Future experiments may determine whether mutant oocytes that survive postnatally retain meiotic errors and are subsequently either eliminated prior to fertilization via the spindle checkpoint or alternatively, survive to produce some level of aneuploidy in fertilized oocytes.Cytological analysis of Mei11Dmc1 provide proof that dominant mutations can arise in a key meiotic recombination gene such as Dmc1 and cause infertility in mice. Because of the high conservation in the process of meiosis, it is highly likely this could occur in humans with similar consequences . What makes such a mutation particularly insidious is that it is transmissible through populations via females, who would not manifest any apparent problems until well into their reproductive lifespans.Our genetic and functional studies of http://www.emdbiosciences.com) to generate a protein linked to a histidine tag on the amino terminus. The proteins were overexpressed in E. coli BL21 (DE3) and purified using the following series of chromatographic steps: Ni-NTA-agarose , heparin , MonoQ, and Superdex 200. The proteins were concentrated and stored in buffer  at −80 °C.Human wt and A272P mutant cDNAs were cloned into the pET-15b vector : 1, 100-mer, TGTGGAATGCTACAGGCGTTGTAGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGG; 2, 100-mer, CCACCCTCATTTTCAGGGATAGCAAGCCCAATAGGAACCCATGTACCGTAACACTGAGTTTCGTCACCAGTACAAACTACAACGCCTGTAGCATTCCACA; 3, 60-mer AATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATT.These oligonucleotides were synthesized by MWG Biotech (32P)-ATP using standard labeling procedures. The labeled strand was then annealed to the complementary strand (oligonucleotide 2) to create blunt-ended dsDNA and gel purified in 7% polyacrylamide, 1× TAE buffer. Finally, all the duplexes were purified using ion exchange columns . The single-stranded and the double-stranded 100-mer oligonucleotides were then mixed with either wt or A272P HuDMC1 in reactions containing 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 10% glycerol, and 1 mM MgCl2. The reaction mixtures were incubated at 37 °C for 10 min, and products were analyzed by electrophoresis in 8% polyacrylamide gels in 1× TAE buffer at 5 V/cm for 3 h. The formation of nucleoprotein complexes was quantitated using a BAS 2500 Bio-imaging Analysis System .Oligonucleotide 1 was 5′ end-labeled with . hDMC1 and/or A272P hDMC1 were preincubated with the 60-mer oligo for 5 min, followed by incubation with dsDNA for 15 min at 37 °C. When both HuDMC1 and A272P hDMC1 were incubated together, 100 μg/ml BSA was added to the reaction mixture. Reactions were stopped by the addition of 0.5% SDS and 1 mg/ml proteinase K and incubation for 15 min at 37 °C. Products were resolved on 1% agarose gels in 1× TAE containing 3 mM MgCl2. Gels were dried to DE81 paper and analyzed with the BAS 2500 Bio-imaging Analysis System.Supercoiled pUC18 plasmid DNA was purified by CsCl banding. Oligonucleotide 3  with homology to the pUC18 plasmid was radiolabeled and mixed with supercoiled pUC18 (8 μM bp). Reaction mixtures contained 1 mM DTT, 20 mM Tris-HCl (pH 7.4), 2.5 mM MgClprod,i / Ppitotal,i), where prod,iPpi are pixels per inch of oligonucleotide ssDNA in supercoiled dsDNA (plasmid), total,iPpi are the sum of pixels per inch of free oligonucleotide ssDNA and oligonucleotide ssDNA in supercoiled dsDNA, and A is the molar ratio of oligonucleotide ssDNA with respect to dsDNA in the reaction mixture.The amount of D-loop relative to dsDNA was calculated using this formula: % dsDNA in D-loop = A × 100 ×  mutation. Sgdp, like Mei11, also causes male-specific infertility, but not due to meiotic arrest. At first, only the Sgdp phenotype was seen and characterized histologically. Afterwards, in the course of mapping Sgdp, males were phenotyped on the basis of testis size, an autosomal dominant pattern of inheritance was evident, and linkage to Chr 15 was obtained [Sgdp, but of Mei11. We cannot determine whether Sgdp and Mei11 were both present in the original pedigree, or if Mei11 arose thereafter. Initial genetic mapping indicated that the Mei11 mutation was segregating with B6 microsatellite alleles on distal Chr 15. For high-resolution genetic mapping, C3H-Mei11/+ females were backcrossed to C3H, and 810 offspring were genotyped for the presence of crossovers in the 7.5 Mb Mei11 critical region, between markers D15Mit68 and D15Mit107. Recombinant males were scored for the mutant phenotype by checking for presence/absence of epididymal sperm or examining testis histopathology. Recombinant daughters were backcrossed to C3H for phenotyping of recombinant sons.The stock of mice containing the Mei11 mutant allele was identified using the forward primer 5′ TTGTGACCAATCAAATGACAC 3′ and the common reverse primer listed above. The −Dmc1 allele was genotyped as described [The wt allele was identified using the forward primer 5′ TTGTGACCAATCAAATGACAG 3′ and the common reverse primer 5′ CCTAGGATCATCCCCCAAGT 3′. The http://www.invitrogen.com).Total RNA was isolated from adult mouse testes using RNA midi-prep columns (Qiagen). cDNA was generated from 2 mg of randomly primed RNA using Superscript II ; mouse DMC1 monoclonal ; rabbit anti-phosphorylated H2AX (γH2AX), serine 139 ; rabbit anti-STAG3 ; rabbit anti-MLH3 ; rabbit anti-RPA , and human anti-CREST . All secondary antibodies conjugated with either Alexa Fluor 488 or Alexa Fluor 594  were used at a dilution of 1:1,000. All images were taken with a 100× objective lens under immersion oil.The methodology used for surface spreading and immunolabeling of spermatocyte and oocyte and meiotic chromosomes was as described ,51. Primst RAD51 ); rabbitParaffin-embedded ovaries (see above) were entirely serial sectioned at 6 μm. Follicle counts were carried out under standard light microscopy, with every tenth section counted. Ovarian follicles were scored and categorized as primordial, primary, secondary, early antral, and antral according to previously described morphological criteria . Only thFigure S1Mei11. Schematic representation of mouse Chr 15, showing the relative location of MIT markers used in genetic mapping  and the number of recombinants/total backcross progeny genotyped.(A) Positional cloning of Dmc1 RT-PCR products. Noncoding strand is shown. Mei11 contains two adjacent nucleotide changes, resulting in double peaks in the heterozygote .(B) Sequence traces of Mei11, which encodes the amino acid immediately proximal to the L2 loop.(C) DMC1 protein structure. A, B, L1, and L2 represent amino acid sequences encoding Walker Motif A, Walker Motif B, and disordered loop regions L1 and L2, respectively. The asterisk indicates the location of the A272 > P mutation in DMC1(46 KB PDF)Click here for additional data file.Figure S2−Dmc1; Mei11Mei11, Dmc1.Wt (A) and mutant  spermatocyte surface–spread nuclei were colabeled with antibodies against SYCP3 (red) and RAD51 (green). +, wt; −, (120 KB PDF)Click here for additional data file.Figure S3. +, wt; −, −Dmc1; Mei11Mei11, Dmc1.Surface-spread spermatocyte chromosomes were colabeled with antibodies against STAG3  and DMC1(113 KB PDF)Click here for additional data file.Figure S4y-axis) from both ovaries of 6-wk-old mutant and wt control littermates as described in Materials and Methods. The types of follicles are indicated on the x-axis. Mei11 = B6− Mei11Dmc1/+ ; Mei11/− = B6− Mei11/Dmc1−Dmc1 ; wt = C57BL/6J . Error bars are standard error of the mean.The graph shows ovarian follicle counts ((9 KB PDF)Click here for additional data file.Figure S5Genotype abbreviations are as in (A–B) Mutant oocytes with >40 meiotic cores showing a lack of synapsis.(C) Wt oocyte nucleus with fully synapsed chromosomes.(D) Core fragmentation in mutant as suggested by lack of CREST staining (white arrow denotes fragmented core lacking centromeric CREST staining).(E) Persistent DSBs as identified by persistent RAD51 foci.(140 KB PDF)Click here for additional data file.Figure S6Mei11 proteins, with clones and yeast strains constructed as described in Materials and Methods. All strains were grown on SD-Leu/Trp/His/Ade agar plates. 1–2: DMC1 bait/DMC1Mei11 prey; 3–4: DMC1 bait/DMC1prey; 5–6: DMC1Mei11 bait/DMC1Mei11 prey.Shown are yeast two-hybrid interactions of mouse wt and DMC1(37 KB PDF)Click here for additional data file.Figure S7Mei11 (A272P). HuDMC1 and A272P were incubated together in the indicated ratios  with ssDNA, followed by the addition of dsDNA. For mock treatments, an identical volume of A272P-free buffer was added to the reaction .(A) The ability of human DMC1 (HuDMC1) to form D-loops was analyzed in the presence of varying amounts of HuDMC1(B) Graphs of data from experiments of the type shown in (A). These represent an average of four independent experiments.(38 KB PDF)Click here for additional data file.Table S1(19 KB PDF)Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) accession number for the mouse Dmc1 transcript is NM_010059.The GenBank (


We show that the Tol2 transposon can efficiently integrate DNA sequences larger than 10 kb into human cells. We characterize minimal sequences necessary for transposition (miniTol2) in vivo in zebrafish and in vitro in human cells. Both the 8.5-kb Tol2 transposon and 5.8-kb miniTol2 engineered elements readily function to revert the deficiency of fumarylacetoacetate hydrolase in an animal model of hereditary tyrosinemia type 1. Together, Tol2 provides a novel nonviral vector for the delivery of large genetic payloads for gene therapy and other transgenic applications.Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrate genomes but exhibit diminished activity when cargo exceeds 8 kilobases (kb). This size restriction limits their molecular genetic and biotechnological utility, such as numerous therapeutically relevant genes that exceed 8 kb in size. Furthermore, a greater payload capacity vector would accommodate more sophisticated  Sleeping Beauty (SB) and the naturally occurring fish transposon, Tol2. The authors explore and systematically unlock some of the potential of Tol2, characterizing a minimal set of transposon sequences required for gene transfer by the Tol2-encoding enzyme, transposase. The authors further demonstrate full activity of this “mini” element in human tissue culture cells and in the treatment of a mouse model of tyrosinemia. Tol2 demonstrates high cargo-capacity, readily transferring large  DNA sequences, an ability that opens the door to an array of molecular genetic approaches in vertebrates previously difficult or impossible using prior tools.Mobile genetic elements (transposons) are effective vehicles for the delivery of foreign DNA for gene therapy and gene discovery applications. Their utility in vertebrates has been, however, limited to relatively few known elements with high activity, including the engineered element  Tol2 [mariner transposons Sleeping Beauty (SB) [Frog Prince [PiggyBac [Sleeping Beauty transposable element for molecular genetic applications in vertebrates. While exceptionally active in higher vertebrates [(overexpression inhibition) [Tol2 is widely used as a transgenesis tool in the zebrafish Danio rerio due to its high level of activity in this organism's germline [Tol2 has not been previously reported, nor have the transposon sequence requirements for Tol2-based transposition been previously well characterized.The natural medaka fish hAT gene family element Tol2 –3, the euty (SB)  and Frogg Prince , and thePiggyBac  representebrates , the SB ibition) ,9. The agermline ,11. HoweTol2 vectors in zebrafish, we noticed an unusually high number of GFP-positive cells in embryos injected with Tol2 transposon and synthetic transposase mRNA derived from an updated Tol2 transcription vector , with an average founder fish transmitting an estimated five independent insertion events (unpublished data).While testing green fluorescent protein (GFP)-marked n vector , suggestnjection B. This mtetramer ,15 and m a dimer . We alsoTol2 activity assay using zebrafish embryos. We used this assay to address a significant drawback of Tol2—minimal sequences required for transposition had not been previously defined. The commonly used Tol2 vector [Tol2 vector and tested them in our zebrafish somatic transposition assay (Tol2 DNA and 202 nucleotides of Tol2 3′ DNA (“miniTol2”). A further truncation of the Tol2 sequences (“Tol2 clipped”) by PCR inactivated the element, however, indicating that there are sequence requirements beyond the minimal 17-base inverted terminal repeats. The miniTol2 vector was subsequently tested in human cells in vitro  versus the presumed size of the SB progenitor (2 kb) might explain the surprisingly high activity that was observed for the 5-kb Tol2 element in human cells  have been reported for integrating viral vectors including retroviruses and lentiviruses . The SB an cells A. We testhe 5-kb C elementTol2 an excellent candidate for molecular medicine applications. We therefore tested whether Tol2 can be used for gene delivery in live animals. We first generated a Tol2 transposon encoding a firefly luciferase expression cassette to facilitate in vivo imaging of live animals. We then injected 5 μg of pTol2/luc with varying amounts of pCMV-Tol2 to achieve gene transfer into mouse liver using a rapid, high-volume (hydrodynamic) injection method [Large cargo-capacity and gene transfer efficiency in human cells make n method ,22. LuciTol2 to correct a mouse model of hereditary tyrosinemia type 1 (HT1). HT1 is caused by deficiency of fumarylacetoacetate hydrolase (FAH) leading to the accumulation of fumarylacetoacetate, which is toxic to hepatocytes. HT1 patients can be treated with 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3 cyclohexanedione (NTBC), a compound that blocks an enzyme upstream of FAH, thus preventing toxicity [Tol2 in this gene therapy application, FAH knockout mice maintained on NTBC were treated by hydrodynamic injection of either standard or minimal (mini) versions of pTol2/FAHIL, a transposon that encodes both murine FAH and firefly luciferase as products of a single transcription unit  and Tol2-R1 (5′-CAGACTAGTCTACTCAAAGTTGTAAAACC-3′) and the pBluescript-based Tol2 transposase vector described by Parinov et al. (2004) as template. The PCR fragment was first cloned into pCRIItopo vector , resulting in pDB595. For RNA expression, the Tol2 ORF was subcloned from pDB595 as BamHI-SpeI fragment into BglII-SpeI digested transcription vector pT3TS [ATATGCGGCCGCCACCATGGAGGAAGTATG-3′) and M13R primers, cloned into pCR4topo vector (Invitrogen), and then a Tol2 NotI fragment was excised and cloned into the NotI site of pCMV-bgal vector  in the forward orientation. The SB10 ORF [The or pT3TS , resultit al. 200 as templTo construct pTol2/S2EF1a-GM2 (pDB591), the S2EF1a-GM2 expression cassette  was clonTol2 transposition in zebrafish somatic tissues, two strategies were used. Tol2 sequences between SwaI and XcmI sites of pDB591 were deleted to make pDB665 (delta exon1), and sequences between XcmI and EcoO109I sites were deleted to make pDB664 (delta repeats). Also, S2EF1a-GM2 cassette was cloned into pGEM-T/Tol2 in reverse orientation between the following restriction enzyme sites: SwaI and EcoRV , EcoRV and HindIII , and SwaI and HindIII . To make a “clipped” version of Tol2 transposon, S2EF1a-GM2 expression cassette was amplified from pDB371 [CAGAGGTGTAAAGTACTTGAGTAATTTTACTTGATTACTGTACTTAAGTATGCATGCAAGCTAGTACAAGACG-3′) and Tol2-nru-R (5′-CAGAGGTGTAAAAAGTACTCAAAAATTTTACTCAAGTGAAAGTACAAGTACTTATCGCGATGATAATCAGCCATACC-3′). The PCR fragment was cloned into pCR4topo vector (Invitrogen) and sequenced.To define minimal sequences required for m pDB371  using prTo express FAH and firefly luciferase in mouse, bicistronic expression cassette consiting of miniCAGGS promoter, mouse FAH cDNA, encephalomyocarditis virus internal ribosome entry site, firefly luciferase, and rabbit β-globin polyadenylation sequences was subcloned as an NheI-PmeI fragment from pKT2/FAHIL  into EcoRV-digested pGEM-T/Tol2  . Two days later, the cells were trypsinized and 3 × 104 cells were plated onto 10-cm plates with complete medium supplemented with 600 μg/ml Geneticin . After 10 d of selection, the cells were stained with methylene blue and counted. Three plates of cells were transfected for each data point using different transposon DNA preps. For Tol2/SB activity comparison (Tol2 elements (http://rsb.info.nih.gov/ij). The number of colonies obtained in transfection including transposase was divided by the number of colonies obtained in control transfection to compute gene transfer efficiency. The efficiency of miniTol2/SVneo was set as 100%.A colony-forming assay was conducted as described . Brieflymparison B, colonielements C, coloniACTGGGCATCAGCGCAATTCAATTG-3′) and Tol2-F7 (5′-AGCAGGATAAAACCTTGTATGCATT-3′) for the first round and Tol2-R4 (5′-ATAATACTTAAGTACAGTAATCAAG-3′) and Tol2-F8 (5′-CTCAAGTAAGATTCTAGCCAGATAC-3′) for the nested reaction. For iPCR of cell clones, Neo-resistant colonies were picked and amplified 2 wk after tranfection with pDB625 and pCMV-Tol2. Genomic DNA was extracted, digested with NheI, SpeI, and XbaI and ligated in a dilute reaction. The first round of iPCR was performed with Tol2-R3 and Tol2-F7, and a second with Tol2-R4 and Tol2-F7. The PCR products were isolated by gel electrophoresis, and sequenced using primers Tol2-R4 or Tol2-F7. For cloning of miniTol2 insertion sites, HT1080 cells were transfected with miniTol2SVneo and pCMV-Tol2 and grown for 4 d without selection. Genomic DNA was extracted and digested with AflIII (which cuts the vector twice outside of the transposon and leaves incompatible ends) SpeI and XbaI, ligated in a dilute reaction, and one round of PCR was performed with Tol2-R3 and Tol2-F8. The resultant products were used in a Topo-cloning reaction (Invitrogen), and two insertions were identified by sequencing using M13-F and M13-R primers. For the mouse insertion sites, genomic DNA was extracted from each of five liver lobes of one FAH-deficient mouse 2 mo after injection with pTol2-FAHIL. The DNA was digested with NheI, BamHI, XhoI, and BsiWI, which do not cut vector sequences, blunted with Klenow fragment, and ligated in a dilute reaction. Two rounds of iPCR were performed and sequenced as described above for cell clones.DNA was isolated from cultured cells or mouse liver using the Puregene DNA purification system . DNA was digested and used in an adapted inverse PCR (iPCR) protocol . Tol2-spSB/Tol2 comparison in the GFP fluorescence analysis  mice , 18 to 2At various times after injection of DNA, animals were anesthetized by intraperitoneal injection of 160 mg/kg ketamine plus 0.8 mg/kg acepromazine and 0.08 mg/kg butorphanol. One hundred milliliters of 28.5 mg/ml luciferin substrate was then injected intraperitoneally. At 4 to 5 min after injection of substrate, the live anesthetized mice were imaged for 1 s to 2 min using an intensified, charge-coupled device camera  as described . Raw val


Escherichia coli is used today as the microbial factory for production of plasmid DNA vaccines. To avoid hazardous antibiotic resistance genes and endotoxins from plasmid systems used nowadays, we have developed a system based on the food-grade Lactococcus lactis and a plasmid without antibiotic resistance genes. We compared the L. lactis system to a traditional one in E. coli using identical vaccine constructs encoding the gp120 of HIV-1. Transfection studies showed comparable gp120 expression levels using both vector systems. Intramuscular immunization of mice with L. lactis vectors developed comparable gp120 antibody titers as mice receiving E. coli vectors. In contrast, the induction of the cytolytic response was lower using the L. lactis vector. Inclusion of CpG motifs in the plasmids increased T-cell activation more when the E. coli rather than the L. lactis vector was used. This could be due to the different DNA content of the vector backbones. Interestingly, stimulation of splenocytes showed higher adjuvant effect of the L. lactis plasmid. The study suggests the developed L. lactis plasmid system as new alternative DNA vaccine system with improved safety features. The different immune inducing properties using similar gene expression units, but different vector backbones and production hosts give information of the adjuvant role of the silent plasmid backbone. The results also show that correlation between the in vitro adjuvanticity of plasmid DNA and its capacity to induce cellular and humoral immune responses in mice is not straight forward.For reasons of efficiency  Escherichia coli, with its concomitant benefits and drawbacks, has been the production host of choice. The benefits include a high DNA yield and well-established procedures for down-stream processing of the plasmid [E. coli contains highly immunogenic endotoxins, or lipopolysaccharides (LPS), in its outer membrane. Because of the net negative charge of both LPS and DNA, these molecules may be co-purified by the ion exchange principle used in the purification of plasmid DNA [E. coli vaccine plasmids are typically maintained during growth by plasmid-encoded antibiotic resistance and addition of antibiotics to the growth medium. Thus, antibiotics may be contaminants in the purified DNA vaccines with the potential of inducing allergic responses in disposed individuals [Lactococcus lactis with improved safety properties concerning the microbial production host and the genetic content of the plasmid [L. lactis is attractive for production of vaccines due to its food grade status. The developed plasmid is free of antibiotic resistance genes and is based entirely on L. lactis genes; the minimal theta type plasmid replicon [hom-thrB operon [L. lactis host strain containing an internal deletion in the hom-thrB operon on the chromosome. Thus plasmid complementation relieves the strains requirement for threonine. Other food grade genetic markers have been developed in L. lactis to meet the high safety demands of optimised and recombinant starter cultures for use in the manufacture of dairy products. An example is the use of suppressible pyrimidine auxotrophs where suppressor tRNA is encoded by the plasmid and allows read through of the pyrF gene containing an amber mutation [L. lactis for plasmid DNA production is new. We have investigated the use of the new L. lactis-based plasmid as a potential HIV-1 DNA vaccine by including a eukaryote expression unit encoding the gp120 surface molecule from the primary CCR-5-trophic HIV-1BX08 [L. lactis genes are relatively AT rich compared to the more GC rich E. coli genes and thus may contain fewer putative ISS.Genetic immunization or DNA vaccination has initiated a new era of vaccine research. The technology involves the inoculation of plasmid DNA into a living host to elicit an immune response to a protein encoded on the plasmid . The pot plasmid . Howeversmid DNA , althougividuals . Furtherividuals . We have plasmid . As hostreplicon  and the B operon  encodingmutation . A selecmutation . Both foIV-1BX08 . AlthougIV-1BX08 ,15, the IV-1BX08 -19, whicL. lactis and compare it to a traditional one using same vaccine cassette. The developed system shows potential as a new plasmid production system, but gives also information about the adjuvant role of the silent plasmid backbone in DNA vaccination.The goal of this study was to develop an alternative host vector system based on E. coli expression plasmid WRG7079  contains the kanamycin resistance gene, a ColE1-based origin of replication and a eukaryotic expression unit harboring the cytomegalo virus (CMV) promoter and enhancer region, Intron A, the tissue plasminogen activator (tPA) secretion signal, a polylinker, and the polyadenylation signal from bovine growth hormone (BGHpA). The synthetic gp120 gene with human codons from the primary, CCR5-tropic, clade B HIV-1BX08 has been cloned into WRG7079 [CGACTCTCGAGCGTTCTATCGACTCTCGAGCGTTCTCACTCTCGAGCGTTCTCGCTAGACGTTAGCGTTCAACGTTGA) containing 12 CpGs (bold) including human and mouse ISS motifs [L. lactis pJAG5 cloning vector [hom-thrB auxotroph marker, the minimal theta type replicon, and a polylinker region for cloning  and to the 3' end of the BGHpA (5'-CGGGGTACCCTGTGGATAACCGTATTACCG-3'). Similarly, we isolated the gp120 expression cassette including the 80 bp CpG motif using PCR, the same primers and pEC120CpG as template DNA. The primers introduced terminal KpnI restriction sequences (underlined). The gp120 expression units were ligated to KpnI-treated pJAG5 and electroporated into competent L. lactis MG1614ΔhomthrB. PCR and DNA sequencing confirmed the correct structure of the chimeric pJAG5 vectors. The resulting 7.8 kb vaccine plasmids were named pLL120 and pLL120CpG.The S motifs , was intg vector . PJAG5 coduction . The HIVE. coli based plasmids were produced and purified using Qiagen Midiprep according to the manufacturer . The L. lactis-based plasmids were purified similarly but with modifications as follows. A 100 ml (OD600 = 3) culture was harvested and washed in 20 ml TE buffer containing 7% sucrose (STE). Cells were resuspended in 4 ml STE containing 20 mg/ml lysozyme (Sigma) and 0.1 mg/ml RNase H (Sigma) and incubated at 37°C for 60 min. Four ml NaOH-SDS buffer (#P2 from Qiagen) was added and incubated for 5 min at room temperature. Four ml of ice cold potassium acetate buffer (#P3 from Qiagen) was added and mixed before incubated 30 min on ice and centrifuged at 20,000 g at 4°C for 1 h. The clear supernatant was applied to midi cartridges and thereafter handled as suggested (Qiagen). The quality of the PBS dissolved DNA was analysed by agarose gel electrophoresis and evaluation of the A260/A280 ratio.The BX08 gene, the human HEK293 kidney fibroblast cell line  was transfected with pLL120, pLL120CpG, pEC120, and pEC120CpG, respectively, using Effectene transfection kit (Qiagen). Radioimmunoprecipitation (RIPA) of 35S-met and 35S-cys labelled protein using gp120 specific antibodies was done as described [To examine eukaryotic expression the syn.gp120ad libitum and artificial light 12 h per day. The acclimatization period before immunization was 5 days. Mice were immunized intramuscularly (i.m.) in Tibialis anterior with 50 μl of 2 mg/ml plasmid in endotoxin-free PBS buffer using pLL120, pLL120CpG, pEC120, and pEC120CpG, respectively. The four vaccine constructs were tested on four groups of 10 mice each (n = 10). A group of 5 mice received PBS buffer alone and served as a negative control. Mice were vaccinated at week 0, 9 and 15. Blood samples were drawn one day prior to the first immunization as a pre-immune control serum (day 0), and thereafter every third week. The experiment was terminated at week 19.6–7 weeks-old BALB/c mice were purchased from Bomholdtgaard  and kept in groups of five per cage with food and water o-phenylenediamine in 11 ml water plus 4 μl hydrogen peroxide . The reaction was terminated after 30 min by 150 μl/well of 1 M H2SO4. The optical density (OD) of wells was measured at 492 nm using a microplate photometer. Anti-HIV-gp120 IgG titers were expressed as the reciprocal of the plasma dilution resulting in an OD492 nm value of 0.500.Mouse anti HIV-1 gp120 antibodies were measured by indirect ELISA. Wells of polystyrene plates  were coated for 2 days at room temperature with HIV-1 IIIB recombinant gp120 (Intracel) at 0.2 μg/100 μl of carbonate buffer, pH 9.6. Before use the plates were blocked for 1 h at room temperature with 150 μl/well of washing buffer  plus 2% BSA and 2% skim milk powder. After 3 × 1 min washings, mouse plasma was added at 100 μl/well diluted in blocking buffer and the ELISA plates were incubated for 90 min at room temperature using a microtiter plate shaker. The standard curve was made from a pool of plasma consisting of BX08 gp120 positive mice sera [BX08. A dilution series of plasma samples from each mouse was incubated for 2 h at room temperature. Wells were washed five times in PBS-Tween-20 and incubated with HRP-conjugated Rat anti-mouse IgG1 or -IgG2a antibodies  both diluted 1:1000 in dilution buffer  and incubated for 1 h at room temperature. Wells were washed five times as above and once using distilled water before colorimetric development using 100 μl o-phenylenediamine substrate solution . The reaction was stopped with 150 μl 1 M H2SO4 and absorbance measured at 490 nm.IgG1 and IgG2a were analysed by capture gp120 sandwich ELISA ,25. Brie7 cell/ml. Spleens from individual mice were homogenised and transferred to a round-bottom 96-well plate (2 × 105 cells per well). Wells, containing a total volume of 200 μl RPMI1640 with 10% FCS, were incubated for 6 h at 37°C with 50 U/ml of IL-2 , monensin (3 μM)  with and without a 15-mer peptide derived from the V3-loop of HIV-1BX08 containing a conserved murine H-2Dd restricted CTL epitope (IGPGRAFYTT). Wells were washed and cells re-suspended in 50 μl medium containing fluorescent antibodies for the relevant surface markers, CD4-FITC, CD8-PECy7, and CD44-APC (Pharmingen) and incubated for 20 min in the dark at 4°C. Then 100 μl monensin (3 μM) in PBS was added before centrifugation at 400 × g for 5 min. Cells were resuspended in 100 μl monensin (3 μM) in PBS and 100 μl 2% paraformaldehyde  and incubated for 30 min in the dark at 4°C. Cells were pelleted and resuspended in 100 μl PBS solution containing 3 μM monensin and 1% BSA. Cells were pelleted and resuspended in 200 μl PBS containing 0.5 % Saponin (Sigma) and incubated in the dark for 10 min at room temperature. Cells were pelleted and incubated for 20 min at 4 °C with 50 μl 0.5 % saponin solution containing PE conjugated anti-IFN-γ antibodies (Pharmingen) to stain for intracellular IFN-γ. Cells were washed twice with 100 μl 0.5 % saponin/PBS solution before addition of a fixing solution containing 2% paraformaldehyde. Fixed cells were analysed on a BD LSR-II flowcytometer (Becton Dickenson) using the BD FACS Diva software.Mouse spleens were removed aseptically at week 19 and gently homogenized to single cell suspension, washed 3 times in cold RPMI1640  supplemented with 10% fetal calf serum (FCS) (Invitrogen) and resuspended to a final concentration of 5 × 10d) stimulator cells at a ratio of 10:1 in medium supplemented with 5 × 10-5 M β-mercaptoethanol. For assay of cytolytic CTL response to HIV-1BX08, P815 target cells were pulsed with 20 μg/ml of the HIV-1BX08 V3 peptide SIHIGPGRAFYTTGD containing the H-2Dd restricted CTL epitopeCorbet, 2. After stimulation splenocytes were washed three times with RPMI1640 supplemented with 10% FCS and resuspended to a final concentration of 5 × 106 cells/ml. Cell suspension (100 μl) was added in triplicate to U-bottom 96-well microtiter plates and a standard 4 h 51Cr-release assay performed as described [For assay of cytotoxic T lymphocytes (CTL) the splenocytes were incubated 5 days with mitomycin-C treated (50 μg/ml for 1 h) and CTL epitope peptide loaded mouse P815  were treated with RPMI1640 medium, plasmid (3 ug/well) or plasmid DNA treated with DNase I  using 1 U DNase per 1 ug DNA for 1 hr at 37°C followed by inactivation at 75°C for 10 min. The synthesis of IL-6 and IFN-γ in supernatants of cells cultured for 0, 1, 2, and 3 days was measured by sandwich ELISA kits .Splenocytes were prepared from spleens of non-immunized 6–12 weeks old female BALB/c mice. Cells were cultured in 96-well U-bottom plates at 37°C and 5% COL. lactis (pLL120 and pLL120CpG) and E. coli (pEC120 and pEC120CpG) based vectors are shown in Fig. L. lactis vector backbone had a lower GC content (37%) than the E. coli vector (48%) and contained two copies of a known immune stimulatory mouse CpG motifs (GACGTT), while the E. coli vector backbone only contained one CpG motif. Even though the synthetic gp120 gene is very GC rich (56%) because of the humanized codons, it did not contain classical ISS motifs, but did harbour three human CpG motifs (two copies of GTCGTT and one copy of CTCGAG). The added CpG minigene contained 12 CpG dinucleotides giving rise to six consensus ISS CpG motifs (5'-two purines-CpG-two pyrimidines-3') including two copies of the mouse ISS CpG motifs (GACGTT and AACGTT) [The characteristics of the  AACGTT) . Thus, wBX08 gene using the L. lactis or E. coli-based vectors was examined by RIPA of 35S-labelled supernatant proteins from transfected HEK293 cells and purified IgG from a pool of HIV sero-positive patients. 35S-labelled supernatant from untransfected cells served as a negative control and was also precipitated but did not produce a protein with a molecular weight of 120 kDa . All IgG1 and IgG2a ratios were above 1 and below 1.5, which is indicative of a mixed Th1:Th2 response with a slight Th2 predominance   Figs.  and 6. Amid Fig. . Additiotor Fig. .+ T-cells . However, higher copy number plasmids were also tested in L. lactis but showed lower segregational stability or a less favourable distribution between supercoiled and non-supercoiled plasmid forms (data not shown).Plasmid DNA purification from Gram-positive bacteria is complicated due to the presence of a thick peptidoglycan cell wall. Purification of ocedures  includinBX08 gp120 expression using the L. lactis pJAG5 backbone as delivery vehicle. The difference in nucleotide content and/or difference in methylation pattern in L. lactis DNA could potentially influence the expression and/or the immune response. However, the intensity in RIPA of gp120 produced from cells transfected with pLL120 and pLL120CpG was similar to that of cells transfected with E. coli vectors. This illustrates that the backbone vector did not affect the level of expression, which may not be surprising since both vectors contain the same gp120 expression unit. The reasons for a somewhat lower in vitro expression from both vectors when the ISS CpG unit is included are not known but could be partly due to DNA methylation and gene silencing [The transfection of human HEK293 cells demonstrated HIV-1ilencing ,28.E. coli plasmids. Moreover the IgG1/IgG2a ratios obtained with the L. lactis and the E. coli-based vectors were not statistically different in this mouse model. Thus, the induced Th1/Th2 balance determined by the IgG1/IgG2a surrogate marker was similar for the L. lactis and E. coli based-vector, respectively. Interestingly, both antibody titers and IgG1/IgG2a ratios were largely unaffected by the introduced CpG minigene supposed to act as a Th1 adjuvant via the Toll-like receptor 9 [The usefulness of pJAG5 to carry the eukaryotic gp120 expression unit and induce an antibody response to the encoded gp120 was evaluated by naked DNA immunization. Mice serum showed IgG antibodies specific for gp120 and the induction followed the same kinetics and potency as that obtained using the corresponding ceptor 9 -33. It cceptor 9 ,35. Thus+ T-cells were activated less by the L. lactis-based vectors than by the E. coli-based vectors as measured by both IC-FACS and CTL activity. In previous studies of synthetic HIV-1 envelope genes from strains BX08 and MN no correlation was found between the CTL response and the expression levels, secreted or membrane-bound antigens, mode of DNA delivery, T helper cell response, or antibody response [L. lactis-based vector could be due to a lower adjuvant effect on CD8+ T cells exerted by differences in ISS CpG motifs since the introduction of the CpG unit showed a tendency to increase the CTL activities within each group of mice  declare that they have no competing interests.in vitro splenocyte assay testing the immune stimulatory role of the plasmids. AFO supervised the immunological studies, the design of the vaccine plasmids and helped to draft the manuscript. SMM gave helpful input to the genetic design of the plasmids. JG made the DNA vaccine constructions, vaccine production and drafted the manuscript.GJG carried out the immunological studies together with JG. MT made the


Met (tRNAiMet). Here we show that Trf4p is the catalytic subunit of a new poly(A) polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p. Comparison of native tRNAiMet with its in vitro transcribed unmodified counterpart revealed that the unmodified RNA was preferentially polyadenylated by affinity-purified Trf4 complex from yeast, as well as by complexes reconstituted from recombinant components. These results and additional experiments with other tRNA substrates suggested that the Trf4 complex can discriminate between native tRNAs and molecules that are incorrectly folded. Moreover, the polyadenylation activity of the Trf4 complex stimulated the degradation of unmodified tRNAiMet by nuclear exosome fractions in vitro. Degradation was most efficient when coupled to the polyadenylation activity of the Trf4 complex, indicating that the poly(A) tails serve as signals for the recruitment of the exosome. This polyadenylation-mediated RNA surveillance resembles the role of polyadenylation in bacterial RNA turnover.Eukaryotic cells contain several unconventional poly(A) polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A) tails of nuclear messenger RNA precursors. The yeast protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNA A new molecular surveillance mechanism is uncovered in eukaryotes, in which incorrectly folded tRNAs are polyadenylated and then targeted for degradation The addition of a tract of poly(A) to the 3′ ends of most eukaryotic messenger RNA precursors (pre-mRNAs) is an important step in their processing. Poly(A) tails have multiple functions: they facilitate the export of mRNAs from the nucleus, increase the efficiency of translation initiation, and stabilize the mRNAs in the cytoplasm. 3′ End processing is carried out by a multiprotein machinery and is coupled to transcription by RNA polymerase II and to the other RNA-processing reactions –6. PolyaSchizosaccharomyces pombe rf4-TAP; ) of the  primer . We identified five proteins A: Trf4p;To confirm the polypeptide composition of the Trf4p-containing complex, we affinity-purified complexes from yeast strains carrying Air1p-TAP, Air2p-TAP, and Mtr4p-TAP. All the “reverse tagged” complexes showed polyadenylation activity C. WesteriMet in vivo, we decided to test unmodified tRNAiMet and native tRNAiMet as substrates for the Trf4 complex . The affinity-purified Trf4-bac alone showed no poladenylation activity on unmodified tRNAiMet -tRNA with Rrp6-TAP resulted in removal of the poly(A) tails only (To test whether the presence of a poly(A) tail is sufficient to mediate tRNA degradation we carried out uncoupled exosome assays with pre-adenylated tRNAS. cerevisiae. Unlike the other newly described PAPs, which are cytoplasmic, Trf4p is located in the nucleus 6 tag were purified on Ni2+-NTA agarose (Sigma) according to the manufacturer's instructions.6 affinity tag at both ends. Proteins were expressed with the BAC-To-BAC expression system (Invitrogen) according to the manufacturer's protocol. A total of 1 × 106 Sf9 cells were seeded per 35-mm well of a six-well plate in 2 ml of Sf-900 II SFM  containing penicillin/streptomycin and transfected with approximately 5 μl of bacmid DNA. After 48 h at 27 °C, the virus was used to infect 2 × 106 cells that had been seeded on a 10-ml plate. After 4–5 d, 2 ml of the newly produced virus was added to a 30-ml suspension culture with 1 × 106 Sf9 cells/ml. The virus was collected 3–5 d later and used to infect 100-ml cultures. After 4–5 d the cells were harvested and washed with 1× PBS. The pellet was resuspended in approximately two packed volumes of lysis buffer  and incubated on ice while shaking for 30 min. The lysate was centrifuged for 30 min at 10,000 rpm at 4 °C. The supernatant containing recombinant proteins carrying a [His]6 affinity tag was then incubated with an appropriate amount of Ni2+-NTA resin on a rotor arm in the cold room for approximately 1 h. Proteins attached to the Ni2+-NTA beads were either batch-purified or applied to a column and washed with lysis buffer. Elution was done with lysis buffer containing 250 mM imidazole, and proteins were analyzed by SDS-PAGE followed by Western blot analysis.The open reading frame of Trf4p was cloned into the StuI and XhoI sites of the pFASTBAC-vector HTb that contains a [His]Protein concentrations were determined with a commercial protein assay kit , and protein amounts in SDS-PAGE were estimated by comparison to BSA standards.E. coli strain M15pREP4 . The resulting C-terminal Trf4p fragment contained a [His]6 tag fusion on its C-terminus. The protein was expressed according to the manufacturer (Qiagen) and affinity-purified on Ni2+-NTA agarose (Sigma) under denaturing conditions as described. Approximately 100 μg of purified protein was used for three injections into a rabbit . Mtr4 antibodies were kindly provided by Dr. Patrick Linder (University of Geneva). The Anti-TAP antibody against the C-terminal region of the TAP-tag were from Open Biosystems.A C-terminal portion of Trf4p (aa 356–584) was cloned into the pQE-9 vector and expressed in the iMet and 80% enriched fractions of tRNAAla were prepared from S. cerevisiae by counter-current distribution ATP , and 5 μM (A)15 or unmodified tRNAiMet.Recombinant yeast Pap1p [2 DE-81 paper. The filters were washed 3× 10 min in 0.3 M ammonium formate/10 mM Na-pyrophosphate, and the incorporated radioactivity was measured in a scintillation counter.The specific activities of poly(A) polymerases were measured in 10-μl reaction mixtures containing 20 mM Tris-Cl (pH 8.4), 40 mM KCl, 5 mM MgClst Pap1p  or Trf4p2, 25 mM Tris-HCl (pH 7.9), 20 mM KCl, 10% glycerol, 0.01 mM EDTA, 0.1 mg/ml BSA, 1 mM DTT, 0.02% Nonidet P-40, 5U of RNA guard (Promega), and 100 ng of heparin. Reactions were incubated at 30 °C for 30 min and stopped by the addition of 25 mM EDTA. The RNA was precipitated by adding 0.1 volumes of 3 M ammonium acetate and three volumes of ethanol. Pellets were resuspended in 6 μl of formamide loading buffer and separated on denaturing polyacrylamide gels. The polyadenylated population of tRNAs was purified as described [Polyadenylation of tRNA for uncoupled exosome assays was done in 50-μl reaction volumes containing 1 μg of recombinant Pap1p in the presence 100 ng of 5′-end-labeled tRNA, 50 μM ATP, 5 mM MgClescribed .Exosome assays were carried out under the same conditions used for polyadenylation assays except that the reactions contained 1 mM ATP and 2 mM DTT. To prevent loss of small degradation products during RNA precipitation, the reactions were stopped by the addition of an equal volume of gel loading buffer containing 80% formamide and separated by electrophoresis without prior precipitation.http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) or other nonredundant or EST databases at the Swiss EMBnet node [http://www.ch.embnet.org/software/BottomBLASTadvanced.html) or at the NCBI (http://www.ncbi.nlm.nih.gov/BLAST/).BLAST searches were conducted in the genome databases  on native and unmodified tRNAiMet. The 5′-end-labeled tRNAs were incubated with 50 ng of Trf4 complex, 50 ng of Pap1p, or 50 ng of E. coli PAP for the times indicated and separated on a 10% gel. The migration position of the input tRNA is indicated by an arrow. Lane I on each gel contained input tRNA alone.Polyadenylation activity of Pap1p, Trf4p-TAP, and (871 KB PDF).Click here for additional data file.Figure S2(A) Polypeptide composition of the affinity-purified TAP-tagged Rrp6p (Rrp6-TAP). The purification was done as described in (B) Rrp6p-TAP eluates contain residual amounts of Mtr4p. Western blot analysis of Rrp6-TAP with antibodies directed against Mtr4p, Trf4p, and the C-terminus of the TAP-tag. Affinity-purified fractions of Mtr4p (Mtr4-TAP) were used as a control.iMet. The 5′-end-labeled in vitro transcribed tRNAiMet was incubated with 100 ng of Rrp6p-TAP complex in the presence of 1 mM ATP (lanes 2–5) or without ATP (lanes 6–9). Reactions were stopped after 10, 30, 60, or 120 min by the addition of one volume of formamide loading buffer and analyzed by electrophoresis on a 15% gel. The position of the input RNA is indicated by an arrow.(C) The Rrp6p-TAP complex alone does not efficiently degrade unmodified tRNA(1.2 MB PDF).Click here for additional data file.Figure S3iMet was polyadenylated with recombinant yeast Pap1p; the polyadenylated products were eluted from polyacrylamide gel and labeled at their 3′ ends with α-32P cordycepin triphosphate . The poly(A)-tRNAiMet was then incubated with 100 ng of Rrp6p-TAP complex (lanes 2–6) or with buffer A (lanes 7–11) for 2, 15, 30, 45, or 60 min, stopped by the addition of one volume of formamide loading buffer, and analyzed by electrophoresis on a 15% gel.In vitro transcribed tRNA(750 KB PDF).Click here for additional data file.Figure S4Western blot analysis of 100 ng of Trf4-TAP and Mtr4-TAP complexes purified on IgG Sepharose at low salt (L) and high salt (H) conditions. Lane W on each gel shows samples eluted by 1 M NaCl from the affinity columns prior to TEV protease treatment.(337 KB PDF).Click here for additional data file.Table S1“Start” and “stop” indicate regions of proteins expressed in particular clones. Numbers in brackets indicate that the clone extended beyond the open reading frame. Global predicted biological score (PBS) is a confidence score assigned to each interaction by Hybrigenics, which predicts the occurrence of an interaction in vivo .(27 KB DOC).Click here for additional data file.http://www.sgc.ox.ac.uk/) accession numbers for genes and gene products discussed in this paper are Air1p (SGDID:S000001341), Air2p (SGDID:S000002334), Hul4p (SGDID:S000003797), Mtr4p (SGDID:S000003586), Pap1p (SGDID:S000001710), Prp16p (SGDID:S000001794), RRP44 (SGDID:S000005381), Rrp6p (SGDID:S000005527), Tad1p (SGDID:S000003212), Trf4p (SGDID:S000005475), Trf5p (SGDID:S000005243), and TRM6 (SGDID:S000005006). The Swiss-Prot/EMBL (http://www.ebi.ac.uk/swissprot/) accession numbers for genes and gene products discussed in this paper are Cid1 , Cid13 , GLD-2 , GLD-3 , PAP , Pap1p , PAPOLA , Trf4 , and Trf5 .The Structural Genomics Consortium (


TP53 and in ATM and CHEK2 predispose to cancer. We have therefore examined the effects of 53BP1 single nucleotide polymorphisms  and the novel 53BP1 6bp deletion (1347_1352delTATCCC) on breast cancer risk.The TP53-binding protein (53BP1) has been shown to influence TP53-mediated transcriptional activation, thus playing a pivotal role in DNA damage signalling. Genetic aberrations in Allelic discrimination was performed to investigate the frequencies of 53BP1 D353E, G412S, and K1136Q and of 1347_1352delTATCCC in 353 patients with breast cancer and 960 control individuals.53BP1 1347_1352delTATCCC, leading to the loss of an isoleucine and a proline residue, showed a nonsignificant inverse association with breast cancer risk .No significant association of 53BP1 D353E, G412S, or K1136Q with breast cancer risk was detected. The lack of association casts doubt on the putative effects of D353E, G412S, and K1136Q on breast cancer risk. Investigating a larger study cohort might elucidate the influence of the 6bp deletion 1347_1352delTATCCC. Studying the functional effect and the impact of this variant on the risk of other cancers may be revealing. In resp53BP1 6bp deletion (1347_1352delTATCCC) showing an inverse association with breast cancer risk (age-adjusted odds ratio (OR) = 0.61, 95% confidence interval (CI) = 0.22 to 1.68), lacking significance (P = 0.34).This is the first study to investigate the effects of the 53BP1 single nucleotide polymorphisms (SNPs) D353E (1059C>G), G412S (1234G>A), and K1136Q (3406A>C) on breast cancer risk, analysing 353 German patients with breast cancer and 960 controls. 53BP1 D353E, G412S, and K1136Q showed no association with breast cancer risk. In addition, we detected a novel, very rare 53BP1 SNPs ) by DNA sequencing. Sequencing primers are available upon request. The initial analysis included 53BP1 exons 9, 11, and 17, harbouring three reported nonsynonymous polymorphisms . When sequencing exon 11, we additionally detected the 6bp deletion 1347_1352delTATCCC. All validated variants were chosen for further analyses using a large cohort of breast cancer patients.A randomly chosen set of 23 German patients with familial breast cancer was initially screened for annotated BRCA1 and BRCA2 mutations. In accordance with the German Consortium for Hereditary Breast and Ovarian Cancer, they were classified into six categories based on family history: (A1) families with two or more breast cancer cases including two cases with onset below the age of 50 ; (A2) families with at least one male breast cancer case (0.9%); (B) families with at least one breast cancer and one ovarian cancer case (16.2%); (C) families with at least two breast cancer cases including one case diagnosed before the age of 50 (33.5%); (D) families with at least two breast cancer cases comprising two cases diagnosed after the age of 50 (5.5%); (E) single cases of breast cancer diagnosed before the age of 35 (4.6%) [The breast cancer patients were 353 unrelated German women  who were negative for 5 (4.6%) . They we5 (4.6%) . The stuPCR amplification and sequencing were performed as previously described . Conditi53BP1 1347_1352delTATCCC was analysed using the MGB Eclipse™ Probe System by Epoch Biosciences . Allelic discrimination was carried out as recommended by the manufacturers using the following probes: D353E: assay-on-demand C_2944794_10; G412S: VIC-ACTTCAAAGTGGTGAACC, FAM-AACTTCAAAGTAGTGAACC; K1136Q: VIC-GGAGTACTAATAAGGAAA, FAM-CGGAGTACTAATCAGGAAA; 1347_1352delTATCCC: FAM-CACTTCATCCCAT; TET-CACTTCCTATCCCATC. Primers and probes were designed based on GenBank NM_005657 (NCBI) and are available on request. More than 5% of the genotyping results were confirmed by sequencing, and genotype distributions were consistent with Hardy–Weinberg equilibrium.53BP1 polymorphisms D353E, G412S, and K1136Q were analysed using TaqMan allelic discrimination. TaqMan assays were performed in a reaction volume of 10 μl comprising 5ng of genomic DNA, each probe at 50 nM, each primer at 225 nM, and 1× Universal Master Mix with the following amplification conditions: 2 min at 50°C, 10 min at 95°C and 35 to 45 cycles at 92°C for 15 s and 60°C for 1 min. Amplification products were measured and analysed with the ABI Prism 7900 HT sequence detection system and the SDS software . TaqMan probes and primers were provided by the assay-on-demand and assay-by-design services, respectively (Applied Biosystems). Calculations of Hardy–Weinberg equilibrium, genotype-specific OR, and 95% CI were carried out using a tool offered by the Institute of Human Genetics, Technical University Munich, Munich, Germany . Age-adj53BP1 are excellent candidates for cancer susceptibility. We investigated the impact of three nonsynonymous amino acid exchanges in 53BP1 on breast cancer risk. 53BP1 G413S and K1136Q represented promising candidate SNPs, resulting in the replacement of a nonpolar by a polar amino acid. Genotype frequencies of the three 53BP1 polymorphisms between breast cancer cases and control samples were similar, showing no significant association with breast cancer risk  declare that they have no competing interests.All authors listed contributed to the production of this manuscript: RK, PB, BW, and RKS provided genomic DNAs of cases studied and helped to draft the manuscript. BB and KH participated in the design and coordination of the study and critically revised the manuscript. BF and JLB performed statistical analyses. BF carried out the SNP genotyping and wrote the manuscript. All authors read and approved the final manuscript.