Pmp22, a member of the junction protein family Claudin/EMP/PMP22, plays an important role in myelin formation. Increase of pmp22 transcription causes peripheral neuropathy, Charcot-Marie-Tooth disease type1A (CMT1A). The pathophysiological phenotype of CMT1A is aberrant axonal myelination which induces a reduction in nerve conduction velocity (NCV). Several CMT1A model rodents have been established by overexpressing pmp22. Thus, it is thought that pmp22 expression must be tightly regulated for correct myelin formation in mammals. Interestingly, the myelin sheath is also present in other jawed vertebrates. The purpose of this study is to analyze the evolutionary conservation of the association between pmp22 transcription level and vertebrate myelin formation, and to find the conserved non-coding sequences for pmp22 regulation by comparative genomics analyses between jawed fishes and mammals.pmp22 over-expression medaka fish line was established. The transgenic fish had approximately one fifth the peripheral NCV values of controls, and aberrant myelination of transgenic fish in the peripheral nerve system (PNS) was observed. We successfully confirmed that medaka fish pmp22 has the same exon-intron structure as mammals, and identified some known conserved regulatory motifs. Furthermore, we found novel conserved sequences in the first intron and 3'UTR.A transgenic pmp22 transcription increases. This result indicates that an adequate pmp22 transcription level is necessary for correct myelination of jawed vertebrates. Comparison of pmp22 orthologs between distantly related species identifies evolutionary conserved sequences that contribute to precise regulation of pmp22 expression.Medaka fish undergo abnormalities in the PNS when  In Charcot-Marie-Tooth disease type1A (CMT1A) patients, the majority have a heterozygous tandem duplication of chromosome 17p11.2-p12, a 1.5 Mbp region that includes pmp22, and the patients undergo slow NCV and distal muscle weakness [pmp22 transcription is likely to be tightly regulated to prevent over-expression. Understanding the regulatory mechanisms of this gene might improve treatment of CMT1A patients.Peripheral myelination is important for rapid body movement and sensing several environmental stimuli . The pmpMP/PMP22 ,4. It isweakness ,5. Thus,pmp22 have been reported. Transgenic rodent studies have shown the promoter regions and sequence conservation of the gene [in vitro studies have identified regulatory motifs for Schwann cell specific expression of pmp22 [pmp22 expression might be found by comparative genomics analyses.Promoter regions and some regulatory motifs of mammalian the gene ,7. Some of pmp22 ,9. HowevThe comparative genomics approach between jawed fishes and mammals facilitates the identification of novel conserved motifs ,11. Puffpmp22 transcription level is necessary for correct myelination in all jawed vertebrates. To address this issue, we established a transgenic medaka fish line overexpressing pmp22 and analyzed aberrations in their PNS. Although zebrafish is more universally used for experiments, we focused on medaka fish [pmp22 has an important role in myelin formation not only in mammals but also in fishes, and shows that the gene structure and some regulatory motifs are conserved among jawed vertebrates.In the present vertebrate phylogeny, the myelin sheath is seen only among jawed vertebrates ,15. It iaka fish , becauseaka fish , and thupmp22 ortholog in medaka fish, a phylogenic tree of the EMP/PMP22 family was drawn based on the amino acid sequence alignment . The medaka fish EST sequence AM313848, which maps to the syntenic region of LG19, clustered with mammalian PMP22 sequences. The transmembrane regions, N41 glycosylation site (NXS/T) and C terminus ER-retention/retrieval signal (LRKRE) [pmp22 in medaka fish (ol_pmp22) and the other gene has been destined for degradation or neo-functionalization. Thus, we focused on the ol_pmp22 candidate encoded on LG19. Ol_pmp22 endogenous transcription was detected in multiple tissues of adult fish   are consol_pmp22 translation start codon and the FLAG tagged ol_pmp22 coding region with a polyadenylation site were inserted into the promoter and downstream of the FRT flanked region, respectively . We observed distinct GFP fluorescence from 3 day postfertilization (dpf) . The transgene, FLAG tagged ol_pmp22, was transcribed in the pmp22 fish, and transcribed at a low level in the GFP fish  with 100 μA stimulation, suggesting that the spikes were generated by electrical excitability of neurons Figure . We obsepmp22 over-expression causes weak swimming ability in medaka fish.To test kinematic abnormalities, we observed medaka fish swimming in running water. Medaka fish usually swim against the current to keep their position. However, in a rapid current, the fish were swept downstream while swimming against the current. We measured flow distances of the wild type, GFP fish and pmp22 fish per time in rapid water current (0.25 m/s). The pmp22 fish had a tendency to be swept away faster than the control lines Figure , indicatpmp22 over-expression is important for keeping fast peripheral NCV and maintaining the myelin sheath in jawed vertebrates.From external observations, there were not obvious morphological and developmental abnormalities between the controls and pmp22 fish. However, aberrations were observed in cross sections of pmp22 fish motor nerve fibers by electron microscopy Figure . The pmppmp22 genes have alternative untranslated first exons, 1A and 1B [ol_pmp22 EST sequences always have the same first exon. To confirm whether ol_pmp22 has a single first exon or alternative first exons, we constructed the EGFP expression constructs regulated by several regions upstream of the ol_pmp22 translation start codon, and analyzed the EGFP fluorescence patterns by a transient expression assay. The constructs, p11-0, p6.5-0, p3-0, p6.5-3, and p11-5, contain 11 kbp, 6.5 kbp, 3 kbp, 6.5 kbp to 3 kbp followed by 0.5 kbp, and 11 kbp to 5 kbp upstream of the translation start codon, respectively . The transcription start site (TSS) of the ol_pmp22 1A transcript is located 5,193 bp upstream of the translation start codon. Then we observed EGFP fluorescence only in Schwann cells of the p11-5 fish . We scanned for four known conserved mammalian motifs in the promoter region of ol_pmp22 exon1A by a motif finding program [pmp22 exon1A [pmp22 1A transcription, and identified the important residues of this motif for DNA-protein interaction (CCAT), located at 3 bp to 6 bp downstream of the motif [ol_pmp22 exon1A , however the important residues are not fully conserved (CAAA), which is the sequence at the corresponding position in mammals. In the promoter region of human pmp22 (hs_pmp22), CREB is identified as the silencer at 1.7 kbp upstream of the 1A TSS [pmp22 (mm_pmp22) exon1A and ol_pmp22 exon1A, respectively.First, in the  program . Althoug2 exon1A . The rathe motif . The FP3e 1A TSS . We idenpmp22 in jawed vertebrates, we performed comparative genomics analyses. Among hs_, mm_ and ol_pmp22, the multiple alignment of genomic sequences revealed some conserved sequences in the non-coding regions   Figure , 10A. Th program . We triepmp22 expression causes CMT1A in humans and rodents. CMT1A patients, which have a heterozygous tandem duplication of a 1.5 Mbp region that includes hs_pmp22, have reduced peripheral NCV, aberrant myelination and distal muscle atrophy [pmp22 over-expression, have been established to understand and treat this neuropathy [hs_pmp22, the degree of the CMT1A phenotype depends on the hs_pmp22 transcription level associated with YAC copy number [pmp22 over-expression reveals that demyelination occurs only when pmp22 is highly expressed in adult, and that the neuropathic phenotype is reversible [in vivo and in vitro studies show that progesterone promotes pmp22 1A transcription in Schwann cells [pmp22 transcription than wild type rats, is used for the progesterone antagonist test. The progesterone antagonist reduces pmp22 expression level. The treated CMT1A rats show higher motor performance than controls with the relief of muscle atrophy and sciatic nerve demyelination [pmp22 transcription. The ascorbic acid treated C22 mice have more activity, correct myelination and a longer life span than control neuropathy mice [pmp22 transcription. Recently, another approach for the treatment of this neuropathy has been reported. In the C22 Schwann cells, there are large aggresomes, including PMP22 and ubiquitin, at perinuclear regions [Increase of  atrophy ,5. CMT1Auropathy -32. In my number ,34. The versible . Rat in nn cells ,37. A CMlination . Ascorbithy mice . The pri regions . Also, t regions .pmp22. To analyze the relationship between pmp22 transcription level and peripheral myelination in medaka fish, pmp22 fish were established. Compared with control lines, ol_pmp22 overexpressing fish, having approximately two-fold higher transcription of ol_pmp22, have one fifth peripheral NCV and aberrant PNS myelination, in spite of the transgene lacking intron 2–4 and the region downstream of the coding regions  has been identified as the silencer responding to low cyclic AMP levels [mm_ and ol_pmp22 exon1A promoter region, respectively. However, it is unclear whether both of these CREB sites are functional.Our bioinformatics analyses found that s Figure . The FP32 exon1A ,24. ThisP levels . A CREB P levels  were foupmp22 1A transcription is activated by the zinc finger transcription factor EGR2, which is the major positive regulator for essential myelin genes [p0, mbp, mag, prx and dhh, but not pmp22 [ol_pmp22 region, the EGR2 binding motif was not found in our analyses. In a transgenic mouse study, over-expression of POU-domain protein POU3f1 downregulates pmp22 transcription [ol_pmp22 (data not shown); however it is unknown which sites are functional. MicroRNAs miR-9 and miR-29a inhibit pmp22 translation [pmp22, but not among amphibian and jawed fish pmp22.In mammalian Schwann cells, in genes . The dirot pmp22 ,55. In tcription . There anslation ,58. The pmp22 gene has highly conserved sequence in the 3'UTR, the length of which is about 70 bp, and includes an mRNA destabilization signal  and ip167 (5'-ATGAGTCCGCTGATGAGCAC-3'). Manipulation of fish was performed following both national and institutional guidelines.All fish lines were maintained at around 26°C under a 14 h light and 10 h dark cycle. Collected eggs were grown with 0.3% sea salt water. GFP transgenic fish were screened by fluorescent microscopy observation at embryonic and juvenile stages. Genotyping to select Fish genomic DNA was prepared by phenol-chloroform and ethanol precipitation. Plasmids were prepared with the QIAprep Spin Miniprep Kit . Total RNA was extracted by ISOGEN . First strand cDNA was synthesized by using the First-Strand cDNA Synthesis Kit .NcoI/XbaI digested EGFP coding fragment was obtained from a pEGFP vector  and inserted into the NcoI/XbaI digested pGL3-basic vector .To construct the EGFP basic vector, an ol_pmp22 was amplified by PCR with HdrR genomic DNA as the template and primers ip097 (5'-ATACGCGTATCATTTTCAAGGTCACTGGG-3') and ip098 (5'-AAGGATCCTTCACGGCCAGTCTAAGAAGAGAGGAGACA-3'). To construct the p11-0 EGFP expression vector, the amplified 11 kbp DNA fragment was digested with MluI/BamHI, and inserted into the MluI/BglII digested EGFP basic vector. The amplified 11 kbp DNA was digested with BamHI and partially digested with BglII, to obtain 6.5-0, 3-0 and 6.5-3 fragments. To construct the p6.5-0 and p3-0 vector, the fragments were inserted into the BglII digested and BAP treated EGFP basic vector. The 0.5 kbp fragment including the splice accepter sequence was obtained by PCR amplification of genomic DNA using primers ip119 (5'-TAAGATCTCTTTGCTCTGGGTTAAAGTT-3') and ip098, digested with BglII/BamHI and inserted into the BglII digested and BAP treated EGFP basic vector. To construct the p6.5-3 EGFP expression vector, the 6.5-3 fragment was inserted into the BglII digested and BAP treated EGFP vector containing the 0.5 kbp region upstream of the translation start codon. To construct p11-5, the 6 kbp fragment including ol_pmp22 exon1A and its upstream region was obtained by PCR amplification of genomic DNA using primer ip097 and ip134 (5'-TTTGGATCCAGTGAGGCTCTACCTGCTCG-3'), digested with MluI/BamHI and inserted into MluI/BglII digested EGFP basic vector.The 11 kbp region upstream of the translation start codon of pmp22 over-expression line. The FLAG-ol_pmp22 fragment was obtained by RT-PCR of medaka 8 dpf whole embryos using primers ip017 (5'-CCATGGATTACAAAGACGACGACGACAAAATGCTGATCTTA-3') and ip018 (5'-TCTAGATCATTCTCGCTTCCT-3'). The PCR product was digested with NcoI/XbaI and inserted into the NcoI/XbaI digested pGL3-basic vector. The fragment of BGHp(A) was amplified by PCR with phrGFPII-1  as template and primers ip041 (5'-GGTCTAGATTCCCTTTAGTGAG-3') and ip042 (5'-GCGGATCCGGCGCCCCAGCATG-3'), digested with XbaI/BamHI and inserted into the XbaI/BamHI digested EGFP basic vector. To construct the FRT-flanked EGFP-BGHp(A) vector, the fragment was obtained by PCR amplification with the EGFP-BGHp(A) vector as template and primers ip044 (5'-CGAGATCTGAAGTTCCTATTCCGAAGTTCCTATTCTCTA GAAAGTATAGGAACTTCAAGCTTGGCATTCCGGTACTG-3') and ip045 (5'-AGAGCGCTCGAGGGCCCGTCTCCCATGAAGTTCCTATACTTTCTAGAGAA TAGGAACTTCGGAATAGGAACTTCGAATTCCGACGATAGTCATGCCC-3'), digested with BglII/Eco47III and inserted into the BglII/Eco47III digested pGL3-basic vector. An NcoI/Eco47III digested FLAG-ol_pmp22 fragment was obtained, and it was inserted into the BsmBI/Eco47III digested FRT-flanked EGFP-BGHp(A) vector. This vector was digested with MluI/BglII and ligated with the MluI/BamHI digested 11 kbp PCR product of the region upstream of the translation start codon  – FLAG-ol_pmp22 vector was constructed for establishing the 2, 0.2 mM NaHCO3, pH 7.3). The solution  with either plasmid DNA (30 ng/μl) or mRNA (400 ng/μl) was injected into the cytoplasm of one – cell stage eggs through the chorion. For in vitro capped mRNA synthesis, template DNA including the T7 promoter and FLP recombinase coding sequence were amplified by PCR with pOG44 as template and primers ip131 (5'-ATTAATACGACTCACTATAGGGCTCTCCACAGGTGTCCACTC-3') and ip132 (5'-ACCCGCACATACAGCTCACT-3'). Flp mRNA was synthesized by using the mMESSAGE mMACHINE T7 kit  and polyadenylated by the Poly (A) Tailing kit (Ambion). A pressure injector, FemtoJet , was used with borosilicate glass capillaries GC100F-10 .Medaka fish fertilized eggs were collected immediately after spawning and placed into cold Yamamoto's solution  (pH 7.4) at 4°C for several hours. Fixed samples were decalcificated by 6% sucrose at 4°C overnight. Infiltration and embedding in glycol methacrylate were performed with Technovit 8100 . Embedded samples were sliced horizontally (10 μm) by a rotary microtome, Leica RM2165  equipped with a disposable microtome blade N35 , stretched with distilled water on slide glasses, and dried at room temperature (RT) overnight. After soaking with PBS, samples were incubated with 0.1 mg/ml proteinase K at 37°C for 15 min, rinsed with PBS, incubated with blocking solution (1% horse serum in PBS with 0.5% Tween 20) at RT for 1 h, incubated with 1/500 anti-GFP monoclonal antibody  in blocking solution at 4°C overnight, rinsed with PBS with 0.5% Tween 20, incubated with 1/500 Alexa fluor 488 conjugated anti-mouse IgG antibody  in blocking solution at RT for 1 h, and rinsed with PBS. Bright-field and fluorescence images were observed using a confocal microscope FV1000  and data were obtained by Fluoview ver 1.6b software (Olympus).Total RNA was prepared from whole bodies of p6.5-3 injected fish at 14 dpf. The SMART RACE cDNA Amplification kit (Clontech) and SuperScript II Reverse Transcriptase (Invitrogen) were used for first strand cDNA synthesis. RACE PCR was performed by using the Advantage 2 PCR kit (Clontech) with primer ip128 (5'-TAGGTCAGGGTGGTCACGAGGGT-3').ol_pmp22 transcript, ip154 (5'-GCCCCACCAGAGCGTAAATAC-3') and ip155 (5'-CATCGTACTCCTGCTTGCTGAT-3') for beta-actin[The target mRNA quantification was performed by using the Power SYBR Green PCR Master Mix . The following primers were used: ip150 (5'-GCTCTCTGATGATTCTTGCTTTTG-3') and ip151 (5'-AGGAGCAGAATGTCCAGAATGC-3') for the 1A transcript, ip152 (5'-CTACTGCAGGTCCACTCTTTTGG-3') and ip153 (5'-GGCCAGTTTCAAGCAAATCG-3') for the 1B transcript, ip160 (5'-TCCTCTTCTTCTGTCAGCTCTTCA-3') and ip161 (5'-GCTCCGCTCATCACAAACAA-3') for total eta-actin. Relativ2/5% CO2) physiological solution, artificial cerebrospinal fluid (ACSF) at 4°C, containing: 126 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 26 mM NaHCO3, 1,25 mM NaH2PO4, 2 mM CaCl2 and 10 mM D-glucose. The sample was attached to a 64 channel-electrode dish perfused with ACSF (3 ml/min). Peripheral nerve fibers were stimulated with 0.1 Hz, 60 or 100 μA, and population spikes of fish nerve fibers at 300 μm, 600 μm, and 900 μm distance between two channels were recorded in the perfusion of ACSF. The recording was stored in Windows XP hardware and data analysis was performed with Mobius software of the MED64 system . Statistical analysis was performed by Student's t-test.The electrical excitability of fish nerve was measured according to Imamura Y., 2008 . SagittaThe medaka fish swimming ability test was performed in running water (0.25 m/s) in a straight horizontal watercourse  at room temperature. Adult male fish were used. The volume of water flow from a 15 mm diameter hose was 188 ml per second. Medaka fish were put into a decelerated water current in a net for 30 seconds and gently released into the rapid water current. The movie of a top view of fish swimming was acquired by a digital video camera, NV-GS250-S . A picture of each time point was obtained by MotionDV STUDIO 5.3J LE for DV software (Matsushita), and analyzed by the scion image software . Statistical analysis was performed by Student's t-test.4 in phosphate buffer (pH 7.4) for 2 h. Then samples were dehydrated: 50% ethanol for 10 min, 60% ethanol for 10 min, 70% ethanol for 10 min, 80% ethanol for 10 min, 90% ethanol for 10 min, 99% ethanol for 10 min and 100% ethanol for 20 min twice. Dehydrated samples were transferred to propylene-oxide for 20 min twice, 1:1 propylene-oxide:epoxyresin for 1 h, 1:3 propylene-oxide:epoxyresin for 1 h and epoxyresin overnight. For embedding, samples were left at 60°C with fresh epoxyresin three overnight. Ultra thin sections (80 nm) were obtained on an Ultra microtome EM UC6 , counterstained by Reynolds method and examined on an H-7650 . Medaka fish motor nerve fibers were determined on the locations according to the studies for zebrafish motor nerve fibers [Adult trunks were removed along with the bones and cut into 1–2 mm pieces. Samples were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h, washed in 0.1 M phosphate buffer (pH 7.4) for 20 min 5 times and postfixed in 1% OsOe fibers ,65.pmp22 was performed by LAGAN [Sequences of the EMP/PMP22 family were obtained by BLAST and BLAT, using human amino acid sequences of the family as the queries. For phylogenic analysis, mRNA sequences were translated into amino acid sequences. The analyses for conserved motifs were performed by the scan for matches , Weeder by LAGAN  and presby LAGAN .pmp22: peripheral myelin protein 22; CMT1A: Charcot-Marie-Tooth disease type1A; NCV: nerve conduction velocity; PNS: peripheral nerve system; EMP: epithelial membrane protein; UTR: untranslated region; LG: linkage group; FRT: FLP recognition target; EGFP: enhanced green fluorescent protein; dpf: day postfertilization; TTX: tetrodotoxin; EST: expression sequence tags; TSS: transcription start site; CREB: cyclic AMP response element binding; YAC: yeast artificial chromosome; ACSF: artificial cerebrospinal fluid; PCR: polymerase chain reaction.JI carried out the transgenic, molecular, electron microscopic, kinematic and bioinformatics studies; and participated in the design and drafted the manuscript. MS carried out the bioinformatics studies, participated in the design and helped to draft the manuscript. YI carried out the electrophysiological studies. TD and KF carried out the molecular studies and participated in the design. SY and YK participated in the design and coordination. TK carried out the molecular and kinematic studies, participated in the design and helped to draft the manuscript. All authors read and approved the final manuscript.Endogenous expression of ol_pmp22 in adult fish. Total RNA prepared form several tissues of adult fish was used as template for RT-PCR . The primers were ip008 (5'-GGAATCATCCTGCTGCACAT-3') and ip009 (5'-GGGTTGCAGTTAAGGTTACCG-3'). ey: eye, br: brain, li: liver, in: intestine, pa: pancreas, ga: gallbladder, ki: kidney, he: heart, fi: fin, ov: ovary, te: testis, M: 100 bp marker.Click here for fileThe construct for establishing the pmp22 fish. Amplified FRT flanked EGFP with a BGH polyadenylation site was digested with BglII/Eco47III and inserted into the BglII/Eco47III digested pGL3-basic vector (pFRT-EGFP-BGHp(A)-FRT). The 11 kbp region upstream of ol_pmp22 translation start codon was inserted into the plasmid digested with MluI/BglII. The FLAG tagged ol_pmp22 coding sequence with an SV40 polyadenylation site was inserted into the plasmid digested with BsmBI/Eco47III.Click here for fileGFP fluorescence of GFP fish. The pictures show fluorescent microscopy observations, A-O, and light microscopy, A'-O', of GFP fish. Embryonic stages, 0 dpf , 1 dpf , 2 dpf , 3 dpf , 4 dpf , 5 dpf , 6 dpf  and 7 dpf , are shown. Distinct fluorescence was observed from 3 dpf. In embryonic stages, strong GFP fluorescence was observed in nervous system, branchial arches  and olfactory epithelium . Dorsal view of the anterior , left side view of the middle, , left side view of the tail , and ventral view of the anterior  of 10 dpf are shown. ey: eye, mb: midbrain, hb: hindbrain, sp: spinal cord, lsc: longitudinal fissure of cerebrum, li: liver, ki: kidney, in: intestine, yo: yolk, fi: fin fibroblast, ba: bulbous arteriosus, gi: gill, ph: pharynx. Bars: 200 μm.Click here for fileValues of mRNA quantification of the total, 1A and 1B transcription levels. Total RNA was prepared from mixed extract of some fish at 4, 6, 8, 14 and 30 dpf. The upper table shows the threshold cycles of the beta-actin, internal control, and ol_pmp22 mRNA. The lower table shows the relative quantification of total, 1A and 1B transcript defining the average of each 4 dpf transcription level as 1.0. The graph is shown in Figure Click here for file


Methylation of residues in histone tails is part of a network that regulates gene expression. JmjC domain containing proteins catalyze the oxidative removal of methyl groups on histone lysine residues. Here, we report studies to test the involvement of Jumonji domain-containing protein 6 (Jmjd6) in histone lysine demethylation. Jmjd6 has recently been shown to hydroxylate RNA splicing factors and is known to be essential for the differentiation of multiple tissues and cells during embryogenesis. However, there have been conflicting reports as to whether Jmjd6 is a histone-modifying enzyme.Jmjd6-knockout cells indicate that Jmjd6 is not involved in the demethylation of these histone lysine residues. This is further supported by overexpression of enzymatically active and inactive forms of Jmjd6 and subsequent analysis of histone methylation patterns by immunocytochemistry and western blot analysis. Finally, treatment of cells with RNase A and DNase I indicate that Jmjd6 may preferentially associate with RNA/RNA complexes and less likely with chromatin.Immunolocalization studies reveal that Jmjd6 is distributed throughout the nucleoplasm outside of regions containing heterochromatic DNA, with occasional localization in nucleoli. During mitosis, Jmjd6 is excluded from the nucleus and reappears in the telophase of the cell cycle. Western blot analyses confirmed that Jmjd6 forms homo-multimers of different molecular weights in the nucleus and cytoplasm. A comparison of mono-, di-, and tri-methylation states of H3K4, H3K9, H3K27, H3K36, and H4K20 histone residues in wildtype and Taken together, our results provide further evidence that Jmjd6 is unlikely to be involved in histone lysine demethylation. We confirmed that Jmjd6 forms multimers and showed that nuclear localization of the protein involves association with a nucleic acid matrix. Covalent histone modifications play an important role in regulating chromatin structure, gene expression and epigenetic inheritance Until recently, histone methylation was thought to be irreversible. Two types of enzymatic mechanisms for the reversal of histone methylation marks have now been identified. The first histone demethylase identified was the nuclear amine oxidase KDM1 (LSD1), which catalyzes demethylation of H3K4 and H3K9 by employing a flavin dependent reaction Jumonji domain containing gene 6 (Jmjd6) Jmjd6 in mice and zebrafish have demonstrated its essential role in cell differentiation and development Jmjd6 knockout mice die around birth due to severe cardiopulmonary malformations. These include ventral septal defects, the formation of a double-outlet right ventricle, and hypoplasia of the pulmonary artery that resemble the human congenital syndrome Tetralogy of Fallot Jmjd6-deficient mice ranging from mild retinal defects to ectopic induction of rudimental eye anlagen in nasal cavities Jmjd6 knockout embryos are impaired in the release of pro- and anti-inflammatory cytokines upon stimulation with LPS Jmjd6 inactivation in multiple tissues and cells, and the role of other JmjC domain containing proteins in regulating histone methylation, we analyzed if Jmjd6 functions as a JmjC-domain containing histone lysine demethylase.We have been interested in the function of the Jmjd6 wildtype and knockout cells as well as under Jmjd6 overexpressing conditions. We report that Jmjd6 is not involved in lysine demethylation of H3K4, H3K9, H3K27, H3K36, and H4K20 residues. Instead, we provide evidence that Jmjd6 may preferentially be associated with RNA/RNA complexes which further supports its proposed role in the regulation of RNA splicing.Using a novel monoclonal antibody for Jmjd6, we investigated its nuclear localization and distribution under various cell culture and growth conditions in different human cell lines and primary mouse embryonic fibroblasts (MEFs). Jmjd6 was found to be located in the nucleoplasma outside of regions containing heterochromatic DNA. This intracellular location was stable and prominent during the interphase of the cell cycle. However, during mitosis Jmjd6 disappears from the nucleus and condensates again in the characteristic fine dotted nuclear pattern of the interphase during the late anaphase and telophase of the cell cycle. Occasionally Jmjd6 was observed in nucleoli indicating that it may be capable of shuttling in and out of this nuclear compartment. Using western blot analysis and immunofluorescence imaging, we compared histone methylation statuses in Jmjd6 knockout context Jmjd6−/− and Jmjd6+/+ embryonic fibroblast cells  that shows a high specificity for Jmjd6 staining  is a nuclear protein   in (YFP) . Beside in (YFP) . Fusionsin (YFP) .N- and C-terminal epitopes in Jmjd6, respectively. For detection of YFP fusion tags the anti-green fluorescent protein (GFP) antibody AB-290 was used -YFP transfected cells -YFP fusion construct  and of histone H4 at arginine 3 (H4R3) analysis  and by ianalysis  and S7. in vivo, and of LUC7L2 in vitroin vivo is needed for nuclear localization and function.Most recently, we identified Jmjd6 to interact with splicing regulatory proteins and to modify some of these by posttranslational hydroxylation 0 through serum starvation or contact inhibition.Chemicals and restriction enzymes were from Sigma  and New England Biolabs , respectively. DMEM cell culture medium was from GIBCO , FCS Gold foetal calf serum from PAA Laboratories . The human A549  and HEK 293-T  cell lines were obtained from the German Collection of Microorganisms and Cell Cultures . For preperation of nuclear cell extracts from transfected HEK 293-T cells the NE-PER nuclear and cytoplasmatic extraction reagents have been used together with the Thermo Scientific Halt Protease Inhibitor Cocktail . Standard cell culture conditions were used for A549 and HEK 293-T cell lines, harvesting and cell culture of mouse embryonic fibroblasts (MEFs) was described previously BamHI/NheI. The Jmjd6-(AxA) variant containing an inactivated JmjC-domain was obtained by replacing the JmjC domain and C-terminal part of Jmjd6 using the product of primer pair 2. The forward primer contains two mutations to inactivate the HxD motif of Jmjd6 . The PCR product was digested with PfIMI and XhoI and exchanged against the same fragment of Jmjd6, gaining full-length Jmjd6-(AxA). A YFP expression vector was generated by amplifying full-length YFP with primer pair 3 and cloning it into pCMV-Tag1  using NheI/XhoI. Jmjd6-YFP deletion constructs were obtained by amplifying parts of Jmjd6 from clone IRAV D0197p968D6 using primer pairs 4 to 11 and cloning them into the YFP expression vector using NotI/SalI. Correct reading frames of all constructs were confirmed by sequencing before usage.Sequences of primers used for the cloning of Jmjd6 fusion, reporter and deletion constructs are given in Cells and cell lines were transfected with these constructs using Nanofectin  or using FuGENE HD transfection reagent  according to the manufacturers instructions.Jmjd6-knockout MEFs. Positive antibody producing cells were further selected in two rounds of subcloning.Generation of a monoclonal antibody against Jmjd6 was performed as described Epitope mapping was performed on a spot-membrane covering Jmjd6 in fifteenmers, each shifted by three amino acids. The membrane was blocked for 1 h in skim milk and incubated with the primary antibody in Genosys blocking buffer  1∶10 in TBS over night according to manufactures instructions. The membrane was washed 2×20 min in TBS-T  and incubated for 1 h with a horseradish peroxidase (HRP) conjugated secondary antibody in Genosys blocking buffer. After washing the membrane twice in TBS-T, it was imaged with ECL plus  for chemifluorescence.Antibodies used for immunocytochemistry are listed in For RNase A or DNase I treatment, cells were incubated in 0.1% Triton X-100 for 15 sec, washed twice and incubated with RNase A  or DNase I  for 15 min and washed twice with PBS prior to fixation.2, 10 mM KCl, 0.2% NP-40, 0.2% 2-mercaptoethanol, proteasome inhibitor, and kept on ice for 20 min. After centrifuging at 2000 g for 5 min, the cytosolic fraction was collected as supernatant. The pellets were resuspended in Nucleusbuffer , kept on ice for 20 min and centrifuged again (2000 g for 5 min). The supernatant was collected as nuclear fraction. Nuclear extracts were prepared from transfected HEK 293-T cells using the NE-PER reagents from Pierce Biotechnology  according to manufactures instructions.Cells were washed in PBS, scrapped in Lysisbuffer  for 25 min at 100 V using blotting buffer  for 1 h and incubated with the first antibody in Western milk over night. After washing twice with TBS-T for 20 min, the membrane was incubated with an appropriate HRP conjugated secondary antibody in Western milk, washed again (2×20 min in TBS-T) and imaged using ECL plus for chemifluorescence. Experiments were performed in triplicate.Figure S1Western blot analysis of wildtype and Jmjd6 knockout MEFs using different anti-Jmjd6 antibodies. This panel shows western blots with total cell lysates from wildtype and Jmjd6-KO MEFs using four different antibodies against Jmjd6. AB-32740, AB-28349, AB-28348, and AB-11632 were evaluated. Red arrowheads mark bands not present in Jmjd6-knockout lanes. All lanes contain the same amount of protein and are from the same sample. Beta-actin served as a loading control for equal amounts of protein on the gel.(1.57 MB PDF)Click here for additional data file.Figure S2Epitope-mapping of AB-11632 and AB-10526. A spot membrane covering the full length Jmjd6 protein in fifteenmers, each shifted by three amino acids, was incubated with AB-11632 and AB-10526. (A) AB-11632 binds to seven fifteenmers and six thereof show high intensity. The core epitope covers the amino acids DWTRHN. (B) AB-10526 binds to seven fifteenmers and five thereof show high intensity. The core epitope covers the amino acids GDG.(0.07 MB PDF)Click here for additional data file.Figure S3Jmjd6 staining pattern in different cell lines and Jmjd6 distribution over time after releasing A549 cells from a G0-block. (A) Comparison of Jmjd6 nuclear expression pattern between murine MEFs and the human cell lines A549 and HEK 293-T. Cells were stained with Hoechst DNA stain (left panel) and for Jmjd6 using the anti-Jmjd6 mAB328 antibody (right panel). (B) Vertical image pairs correspond to a time point 6 h, 24 h, 48 h, or 72 h after releasing A549 cells from a G0-block. The large images show Jmjd6 distribution in green, the small insets the corresponding Hoechst DNA stain in blue. Red arrowheads indicate nuclei with deviant intranuclear Jmjd6 distribution. Shown is one representative experiments of at least at least three performed.(4.22 MB PDF)Click here for additional data file.Figure S4Single cell analysis of H3K9 and H4K20 methylation effects caused by Jmjd6 deficiency or overexpression. The top two rows show immunofluorescence images of Hoechst DNA stain (blue), Jmjd6 (green), and a histone lysine methylation state specific antibody (red) from wildtype and Jmjd6-KO MEFs . The bottom row shows immunofluorescence images of Hoechst DNA stain (blue), Jmjd6 (red), and a histone lysine methylation state specific antibody (green) in Jmjd6-Halo overexpressing A549 cells . Yellow arrowheads indicate transfected cells. Figure (A) shows results for H3K9me1, (B) for H3K9me2, (C) for H4K20me1, and (D) for H4K20me3. All experiments shown were performed at least at least three times with similar results.(0.76 MB PDF)Click here for additional data file.Figure S5Immunofluorescence analysis of H3K4 and H3K36 methylation effects caused by Jmjd6 deficiency or overexpression. The top two rows show immunofluorescence images of Hoechst DNA stain (blue), Jmjd6 (green), and a histone lysine methylation state specific antibody (red) from wildtype and Jmjd6-KO MEFs . The bottom row shows immunofluorescence images of Hoechst DNA stain (blue), Jmjd6 (red), and a histone lysine methylation state specific antibody (green) in Jmjd6-Halo overexpressing A549 cells . Yellow arrowheads indicate transfected cells. Figure (A) shows results for H3K4me1, (B) for H3K4me2, (C) for H3K4me3, (D) for H3K36me1, (E) for H3K36me2, and (F) for H3K36me3. The experiments were performed at least three times with similar results.(1.22 MB PDF)Click here for additional data file.Figure S6Immunofluorescence analysis of H3K27 methylation effects caused by Jmjd6 deficiency or overexpression. The top two rows show immunofluorescence images of Hoechst DNA stain (blue), Jmjd6 (green), and a histone lysine methylation state specific antibody (red) from wildtype and Jmjd6-KO MEFs . The bottom row shows immunofluorescence images of Hoechst DNA stain (blue), Jmjd6 (red), and a histone lysine methylation state specific antibody (green) in Jmjd6-Halo overexpressing HEK 293-T cells . Yellow arrowheads indicate transfected cells. Figure (A) shows results for H3K27me1, (B) for H3K27me2, and (C) for H3K27me3. The experiments were performed three times with similar results.(0.85 MB PDF)Click here for additional data file.Table S1Primer pairs used for cloning of Jmjd6 fusion/reporter proteins. Sequences of primers used for the cloning of Jmjd6 fusion, reporter and deletion constructs.(0.04 MB PDF)Click here for additional data file.Table S2Antibodies used in Western blotting and immunofluorescence imaging. List of antibodies used for western blotting and immunocytochemistry.(0.06 MB PDF)Click here for additional data file.


Among a total of 9.4×106 40-mer short reads, more than 98% appeared to be human, while 0.85% were identified as A/H1N1/2009 (A/Nagano/RC1-L/2009(H1N1)). Suspected bacterial reads such as Streptococcus pneumoniae and other oral bacteria flora were very low at 0.005%, and a significant bacterial infection was not histologically observed. De novo assembly and read mapping analysis of A/Nagano/RC1-L/2009(H1N1) showed more than ×200 coverage on average, and revealed nucleotide heterogeneity on hemagglutinin as quasispecies, specifically at two amino acids (Gly172Glu and Gly239Asn of HA) located on the Sa and Ca2 antigenic sites, respectively. Gly239 and Asn239 on antigenic site Ca2 appeared to be minor amino acids compared with the highly distributed Asp239 in H1N1 HAs. This study demonstrated that de novo sequencing can comprehensively detect pathogens, and such in-depth investigation facilitates the identification of influenza A viral heterogeneity. To better characterize the A/H1N1/2009 virus, unbiased comprehensive techniques will be indispensable for the primary investigations of emerging infectious diseases.Pandemic 2009 influenza A virus (A/H1N1/2009) has emerged globally. In this study, we performed a comprehensive detection of potential pathogens by  Fatal cases from A/H1N1/2009 viral infection were summarized in a report by Gill et al.In April 2009, an H1N1 triple-reassortant swine influenza virus (A/H1N1/2009) was detected in humans with febrile respiratory illness in North America th century The genome of influenza A virus (family Orthomyxoviridae) consists of 8 single-stranded negative sense RNA molecules spanning approximately 13.5 kb. The segments range in length from 890 to 2341 nucleotides (nt) and encode a total of 11 proteins http://www.immuneepitope.org/) http://www.fludb.org/brc/homeExtraPage.do?decorator=influenza&extraPage=separate) Influenza A virus can evade antibodies specific to its attachment protein, hemagglutinin (HA), by the accumulation of amino acid substitutions in HA epitopes et al. and Shen et al. reported that a structural comparison of HAs could predict probable future antigenic changes during the evolution of A/H1N1/2009 in the human population To better predict a future pandemic of influenza A virus, the characterization of possible antigenic drift will be indispensable. Igarashi et al. demonstrated the direct detection of potential pathogens, including influenza virus, using de novo pyrosequencing et al. demonstrated the discrimination of quasispecies in mixed HA subtype infections of influenza A virus using the same pyrosequencing approach In addition to this prediction, extensive investigations on viral quasispecies will be required to reveal the actual appearance of those antigenic changes. Nakamura de novo sequencing using total RNAs extracted from an autopsy lung of a patient infected with A/H1N1/2009, and detected potential pathogens such as bacteria in addition to A/H1N1/2009. Extensive DNA sequencing using the Illumina Genome Analyzer II (GA II) revealed that quasispecies for the HA sequence were generated in single patient. Such heterogeneity demonstrated the antigenic drift of HA, implying the existence of a mechanism to escape pre-existing human immunity to the virus.Here, we performed de novo sequencing of double-stranded cDNA from total RNA extracted from the autopsy lung of a patient infected with the A/H1N1/2009 virus (A/Nagano/RC1/2009(H1N1)) in August 2009 in Japan. The patient was found to be positive for A/H1N1/2009 by real-time reverse transcriptase-polymerase chain reaction (RT-PCR); histopathological findings were also reported 6 40-mer reads from the cDNA library . Therefore, the presence of P. acnes could be the result of contamination at some point from the autopsy to the preparation of the cDNA library. In addition to P. acnes, Escherichia coli and Acinetobacter baumannii were frequently detected as possible contaminants. Suspected bacterial pathogens were identified as Streptococcus pneumoniae and Porphyromonas gingivalis. Specific PCR using 16S-rDNA and the lytA gene was performed for further verification of the presence of S. pneumoniae (data not shown). Although S. pneumoniae was not sufficiently abundant to conclude a coinfection with A/H1N1/2009, the severity of the A/H1N1/2009 infection could be correlated with S. pneumoniae, as reported by Palacios et alStreptococcus sp., generally constitute the normal human oral flora.Regarding the bacterial hits, species classification was determined based on the results of a BLASTN search against the nt database . The mosde novo assembler. The resultant contigs generated using Euler-SR had longer extended sequences than those generated using Velvet (data not shown); thus, all further analyses were performed using the contigs generated using Euler-SR  for the 8 segments, the 40-mer short reads were aligned to the sequence of A/Tronto/T0106/2009(H1N1), which was found to be the most similar to the A/H1N1/2009 virus using a BLASTN search. The coverage plot curves were not flat throughout the segments. Intriguingly, both ends of segment 1 (encoding PB2), the 3′–end of segment 3 (encoding PA), and approximately 700 nt of segment 8 (encoding NS) showed significant abundant coverage greater than ×1000.172Glu and the other two alterations cause Gly239Asn  was located on the Sa antigenic site  in Germany and A/Catalonia/S1207/2009(H1N1) in Spain (data not shown). One intriguing hit was to A/Pennsylvania/14/2009(H1N1) isolated in the US, whose HA sequence has an Xaa amino acid at position 172 due to the presence of the heterogeneous nucleotide R (A or G) (The HA Gly(A or G) . This de239Asn was located on the Ca2 antigenic site that contributes to binding with the host's sialic acid receptor Furthermore, HA GlyS. pneumoniae could contribute to the severity and fatality of infection with the A/H1N1/2009 virus S. pneumoniae coinfection has been reported to play a crucial role in the severity of influenza virus infection in some cases In this study, we demonstrated the detection of potential pathogens using a next-generation DNA sequencer. We speculated that, in addition to influenza A virus, additional potential pathogens such as de novo assembly, suggesting the importance of this result in terms of being able to evaluate uncharacterized emerging infectious agents using an unbiased sequencing technique at the outbreak of a pandemic. Indeed, this study demonstrated that whole contigs can be identified as A/H1N1/2009, but not seasonal H1N1 or other subtypes  were determined using qRT-PCR, but this was not always observed in autopsy samples from other patients (less than β-actin) (data not shown). Such abundant read sequencing enabled us to obtain almost full-coverage contig sequences for the viral segments using subtypes .The read coverage profile generated by mapping was very indicative for segment 1 encoding PB2 . Both ende novo sequencing revealed the presence of A/Nagano/RC1-L/2009(H1N1) HA quasispecies in the autopsy sample (172Glu in the HA Sa antigenic site in A/Nagano/RC1-L/2009(H1N1), and this appears to be a very rare event among A/H1N1/2009 viruses to date.Contrary to the viral sequences obtained for A/Nagano/RC1/2009(H1N1) isolated from passaging in MDCK cells, y sample . Despite239Asn in the HA Ca2 antigenic site of A/Nagano/RC1-L/2009(H1N1). This antigenic site plays a crucial role in conferring specificity to galactose of the human α2-6 sialylated glycan receptor We also observed another substitution of GlyThus far, amino acid substitutions in the HAs of A/H1N1/2009 have been identified compared with seasonal H1N1 HAs. Homology-based structural investigations et al. predicted possible substitutions and these included the two amino acid substitutions presented here (Gly172Glu and Asp239Gly) et al. suggested that host-driven antigenic drift based on evolutionary trends appeared to favor Asp239 (corresponding to Asp225 in the mature HA) in swine HAs and the 1918 pandemic, while Asp204 (corresponding to Asp190 in the mature HA) was favored in seasonal H1N1 HAs a posteriori. Furthermore, recent study has shown that receptor-binding avidity can influence antigenic drift Very suggestive reports predicted the possible future antigenic drift of A/H1N1/2009 viruses from viral sequence and structural comparative analyses de novo sequencing can comprehensively detect pathogens, and such in-depth investigation facilitates the identification of influenza A viral heterogeneity during infection. The possibility of mixed infections with variants remains to be elucidated in this case, but worldwide sequencing efforts suggest that quasispecies of the A/H1N1/2009 virus evidently appear and are observed. To better characterize the currently emerging A/H1N1/2009 virus and prevent worse pandemics in the near future, unbiased de novo sequencing techniques will be indispensable for the primary investigations of emerging infectious diseases.In conclusion, this study demonstrated that The study protocol was approved by the institutional medical ethical committee, National Institute of Infectious Diseases, Japan , and the study was conducted according to the Declaration of Helsinki Principles. In the autopsy case, a written consent for autopsy was obtained from relatives.Information for the patient was previously reported 7 clusters for one lane, the general procedure as described in the standard recipe (Illumina) was performed as follows: template hybridization, isothermal amplification, linearization, blocking, denaturation, and hybridization of the sequencing primer (Illumina). All sequencing runs for 40 mers were performed with GA II using the Illumina Sequencing kit (ver. 3). Fluorescent images were analyzed using the Illumina base-calling pipeline 1.4.0 to obtain FASTQ formatted sequence data.An approximately 300-bp length cDNA library was prepared using a genomic DNA sample prep kit , and DNA clusters were generated on a slide using a Cluster Generation kit (ver. 2) on an Illumina cluster station (Illumina), according to the manufacturer's instructions. To obtain ∼1.0×10The obtained DNA sequences were investigated using a BLAST search as shown in de novo assembly, all obtained 40-mer reads were trimmed based on the phred quality value obtained using the Euler-SR qualitytrimmer command Prior to easyrun Perl-command. The consensus sequences were extracted as a “cns.fq” file for each segment, and deposited in the DNA Data Bank of Japan , and AB538394 for segment 4 encoding the HA-Minor sequence). Read coverage at every nucleotide was obtained using Maq software (ver. 0.7.1) with the pileup command. Read alignment for the validation of SNPs was performed using the MapView graphical alignment viewer To obtain consensus sequences for the respective 8 segments of influenza A virus, 40-mer short reads were aligned to A/Tronto/T0106/2009(H1N1) sequences (gb|CY045951.1 – .8) as reference sequences with Maq software (ver. 0.7.1) 5′–CGAACAAAGGTGTAACGGCAGCAT–3′; HA-Major-specific reverse primer: pdmFlu-HA-R_Major, 5′–ATAGTTCATTCTCCCTTCTTGACC–3′; HA-Minor-specific reverse primer: 5′–ATAGTTCATTCTCCCTTCTTGATT–3′), and the SuperScript III Platinum SYBR Green One-Step qRT-PCR kit with ROX (Invitrogen), and analyzed using the ABI PRISM 7900HT Real-time PCR System . The following qRT-PCR program was used: RT reaction, 50°C for 3 min; initial denaturation, 95°C for 5 min; 2 steps of amplification (×40 cycles), 95°C for 15 sec and 60°C for 30 sec. The human β-actin gene was used as the internal control. PCR products were resolved by 5% polyacrylamide gel electrophoresis, followed by Sanger sequencing using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems).qRT-PCR was performed using 100 ng of total RNA, HA variant-specific primers Click here for additional data file.Text S2De novo assembly of the influenza A virus using Euler-SR v1.0 (0.04 MB RTF)Click here for additional data file.


Cyclin/cdk complexes are generally inhibited by cyclin-dependent kinase inhibitors(ckis), some of which are induced by wild-type p53. The aims of this study were: to investigate levels of cyclin D1 expression in transitional cell carcinoma (TCC) of the bladder; to correlate these results with data concerning the expression of p53, waf1, pRb and Ki67; and to determine whether cyclin D1 expression could predict clinical outcome. Paraffin-sections from 150 newly diagnosed bladder tumours  were stained for cyclin D1 using immunohistochemistry and a cyclin D1 index assigned. These results were correlated with data relating to the expression of p53 and waf1 by the same tumours. A representative subset of 54 tumours  was also stained for Ki67 and 55 were stained for pRb. The clinical course of each patient was recorded and multivariate analyses of risk factors for tumour recurrence, stage progression and overall survival were performed. Positive staining for cyclin D1 was found in 83% of tumours. The staining pattern varied between tumours with nuclear, cytoplasmic or a combination of the two evident in different tumours. 89% of Ta/T1 and 74% of T2–T4 tumours showed nuclear staining with or without cytoplasmic staining. The median value for cyclin D1 staining was significantly higher in Ta/T1 tumours (41%) compared with T2–T4 tumours  with 26% of muscle-invasive tumours demonstrating absent staining. In addition, the median value for cyclin D1 staining was significantly higher in G1/G2 tumours (43%) compared with G3 tumours . There was a significant positive correlation between expression of cyclin D1 and waf1 expression (P< 0.0001) as well as pRb expression but not between cyclin D1 expression and expression of p53. Ki67 expression was significantly associated with increasing tumour stage (P< 0.005) and histological grade (P< 0.05) but did not correlate with cyclin D1 expression. A cyclin D1 index ≥ 8% was associated with significantly better survival in those patients with muscle-invasive disease (T2–T4). In addition, there was a significantly higher progression rate for those patients with Ta/T1 disease whose tumours demonstrated cytoplasmic cyclin D1 staining. These results indicate that cyclin D1 expression is significantly higher in low-stage, well differentiated bladder tumours and strongly correlates with waf1 expression. In a multivariate analysis, cyclin D1 expression is an independent prognostic indicator of survival in those patients with muscle-invasive disease. © 2001 Cancer Research Campaign http://www.bjcancer.comNormal cell proliferation is closely regulated by proteins called cyclins. One of these, cyclin D1, in combination with its corresponding cyclin-dependent kinase (cdk), is essential for G


B. subtilis is an important organism in the biotechnological application. The efficient expression system is desirable in production of recombinant gene products in B. subtilis. Recently, we developed a new inducible expression system in B. subtilis, which directed by B. subtilis maltose utilization operon promoter Pglv. The system demonstrated high-level expression for target proteins in B. subtilis when induced by maltose. However, the system was markedly repressed by glucose. This limited the application of the system as a high-expression tool in biotechnology field. The aim of this study was to further improve the Pglv promoter system and enhance its expression strength.glv. The transcription level from four mutants was increased. Production of β-Gal from the mutants reached the maximum 1.8 times as high as that of wildtype promoter. When induced by 5% maltose, the production of β-Gal from two mutants reached 14.3 U/ml and 13.8 U/ml, 63.5% and 57.5% higher than wildtype promoter (8.8 U/ml) respectively. Thus, site-directed mutagenesis alleviated the repression of glucose and improved the expression activity. To further improve the promoter system, the B. subtilis expression host was reconstructed, in which B. subtilis well-characterized constitutive promoter P43 replaced the promoter of the glv operon in B. subtilis chromosome through a double crossover event. The β-galactosidase production from the improved system (21.1 U/mL) increased compared to that from origin system. Meanwhile, the repression caused by glucose was further alleviated.Here, site-directed mutagenesis was facilitated to enhance the expression strength of Pglv-M1 through site-directed mutagenesis, which demonstrated high expression strength and alleviated the repression caused by glucose. Moreover, we alleviated the repression and enhanced the expression activity of the Pglv-M1 promoter system via reconstruction of the B. subtilis host. Thus, we provided a valuable expression system in B. subtilis.In this study, we obtained a mutated promoter P B. subtilis is an important organism in the biotechnological application, regarding its non-pathogenic and well-characterized biochemical and physiological property . In bri and . InApaI and EcoRI was carried out as described previously [Southern blot analysis of chromosomal DNA digested with eviously . Probe lReducing sugar was measured by the dinitrosalicylic method (DNS)  using glThe concentration of glucose was determinate as previously described The authors declare that they have no competing interests.MMY initiated and coordinated the project. WWZ and MMY were responsible for site-directed mutagenesis and its analysis. WWZ and YLC performed the batch cultivation. MMY and Y-S Gong performed construction of expression system. All authors wrote the paper and approved the final version of the manuscript.B. subtilis 1A747 harboring different plasmids when cultured in LBThe growth curves from . (black diamond) represents OD595 from B. subtilis 1A747 harboring pLJ-7; (black square) represents OD595 from B. subtilis 1A747 harboring pJRINM1; (black triangle) represents OD595 from B. subtilis 1A747 harboring pJRINM2; cross (x) represents OD595 from B. subtilis 1A747 harboring pJRINM3; asterisk (*) represents OD595 from B. subtilis 1A747 harboring pJRINM4.Click here for fileB. subtilis 1A747 harboring different plasmids when cultured in LB supplemented with 5% maltoseThe growth curves from . (black diamond) represents OD595 from B. subtilis 1A747 harboring pLJ-7; (black square) represents OD595 from B. subtilis 1A747 harboring pJRINM1; (black triangle) represents OD595 from B. subtilis 1A747 harboring pJRINM2; cross (×) represents OD595 from B. subtilis 1A747 harboring pJRINM3; asterisk (*) represents OD595 from B. subtilis 1A747 harboring pJRINM4.Click here for fileThe growth curves from B. subtilis 1A747 harboring different plasmids when cultured in LB supplemented with 5% maltose plus 5% glucose. (black diamond) represents OD595 from B. subtilis 1A747 harboring pLJ-7; (black square) represents OD595 from B. subtilis 1A747 harboring pJRINM1; (black triangle) represents OD595 from B. subtilis 1A747 harboring pJRINM2; cross (×) represents OD595 from B. subtilis 1A747 harboring pJRINM3; asterisk (*) represents OD595 from B. subtilis 1A747 harboring pJRINM4.Click here for filePlasmids used in this study.Click here for filePrimers and oligonucletides used in this study.Click here for file


Two amino acids (lysine at position 627 or asparagine at position 701) in the polymerase subunit PB2 protein are considered critical for the adaptation of avian influenza A viruses to mammals. However, the recently emerged pandemic H1N1 viruses lack these amino acids. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of lysine at position 627 and confers efficient viral replication to pandemic H1N1 viruses in mammals. Moreover, a basic amino acid at position 591 of PB2 substantially increased the lethality of an avian H5N1 virus in mice. We also present the X-ray crystallographic structure of the C-terminus of a pandemic H1N1 virus PB2 protein. Arginine at position 591 fills the cleft found in H5N1 PB2 proteins in this area, resulting in differences in surface shape and charge for H1N1 PB2 proteins. These differences may affect the protein's interaction with viral and/or cellular factors, and hence its ability to support virus replication in mammals. Influenza viruses that originate from avian species likely have to acquire adapting amino acid changes to replicate efficiently in mammals. Two amino acid changes in the polymerase PB2 protein—a glutamic acid to lysine change at position 627 or an aspartic acid to asparagine change at position 701—are known to allow influenza viruses of avian origin to replicate efficiently in mammals. Interestingly, the pandemic H1N1 viruses (which possess an avian-like PB2 gene) do not encode the ‘human-type’ amino acids PB2-627K and PB2-701N. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of PB2-627K and allows efficient replication of highly pathogenic H5N1 and pandemic H1N1 viruses in mammalian species. We also present the X-ray crystal structure of the C-terminal portion of a pandemic H1N1 PB2 protein. The basic amino acid at position 591 fills a distinctive cleft found in the PB2 proteins of H5N1 viruses. We also speculate on the biological significance of the altered surface of the H1N1 PB2 protein. Reverse genetics approaches demonstrated a critical role of the UT3006 PB2 segment in facilitating more efficient growth of UT3006-CkUT3091 reassortants in NHBE cells  replicated more efficiently than an avian H5N1 virus  in normal human bronchioepithelial (NHBE) cells , despiteBE cells . AlthougBE cells , althougBE cells . In contBE cells . The mut6 or 105 PFU of virus] lost more than 25% of their pre-infection weight and had to be euthanized on day 4 or 5 post-infection (2–104 PFU), most animals infected with CkUT3091 virus survived (1 PFU), infected animals did not experience significant weight loss and survived (50 was 104.3 PFU for CkUT3091 and 50 PFU for the mutant possessing PB2-Q591K (CkUT3091-PB2-Q591K). These findings demonstrated that the glutamine-to-lysine mutation at position 591 of PB2 increased the virulence of an avian H5N1 influenza virus in mice.Next, we tested CkUT3091 and its mutant possessing the PB2-Q591K substitution (CkUT3091-PB2-Q591K) for their virulence in mice. All mice infected intranasally with high doses of virus , PB2-591K/627E , or PB2-591R/627E (found in pandemic H1N1 viruses). However, the combination of PB2-591R/K and PB2-627K does not seem to further enhance replication in mammalian cells  probably favors the former possibility, since it is less likely that they would both enhance interaction with the same host protein, but the positive charge introduced by either change could easily disrupt interaction with an inhibitory host protein.A previous study A serine at position 590 of PB2 is found in pandemic H1N1 viruses (but not consistently in older human H1N1 isolates), and this residue (PB2-590S) has been suggested to play a role in the enhanced replicative ability of pandemic H1N1 viruses in mammals http://www.promedmail.org/pls/otn/f?p=2400:1001:19224::NO::F2400_P1001_BACK_PAGE,F2400_P1001_PUB_MAIL_ID:1010,79432), this mutation did not appear to increase the severity of disease, and does not seem to spread in human populations, consistent with our conclusion that the PB2-627K mutation does not provide a significant replicative advantage to pandemic H1N1 viruses. Based on findings with H5N1 influenza viruses, it was feared that the introduction of PB2-627K into pandemic H1N1 viruses could increase the pathogenicity of the pandemic viruses. However, our data and recent findings by others Our study identifies a new marker (PB2-591R or K) for influenza virus adaptation in mammals that compensates for the lack of PB2-627K. This finding provides an explanation for the efficient replication of pandemic H1N1 viruses (which possess PB2-591R) in mammals. Our X-ray crystal structure of the C-terminal portion of a pandemic H1N1 PB2 protein reveals changes in surface shape and charge created by PB2-591R which may prevent an inhibitory host factor as suggested by Mehle & Doudna 2.Human embryonic kidney (293 or 293T) cells were maintained in Dulbecco's modified essential medium (DMEM) containing 10% fetal calf serum and antibiotics. Madin-Darby canine kidney (MDCK) cells were maintained in Eagle's minimal essential medium (MEM) containing 5% newborn calf serum. Normal human bronchioepithelial cells (NHBE) were obtained from Lonza  and maintained in serum-free and hormone-supplemented growth medium according to the manufacturer's instructions. All cells were incubated at 37°C with 5% COAll influenza viruses used in this study were amplified in MDCK cells. All viruses were stored at −80°C until their use in experiments. The titers of stock viruses were determined by plaque assays in MDCK cells (for H1N1 viruses) or NHBE cells (for H5N1 viruses). All experiments with H5N1 viruses were performed in enhanced biosafety level 3 (BSL3) containment laboratories at the University of Wisconsin-Madison, which are USDA approved for such use by the CDC and the U.S. Department of Agriculture, or in BSL3 containment laboratories at the University of Tokyo , which are approved for such use by the Ministry of Agriculture, Forestry and Fisheries, Japan.The cDNAs of the A/chicken/Indonesia/UT3091/05 , A/Indonesia/UT3006/05 , and A/California/04/09  viruses were synthesized by reverse transcription as previously described Mutations in the PB2 genes of CkUT3091 or Cal04 viruses were generated by PCR amplification of the respective RNA polymerase I PB2 construct with primers possessing the desired mutations (primer sequences will be provided upon request). All constructs were sequenced to ensure the absence of unwanted mutations.To assess the viral polymerase activity, we performed luciferase activity-based minireplicon assay as described previously 2. At the indicated times post infection, the virus titers in the cell culture supernatant were determined by plaque assays in NHBE cells. For the determination of viral growth kinetics in MDCK cells, cells were infected at an m.o.i. of 0.001; at the indicated time points, virus titers were determined by plaque assays.NHBE cells (Lonza) were infected with the indicated viruses at a multiplicity of infection (m.o.i) of 0.0005. One hour later, the viral inoculum was removed and replaced with bronchial epithelial growth medium  containing bovine pituitary extract , hydrocortisone (0.5 µg/ml), human epidermal growth factor , epinephrine (0.5 µg/ml), transferrin (10 µg/ml), insulin (5 µg/ml), triiodothyronine (6.5 ng/ml), bovine serum albumin – fatty-acid free , retinoic acid , gentamycin (30 µg/ml) and amphotericin B (15 ng/ml). Infected cells were incubated at 37°C and 5% CO1–106 PFU (50 µl) of CkUT3001 or CkUT3091-PB2-Q591K, or with 103 or 106 PFU of Cal04 possessing wild-type or mutant PB2 protein. Body weight and survival were monitored daily for 14 days; mice with body weight loss of more than 25% of pre-infection values were euthanized. For virus titration in organs, mice were infected intranasally with 105 PFU of virus and euthanized on days 3 and 6 postinfection.Six-week-old female BALB/c mice  were used in this study. Baseline body weights were measured prior to infection. Four mice per group were anesthetized with isoflurane and intranasally inoculated with 106 plaque-forming units  of the mixture of Cal04 and mutant virus at a 1∶1 ratio . One day after infection, three naive ferrets were each placed in a cage adjacent to an inoculated ferret (contact ferrets). Hence, we tested three pairs of infected/contact ferrets for each virus. Nasal washes from ferrets were collected on days 1, 3, 5, and 7 post-infection or co-housing and titrated for virus titers in MDCK cells. Samples with greater than 1×102 pfu/ml of virus were subjected to sequence analysis as described below. All animal experiments were carried out in accordance with institutional guidelines.We used six-to-seven-month-old female ferrets , which were serologically negative by hemagglutination inhibition (HI) assay for currently circulating human influenza viruses (including pandemic H1N1 viruses). The animals were housed in adjacent transmission cages that prevent direct and indirect contact between animals but allow spread of influenza virus through the air. Three ferrets were intranasally inoculated with 108 molecules/µl, emulsion PCR was performed on four pools containing 14 MID-tagged amplicons each.Viral RNA was extracted from nasal wash samples and twice from the inoculum (to verify the 1∶1 ratio of the two test viruses). The cDNAs were synthesized as previously described www.454.com). An average of 2221 sequence reads was obtained for each sample (range: 705–5102). All sequences from a single time point were aligned to the PB2 sequence of A/California/04/09 (Roche/454 AVA software). The frequencies of wild-type and mutant amino acids at residues 591, 627, and 701 were tabulated from the aligned sequences. To confirm the pyrosequencing results, independent PB2 sequences from 37 of the 42 samples were generated using alternative sets of PCR primers and pyrosequenced using the Roche/454 FLX chemistry.The resulting pools were pyrosequenced in individual regions of a 70×75 PicoTiterPlate with a 16-well gasket on a Roche/454 GS FLX instrument following protocols provided by the manufacturer  Codon engineered synthetic genes for the PB2 gene of influenza A virus strains A/Vietnam/1203/04 (H5N1) (AAT73550) and A/Mexico/InDRE4487/09 (H1N1) (ACQ73384) were designed in Gene Composer  using a combined Our experiments in mice, ostriches, and ferrets followed the University of Tokyo's Regulations for Animal Care and Use and the University of Wisconsin-Madison's Animal Care and Use Protocol. They were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo  and the Animal Care and Use Committee of the University of Wisconsin-Madison (protocol number V00806), which acknowledged and accepted both the legal and ethical responsibility for the animals, as specified in the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, 2006 (Japan) and in the Animal Welfare Act and associated Animal Welfare Regulations and Public Health Service Policy (USA).Figure S1Alignment of PB2 protein sequences of influenza A viruses isolated from ostriches, emus, and rheas. Shown is the region covering amino acid positions 591, 627, and 701.(0.44 MB TIF)Click here for additional data file.Figure S2in vitro. 293 cells were transfected with plasmids for the expression of a virus-like RNA encoding luciferase, and with plasmids encoding the Cal04 PB1, PA, NP, and wild-type or mutant PB2 proteins. Forty-eight hours later, luciferase activity was assessed.Polymerase activity of Cal04 PB2 variants (0.10 MB TIF)Click here for additional data file.Figure S3Growth curves of wild-type and mutant Cal04 viruses in MDCK cells. Cells were infected with wild-type or mutant Cal04 viruses at an m.o.i. of 0.001. At the indicated times post-infection, the virus titers in the cell culture supernatant were assessed by plaque assays. Experiments were carried out in triplicate.(0.10 MB TIF)Click here for additional data file.Figure S45 PFU (A) or 104 PFU (B) of the indicated viruses and assessed daily for weight losses. For mice infected with the PB2-627K or PB2-701N variants, one mouse each had to be euthanized on day 5 or 6, respectively, due to body weight loss of more than 25% of the respective pre-infection body weight.Virulence of wild-type and mutant Cal04 viruses in mice. BALB/c mice were infected with 10(0.17 MB TIF)Click here for additional data file.Table S1Biological features of PB2 variants in mice(0.03 MB DOC)Click here for additional data file.Table S2Competitive transmission studies in ferrets for wild-type and mutant pandemic H1N1 viruses(0.04 MB XLS)Click here for additional data file.


RPS6KB1), located at 17q23, is amplified and overexpressed in 10–30% of primary breast cancers and breast cancer cell lines. p70S6K is a serine/threonine kinase regulated by PI3K/mTOR pathway, which plays a crucial role in control of cell cycle, growth and survival. Our aim was to determine p70S6K and PI3K/mTOR/p70S6K pathway dependent gene expression profiles by microarrays using five breast cancer cell lines with predefined gene copy number and gene expression alterations. The p70S6K dependent profiles were determined by siRNA silencing of RPS6KB1 in two breast cancer cell lines overexpressing p70S6K. These profiles were further correlated with gene expression alterations caused by inhibition of PI3K/mTOR pathway with PI3K inhibitor Ly294002 or mTOR inhibitor rapamycin.The 70 kDa ribosomal protein S6 kinase (RPS6KB1. Furthermore, 17 genes including VTCN1 and CDKN2B showed overlap with genes differentially expressed after PI3K or mTOR inhibition. The gene expression signatures responsive to both PI3K/mTOR pathway and p70S6K inhibitions revealed previously unidentified genes suggesting novel downstream targets for PI3K/mTOR/p70S6K pathway.Altogether, the silencing of p70S6K altered the expression of 109 and 173 genes in two breast cancer cell lines and 67 genes were altered in both cell lines in addition to Since p70S6K overexpression is associated with aggressive disease and poor prognosis of breast cancer patients, the potential downstream targets of p70S6K and the whole PI3K/mTOR/p70S6K pathway identified in our study may have diagnostic value. RPS6KB1, which is located at 17q23 and is amplified and overexpressed in 10–30% of breast cancer cell lines and primary breast cancers : siRNA 1 , siRNA 2  and siRNA 3 (siRNA ID#: 110802): sense strand 5'-GGUCAUGUGAAACUAACAGtt-3', antisense strand 5'-CUGUUAGUUUCACAUGACCtt-3') purchased from Ambion® . Transfections were performed using the "Transfecting Stealth™ RNA or siRNA into Mammalian Cells Using Lipofectamine™ 2000" protocol according to the manufacturer's recommendations . 150,000 cells per well were plated in 2.5 ml of culture medium onto a 6-well plate 24 hours before the siRNA transfections. For BT-474 cell line, 7.5 μl of Lipofectamine2000 transfection reagent (Invitrogen) and 50 pmol of RPS6KB1 siRNA were used. For MCF-7 cell line, the conditions were 7.5 μl of Lipofectamine2000 and 200 pmol of siRNA. After four hours of transfection, the cell culture medium was replaced with fresh medium and the cells were incubated altogether for 72 hours. The cells from two parallel wells were pooled and the total RNAs of BT-474 and MCF-7 breast cancer cell lines were isolated using Qiagen RNeasy Mini Kit . The quality of the RNA was assessed by 2100 Bioanalyzer  to control the integrity of the samples before microarray hybridizations.BT-474 and MCF-7 breast cancer cell lines with high-level amplification and overexpression of ® reagent  according to the manufacturer's instructions. After the Trizol extraction, the RNAs were purified using Qiagen's  RNeasy column purification to remove remnants from Trizol extraction. Then, all the RNAs were run by 2100 Bioanalyzer (Agilent) before microarray hybridizations.Five breast cancer cell lines, BT-474, MCF-7, MDA-MB-361, MDA-MB-436, and SK-BR-3, were treated with 50 μM of PI3K inhibitor Ly294002  and 100 nM of mTOR inhibitor rapamycin  and the cells were harvested at 24 h and 48 h time points. The cell lines were purchased from ATCC and cultured according to the recommended conditions. The RNA was extracted using TRIzolRPS6KB1 siRNAs. Additionally, phosphorylation of p70S6K was determined in Ly294002- and rapamycin-treated cells to study whether the inhibitors blocked the phosphorylation of p70S6K Thr389. The inhibitor- and siRNA-treated cells harvested at the time points were lysed with 150 μl of boiling hot SDS lysis buffer  per well on a 6-well plate. The downregulation of p70S6K was detected with rabbit p70S6 kinase antibody  in 1:1000 dilution and rabbit β-actin antibody  was used to detect the equal loading of samples. The effect of the inhibitors on p70S6K phosphorylation was studied using rabbit p-p70S6 kinase antibody for Thr389 phosphorylation (Santa Cruz Biotechnology) in 1:1000 dilution. The total protein amount of p70S6 kinase was studied as above. We also performed protein-level validation of the microarray results with the following antibodies: Akt , mTOR , and eIF4G1 . The detection of the proteins was performed using anti-rabbit secondary antibody  and chemiluminescence by ECL detection kit (Amersham Biosciences).Western immunoblotting was carried out to study the protein levels of p70S6K, AKT, mTOR and eIF4G1 after the perturbation of the breast cancer cells with PI3K/mTOR pathway inhibitors or RPS6KB1 siRNAs induced apoptosis, caspase-3 activity was measured from 30 or 40 μg of protein from each inhibitor- and siRNA-treated cell lines using colorimetric caspase-3 activity (Ac-DEVD-pNa cleavage) assay as described previously [Apoptosis and cell cycle assays were performed for inhibitor-treated breast cancer cell lines and their controls to evaluate biological response to PI3K/mTOR pathway inhibitors. To study whether the inhibitors or eviously . AdditioRPS6KB1 siRNA-suppressed BT-474 and MCF-7 cell lines were hybridized against their corresponding control cell lines transfected with siRNA Scramble Duplex  using identical conditions as with RPS6KB1 siRNA. Five-hundred nanograms of total RNA was labeled according to the manufacturer's recommendations (Agilent Technologies). The RNAs were hybridized on Agilent's Human 4x44K Oligo Microarrays containing 45,220 features. Two technical replicates were performed for each hybridization and the logarithmic transformed (base 10) mean gene expression ratios were taken for further analysis. Pre-processing of the data was performed by Feature Extraction software . Only those genes, for which the mean signal log 10 ratio from inhibitor-treated cell lines or the two siRNA-suppressed replicates was ≥ 0.3 or ≤ -0.3 with p values ≤ 0.05 representing two-fold up- or down-regulation, respectively, were considered differentially expressed. The data from the inhibitor-treated and the siRNA-transfected samples were analyzed separately due to different processing of the samples. All the raw data is available at CanGEM (please see Availability & requirements for more information).Microarray analyses from inhibitor-treated cell lines were performed using Agilent's Human 1A Oligo Microarrays containing 17,986 genes or transcripts. The inhibitor-treated samples were hybridized against the corresponding untreated cell line harvested at 24 h and 48 h time points. The labeling was performed from 20 μg of total RNA using direct labeling method according to the manufacturer's instructions (Agilent Technologies). The RNAs extracted from the To further study the genes that were identified as differentially expressed due to PI3K/mTOR pathway inhibition across all treated breast cancer cell lines, SOM clustering algorithm with component plane presentation was used to analyze and visualize the differences between the drug-treated cell lines ,44. OnlyTo highlight functionally important biological responses to PI3K/mTOR pathway inhibitors, the representation of gene ontology classes among differentially expressed genes (SD>3) in inhibitor-treated breast cancer cell lines was explored using The Gene Ontology Categorizer . First, To study whether other small molecules would cause similar transcriptional alterations in human cell lines, the inhibitor-perturbed gene expression data was downloaded into the Connectivity Map, which is a web-based catalogue of gene expression data from chemically treated cultured human cells . The AgiGO: gene ontology; mTOR: mammalian target of rapamycin; p70S6K: 70 kDa ribosomal protein S6 kinase; PI3K: phosphoinositide-3-kinase; SOM: self-organizing mapCanGEM: Ensembl: Broad Institute, Connectivity Map: OM designed and supervised the study. HH performed the RNAi experiments, inhibitor treatments and Western assays. AN and JK carried out the apoptosis analyses. HH, MS and SH carried out the bioinformatics analysis of the microarray data. AK was involved in setting up the study. HH and OM wrote the manuscript. All the authors have read and accepted the final version of the manuscript.RPS6KB1 siRNA treatments in breast cancer cell lines.Differentially expressed genes after Click here for fileDifferentially expressed genes after inhibitor treatments in breast cancer cell lines.Click here for fileEffects of Ly294002 and rapamycin treatments on cell cycle arrest in breast cancer cell lines.Click here for fileCommon differentially expressed genes by inhibitor and siRNA treatments in BT-474 and MCF-7.Click here for fileEnriched gene ontology (GO) classes after Ly294002 and rapamycin treatments in breast cancer cell lines.Click here for file


The Type IIS restriction endonuclease BtsI recognizes and digests at GCAGTG(2/0). It comprises two subunits: BtsIA and BtsIB. The BtsIB subunit contains the recognition domain, one catalytic domain for bottom strand nicking and part of the catalytic domain for the top strand nicking. BtsIA has the rest of the catalytic domain that is responsible for the DNA top strand nicking. BtsIA alone has no activity unless it mixes with BtsIB to reconstitute the BtsI activity. During characterization of the enzyme, we identified a BtsIB mutant R119A found to have a different digestion pattern from the wild type BtsI. After characterization, we found that BtsIB(R119A) is a novel restriction enzyme with a previously unreported recognition sequence CAGTG(2/0), which is named as BtsI-1. Compared with wild type BtsI, BtsI-1 showed different relative activities in NEB restriction enzyme reaction buffers NEB1, NEB2, NEB3 and NEB4 and less star activity. Similar to the wild type BtsIB subunit, the BtsI-1 B subunit alone can act as a bottom nicking enzyme recognizing CAGTG(-/0). This is the first successful case of a specificity change among this restriction endonuclease type. Restriction-Modification (R-M) systems mostly occur naturally in bacteria and serve as defensive systems against invading DNA, particularly bacteriophages. The restriction endonuclease digests invading DNA while the DNA methyltransferase component(s) (cognate methyltransferase(s)) modifies the host genome and protects the host DNA from restriction endonuclease digestion Bacillus thermoglucosidasius . BtsI REase consists of two subunits BtsIA and BtsIB, and the modification system also contains two methyltransferases (M1.BtsI and M2.BtsI) GCAGTG(2/0))GCAGTG(2/0), which means that BtsI cuts 2 nt after the recognition sequence on the top strand and right after the recognition sequence on the bottom strand.BtsI (Genbank: CS808224.1), a Type IIS REase, was isolated from CTGAAG(16/14)) has been engineered to Eco57MI (CTGRAG(16/14)) by random mutagenesis followed by challenges with Eco57I (CTGAAG(16/14) and GsuI(CTGGAG(16/14)) GAACNNNNNNTCC(12-13/7)) a Type IIG-like REase with two recognition domains, can acquire new recognition and digestion specificities by domain swapping with another similar Type IIG REase Numerous efforts have been made to engineer REases for altered specificity but with rather few successful examples. Several groups reported success in altering the specificity of Type IIG REases. For example, Eco57I  compared with the wild type GCAGTG(-/0). When combined with the A subunit, BtsIB (R119A) can recognize and cleave the following sequence CAGTG(2/0). This specificity change is complete and shows little preference for any other specific sequences. To the best of our knowledge, this essentially complete specificity change of BtsI is the first successful example of such in REases other than Type IIG. The new enzyme BtsI(B:R119A) was named as BtsI-1. Correspondingly, the BtsIB(R119A) was named as Nb.BtsI-1.Here, we describe a BtsIB mutant (BtsIB:R119A) with altered specificity. After careful examination, the new specificity is one base pair shorter than the wild type enzyme. The new recognition sequence is During the process of BtsIB subunit mutagenesis aiming to investigate the effect of a single amino acid change on its protein function, one BtsIB mutant (R119A) was found to have a different digestion pattern from the wild type BtsIB. To avoid contamination from the host bacteria, we used the IMPACT system to purify BtsIB(R119A) and BtsIA to near homogeneity, as shown in To compare the specific activity of BtsIB and BtsI-1B, we used same amount of each enzyme  to digesCAGTG”. To confirm our prediction, BtsI-1was used to digest different plasmids including pUC19, pBR322 and Litmus28i. As shown in GCAGTG”, BtsI-1 no longer recognizes the first “G” which strongly suggests that Arg119 plays an important role for the first “G” recognition.To identify the recognition sequence of BtsI-1, we analyzed the pUC19 fragments digested by BtsI-1with REBpredictor CAGTG(2/0), which is not recognized by wild type BtsI, whose cutting site is GCAGTG(2/0). However, the 3′ 2-base overhang after digestion remains unchanged.To determine the cutting sites of BtsI-1, we cut pUC19 with BtsI-1 for 1 hour and subjected the substrate to run-off sequencing. Primers flanking one recognition sequence allowed sequencing from both directions, and the resulting sequences were aligned with pUC19 . After aWe explored whether substitution of other amino acid residues at position 119 of B subunit would also alter BtsI sequence recognition. R119G, R119H, R119I, R119L, R119M, R119N, R119S, R119T, R119V, R119W and R119Y showed cleavage patterns similar to the R119A mutant. It is interesting to note that BtsI(B:R119K) retained the cleavage specificity of wild type BtsI at low concentration . Considering this, we digested phiX174 RFI with BtsI-1B. As shown in Engineering new REase specificities has been limited mainly due to two reasons: 1) the complexity and extended nature of the binding interaction between REases and DNA: ∼15-20 hydrogen bonds, numerous van der Waals contacts GCAGTG; however, there is no crystal structure currently available, and there are no other restriction enzymes that recognize HCAGTG (H = A or C or T) to offer insight as to how R119 recognizes the first G. Crystallization of BtsI and BtsI-1 with DNA would be an essential step for further understanding.The typical PD-EXK motif of BtsIB is at P57, D58 and E72. R119 is distant from those residues on the polypeptide chain, yet it still can affect the catalytic site and apparently is involved in the recognition of the first G in The star activity for wild type BtsI is so severe that it is impossible to overexpress BtsIA and BtsIB subunits in the same strain even with M1.BtsI and M2.BtsI protection. However, it is interesting to note that BtsI-1 has decreased star activity. BtsI-1 is the first REase other than Type IIG with a complete specificity change. It provides a new direction for future research into changing the specificity of REases. As specificity engineering cases accumulate, understanding the mechanisms of the specificity determination will increase.All reagents, strains and vectors, unless stated otherwise, were from New England Biolabs, Inc. E. coli strain with the methylase protection ER2566(pACYC184-Bts1M1M2) containing the T7 RNA polymerase, controlled by the lac operon. The bacteria were induced by IPTG for protein expression and lysed via sonication. After centrifugation, the supernatant was loaded onto a chitin column, which was equilibrated with chitin column buffer . After washing with the same column buffer, the column was incubated with Cleavage Buffer  at 4°C overnight. The protein was then eluted. The pure BtsIA protein was dialyzed against the storage buffer .BtsIA and BtsIB genes were cloned in pUC19 and BtsIM1M2 was cloned in pACYC184, as previously described For BtsIB(R119A), the gene was fused with Mxe intein at C-terminal and cloned into pBAD241, a derivative vector from pBAD24, with some changes in the multiple cloning sites. After expression, the BtsIB(R119A) was purified with the same procedure as the BtsIA. For BtsIB(R119K), the protein was expressed in ER2984 and partially purified through Heparin Column .All sequencing procedures were performed as previously describedE. coli ER2984(pACYC184-BtsIM1M2) for expression.By using site-directed mutagenesis with Vent polymerase and inverse PCR, charged or polar residues  were individually mutated to Ala one residue at a time, and each Tyr residue was mutated to Phe. After DpnI digestion, the PCR products carrying the mutated sequences were transformed into a methyltransferase-protected (M1.BtsI and M2.BtsI) bacterial strain 2, 1 mM dithiothreitol (pH 7.9 @ 25°C); NEB2∶ 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol (pH 7.9 @ 25°C); NEB3∶ 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol (pH 7.9 @25°C); NEB4∶ 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol (pH 7.9 @ 25°C).To examine the effect of the mutated BtsIB, 2 µl crude extract or purified protein containing the mutated subunit was mixed with 1 µl purified BtsIA subunit, either at molar ratio of 1∶1 or with constant excess amount of A subunit, to reconstitute the full endonuclease activity of each BtsI mutant. The mixture of total 30 µl reaction was incubated with pUC19 (0.6 µg) in NEB1, NEB2, NEB3 or NEB4 for 1 hour at 55°C; then, the reaction was resolved on an agarose gel. The composition of 4 different reaction buffers as following: NEB1∶ 10 mM Bis Tris Propane-HCl, 10 mM MgCl


Plasmodium falciparum apical membrane antigen-1 (AMA1) is a leading malaria vaccine candidate antigen that is expressed by sporozoite, liver and blood stage parasites. Since CD8+ T cell responses have been implicated in protection against pre-erythrocytic stage malaria, this study was designed to identify MHC class I-restricted epitopes within AMA1.P. falciparum AMA1 was highly immunogenic when administered to healthy, malaria-naive adult volunteers as determined by IFN-γ ELISpot responses to peptide pools containing overlapping 15-mer peptides spanning full-length AMA1. Computerized algorithms (NetMHC software) were used to predict minimal MHC-restricted 8-10-mer epitope sequences within AMA1 15-mer peptides active in ELISpot. A subset of epitopes was synthesized and tested for induction of CD8+ T cell IFN-γ responses by ELISpot depletion and ICS assays. A 3-dimensional model combining Domains I + II of P. falciparum AMA1 and Domain III of P. vivax AMA1 was used to map these epitopes.A recombinant adenovirus serotype 5 vector expressing Fourteen 8-10-mer epitopes were predicted to bind to HLA supertypes A01 (3 epitopes), A02 (4 epitopes), B08 (2 epitopes) and B44 (5 epitopes). Nine of the 14 predicted epitopes were recognized in ELISpot or ELISpot and ICS assays by one or more volunteers. Depletion of T cell subsets confirmed that these epitopes were CD8+ T cell-dependent. A mixture of the 14 minimal epitopes was capable of recalling CD8+ T cell IFN-γ responses from PBMC of immunized volunteers. Thirteen of the 14 predicted epitopes were polymorphic and the majority localized to the more conserved front surface of the AMA1 model structure.This study predicted 14 and confirmed nine MHC class I-restricted CD8+ T cell epitopes on AMA1 recognized in the context of seven HLA alleles. These HLA alleles belong to four HLA supertypes that have a phenotypic frequency between 23% - 100% in different human populations. The sterile protective immunity to malaria induced in humans by immunization with irradiated sporozoites is thought to be mediated by CD4+ and CD8+ T cells responding to malaria peptides expressed on the surface of hepatocytes or antigen presenting cells by secreting interferon-gamma (IFN-γ) and/or by cytotoxic responses, although anti-sporozoite antibodies may contribute -5. Many Plasmodium and has traditionally been regarded as a blood stage antigen, since it is required for the invasion of red blood cells. The final 3 D model was generated by combining Domains I and II of the P. falciparum AMA1 model with Domain III of the P. vivax AMA1 model, omitting Domains I and II of the P. vivax AMA1 model. Accessibility of the amino acid residues was determined by the same software.The The five volunteers used in this study expressed a total of seven supertypes Table , A01, A0The 91 15-mers contained within peptide pools Ap1, Ap3, Ap4, Ap7, Ap8, Ap10 and Ap11 were tested individually by ELISpot assay using PBMC from 1 month post immunization. Sixteen peptides were recognized by one or more of the five volunteers at levels > 40 sfc/m, and assigned numbers of 1-16  were classified as strong binders (<500 nM). Of these 14 minimal epitopes, two were predicted to be restricted by HLA-A*0101 and one by HLA-A*3002 (A01 supertype); two by HLA-A*0201 and two by HLA-A*6802 (A02 supertype); two by HLA-B*0801 (B08 supertype); four by HLA-B*1801 and one by HLA-B*4402 (B44 supertype). Epitopes predicted to bind to supertypes A03, B27 and B58 present in the volunteers were not identified.PBMC available from bleeds at 4 months had similar activity to those at 1 month for v001 and v002 and were, therefore, used for the evaluation of epitopes E1, E2, E3, E4, E6, E8, E9, E12 and E14. ELISpot activity of these predicted minimal epitopes compared favorably to the ELISpot activity of parent peptide pool activity for all nine Table .PBMC from the same time points were used in ICS assays to measure total IFN-γ from CD4+ and CD8+ T cell subsets in response to selected minimal epitopes and parent peptide pools Figure . These aThe pattern of multifunctional CD8+ T cell responses (defined as cells expressing two or more cytokines) recalled by each minimal epitope Figure  were genThe feasibility of using pooled epitopes to evaluate CD8+ T cell responses to AMA1 was investigated to establish that non-binding epitopes did not block specific HLA-restricted binding. Three volunteers who consistently gave high responses to peptide pools were tested with four different stimulants: (1) Ap1-Ap12: all 153 15-mer peptides at 1.25 μg/mL; (2) AMA1-14e: the 14 predicted epitopes mixed and tested at 10 μg/mL; (3) 15-mer peptide pools at 10 μg/ml that were strongly recognized by these volunteers; (4) The predicted epitope at 10 μg/ml within those selected 15-mer peptide pools previously shown to have ELISpot activity. As shown in Figure CD8+ T cell depletion reduced ELISpot activity to AMA1-14e by about 85% - 95% in the two volunteers tested, v001 and v005, and AMA1-14e much more strongly induced CD8+ than CD4+ T cell IFN-γ by ICS assay. In contrast, Ap1-Ap12 induced both CD4+ and CD8+ T cell responses, whereas AMA1 recombinant protein only recalled CD4+ T cell responses  the predicted epitope recalled IFN-γ responses as effectively as the parent peptide pool, (2) the responses of the parent 15-mer peptide were CD8+ T cell-dependent by ELISpot assay, and (3) the epitopes themselves stimulated IFN-γ production by CD8+ T cells on ICS assay.To map the epitopes, low to mid level HLA typing was performed to determine the HLA A and B supertype for each of five immunized volunteers. Subsequently, all possible 8-10 amino acid sequences from 15-mer AMA1 peptides inducing recall responses on ELISpot assay were evaluated Using this approach, sixteen 15-mer peptides were down-selected from the peptide pools on the basis of stimulating positive recall responses by ELISpot assay from the immunized volunteers. NetMHC predicted 14 class I 8-10-mer epitopes lying within these peptides for the five volunteers. Nine of these were shown by ELISpot assay to be active , eliciting IFN-γ responses comparable to those elicited by the parent peptide pools, while four could not be adequately assessed as the PBMC eliciting strong recall responses for these volunteers were no longer available , and one induced low recall responses relative to the parent peptide pool (E10).To further evaluate the predicted epitopes, ELISpot depletion assays were conducted by stimulating PBMC from four volunteers with three parent peptide pools  or with five individual 15-mer peptides from those pools . The 15-mers contained, respectively, the epitopes E1, E3, E4, E6 and E14. CD8+ T cell depletion reduced responses in five tests of the three peptide pools by 65%, 97%, 96%, 100% and 100%, respectively, and to the five 15-mers by 56-100% (mean of 88%). In contrast, depleting CD4+ T cells increased activity in Ap1 and two of the three peptides it contained as well as one peptide in Ap10, and reduced the ELISpot activity recalled by the remaining peptides by only 18- 37% (mean of 30%), strongly supporting the contention that the selected 15-mers contained minimal class I epitopes.ICS assays performed using six minimal epitopes showed that four could recall IFN-γ responses from CD8+ T cells , while two could not (E10 and E11). In contrast IFN-γ responses from CD4 cells were minimal or absent when stimulating with the same minimal epitopes.In summary, of the nine epitopes active by ELISpot , subsequent ELISpot depletion assays support the identification of E1, E3, E4, E6 and E14 and ICS assays the identification of E3, E6, E8 and E14, while E2, E9 and E12 could not be adequately assessed due to PBMC supplies. Four additional epitopes  have yet to be confirmed as class I-restricted by testing for reductions in ELISpot responses on CD8+ T cell depletion or by testing for CD8+ T cell responses on ICS assay. The remaining epitope, E10, recalled only 10% of the response recalled by the parent peptide pool on ELISpot assay and was not active on ICS assay, and thus may not be correctly identified.While the focus of these experiments was induction of IFN-γ in ELISpot or ICS assays, four epitopes  demonstrated recall of multifunctional responses  that might, due to the higher IFN-γ secretion levels in multifunctional T cells, contribute disproportionately to the overall IFN-γ response at 1 month following immunization, acting as more potent immune effectors.The 14 putative minimal class I epitopes were combined into a reagent pool, denoted AMA1-14e, and this pool was shown to be an efficient stimulant for recalling T cell responses to AMA1. The AMA1-14e pool stimulated responses equivalent to parent 15-mer peptides and peptide pools, and these responses were CD8+ T cell-dependent by ELISpot depletion assay. Future trials with the NMRC-M3V-Ad-PfCA vaccine should reveal the usefulness of the AMA1-14e reagent. A previous study using DNA plasmids containing five malaria candidate vaccine genes relied on using HLA-matched peptides; an HLA-The 14 epitopes identified in this study were characterized with regard to the coverage provided for the HLA diverse human population. The epitopes were restricted by four of the seven HLA supertypes expressed by the volunteers: A01, A02, B08 and B44. MHC class 1 molecules from the other supertypes identified in the volunteers, A03, B27 and B58, may also have recognized AMA1 CD8+ T cell epitopes. Such epitopes binding to these allelic supertypes were predicted by NetMHC, but were not located within the 15-mers recalling the strongest responses by ELISpot assay, and thus were not synthesized or tested. A01, A02, B08 and B44 supertypes cover approximately 100% of Caucasian populations and 27% of US African Americans . TherefoP. falciparum AMA1 are relatively conserved among Plasmodium species [The structures of Domains I, II and III of  species ,28, but  species -65, refl species ,26,66. M species ,43. The A model of the crystal structure of AMA1 was generated to allow localization of ten of the 14 epitopes within the tertiary structure Figure . This is197, one of the variable residues in E6 , while seven of the 10 remaining epitopes are HLA-A1/A2-restricted. The signal sequence and prodomain are cleaved off first during merozoite release and red cell invasion during wThis study identified fourteen putative minimal CD8+ T cell-dependent epitopes within AMA1 that are restricted by four HLA supertypes that together are expressed by 100% of Caucasians, but only 27% of African Americans. Nine epitopes were confirmed using ELISpot or ELISpot and ICS assays. Some of these clustered on the front face of AMA1, while others were more randomly distributed but still mostly surface accessible. When the 14 epitopes were mixed together, they recalled CD8+ T cell responses in volunteers with different HLA supertypes providing a reagent useful for measuring CD8+ T cell responses in genetically different populations.Further testing of the NMRC-M3V-Ad-PfCA vaccine in volunteers representing different HLA A and B supertypes would provide the opportunity to identify additional class I epitopes. This could support the design of a chimeric AMA1 molecule able to elicit broadly protective responses among an HLA diverse population. Determining the potential contribution of these epitopes for inducing protective responses awaits future testing of the NMRC-M3V-Ad-PfCA vaccine in challenge studies.JTB, CRK and DLD are inventors listed on U.S. Patent No., U.S. Patent No. 2009-0148477 A1, and international patent application PCT/US06/33982, titled "Adenoviral Vector-based Malaria Vaccines"; JTB, CRK, TLR and DLD are inventors listed on U.S. Patent Application 12/522,335, and international patent application PCT/US08/50565 titled "Adenoviral Vector-based Malaria Vaccines". JTB, GenVec, Inc. contributed to the design of the study, interpretation of data, editing of drafts and final manuscript approval but he did not contribute to protocol development or study execution.MS and TLR designed the research; TLR, DLD, JB and CRK designed the vaccine; HG, JL, EA, GB, MB, and RS performed ELISpot assays; SM and FF performed the ICS assays; YK, BP and AS used NetMHC to predict epitopes; CO, CT, DR, and IC were investigators in the clinical trial; BF and ER performed the crystal localization studies; MS, YK, DLD, TLR and MRH wrote the paper. All authors read and approved the final manuscript.