Arabidopsis H3 variants in order to find core properties of this class of histones. Here we present genome-wide maps of H3.3 and H3.1 enrichment and the dynamic changes of their profiles upon cell division arrest. We find H3.3 enrichment to positively correlate with gene expression and to be biased towards the transcription termination site. In contrast with H3.1, heterochromatic regions are mostly depleted of H3.3. We report that, in planta, dynamic changes in H3.3 profiles are associated with the extensive remodeling of the transcriptome that occurs during cell differentiation. We propose that H3.3 dynamics are linked to transcription and are involved in resetting covalent histone marks at a genomic scale during plant development. Our study suggests that H3 variants properties likely result from functionally convergent evolution.In animals, replication-coupled histone H3.1 can be distinguished from replication-independent histone H3.3. H3.3 variants are enriched at active genes and their promoters. Furthermore, H3.3 is specifically incorporated upon gene activation. Histone H3 variants evolved independently in plants and animals, and it is unclear whether different replication-independent H3.3 variants developed similar properties in both phyla. We studied  Histone proteins are assembled into nucleosomes to build the skeleton of chromosomes. Beyond their role as DNA scaffold, histones participate in the regulation of gene activity. Studies in animals have shown that the deposition of two different histone H3 variants, H3.1 and H3.3, requires distinct pathways and results in distinct profiles throughout the genome. H3 variants evolved independently in plants and animals. Hence, H3 variants' properties shared by plants and animals would reflect core functions that have been selected during evolution. Our study indicates that these core properties include the high enrichment of H3.3 at active genes and a relative low deposition of H3.3 over regions deprived of genes or with inactive genes. In contrast with H3.1, H3.3 incorporation is dynamic and accompanies global changes of gene activity at major developmental transitions. We anticipate that the dynamic link between H3.3 variants and transcription enables remodeling of histone modifications that contribute to developmental transitions. Histones are not static scaffolding proteins but dynamic actors involved in many aspects of chromatin related functions. They are targets of chromatin modifiers that deposit covalent modifications on histone tails and thereby influence chromatin properties and affect transcriptional and translational activities. Histones H3 can be subdivided into several classes. In addition to the centromeric variant CENH3 Drosophila H3.3 is enriched in euchromatic regions, at loci of active gene expression Drosophila, induction of gene expression leads to enrichment of H3.3 To date, many studies in animal species show that H3.3, in contrast to H3.1, is distinctly distributed along the genome. Drosophila and mammals In mammals, H3.3 enrichment has also been detected over telomeres, repressed genes and pericentric heterochromatin, resulting from deposition by distinct chaperone complexes Based on DNA sequences and gene structure, it is clear that H3.1 and H3.3 have evolved separately in animals and plants Arabidopsis H3.3 and H3.1 enrichment in chromatin and clarify their specificities regarding genomic features and cell cycle regulation.In contrast to animals, the knowledge about plant histone H3 is limited. Although it is likely that plant H3.1 expression is coupled to the cell cycle as in animals Arabidopsis genome contains three H3.3 genes encoding the same protein and we tagged HTR5 (HISTONE THREE RELATED 5), the most highly expressed gene of the family HTR13, one of the five genes encoding the unique H3.1 protein in ArabidopsisTo generate genome-wide maps of H3 variant localization, we performed Chromatin-Immunoprecipitation followed by deep sequencing (ChIP-Seq). We detected the localization of fusion proteins between a green fluorescent protein (GFP) tag and histone variants expressed under their endogenous promoter in transgenic plants. As a positive control, we used an H3 antibody recognizing the C-terminal part of H3, while an anti-IgG antibody was used as a negative control. The Arabidopsis nuclear chromosomes , defined in a previous study combining twelve different covalent histone marks CS1 and CS2 are predominantly associated with genes These results motivated the analysis of the H3 variant distribution over general genomic features including protein-coding genes, TEs and inter-annotation regions (IRs) . The medDrosophila and mammals In summary, the even distribution of H3.1 over all genomic features, similar to H3, suggests that H3.1 serves as a rather static chromatin backbone. In contrast, H3.3 appears to be more associated with active genes (CS1) than with repressed ones (CS2) and is depleted in regions of constitutive heterochromatin (CS3), including centromeres and TEs. H3.3 deposition at active genes appears to be conserved in yeast, We next investigated the enrichment profiles of the H3 variants over protein-coding genes and their flanking intergenic sequences in dividing tissue . To reveAccording to our results on CS1 and CS2 , H3.3 miIn agreement with our observations, plant H3.3 is associated with several histone marks, which are correlated with active gene expression Arabidopsis RNAPII enrichment over genes using data published previously Animal H3.3 enrichment shows a positive correlation to gene expression C. elegans H3.3 In summary, plant H3.3 enrichment positively correlates with gene expression and gene length and appears to gradually increase towards the distal gene end, reaching a maximum immediately upstream of the TTS. This profile appears to be similar to that reported for Drosophila and mammals is not limited to the coding regions but is also high on cis-regulatory elements, repressed genes and telomeres Arabidopsis genome lacks long distance acting enhancers, which are common in mammals and Drosophila.While plant and animal H3.3 obviously share similar features, we observed important differences as well. H3.3 enrichment in Arabidopsis genome . The SAM and the primordia comprise dividing cells s genome . Amongstll cycle . TogetheTo investigate H3.3 and H3.1 dynamics during the developmental transition from primordia to differentiated leaves, we selected two subsets of genes, according to their higher expression levels (at least five-fold) in either dividing or non-dividing tissue. Having gene sets that were preferentially expressed in either of the two tissue types, we could examine the changes in H3 variant levels that accompanied repression  and induWe conclude that the repression of gene expression during leaf differentiation is linked with a decrease in the H3.3 level, but not H3.1 level, suggesting that H3.3 may contribute to developmental transitions. Differentiation also requires the induction of gene expression, which correlates with gain of H3.3 enrichment at the 3′ end of some genes. H3.1 enrichment on the other hand, is not significantly affected by developmental transitions and appears to be a relatively stable chromatin component. This property would support a role of H3.1 in propagation of epigenetic patterns of histone modification through division, in agreement with the preference of H3.1 over H3.3 enrichment at heterochromatic regions, which need to be maintained in a transcriptional silent state.Arabidopsis H3.3 is dynamically deposited over gene bodies and its enrichment is linked to gene expression. Thus, the incorporation specificities observed for H3.3 and H3.1 are largely similar between animals and plants, suggesting a functional convergence during evolution of H3 variants in eukaryotes. Whether this convergence was driven by the conservation of the distinct mechanisms that incorporate H3.1 and H3.3 remains to be established since we currently lack biochemical characterization of histone H3 chaperones in plants.There are remarkable similarities between H3 variants in animals and plants, yet phylogenetic analyses indicate that amongst metazoa and plantae, H3.1 and H3.3 variants evolved independently. However, both share similar features such as specific amino acid changes at positions 31 and 87 and the absence of introns in H3.1 Arabidopsis remains unclear.Our study outlines a specific enrichment of H3.3 culminating towards the 3′ end of genes, a phenomenon that might be linked with gene length. Noteworthy, active marks such as H3K4me3 and H3K4me36 are enriched towards the 5′ part of genes, in contrast to H3.3. Although we observe a correlation with RNAPII, the origin and significance of H3.3 enrichment at the 3′ end of genes in in planta. We find that gene expression changes during differentiation are reflected in H3.3 enrichment. This dynamic replacement of H3.3 potentially allows covalent histone marks present in the chromatin of dividing cells to be remodeled in order to allow repression or expression of a new repertoire of genes that participate to the differentiation program. Hence, H3 variant replacement might serve as a mechanism that enables reprogramming at developmental transitions by globally facilitating dynamics of covalent marks.Our study addresses genome-wide dynamics of H3.3 and H3.1 enrichment during differentiation We used homozygous transgenic plants constructed by Mathieu Ingouff Nuclei enrichment was performed as previously described http://www.illumina.com/applications/sequencing/.ilmn). For mRNA-Seq libraries, briefly, 10 µg of total RNA was purified to yield poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were copied into first strand cDNA using Superscript II Reverse Transcriptase (Invitrogen) and random primers. This was then followed by second strand cDNA synthesis using DNA Polymerase I and RNaseH. These cDNA fragments then underwent an end repair process, the addition of a single ‘A’ base, and then ligation of adapters specific for sequencing flow cell. These products were then purified by gel excision and enriched by PCR with Phusion polymerase (Fermentas) to create the final cDNA library. This library was validated by loading 1 µl of the re-suspended constructs onto an Agilent Technologies 2100 Bioanalyzer DNA-1000 microfluidic chip. The final products showed a distinct band at 200–300 bp and were subsequently sequenced on an Illumina Genome Analyser IIx.mRNA-Seq and ChIP-Seq libraries of template molecules suitable for high-throughput sequencing were constructed according to the guide lines described in the Illumina website . This library was validated by loading 1 µl of the re-suspended constructs onto an Agilent Technologies 2100 Bioanalyzer DNA-1000 microfluidic chip. The final products showed a distinct band at 200–300 bp and were subsequently sequenced on an Illumina Genome Analyser IIx.Arabidopsis genome (TAIR9) using CASAVA v1.7. The number of mapped reads varied from 228 k to 11.9 M for the first biological replicates, and from 15.1 M to 20.7 M for the second (The ChIP-Seq reads were mapped onto the e second .http://genome.gis.a-star.edu.sg/).These files were then converted to 10 bp density WIG files using MACS v1.4.0 The mRNA-Seq reads were mapped onto TAIR9 using recommended settings of TopHat v1.2.0 http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/).The quality of the mapped reads was assessed using FastQC v.0.9.0 (http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession number GSE36631.The raw reads and processed files from both ChIP-Seq and mRNA-Seq experiments have been deposited in the NCBI Gene Expression Omnibus (GEO)  regions used in For the scatterplots of All the data used in the boxplots and scatterplots are available in ftp://ftp.arabidopsis.org/home/tair/Software/UpdateCoord/”. Based on the authors suggestion, the number of sites per matrix was limited to 200,000 following the “restriction” procedure (http://www.athamap.de/restriction_scores.php), giving ∼4.7 millions sites. The 2,390,614 non-exonic sites were then used to generate an average profile as described above  and presented in Predicted Transcription Factor Binding Sites (TFBS) data from AthaMap database Figure S1Genome browser snapshot of 31 kb on chromosome 3. Profiles of H3 (blue), H3.1 (orange), H3.3 (green) and IgG (grey) obtained from two independent biological replicates in the two tissue types. The region includes several protein-coding genes  having the following average FPKM values per condition (div/nonDiv): AT3G14820 (0/0), AT3G14830 (18.6/19.7), AT3G14840 (19.8/38.2), AT3G14850 (1.7/1.1), AT3G14855 (0/0), AT3G14860 (12.0/13.9), AT3G14870 (1.4/3.7). Vertical dashed grey lines correspond to the TTS of the genes.(EPS)Click here for additional data file.Figure S2Genomic distribution of H3.3 and H3.1 enrichment in non-dividing tissue. (A–D) Replicate of (EPS)Click here for additional data file.Figure S3Genomic distribution of H3.3 and H3.1 enrichment over Transcription Factor Binding Sites (TFBS). Profiles of H3.3 (green), H3.1 (orange), H3 (blue) and IgG (dashed grey) over non-exonic TFBS extracted from AthaMap (EPS)Click here for additional data file.Figure S4Enrichment profiles of marks and RNAPII over genes. (A) Average profile of H3 enrichment in dividing cells over the protein-coding genes grouped according to their expression levels into six different subsets see . (B) Ave(EPS)Click here for additional data file.Figure S5H3.3 and H3.1 enrichment over genes in non-dividing tissue. (A) Average profile of H3.3 (green), H3.1 (orange), H3 (blue) and IgG (dashed grey) enrichment over gene bodies . (B–E) Average profile of H3.3 (B), H3.1 (C), H3 (D) and IgG (E) enrichment over the protein-coding genes grouped according to their expression levels into six different subsets .(EPS)Click here for additional data file.Figure S6Correlation of H3.1 and H3.3 enrichment with gene length. Average profile of H3.3 (A), H3.1 (B), H3 (C), IgG (D) from dividing tissue over the protein-coding genes grouped according to their length into five different subsets . Non-dividing profiles (not shown) are almost identical.(EPS)Click here for additional data file.Figure S7Dynamics of H3.3 and H3.1 enrichment during development over different sets of genes. Average profile of H3.3 (green) and H3.1 (orange) enrichment in dividing tissue (plain line) compared to non-dividing tissue (dashed line) over genes that are cell cycle regulated according to CycleBase (A), genes that are ON (FPKM>3) in dividing tissue and OFF (FPKM<1) in non-dividing tissue (B) and vice-versa (C) and genes that present similar expression in both tissue types and are either highly expressed (D), or have low expression levels (E). (F–G) Snapshots of the UCSC genome browser showing H3 (blue), H3.1 (orange), H3.3 (green), IgG (grey) and mRNA-Seq (black) over representative protein-coding genes  more expressed in dividing tissue than non-dividing tissue (F) or the opposite (G). The corresponding FPKM values per condition (div/nonDiv) are: AT3G14600 (126.9/422.4), AT3G14610 (1.3/2.4), AT3G14620 (8.7/3.3), AT1G20440 (1768.8/134.5), AT1G20450 (503.1/94.9). Horizontal dashed grey lines correspond to half the scale of each graph.(EPS)Click here for additional data file.Table S1Overview of sequencing libraries analyzed.(DOC)Click here for additional data file.Table S2www.cyclebase.org with ranks from 1 to 20,945 reflecting the magnitude of cell-cycle dependent regulation. FPKM values are mean values obtained from the HTR5::GFP and HTR13::GFP mRNA-Seq libraries.Expression of the top-most cell-cycle dependent and independent genes in dividing and non-dividing tissue. CycleBase ranks were extracted from (DOC)Click here for additional data file.Table S3Tab-delimited file containing FPKM values for each TAIR9 annotation. The last column contains the information whether the gene was kept or not for the downstream analyses based on expression variation between replicates from the same tissue see .(GZ)Click here for additional data file.Table S4Tab-delimited file containing the average signal of all datasets used in the boxplots and scatterplots over each annotation , inter-annotation and CS segment.(GZ)Click here for additional data file.


Y. enterocolitica biotype (BT) 1A strains are often isolated from human clinical samples but their contribution to disease has remained a controversial topic. Variation and the population structure among the clinical Y. enterocolitica BT 1A isolates have been poorly characterized. We used multi-locus sequence typing (MLST), 16S rRNA gene sequencing, PCR for ystA and ystB, lipopolysaccharide analysis, phage typing, human serum complement killing assay and analysis of the symptoms of the patients to characterize 298 clinical Y. enterocolitica BT 1A isolates in order to evaluate their relatedness and pathogenic potential.adk, argA, aroA, glnA, gyrB, thrA, trpE) conducted on 43 of the strains discriminated them into 39 MLST-types. By Bayesian analysis of the population structure (BAPS) the strains clustered conclusively into two distinct lineages, i.e. Genetic groups 1 and 2. The strains of Genetic group 1 were more closely related (97% similarity) to the pathogenic bio/serotype 4/O:3 strains than Genetic group 2 strains (95% similarity). Further comparison of the 16S rRNA genes of the BT 1A strains indicated that altogether 17 of the 71 strains belong to Genetic group 2. On the 16S rRNA analysis, these 17 strains were only 98% similar to the previously identified subspecies of Y. enterocolitica. The strains of Genetic group 2 were uniform in their pathogenecity-related properties: they lacked the ystB gene, belonged to the same LPS subtype or were of rough type, were all resistant to the five tested yersiniophages, were largely resistant to serum complement and did not ferment fucose. The 54 strains in Genetic group 1 showed much more variation in these properties. The most commonly detected LPS types were similar to the LPS types of reference strains with serotypes O:6,30 and O:6,31 (37%), O:7,8 (19%) and O:5 (15%).A subset of 71 BT 1A strains, selected based on their varying LPS patterns, were subjected to detailed genetic analyses. The MLST on seven house-keeping genes (Y. enterocolitica BT 1A represent more than one subspecies. Especially the BT 1A strains in our Genetic group 2 commonly showed resistance to human serum complement killing, which may indicate pathogenic potential for these strains. However, their virulence mechanisms remain unknown.The results of the present study strengthen the assertion that strains classified as  Yersinia enterocolitica species has six biotypes (BTs) of which five  contain pathogenic strains. Y. enterocolitica ssp. enterocolitica consists mainly of the strains of BT 1B, which are considered highly virulent. Low-virulent ssp. palearctica encompasses BTs 2–5 and 1A. Since BT 1A strains lack most of the classical virulence markers, this biotype is often considered non-pathogenic. Nevertheless, BT 1A strains are commonly isolated from patients with diarrhoea. Reports supporting the pathogenicity of some BT 1A strains comprise clinical data[ystB encoding heat-stable Yersinia enterotoxin B whereas they usually lack the ystA gene found from Y. enterocolitica 4/O:3 strains. Yersinia enterotoxins A and B are homologues to enterotoxins found in enterotoxigenic E. coli (ETEC) and Vibrio cholerae non-O1 strains[Y. enterocolitica strain that produces heat-stable enterotoxin compared to the infection with a knock-out mutant[Y. enterocolitica BT 1A strains possess the ystB gene[cal data and cellcal data-10. A vi strains. Higher t mutant. A majorstB gene and somestB gene,15.Y. enterocolitica biotypes[Y. enterocolitica ssp. enterocolitica BT 1B strain 8081[Y. enterocolitica BT 1A strains could be divided into two main clusters[The BT 1A strains are genetically the most heterogeneous of all the biotypes-19. Theybiotypes. It has ain 8081. Likewisclusters,21-25. Hclusters. Notableclusters.Y. enterocolitica detected among Yersinia isolates from human clinical stool samples in Finland in 2006[yst-PCR, lipopolysaccharide (LPS) analysis, sensitivity to five yersiniophages and serum killing assay. MLST results were analysed with BAPS  program, genetic and phenotypic characteristics of the BT 1A strains were compared and statistical analysis was applied to assess their correlation with the symptoms of the patients.BT 1A was the predominant biotype of  in 2006, as also in 2006. Of the  in 2006. We hypoY. enterocolitica BT 1A strains were discriminated into 39 MLST types and the 10 4/O:3, 3/O:3 or 2/O:9 strains were discriminated into four different MLST types. The genetic diversity indexes for the genes used in MLST were 0.86 (adk), 0.93 (argA), 0.93 (aroA), 0.83 (glnA), 0.82 (gyrB), 0.94 (thrA) and 0.89 (trpE). Bayesian analysis of the MLST sequences divided the BT 1A strains into two distinct genetic clusters, which were clearly separated from the tight cluster formed by the strains of BT’s 2–4 and from the BT 1B strain (8018)  were compared to each other, the BT 1A Genetic group 2 strains were 95–96% similar to BT 1A Genetic group 1, bio/serotype 4/O:3 and 2/O:9, as well as to Y. enterocolitica BT 2-4/O:3 or O:9 strains  revealed BT 1A Genetic group 2 strains to have over 99% similarity among themselves, 98–99% similarity to Genetic group 1 strains and 98% similarity to ystA positive in PCR, but 98% were ystB positive. All five of the BT 1A Genetic group 2 strains were both ystA and ystB negative in PCR. The 4/O:3, 3/O:3 and 2/O:9 strains were all ystA positive and ystB negative in PCR. When also the BT 1A strains that were not included in the MLST analysis were tested for ystA and ystB, 12 further strains were found to be negative in ystB PCR. They were also subjected to 16S rRNA gene sequencing and were found to be part of BT 1A Genetic group 2 , with each containing several subtypes  in silver-stained DOC-PAGE gels, the 298 es Table. The subes Table. Three oYersinia specific bacteriophages (TablePhage sensitivity of the strains was tested using five es Table. Most ofY. enterocolitica serotype O:3 strains (TableResistance to human serum complement-mediated killing was most common (99%) in the LPS subtype A3 strains, which included the known pathogenic ns Table. Of the ns Table. Among tns Table.The symptoms  of patients with BT 1A did not differ significantly when the statistical analyses were based on the genetic grouping or serum resistance of the BT 1A isolates. The patients with isolates belonging to different LPS-groups were symptomatic, but due to the small amount of patients in analyses, no significant statistical inference could be made.Y. enterocolitica BT 1A[Y. enterocolitica BT 1A strains. The genetic similarity of this group to Genetic group 1 was 95–96% based on the MLST sequences and 98–99% based on the 16S rRNA gene sequences. All the 17 strains determined to belong to Y. enterocolitica BT 1A Genetic group 2 were ystB negative in PCR and were resistant to the five tested yersiniophages. Additionally, none of them fermented fucose, as determined in our previous study[The strains previously identified by phenotypic tests to belong to ca BT 1A formed tus study. LikewisY. intermedia, Y. mollaretii, Y. aldovae and Y. bercovieri than to Y. enterocolitica 16S rRNA sequences. However, a previous study indicated that the use of MLST of house-keeping genes determined genetic relatedness among Yersiniae better than 16S rRNA[Y. enterocolitica based on the comparison of whole genomes using DNA microarray. It is likely that the genetic difference between the two phylogenetic groups of Y. enterocolitica BT 1A discovered in the present study may also be high enough to justify designation of different subspecies or even species. Although further analyses would be needed for species designation, our data add insight into the phylogeny of the genus Yersinia, which is continuously evolving: three novel Yersinia species, Y. entomophaga, Y. pekkanenii and Y. nurmii were described as recently as 2010[Remarkably, the 16S rRNA sequences of BT 1A Genetic group 2 were more similar to 16S rRNA. Studies16S rRNA. Neverth16S rRNA have alr as 2010-33.Y. enterocolitica BT 1A strains are reported based on the sequence analysis of house-keeping genes, but similar results indicating the existence of two main clusters of BT 1A strains have been obtained with other molecular methods, such as ribotyping and REP-ERIC[gyrB-RFLP[gyrB genes of representative two clonal groups of BT 1A strains were only 97% similar. When we compared these gyrB sequences to our data, sequences DQ140396 and DQ140397[ystB gene correlated with the clonal groups, except in one strain[ystB gene in PCR test does not always correlate with the phylogenetic lineages, since our study also found six strains without the ystB gene in BT 1A Genetic group 1. However, only the use of hybridization analysis or sequencing would confirm the PCR results. In a recent study of the whole genome sequences no evident structural difference was found with ystB-positive BT 1A/O:5 and BT 1A/O:36 strains[This is the first time that two phylogenetic clusters of REP-ERIC, gyrB-RFyrB-RFLP, AFPL[1yrB-RFLP, MLEE[2yrB-RFLP,24 and, yrB-RFLP. Gulati yrB-RFLP found inDQ140397 were clue strain. The lac strains. TherefoY. enterocolitica strains. This method offered a practical substitute for O-serotyping, since there are no commercial O-specific antisera available for numerous Y. enterocolitica serotypes. The results were consistent with earlier O-serotyping of the BT 1A strains using available commercial antisera[Y. enterocolitica 4/O:3 strains are able to resist serum killing by YadA- and Ail-mediated binding of the serum complement regulatory proteins factor H and C4 binding protein[yadA gene and only one strain had the ail gene[We used DOC-PAGE based classification of LPS to subtype our antisera which deantisera. In the antisera. Pathoge protein-38. The ail gene. Thus thAlthough the potential pathogenicity of BT 1A strains remains controversial, there are a few studies that show an association to disease. For instance, BT 1A/O:6,30 was associated with spondyloarthropaties of patients in England and South-Wales. Also, iY. enterocolitica BT 1A represent more than one subspecies. BT 1A Genetic group 1 consisted of strains with a variety of pathogenicity-related properties, whereas all 17 strains of BT 1A Genetic group 2 lacked the ystB gene, belonged either to the same LPS subtype or were rough, were all resistant to the five tested yersiniophages and were largely resistant to serum complement killing. Furthermore, none of them fermented fucose. Although several studies have been conducted to reveal the significance of the BT 1A strains in causing disease, indisputable results have not been obtained. This study shows, however, that BT 1A is a very heterogenous group of strains, some of which might be potential pathogens. Therefore, better understanding of the genetic and phenotypic variability and clustering of these strains, as achieved in our study, would be crucial in determining the pathogenic role of the strains belonging to the defined clusters.The results of our study present strong evidence that strains classified as Y. enterocolitica strains isolated in 2006 from human samples[Altogether 298 BT 1A, 75 bioserotype 4/O:3, two 3/O:3, five 2/O:9 and two non-biotypable  samples were utiY. enterocolitica strains (43 BT 1A and 10 BT’s 2–4 strains) that represented various LPS patterns. Additionally, two reference strains, NCTC11174 (O:9) and NCTC11176 (O:3), were included in the analysis. Genomic DNA was extracted using Jetflex Genomic DNA purification kit . Fragments of seven house-keeping genes  were amplified by PCR. For the adk, argA, aroA, glnA, thrA and trpE genes, the primers available in the MLST database for Y. pseudotuberculosis at the ERI, University College Cork, were used (http://mlst.ucc.ie/mlst/dbs/Ypseudotuberculosis/documents/primersPseudotuberculosis_html). The gyrB gene amplification was done with the primers described earlier[Y. enterocolitica strains obtained in the study were deposited to EMBL/GenBank database under the accession numbers HE803367- HE803737.MLST analysis was done on 53  earlier. The 25  earlier,41. The Population genetic analyses were performed using BAPS  software-44 with Mosaicism is defined as sequence types composed of sequence characteristic of more than one BAPS group. Significance of admixture or mosaicism was determined for each sequence type using the threshold p < 0.05.Maximum likelihood tree was constructed by using the concatenated sequences under the general time-reversible model as implemented in the MEGA5 software.Y. enterocolitica BT 1A strains with the primers FD1mod[Y. enterocolitica BT 1A strains obtained during this study were deposited to the EMBL/GenBank database under the accession numbers HE803738 - HE803765. Eight of the BT 1A strains were sequenced during our previous studies and have accession numbers FM958217 - FM958223 and FN812721[16S rRNA gene sequencing was obtained for 36 s FD1mod, pHr, pDs FD1mod in condis FD1mod. The seqs FD1mod. The 16SystA gene specific PCR the forward primer 3-ATC GAC ACC AAT AAC CGC TGAG −5 and reverse primer 3- CCA ATC ACT ACT GAC TTC GGCT −5 were used for 38 Y. enterocolitica BT 1A strains. For the ystB gene specific PCR the forward primer 3-GTA CAT TAG GCC AAG AGA CG −5[Y. enterocolitica BT 1A strains. Chromosomal DNA was used as a template; the conditions for PCR amplification were as described earlier[For the GA CG −5 and the GA CG −5 were use earlier.Y. enterocolitica BT 1A strains were prepared by the small scale proteinase K method as described earlier[600 was determined, the bacteria were then pelleted by centrifugation, and the pellet was re-suspended in DOC lysis buffer  in a volume adjusted according to the density of the culture . The suspension was heated to 100°C for 10 min and then 2–4 μl of proteinase K (20 mg/ml) was added and the suspension was incubated overnight at 60°C. An aliquot of 10 μl was loaded on the gel and analysed in 12% DOC-PAGE and the LPS bands were visualized by silver staining as described earlier[Y. enterocolitica and Y. enterocolitica –like bacteria has been described elsewhere[LPS samples of 298  earlier. Briefly earlier. The DOClsewhere. BrieflyYersinia expressing the outer core hexasaccharide in LPS; PY100 that infects a broad range of Yersinia strains[Y. enterocolitica serotype O:3 O-PS as receptor[Y. enterocolitica serotype O:3 grown at RT ; and ф80–18 that is a serotype O:8 O-PS specific phage[Y. enterocolitica BT 1A strains, a 40 μl aliquot from a bacterial culture grown for 14–16 h at RT or 37°C with shaking in LB was mixed with 3.5 ml of molten 0.4% soft agar adjusted to 50°C, mixed briefly and poured on an LA plate. After the soft agar had solidified, 10 μl drops of different phage suspensions (~105 plaque forming units ml-1) were pipetted onto the surface and the plates were incubated at RT or 37°C 14–16 h. Phage sensitivity was scored as a clear lysis zone in the soft agar.The following bacteriophages were used in the typing scheme: фR1–37,58 that  strains; фYeO3–1receptor,61; ϕR1-ic phage. For altYersinia antibodies. Sera were pooled and stored in aliquots at −70°C. The killing assay for 298 Y. enterocolitica BT 1A strains was performed essentially as described previously[-1 MgSO4 × 7H2O, 2 g l-1 citric acid, 10 g l-1 K2HPO4, 3.5 g l-1 NaNH4HPO4 × 4H2O and 1 mg l-1 vitamin B1, supplemented with 0.2% glucose, 0.2% casamino acids and 2.5 mM CaCl2) at 37°C without shaking. The cultures were diluted 1:1000–5000 into PBS to obtain a suspension of ca. 105 cfu/ml and 10 μl of the suspension was mixed with 20 μl of normal human serum (NHS) or heat-inactivated serum . After 60 min incubation at 37°C, the complement reaction was stopped by transferring the tubes on ice and the addition of 70 μl of ice-cold BHI. Aliquots of 20 μl were cultured on LA-plates and the surviving bacteria were counted after 48 hr incubation at RT. The serum bactericidal effect was calculated as the survival percentage taking the bacterial counts obtained with bacteria incubated in HIS as 100%. The survival was scored as follows: >50% survival, +++; 5–50% survival, ++; and 0.01–5% survival, +; and no colonies, 0.Blood was obtained from healthy human donors who were devoid of anti-eviously. BrieflyY. enterocolitica BT 1A isolate, who had answered a questionnaire about the symptoms[We compared the symptoms of diarrhoea, vomiting, fever, abdominal pain and blood in stools among 98 patients with a symptoms and had Informed consent was obtained from the patients who participated in the questionnaire study. The study was approved by the Ethics Committee of National Institute for Health and Welfare (THL). The voluntary healthy blood donors whose sera were used in serum-killing assay gave their verbal consent. They were informed of the details of the study and their blood samples were pooled and used for the study without an individual being identified.The authors’ declare that they have no competing interests.LMS conducted the MLST work, combined all the results together and drafted the manuscript. KJ contributed to the genomic analyses. ST and MS conducted and analyzed the LPS, serum resistance and phage typing assays. EH and MK analysed the clinical data and JC did the BAPS and phylogenetic analysis of the MLST data. AS and KH participated in planning of the work, analyzing the results and writing the article. All authors read and approved the final manuscript.Neighbour-joining tree based on seven concatenated MLST genes (4580 bp). Neighbour-joining bootstrap confidence values over 75% (1000 replicates) are given in the branches. BT 1A strains were ystB positive in PCR and had positive reaction in fucose fermentation unless otherwise indicated. sr=serum resistance; pt= phage type, which encodes reaction to 5 phages . Strains sequenced in the present study are marked bold. In addition, the following GenBank sequences were used: Y. enterocolitica 8081 (AM286415), Y. aldovae ATCC 35236 (ACCB00000000), Y. kristensenii ATCC 33638: (ACCA00000000), Y. intermedia ATCC 29909 (AALF00000000), Y. frederiksenii ATCC 33641 (AALE00000000), Y. mollaretii ATCC 43969 (AALD00000000), Y. bercovieri ATCC 43970 (AALC00000000), Y. rohdei ATCC 43380 (ACCD00000000) and Y. ruckeri ATCC 29473 (ACCC00000000).Click here for fileAnalysis of Y. enterocolitica LPS by DOC-PAGE and silver staining. The picture is compiled of gel images with different LPS types as indicated above the lanes by the LPS type codes that are explained in the text box. Please note that LPS types A2, B1c, B1d, B2a, B2c and B4 are not shown. The gel regions where O-PS and lipid A (LA) plus core migrate are indicated by arrows.Click here for file


N-formylkynurenine (NFK), of W365 in the CP43 subunit. The yield of this modification increases under light stress conditions, in parallel with the decrease in oxygen evolving activity. In this work, we show that this modification, NFK365-CP43, is present in thylakoid membranes and may be formed by reactive oxygen species produced at the Mn4CaO5 cluster in the oxygen-evolving complex. NFK accumulation correlates with the extent of photoinhibition in PSII and thylakoid membranes. A modest increase in ionic strength inhibits NFK365-CP43 formation, and leads to accumulation of a new, light-induced NFK modification (NFK317) in the D1 polypeptide. Western analysis shows that D1 degradation and oligomerization occur under both sets of conditions. The NFK modifications in CP43 and D1 are found 17 and 14 Angstrom from the Mn4CaO5 cluster, respectively. Based on these results, we propose that NFK is an oxidative modification that signals for damage and repair in PSII. The data suggest a two pathway model for light stress responses. These pathways involve differential, specific, oxidative modification of the CP43 or D1 polypeptides.Light stress in plants results in damage to the water oxidizing reaction center, photosystem II (PSII). Redox signaling, through oxidative modification of amino acid side chains, has been proposed to participate in this process, but the oxidative signals have not yet been identified. Previously, we described an oxidative modification,  Re-insertion of a new D1 subunit into the partially disassembled PSII complex and reassembly completes the repair cycle Light stress causes protein damage and suboptimal photosynthetic rates in PSII N-formylkynurenine (NFK)   plays a ne (NFK) . NFK hasRecently, we described a light-induced modification, NFK , resulti1O2) 3) •) 23chl) or by reactions at the Mn4CaO5 cluster (3chl) and donor side (Mn4CaO5) reactions.NFK results from the reaction of the Trp side chain with several types of ROS, including singlet oxygen  −2 s−1 did not significantly decrease oxygen evolution rates  samples were not significantly inhibited at a chlorophyll concentration of 1 mg/mL (NFK has a unique absorption at 318 nm . The HPL363AP(W*)LEPLRGPNGLDLSR379), confirming our earlier result 363AP(W*)LEPLR370 ADIINR323 in D1  and Na2EDTA (1 mM). Peptide B was observed under both sets of conditions  2+ (0.74 Å) 2+ (ionic radius  = 0.99 Å) To rule out the possibility of a non-specific cation-binding site as inducing the fraction B modification, the effect of 2 mM tetra-methyl ammonium chloride (TMA) was assessed. TMA has a nearly three-fold larger ionic radius (2.9 Å) 2), and 36.4 mM (1 mM Na2EDTA) when peptide B was observed in the light.We conclude that the small ionic strength increase underlies the observation of fraction B. The ionic strength of the SMN buffer, prior to the addition of salts, is calculated to be 34.9 mM. The ionic strength increased to 36.9 mM (2 mM NaCl or TMA), 35.4 mM LR24 and the other identified as the D2 polypeptide 14DLFDSMDD(W*)LR24 LR24  and S2. 363AP(W*)LEPLR370 in CP43 and is observed in oxygen-evolving PSII and TM, but not in TW PSII. Fraction A showed no detectable light-induced increase in any sample that we examined. Fraction C, corresponds to 363AP(W*)LEPLRGPNGLDLSR379 in CP43, and is observed in oxygen evolving PSII, TW PSII, and TM. Fraction C showed a light induced increase in only one sample, oxygen-evolving PSII. Fraction B corresponds to 313VINT(W*)ADIINR323 in D1. It was observed only in oxygen-evolving PSII, after illumination and under conditions of higher ionic strength.In this work, three NFK containing peptides, originating from the donor side of PSII, are identified. Fraction A, corresponds to T. vulcanus4CaO5 cluster; NFK 317-D1 is 14 Å from the cluster. 680 and YZ. YZ is an electron transfer intermediate during the water oxidizing reaction  significantly decreased activity.Reversible, light induced structural changes in the degree of spinach TM stacking (grana) have been observed by electron microscopy In this work, we found the interesting result that small ionic strength increases had a dramatic effect on the pattern of NFK modifications. However, this change did not alter the degradation pattern of D1 as assessed with a C-terminal antibody. Although the D1 degradation pattern was not changed, the alternative D1-NFK modification to Trp-317 was induced by increasing ionic strength. The ionic strength effect may cause conformational changes in the extrinsic loops. Lowering of the thylakoid lumen pH during excess light involves protein conformational changes that may be necessary in non-photochemical quenching 2+ efflux into the stroma occurs during the transition from dark to light conditions 363AP(W*)LEPLR370 peptide (fraction A) and the 363AP(W*)LEPLRGPNGLDLSR379 (fraction C) peptide were observed in intact PSII and TM. However, in TW PSII, the shorter CP43 peptide 363AP(W*)LEPLR370 was not detected. These results can be attributed to different conformations of the CP43 loop region. These conformational changes may be important in control of photoinhibitory responses in the chloroplast, where changes in the proton motive force can occur during illumination.Concomitant MgSynechocystis 6803) did not affect the steady-state rate of oxygen evolution under normal light-saturated conditions −2 s−1) The signaling pathways for induction and control of D1 turnover are not known. Oxidative PTMs of aromatic amino acids have been proposed to participate in signaling. The NFK modifications identified here may function as these signals. We showed previously that substitutions at Trp-365  stimulates repair by the alternative pathway (the “D1” pathway). These results provide new insight into redox signaling in oxygenic photosynthesis.N-morpholino)-ethanesulfonic acid (MES)-NaOH (pH 6.0), 150 mM NaCl, 4 mM MgCl2•H2O. The final resuspension was in 50 mM MES-NaOH (pH 6.0), 400 mM sucrose, 15 mM NaCl (SMN buffer). Chlorophyll 3Fe(CN)6 and recrystallized 1 mM 2,6-dichlorobenzoquinone (DCBQ). Activity rates of PSII membranes and TM were ≥600 and ≥130 µmol O2 mg chl−1 h−1, respectively.Spinach PSII membranes were isolated as described 4CaO5 cluster were removed with a 800 mM tris(hydroxymethyl)aminomethane (Tris)-NaOH, pH 8.0 wash performed for 45 minutes at room temperature in the light The 18- and 24-kDa extrinsic subunits were removed from PSII membranes with a 2 M NaCl wash −2 s−1 as measured with a Li-Cor  Light Meter (model LI-189 with a ∼8 cm diameter sensor). The illumination was performed for two hours. Controls were kept in the dark at room temperature (∼25°C). Samples were either un-treated or treated with 2 mM NaCl , 0.15 mM ZnCl2 , 1 mM disodium-ethylenediaminetetraacetic acid (Na2EDTA) , or 2 mM tetra-methylammonium chloride (TMA)  by addition just prior to the dark or light incubation. The ionic strength of the SMN buffer alone, prior to the addition of salts, was 34.9 mM. The ionic strength increased to 36.9 mM (2 mM NaCl and 2 mM TMA), 35.4 mM (0.15 mM ZnCl2), and 36.4 mM (1 mM Na2EDTA) with the additional salts. Oxygen evolution was assayed every 30 minutes.Photoinhibition experiments were performed on spinach TM, PSII membranes, and TW PSII membranes L-tryptophan , L-kynurenine (Sigma-Aldrich), and NFK 2O. The NFK-containing peptide optical spectra were derived from the chromatogram through the use of a Beckman  System Gold® HPLC, equipped with a 125 solvent module, a 168 photodiode array detector , and 32 Karat Software, version 7.0.Optical spectra of model compounds tryptophan, NFK, and kynurenine were recorded at room temperature from 200–750 nm on a Hitachi (U3000) spectrophotometer In-situ trypsin  digestion of TM, PSII, and TW PSII was conducted as described SDS-PAGE of PSII membranes was performed as described PSII tryptic peptides were analyzed as described Figure S1Representative MS/MS spectra of NFK modifications in CP43 (A and C) and D1 (B) proteins. The peaks in blue represent the b-fragments. The peaks in red represent the y-fragments. The NFK modified W is indicated in the corresponding sequences. This residue carries the +32 m/z masss shift, which was unambiguously assigned to Trp-365 in CP43 (A and C) and Trp-317 in D1 (B).(DOCX)Click here for additional data file.Table S1#NOD, not observed in dark; NODL, not observed in dark or light.(DOCX)Click here for additional data file.Table S2For the identification of peptides, filter criteria were set to warrant a false discovery rate of less than 1% on the peptide level. In each of the three independent LC-MS/MS runs of the four fractions, more than 20000 MS/MS spectra were recorded. For fraction A–C, between 3500–5000 spectra were assigned to peptides from 50–80 proteins from S. oleracea. In fraction D only, 1200 spectra could be assigned to peptides of about 50 proteins.(DOCX)Click here for additional data file.


In addition, antigen-antibody interaction-based method cannot be used for the detection of influenza viruses with major antigenic change. In this study, we established the use of fluorescent immunochromatography (FLIC) to detect a broad spectrum of H5 subtype influenza A viruses. This method has improved sensitivity 10–100 fold higher than traditional IC because of the use of fluorescent conjugated beads. Our Type-E FLIC kit detected all of the H5 subtype influenza viruses that were examined, as well as recombinant hemagglutinin (HA) proteins (rHAs) belonging to the Eurasian H5 subtype viruses and the Type-N diagnosed North American H5 subtype influenza A viruses. Thus, this kit has the improved potential to detect H5 subtype influenza viruses of different clades with both Type-E and Type-N FLIC kits. Compared with PCR-based diagnosis, FLIC has a strong advantage in usability, because the sample preparation required for FLIC is only mix-and-drop without any additional steps such as RNA extraction. Our results can provide new strategies against the spread and transmission of HPAI H5N1 viruses in birds and mammals including humans.Immunochromatography (IC) is an antigen-detection assay that plays an important role in the rapid diagnosis of influenza virus because the protocol is short time and easy to use. Despite the usability of IC, the sensitivity is approximately 10 Orthomyxoviridae. Seasonal outbreaks of influenza present global health problems involving morbidity, mortality, and economic losses. Influenza A viruses are classified into 16 HA and 9 NA subtypes, based on 2 viral surface proteins–hemagglutinin (HA) and neuraminidase (NA). Almost all possible combinations of HA and NA have been isolated from aquatic birds, poultry, and other avian species. In 1997, highly pathogenic avian influenza (HPAI) H5N1 viruses were the cause of the first lethal human infection in Hong Kong HA gene that was derived from A/goose/Guangdong/1/96-like virus  and 7 other viral gene segments derived from A/teal/Hong Kong/W312/97-like virus  HA gene from isolated HPAI H5N1viruses formally identified over 30 distinct sub-clades of the virus Influenza is a highly contagious respiratory disease of humans, caused by negative-strand RNA viruses of the family A recent pandemic of A/H1N1pdm in 2009 occurred following reassortment between 2 swine influenza viruses, triple-reassortant swine influenza virus and Eurasian-lineage swine influenza virus Fluorescent immunochromatography; FLIC). Although the FLIC strip has to be scanned by a fluorescent reader, the sensitivity is significantly improved. In addition, our combination of antibodies can detect a broad spectrum of H5 subtype influenza A viruses. Thus, FLIC is expected to provide new strategies for initial diagnosis of the human transmission of HPAI H5N1 viruses to humans.Immunochromatography (IC), an antigen-detection assay completed within 20 minutes, is an important rapid test for clinical diagnosis and surveillance of influenza viruses Table. 1). MDCK cells were infected with 3 H5N1 subtype avian influenza viruses: A/whooper swan/Mongolia/3/2005 , A/whooper swan/Hokkaido/1/2008 , and A/duck/Hokkaido/Vac-3/2007  at a multiplicity of infection (MOI) of 4. To check cross reactivity, cells were also infected with influenza viruses of 3 other subtypes: A/WSN/1933 , A/Aichi/2/1968 , and B/Mass/3/1966 (Mass) at similar MOI. Infected cells were stained with candidate antibodies  and ELISA . Both 8C1 and 5H7 interacted with all 4 rHAs: A/Vietnam/1194/2004 , A/Indonesia/5/2005 , A/bar-headed goose/Qinghai/1A/2005 , and A/Anhui/1/2005 . On the other hand, 14A7 and 7A5-1 detected rHA from VN1194 and QH1A, but not IDN5 and AH1.ELISA was then conducted with recombinant HA protein (rHA) of H5 subtype viruses as the target (Table. 2). Although Type-E could detect all 13 rHAs belonging to the Eurasian type of both GS/GD/1-lineage and outlier virus, 2 North American type rHAs could not be detected. On the other hand, the reactivity of Type-N to GS/GD/1-lineage viruses was limited to clades 1, 2.1, 2.2, and these sub clades, but it could detect rHAs of Eurasian type outlier virus and North American type virus.To determine the viability of these 4 antibodies in IC, we conjugated these antibodies to colloidal gold. First antibody combination, named Type-E, consisted of 8C1 as the immobilized antibody and 5H7 as the colloidal gold conjugated antibody. Second antibody combination, named Type-N, consisted of 7A5-1 as the immobilized antibody and 14A7 as the colloidal gold antibody. We tested 15 rHAs of H5 subtypes at a concentration of 100 or 10 ng/reaction with colloidal gold Type-E and Type-N kits (Table. 3). Type-E detected all 3 viruses with a sensitivity between 105 and 106 pfu (per 100 μl sample solution), and Type-N detected MNG3 and Vac-3 with a sensitivity of about 105 pfu (per 100 μl sample solution), but not HKD08. Interestingly, 5H7 could not detect Vac-3 under the condition of IFA, but Type-E, which consisted of 5H7 as the colloidal gold conjugated antibody, could detect Vac-3, indicating that 5H7 is more suitable for detection of HA proteins of Eurasian type outlier under the conditions of IC than IFA.To evaluate the ability of IC to detect virus, 3 H5N1 viruses, MNG3, HKD08 and Vac-3, were tested by using Type-E and Type-N kits at serial concentrations (6 50% tissue culture infectious dose (TCID50) of 7 strains of H5N1 viruses and 2 strains of H5N2 viruses (Table. 4). Type-E could detect all 7 Eurasian type viruses both GS/GD/1-lineage and outlier, but not 2 North American type viruses. On the other hand, FLIC Type-N detected only 5 strains: MNG3, Vac-3, A/Muscovy duck/Vietnam/OIE-559/2011 , and 2 strains belonging to the North American type. Taken together, our results indicated that the set of Type-E could detect all Eurasian type viruses we tested but not any North American type viruses, and that the pair of Type-N detected a part of GS/GD/1-lineage , Eurasian type outlier, and all North American type viruses we tested , the sensitivity of FLIC is several 10 times higher than that of IC.The sensitivity of FLICs was calculated using the exponential approximation of viral titer (pfu/mL) and the rate of signal/background (S/B). The cut-off S/B value was set at 1. The amount of viral titer at the intersection point of the exponential approximation and S/B  = 1 was defined as the limit of detection (LOD). The LODs of FLICs for detection of H5N1 viruses are described in 7 TCID50 of other subtype viruses, including HPAI H7N7 virus (A/chicken/Netherlands/2586/2003), were tested (Table. 6). In addition, 1 μg/mL of 15 rHA of other subtypes, including Spanish influenza virus (A/BrevigMission/1/1918) and H1N1pdm , were also tested (Table. 7). All of the viruses and rHAs were not detected with any positive signals, meaning the FLICs maintained the specificity with the high sensitivity.Improvement in sensitivity may cause a decrease in the specificity of the system. To confirm the specificity of FLICs, 10Clinical diagnostic tests for influenza viruses in outpatient departments or clinics are typically based on IC detection of influenza virus antigens and3 pfu or 2.63×103 pfu (per 100 μl sample solution), respectively, and these are almost equal to the LODs of commercial IC kits detecting viral NP protein (102-104 TCID50/reaction) In this study, we succeed in improving IC by using antibodies conjugated with fluorescent beads and a specific IC reader. The sensitivity of FLIC was several 10 times higher than that of colloidal gold IC and6, indiIn 2013, World Health Organization has defined new 4 phases  of the pandemic stage of a disease Our combination of antibodies against H5 HA detected many kinds of HA proteins. Type-E could detect all influenza viruses and rHAs belonging to the Eurasian type of both GS/GD/1-lineage and outlier. Because avian influenza viruses of Eurasian type play an important role in the human infection by avian influenza viruses, the Type-E would be useful for clinical diagnosis and surveillance in Asian countries. The antisera against clade 2.3.2 viruses very weakly interact with clade 2.3.4 viruses, but that against clade 2.3.4 viruses could not detect clade 2.3.2 virus et al. reported Type-N could detect all low pathogenic avian influenza H5 virus (LPAI H5 virus) and rHAs which we tested. All LPAI H5 viruses in poultry are notifiable to Office International des Epizooties  We have previously reported another method for rapid diagnosis, super high-speed quantitative PCR (SHRT-PCR) All animal care and experimental procedures were performed according to the guidelines established by the Tokyo Metropolitan Institute of Medical Science Subcommittee on Laboratory Animal Care; these guidelines conform to 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, Japan, 2006. All protocols were approved by the Committee on the Ethics of Animal Experiments of the Tokyo Metropolitan Institute of Medical Science (Permit Number: 11–065). All efforts were made to minimize the suffering of the animals.2.Madin–Darby canine kidney cells  were maintained in Dulbecco's modified Eagle's medium (D-MEM) supplemented with 10% fetal calf serum (FCS) and penicillin-streptomycin solution. RK-13 cells (ATCC) were maintained in modified Eagle's medium (MEM) supplemented with 5% FCS and penicillin-streptomycin solution. Cells were grown in an incubator at 37°C under 5% COAll influenza A virus strains used in this study are listed in All rHAs except QH1A rHA were obtained from Sino Biological Inc. .g for 30 min. His-tagged (6×) QH1A rHA protein was then purified by using Ni-NTA Agarose  according to the manufacturer's instruction.QH1A rHA was generated by recombinant vaccinia virus (rVV) 4 cells/well with IL-6 (0.25 U/mL). Hybridomas were cloned by limited dilution and several clones were picked up. The antibodies were purified from the culture supernatant by protein-A sepharose  in the accompanying manuscript.8C1, 5H7, and 14A7 were established by GANP mice with Balb/c background For IFA, MDCK cells were inoculated with at MOI 4 with corresponding viruses for 30 min and washed with PBS several times. Fresh medium with 10% of FCS (without trypsin) was added to each well and cells were incubated at 37°C. At 6 h post infection, cells were fixed in 4% paraformaldehyde. Fixed cells were treated with each antibody for 1 h. Cy3-conjugated anti-mouse antibody  was used as a secondary antibody. Cells were observed by a fluorescence microscope .For ELISA, 100 ng of target rHA protein was immobilized to F96 MAXISORP NUNC-IMMUNO PLATE  and plates were incubated overnight at 4°C. The immobilized plate was blocked with 150 mL of 1% BSA/PBS for 2 h at room temperature. Diluted antibody was added and incubated for 1 h at room temperature. HRP-conjugated anti-mouse IgG specific antibody was used as secondary antibody. OPD was added as a substrate for HRP, and absorbance was measured at 492 nm by ARVO™ X2 .Detection antibodies (5H7 and 14A7) were conjugated with fluorescent latex beads . 1 ml of latex beads solution (0.5% in 10 mM HEPES-NaOH (pH 7.0) was combined into 300 μl of water soluble carbodiimide (1 mg/ml in 10 mM HEPES-NaOH (pH 7.0), 346–03632, DOJINDO Molecular technologies, Inc., Kumamoto, Japan), and incubated for 15 min at room temperature. Antibody (0.3 mg) was added, and incubated for 1 h at room temperature. Centrifuged and discard the supernatant. Latex beads were blocked with 1% BSA solution for 1 h at room temperature. Centrifuged and discard the supernatant. Latex beads were washed with wash buffer ), resuspended into reaction buffer ), and incubated for 48 h at 37°C. The fluorescent reagent was 2-indol- 2-ylidene]-penta-1,3-dienyl]-1,1-dimethyl-3-(3-methyl-butyl)-1H-benzo [e] indolium hexafluorophosphate. The absorption maximum and fluorescent maximum were 683 nm and 701 nm, respectively.Sample solutions (10 μl) were suspended in 90 μL of FLIC dilution buffer (50 mM Tris-HCl (pH = 7.2), 150 mM NaCl, 1% Trition X-100), and dropped on the sample pad of the IC strip . The flu


Familial Mediterranean fever (FMF) is an autosomal-recessive disorder characterized by recurrent attacks of fever and serositis common in eastern Mediterranean population. Over 160 mutations have been identified in MEFV gene responsible for FMF. The most common mutations in MEFV gene are E148Q, M694I, M694V, V726A and M680I. The distribution pattern of MEFV mutation along the Mediterranean Sea is not uniform; eastern populations have the highest number of carriers (20-39%), whereas western Mediterranean populations are practically unaffected.The aim of this study is to determine the carrier rate in healthy Macedonian, Serbian, Slovene, Bosnian and Hungarian population.We screened 100 healthy subjects from all 5 populations. Exon 10 was PCR amplified and screening was performed with dHPLC. All amplicons with detected nucleotide changes were subsequently sequenced with ABI prism 310 genetic analyzer. Amplicons of exon 2 were directly sequenced.Heterozygous mutations were found in 4% of apparently healthy Hungarians, 7% of Slovenians, 8% of Bosnians, 11% of Serbians and in 16% of apparently healthy Macedonians. Mutations found in Hungarian population were as follows: V726A (1), K695R (3). Mutations found in Slovenian population were: V726A (1), K695R (5) and E148Q (1). Mutations found in Bosnian population were: V726A (1), K695R (6) and F756C (1). Mutations found in Serbian population were: E148Q (6), K695R (5). Mutations found in Macedonian population were as follows: E148Q (8), K695R (7) and M694V (1).We found higher than expected carrier rate in all populations, from 4% to 16%. It is interesting to note that more than half (60%) of detected carriers in all analyzed populations has K695R mutation.None declared


An epidemic caused by influenza A (H7N9) virus was recently reported in China. Deep sequencing revealed the full genome of the virus obtained directly from a patient’s sputum without virus culture. The full genome showed substantial sequence heterogeneity and large differences compared with that from embryonated chicken eggs. Recently, a novel influenza A (H7N9) virus infected humans in China (www.ncbi.nlm.nih.gov/blast) with parameters −e 1e−5 −F T (−e 1e−5 for selection of highly similar reads and −F T for masking the low-complexity reads) after filtering of the sequence adapters and RT-PCR primers. No assembly was performed before alignment. We obtained 19,177 reads aligned to influenza A viruses.We collected a sputum specimen from a 54-year-old woman with fever, cough, sputum production, and pneumonia. Influenza A (H7N9) virus was detected in the specimen by specific real-time reverse transcription PCR (RT-PCR). The specimen was then processed with a viral particle–protected nucleic acid purification method (http://www.dnastar.com). The novel influenza A (H7N9) virus A/Anhui/1/2013was selected as the reference. With 80% minimum sequence similarity tolerance and 12 bp minimum match size, those 19,177 reads were assembled into 439 contigs. The top 8 contigs covered by the most reads corresponded to the 8 genome segments of the novel influenza A (H7N9) virus. The other contigs did not align to the reference virus, which might have resulted from sequencing or assembling errors. Calculating the consensus sequence, we obtained the genome of the influenza A (H7N9) virus directly from the sputum specimen of this patient. Further RT-PCR and Sanger sequencing confirmed the quality of the assembled subtype H7N9virus genome. Sequences were deposited in GenBank under accession nos. KF226105–KF226120 and KF278742–KF278749.We then conducted a reference-guided assembly based on the 19,177 reads by the Seqman program in the DNAStar software package version 7.1 (The influenza A (H7N9) genome that we report varies from that obtained by Sanger sequencing after passage in the allantoic sac and amniotic cavity of 9–11-day-old specific pathogen–free embryonated chicken eggs for 48–72 hours at 35°C . In the The influenza A (H7N9) genome also demonstrates significant intraspecimen heterogeneity. Deep sequencing revealed that the average coverage  of the 8 genes was quite inhomogeneous. Average coverage (± SD) was highest for neuraminidase (NA) (131.94 ± 30.25) and second highest for NP (130.41± 27.01). The average coverages of PB2, PB1, PA, matrix protein, and hemagglutinin were 99.89 (± 22.49), 95.35 (± 21.34), 43.35 (± 14.13), 53.73 (± 17.67), and 69.82 (± 19.02), respectively. Average coverage was lowest for NS (27.73± 11.31). Besides the gene abundance, the genome sequence of influenza A (H7N9) virus also demonstrated heterogeneity (the heterozygous peak threshold 80%). In total, 22 positions were confirmed by PCR and Sanger sequencing to be heterogeneous . In the Compared with the reference influenza A (H7N9) virus strain A/Anhui/1/2013, the influenza A (H7N9) virus demonstrated prominent sequence differences . In partwww.megasoftware.net) was used to construct the phylogenetic trees on the basis of the nucleotide sequences of all influenza A (H7N9) viruses in the Global Initiative on Sharing All Influenza Data (GISAID) database (www.megasoftware.net/); then, to construct the phylogenetic trees with 1,000 bootstrap replicates, we used the minimum-evolution method. Results suggested that all 8 genome segments are closely related to the available influenza A (H7N9) virus strains. MEGA5.0  viruses isolated in China in 2013 to closely investigate the relationships between this virus and available subtype H7N9 genomes isolated during epidemics. However, the phylogenetic topologies based on different gene segments were not consistent , suggestUsing deep sequencing technologies, we derived the full-length genome of the novel influenza A (H7N9) virus directly from the sputum specimen of a patient, without conducting virus culture. The full genome revealed substantial sequence heterogeneity within the specimen, obvious sequence variations from that obtained from embryonated chicken eggs, and prominent differences from the available influenza A (H7N9) strains, most of which were sequenced after culture.Phylogenetic trees of the influenza A (H7N9) viruses isolated in China in 2013, based on gene segments.


Heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C) is a core component of 40S ribonucleoprotein particles that bind pre-mRNAs and influence their processing, stability and export. Breast cancer tumor suppressors BRCA1, BRCA2 and PALB2 form a complex and play key roles in homologous recombination (HR), DNA double strand break (DSB) repair and cell cycle regulation following DNA damage.PALB2 nucleoprotein complexes were isolated using tandem affinity purification from nuclease-solubilized nuclear fraction. Immunofluorescence was used for localization studies of proteins. siRNA-mediated gene silencing and flow cytometry were used for studying DNA repair efficiency and cell cycle distribution/checkpoints. The effect of hnRNP C on mRNA abundance was assayed using quantitative reverse transcriptase PCR.BRCA gene expression and HR-based DNA repair. They also suggest the existence of an RNA regulatory program at sites of DNA damage, which involves a unique function of hnRNP C that is independent of the 40S ribonucleoprotein particles and most other hnRNP proteins.We identified hnRNP C as a component of a nucleoprotein complex containing breast cancer suppressor proteins PALB2, BRCA2 and BRCA1. Notably, other components of the 40S ribonucleoprotein particle were not present in the complex. hnRNP C was found to undergo significant changes of sub-nuclear localization after ionizing radiation (IR) and to partially localize to DNA damage sites. Depletion of hnRNP C substantially altered the normal balance of repair mechanisms following DSB induction, reducing HR usage in particular, and impaired S phase progression after IR. Moreover, loss of hnRNP C strongly reduced the abundance of key HR proteins BRCA1, BRCA2, RAD51 and BRIP1, which can be attributed, at least in part, to the downregulation of their mRNAs due to aberrant splicing. Our results establish hnRNP C as a key regulator of  Its two isoforms, hnRNP C1 and C2, form a (C1)DNA double strand breaks (DSBs) occur due to both endogenous damage and exogenous genotoxic insults. Homologous recombination (HR) and non-homologous end joining (NHEJ) are two main modes of double strand break repair (DSBR) HR is an extremely complex and highly regulated process. The initiation of HR requires the processing of DSBs to generate a long single-stranded DNA (ssDNA) overhang, a step termed resection, and the binding of the ssDNA by the recombination enzyme RAD51 to form a nucleoprotein filament capable of searching for and invading a homologous template Tandem affinity purifications of epitope-tagged PALB2 has led to the identification of BRCA1 and MRG15 as additional components of the PALB2/BRCA2 complex To identify proteins that interact with PALB2 in chromatin, we purified FLAG-HA-double tagged PALB2 from micrococcal nuclease (MNase)-solubilized nuclear fractions of HeLa S3 cells stably expressing the tagged protein. As show in Unexpectedly, hnRNP C was found to be a relatively abundant component of the complexes, indicating that it may either directly interact with PALB2 or its above-noted partner proteins, or reside on the same nucleosomes with PALB2. Another possibility is that hnRNP C and PALB2 may exist on the same small, residual segment(s) of non-nucleosomal DNA or RNA that might persist even after the extensive digestion by MNase. Importantly, other components of the 40S hnRNP particle were not identified in the PALB2/BRCA nucleoprotein complex, indicating that the binding is specific to hnRNP C and that hnRNP C has functions outside of the 40S particle. Again, no difference in hnRNP C abundance was observed in the complexes purified after DNA damage .To test whether hnRNP C and PALB2 interact with each other, we immunoprecipitated (IPed) endogenous PALB2 or hnRNP C from whole cell lysates, but no co-IP of the other protein was detected (not shown). We also overexpressed and IPed GFP- or FLAG-HA-double tagged versions of hnRNP C from whole cell lysates but also failed to detect any co-IP of PALB2 (not shown). Thus, it is unlikely that hnRNP C and PALB2 interact in cell lysates in a significant manner. To confirm the association of endogenous PALB2 and hnRNP C in the chromatin fraction and further test if the association is DNA- or RNA-mediated, we digested the insoluble nuclear materials from U2OS cells with either DNase I or RNase A and then IPed PALB2 from the solubilized fractions. As shown in The existence of hnRNP C in the chromatin-bound PALB2/BRCA complex raises the immediate question whether it functions in HR, a process in which PALB2 and BRCA1/2 play critical roles. To address this question, we used DR-U2OS cells stably integrated with a single copy of a GFP direct repeat HR reporter  [13], , anGiven the important role of hnRNP C in HR and DSBR pathway choice, we analyzed the effect of hnRNP C loss on cell cycle distribution and progression before and after IR by 5-bromo-2′-deoxyuridine (BrdU) incorporation. In addition to a non-targeting control siRNA, a PALB2 siRNA was also used as a reference for DNA damage-induced cell cycle checkpoints, as PALB2 plays important roles in both S phase and G2/M checkpoints Six hours post IR (10Gy), control cells showed a marked reduction of DNA synthesis that was especially pronounced in late S phase population , indicatSince PALB2 and BRCA proteins play important roles in the G2/M checkpoint maintenance, we further analyzed the mitotic index of the cells before and after IR. As shown in To test the importance of hnRNP C in the overall DSB repair efficiency after IR, hnRNP C-depleted cells were irradiated and analyzed for the induction and persistence of phosphorylated histone H2A.X (γH2A.X), a marker of DSBs, post damage. At 1 hr post IR, hnRNP C-depleted cells showed similar induction of γH2A.X compared with control cells , indicatWe further asked if the protein is recruited to DSBs following DNA damage by IF. In un-irradiated cells, hnRNP C showed a largely pan-nuclear staining pattern with varying numbers of distinct foci . InteresBeing an RNA-binding protein, hnRNP C may also be able to bind ssDNA, which plays essential roles in DNA damage signaling and the initiation of HR The limited and RNA-dependent localization of hnRNP C to DNA damage sites makes it unlikely that the protein directly participates in HR by binding to ssDNA intermediates generated during the process. Thus, we asked if hnRNP C regulates the expression of key HR genes. Interestingly, we observed in hnRNP C knockdown cells strongly reduced protein levels of BRCA1, BRCA2, RAD51 and BRIP1 . For BRCBRCA1, BRCA2, PALB2, RAD51, BARD1 and BRIP1 in control and hnRNP C-depleted cells. As shown in BRCA1, BRCA2, RAD51 and BRIP1 mRNAs, while the PALB2 messenger was slightly upregulated. We noticed that transfection of the control siRNA caused modest but consistent decreases in RAD51, BARD1 and particularly BRIP1 mRNA levels, indicating that a sequence-independent effect of siRNA transfection may be responsible for a fraction of the reduction observed for these genes. Nevertheless, the effect of the hnRNP C-specific siRNAs was significantly stronger.Next, we measured mRNA amounts of Alu elements was found in BRCA1, BRCA2, RAD51 and BRIP1 mRNAs following hnRNP C depletion . Taken together, our results demonstrate that hnRNP C directly binds to transcripts of above key HR genes and regulates their splicing and functionality.Finally, we asked if hnRNP C may directly bind the transcripts of the above HR genes and regulate their splicing. To this end, we analyzed the newly generated high resolution iCLIP and RNA-Seq data epletion  but not In this study, we found a significant presence of hnRNP C in PALB2-containing nucleoprotein complexes. The association between hnRNP C and PALB2 appeared to be indirect and instead mediated by RNA . hnRNP CThe pathway choice in DSBR is a competitive process in which the commitment to one mechanism at a given break precludes others. In general, SSA should increase following BRCA2 and/or PALB2 loss as reported before BRCA and related genes may be complex. As a chaperone of the transcriptome, hnRNP C presumably influences the expression of a large number of genes and therefore may indirectly affect the abundance of BRCA gene products through other factors involved in transcription, RNA splicing and stability, or protein synthesis, posttranslational modification and degradation, etc. However, analysis of iCLIP and RNA-Seq data revealed direct binding of hnRNP C to transcripts of all of the 6 genes tested and that loss hnRNP C resulted in exonization of intronic Alu sequences in the four genes whose mRNA amounts were affected (The mechanism(s) by which hnRNP C regulates the expression of the affected  and S5, BRCA1 promoter Similar to our findings, a recent whole genome screen for HR regulatory genes by Adamson et al. identified RBMX (hnRNP G) as a novel factor that positively regulates HR and resistance to DNA damage Brca1, Brca2 and Rad51 in mice all result in embryonic lethality 2O2) Since knockouts of hnRNP C undergoes a dynamic relocalization after ionizing radiation and localizes to a subset of DNA damage sites , indicat2 at 37°C. HeLa S3 cells were used for PALB2 purification and were grown in Minimum Essential Medium Eagle (MEM)  supplemented with 5% FBS in spinner flasks in a 37°C warm room.DR-U2OS as described before For general siRNA knockdown experiments, cells were plated at ∼60% confluence in 6 cm dishes and siRNAs were transfected using Lipofectamine RNAiMax (Invitrogen) at 5 nM final concentration. Unless otherwise mentioned, cells were trypsinized and reseeded into 6-well (for Western and FACS) or 12-well plates (for IF) 48 hr after transfection and various analyses were carried out another 24 hr later. The sense strand sequences of siRNAs used are: control, UUCGAACGUGUCACGUCAAdTdT; hnRNP C-629, CAACGGGACUAUUAUGAUAdTdT; and hnRNP C-920, GUAGAGAUGAAGAAUGAUAdTdT.2, 0.3 M sucrose, 0.1% Triton X100, 10 mM NaF, 1 mM sodium orthovanadate, with Complete® EDTA-free protease inhibitor tablet) containing MNase at a final concentration of 3 u/µl. The nuclei were digested by rocking at room temperature for 90 min. The reactions were stopped by adding EGTA and EDTA to 5 mM each, and supernatants containing solubilized chromatin were collected by centrifugation at 5,000 rpm for 10 min. Nucleoprotein complexes containing the FLAG-HA double tagged PALB2 were isolated by tandem affinity purification as previously described HeLa S3 cells harboring the empty vector or stably expressing tagged PALB2 were described before For analysis of DNA fragment size after MNase digestion, solubilized fractions were treated first with 5 µg/100 µl RNase A at 37°C for 30 min and then with 100 µg/ml proteinase K in the presence of 0.5% SDS at 55°C overnight. Digested samples were extracted with phenol/chloroform, and then analyzed on a 1.5% agarose gel.The anti-PALB2 M10 and M11 antibodies used in this study were raised in rabbits against GST-fusions of 1–120aa and 601–880aa of human PALB2, respectively, and affinity purified. The rabbit BRIP1 antiserum is a gift from Dr. Sharon Cantor (University of Massachusetts). Other antibodies used are as follows: hnRNP C , γH2AX , BRCA1 , BRCA2 , RAD51 , BARD1 , RAP80 , CtIP , NBS1 , DNAPK-cs , 53BP1 , MCM10 , CDC6 , CDC45 , α-Tubulin .g for 10 min at 4°C. Supernatants were collected and protein concentration was measured using Bradford's assay (Bio-Rad). Equal amounts (15–20 µg) of proteins were subjected to SDS-PAGE. Proteins were then immobilized onto nitrocellulose membranes followed by incubations in primary and secondary antibodies. Detection was carried out using Enhanced Chemiluminescence (ECL) detection system .For Western analysis, cells were lysed in NETNG400  buffer for 10 min with mixing at 4°C. Lysates were clarified by centrifugation at 21,000×6 cells were used for each IP. The digestion was for 45 min at room temperature with 50 units of DNase I or 30 µg of RNase A in a volume of 300 µl, and the IP was carried out for overnight at 4°C.For immunoprecipitation of endogenous PALB2 from nuclease-released nuclear fractions, low salt-resistant nuclear structures were prepared from DR-U2OS cells with MNase buffer I, digested with DNase I or RNase A in MNase buffer II, and the released proteins (supernatants) were subjected to IP with the anti-PALB2 M11 antibody. Approximately ∼4×106 cells per dish) were transfected with siRNAs using Lipofectamine RNAiMax. 24 hr later, cells were split into 6-well plates . On the next day, cells were co-transfected with the same siRNA, pCBAsce (750 ng) and hnRNP C expression vectors (750 ng) using Lipofectamine 2000. Medium was refreshed 8 hr later and GFP positive cells were counted 72 hr post co-transfection. DNA repair assays shown in In experiments shown in Cells were seeded into 12-well plates containing 15 mm #1 round coverslips the day before treatment or analyses. Briefly, cells were fixed with 3% (w/v) paraformaldehyde (in PBS with 300 mM sucrose) for 10 min at room temperature, permeabilized with 0.5% Triton X-100 (in PBS) and then sequentially incubated with primary and secondary antibodies (diluted in PBS containing 5% goat serum) for 1 hr each at 37°C. Each of the above steps was followed by three PBS washes. After staining, coverslips were mounted onto glass slides with VECTASHIELD mounting medium with DAPI (Vector Labs) and observed using a Nikon Eclipse Ti fluorescent microscope.2, 50 mM NaCl, 0.5% Triton X-100) for 4 min and then treated with 100 µg/ml of RNase A (Sigma) for 10 min at room temperature. Cells were then fixed with 3% paraformaldehyde and stained as described above.For analyzing the RNA dependence of hnRNP C nuclear localization, cells were first permeabilized with CSK buffer  with 10 µM BrdU for 10 min prior to harvest by trypsinization. Collected cells were washed with PBS and fixed with 10 volumes of ice-cold 70% ethanol and placed at −20°C overnight. Cells were then stained using FITC-conjugated anti-BrdU  following manufacturer's instructions. After staining, cells were pelleted and resuspended in 0.5 ml of PBS solution containing 0.02% (w/v) propidium iodide  and 200 µg/ml RNase A, and incubated for 15 min at 37°C. Finally, cells were analyzed by FACS on a Beckman Coulter FC-500 flow cytometer and using the CXP software. To measure mitotic index, cells plated in 10 cm dish were transfected with siRNAs for 48 hr and split into 6-well plates at 750,000 cells per well. On the next day, cells were irradiated and collected at indicated time points. When nocodazole was used, it was added 1 hr after radiation to a final concentration of 100 ng/ml. Harvested cells were fixed with 70% ethanol overnight and stained with anti-phosphorylated histone H3 (pSer10)  following standard protocols. Following the staining cells were incubated with PI and then analyzed by FACS.Total RNAs were extracted using RNeasy Plus Mini kit (QIAGEN), and cDNAs were generated using the SureScript® III First Strand Synthesis System (Invitrogen). Real-time PCR was performed using Brilliant II SYBR® Green qPCR Master Mix (Agilent Technologies) on a MX3005P system (Stratagene) using the following parameters: 15 min initial heating (denaturation and hot start enzyme activation) at 95°C, 40 cycles of amplification (95°C for 10 sec and 60°C for 30 sec) followed by melting curve measurement. Data presented are relative mRNA levels normalized to that of RPLP0, with the value in the control group (transfection reagent only) set as 1. Experiments were performed in triplicates for at least 3 times.ATCAACGGGTACAAACGAGTCCT, AGGCAGATGGATCAGCCAAGAAG; BRCA1- GAATGGATGGTACAGCTGTGTG, ATGGAAGCCATTGTCCTCTGTC; BRCA2- GCCACTTTCAAGAGACATTCAACA, GTACAGTCTTTAGTTGGGGTGGA; PALB2- TGTGATGCTGTACTGTCTTCCTC, GCAATTGTTCCAGAAGTCAAGAT; RAD51- TGTTTGGAGAATTCCGAACTG, GTCAATGTACATGGCCTTTCCT; BARD1- ATTGCTGCTACCAGAGAAGAATG, ACAGCCCACTGCCTATAAGTACA; and BACH1- CAGAAAGGAGAAAAATGATCCAG, CTTTGTTTGTTTGTTGAAAGTTGG.Primers were synthesized by Sigma. The sequences of the primers are as follows: RPLP0- Figure S1Construction and expression of an siRNA-resistant form of hnRNP C cDNA expression vector. A. Silent mutations introduced into the target sequence of the hnRNP C siRNA (RNPC-629). Shown on top is the sequence of the sense primer used for mutagenesis containing 4 silent mutations that would render the cDNA resistant to the siRNA. The bottom sequence is of the wt cDNA with the siRNA target sequence shown in red. The corresponding protein sequence is also shown. B. The modified hnRNP C expression vector was co-transfected, in parallel with the empty vector and the wt expression vector, with pCBASce into DR-U2OS cells. Cells were fixed 48 hr after transfection and IF was conducted using the indicated antibodies.(PDF)Click here for additional data file.Figure S2Effect of hnRNP C depletion on cell cycle distribution before and after IR. DR-U2OS cells were treated with control, PALB2 or hnRNP C siRNAs for 72 hr and then subjected to 10 Gy of IR. Cells were labeled with BrdU either before (A) or at 6 and 16 hr post IR , and cell cycle profiles were analyzed by anti-BrdU staining and FACS. Cells in S, G1 and G2/M phases were indicated by upper, lower left and lower right boxes, respectively. Numbers in the boxes indicate the percentages of cells in the corresponding phases. In the left panel of B, early S and late S phase cells are indicated by "ES" and "LS" and separated by an arbitrary dotted line.(PDF)Click here for additional data file.Figure S3Comet assay of hnRNP C-depleted cells after IR. A. DR-U2OS cells were treated with control or hnRNP C (1∶1 mix of 629 and 920) siRNAs for 72 hr and then subjected to 10 Gy of IR. Cells were harvested at indicated time points following IR and subjected to alkaline comet assay (Trevigen) following manufacturer's instructions. B. Number (in percentage) of cells with comet tails in a representative experiment. C. Mean length of comet tails in a representative experiment. D–G. Length distribution of comet tails in a representative experiment. Comet measurements were carried out using the Image J software, and approximately 100 comets were measured for each condition.(PDF)Click here for additional data file.Figure S4Reduced abundance and impaired focus formation of BRCA1 and RAD51 in hnRNP C-depleted cells. Control treated or hnRNP C-depleted DR-U2OS cells were subjected to 10 Gy of IR. Cells were fixed at indicated time points and stained for BRCA1 (A) or RAD51 (B) together with γH2A.X. The antibody used were anti-BRCA1 , anti-RAD51  and anti-γH2A.X .(PDF)Click here for additional data file.Figure S5Binding of hnRNP C to transcripts of HR genes. A. Genome browser view of PALB2 and BARD1 genes displaying RNA-Seq data  from control and hnRNP C knockdown HeLa cells, that were independently transfected with two different siRNAs (KD1 and KD2), as well as hnRNP C iCLIP data . RefSeq transcript annotations (blue) and Alu elements in antisense orientation to the shown strand (orange) are depicted below. No Alu exonization events were found in these two genes. B. "Weblogo" showing the base composition at the hnRNP C crosslink sites (position 0) within BRCA1, BRCA2, PALB2, RAD51, BARD1 and BRIP1 gene transcripts as well as the surrounding sequence. The y-axis indicates the informational content for each position in bits. The graph shows the aggregate of all the crosslink sites in the 6 genes.(PDF)Click here for additional data file.


Escherichia coli. Most current protocols rely on commercial kits or isolation of BACs, their transfer between different host strains, and their restriction.Promoter-specific expression of foreign DNA in transgenic organisms often relies on bacterial artificial chromosomes (BACs). This approach requires modification and subcloning of BAC-DNA by recombineering technologies in In this report we present a 2-step protocol for efficient modification and subcloning of DNA from bacterial artificial chromosomes using the non-commercial plasmids pKM208 and pTP223, distributed from addgene.com. A targeting cassette was successfully integrated into a BAC and 42 kb of this construct were subcloned. Both a plasmid-derived substrate with longer homology arms and a PCR-generated substrate with short homology arms (50 bp) were used for recombination. pKM208 and pTP223 contain all required genes for recombineering, but differ in their antibiotic resistance genes. This makes the system independent of the selection markers on the DNA molecules targeted for recombination.in vivo without restriction and ligation. Furthermore, both steps are performed in the same host eliminating the need to isolate BAC DNA and to use different bacterial strains.The time and cost saving protocol presented here compares favorably to currently used systems. Using non-commercial plasmids, it allows targeted modification and cloning of large DNA (> 40 kb) fragments  E. coli. They can carry up to 300 kb of foreign DNA. The large insert size has made BACs an important source for the generation of transgenic organisms, because they carry entire genes including their regulatory elements. This can be used to express foreign genes in a spatiotemporal pattern, which mimics endogenous gene expression [E. coli strains to achieve high clonal stability and low rate of chimerism.Bacterial artificial chromosomes (BACs) are low copy plasmids based on the F-plasmid of pression -4. BACs in vivo manipulation of DNA molecules is called recombinogenic engineering or simply recombineering [Methods to introduce genetic modifications into BACs such as insertions, deletions, or point mutations are primarily based on homologous recombination between the BAC and targeting molecules and do not require restriction endonucleases or DNA ligases. The most popular approach for the ineering ,6. Two mineering . The secineering . The exoineering .E. coli [Serratia, Shigella, Yersenia, Salmonella and Pseudomonas [® system from Gene bridges. The second one consists of a publically available system developed by Copeland and coworkers [Recombineering systems have been used to engineer the chromosome of E. coli ,8,10-12,udomonas -17, and udomonas ,19. For Math5  in the BAC RP23-328P3 by a 2-Neo CreERTcassette and subsequent subcloning of the novel 42 kb transcription unit by gap repair  in an easy 2-step recombineering protocol. Both plasmids contain the open reading frames for Redα, Redβ and Redγ, but differ in the origins of replication and the selection markers. The low copy plasmid pKM208 has the temperature-sensitive origin pSC101 and confers ampicillin (Ap) resistance, whereas the high copy plasmid pTP223 confers resistance to tetracycline (Tc) . Both pl600 nm of 0.5 in the presence of chloramphenicol (Cm) (12.5 μg/ml) on a shaker at 37°C. After centrifugation at 6,000 rpm for 10 min, cells were resuspended in 20 ml precooled deionized water. After another round of centrifugation, cells were resuspended in 50 μl precooled deionized water and electroporated with 100 ng of pKM208 ([A 20 ml culture from a single BAC carrying host colony was grown to an ODcol (Cm) .5 μg/ml 600 nm of 0.2 - 0.3, cultures were split into two 25 ml cultures. One culture was supplemented with IPTG to a final concentration of 1 mM, whereas the other culture served as a negative control. After 1 h of shaking, cells containing pKM208 were heat-shocked for 15 min at 42°C and kept on ice to stimulate subsequent gene replacement [BAC hosts carrying pKM208 were grown at 30°C in 50 ml LB broth, containing Cm 12.5 μg/ml) and Ap (100 μg/ml), whereas BAC hosts carrying pTP223 were grown at 37°C in the presence of Cm (12.5 μg/ml), kanamycin (Km) (25 μg/ml) and Tc (10 μg/ml). After they reached an OD.5 μg/ml 2-Neo was constructed by cloning a PCR product containing the 2-Neo CreERTfrom pl451-CreERT2 into pMECA [2-Neo CreERTcassette was flanked by two homology arms derived from the Math5 gene of the BAC RP23-328P3. After validation by sequencing, a 5,107 bp long fragment, containing 2-Neo CreERTand flanking 708 bp and 372 bp long homology arms, was released by restriction with DraI  and gel-purified using peqGOLD Gel Extraction Kit .The plasmid pMath5-CreERTto pMECA . This CrTGTCTCCCACAGTACCTAACATAGGATCTTACAGAGTAGACACACATGATGTTTAAACGATACGCGAGCGAACGTG-3' and pBR322-Math5-L 5'-CCTGCAGCACAGCCTCCAACAGTTTTCAGTCTACTCATCTTTCCTAGTATGTTTAAACTTAGACGTCAGGTGGCAC-3' . After DpnI (MBI Fermentas) restriction, the PCR product was gel-purified using peqGOLD Gel Extraction Kit (Peqlab) and eluted with deionized water.To generate the targeting cassette for subcloning, a PCR was performed with Phusion-Polymerase  using pBR322 as template and the primers pBR322-Math5-R 5'-The gene targeting DNA (300 ng) was mixed with 50 μl of cell suspension and put into a precooled 0.1-cm electroporation cuvette and electroporated with a Gene Pulser (Bio-Rad) at 1.8 kV, 200 Ohm, 25 μF for ~ 4.5 ms. Cells were resuspended in 1 ml LB broth, incubated for 1 h at 37°C on a shaker, and then plated on selective LB-agar plates.2-Neo CreERTcassette, using the targeting cassette specific primer 5'-CTCCTGTCATCTCACCTTGC-3' and the BAC specific primer Math5_90718rev 5'-CCGGACGCATTCAACATCAC-3' (expected product size: 1.6 kb). For further validation, additional junction PCRs were performed. Primers were as follows: Math5_90718rev and 5'-GGCCCGCGCTGGAGTTTC-3' (3.9 kb), Math5_90718rev and 5'-CCACTGCGGGCTCTACTTC-3' (2.9 kb), as well as with the primers 5'-CCTGACACCCTTTTAGTTAAG-3'and 5'- CCATGAGTGAACGAACCTGGTCG-3' (2.6 kb). For gap repair mediated subcloning, DNA from positive clones was purified by alkaline lysis and analyzed by restriction with MssI (MBI Fermentas).Recombinant clones were screened by a junction PCR for probing the insertion of the To establish an easy, cheap, and fast protocol for recombination-based modification of BACs, we assessed the non-commercial plasmids pKM208 and pTP223. Both plasmids have been successfully used for engineering of bacterial chromosomes ,12,16, b2-Neo CreERTcassette with flanking homology arms of 708 bp and 372 bp . 24 colonies from induced cells were analyzed by a plasmid preparation followed by restriction with MssI. This resulted in 2 putative positive clones, which were confirmed by restriction using HindIII (Figure DH10B cells containing the modified BAC and pTP223 were transformed with the PCR-generated pBR322-derived targeting cassette to subclone a 42 kb fragment containing the modified transcription unit (Since spontaneous loss of pTP223 has been reported , we testWe report a protocol for the modification and subcloning of large BAC-DNA by the use of the non-commercial plasmids pKM208 and pTP223. The protocol sidesteps restriction and gel-purification of large DNA fragments. Furthermore, no BAC isolation and transformation into other strains like DY380, EL250 and EL350 is required . Both plHere, we used pKM208 for targeting the BAC with a plasmid-derived substrate and pTP223 for subcloning. This was done to demonstrate the potential of both plasmids to promote homologous recombination using BACs as template. In future studies, only one of the two plasmids is sufficient. The choice of the plasmid solely depends on the selection marker, which should be different from those present in the BAC and the DNA substrate for homologous recombination. The availability of two different selection markers renders the system very flexible and versatile. Finally, selection for these markers in the media will ensure recombineering potential in all cells of the culture.The authors declare that they have no competing interests.HH conceived and carried out the experiments and drafted the manuscript. HGN discussed the results with HH and helped to draft the manuscript. Both authors read and approved the final manuscript.