SOX1) inhibits cervical and liver tumorigenesis by interfering with the Wnt/β-catenin signaling pathway. However, the role of SOX1 in NPC remains unclear. This study investigates the function of SOX1 in NPC pathogenesis.Aberrant activation of the Wnt/β-catenin signaling pathway is an important factor in the development of nasopharyngeal carcinoma (NPC). Previous studies have demonstrated that the developmental gene sex-determining region Y (SRY)-box 1  contains supplementary material, which is available to authorized users. Nasopharyngeal carcinoma (NPC) is the most common head and neck cancer in southern China, Southeast Asia, the Arctic and the middle/northern regions of Africa. The incidence of NPC in southern China is approximately 25–50 per 100,000 persons each year, which is 100-fold higher than that in Western countries. The pooINK4a promoter leads to decreased expression of its protein in NPC, further promoting tumorigenesis[CDH13, DLEC1, CHFR and CDH1 has been implicated in tumorigenesis[SOX1 promoter is involved in the development of NPC remains to be elucidated.The family of sex-determining region Y (SRY)-box (SOX) proteins is a group of transcription factors that contain a highly conserved high-mobility group (HMG) DNA-binding domain. SOX family members play crucial roles in both embryonic and postnatal development and in stem cell regulation–8. Moreoigenesis, 18. Addigenesis, 20. HowCCND1, MYC and Cdx-1[The canonical Wnt signaling pathway is involved in various biological processes, including embryonic development, cell proliferation and stem cell maintenance. Moreoveand Cdx-1. Previouand Cdx-1, 24. Addand Cdx-1. TherefoIn this study, we demonstrate that depletion of SOX1 is responsible for the malignant phenotype of NPC. We show that recovery of SOX1 expression leads to a down-regulation of β-catenin that is independent of proteasomal activity. These new data show that SOX1 decreases β-catenin activity and reverses the malignant phenotype of NPC.SOX1 promoter methylation. We determined the methylation status of the NPC cell lines by quantitative methylation-specific PCR (qMS-PCR). Hypermethylation was confirmed in the NPC cell lines that showed down-regulated SOX1 expression, whereas methylation was almost absent in NP69 cells  and immunofluorescence (IF) analysis . These results suggest that SOX1 impairs tumor growth in NPC cells in vivo.Next, we examined the influence of SOX1 on NPC cells tumor formation  Figure A. The tu Figure B and tum Figure C were siin vitro were further investigated by overexpressing SOX1 in either CNE2 or HONE1 cells. As shown in Figure p <0.01) and HONE1  cells. Meanwhile, colony-forming ability in soft agar was also decreased in these cells . We also found that overexpression of SOX1 in HONE1 cells enhanced SA-β-gal staining  and an immortalized normal nasopharyngeal epithelial cell line (NP69) were used in this study. All cells were obtained from the State Key Laboratory of Oncology in South China. CNE1 and CNE2 were cultured in RPMI-1640  containing 10% fetal calf serum (Gibco). HONE1, SUNE1, 58 F and HK1 were cultured in RPMI-1640 containing 10% fetal bovine serum . NP69 cells were propagated in defined keratinocyte-serum-free medium  supplemented with growth factors. Virus-converted cells with and without stable SOX1 overexpression  were maintained in incomplete medium with 1.5 μg/ml puromycin. All cells were maintained at 37°C in a humidified atmosphere containing 5% COqMS-PCR was performed using the EZ DNA Methylation-Gold™ Kit  according to the manufacturer’s protocol. The primer sequence for qMS-PCR has been described. MethylaRNA was isolated from each tissue or cell sample using Trizol reagent  according to the manufacturer’s protocol. A 2-μg aliquot of total RNA from each sample was used for cDNA synthesis with SuperScript® III RT (Invitrogen) and an oligo-dT primer. PCR was performed at 95°C for 5 min, then 30 cycles  with a final extension of 10 min at 72°C. The primer sequence for SOX1 was as follows: forward 5′-AAGGTGAAGGTCGGAGTCAAC-3′, reverse 5′-GGGGTCATTGATGGCAACAATA-3′. Real-time PCR was performed using Platinum® SYBR® Green qPCR SuperMix (Invitrogen) and analyzed on a CFX96 Real-Time System . Glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as an internal control. The difference of real-time PCR cycle number  between the target gene and GAPDH was quantified using the ΔΔCT method.The WB procedure was performed as described previously with somThe IF procedure was performed as described previously with somImmunoprecipitation was conducted according to a previous report. PolycloThe colony formation assay was performed according to the procedure reported previously. Briefly5 cells) in SFM was added to the upper chamber. After incubation for 24 h , the membrane was fixed with 2% methanol and stained with crystal violet. Pictures were taken under a microscope and the stained cells in the lower side of the membrane were counted.For the wound-healing assay, CNE2-EV&SOX1 and HONE1-EV&SOX1 cells were seeded in a 6-well plate at 90% confluence. The day before the assay, cells were starved with serum-free medium overnight. A sterile 200-μl pipet tip was used to scratch three separate wounds through the cells moving perpendicular to the line drawn with a marker on the bottom of the dish. Then, cells were gently rinsed with PBS and 1.5 ml of media was added. Pictures were taken using a phase contrast microscope at 0, 12, 16 and 20 h. For the transwell migration assay, 800 μl of normal medium was added to the lower chamber in a 24-well plate, and 200 μl cell suspension (1 × 105) transfected with empty vector or SOX1-expressing plasmid were injected subcutaneously into the left and right flanks of 6-week-old nude mice . The tumor volume was calculated as 0.5 × L1 × (L2)2, where L1 is the long axis and L2 is the short axis of the tumor. The developing tumors were observed every third day. After 20 days, the mice were sacrificed at the end of follow-up. The animal study protocol was approved by the Institutional Animal Care and Use Committee.CNE2 cells  was used according to the manufacturer’s instructions. Briefly, cells were fixed with fixation buffer for 10 min after rinsing three times in PBS for 5 min at RT. The cells were stained with staining buffer at 37°C overnight covered with plastic wrap. The cells were rinsed three times in PBS for 5 min at RT and observed under a microscope.Cells were harvested by digestion, washed with pre-chilled PBS, fixed with 70% ethanol at 4°C for 30 min, then stained with propidium iodide  for 30 min at 4°C in the dark. Stained cells were subjected to flow cytometry analysis to measure the sub-G1 fraction.Sphere formation assays were performed as per a previous report. Cells wSoft agar colony formation assays were conducted according to a previous report. Brieflyp <0.05. Error bars represent the standard deviation of the mean (SD), and each experiment was completed at least twice with samples in triplicate.Statistical analyses were performed using the SPSS software, version 16.0 (SPSS Inc.) or with GraphPad Prism 5.0 . The unpaired Student’s t test was used to perform a statistical comparison between two groups. The ANOVA test was used when performing multiple comparisons. The level of significance was set at in vitro. (A) The wound-healing assay performed in CNE2 cells overexpressing SOX1. (B) Morphology of differentiated CNE2 cells induced by SOX1 was observed under microscopy. (C) WB was used to detect the cell surface markers related to cell differentiation in CNE2 cells with or without SOX1 overexpression. . (TIFF 7 MB)Additional file 1: Figure S1: Ectopic expression of SOX1 represses CNE2 cells migration and induces cell differentiation Additional file 2: Figure S2: SOX1 reduces epithelial–mesenchymal transition in CNE2 cells. (A) Presence of the EMT-related proteins E-cadherin and Vimentin were detected via IF in CNE2 cells. Blue, DAPI; Red, E-cadherin; Green, Vimentin. (B) EMT-related markers were detected by WB in CNE2 cells with forced SOX1 expression. GAPDH served as an internal control. (TIFF 2 MB)p < 0.01, Student’s t test) and HONE1 . (C) The colony formation ability in soft agar dramatically decreased upon SOX1 overexpression, from 19.3 ± 4.5 to 5.3 ± 1.5 each 20× field in CNE2 and from 20.7 ± 5.0 to 7.7 ± 2.1 each 20× field in HONE1, both *p < 0.05, Student’s t test. Quantitative data were shown as the mean ± SD from three independent experiments. . (TIFF 9 MB)Additional file 3: Figure S3: Overexpression of SOX1 negative regulated the stem cell ability of NPC cells.  The sphere formation ability reduced significantly in both CNE2 (from 64.5 ± 9.95 to 32 ± 10.23 per 500 cells, **


The samples were analyzed for the contents of 28 elements. Sources of PM2.5 were identified and the contribution of each source to the PM2.5 concentration was assessed using an enrichment factor (EF) analysis, a principal component analysis (PCA), and multi-linear regression analysis (MLRA). In the cold season (January–March and October–December 2011–2012), the mean ambient concentration of PM2.5 in Racibórz was 48.7 ± 39.4 µg·m−3, which was much higher than at other suburban or rural sites in Europe. Additionally the ambient concentrations of some toxic PM2.5-bound elements were also high, i.e., the mean ambient concentrations of PM2.5-bound As, Cd, and Pb were 11.3 ± 11.5, 5.2 ± 2.5, and 34.0 ± 34.2 ng·m−3, respectively. In the warm season (April–September 2011–2012), the PM2.5 and PM2.5-bound element concentrations in Racibórz were comparable to the concentrations noted at other suburban  sites in Europe. Our findings suggest that elemental composition and concentrations of PM2.5 in Racibórz are mainly influenced by anthropogenic emissions, i.e., the energy production based on coal and biomass combustion, traffic, and industry.Twenty-four-hour samples of fine ambient particulate matter (PM There is a limited number of appropriate PM2.5 sampling points in Central and Eastern Europe  affects the environment most extensively. It contributes to a variety of health effects and climate changes ,6,7,8,9.n Europe , which s2.5. The two cities, located approximate 60 km north of Racibórz, are typical in terms of atmospheric air pollution by PM. Both in Zabrze and in Katowice very high mass contributions of PM2.5 in total PM are observed, particularly in winter [An interesting contrast can be noticed between the origin of ambient particulate matter in Western Europe and in Poland, especially in the highly urbanized and industrialized Silesia region (one European PM pollution hotspot), where municipal and industrial emissions and emissions from energy production are greater than traffic emissions. The structure of air pollution in Poland is representative of Eastern and Middle Europe countries, where the energy production relies on the combustion of fossil fuels . Zabrze and Katowice (Poland) are well monitored areas in terms of PM concentrations and elemental composition of PMn winter ,17,18,19n winter ,21.2.5 has not been carried out in any suburban areas located in a European hotspot yet. Still, some research shows that PM–related exposure among Racibórz residents is the same as or sometimes even higher than that found among the inhabitants of city centers [In European rural areas most of PM comes from long-distant pollutant transport and natural sources. Its concentration is generally lower than in city centers and rarely causes any air quality problems ,61,62,63 centers . 2.5 in a suburban area located in southern Poland, based on statistical analysis of a two-year data series of elemental composition of PM2.5.The purpose of this work is to identify the souces of PM2.5 samples were collected by means of a PNS  Atmoservice sampler  equipped with a PM2.5 head (PM inlet with PM2.5 sharp-cut cyclone). The air flow rate was 2.3 m3·h−1. The PM2.5 samples were collected on 47-mm diameter PTFE membranes . Before and after the exposure, the filters were conditioned in a weighing room  and weighed twice  on a Mettler Toledo AT microbalance (with a resolution of 2 µg) using a Haug U-ionizer . The adopted method of PM sampling and the subsequent gravimetric analysis of the PM were compliant with EN 14907/2005 (Standard gravimetric measurement method for the determination of the PM2.5 mass fraction of suspended particulate matter) and EN 12341/1998 . The weighing accuracy, determined as three standard deviations from the mean obtained from ten weighings of a blank filter (conditioning performed every 48 h), was 20.5 µg.Twenty-four-hour PM2 in the whole area, and about 700 people/km2 in Racibórz itself. It is 3–5 times less than in other cities and towns in southern Poland. The majority of the inhabitants live in Racibórz. Low buildings with individual heating systems  dominate the landscape. The building development is compact in the center of Racibórz and scattered in the vicinity of the sampling site. The mean annual temperature in the region is +8.0 °C. January is the coldest month (−2.1 °C), whereas July is the warmest one (+18.0 °C). During the year, there are 100–110 frost days and 170 days with precipitation (including 45 snowy days) in the region. South winds prevail in autumn and winter and north winds are common in spring and summer. Calms are frequent.The samples were collected between 1 January 2011 and 26 December 2012 in Racibórz, a town located in the south-western part of the Śląskie (Silesian) Province in south Poland, close to the Czech-Polish border. The sampling point (50°5′ N and 18°14′ E) was located approximate 6 km south of the center of Racibórz. The Racibórz area is an agricultural region , where f2.5 samples  were analyzed for their elemental content by means of energy dispersive X-ray fluorescence (EDXRF) on an Epsilon 5 spectrometer . The apparatus was equipped with an X-ray tube with a side window , a system of nine secondary targets , and a Ge(Li) detector . The measurements were conducted under vacuum condition. The analysis, including the run of the whole program of the changes of targets and X-ray tube settings  lasted 2400 s. The concentrations of particular analytes were derived by comparing the results with calibration curves. The curves were determined by measuring thin-layer standards  and corrected for possible matrix effects according to a standard procedure saved in the spectrometer software.The PM−2.The NIST standard reference material (SRM2873) was measured weekly. Except 52% and 39% recoveries of V and Co, the recovery of each element was between 85% (As) and 120% (Pb) of the certified value. Ten blanks (PTFE membranes) were used to determine the detection limits for the procedure. Each blank underwent the entire EDXRF procedure devised for a regular sample 30 times; a detection limit for an element was the standard deviation from the 300 results obtained for this element. The detection limits were between 0.2 (Se) and 21.3 (Si) ng·cm2.5 and twenty-four-hour PM2.5-bound elements were divided at first into two sets and averaged. The measurement period was used as the division criterion. The results were therefore averaged for two periods: the cold/heating season  and the warm/non-heating season . A nonparametric technique known as the Mann-Whitney U test was used to test the significance of inter-season variation in PM2.5 and PM2.5-bound element concentrations PM and (xC/refC)soil are proportions of these concentrations in PM and in the soil. The enrichment factor xC concentrations are related to the SiC concentration of Si. Consequently, SiEF = 1. The element concentrations of the Earth’s upper continental crust soil were taken from the study [In the paper, the observed he study . xEF with a value up to 10 indicates the crustal origin of the element x [xEF suggests a strong anthropogenic effect on the concentrations of x. In this study, an lement x ,19,20,21Further data analysis consisted in applying the methodology described by Thurston and Spengler . The met2.5-bound element concentrations. Subsequently, values of the new variables—principal components —were used in the MLRA. All the calculations were performed using Statistica 8.0 software .In the first step, a principal component analysis (PCA) was applied to the 28 × 320 data matrix representing the twenty-four-hour PM2.5 concentrations in Racibórz ranged from 3.2 µg·m−3 to 209.5 µg·m−3  [−3; annual mean value) [The twenty-four-hour PM5 µg·m−3 . The mean value)  and the n value) . 2.5 concentrations clearly indicate a poor quality of air as far as pollution with fine PM in the cold (heating) season in Racibórz is concerned  season in 2011–2012, less than 11% of the twenty-four-hour PM2.5 concentrations exceeded 25 µg·m−3. A distinct seasonality of the concentration of PM2.5 and other air pollutants has already been observed in other parts of Poland [2.5 concentrations was characterized by a maximum in the cold period and a minimum in the warm period (2.5 concentrations noted in the cold and warm season (p ≤ 0.001); the mean PM2.5 concentration in the cold season (48.7 µg·m−3) was about 3.5 times higher in comparison with the warm season (13.9 µg·m−3).The twenty-four-hour PMoncerned . Only 34f Poland ,69,70 anf Poland ,63,71. Tm period . The nonm season  were sta2.5 concentrations [2.5 concentrations were also affected by the incoming air masses from distant areas [In the cold season, Racibórz residents increased combustion activities for heating purposes, which generally contributed to the increased PMtrations ,70. In wtrations ,73. The trations ,70. In Rnt areas ,70.2.5 concentration in Racibórz did not differ much from the PM2.5 concentrations recorded in other cities of southern Poland such as Zabrze , Katowice  [2.5 = 36.0 µg·m−3) [2.5 = 25.2 µg·m−3) and Złoty Potok  [2.5 concentrations in other parts of the country, such as Diabla Góra  [2.5 = 17.1 µg·m−3) [2.5 = 16.1 µg·m−3) [2.5 = 10.7 µg·m−3) in central Poland [2.5 concentration in Racibórz was also high as compared to the values registered at various European stations situated in suburbs, such as Košetice  [2.5 = 5.1 µg·m−3), Waldhof , Harwell , Cabauw-Zijdeweg  and Ispra  [2.5 concentrations registered at industrial sites , 2001, PM2.5 = 24.0 µg·m−3) [2.5 = 35.9 µg·m−3) in Europe [In 2011–2012, the mean PM µg·m−3) , Wrocław µg·m−3) , Trzebin µg·m−3) . This co µg·m−3)  and Szcz µg·m−3)  in north µg·m−3)  and Wars µg·m−3) , Hyytiäl µg·m−3) . It was  µg·m−3)  or traffn Europe .2.5-bound elements ranged from 0.8 ng·m−3 (Se) to 2364.3 ng·m−3 (Cl) and from 0.3 ng·m−3 (Se) to 924.4 ng·m−3 (S) in the cold and the warm seasons (2011–2012), respectively  and S (1201.7 ng·m−3) in Racibórz (averaged for the entire measurement period) were generally much higher than at other European locations, such as Mount Cimone  [The mean concentrations of the PM  , Zürich    and Mene  . As the   ,70. This2.5-bound Cl and S, the concentrations of other  elements could be affected by coal and biomass combustion in Racibórz during the heating season. This is indicated by evidently greater values of EF for such elements as compared with the non-heating season  than in other regions of Europe [Similarly to the PMg season , and by f Europe ,22,23,702.5-bound elements recorded in Racibórz in 2011–2012, just like the mean PM2.5 mass concentration, are comparable to the values reported in different cities in southern Poland [The mean ambient concentrations of most PMn Poland ,70,79. On Poland ,24,70 ann Poland ,71,78,80−3, 5 ng·m−3, 20 ng·m−3, and 0.5 µg·m−3, respectively) [The International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) classified As, Cd, Ni and their compounds, as well as Cr(VI) within Group 1 (carcinogenic to humans). The Pb compounds are included in Group 2A (probably carcinogenic to humans). In 2011–2012, the ambient concentrations of As, Cd, Ni, and Pb in Racibórz did not exceed the permissible values of annual concentrations established by the European Commission (6 ng·mctively) . However2.5 and the contribution of these sources to the concentrations of PM2.5 based on the PCA and MLRA results. 2.5 in Racibórz. For a better interpretation of the PCA results, the enrichment factors (EFs) for PM2.5-bound elements . Their variances were within the range of 4.7%–37.9%. 2.5  were located in both directions , biomass  and various waste types  for heating. This source was assigned with FF_BB_WI symbol, i.e., fossil fuels (FF) and biomass (BB) combustion, and waste incineration  with PC1: Zn, K, Br, Rb, As, Pb, Cl, S, Na, Cd, Mn, Co, Se, V, Sb, Mg, and Fe. The mean ambient concentrations of all these elements (except Fe) were statistically significant and evidently higher in the heating (cold) season than in the non-heating (warm) season . High corections . The poltion WI; . 2.5 concentrations in Racibórz were the greatest in the S-E and E directions (about 30%) and slightly smaller in the S and N-E . Mean mass percentages of FF_BB_WI in the PM and N-E . GeneralThe EFs calculated for each of the 17 elements correlated with PC1 were much higher in the heating season than in the non-heating season , which i2.5 concentrations was low (16.3%–19.2%) in the entire analyzed period. PC3 was the most strongly correlated with Sr, Mo, and V. In 2011–2012, those three elements had statistically higher concentrations in the heating (cold) season  compared to PC1 . The var) season . Their E) season . The con) season . 2.5 whose efficiency changed with air temperature. Its contribution to the concentrations of PM2.5 in Racibórz did not depend on the direction of air masses . The lowest percentage of PC5 in PM2.5 was observed on cold (heating) season days. The values for the days analyzed in the warm season were much higher. Generally, a mean mass content of PC5 in the concentrations of PM2.5 did not depend on the direction of the incoming air masses (nonexh_TRexh). PC4 was classified in the same way. It was the most strongly correlated with Co and Al, which suggested that tire and road wear particles were some of the identified sources of PM2.5 in Racibórz [2.5 occurred during the warm season rather than in the cold part of the year. Besides, the greatest percentages of PC4 in PM2.5 were determined for the western and northern directions  in the PM2.5 mass in Racibórz was calculated in a simplified way in the previous study [Cu and Mo were the most strongly correlated with PC5. Their concentrations were the highest at the inflow of air masses from the center of Racibórz and other cities of the Silesian agglomeration (S-E and E). High concentrations of these elements were also observed at the inflow of air masses from the Czech Republic and Germany , especially from the Moravian-Silesian Region S; . Cu and r masses . This fiRacibórz . Yet, inrections , where tus study . Its mea2.5 in Racibórz , or oxidants used for oxidizing the metal stock admixtures and transforming them into slag (such as Fe or Mn ores). Sc and other rare earth elements are broadly used in metallurgy as alloying additions improving the properties of admixture metals, as permanent magnets or polishing pastes. In the case of a majority of elements correlated with PC2, relatively high concentrations (visibly higher for Cr and Ni) were observed when the air masses were transported from the N-E, where the most industrialized cities of the biggest urban and industrial center of Poland  are located, and in the S direction (Moravian–Silesian Region), where the biggest European steel works is situated —the one dominant industrial source in the N-E direction. The percentage of the last extracted principal component (PC2) was independent of the direction of incoming air masses or the season . Cr and 2.5 found at the quasi-rural site in that area differs significantly from the PM2.5 at other similar areas located in various parts of Europe. Differences are observed in both the concentrations and elemental compositions of PM. The obtained results indicate that sources originate from the common use of solid fuels for energy production in Poland. Nearly 17% of the PM2.5 in the air in Racibórz comes from the hard and brown coal combustion in power plants and large industrial heat and power plants located in various parts of Poland and abroad. In our study we found that 22% of the PM2.5 is associated with the combustion of the coal and biomass mixture (and most probably home waste) at local small boiler rooms and home furnaces. In other European regions, traffic emissions are currently the primary source of air pollution . In Racibórz, the contribution of traffic related pollutants to the concentration of PM2.5 does not exceed 30%. Considering the elemental composition of PM2.5 alone is insufficient to determine whether the majority of emissions comprise PM particles of mechanical origin (non-exhaust emission) or particles/particle precursors from fuel combustion (exhaust emission). In southern Poland, the industrial emissions exert a significant effect on PM2.5 concentrations. Steelworks, non-ferrous metal works and other industrial plants located outside the Racibórz area,  are responsible for 16% of the PM2.5 mass in Racibórz.Southern Poland, where Racibórz is located, is one of the most urbanized and polluted “hot spot” areas in Europe. The PMLong-distance transport of PM is known to be associated with adverse health effects and impacts related to climate change. Source identification of PM-bound elements via principal component analysis and monitoring of PM concentrations are critical tools used to evaluate their potential impacts and effectiveness of control measures. Regionally in Poland and locally in Raciborz the combustion of solid fuels has been identified as primary anthropogenic contributors to air pollution. While total elimination of many anthropogenic sources is not possible, important findings of this study can be used to help inform the development of realistic management policies and cost-effective methods to improve air quality. It is our opinion that simply reducing the combustion of coal, biomass, and wastes in domestic ovens in the Raciborz region would benefit local and regional air quality.


Histone variants establish structural and functional diversity of chromatin by affecting nucleosome stability and histone-protein interactions. H3.3 is an H3 histone variant that is incorporated into chromatin outside of S-phase in various eukaryotes. In animals, H3.3 is associated with active transcription and possibly maintenance of transcriptional memory. Plant H3 variants, which evolved independently of their animal counterparts, are much less well understood.Arabidopsis at mono-nucleosomal resolution using native chromatin immunoprecipitation. This results in the precise mapping of H3.3-containing nucleosomes, which are not only enriched in gene bodies as previously reported, but also at a subset of promoter regions and downstream of the 3′ ends of active genes. While H3.3 presence within transcribed regions is strongly associated with transcriptional activity, H3.3 at promoters is often independent of transcription. In particular, promoters with GA motifs carry H3.3 regardless of the gene expression levels. H3.3 on promoters of inactive genes is associated with H3K27me3 at gene bodies. In addition, H3.3-enriched plant promoters often contain RNA Pol II considerably upstream of the transcriptional start site. H3.3 and RNA Pol II are found on active as well as on inactive promoters and are enriched at strongly regulated genes.We profile the H3.3 distribution in In animals and plants, H3.3 organizes chromatin in transcribed regions and in promoters. The results suggest a function of H3.3 in transcriptional regulation and support a model that a single ancestral H3 evolved into H3 variants with similar sub-functionalization patterns in plants and animals. Drosophila and mammalian cells revealed specific incorporation into the gene body of active genes, into promoter regions of both active and inactive genes, as well as into regulatory elements[Histones are abundant in most eukaryotic cells where they package DNA into chromatin. Dimers of histones H2A, H2B, H3 and H4 assemble into the histone octamer core to organize 147 bp of DNA into nucleosomes, the basic building blocks of chromatin. In recent years, much has been learned about how posttranslational modifications of histones affect chromatin, such as modifying inter-nucleosomal contacts or nucleosome stability. It is now also well established that incorporation of histone variants can result in formation of chromatin with particular properties. The seqelements, supportelements. Unexpecelements,12, and elements. H3.3 iselements. RecentlArabidopsis H3.3 differs from H3.1 at positions 31, 87 and 90 but also at some additional positions[Arabidopsis [Arabidopsis H3.3 has the same epigenomic properties as the animal H3.3 variant. Recently, the genome-wide distribution of the Arabidopsis H3.3 protein At4g40040 was reported and, similar to animal H3.3, was preferentially found in the bodies of transcribed genes[Replication-coupled and replication-independent (replacement) H3 histone variants evolved independently in animals, plants, basidiomycetes, and alveolates. Similarositions. There a5g10980),17, whic5g10980),18. Howeed genes,20. In cArabidopsis genome, we generated Arabidopsis lines expressing H3.3 tagged by yellow fluorescence protein . Among several transformed lines that were phenotypically indistinguishable from wild-type control plants . We next grouped all the genes into four bins according to their relative transcript abundance, and for each group averaged H3.3 profiles were plotted along genes . Therefore, H3.3 incorporation occurs with the highest frequency at the TTSs of genes. In total, we identified 1,891 genes with only promoter-associated H3.3 nucleosomes, 10,447 genes with only TTS-associated H3.3 nucleosomes, and 3,012 genes with both promoter- and TTS-associated H3.3 nucleosomes  during the transcription of genes[Arabidopsis the role of H3.3 in nucleosomes would also be coupled to Pol II activity. Indeed, we found that in Arabidopsis H3.3 co-localizes with Pol II in the nucleus , consistent with the general trend shown in Figure P-value <2.2 × 10-16, one-tail Wilcoxon test). Therefore, H3.3 presence around TTSs is a better predictor for high transcript abundance than H3.3 presence at promoters.We next asked whether H3.3 enrichment at promoters and around TTSs was similarly related to transcription. Genes with H3.3 enriched only around TTSs and genes with H3.3 enriched at both promoters and around TTSs had significantly higher transcript abundance than the genome-wide median . In contrast, genes with H3.3 enriched only around the TTS had significantly larger expression entropies than the genome-wide median . Genes with H3.3 enriched at both promoters and around TTSs had reduced expression entropies, which were, however, much larger than those of genes with H3.3 enriched only at their promoters. These results support our hypothesis that genes with H3.3 enriched at their promoters are subject to strong transcriptional regulation.In budding yeast, Pol II was found upstream of inactive genes that could be rapidly activated upon exit from the stationary phase. We askeing data,28 . Therefore, H3.3 and mCG appear to exclude each other at promoters.Furthermore, genes with H3.3 enriched at promoters lack almost any mCG, especially in the -800 to -200 bp window where H3.3 is highest Figure C. In conIn summary, H3.3 enrichment in nucleosomes does not strongly correlate with mCG in gene bodies and is negatively correlated with mCG at promoters.Arabidopsis promoters can be categorized into three major groups depending on the core promoter element, that is, TATA, GA and coreless[P-value = 0.087, Wilcoxon’s rank test, two-tail). These results show that GA promoters are special because they are targeted by H3.3 incorporation regardless of transcriptional activity.coreless. Followiin vivo DNA bound by H3.3 nucleosomes might be more accessible than DNA bound by H3.1 nucleosomes. To test this hypothesis, we compared the accessibility of DNA[Mammalian H3.3 nucleosomes greatly impair higher-ordered chromatin folding, suggesty of DNA around Hin vitro[per se does not affect stability of mononucleosomes[H3.3 is a histone variant that differs only in four or five amino acids from the canonical H3.1 but it can have profound effects on chromatin functionality. Earlier studies had suggested that animal H3.3-containing nucleosomes isolated from native chromatin are less stable in vitro but moreleosomes. InsteadArabidopsis replacement histone variant H3.3 and found that in plants H3.3 is located in gene bodies as well and shows a positive correlation with transcriptional activity, which is consistent with earlier reports[Drosophila[in vitro after eviction of a single H2A/H2B dimer while the H3/H4 tetramer remains associated with the DNA[per se might not be sufficient to cause H3 replacement. This is consistent with the non-uniform and specific H3.3 patterns along gene bodies. Our results in Arabidopsis revealed a strong 3′ bias along gene bodies when examining plant H3.3 patterns by normalizing either to input or histone density, similar to earlier observations[per se but appears to be restricted to specific phases of the transcription process.H3.3 incorporation is generally thought to be associated with the transcription initiation and/or elongation activities of Pol II in animals and is highest in gene bodies,11,43-46 reports,20. In crosophila,8 to 3′-rosophila,10. Althrosophila. It is n the DNA,48. Comprvations,20. Our Arabidopsis, H3K4me3 is found proximal to TSSs[Arabidopsis H3.3 is highly enriched for H3K36me2[Arabidopsis[Arabidopsis that H3.3 localization extends considerably beyond the TTS indicates an even stronger connection between H3.3 and Pol II transcription termination.In animals, H3.3 levels correlate with transcriptionally active, Ser-5 phosphorylated Pol II, transcription initiation site-related mono- and tri-methylation of histone 3 lysine 4 , and transcription elongation-related tri-methylation of histone 3 lysine 36 (H3K36me3). In Arab to TSSs where H3H3K36me2, the hisabidopsis. This suArabidopsis, we found H3.3 at promoters of both active and inactive genes. It is possible that H3.3 is incorporated at promoters independently of transcription or that it is a footprint of past transcription activity of the gene. Since we could also find Pol II associated with H3.3-enriched promoters, it is possible that RNA Pol II promoter occupation or transcription caused local H3.3 incorporation. Indeed, transcription of promoter-associated short RNAs is more ubiquitous than initially thought during transcription activation[In addition to gene bodies, we found H3.3 also in plant promoters. H3 replacement at active promoters has been reported for mammals, and it tivation,55. H3.3tivation and can tivation. Alternativation-52 to fativation. The inctivation, suggesttivation,56 or prtivation. Our datArabidopsis and animal H3.3 incorporation patterns is consistent with a general association of H3.3 with several eukaryotic chromatin remodeling processes. The presence of H3.3 on active as well as on many inactive plant promoters of strongly regulated genes suggests a function of H3.3 in transcriptional regulation.Animal and plant H3 variants evolved independently,57, but Arabidopsis  accession Columbia-0 plants. To produce 35S:H3.3-YFP lines the cDNA of HTR4 (At4g40030) was fused to the cauliflower mosaic virus (CaMV) 35S promoter at the amino terminus and the YFP cDNA sequence at the carboxyl terminus, and the fusion construct was inserted into the binary vector pCambia1380. Cloning and amplification of the plasmid was done in Escherichia coli DH5α. The plasmid was transformed into Agrobacterium tumefaciens (strain C58C1) and then transformed into Arabidopsis using the floral dip method. Transformants were selected on Murashige and Skoog medium agar plates containing hygromycin. Experimental plants were grown on soil at 21°C in dark (16 h) and 20°C in light (8 h). Plant age was recorded as days after imbibed seeds were sown on soil and transferred to the growth chamber. Leaves (leaf number 6 from about five plants per sample) were harvested after 35 days at zeitgeber time 7 , and frozen in liquid nitrogen. Note that cell division and expansion had ceased at this developmental stage in the harvested leaves. The experiment was performed with three independent biological replicates.All experiments used PP2A.Expression analysis of the H3.3 transgene was performed as described using ge35S:H3.3-YFP seedlings were ground and the powder was extracted with Buffer M (10 mM Tris-(hydroxymethyl)-aminomethan pH 7.5; 0.5% IGEPAL CA 630; 1% Triton X-100; EDTA free protease inhibitor cocktail (Roche)) plus 150 mM NaCl for 10 minutes at 4°C. The suspension was centrifuged at 16,100 × g at 4°C for 10 minutes. The pellet was subsequently extracted using Buffer M containing 500 mM NaCl, centrifuged again, and extracted once more with Buffer M containing 2 M NaCl. Extracted proteins were separated using SDS-PAGE. Total protein was transferred to PVDF-membrane . The H3.3-YFP fusion protein was detected using anti-GFP antibody  and horseradish peroxidase (HRP)-coupled anti-mouse antibody , and was visualized using Immun-Star HRP Substrate .For protein immunoblots, 50 mg of frozen 35S:H3.3-YFP line were germinated and grown for 3 days in Petri dishes on wet filter paper. For visualizing nuclear DNA in live cells, 1 μM of DRAQ5  was applied to Arabidopsis roots for 5 to 10 minutes with vacuum to facilitate penetration. DRAQ5 stain and YFP signals in roots were consecutively analyzed using a Zeiss 710 confocal laser scanning microscope.Seeds of the 4, 5 mM EGTA, pH 6.9). Root tips were digested for 10 minutes at 37°C with a PCP enzyme mixture  dissolved in MTSB) and squashed in a drop of MTSB buffer. Immunostaining was performed as described[35S:H3.3-YFP seedlings were grown on Murashige and Skoog medium for 5 days before YFP signals in roots were analyzed using a Zeiss 710 confocal laser scanning microscope .For immuno-staining, seedlings were fixed for 20 minutes with ice-cold 4% (w/v) paraformaldehyde in MTSB buffer ) for 15 minutes at 4°C. The homogenate was filtered through Miracloth , and a nuclei pellet was collected by centrifugation for 10 minutes at 1,500 × g at 4°C. Isolated nuclei were washed once in MNase buffer ), treated with 1.3 μl of RNase A, 30 μg/μl  and used for Micrococcal Nuclease  digestion for 4 minutes  in MNase buffer. The reaction was stopped with 10 mM EDTA. After a centrifugation the supernatant was collected as phase 1 chromatin preparation. The pellet was resuspended in buffer S2 ) for 30 minutes. After centrifugation the supernatant was collected as phase 2 chromatin preparation. The two phases of chromatin preparations were combined and the NaCl concentration was adjusted to 50 mM. The majority of the chromatin was of mononucleosome size (data not shown). Histone H1 was depleted by incubating the chromatin preparation with Sephadex C25-CM resin  for 1 h at 4°C[2O, Sigma-Aldrich) and collected with Dynabeads Protein A (Invitrogen). After washing, beads were re-suspended in TE buffer , and DNA was extracted using phenol-chloroform extraction and ethanol/salt precipitation. Cross-linked ChIP was performed as described[Native ChIP was performed as described with minescribed. ChIP waqPCR was performed using the ChIP-recovered DNA as template using specific primers and probes  followed by purification using MinElute PCR Purification kit .qPCR was performed for six genomic fragments before and after amplification to control for amplification bias (data not shown). Amplified ChIP DNA was fragmented, labeled and hybridized to Affymetrix AGRONOMICS1 escribed.P-values were calculated using Wilcoxon’s signed rank test.Background correction and normalization were performed as described previously. ChIP-chSupplementary raw data are available in ArrayExpress, accessibp: base pair; ChIP: chrommatin immunoprecipitation; MNase: micrococcal nuclease; PCR: polymerase chain reaction; Pol II: RNA Polymerase II; qPCR: quantitative PCR; TSS: transcription start site; TTS: transcription termination site; YFP: yellow fluorescence protein.The authors declare that they have no competing interests.HS, WG, LH designed the research; HS, MN, LB, IM, TW performed research; HS, AS, LH analyzed data; HS, WG, LH wrote the paper. All authors read and approved the final manuscript.Figure S2. ChIP-qPCR confirmation for H3.3 incorporation. Figure S3. H3.3 is present at transcribed genes and has a strong 3′ bias. Figure S4. Schematic representations of positional relation between test genes and amplicons for qPCR. Figure S5. H3.3 incorporation profiles for genes with differential H3.3 nucleosome association. Figure S6. Genes with H3.3 nucleosomes in the promoter are strongly regulated upon environmental stress. Figure S7. H3K27me3 profiles of Polycomb group target genes and genes with H3.3 in promoters. Figure S8.Arabidopsis H3.3 does not colocalize with H2A.Z. Figure S9. Identification of nucleosomes.Generation of H3.3-YFP expressing plants. Click here for fileTable S2. Primers and Universal Probes (Roche) for qPCR used in this study.List of genes with H3.3 nucleosomes in the promoter, around the TTS or both in promoters and around TTSs and list of all H3.3 nucleosomes. Click here for file


Clostridium sp. strains were isolated from soil and sediment collected from the Kurdistan region of Iraq. All three isolates were found to harbor putative prophages, with a CRISPR-Cas system found in strains C105KSO13 and C105KSO14.Three  Clostridium sp. strains  were isolated from sediment and soil samples from the Kurdistan region of Iraq. Motility assays revealed that only C105KSO14 and C105KSE15 were motile. The strains were isolated by adding ~1 g of soil, with 10 ml of fastidious anaerobic broth (FA) , supplemented with 250 µg·ml−1 cycloserine, 8 µg·ml−1 cefoxitin, and 0.1% sodium taurocholate. Cultures were incubated for 10 days in a MiniMACS anaerobic chamber at 37°C, prior to subculturing on Brazier’s cycloserine, cefoxitin, and egg yolk agar plates  for 48 h. Colonies were subcultured three times on BHI agar with 7% defibrinated horse blood. Bacterial genomic DNA was prepared from cultures by using a QIAGEN Genomic-tips kit according to the manufacturer’s instructions and sequenced using MiSeq . One nanogram of genomic DNA was prepared using the Nextera XT DNA sample preparation kit (Illumina) prior to sequencing on the MiSeq platform using the paired-end 2- × 250-bp (Version 2) protocol. Reads were trimmed with Sickle prior to assembly with Spades  and 55 tRNAs; C105KSE14 is 5.48 Mb, with 5,273 predicted ORFs and 57 tRNAs; and C105KSE13 is 3.21 Mb, with 2,982 predicted ORFs and 45 tRNAs.Analysis of 40 single-copy phylogenetic makers with specI  did not Bacillus phage MG-B1, with the second spacer homologous to sequences from Staphylococcus phages MR25 (All 3 isolates encoded between 1 (C105KSE15) and 12 putative prophage-like elements (C105KSE14). Strains C105KSO13 and C105KSO14 encoded a CRISPR-Cas system composed of two CRISPR arrays, with no putative CRISPR system in strain C105KSE15. The arrays in both strains contained multiple spacers ranging from 54 (C105KSO14) to 103 spacers (C105KSO13). Two spacers were found in C105KSO13, one was homologous to a sequence from a ges MR25  and 88 (ges MR25 . Three sFBWL01000001, FBWO01000001, and FBWN01000001, respectively.The draft genome sequences of C105KSO13, C105KSO14, and C105KSO15 have been deposited in DDBJ/ENA/GenBank under the accession numbers,


The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT  repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form. Mec1 ) and Tel1 (yeast orthologue of ATM (ataxia telangiectasia-mutated)) belong to the phosphatidylinositol 3-kinase-related protein kinase (PIKK)Because of the large size of these proteins, ranging from 2,368 amino acids for Mec1 to 4,128 amino acids for DNA-PKcs, these proteins are difficult to be purified in large quantities, thus hindering biochemical and structural analysis. There are a number of low resolution electron microscopy reconstructions available. These include a monomeric ATM with and without DNA bound at 30 Å resolution , 6, DNA-In budding yeasts, Mec1 and Tel1 are apical enzymes in DNA damage checkpoint pathways. They detect DNA damage and temporarily halt cell cycle progression while elevating and activating DNA repair proteins –21. Mec1–22–Mec1·Ddc2 complexes to single-stranded DNA  –35. This–nded DNA . Remarka regions , 41. A sATR-ATRIP complex and Tel1ATMis tightly regulated. Post-translational modifications such as acetylation in ATM . The cell lysate was adjusted to a pH of 7.4 and a conductivity corresponding to that of 200 mm KCl buffer and was clarified by ultracentrifugation at 35,000 rpm for 1 h in a 45 Ti rotor (Beckman Coulter). The supernatant was incubated with IgG beads  for 3 h and subjected to four consecutive washes with buffer HEP250, HEP300, HEP300 supplemented with 10 mm magnesium acetate and 1 mm ATP, and HEP400. Mec1·Ddc2 was cleaved with HRV 3C protease and eluted.The Mec1·Ddc2 complex was expressed and purified as previously described  with som300 . Ammonium sulfate precipitated protein was resuspended in buffer HEP0 and incubated with glutathione S-Sepharose beads for 3 h and subjected to four consecutive washes with buffer HEP100, HEP100 lacking the protease and phosphatase inhibitors hereinafter, HEP100 supplemented with 10 mm magnesium acetate and 1 mm ATP, and HEP100. The GST-tagged Tel1 was cleaved by HRV 3C protease and further purified over a heparin column and eluted in HEP700.The GST-tagged Tel1 was overexpressed in yeast from pBL602 and purified as previously described  with somm HEPES-KOH, pH 7.4, 200 mm KCl, 10% glycerol, 0.1% Tween 20, 0.01% Nonidet P-40, 1 mm EDTA, 1 mm EGTA, and 2 mm DTT using Amicon Ultra 0.5 columns (Merck Millipore) and run over a Superose 6 HR 10/30 gel filtration column (Pharmacia Biotech) at 0.3 ml/min. Tel1 was buffer-exchanged to 50 mm HEPES-KOH (pH 7.4), 200 mm KCl, 10% glycerol, 0.1% Tween 20, 0.02% C12E10, 1 mm EDTA, and 3 mm DTT and run over a Superose 6 Increase 5/150 GL gel filtration column  at 0.1 ml/min. Elution was monitored at 280 nm, and peak fractions were separated on an 8% SDS-PAGE gel to verify protein identity. Migration of the Mec1·Ddc2 complex and Tel1 was compared with that of the gel filtration standards  to estimate the size of the complex.Mec1·Ddc2 was buffer-exchanged to 50 mm Mec1·Ddc2 was incubated with 100 nm GST-Rad53-kd in 10-μl reactions containing 25 mm HEPES (pH 7.6), 100 mm NaCl, 8 mm magnesium acetate, 100 μg/ml BSA, 1 mm DTT, 100 μm ATP, 0.5 μCi of [γ-32P]ATP and 50 nm Dpb11, where indicated. Reactions were allowed to proceed for 10 min at 30 °C and stopped by addition of 4 μl of 5× SDS-PAGE loading dye. Samples were boiled for 5 min, separated on 8% SDS-PAGE gels, dried, and exposed to a phosphor screen .10 nm Mec1·Ddc2 complex was labeled with 32 nm goat anti-Mec1 FATC, yS-20, polyclonal antibody (Santa Cruz Biotechnology) in 10-μl reactions containing 30 mm HEPES-KOH (pH 7.4), 150 mm NaCl, 2.5% glycerol, 0.25 mm EDTA, and 0.5 mm DTT. 20 nm Tel1 was labeled with the same anti-FATC antibody at 40 nm in 25-μl reactions containing 50 mm HEPES-KOH (pH 7.4), 220 mm NaCl, 2% glycerol, 1 mm EDTA, 0.5 mm EGTA, 1 mm DTT, 1 mm MgCl2, 0.02% Tween 20, 0.002% Nonidet P-40. The anti-Mec1, yS-20, antibody has been raised against the highly conserved C-terminal region of Mec1 . The reactions were incubated on ice for 30 min before putting the labeled samples onto EM grids.16 nm Mec1·Ddc2 , 2 μl of 20 nm Tel1 , and antibody-labeled samples at concentrations described above were deposited for 1 min on glow-discharged continuous carbon grids (TAAB Laboratory Equipment). The excess of liquid was blotted, and a 2-μl drop of 2% (v/v) uranyl acetate was added to the grids for 2 min. The excess liquid was blotted and left to air dry. Mec1·Ddc2, Tel1, and the anti-Mec1-labeled complexes of Mec1·Ddc2 were imaged in a Philips CM200 electron microscope operating at 200 kV and equipped with a TVIPS slow scan 4k × 4k CCD camera. Micrographs were manually collected at a nominal magnification of 50,000 with a pixel size of 1.76 Å/pixel corresponding to an electron dose of 20 e/Å2/s. All images were taken at a range of defocus from −2.5 to −3.5 μm. The antibody-labeled Tel1 sample was imaged in an FEI Tecnai F20 electron microscope operating at 200 kV and equipped with an FEI Falcon II CMOS direct electron detection camera. Micrographs were manually collected at a nominal magnification of 62,000 corresponding to a pixel size of 1.65 Å/pixel and an electron dose of 32 e/Å2/s.2 μl of 70 nab initio reconstructions using a standard multivariate statistical analysis/multireference alignment routine in IMAGIC-V , 210 mm KCl, 1% glycerol, 0.02% Tween 20, 0.002% Nonidet P-40, 1 mm EDTA, 0.5 mm EGTA, 1 mm DTT, and 0.5 mm MgCl2. Aliquots of 2 μl of Tel1 at a concentration of 20 nm were applied to glow-discharged holey carbon grids  coated with an additional thin layer of carbon. After 30 s of incubation in a humidity chamber at 4 °C and 100% relative humidity, the grids were blotted for 6 s and flash frozen in liquid ethane using an FEI Vitrobot Mark III. The grids were imaged in an FEI Tecnai F20 electron microscope operating at 200 kV and equipped with an FEI Falcon II CMOS direct electron detection camera. Micrographs were manually collected at a nominal magnification of 62,000 corresponding to a pixel size of 1.65 Å/pixel and an electron dose of 32 e/Å2/s. Images were recorded at a defocus ranging from −1 to −4 μm.Tel1 was diluted in a buffer optimized for cryofreezing while preserving the protein stability and integrity. The final buffer included 50 mAll data were processed in RELION-1.4 . All the4JSV) without the FKBP12-rapamycin binding domain  was docked into the EM density corresponding to the kinase domain of Mec1 and Tel1. A 5.9 Å atomic structure of mTOR (Protein Data Bank code 5FLC) modeled into the cryo-EM map of mTORC1 (EMD-3213) was docked into the EM densities of the Mec1·Ddc2 complex and Tel1. Prior to fitting, the densities corresponding to Raptor and mLST8 subunits and the FKBP protein were removed. The Mec1·Ddc2 and Tel1 three-dimensional maps were then segmented in Chimera  to prevent aggregation. In this study, the protein was purified to high homogeneity with improved buffer conditions (A), and all the experiments have been carried out in physiological salt concentrations (100–200 mm KCl). We analyzed Mec1·Ddc2 on a Superose 6 HR 10/30 size exclusion column, and interestingly, we found that the majority of Mec1·Ddc2 elutes with a retention time corresponding to a complex of ∼700 kDa and is thus consistent with the molecular mass of the Mec1·Ddc2 dimer . Mec1 activity has been measured with in vitro kinase assays. The enhanced phosphorylation of Rad53 (with the active site mutated) and Rpa1 in the presence of Dpb11 was observed for the fraction containing the Mec1·Ddc2 dimer . Therefore, we confirmed that Dpb11 could activate the oligomeric form of Mec1·Ddc2 used in our structural studies. Tel1 was purified to high homogeneity (C), analyzed on a Superose 6 Increase 5/150 GL size exclusion column, and eluted with a retention time corresponding to ∼700 kDa, which is consistent with the molecular mass of a Tel1 dimer .Mec1·Ddc2 was previously shown to form a stable complex of 360 kDa using analytical ultracentrifugation . Howevernditions A, and al720 kDa; B. Mec1 ac2 dimer B, inset.ogeneity C, analyz640 kDa; D.A). Based on the eigenimage analysis in IMAGIC-V  with their corresponding reprojections (RP) generated from the three-dimensional model (B). The final negative stain Mec1·Ddc2 dimer model (C) was obtained from 5,633 particles. The distribution of Euler angles of all particles used in generating the C2 structure (D) covers all angular space. The three-dimensional refinement of the model converged at 22.5 Å (E). The dimeric Mec1·Ddc2 reconstruction (C) is 175 Å tall, 215 Å wide, and 115 Å thick, resembling a pretzel with tubular density encircling multiple cavities. We can clearly distinguish two regions, which have been referred to as the head and arm domain in ATM, DNA-PKcs, and SMG-1 reconstructions . The model reveals multiple interaction sites within the arm and the head domains. The arm of one monomer is intertwined with the arm of another monomer. Weak contacts are also observed between the two opposite head domains, suggesting their auxiliary roles in dimer formation and stabilization.To investigate the structural organization of Mec1·Ddc2, we used single particle negative stained EM to generate the three-dimensional model of the complex. Negative stained EM was selected because of relatively low sample concentration and poor contrast in cryo-EM. We observed two populations of particles that differed significantly in their sizes. The majority (81%) was consistent with the size of Mec1·Ddc2 dimer of 720 kDa A. Based IMAGIC-V , dimerical model B. The fier model C was obttructure D covers t 22.5 Å E. The ditruction C is 175 ructions . Here, tB). The final negative stained three-dimensional model measures 185 Å tall, 230 Å wide, and 105 Å thick .Tel1 samples were initially analyzed using negative stained EM to build an initial model . Single Fig. 3A). An initial data set of 20,519 particles was selected by a combination of manual and autopicking in RELION (B).Tel1 was subsequently vitrified in a buffer that was compatible for cryo-freezing and stability of the protein to perform cryo-EM analysis A. An inin RELION . Multipln RELION B.C) overall resembles the negative stain reconstruction (C). However, it is taller (200 Å versus 185 Å) and narrower (215 Å versus 230 Å) when compared with the negatively stained model. This could be due to the stain procedure, which could cause flattening artifact. The Tel1 structure starkly resembles the overall Mec1·Ddc2 dimer architecture. Each monomer displays the characteristic domains of the PIKKs with the arm and the head region. However, the two head domains in Tel1 are fully separated by 35 Å in the narrowest region and enclose a cavity ∼20 Å long and 125 Å wide. The Tel1 dimer structure displays no obvious steric hindrance for substrates to access the kinase domains.The cryo-EM Tel1 structure C overalltruction C. HoweveA). For antibody-labeled Tel1 data, because of the preferential orientation and the low antibody labeling efficiency, there were insufficient data for reliable two-dimensional classification. However, because of the distinct front view, it is unambiguous that the anti-FATC antibody is located within the globular head domain (B). Thus the FATC domains in both Tel1 and Mec1·Ddc2 are located within the globular domain.The three-dimensional reconstructions of both Mec1·Ddc2 and Tel1 display the head domain that is similar to head regions in other PIKK structures, where the FAT-kinase-FATC domains reside. We have used an antibody against the FATC domain to locate the FATC within Mec1·Ddc2 and Tel1 particle. Individual particles were carefully selected based on visual inspection. Only those representing the anti-FATC-labeled particle with additional density adjacent to it were selected. Subsequently we carried out the reference-free alignment and two-dimensional classification. Comparisons of the class averages with the reprojections generated from the Mec1·Ddc2 reconstruction located an extra density that corresponds to the anti-FATC antibody A. For and domain B. Thus tB and C) further supports that the cradle-shaped density of the head region consist of the FAT, kinase domain, and the FATC domain. Based on our molecular fitting, we could also suggest the possible location of the catalytic and activation loop within the cleft of the Mec1 and Tel1 kinase domain as shown for mTOR (D). The elongated tubular arm domains agree with the shape and the dimensions of the HEAT repeats, which dominate both the N-terminal domains of Tel1 and Mec1, as well as the majority of Ddc2. Upon fitting all domains of the full-length mTOR into the Mec1·Ddc2 model, we have observed an unassigned density extending from the N-terminal HEAT repeats of Mec1. This density also forms an elongated and tubular arm, indicating that it is a natural extension of the N-terminal Mec1 and therefore might correspond to the Ddc2 subunit. These tubular arm domains intertwine and form the dimer interface.Fitting the crystal structure of the C-terminal mTOR  and the for mTOR D. The elC and 3C). Monomeric Mec1·Ddc2 has a molecular mass of 360 kDa compared with 320 kDa of Tel1 (A); thus, it is likely that one of the tubular densities in the Mec1·Ddc2 reconstruction  accounts for Ddc2, as also concluded from our molecular fitting. Another striking difference between these two structures is the orientation of the head regions within the dimer. The two kinase domains are fully separated in Tel1, thereby suggesting no impediment for substrate recruitment in this model. On the other hand, the individual kinase domains in Mec1 are in close proximity, enclosing only a small cavity between the two domains.Mec1·Ddc2 and Tel1 structures display a similar dimer arrangement with the dimer interface formed by the N-terminal HEAT repeats of Tel1 and Mec1, as well as Ddc2 in Mec1·Ddc2 . The oveFigs. 2 of Tel1 A; thus, Mec1 and Tel1 serve as key regulators of the DNA damage response. Together with other members of the PIKK family, they share a conserved domain arrangement of the N-terminal arrays of HEAT repeats followed by the C-terminal globular kinase flanked with the FAT and FATC domains.ATR-ATRIPand Tel1ATM. Both models adopt a similar architecture with the head region comprising the FAT, kinase, and FATC domains, as well as the curved arm region consisting of large stretches of the N-terminal HEAT repeats. Our models are consistent with the 6.6 Å crystal structure of DNA-PKcs (B) into the EM density of the Mec1·Ddc2 dimer, there is a remaining unassigned density extending from the N-terminal HEAT repeats of Mec1 that could accommodate the Ddc2 subunit. Then similar to DNA-PKcs and SMG-1, Ddc2 could possibly fold back into Mec1, positioning its N terminus in close proximity to the head region of Mec1. All PIKKs thus stabilize their C-terminal catalytic domains by using extended HEAT repeats, either within the same polypeptide chain as in Tel1ATM and DNA-PKcs or by merging with an additional polypeptide chain such as Ddc2ATRIP.Our EM reconstructions of Mec1·Ddc2 and Tel1 reveal their dimeric architecture and provide the first structural insights supporting earlier biochemical findings on Mec1·Ddc2DNA-PKcs , as wellDNA-PKcs . The DNADNA-PKcs , it mighDNA-PKcs B into thB, inset). The oligomerization of ATR-ATRIP through the ATRIP coiled-coil region is required for localization into foci of radiation-induced damage sites, as well as stalled replication forks (B) locates HEAT repeats at the dimeric interface. It is also seen in Tel1 structure that the HEAT repeats form the major dimer interface at the arm region (C). Similar findings have also been observed in the EM structures of mTORC1 (1/S and intra-S checkpoints (D). In mTORC1, the two kinase domains are located similarly to that of Mec1 and Tel1, although the active sites are facing away from the dimer interface (B–D). It is possible that, depending on the number of HEAT repeats, different forms of dimerization exist for the PIKKs. Alternatively, the different dimeric forms observed in our work and in mTORC1 could represent different functional states of the PIKKs.Dimers of Mec1·Ddc2 and Tel1 represent a PIKK in a functional form that exhibits basal kinase activity prior to activation B, inset.on forks , 38. Ddcon forks B locatesm region C. Similaf mTORC1  and TORCf mTORC1 , and mutckpoints . Interesckpoints  also shockpoints D. In mTOnterface , B–D. Itin vitro.A number of the PIKKs were reported to autophosphorylate via residues in the C-terminal domain as a molecular switch for their kinase activity , 62, 63.Our data here suggest a putative model for Mec1 and Tel1. Although dimerization through these HEAT repeats brings the kinase domains of Mec1·Ddc2 in close proximity, perhaps suggesting that activation may require the separation of these domains, in Tel1, the kinase domains are separated by 35 Å. Therefore, activation of these PIKKs may proceed through a more complex mechanism rather than a simple separation of the kinase domains. There are certain mechanistic advantages to maintaining the dimer through the HEAT repeats anchor while allowing possible repositioning and activation of the kinase domains. A dimer could increase the interaction surface with cofactors or support cooperativity to allow a more efficient recruitment. Furthermore, a complete dissociation of dimers to monomers would imply that to deactivate Mec1 or Tel1 or to recycle it for other activities, reassociation into dimers would be required. A tethered dimer could ensure that it can be readily recycled between an inactive and an active state, allowing rapid multiple rounds of controlled activation/deactivation. However, understanding the exact activation mechanisms requires structural and biochemical data on a reconstituted complex involving the PIKK bound to its activator.M. S., V. C. D., P. H. W., E. T., S. H., and D. B. designed and performed the experiments, and M. S., V. C. D., P. H. W., P. M. B., and X. Z. analyzed the data. P. M. B. and X. Z. conceived and designed the study. M. S. and V. C. D. performed the majority of electron microscopy, and M. S. did the antibody labeling studies, while D. B. collected the initial negative stain EM data for the Mec1·Ddc2 complex. P. H. W., E. T., A. V. M., and S. H. purified the proteins and performed biochemical analysis. M. S., V. C. D., X. Z., and P. M. B. wrote the manuscript.


Both results of specificity analysis and thermal stability determination add support for reliability of the following DAS-ELISA with a minimum detection limit of 0.005 μg·mL−1 and a working range 0.010–1.0 μg·mL−1. The linear curve displayed an acceptable correlation coefficient of 0.9976. These results indicated promising applications of nanobodies for detection of Cry1Ac toxin with biotin-streptavidin based DAS-ELISA system.Nanobodies are the smallest natural fragments with useful properties such as high affinity, distinct paratope and high stability, which make them an ideal tool for detecting target antigens. In this study, we generated and characterized nanobodies against the Cry1Ac toxin and applied them in a biotin-streptavidin based double antibodies (nanobodies) sandwich-ELISA (DAS-ELISA) assay. After immunizing a camel with soluble Cry1Ac toxin, a phage displayed library was constructed to generate Nbs against the Cry1Ac toxin. Through successive rounds of affinity bio-panning, four nanobodies with greatest diversity in CDR3 sequences were obtained. After affinity determination and conjugating to HRP, two nanobodies with high affinity which can recognize different epitopes of the same antigen (Cry1Ac) were selected as capture antibody (Nb61) and detection antibody (Nb44). The capture antibody (Nb61) was biotinylated  Bacillus thuringiensis [Bt toxins are known as insecticidal crystal proteins produced by Gram-positive spore-forming soil bacterium ngiensis . They hangiensis ,3. Crystngiensis ,5,6. Howngiensis ,8,9. Prengiensis . Others ngiensis . It is eTo detect GM crops and their products, numerous methods have been developed primarily based on DNA and proteins , such asAs a result of the development of bioengineering technology, single-domain antigen-binding fragments, known as VHHs or nanobodies consist of about 120 amino acids ,19 and hIn this study, we firstly generated and characterized nanobodies with high affinity, specificity and stability against the Cry1Ac of Bt toxins by using phage display technology and other analytical methods. The expressed anti-Cry1Ac nanobodies were purified and modified so as to be applied in a DAS-ELISA assay. This system showed a promising future in detection of Cry1Ac toxin.9 colonies, with an insertion rate of 100% according to PCR analysis by randomly choosing 24 colonies . This procedure is based on the principle that nanobodies resided in supernatant of the cells disrupted by osmotic shock can bind to the corresponding antigen directly ,25. 19 cE. coli WK6 electrocompetent cells respectively from TG1 strain by conventional method [C-terminally His6-tagged proteins was permitted by display vector pMECS. WK6 strain has the ability to suppress the amber stop codon between gene III and VHH gene on pMECS while TG1 does not have the property, which is one of the reasons why the recombinant phagemid need to be transformed from TG1 to WK6 strain without doing subcloning step. SDS-PAGE analysis were performed after purifying soluble Nbs with NI-NTA Superflow Sepharose columns, which showed a band corresponding approximately to 15 kD molecular weight  were transformed into l method . In thisr weight A, demonsDK ranging from 1.80 × 10−8 to 6.89 × 10−9 nM  experiment presented, three of the anti-Cry1Ac Nbs showed weak alkalinity with theoretical isoelectric point (pI) at 7.84–8.66 and one (Nb61) possessed weak acidity with pI at 5.5 B. The af 10−9 nM B. To sumNanobodies were conjugated to horseradish peroxidase (HRP) as signal indicators in color reactions. On the other hand, by taking advantage of these Nb-HRPs, it can be known whether Nbs recognize different epitopes of the same antigen (Cry1Ac). After performing this process, 2 nanobodies (Nb44 and Nb61) against Cry1Ac with high affinity were selected as capture antibody (Nb44) and detection antibody (Nb61), respectively.DK = 6.89 × 10−9 nM), was selected to be labeled with Biotin in vivo. In order to obtain biotinylated nanobody, VHH genes were sub-cloned into plasmid pBAD which contained a Biotin Acceptor Domain (BAD) and then were co-transformed into WK6 cells with plasmid BirA (encoding biotin protein ligase) for the expression of biotin-conjugating Nb (Cry1Ac Nb44-Biotin) which were further purified with Streptavidin-Mutein Matrix and it was produced at a final yield of 1.0 mg·L−1 culture.In this study, the capture antibody was immobilized on wells coated with streptavidin matrix for capturing Cry1Ac in corresponding reagent. Thus, Nb44, with the highest affinity (The specificity of the proposed nanobodies (Nb44 and Nb61) was examined by ELISA A. The reAs shown in As shown in −1. The linear equation was calculated as (1) with an acceptable correlation coefficient of 0.9976 (R2), and the minimum detection limit  sandwich ELISA was shown in ethod in ) was 0.0The measured recoveries of Cry1Ac from the spiked samples are shown in dT primer was obtained from Thermo Scientific . Restriction enzymes Pst I, Not I, Nco I and BstE II were provided from NEB . Mouse anti-HA tag antibody was purchased from Covance . Anti-mouse IgG-alkaline phosphatase, Bis phosphate and His-Select column were purchased from Sigma-aldrich . D-biotin and horseradish peroxidase (HRP) were purchased from Bio Basic Inc. . Streptavidin Mutein Matrix was purchased from Roche . Microtitre plate was purchased from Thermo Scientific NUNC . BeaverNano™ Streptavidin Matrix Coated 96-Well plates were provided by BeaverNano technology company . VCSM13 helper phages, TG1 cells, WK6 cells, plasmid pBAD and plasmid pBirA were obtained from Prof. Serge Muyldermans’s lab . All other reagents were of analytical grade and used as received.Cry toxins this study used were purchased from YouLong Bio. Co. Ltd.  and dissolved in 1 × PBS, stored at −80 °C. Freund’s complete adjuvant and Freund’s incomplete adjuvant were purchased from Sigma-Aldrich . Fast Track 2.0 Kit was provided by Invitrogen  and OligoA healthy young dromedary camel was immunized by weekly injections of soluble and pure antigen (Cry1Ac)  mixed with Freund’s incomplete adjuvant, to stimulate antigen-specific B cells to express nanobodies. In addition, after 6 times, lymphocytes were extracted from 100 mL peripheral blood collected from the dromedary for constructing the library.Pst I and Not I. After being digested by Pst I and Not I restriction enzymes , the amplified second PCR products were unidirectionally ligated into Pst I-, Not I- and Xba I-digested pMECS to recover pMECS-VHH [Escherichia coli TG1 electrocompetent cells at 1.7 kV followed by addition of 1 mL SOC media and shaking at 37 °C for 1 h. Finally, the library was stored in 15% glycerol at −80 °C until use [PBLs  were isolated from an immunized dromedary camel, by density gradient centrifugation with Ficoll-paqueTM plus . Then by using Fast Track 2.0 Kit , total RNA was extracted from the purified PBLs and its concentration was calculated then. The variable regions of heavy-chain immunoglobulins (VHH) were amplified by nested PCR for the purpose of avoiding contaminating with VH genes. PCR conditions were: 1 cycle at 94 °C for 7 min, 30 cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min and finally 1 cycle of 72 °C for 10 min. The secondary PCR amplification was done with templates near 700 base pairs (bp) from the first PCR products and used the same protocol as first PCR but reduced to 17 cycles with primers contained restriction sites ntil use . Meanwhi−1, the second round was 100 mg·mL−1, respectively) in coating buffer  was used as antigen to coat onto microtiter plates of 96-wells at 4 °C overnight, with 0.1 M NaHCO3 (pH 8.2) used as control. Wells were incubated with phages of the displayed library with PBS for 1 h at room temperature after blocking with 0.1% casein in phosphate-buffered saline (PBS) for 2 h. The binding phages were eluted with 100 mM triethylamine for 10 min and then immediately mixed with 1.0 M Tris-HCl (pH 7.4) for the purpose of neutralizing. Fresh exponentially growing culture of E. coli TG1 cells (OD600 = 0.4–0.6) were infected with the eluted phages and incubated at 37 °C in constant temperature incubator (without shaking) for 30 min to allow for optimal infection [VCSM13 was used as helper phages to infect a representative fraction of the VHH library cultured for bio-panning . Cry1Ac nfection . Phage pThe process above represented one round of bio-panning and after 2–4 rounds, the Cry1Ac-specific phages enriched.2PO4 and 0.72 M K2HPO4) containing 100 μg·mL−1 ampicillin. After 3 h of culturing, IPTG (1 mM) was added to induce the expression of nanobodies during overnight growing at 28 °C. Then cells collected were resuspended into 200 μL TES  for shaking 2 h at 4 °C, after which 300 μL TES/4 were added for shaking another 2 h at 4 °C. Next, the supernatant was collected and added into the plate wells which were coated with Cry1Ac toxin (2 μg·mL−1) in advance and for 1 h incubating. Then mouse anti-HA tag antibody and anti-mouse IgG-alkaline phosphatase were added for 1 h incubating successively. Finally, chromogenic solution containing Bis phosphate (pNPP) was added and after minutes of waiting the absorbance in 405 nm can be read with an ELISA reader .PE-ELISA is based on the principle that nanobodies resided in supernatant of the cells disrupted by osmotic shock and can bind to corresponding antigen directly. To obtain positive clones, 95 individual clones were picked randomly from rounds of panning for PE-ELISA. Each of these picked clones were cultured in 1 mL Terrific Broth  and 2 mM MgCl2. Expression of the nanobodies was induced subsequently by adding IPTG to a final concentration of 1 mM when the optical density (OD) was between 0.6 and 0.9 at 600 nm, and cultures were further grown for 16 h with shaking (220 rpm) at 28 °C. Then the cells collected from the 330 mL overnight culture were resuspended into 4 mL TES  for shaking 2 h at 4 °C, after which 8 mL TES/4 were added for shaking another 2 h at 4 °C. The supernatant containing the periplasmic proteins was collected and using His-Select columns , soluble Nb with His-tags were purified by Immobilized Metal Affinity Chromatography (IMAC). His-tagged proteins were eluted with gradient concentration of imidazole (pH 7.0) after washing 5 times with PBS. The purity of the eluted proteins was checked by 15% SDS-PAGE.Nanobodies were expressed by transforming the selected VHH genes in pMECS into ® HT system  as previously described [−1) were immobilized on PlexArray® Nanocapture® Sensor Chips . Then, to take note of the signals, different concentrations of  were injected with a flow rate of 3.0 μL·s−1. Finally, a regeneration step was performed after the affinity binding reaction. Briefly, the sensor chips were regenerated with 0.05 mol·L−1 NaOH for 2 min, and then PBS buffer was injected over the sensor surface for about 5 min. All signals were recorded as sensorgrams, and steps were performed in triplicate at 25 °C.The affinities of selected anti-Cry1Ac Nbs were measured by the kinetic analysis by Surface Plasmon Resonance (SPR) experiment with PlexArrayescribed ,25. Firs4 (0.1 M) was mixed with 200 μL horseradish peroxidase (HRP)  (5 mg/mL) for 30 min incubation at 4 °C, after which an equal volume of ethylene glycol (2.5%) were added and incubated for 30 min at room temperature. Then 1 mL Cry1Ac-specific nanobodies (1 mg·L−1) were added, and after an overnight incubation at 4 °C in the dark, 20 μL Sodium borohydride (5 mg·mL−1) were mixed into it for 3 h at 4 °C and dialyzed into PBS. Nanobodies failed to be conjugated to HRP were removed by ultrafiltration and free HRP were eliminated by saturated ammonium sulfate. Generally, ammonium sulfate precipitation was started by adding the same volume of saturated ammonium sulfate to the mixture dropwise, while mixing it gently. After 1 h at 4 °C, the mixture was centrifuged at 3000 rpm for 30 min and then the precipitate was washed twice by using 50% saturated ammonium sulfate and dissolved in 0.15 M PBS (pH 7.4). Then the solution was dialyzed into PBS (pH 7.4) for the purpose of eliminating ammonium ion, after which the supernatants (namely Nb-HRPs) of the mixture were collected by centrifugation.The purified anti-Cry1Ac Nbs were conjugated to HRP as follows: 100 μL fresh NaIONco I and BstE II as restriction sites and the recombinant plasmid was co-transfected into WK6 competent cells with plasmid pBirA. 50 μM of d-biotin were added into medium 30 min before adding 1 mM IPTG to induce expression of VHH-BAD fusion proteins. By osmotic shock protocol, periplasmic proteins were extracted. Streptavidin Mutein Matrix was used for purifying biotinylated nanobodies (BiNb) eluted with 6 mM D-biotin solution.Genes encoding anti-Cry1Ac VHHs were sub-cloned into plasmid pBAD by using −1 different Cry1 toxins in 0.1 M NaHCO3 (pH 8.2) were coated on plate wells overnight at 4 °C respectively, blocked with 200 mL per well of 1% BSA at room temperature for 2 h. Then Cry1Ac Nb44 and Nb61 (10 μg·mL−1) were added into the plate wells at room temperature for 1 h incubating. Following steps were undertaken as described in PE-ELISA.In order to characterize the specificity of purified Nb44 and Nb61, 4 kinds of different Cry1 toxins  were chosen for ELISA. Firstly, 5 μg·mL−1 of Cry1 Ac toxin. NaHCO3  was the blank control of each sample. 100 μL (2 μg·mL−1) of Cry1Ac Nb44 and Nb61 solution were added into corresponding wells to incubate for 1 h after the ELISA plate was blocked with 1% skim milk for 2 h. The rest of the experiments were the same as that described in PE-ELISA and ELISA for nanobody specificity detection. Meanwhile, these nanobodies (1 mg·mL−1) exposed to different conditions for different length of time were checked by 15% SDS-PAGE.To determine the thermal stability, different samples (Nb44 and Nb61) under different conditions were used for performing ELISA. The activity of Nb44 and Nb61 stored at −20 °C was regarded as 100%. Specifically, Cry1Ac Nb44 and Nb61 diluted in PBS was incubated at 37 °C, 60 °C and 90 °C respectively for 1 h, 2 h and 3 h with the no-treatment group of nanobodies stored at −20 °C being used as control, after which they were introduced to an ELISA plate coated with 5 μg·mL−1 of biotinylated nanobody Cry1Ac Nb61 was added into plate coated with streptavidin matrix and incubated at room temperature for 1 h. Then different concentration gradients of Cry1Ac toxin  were added for 2 h after blocking the plate with 5% BSA for 1 h. Cry1Ac Nb44 conjugated with HRP were diluted with 5% BSA to 1 μg·mL−1 and incubated with Cry1Ac toxin for 1 h. Finally, the chromogenic reaction was measured by automatic micro-plate reader at 450 nm.Based on streptavidin-biotin system, directional double antibodies (nanobodies) sandwich ELISA was performed. 1 μg·mL−1), and then shaken with 1 mL of protein extraction solution containing 0.1 M PBS pH 7.4, containing 0.1% BSA and 0.05% Tween-20. The suspension was centrifuged for 15 min at 10,000 g after shaking at room temperature for 4 h [The new DAS-ELISA method was assessed by performing Cry1Ac toxin determination in corn samples and soybean samples. 1 g of corn samples and soybean samples were spiked with Cry1Ac toxin at five concentration levels (0.05, 0.1, 0.25, 0.5 and 1.0 mg·kg for 4 h . Then thIn conclusion, we have successfully isolated a number of novel nanobodies from a high quality phage display library which have high stability and specific binding affinity to different epitopes of Cry1Ac toxin. Phage display and application of these nanobodies could be a promising approach for the detection of Cry1Ac toxin in agricultural as well as environmental samples. Except for streptavidin-biotin based ELISA, Nbs can be combined with biosensors such as electrochemical impedance spectroscopy and flow injection chemiluminescence, which promise to generate quicker and more sensitive results. By taking advantage of nanobodies, we will put more efforts to develop the immunoassay for sensitive and selective detection in GMOs.


Bispora sp. MEY-1, as the microbial source for glucoamylase gene exploitation.Glucoamylase is an exo-type enzyme that converts starch completely into glucose from the non-reducing ends. To meet the industrial requirements for starch processing, a glucoamylase with excellent thermostability, raw-starch degradation ability and high glucose yield is much needed. In the present study we selected the excellent Carbohydrate-Activity Enzyme (CAZyme) producer, gla15) was cloned from Bispora sp. MEY-1 and successfully expressed in Pichia pastoris with a high yield of 34.1 U/ml. Deduced GLA15 exhibits the highest identity of 64.2% to the glucoamylase from Talaromyces (Rasamsonia) emersonii. Purified recombinant GLA15 was thermophilic and showed the maximum activity at 70°C. The enzyme was stable over a broad pH range (2.2–11.0) and at high temperature up to 70°C. It hydrolyzed the breakages of both α-1,4- and α-1,6-glycosidic linkages in amylopectin, soluble starch, amylose, and maltooligosaccharides, and had capacity to degrade raw starch. TLC and H1-NMR analysis showed that GLA15 is a typical glucoamylase of GH family 15 that releases glucose units from the non-reducing ends of α-glucans. The combination of Bacillus licheniformis amylase and GLA15 hydrolyzed 96.14% of gelatinized maize starch after 6 h incubation, which was about 9% higher than that of the combination with a commercial glucoamylase from Aspergillus niger.A glucoamylase gene (A. niger, GLA15 represents a better candidate for application in the food industry including production of glucose, glucose syrups, and high-fructose corn syrups.GLA15 has a broad pH stability range, high-temperature thermostability, high starch hydrolysis capacity and high expression yield. In comparison with the commercial glucoamylase from  It has two homopolysaccharide types: amylopectin and amylose. Amylopectin is an α-1,4-linked d-glucose by glucoamylase. Currently, Aspergillus niger glucoamylase is widely used in industry. It has the temperature and pH optima at 60–65°C and 4.0–4.5, respectively, and retains only 10% of initial activity after incubation at 70°C for 30 min Mass production of glucose from starch is a two-stage process, involving α-amylase and glucoamylase. In the first step, starch slurry is gelatinized, followed by liquefaction rapidly by a thermostable α-amylase at 95–105°C and pH 6.0–6.5 Clostridium thermohydrosulfuricumClostridium thermosaccharolyticumThermoanaerobacter tengcongensis MB4 SulfolobusThermoplasmaPicrophilus and MethanococcusTalaromycesThermomycesChaetomiumGreat efforts have been attempted to improve the performance of glucoamylase, including screening of glucoamylase-producing thermotolerant and thermophilic organisms Bispora sp. MEY-1 is an excellent Carbohydrate-Activity Enzyme (CAZyme) producer Bispora sp. MEY-1 and expressed the gene product in Pichia pastoris at high level. The recombinant glucoamylase (GLA15) showed better thermostability and pH stability than all other fungal counterparts reported before, and had wide substrate specificity and great hydrolysis capacity.Bispora sp. MEY-1  was cultivated in wheat bran medium Escherichia coli Trans1-T1 and the plasmid pEASY-T3 from TransGen  were used for gene cloning and sequencing. Pichia pastoris GS115 and plasmid pPIC9  were used for gene expression. P. pastoris expression media were prepared as described in the Pichia expression kit manual (Invitrogen).d-maltopentaoside (p-NPG5), amylose, amylopectin, glycogen, soluble starch, dextran and isomaltose were purchased from Sigma-Aldrich . The Genome walking kit and restriction endonucleases were from TaKaRa . T4 DNA ligase and endoglycosidase H (Endo H) were from New England Biolabs . All other chemicals were of analytical grade and commercially available.Maltooligosaccharides, 4-nitrophenyl-α-5′-TGGGGHCGTCCDCARMGNGAYGG-3′) and Gamy-R (5′-CCACTGRTARAYNGCRTCRTA-3′) was designed and used to clone the core region of glucoamylase gene. The PCR products were cloned into the vector pEASY-T3 and sequenced. Based on the known sequences, six specific primers were designed to obtain the 5′ and 3′ flanking regions (data not shown). The gene fragments were assembled and subject to sequence analysis.Based on the conserved motifs WGRPQRDG and YDAV(I/L)YQW of glucoamylases of GH 15, a degenerate primer set Gamy-F  according to the manufacture's instructions. Reverse transcription was carried out with the First Strand cDNA Synthesis Kit . The full-length cDNA of the glucoamylase gene was amplified by PCR primers GAΙF (5′-ATGCAAGTATCAACTTGCCTATTTGCGCTATG-3′) and GAΙR (5′-TTACTGCCAACTATCATTCACCTCAGCAACCCC-3′). The PCR product was cloned into vector pEASY-T3 and sequenced.Total RNA was isolated from the mycelia of GAATTCGCTCCAAGGCAGGAAAATCTGTTACGGCG-3′5′-CTA and GamyR GCGGCCGCTTACTGCCAACTATCATTCACCTCAGCAACCCC-3′5′-GAC . The PCR products were digested with EcoRΙ and NotI, isolated by gel electrophoresis, purified, and cloned into EcoRΙ/NotI digested pPIC9 to construct the recombinant plasmid pPIC9-gla15. The recombinant plasmid was linearized with BglII and transformed into P. pastoris GS115 competent cells by electroporation. Positive transformants were screened based on the glucoamylase activities as described below. The positive transformant with highest glucoamylase activity was selected for further fermentation in 1 l conical flasks containing 400 mL of BMGY at 30°C with an agitation rate of 250 rpm for 48 h. Cells were harvested by centrifugation at 5,000× g, 4°C for 10 min and resuspended in 200 mL of BMMY containing 0.5% (v/v) methanol for induction at 30°C. Culture samples were taken every 24 h for enzyme activity assay.The gene fragment coding for the mature protein without the signal peptide sequence was amplified by PCR with the expression primers GamyF g, 4°C, 10 min), and then concentrated by ultrafiltration using the vivaflow 50 ultrafiltration membrane with a molecular weight cut-off of 10 kDa . The supernatant was loaded onto a HiTrap Desalting column pre-equilibrated with buffer A . The crude enzyme was collected and applied to a HiTrap SP XL column pre-equilibrated with buffer A. Proteins were eluted with a linear gradient of 0–0.7 M NaCl in the same buffer. Fractions containing glucoamylase activity were collected and confirmed by SDS-PAGE.Fermentation broth was collected by centrifugation , and 10 µl of appropriately diluted enzyme. After incubation at 70°C for 10 min, the reaction was terminated by boiling at 100°C for 5 min. The mixture was then centrifuged at 12,000× g, 4°C for 2 min, and 140 µl of the supernatant was combined with 2.1 ml of a commercial reagent consisting of glucose oxidase and catalase  at 37°C for 30 min to dye. The absorption was measured at 520 nm, and the enzyme activity was calculated using glucose as a standard. One unit of glucoamylase activity was defined as the amount of enzyme that liberates 1 µmol of glucose per min under assay conditions .Glucoamylase activity was determined with maltose as the substrate. The reaction mixture contained 140 µl of substrate solution  and 0.10 U of GLA15 were incubated at 60°C for various durations. Samples were collected, boiled for 5 min to terminate the reaction, and spotted on the TLC plate . n-butanol, acetic acid and water at the ratio of 2∶1∶1 (vol/vol/vol) was used as the solvent. The plate was air dried and soaked in ethanol containing 5% concentrated H2SO4. The plate was then dried and placed in an oven at 110°C for 5 min.Aliquots of 1H-NMR spectrum was observed with a Bruker AVANCE DRX-500 NMR spectrophotometer .To identify the anomeric configuration of the hydrolysis product by GLA15 hydrolysis, 300 µl of 1% (wt/vol) maltotriose was incubated with 0.13 U of GLA15 for different intervals. The Bacillus licheniformis  and incubation at 90°C for 2 h with agitation (150 rpm). Then the pH was adjusted to 4.0, and the mixture was further incubated at 70°C with addition of 10 U of GLA15 for 2, 4, 6 and 8 h. The commercial glucoamylase CGA from A. niger (Longda) was treated as the control at its temperature optimum (60°C). Each experiment had triplicate, and the glucose content was determined as described above. The saccharification efficiency of GLA15 (%) was calculated as the amount of glucose produced×100/the amount of starch.Complete hydrolysis of starch involves liquefaction and saccharification steps, which are catalyzed by α-amylase and glucoamylase, respectively Glucose solution (30% wt/vol) in 100 mM sodium acetate buffer (pH 4.0) was incubated with various amounts of GLA15  at 60°C for 150 h with agitation of 100 rpm. The reverse reaction products were analyzed using high performance anion exchange chromatography-pulsed amperometric detector (HPAEC-PAD). The samples were eluted in 100 mM NaOH for 15 min.Bispora sp. MEY-1 glucoamylase gene (gla15) was deposited in the GenBank database under accession number KJ866875.The nucleotide sequence for the cDNA of Bispora sp. MEY-1 with degenerate primers Gamy-F and Gamy-R. The full-length 2001-bp gla15 was obtained by TAIL-PCR and sequence assembly. The cDNA of gla15 was then obtained via RT-PCR. The cDNA of gla15 was 1827 bp in length, and encoded 608 amino acids. SignalP  A gene fragment of 704 bp was cloned from the genome of gla15 shows the highest identity and similarity of 64.2% with a glucoamylase from Talaromyces (Rasamsonia) emersoniiThe deduced amino acid sequence of P. pastoris GS115. After 60 h growth in conical flasks at 30°C, the glucoamylase activity and protein content in the culture supernatant were determined to be 34.1 U/ml (pH 4.0 and 70°C for 10 min with soluble starch as the substrate) and 0.121 mg/ml, respectively. Recombinant GLA15 was purified to electrophoretic homogeneity with a yield of about 27.4%. SDS-PAGE analysis showed that the molecular mass of GLA15 was about 80 kDa .The cDNA fragment of mature GLA15 without the signal peptide-coding sequence was amplified by PCR and cloned into the pPIC9 vector for heterologous expression in t 80 kDa , which iThe pH optimum of GLA15 for the glucoamylase activity was pH 4.0 at 70°C, and more than 60% of the maximum activity was retained at pH 2.0–5.0 . GLA15 w2+, Ag+, Hg2+ and SDS at the concentration of 5 mM .GLA15 hydrolyzed maltooligosaccharides effectively as well as maltopolysacchardes. With maltooligosaccharides (G3–G7) as the substrates , the enz1H-NMR techniques were employed to determine the cleavage mode of GLA15 and glucose configuration, respectively. As shown in p-NPG1–3 and glucose were observed in TLC plate when incubated p-NPG5 and GLA15 at 60°C for 10 min. p-NPG4 might be mixed with glucose and thus undetectable as reported on the glucoamylase of Sulfolobus solfataricusp-NPG5 was completely degraded into glucose and p-NPG1. The result indicated that GLA15 mainly attacked the α-1,4-glycosidic linkages from the non-reducing end to release glucose as the final products. 1H-NMR spectrum  and a little proportion of maltose . The resA. niger or closely related species are the most widely used in the saccharification stage of starch industry because of the beneficial properties for industrial bioprocesses such as high productivity and the ability to convert starch completely to glucose A. niger glucoamylase only retained 10% activity after incubation at 70°C for 30 min T. emersonii glucoamylase only retained stable at 65°C C. thermophilum lost 20% activity after incubation at 70°C for 1 h A. niger glucoamylase with temperature optimum at 75°C was only stable at 40°C A. niger and Aspergillus niveus (pH 4.0–9.5) At present, the glucoamylases from P. pastoris with a high yield of 34.1 U/ml, significantly higher than that of glucoamylases from C. thermosaccharolyticum (1.42 U/ml) Sulfolobus solfataricus (2.08 U/ml) Thermomyces lanuginosus (7.3 U/ml) Glucoamylases are biotechnologically very important as they are used industrially in large amounts. Researchers have identified and characterized various glucoamylases from archaea A. niger glucoamylase (87.37%). This high conversion rate might be ascribed to the relatively broader substrate specificity, including maltooligosaccharides, polysaccharides, and even raw starch. Most fungal glucoamylases have preference for large molecules, such as starch, amylopectin and glycogen, rather than small maltooligosaccharides d-glucose from the non-reducing end of the substrate, while α-glucosidase releases α-d-glucose Aspergillus awamori glucoamylase, equivalent to Pro58 in GLA15, is another key residue related to substrate binding. Its substitution with Pro not only reduced the conformational unfolding, but also favored the α-1,4 binding mode at the active site A. awamori glucoamylase changed the conformation around the catalytic cavity by making helix-4 more stable, which disfavored its binding to α-1,6-linked substrate and resulted in higher glucose yields A. awamori glucoamylase for isomaltose hydrolysis significantly increased when substituted Tyr175 (Phe203 in GLA) with Phe Industrial saccharification typically results in 96% maximum conversion to glucose To be economical, high concentrations of starch (up to 30%) are generally used in the industrial process, which may result in high concentrations of glucose and formation of glucose reversion products Table S1The product compositions of maltooligosaccharides and isomaltose hydrolyzed by GLA15.(DOC)Click here for additional data file.


Halorubrum H3 strain was isolated from a water and silt sample from Burlinskoye Lake . According to 16S rRNA sequences, this strain is most closely related to Halorubrum saccharovorum. The completely sequenced and annotated genome is 3,282,373 bp and contains 3,237 genes.A  Halorubrum H3 strain was isolated from a water and silt sample taken from Burlinskoye . The water temperature was 20°С at the time of sampling.The studied Halorubrum includes extremely halophilic archaeal species that are widely distributed in high-saline environments and are a major component of the microbial community of hypersaline environments (3 to 5 M) (–The genus  to 5 M) . The gen to 5 M) –6.Halorubrum H3 culture was cultivated in liquid medium containing 1% tryptone, 0.5% yeast extract, and 3.5 M NaCl. Eight milliliters of cell culture was pelleted by centrifugation and resuspended in 75 µl of H2O by intense pipetting. DNA was isolated using the DNA purification kit (Fermentas). The Ion PGM Template OT2 400 kit was used to create libraries for genome sequencing. Genome sequencing was performed on an Ion Torrent platform (Applied Biosystems) with Ion 316 chip using the Ion PGM sequencing 400 kit at the Siberian Branch of the Russian Academy of Sciences (SBRAS) Sequencing Center.De novo assembly of short reads into contigs was performed using SPAdes 3.1.0. Contigs <1,000 bp were deleted. A total of 189 contigs yielded a genome sequence 3,282,373 bp long, with a G+C content of 67.57%. Open reading frame (ORF) prediction and automatic annotation were performed using the NCBI Prokaryotic Genome Annotation Pipeline (http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html). The complete genome sequence contains 3,237 genes, 2,337 coding sequences (CDSs), 3 rRNAs , 46 tRNAs, and 1 noncoding RNA (ncRNA).Halorubrum type strains were found using the StrainInfo (http://www.straininfo.net) and GenBank (http://www.ncbi.nlm.nih.gov/nucleotide) databases. According to phylogenetic analysis, the Halorubrum H3 strain is most closely related to Halorubrum saccharovorum.Phylogenetic analysis was performed using 16S rRNA sequences with the unweighted pair group method using average linkages (UPGMA) algorithm implemented in MEGA version 6. The 16S rRNA sequences of Halorubrum H3 has been deposited in GenBank under the accession number JNFH00000000. The 189 contigs have been deposited under the accession numbers JNFH02000001 to JNFH02000189.The draft genome sequence for