SDS PAGE

Sub-cellular fractionation of THP-1 macrophages was performed after lysis with hypotonic buffer. THP-1 macrophages after appropriate treatment were allowed to swell for 10 min in hypotonic buffer (1 0 mM NaCl, 1.5 mM MgCh, 10 mM Tris-HCl, pH 7 .5) followed by homogenization with 50 strokes using a Dounce homogenizer. More than 90% cellular lysis was ensured by visualizing under a light microscope, and immediately after lysis, the mitochondrial membranes were stabilized by addition of 2.5x mitochondrial stabilization buffer (525 mM mannitol, 175 mM sucrose, 12.5 mM Tris-HCl, 2.5 mM EDTA, pH 7.5) to a final concentration of 1x. The homogenate was centrifuged at 1300 x g for 15 min to isolate the nuclear fraction. The post-nuclear fraction was further centrifuged at 17,000 x g for 15 min in an ultracentrifuge (Beckman Optima XL-1 OOK ultracentrifuge) to isolate the mitochondria. The post-mitochondrial supernatant was centrifuged at 100,000 x g for 1 h to obtain the membranous fraction as a pellet and the supernatant obtained was the cytosol. The homogeneity of the obtained fractions was determined by probing for fraction specific proteins by Western blotting

Sub-cellular fractionation

Heterologus expression of various proteins was done in BL21 (A.DE3) strain of E. coli. Bacterial cells were transformed with the desired construct and grown in Super broth (pH 7.2) containing 100 J.lg/ml ampicillin, at 37 °C with continous shaking in a gyratory shaker at 225 rpm. The cultures were induced, at ~oo of 2.0, with I mM IPTG, and harvested two hours later by centrifugation at 4000 g, at 4 °C, for 20 min .. The recombinant proteins were purified from the inclusion bodies using the procedure described by Buchner et al ( 1992). The total cell pellet from a liter of culture was homogenized in 180 ml of inclusion bodies washing buffer containing 8 ml of freshly prepared lysozyme solution (5 mg/ml). The suspension was incubated at room temperature for 1 hr with intermittent shaking. Added 20 ml each of 5M NaCI and 25% Triton X-100 were added to the suspension and incubated at room temperature for 30 min. with vigorous shaking. The suspension was centrifuged at 13,000 g at 4 °C, for 50 min. and the pellet was resuspended, in the washing buffer containing 1% Triton X-1 00, using a polytron homogenizer and centrifuged at 13,000 g for 50 min. The pellet was washed four times with washing buffer without Triton . X-100. The pellet containing inclusion bodies was solubilized in 6 M guanidine hydrochloride by incubating for 2 hours at room temperature. The solubilized protein was centrifuged at 50,000 g, at 4 °C, for 30 min. and the protein concem.ration was adjusted to 10 mg/ml in the supernatant with 6 M guanidine hydrochloride. The denatured protein thus obtained was reduced by adding 65 mM dithioerythritol and incubated at room temperature for 2 h. To renature, the protein was diluted 1 00-fold in the refolding buffer and incubated at 10 °C for 48 h without stirring or shaking. Renatured material, after dialysis in 20 mM MES buffer, pH 5.0 containing 100 mM urea, was loaded on a S-Sepharose column, and the protein bound to the column was eluted with a 0-1 M NaCI gradient in 20 mM MES on an FPLC system (Pharmacia). The fractions containing the desired protein were pooled and concentrated, and the protein was further purified to homogeneity by gel filtration chromatography on a TSK 3000 column (LKB) in PBS, pH 7.4.

Isolation and Purification of Proteins from the Inclusion Bodies

Sf9 cells infected with AcNPV, VI, V2, V3, or V4 were harvested 72 h pi, washed twice with 10 mM PBS, air dried on a slide and fixed in chilled methanol for 15 min. Cells were incubated with MA-451 culture supernatant and N-terminal anti-peptide serum ( 1 :500) at 37°C for 1 h, washed with PBS and further incubated at 37oc for 1 h with I :50 dilution of anti-mouse FITC or I :2000 anti-rabbit FITC. Slides were washed extensively, mounted in 90% glycerol in PBS (50 mM, pH 7.4) and examined under Optiphot fluorescent microscope (Nikon, Tokyo, Japan).

Immunocytochemical Localization of Recombinant Proteins in Infected Cells

lysed directly in 1. 5 ml of solution D ( 4 M guanidium thiocyanate, 25 rnM sodium citrate, pH 7.0, 0.5 % sarcosyl and 0.1 M 2-mercaptoethanol ) . For every 2 ml of the lysate, 0.2 ml of chloroform was added, followed by vigorous mixing for 15 seconds, and incubation on ice for 15 minutes. The lysate was spun at 12, OOOg, at 4 °c for 15 mins. , and the aqueous phase transferred to another tube. RNA was precipitated with an equal volume of isopropanol and incubation at -2o0c for 45 mins. The samples were then spun at 12,000g for 15 mins. at 4°c, and the supernate discarded. The RNA pellet was washed twice with 75 % ethanol. Finally, the pellet was dried briefly under vacuum for 10 -15 mins. and dissolved in 0.5 % SDS. All chemicals and glassware used for handling RNA were treated with diethylpyrocarbonate ( DEPC ) .

Total RNA was isolated from cultured mammalian cells by the method of Chornczynski and Sacchi ( 1987 ), with slight modifications. Briefly, cells from a 3.5 ern petri-dish were

Isolation of RNA

All computer software facilities were provided by the NII computer centre.

Computer software.

stranded DNA. The reaction was carried out at 37°C for 1 h. The reaction mixture contained 100 ng Bgl II digested VR1020 vector, SAP (0.5 U) and 1 fll lOX SAP buffer (20 mM Tris-HCl, pH 8.0, 10 mM MgCh) in 10 f.!l oftotal reaction volume. The reaction was stopped by inactivating the enzyme at 65°C for 15 min. The digested bmZP1 eDNA was ligated with SAP treated VR1 020 at vector : insert ratio of 1:10 in a 10 fll reaction volume for 16 h at l6°C. The reaction mixture contained 10 ng VR1020 vector, 26 ng bmZPl insert, 1 fll lOX ligase quffer (30 mM Tris-HCl, pH 7.8, 10 mM MgCh, 10 mM DTT and 1 mM ATP), lfll T4 DNA ligase (20 U) in a total reaction volume of 10 fll. The ligation product was used for transformation of DH5a competent cells as described previously. Transformants were selected on LB plates containing 50 f.!g/ml Kanamycin (Kan). Similarly, the inserts corresponding to dZP3, rG and dZP3-rG fusion were digested with Bgl II restriction enzyme, gel purified and cloned in VR1020 vector, except that the ligation product of dZP3-rG fusion with VR1020 was transformed into JM109 competent cells

The insert corresponding to bmZP1 was released from the pPCR-Script-bmZPl clone by Bgl II restriction and purified on the agarose gel. VR1020 vector was similarly digested and gel purified. To prevent self-ligation, the digested vector was treated with Shrimp Alkaline Phosphatase (SAP), which removes 5'-phosphate from the termini of double

Cloning in VRl 020 mammalian expression vector

Meancorpuscularhaemoglobinconcentration(MCHC)istheaverageHbconcentrationperunitvolume(100)ofpackedredcells(W/V).Henceitisexpresseding/1whichisthesameaspercent(%).ItiscalculatedbythefollowingformulaHbMCHC=—......x100(g/dl)PCV

MCHC

MeancellVolume(MCV).Itisexpressedinfentolitres(1fentolitreorflisequivalentto10'151)andcalculatedby thefollowingformula:PCVMCV=.....................x10(fl)RBC8.10.6.2.MCHMeancellhaemoglobin(MCH)=AverageweightofHbinanerythrocyte.Itisexpressedinpicograms(pg)whichisequivalentto10"12g.Itiscalculatedbythefollowingformula:HbMCH=-----------------x10(ppg)RBC

MCV

RedBloodcellsindices

micro-haematocrittubewasfilledto100mmwithanticoagulatedblood.Oneendofthetubewassealedwithsealingwaxandthetubewasthenkeptinaverticalpositioninaglassbeakerstuffedwithcotton.Afteronehour,lengthoftheplasmacolumnwasmeasuredwitha rulergraduatedin0.5mm.

ESRwasdeterminedbythemicromethodbecausethequantityofbloodavailablefromindividualfishwasinsufficienttoadoptanymacromethod.Anon-heparinised

ErythrocytesedimentationRate(ESR)

BloodwascollectedfromtheheartbycardiacpunctureusinganRBCpipette.ItwasdilutedwithHayem’sfluidintheratioof1:200.Thecontentswereshakenwell.AdropofthedilutedbloodwasplacedinaNeubauerdoublehaemocytometer(Germany)countingchamberandtheredbloodcellcountpercubicmmwascalculated

Redbloodcellcount(RBC

Thepackedcellvolumeorhaematocritisthevolumeoccupiedbythepackedredcells,afteravolumeofanticoagulatedvenousbloodisfullycentrifuged.Thevolumeofpackedcellisexpressedasapercentageoftheoriginalvolumeoftheblood.ThePCVisusedtoestimatehaematologicalindices,includingthemeancellhaemoglobinconcentration(MCHC)andmeancorpuscularvolume(MCV).PCVdetermination followedthemethodsofBlaxhallandDaisley(1973).Thehaematocritvaluewasdeterminedbycentrifuging(3000rpm)aknownvolume ofincoagulantbloodkeptinWintrobe’stubes

PackedCellVolume(PCV)orHaematocrit(Ht)

HaemoglobinwasdeterminedbySahlimethod.HaemoglobinisconvertedtoacidhaematinbytheactionofHC1.Theacidhaematinsolutionisfurtherdilutedwiththeaciduntilitscolormatchesexactlythatofthepermanentstandardofthecomparatorblock.TheHbconcentrationisreaddirectlyfromthecalibrationcurve.

Haemoglobin(Hb)determination

BloodwastakenbyheartpunctureusingMS222astheanaesthetic.Nofishwasusedmorethanonce.

CollectionofBlood

HaematologicalAnalysis

The cells were assayed for Luciferase gene expression using Luciferase Assay kit (Promega, U.S.A.). After transfection, the cells were washed twice with PBS and then lysed by adding reporter lysis buffer provided in the kit. The cell lysate was collected from individual wells in eppendorf tubes, the cells were twice freeze-thawed in liquid N2 and then centrifuged at 13,000 rpm for 10 min at 40C. The supernatant was transferred to a fresh tube. 20¢ of cell extract was mixed with lOOp! of luciferase assay reagent that was kept at room temparature. The activity was determined using a luminometer (Packard lumicount, U.S.A.

Luciferase assay

normalized to the optical density at day 0 for the appropriate cell type. Growth curve was determined twice

Growth curves were prepared for various cell lines using the modified method adopted by Serrano et al, 1997. Briefly, 10, 000 cells were seeded in a 24 well plate in quadruples. At the indicated times, cells were washed once with PBS and fixed in 10% formalin for 20 minutes and rinsed with distilled water. Cells were stained with 0.05% crystal violet for 30 minutes, rinsed extensively and dried. Cell associated dye was extracted with 1.0ml acetic acid. Aliquots were diluted 1:4 with water and transferred to 96 well microtitre plates and the optical density at 590nm determined. Values were

Growth Curves:

Inverse PCR is a technique to amplify unknown regions flanking the site of transposon insertion using the primers designed from the known sequence from one end of the transposon element. Genomic DNA was digested with a 4-base recognition restriction enzyme, Sau3A1 followed by intramolecular ligation set up at high dilutions. These ligated molecules were then used as templates for the PCR performed with a pair of divergently-oriented primers designed from one end of the transposon named AH1-AH2. The PCR product thus obtained was sequenced with the same set of primers to identify the junction sequence at the site of transposon insertion and hence the identity of the gene disrupted in each case. Typical PCR conditions used were as follows:- Annealing 55°C 2 min Elongation 72°C (1 min/kb of DNA template to be amplified) Denaturation 95°C 2 min After 30 cycles of PCR, the final elongation step was carried out again for 10 min at 72°C

Inverse PCR

To quantitate the P1 phage lysate preparations, titration was done using a P1-sensitive indicator strain such as MG1655. 100 μl each of serial dilutions of the phage (typically 10−5, 10−6) were mixed with 0.1 ml of the fresh culture grown in Z-broth. After 15 min adsorption at 37°C without shaking, each mixture was added in a soft agar overlay to Z agar plates, and incubated overnight at 37°C. The phage titre was calculated from the number of plaques obtained on the plates

To 0.3 ml of infection mixture, 10 ml of Z-broth was added and incubated at 37°C with slow shaking until growth followed by the visible lysis of the culture occurred (in ~ 4-6 h). The lysate was treated with 1 ml of chloroform, centrifuged and the clear lysate was stored at 4°C with chloroform

Broth method

0.3 ml of overnight culture of the donor strain in Z-broth was mixed with 107 plaque forming units (pfu) of a stock P1 lysate prepared on strain MG1655. Adsorption was allowed to occur at 37°C for 15 min and the lysate was prepared in the following ways

Phage P1 lysate preparation

Rhod-2 acetoxymethyl ester is a fluorescent long wavelength calcium indicator, where the AM ester forms are cationic, resulting in a potential driven uptake into the mitochondria making them selective detectors of mitochondrial calcium. Log phase cultures were taken and dead cells pelleted at 129 x g for 5 min at RT. The live cells were washed twice with Kreb' s buffer (118mM Sodium chloride, 5.4mM Potassium chloride, 1.2mM Magnesium chloride, 1.2mM Potassium dihydrogen phosphate, 25mM Sodium hydrogen phosphate, llmM glucose, 1.5mM Calcium chloride, pH 7.4) by centrifugation at 1258 x g for 5 min to wash off all traces of medium and FBS. Washed cells were loaded with 1:1 mixture of Rhod -2 AM (1p.g/p.L stock solution prepared in DMSO) and 20%w/v Pluronic F127 for 1h at RT in the dark with shaking. Excess dye was removed by one wash with Kreb' s buffer followed by incubation of cells at RT for a further 30min for complete hydrolysis of the dye trapped in the mitochondria. Fluorescence intensities of the stained cells were measured fluorimetrically at excitation of 530nm and emission of 576nm or alternatively acquired by flow cytometer.

Assay for measuring mitochondrial calcium

The DNA fragments eluted from the agarose gel or purified PCR products were cloned into pGEM-T easy vector which allows efficient sequencing using the common sequencing primers T7 and SP6. SOng of the vector was used with 1J..lL of T4 DNA ligase in a 10J..lL reaction volume. The reaction was allowed to proceed at 4 °C for 16h followed by transformation into DHS-a strain of E coli following standard protocols. The transformed cells were plated onto LB-agar plates containing appropriate ampicillin (100J..lg/mL) and blue-white selection reagent (40J..lL/plate). The plate was incubated at 37°C for 12 hrs, following which white colonies were picked up for screening for the presence of the gene of interest.

Sub-cloning of PCR products into pGEM-TEasy Vecto

Polyphosphates from yeast cells were extracted with phenol-chloroform solutionas described previously(Neefand Kladde,2003). Cells grown eitherovernight or to logarithmic phase in YPDmediumwere harvested by centrifugation at 1,700 rpm for 5 min. Cells were washed with 10 ml sterile MQ water and resuspendedin ice-cold 500 μl20%trichloro acetic acid (TCA) solution to the final cell densityof 100 OD. Cell suspension was transferred toa1.5 ml microcentrifuge tube. After incubation at room temperature for 5 min, cells were harvested by centrifugation at 12,000 g for 10 minat 4 ̊C and resuspendedin 250-350 μlpolyphosphate extraction buffer.Equal volume of phenol-chloroform (25:24) was addedtothe microcentrifuge tube and aqueous phase was extracted by centrifugation at 12,000 g for 8 minat room temperature. Top aqueous layer was collected with a 200 μl tip. Aqueous layer extraction was repeated once more after removal of DNA with chloroform.After centrifugation,aqueous phasecontaining RNA and polyphosphates wascollected,RNA was quantified at A260nmandstored at -20 ̊C

Polyphosphate extraction

hybridizations from biological replicates for each sample. Data was extracted with Feature Extraction software v 10.5 (Agilent) and normalizedwith GeneSpring GX v 11.0.1 (Agilent) software using the recommended Percentile shift Normalization to 75th percentile. Raw Data sets for this study are available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=

Log-phase wild-type and Cgyps1∆cells were grown in YNB and YNB-pH 2.0 medium. After 1 h incubation, yeast cells were collected, washed and were stored in RNAlater at -80°C. These frozen samples were sent to Genotypic Technology Ltd., Bangalore(http://www.genotypic.co.in) whichprovides services of global gene analysis on Agilent platform. A 8x15k GE array comprised of 60mer oligonucleotidesfor a total of C. glabrata5503 genes was used wherein average number of replicates for each probe was three. Labeling was done in single color and data is the average of two

Microarray analysis

For RNA experiments, all solutions were prepared in RNase free diethylpyrocarbonate (DEPC) water. Microcentrifuge tubes and tips used for RNAworkwere autoclaved twice and driedat 70 ̊C for overnight before use. Non-autoclavable plastic items were wiped with Ambion RNAseZap to remove RNAse contamination, if any. RNA was extracted from C.glabrata cells usingacid phenol extraction method. C. glabratacells were harvested at 2,500g for 5 minat 4 ̊C, resuspended in 1 ml ice-cold DEPC water and were transferred toa2 ml microcentrifuge tube. Cells were spun down at 6,000g for 3 minat 4 ̊C and resuspended in 350 μl AE solution. Next, 50 μl SDS and 400 μl acid phenol(pH 4.5)solutions were added to thetube and mixed well by vortexing. The tube wasincubated at 65 ̊C for 15 min with continuous mixing. After incubation, tube was kept on ice for 5 minand centrifuged at 7,500g for 5 minat 4 ̊C. Aqueous phase was transferred toa new 1.5 ml microcentrifuge tube and RNAwasextracted with an equal volume of chloroform. Total RNA was precipitated at room temperature with 1/10thvolume of 3 M sodium acetate (pH 5.2) and 2.5 volumesof chilledabsolute ethanol for 20 min. Precipitated RNA was collected by centrifugation at 7,500g for 5 minat 4 ̊C. RNA pellet was washed with chilled 70% ethanol and resuspended in 50 μl nuclease-free water. RNA concentration was determined by measuring absorbance at 260 nm. Quality of extracted RNA was examined by gel electrophoresis on 0.8% agarose gel prepared in DEPC-treated TAE buffer

RNA extraction

Alipophilic styryl dye,FM4-64,is a vital stain which istakenupby cells viaendocytosis through plasma membrane(Vida and Emr, 1995). Therefore, it fluorescesonly in live cells. Importantly, neitherfixed cells canbe stained with FM 4-64norcells canbe fixed afterFM 4-64staining. For vacuole staining, single colony of the test strain grown onYPD plate was inoculated in 10 ml YPD medium for overnight. 100 μlovernight culture was inoculated in fresh YPD medium and incubated at 30ºC for 3 hto obtain log-phase cells. C. glabratacells from 1 ml log-phase culture were harvested at 4,000 rpm for 5 minin a table top centrifuge. Supernatant was aspirated out,cells were resuspended in 50 μl YPD medium and 1 μl FM 4-64 (16 μM final concentration) was added.C. glabratacells were incubated in a 30ºC water bath for 30 min. 1 mlYPD medium was added and cells were harvested at 4,000 rpm for 5 minin a table-top centrifuge. After discarding supernatant,C. glabratacells were washed with fresh YPD medium and resuspended in 1 ml YPD medium. C. glabratacells were incubated at 30ºC for 90 min, washed with 1 mlsterile water and were resuspended in 50 μl YNB medium. Labeled C. glabratacells were observed underfluorescence microscope in red filter(730nm)

Stainingof C. glabratavacuoleswith FM4-64

DNA staining solution

Fixative

For cell cycle analysis by flow cytometry

Benzamidine

Immunofluorescence assay was carried out as described by Bhattacharyyaet al., 2010.Adherent cells weregrown either on cover slips. After treatment, cells were fixed with 3.7% paraformaldehyde solution in PBS for 15 min and permeabilised with 0.5% Triton X-100 at room temperature for 10 min, followed by blockingin PBS containing 2% BSA for 1h. Post blocking, cells were incubated with a primary antibody in PBS (1:200 to 1:500) for 2 h. After washing, cells were incubated with fluorescent-conjugated secondary antibodyin PBS(Alexa Fluor 488 or 594 goat anti-rabbit or antii-mouse, 1:1000) for 30 min. After final wash with PBS, nuclei were counterstained with DAPI containing mounting medium (Vectashield, USA). All the steps were performed at room temperature, unless otherwise stated. Images were obtained using either the laser scanning confocal LSM510 (Carl Zeiss, Oberkochen, Germany) or fluorescence inverted (Olympus 1X51, Tokyo, Japan) microscopes

Immunofluorescence

After the RNA samples were resolved, the gel was placed in a tray and incubated for 5-10 min in DEPC treated water followed by 10X SSC buffer. A nylon membrane (N+Hybond, GE Life Sciences) was cut to the size of the gel and it was rinsed in 10X SSC buffer for 5 min. In the same tray, 10X SSC buffer was filled and a gel running boat (15 cm length) was placed in an inverted position. Whatmannumber 3 filter paper (wick) was cut to an appropriate size and placed on the inverted boat in such a way that the longer edges of the paper should touch the buffer. The gel was placed upside down on the wick and care was taken not to allow any air bubbles between the gel and wick. On top of the gel, three Whatman number 3 papers that were cut to the size of gel were placed by avoiding the air bubbles. A bundle of blotting sheets was placed on top of this, on which appropriate weightwas placed and was allowed the transfer to take place for 15 –16 h

Capillary transfer of RNA on to the membrane

A bacterial clone expressing the recombinant protein was grown overnight in 5ml of LB medium with 50ug/ml ampicillinand the next day culture was transferred into 500ml of LB medium with 50 ug/ml ampicillin. Bacterial culture was allowed to grow at 37°C until reached an OD600 of 0.6-0.8 and then induced with IPTG (1 mM) for 4 h at 37°Cor overnight at 16°C to induce fusion protein expression. Subsequently, bacterial culture was centrifuged at 4000 RPM for 10 minutes (At this point bacterial cell pellets can be stored at -20° C for later use). The bacterial cell pellet was washed once with PBS and resuspended in 10ml of lysis buffer (1X NETN and protease inhibitors) followed by sonication (45 sec. pulse was given three times). The cell lysate was centrifuged for 10 minutes at 14000 RPM. The supernatant containing the GST fusion proteins was added to the 200μl of 50% GST/MBP beads. After 2 hours of incubation at 4°C, beads were washed three times with 1 ml lysis buffer. Purified proteins were eluted by using GST/MBP elution buffer. The recombinant proteins were analyzed by SDS-PAGE followed by either Coomassie staining orwestern blotting

GST/MBP recombinant protein purification from bacteria

Intracellular iron content in different Xanthomonas oryzaepv. oryzicolastrains was measured by using atomic absorption spectroscopy as described previously with few modifications (Velayudhan et al., 2000). For estimation of intracellular iron, different strains of Xanthomonas oryzaepv. oryzicolawere grown overnight in 3 ml PS media with appropriate antibiotics for differentially marked strains. 0.2% of the overnight grown culture was inoculated in 250 ml PS medium alone or PS plus 2, 2’-dipyridyl for iron stravation, and grown to an OD600 of 1.2. Cells were then pelleted down by centrifuging at 7000 g for 10 min, and washed twice with phosphate buffer saline (PBS). After washing, cells were lyophilized, and their dry weights were determined. Lyophilized cells were then dissolved in 30% nitric acid at 80ºC for 12 h and diluted 10-fold with miliQ water. Iron content was determined by atomicabsorption spectroscopy using ICP-OES (JY 2000 sequential Inductively Coupled Plasma Optical Emisson spectrometer,Jobin Yvon, Horiba, France). Iron level was quantified against aqueous standard of iron traceable to NIST (National institute of standards and technology, India)

Estimation of intracellular iron content

confirmed through PCR (by using primers SC11 and SC10) and squencing. Double mutant was complemented for DSF production by cloning whole rpfFgene of Xoo, cloned in HindIII and EcoRI sites of pHM1 (a broad host range vector for Xanthomonas) to get pSC6 plasmid. The resultant pSC6 plasmid was introduced into double mutant by electroporation

To obtain the insertional nonpolar mutant in the motA (encodes flagellar motor stator protein)andfliC (flagellin)genes, a 321 bp internal fragment of the motAgene and a 450 bp internal fragment of fliC containing the EcoRI and XbaI site were amplified using respective primer listed in Table 2.2. These fragments were cloned in pk18mob suicide vector, in which the lacZpromoter drives the expression of downstream gene (Schäfer et al., 1994; Windgassen et al., 2000), to obtain pRR1 and pRR2,respectively (Table 2.2). The resulting plasmid (pRR1& pRR2) was introduced into XooBXO43 strain by electroporation. Single Kmrrecombinants were selected on PSA plate containing kanamycin. Insertion of the pK18mob vector in motA andfliCgene was confirmed with PCR and sequencing. To further confirm the mutation in the flagellar genes, we did swimming motility assay on 0.1% peptone-sucrose agar (PSA). Swimming plate assay indicated that both motA and fliCmutant of Xoowas deficient in motility. Further, to obtain motAand fliC insertional knock out mutants in rpfFbackground, we cloned spectinomycin cassette obtained from pUC1318Ω plasmid, into the HindIII site of pRR1 and pRR2 plasmid to obtain pRR3 and pRR4. The resulting plasmid (pRR3& pRR4) were transformed in rpfF. Single specrrecombinants were selected on PSA plate containing kanamycin and spectinomycin. Insertion of the vector was further confirmed by PCR and sequencing. T2SSrpfFdouble mutant was constructed by transforming the plasmid with rpfF::Tn7Kanamycin cassette in the T2SS (xpsF) mutant background, and Kmrrecombinants were selected on PSA plates containing kan

Construction of mutants in X. oryzaepv. oryzae

3.0–5.0, phosphate buffer for pH 6.0–8.0 and Tris-HCl buffer for pH 9.0) were used. •pH stability: The pH stability of the selected tannases was examined in the range of 3.0–9.0 by incubating the enzyme samples for 6 h in different buffers. Tannase activity was estimated under standard assay conditions. •Temperature tolerance: Temperature tolerance of the tannases was examined by assaying their activity at different temperatures in the range of 20 to 80ºC. •Temperature stability: Temperature stability of the tannases was determined by incubating them in the temperature range of 20 to 70 ºC for 6 h. After the incubation tannase activity (%) was determined under standard assay conditions. •Organic solvent stability: In order to determine the suitability of the selected tannases for organic synthesis, their stability was determined in different organic solvents. Experimentally, 10 mg of each of the crude lyophilized tannase from the selected cultures were mixed with 1.0 ml of the following organic solvent: a) Hexane b) Methanol c) Propanol d) Isoamyl alcohol e) Petroleum ether f ) Chloroform The mixture was incubated for 6 h at optimal temperature and the organic solvents were then decanted and the residues were dried in a vacuum desiccator. These dried samples were dissolved in 1.0 ml of citrate phosphate buffer (50 mM, pH 5.0) and the tannase activity was determined under standard assay conditions. The tannase activity thus obtained from each culture were compared with initial tannase activity. Finally, on the basis of tannase titres produced per ml and desirable biochemical properties, the best tannase producer was selected for further investigations

The tannases obtained (at high titres) from selected cultures were evaluated for the following important biochemical properties. 1. pH tolerance and stability 2. Temperature tolerance and stability 3. Organic solvent stability •pH tolerance: pH-tolerance of the selected tannases was examined in the range of 3.0–9.0. Buffers (0.05 M) of different pH (citrate phosphate for pH

Preliminary biochemical characterization of tannases from the potent tannase producers

PCR-positive transformants were inoculated in 10 ml YPD medium, allowedto grow for 12 handgenomic DNAwas isolated.Another round of PCR was performed using genomic DNA asatemplate toconfirm the gene deletion

A homologous recombination-based strategy was used to disrupt C. glabraraORFs witha cassette containing the nat1gene, which codes for nourseothricin acetyltransferase and imparts resistance to nourseothricin. Briefly, 5’-and 3’-UTR region (nearly 500-700 bp) of the gene to be deleted were amplified by PCR using wild-type genomic DNA as template. Both 5’-and 3’-UTR amplified products were fused to one half each ofthenat1gene amplified from theplasmid(pRK625). The two nat1-amplified fragments share about 300-350 bp complimentary region. To obtain fusion products, primers were designed insucha way thatthereverse primer for 5’-UTR and the forward primer for 3’-UTRof the gene of interestshare 20 bp complimentary region with the forwardprimer for 5’nat1fragment andthereverse primer for 3’nat1fragment amplification, respectively. The fused PCR products were co-transformed in to the wild-type strain and transformants were plated on YPD-agar plates. Plates were incubated at 30°C for 16 h to allow the homologous recombination between nat1fragments,and 5’-and 3’-UTR atthegenomic loci. Post incubation,cells were replica plated ontoYPD-agar plate supplemented with 200 μg/ml nourseothricin and incubated for another 24 h. Nourseothricin-resistant colonies were purified and verified for gene disruption viahomologous recombination by PCR using appropriate set of primers

Generation of C. glabratadeletion strains

PCR-positive transformants were inoculated in 10 ml YPD medium, allowedto grow for 12 handgenomic DNAwas isolated.Another round of PCR was performed using genomic DNA asatemplate toconfirm the gene deletion

A homologous recombination-based strategy was used to disrupt C. glabraraORFs witha cassette containing the nat1gene, which codes for nourseothricin acetyltransferase and imparts resistance to nourseothricin. Briefly, 5’-and 3’-UTR region (nearly 500-700 bp) of the gene to be deleted were amplified by PCR using wild-type genomic DNA as template. Both 5’-and 3’-UTR amplified products were fused to one half each ofthenat1gene amplified from theplasmid(pRK625). The two nat1-amplified fragments share about 300-350 bp complimentary region. To obtain fusion products, primers were designed insucha way thatthereverse primer for 5’-UTR and the forward primer for 3’-UTRof the gene of interestshare 20 bp complimentary region with the forwardprimer for 5’nat1fragment andthereverse primer for 3’nat1fragment amplification, respectively. The fused PCR products were co-transformed in to the wild-type strain and transformants were plated on YPD-agar plates. Plates were incubated at 30°C for 16 h to allow the homologous recombination between nat1fragments,and 5’-and 3’-UTR atthegenomic loci. Post incubation,cells were replica plated ontoYPD-agar plate supplemented with 200 μg/ml nourseothricin and incubated for another 24 h. Nourseothricin-resistant colonies were purified and verified for gene disruption viahomologous recombination by PCR using appropriate set of primers

Generation of C. glabratadeletion strains

Dishes containing adherent cultures were placed on ice and washed once with cold PBS. Cells were scraped in 1x sample buffer on ice. Samples were sonicated in a probe sonicator (Misonix ultra sonic liquid processor, S-4000) 3 times for 15 sec each to complete cell lysis and shear DNA to reduce viscosity. Equal volumes of lysates were loaded and resolved using 12% SDS-PAGE. Samples were stored at -80ºC if necessary. Protein transfer onto PVDF membrane (GE Healthcare) was carried out at 200mA constant current for 2 h by placing the transfer tank in ice. Membranes were blocked with appropriate blocking buffer according to manufacturer’s instructions provided for each antibody. Preferentially TBST with 5% non-fat dry milk was used for blocking and for antibody dilutions. Briefly, non-specific interactions were blocked with blocking buffer (TBST+5%non-fat dry milk) for 1 h at room temperature. Membranes were probed with appropriate primary (overnight at 4ºC) and HRP-conjugated secondary antibodies (2 h at room temperature). Proteins were detected using ECL detection system. Chemiluminiscence was detected using the LAS4000 (GE Healthcare) or FlourChem E (Protein Simple) documentation system. Densitometry analysis of bands was done using ImageJ documentation software (Fiji) or the multiplex band analysis tool in AlphaView software (Protein Simple)

Western blot analysis

ZFIN_ZDB-GENO-050809-10

ZFIN_ZDB-GENO-100513-10

ZFIN_ZDB-GENO-101227-10

ZFIN_ZDB-GENO-101227-10

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HON. MR. REESOR—There are several other provisions in the proposed Constitution which seem to be ambiguous in their meaning, and before discussion upon them it would be well to have them fully explained. In the eleventh clause of the twenty-ninth resolution, for instance, it is declared that the General Parliament shall have power to make laws respecting ” all such works as shall, although lying wholly within any province, be specially declared by the acts authorizing them to be for the general advantage.” It would appear from this, that works like the Welland canal, which yield a very large revenue, will be given over to the General Government; and this being the case, surely this is a sufficient setoff, five times over, for the railways given by New Brunswick, without the annual subsidy proposed to be given to that province of $63,000. HON. MR. MACPHERSON—The cost of these works forms part of the public debt of Canada, which is to be borne in part by the Lower Provinces under the Confederation. HON. MR. CAMPBELL—The honorable gentleman will see that there are some works which, although local in their geographical position, are general in their character and results. Such works become the property of the General Government. The Welland canal is one of them, because, although it is local in its position, it is a work in which the whole country is interested, as the chief means of water communication between the western lakes and the sea. Other works, in the Lower Provinces, may be of the same character, and it is not safe to say that because a certain work lies wholly in one province, it is not to belong to the General Government. HON. MR. REESOR—I do not object to the General Government having the control of these works. It is, I believe, a wise provision to place them under such control. But I do say that it is unfair that an express stipulation should be made to pay one province a large sum per annum for certain works, while, at the same time, we throw in our public works, such as the Welland and St. Lawrence canals, without any consideration whatever. This, I think, is paying quite too much for the whistle. Then the answer of the Commissioner of Crown Lands about the export duty on minerals in Nova Scotia is not at all satisfactory. Whatever dues may be levied on minerals in Canada—and Canada, although it may contain no coal, is rich in gold, silver, copper, iron, and other ores—in the shape of a royalty or otherwise, go to the General Government, while in Nova Scotia they accrue for the benefit of the Local Government. HON. MR. ROSS—NO, they will not go to the General Government. HON. MR. REESOR—Well, there is nothing to the contrary in the resolutions, and you may depend upon it that whatever revenues the General Government may claim, under the proposed Constitution, will be fully insisted upon.

8

Local works naturally fell within the scope of local governments, and would undoubtedly be under the immediate influence of the municipal councils, but all the works of a really public character would be under the General Legislature; such, he meant, as were connected with the general policy of the whole country.

Lines of steam or other ships, railways, as well as canals and other works connecting any two or more of the Provinces together, or extending beyond the limits of any Province, would be under the control of the General Government.

a Governor General, who should be appointed by our Gracious Sovereign.

Go to Control Panel > Uninstall a Program > Turn Windows features on or off to turn off Hyper-V.

sity faculty member) describes her temporomandibular joint disorder in this way: "It's like a shadow that throws the other parts of my life into brighter contrast. You see my brights are brighter, 'cause I have this darkness hovering all around the edges" (1992:129). Weapon or demon metaphors are often used to describe pain (see also Good 1992). Scarry's respon- dents refer to pain as a "knife, or bones that cut through

God

and may even cause insanity.

Cindy A. Buckmaster: Animal research Is a labor of love for animals and people

You gotta go for what you knowMake everybody see, in order to fight the powers that beLemme hear you sayFight the Power

Some blocks in my neighborhood are getting downright spooky – front yards are filling with spider webs and tombstones, and ghosts peek through the bushes. Along with the piles of pumpkins and inevitable candy corn appearing in the supermarket, they are a reminder that Halloween is just around the corner. Americans celebrate Halloween on October 31 by trick-or-treating, displaying jack-o’-lanterns (carved pumpkins) on their porches or windowsills, holding costume parties, and sharing scary stories

10 Oct 2016 22:13:49 UTC

Reid, Clinton supporters hit Trump over Nevada pronunciation Published October 06, 2016 FoxNews.com Facebook0 Twitter0 livefyre9791 Email Print Now Playing What's Trump doing to prepare for 2nd presidential debate? Never autoplay videos Supporters for Democratic presidential nominee Hillary Clinton and Sen. Harry Reid attacked Donald Trump after the Republican presidential nominee told his supporters about the “correct” way to pronounce Nevada. Trump, during a rally Wednesday in Reno, insisted the correct way to pronounce the name of the Silver State was “Neh-VAH-da.” He declared that “nobody says it the other way.” Clinton supporters and Reid, a Democrat from Nevada, both used the moment to assail Trump. American Bridge immediately put up a video declaring that Trump was “looking like an idiot” for getting the name wrong. A statement from Reid declared that Trump’s stop in Reno was “disastrous.” "If Donald Trump wants to come down from the penthouse his daddy bought him to lecture us on how to say Nevada, he could at least pronounce it correctly,” Reid said in a statement. "Instead, Trump told us we pronounce the name of our state wrong minutes before he refused to take a position on Yucca Mountain. Predictions Map See the Fox News 2016 battleground prediction map and make your own election projections. See Predictions Map → “I have news for Donald: it's pronounced Nev-AD-a and Yucca Mountain is dead.” Trump made a stop at the International Church of Las Vegas and the International Christian Academy before his rally in Reno. He said the Pledge of Allegiance with schoolchildren at the school. He also visited with Hispanic business leaders at a Mexican restaurant before departing for northern Nevada. Fox News’ Chad Pergram and the Associated Press contributed to this report.

Without the tax-related reduction, Mylan’s profits on the EpiPen two-pack were about 60% higher than the figure given to Congress, or $166, it said in a new regulatory filing to the Securities and Exchange Commission Monday.

While we have yet to receive any definitive answer as to exactly what group hacked the DNC, the vast majority of experts believe that the attacks came from Russia or Russian-backed actors.

And research on both people and animals suggests the reason is that a brain injury can disrupt circuits that normally dampen the response to a frightening event.

Because many of the theories deal with issues of power, students on the margin for particular reasons--ethnicity, class, ability--;ire often more receptive to the basic ideological premises of these theories than are their more privileged peers, who sometimes respond to theories such as gender and class as using the master's tools to dismantle the tnaster's house.

Matthew S. MacLennan