Preparation of cell extracts

These experiments were undertaken in the laboratories of Dr. Sylvie Rimsky and Dr. Malcolm Buckle at the Ecole Normale Superioure, Cachan, Paris (France)

Methods for SELDI (Surface Enhanced Laser Desorption/Ionization)

A single plaque of λ contains approximately 105-106 pfu/ml. The method of propagation of λ from a single plaque was as follows. The contents of a single isolated plaque were drawn into a 1-ml pipette tip and dispensed into 0.2 ml of LB broth. After addition of a drop of chloroform, the contents were vortexed and centrifuged. The clear supernatant was mixed with 50 μl of λ-sensitive cells and incubated for 20 min at room temperature for adsorption. 10 ml of Z-broth supplemented with 5 mM MgSO4 was then added to the infection mixture, and incubated at 37°C with shaking until lysis. The lysate thus obtained usually contained 109pfu/ml

Preparation of λ lysate by propagation from a single isolated plaque

The method used was essentially the same as that described for preparation of P1 lysate, except that the λ-sensitive C600 cells used for infection were grown in LB broth containing 0.4% maltose and 10 mM MgSO4. The lysate thus prepared was checked for supE+revertants by plaquing on both supE (C600) and supE+strains (MG1655) using various dilutions of the lysate. To be used for experimental purposes, a phage titre of the order of 1010 to 1011 on the supE strain and around four orders of magnitude lower on the supE+strain, indicating very less frequency of supE+ revertants in the lysate is ideal

Preparation of lambda (λ) lysates

30nm corresponds only to the intermediate. Since equal number of cells was taken for estimation, the height of the peak was taken as a measure of the ergosterol content

Ergosterol content was measured as described by Arthington-Skaggs et al. (1999) with slight modifications. Briefly, equal number of cells were harvested and washed with PBS to remove media and FBS. They were resuspended in 3mL of 25% alcoholic KOH (25g KOH, 35mL sterile distilled water brought to lOOmL with ethanol) and vortexed for one minute. The cell suspension was transferred to a glass vial and incubated at 85°C for 1 hr. The vial was cooled to room temperature followed by the addition of 1mL sterile distilled water and 3mL of n-heptane. The vial was vortexed for 3 mins after which the heptane layer was collected. It was diluted 5 times in absolute ethanol and transferred to a cuvette. A spectrum was recorded between 220nm and 300nm. The peak at 281.5nm corresponds to ergosterol along with some intermediates.

Spectrophotometric estimation of ergosterol

Plasmid DNA was isolated at small scale using QIAprep Miniprep kit according to manufacturer's protocol. Briefly, 5mL of overnight E. coli culture was pelleted and resuspended in 250J..LL Buffer P1(50mM Tris-Cl, pH8.0; 10mM EDTA and 100p.g/mL RNase A). To this, 250 J..LL of Buffer P2(200mM NaOH and 1 %w /v NaOH) was added and mixed thoroughly by inverting the tube 4-6 times. 350 IlL of Buffer N3 (proprietary) was added and mixed immediately and thoroughly by inverting the tube 4-6 times. This was followed by centrifugation at 13000 x g for 10min at RT. The supernatant was applied to a QIAspin column and centrifuges at 13000 x g for 30-60s. The column was washed with 0.5mL Buffer PB followed by 0.75 mL Buffer PE. Residual wash buffer was removed by centrifugation for an additional 60s. The plasmid DNA bound to the column was eluted using the elution buffer, Buffer EB (10mM Tris-Cl, pH 8.5) provided with the kit, or alternatively with nuclease-free water. The concentration of the obtained DNA was estimated by measuring the absorbance at 260nm (A26o) and using the known formula: DNA concentration= A260 X SOX dilution factor

Miniprep to isolate plasmids

cellswere collected and washed with chilled sterile water.1 OD600cells were resuspendedin 20 μl chilled10%TCA solution containing 8 mM EDTA (pH 8.0) and incubated at room temperature for 15-20 min.Followingincubation, cellsuspension was centrifuged at 12,000 rpm for 5 minat 4 ̊Cand supernatant was transferred to a fresh 1.5 ml microcentrifuge tube. 10 μl of this supernatant fraction was diluted 75-foldwith ATPassay mix dilution buffer provided with the kit. 50 μl of diluted suspension was added to anequal volume of ATPassay mix (Sigma # FLAAM) which containedfirefly luciferase and luciferin with MgSO4, EDTA, DTT and BSA inTricine buffer.Luminescence was measured inluminometer (Varioskan flash-3001,Thermo Scientific). Total ATP was quantified usingpurified ATP as the standardand expressed in moles/OD cells

ATPconcentrationin yeast cells was measuredby luminometricluciferase-luciferinbased assayusingATPbioluminescent kit(Sigma # FLAA).Briefly, log-phase yeast

Determination of intracellular ATPlevels

Estimation of total glycogen in cells was performed asdescribed previously (Parrou et al., 1997) with slightmodifications.Briefly, YPD medium-grown C. glabratacells were harvested, washed once with 1 ml ice-cold waterandresuspendedin 250 μl sodium carbonate(0.25 M)solution. After incubation at95 ̊C for 4 hin water bath with occasional stirring, cell suspension was cooled and pH of the suspension was adjusted to 5.2 by adding 150 μl 1 M acetic acid. Tothis suspension,600 μl 0.2M sodium acetatewas added and cell suspension was incubated with 1-2 U/ml of α-amyloglucosidase from A.niger(Sigma #A7420)at 57 ̊C for overnight with constant agitation.Resultant glucose liberated by α-amyloglucosidase digestion was collected in the supernatant fraction and quantifiedby phenol-sulphuric acid methodof carbohydratedetermination.For quantification, commercially available purified glucose was used as a standard and total glycogen incells was expressed as μg/2 x 107cells tonormalizeagainstcell density

Estimation of glycogenlevels

Trehalose from C. glabratacells was extracted by trichloro acetic acid (TCA)solutionas described previously (Lillie et al.,1980). Cells grown in YPDmediumwere collected at different time pointsof growth and washed thrice with ice-cold sterile water. Cells were immediatelystored at-20 ̊Ctill further use.For trehalose isolation, 10-20 OD600cells were thawed in 500 μl TCA (0.5 M) solutionon ice and incubated at room temperaturefor 1 h.Supernatant fraction was collected by sedimenting cells at 14,000 rpm for 5 minat 4 ̊C.TCA extractionwas repeated withcells once more and the resultingsupernatant was mixed with the earlier fraction.Extractedtrehalose was measuredby phenol-sulphuric acid methodof carbohydratedeterminationwithcommercially available purified trehalose(Becton, Dickinson and Co.) as a standard.Total trehalosecontent was normalized to the cell densityand expressed as μg/2 x 107cells

Estimation of trehalosecontent

Estimation oftrehalose, glycogen and ATPlevels

QIAGEN QIAquick Gel extraction kit containing required buffers, spin columns and collection tubes was used to extract and purify DNA from agarose gels. Digested DNA sample was resolved on 1-1.2% agarose gel and gel piece containing desired fragment was cut ona UV-transilluminator. DNA fragment was purified as per the kit manufacturer’s instructions

Gel extraction of DNA

To prepareelectrocompetent cells, a single colony of E. coli BW23473 strain from a freshly-streaked LB agar plate was inoculated in 50 ml LB liquid medium. Culture was incubated at 37°C for 14 h with continuous shaking at 200 rpm. 25 mlovernight-grown E. coliBW23473 culture was transferred to 500 ml LB liquid medium and incubated at 37°C till the OD600 reached to 0.4. Post incubation, cultures were transferredto ice and centrifuged at 1,000g for 15 minat 4°C. Cells were washed twice with 500 ml ice-cold sterile water, three times with 250 ml ice-cold 10% glycerol solution and resuspended in 1 ml 10% glycerol solution. After absorbance measurement, cell suspension was normalized to final cell density of 3X1010cells/ml and dispensed in 50 μl aliquots to sterile ice-cold 1.5 ml microcentrifuge tubes. Aliquots were snap frozen in liquid nitrogen and stored at -70ºC

Preparation of E. coliBW23473electrocompetent cells

Secretory alkaline phosphatase (SEAP) assay: For SEAP assay, the culture supernatant was analyzed for SEAP activity essentially as per the Clontech kit protocol (Palo Alto, CA). Briefly, cells were transiently co-transfected with Lipofectamine 2000 transfection reagent, 0.5 μg of required plasmid DNA(s) with the protein of interest or empty vector, 0.5 μg of reporter plasmid containing NF-κB binding site cloned upstream of heat-stable SEAP (designated asNF-κB-SEAP)and 0.5 μg of green fluorescence protein (GFP) expression plasmid (Clonetech) in Opti-MEM media.After 6 h of transfection, cells were washed and cultured for 12 h in complete media, followed by treatment with different inducers. GFP positive cells were then counted to ensure similar transfection efficiency. At the end of treatment, cell culture-conditioned medium was harvested and 25 μl of medium was mixed with 20 μl of 5X buffer (0.5 M Tris, pH 9 and 0.5% bovine serum albumin) in a total volume of 100 μl in a 96-well plate followed by incubation at 65°C for 30 min. The plate was chilled on ice for 2 min and 50 μl of 1 mM 4-methylumbelliferylphosphate (MUP, substrate) was added to each well and incubated at 37 °C for 2 h. The activity of SEAP was assayed on a 96-well fluorescent plate reader (Fluoroscan, Lab Systems, MA) with excitation set at 360 nm and emission at 460 nm. The average number (± SD) of relative fluorescent light units for each transfection was then determined and reported as fold activation with respect to empty SEAP-transfected cells.Luciferase (Luc) assay:The cell pellet was lysed and extract was analysed as per Promega kit protocol.Briefly, cells wereco-transfected with Lipofectamine with 0.5 μg of reporter plasmid containing p53 binding site cloned upstream of luciferase (designated as p53-luciferase) and 0.5 μg of GFP constructs. After 6 h of transfection, cells were washed and cultured for 12 h, followed by treatment with different inducers of apoptosis. GFP positive cells were then counted.Cellswere pelleted down and lysed using the lysis buffer. The samples were freeze-thawed twice by storing them at -70oC to ensure total lysis. The supernatant,obtained by centrifuging the same at 11,000 rpm for 2 min was transferred to a fresh tube. About 100 μl of the substrate (Firefly luciferin, Promega) was added to the supernatant and light emission wasmeasured in luminometer by using a delay time of 2 sec andread time of 10 sec.The values were calculated as fold of activation over vector-transfected value

Reporter gene transcription assays

apparatus.Membranes were neutralised in 2X SSC, and denatured plasmids were cross-linked to Hybond-N+membranes usingaUV cross linker at 2000 J/inch square energy for 2 min

+610),end 5’ETS (+611 to +952),25S (+5270 to +5630) and NTS2(gifted byDr. Susan J Baserga) (Gallagheret al., 2004); ACT1 cDNA (+175 to +701) cloned into TOPO vector; pUC12 plasmid containing 5S rDNA construct (giftedby Dr. Purnima Bhargava). Empty TOPO plasmid and genomic DNA extracted from wild type yeast were used as controls. Plasmids and gDNA were extracted, quantitated and denatured in alkaline denaturing solution. 10 μg of each plasmid and gDNAin replicates weredenaturedin alkali, blotted on a Hybond N+membrane using a 96-well Dot Blot

The following plasmids (Table 2.2)were used as probes to detect the transcribed RNA (Fig. 2.1). The TOPOplasmids containing rDNA start (+1 to +177), 5’ETS (+351 to

Dot blot membrane preparation

Cells were transfectedwith various combinations of plasmids and treated with cycloheximide (50ug/ml) 24hrs.post-transfection. Cells were harvested at different time points, and the protein levels were determined by using the standard protocol for western blotting/immunoblotting

Cycloheximide-chase assay

concentarion in the samples were determined based on their peak area against standard oxalic acid plot.For GC-MS analysis, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) derivatization was performed with the dried HPLC fraction of samples as well as standards as described previously (Šťávová et al., 2011). Briefly, 200 μl BSTFA, and 100 μl hexane were added to the sample, and incubated at 50 °C for 70 min. GC analyses were performed using a Shimazdu GP 2010 plus instrument equipped with an autosampler, and a split injector.Separations were accomplished using a 30-m long DB-5 capillary column, 0.25 mm internal diameter (I.D.) at a constant helium flow rate of 1.5 mL/min. Samples (10 μL) were injected with a split ratio of 10 into the column at 100 °C. The final column temperature program started at 100 °C and attained final temperature 280°C with a gradient increase of 5 °C/min. The MS data (total ion chromatogram,TIC) was acquired in the full scan mode (m/z of 50–500) at a scan rate of 1000 amu using the electron ionization (EI) with an electron energy of 70 eV. The acquired spectrum was searched against standard NIST-05 library

Xanthomonas oryzaepv. oryzicola strains were grown overnight in PS medium supplemented with appropriate antibiotics. 0.2 % of the overnight grown culture was reinoculated in 250 ml of fresh PS medium supplemented with 50 μM 2,2’-dipyridyl, and allowed to grow till OD600reached 1. Cultures were centrifuged to obtain cell free culture supernatant, concentrated on vaccum evaporator, and freeze dried at regular time intervals to remove the water completely. Oxalic acid was estimated from the dried supernatant by using Agilent 1100 series HPLC system as described previously with slight modifications (Ding et al., 2006). In brief, dried supernatant fractions of different cultures were dissolved in mobile phase of pH 2.7, and allowed to stand for 3 h for the precipitation of humic substances. These samples were filtered through membrane filter (porosity, 0.45 μm), and 20 μl volume of the filtrate was injected into the Agilent C18 (4.6 mm× 250 mm× 5 μm) column. The mobile phase used was 10 mM KH2PO4-CH3OH (95:5, pH 2.7), and the samples were separated by isocratic elution at 0.8 mL/min at 26°C temperature. Standard oxalic acid was detected in similar way in mobile phase (pH 2.7 at 210 nm) with retention time (RT) of 6.7 min. Likewise, oxalic acid in the test samples were also detected at 210 nm with RT 6.7

Oxalic acidestimation from culture supernatant of different strains of X. oryzaepv. oryzicola by HPLC and GCMS analysis

fluorophore-conjugated phalloidin (Alexa Fluor 488 or rhodamine) for 45 min, followed by DAPI staining for 2 min. Cover slips were mounted onto glass slides using Fluoromount G (Southern Biotech) or Vectashield (VectorLabs),and imaged using an LSM 710 laser confocal-anisotropymicroscope (Zeiss, Zen acquisition software, 40x 1.3 N.A. objective) for 15 min spreading,or LSM 510laser confocal microscope (Zeiss, LSM acquisition software, 63x 1.4 N.A. objective) for 24 hspreading.To measure the cell spread area for serum depleted cells (0.2% serum), images captured at identical zoom settings were analyzed using the Image J software (NIH)as follows:thresholdvalues were set to define the cell edge and amaskwas then createdfor each cellto get the totalcellarea(with arbitrary units)within the mask.For 15 min spreading (10% serum) and 24 h spreading, the exact spread area was calculated based on pixel dimensions during image acquisition

Cell spreading assays were done as described previously (Balasubramanianet al., 2010).Briefly, cells were cultured in complete medium (10% FBS) or subjected to serum starvation (0.2% FBS for 12-14 h), trypsinized, counted using a hemocytometer, and held in suspension in the same medium containing 1% methylcellulose at 37°Cand 5% CO2in a 50 mL tube in a slanted position. After 90 min, cells werepelleted down at 1350 rpm for 8 min at 4°Cand the supernatant was discarded leaving 5 mL at the bottom. Cells were dislodged or displaced gently without touching the walls of the tubes, which might lead to activation of adhesion dependent signalling. Now, cells were washed once with excess of cold media (4°C)and one more time with warm media (37°C). A day before the experiments coverslips were coated with fibronectin (2 μg/mL)overnight at 4°Cand washed once with PBS before plating the cells. Cell suspension equivalent to 105cells was plated onto each fibronectin (2 μg/mL) coated coverslip and allowed to spread for 15 min or24 hin serum depleted (0.2% FBS) or complete medium. At each time point, unadhered cells were washed off with PBS and the coverslips were fixedwith 3.5-4% paraformaldehyde for 20 minat room temperature. After fixation coverslips are washed thrice with PBS and cells were permeabilized with 0.1% Triton X-100 for 5 min,followed by blocking with 5% BSAfor 30 min at room temperature. Fixed cells were stained with

Cell spreading

RRID:ZFIN_ZDB-ALT-001205-14

A geographical line has been drawn across the Union, and all the States north of that line have united in the election of a man to the high office of President of the United States, whose opinions and purposes are hostile to slavery. He is to be entrusted with the administration of the common Government, because he has declared that that “Government cannot endure permanently half slave, half free,” and that the public mind must rest in the belief that slavery is in the course of ultimate extinction.

The whole of the clauses which refer to the latter are as complete as the most ardent supporters of union could desire, tempered by the lew exceptions by means of which the provinces have wished to shelter their local institutions from attack.

The 34th paragraph of the 29th clause of the scheme reads thus: ” The establishment of a General Court of Appeal for the Federated Provinces.” What is the object—what will be the character of the tribunal?

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There is a provision that the nomination of the judges of the superior courts shall be vested in the General Government, but it would seem that the constitution of the courts is to be left to the local governments ; and I put the question, What does this mean ? Do you mean that the local governments are to establish as many courts as they please, declare of how many judges they will be composed, and that the General Government will have to pay for them ? Is a local government to say, here is a court with three judges ; we want five, and those five must be appointed and paid by the General Government ?

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There is one currency here, another in Newfoundland, another in Prince Edward Island, and so on. The shilling and pound of this province are different from the shilling and pound of Newfoundland and those of the other Maritime Provinces. But, with Confederation, all these matters would be placed under the control of our central legislature; the currency would become uniform throughout, and capital might be everywhere invested without obstacle. So also it will be with respect to the rights of authors, patents for mechanical inventions, &c.

10

The control of property and civil rights, the administration of justice, including the constitution, maintenance, and organization of the courts of civil jurisdiction, and the procedure in civil matters, were also left to the local legislatures. From the peculiar position of Lower Canada it was felt impossible to confide the matter of civil law to the General Legislature. The principles upon which the civil law of Lower Canada were founded differed entirely from those of the English law. Under it property was secured, and civil rights of every kind maintained, and the people had no particular wish to see it changed, especially at this moment, when the work of codifying and simplifying it was about completed, and when they knew that within the next three or four months they would have it put into their hands in one volume. He thought it was undesirable to do away with that law, which had been beneficial to the country and under which it had prospered. It was necessary to have it left to the local Legislature, because all in Lower Canada were unwilling to have substituted another law with which they were unacquainted.

It was desirable the General Government should have the control of the medium through which the trade and commerce of the country was carried on, and that in the establishment of banks, the issue of paper money and in offering to the public the paper representative of their labor, in whatever part of the country, there should be the same legislative security for the people

We want to end gender inequality, and to do this, we need everyone involved

We want to try and make sure that it’s tangible.

She said I am the one who will dance on the floor in the round