Co-Immunoprecipitation (Co-IP)

in 5% fat free milk solution in TBST (1:7000) for 45 min at room temperature and then washed thrice.The detection of signal was performed with ECL detection reagent (Amersham Biosciences) followed by detection of signal either on X-ray film (Hyperfilm-ECL, Amersham Biosciences)or in a chemidoc system (Proteinsimple, California, USA).The blot was reprobed with anti-tubulinor anti-GAPDHantibody to ensure equal loading of extracted protein

Materials and Methods472.2.7 Estimationof protein concentration in cellular lysatesBradford method(Bradford, 1976)was used to determine the quantity of protein in various samplesin a 96-well plate. Bradford’s reagent was prepared by diluting Bradford dye with water in the ratio of 1:5.For estimating the concentration of protein in a particular sample, 50μl volume reactionwas set and200μl of freshly prepared Bradford’s reagent was added. The complex givesa purplish colorwhose intensity is proportional to the amount of protein present in the sample. A standard curve was also generated using increasing concentrations of BSA (50 μg/ml, 100 μg/mland 200μg/ml).Cell lysatesof test samples werediluted to1:50 in the same volume.Each sample (including blank and standards) was taken in duplicates.The concentration of protein was measuredusing the ELISA reader at 570 nm. The unknown protein concentration (X) was calculated as follows:where,OD1& OD2: Optical densities of Standard (Std) 1 & Standard (Std) 2, respectively.BSA: Bovine serum albuminX×50 (dilution factor)/1000 = YConcentration of unknown protein (μg/μl) = Y × OD2.2.8 Immunoblotting(Western Blotting)Immunoblotting was performed as essentailly described by Lee (Lee, 2007). Equal amounts of protein were resolved on a denaturating SDS-polyacrylamide gel (8-12%). After completion of the run, the gel was transferred onto PVDF membrane and placed in the blotting cassette. The cassette wasthenput intothe mini transblot apparatus and transfer was done for 2-3 hours at a constant voltage of 80 V, depending on the size of the protein. Post transfer, membrane was rinsed in TBS containing 0.1% Tween-20 (TBST) and blocked with 5% non-fat milk in TBST for 1 h at 37ºC,on a gentle shaking rotator. After blocking, membrane wasrinsed thrice in TBST and incubated with primary antibodydiluted in TBST (ranging from 1:1000 to 1:10000, depending upon antibody used) for either3h at room temperature or overnight in the cold room.The membrane was then washed thrice with TBST and incubated withhorseradish peroxidase(HRP)-conjugated secondary antibody diluted

Immunoblotting(Western Blotting)

Bradford method(Bradford, 1976)was used to determine the quantity of protein in various samplesin a 96-well plate. Bradford’s reagent was prepared by diluting Bradford dye with water in the ratio of 1:5.For estimating the concentration of protein in a particular sample, 50μl volume reactionwas set and200μl of freshly prepared Bradford’s reagent was added. The complex givesa purplish colorwhose intensity is proportional to the amount of protein present in the sample. A standard curve was also generated using increasing concentrations of BSA (50 μg/ml, 100 μg/mland 200μg/ml).Cell lysatesof test samples werediluted to1:50 in the same volume.Each sample (including blank and standards) was taken in duplicates.The concentration of protein was measuredusing the ELISA reader at 570 nm. The unknown protein concentration (X) was calculated as follows:where,OD1& OD2: Optical densities of Standard (Std) 1 & Standard (Std) 2, respectively.BSA: Bovine serum albuminX×50 (dilution factor)/1000 = YConcentration of unknown protein (μg/μl) = Y × OD

Estimationof protein concentration in cellular lysates

Materials and Methods462.2.5 Cell proliferation assayThe method described earlier by Gilliesand co-workerswas slightly modified and followed (Gillies et al.,1986). Briefly, parentaland profilin-stable cells were seeded in triplicates at a density of 20,000 cells per well of a 24-well culture plates. Each day after seeding, cells were washed with PBS and stained with 0.2% crystal violet in 2% ethanol for 15 minutes. Vigorous washing was done with PBS to remove excess dye. Crystal violet dye was then eluted using 1% SDS solution with extensive pipetting and diluted 10 fold. Absorbance of the extracted dye was then determined at 570 nm in a spectrophotometer. Absorbance data based on triplicate set of samples for each experimental condition were then averaged for each time point to generate a growth curve.2.2.6 Preparation of whole cell, cytoplasmic and nuclear lysatesIn order to extract the total cell homogenate, the culture media was removed and cells were washed with ice cold PBS. The cells were then gently scrapped and pelleted by centrifugation at 3000 rpm for 3 minutes at 4°C. The cell extraction buffer was added to the cell pellet and placed on rotor kept in cold room for 30 minutes for cell lysis. Lysed cellswere then centrifuged at 13000 rpm for 10min at 4°C. The supernatant was collected as cellular lysate. Protein concentration was estimated using Bradfordassay, described below.For a typical Western blot assay, 30-70μg of protein was loaded on theSDS-PAGE.For the preparation of cytoplasmic lysate,ice-cold hypotonic cytoplasmic extract buffer was added in the cell pellet andgently mixed with the pipette in a microfuge tube. The cell suspension was incubated on ice for 30 minto allow them to swell.After incubation, freshly prepared 10% NP-40was added andvortexed vigorously for 15 seconds torupture the plasma membrane. The contents were then centrifuged at 13000 rpmfor a minuteat 4°C and supernatant containing the cytoplasmic lysate was transferred to another pre-chilled microfuge tube and stored at –70°C.The pellet was then further processed for extraction of nuclear lysate.For this, ice-cold nuclear extractbuffer was added to the pellet and incubated on ice for 45 min with intermittent vortexingafter every 10 min of incubation. Finally, cell suspension wascentrifuged for 5 min at 14000rpm. The supernatant containing nuclear lysatewas stored at –70°C for further experiment

Preparation of whole cell, cytoplasmic and nuclear lysates

The method described earlier by Gilliesand co-workerswas slightly modified and followed (Gillies et al.,1986). Briefly, parentaland profilin-stable cells were seeded in triplicates at a density of 20,000 cells per well of a 24-well culture plates. Each day after seeding, cells were washed with PBS and stained with 0.2% crystal violet in 2% ethanol for 15 minutes. Vigorous washing was done with PBS to remove excess dye. Crystal violet dye was then eluted using 1% SDS solution with extensive pipetting and diluted 10 fold. Absorbance of the extracted dye was then determined at 570 nm in a spectrophotometer. Absorbance data based on triplicate set of samples for each experimental condition were then averaged for each time point to generate a growth curve

Cell proliferation assay

Wound healing assay(Liang et al., 2007) was employed to study the difference in migration rates ofprofilin-stable compared to parentalcells. Briefly, cells were cultured upto 90-95% confluent monolayer and a scratch was created through the cell monolayer with sterile needle. Cell debris was then removed by washing with PBS before adding the media. Images of the open gap created by this “wound” were then captured at three random locations immediately (0 h) and then at the same locations after regular interval using phase contrast microscopy untilthey are closed by migrating cells. Captured images were then used to quantify wound closure by the percentage change in the wound area per unit time and averaged for three locations for each experimental condition. During the course of the experiment, cells were maintained in 0.1% FBS containing DMEM media to ensure that wound closurewas due to the migration of cells rather thandivisonof cells

Wound healing assay

Briefly, cells were cultured on 35 mm culture dishes (Corning) and were transfected with pcDNA3.1 (+) empty vector or pcDNA3.1 (+) profilin-1 clone (encodes for neomycin resistance for selection in mammalian cells) using Lipofectamine 2000 reagent. The following day, cells were trypsinized and plated in four 10 cm culture dishes separately for each cell clones in selective media containing 800μg/ml of Geneticin, G418 (whose concentration was obtained from kill curve based on MTT assay). Over the time,it will select only those cells that have stably incorporated the plasmid into their genomic DNA. Cells were selected in G418 for about two weeks until colonies appeared. Meanwhile, cells were regularly washed with sterile PBSto remove dead cells from culture dishes. Colonies were picked using a pipettewith a sterile tip by loweringitto the surface of the colony of interest, followed by gentle scraping and rapid sucking.About 20 colonies were picked, followed by dilution plating in 96-well plates until single cell per well was obtained to establish a pure colony. Once colonies have reached to fair confluency, they were further expanded by dilution plating and screened for stable expression of profilinby immunoblotting and semi-quantitative RT-PCR. Once the clonal cell line is established, aliquots were freezed for future use and rest were maintained in lower concentration of G418 with proper track of passage number

Generation of profilin stable MDA-MB-231 cells

Transfection of plasmid DNAin cellswas performed using Lipofectamine 2000 reagent as per manufacturer’s protocolprovided with the reagent. Briefly, 0.5 to 1 million cells were seeded in a 60mm or 100mm tissue culture dish. After 12h of seeding,transfections were performed. 6-12 μg DNA was mixed in 500-1500 μl of Opti-MEM in one polypropylene tube and simultaneously, 15-30 μl of Lipofectamine 2000 was mixed in similar volumes of Opti-MEM in another tube and incubated at room temperature for 10minutes. Opti-MEM containingDNA and Lipofectamine 2000 were then mixed and incubated for 30 minutes at room temperature for the formation of DNA-lipid complex. Meanwhile, the cells were washed with sterile PBS and 4-10ml of Opti-MEM was addedin the plate. DNA-lipid complexes were then added to each dish for 6h. After that, the medium containing complexes was removed and complete medium (DMEM containing FBS) was added. Expression of transgene was evaluated 24-48hafter transfectioneitherby immunoblottingor immunofluorescence or by RT-PCR followed by PCR

Transient transfection in adherent cells

Maintenance of cell lines

Inpresent thesis, various cell lines have been used as mentionedearlier. Cells were either cultured in DMEM or RPMI medium containing 10% fetal bovine serum (FBS)along with antibiotics such as penicillin (100 U/ml), and streptomycin (100 μg/ml).In general, cells were grownin tissue culture T-75 flaskupto 85-90% confluency. Cells are washedwith PBS, followed by trypsinization with 0.05% Trypsin EDTA solution. Cells were detachedfrom the surfaceeither by gentle tapping or gentlepipettingor incubated for 5 minutes at 37°C. Culture medium containing serum was then added to inactivate trypsin. After careful mixing, cells were transferred to a 15 ml tube and centrifuged at 800 rpm for 5minutes. The cell pellet wasre-suspended in a fresh culture media containing FBS. The cell viability was checked by trypan blue staining, followed bycounting in Neubauer cell-counting chamber. Appropriate number of cells wasthen either sub-culturedin the ratio of 1:4 to 1:6or seeded in culture dishes as per the experimental requirements.Cells were maintained in humidified incubator at 37ºC in 5% CO2-95% air, throughout the experiment

Extraction buffer

MTT reagent

For Cytotoxicity assays

6XEMSA sample loading dye

5X EMSA buffer

Native EMSA PAGE

10XBinding buffer

For Electrophoretic Mobility Shift Assay (EMSA)

For preparation of Ultra competent cells

Inoue buffer

6X DNA loading dye

Agarose gel

TAE

For DNA electrophoresis

Nuclear lysis buffer (without protease inhibitors

Cytoplasmic extraction buffer (without protease inhibitors)

For Cell fractionation

Blocking buffer: 2% BSA

Permeabilisation buffer: 0.2% Triton X100

4% Formaldehyde fixative

For Immunofluorescence(IF)

Stripping buffer

Blocking buffer

TBS-T

Transfer buffer

(f) Running buffer

(e) Stacking polyacrylamide gel

(d) Resolvingpolyacrylamide gel

(c) 6X Protein loading buffer (Lammeli buffer)

(b) Celllysis buffer B(For IB)

Cell lysis bufferA(For IP)

II. For Immunoprecipitation(IP)and Immunoblotting(IB)

Table 2.1: Commonly used buffers and solutionsI. General buffers(a)Phosphate Buffered Saline (PBS)

(b) Tris Buffered Saline (TBS)

For DNA isolation and purification, various kitssuch as Miniand midi-prep plasmid isolation, Gel extraction, PCR purification,etc., wereprocured fromQiagen(Hilden, Germany) or HiMedia(India). For RNA extraction, TRIzol wasobtained from Gibco BRL(Grand Island, NY). cDNA was made from RNA byeither Reverse transcriptase (SuperScript III, Invitrogen) or One step Access RT-PCR kit (Promega, Madison, WI). Reagents for PCR such as PCR 10X buffer, dNTPs, MgCl2, Taqpolymerase or AccuTaq were obtained from Fermentas or Sigma Aldrich. Recombination enzymes such as Restriction Endonucleases and DNA ligaseused for recombinant DNA experiments (Bam-H1, Hind-III, Xho-I, Eco-RI, Not-I, and Sal-I) were obtained from New England Biolabs(Ipswich, MA, USA). Oligonucleotidesusedfor various Gel shift assays viz.AP-1, NF-κB, p53 and Sp-1 were commercially synthesizedfrom XCelris(Ahmedabad, India).For protein extraction, protease inhibitors such as aprotinin, leupeptin, PMSF, NaF, NaVO4,etc. were obtained from Sigma Aldrich.Bradford reagent for estimation of protein concentration wasobtained from Bio-Rad(Rockford Illinois, USA).ForImmunoblotting, PVDF membrane, X-ray films andchemi-luminiscentdetection reagent (ECL prime) were obtained from GE Healthcare(Little Chalfont, UK). For Immunofluorescence, vectashield-mountingmedium with DAPIand Propidium Iodide (PI)were obtained from Molecular Probes, Invitrogen.For detection of cytotoxicity, MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) dye, SDS and DMF (Dimethylformamide) wereobtained from SigmaAldrich. Live and dead cell assay kit was obtained from Molecular Probes.Various chemicals required for preparation of regular buffers and solutionsviz. Tris, Glycine, SDS, Sodium Chloride, Potassium Chloride, HEPES, Disodium Phosphate, Nonidet P-40, Tween 20, TritonX100, Formaldehyde, Glycerol, Agarose, Acrylamide,Bis-acrylamide,APS, TEMED, BSA,etc. were obtained from SigmaAldrich.The procedure of preparation of buffers and reagents usedin the present studied are described below:

Reagents and Buffers

Wild type or H133S mutant of profilin-1 witheither FLAG or un-tagged werecloned in pcDNA3.1 (+).Mdm2 gene upstreampromoter region having p53 binding site was cloned in pLUC vector (designated as p53-Luc). The constructs of NF-κB-SEAP, p65 (RelA), wild type and dominant negativeIKKβ(IKKβ-WT and IKKβ-DN, respectively)were a kind gift fromProf.Bharat B. Aggarwal (M. D. Anderson Cancer Center,Houston, TX). The constitutive active mutant of IKKβ, in which two serine residues are mutated to glutamic acid, at position 177 and 181 (referred as IKKβ-EE or IKKβ-CA) was gifted byProf. GourisankarGhosh (University of California, San Diego, USA).FLAG or Myc tagged Full length andtruncationmutants of PTEN wereprovided by Dr.M.Subba Reddy (CDFD, Hyderabad).For p53 gene knockdownstudies, TP53 mission shRNA were obtained from Sigma Aldrich (St Louis, MO, USA). For PTEN silencing, retroviral vector based PTEN shRNA (shRNA#1-AGGCGCTATGTGTATTATTAT; shRNA#2-CCACAGCTAG-AACTTATCAAA; shRNA#3-CCACAAATGAAGGGATATAAA)wasgifted by Dr. M.Subba Reddy (CDFD, Hyderabad)

Plasmids

obtained from Gibco, Invitrogen(Carlsbad, CA, USA). For cell culture transfections, Lipofectamine-2000 and Opti-MEM were alsoobtainedfrom Life Sciences, Invitrogen(Carlsbad, CA, USA).Commonly used chemicals in cell culture based experiments such asall-trans retinoic acid (ATRA), arabinoside cytosine (AraC),carbobenzoxy-Leu-Leu-Leucinal (MG-132), cycloheximide (CHX),DMSO, doxorubicin, hydrogen peroxide (H2O2),lipopolysaccharide (LPS, Escherichia coli055:B5), okadaicacid (OA), oleandrin,paclitaxel, phorbolmyristate acetate (PMA), vinblastine and vincristine wereobtained from SigmaAldrichChemicals.Benzofuran was synthesized as reported earlier (Manna et al., 2010).Recombinant human TNFα, IL-1and IL-8 were obtained from PeproTech Inc.(Rocky Hill, NJ, USA).Growth media for bacteria culture,Luria Broth (LB) and Agar were obtained from HiMedia laboratories (Mumbai, India). Bacterial strain DH5was used to make ultra-competent cells for transformation and plasmid isolation. Antibiotics, such as Ampicillin and Kanamycin used for selection of transformed colonies and culture were obtained from Sigma AldrichChemicals

The cell lines used in the present study, HuT-78 (human T-cell lymphoma), MDA-MB-231 (human breast cancer) and MDA-MB-468 (human breast cancer) were obtained from American Type culture collection (Manassas, VA, USA). Human colon carcinoma cell lines HCT-116 (wild-type, p53+/+) and HCT-116 (null, p53-/-) were a kind gift fromProf. B. Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD). Cells were cultured in DMEM or RPMI medium containing 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were maintained in humidified incubator at 37ºC in 5% CO2-95% air. Media for mammalian cell culture (DMEM and RPMI),fetal bovine serum (FBS)and other reagentsused in cell culture such as, PBS, Trypsin-EDTA, Antibiotic-antimycotic, Freezing medium, Geniticin, L-Glutamine, HEPES, etc. were

The following antibodies were used in the present study:Primary antibodies against GAPDH (anti-rabbit), FLAG (anti-mouse), Immunoglobulin (IgG, anti-rabbit or anti-mouse),profilin-1 (anti-rabbit), tubulin (anti-mouse) and ubiquitin (anti-rabbit) were obtained from Sigma Aldrich Chemicals(St Louis, MO, USA). Antibodies againstAKT (anti-rabbit), cleaved caspases-3, 8 and 9 (anti-rabbit),HA-tag(anti-rabbit), Myc-tag (anti-rabbit), p21 (anti-rabbit), phospho-p53 (anti-mouse), PTEN (anti-mouse), phospho-AKT (Ser473; anti-rabbit), phospho-GSK-3β (Ser9; anti-rabbit), phospho-IKKα/β (Ser177/181; anti-rabbit), phospho-IκBα (Ser32; anti-rabbit), and phospho-p65 (Ser276; anti-rabbit) were obtained from Cell Signaling Technologies(Danvers, MA, USA), whereas antibodies for cox-2 (anti rabbit), c-Rel (anti-rabbit), ICAM-1 (anti-rabbit), IKKα/β (anti rabbit), IκBα (anti-rabbit), Mdm2 (anti-rabbit), PARP-1/2 (anti-rabbit), Rel-B (anti-rabbit), p50 (anti-rabbit), p53 (anti-mouse), p65 (anti-rabbit) were obtained from Santa Cruz Biotechnology(Santa Cruz, CA, USA).HRP (Horse radish peroxidase)-conjugated secondary antibodies (anti mouse and anti-rabbit) were obtained from Bangalore Genie(Peenya, India). For immuno-fluorescencestudies, secondary antibodiesconjugated toAlexa Fluor (488 and 594, anti-mouse and anti-rabbit) were obtained from Molecular Probes, Invitrogen(Eugene, OR, USA)

Antibodies

Cell cultureand Media

Manipal

Manipal

Manipal University

For each strain calls from a single colony, picked with a microtip from the appropriate medium were suspended in 10 μL zymolyase cocktail and incubated at 37ºC for 90 min. 2 μL of zymolyase-treated cell suspension was used as the template in a 25 μL PCR reaction

was fused to the 5’ region of the nat1gene. The 3’ region of the nat1gene was in turn fused to the 5’ region of UBC11, the gene that is downstream of RPA43in the genome. These two fragments were transformed into the NOY222 rpa34strain using yeast transformation kit (Clontech) and selected on nourseothricin(NAT) 200 μg/mL to identify strains carrying mutant RPA43generated by homologous recombination (Fig. 2.4) containing YPD. Colony PCR was performed using primers that amplify the merged region of RPA43fragmentand natgene to verify the site of insertion

Yeast colony PCR

To conduct yeast transformation competent cells were made from relevant haploid yeast strain using Frozen EZ Yeast Transformation II Kit from Zymo Research Corporation, according to the manufacturer’s instructions.To conduct plasmid shuffling in the NOY222, NOY222rpa34Δ and NOY222rpa34ΔRPA43S/322/323/325/A strainsto generate native and S/A mutant versions of the RPA190gene, the competent cells were transformed with either pRS314RPA190 or pRS314 RPA190S1413/1415/1417/A harbouring wild type a mutant versions of RPA190 generespectively,and transformants were selected by growing them on yeast nitrogen base containing leucine, G418 (200 g/mL) and canavanine (6 g/mL) but lacking uracil. The resulting strains have chromosomal deletions of RPA34 and RPA190 and harbour either wild type or mutant versions of RPA190on pRS314 plasmids

genomic mutation on the A43 protein coding gene RPA43was inserted in the wild type BY4741 and NOY222 rpa34strains. Plasmid pGP5 RPA43, gifted by Dr.Herbert Tschochner, and a plasmid harbouring the nourseothricin N-acetyltransferase (nat1) gene(Goldstein and McCusker, 1999)were used for a PCR based site directedmutagenesis to create a DNA fragment to generate a genomic mutant of RPA43 (Fig

with 0.01% NP 40 in water, andwere diluted serially and plated on rich medium (YPD). RPA34in the genome was replaced with kanMx4that provides resistance toG418 and RPA190gene deletion was done by replacing it with URA3gene that supports growth of the strain in the absence of uracil in the medium. pNOY20, a shuffle plasmid harbours a LEU gene which supports yeast growth in the absence of uracil. Therefore germinated spores were streaked on a selection mediumlacking leucine and uracil and containing 200 g/mLG418to select the strain containing double mutation. The genotype of the strain was confirmed by growing the straindifferent combinations of media such as SC-Ura, SC-Leu, SC-Trp, SC-Met and SC-His. The procedure to generate RPA190 genome deletion in the background of rpa34Δ(NOY222 rpa34) was conducted by a colleague, Ms. Sitalakshmi Thampatty, in thelaboratory

TheS. cerevisiaestrains used were BY4741 rpa34and NOY222 (Table 2.1) (Gerberet al., 2008).NOY222 harbours agenomic deletion of RPA190 with the RPA190encoded on pNOY20, a shuffle plasmid,as the genomic deletion is lethal. BY4741 rpa34(MATa), and NOY222(MATα)were mated by mixing these two strains and patched on the YPD agar. The resulting diploids were sporulated on yeast sporulating medium at 25°Cfor 2-3 weeks.Thespores were treated with 5U zymolyase and sonicated briefly in a bath sonicator to release the spores. The free spores were washed

Generation of yeast mutants

The pyrophosphorylation reaction was performed with proteins on beads in presence ofIP7 reaction buffer(Werneret al., 2010). 10X IP7 reaction buffer (Section2.1.6.7)was prepared, aliquoted andstored at 4°C. For the reaction, 30 μLof purified protein on GSH beads(1:1 beads in PBS suspension), 3.5μLof 10X buffer and 1 μCi of 5[β-32P]IP7were added, and made up to afinal volumeof 35 μL, and incubated at 37°C for 15 min. A 50 μLreaction was performed for proteins with low expression levels such as A190. To the reaction mix, 4X LDS sample buffer (Invitrogen) was added to a final concentration of 1X and incubated at 95°C for 5 min. The reaction mix was centrifuged at high speed and resolved on a 4%-12% gradient gel by Nu-PAGE(Invitrogen) using 1X MES buffer (Invitrogen). Proteins were transferred to a Hybond-P membrane (GE Lifescience) and the radiolabelled proteins were detected using a phosphorimager (Fuji Film FLA-9000). The membrane was blocked with 5% non-fat dry milk (Rockland)in 1X PBST(pH 7.4)for 2 h at room temperature followed by washes with 1X PBST at room temperature for 10 min three times.Proteins were detected by western blot using a rabbit anti-GST antibody. 1:5000 dilution of anti-GST antibody in 1X PBST containing 0.2% BSA, was added and incubated overnight at 4°C on a rotating platform. The membrane was washed in 1X PBST for 10 min three times, followed by incubation with HRP conjugated goat anti-rabbit IgG at 1:20,000 dilution in 5% non-fat dry milk (Rockland)in 1X PBST, for 1 h at room temperature. Membrane was washed with 1X PBST at room for 10 min three times.Protein bands were detected by using Super Signal West pico chemiluminiscence substrate (Perkin Elmer)

IP7-mediated phosphorylation reaction

After the HPLC run, vials numbered from 50 to 65 were surveyed using a Geiger counter, 4 vials with high counts were pooled into one vial and 5 μL of this 4 mL solution was measured in a liquid scintillation counter (Perkin Elmer). To the remaining solution, 800 μL of 50% ammonia solution was added to neutralise the pH and the tube was kept on ice. In a 50 mL conical tube, 45 mL of chilled water was taken, to which the neutralised IP7solution was added and kept on ice. A Sep-Pak cartridge [Waters, WAT020545] was equlibrated with 10 mL of ice cold deionized water using a 10 mL syringe. The Sep-Pak cartridge was attached to a 60 mL syringe and 50 mL of diluted IP7 solution was passed slowly through ot so that IP7would bind to the Sep-Pak column. The cartridge was washed with 8 mL of chilled water, followed by chilled 8 mL 0.2 M triethylammonium bicarbonate solution pH 8.5 (4 mL of 1.5 M triethylammonium bicarbonate, pH 8.5 + 26 mL chilled water). The bound IP7 was eluted in 4 mL of 1.5 M triethylammonium bicarbonate solution, pH 8.5, into three 1.5 mL microfuge tubes. The eluted IP7 was concentrated in a vacuum concentrator (Scanvac) at 2000 rpm, 25°C, to obtain 30-50 μL of a 1-2 μCi/μL solution of radiolabelled IP7

Purification of IP7 by anion exchange cartridge

of ice cold deionized water (water was kept in the cold room overnight) was added to a 250 mL volumetric flask which was placed on ice. To the water, 52.1 mL of triethylamine solution (~7.2 M, Cat. No-T088, Sigma-Aldrich) was added with the help of a glass measuring cylinder, and mixed well. The volume of the solution was made up with ice cold water till the mark on the volumetric flask, and the flask was mixed well. A pH meter (Eutech instruments 510) was calibrated using pH 7.0 and pH 10.0 solutions (Eutech instruments 510). Approximately 100 mL of 1.5 M triethylamine solution from the volumetric flask was added to a 250 mL conical flask that was placed on ice. A magenetic bead was placed in the flask and the ice bucket was placed on the magnetic stirrer. The pH measuring probe was immersed into the solution, and CO2was bubbled through the triethylamine solution and stirred until the pH reached 8.5. The conical flask was covered with paraffin film and kept on ice until the solution wasused (within 1-4 h)

During the HPLC run,a triethylammonium bicarbonatesolution(1.5 M) was prepared. Triethylamine and water mixture generates heat, and therefore, approximately 100 mL

Preparation triethylammonium bicarbonate solution

On the HPLC system (Waters Empower Software), the instrument method was set to the programme ‘tritium small coloumn’ and the method set was set to ‘tritium small column’. The ‘set up’ icon was selected and once the flow rate was 1 mL/min, the ‘prepare’ icon was selected. A gradient was generated by mixing buffer A and bufferB as described below (Section 2.1.6.6). The injector was moved to the load position and sample was injected using a 1 mL syringe. The injector was moved to the inject position and the ‘inject’ icon was pressed immediately. On the fraction collector, the ‘run’ button was pressed immediately. The syringe was rinsed 5 times with buffer A

HPLC set upA strong anion exchange partisphere SAX 4.6 mm diameter and 125 mm length column (Whatman) was fixed to the HPLC system (Waters 515 pumps). The column was equilibrated with buffer A (1 mM EDTA) (Section 2.1.6.6) overnight at 100 μL/min flow rate.Before starting the HPLC run, the fraction collector (BioRad 2110) was prepared by placing the outlet tube to vial 1 on the fraction collector. Fresh FACS tubes were placed at fraction numbers 40-65 on the fraction collector. The flow rate on the fractioncollector was set to 1 mL/min and it was kept ready.The SAX column was allowed to equilibrate (warmup programme) with Buffer A by slowly increasing theflow rate from 0.1 to 1 mL/min over a period of 70 min

Purification of radiolabelled IP7by HPLC method

The reaction mix was mixed well using a 200 μLpipette and 20μLeach was dispensed into eight 1.5 mL microfuge tubes. To this reaction mix 5μLof purified hexa histidine tagged IP6K1 enzyme (2-3 μg) was added. Tubes were placed in an acrylic box and incubated at 37°C overnight in a hybridization oven. The next morning, reactions were pooled into two 1.5 mL microfuge tubes containing 100 μLeach.100μLof 0.6 M perchloric acid was added to neutralise the reaction, the tubes were kept on ice for 1 min, and 33.5 μL of 1 M potassium carbonate with 5 mM EDTA was added and mixed by gentle tapping. CO2was liberated leaving a white precipitate, while tubes were kept open on ice for 1 h with gentle tapping at 15 min intervals.Tubes were centrifuged at 12000 x gfor 2-5 min and the supernatant from each tube was pooled into a new 1.5 mL microfuge tube

to hold 1.5 mL microfuge tubes.Acrylic shield was used through out to block the β radiation

Synthesis of 5[β-32P]IP7was conducted as described earlier (Azevedoetal., 2010). An acrylic box was placed at a 37°C in a hybridization oven before settingup the reaction

Synthesis of radiolabelled 5-IP7

GST fusions of yeast RNA Pol I subunits were purified as described in (Werneret al., 2010). Yeaststrainsover expressing GST tagged RNA Pol I proteins were grown overnight at 30°C in 10 mL of SC-Ura medium with 2% glucose medium. Cells were pelleted, washed in SC-Ura with galactose. Protein expression was induced by transferring the entire pellet into200 mL of SC-Ura with 2% galatose to give a final OD600of 0.8-1.0. For proteins A190 and A43 that express at very low levels, the overnight culture volume and induction volume were doubled. Cells were cultured at 30°C harvested at 3.0-5.0 OD and washedwith ice cold water. The cell pellet was suspended in 5 mL of ice cold Buffer A (Section 2.1.6.7), 750 μL ofcell suspensions were aliquoted into 1.5 mL microfuge tubes and to this 500 μL glass beads were added. Cells were lysed by bead beating using a vortex mixer (VortexGenei -2 with mix-mate attachment), and the lysate was centrifuged at high speed for 15 min at 4°C. Supernatants were dispensed into a 15 mL conical tube and Triton X-100 was added to a final concentration of 1%. Pre-swollen glutathionebeads were washed in Buffer B(Section 2.1.6.7)from which 200 μL of 1:1 bead suspension was added to approximately 5 mL of A34 and A43 expressing cell lysate and 100 μL of 1:1 bead suspension was added to 5 mL of A190 cell lysate and incubated for 2 h at 4°C on a rotary mixer. Lysates were centrifuged at 5000 xgfor 2 min and the beads were washed with ice cold Buffer C (Section 2.2.6.6) twice. Beads were further washed with ice cold Buffer B followed by ice cold 1X phosphate buffered saline (PBS) twice. Beads were suspended in an equal volume of 1X PBS with protease inhibitor cocktail (Sigma)

Protein expression and purification from yeast

recovery, cells were centrifuged at 2,500x gfor 4 min. The medium supernatant was discarded and cells were resuspended in 200 μLfresh sterile LB medium. Cells were plated on LB agar medium containing appropriate antibiotics. Plates were incubated at 37°C for 12-16 hto allow growh of individual colonies

E. coli DH5α strain was transformed with yeast plasmids carrying appropriate inserts that express S.cerevisiaeproteins (Sambrook and Russell, 2001). Ultracompetent cells stored at -80°C were thawed on ice for 5-10 min. 20μLligated plasmid was added to 100 μLultracompetent cells,and the cells were incubated on ice. After 30 min, competent cells were subjected to heat shock at 42°C for 90 seconds. Cells were immediately transferred to ice for 2-3 min. Next, 900 μLLB medium was added and cells were allowed to recover for 45 min on a shakerincubator set at 37°C. After the

Bacterial transformation

A single colony of E. coli DH5α strain was inoculated in 10 mL LB medium and incubated at 37°C overnight. Overnight culture was subculturedin 250 mL of LB medium of about 0.1 OD and incubated at 18°Cfor36 htillthe OD600reached approximately0.5. Cultures were centrifuged at 2,500 x gfor 10 min at 4 ̊C and harvested cells were washed gently with 80 ml ice-cold Inoue transformation buffer(Section 2.1.6.2). Cells were collected by centrifugation at 2,500x g for 10 min at 4 ̊C and gently resuspended in 20 mLice-cold Inoue transformation buffer. To this cell suspension, 1.5 ml sterile DMSO was added and swirled gently. The cell suspension waskept on ice for 30min,and 100 μLvolume was aliquoted into pre-chilled sterile microcentrifuge tubes. Cells were immediately snap-frozen in liquid nitrogen and stored at -80°C

Preparation of E. coli DH5α ultracompetent cells

and A190middle (557-1100 a.a)fragments were amplified from the plasmid pRS314RPA190gifted byDr. Herbert Tschochner with primers containing BamHI and XhoI (Table 2.4).The mutant versions of A34 and A190 fragments were generated by overlap extension PCR based method. p416GPD GST-RPA43 used in ChIPwas generated by extracting the GST-RPA43fragmentsfrom pYesGex6p2 GST-RPA43by restriction digestion with HindIII and XhoIfollowed by ligation into p416GPD. Clones were verified by bacterial colony PCR, sequencing and western blot. For colony PCR a small amount of bacterial colony was taken with the help of tooth pick and touch the bottom of the 0.2 mL PCR tube to which PCR reaction mix was added and performed PCR

The nomenclature system for RNA Pol I subunits, gene and protein name is given in Table 2.4 and The S.cerevisiaeRNA Pol I subunits Uaf30, A34.5, A43 full length and fragmentsof A135 (1-112 a.a) and A190 (1101-1664 a.a) were PCR amplified from genomic DNA of the wild-type strain using high fidelity DNA Taq polymerase, with primers carrying restriction sites for BamHI and NotI or BamHI and XhoI (Table 2.4). Amplified fragments were cloned downstream of the GAL4promoter in the pYesGex6p2 plasmid (Werneret al., 2010).These plasmids were generated by a colleague,Mr. Unnikannan CP,in the laboratory. Fragments and mutant versions of full length RNA Pol I subunits A34.5 and A43 weregenerated using above plasmids as templates (Table 2.3).A190 N-terminal (1-556 a.a)Calculation of adjusted input-Volume of cell lysate: 500 uL-Volume set aside as input: 10 uL, eluted into a final volume of 40 uL-Volume of lysate taken for IP: 490 uL; eluted into 100 uL after IP, of which 90 uL was eluted into a final volume of 40 uL. Therefore, volume of lysate for IP corresponds to (90/100)*490 = 440 uL (approx. 500 uL)1μLof input sample, and 3 μLof immunoprecipitated sample was taken from40μLof eluted volumes, for q-PCR reaction.The input sampletaken for q-PCR = 1/40x10 uL= 0.25 uLThe immunoprecipitated DNAtaken for q-PCR = 3/40x 500=37.5uLTherefore, the Ct values obtained for input samples (Ct input) were from 0.25 μL out of 37.5 uLoftotal lysateAdjusted Ct=[Ct(Input)-log2(input dilution factor)]The dilution factor forinput sample was 37.5/0.25 = 150Adjusted Ct = [Ct(Input)-log2(150)] = Ct(Input)-7.23Table. 2.6 The gene name and correspondingprotein name for RNA Pol Isubunits.RPA represents RNA polymerase I and protein name starts with A which represents that the subunit is present in RNA pol I

Cloning of S.cerevisiaeORFs

Calculation of adjusted input-Volume of cell lysate: 500 uL-Volume set aside as input: 10 uL, eluted into a final volume of 40 uL-Volume of lysate taken for IP: 490 uL; eluted into 100 uL after IP, of which 90 uL was eluted into a final volume of 40 uL. Therefore, volume of lysate for IP corresponds to (90/100)*490 = 440 uL (approx. 500 uL)1μLof input sample, and 3 μLof immunoprecipitated sample was taken from40μLof eluted volumes, for q-PCR reaction.The input sampletaken for q-PCR = 1/40x10 uL= 0.25 uLThe immunoprecipitated DNAtaken for q-PCR = 3/40x 500=37.5uLTherefore, the Ct values obtained for input samples (Ct input) were from 0.25 μL out of 37.5 uLoftotal lysateAdjusted Ct=[Ct(Input)-log2(input dilution factor)]The dilution factor forinput sample was 37.5/0.25 = 150Adjusted Ct = [Ct(Input)-log2(150)] = Ct(Input)-7.23

PCR reactions were set up with the immunoprecipitated DNA by using the primers thatamplify the 5’ETSand promoter regions. Real-time PCR was performed using Mesa Green 2X PCR MasterMix (Eurogentec)in a 20 μL reactionvolume using 1 μL from the input sample and 3 μL from the immunoprecipitated samplein an RT-PCR mechine (Applied Biosystems). Ct values of the immunoprecipitated samples were normalised to the adjusted Ct values of the inputsample, andthedata were plotted as ‘immunoprecipitated DNA as a percentage of input DNA (% Input)’, as described in (Haringet al., 2007; Livak and Schmittgen, 2001).Calculation of %InputThe amount of DNA taken for q-PCR analysis is inversely proportional to the Ct value% Input = 2-Ct(IP) / 2-Ct(Input)x 100, (% of gDNA immunoprecipitated from the total amount of gDNA taken)Ct(IP) = Ctvalue of immunoprecipitated DNACt(Input) = Ctvalue of Input DNAIn this experiment,10 μL of this lysate was taken as input and this has to be adjusted to the total volume of the lysate. Therefore, the formula would be,

Quantitative PCR (q-PCR) reaction

Immunoprecipitation of chromatin was performed by incubating the lysate with 3 μg of anti-GST antibody overnight at 4°C followed by50 μL of 1:1 suspension of Protein A beads for 4 h. Beads were washedtwice each in wash buffer I, wash buffer II, and TE buffer(Section 2.1.6.5). Each wash was performed for 15 min at 4oC by rotating on a Lab-net end over mixer. Chromatin was eluted in 100 μL of elution buffer(Section 2.1.6.5) by rigorous mixing on a Thermo mixer (Eppendorf) for 30 min at 65oC. 90 μLof this eluted sample was incubated overnight at 65oCto reverse the cross linking. 10 μLlysate taken as input was diluted to 90 μLwith elution buffer and incubated overnight at 65oC to reverse the crosslinking.DNA was extractedfrom the input and immunoprecipitated samplesintoafinal volumeof40μLusing aPCR purification kit (Qiagen)

Yeast strains carrying p416GPD GST-RPA43were grown in SC-Ura medium overnight and sub cultured at 0.2 OD600.45 mL of mid-log phase yeast cultures were subjected to cross linking with 1% formaldehyde for 15 min at room temperature(Szijgyartoet al., 2011). Cross linking was quenched by adding glycine to a final concentration of 0.1 M. Cells were washed in ice cold Tris-buffered saline and were lysed in 500 μL of ice cold lysis buffer (Section 2.1.6.5) by bead beating. Chromatin was fragmented using a Diagenode bath sonicator, 15 sec on time and 30 sec off time, for 15 min. Cell lysates were centrifuged at high speed and the supernatant was pre-cleared with 3 μg of normal rabbit IgG followed by30 μL of 1:1 suspension ofProtein A beads. Supernatant was collected and 10 μL of this lysate was taken as input

Chromatin immunoprecipitation(ChIP)

The desired S.cerevisiae strain was grown overnight in YPD liquid medium and yeast cells were harvested by centrifugation at 2,500x gin 15 mL polypropylene tubes. Yeast cells were washed with PBS, suspended in 500 μL lysis buffer (Section 2.1.6.2) and were transferred to a 1.5 mL microcentrifuge tube. Yeast cells were incubated for 15 min on a thermomixer set at 65 ̊C and 750 rpm.After incubation, a volume equivalent to 500 μLof glass beads (0.5 mm) were added and cells were lysedon a beating apparatus by mixing three times for 45 seconds each with intermittent cooling on ice to prevent overheating.Cell lysates were centrifuged at 12,000 x gfor 5 min and upper aqueous phase was transferred carefully to a new 1.5 mLmicrocentrifuge tube, to which 275 μL of 3M sodium acetate was added. To this solution, 500 μL of chloroform was added, mixed well, and centrifuged at 12,000 x gfor 5 min at 4°C (this step was repeated twice). Supernatant was transferred to a new 1.5 mLmicrocentrifuge tubeand500 μL of isopropanolwas added and mixed well by inverting the tube 3-4 times. To precipitate genomic DNA, the suspension was centrifuged at 12000 x gfor 15minat 4°C. Precipitated genomic DNA was washed with 70% ethanol and dried at room temperature. The genomic DNA pellet was dissolved either in 100μLof Sigma molecular biology grade water or TE buffer supplied with Qiagen plasmid mini prep kit, andadd 1 μL of RNase solution (30 mg/mL) was added to this and incubated at 37oC for 1 h. The extracted genomic DNA was checked for integrityon a 0.8%agarose gel by electrophoresis and stored at -20oC

Genomic DNA isolation by glass bead lysis method

After 14-16 h incubation, hybridization buffer was decanted to a radioactive liquid waste container. Membranes were washed twice with 2X SSC (saline-sodium citrate) containing 0.1% SDS for 15 min at 55°C followed by two washes with 1X SSC containing 0.1% SDS for 15 min at room temperature. Post washes, membranes were rinsed with 1X SSC buffer at room temperature exposed to a phosphorimager screen for 1 h and scanned using a phosphorimager (Fuji Film FLA-9000). The data were analysed by densitometry using Fuji Film Multigauge software V3.11 and graphs were plotted using GraphPad Prism5 software.Note:Depending on signal saturation or non-specificity, high stringency washes were performed starting from 0.5X SSC followed by 0.2X SSC or 0.1X SSC wash buffers containing 0.1% SDSat room temperature

Post-hybridization washes

Yeast were grown in appropriate medium while the logarithmicphase and 1 OD600cells wereharvested and chilled on an ice(Elion and Warner, 1986). Allsubsequent steps,unless specified, werecarried out at4°C or on ice. Cells were collected by centrifugation at 2500 gfor 6min and washed with2 mLof TMN (Section 2.1.6.4). Cells were suspended in 1 mL of ice cold permeabilization buffer (Section 2.1.6.4) and incubated for 15 min.Cells were pelleted and incubated with 100μLof transcription assay buffer (Section 2.1.6.4) containingradiolabelled [α-32P]UTP. After incubation for 10 min at30°C and 300 rpm in a shaking drybath (Eppendorf),1mLof cold TMN containing1 mM nonradioactive UTP was added, the cells were collected by centrifugation, and RNA was prepared by the hot phenol methodas described in Section 2.2.8. Equal counts of labelled RNA were used for hybridization. The membrane was pre incubated with 50 mL of hybridization buffer for 20-30 min, and hybridization was performed with labelled RNA at 65°C for 15 h in 20 mL of hybridization buffer

Transcription run on analysis

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

Cellswere grown in YPD to an OD600of 0.5-0.7. Cells equivalent to 1OD600were washed with synthetic complete medium without uacil twice and suspended in SC-Ura containing 3 μCi/mLof [14C]uracil for 5 min. Cells were pelleted and washed with SC-Ura medium twice and suspended in 0.5 mLof AE solution(Section 2.1.6.2). 1OD600of this cell suspension was counted in a liquid scintillation counter (Perkin Elmer-Tricarb 2900). The cpm values obtained were and converted into moles based on thespecific activity of [14C] uracil and plotted using GraphPad Prism5

Uracil uptake assay

labelled RNAs to N+Hybond membrane (GE Life Science), the radiolabelled rRNA was detected using a phosphorimager (Fuji Film FLA-9000)

Overnight grown yeast were sub-cultured at 0.2 OD600and growntill 0.8 OD600.Cells equivalent to 1 OD600were harvested and were washed in SC-Ura medium, suspended in 1 mL of SC-Ura medium containing 3μCi/mL of [14C]uracil and pulselabelledfor 15 min at30°C.After incubation the cells were pelleted and a chase was performed by adding SC medium containing 240 mg/L unlabelled uracil. Samples were taken at 0, 1, 5, 10and 20min after the chase, centrifuged at 12000 x gfor 1 min at 4°C, and total RNA was isolated from cells by the hot-phenol techniquedescribed in Section 2.2.8.Equal total RNA was loaded on a 1.2% formaldehyde-agarose gel. After transfer of

Pulse-chase analysis of rRNA synthesis

Overnight grown yeast were sub-cultured at 0.2 OD600and growntill 0.8 OD600. Cells equivalent to 1 OD600were harvested and washed twice with SC-Uramedium to remove any residual uracil from the cells. Cells were incubated in SC-Ura medium containing 3 μCi/mL [14C]uracil for 1, 5, 10 and 20 min, and RNA was extracted as described in Section 2.2.8. Equal total RNA was resolved on a formaldehyde agarosegel and transferred to an N+Hybond membrane (GE Life Sciences). Radiolabeled rRNA was detected using a phosphorimager scanner (Fujifilm FLA-9000)

[14C]uracillabelling of total RNA

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 mixture of 40 mL of DEPC treated water and 0.6 gof agarose was meltedby boiling. After cooling down the temperature to 55°C, 8.4 mL of formaldehyde (final concentration 2.2 M)and 5 mL of 10X MOPS were added, mixed well and poured into a boat to cast a gel

Preparation of formaldehyde agarose gel(50 mL) and RNA sample

Yeast were grown till mid-log phase 0.6-0.8 OD600in an appropriate medium and 10 mL of culture was pelleted at 2500 x g. Cells were suspended in 350μLof AEbuffer,mixed with 50μLof 10% sodium dodecyl sulphate and 400μLacid phenol(pH4.3) and immediately shaken vigorously on a dry bath (Eppendorf)at 65°C for 15 min.The tube was then quickly chilled on iceand centrifuged at 12000 x gfor 15 min to separate the aqueous phase from the phenol. After centrifugation, the aqueous phase was transferred to a new tube and extracted with an equal volume of chloroform.RNA was precipitated by adding 50 μLof 3 M sodium acetate (pH 5.3) and equal volume of 100% ethanol followed by incubation at -20°C for 2 h, andcentrifugation at maximum speed for 30 min at 4°C. The pellet obtained was washed in 70% ethanol, dried at room temperature anddissolved in an appropriate volume of DEPC-treated water. The concentration of RNA was estimated by measuring A260using a Nano Drop Spectrophotometer (ND1000). To monitor different classes of rRNA levels, 10 μg of total RNA from each strain was resolved on a 1.2% formaldehyde-agarose gel

RNA extraction by hot-phenol method

placed on ice.The samples were centrifuged at 10,000x gfor 10 min at 4°C and the supernatant was extracted by avoiding the glass beads. Absorbance of thelysatewas measuredat 260 nm, and it was divided into 200 μL aliquots and frozen at -80°C. No difference in the ribosome profiles were detected between thesesamples and fresh samples analysed immediately.Polysomes were analysed by centrifugation through a 10-50% sucrose continuous gradient. The gradient was prepared by pipetting, gradient buffers (Section 2.1.6.3), 5 mLof each layer onto the bottom of an 11 mLopen top centrifuge tube, covering the tube with paraffin filmand by placing it horizontally on a flat surface at 40C for 2 h. The ribosome sample (cell lysate) was loaded on top, and gradient was centrifuged at 100,000x gat4°C for 6 h in an SW41 rotor (Beckman). Anamount of lysate equivalent to 10 A260units was loaded on the gradient. Ribosome levels were measured by the gradient analysis with an ISCO UV-6gradient collector with continuous monitoring atA254.Polysomepurification was performed by layering the cell lysate on 37% sucrose solution in an 11 mLopen top centrifuge tube, and centrifuged at 100,000x gfor 14 h in an SW41 rotor(Beckman). The pellet was suspended in 50 to 100 μL of lysis buffer. An amount oflysate equivalent to 0.7A260unitswas loaded on the gradient. Ribosome levels were measured by gradient analysis with an ISCO UV-6gradient collector with continuous monitoring atA254.Ribosome subunits were analysedby centrifugation through a 10-30% sucrose continuous gradient. The gradient was prepared by pipetting 2.2 mLof each layer onto the bottom of a 5 mLopen top centrifuge tube, sealing it with paraffin filmand placing it horizontally on a flat surface at 4°C for 2 h.The ribosome sample wasloaded on top, and gradient was centrifuged at 100,000x gand 40C for 4.5 hin an SW55rotor (Beckman). Ribosome subunit levels were measured by gradient analysis with an ISCO UV-6gradient collector with continuous monitoring atA254.Ribosome profiles with yeast cell lysates and purification of ribosomes from yeast were performed with the help of Mr. Aluri Srinivasand Dr. Umesh Varshney

The method to analyse ribosome profiles was adapted from (Leeet al., 1992). Yeast cells were grown to 0.2 to 0.8 OD600at 30°C in 200mLof YPD,and cycloheximide was added to this media at 50μg/mLfinal concentration. The culture was placedand mixed continuously onanice and salt mixture for 2-5 min and centrifuged immediately at 4,000 x g. The culture was not allowed to stay for a longer time on the ice and salt mixture to avoid freezing. The cell pellet wassuspended in 1mLof lysisbuffer (Section 2.1.6.3),andtransferred to a2 mL microfuge tube, to which1mLglass beads (0.45-0.6mm diameter)were added, and lysed by bead beating for 10 min with intervals (30 sec on time and 1min off time).During the 1 min off time, tubes were

Ribosome profiles

Yeast weregrown in YPD (Difco) overnight,and sub-cultured at 0.2 OD600. Cells were harvested at 0.6-0.8 OD600. 1 OD600of each culture was used for the labelling. Cells were washed in SC-Metmedium twice, suspended in SC-Metmedium containing 25μCi/mLof 35S Met-Cys and pulsed for 15 min. Cells were washed twice in methionine-free medium and suspended in 300Lof Tris-saline. Cell suspension was counted in a liquid scintillation counter (Perkin Elmer-Tricarb 2900). The cpm values obtained were plotted using GraphPad Prism5

35S-Met uptake assay

growntill the OD600reached 0.8-1.0. Cells equivalent to 1OD600of each culture was taken for the labellingof total protein. Cells were washed in methionine-free synthetic complete medium(SC-Met) twice, suspended in SC-Met mediumcontaining 25μCi/mLof 35S Met-Cys twin label mixand incubatedfor 1min, 5 min and 15min. Cells were washed twice in ice-cold SC-Met medium twice and suspended in 500 μL of Tris-salinecontaining protease inhibitor cocktail.To this,300 μL ofglass beads (0.45-0.6mm diameter)were added and cells were lysed for 10 min by bead beating (with intervals of 1 min on time and 30 sec off time).The lysate was centrifuged at high speed for 15 minat 4°C. To the supernatant,sodium deoxycholate was added to a final concentration of 0.1 mg/mLand incubated on ice for 30 min. To this solution, 20% trichloroacetic acid was addedto a final concentration of 6%, incubated for 1 h on ice, and centrifugedat high speedfor 20minat 4°C. The pellet was suspended in 300 μL of Tris-saline and counted inaliquid scintillation counter (Perkin Elmer-Tricarb 2900). The cpm values obtained were plotted using GraphPad Prism5

Wild type and knock out yeast strains were grown in YPD (Difco) whereas synthetic complete medium without uracil was used for the KCS1 complementedstrains. Overnight grown yeast were subcultured in appropriate medium at 0.2 OD600and

Protein synthesis analysis

Analysis of sensitivity to translation inhibitors was conducted in theDDY1810 S. cerevisiaestrain background, whichdoes not contain the kanrselection marker. Sensitivity to 6-azauracil (6AU) was monitored in the DDY1810, BY4741 or NOY222 strain backgrounds (Table 2.1). As uracil is a competitive inhibitor of 6AU, the plasmid p416GPD, carrying the URA3gene (Mumberget al., 1995)was introduced into BY4741-derived strainswhereas DDY1810 derived yeast strains were supported by the pYesGex plasmid carrying the URA3gene.Yeast strains were grown in YPD or SC-Ura medium,for 14-16 h at 30°C under continuous shaking at 200 rpm. Cultures were diluted to 0.25OD600, followed by 5 fold serial dilutions, and 3μL of each dilution was spotted on a YPD-agar plate containing the translation inhibitors G418 (8 μg/mL), paromomycin (100μg/mL or 200μg/mL), or hygromycin B (8 μg/mL), or an SC-Ura agar plate, containing 6AU 50 μg/mL or100 μg/mL and growth was monitored at 30°C or 37°C for 2-3 days. To perform an analysis of 6AU sensitivity with yeast carrying pYesGex6p2 plasmid, cells were grown overnight in SC-Uramedium and the serial dilutions were plated on SC-Ura medium containing 6AU, with galactose instead of glucose to express proteins under the GAL4promoter

Drug sensitivity analysis

To monitor yeast cell death 1 OD600equivalent cellsfrom mid-log and overnight growncultures were pelleted, washed in PBS and the cell pellet was suspended in 100 μL of PBS. 20 μLof 0.4% trypan bluesolution was added to 20 μLof cell suspension and incubated for 10 min. 20 μL of this suspension was placed on a slide, covered with a cover slip, and cell death was measured by scoring dead cells that take up the dye.To monitor cell viability, cells equivalent to 10-5OD600 from mid-log and stationary phase cultures were plated on YPD-agar, incubated at 30°C for 48 h, and colonies were counted to extrapolate viable cell count per OD600

To determine yeast cell mass, cells equivalent to 5 OD600were harvested from mid-log and overnight growncultures, and washed twice with PBS. Cell pellets were dried at 50°C for 20 minand the dry weight of yeast was measured. To assess the cell number, cells in mid-log or stationary phase were counted using a Neubauer chamber and the number of cells present in 1 OD600was calculated

Cell mass, cell numberand viability assessment

For growth analysis of S. cerevesiae strain, single colony was inoculated in appropriate broth medium and grown over night. This culture was used to inoculate the test medium to an initial OD600 of 0.1. Cultures were transferred to a shaker incubator set at 30°C and 200 rpm. Absorbance of cultures was measured using Ultraspec 2100 pro UV/visible spectrophotometer (Amersham Biosciences) at 600 nm at regular time-intervals till 72 h. Absorbance values were plotted with respect to time and the generation time was calculated from the logarithmic phase of the growth curve, by plotting A600vs.timeon a semi-logarithmic scale, using GraphPad Prism5 for curve fitting analysis

Growth analysis and determination of generation time

S. cerevisiae strains were routinely grown either in rich YPD mediumorsyntheticcomplete medium (SC)(Section 2.1.5.1) at 30°C with continuous shaking at 200 rpm unless otherwise stated. In general, S. cerevisiae frozen glycerol stocks were revived on 2% YPD medium by streaking and allowed to grow for 1-2 days. S. cerevisiae strains harbouringaplasmid containingthe URA3geneas the auxotrophy selectionmarker were revived on synthetic complete medium lacking uracil (SC-Ura).To prepare liquid cell culture, a single colony of each S. cerevisiae strain was inoculated either in YPD or SC-Ura medium and grown for 14-16 h. S. cerevisiae strains streaked on plates were sealed with paraffin film (parafilm M) and stored at 4°C for a maximum period of 2 weeks.Protein over expression in yeast was carried in presence of galactose instead of glucose as the carbon source, as the plasmid pYesGex 6p2 carries the GALpromoter under which yeast proteins were expressed

Strains and culture conditions

0.83mL1.5 M Tris-HCl,pH 6.8 50μL10% SDS 50μL10% Ammonium persulfate (APS)8 μLN,N,N′,N′-Tetramethylethylenediamine (TEMED)Resolving gel mix (12%) (20 ml)6.6 mLH2O 8 mL 30% acrylamide:bisacrylamide (29:1) mix 5 mL1.5 M Tris-HCl,pH 8.8 200 μL10% SDS 200 μL10% Ammonium persulfate (APS)8 μLN,N,N′,N′-Tetramethylethylenediamine (TEMED)

Whole cell lysis buffer for yeast (Homogenizing buffer) 50 mM Tris-HCl,pH 7.52 mM EDTA yeastprotease inhibitor cocktail SDS-PAGE 30% Acrylamide solution 29 g acrylamide 1 g bis-acrylamide dissolved in 100 mLH2O. 10% sodium dodecyl sulfate (SDS) 10 g SDS in 100 mLH2O Stacking gel mix (6%)(5 mL)3.4mLH2O 0.63mL 30% acrylamide:bisacrylamide (29:1) mix

Buffers for SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis)

20 mM HEPES500 mM NaCl 2 mM EDTA1% Triton-XYeast protease inhibitor cocktail and phosphatase inhibitor cocktail (added fresh to the buffer C)IP7 reaction buffer(10X)250 mM HEPES,pH 7.4500 mM NaCl60 mM MgCl210 mM DTT (1 M stock was made separately, aliquoted into 100 μL and stored at -20oC).10X buffer was made and stored at 4oC. An appropriate amount was added to the reaction mix to get a final concentration of 1X.DTT was added fresh to the reaction buffer just before use

Buffer C

2 mM EDTA5 mM DTT1% Triton-XYeast protease inhibitor cocktail and phosphatase inhibitor cocktail (added fresh to the buffer B)

20 mM HEPES pH 6.8100 mM NaCl

Buffer B

20 mM HEPES pH 6.8100 mM NaCl2 mM EDTA5 mM DTTYeast protease inhibitor cocktail and phosphatase inhibitor cocktail (added fresh to the buffer A)

Buffer A

Buffers for protein purification and IP7reaction

Buffer A1 mM EDTAin HPLC grade water (Fisher Scientific)Buffer B 1 mM EDTA(NH4)2HPO41.3 M, pH 3.8171.6 g of (NH4)2HPO4was dissolved in 750 mL of HPLC grade water. pH was adjusted to 3.8 with 75 mL of H3PO4by continuous stirring and the volume was made upto 1000 mL.Both buffers were filtered througha0.22 μm filter (Millipore) using vacuume filter apparatus (Tarsons) and degassing was performed atleast for 20 min using a vacuume pump

Buffers for IP7 purification

Wash buffer II10 mM Tris-HCI,pH 8.01 mM EDTA250 mM LiCl0.75% NP-400.75% sodium deoxycholateProtease inhibitor cocktailElutionbuffer II50 mM Tris-HCl,pH 8.0 10 mM EDTA 1% SDS

Lysis buffer50 mM HEPES,pH 7.5140 mM NaCl1% Triton X-1000.1 % sodium deoxycholate1 mM EDTAProtease inhibitor cocktail (added fresh)Wash buffer I50 mM HEPES,pH 7.5500 mM NaCl1% Triton X-1000.1 % sodium deoxycholate1 mM EDTAProtease inhibitor cocktail

Buffers for chromatin immunoprecipitation

Volume was adjusted with water to 1 L and solution was sterilized by autoclaving.Pre-hybridization/hybridization buffer (Modified Church and Gilbert buffer)0.5 M phosphate buffer (134g of Na2HPO4.7H2O,4 mL of 85%H3PO4), pH7.27% (w/v) SDS10 mM EDTA Volume was adjusted to 1 L with DEPC treated sterile water. Buffer was aliquoted into 50 mL RNase free conical tubes (Corning) and stored in -20oC.Post hybridization wash buffersWash buffer 1 2X SSC 0.1% SDS Wash buffer 2 1X SSC 0.1% SDSWash buffer 3 0.5X SSC0.1% SDSBuffers were prepared with sterile DEPC treated water

TMN buffer10mM Tris-HCl, pH 7.45 mM MgCl2100 mM NaCl Permeabilization buffer950 μLof coldwater50 μLof 10% (wt/vol)sodium N-lauroyl sarcosineTranscription assay buffer(100 μL)50mM Tris-HCl, pH 7.4100mMKCl5mM MgCl21mM MnCl22 mM dithiothreitol 0.5mM ATP 0.25 mM GTP0.25mM CTP10mM phosphocreatine2.4 units creatine phosphokinase100μCi [α-32P] UTP (3,000Ci/mmol)Alkaline denaturing solution for DNA for membrane preparation0.5 M NaCl 0.25 M NaOH Volume was adjusted to 20 mLwith sterile water. Saline Sodium Citrate (SSC) buffer (20X) 3.0 M Sodium chloride 0.3 M Sodium citrate

Buffers fortranscription run on analysis

50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC37% sucrose solution

100mM NaCl30mM MgCl250μg/mLcycloheximide 200μg/mL heparin All the components were made in DEPC treated water.Gradient buffer10% sucrose gradient buffer50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC10% sucrose solutionTo analyse individual ribosome subunits, MgCl2 was eliminated from the gradient buffer.30% sucrose gradient buffer50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC30% sucrose To analyse individual ribosome subunits, MgCl2 was eliminated from the gradient buffer.50% sucrose gradient buffer50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC50% sucrose To analyse individual ribosome subunits, MgCl2 was eliminated from the gradient buffer.37% sucrose gradient buffer

Lysis buffer10mM Tris, pH7.4

Buffers for ribosome and polysome analysis

30%GlycerolMade in 100 mL.RNA sample loading buffer (10X)50% glycerol10mM EDTA 0.025% Bromophenol blue 0.025% Xylene cyanolInoue transformation buffer, pH 6.7(125 mL, prepared just before use)10 mM PIPES 15 mM CaCl2.2H2O 250 mM KCl 55 mM MnCl2.4H2O (1.361 g is dissolved in 10 mL of water separately)PIPES(0.307 g), CaCl2.2H2O (0.275 g) and KCl (2.325 g)were added to 80 mL ofsterile water while mixing with a magnetic stirrer and the pH was adjusted to 6.8with 1 N KOH. After attaining the appropriate pH, MnCl2solution wasadded slowly in aliquotes of 300 μL over 10 min,while stirring to avoidabrown precipitate.MOPS buffer(10X)0.2 M MOPS, pH 7.220 mM CH3COONa10 mM EDTABuffer was made in DEPC treated waterYeast transformation reagents1 M Lithium acetate 50% Polyethylene glycol2 mg/mLSalmon sperm carrier DNA Dimethyl sulfoxide (DMSO) Zymolyase cocktail buffer for yeast colony PCR 2.5 mg/mLZymolyase (ZymoResearch)1.2 M SorbitolZymolyase buffer was prepared in 1X PBS

Yeast lysis buffer for genomic DNA extraction50 mM Tris-HCl,pH 8.010 mM EDTA 150 mM NaCl 1% Triton-X 1% SDSAE buffer for RNA extraction50 mMSodium acetate,pH 5.31 mMEDTA,pH 8.0Solution was made in DEPC treated water. 0.2%diethyl pyrocarbonate (DEPC)was added to the water and stirred for 12 h. To remove DEPC,water was autoclaved twice. DNA sample loading buffer (6X)15.25 mg Bromophenol blue15.25 mg Xylene cyanol

Buffers for extraction and analysis of genomic DNA and RNA

EDTA (pH 8.0)186.1 g of EDTA.2H2O was dissolved into 800 mL of water stirredvigorously and the pH was adjusted with NaOH pellets. When the pH of the solution reached8.0 EDTA dissolvedcompletely and was made upto 1000 mL with water.Tris-HCl buffer (1M)121.1 g of Tris base was dissolved in 800 mLof water and pH was adjusted to 7.2 using concentrated HCl Tris-EDTA (TE) buffer 10 mM Tris-HCl, pH 8.01 mM EDTA Tris-Acetic acid EDTA (TAE) buffer 40 mM Tris base 1mMEDTApH was adjusted to 8.4with glacial acetic acid. TAE buffer was prepared as a 50X stock solution and used at 1Xconcentration.Tris-Saline20 mM Tris-HCl, pH 7.20.9% NaCl

PhosphateBuffered Saline (PBS) 137 mM NaCl 2.7 mM KCl 10 mM Na2HPO42 mM KH2PO4pH was adjusted to 7.3 using HCl and NaOH beforeautoclaving. PBS was prepared as a 10X stock solution and diluted to 1X concentration before autoclaving

Common buffers

Buffers and solutions

0.5% Yeast Extract 1% Tryptone 1% NaClLB-ampicillin plates LB medium 100 μg/mL ampicillin Media and solutions were sterilized either by routine autoclaving at 121°C and 15 psi for 20 min or by filtration through membrane of 0.22 μm porosity.For yeast and bacterial growth, plates were preparedby adding 2% to the medium before autoclaving

Luria-Bertani (LB) medium forbacterialgrowth

Yeast synthetic complete medium without leucine(SC-Leu)0.67% Yeast Nitrogen Base without amino acids 76mg/L His76mg/L Ura76 mg/mL Trp76 mg/mL Met2% DextroseYeast sporulating medium1% Potassium acetate0.05% Dextrose

Yeast extract Peptone Dextrose (YPD)1% Yeast extract2% Peptone 2% Dextrose Yeast synthetic complete medium(SC)0.67% Yeast Nitrogen Base with amino acids 2% Dextrose1.92 g/LYeast Synthetic Drop-Out media supplement without Uracil76 mg/L uracilYeast synthetic complete medium without histidine(SC-His)0.67% Yeast Nitrogen Base without amino acids 1.92 g/L Yeast Synthetic Drop-Out media supplement without histidine2% DextroseYeast synthetic complete medium without uracil(SC-Ura)0.67% Yeast Nitrogen Base without amino acids 1.92 g/LYeast Synthetic Drop-Out media supplement without Uracil2% DextroseYeast synthetic complete medium without methionine(SC-Met)0.67% Yeast Nitrogen Base without amino acids 380mg/L Leu76 mg/L His76mg/L Ura2% DextroseYeast synthetic complete medium without tryptophan(SC-Trp)0.67% Yeast Nitrogen Base without amino acids 380mg/L Leu76mg/L His76mg/L Ura76 mg/L Met2% Dextrose

Yeast media(Media composition was followed as described by Sigma product data sheet)

Media

Taq polymerase was from ThermoScientific. Plasmid DNA purification, PCR purification, gel extraction and reaction clean up kits were procured from Qiagen. Medium components for growth of S. cerevisiae,namely, YPD, yeast nitrogen base, and yeast nitrogen base without ammonium sulphate were purchasedfrom BD (Becton, Dickinson and Company, USA).Yeastsyntheticdropoutmediasupplementwithouturacil/histidinewereobtainedfromSigma-Aldrich

Agarose, phenol, dimethyl sulphoxide (DMSO), sodium acetate, sodium chloride, sodium carbonate, sodium bicarbonate, sodium dodecyl sulphate (SDS), formamide, calcium chloride, ethylenediaminetetraacetic acid (EDTA), glycerol, polyethylene glycol, ammonium persulphate, N,N,N′,N′-Tetramethylethylenediamine (TEMED), acrylamide,dithiothreitol (DTT),bis-acrylamide, chloroform, formaldehyde, lithium chloride, lithium acetate,isopropanol, nuclease free water, diethylpyrocarbonate (DEPC), Tween-20, acid washed glass beads, trisodium citrate dehydrate, β-mercaptoethanol, 0.4% trypan blue solution, yeast protease inhibitor cocktail, magnesium chloride, manganese chloride and phosphatase inhibitors were purchased from Sigma-Aldrich Chemicals. Agar, uracil, leucine, lysine, histidine, tryptophan, methionine, yeast extract, peptone, tryptone, and sorbitol were obtained from HiMedia. Dextrose, sucrose, potassium chloride, sodium hydroxide, hydrochloric acid, Tris and glycine were from Fisher Scientific. [14C]-labelled uracil was from Ogene Systems.γ[32P]ATP, [35S]Met/Cysin vivoprotein twin label mix, α[32P]UTP and Taq DNA polymease were from JONAKI/BRIT,Ultimaflow liquid scintillation fluid was obtained from Perkin-Elmer.Hybond-N+and Hybond-P membranes for nucleic acid and protein transferrespectively, and protein A beads were purchased from GE Life Science. NuPAGE gradient gels, MES running buffer and 4X LDS sample buffer were purchased from Invitrogen. Super Signal West pico chemiluminiscent substrate was from Thermo Scientific. Different restriction enzymes used for cloning and knock-out generation were purchased from New England Biolabs (NEB). High-fidelity Phusion

Chemicals, kits and culture medium components

Oligonucleotides used in this study were designed manually by examining the relevant DNA sequences. Oligonucleotides were commercially synthesised at MWG Biotech Pvt. Ltd., Bangalore, Indiaor Ocimum biosolutions, Hyderabad, India. Oligonucleotides used inthis study are listed in Table 2.4 and 2.5

Oligonucleotides

Antibodies used in this study are listed in Table 2.3

Antibodies

All S. cereviseae and bacterial strains and plasmids used in this study are listed in Table 2.1and 2.2

Strains and plasmids

Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad, India

Swarna Gowri Thota

Role of inositol pyrophosphates in yeast physiology

1. The composition of different types of gels run during the course of this study is

Media

StrainsThe

Centre for DNA Fingerprinting and Diagnostics Hyderabad 500001, India

Suhail Yousuf

Characterization and functional studies on FadR like proteins from M. tuberculosis

Luciferase assay was performed using luciferase assay systems (Promega #1500). Cells were transfected with shRNA. Following 24 hrs.of shRNA transfection, cells were transfected separately with FOP-Flash and TOP-Flashvectors in the absence and presence of Wnt3a plasmids. After 24 hrs, media was removed, and the cells were rinsed twice with 1X PBS. After removing the final wash, the cells were incubated with lysis buffer (1X lysis reagent: CCLR; 20μl/well for a96-wellplate, or 400 μl/60mm culture dish, or 900μl/100mm culture dish). Cells were collected in a microcentrifuge tube and were centrifuged at 14000 rpm for 10 minutes. The cell lysate (supernatant) was transferred to a new tube. 20μl of cell lysate was mixed with 100μl of Luciferase Assay Reagent(LAR), and the amount of light produced was measuredin luminometer by usinga delay time of 2 sec and a read time of 10 sec.

Luciferase reporter assay

44KDa to 670 KDa; Ovalbumin (44KDa), Conalbumin (75KDa), Aldolase (158KDa), Ferritin (440KDa), Thyroglobulin (669KDa)] purchased fromGE Healthcare Life Science. Blue dextran was used for the void volume of the column. The molecular weight of the protein complex fractions was calculated from the plot. The different fractions that were collected as eluates from the column were concentrated,and the presence of WWP2 or WWP1 as a monomer or multimer was identified by western blotting using specific antibodies for WWP2 and WWP1